ZA200607739B

ZA200607739B - Cellular permissivity factor for viruses, and uses thereof

Info

Publication number: ZA200607739B
Application number: ZA200607739A
Authority: ZA
Inventors: Calvert Jay Gregory; Shields Shelly Lynn; Slade David Ewell; Welch Siao-Kun Wan
Original assignee: Pharmacia & Upjohn Co Llc
Priority date: 2004-04-23
Filing date: 2006-09-15
Publication date: 2008-05-28
Also published as: TNSN06343A1; CN101031318B; CN101031318A

Description

Cellular Permissivity Factor for Viruses, and Uses Thereof

FIELD OF THE INVENTION

The present invention provides methods and compositions related to the generation of host cells permissive for virus growth for viruses of the family Asfarviridae and Arteriviridae.

BACKGROUND OF THE INVENTION

Asfarviridae

Asfarviridae is a family of icosohedral enveloped viruses whose genome consists of a single molecule of linear double-stranded DNA of about 150000-190000 nucleotides long. The name of the family is derived from African Swine Fever And Releted +

Viruses. African Swine Fever Virus (ASFV) is the type species of the Asfivirus genus ) and is the sole member of the family. Recently, porcine CD163 polypeptide has been surmised by implication to be the cellular receptor for African swine fever virus (ASFV) (Sanchez-Torres et al., 2003) Arteriviridae

Viruses of the family of Arteriviridae includes equine arteritis virus (EAV), lactate dehydrogenase-elevating virus (LDV) and simian hemorrhagic fever virus (SHEV). The Arterivirus having the greatest economic importance is Porcine

Reproductive and Respiratory Syndrome Virus (PRRSV).

PRRSV

Porcine Reproductive and Respiratory Syndrome (PRRS) is one of the most economically important diseases of swine. The syndrome appeared almost simultaneously in North American and in Western Europe in the late 1980s, and has since spread to become endemic in the major swine producing nations of Europe,

Asia, and the Americas. The etiologic agent of PRRS is a virus that has been designated PRRS virus or PRRSV. For both European and North American PRRS, the disease is characterized by reproductive failure in sows and gilts (late term abortions, still births, and mummies), high mortality among nursery pigs, and respiratory disease in swine of all ages. The disease has been the subject of recent reviews (Mengeling and Lager, 2000; Murtaugh et al., 2002; Nodelijk, 2002;

Plagemann, 2003).

In the pig, PRRSV infection is limited to a subset of cells of the monocyte/macrophage lineage. Fully differentiated porcine alveolar macrophage (PAM) cells are the primary target cells for viral replication (Duan et al., 1997a; Duan et al., 1997b). Immortalization of PAM cells is technically challenging, and when successful, has resulted in cell lines that are not permissive for PRRS virus growth (Weingartl et al., 2002). PRRS virions are specifically bound by macrophages and internalized in clathrin-coated pits by endocytosis. Release from endocytic vesicles requires acidic pH (Nauwynck et al., 1999). Initial binding of virions is mediated by interaction of the viral matrix protein with heparin sulfate glycosaminoglycans (Delputte et al., 2002). Internalization can be facilitated by a 210 or 220 kDa membrane glycoprotein, as incubation of PAM cells with monoclonal antibodies to this polypeptide block infection with PRRS virus (Duan et al., 1998; Wissink et al., 2003). The 210 kDa glycoprotein has recently been identified as sialoadhesin, a member of siglec family of sialic acid binding immunoglobulin-like lectins (Pensaert et al., 2003). Transfection of the non-permissive PK-15 (porcine kidney) cell line with porcine sialoadhesin conferred the ability to internalize PRRSV particles, but there remained an apparent block at the uncoating stage, as virions entered into cellular vesicles but did not undergo nucleocapsid disintegration and vesicle membrane fusion. Viral genes were not expressed, and the transfected PK-15 cells were not rendered permissive for the PRRS virus (Vanderheijden et al., 2003). To our knowledge, transfection with sialoadhesin has not been shown to be sufficient to convert any PRRSV non-permissive cell line to a PRRSV-permissive phenotype.

Apart from primary porcine cells of the monocyte/macrophage lineage, the only other cell type known to be permissive for the growth of PRRSV in cell culture is the immortalized monkey kidney cell line MA-104(Chladek et al., 1998) and derivatives such as MARC-145 (Kim et al., 1993) and CL-2621. It is not known why this one particular cell line is permissive, yet other mammalian cell lines are not. The

PRRS virus binds specifically to a number of different cell types, but does not initiate infection (Kreutz, 1998; Therrien et al., 2000). In MARC-145 cells, the internalization of the virus by endocytosis and subsequent uncoating in low pH vesicles seems to mimic similar events in PAM cells (Kreutz and Ackermann, 1996).

However, a number of monoclonal antibodies that bind to porcine sialoadhesin fail to detect a homologous protein on the surface of MARC-145 cells (Duan et al., 1998;

Wissink et al., 2003), suggesting that MARC-145 cells may use a divergent member of the same protein family or a different receptor altogether. _2-

Current PRRSV vaccines are propagated on simian cell lines, which is a potentially dangerous activity. The use of simian cell lines for vaccine production has the potential to introduce primate viruses of significance into swine lines intended for xenotransplant purposes. Because swine are being increasingly explored as a source of xenotransplanted organs for humans, the introduction of primate cell lines to swine populations may ultimately pose a risk to humans receiving xenotransplanted organs.

Thus, it would be prudent to avoid the use of simian cell lines in swine vaccine preparations. It would be therefore desirable to identify or generate non-simian cells or cell lines capable of supporting PRRSV replication. Towards this goal, it is essential to identify the gene product(s) which may be responsible for conferring the permissivity for PRRSV replication as seen in certain simian cells lines as well as

PAM cells. Once such gene products are identified, non-permissive cells might be rendered permissive by transfection of the essential gene into them, thereby affording a wider array of production lines for a vaccine.

One lab has reported that the tetraspanin protein CD151 from MARC-145 cells, when transfected into non-permissive BHK-21 cells, confers permissivity to the

PRRS virus (Kapil and Shanmukhappa, 2003; Shanmukhappa and Kapil, 2001). This observation has yet to be confirmed by an independent lab.

We describe here an unrelated polypeptide, which when transfected into non- permissive cells, confers permissivity to the PRRS virus.

References Cited

Chladek, D. W., Harris, L. L., and Gorcyca, D. E. Method of growing and attenuating a viral agent associated with mystery swine disease. Boehringer Ingelheim Animal

Health, Inc. 677,585[US 5,840,563], 1-24. 11-24-1998. USA. 7-9-1996.

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Arch. Virol. 145, 659-688.

Delputte,P.L., Vanderheijden,N., Nauwynck,H.J., and Pensaert, M.B. (2002).

Involvement of the matrix protein in attachment of porcine reproductive and respiratory syndrome virus to a heparinlike receptor on porcine alveolar macrophages.

J. Virol. 76, 4312-4320.

Duan, X., Nauwynck,H.I., and Pensaert,M.B. (1997a). Effects of origin and state of differentiation and activation of monocytes/macrophages on their susceptibility to porcine reproductive and respiratory syndrome virus (PRRSV). Arch. Virol. 142, 2483-2497. Duan,X., Nauwynck,H.J., and Pensaert, M.B. (1997b). Virus quantification and identification of cellular targets in the lungs and lymphoid tissues of pigs at different time intervals after inoculation with porcine reproductive and respiratory syndrome virus (PRRSV). Vet. Microbiol. 56, 9-19.

Duan, X.B., Nauwynck,H.J., Favoree, H.W, and Pensaert M.B. (1998). Identification of a putative receptor for porcine reproductive and respiratory syndrome virus on porcine alveolar macrophages. J. Virol. 72, 4520-4523.

Graversen,J.H., Madsen, M., and Moestrup,S.K. (2002). CD163: a signal receptor scavenging haptoglobin-hemoglobin complexes from plasma. [Review] [19 refs].

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Kapil, S. and Shanmukhappa, K. Host susceptibility factor(s) for porcine reproductive and respiratory syndrome virus and uses in swine breeding, as a target for antiviral compounds, and development of a non-simian recombinant cell line for propagation of the virus. none. US 2003/0186236 Al, 1-45. 10-2-2003. USA. 1-28-2002.

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Kim,H.S., Kwang,J., Yoon,LJ., Joo,H.S., and Frey M.L. (1993). Enhanced replication of porcine reproductive and respiratory syndrome (PRRS) virus in a homogenous subpopulation of MA-104 cell line. Arch. Virol. 133, 477-483.

Kreutz, L.C. (1998). Cellular membrane factors are the major determinants of porcine reproductive and respiratory syndrome virus tropism. Virus Res. 53, 121-128.

Kreutz,L.C. and Ackermann,M.R. (1996). Porcine reproductive and respiratory syndrome virus enters cells through a low pH-dependent endocytic pathway. Virus

Res. 42, 137-147.

Mengeling, W.L. and Lager,K.M. (2000). A brief review of procedures and potential problems associated with the diagnosis of porcine reproductive and respiratory syndrome. Veterinary Research 31, 61-69. Meulenberg,J.J.M. (2000). PRRSV, the virus. Veterinary Research 31, 11-21.

Murtaugh,M.P., Xia0,Z.G., and Zuckermann,F. (2002). Immunological responses of swine to porcine reproductive and respiratory syndrome virus infection [Review].

Viral Immunology 75, 533-547.

Nauwynck,H.J., Duan X., Favoreel, H.W., Van Oostveldt,P., and Pensaert, M.B. 40 (1999). Entry of porcine reproductive and respiratory syndrome virus into porcine alveolar macrophages via receptor-mediated endocytosis. J. Gen. Virol. 80, 297-305.

Nodelijk,G. (2002). Porcine Reproductive and Respiratory Syndrome (PRRS) with special reference to clinical aspects and diagnosis - A review [Review]. Vet. Quart. 24, 95-100.

Pensaert, M., Nauwynck, H., and Vanderheijden, N. Nucleic acid encoding polypeptide involved in cellular entrance of the PRRS virus. Akzo Nobel N.V. and

Universiteit Gent. WO 03/010200 A2, 1-24. 2-6-2003. 7-18-2002.

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Philippidis,P., Mason,J.C., Evans, B.J., Nadra,L, Taylor K.M., Haskard,D.O., and

Landis,R.C. (2004). Hemoglobin scavenger receptor CD163 mediates interleukin-10 release and heme oxygenase-1 synthesis - Antiinflammatory monocyte-macrophage responses in vitro, in resolving skin blisters in vivo, and after cardiopulmonary bypass surgery. Circulation Research 94, 119-126.

Plagemann,P.G.W, (2003). Porcine reproductive and respiratory syndrome virus:

Origin hypothesis. Emerging Infectious Diseases 9, 903-908. Ritter,M., Buechler,C., Langmann,T., and Schmitz,G. (1999). Genomic organization and chromosomal localization of the human CD163 (M130) gene: a member of the scavenger receptor cysteine-rich superfamily. Biochemical & Biophysical Research

Communications 260, 466-474.

Sanchez-Torres,C., Gomez-Puertas,P., Gomez-del-Moral,M., Alonso,F., Escribano,J.M., Ezquerra,A., and Dominguez,J. (2003). Expression of porcine CD163 on monocytes/macrophages correlates with permissiveness to African swine fever infection. Arch. Virol. 148, 2307-2323.

Shanmukhappa,K. and Kapil, S. (2001). Cloning and identification of MARC-145 cell proteins binding to 3'UTR and partial nucleoprotein gene of porcine reproductive and respiratory syndrome virus. Adv. Exp. Med. Biol. 494, 641-646. Snijder,E.J. and Meulenberg,J.J.M. (2001). Arteriviruses. In Fields Virology,

D.M.Knipe, P.M.Howley, D.E.Griffin, M.A Martin, R.A.Lamb, B.Roizman, and

S.E.Straus, eds. (Philadelphia: Lippincott Williams & Wilkins), pp. 1205-1220.

Therrien,D., St Pierre,Y., and Dea,S. (2000). Preliminary characterization of protein binding factor for porcine reproductive and respiratory syndrome virus on the surface of permissive and non-permissive cells. Arch. Virol. 145, 1099-1116.

Vanderheijden,N., Delputte,P.L., Favoreel H.W., Vandekerckhove,J., Van Damme, ]., van Woensel,P.A., and Nauwynck,H.J, (2003). Involvement of sialoadhesin in entry of porcine reproductive and respiratory syndrome virus into porcine alveolar macrophages. J. Virol. 77, 8207-8215.

Weingart] H.M., Sabara,M., Pasick,J., van Moorlehem,E., and Babiuk,L. (2002).

Continuous porcine cell lines developed from alveolar macrophages - Partial characterization and virus susceptibility. J. Virol. Methods 104, 203-216.

Wissink,E.H.J., van Wijk,H.A.R., Pol] M.A., Godeke,G.J., van Rijn,P.A., 40 Rottier,P.J.M., and Meulenberg,J.J.M. (2003). Identification of porcine alveolar macrophage glycoproteins involved in infection of porcine respiratory and reproductive syndrome virus. Arch. Virol. 148, 177-187.

SUMMARY OF THE INVENTION

The invention includes a method of facilitating infection of one or more cells by a virus that is selected from the group consisting of Arteriviridae and Asfarviridae, which comprises the step of directing increased expression of a CD163 polypeptide within said cell. In a preferred embodiment the CD163 is membrane bound. In one embodiment the virus is selected from the group consisting of Arteriviridae. In a preferred embodiment the virus is PRRSV. In another embodiment said virus is equine arteritis virus (EAV). In yet another embodiment said virus is African Swine

Fever virus (ASFV).

Increased expression of a CD163 polypeptide may be accomplished by methods such as introduction of exogenous nucleic acids encoding CD163 polypeptides such methods include but are not limited to transfection, electroporation and fusion with a carrier of a polynucleotide comprising a polynucleotide encoding a

CD163 polypeptide. Increased expression may also be accomplished by induction of expression of endogenous CD163 by chemical treatment.

The method may render previously non PRRSV-permissive cells PRRS permissive . The method may also include rendering one or more cells that previously did not express a CD163 polypeptide into cells that are induced to express a CD 163 polypeptide.

The cells in a preferred embodiment are animal cells. They may be vertebrate or invertebrate cells. The cells may be mammalian. The cells or cell line may be an insect cell line. The cells may be BHK21 cells. The cells may be derived from porcine kidney cells. The cells or cell line may be derived from feline kidney cells.

The cells or cell line may be but are not limited to BHK-21, NLST-1, NLFK-1, Vero or RL cells. The PRRSV may be or the European.or North American genotype.

As noted above, increased expression of a CD163 polypeptide may be accomplished by methods which include but are not limited to: transfection, electroporation and fusion with a carrier of a polynucleotide comprising a polynucleotide encoding a CD163 polypeptide. Any CD163 polypeptides are contemplated. Those containing a transmembrane region are preferred. Exemplary

CD163 polypeptides are selected from the group consisting of the polynucleotides listed below.

One such polynucleotide comprises a polynucleotide encoding a polypeptide having at least 70% 71%, 72%, 73%, 74%, 75%, 76%, 11%, 18%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 93%, 94%, 95%, 96% 97% 98%, 99%, identity with SEQ ID NO: 2.

One such polynucleotide comprises a polynucleotide encoding a polypeptide differing from SEQ ID NO: 2 by no more than 20 conservative amino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding a polypeptide differing from SEQ ID NO: 2 by no more than 10 conservative amino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding a polypeptide comprising SEQ ID NO: 2. .

One such polynucleotide comprises a polynucleotide with the sequence set forth in SEQ ID NO:1

One such polynucleotide comprises a polynucleotide encoding a polypeptide that has at least 99% identity to a polypeptide set forth in SEQ ID NO: 14

One such polynucleotide comprises a polynucleotide encoding a polypeptide differing from SEQ ID NO: 14 by no more than 15 conservative amino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding a polypeptide differing from SEQ ID NO: 14 by no more than 10 conservative amino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding a polypeptide comprising SEQ ID NO: 14.

One such polynucleotide comprises a polynucleotide with the sequence set forth in SEQ ID NO: 13.

One such polynucleotide comprises a polynucleotide encoding a polypeptide differing from SEQ ID NO: 24 by no more than 2 conservative amino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding a polypeptide comprising SEQ ID NO: 24.

One such polynucleotide comprises a polynucleotide with the sequence set forth in SEQ ID NO: 23.

One such polynucleotide comprises a polynucleotide encoding a polypeptide having at least 96% 97% 98%, or 99%, identity to a polypeptide set forth in SEQ ID

NO: 27.

One such polynucleotide comprises a polynucleotide encoding a polypeptide differing from SEQ ID NO: 27 by no more than 20 conservative amino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding a polypeptide differing from SEQ ID NO: 27 by no more than 10 conservative amino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding a polypeptide comprising SEQ ID NO: 27.

One such polynucleotide comprises a polynucleotide with the sequence set forth in SEQ ID NO: 26. : One such polynucleotide comprises a polynucleotide encoding a polypeptide that has at least 98%, 99% identity to a polypeptide set forth in SEQ ID NO: 32

One such polynucleotide comprises a polynucleotide encoding a polypeptide differing from SEQ ID NO: 32 by no more than 15 conservative amino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding a polypeptide . differing from SEQ ID NO: 32 by no more than 10 conservative amino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding a polypeptide comprising SEQ ID NO: 32.

One such polynucleotide comprises a polynucleotide with the sequence set forth in SEQ ID NO: 31.

One such polynucleotide comprises a polynucleotide encoding a polypeptide that has at least 95%, 96%, 97%, 98%, 99% identity to a polypeptide set forth in SEQ

IDNO:34

One such polynucleotide comprises a polynucleotide encoding a polypeptide differing from SEQ ID NO: 34 by no more than 15 conservative amino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding a polypeptide differing from SEQ ID NO: 34 by no more than 10 conservative amino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding a polypeptide comprising SEQ ID NO: 34.

One such polynucleotide comprises a polynucleotide with the sequence set forth in SEQ ID NO: 33.

IDNO: 36

One such polynucleotide comprises a polynucleotide encoding a polypeptide differing from SEQ ID NO: 36 by no more than 15 conservative amino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding a polypeptide differing from SEQ ID NO: 36 by no more than 10 conservative amino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding a polypeptide comprising SEQ ID NO: 36.

One such polynucleotide comprises a polynucleotide with the sequence set forth in SEQ ID NO: 35.

One such polynucleotide comprises a polynucleotide encoding a polypeptide that has at least 90%. 91%, 92%, 93%, 94%, 95%. 96%, 97%, 98%, 99% identity to a polypeptide set forth in SEQ ID NO: 42

One such polynucleotide comprises a polynucleotide encoding a polypeptide differing from SEQ ID NO: 42 by no more than 15 conservative amino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding a polypeptide differing from SEQ ID NO: 42 by no more than 10 conservative amino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding a polypeptide comprising SEQ ID NO: 42.

One such polynucleotide comprises a polynucleotide with the sequence set forth in SEQ ID NO: 41.

One such polynucleotide comprises a polynucleotide encoding a polypeptide that has at least 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to a polypeptide set forth in SEQ ID NO: 44

One such polynucleotide comprises a polynucleotide encoding a polypeptide differing from SEQ ID NO: 44 by no more than 15 conservative amino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding a polypeptide differing from SEQ ID NO: 44 by no more than 10 conservative amino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding a polypeptide comprising SEQ ID NO: 44.

One such polynucleotide comprises a polynucleotide with the sequence set forth in SEQ ID NO: 43

ID NO: 46

One such polynucleotide comprises a polynucleotide encoding a polypeptide differing from SEQ ID NO: 46 by no more than 15 conservative amino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding a polypeptide differing from SEQ ID NO: 46 by no more than 10 conservative amino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding a polypeptide comprising SEQ ID NO: 46.

One such polynucleotide comprises a polynucleotide with the sequence set forth in SEQ ID NO: 45.

One such polynucleotide comprises a polynucleotide encoding a polypeptide that has at least 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 %identity to a polypeptide set forth in SEQ ID NO: 48

One such polynucleotide comprises a polynucleotide encoding a polypeptide differing from SEQ ID NO: 48 by no more than 15 conservative amino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding a polypeptide differing from SEQ ID NO: 48 by no more than 10 conservative amino acid substitutions.

One such polynucleotide comprises a polynucleotide encoding a polypeptide comprising SEQ ID NO: 48.

One such polynucleotide comprises a polynucleotide with the sequence set forth in SEQ ID NO: 47.

The method of facilitating infection described above may further comprise the step of producing a culture of virus.

The invention further comprises the culture isolated by the method described above.

Any of the methods described above may further comprise the step of producing a PRRS or other viral vaccine. The vaccine may be killed.or live attenuated.

The invention also comprises a cell or cell line wherein the ability of one or more cells to be infected by a virus selected from the group consisting of Arteriviridae and Asfarviridae has been modified by directing increased expression of a CD163 polypeptide within said cells.

In a preferred embodiment the CD163 polypeptide comprises a transmembrane region. In one embodiment the virus is selected from the group consisting of

Arteriviridae. In a preferred embodiment the virus is PRRSV. In another embodiment said virus is equine arteritis virus (EAV). In yet another embodiment said virus is

African Swine Fever virus (ASFV).

The cell or cell line of the invention may have been previously PRRSV non- permissive and is rendered PRRSV permissive by directing increased expression of a

CD163 polypeptide within said cell or cell line.

The cell or cell line of the invention includes cells or cell lines that did not express a CD163 polypeptide and is induced to express a CD 163 polypeptide.

The cells in a preferred embodiment are animal cells. They may be vertebrate orinvertebrate cells. The cells may be mammalian. The cell or cell line may be an insect cell or cell line. The cells may be BHK21 cells. The cells may be derived from porcine kidney cells. The cell or cell line may be derived from feline kidney cells.

The cells may be, but are not limited to, BHK-21, NLST-1, NLFK-1, Vero or RL cells. The PRRSV may be North American or European.

The invention includes a method for measuring the propensity of a test cell or cell line to allow infection by a virus selected from the group consisting of Arteriviridae and Asfarviridae comprising: a) providing a sample containing nucleic acids from the test cell or cell line; b) determining the amount of polynucleotide encoding a CD163 polypeptide or its complement in said sample; wherein an increased amount of polynucleotide encoding a CD163 polypeptide relative to a control sample derived from a control cell or cell line known not to support the growth of said virus indicates a propensity of the test cell or cell line to support the replication of said virus.

In one embodiment the virus is selected from the group consisting of

Arteriviridae. In a preferred embodiment the virus is PRRSV.In another embodiment said virus is equine arteritis virus (EAV). In yet another embodiment said virus is

African Swine Fever virus (ASFV).

The amount of polynucleotide encoding a CD163 polypeptide may be determined by hybridization,

The amount of polynucleotide encoding a CD163 polypeptide may be determined by PCR.

The invention also includes a method for measuring the propensity of a test cell or cell line to allow infection by a virus selected from the group consisting of

Arteriviridae and Asfarviridae comprising: (a) providing a sample containing polypeptides from the test cell or cell line; (b) determining the amount of CD163 polypeptide in said sample; wherein an increased amount of a CD 163 polypeptide relative to a control sample derived from a control cell or cell line known not to support the growth of said virus indicates a propensity of the test cell or cell line to support the replication of said virus.

In one embodiment the virus is selected from the group consisting of

African Swine Fever virus (ASFV).

In one embodiment the deterermining is accomplished by contacting a CD163 polypeptide with an antibody specific for the CD163 polypeptide, under conditions wherein the antibody binds the CD163 polypeptide.

The invention includes a method for measuring the propensity of a pig to be infected by a virus selected from the group consisting of Arteriviridae and

Asfarviridae comprising: a) providing a sample containing nucleic acids from the pig to be tested; b) determining the amount of polynucleotide encoding a CD163 polypeptide or its complement in said sample; wherein an increased amount of polynucleotide encoding a CD163 polypeptide relative to a control sample derived from a pig known to be resistant to said virus infection indicates a propensity of the pig to be tested to be infected by said virus.

In one embodiment the virus is selected from the group consisting of

African Swine Fever virus (ASFV).

In one embodiment the determining is accomplished by hybridization. In another embodiment the determining is accomplished by PCR.

The invention also includes a method for measuring the propensity of a pig to be infected by a virus selected from the group consisting of Arteriviridae and

Asfarviridae comprising: (a) providing a sample containing polypeptides from pig to be tested; (b) determining the amount of CD163 polypeptide in said sample; wherein an increased amount of a CD163 polypeptide relative to a control sample derived from a pig known to be resistant to said virus infection indicates a propensity of the pig to be tested to be infected by said virus.

In one embodiment the virus is selected from the group consisting of

Arteriviridae. In a preferred embodiment the virus is PRRSV. In another embodiment said virus is equine arteritis virus (EAV). In yet another embodiment said virus is African Swine Fever virus (ASFV).

‘The invention also includes an isolated polypeptide wherein the polypeptide is selected from the group consisting of the polypeptides described below.

Therefore the invention also includes an isolated polypeptide having at least 70% 71%, 712%, 13%, 14%, 15%, 716%, 11%, 18%, 19%, 80%, 81%, 82%, 83%, s 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 93%, 94%, 95%, 96% 97% 98%, 99%, identity with SEQ ID NO: 2.

One such polypeptide is a polypeptide differing from SEQ ID NO: 2 by no more than 20 conservative amino acid substitutions.

One such polypeptide is a polypeptide differing from SEQ ID NO: 2 by no more than 10 conservative amino acid substitutions.

One such polypeptide comprises SEQ ID NO: 2.

Therefore the invention also includes an isolated polypeptide having at least 99%, identity to a polypeptide set forth in SEQ ID NO: 14

One such polypeptide is a polypeptide differing from SEQ ID NO: 14 by no more than 15 conservative amino acid substitutions.

One such polypeptide is a polypeptide differing from SEQ ID NO: 14 by no more than 10 conservative amino acid substitutions.

One such polypeptide comprises SEQ ID NO: 14.

Therefore the invention also includes an isolated polypeptide differing from

SEQ ID NO: 24 by no more than 2 conservative amino acid substitutions.

One such polypeptide comprises SEQ ID NO: 24.

Therefore the invention also includes an isolated polypeptide having at least 96% 97% 98%, or 99%, identity to a polypeptide set forth in SEQ ID NO: 27.

One such polypeptide is a polypeptide differing from SEQ ID NO: 27 by no more than 20 conservative amino acid substitutions.

One such polypeptide is a polypeptide differing from SEQ ID NO: 27 by no more than 10 conservative amino acid substitutions.

One such polypeptide is a polypeptide comprising SEQ ID NO: 27.

Therefore the invention also includes an isolated polypeptide having at least 98%, 99% identity to a polypeptide set forth in SEQ ID NO: 32

One such polypeptide is a polypeptide differing from SEQ ID NO: 32 by no more than 15 conservative amino acid substitutions.

One such polypeptide is a polypeptide differing from SEQ ID NO: 32 by no more than 10 conservative amino acid substitutions.

One such polypeptide is a polypeptide comprising SEQ ID NO: 32.

Therefore the invention also includes an isolated polypeptide having at least 95%, 96%, 97%, 98%, 99% identity to a polypeptide set forth in SEQ ID NO: 34

One such polypeptide is a polypeptide differing from SEQ ID NO: 34 by no more than 15 conservative amino acid substitutions.

One such polypeptide is a polypeptide differing from SEQ ID NO: 34 by no more than 10 conservative amino acid substitutions.

One such polypeptide is a polypeptide comprising SEQ ID NO: 34,

Therefore the invention also includes an isolated polypeptide having at least 95%, 96%, 97%, 98%, 99% identity to a polypeptide set forth in SEQ ID NO: 36

One such polypeptide is a polypeptide differing from SEQ ID NO: 36 by no more than 15 conservative amino acid substitutions.

One such polypeptide is a polypeptide differing from SEQ ID NO: 36 by no more than 10 conservative amino acid substitutions.

One such polypeptide is a polypeptide comprising SEQ ID NO: 36.

The invention also includes an isolated polypeptide having at least 95%, 96%, 97%, 98%, 99% identity to a polypeptide set forth in SEQ ID NO: 38

One such polypeptide is a polypeptide differing from SEQ ID NO: 38 by no more than 15 conservative amino acid substitutions.

One such polypeptide is a polypeptide differing from SEQ ID NO: 38 by no more than 10 conservative amino acid substitutions.

One such polypeptide is a polypeptide comprising SEQ ID NO: 40

Therefore the invention also includes an isolated polypeptide having at least 95%, 96%, 97%, 98%, 99% identity to a polypeptide set forth in SEQ ID NO: 40

One such polypeptide is a polypeptide differing from SEQ ID NO: 40 by no more than 15 conservative amino acid substitutions.

One such polypeptide is a polypeptide differing from SEQ ID NO: 40 by no more than 10 conservative amino acid substitutions.

One such polypeptide is a polypeptide comprising SEQ ID NO: 40.

. Therefore the invention also includes an isolated polypeptide having at 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to a polypeptide set forth in

SEQ ID NO: 42

One such polypeptide is a polypeptide differing from SEQ ID NO: 42 by no more than 15 conservative amino acid substitutions.

One such polypeptide is a polypeptide differing from SEQ ID NO: 42 by no more than 10 conservative amino acid substitutions.

One such polypeptide is a polypeptide comprising SEQ ID NO: 42.

Therefore the invention also includes an isolated polypeptide having at least 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to a polypeptide set forth in SEQ ID NO: 44

One such polypeptide is a polypeptide differing from SEQ ID NO: 44 by no more than 15 conservative amino acid substitutions.

One such polypeptide is a polypeptide differing from SEQ ID NO: 44 by no more than 10 conservative amino acid substitutions.

One such polypeptide is a polypeptide comprising SEQ ID NO: 44.

Therefore the invention also includes an isolated polypeptide having at least 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to a polypeptide set forth in SEQ ID NO: 46

One such polypeptide is a polypeptide differing from SEQ ID NO: 46 by no more than 15 conservative amino acid substitutions.

One such polypeptide is a polypeptide differing from SEQ ID NO: 46 by no more than 10 conservative amino acid substitutions.

One such polypeptide is a polypeptide comprising SEQ ID NO: 46.

Therefore the invention also includes an isolated polypeptide having at least 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to a polypeptide set forth in SEQ ID NO: 48

One such polypeptide is a polypeptide differing from SEQ ID NO: 48 by no more than 15 conservative amino acid substitutions.

One such polypeptide is a polypeptide differing from SEQ ID NO: 48 by no more than 10 conservative amino acid substitutions.

The invention also includes an isolated CD 163 polynucleotide wherein said polynucleotide is selected from the group consisting of the polynucleotides enumberated below.

Therefore the invention also includes an isolated polynucleotide comprising: (a) a polynucleotide sequence set forth in SEQ ID NOs: 1 or § (b) a polynucleotide that encodes a polypeptide that has at least 70%, 71%, 72%, 73%, 74%, 15%, 16%, 11%, 18%, 19%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 93%, 94%, 95%, 96% 97% 98%, or 99%, identity and or similarity to a polypeptide set forth in SEQ ID NO: 2 (c) a polynucleotide encoding a polypeptide of SEQ ID: 2, (d) a polynucleotide which is the complement of any of (a), (b) or (c).

Therefore the invention also includes an isolated polynucleotide comprising: (a) a polynucleotide sequence set forth in SEQ ID NOs: 12 or 13 (b) a polynucleotide that encodes a polypeptide that has at least 99%, identity and or similarity to a polypeptide set forth in SEQ ID NO: 14 (c) a polynucleotide encoding a polypeptide of SEQ ID: 14, (d) a polynucleotide which is the complement of any of (a), (b) or (c).

Therefore the invention also includes an isolated polynucleotide comprising: (a) a polynucleotide sequence set forth in SEQ ID NOs: 22 or 23 (b) a polynucleotide encoding a polypeptide of SEQ ID: 24, (c) a polynucleotide which is the complement of any of (a) or (b).

Therefore the invention also includes an isolated polynucleotide comprising: (a) a polynucleotide sequence set forth in SEQ ID NOs: 25 or 26 (b) a polynucleotide that encodes a polypeptide that has at least 96% 97% 98%, or 99%, identity and or similarity to a polypeptide set forth in SEQ ID NO: 27 (c) a polynucleotide encoding a polypeptide of SEQ ID: 27, (d) a polynucleotide which is the complement of any of (a), (b) or (c).

Therefore the invention also includes an isolated polynucleotide comprising: (a) polynucleotide sequence set forth in SEQ ID NOs: 30 or 31 (b) a polynucleotide that encodes a polypeptide that has at least 98% or 99%,, identity and or similarity to a polypeptide set forth in SEQ ID NO: 32 (c) a polynucleotide encoding a polypeptide of SEQ ID: 32, (d) a polynucleotide which is the complement of any of (a), (b) or (c).

Therefore the invention also includes an isolated polynucleotide comprising: (a) polynucleotide sequence set forth in SEQ ID NOs: 33 (b) a polynucleotide that encodes a polypeptide that has at least 95% 96%, 97%, 98% or 99%,, identity and or similarity to a polypeptide set forth in SEQ ID NO: 34 (c) a polynucleotide encoding a polypeptide of SEQ ID: 34, (d) a polynucleotide which is the complement of any of (a), (b) or (c).

Therefore the invention also includes an isolated polynucleotide comprising: (a) polynucleotide sequence set forth in SEQ ID NOs: 35 (b) a polynucleotide that encodes a polypeptide that has at least 95% 96%, 97%, 98% or 99%,, identity and or similarity to a polypeptide set forth in SEQ ID NO: 36 (c) a polynucleotide encoding a polypeptide of SEQ ID: 36, (d) a polynucleotide which is the complement of any of (a), (b) or (c).

Therefore the invention also includes an isolated polynucleotide comprising: (a) polynucleotide sequence set forth in SEQ ID NOs: 37 (b) a polynucleotide that encodes a polypeptide that has at least 95% 96%, 97%, 98% or 99%.,, identity and or similarity to a polypeptide set forth in SEQ ID NO: 38 (c) a polynucleotide encoding a polypeptide of SEQ ID: 38, (d) a polynucleotide which is the complement of any of (a), (b) or (c).

Therefore the invention also includes an isolated polynucleotide comprising: (a) polynucleotide sequence set forth in SEQ ID NOs: 39 (b) a polynucleotide that encodes a polypeptide that has at least 95% 96%, 97%, 98% or 99%,, identity and or similarity to a polypeptide set forth in SEQ ID NO: 40 (c) a polynucleotide encoding a polypeptide of SEQ ID: 40, (d) a polynucleotide which is the complement of any of (a), (b) or (c).

Therefore the invention also includes an isolated polynucleotide comprising: (a) polynucleotide sequence set forth in SEQ ID NOs: 41 (b) a polynucleotide that encodes a polypeptide that has at least 95% 96%, 97%, 98% or 99%,, identity and or similarity to a polypeptide set forth in SEQ ID NO: 42 (c) a polynucleotide encoding a polypeptide of SEQ ID: 42, (d) a polynucleotide which is the complement of any of (a), (b) or (c).

Therefore the invention also includes an isolated polynucleotide comprising: (a) polynucleotide sequence set forth in SEQ ID NOs: 43

(b) a polynucleotide that encodes a polypeptide that has at least 95% 96%, 97%, 98% or 99%,, identity and or similarity to a polypeptide set forth in SEQ ID NO: 44 (c) a polynucleotide encoding a polypeptide of SEQ ID: 44, (d) a polynucleotide which is the complement of any of (a), (b) or (c).

Therefore the invention also includes an isolated polynucleotide comprising: (a) polynucleotide sequence set forth in SEQ ID NOs: 45 (b) a polynucleotide that encodes a polypeptide that has at least 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98% or 99%, identity and or similarity to a polypeptide set forth in SEQ ID NO: 46 (c) a polynucleotide encoding a polypeptide of SEQ ID: 46, (d) a polynucleotide which is the complement of any of (a), (b) or (c).

The invention also includes an isolated polynucleotide comprising: (a) polynucleotide sequence set forth in SEQ ID NOs: 47 (b) a polynucleotide that encodes a polypeptide that has at least 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98% or 99%,, identity and or similarity to a polypeptide set forth in SEQ ID NO: 48 (¢) a polynucleotide encoding a polypeptide of SEQ ID: 49, (d) a polynucleotide which is the complement of any of (a), (b) or (c).

Therefore the invention also includes a CD163 polypeptide in which the transmembrane region is deleted.

Therefore the invention also includes a polynucleotide encoding a CD163 polypeptide in which the transmembrane region is deleted.

In addition to the foregoing, the invention includes, as an additional aspect, all embodiments of the invention narrower in scope in any way than the variations specifically mentioned above.

Brief Description of the Sequence Listings

SEQ ID NO: 1—DNA sequence encoding porcine susCD163v1

SEQ ID NO: 2—predicted amino acid sequence of porcine susCD163v1

SEQ ID NO: 3-- cDNA sequence, Genbank accession number AJ311716

SEQ ID NO:4---predicted amino acid sequence derived from Genbank accession number AJ311716

SEQ ID NO: 5—cDNA sequence of susCD163v1 containing flanking (non-coding) sequence.

SEQ ID NO: 6-11---primer sequences

SEQ ID NO:12 ---cDNA sequence encoding porcine susCD163v2 containing flanking (non-coding) sequence.

SEQ ID NO:13 --cDNA sequence encoding porcine susCD163v2

SEQ ID NO:14 predicted amino acid sequence of porcine susCD163v2

SEQ ID NO:15-16—--primer sequences

SEQ ID NO:17---cDNA sequence encoding human CD163v2 containing flanking (non-coding) sequence.

SEQ ID NO:18--- cDNA sequence encoding human CD163v2

SEQ ID NO:19--- predicted amino acid sequence of human CD163v2

SEQ ID NO:20-21---primer sequences

SEQ ID NO:22--cDNA sequence encoding murine CD163v2 containing flanking (non-coding) sequence.

SEQ ID NO:23-— cDNA sequence encoding murine CD163v2

SEQ ID NO:24--- predicted amino acid sequence of murine CD163v2

SEQ ID NO:25-~cDNA sequence encoding murine CD163v3 containing flanking (non-coding) sequence.

SEQ ID NO:26--- cDNA sequence encoding murine CD163v3

SEQ ID NO:27--- predicted amino acid sequence of murine CD163v3

SEQ ID NO:28-29---primer sequences

SEQ ID NO:30---cDNA sequence encoding MARC-145 CD163v3 containing flanking (non-coding) sequence.

SEQ ID NO:31--- cDNA sequence encoding MARC-145 CD163v3

SEQ ID NO:32--- predicted amino acid sequence of MARC-145 CD163v3

SEQ ID NO:33-cDNA sequence encoding Vero cell CD163v2 transcript

SEQ ID NO:34-- predicted amino acid sequence of Vero cell CD163v2

SEQ ID NO:35-- cDNA sequence encoding Vero cell CD163v3 transcript

SEQ ID NO:36-- predicted amino acid sequence of Vero cell CD163v3

SEQ ID NO:37-- cDNA sequence encoding Vero cell CD163v4 transcript

SEQ ID NO:38-- predicted amino acid sequence of Vero cell CD163v4

SEQ ID NO:39-- cDNA sequence encoding Vero cell CD163v5 transcript

SEQ ID NO:40-- predicted amino acid sequence of Vero cell CD163v5

SEQ ID NO:41-- cDNA sequence encoding Vero cell CD163v6 transcript

SEQ ID NO:42-- predicted amino acid sequence of Vero cell CD163v6

SEQ ID NO:43-- cDNA sequence encoding Vero cell CD163v7 transcript

SEQ ID NO:44-- predicted amino acid sequence of Vero cell CD163v7

SEQ ID NO:45-- cDNA sequence encoding canine CD163v2 transcript.

SEQ ID NO:46-- predicted amino acid sequence of canine CD163v2

SEQ ID NO:47-- cDNA sequence encoding canine CD163v3 transcript.

SEQ ID NO:48-- predicted amino acid sequence of canine CD163v3

Brief Description of the Drawings

Figure 1 Schematic comparison of susCD163v1 with AJ311716

Figure 2 Amino acid Sequence alignment of susCD163v1l (SEQ ID NO: 2) with

AJ311716 (SEQ ID NO: 4)

Figure 3 Nucleotide Sequence alignment of susCD163v1 with AJ311716

Figure 4 Generation of DNA fragments and ligation to place CD163 directly behind the RSV promoter. Plasmids were digested with either Dralll or Drdl, followed by a blunting reaction with Klenow enzyme. After clean up, the plasmids were digested with Notl. Gel purification yielded DNA fragments that were subsequently ligated utilizing the cohesive NofI termini. Promoters from RSV (pRSV) and SV40 (pSV40) are indicated with arrows.

Figure 5 Map of pCDNA3.1 Directional V5/His/TOPO cloning vector

Figure 6 Three BHK/CMV/v1 cell lines, #3, #5, and #12 and a non-permissive BHK cell line were infected with PRRSV isolate P129 and stained with SDOW17-FITC.

Panel A shows a non-permissive BHK21 cell clone. Panel B shows BHK/CMV/v1 clone #3. Panel C shows BHK/CMV/v1 clone #5. Panel D shows BHK/CMV/v1l clone #12.

Figure 7 Three BHK/RSV/v1 cell lines, #2, #3, and #4 were infected with PRRSV isolate P129 and stained with SDOW 17-FITC. Panel A shows BHK/RSV/v1 clone #2. Panel B shows BHK/RSV/v1 clone #3. Panel C shows BHK/RSV/v1 clone #4.

Figure 8 Feline kidney cell lines stably expressing porcine CD163v1, showing

PRRSYV plaques. Cell lines NLFK-CMV-susCD163v1-G4F and NLFK-CMV- susCD163v1-G4L, both at passage 4, were infected with the P129 isolate of North

American PRRSYV and incubated for 6 days. Monolayers were fixed with 80% acetone and stained with monoclonal antibody SDOW17-FITC. Phase contrast microscopy (right) shows localized regions of viral CPE (plaques), while FA detection (left) shows co-localized viral nucleocapsid antigen.

Figure 9 Four FK/RSV/v1 cell lines, #1, #2, #3, and #4 were infected with PRRSV isolate P129 and stained with monoclonal antibody SDOW 17-FITC. Panel A shows

FK/RSVvl #1 cell clone. Panel B shows FK/RSV/v1 clone #2. Panel C shows

FK/RSV/v1 clone #3. Panel D shows FK/RSV/v1 #4.

Figure 10 PK-CMV-susCD163v1-A10 cells at passage 19, infected with PRRSV isolate P129. Left: The monolayer was fixed with 80% acetone and stained with

FITC-conjugated monoclonal antibody SDOW17 (Rural Technologies Inc), which is specific for PRRSV nucleocapsid. Right: The same well under bright field illumination, showing cell distribution.

Figure 11 BHK-CMVScript-susCD163v2-A9 at passage 17 infected with PRRSV isolate P129. The monolayer was fixed with 80% acetone and stained with FITC- conjugated monoclonal antibody SDOW 17 (Rural Technologies Inc), which is specific for PRRSV nucleocapsid.

Figure 12 Three representative examples of the BHK/RSV/v2 cell lines. The cells were infected with PRRSV isolate P129 and subsequently stained with SDOW17-

FITC. Panel A shows cell line BHK/RSV/v2 #1, panel B shows cell line

BHK/RSV/v2 #34, and panel C shows cell line BHK/RSV/v2 #47.

Figure 13 FK-cDNA3.1D-humCD163v2-A6 at passage 15 infected with PRRSV isolate P129. The monolayer was then fixed with 80% acetone and stained with FITC- conjugated monoclonal antibody SDOW17 (Rural Technologies Inc), which is specific for PRRSV nucleocapsid.

Figure 14 The amount of progeny PRRSV produced by four recombinant cell lines stably expressing susCD163v1, and by MARC-145 cells, was determined in a growth curve experiment using the NVSL 94-3 isolate of PRRSV. Samples harvested at 12- hour intervals were titrated on MARC-145 cell monolayers.

Figure 15 Flow cytometry analysis of PRRSV infection in the presence of CD163 specific antibody. BHK-21 cells expressing MARC-145 CD163 from transient transfection were incubated with either CD163 specific antibody or normal goat IgG (NGS) and infected with a GFP-expressing PRRSV. Each data point represents the results of triplicate wells.

Figure 16 Flow cytometry analysis of PRRSV infection in the presence of CD163 specific antibody. NLFK cells stably expressing human CD163 were incubated with either CD163 specific antibody or normal goat IgG (NGS) and infected with a GFP expressing PRRSV. At 24 hours post infection the percentage of GFP expressing infected cells was determined. Each data point represents the result from a single well of cells

Figure 17. Graphical depiction of six altemative splicing variants of CD163 mRNA recovered from Vero cells. The six variants differ in the presence or absence of three exons, designated E6, E105, and E83. Exons E6 and E105 have lengths that are multiples of three, and therefore do not result in a change in reading frame when absent.

In contrast, the absence of E83 results in a shifted reading frame and an alternative amino acid sequence at the carboxy terminus of the protein (indicated by a hatched pattern in the figure). The hydrophobic transmembrane (TM) region is encoded within

E105.

Figure 18. PK-RSVScript-susCD163v2 #9 cells infected with PRRSV isolate P129.

Undiluted supernatant from PRRSV isolate P201 infected PAMs was used to infect

PK-RSVScript-susCD163v2 #9 cells. After two days of incubation the cells were fixed and stained with monoclonal antibody SDOW17 as described in Example 11.

Figure 19. FK-RSVScript-susCD163v2 #51 cells infected with PRRSV isolate P129. Undiluted supernatant from PRRSYV isolate P201 infected PAMs was used to infect

FK-RSVScript-susCD163v2 #51 cells. Two days post infection the cells were acetone fixed and stained with monoclonal antibody SDOW 17 as described in

Example 11.

Figure 20. Infection of PK-RSVScript-susCD163v2 clone #9 cells with PRRSV isolate P201. Panel A shows a monolayer of cells infected with PRRSV P201 at passage 1, twenty-four hours post infection. Panel B shows a monolayer of cells 2 days post infection with cell free supernatant PRRSV P201 at passage 10.

Figure 21. NLFK parent cells and one subclone of FK-cDNA3.1D-humnCD163v2-A6 were examined for the CD163 expression. Cells were fixed in 80% acetone and reacted with Goat anti-human CD163 (R&D System at 1:200) for one hour following by washing with PBS. For visualization, donkey anti-Goat IgG conjugated with FITC (Biodesign Inc at 1:100) were used. No specific fluorescence was detected in the

NLFK parent cells as shown in Figure 21A. The majority of the FK.A6.A2 subclone showed good fluorescent staining indicating the presence of CD163 (Figure 21B).

DETAILED DESCRIPTION OF THE INVENTION

General Definitions

Cells and cell lines can be either virus permissive” or “virus non-permissive”.

For example, a cell or cell line that is virus permissive is capable of allowing virus infection, subsequent replication and virus production. A cell or cell line that is virus non-permissive is incapable of allowing virus infection, subsequent replication and virus production. A cell line that is already somewhat permissive may be rendered more permissive by the methods of the invention.

Arteriviridae refers to a family of enveloped, positive-stranded RNA viruses belonging to the order Nidovirales. The family includes lactate dehydrogenase- elevating virus (LDV) of mice, equine arteritis virus (EAV), simian hemorrhagic fever 15s virus (SHFV), and PRRSV.

Asfarviridae is a family of icosohedral, enveloped viruses whose genomes consist of single molecules of linear double-stranded DNA about 150000-190000 nucleotides long. The name of the family is derived from African Swine Fever And

Releted Viruses. African Swine Fever Virus (ASFV) is the type species of the Asfivirus genus and is the sole member of the family.

The term “PRRSV” or PRRS virus refers to both European and North

American PRRS virus genotypes. Within each genotype, isolates typically share 85% or higher nucleotide identity. Between genotypes, however, the level of sequence identity is only about 60%.

The PRRS virus is a member of the family Arteriviridae. The genome of the arteriviruses is single-stranded RNA of positive polarity between 12 and 16 kb in length, capped at the 5’ end and polyadenylated at the 3’ end. Over two-thirds of the genome is dedicated to open reading frames (ORFs) 1a and 1b, which encode the non- structural functions of the virus. ORF1b is an extension of ORF1a, and is the result of aribosomal frameshift. ORFs 1a and Ib are translated directly from the genomic

RNA. These large polypeptide products are cleaved by viral proteases to yield 12 or 13 discrete smaller peptides. The remaining ORFs, which encode viral structural proteins, are expressed from a series of 3’ co-terminal subgenomic RNAs (sgRNAs).

The sgRNAs are produced by discontinuous transcription of negative-stranded RNA, such that a common 5’ leader sequence becomes fused to each transcript. The major structural proteins are the nucleocapsid (N, encoded by ORF7), the matrix protein (M, encoded by ORF6), and the major envelope glycoprotein (GPS, encoded by ORFS).

The remaining proteins, GP4 (ORF4), GP3 (ORF3), GP2 (ORF2a), and E (ORF2b) are minor structural components of the virion, whose functions have not yet been elucidated. The molecular biology of PRRSV has been the subject of recent review articles (Dea et al., 2000; Meulenberg, 2000; Snijder and Meulenberg, 2001).

As used herein, the term “CD163 polypeptide” means a protein encoded by a mammalian CD163 gene, including allelic variants containing conservative or non- conservative changes. A cDNA sequence that encodes a porcine CD163 polypeptide has been reported (Genbank accession number AJ311716). A murine CD163 polypeptide has also been reported (Genbank access number AF274883), as well as multiple human variants, exemplified by Genbank access numbers AAHS1281and

CAAS80543. We report herein polynucleotides that encode porcine, human, murine, canine, and african green monkey CD163 polypeptides and which comprise the sequences set forth in SEQID NO: 1, §, 12 13, 17, 18, 22, 23, 25, 26, 30, 31 33, 35, 37, 39, 41, 42, 43, 45 and 47. A “CD163 polypeptide” is a member of the scavenger receptor cysteine-rich (SRCR) family of transmembrane glycoproteins, and is thought to be expressed exclusively on monocytes and macrophages. One identified role of

CD163 is to inhibit oxidative tissue damage following hemolysis by consuming hemoglobin:haptoglobin complexes by endocytosis. The subsequent release of interleukin-10 and synthesis of heme oxygenase-1 results in antiinflammatory and cytoprotective effects (Philippidis et al., 2004; Graversen et al., 2002). The human CD163 gene spans 35 kb on chromosome 12, and consists of 17 exons and 16 introns.

A number of isoforms of the CD163 polypeptide, including membrane bound, cytoplasmic and secreted types, are known to be generated by alternative splicing (Ritter et al., 1999). Isoforms that comprise a transmembrane domain are particularly preferred.

A transmembrane domain is characterized by a polypeptide segment of a larger sequence that is exposed on both sides of a membrane. The cytoplasmic and extracellular domains are separated by at least one membrane-spanning segment that traverses the hydrophobic environment of the lipid bilayer. The membrane- spanning segment is composed of amino acid residues with nonpolar side chains, usually in the form of an alpha helix. Segments that contain about 20-30 hydrophobic residues are long enough to span a membrane as an alpha helix, and they can often be identified by means of a hydropathy plot. The predicted transmembrane domain of SEQ ID NO:2 and 14 are indicated by bolding in the specification. To determine whether other

CD163 sequences contain a similar sequence feature is easily determined by inspection of the sequence or hydropathy plots. SEQ ID NOS: 37-40 are representative of variant CD163 proteins which do not contain a transmembrane domain and their encoding nucleic acids.

As used hereinafter, "polynucleotide" generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or

DNA or modified RNA or DNA. "Polynucleotides” include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double- stranded regions, single- and double-stranded RNA, RNA that is mixture of single- and double-stranded regions, and hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, "polynucleotide" refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term "polynucleotide" also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications may be made to DNA and RNA; thus, "polynucleotide" embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. "Polynucleotide" also embraces relatively short polynucleotides, often referred to as oligonucleotides.

As used hereinafter, "polypeptide" refers to any peptide or protein comprising amino acids joined to each other by peptide bonds or modified peptide bonds. "Polypeptide" refers to both short chains, commonly referred to as peptides, oligopeptides or oligomers, and to longer chains, generally referred to as proteins.

Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. "Polypeptides" include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature.

Modifications may occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present to the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides may result from post-translation natural processes or may be made by synthetic methods. Modifications or modified forms include acetylation, acylation,

ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination (see, for instance, Proteins-Structure and Molecular Properties, 2nd Ed., T. E.

Creighton, W. H. Freeman and Company, New York, 1993; Wold, F., Post- translational Protein Modifications: Perspectives and Prospects, pgs. 1-12 in

Postranslational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic

Press, New York, 1983; Seifter ct al., "Analysis for protein modifications and nonprotein cofactors”, Meth Enzymol (1990) 182:626-646 and Rattan et al., "Protein

Synthesis: Post-translational Modifications and Aging", Ann NY Acad Sci (1992) 663:4842).

As used hereinafter, "isolated" means altered by the hand of man from the natural state. If an "isolated" composition or substance occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living animal is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated", as the term is employed herein. Therefore “isolated” as used herein and as understood in the art, whether referring to “isolated” polynucleotides or polypeptides, is taken to mean separated from the original cellular environment in which the polypeptide or nucleic acid is normally found. As used herein therefore, by way of example only, a transgenic animal or a recombinant cell line constructed with a polynucleotide of the invention makes use of the “isolated” nucleic acid. Specifically excluded from the definition of isolated polynucleotides of the invention are entire isolated chromosomes from native host cells from which the polynucleotide was originally derived.

In the disclosure to follow we will often make use of the term “identity” or similarity as applied to the amino acid sequences of polypeptides. Percent amino acid sequence “identity” with respect to polypeptides is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the residues in the target sequences after aligning both sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Percent sequence identity is determined by conventional methods. For example, BLASTP 2.2.6 [Tatusova TA and TL Madden, “BLAST 2 sequences- a new tool for comparing protein and nucleotide sequences.” (1999) FEMS Microbiol Lett. 174:247-250.]

Briefly, as noted above, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 0.1, and the “blosum62” scoring matrix of Henikoff and Henikoff (Proc. Nat. Acad. Sci.

USA 89:10915-10919. 1992).

The percent identity is then calculated as:

Total number of identical matches x 100 [length of the longer sequence + number of gaps introduced into the longer sequence to align the two sequences]

Percent sequence “similarity” (often referred to as “homology”) with respect to a polypeptide of the invention is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the residues in the target sequences after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity (as described above), and also considering any conservative substitutions as part of the sequence identity.

Tota] number of identical matches and conservative substitutions Xx [length of the longer sequence +number of gaps introduced into the longer sequence to align the two sequences)

Amino acids can be classified according to physical properties and contribution to secondary and tertiary protein structure. A conservative substitution is recognized in the art as a substitution of one amino acid for another amino acid that has similar properties.

Exemplary conservative substitutions are set out in Table 1, 2, and 3 below.

Table 1

Conservative Substitutions I

SIDE CHAIN

CHARACTERISTIC AMINO ACID

Aliphatic

Non-polar GAP

ILV

Polar - uncharged CSTM

NQ

Polar - charged DE

KR

Aromatic HFWY

Other NQDE

Alternatively, conservative amino acids can be grouped as described in Lehninger, [Biochemistry, Second Edition; Worth Publishers, Inc. NY:NY (1975), pp.71-77] as set out in Table 2, immediately below

Table 2

Conservative Substitutions IT

SIDE CHAIN

Ss CHARACTERISTIC AMINO ACID

Non-polar (hydrophobic)

A. Aliphatic: ALIVP

B. Aromatic: FW

C. Sulfur-containing: M

D. Borderline: G

Uncharged-polar

A. Hydroxyl: STY

B. Amides: NQ

C. Sulfhydryl: C

D. Borderline: G

Positively Charged (Basic): KRH

Negatively Charged (Acidic): DE

As still another alternative, exemplary conservative substitutions are set out in Table 3, immediately below.

Table 3

Conservative Substitutions III

Original Residue Exemplary Substitution

Ala (A) Val, Leu, lle

Arg (R) Lys, Gln, Asn

Asn (N) Gln, His, Lys, Arg

Asp (D) Glu

Cys(O) Ser

Gln (Q) Asn

Glu (E) Asp

His (H) Asn, Gln, Lys, Arg

Ile (D Leu, Val, Met, Ala, Phe,

Leu (L) Re, Val, Met, Ala, Phe

Lys (K) Arg, Gln, Asn

MetM) Leu, Phe, Ile

Phe (F) Leu, Val, Ile, Ala

Pro (P) Gly

Ser (S) Thr

Thr (T) Ser

Trp (W) Tyr

Tyr (Y) Trp, Phe, Thr, Ser

Val (V) Ile, Leu, Met, Phe, Ala

Methods Directed to the Production of Virus and Host Cells of the

Invention

The invention provides a method of modifying production a virus that is a member of the family Arteriviridae and Asfarviridae in a cell comprising the step of directing said cell to express a CD163 polypeptide. This may include rendering a virus non-permissive cell into a virus permissive cell, or may involve rendering a cell more permissive to the virus.

In one embodiment, the virus that is a member of the family Arteriviridae or

Asfarviridae is selected from the group consisting of LDV of mice, equine arteritis virus (EAV), simian hemorrhagic fever virus (SHFV), PRRSV of swine and ASFV of swine.

In a preferred embodiment the virus is PPRSV.

The invention further provides a method of preparing a culture of a virus that is a member of the family Arzeriviridae or Asfarviridae comprising the steps of: providing a cell line; directing said cell line to express a CD163 polypeptide; infecting said cell line with virus; and causing said cell line to produce viral progeny.

In one embodiment, the virus that is a member of the family Arteriviridae is selected from the group consisting of LDV of mice, equine arteritis virus (EAV), simian hemorrhagic fever virus (SHFV), PRRSV of swine and ASFV of swine.

In a preferred embodiment the virus is PRRSV.

All of the above methods utilize cells and cell lines expressing a CD163 polypeptide. CD163 may be facilitated or increased by methods that involve the introduction of exogenous nucleic acid into the cell. Such a cell may comprise a polynucleotide or vector in a manner that permits expression of an encoded CD163 polypeptide.

Polynucleotides that encode CD163 may be introduced into the host cell as part of a circular plasmid, or as linear DNA comprising an isolated protein-coding region, or in a viral vector. Methods for introducing exogenous nucleic acid into the host cell well known and routinely practiced in the art include transformation, transfection, electroporation, nuclear injection, or fusion with carriers such as liposomes, micelles, ghost cells, and protoplasts. Host cell systems of the invention include invertebrate and vertebrate cells systems. Hosts may include, but are not limited to, the following: insect cells, porcine kidney (PK) cells, feline kidney (FK) cells, swine testicular (ST) cells, African green monkey kidney cells (MA-104,

MARC-145, VERO, and COS cells), Chinese hamster ovary (CHO) cells, baby hampster kidney cells, human 293 cells, and murine 3T3 fibroblasts. Insect host cell culture systems may also be used for the expression of CD163 polypeptides. In another embodiment, the CD163 polypeptides are expressed using a drosophila expression system.

The choice of a suitable expression vector for expression of the

CD163polypeptides will of course depend upon the specific host cell to be used, and is within the skill of the ordinary artisan. Examples of suitable expression vectors include pSport and pcDNA3 (Invitrogen), pCMV-Script (Stratagene), and pSVL (Pharmacia Biotech). Expression vectors for use in mammalian host cells may include transcriptional and translational control sequences derived from viral genomes. Commonly used promoter sequences and modifier sequences which may be used in the present invention include, but are not limited to, those derived from human cytomegalovirus (CMV), Rous sarcoma virus (RSV), Adenovirus 2, Polyoma virus, and Simian virus 40 (SV40). Methods for the construction of mammalian expression vectors are disclosed, for example, in Okayama and Berg (Mol. Cell. Biol. 3:280 (1983)); Cosman et al. (Mol. Immunol. 23:935 (1986)); Cosman et al. (Nature 312:768 (1984)); EP-A-0367566; and WO 91/18982.

Because CD163 sequences are known to exist in cells from various species, the endogenous gene may be modified to permit, or increase, expression of the CD163 polypeptide. Cells can be modified (e.g., by homologous recombination) to provide increased expression by replacing, in whole or in part, the naturally occurring CD163 promoter with all or part of a heterologous promoter, so that the cells express CD163 polypeptide at higher levels. The heterologous promoter is inserted in such a manner that it is operatively linked to endogenous CD 163 encoding sequences. [See, for example, PCT International Publication No. WO 94/12650, PCT International

Publication No. WO 92/20808, and PCT International Publication No. WO 91/09955.] It is also contemplated that, in addition to heterologous promoter DNA, amplifiable marker DNA (e.g., ada, dhfr, and the multifunctional cad gene, which encodes for carbamyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase) and/or intron DNA may be inserted along with the heterologous promoter DNA. If linked to the CD163 coding sequence, amplification of the marker

DNA by standard selection methods results in co-amplification of the CD163 coding sequences in the cells.

CD163 expression may also be induced by chemical treatment. Phorbol esters, especially phorbol myristyl acetate (PMA), activate one or more isozymes of the ubiquitous membrane receptor, protein kinase C (PKC) and are particularly preferred means of increasing CD163 expresssion. Other methods of intracellular calcium mobilization are also contemplated.

Vaccine Production

The methods described above may be used to produce any virus that is a member of the family Arteriviridae or Asfarviridae for the purpose of vaccine production or diagnostics.

In one embodiment the virus that is a member of the family Arteriviridae is selected from the group consisting of LDV of mice, equine arteritis virus (EAV), simian hemorrhagic fever virus (SHFV), and PRRSV of swine.

In a preferred embodiment the virus is PRRSV.

Vaccine Production

The methods described above may be used to produce virus for the purpose of vaccine production or diagnostics.

Killed (inactivated) or live vaccines can be produced. Therefore, to make a live vaccine, a viral isolate, or an attenuated or mutated variant thereof, is grown in cell culture. The virus is harvested according to methods well known in the art. The virus may then be concentrated, frozen, and stored at -70°C, or freeze-dried and stored at 4°C. Prior to vaccination the virus is mixed at an appropriate dosage, (which is from about 10 to 10° tissue culture infectious doses per ml (TCIDso/ml)), with a pharmaceutically acceptable carrier such as a saline solution, and optionally an adjuvant.

The vaccine produced might also comprise an inactivated or killed vaccine comprising a virus grown by the methods of the invention. The inactivated vaccine is made by methods well known in the art. For example, once the virus is propagated to high titers, it would be readily apparent by those skilled in the art that the virus antigenic mass could be obtained by methods well known in the art. For example, the virus antigenic mass may be obtained by dilution, concentration, or extraction. All of these methods have been employed to obtain appropriate viral antigenic mass to produce vaccines. The virus is then inactivated by treatment with formalin, betapropriolactone (BPL), binary ethyleneimine (BEI), or other methods known to those skilled in the art. The inactivated virus is then mixed with a pharmaceutically acceptable carrier such as a saline solution, and optionally an adjuvant. Examples of adjuvants include, but not limited to, aluminum hydroxide, oil-in-water and water-in- oil emulsions, AMPHIGEN, saponins such as QuilA, and polypeptide adjuvants including interleukins, interferons, and other cytokines.

Inactivation by formalin is performed by mixing the viral suspension with 37% formaldehyde to a final formaldehyde concentration of 0.05%. The virus- formaldehyde mixture is mixed by constant stirring for approximately 24 hours at room temperature. The inactivated virus mixture is then tested for residual live virus by assaying for growth on a suitable cell line.

Inactivation by BEL is performed by mixing the viral suspension of the present invention with 0.1 M BEI (2-bromo-ethylamine in 0.175 N NaOH) to a final BEI concentration of 1 mM. The virus-BEI mixture is mixed by constant stirring for approximately 48 hours at room temperature, followed by the addition of 1.0 M sodium thiosulfate to a final concentration of 0.1 mM. Mixing is continued for an additional two hours. The inactivated virus mixture is tested for residual live virus by assaying for growth on a suitable cell line.

Virus permissive cells that have been directed to express CD163 can also be used to quantify live virus. Two common methods, which are well known to those s skilled in the art, are the plaque assay and the limiting dilution assay.

CD163-expressing cell lines of the present invention can be used to grow virus for the purpose of producing viral antigen for diagnostic kits. For example lysates from infected cells (with optional purification of viral particles or extraction of selected viral proteins) may be coated on ELISA plates in order to detect and quantify antibodies to the virus in swine sera.

Live or inactivated virus grown in CD163-expressing cells can be used after optional separation of the viral proteins to immunize animals in order to generate polyclonal, monospecific or monoclonal antibodies. These in turn can be used as the basis of diagnostic assays for the detection and quantification of virus in swine serum and other biological samples.

Assays of the Invention

The invention provides methods for determining the propensity of an animal to be infected by a virus that is a member of the family Arteriviridae or Asfarviridae or of a cell line to support the replication of a virus that is a member of the family Arteriviridae or Asfarviridae. Samples from either source are obtained and assayed for expression of CD163. The level of CD163 gene expression can be compared with levels of controls known not to support replication of the virus.

In the case of an animal, samples can be any sample comprising sample nucleic acid molecules or proteins, and obtained from any bodily tissue expressing CDI163, including, but not limited to, alveolar macrophages, cultured cells, biopsies, or other tissue preparations. The level of expression can be assessed at either or both the level of messenger RNA or protein produced. In a preferred embodiment the member of the virus of the family Arteriviridae or Asfarviridae is selected from the group consisting of LDV of mice, equine arteritis virus (EAV), simian hemorrhagic fever virus (SHFV), PRRSV of swine, and ASFV of swine.

Nucleic Acid based Assays

Methods of determining CD163 levels may be nucleic acid based as noted above. CD163-derived nucleic acids may be in solution or on a solid support. In some embodiments, they may be employed as array elements in microarrays alone or in combination with other array element molecules. Nucleic acid based methods generally require the isolation of DNA or RNA from the sample and subsequent hybridization or PCR amplification using sprecific primers derived from any known

CDI163 encoding sequences in the art or those specifically disclosed as SEQ ID NO: 1, 3,5,12,13,17, 18,22, 23, 25, 26, 30, 31 33, 35, 37, 39, 41, 43, 45, and 47. DNA or

RNA can be isolated from the sample according to any of a number of methods well known to those of skill in the art. For example, methods of purification of nucleic acids are described in Tijssen, P. (1993) Laboratory Techniques in Biochemistry and

Molecular Biology: Hybridization With Nucleic Acid Probes, Part I. Theory and

Nucleic Acid Preparation, Elsevier, New York, N.Y. In one preferred embodiment, total RNA is isolated using the TRIZOL total RNA isolation reagent (Life

Technologies, Inc., Gaithersburg Md.) and mRNA is isolated using oligo d(T) coluran chromatography or glass beads. When sample nucleic acid molecules are amplified it is desirable to amplify the sample nucleic acid molecules and maintain the relative abundances of the original sample, including low abundance transcripts. RNA can be amplified in vitro, in situ, or in vivo (See Eberwine U.S. Pat. No. 5,514,545).

It is also advantageous to include controls within the sample to assure that amplification and labeling procedures do not change the true distribution of nucleic acid molecules in a sample. For this purpose, a sample is spiked with an amount of a control nucleic acid molecule predetermined to be detectable upon hybridization to its complementary arrayed nucleic acid molecule and the composition of nucleic acid molecules includes reference nucleic acid molecules which specifically hybridize with the control arrayed nucleic acid molecules. After hybridization and processing, the hybridization signals obtained should reflect accurately the amounts of control arrayed nucleic acid molecules added to the sample.

Prior to hybridization, it may be desirable to fragment the sample nucleic acid molecules. Fragmentation improves hybridization by minimizing secondary structure and cross-hybridization to other sample nucleic acid molecules in the sample or noncomplementary nucleic acid molecules. Fragmentation can be performed by mechanical or chemical means.

Labeling

The sample nucleic acid molecules or probes may be labeled with one or more labeling moieties to allow for detection of hybridized arrayed/sample nucleic acid molecule complexes. The labeling moieties can include compositions that can be detected by spectroscopic, photochemical, biochemical, bioelectronic, immunochemical, electrical, optical, or chemical means. The labeling moieties include radioisotopes, such as (32)P, (33)P or (35)S, chemiluminescent compounds, labeled binding proteins, heavy metal atoms, spectroscopic markers, such as fluorescent markers and dyes, magnetic labels, linked enzymes, mass spectrometry tags, spin labels, electron transfer donors and acceptors, and the like. Preferred fluorescent markers include Cy3 and CyS5 fluorophores (Amersham Pharmacia

Biotech, Piscataway N.J.).

Hybridization

The nucleic acid molecule sequence of SEQ ID NO: 1, 3, 5, 12, 13, 17, 18, 22, 23, 25, 26, 30, 31, 33, 35, 37, 39, 41, 43, 45 and 47 or other CD163 encoding sequences in the art and fragments thereof can be used in various hybridization technologies for various purposes. Hybridization probes may be designed or derived from any mammalian CD163 sequence but may make use of those sequences disclosed in SEQ ID NO: 1, 3, 5, 12, 13, 17, 18, 22, 23, 25, 26, 30, 31, 33, 35, 37, 39, 41, 43,45 and 47. Such probes may be made from a highly specific region or from a conserved motif, and used in protocols to quantify CD163 message, allelic variants, or related sequences. The hybridization probes of the subject invention may be DNA or

RNA and may be derived from any mammalian CD163 sequence known in the art or from those sequences disclosed herein as SEQ ID NO: 1, 3, 5, 12, 13, 17, 18, 22, 23, 25, 26, 30 or from genomic sequences including promoters, enhancers, and introns of the mammalian gene. Hybridization or PCR probes may be produced using oligolabeling, nick translation, end-labeling, or PCR amplification in the presence of the labeled nucleotide. A vector containing the nucleic acid sequence may be used to produce an mRNA probe in vitro by addition of an RNA polymerase and labeled nucleic acid molecules. These procedures may be conducted using commercially available kits such as those provided by Amersham Pharmacia Biotech.

The stringency of hybridization is determined by the G+C content of the probe, salt concentration, and temperature. In particular, stringency can be increased by reducing the concentration of salt or raising the hybridization temperature. In solutions used for some membrane-based hybridizations, additions of an organic solvent such as formamide allows the reaction to occur at a lower temperature. Hybridization can be performed at low stringency with buffers, such as 5xSSC with 1% sodium dodecyl sulfate (SDS) at 60°C., which permits the formation of a hybridization complex between nucleotide sequences that contain some mismatches. Subsequent washes are performed at higher stringency with buffers such as 0.2xSSC with 0.1% SDS at either 45°C (medium stringency) or 68°C (high stringency). At high stringency, hybridization complexes will remain stable only where the nucleic acid sequences are almost completely complementary. In some membrane-based hybridizations, preferably 35% or most preferably 50%, formamide can be added to the hybridization solution to reduce the temperature at which hybridization is performed, and background signals can be reduced by the use of other detergents such as Sarkosyl or

Triton X-100 and a blocking agent such as salmon sperm DNA. Selection of components and conditions for hybridization are well known to those skilled in the art and are reviewed in Ausubel (supra) and Sambrook et al. (1989) Molecular Cloning,

A Laboratory Manual, Cold Spring Harbor Press, Plainview N.Y.

Exemplary highly stringent hybridization conditions are as follows: hybridization at 42°C in a hybridization solution comprising 50% formamide, 1%

SDS, 1M NaCl, 10% Dextran sulfate, and washing twice for 30 minutes at 60°C in a wash solution comprising 0.1 X SSC and 1% SDS. It is understood in the art that conditions of equivalent stringency can be achieved through variation of temperature and buffer, or salt concentration as described Ausubel, et al. (Eds.), Protocols in

Molecular Biology, John Wiley & Sons (1994), pp. 6.0.3 to 6.4.10. Modifications in hybridization conditions can be empirically determined or precisely calculated based on the length and the percentage of guanosine/cytosine (GC) base pairing of the probe.

The hybridization conditions can be calculated as described in Sambrook, et al., (Eds.), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory

Press: Cold Spring Harbor, New York (1989), pp. 9.47 to 9.51.

Hybridization specificity can be evaluated by comparing the hybridization of specificity-control nucleic acid molecules to specificity-control sample nucleic acid molecules that are added to a sample in a known amount. The specificity-control arrayed nucleic acid molecules may have one or more sequence mismatches compared with the corresponding arrayed nucleic acid molecules. In this manner, it is possible to determine whether only complementary arrayed nucleic acid molecules are _38-

hybridizing to the sample nucleic acid molecules or whether mismatched hybrid duplexes are forming is determined.

Hybridization reactions can be performed in absolute or differential hybridization formats. In the absolute hybridization format, nucleic acid molecules from one sample are hybridized to the molecules in a microarray format and signals detected after hybridization complex formation correlate to nucleic acid molecule levels in a sample. In the differential hybridization format, the differential expression of a set of genes in two biological samples is analyzed. For differential hybridization, nucleic acid molecules from both biological samples are prepared and labeled with different labeling moieties. A mixture of the two labeled nucleic acid molecules is added to a microarray. The microarray is then examined under conditions in which the emissions from the two different labels are individually detectable. Molecules in the microarray that are hybridized to substantially equal numbers of nucleic acid molecules derived from both biological samples give a distinct combined fluorescence (Shalon et al.; PCT publication W095/35505). In a preferred embodiment, the labels are fluorescent markers with distinguishable emission spectra, such as Cy3 and CyS5 fluorophores.

After hybridization, the microarray is washed to remove nonhybridized nucleic acid molecules and complex formation between the hybridizable array elements and the nucleic acid molecules is detected. Methods for detecting complex formation are well known to those skilled in the art. In a preferred embodiment, the nucleic acid molecules are labeled with a fluorescent label and measurement of levels and patterns of fluorescence indicative of complex formation is accomplished by fluorescence microscopy, preferably confocal fluorescence microscopy.

In a differential hybridization experiment, nucleic acid molecules from two or more different biological samples are labeled with two or more different fluorescent labels with different emission wavelengths. Fluorescent signals are detected separately with different photomultipliers set to detect specific wavelengths. The relative abundances/ expression levels of the nucleic acid molecules in two or more samples are obtained.

Typically, microarray fluorescence intensities can be normalized to take into account variations in hybridization intensities when more than one microarray is used under similar test conditions. In a preferred embodiment, individual arrayed-sample nucleic acid molecule complex hybridization intensities are normalized using the intensities derived from internal normalization controls contained on each microarray.

Polypeptide Based Assays

The present invention provides methods and reagents for detecting and i quantifying CD163 polypeptides. These methods include analytical biochemical methods such as electrophoresis, mass spectroscopy, chromatographic methods and the like, or various immunological methods such as radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, western blotting, affinity capture mass spectrometry, biological activity, and others described below and apparent to those of skill in the art upon review of this disclosure. lmmunoassays

The present invention also provides methods for detection of CD163 polypeptides employing one or more anti- CD163 antibody reagents (i.e., immunoassays). As used herein, an immunoassay is an assay that utilizes an antibody (as broadly defined herein and specifically includes fragments, chimeras and other binding agents) that specifically binds a CD163 polypeptide or epitope.

A number of well-established immunological binding assay formats suitable for the practice of the invention are known (see, e.g., U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168). See, e.g., Methods in Cell Biology Volume 37:

Antibodies in Cell Biology, Asai, ed. Academic Press, Inc. New York (1993); Basic and Clinical Immunology 7th Edition, Stites & Terr, eds. (1991); Harlow and Lane, supra [e.g., Chapter 14], and Ausubel et al., supra, [e.g., Chapter 11]. Typically, immunological binding assays (or immunoassays) utilize a "capture agent" to specifically bind to and, often, immobilize the analyte to a solid phase. In one embodiment, the capture agent is a moiety that specifically binds to a CD163 polypeptide or subsequence, such as an anti- CD163 antibody.

Usually the CD163 gene product being assayed is detected directly or indirectly using a detectable label. The particular label or detectable group used in the assay is usually not a critical aspect of the invention, so long as it does not significantly interfere with the specific binding of the antibody or antibodies used in the assay. The label may be covalently attached to the capture agent (e.g., an anti-

CD163 antibody), or may be attached to a third moiety, such as another antibody, that specifically binds to the CD163 polypeptide.

The present invention provides methods and reagents for competitive and noncompetitive immunoassays for detecting CD163 polypeptides. Noncompetitive immunoassays are assays in which the amount of captured analyte (in this case

CD163) is directly measured. One such assay is a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on the CD163 polypeptide. See, e.g., Maddox et al., 1983, J. Exp. Med., 158:1211 for background information. In one "sandwich" assay, the capture agent (¢.g., an anti-

CD163 antibody) is bound directly to a solid substrate where it is immobilized. These immobilized antibodies then capture any CD163 polypeptide present in the test sample. The CD163 polypeptide thus immobilized can then be labeled, i.e., by binding to a second anti-CD 163 antibody bearing a label. Alternatively, the secondCD163 antibody may lack a label, but be bound by a labeled third antibody specific to antibodies of the species from which the second antibody is derived. The second antibody alternatively can be modified with a detectable moiety, such as biotin, to which a third labeled molecule can specifically bind, such as enzyme- labeled streptavidin.

In competitive assays, the amount of CD163 polypeptide present in the sample is measured indirectly by measuring the amount of an added (exogenous) CD163 polypeptide displaced (or competed away) from a capture agent (e.g., CD163 antibody) by the CD163 polypeptide present in the sample. A hapten inhibition assay is another example of a competitive assay. In this assay CD163 polypeptide is immobilized on a solid substrate. A known amount of CD163 antibody is added to the sample, and the sample is then contacted with the immobilized CD163 polypeptide. In this case, the amount of anti- CD 163 antibody bound to the immobilized CD163 polypeptide is inversely proportional to the amount of CD163 polypeptide present in the sample. The amount of immobilized antibody may be detected by detecting either the immobilized fraction of antibody or the fraction of the antibody that remains in solution. In this aspect, detection may be direct, where the antibody is labeled, or indirect where the label is bound to a molecule that specifically binds to the antibody as described above.

Other Antibody-based Assay Formats

The invention also provides reagents and methods for detecting and quantifying the presence of CD163 polypeptide in the sample by using an immunoblot (Western blot)

format. Another immunoassay is the so- called "lateral flow chromatography." In a non-competitive version of lateral flow chromatography, a sample moves across a substrate by, e.g., capillary action, and encounters a mobile-labeled antibody that binds the analyte forming a conjugate. The conjugate then moves across the substrate and encounters an immobilized second antibody that binds the analyte. Thus, immobilized analyte is detected by detecting the labeled antibody. In a competitive version of lateral flow chromatography a labeled version of the analyte moves across the carrier and competes with unlabeled analyte for binding with the immobilized antibody. The greater the amount of the analyte in the sample, the less the binding by labeled analyte and, therefore, the weaker the signal. See, e.g., May et al., U.S. Pat.

No. 5,622,871 and Rosenstein, U.S. Pat. No. 5,591,645.

Depending upon the assay, various components, including the antigen, target antibody, or anti-cathepsin S antibody, may be bound to a solid surface or support (i.e., a substrate, membrane, or filter paper). Many methods for immobilizing biomolecules to a variety of solid surfaces are known in the art. For instance, the solid surface may be a membrane (e.g., nitrocellulose), a microtiter dish (e.g., PVC, polypropylene, or polystyrene), a test tube (glass or plastic), a dipstick (e.g. glass,

PVC, polypropylene, polystyrene, latex, and the like), a microcentrifuge tube, or a glass or plastic bead. The desired component may be covalently bound or noncovalently attached through nonspecific bonding. "A wide variety of organic and inorganic polymers, both natural and synthetic may be employed as the material for the solid surface. Illustrative polymersinclude polyethylene, polypropylene, poly (4- methylbutene), polystyrene,polymethacrylate, poly(ethylene terephthalate) , rayon, nylon, poly(vinylbutyrate), polyvinylidene difluoride (PVDF), silicones, polyformaldehyde, cellulose, cellulose acetate, nitrocellulose, and the like. Other materials which may be employed, include paper, glasses, ceramics, metals, metalloids, semiconductive materials, cements or the like.

In addition, substances that form gels, such as proteins (e. g., gelatins), lipopolysaccharides, silicates, agarose and polyacrylamides can be used. Polymers that form several aqueous phases, such as dextrans, polyalkylene glycols or surfactants, such as phospholipids, long chain (12-24 carbon atoms) alkyl ammonium salts and the like are also suitable. Where the solid surface is porous, various pore sizes may be employed depending upon the nature of the system.

Mass Spectrometry

The mass of a molecule frequently can be used as an identifier of the molecule.

Therefore, methods of mass spectrometry can be used to identify a protein analyte.

Mass spectrometers can measure mass by determining the time required for an ionized analyte to travel down a flight tube and to be detected by an ion detector. One method of mass spectrometry for proteins is matrix-assisted laser desorption ionization mass spectrometry ("MALDI"). In MALDI the analyte is mixed with an energy absorbing matrix material that absorbs energy of the wavelength of a laser and placed on the surface of a probe. Upon striking the matrix with the laser, the analyte is desorbed from the probe surface, ionized, and detected by the ion detector. See, for example,

Hillenkamp et al., U.S. Pat. No. 5,118,937.

Other methods of mass spectrometry for proteins are described in Hutchens and Yip, U.S. Pat. No. 5,719,060. In one such method referred to as Surfaces

Enhanced for Affinity Capture ("SEAC") a solid phase affinity reagent that binds the analyte specifically or non-specifically, such as an antibody or a metal ion, is used to separate the analyte from other materials in a sample. Then the captured analyte is desorbed from the solid phase by, e.g., laser energy, ionized, and detected by the detector.

Nucleic Acids of the Invention

The examples disclose our discovery of several novel CD163 polynucleotides.

The invention includes these novel CD163 polynucleotides. The present invention provides several isolated novel polynucleotides (e.g., DNA sequences and RNA transcripts, both sense and complementary antisense strands, both single and double- stranded, including splice variants thereof, which encode novel CD163 polypeptides.

We report herein isolated novel polynucleotides which encode porcine, murine, human, canine, and african green monkey CD163 polypeptides and which comprise the sequences set forth in SEQ ID NO: 1, 5, 12 13, 22, 23, 25, 26, 30 31, 33, 35, 37, 39, 41, 43, 45, and 47.

It should be recognized that by disclosing SEQ ID NOs: 1, 5, 12 13, 22, 23, 25, 26,3031, 33, 35,37, 39, 41, 43, 45, and 47 it provides one skilled in the art a multitude of methods of obtaining these sequences. By way of example, it would be possible to generate probes from the sequence disclosed in SEQ ID NOs: 1, 5, 12 13, 22,23, 25, 26, 30 31, 33, 35, 37, 39, 41, 43, 45, and 47 and screen porcine, murine,

human, canine, and african green monkey cDNA or genomic libraries and thereby obtain the entire SEQ ID NO: 1, 3, 5, 12, 13, 17, 18, 22, 23, 25, 26, 30, 31, 33, 35, 37, 39, 41, 43, 45 and 47, or its genomic equivalent. Sambrook, ef al., (Eds.), Molecular

Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press: Cold Spring

Harbor, New York (1989). Also by way of example, one skilled in the art would immediately recognize that given the sequence disclosed in SEQ ID NO: 5, 12 13, 22, 23, 25, 26, 30 31, 33, 35, 37, 39, 41, 43, 45, and 47it is then possible to generate the appropriate primers for PCR amplification to obtain the entire sequence represented by these sequences. (See e.g., PCR Technology, H. A. Erlich, ed., Stockton Press,

New York, 1989; PCR Protocols: A Guide to Methods and Applications, M. A. Innis,

David H. Gelfand, John J. Sninsky, and Thomas J. White, eds., Academic Press, Inc.,

New York, 1990.)

DNA polynucleotides of the invention include cDNA, and DNA that has been chemically synthesized in whole or in part and is also intended to include allelic variants thereof. Allelic variants are modified forms of a wild type gene sequence, the modification resulting from recombination during chromosomal segregation, or exposure to conditions which give rise to genetic mutation. Allelic variants, like wild type genes, are naturally occurring sequences (as opposed to non-naturally occurring variants which arise from in vitro manipulation).

DNA sequences encoding the novel CD163 polypeptides are set forth in SEQ

ID NOs: 5, 12 13, 22, 23, 25, 26, 30 31, 33, 35, 37, 39, 41, 43, 45, and 47. The worker of skill in the art will readily appreciate that the DNA of the invention comprises a double stranded molecule, for example the molecule having the sequence set forth in SEQ ID NO: 5, 12 13, 22, 23, 25, 26, 30 31, 33, 35, 37, 39, 41, 43, 45, and 47 along with the complementary molecule (the “non-coding strand” or “complement”) having a sequence deducible from the sequence of SEQ ID NOs: 5, 12 13, 22, 23, 25, 26, 30 31, 33, 35, 37, 39, 41, 43, 45, and 47 according to Watson-

Crick base pairing rules for DNA. Also contemplated by the invention are other polynucleotides encoding for the porcine, murine and african green monkey

CD163polypeptides of SEQ ID NO: 2, 14, 24, 27 and 32, 34, 36, 38, 40, 42, 44, 46, 48 which differ in sequence from the polynucleotide of SEQ ID NOs: 1, 3, 5, 12, 13, 17, 18, 22, 23, 25, 26, 30, 31, 33, 35, 37, 39, 41, 43, 45 and 47 by virtue of the well- known degeneracy of the universal genetic code, as is well known in the art. The present invention, therefore, contemplates those other DNA and RNA molecules that, on expression, encode the polypeptides of SEQ ID NO: 2, 14, 24, 27 and 32. Having identified the amino acid residue sequence encoded the porcine CD163 polypeptide, and with the knowledge of all triplet codons for each particular amino acid residue, it is possible to describe all such encoding RNA and DNA sequences. DNA and RNA molecules other than those specifically disclosed herein characterized simply by a change in a codon for a particular amino acid, are, therefore, within the scope of this invention.

A table of amino acids and their representative abbreviations, symbols and codons is set forth below in the following Table 4.

Table 4

Aminoacid | Abbrev. | Symbol [ ~~ Codon(s) |]

Cysteine | Cys | € Jue Jou | | | — | 1

Asparticacid | Asp | D Jgac eau | I

Clutamicacid | Gln | E J6AA gag | [| [TT

Phenylalanine | Phe | F Juuc Jouw | [

Glyeine ~~ | Gly [| 6 J6GA JooCc 666 joGu [| | —

Histidine ~~ | His [| H JcAc Jeau | | 1 ] =

Methionine | Met | ™M Jue | | [TT

Proline | Pro [| P OCA lccC JocG ecu

Glutamine | Gln | Q CAA cag | | | 1 ]

Threonine | The | T ACA |acC ACG JacU

Valine | val | Vv JGuA JoUC BUG Guu

Tyrosine ~~ [ Tye |v Joac au | —

As is well known in the art, codons constitute triplet sequences of nucleotides in mRNA and their corresponding cDNA molecules. Codons are characterized by the base uracil (U) when present in an mRNA molecule but are characterized by thebase thymidine (T) when present in DNA. A simple change in a codon for the same amino acid residue within a polynucleotide will not change the sequence or structure of the encoded polypeptide. It is apparent that when a phrase stating that a particular 3 nucleotide sequence “encode(s)” any particular amino acid, the ordinarily skilled artisan would recognize that the table above provides a means of identifying the particular nucleotides at issue. By way of example, if a particular three-nucleotide sequence encodes theonine the table above discloses that the posible triplet sequences are ACA, ACG, ACC and ACU (ACT if in DNA).

The invention includes therefore, an isolated polynucleotide comprising: (a) a susCD163v1 polynucleotide sequence set forth in SEQ ID NOs: 1, and 5 (b) a polynucleotide that encodes a polypeptide that has at least 70%, 71%, 72%, 73%, 74%, 15%, 716%, 11%, 18%, 719%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 93%, 94%, 95%, 96% 97% 98%, or 99%, identity and/or similarity to a polypeptide set forth in SEQ ID NO: 2 (c) a-polynucleotide encoding a polypeptide of SEQ ID: 2, (d) a polynucleotide which is the complement of any of (a), (b) or (¢).

The invention also includes an isolated polynucleotide comprising: (a) a susCD163v2 polynucleotide sequence set forth in SEQ ID NOs: 12 or 13 (b) a polynucleotide that encodes a polypeptide that has at least 99%, identity and/or similarity to a polypeptide set forth in SEQ ID NO: 14 (c) a polynucleotide encoding a polypeptide of SEQ ID: 14, (d) a polynucleotide which is the complement of any of (a), (b) or (c).

The invention also includes an isolated polynucleotide comprising: (a) a murine CD63v2 polynucleotide sequence set forth in SEQ ID NOs: 22 or 23 (b) a polynucleotide encoding a polypeptide of SEQ ID: 24, (c) a polynucleotide which is the complement of any of (a) or (b).

The invention also includes an isolated polynucleotide comprising: (a) a murine CD163v3 polynucleotide sequence set forth in SEQ ID NOs: 25 or 26 (b) a polynucleotide that encodes a polypeptide that has at least 96% 97% 98%, or 99% identity and/or similarity to a polypeptide set forth in SEQ ID NO: 27 (c) a polynucleotide encoding a polypeptide of SEQ ID: 27, (d) a polynucleotide which is the complement of any of (a), (b) or (c).

The invention also includes an isolated polynucleotide comprising: (a) an african green monkey CD163v2 polynucleotide sequence set forth in SEQ ID

NOs: 30 or 31 (b) a polynucleotide that encodes a polypeptide that has at least 98% or 99% identity and/or similarity to a polypeptide set forth in SEQ ID NO: 32 (¢) a polynucleotide encoding a polypeptide of SEQ ID: 32, (d) a polynucleotide which is the complement of any of (a), (b) or (c).

The invention also includes an isolated polynucleotide comprising: (a) a polynucleotide sequence set forth in SEQ ID NO: 33 (b) a polynucleotide that encodes a polypeptide that has at least 95% 96%, 97%, 98% or 99% identity and/or similarity to a polypeptide set forth in SEQ ID NO: 34 (c) a polynucleotide encoding a polypeptide of SEQ ID: 34, (d) a polynucleotide which is the complement of any of (a), (b) or (c).

The invention also includes an isolated polynucleotide comprising: (a) a polynucleotide sequence set forth in SEQ ID NO: 35 (b) a polynucleotide that encodes a polypeptide that has at least 95% 96%, 97%, 98% or 99% identity and/or similarity to a polypeptide set forth in SEQ ID NO: 36 (c) a polynucleotide encoding a polypeptide of SEQ 1D: 36, (d) a polynucleotide which is the complement of any of (a), (b) or (c).

The invention also includes an isolated polynucleotide comprising: (a) a polynucleotide sequence set forth in SEQ ID NO: 37 (b) a polynucleotide that encodes a polypeptide that has at least 95% 96%, 97%, 98% or 99% identity and/or similarity to a polypeptide set forth in SEQ ID NO: 38 (c) a polynucleotide encoding a polypeptide of SEQ ID: 38, (d) a polynucleotide which is the complement of any of (a), (b) or (c).

The invention also includes an isolated polynucleotide comprising: (a) a polynucleotide sequence set forth in SEQ ID NO: 39 (b) a polynucleotide that encodes a polypeptide that has at least 95% 96%, 97%, 98% or 99% identity and/or similarity to a polypeptide set forth in SEQ ID NO: 40 (c) a polynucleotide encoding a polypeptide of SEQ ID: 40, (d) a polynucleotide which is the complement of any of (a), (b) or (c).

The invention also includes an isolated polynucleotide comprising: (a) a polynucleotide sequence set forth in SEQ ID NO: 41 (b) a polynucleotide that encodes a polypeptide that has at least 95% 96%, 97%, 98% or 99% identity and/or similarity to a polypeptide set forth in SEQ ID NO: 42 (c) a polynucleotide encoding a polypeptide of SEQ ID: 42, (d) apolynucleotide which is the complement of any of (a), (b) or (c).

The invention also includes an isolated polynucleotide comprising: (a) a polynucleotide sequence set forth in SEQ ID NO: 43

(b) a polynucleotide that encodes a polypeptide that has at least 95% 96%, 97%, 98% or 99% identity and/or similarity to a polypeptide set forth in SEQ ID NO: 44 (c) a polynucleotide encoding a polypeptide of SEQ ID: 44, (d) a polynucleotide which is the complement of any of (a), (b) or (c).

The invention also includes an isolated polynucleotide comprising: (a) a polynucleotide sequence set forth in SEQ ID NO: 45 (b) a polynucleotide that encodes a polypeptide that has at least 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98% or 99% identity and/or similarity to a polypeptide set forth in SEQ ID NO: 46 (c) a polynucleotide encoding a polypeptide of SEQ ID: 46, (d) a polynucleotide which is the complement of any of (a), (b} or (c).

The invention also includes an isolated polynucleotide comprising: (a) a polynucleotide sequence set forth in SEQ ID NO: 47 (b) a polynucleotide that encodes a polypeptide that has at least 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98% or 99% identity and/or similarity to a polypeptide set forth in SEQ ID NO: 48 (c) a polynucleotide encoding a polypeptide of SEQ ID: 49, (d) a polynucleotide which is the complement of any of (a), (b) or (c).

The polynucleotide sequence information provided by the invention makes possible large-scale expression of the encoded polypeptide by techniques well known and routinely practiced in the art. Polynucleotides of the invention also permit identification and isolation of polynucleotides encoding related porcine CD163v1 polypeptides, such as human allelic variants and species homologs, by well-known techniques including Southern and/or Northern hybridization, and the polymerase chain reaction (PCR).

Knowledge of the sequence of a any of the CD163 sequences disclosed herein also makes possible through use of Southern hybridization or polymerase chain reaction (PCR), the identification of genomic DNA sequences encoding CD163 regulatory sequences, such as promoters, operators, enhancers, repressors, and the like.

As noted in the section above entitled “Assays of the Invention” polynucleotides of the invention are also useful in hybridization assays to detect the capacity of cells to express CD163, or to measure levels of CD163 expression.

Polynucleotides of the invention may also be the basis for diagnostic methods useful for determining the susceptibility of an animal to virus infection as described above.

The disclosure herein of the full-length polynucleotides encoding a

CD163polypeptide makes readily available to the worker of ordinary skill in the art fragments of the full length polynucleotide. The invention therefore provides unique fragments of the CD163 encoding polynucleotides comprising at least 15 through the length of the full-length sequence (including each and every integer value between) consecutive nucleotides of a polynucleotide encoding a CD163 disclosed herein.

Because polynucleotides of the invention (including fragments) comprise sequences unique tothe particular CD163-encoding polynucleotide sequence, they therefore would hybridize under highly stringent or moderately stringent conditions only (i.c., “specifically”) to polynucleotides encoding the various CD163 polypeptides.

Sequences unique to polynucleotides of the invention are recognizable through sequence comparison to other known polynucleotides, and can be identified through use of alignment programs routinely utilized in the art, ¢.g., those made available in public sequence databases. Such sequences also are recognizable from Southern hybridization analyses to determine the number of fragments of genomic DNA to which a polynucleotide will hybridize. Polynucleotides of the invention can be labeled in a manner that permits their detection, including radioactive, fluorescent, and enzymatic labeling.

One or more unique fragment polynucleotides (or other CD163 polynucleotides as discussed above) can be included in kits that are used to detect the presence of a polynucleotide encoding for CD 163, or used to detect variations in a polynucleotide sequence encoding for CD163.Also made available by the invention are anti-sense polynucleotides that recognize and hybridize to polynucleotides encoding CD163. Full length and fragment anti-sense polynucleotides are provided.

Fragment anti-sense molecules of the invention include (i) those that specifically recognize and hybridize to the CD163 variants disclosed herein (as determined by sequence comparison of DNA encoding CD163s to DNA encoding other known molecules). Identification of sequences unique to the novel CD163-encoding polynucleotides can be deduced through the use of any publicly available sequence database, and/or through the use of commercially available sequence comparison programs. The uniqueness of selected sequences in an entire genome can be further verified by hybridization analyses. After identification of the desired sequences, isolation through restriction digestion or amplification using any of the various polymerase chain reaction techniques well known in the art can be performed. Anti- sense polynucleotides are particularly relevant to regulating expression of CD163 by those cells expressing CD163 mRNA.

Antisense nucleic acids (preferably 10 to 20 base pair oligonucleotides) capable of specifically binding to CD163 expression control sequences or CD163

RNA are introduced into cells (e.g., by a viral vector or colloidal dispersion system such as a liposome). The antisense nucleic acid binds to the porcine CD163 target nucleotide sequence in the cell and prevents transcription or translation of the target sequence. Phosphorothioate and methylphosphonate antisense oligonucleotides are specifically contemplated for therapeutic use by the invention. The antisense oligonucleotides may be further modified by poly-L-lysine, transferrin polylysine, or cholesterol moieties at their 5’ end. Suppression of porcine CD163 expression at either the transcriptional or translational level is useful to generate cellular or animal models for diseases characterized by aberrant porcine CD163 expression or as a therapeutic modality.

As noted above in more detail, the nucleic acids of the invention include vectors comprising a polynucleotide of the invention. Such vectors are useful, e.g., for amplifying the polynucleotides in host cells to create useful quantities thereof. In other embodiments, the vector is an expression vector wherein the polynucleotide of the invention is operatively linked to a polynucleotide comprising an expression control sequence. Such vectors are useful for recombinant production of polypeptides of the invention.

Also as noted above the invention provides host cells that are transformed or transfected (stably or transiently) with polynucleotides of the invention or vectors of the invention. As stated above, such host cells are useful for the production of virus and the production of vaccines.

The invention also provides isolated CD163 polypeptides encoded by a novel polynucleotide of the invention.

Polypeptides of the Invention

The examples disclose our discovery of several novel CD163 polypeptides. The invention includes these novel CD163 polypeptide which are set forth in SEQ ID NOs: 2, 14, 19, 24, 27, 32, 34, 36, 38, 40, 42, 44, 46, and 48.

The invention includes therefore, an isolated polynucleotide comprising a susCD163v1 polypetide with the sequence set forth in SEQ ID NO: 2.

The invention also includes a polypeptide that has at least 70%, 71%, 72%, 73%, 14%, 15%, 16%, 17%, 18%, 19%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 93%, 94%, 95%, 96% 97% 98%, or 99%, identity and/or similarity to a polypeptide set forth in SEQ ID NO: 2.

The invention includes therefore, an isolated polynucleotide comprising a susCD163v2 polypetide with the sequence set forth in SEQ ID NO: 14.

The invention also includes a polypeptide that has at least 99%, identity and or similarity to a sus CD163v2 polypeptide set forth in SEQ ID NO: 14.

The invention also includes a murine CD163v2 polypeptide having the sequence set forth in SEQ ID: 24.

The invention also includes a murine CD163v3 polypeptide having the sequence set forth in SEQ ID: 27.

The invention also includes at least a polypeptide having 96% 97% 98%, or 99%, identity and/or similarity to a polypeptide set forth in SEQ ID NO: 27.

The invention also includes a polypeptide having the sequence set forth in

SEQ ID: 32.

The invention also includes a polypeptide that has at least 98% or 99%, identity and/or similarity to a polypeptide set forth in SEQ ID NO: 32.

The invention also includes a polypeptide having the sequence set forth in SEQ ID: 34.

The invention also includes a polypeptide that has at least 95%, 96%, 97%, 98%, 99% identity and/or similarity to a polypeptide set forth in SEQ ID NO: 34.

The invention also includes a polypeptide having the sequence set forth in SEQ ID: 36.

The invention also includes a polypeptide that has at least 95%, 96%, 97%, 98%, 99% identity and/or similarity to a polypeptide set forth in SEQ ID NO: 36.

The invention also includes a polypeptide having the sequence set forth in SEQ ID:

The invention also includes a polypeptide that has at least 95%, 96%, 97%, 98%, 99% identity and/or similarity to a polypeptide set forth in SEQ ID NO: 39.

The invention also includes a polypeptide having the sequence set forth in SEQ ID: 40.

The invention also includes a polypeptide that has at least 95%, 96%, 97%, 98%, 99% identity and/or similarity to a polypeptide set forth in SEQ ID NO: 40.

The invention also includes a polypeptide having the sequence set forth in SEQ ID: 42.

The invention also includes a polypeptide that has at least 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99% identity and/or similarity to a polypeptide set forth in SEQ

ID NO: 42.

The invention also includes a polypeptide having the sequence set forth in SEQ ID: 44.

ID NO: 44.

The invention also includes a polypeptide having the sequence set forth in SEQ ID: 46.

The invention also includes a polypeptide that has at least 50%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99% identity and/or similarity to a polypeptide set forth in SEQ

ID NO: 46.

The invention also includes a polypeptide having the sequence set forth in SEQ ID: : 48.

ID NO: 48.

Polypeptides of the invention may be isolated from natural cell sources or may be chemically synthesized, but are preferably produced by recombinant procedures involving host cells of the invention. Use of mammalian host cells is expected to provide for such post-translational modifications (e.g., glycosylation, truncation,

lipidation, and phosphorylation) as may be needed to confer optimal biological activity on recombinant expression products of the invention. Glycosylated and non- glycosylated forms of the novel CD163 polypeptides are embraced.

Overexpression in eukaryotic and prokaryotic hosts as described above facilitates the isolation of CD163 polypeptides. The invention therefore includes isolated CD163 polypeptides as set out in SEQ ID NOs: 2, 14, 19, 24, 27 32, 34, 36, 38, 40, 42, 44, 46, 48 and variants and conservative amino acid substitutions therein including labeled and tagged polypeptides.

The invention includes novel CD163 polypeptides that are “labeled”. The term "labeled" is used herein to refer to the conjugating or covalent bonding of any suitable detectable group, including enzymes (e.g., horseradish peroxidase, beta - glucuronidase, alkaline phosphatase, and beta-D-galactosidase), fluorescent labels (e.g., fluorescein, luciferase), and radiolabels (e.g., 1*C, "1 *°H, *P, and **S) to the compound being labeled. Techniques for labeling various compounds, including proteins, peptides, and antibodies, are well known. See, e.g., Morrison, Methods in

Enzymology 32b, 103 (1974); Syvanen et al., J. Biol. Chem. 284, 3762 (1973);

Bolton and Hunter, Biochem. J. 133, 529 (1973). The termed labeled may also encompass a polypeptide which has covalently attached an amino acid tag as discussed below.

In addition, the novel CD163 polypeptides of the invention may be indirectly labeled. This involves the covalent addition of a moiety to the polypeptide and subsequent coupling of the added moiety to a label or labeled compound that exhibits specific binding to the added moiety. Possibilities for indirect labeling include biotinylation of the peptide followed by binding to avidin coupled to one of the above label groups. Another example would be incubating a radiolabeled antibody specific for a histidine tag with a CD163s polypeptide comprising a polyhistidine tag. The net effect is to bind the radioactive antibody to the polypeptide because of the considerable affinity of the antibody for the tag.

The invention also embraces variants (or analogs) of the novel CD163 protein.

In one example, insertion variants are provided wherein one or more amino acid residues supplement a novel CD163 amino acid sequence. Insertions may be located at either or both termini of the protein, or may be positioned within internal regions of the novel CD163 protein amino acid sequence. Insertional variants with additional residues at either or both termini can include for example, fusion proteins and proteins including amino acid tags or labels. Insertion variants include novel CD163 polypeptides wherein one or more amino acid residues are added to a CD163 acid sequence, or to a biologically active fragment thereof.

Insertional variants therfore can also include fusion proteins wherein the amino and/or carboxy termini of the novel CD163 polypeptide is fused to another polypeptide. Various tag polypeptides and their respective antibodies are well known in the art. Examples include poly-histidine (poly-his) or poly-histidine- glycine (poly-his-gly) tags; the influenza HA tag polypeptide and its antibody 12CAS5 [Field et al., Mol. Cell. Biol., 8:2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan et al., Molecular and

CellularBiology, 5:3610-3616 (1985)], and the Herpes Simplex virus glycoprotein

D (gD) tag and its antibody [Paborsky et al., Protein Engineering, 3(6):547-553 (1990)]. Other tag polypeptides include the Flag -peptide [Hopp et al.,

BioTechnology, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin et al.,

Science, 255:192-194 (1992)]; an alpha -tubulin epitope peptide [Skinner et al.,

J. Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA, 87:6393-6397(1990)]. In addition, the CD163 polypeptide can be tagged with enzymatic proteins such as peroxidase and alkaline phosphatase.

In another aspect, the invention provides deletion variants wherein one or more amino acid residues in a novel CD163 polypeptide is removed. Deletions can be effected at one or both termini of the novel CD163 polypeptide, or with removal of one or more residues within the novel CD163 amino acid sequence. Deletion variants, therefore, include all fragments of the novel CD163 polypeptide.

CD163 polypeptides contain a transmembrane or membrane anchor region. It should be recognized that such transmembrane domains are useful when expressed in the context of a heterologous protein to aid in the targeting of the heterologous protein to membranes. It should also be recognized that it may be advantageous to delete some transmembrane domains to enhance the purification or solubility of the protein.

Transmembrane deleted variants of CD163 and polynucleotides encoding them are of potential value as antiviral therapeutics. Such variants are specifically disclosed here as SEQ ID NOs: 37-40.

The present invention also includes variants of the aforementioned polypetides, that is, polypeptides that vary from the reference sequence by conservative amino acid substitutions,

Exemplary conservative substitutions are set out in Tables 1, 2 and 3 in the 5s section above entitled “Definitions”.

In those situations where it is preferable to partially or completely isolate the novel CD163 polypeptides, purification can be accomplished using standard methods well known to the skilled artisan. Such methods include, without limitation, separation by electrophoresis followed by electroelution, various types of chromatography (immunoaffinity, molecular sieve, and/or ion exchange), and/or high pressure liquid chromatography. In some cases, it may be preferable to use more than one of these methods for complete purification.

Purification of novel CD163 polypeptides can be accomplished using a variety of techniques. If the polypeptide has been synthesized such that it contains a tag such as Hexahistidine (CD 163 /hexaHis) or other small peptide such as FLAG (Eastman Kodak Co., New Haven, Conn.) or myc (Invitrogen, Carlsbad, Calif.) at either its carboxyl or amino terminus, it may essentially be purified in a one-step process by passing the solution through an affinity column where the column matrix has a high affinity for the tag or for the polypeptide directly (i.e., a monoclonal antibody specifically recognizing CD163). For example, polyhistidine binds with great affinity and specificity to nickel, thus an affinity column of nickel (such as the

Qiagen Registered TM nickel columns) can be used for purification of CD163 /polyHis. (See for example, Ausubel et al., eds., Current Protocols in Molecular

Biology, Section 10.11.8, John Wiley & Sons, New York [1993]).

Even if the novel CD163 polypeptide is prepared without a label or tag to facilitate purification, the novel CD163 of the invention may be purified by immunoaffinity chromatography. To accomplish this, antibodies specific for CD163 polypeptides must be prepared by means well known in the art.

Antibodies generated against the novel CD163 polypeptides of the invention can be obtained by administering the polypeptides or epitope-bearing fragments, analogues, or cells to an animal, preferably a non-human, using routine protocols. For preparation of monoclonal antibodies, any technique known in the art that provides antibodies produced by continuous cell line cultures can be used. Examples include various techniques, such as those in Kohler, G. and Milstein, C., Nature 256: 495- 497 (1975); Kozbor et al., Inmunology Today 4: 72 (1983); Cole et al., pg. 77-96 in

Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985).

Where the novel CD163 polypeptides are prepared without a tag attached, and no antibodies are available, other well-known procedures for purification can be used. Such procedures include, without limitation, ion exchange chromatography, molecular sieve chromatography, HPLC, native gel electrophoresis in combination with gel elution, and preparative isoelectric focusing ("Isoprime”machine/technique,

Hoefer Scientific). In some cases, two or more of these techniques may be combined to achieve increased purity.

It should be understood that the definition of polypeptides of the invention is intended to include polypeptides bearing modifications other than insertion, deletion, or substitution of amino acid residues. By way of example, the modifications may be covalent in nature, and include for example, chemical bonding with polymers, lipids, other organic, and inorganic moieties.

Antibodies

Also comprehended by the present invention are antibodies (e.g., monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, bifunctional/bispecific antibodies, humanized antibodies, human antibodies, and complementary determining region (CDR)-grafted antibodies, including compounds which include CDR sequences which specifically recognize a polypeptide of the invention) specific for novel CD163 or fragments thereof.

The term “specific for,” when used to describe antibodies of the invention, indicates that the variable regions of the antibodies of the invention recognize and bind CD163s polypeptides exclusively (i.e., able to distinguish CD163s polypeptides from other known polypeptides by virtue of measurable differences in binding affinity, despite the possible existence of localized sequence identity, homology, or similarity between the novel CD163 and such polypeptides). It will be understood that specific antibodies may also interact with other proteins (for example, S. aureus protein A or other antibodies in ELISA techniques) through interactions with sequences outside the variable region of the antibodies, and in particular, in the constant region of the molecule. Screening assays to determine binding specificity of an antibody of the invention are well known and routinely practiced in the art. For a comprehensive discussion of such assays, see Harlow et al. (Eds), Antibodies A Laboratory Manual;

Cold Spring Harbor Laboratory; Cold Spring Harbor, NY (1988), Chapter 6.

Antibodies that recognize and bind fragments of the CD163s polypeptides of the invention are also contemplated, provided that the antibodies are, first and foremost, specific for novel CD163 polypeptides. Antibodies of the invention can be produced using any method well known and routinely practiced in the art. Non-human antibodies may be humanized by any methods known in the art. In one method, the non-human CDRs are inserted into a human antibody or consensus antibody framework sequence. Further changes can then be introduced into the antibody framework to modulate affinity or immunogenicity.

Antibodies of the invention are useful for, diagnostic purposes to detect or quantitate CD163s, as well as purification of CD163s. Kits comprising an antibody of the invention for any of the purposes described herein are also comprehended. In general, a kit of the invention also includes a control antigen for which the antibody is immunospecific

The present invention is further illustrated, but not limited, by the following examples.

Example 1: Transient transfection with porcine C163 confers permissivity to

PRRS virus infection to a non-permissive cell line. Total mRNA from primary porcine alveolar macrophage cells was used to construct a cDNA library in the plasmid pCMV-Sport6.1 (Invitrogen), with the cDNA cloned between the EcoRV and

Notl sites. A member of this library, when isolated and transiently transfected into the

BHK-21 (baby hamster kidney) cell line, conferred a PRRS-permissive phenotype.

Cells were grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 5% fetal bovine serum (FBS) in a 5% CO, atmosphere at 37°C. Cell cultures were transiently transfected using 10.0 uL of Lipofectamine 2000 (Invitrogen) and 2.0ug of plasmid. A duplicate monolayer was transfected with negative control plasmid pPAMB. This plasmid is pPCMV-Sport6.1 lacking an insert. Transfection efficiency was monitored with a plasmid expressing green fluorescent protein (GFP).

Approximately 24 hours post-transfection, monolayers were infected with either North

American (isolate P129) or European (isolate 96V 198) genotypes of PRRS virus. For detection of PRRS replication, the monolayers were fixed using 80% acetone approximately 24 hours post-infection and incubated for approximately 1 hour with

FITC-conjugated monoclonal antibody SDOW17 (Rural Technologies Inc.). This monoclonal antibody is specific for PRRS viral nucleocapsid expressed from open reading frame 7. A Nikon TE 300 inverted fluorescent microscope with a 10x objective was used to photograph a monolayer containing FITC positive cells and a negative control monolayer.

It was confirmed that transfected cells became permissive to both the North

American (isolate P129) and European (isolate 96V 198) genotypes of PRRSV.

Expression of viral genes could be detected in many of the transfected BHK cells, and progeny virus was readily detectable in the supernatant. Control transfections using vector without insert or irrelevant plasmids did not confer permissivity.

Sequencing of the insert in the functional plasmid, using the Big Dye

Terminator Version 1.0 Sequence Reaction kit (Applied Biosystems, Foster City, CA) and the Applied Biosystems 3730 DNA Analyzer (Applied Biosystems), revealed a gene that was highly homologous to the published porcine CD163 gene cDNA (Genbank accession number AJ311716). The cDNA we identified contained additional 5’ and 3’ untranslated regions relative to AJ311716, and the open reading frame differed in three ways: (1) a 738 bp internal deletion near the 5° end, (2) a 15 bp extension of the 5’ end to an upstream ATG codon, and (3) sixteen nucleotide changes predicted to cause 10 amino acid changes. Nucleotide sequence identity between the sequences was 99.4%. Alignments of the newly discovered porcine CD163 sequence with the previously reported sequence AJ311716 are shown in figures 1 and 2. The novel porcine CD163 variant was designated “susCD163v1”.

SEE [ow gtaataatac aagaagattt aaatgggcat aaaaccttgg aatggacaaa ctcagaatgg 60 SEQ ID tgctacatga aaactctgga tctgcagacc tgaaactgag agtggtagat ggagtcactg 120 NO:5 aatgttcagg aagattggaa gtgaaattcc amggagaatg gggaacaatc tgtgatgatg 180 gctgggatag tgatgatgce getgtggeat gtaagcaact gggatgtcea actgetgtca 240 ctgccattgg tcgagttaac gocagtgagg gaactggaca catttggett gacagtgttt 300 cttgccatgg acacgagtct gectcototgge agtgtagaca ccatgaatgg ggaaagcatt 360 atigcaatca taatgaagat gctggtgtga catgttctga tggatcagat ctggaactga 420 taggaaaagt gtgtgataga agctggggac tgaaagaagc tgatgtggtt tgcaggcage 540 tgggatgtgg atctgeactc aaaacatcat atcaagttta ttccaaaacc aaggcaacaa 600 acacatggct gtttgtaagc agctgtaatg gaaatgaaac ttetctttgg gactgcaaga 660 attggcagtyg gggtggactt agttgtgatc actatgacga agccaaaatt acctgctcag 720 cccacaggaa acccaggety gttggagggg acattccctg ctotggtcgt gttgaagtac 780 aacatggaga cacgtggggc accgtctgtyg attctgactt ctctctggag geggecageg 840 tgctgtgcag ggaactacag tgeggeactg tggtttccct cctgggggga getcactttg 900 gagaaggaag tggacagatc tgggctgaag aattccagtg tgaggggeac gagtcccace 960 tttcactctg cccagtagca ccccgecetg acgggacatg tagccacage agggacgteg 1020 gegtagtctg ctcaagatac acacasatcC gcttggtgaa tggcaagacce ccatgtgaag 1080 gaagagtgga gctcaacatt cttgggtcct gggggtccct ctgcaactct cactgggaca 1140 tggaagatgc ccatgtttta tgccageage ttaaatgtgg agttgocctt tctatecccgg 1200 gaggagcacc ttttgggaaa ggaagtgage aggtctggag gcacatgttt cactgcactg 1260 ggactgagaa gcacatggga gattgttccg tcactgetct gggegcatca ctetgttott 1320 cagggcaagt ggcctctgta atctgctcag ggaaccagag tcagacacta tcccegtgea 1380 attcatcatce ctcggaccca tcaagctcta ttatttcaga agaaaatggt gttgcctgca 1440 tagggagtgg tcaacttcge ctggtcgatg gaggtggtcg ttgtgctggg agagtagagg 1500 tctatcatga gggctcoctgg ggcaccatct gtgatgacag ctgggacctg aatgatgcce 1560 atgtggtgtg caaacagctg agctgtggat gggccattaa tgecactggt tctgetcatt 1620 ttggggaagg aacagggccc atttggetgg atgagataaa ctgtaatgga aaagaatctc 1680 atatttggca atgccactca catggttggg ggcggcacaa ttgcaggcat aaggaggatg 1740 caggagicat ctgctecggag ttcatgtcte tcagactgat cagtgaaaac agcagagaga 1800 cctgtgecagg gegectggaa gttttttaca acggagettg gggcagegtt ggcaagaata 1860 gcatgtctce agccacagtg ggggtggtat gcaggeaget gggctgtgca gacagagggg 1920 acatcagcce tgcatcttca gacaagacag tgtccaggea catgtgggtg gacaatgtte 1980 agtgtcctaa aggacctgac accctatgge agtgcccatc atctccatgg aagaagagac 2040 tggccagece ctcagaggag acatggatca catgtgccaa caaaataaga cttcaagaag 2100 gaaacactaa ttgttctgga cgtgtggaga tctggtacgg aggtteectgg ggcactgtgt 2160 gtgacgactc ctgggacctt gaagatgcetc aggtggtgtg ccgacagetg ggctgtgget 2220 cagctttgga ggcaggaaaa gaggcegcat ttggccaggg gactgggccc atatggctca 2280 atgaagtgaa gtgcaagggg aatgaaacct ccttgtggga ttgtcctgcc agatcctggg 2340 gccacagtga ctgtggacac aaggaggatg ctgctgtgac gtgttcagaa attgcaaaga 2400 gccgagaatc cctacatgcc acaggtcget catcttttgt tgcacttgea atctttgggg 2460 tcattectgtt ggectgtctc atcgcattee tcatttggac tcagaagcga agacagagge 2520 agcggctetc agttttcotca ggaggagaga attctgtcca tcaaattcaa taccgggaga 2580 tgaattcttg cctgaaagca gatgaaacgg atatgctaaa tccctcagga gaccactetg 2640 aagtacaatg aaaaggaaaa tgggaattat aacctggtga gttcagcctt taagatacct 2700 tgatgaagac ctggactatt gaatgagcaa gaatctgcct cttacactga agattacaat 2760 acagtcctct gtctcetggt attccaaaga ctgctgttga atttctaaaa aatagattgg 2820 tgaatgtgac tactcaaagt tgtatgtaag actttcaagg gcattaaata aaaaagaata 2880 ttgctgaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2930 te ® atagacaactcagastautostacate Cott go Ren SEQID

21V V DG V T B C 8 PIA MEA air

61 gtggtagatggagtcactgaatgttcaggaagattggaagtgaaattccaaggagaatag NO:1 4G T I C DD 8 WD SDD AA AVA ATCIKTG QQHL

122 Sp Terre Ic g9gatagtaatgatgecgetgtageatgtaageaacty and 2 181 ggatgtccaactgetgt cactgecattggtcgagt taacgccagtgagggaactggacac

811 W L DS V 8 CHG HUE SAULWNAOQT CT RH

A 502 241 atttggcttgacagtgtttcttgecatggacacgagtorgotetetggcagtgtagacac 101H BR ¥ G K H Y C NH NEDA ARAGUV TC SD 301 catgaatggggaaagcattattgcaatcataatgaagatgctggtgtgacatgttctgat 1216 8S DL EL RL KQ@@GQ@ 8 HCAGT VE 361 ggatcagatctggaactgagacttaaaggtggaggcagceccactgtgetgggacagtggag 141 V BE I Q KL V G K V C DR 8 WGUL K E A 421 gtggaaattcagaaactggtaggaaaagtgtgtgatagaagctggggactgaaagaagcet 61D Vv v CC R QQ L GC GS AL KT S88 Y Q VY 481 gatgtggtttgcaggcagctgggatgtggatctgcactcaaaacatcatatcaagtttat 181 S K T K A T NT WULF V 8 8 CNGNZBEBT

S41 tccaaaaccaaggcaacaaacacatggctgtttgtaagcagctgtaatggaaatgaaact 2018S L WD CKNWXOQWGUA3L SS CDHYDE 601 tcteoctttgggactgcaagaattggcagtggggtggacttagttgtgatcactatgacgaa 221A K I T C S A HRKUPRULUVGGUDTI POC 661 gccaaaattacctgctcagcccacaggaaacccaggctggttggaggggacatteeetge 2418 @ R V BR V Q H 6G DTW GT V CD S DF 721 tctggtcgtgttgaagtacaacatggagacacgtggggcaccgtcetgtgattctgactte 26S LL. BAAS VL CRUZ EBELAGQCGCGT VV § L 781 tctctggaggcggccagegtgotgtgeagggaactacagtgeggeactgtggtttoecte 281. GG 3 AH FP G R G S§ GG Q I Ww A ERP QC 841 ctggggggagctcactttggagaaggaagtggacagatctgggctgaagaattccagtgt

E @ HH BR SS HL 8 L CP V A PRP DGTUZC 901 gaggggcacgagtcccacctttcactetgeccagtagcaccecgecctgacgggacatgt 3218s H S R D V 66 V Vv ¢C 8s R ¥Y T Q I R L V N 961 agccacagcagggacgtcggogtagtcetgetcaagatacacacaaatcegettggtgaat 341 GCG K T P C ERE GG R V BL N I L G 8 W GG 8 L 1021 ggcaagaccccatgtgaaggaagagtggagcetcaacattcttgggtectgggggteccte 361 C N 8 H WDM EDA HUYVL CQ QL K C @G 1081 tgcaactctcactgggacatggaagatgcccatgttttatgecagcagettaaatgtgga 381 V A L 6§ IT P GG G A PPG KG SE Q V WR 1141 gttgccctttctatcecgggaggageaccttttgggaaaggaagtgageaggtctggagyg 401 H M F H C T G T B K HR M GG DCS V T A L 21201 cacatgtttcactgcactgggactgagaagecacatgggagattgttecogtcactgetctg 421 G A § L C 8 8S G Q V A BB V I C 8 G N Q S 1261 ggegeatcactctgttettcagggcaagtggectetgtaatctgetcagggaaccagagt 441 Q T . S$ P C N 8 8 8 Ss D P 8 8 8 I I 8 BE 1321 cagacactatccccgtgcaattcatcatcctcggacccatcaagetctattatttoagaa 461 8B N G6 V A C I G 8 G QL RL V D G3 @& G R 1381 gaaaatggtgttgcctgcatagggagtggtcaacttegectggtogatggaggtggtegt 481 C A G R V R V Y H B G 8 WG3@TTIOCTUDTUD 8 1441 tgtgctgggagagtagaggtctatcatgagggctoctggggeaccatctgtgatgacage

S01W D L N D A BEV V CK QL 8 CG WATIN 1501 tgggacctgaatgatgcccatgtggtgtgcaaacagctgagetgtggatgggccoattaat

S522 A T G S A BE F G EG TGP I WL DUE ETIN 1561 gccactggttcetgcetcattttggggaaggaacagggecccatttggetggatgagataaac

S41 C N G K E 8S H I W Q CH S HG W 3@UR HN 1621 tgtaatggaaaagaatctcatatttggcaatgccactcacatggttgggggeggcacaat

S61C R H K ED AG VI CS EF MB L RL I 1681 tgcaggcataaggaggatgcaggagtcatctgeteggagtteatgtctetcagactgate

S81 Ss E N S R ET C A GR L BE V P Y NG A W 1741 agtgaaaacagcagagagacctgtgcagggegectggaagttttttacaacggagettgg 6016 8s Vv @G K N 8 M 8 P AT V 6 V V CR QL 1801 ggcagcgttggcaagaatagcatgtctccagecacagtgggagtggtatgeaggeagetg 621 G C A D R G D I 8 P A 8S 8S D KT V § R H 1861 ggctgtgcagacagaggggacatcagccetgeatcttcagacaagacagtgtecaggeac 641 M W V D N V Q C P K GG P D TUL W Q C P § 1921 atgtgggtggacaatgttcagtgtcctaaaggacctgacaccctatggcagtgeccatca 661 S P W X K RL A 8 P 8 EE TW I T CAN 1981 tctccatggaagaagagactggccagcccctcagaggagacatggatcacatgtgccaac 681 K I RL Q B GG N T NC 8 @ R V BE I W Y 6G 2041 aaaataagacttcaagaaggaaacactaattgttctggacgtgtggagatctggtacgga 701 G 8S W G T V C DDS W DIL EDO AGQV V C 2101 ggttcctggggcactgtgtgtgacgactcctgggaccttgaagatgetcaggtggtgtac 721R Q L G C G 8S AL E A GG K B A A F G @Q @ 2161 cgacagctgggetgtggetcagetttggaggecaggaaaagaggecgeatttggecaggag 741 T G P I W L N B V K C K @ N BT 8 L W D 2221 actgggcccatatggcetcaatgaagtgaagtgcaaggggaatgaaacctecttgtgggat 761 C P AR 8S W G H S D CG H KE EUDA AA BAUVT 2281 tgtcctgecagat cctggggccacagtgactgtggacacaaggaggatgetgctgtgacy 781 C § E I A K S R E 8 L H AT GR 8 8 F V 2341 tgttcagaaattgcaaagagccgagaatcectacatgeocacaggtcgctcatettttgtt 80lA L RAR I P 6 V I L L ACTILTIA ATFULTIMWWT 2401 gcacttgcaatctttggggtcattcetgttggcctgtetcatcgecattcctcatttggact 8219 K R R Q R Q RL 86 VP 8 G G BE N 8 V H 2461 cagaagcgaagacagaggcagcggctctcagtttt ctcaggaggagagaattctgtceat 841 Q I Q Y R BR M N S CL K A DETUDMTLN 2521 caaattcaataccgggagatgaattcttgcctgaaagcagatgaaacggatatgctaaat

B61 P 8S G D H S BR V Q 2581 ccctcaggagaccactctgaagtacaa 1 MDKLRMVLHE NSGSADLKLR VVDGVTECSG RLEVKPQGEW GTICDDGWDS SEO ID s1 DDAAVACKQL GCPTAVTAIG RVNASEGTGH IWLDSVSCHG HESALWQCRH

101 HEWGKHYCNH NEDAGVTCSD GSDLELRLKG GGSHCAGTVE VEIQKLVGKV E 151 CDRSWGLKBA DVVCRQLGCG SALKTSYQVY SKTKATNTWL FVSSCNGNET 201 SLWDCKNWQW GGLSCDHYDE AKITCSAHRK PRLVGGDIPC SGRVEVQHGD 251 TWGTVCDSDP SLEAASVLCR ELQCGTVVSL LGGAHPGEGS GQIWAEEFQC 301 EGHESHLSLC PVAPRPDATC SHSRDVGVVC SRYTQIRLVN GKTPCEGRVE 351 LNILGSWGSL CNSEWDMEDA HVLCQQLKCG VALSIPGGAP FGKGSEQVWR 401 HMPHCTGTEK HMGDCSVTAL GASLCSSGQV ASVICSGNQS QTLSPCNSSS 451 SDPSSSIISE RNGVACIGSG QLRLVDGGGR CAGRVEVYHE GEWGTICDDS 501 WDLNDAHVVC KQLSOGWAIN ATGSAHFGEG TGPIWLDEIN CNGKESHIWQ 551 CHSHGWGRHN CRHKEDAGVI CSEFMSLRLI SBNSRETCAG RLEVFYNGAW 601 GSVGKNSEMSP ATVGVVCRQL GCADRGDISP ASSDKTVSRH WWVDNVQCPK 651 GPDTLWQCPS SPWKKRLASP SEETWITCAN KIRLQBEGNTN CSGRVEIWYG 701 GSWGTVCDDS WDLEDAQVVC RQLGCGSALE AGKRAAFGQG TGPIWLNEVK 751 CKGNBTSLWD CPARSWGHSD CGHEEDAAVT CSEIAKSRES LHATGRSSFV 801 ALAIPGVILL ACLIAFLIWT QKRRQRQRI:S VFSGGENSVH QIQYREMNSC 851 LKADBTDVMLN PSGDHSEVQ

Example 2: Construction of plasmid pCMVsusCD163v1

Construction of the plasmid pCMVsusCD163v1 was performed as follows. The functional clone identified in the primary porcine macrophage cDNA library as 5s conferring PRRSV permissivity served as template for PCR amplification of the

CD163 insert, including the 5° and 3’ untranslated regions, using the primers 5’'DS-

CD163 (SEQ ID NO:6) (5’-CGGAATTCCGCGGATGTAATAATACAAGAAGA- 3’) and 3’CD163 (SEQ ID NO:7) (5’CCGCTCGAGTAGTCCAGGTCTTCATCAAGGTATCTT-3’). Primer 5’DS- CD163 incorporates a Sacll restriction site at the 5’ end of the CD163 insert, while primer 3’CD163 incorporates an Xhol restriction site at the 3’ end of the insert (underlined). Reactions containing 190ng of plasmid template were amplified using

Platinum Pfx DNA polymerase (Invitrogen cat # 11708-013) following the manufacture’s instructions. Reactions were heated to 94° for 2 minutes then cycled 35 times through 94° for 20 seconds, 55° for 30 seconds, and 68° for 3.5 minutes followed by a terminal extension at 72° for 7 minutes. The resulting PCR products were purified using the Qiaquick PCR purification kit (Qiagen cat # 28104), digested with restriction enzymes Sacll and Xhol, and the resulting fragments were gel purified using the Qiaquick Gel Extraction kit (Qiagen cat # 28704). The CD163 PCR fragment was then ligated into the plasmid pCMV-Script (Stratagene cat # 212220) prepared to accept the digested PCR fragment by digestion with Sacll and Xhol followed by gel purification as described above. The ligated material was transformed into E. coli strain DH5a and recombinants were selected by growth in 50pg/ml kanamycin and identified by restriction analysis. The resulting plasmid, “pCMVsusCD163v1”, contains the internally deleted porcine CD163 insert described in Example 1 under the transcriptional control of the eukaryotic CMV promoter and the neomycin/kanamycin resistance gene under the control of both eukaryotic and prokaryotic promoters.

Example 3: Construction of the pRSV-Script expression vector and pRSVsusCD163v1

The plasmid pRc/RSV (Invitrogen) was used as a template for PCR amplification of the RSV promoter. RSV promoter sequence was contained within nucleotides 209 through 604 of pRc/RSV. Forward primer PCIRSVLTR (SEQ ID

NO:8) (5’-ACACTCGACATGTCGATGTACGGGCCAGATATACGCGT-3’) and reverse primer VSRRTLSAC (SEQ ID NO: 9) (S’TTCCTTACAGAGCTCGAGGTGCACACCAATGTGGTGAA -3’) were synthesized. Restriction endonuclease Pci I and Sac I recognition sites (underlined) were incorporated into the 5’ and 3’ primers, respectively, for future cloning. PCR was performed using the HotMaster Taq DNA Polymerase kit (Eppendorf) following the manufacturer’s instructions. The reactions contained 0.9ng of pRc/RSV plasmid template and 0.3uM of each primer described above. The reactions were heated to 94° for 2 minutes then cycled 30 times through 94° for 20 seconds, 52° for 10 seconds, and 65° for 1 minute. The resulting PCR fragment was digested with restriction enzymes Pci I and Sac I, gel purified, and cloned into the plasmid pCMV-

Script (Stratagene) that had been similarly digested to remove the CMV promoter sequence. The final construct placed the RSV promoter immediately upstream of the multiple cloning site, and was named “pRSV-Script”.

The susCD163v1 insert was cloned behind the RSV promoter as follows. The susCD163v1 sequence was excised from plasmid pCMVsusCD163v1 by restriction digestion (Kpn I and Sac II) and gel purified. This fragment was ligated into pRSV-

Script which had also been digested with the same enzymes and gel purified. The ligation mixture was transformed into DH5a. E. coli and transformants selected using kanamycin at 50ug/ml. The clone contained the correct insert was designed “pRSVsusCD163v1”.

Example 4: Cloning and characterization of a longer variant of porcine CD163 cDNA

Based on the porcine CD163v1 sequence, a forward primer 5’CD163Notllong (SEQ ID NO:10) (5’CGGTCCGGAGCGGCCGCGATGTAATAATACAAGAAGATTTAAATGG-3’) and a reverse primer 3'CD163Kpnl (SEQ ID NO:11)

(5'CGGTTGGTACCCAGCAATATTCTTTTTTATTTAATGCC-3") were designed using the Lasergene PrimerSelect program (DNASTAR Inc., Madison WI) for amplification of a full-length porcine CD163 gene. Restriction endonuclease sites for

Not 1 and Kpn I (underlined) were included in 5° and 3’ primers, respectively, to allow s for convenient cloning. Total cellular RNA was prepared from primary alveolar macrophages (PAM) harvested from lung lavages of healthy pigs. RNA preparation was done using the RNeasy mini kit (Qiagen, Valencia, CA). RT-PCR reactions were prepared using the SuperScript one-step RT-PCR for Long Templates kit (Invitrogen,

Carlsbad, CA) and RT-PCR parameters were set as follows: 50°C for 30 min, 94°C for 2 min, (94°C 30 sec, 55°C 30 sec and 68°C 4 min) for 35 cycles, 72°C for 10 min.

PCR products were analyzed on 0.8% SeaKem GTG agarose gels. RT-PCR products of various sizes were cut from agarose gels and DNA was extracted using the

GeneClean kit (QBiogene). These RT-PCR products were cloned into the pCR2.1-

TOPO cloning vector (Invitrogen). Clones were analyzed by restriction enzyme digestion for the presence of an insert. Colonies containing inserts were sequenced using Big Dye Terminator Version 1.0 Sequence Reaction kit (Applied Biosystems,

Foster City, CA) and Applied Biosystems 3730 DNA Analyzer (Applied Biosystems) to confirm sequence authenticity. Sequences were edited and assembled using the

Lasergene EditSeq and SeqMan programs (DNASTAR Inc., Madison WI). One plasmid with a large insert was designated “pCRsusCD163v2” (pCR2.1 containing porcine CD163 variant 2 which we have designated SEQ ID NO:12). The coding sequence contained within SEQ ID NO:12 is reproduced below and is designated SEQ

ID NO:13. Sequence analysis showed that this porcine CD163 encodes an amino acid sequence of 1115 amino acids which we have designated SEQ ID NO:14. When compared to the porcine CD163 sequence in GenBank (Accession No. AJ311716), our CD163v2 sequence is 98.9% identical at the amino acid level. CD163v2 also has an additional 5 amino acid residues at the extreme 5’ end, extending the open reading frame to an in-frame upstream ATG initiation codon (as in the porcine CD163v1 sequence described in example 1). Porcine CD163 is 84.3% identical to human

CD163 (GenBank Accession No. Z22968), and 73.7% identical to mouse CD163 (GenBank Accession No. AF274883) at the amino acid level. The predicted signal sequence and transmembrane region of SEQ ID NO:14 are indicated by underlining and bolding respectively.

To determine whether other CD163 sequences contain similar sequence features is easily determined by inspection of the sequence. gtaataatac aagaagattt aaatggcata aaaccttgga dtggacaaac tcagaatggt 60 SEQ ID gctacatgaa aactctggat ctgcagactt tagaagatgt tctgeccatt taagttectt 120 NO:12 cacttttgct gtagtcgcetg ttcetcagtge ctgcttggte actagttctc ttggaggaaa 180 agacaaggag ctgaggctaa cgggtggtga aaacaagtge tctggaagag tggaggtgaa 240 agtgcaggag gagtggggaa ctgtgtgtaa taatggctgg gacatggatg tggtctctgt 300 tgtttgtagg cagctgggat gtccaactge tatcaaagcc actggatggg ctaattttag 360 tgcaggttct ggacgcattt ggatggatca tgtttcttgt cgagggaatg agtcagctct 420 ctgggactgc aaacatgatg gatggggaaa gcataactgt actcaccaac aggatgctgg 480 agtaacctgc tcagatggat ctgatttaga gatggggctg gtgaatggag gaaaccggtg 540 cttaggaaga atagaagtca aatttcaagg acggtgggga acagtgtgtg atgataactt 600 caacataaat catgcttcetg tggtttgtaa acaacttgaa tgtggaagtg ctgtcagttt 660 ctctggttca gectaattttg gagaaggttc tggaccaatc tggtttgatg atcttgtatg 720 caatggaaat gagtcagctc tctggaactg caaacatgaa ggatggggaa agcacaattg 780 cgatcatgct gaggatgctg gagtgatttg cttaaatgga gcagacctga aactgagagt 840 ggtagatgga gtcactgaat gttcaggaag attggaagtg aaattccaag gagaatgggg 900 aacaatctgt gatgatggct gggatagtga tgatgccget gtggcatgta agcaactggg 960 atgtccaact gectgtcactg ccattggtcg agttaacgcc agtgagggaa ctggacacat 1020 ttggcttgac agtgtttett gecatggaca cgagtetget ctetggeagt gtagacacca 1080 tgaatgggga aagcattatt gcaatcatga tgaagatgct ggtgtgacat gttctgatgg 1140 atcagatctg gaactgagac ttaaaggtgg aggcagccac tgtgctggga cagtggaggt 1200 ggaaattcag aaactggtag gaaaagtgtg tgatagaagc tggggactga aagaagctga 1260 tgtggtttge aggcagetgg gatgtggatc tgcactcaaa acatcatatc aagtttatte 1320 caaaaccaag gcaacaaaca catggctgtt tgtaagcagc tgtaatggaa atgaaacttc 1380 tctttgggac tgcaagaatt ggcagtgggg tggacttagt tgtgatcact atgacgaagc 1440 caaaattacc tgctecagecce acaggaaacc caggetggtt ggaggggaca ttecctgete 1500 tggtcgtgtt gaagtacaac atggagacac gtggggcacc gtctgtgatt ctgacttete 1560 tctggaggeg gccagegtge tgtgcaggga actacagtge ggcactgtgg tttecctect 1620 ggggggagct cactttggag aaggaagtgg acagatctgg gcotgaagaat tccagtgtga 1680 ggggcacgag tcccaccttt cactctgecc agtagcaccee cgecectgacg ggacatgtag 1740 ccacagcagg gacgtcggcg tagtctgetc aagatacaca caaatccget tggtgaateg 1800 caagacccca tgtgaaggaa gagtggaget caacattcett gggtectggg ggtcecctctg 1860 caacictcac tgggacatgg aagatgccca tgttttatge cagcagetta aatgtggagt 1920 tgccetttet atccogggag gagcaccttt tgggaaagga agtgagcagg tctggaggca 1980 catgtttcac tgcactggga ctgagaagca catgggagat tgttccgtca ctgctctggg 2040 cgcatcactc tgttcttcag ggcaagtgge ctctgtaate tgctcaggga accagagtca 2100 gacactatct cegtgcaatt catcatcctc ggacceatca agctctatta tttcagaaga 2160 aaatggtgtt gcctgcatag ggagtggtca acttcgectg gtcgatggag gtggtogttg 2220 tgctgggaga gtagagqtct atcatgaggg ctcetgggge accatctgtg atgacagetg 2280 ggacctgaat gatgcccatg tggtgtgcaa acagctgage tgtggatggg ccattaatge 2340 cactggttct gctcattttg gggaaggaac agggeccatt tggetggatg agataaactg 2400 taatgocaaaa gaatctcata tttggcaatg ccactcacat ggttggggge ggcacaattg 2460 caggcataag gaggatgcag gagtcatctg ctcagagttc atgtctctga gactgatcag 2520 tgaaaacagc agagagacct gtgcagggcg cctggaagtt ttttacaacg gagcttggag 2580 cagcgttgge aggaatagca tgtctccage cacagtgggg gtggtatgca ggcagetggg 2640 ctgtgcagac agaggggaca tcagccctgc atcttcagac aagacagtgt ccaggeacat 2700 gtgggtggac aatgttcagt gtcctaaagg acctgacaca ctatggcagt gcccatcatc 2760 tceatggaag aagagactgg ccagccecctc agaggagaca tggatcacat gtgccaacaa 2820 aataagactt caagaaggaa acactaattg ttctggacgt gtggagatct ggtacggagg 2880 ttcctgggge actgtgtgtg acgactcctg ggaccttgaa gatgctcagg tggtgtgeeg 2940 acagctgggc tgtggctcag ctttggaggc aggaaaagag gccgcatttg gecaggggac 3000 tgggcccata tggctcaatg aagtgaagtg caaggggaat gaaacctcoct tgtgggattg 3060 tcctgccaga tcctggggece acagtgactg tggacacaag gaggatgctg ctgtgacgtg 3120 ctcagaaatt gcaaagagcc gagaatccct acatgccaca ggtcgctcat cttttgttge 3180 acttgcaatc tttggggtca ttctgttggc ctgtctcatc geattcctceca tttggactca 3240 gaagcgaaga cagaggcagc ggctctcagt tttctcagga ggagagaatt ctgtccatca 3300 aattcaatac cgggagatga attcttgcct gaaagcagat gaaacggata tgcetaaatcco 3360 ctcaggagac cactctgaag tacaatgaaa aggaaaatgg gaattataac ctggtgagtt 3420 cagectttaa gataccttga tgaagacctg gactattgaa tgagcaagaa tctgectett 3480 acactgaaga ttacaataca gtcctctgte tcctggtatt ccaaagactg ctgetgaatt 3540 tctaaagaat agattggtga atgtgactac tcaaagttgt atgtaagact ttcaagggca 3600 ttaaataaaa aagaatattg ctg 3623 1M D KL RM VL HENS G S88 ADV FRTZRC SEQ ID 1 atggacaaactcagaatggtgctacatgaaaactctggatctgcagactttagaagatgt 2S A HL 8 §$S FTF AV YV AVL 8A CULV 61 tctgcccatttaagttecttcacttttgetgtagtcecgetgttetecagtgectgettggte NO:13 42 T §s §s L 6G G XK D K EL RL TG GUE ENIZKZC 121 actagtictcttggaggaaaagacaaggagctgaggctaacgggtggtgaaaacaagtge and14 618 G R V BE V KV QB EW GT V CNNG W 181 tctggaagagtggaggtgaaagtgcaggaggagtggggaactgtgtgtaataatggectgg 81D M D V V 8 V V CR QL 6 C PTA I K A 241 gacatggatgtggtctetgtigtttgtaggcagectgggatgtccaactgetatcaaagee 102 T 6 W A N F S A BG 8S G R XI W MDH V ss C 301 actggatgggctaattttagtgcaggttetggacgeatttggatggateatgtttcttgt 322 R @ N E 8S A LL WD C K HD GW G K HN C 361 cgagggaatgagtcagctctctgggactgcaaacatgatggatggggaaagcataactgt 141 T H Q Q D A @ V T C 8S D G § DL B M G L 421 actcaccaacaggatgctggagtaacctgctcagatggatctgatttagagatgggactg 161 V N G 8 N R CL @ RTI BV X F Q GR W G 481 gtgaatggaggaaaccggtgcttaggaagaatagaagtcaaatttcaaggacggtgggga l8l1T Vv ¢C D D NF NINUHMD ASV V CIKOQTULE 541 acagtgtgtgatgataacttcaacataaatcatgcttctgtggtttgtaaacaacttgaa 200 C G 8s A V S P S G8 8 A NF GG EG S G P I 601 tgtggaagtgctgtcagtttctctggttcagctaattttggagaaggttctggaceaate 221 W P D DL V CNUG GNU BS AL WNUZ CI KIUBHE 661 tggtttgatgatcttgtatgcaatggaaatgagtcagetctetggaactgcaaacatgaa 241 G W G KH NCD HAZ BDA AG GV ICLNSG 721 ggatggggaaagcacaattgcgatcatgcotgaggatgetggagtgatttgettaaatgga 261A D L X L RV VD GV TU ETC CSGURULTEUV 781 gcagacctgaaactgagagtggtagatggagtcactgaatgttcaggaagattggaagty 281 K FP Q G E W G T I C DD G@WD 8S DD A RA 841 aaattccaaggagaatggggaacaatctgtgatgatggctgggatagtgatgatgecoget 30lV A CK QL G@GCUPTAVTA ATIUGT RV XN A 901 gtggcatgtaagcaactgggatgtccaactgctgtcactgecattggtcgagttaacgee 3218 B 6G TT @G HI WL DS V 8 CH GHE 8 A 961 agtgagggaactggacacatttggcttgacagtgtttettgecatggacacgagtctget

$/011502 i M1 L W Q C R H H B WA XK HY CMNJBHTDE D A 1021 ctctggcagtgtagacaccatgaatggggaaagcattattgcaatcatgatgaagatgcet 361 6 Vv TT ¢C 8 D GG $s DL EL RL KG GG 8 H 1081 ggtgtgacatgttctgatggatcagatctggaactgagacttaaaggtggaggcagccac 381 C A GT V B V EI Q KL VG K V CDR 8 1141 tgtgctgggacagtggaggtggaaattcagaaactggtaggaaaagtgtgtgatagaage 401 W 6 L K B A PD V V ¢C R QL GCG SAUL K 1201 tggggactgaaagaagctgatgtggtttgcaggeagetgggatgtggatctgcactcaaa 421 T § Y Q V Y 8 KT KA T NT WL PF V § S 1261 acatcatatcaagtttattccaaaaccaaggcaacaaacacatggctgtttgtaagcage 441 ¢ ¥ G N BE T 8 L W D C K N W Q WG GG L S8 1321 tgtaatggaaatgaaacttctctttgggactgcaagaattggcagtggggtggacttagt 461 C D H Y D BEB A K I T C 8 AH R KP RL V 1381 tgtgatcactatgacgaagccaaaattacctgctcagcccacaggaaacccaggetggtt 481 G G6 Db I P C¢C 8S G R V B V Q H G DT WG T 1441 ggaggggacattccctgetctggtegtgttgaagtacaacatggagacacgtggggeace $01 Vv ¢ pp S$ D FP SL EA A S5 VL CRZETLZQTZC 1501 gtctgtgattctgacttctctctggaggeggecagegtgetgtgeagggaactacagtge 5213 T Vv V 8S L L GG G A HF GG E G 8 GG Q I WwW 1561 ggcactgtggtttccctectggggggagctcactttggagaaggaagtggacagatetgg

S541 A BRB B F Q CC BE G HE 8 HL SL CP VV A P 1621 gctgaagaattccagtgtgaggggcacgagtcccacctttcactctgeccagtageacee

S61 R P D G T €C S H S RD V GV V CSU RYT 1681 cgccctgacgggacatgtagcecacageagggacgtcggegtagtetgetcaagatacaca 581 Q I R LL V N G K T P C BR G R V EL N I IL 1741 caaatccgcettggtgaatggcaagacccecatgtgaaggaagagtggagctcaacattett 601 G § W G 8 L C N 8S H WD MZ BDA AUHUVYLSUC 1801 gggtcctgggggtccectctgceaactctcactgggacatggaagatgeccatgttteatge 621 0 Q L K C G6 V A L 8S I P G G@G A P FP GG K G6 1861 cagcagcttaaatgtggagttgccctttctatcccgggaggagcaccttttgggaaagga 64 8 E Q V W R HM P H CT GG TTUE EI KU HMSGTD 1921 agtgagcaggtctggaggcacatgtttcactgcactgggactgagaagcacatgggagat 661 C 8 V T A L 3 A 8 LL C 8 8 G Q V A 8 V I 1981 tgttcegtcactgctctgggogecatcactetgttetteagggeaagtggectctgtaate 681 C § G FR Q § Q T L 8 » C BH 8 S § S D PB 8 2041 tgctcagggaaccagagtcagacactatctccgtgcaattcatcatceteggacccatca 71 Ss 8 I I 8 BE BE N BG V A C I G6 S 6 Q L RL 2101 agctctattatttcagaagaaaatggtgttgcotgcatagggagtggtcaacttogectg 721V D G 6G G R C A G RV BE V Y HEG GS W Gg 2161 gtcgatggaggtggtcegttgtgetgggagagtagaggtotatcatgagggetectgggge 741 T I € DD 8 WD UL NDABRUV V CK QUL 8 2221 accatctgtgatgacagctgggacctgaatgatgececatgtggtgtgcaaacagetgage 761 C G W A I NA TG 8 AHF Ad EGTG TP I 2281 tgtggatgggccattaatgccactggttetgetcattttggggaaggaacagggeccatt 781 W L D E I N C N G K E 8 HI W Q CH 8 H 2341 tggctggatgagataaactgtaatggaaaagaatctcatatttggcaatgecactcacat 8013 W G R H N CR HK EDA AG GV TICSTEF 2401 ggttgggggeggcacaattgcaggeataaggaggatgcaggagtcatctgeotcagagtte 821M 8 L RL I S EN S RE TCA AGT RTLTE EBV 2461 atgtctctgagactgatcagtgaaaacagcagagagacctgtgcagggegectggaagtt

B41 F Y N G R W G S V G R N SS M 8 P A T V G&G 2521 ttttacaacggagcttggggcagegttggeaggaatagecatgt ctccagecacagtggyg

B61 V V CR Q L 6G C A DRG GUDTI SSP AS 8 D 2581 gtggtatgcaggcagetgggetgtgeagacagaggggacatcagecctgeatcttcagac 88 XK T V S R HM W V DN V Q CP KG PDT 2641 aagacagtgtccaggcacatgtgggtggacaatgtteagtgtcctaaaggacctgacaca 901 L W Q C P 8 8S P W K KR L A S P 8 EE T 2701 ctatggcagtgcccatcatctcecatggaagaagagactggecagecect cagaggagaca 921 W I T C A NK ZT RL Q EG N TNUZCGS G R 2762 tggatcacatgtgccaacaaaataagacttcaagaaggaaacactaattgttctggacgt 941 V E I W Y G GG 8 W GG T V C DD S WD TUL R 2821 gtggagatctiggtacggaggttectggggeactgtgtgtgacgactcectgggaccttgaa 961 D A Q V V C R QL G6 C G SS A L E A G K EB 2881 gatgctcaggtggtgtgecgacagetgggetgtggct cagetttggaggeaggaaaagag 981A A P G Q G T G P I WL NEV XK CK GN 2941 gecgeattiggecaggggactgggoccatatggctcaatgaagtgaagtgecaaggggaat 1001 E T S L W D C P AR 8 WG HS DO CGH K 3001 gaaacctccettgtgggattgtectgecagatcctggggccacagtgactgtggacacaag 1021 E D A A V T C 8 EI A K 8S R E S L H A T 3061 gaggatgctgetgtgacgtgeotcagaaattgeaaagagecgagaatcectacatgecaca 1041 G R SS §s F V AL A I P 6G V I L L A CL I 3121 ggtcgctcatcttttgttgoacttgecaatccttggggtcattetgttggectgteteate 1061 A F L I W T Q KR R QR QURUL SVP SS G 3181 gcattcctcatttggactcagaagcgaagacagaggcageggcteteagttttctcagga 1081 G BE N 8 V H Q I Q YR E MNS CULKUA AD 3241 ggagagaattctgtccatcaaattcaataccgggagatgaattcttgectgaaageagat 1101 B T D M L N P S G D H 8 B V Q 3301 gaaacggatatgctaaatccctcaggagaccactcetgaagtacaa 1 MDELRMVLEE NSGSADFRRC SAHLSSFTFA VVAVLSACLV TSSLGGKDKR SEO ID 51 LRLTGGENKC SGRVBVKVQE EWGTVCNNGW DMDVVSVVCR QLGCPTAIKA [SEQDD

151 SDGSDLEMGL VNGGNRCLGR IRVKFQGRWG TVCDDNFNIN HASVVCKQLE 201 CGSAVSPSGS ANPGEGSGPI WFDDLVCHGN ESALWNCKHB GWGKHNCDHA 251 EDAGVICLNG ADLKLRVVDG VTECSGRLEBV KFQGEWGTIC DDGWDSDDAA 301 VACKQLGCPT AVTAIGRVNA SEGTGHIWLD SVSCHGHRSA LWQCRHHEWG 351 KHYCNHDEDA GVTCSDGSDL ELRLKGGGSH CAGTVEVBIQ KLVGKVCDRS 401 WGLKEADVVC RQLGCGSALK TSYQVYSKTK ATNTWLFVSS CNGNETSLWD 451 CKNWQWQGLS CDHYDRAKIT CSAHRKPRLV GGDIPCSGRV BVQHGDTWAT 501 VCDSDPSLEA ASVLCRBLQC GTVVSLLGGA HPGRGSGQIW ABEFPQCEGHE 551 SHLSLCPVAP RPDGTCSHSR DVGVVCSRYT QIRLVNGKTP CEGRVELNIL 601 GSWGSLCNSH WDMEDAHVLC QQLKCGVALS IPGGAPPGKG SEQVWRHMPH 651 CTGTEKHMGD CSVTALGASL CSSGQVASVI CSGNQSQTLS PCNSSBSDPS 701 SSTIISRENGV ACIGSGQLRL VDGGGRCAGR VEVYHEGS8WG TICDDSWDLN 752 DAHVVCKQLS CGWAINATGS AHFGEGTGPI WLDEINCNGK ESHIWQCHSH 801 GWGRHNCRHK EDAGVICSEF MSLRLISENS RETCAGRLEV FYNGAWGSVG 851 RNSMSPATVG VVCRQLGCAD RGDISPASSD KTVSRHMWVD NVQCPKGPDT 901 LWQCPSSPWK KRLASPSEET WITCANKIRL QEGNTNCSGR VEIWYGGSWG 951 TVCDDSWDLE DAQVVCRQLG COSALEAGKE AAFGQGTGPI WLNEVKCKGN 1001 BTSLNDCPAR SNGHSDCUHK EDAAVTCSEI AKSRESLHAT GRSSPVALAT 1051 FGVILLACLI AFLIWTQKRR QRQRLSVPSG GENSVHQIQY REMNSCLKAD 1102 BTDMLNPSGD HSEVQ

Sus CD163 v2 in pCRsusCD163v2 was liberated from pCR2.1 vector after restriction enzymes Kpn I and Not I digestion and gel purification. Recipient vector

PCM V-script was also cut with the same restriction enzyme pair and allowed for directional cloning of susCD163v2 into the pCMV-script. After ligation of susCD163 v2 with pCMV-script, the ligated mixture was used to transform STBL 2 E. coli cells (Invitrogen). One transformant was found to contain the CD163 gene by restriction enzyme digestion analysis and was designed pCMV-script susCD163v2 clone#3.

Example S: Preparation of a RSV promoter based expression system by direct ligation and transfection method.

A non-cloning based procedure to generate microgram quantities of linear DNA suitable for use in generating stable cell lines expressing CD163 from an RSV promoter was developed (figure 4). The procedure involves the isolation and ligation of two pieces of DNA, one containing the neomycin gene and RSV promoter cassette derived from pRSV-script, and the other containing the susCD163v2 coding sequence from pCMVsusCD163v2. Vector plasmid pRSV-Script was linearized with Dralll upstream of the neomycin gene, and bunted with the Klenow fragment of E. coli DNA polymerase. This plasmid was then digested with Nofl immediately downstream of the RSV promoter. The pCMVsusCD163v2 clone was digested in the vector sequence downstream of the CD163 insert with Drdl, and blunted with Klenow fragment of DNA polymerase. The CD163 coding sequence was liberated from the vector with a NotI located immediately upstream of the CD163 coding sequence. For each plasmid digestion the appropriate fragments were purified from agarose gels. A large-scale ligation reaction was performed as follows. Approximately 20 Jig of each

DNA fragment was incubated in a volume of 600 uL with 15 units of T4 DNA ligase.

The reaction was incubated at room temperature for 20 minutes, at which time an aliquot was removed and the reaction frozen on dry ice. Agarose gel analysis of the aliquot revealed that a significant amount of non-ligated DNA remained, so another 15 units of ligase was added and incubated for another 10 minutes at room temperature. Following ligation, a linear piece of DNA containing all of the appropriate clements was purified by agarose gel electrophoresis. Ligation of the two

DNA fragments via the cohesive Not I termini resulted in the placement of the 5’ sequences of the CD163 gene downstream of the RSV promoter, allowing for directed expression of CD163 in mammalian cells. Once isolated, the purified DNA was used to transfect various mammalian cell lines.

Example 6: Cloning and characterization of human CD163 cDNA

Based on a known human CD163 cDNA sequence (GenBank Accession No.

BCO051281), a forward primer HuS'Not (SEQ ID NO: 15) (3’CACCGCGGCCGCGAAGTTATAAATCGCCACCATGAGCAAACTCAGAAT

GG-3’) and a reverse primer Hu3’Kpn (SEQ ID NO: 16) (5’-TGCTCCGGTACCTAGTCCAGGTCTTCATCAAGGTATCTTA-3’) were designed using the PrimerSelect program. Restriction sites for NofI and Kpnl (underlined) were incorporated into the 5° and 3’ primers, respectively, to facilitate cloning into expression vectors. The sequence CACC was added to the 5’ end of the 5’ primer to allow directional cloning into the pPCDNA3.1D/V5/His/TOPO vector (Cat. No. K49001, Invitrogen, see figure 6). Human CD163 cDNAs were amplified from RNA extracted from the U937 cell line after stimulated with phorbol 12- myristate 13-acetate (100ng/ml) for 3 days. Total cellular RNA was prepared using the RNeasy kit (Qiagen). RT-PCR reactions and sequencing methods were the same as described in Example 4. PCR products were separated on 0.8% SeaKem agarose gel and extracted from the gel using the GeneClean kit. PCR products were cloned directionally into the pCDNA3.1D/V5/His/TOPO vector following the manufacturer’s instructions. Two clones with large inserts were sequenced. Sequencing and sequence analysis methods were described in Example 4. A clone with a correct insert was designed “pcDNA3.1D-humCD163v2” and we have designated the sequence of the insert SEQ ID NO: 17

005/011502

The CD163 open reading frame in pPCDNA3.1D-humCD163v2 is 1121 residues in length (designated SEQ ID NO: 18 which encodes SEQ ID NO:19 disclosed below), and is 100% identical to Genbank Z22968 (a human CD163 cDNA of the same length). Our human CD163v2 sequence is also 100% identical to

Genbank BC051281 and Z22969 (splice variants of human CD163) except that 42 nonhomologous residues in the two Genbank sequences replace the seven carboxy- terminal residues of our sequence. This difference is due to the presence of an 83- nucleotide exon in BC051281 and Z22969, and the resulting frame shift at the 3’ end of the exon. (Law, S.K., Micklem, K.J., Shaw, J.M., Zhang, X.P., Dong, Y., Willis,

A.C. and Mason, D.Y. (1993) A new macrophage differentiation antigen which is a member of the scavenger receptor superfamily. European Journal of Immunology 23 (9), 2320-2325). atgagcaaac tcagaatggt gctacttgaa gactctggat ctgctgactt cagaagacat 60 SEQID tttgtcaacc tgagtccctt caccattact gtggtcttac ttctecagtge ctgttttgte 120 NO:17 accagttctc ttggaggaac agacaaggag ctgaggctag tggatggtga aaacaagtgt 180 ) agcgggagag tggaagtgaa agtccaggag gagtggggaa cggtgtgtaa taatggctgg 240 agcatggaag cggtctctgt gatttgtaac cagctgggat gtccaactgc tatcaaagcce 300 cctggatggg ctaattccag tgcaggttct ggacgcattt ggatggatca tgtttettgt 360 cgtgggaatg agtcagctct ttgggattgce aaacatgatg gatggggaaa gcatagtaac 420 tgtactcacc aacaagatgc tggagtgacc tgctcagatg gatccaattt ggaaatgagg 480 ctgacgegtg gagggaatat gtgttctgga agaatagaga tcaaattcca aggacggtygg 540 ggaacagtgt gtgatgataa cttcaacata gatcatgcat ctgtcatttg tagacaactt 600 gaatgtggaa gtgctgtcag tttetcetaggt tcatctaatt ttggagaagg ctcotggacca 660 atctggtttg atgatcttat atgcaacgga aatgagtcag ctctctggaa ctgcaaacat 720 caaggatggg gaaagcataa ctgtgaceat getgaggatg ctggagtgat ttgctcaaag 780 ggagcagatc tgagcctgag actggtagat ggagtcactg aatgttcagg aagattagaa 840 gtgagattcc aaggggaatg ggggacaata tgtgatgacg gctgggacag ttacgatgct 900 gctgtggcat gcaagcaact gggatgtcca actgoegtca cagccattgg tcgagttaac 960 gccagtaagg gatttggaca catctggctt gacagegttt cttgecaggg acatgaacct 1020 gctgtctgge aatgtaaaca ccatgaatgg ggaaagcatt attgcaatca caatgaagat 1080 gctggcgtga catgttctga tggatcagat ctggagctaa gacttagagg tggaggcagce 1140 cgetgtgetg ggacagttga ggtggagatt cagagactgt tagggaaggt gtgtgacaga 1200 ggctggggac tgaaagaagce tgatgtggtt tgcaggeage tgggatgtgg atctgecactc 1260 aaaacatctt atcaagtgta ctccaaaatc caggcaacaa acacatgget gtttctaagt 1320 agctgtaacg gaaatgasac ttctetttgg gactgcaaga actggcaatg gggtggactt 1380 acctgtgatc actatgaaga agccaaaatt acctgetcag cccacaggga acccagactg 1440 gttggagggyg acattccctg ttctggacgt gttgaagtga agcatggtga cacgtgggge 1500 tccatctgtg attcggactt ctctctggaa gotgccageg ttetatgeaq ggaattacag 1560

PCT/US2005/011502 tgtggcacag ttgtctctat cctgggggga goetcactttg gagagggaaa tggacagate 1620 tgggctgaag aattccagtg tgagggacat gagtcccatce tttcactctyg cccagtagea 1680 cccecgeccag aaggaacttg tagecacage agggatgttg gagtagtctg ctcaagatac 1740 acagaaattc gcttggtgaa tggcaagacc ccgtgtgagg geagagtgga gctcaaaacg 1800 cttggtgcct ggggatccct ctgtaactct cactgggaca tagaagatge ccatgttctt 1860 tgccagcage ttaaatgtgg agttgccctt tctaccccag gaggageacg ttttggaaaa 1920 ggaaatggtc agatctggag gcatatgttt cactgcactg ggactgagca gcacatggga 1980 gattgtccetg taactgectct aggtgcttca ttatgtcctt cagagcaagt ggcotetgta 2040 atctgctcag gaaaccagtc ccaaacactg tcctcgtgca attcatcgte tttgggecca 2100 acaaggcocta ccattccaga agaaagtgcet gtggoctgea tagagagtgg tcaacttcge 2160 ctggtaaatg gaggaggtceg ctgtgetggg agagtagaga tctatcatga gggectccotgg 2220 ggcaccatct gtgatgacag ctgggacctg agtgatgcce acgtggtttg cagacagetg 2380 ggctgtggag aggccattaa tgccactggt tetgctcatt ttggggaagg aacagggecce 2340 atctggctgg atgagatgaa atgcaatgga aaagaatccc gecatttggca gtgecattca 2400 cacggctggg ggcagcaaaa ttgcaggcac aaggaggatg cgggagttat ctgctcagaa 2460 ttcatgtcte tgagactgac cagtgaagcc agcagagagg cctgtgeagg gegtctggaa 2520 gttttttaca atggagcttg gggcactgtt ggcaagagta gcatgtctga aaccactgtg 2580 ggtgtggtgt geaggcagct gggctgtgca gacaaaggga aaatcaaccce tgeatcttta 2640 gacaaggcca tgtccattce catgtgggtg gacaatgttc agtgtccaaa aggacctgac 2700 acgetgtggc agtgcccatc atctccatgg gagaagagac tggocagcecoc cteggaggag 2760 acctggatca catgtgacaa caagataaga cttcaggaag gacccacttc ctgttctgga 2820 cgtgtggaga tctggcatgg aggttcctgg gggacagtgt gtgatgacte ttgggacttg 2880 gacgatgotc aggtggtgtg tcaacaactt ggetgtggtc cagctttgaa agcattcaaa 2940 gaagcagagt ttggtcaggg gactggaccg atatggctca atgaagtgaa gtgcaaaggg 3000 aatgagtctt ccttgtogga ttgtcectgce agacgetggg gccatagtga gtgtgggcac 3060 aaggaagacg ctgcagtgaa ttgcacagat atttcagtgc agaaaacccc acaaaaagce 3120 acaacaggtc gctcatcceg tcagtcatcc tttattgcag tcgggatcct tggggttgtt 3180 ctgttggcca ttttegtoge attattcttc ttgactaaaa agcgaagaca gagacagcgg 3240 cttgcagttt cctcaagagg agagaactta gtccaccaaa ttcaataccg ggagatgaat 3300 tcttgectga atgcagatga tctggaccta atgaattcoct caggaggcca ttctgageca 3360 cactgaaaag gaaaatggga atttataacc cagtgagttc agcctttaag ataccttgat 3420 gaagacctgg acta 3434 1M 8S KL RMV LL EUDSG SA ADTPTURTZ RH SEQID 1 atgagcaaactcagaatggtgctacttgaagactctggatctgcetgacttcagaagacat 21 F V NL 8 P FTITVVLULULJSA ACTFEFV 61 tttgtcaacctgagtccottcaccattactgtggtcttacttcteagtgectgttttgte NO:18 41T 8 S L 6G 6 TD KEULURULVYVDGENIKC 121 accagttctettggaggaacagacaaggagctgaggctagtggatggtgaaaacaagtgt and19 618 G R V B V XK V Q ER W GT V CNNUG W 181 agcgggagagtggaagtgaaagtccaggaggagtggggaacggtgtgtaataatggetgg 8168 M BE A V 8S VI CNQULGUCUPTA ATITZK A 241 agcatggaagcggtctctgtgatttgtaaccagetgggatgtecaactgetatcaaagee 101 P G W A NS 8 AG 8 G RTI WMUDUHVYVYS C 301 cctggatgggctaattccagtgcaggttotggacgeatttggatggatcatgtttottgt i21R G N BR 8 AL WwW DPD C K HD GWG KUH 8 N

T/US2005/011502 361 cgtgggaatgagtcagctctttgggattgcaaacatgatggatggggaaagcatagtaac 141C T H Q Q DAG V TCS DG 8 NUL RB MR 421 tgtactcaccaacaagatgctggagtgacctgctcagatggatccaatttggaaatgagg 161L T R G 83 N M Cc §s G R I E I K F Q G R W 481 ctgacgcgtggagggaatatgtgttctggaagaatagagatcaaattocaaggacggtag 1816 T V C D OD N F NI DHA SS VI CRIOQDUDL 541 ggaacagtgtgtgatgataacttcaacatagatcatgcatctgtcatttgtagacaactt

E ¢C @ 8s A V 8 F 8s GS SS NP? G BG s§ a Pp 601 gaatgtggaagtgctgtcagtttctctggttcatctaattttggagaaggctetggacea 221 I WwW P DD LI CNG GNU EBSA AULWNTZ CIKH 661 atctggtttgatgatcttatatgcaacggaaatgagtcagectctctggaactgcaaacat 241 Q GW G K HN CDPD HAZE EDA AGVYV ICSK 721 caaggatggggaaagcataactgtgaccatgctgaggatgctggagtgatttgeteaaag 2616 A DL 8 L RL VDGGVYV TZ ECS GURL E 781 ggagcagatctgagcctgagactggtagatggagtcactgaatgttcaggaagattagaa 281 Vv R P 0 G BR W GG TTI CDUDG WD S Y D A 841 gtgagattccaaggggaatgggggacaatatgtgatgacggctgggacagttacgatget 301A V AR C K QL GG CPT AVTA ATIGT RUVN 901 gctgtggcatgcaagcaactgggatgtccaactgeegtcacagecattggtegagttaac 321A § K 6G F 6G HI Ww LD S88 V 8 CQ GUHZEP 961 gccagtaagggatttggacacatctggcttgacagegtttcottgocagggacatgaacct 341A V W Q C X H HE WG KH Y CNUB RNTED 1021 gectgtctggcaatgtaaacaccatgaatggggaaagcattattgcaatcacaatgaagat 361A 6 V T ¢C 8 DG 8 DL BEL RL RGG GG 8 1081 gctggcgtgacatgttctgatggatcagatctggagetaagacttagaggtggaggcage 3831R ¢ A GT V EVETIOGQQRULULUG GZ KT YVCDR 1141 cgetgtgctgggacagttgaggtggagattcagagactgttagggaaggtgtgtgacaga 401 G W @ L K E A D V V CR QL GG CG 8S A L 1201 ggctggggactgaaagaagctgatgtggtttgeaggcagetgggatgtggatetgoacte 421 XK T 8 Y Q V Y 8S K I Q A T N T W L P L Ss 1261 aaaacatcttatcaagtgtactccaaaatccaggcaacaaacacatggetgtttetaagt 441 S C N @ N E T 8S L W D C K N W Q WG G6 L 1321 agctgtaacggaaatgaaacttctctttgggactgcaagaactggcaatggggtggactt 461 T C D H Y BE E A K I T C 8 A H R BR P R IL 1381 acctgtgatcactatgaagaagccaaaattacctgetcoagceccacagggaacccagactg 481 V 6 6 D I P C S 6G R V BE V K HG DTMWG 1441 gttggaggggacattcoctgttetggacgtgttgaagtgaageatggtgacacgtgggge 5018 I €C D 5 DP SL EA AS V L CRE L Q 1501 tccatctgtgattceggacttctetctggaagetgecagegttctatgeagggaattacag 521 Cc 6 T V V 5 I L G G A H F G B G N G Q I 1561 tgtggcacagttgtctctatcctggggggagetcactttggagagggaaatggacagate 541 W A B BEB FP Q CEG HE S8 HL §L CP V A 1621 tgggctgaagaattccagtgtgagggacatgagtcccatctttcactctgoccagtagea

S561 P R P BE G T C S H 8 RD V GV V CSUR RY 1681 ccccgccoagaaggaacttgtagccacagecagggatgttggagtagtctgetcaagatac 581 T E I RL V NG KT P CRB G RV ETLIKT 1741 acagaaattcgctrggtgaatggcaagaccccgtgtgagggcagagtggagctcaaaacg 601 L G A WW G S LL CN 8 HW DI EDAZENUVL 1801 cttggtgcctggggatccctetgtaactctcactgggacatagaagatgeceatgttett 621 C Q Q L K C G VAL 8 TUPGGATRTFGK 1861 tgccagcagcttaaatgtggagttgecctttctaccccaggaggagcacgttttggaaaa 6413 N G @Q I W R HM PF HCTGTUEBEOQUHNG 1921 ggaaatggtcagatctggaggeatatgtttcactgcactgggactgagcagcacatggga 661 D C P VT A L GA § L C P 8 BE QV A SV 1981 gattgtcctgtaactgctctaggtgettcattatgtecttcagagcaagtggectetgta 681 I C 8S GN Q $s Q TL S S$ CN S88 S 8 TL @ P 2041 atctgctcaggaaaccagtcccaaacactgtectogtgeaatteategtetttgggecea 700T R P T I P EE S A VA CIES SGT QTELR 2101 acaaggcctaccattccagaagaaagtgetgtggectgeatagagagtggtcaacttcge 7201 L. V N @ 6 @ R C A G R V E I Y HE G 8 W 2161 ctgctaaatggaggaggtegetgtgctgggagagtagagatctatcatgagggetectgg 741 G T I C D D S WD UL SD AUHUV VCR RUGQTL 2221 ggcaccatctgtgatgacagctgggacctgagtgatgeccacgtggtttgecagacagetg 761 G6G C G E A I NA TG S A HP GE GT GG P 2281 ggctgtggagaggccattaatgecactggttetgeteattttggggaaggaacagggece 761 1 W L D EM K C N G XK E 8S RI WQOCH 8 2341 atctggctggatgagatgaaatgcaatggaaaagaatcccgeatttggcagtgecattca 801H G W 6G Q Q NCR HKU ED AGU V I C 8S E 2401 cacggctgggggcagcaaaattgcaggcacaaggaggatgegggagttatctgetcagaa 821 F M 686 L R L T 8S E A 8 R BE A CA GR L E 2461 ttecatgtctctgagactgaccagtgaagecageagagaggectgtgcagggegtctggaa 841 V F Y N G A W GT V GK S S MS ETTV 2521 gttttttacaatggagettggggcactgttggcaagagtageatgtetgaaaccactgty 861G V V C R QL 6 C AD KG XK INUPATZGEL 2581 ggtgtggtgtgcaggcagetgggetgtgcagacaaagggaaaatcaaccctgcatcttta 881 D K A M 8 I PM WV DNJVGQU CUP XKGTZ PD 2641 gacaaggccatgtccattcccatgtgggtggacaatgt tcagtgtccaaaaggacctgac

S01 T L W Q C P S S P WEI KT RTULATSTPTSTEE 2701 acgctgtggcagtgcccatcatctecatgggagaagagactggecagecect cggaggag 921 T W I T ¢C DN XK I RL QE G PTS C 8 G 2761 acctggatcacatgtgacaacaagataagacttcaggaaggaccoacttcetgttctgga 941 R V E I # H 6G G S # GT V C DDS WD L 2821 cgtgtggagatctggcatggagg toctgggggacagtgtgtgatgactcttgggacttg

S2005/011502 %€1D OD A Q VV 6cQ QL dC GP ATLTEKAMATFCK 2881 gacgatgctcaggtggtgtgtcaacaacttggetgtggtccagctttgaaagcattcaaa 981 E A E P G Q GG T 6 P I W LL N E V X C K G 2941 gaagcagagtttggtcaggggactggaccgatatggcetcaatgaagtgaagtgecaaaggg 1001 ¥N B § Ss LL, # DC P A RR WG H S EC GH 3001 aatgagtcttccttgtgggattgtectgecagacgcetggggccatagtgagtgtgggecac 1021 K E D A A V N C TD II 8 V @Q K T P Q KX A 3061 aaggaagacgctgcagtgaattgcacagatatttcagtgcagaaaaccccacanaaaagcec 1041 T T G R &§ 8 R Q 8 8 P I A V G I L G Vv V 3121 acaacaggtcgctcatcccgtcagtcateoctttattgcagtegggatecttggggttgtt 1061 L L A I FP V A L F PL T K X R R QR QR 3181 ctgttggccattttegtegecattattcttocttgactaaaaagcgaagacagagacagegg 1081 L A V 8S 8 R G E NL V H Q I Q YREMN 3241 cttgcagtttcctcaagaggagagaacttagtccaccaaattcaatacecgggagatgaat 1101 8 ¢ LL. N AD DL DL MNUG SS GH S88 E P 3301 tcttgcctgaatgcagatgatctggacctaatgaattectcaggaggccattcotgagcea 1121 KH 3361 cac 1 MSKLRMVLLE DSGSADFRRH PVNLSPFTIT VVLLLSACFV TSSLGGTDKE SEQ ID 51 LRLVDGENKC SGRVEVKVQE BWGTVCNNGW SMEAVSVICN QLGCPTAIKA 101 PGWANSSAGS GRIWMDHVSC RGNBSALWDC KHDGWGKHSN CTHQQDAGVT 151 CSDGENLEMR LTRGGNMCSG RIETIKPQGRW GTVCDDNFNI DHASVICRQL NO:19 201 ECGSAVSFSG SSNFGEGSGP IWFDDLICNG NESALWNCKH QGWGKHNCDH 251 AEDAGVICSK GADLSLRLVD GVTBCSGRLE VRFQGEWGTI CDDGWDSYDA 301 AVACKQLGCP TAVTAIGRVN ASKGPGHIWL DSVSCQGHEP AVWQCKHHEW - 351 GKHYCNHNED AGVTCSDGSD LELRLRGGGS RCAGTVEVEI QRLLGKVCDR 401 GWGLKBEADVV CRQLGCGSAI: KTSYQVYSKI QATNTWLFLS SCNGNBTSLW 451 DCXNWQWGGL TCDHYERAKI TCSAHREPRL VGGDIPCSGR VBVKHGDTWG 501 SICDSDPSLE AASVLCRELQ CGTVVSILGG AHFGERGNGQI WABRPQCEGH 551 ESHLSLCPVA PRPEGTCSHS RDVGVVCSRY TEIRLVNGKT PCBGRVELKT 6C1 LGAWGSLCNS HWDIEDAHVL CQQLKCGVAL STPGGARFGK GNGQINRHMF 651 HCTGTEQHMG DCPVTALGAS LCPSEQVASV ICEGNQSQTL SSCNSSSLGP 701 TRPTIPERSA VACIEBSGQLR LVNGAGRCAG RVEIYHBGSW GTICDDSWDL 751 SDAHVVCRQL GCGBAINATG SAHFGBGTGP IWLDBMKCNG KBSRIWQCHS 801 BGWGQQNCRH KEDAGVICSE PMSLRLTSEA SREACAGRLE VFYNGANGTV 851 GKSSMSETTV GVVCRQLGCR DKGKINPASL DKAMSIPMWV DNVQCPKGPD 901 TLWQCPSSPW EKRLASPSER TWITCDNKIR LQRGPTSCSG RVEIWHGGSW 951 GTVCDDSUDL DDAQVVOQQL GCGPALKAPK EARPGQGTGP INLNEVKCKG 1001 HESSLWDCPA RRWGHSECGH KEDAAVNCTD ISVQKTPQXA TTGRSSRQSS 1051 FIAVGILGVV LLATPVALFF LTKXRRQRQR LAVSSRGENL VHQIQYREMN 1101 SCLNADDLDL MNSSGGHSEP H

Example 7: Cloning and characterization of murine CD163

Based on the murine CD163 sequence in GenBank (AF274883), a forward primer Mus-new5’ (SEQ ID NO: 20) (5'-

CACCGCGGCCGCCACACGGAGCCATCAAAATCATCAA-3’) and a reverse primer Mus-new3’ (SEQ ID NO:21) (5’-

GGTACCGCGAACAAGCAAACCAATAGCAATATTGTTTAATTCCCTC-3’) were designed using the PrimerSelect program. Restriction endonucleases sites for No and

Kpnl were included in 5° and 3” primers, respectively, to allow future cloning into other expression vectors. Mouse peritoneal macrophages were harvested from mice 2 days after injecting thioglycollate medium into the peritoneal cavity. Total cellular RNA was prepared from peritoneal macrophages using the RNeasy kit. RT-PCR reactions and

RT-PCR parameters were the same as described in Example 4, except the annealing temperature was increased to 60°C and extension temperature increased to 72°C. The

PCR product was purified on a 0.8% SeaKem agarose gel and directionally cloned into

PCDNA3.1D/V5/His/TOPO according to the manufacturer's instructions. Several clones with large mserts were identified for further analysis. A plasmid containing an insert (SEQ ID NO: 22) with a murine CD163 that encodes a protein of the same length as (1121 amino acids SEQ ID NO:24 ) and differs from Genbank AF274883 by only two amino acids (99.8% identity) was designated “pCDNA3.1D-murCD163v2",

Another plasmid, “pCDNA3.1D-murCD163v3 was generated which contained an insert (SEQ ID NO: 25) containing a murine CD163 coding sequence (SEQ ID NO: 26) which encodes a protein of 1159 amino acids in length (SEQ ID NO: 27). It differs from AF274883 by only 3 amino acids within the first 1107 residues (99.7% identity), but the sequences diverge completely beginning at residue 1108. This is due to an insertion of 82 nucleotides in the cDNA, and a concomitant shift in reading frame downstream of the insertion. As a result, murine CD163v3 contains 52 amino acids at its carboxy-terminus that are not homologous to the 14 carboxy-terminal residues of murine CD163v2. These two alternative versions of “full length” murine CD163 are most likely splice variant of the same gene, as has been described for human CD163 (Law, S.K., Micklem, K.J., Shaw, J.M., Zhang, X.P., Dong, Y., Willis, A.C. and Mason,

D.Y. (1993) A new macrophage differentiation antigen which is a member of the scavenger receptor superfamily. European Journal of Immunology 23 (9), 2320-2325). gctttggaat gggtggacac agaatggttc ttcttggagg tgctggatet cctggttgta 60 SEQ ID aaaggtttgt ccatctaggt ttctttgttg tggctgtigag ctcacttctc agtgoctctg 120 NO:22 ctgtcactaa cgctcctgga gaaatgaaga aggaactgag actggegggt ggtgaaaaca 180 actgtagtgg gagagtggaa cttaagatcc atgacaagtg gggcacagtg tgcagtaacg 240 gctggagcat gaatgaagtg tecgtggttt gccagcagct gggatgcecca acttctatta 300 aagcccttgg atgggctaac tecagegccg gcotctggata tatctggatg gacaaagttt 360 cttgtacagg gaatgagtca getetttggg actgcaaaca tgatgggtgg ggaaagcata 420 actgtaccca tgaaaaagat gctggagtga cctgctcaga tggatctaat ttggagatga 480 gactggtgaa cagtgoggge caccgatgct taggaagagt agaaataaag ttccagggaa 540 agtgggggac ggtgtgtgac gacaacttca gcaaagatca cgcttctgtg atttgtaaac 600 agcttggatg tggaagtgee attagtttcot ctggctcage taaattggga getggttotg 660 gaccaatctg gctegatgac ctggcatgca atggaaatga gteagctctc tgggactgea 720 aacaccgggg atggggcaag cataactgtg accatgcetga ggatgteggt gtgatttget 780 tagagggagc agatctgagce ctoagactag tggatggagt gtccagatgt tcaggaagat 840 tggaagtgag attccaagga gaatggggga cogtgtgtga tgataactgg gatctcoggg 900 atgcttctgt ggtgtgcaag caactgggat gtccaaoctgc catcagtgec attggtcegag 960 ttaatgccag tgagggatct ggacagattt ggottgacaa catttcatge gaaggacatg 1020 aggcaactct ttgggagtgt aaacaccaag agtggggaaa gecattactgt catcatagag 1080 aagacgctgg cgtgacatqt tctgatggag cagatctgga acttagactt gtaggtggag 1140 gcagtcgctg tgctggeatt gtggaggtgd agattcagaa gotgactggg aagatgtgta 1200 gccgaggctg gacactggea gatgcggatg tggtttgeag acagettgga tgtggatctg 1260 cgcttcaaac ccaggctaag atctactcta aaactgggge aacaaatacg tggctcttte 1320 ctggatcttg taatggaaat gaaactactt tttggcaatg caaaaactgg cagtggggcg 1380 gectttoctg tgataatttc gaagaagcca aagttacctg ctcoaggccac agggaaccca 1440 gactggttgg aggagaaatc ccatgctctg gtegtgtgga agtgaaacac ggagacgtgt 1500 ggggctcagt ctgtgatttt gacttgtetc tggaagetge cagtgtggtg tgcagggaat 1560 tacaatgtgg aacagtcgtc tctatcctag ggggagcaca ttttggagaa ggaagtggac 1620 agatctgggg tgaagaattc cagtgtagtg gggatgagtc ccatctttca ctatgctcag 1680 tggcgeccce gctagacaga acttgtacce acageaggga tgtcagcegta gtctgctcac 1740 gatacataga tattcgtctg gcaggegdcg agtcecteoctg tgagggaaga gtggagetca 1800 agacactcgg agectggggt cccoctetgea gttctcattg ggacatggaa gatgctceatg 1860 tcttatgtca gcagctgaag tgtggggttg cccaatctat tccagaagga gcacattttg 15920 ggaaaggagc tggtcaggtc tggagtcaca tgttccactg cactggaact gaggaacata 1980 taggagattg cctcatgact gctctgggtg cgccgacgtg ttccgaagga caggtggect 2040 ctgtcatctg ctcaggaaac caatcccaga cactattgece atgtagttca ttgtctccag 2100 tccaaacaac aagctctaca attccaaagg agagtgaagt tccctgceata gcaagtggece 2160 agcttcgett ggtaggtgga ggtggteget gegetggaag agtggaggtce taccacgagg 2220 getcttgggg caccgtetgt gatgacaatt gggatatgac tgatgccaat gtggtgtgea 2280 agcagctgga ctgtggegtg gecaattaacg ccactggetc tgcottactto ggggaaggag 2340 caggagctat ctggctagac gaagtcatct gcactgggaa agagtctcat atttggcagr 2400 gccattcaca tggctgggga cgccataact gcaggcacaa agaagatgca ggtgttatct 2460 gotccgagtt catgtctctg aggctgacca acgaagccca caaagaaaac tgcacaggtc 2520 gccttgaagt gttttacaat ggtacatggg gcagtattgg cagtagcaat atgtctccaa 2580 ccactgtggg ggtggtgtge cgtcagcectgg getgtgcaga caacgggact gtgaaaccca 2640 taccttcaga caagacacca teocaggccca tgtgggtaga tcgtgtgeag tgtccaaaag 2700 gagttgacac tttgtggcag tgcccctcgt caccttggaa acagagacag gccageccct 2760 cctceccagga gtectggatce atctgtgaca acaaaataag actccaggaa gggcatacag 2820 actgttctgg acgtgtggag atctggcaca aaggttcctg gggaacagtg tgtgatgact 2880 cctgggatct taatgatget aaggtitgtat gtaagcagtt gggctgtggc caagctgtga 2940 aggcactaaa agaagcagca tttggtccag gaactgggec catatggctc aatgaaatta 3000 agtgtagagg gaatgagtct tccctgtggg attgtectge caaaccgtgg agtcacageg 3060 actgtgggca caaagaagat gcttccatee agtgectccc aaaaatgact tcagaatcac 3120 atcatggcac aggtcaccce accctcacgg cactcttggt ttgtggagee attctattgg 3180 tcctectecat tgtcttcctc ctgtggactc tgaagcgacg acagattcag cgacttacag 3240 tttcctcaag aggagaggtc ttgatacatc aagttcagta ccaagagatg gattcaaagg 3300 cdgatgatct ggacttgctg aaatcctcgg gggtcattca gaggcacact gagaaggaaa 3360 atgataattt ataatccact gaggttggag tttaagaagc cttgacagga cagccagcta 3420 aatggaacaa gagcccaggc aacgcacgga tgaccacagc tgcatcttca tgcagtcott 3480 tgtttcetgg aactctgctg aacctgecaaa aaccatattt gtgaatgtga ccacttaata 3540 gagatgggag actttt 3556

1M GG G H R MV LL GGA AGS P GC KR PF SEQ ID 1 atgggtggacacagaatggttcttcttggaggtgctggatctcctggtigtaaaaggttt 21 vV HL a F F V V AV § SL L 8 AR § A VT 61 gtccatctaggtttetttgttgtggetgtgagctcacttctcagtgectetgetgtcact NO:23and 41 FN A P 3 BM KK EL RL A GG ENUNTC S 121 aacgctcctggagaaatgaagaaggaactgagactggegggtggtgaaaacaactgtagt 24 61@ R V BE LL KI HD KW GT V ¢C 8S NG WNW § 181 gggagagtggaacttaagatccatgacaagtggggcacagtgtgcagtaacggctggage 81M N B V 8 VV CC Q QL @C PT SI KAUL 241 atgaatgaagtgtccgtggtttgccagcagctgggatgeccaacttectattaaagecctt 101G W A N 8 8S A G 8 @G Y I W MD XK V 8 CT 301 ggatgggctaactccagegccggetcetggatatatctggatggacaaagtttcttgtaca 122 G N BE 8 AL ¥ DD ¢C RK H D GG WG KHNUCT 361 gggaatgagtcagctctttgggactgcaaacatgatgggtggggaaagecataactgtace 141 H E K D A @VeTCSDGS NLU EMMUZBRTILYV 421 catgaaaaagatgctggagtgacctgctcagatggatctaatttggagatgagactggtyg 161 N 8S A G H R CL GR V BEI XK P Q G K WG 481 aacagtgcgggccaccgatgcttaggaagagtagaaataaagttccagggaaagtaggdg 181 T V C DD NP 8 KD HAS VI CIKOQULG 541 acggtgtgtgacgacaacttcagcaaagatcacgcttcetgtgatttgtaaacagettgga 201C G 8S AI 8 F 8G 8 A KL @ AG ss G PI 601 tgtggaagtgccattagtttctotggetcagetaaattgggagctggttetggaccaate 221 Ww LL D DL AC NG NUE ESB A L WD CIKHBHR 661 tggctcgatgacctggcatgcaatggaaatgagtcagetctotgggactgcaaacacegg 241 G W G K H N CC DH ARDUV GG VI CL EG 721 ggatggggcaagcataactgtgaccatgctgaggatgtcggtgtgatttgettagaggga 261A DL SL RL VDGV 88 R CB GURL EV 781 gragatctgagcctgagactagtggatggagtgtccagatgttcaggaagattggaagtg 281 R F Q G BRB W G T V CD DN WDUL ERDAS 841 agattccaaggagaatgggggaccgtgtgtgatgataactgggatctcegggatgettet 301 v Vv CC KK Q LCP TAI S A JI GR V NA 901 gtggtgtgcaagcaactgggatgtccaactgccatcagtgccattggtogagttaatgee 3218 R GG 8S 6G Q I WwW L D N I S$ C EG HBA AT 961 agtgagggatctggacagatttggcttgacaacatttcatgegaaggacatgaggcaact 341 L W B C K H Q B WG KH Y CHUBEBURETUDA 1021 ctttgggagtgtaaacaccaagagtggggaaagcattactgtcatcatagagaagacgct 3G v T ¢C $8 DD @ A DL BEL RUL UV 3G GG & R 1081 ggcgtgacatgttctgatggageagatctggaacttagacttgtaggtggaggeagtege 381 C A G I V BE V B I Q K LL T @ KM C Ss rR 6G 1141 tgtgctggcattgtggaggtggagattcagaagetgactgggaagatgtgtagecgagge 401 W T L A D A DV V C R QL GG CG 8 A L Q 1201 tggacactggcagatgcggatgtggtttgcagacagecttggatgtggatctgegettcaa 421 T 0 A K I ¥ 8 KT G A T N T WL P P G S§ 1261 acccaggctaagatctactctaaaactggggcaacaaatacgtggetetttectggatct 441 C N G N E T T P W Q C K N W Q W G3 GL Ss 1321 tgtaatggaaatgaaactactttttggcaatgcaaaaactggcagtggggeggectttee 461 C OD N F E E A K VT C §S G HR E P RL V 1381 tgtgataatttcgaagaagccaaagttacctgetcaggccacagggaaccecagactggtt 482 G G6 E I P C 8 G R V BEB V K H GD V W G6 § 1441 ggaggagaaatcccatgctctggtcgtgtggaagtgaaacacggagacgtgtggggctec

S01v ¢C DOD F DL SL B AASV V CRU EBILAOGQSTC 1501 gtctgtgattttgacttgtetctggaagetgecagtgtggtgtgcagggaattacaatgt $521 GG T V V 8 I L G G A H F G E G 8 G Q I WwW 1561 ggaacagtcgtctctatcctagggggagcacattttggagaaggaagtggacagatctgg

S416 E E F Q ¢ $s 6G D B § HL 8 L CS V A P 1621 ggtgaagaattccagtgtagtggggatgagtcccatctttcactatgctecagrggegeece 561 P L OD RT CT HS RD VES VV CSR RUYI 1681 ccgctagacagaacttgtacccacagcagggatgtcagegtagtctgetcacgatacata 561 D I R L A G G B § S C EG R V BEL K TL 1741 gatattcgtctggcaggcggegagtcctectgtgagggaagagtggagctcaagacacte 601 A W @ PL CC § § HW DMETUDA AEHUVL C 1801 ggagcctggggtcccctetgeagttcetecattgggacatggaagatgetcatgtettatgt 621 Q Q L XK C GG V AR Q 8S IT P E G A H F G K G 1861 cagcagctgaagtgtggggtigcccaatctattccagaaggagecacattttgggaaagga 641 A G Q V W S H ¥M F H ¢C T G T RB RBH I G D 1921 gctggtcaggtctggagteacatgttccactgeactggaactgaggaacatataggagat 661C L M T A L GG A PT C S EG Q V A § V I 1981 tgcctcatgactgctctgggtgcgocgacgtgttecgaaggacaggtggectetgteate 681 C 8 GG N Q 8 Q TL L P C 8 8 L § P V QT 2041 tgctcaggaaaccaatcccagacactattgcecatgtagttcattgtctecagtceaaaca 701 T 8 8 T I P K E 8 E V P ¢C I A S G Q L R 2101 acaagctctacaattccaaaggagagtgaagttccctgeatageaagtggecagettege 7212 L V 8 G6 @ @ R CA GR UV EV YHEG GTS SH 2161 ttggtaggtggaggtggtcgetgogetggaagagtggaggtctaccacgagggetettgg 741 G T V CD DN WDMTUDA ANUVVCIKO QL 2221 ggcaccgtctgtgatgacaattgggatatgactgatgccaatgtggtgtgcaageagetg 761 D €C @ V A I N AT GG 5 A Y P GE G A G A

“T/US2005/011502 2281 gactgtggcgtggcaattaacgccactggcetcetgcettacttoggggaaggagcaggaget 781 I Ww L D EV I €¢C TG KE S HIWOQCH 8 2341 atctggctagacgaagtcatctgcactgggaaagagtctcatatttggcagtgecattea 801H G W G R H N ¢C RH K EDAGV I C 8S E 2401 catggctggggacgccataactgcaggcacaaagaagatgcaggtgttatctgetcecgag 821 F M 8 L RL T N E A H K EN CT GR L B 2461 ttcatgtctctgaggetgaccaacgaagcccacaaagaaaactgcacaggtegecttgaa 841 V F Y NG T W GG S$ IT G 8 S$ NMS PT TV 2521 gtgttttacaatggtacatggggcagtattggcagtagcaatatgtctccaaccactgtg 861 G6 Vv V ¢C R Q LL @ C AD NGTUVX PTI P 8S 25B1 ggggtggtgtgccgtcagctgggetgtgcagacaacgggactgtgaaacceataccttca 881 D XK T P 8 R PM WV DIRUV QC PKG VD 2641 gacaagacaccatccaggcccatgtgggtagatcgtgtgcagtgtccaaaaggagttgac 901 T L W Q C P 8S 8S P W K QR Q A 8 P 8S 8 Q 2701 actttgtggcagtgcccoctegtcaccttggaaacagagacaggecageccctocteccag 821 E 8§ W I I C DN KTIRULOQETGHTDC 8 2761 gagtcctggatcatctgtgacaacaaaataagactccaggaagggcatacagactgttet 941 G R V E I W H K GG 8 W 3 T V C DD S WD 2821 ggacgtgtggagatctggcacaaaggttcotggggaacagtgtgtgatgactcctgggat

S61 L N D A K V V C K QL GC 38 QA V KA AIL 2981 cttaatgatgctaaggttgtatgtaagcagttgggctgtggccaagctgtgaaggeacta

S81 K E A A FP G P G T 6G P I WL NBR TI K CR 2941 aaagaagcagcatttggtccaggaactgggecccatatggctcaatgaaattaagtgtaga 1000 @ N E S S L WwW D C P A KP W 8 H 8 D C G 3001 gggaatgagtcttcectgtgggattgtectgecaaaccgtggagtcacagegactgtggg 102 H K E D A S I QQ CL P RK MT 8 B S H H G 3061 cacaaagaagatgcttccatccagtgectcccaaaaatgacttcagaatcacatcatgge 1041 T 6 H P T L T AL L V C GG A I L L V L L 3121 acaggtcaccccaccctcacggcactcettggtttgtggagecattetattggtectacte 1061 I V F L L W T L XK R R @Q I Q RL T V 8 8 3181 attgtcttectcoctgtggactctgaagcgacgacagattcagegacttacagtttectea 1081 R G BE V LL I H Q V Q Y Q BE M D § XK A Db» 3241 agaggagaggtcttgatacatcaagttcagtaccaagagatggattcaaaggeggatgat 1101 L D L L X 8 8 GV I Q RH TUE EIXKXUZE EUDNDUDN 3302 ctggacttgctgaaatcctegggggtcattcagaggcacactgagaaggaaaatgataat 1121 L 3361 tta 1 MGGHRMVLLG GAGSPGCKRF VHLGFFVVAV SSLLSASAVT NAPGEMKRRL SEQ ID 53 RLAGGENNCES GRVELKIHDK WGTVCSNGNS MNEVSVVCQQ LGCPTSIKAL 201 GWANSSAGSG YIWMDKVSCT GNESALWDCK HDGWGKHNCT HEKDAGVTCS 151 DGSNLEMRLV NSAGHRCLGR VEBIKFQGKWG TVCDDNPSKD HASVICKQLG NO:24 201 CGEAISFSGS AKILGAGSGPI WLDDLACNGN ESALWDCKHR GWGKHNCDHA 251 BDVGVICLEG ADLSLRLVDG VSRCSGRLEV RFQGEWGTVC DDNWDLRDAS 301 VVCKQLGCPT AISAIGRVNA SEGSGQINLD NISCEGHEAT LWECKHQEWG 351 KHYCHHREDA GVTCSDGADL ELRLVGGGSR CAGIVEVEIQ KLTGKNCSRG 401 WILADADVVC RQLGCGSALQ TQAKIYSKTG ATNTWLFPGS CNGNETTFWQ 451 CKNWQWGGLS CDNFEEAKVT CSGHREPRLV GGEIPCSGRV EVKHGDVWGS

S01 VCDFDLELEA ASVVCRELQC GTVVSILGGA HFGRGSGQIW GEBPQCSGDE 551 SHLSLCSVAP PLDRTCTHSR DVSVVCSRYI DIRLAGGESS CEGRVELKTL 601 GAWGPLCSSH WDMEDAHVLC QQLXCGVAQS IPEGAHFGKG AGQUWSHMFH 651 CTGTEEHIGD CLMTALGAPT CSBGQVASVI CBGNQSQTLL PCSSLSPVQT 701 TSSTIPKESE VPCIASGQLR LVGGGGRCAG RVEVYHEGSW GTVCDDNWDM 751 TDANVVCKQL DCGVAINATG SAYPGEGAGA IWLDEVICTG KBSHIWQCHS 801 HGWGRHNCRH KEDAGVICSE PMSLRLTNEA HRENCTGRLE VFYNGTWGSI 851 GSSNMSPTTV GVVCRQLGCA DNGTVKPIPS DKTPSRPMWV DRVQCPXGVD 901 TLWQCPSSPW KQRQASPSSQ ESWIICDNKI RLQEGHTDCS GRVEIWHKGS 951 WGTVCDDSWD LNDAKVVCKQ LGCGQAVKAL KEAAPGPGTG PIWLNEIKCR 1021 GNESSLWDCP AKPWSHSDCG EKEDASIQCL, PKMTSRSHHG TGHPTLTALL 1051 VCGAILLVLL IVFLLWTLKR RQIQRLTVSS RGRVLIHQVQ YQEMDSKADD 1101 LDLLKSSGVI QRHTEKENDN L getttggaat gggtggacac agaatggttc ttcttggagg tgectggatct cctggttgta 60 ID NO:25 aaaggtttgt ccatctaggt ttctttgttg tggctgtgag ctcacttctc agtgectctg 120 ctgtcactaa cgctcctgga gaaatgaaga aggaactgag acrggcagggt ggtgaaaaca 180 actgtagtgg gagagtggaa cttaagatcc atgacaagtg gggcacagtg tgcagtaacg 240 gctggageat gaatgaagtg tccgtggttt gocagcagct gggatgecca acttctatta 30C aagcccttgg atgggctaac tccagegceg getetggata tatctggatg gacaaagttt 360 cttgtacagg gaatgagtca gctctttggg actgcaaaca tgatgggtgg ggaaagcata 420 actgtaccca tgaaaaagat gctggagtga cctgctcaga tggatctaat ttggagatga 480 gactggtgaa cagtgcggge caccgatgct taggaagagt agaaataaag ttccagggaa 540 agtgggggac ggtgtgtgac gacaacttca gcaaagatca cgcttctgtg atttgtaaac 600 agcttggatg tggaagtgcc attagtttct ctggctcage taaattggga getggttcetg 660 gaccaatctg gctcgatgac ctggcatgca atggaaatga gtcagctctc tgggactgca 720 aacaccgggg atggggeaag cataactgtg accatgetga ggatgtcoggt gtgatttget 780 tagagggagc agatctgagc ctgagactag tggatggagt gtccagatgt tcaggaagat 840 tggaagtgag attccaagga gaatggggga ccgtgtgtga tgataactgg gatctccoggg 900 atgcttctgt ggtgtgcaag caactgggat gtccaactgc catcagtgce attggtcgag 960 ttaatgccag tgagggatct ggacagattt ggcttgacaa catttcatge gaaggacatg 1020 aggcaactct ttgggagtgt aaacaccaag agtggggaaa gcattactgt catcatagag 1080 aagacgctgg cgtgacatgt tctgatggag cagatctgga acttagactt gtaggtggag 1140 gcagtegetg tgetggeatt gtggaggtgg agattcagaa gcectgactggg aagatgtgta 1200 gccgaggetg gacactggea gatgeggatg tggtttgcag acagettgga tgtggatctg 1260 cgcttcaaac ccaggctaag atctactcta aaactggggce aacaaatacg tggcotcttte 1320 ctggatottg taatggaaat gaaactactt tttggcaatg caaaaactgg cagtggggeg 1380 geocttteotlg tgataatttc gaagaagcca aagttacctg ctecaggccac agggaaccca 1440 ’ gactggttgg aggagaaatc ccatgctctg gtegtgtgga aatgaaacac ggagacgtgt 1500 ggggctceccgt ctgtgatttt gacttgtctce tggaagetge cagtgtggtg tgcagggaat 1560 tacaatgtgg aacagtcgte tctatcctag ggggageaca ttttggagaa ggaagtggac 1620 agatctgggg tgaagaattc cagtgtagtg gggatgagtc ccatctttca ctatgctcag 1680 tggcgeccee getagacaga acttgtacce acagcaggga tgtcagegta gtctgctcac 1740 gatacataga tattcgtctg gcaggeggeg agtcoctccetg tgagggaaga gtggagctca 1800 agacactcgg agectggggt ceccctotgea gttetcattg ggacatggaa gatgctcatg 1860 tcttatgtca gcagctgaag tgtggggttg cccaatctat tccagaagga geacattttg 1920 ggaaaggagc tggtcaggtc tggagtcaca tgttccactg cactggaact gaggaacata 1980 taggagattg cctcatgact gctctgggtg cgccgacgtg ttocgaagga caggtggect 2040 ctgtcatctg ctcaggaaac caatcccaga cactattgcc atgtagttca ttgtctcecag 2100 tccaaacaac aagctctaca attccaaagg agagtgaagt tccctgeata geaagtggee 2160 agcttegett ggtaggtgga ggtggteget gegectggaag agtggaggte taccacgagg 2220 geteottgggg caccgtetgt gatgacaatt gggatatgac tgatgccaat gtggtgtgca 2280 agcagctgga ctgtggcegtyg gcaattaacg ccactggctc tgcttacttc ggggaaggag 2340 caggagctat ctggctagac gaagtcatct gcactgggaa agagtctcat atttggcagt 3400 gccattceaca tggetgggga cgecataact gcaggcacaa agaagatgea ggtgttatct 2460 gctccgagtt catgtetcetg aggctgacca acgaagccca caaagaaaac tgcacaggtc 2520 gccttgaagt gttttacaat ggtacatggg gcagtattgg cagtagcaat atgtctccaa 2580 ccactgtgge ggtggtgtgc cgtcagetgg getgtgecaga caacgggact gtgaaaccca 2640 taccttcaga caagacacca tccaggccca tgtgggtaga togtgtgcag tgtccaaaag 2700 gagttgacac tttgtggcag tgeccctegt caccttggaa acagagacag gecagcccct 2760 cctcocccagga gtcctggatce atctgtgaca acaaaataag actccaggaa gggcatacag 2820 actgttctgg acgtgtggag atctggcaca aaggttcctg gggaacagtg tgtgatgact 2880 cctgggatct taatgatgct aaggttgtat gtaagcagtt gggctgtgge caagctgtga 2940 aggcactaaa agaagcagca tttggtccag gaactgggcc catatggcetc aatgaaatta 3000 agtgtagagg gaatgagtct tccctgtggg attgtcoctge caaaccgtgg ag cacageg 3060

. S$/011502 actgtgggca caaagaagat gcttccatcc agtgectcece caaaatgact tcagaatcac 3120 atcatggcac aggtcacccc accctcacgg cactcttggt ttgtggagcc attctattgg 3180 tcctocteat tgtettecte ctgtggactc tgaagegacg acagattcag cgacttacag 3240 tttoctcaag aggagaggtc ttgatacatc aagttcagta ccaagagatg gattcaaagyg 3300 cggatgatct ggacttgctg aaatcctcgg aaaattccaa caattcatat gattttaatg 3360 atgatggact gacatctttg tctaaatatc ttcctatttc tggaattaaa aaggggtcat 3420 tcagaggcac actgagaagg aaaatgataa tttataatcc actgaggttg gagtttaaga 3480 agccttgaca ggacagccag ctaaatggaa caagagccca ggcaacgcac ggatgacoac 3540 agctgcatct tcatgcagte ctttgtttce tggaactctg ctgaacctgc aaaaaccata 3600 tttgtgaatg tgaccactta atagagatgg gagactttt 3639 1M G GH RM VL L GGA AGS PG CKRF ID NO:26 1 atgggtggacacagaatggttcttcttggaggtgetggatctoetggttgtaaaaggttt ° 22 V HL GF F VV AV S SLL 8 AS A VT 61 gtccatctaggtttctttgttgtggotgtgageteacttatcagtgoototgetgteact and 27 41X A P G EM K XK EL RL AGG BNNTUC S 121 aacgctcctggagaaatgaagaaggaactgagactggegggtggtgaaaacaactgtagt 61G R V EL K I HD KW @ TV C8 NG W 8 181 gggagagtggaacttaagatccatgacaagtggggcacagtgtgcagtaacggctggage 81M N BE V 8 V VC Q ¢ LL GG CP TS I KAL 241 atgaatgaagtgtccgtggtttgccageagectgggatgeccaacttctattaaagceett 1013 W A N 8S 8S A G 8 @ Y I WM D KV 6 CT 301 ggatgggctaactccagecgeceoggctctggatatatctggatggacaaagtttettgtaca 121 3 N BR 8 A IL W D €C XK HD G W G K HN CT 361 gggaatgagtcagctctttgggactgcaaacatgatgggtggggaaagcataactgtace 141H BE XK D A G VT C §$ DG S NL EMR L V 421 catgaaaasagatgctggagtgacctgctcagatggatctaatttggagatgagactggtyg 161 N 8 AG HR CL GG R V EI KP Q G K NG 481 aacagtgcgggccaccgatgcttaggaagagtagaaataaagttccagggaaagtggggy 18lT Vv C OD D N P 8 XD HA S VI CX QL a 541 acggtgtgtgacgacaacttcagcaaagatcacgettotgtgatttgtaaacagettgga 2001C G 8A I 8 F 8 GG 8 A KL G A G 8 @ pr 1 601 tgtggaagtgccattagtttctcotggctcagctaaattgggagctggttctggaccaatce 221 W L D DL A CN G NBR S A IL W D CK HR 661 tggctcgatgacctggcatgcaatggaaatgagtcagctctcotgggactgcaaacaccgg 241 G W G K H N CDH AEBEDV GG V I CL EG 721 ggatggggcaagcataactgtgaccatgctgaggatgtcoggtgtgatttgcttagaggga 261A DL § L RL V D G33 V 8 RC 8 6 RL EV 781 gcagatctgagcctgagactagtggatggagtgtccagatgttcaggaagattggaagtg 281 R F Q G E W GT V CC DDNWUDUL RDA A S 841 agattccaaggagaatgggggaccgtgtgtgatgataactgggatctcegggatgettet 301 Vv Vv ¢ K QL GCP TAI 8 A 1 GR V NRA 901 gtggtgtgcaagcaactgggatgtccaactgccatcagtgeccattggtogagttaatgee 321s E 6G S$ GG Q I WL DNI SCE EUGHZER BAT 961 agtgagggatctggacagatttggcttgacaacatttcatgegaaggacatgaggoaact 341 L W E C KX BE Q EWG KH Y CH HREDR 1021 ctttgggagtgtaaacaccaagagtggggaaagcattactgtcatcatagagaagacgct 3616 Vv T €C 8s DP GG ADL BRL RL V G GG 8 R 1081 ggcgtgacatgttctgatggagcagatctggaacttagacttgtaggtggaggcagtege 381C A G6 I V E V E 1 Q KL T G KM C 8S R @G 1141 tgtgetggeattgtggaggtggagattcagaagctgactgggaagatgtgtagecgagge 401 W T LL, AD A DV V CR QL G6 CG S58 ATUL Q 1201 tggacactggcagatgcggatgtggtttgcagacagettggatgtggatctgegettcaa 421 T Q A K I ¥ 8 K T G A T N T W L P P @ § 1261 acccaggctaagatctactctaaaactggggcaacaaatacgtggetcetttoctggatcet 441 C N @ N ET TF W Q C X N W Q WG GUL 8 1321 tgtaatggaaatgaaactactttttggcaatgcaaaaactggeagtggggceggectttce 461 C D N F BE E A XK VT C S G HR EUPR UL V 1381 tgtgataatttcgaagaagccaaagttacctgctcaggecacagggaacccagactggtt 481 G G6 BE I P C 8 G R V EM X HG D V W G 8 1441 ggaggagaaatcccatgotetggtogtgtggaaatgaaacacggagacgtgtgaggetee 501 v ¢C D P DL 8 L BA ASV V CREILAUGQGC 1501 gtctgtgattttgacttgtetctggaagetgeccagtgtggtgtgeagggaattacaatgt 52066 T V V 8 I L GG G A HF G RBG S GQ I Ww 1561 ggaacagtcgtctctatcctagggggagcacattttggagaaggaagtggacagatetgg

S41G B BE F Q C 8 G6 D BE S HL § L C SS V AP 1621 ggtgaagaattccagtgtagtggggatgagtcccatcttteactatgeteagtggegece 561 P L D RT C T H 8 R DV § V V C S R Y I 1681 ccgctagacagaacttgtacccacagcagggatgtcagegtagtctgceteacgatacata 51D I R L A GQ G BE 8 S$ C BR GR V BL K TL 1741 gatattcgtcetggcaggeggecgagtectectgtgagggaagagtggagctcaagacacte

601G A W G P LL C 8s Ss HW DMUEDAHRUVTL C 1801 ggagcctggggtoccctetgecagttctcattgggacatggaagatgetecatgtettatgt 621 Q Q L K C @ V A QQ 8S I P E G A HPF GG K GC 1861 cagcagetgaagtgtggggttgcccaatctatteccagaaggageacat tttgggaaagga 641A G Q V W 8S HM F HCTGTOZ EEUHTIGSTD 1921 gctggtcaggtctggagtcacatgttceactgcactggaactgaggaacatataggagat 661C L MT A LL @ A PT CS EG Q VAS VI 1981 tgcctcatgactgctetgggtgegecgacgtgttcegaaggacaggtggectetgteate 681Cc § 6 N Q 8 Q TLL PCS SLs PV QT 2041 tgctcaggaaaccaatcccagacactattgecatgtagtteattgtctccagtccaaaca 70. T § § T I P XK E 8 EV PC IASG QULR 2101 acaagctctacaattccaaaggagagtgaagttccctgeatagcaagtggecagettege 721 L V 6 8 G G R C A GR V BEB VY HEG S§ W 2161 ttggtaggtggaggtggtcgctgegetggaagagtggaggtctacecacgagggetettgg 7416 T Vv ¢C DD NWDMTUDA ANVYVYV CK QL 2221 ggcaccgtctgtgatgacaattgggatatgactgatgecaatgtggtgtgcaageagetg 761 D ¢C @ V A I N A T G 8 A Y P GG BR G A G A 2281 gactgtggegtggcaattaacgcecactggctetgettactteggggaaggagcaggaget 781 I WwW L D BR V I C T G@G K BE 8 H I WwW Q C H 8 2341 atctggctagacgaagtcatctgcactgggaaagagtctcatatttggecagtgecatteca 80l1H 6G W G R HN CR HKZ BDA AGU VIC CS EB 2401 catggctggggacgccataactgcaggcacaaagaagatgcaggtgttatctgetecgag 821 F M 8S L R LL T N E A HKUENUGOCTG GR RTL B 2461 ttcatgtctctgaggctgaccaacgaagcccacaaagaaaactgcacaggtcegocttgaa 841 V F Y N G T W G 8 I G 8S 8S NM 8 P TT V 2521 gtgttttacaatggtacatggggcagtattggcagtagcaatatgtctecaaccactgtyg 8613 Vv Vv CC R Q L. 66 C AD NG TV KPI P 8 2581 ggggtggtgtgccgtcagctgggctgtgcagacaacgggactgtgaaacccataccttca 881 D K T P 8 R P M W V DR V Q €C P XK G V D 2641 gacaagacaccatccaggceccatgtgggtagatcgtgtgecagtgtccaaaaggagttgac 901 T L W QQ C P 8 8 PW K Q R Q A 8 P 8 8 Q 2701 actttgtggcagtgcccctogtcaccttggaaacagagacaggecageccectocteecag 921 E 8 W I I € D N K I RL Q BE G HTD C 8S 2761 gagtcctggatcatctgtgacaacaaaataagactccaggaagggcatacagactgttet 9491 G R V BR I WwW H K 0 8 W G3 T V C DD SS WD 2821 ggacgtgtggagatctggcacaaaggttoctggggaacagtgtgtgatgactectgggat 961 L N D A K V V CC K QL GCG OQ AV EK ATL 2881 cttaatgatgctaaggttgtatgtaagcagttgggctgtggccaagetgtgaaggcacta 981 K B A AF G P GT 3 PI WILNUETIIZKTCR 2941 aaagaagcagcatttggtccaggaactgggeccatatggotceaatgaaattaagtgtaga 1000 3 N B 8 8 L W D C P A K P WW 8 H 8 DC G6 3001 gggaatgagtcttccctgtgggattgtectgecaaacegtggagtoacagecgactgtggg 1021 H X B D A 8 I Q CL P KMTS BRS HHBRG 3061 cacaaagaagatgcttccatccagtgectccccaaaatgactteagaatcacatoatgge 1041 T 6 H P T L T A L L V CC G6 A I L L V IL L 3121 acaggtcaccccaccctcacggcactcttggtttgtggageccattctattggteoetecte 10611 V P L L W TL KR R QI QR LTV 8 8 3181 attgtcttcctcctgtggactetgaagegacgacagattcagegacttacagtttcctea 1081 R G6 BE V L I H Q V Q Y Q EMD SS KA ADD 3241 agaggagaggtcttgatacatcaagttcagtaccaagagatggattcaaaggcggatgat 1101 L D LL L K § Ss BN S NN B88 YD PF NDTDG 3301 ctggacttgcetgaaatcctcggaaaattccaacaattcatatgattttaatgatgatgga 1121 LL. T § L § K ¥ L P I 8 G I K K G 8 F R G 3361 ctgacatctttgtctasatatcttcctatttctggaattaaaaaggggtcattcagaggce 1141 T L R R K M I I ¥Y ¥N P LL RL BE P K K Pp 3421 acactgagaaggaaaatgataatttataatccactgaggttggagtttaagaagcct 1 MGGHRMVLLG GAGSPGCKRF VHLGFFVVAV SSLLSASAVT NAPGEMKKEL ID NO:27

S1 RLAGGENNCS GRVELKIHDK WGTVCENGWS MNEVSVVCQQ LGCPTSIKAL . 101 GWANSSAGSG YIWMDKVSCT GNBSALWDCK HDGWGKHNCT HEKDAGVTCS 151 LGSNLEMRLV NSAGHRCLGR VEIKFQGKNG TVCDDNFSKD HASVICKQLG 201 CGSAISFSGS AKLGAGSGPI WLDODLACNGM ESALWDCKHR GWGKHNCDHA 251 EDVGVICLEG ADLSLRLVDG VSRCSGRLEV RFQGEWGTVC DDNWDLRDAS 301 VVCKQLGCPT AISAIGRVNA SBGSGQIWLD NISCEGHEAT LWECKHQEWG 351 KHYCHHREDA GVTCSDGADL BLRLVGGGSR CAGIVEVEIQ KLTGKMCSRG 401 WTLADADVVC RQLGCGSALQ TQAKIYSKTG ATNTWLFPGS CNGNRTTFWQ 451 CKNWQWGGLS CDNFEEAKVT CSGHREPRLV GGEIPCSGRV EMKHGDVWGS 501 VCDFDLELEA ASVVCRELQC GTVVSILGGA RPGEGSGQIW GEEFQCSGDE 551 SHLSLCSVAP PLDRTCTHSR DVSVVCSRYI DIRLAGGESS CEGRVELKTL 601 GAWGPLCSSH WDMEDAHVLC QQLKCGVAQS IPEGAHFGKG AGQVWSHMFH 651 CTGTEEHIGD CLMTALGAPT CSBGQVASVI CSGNQSQTLL PCSSLSPVQT 701 TSSTIPKESE VPCIASGQLR LVGGGGRCAG RVEVYHEGSW GTVCDDNWDM 751 TDANVVCKQL DCGVAINATG SAYPGEGAGA IWLDEVICTG KEBSHINQCHS 801 HGWGRHNCRH KEDAGVICSE FMSLRLTNEA HKENCTGRLE VFYNGTWGSI 8s1 GSSMMSPTTV GVVCRQLGCA DNGTVKPIPS DKTPSRPMWV DRVQCPKGVD 901 TLWQCPSSPW KQROASPSEQ BSWIICDNKI RLQEGHTDCS GRVEIWHKGS 951 WATVCDDSWD LNDAKVVCKQ LGCGQAVKAL KEAAFGPGTG PIWLNEIKCR 1001 GNESSLWDCP AKPWEHEDCG HKEDASIQCL PRMTSESHAHG TGHPTLTALL 1051 VOGAILLVLL IVFLLWTLKR RQIQRLTVSS RGEVLIHQVQ YQEMDSKADD 1101 LDLLKSSENS NNSYDFNDDG LTSLSKYLPI SGIKKGSFRG TLRRKMIIYN 1151 PLRLEFKKP

*TIUS2005/011502

Example 8: Cloning and characterization of MARC-145 CD163

A forward primer 5’simianCD163 (SEQ ID NO: 28) (5°’-

CACCGGAATGAGCAAACTCAGAATGG-3’ based on human CD163) and a reverse primer HuCD163-3’Kpn (SEQ ID NO:29) (5°-

TGCTCCGGTACCTAGTCCAGGTCTTCATCAAGGTATCTTA-3’) were used to amplify CD163 cDNA from MARC-145 African Green Monkey kidney cells. Total cellular RNA was prepared from MARC-145 cells using the RNeasy kit. RT-PCR parameters were the same as described in Example 4. RT-PCR products were cloned directionally into the pCDNA3.1D/V5/His/TOPO vector according to the manufacturer’s instruction. Several clones containing large inserts were analyzed.

Clone #25 was designated “pCDNA3.1D-MARC-CD163v2”. This novel CD163 cDNA from MARC-1435 cells is 1116 amino acids in length. When compared to the sequences in GenBank database, the MARC-145 CD163 amino acid sequence is 96.3% identical to human CD163 (Genbank Z22968), 84.7% identical to pig CD163 (Genbank

AJ311716), and 73.9% identical to mouse CD163 (Genbank AF274883). atgagcaaac tcagaatggt gctacttgaa gactctggat ctgctgacgt cagaagacat 60 SEQ ID tttgtcaact tgagtcecctt cactattgct gtggtcttac ttetcegtge ctgttttgte 120 NO 30 accagttcic ttggaggaac aaccaaggag ctgaggctag tggatggtga aaacaagtgt 180 ’ agtgggagag tggaagtgaa aatccaggag gagtggggaa cggtgtgtaa taatggctgg 240 agcatggaag cagtctctgt gatttgtaac cagctgggat gtccaactge tatcaaagcce 300 actggatggg ctaattccag tgcaggttet ggacgcattt ggatggatca tgtttettgt 360 cgtgggaatg agtcagctct ttgggactgc aaacatgatg gatggggaaa gcatagtaac 420 tgtactcacc aacaagatgc tggagtgact tgctcagatg gatccgattt ggaaatgagg 480 ctgacgaatg gagggaatat gtgttctgga agaatagaga tcaaattcca aggacagtgg 540 ggaacagtgt gtgatgataa cttcaacatc aatcatgecat ctgtggtttg taaacaactt 600 gaatgtggaa gtgctgtcag tttetetget tcagectaatt ttggagaagg ctctggacca 660 atctggtttg atgatcttat atgcaacgga aatgagtcag ctetctggaa ctgcaaacat 720 caaggatggg gaaagcataa ctgtgatcat gctgaggatg ctggagtgat ttgctcaaag 780 ggagcagatc tgagcctgag actggtagat ggagtcactg aatgttcagg aagattagaa 840 gtgagattcc aaggagaatg ggggacaata tgtgatgacg gctgggacag tcatgatgct 900 gctgtggcat gcaagcaact gggatgtcca actgctatca ccgecattgg tcgagttaac 960 gccagtgagg gatttggaca catctggett gacagtgttt cttgccaggg acatgaacct 1020 geggtctgge aatgtaaaca ccatgaatgg ggaaagcatt attgcaatca caatgaagat 1080 gctggegtaa catgttotga tggatcagat ctggagctaa gacttagagg tggaggcagc 1140 cgetgigetg ggacagttga ggtggagatt cagagactgt tagggaaggt gtgtgacaga 1200 ggctggggac tgaaagaage tgatgtggtt tgeaggcage tgggatgtgg atctgecactc 1260

* amaacatcct atoaagtata otccasasto caggcaacaa acatgtggct gtttctaagt 1320 agctgtaacg gaaatgaaac ttctctttgg gactgcaaga actggcaatg gggtggactt 1380 acctgtgatc actatgaaga agecaaaatt acctgctcag cccacaggga acccagactg 1440 gttggaggag acattccctg ttetggacge gttgaagtga agcatggtga cacatgggge 1500 tccgtetgtg atteggattt ctctetggaa getgcecageg ttctatgcag ggaattacag 1560 tgtggcacag tcegtctetat cctgggggga getcactttg gagagggaaa tggacagatc 1620 tgggctgaag aattccagtg tgagggacat gagtcccatc tttcactctg cccagtagca 1680 ccccgeccag aaggaacttg tagccacage agggatgttg gagtagtctg ctcaagatac 1740 acagaaattc gcttggtgaa tggcaagacc ccatgtgagg goagagtgga getcaaaacg 1800 cttaatgcct ggggatccect ctgcaactct cactgggaca tagaagatgc ccacgttctt 1860 tgccaacaac ttaaatgtgg agttgccectt tctaccccag gaggageaca ttttggaaaa 1920 ggaaatggtc aggtctggag gcatatgttt cactgcactg ggactgagca gecacatggga 1980 gattgtcctg taactgctet gggtgettca ctatgtcctt cagggcaagt ggcctctgta 2040 atttgetcag gaaaccagte ccaaacactg tcctegtgca attcatcato tctgggecca 2100 acaaggccta ccattccaga agaaagtgct gtggectgea tagagagtag teaacttoge 2160 ttggtaaatg gaggaggtcg ctgtgctggg agagtagaga tttatcatga gggetectag 2220 ggcaccatct gtgatgacag ctgggacctg agcgatgcce acgtggtgtg cagacagotg 2280 ggctgtggag aggccattaa tgccactggt tctgcteatt ttggagaagg aacagggcce 2340 atctggctgg atgagatgaa atgcaatgga aaagaatccc geatttggea gtgecattca 2400 catggctggg ggcagcaaaa ctgcaggcac aaggaggatg caggagttat ctgctcagag 2460 ttcatgtctc tgagactgac cagtgaagece agcagagagg cctgtgeagg gcgtctagaa 2520 gttttttaca acggagcttg gggecagtgtt ggcaggagta acatgtctga aaccactgtg 2580 ggtgtggtgt gcaggcagct ggcctgtgca gacaaaggga aaatcaacce tgcatcttta 2640 gacaaggcca tgtccattce catgtgggtg gacaatgttc agtgtccaaa aggacctgac 2700 acgetgtggc agtgcccatc atctccatgg gagaagagac tggccaggee ctoggaggag 2760 acctggatca catgtgacaa caagatgaga ctacaagaag gacccacttc ctgttctgga 2820 cgtgtggaga tctggcacgg aggttcctgg gggacagtgt gtgatgactc ctgggacttg 2880 aacgatgctc aggtggtgtg tcaacaactt ggetgtggtc cagctttgaa agcattcaaa 2940 gaagcagagt ttggtcaggg gactggaccc atatggctca atgaagtgaa gtgcaaaggg 3000 aatgagtctt ccrtgtggga ttgtcctgec agacgetggg gocacagtga gtgtggacac 3060 daaggaagacg ctgcagtgaa ttgcacagat atttcaacga acaaaacccc acaaaaagcc 3120 acaacaggtc agtcatcect tattgcagtc ggaatccttg gagttgttcet cttggteatt 3180 ttcgtegeat tattcttgac tcaaaagcga agacagagac agcggcttac agtttectea 3240 agaggagaga acttagtcea ccaaattcaa taccgggaga tgaattcttg cctgaatgca 3300 gatgatctgg acctaatgaa ttectcagga ggccattctg aggcacactg aaaagcaaaa 3360 tgggaattta taacccagtg agccttgaag ataccttgat gaagacctgg acta 3414 1 Ss KL RMV LIL ZBD SG SADUVR R H SEQ ID 1 atgagcaaactcagaatggtgctacttgaagactctggatctgetgacgteagaagacat 21F V ¥N L § P 7 T I A VV LUILTULTPRATCTFEV NO: 31 61 tttgtcaacttgagtcccttcactattgctgtggtettacttotecgtgectgttttgte 41T 8 8 L G6 G T T K EL RL V D GE N K C 121 accagttctcttggaggaacaaccaaggagetgaggetagtggatggtgaaaacaagtgt and 32 618 G R VB V XK I Q EEWGTUV C NR NG W

181 agtgggagagtggaagtgaaaatccaggaggagtggggaacggtgtgtaataatggctgg

BlSs M BRA V Ss VI CNQLGOCUPTA ATITZKA 241 agcatggaagcagtctctgtgatttgtaaccagctgggatgtccaactgetatcaaagee 101 T G W AR N S Ss AG 8 GR I WMDH HVS C 301 actggatgggctaattccagtgcaggttctggacgcatttggatggatcatgtttettgt 121 R G N BE 8S A L W D C K HDG WGK KH SN 361 cgtgggaatgagtcagctctttygggactgcaaacatgatggatggggaaagcatagtaac 141 C T HQ Q D AG VT C 8 DG S§ DL EMR 421 tgtactcaccaacaagatgctggagtgacttgctcagatggatccgatttggaaatgagg 161 L T N @G @ N M CC 8s G6 RI E I K PF Q GQ Ww 481 ctgacgaatggagggaatatgtgttctggaagaatagagatcaaattccaaggacagtgg 181G T V C D D N FP N I N H A 8 V V C K QL 541 ggaacagtgtgtgatgataacttcaacatcaatcatgcatctgtggtttgtaaacaactt 200 E C G 8 AV 8 P 8 G § A NF GEG 8 G P 601 gaatgtggaagtgctgteagtttctetggttcagctaattttggagaaggctctggacca 221 I W F D D LL I C N G N BR S A L WN CK H 661 atctggtttgatgatcttatatgcaacggaaatgagtcagcetctctggaactgcaaacat 241 Q G W G KH NC D HAI ETD AGTV ICS K 721 caaggatggggaaagcataactgtgatcatgctgaggatgctggagtgatttgctcaaag 261 G A DL 8 L RL VD GV TUE EG CS SGT RULE 781 ggagcagatctgagcctgagactggtagatggagtcactgaatgttcaggaagattagaa 281 V R F Q G B WG T I C DDG WD S HD a 841 gtgagattccaaggagaatgggggacaatatgtgatgacggctgggacagtcatgatget 30A V AC K QL G CUPTATITA ATIGTZ RYVYVYN 901 gctgtggcatgcaagcaactgggatgtccaactgctatcacegecattggtcgagttaac 321A Ss E G P G HI WL DS V 8 CQ GHEE P 961 gccagtgagggatttggacacatctggettgacagtgtttottgccagggacatgaacct 341A V W Q C K HH E WG KH Y CNUBHNTETD 1021 geggtctggcaatgtaaacaccatgaatggggaaagcattattgcaatcacaatgaagat 361A G6 VT C S$ DG@ S DLEULURTLTERTGTG GG S 1081 gectggegtaacatgttctgatggatcagatctggagctaagacttagaggtggaggcage 381R ¢C A 6G T V §E V E I Q RL L GG KV CDR 1141 cgetgtgetgggacagttgaggtggagattcagagactgttagggaaggtgtgtgacaga 4013 WwW 3 L XK E A D V V CR QL GCG SAID 1201 ggctggggactgaaagaagctgatgtggtttgeaggcagetgggatgtggatctgeacte 421 kK T 8 Y Q V ¥Y § K I Q AT N MNULUPTL 8 1261 aaaacatcctatcaagtatactccaaaatccaggcaacaaacatgtggetgtttetaagt 4418 C N G N BT S L WDC KNWOQUWOGO EG L 1321 agctgtaacggaaatgaaacttctctttgggactgecaagaactggcaatggggtggactt 461 T C D H Y BE BE A K I T C 8S A HREUP PRL 1381 acctgtgatcactatgaagaagccaaaattacctgetcageccacagggaacceagactg 481 Vv @ @ PDP I PC 8 GR V EV KHGUDTUWG 1441 gttggaggagacattccctgttctggacgegttgaagtgaageatggtgacacataggge 501s Vv ¢D ss DPF 8 ILL EAAS VLC CRTETLQ 1501 tcegtctgtgattoggatttototetggaagetgecagegttetatgeagggaattacag $31 C 6 T V V 8 I L 8 GG A HF GE GNGOQTI 1561 tgtggcacagtegtctctatectggggggaget cactttggagagggaaatggacagatc

S41 WW A BR BE P Q CE GHZRS HL SL CP V Aa 1621 tgggctgaagaattccagtgtgagggacatgagtcccatcttteactctgecccagtagea 561 P R P EG T C 8 H 8 RD V G6 V VC SR Y 1681 ccccgcccagaaggaacttgtagecacageagggatgttggagtagtctgetcaagatac 581 T E I RL V N G KT P C E GR VE I K T 1741 acagaaattcgcttggtgaatggcaagaccccatgtgagggcagagtggagctcaaaacg 601 L. N A W GG S L C N S HW DI EDA AETV YL 1801 cttaatgcctggggatcoctctgeaactctcactgggacatagaagatgeccacgttett 621C Q Q L K C G V A L § T P GCG BG A HP G K 1861 tgccaacaacttaaatgtggagttgccctttctaccccaggaggagcacattttggaaaa 641 G N G6 Q V W R H M P H C T GT EB Q HM G 1521 ggaaatggtcaggtctggaggeatatgtttcactgeactgggactgagcagcacatggga 661D C P VT AL G A SL CP 8 G QV A 8 V 1981 gattgtcctgtaactgctctgggtgettcactatgtecttcagggcaagtggectetgta 681 I C 8S 6G N Q S Q T L S 8 C N B8 S § L @G p 2041 atttgctcaggaaaccagtcccaaacactgtcctegtgeaattcatcatctetgggeeca 7010T R P T I P BRE S$ A VA CTIGE ES SGA QL R 2101 acaaggcctaccattecagaagaaagtgetgtggectgeatagagagtggtcaacttege 721 L V N @ 8 6G R C A G R V E I Y HE GOB W 2161 ttggtaaatggaggaggt cgctgtgetgggagagtagagatttat catgagggetectgy 7416 T I ¢ D > 8 W DL SD AUHVYVCRGAGQTL 2221 ggcaccatctgtgatgacagetgggacctgagegatgeceacgtggtgtgcagacagetg 761 G C G E A I N A TG S A HF GEG GTG. UP i 2281 ggctgtggagaggccattaatgccactggttctget cat tttggagaaggaacagggeoe 781 1 W L D BR M K C N G KE 8S RI WOQTZCH 8 2341 atctggctggatgagatgaaatgcaatggaaaagaatccecgeatttggcagtgecattca 80H CG W @ Q Q NC RHEIEKETUDA AGTVYVT ICS SE 2401 catggctgggggcagcaaaactgcaggcacaaggaggatgeaggagttatctgctcagag 821 F M 85S L R LL T 8S E A S R E AC AGU RULE 2461 ttcatgtcetctgagactgaccagtgaagecageagagaggectgtgcagggegtctagaa 841 V FP ¥Y XN @ A WG S$ V GR SNMTGETETTV 2521 gttttttacaacggagcttggggcagtgtiggeaggagtaacatgtctgaaaccactgtg 861G V V C R QL 8 C ADI KGIXTIMNTZPA AT SL 2581 ggtgtggtgtgcaggcagctgggctgtgeagacaaagggaaaatcaacectgcatettta 881 D K A M S I PM W VDNJVOQC CEPI KTGTPD 2641 gacaaggccatgtccattccocaty gggtggacaatgttcagtgtccaaaaggacctgac

) S2005/011502 v 01T L Wa CPS SP WE KFETLARTPS EE 2701 acgctgtggcagtgcccatcatctcecatgggagaagagactggecaggeccteggaggag 921 T W I T ¢€¢ OD N KK M RL Q Ed PT S CC $$ @ 2761 acctggatcacatgtgacaacaagatgagactacaagaaggacccacttcectgttetgga 941 R V EI W HG G S WG TV C DDS WDL 2821 cgtgtggagatctggcacggaggttcetgggggacagtgtgtgatgactectgggacteg 961 N D AQ V v C Q QL GC @ P AL K A PF K 2881 aacgatgctcaggtggtgtgtcaacaacttggctgtggtccagetttgaaagcattcaaa 981 E A E P GG Q 6 T G P I W L NB V XK CK G 2941 gaagcagagtttggtcaggggactggacccatatggcetcaatgaagtgaagtgcaaaggg 1001 N BE 8 9 L WD C P A RR WG GH 8S ECG H 3001 aatgagtcttecttgtgggattgtcctgecagacgetggggecacagtgagtgtggacac 1021 K BE D A A V NC TD I 8 T NI KTP Q K A 3061 aaggaagacgctgcagtgaattgcacagatatttcaacgaacaaaaccccacaaaaagec 1041T T 3G Q 8 $L I AVG ILGVYVUVTLTELV TZ 3121 acaacaggtcagtcatcccttattgeagtcggaatcettggagttgttetottggtoatt 1061 P V A L P LL T Q KR R QR QURUL TV 8S 8 3181 ttcgtegeattattottgactcaaaagecgaagacagagacageggcettacagtttectea 1081 R G B N L V H Q I Q YR B MN 8 CL NA 3241 agaggagagaacttagtccaccaaattcaataccgggagatgaattcettgectgaatgea 11000 D L D L M N 8S 8 8 G H § E A H 3301 gatgatctggacctaatgaattcctcaggaggccattctgaggeacac 1 MSKLRMVLLE DSGSADVRRH FVNLSPFTIA VVLLLRACFV TSSLGGTTKRE SEQ D 51 LRLVDGENKC SGRVEVKIQE EWGTVCNNGN SMEAVSVICN QLACPTAIKA 101 TGWANSSAGS GRIWMDHVSC RGNESALWDC KHDGWGKHSN CTHQQDAGVT 151 CSDGSDLEMR LTNGANMCSG RIEIKFQGON GTVCDDNFNI NHASVVCKQL NO: 32 201 ECGSAVSFSG SANFGEGSGP IWFDDLICNG NESALWNCKH QGWGKHNCDH 251 AEDAGVICSK GADLSLRLVD GVITECSGRLE VRPQGEWGTI CDDGWDSHDA 301 AVACKQLGCP TAITAIGRVN ASBGPGHIWL DSVSCQGHRP AVHQCKHHEW 351 GKHYCNHNED AGQVTCSDGSD LBLRLRGGGS RCAGTVEVEI QRLLGKVCDR 401 GWGLKEADVV CRQLGCGSAL KTSYQVYSKI GATMMWLFLS SCNGNETSLN 451 DCKNWOWGGL TCDHYEEAKI TCSAHREPRL VGGDIPCSGR VEVEKHGDTWG 501 SVCDSDFSLE AASVLCRELQ CGTVVSILGG AHPGEGNGQI WABRFQCBGH 551 ESHLSLCPVA PRPEGTCSHS RDVGVVCSRY TRIRLVNGKT PCERGRVELKT 601 LNAWGSLCNS HWDIEDAHVL CQQLKCGVAL STPGGAHFGK GNGQVWRHMF 651 HCTGTEQHMG DCPVTALGAS LCPSGQVASV ICSGNQSQTL SSCNSSSLGP 701 TRPTIPERSA VACIESGQLR LVNGGGRCAG RVRIYHBGSW GTICDDSWDL 751 SDAHVVCRQL GOGEAINATG SAHFGRGTGP IWLDENKCNG XBSRINQCHS 801 HGWGQQNCRH KEDAGVICSE FMSLRLTSEA SREACAGRLE VFYNGAWGSV 851 GRSNMSETTV GVVCRQLGCA DKGKINPASL DKAMSIPMWV DNVQCPXGPD 901 TLWQCPSSPW EKRLARPSEE TNITCDNEMR LQEBGPTSCSG RVRINHGGSW 951 GTVCDDSWDL NDAQVVCQQL GCGPALKAFK BABPGQATGP INLNEVKCKG 1001 NESSLWDCPA RRWGHSBCGH KEDAAVNCTD ISTNKTPQKR TTGQSSLIAV 1051 GILGVVLLVI FVALPLTQKR RQRQRLTVSS RGENLVHQIQ YREMNSCLNA 1101 DDLDLMNSSG GHSEAH

Example 9. Cloning and characterization of simian CD163 from Vero cells.

A forward primer 5’simianCD163 (SEQ ID NO: 28) (5’-

TGCTCCGGTACCTAGTCCAGGTCTTCATCAAGGTATCTTA-3’) were used to amplify CD163 cDNA from Vero cells. Total cellular RNA was prepared from Vero cells using the RNeasy kit. RT-PCR parameters were the same as described in Example 4. RT-PCR products were cloned directionally into the pCDNA3.1D/V5/His/TOPO vector according to the manufacturer's instruction. Eight clones containing large inserts were sequenced, and six discreet splicing patterns were found. These patterns are depicted graphically in Figure 17.

The six splicing variants differ in the presence or absence of three exons, designated E6, E105, and E83. Omissions of E6 or E105 do not change the reading frame, whereas omission of E83 does. Patterns similar to v2 and/or v3 were also seen in porcine, murine, human, and MARC-145 monkey cells. Patterns v4 and v5 lack the 105-nucleotide exon that encodes the hydrophobic transmembrane region. These cDNAs were unable to render BHK cells permissive to PRRSV infection in a transient transfection assay, probably because the CD163 is secreted rather than remaining membrane bound. Although CD163 molecules lacking a transmembrane region appear to be non-functional as cellular permissivity factors, it is possible that they may have utility either in direct virus neutralization (similar to neutralizing antibodies), or as an immunogen for the induction of anti-CD163 antibodies which would block viral infection in the host animal.

The longest splice variant, v7, contains all three of the exons E6, E105, and E83.

This novel CD163 cDNA from Vero cells encodes a polypeptide that is 1153 amino acids in length. When compared to the sequences in Genbank database, the Vero

CD163v7 amino acid sequence is 95.4% identical to human CD163 (Genbank 722968), 83.7% identical to pig CD163 (Genbank AJ311716), and 72.1% identical to mouse

CD163 (Genbank AF274883). The nucleotide and amino acid sequences of the six splice variants found in Vero cells are provided below (SEQ ID NOS:33-44). 1M 8 KL R MV LL ED S G 8 AD V RR H SEQ ID 1 ATGAGCAAACTCAGAATGETGCTACTTGAAGACTCTGGATCTGCTGACGTCAGARGACAT NO:33 22 P VN IL § PP TIA AV VLLULU RIA BATCGCGTEUV and 34 61 TTTGTCAACTTGAGTCCCTTCACTATTGCTGTGATCTTACTTCTCCGTGCCTGTTTTGTC 41 T 8 8 L GG G T T K BR L RL V D G BR N K C 121 ACCAGTTCTCTTOGAGGAACAACCAAGGAGCTGAGGCTAGTGGATGGTGAAAACAAGTGT 618 G RV BE V XK I QR EWGTUVCNUNGHVW 181 RAGTGGGAGAGTGGAAGTGAAAATCCAGGAGGAGTGAGAAACGATGTATAATAATGGCTGG 81S M E A V 8s V I CN QL GC PTA ATIIZK OA 241 AGCATGGAAGCAGTCTCTATGATTTGTAACCAGCTGGGATGTCCAACTGCTATCAAAGCC 101 T G W A N S§ S A 3G S 6 RI WM DD HV 8 C 301 ACTGGATGGGCTAATTCCAGTGCAGGTTCTGGACGCATTTGGATGGATCATGTTTCTTIGT 121R GN E 8. A L WDC UX HD G W GG KH SN 361 CGTGGGAATGAGTCAGCTCTTTGGGACTGCAAACATGATGGATGGGGAAAGCATAGTAAC 141 C¢C T H Q Q D A G V T €C $ D G § D LL E M R 421 TGTACTCACCAACAAGATGCTGGAGTAACTTGCTCAGATGGATCCGATTTGAAAATGAGG 161 L T N G G N M C 5S @ RI BE I KF Q G Q Ww 481 CTGACGAATGGAGGGAATATATGTTCTAGAAGAATAGAGATCAAATTCCAAGGACAGTGE 181d T V C DP D N P N I N HA § VV CK QL 541 GGAACAGTGTGTGATGATAACTTCAACATCAATCATGCATCTGTGGTTTGTAAACARCTT 2300 E C G 8 AV 5 P 5s @ S ANP GTZET GS G Pp 601 GAATGTGGAAGTGCTGTCAGTTTCTCTGGTTCAGCTAATTTTGGAGAAGGCTCTGGACCA 22) I W F DDL ICNGNETSESA ALTWNDNTGCTE KH 661 ATCTGGTTTGATGATCTTATATGCAACGGAAATGAGTCAGCTCTCTOGAACTGCAAACAT

: 2410 GW GG K HN CD HEAETDA ASG GUY I CS K 721 CAAGGATGGGGAAAGCATAACTGTGATCATGCTGAGGATGCTGGAGTGATTTGCTCAAAG 261G A DL S L RL VDGVTTETCSGRTULE 781 GGAGCAGATCTGAGCCTGAGACTGGTAGATGGAGTCACTGARTGTTCAGGAAGATTAGAR 281V R P Q G EW G T I CDDGWDS HTD A 841 GTGAGATTCCAAGGAGAATGGGGAACAATATGTGATGACGGCTGGGACAGTCATGATGCT 30LA V ACK QLGCPTA ATITA ATILIGTE RTUVYN 901 GCTGTGGCATGCAAGCAACTGGAATGTCCAACTGCTATCACCGCCATTGGTCGAGTTARS 321A S BE G PF G H I W LD SV SCQGHTEP 961 GCCAGTGAGGGATTTGGACACATCTAGCTTGACAGTGTTTCTIGOCAGGGACATGARCCT 341A V W Q C KH H EWG KH TYCNTEHONTED 1021 GCGATCTGGCARTATAAACACCATGAATGGGGAARGCATTATTGCAATCACAATGAAGAT 1A G6 VT CSDGSGLE BLRLTERGG GG a 8 1081 GCTGGCGTAACATGTTCTGATGGATCAGATCTGERGCTAAGACTTAGAGGTGGAGGCAGC 31R C AG TV EVETIOQRTLTLGTE KTVT CDR 1141 CGCTGTGCTAGGACAGTTGAGGTGGAGATTCAGAGACTATTAGGGAAGGTGTATGACAGA 4010 ¥ 6 L X E ADV VCRGQLG GT CGS SATL 1201 GGCTGGGGACTGAAAGAAGCTGATGTGGTTTGCAGACAGCTGGAATATGAATCTGCACTC 421K T S$ Y Q VY SBS KIQATUNMGMWELTPFZTL § 1261 AARACATCCTATCAAGTATACTCCAAAATCCAGGCAACAAACATGTGGCTGTTTCTAAGT 4418 C N @ N ET SL WDCIEKUNTM®WONOGSG G L 1321 AGCTGTAACGGRAATGARACTTCTCTTTGGGACTGCAAGAACTGGCAATGGAATGGACTT 461T ¢ D BE Y EE A K IT CGB8 AUHURTETPR RL 1381 ACCTGTGATCACTATGAAGAAGCCAARATTACCTGCTCAGCCCACAGGGAACCCAGACTG 481V G @ D I P C 8 G RV EV K H GD T W G 1441 GTTOGAGGAGACATTCCCTATTCTGGACGCGTTGAAGTGARGCATGGTGACACATAAAAC 501§ Vv ¢ DS DF SL EBAAGEVTLC CRTETLDQ 1501 TCOGTCTGTGATTCGGATTTCTCTCTGAAAGCTACCAGCITTCTATGCCAGGGAATTACAG 521C 6 T VV 8 I L G GAUHTPEFG GEG GENTGT QI 1561 TGTGGCACAGTCGTCTCTATCCTGGGGACAGCTCACTTTGGAGAGGGAAATGGACAGATC

S41W T E EF Q C E G HES HL EL CTPV A 1621 TGGACTGAAGAATTCCAGTGTGAGGGACATGAGTCCCATCTTTCACTCTGCCCAGTAGCA 51? R P EG T C 8 HS RDVGYV V CST RY 1681 CCCCGCCCAGAAGGAACTTGTAGCCACAGCAGGGATGTTGGAGTAGTCTGCTCAAGATAC

Se1T E I RL VN GK TGPCEGTRTYTETLTEKT 1741 ACAGARATTCGCTTGGTGAATGGCAAGACCCCATGTGAGGGCAGAGTAGAGCTCAAAACG 60LL N A W G § L CN § HW D>T1Z EDA AEHTUYTL 1801 CITAATGCCTGGGGATCCCTCTGCAACTCTCACTGGGACATAGARGATGCCCACATTCTT 621C Q Q L X C 6G V AL S TP GG AHP G K 1861 TGCCAACAACTTARATGTGGAGTTGCCCTTTCTACCCCAGGAQGGAGCACATTTTGGARAR 641G N G Q V ¥ R HM P H CT GT EQ KM G 1921 GGAAATGGTCAGGTCTGGAGGCATATATTTCACTGCACTCAGACTGAGCAGCACATGGGA 661D C P V T AL @G A SL CP? SG QV ATE V 1981 GATTATCCTGTAACTGCTCTGAGTGCTTCACTATGTCCTTCAGIGCAAGTGGCCTCTGTA 6811 C S 6G N Q S Q TL S 8 RENE 8 8 8 1L G P 2042 ATTTGCTCAGGARACCAGTCCCAAACACTATCCTCGCACAATTCATCATCTCTGGGCCCA i 701T R PT I PEESAVACTITESG GAO QTL R 2101 ACARGGCCTACCATTCCAGAAGARAGTGCTGTGGCCTGCATAGAGAGTGGTCAACTTCGC 721L VN GG GR CAGRUYVETITYHE RG SW 2161 TTGGTAAATGAAGGAGGTCOCTATGCTGGGAGAGTAGAGAT TTATCATGAGGGCTCCTGA 7416 T I C DD 8S WDLSDAEHETYUVCROQL 2221 GGCACCATCTGTGATGACAGCTGGGACCTGAGCGATGCCCACOTGGTATGCAGACAGCTA 761G € G BR AI NA TGS AEKTFGA TG BGT GG P 2281 GGCTGTGGAGAGGCCATTAATGCCACTAGTTCTGCTCATTTTGGAGAAGGAACAGAGCCE 7811 W L D BM KC NGJKTETST RTIMWO QTC CHS _85-

PCT/US2005/011502 to 2341 ATCTGGCTUGATGAGATGARATGCARTAGAARAGARTCCCOGCAT I TGGCAGTGCCATTCA 801 H G W G Q Q N CR H K BED A GG V I C 8&8 E 2401 CATGGCTGGGGGCAGCARAACTGCAGGCACAAGGAGGATGCAGGAGTTATCTACTCAGAG 821FP M $$ L RL T § EAS REA ATCA AGT RULE 2461 TTCATGTCTCTGAGACTGACCAGTGAAGCCAGCAGAGAGGCCTGTGCAGGGCGTCTAGAA 841V P Y N G A W G S V GR S NM SS ET T V 2521 GTTTTTTACAACGGAGCTTGGGGCAGTGTTGGCAGGAGTAACATGTCTGAAACCACTGTG 861@ V V C R Q L G C AD KG KI NS ASL 2%81 GGTGTAGTGTGCAGGCAGCTGGUCTGTACAGACAAAGGGARAATCAACTCTGCATCTTTA 881D K A M 68 I PM W VDNVAQCZPKTGTPD 2641 GACAAGGCCATGTCCATTCCCATGTGGGTGGACARTGTTCAGTGTCCAAAAGGACCTGAC

S01 T L WwW Q C P S 8 P W E K R L A RP 8S E B 2701 ACGCTATGGCAGTGCCCATCATCTCCATGGGAGAAGAGACTGACCABGCCCTCGGAGGAG 922 T W I T C D N KMRDLOQTZ RGT PTS SCS G 2761 ACCTGGATCACATGTAACAACAAGATGAGACTACAARGAAGGACCCACTTCCTGTTCTGGA 941 R V BR I WH G6 G S WG TV CDDGS WDL 2821 CGTATGGAGATCTGGCACGGAGGTTCCTGGGGGACAGTATATGATGACTCCTAGGACTTG 961 N D A Q V V CC Q Q L. @ ¢C GG P AL KAU PK 2881 AACGATGCTCAGGTGGTGTATCAACAACTTAGCTGTGGTCCAGCTTTGAAAGCATTCARA 9818 A BP @G Q G T G P I WIL NEJVIEKTCTE BG 2941 GAAGCAGAGTTTEGTCAGGGGACTGGACCCATATGGCTCAATGAAGTGAAGTGCAAAGEE 1001N BE § S L W D C P ARRAN GUH SET CGH 3001 ARTGAGTCTTCCTTGTGGGATTGTCCTGCCAGACGCTGEGGCCACAGTGAGTGTGGACAC 1021K BE D AAV NC T DTI STU RTEKTU® POQK A 3061 AAGGAAGACGCTGCAGTGARTIGCACAGATATTTCAACGCGCAAAACCCCACAAAAAGCC 1041T T 6 Q S §S L I AV G6 I L G V VL L A I 3121 ACARCAGGTCAGTCATCCCTTATTGCAGTCOAAATCCTTAGAGTTGTTCTCTTGGCCATT 1061 F V AL P LT Q XKRRQURU QQURTLTUVS 8 3181 TTCGTCGCATTATTCTTGACTCARRAGCGAAGACAGAGACAGCGACTTACAGTTTCCTCA 1081 R G6 E N L V H Q I Q YR E MN SB CL N A 3241 AGAGGAGMGAACTTAGTCCACCAAAT TCAATACCGOGAGATGAATTCTTGCCTGAATGCA 1101D D L DL M N S § A @G H 8 B A H 3301 GATGATCTGGACCTAATGAATTCCTCAGGAGGCCATTCTGAGGCACAC 1 MEXLRMVLLE DSGSADVRRH FVNLSPFTIA VVLLLRACFV TSSLGGTTKE LRLVDGENKC SEQ ID 61 SGRVEVKIQE EWGTVCNNGW SMERVSVICN QLGCPTAIKA TGWANSSAGS GRIWMDHVSC 121 RGNESALWDC KHDGWGKHSN CTHQQDAGVT CSDASDLEMR LTNGGNMCSG RIEIXFQGOW NOC:3¢ 181 GTVCDDNFNI NHASVVCKQL BCGSAVSPSG SANFGEGSGP IWFDDLICNG NESALWNCKH 241 QGWGKHNCDH AEDAGVICSK GADLSLRLVD GVTECSGRLE VRPQGEWGTI CDDGWDSHDA 301 AVACKQLGCP TAITAIGRVN ASEGFGHIWL DSVSCQOGHEP AVWQCKHHEW GKHYCNHNED 361 AGVTCSDGSG LELRLRGGGS RCAGTVEVEI QRLLGKVCDR GWGLKEADVV CRQLGCGSAL 421 KTSYQVYSKI QATNMWLPLS SCNGNETSLW DCKNWQWGGL TCDEYEEAKI TCSAHREPRL 481 VGGDIPCSGR VEVKHGDTWG SVCDSDFSELE AASVLCRELQ CGTVVSILGG AHFGEGNGQI 541 WTEEFQCEGH ESHLSLCPVA PRPEGTCSHS RDVGVVCSRY TETRLVNGKT PCEGRVELKT 601 LNAWGSLCNS HWDIEDAHVL CQQLKCGVAL STPGGAHPGK GNGQUWRHMF HCTGTEQHMG 661 DCPVTALGAS LCPSGQVASV ICSGNQSQTL SSRNSSSLGP TRPTIPEESA VACIESGQLR 721 LVNGGGRCAG RVEIYHEGSWN GTICDDSWDL SDAHVVCRQL GCGRAINATG SAHFGEGTGP 781 IWLDEMKCNG KESRIWQCHS HGWGQQNCRH KEDAGVICSE PMSLRLTSEA SREACAGRLE 841 VFYNGAWGSV GRSNMSETTV GVVCRQLGCA DKGKINEASL DKAMSIPMWV DNVQCPKGPD

S01 TLWQCPSSPW EKRLARPSEE TNITCDNKMR LQEGPTSCSG RVERINHGGSW GTVCDDSWDL 961 NDAQVVCQQL GCGPALKAFK EARFGQGTGP IWLNEVKCEG NESSLWDCPA RRWGHSBCGH © 1021 KEDMAAVNCTD ISTRKTPQKA TTGOSSLIAV GILGVVLLAI FVALFLTQKR RQRQRLTVES 1081 RGENLVHQIQ YREMNSCLNA DDLDLMNSSG GHSBAH

SEQ ID

1M 8S KL RM V LLEDTGSTGESATDTVT RT RH 1 ATGAGCAAACTCAGAATGGTGCTACTTGAAGACTCTGGATCTGCTGACGTCAGAAGACAT NO:35 20FP V NL § P PP T I A V VULILULULZERATCTPUV and 36 61 TTTGTCAACTTGAGTCCCTTCACTATTACTGTGETCTTACTTCTCCGTGCCTGTTTTOTC 41T 3S S L G G TT KRBULURTULUVDGTEUNZKC 121 ACCAGTTCTCTTOGAGGAACAACCARGGAGCTGAGGCTAGTGGATGGTGAAAACAAGTGT 618 6 R VR V KI 0 BR EWG GTUVCNIENGHW 181 AGTGGGAGAGTOGAAGTGAAAATCCAGGAGGAGTAGGGAACGGTGTGTAATAATGGCTCG

S2005/011502 to B1S M BE A VS VICNOLGTC CT PTRATITK RA 241 AGCATGGAAGCAGTCTCTGTGATTTGTAACCAGCTGCGGATATCCAACTGCTATCAAAGCC 101T @G W A N SS S A G §S 8 RI WMDHUV S C 301 ACTGGATGGGCTAATTCCAGTGCAGGTTCTAGACGCATTTGGATGGATCATGTTTCTTGT 121 R @ N E § AL W D C K HD GG W GG K H 8 N 361 CGTGGGAATGAGTCAGCTCTTTGGGACTACAAACATGATGGATCGAGAAAGCATAGTAAC 141C T HQ QD A GV TCS DOAGSDTILZEMTEPR 421 TGTACTCACCAACAAGATGCTGGAGTAACTTGCTCAGATGGATCCGATTTGGAAATGAGG 161 L T MM @ G N M C § G R I B I K P Q G Q W 481 CTGACGAATGGAGGGAATATGTGTTCTGAAAGAATAGAGATCAAATTCCAAGGACAGTGA 181 G T v C DD N F N I NH AS VV CK QL 541 GGAACAGTGTGTGATGATAACTTCAACATCAATCATGCATCTGTGGTTTGTAAACAACTT 301lE C G 8S AV 8 F S G S A NF GBR G 8S Gg ?p 601 GAATGTGGAAGTGCTGTCAGTTTCTCTGGTTCAGCTAATTTTGGAGAAGGCTCTGGACCA 2211 WwW FP D DL I C NGNUR BS AL WNUCIKBH 661 ATCTGGTTTIGATGATCTTATATGCAACGGAAATAAGTCAGCTCTCTGGAACTGCAAACAT 241 Q G WwW GG K HE N CC DHA BDO AG UV I C SK 721 CAAGGATGGGGARAGCATAACTGTGATCATGCTGAGAATGCTGGAGTGATTTGCTCARRG 26 G A DL 8 L RL VD GV TEU CSE GU RULE 781 GGAGCAGATCTGAGCCTGAGACTOGTAGATGGAGTCACTGAATGTTCAGGAAGATTAGAA 28 V R P Q G B W G8 TTI CDDG WD B HD A 841 GTGAGATTCCAAGGAGAATAGGGGACAATATATGATGACGGCTGAGACAGTCATGATECT 301A V AC K QL @ C PTA I TAI GIR RUVN 901 GCTGTGGCATGCAAGCAACTGGGATGTCCARCTGCTATCACCGCCATTGGTCGAGTTARC 321A 8S BR GG P G HI Ww L DS V 8 C Q G HUE P 961 GCCAGTGAGGGATTTGGACACATCTGGCTTGACAGTGTTTCTTGCCAGGAACATGAACCT 341A V W Q C K HH BE WG K HY CN HNUE ED 1021 GCAGTCTAGCAATGTAAACACCATGAATGGGGAAAGCATTATTGCAATCACAATGAAGAT 361A G VT C 8 P GS JL ELI RUILURTGTG GG G 8 1081 GCTGGCGTAACATGTTCTGATGGATCAGATCTGGAGCTAAGACTTAGAGATGAAGGCAGC 331 R ¢C AG TV E V EI Q RL DL GG KK V CDR 1141 CGCTGTGCTGGGACAGTTIAGGTGGAGATTCAGAGACTGTTAGGCGAAGGTATATGACAGA 401 3 W GL XK BE AD V V CRQL GCG S A L 1201 GGCTGGGGACTGAAMIAAGCTAATATGGTTTGCAGGCAGCTGGIATGTGGATCTGCACTC 421K T § Y Q VY § XK I Q AT N KW UL PUL § 1261 AAAACATCCTATCAAGTATACTCCAAAATCCAGGCAACAAACATGTGGCTGTTITCTAAGT 441 S C N G N E T S L W DC KNW OQ WG GG L 1321 AGCTGTAACGGAAATGAAACTTCTCTTTGGGACTGCAAGAACTGGCAATGGGGTGGACTT 461 T C D H Y B BR A K I T C 86 A HR BR PPR L 1381 ACCTGTGATCACTATGAAGAAGCCAAAATTACCTGCTCAGCCCACAGGGAACCCAGACTG 481 V G @ D I P C § G R VE V K H GDTITMWOGG 1441 GTTGGAGGAGACATTCCCTGTTCTGGACGOGTTOAAGTGAAGCATGCTGACACATAGGGC

S508 v ¢cD 8 DP SL EAAS VL CRTEZLOQ 1501 TCOGTCTGTGATTCGGATTTCTCTCTGGAAGCTACCAGCATTCTATGCAGGGAATTACAG 531 C G6 T Vv V 8 I L G GA HF G RBG NUGUGQTI 1561 TETEACACAGTCATCTCTATCCTGAOGGAAGCTCACTTTGGAGAGGGAAATGGACAGATC 541 W T E BE F Q ¢C E @ HE S$ HL 8§ IL CP V A 1621 TGGACTGAAGAATTCCAGTGTGAGGGACATGAGTCCCATCTTTCACTCTGCCCAGTAGCA

S61 P R P BE G T C S H 8S RD V G V V C SR Y 1681 CCCCACCCAGAAGGRACTTGTAGCCACAGCAGGGATGTTGGAGTAGTCTGCTCAAGATAC 582 T E I R L V N GG KK T P C BRB 6G R V EL K T 1741 ACAGAAATTCGCTTGGTGAATGGCAAGACCCCATGTGAGGGCAGAGTGAAGCTCAAAACG 601 L N A W G 6 L C N 8 HW D I ED A HV L 1801 CTTAATGCCTGGGGATCCCTCTGCAACTCTCACTGGGACATAGAAGATGCCCACGTTCTT 621 C Q Q L K C BG V AL S TP G G A HPF G K 1861 TGCCAACAACTTAAATGTGGAGTTGCCCTTTCTACCCCAGGAGGAGCACATTTTAGGAAAA 641 G N G Q V ¥ R HM FH CT GTTEUGQUHMG 1922 GGAAATGGTCAGATCTGAAGGCATATGTTTCACTGCACTGGGACTGAGCAGCACATGGGA 661 D C P V T AL G A SL C P 8 GQ V A § V 1981 GATTGTCCTGTAACTGCTCTAGGTICTTCACTATGTCCTTCAGGGCAAGTGSCCTCTGTA 681 I CC 8S G6 N Q S Q TL 8 8 C NN S 8 SL G P 2041 ATTTGCTCAGGARACCAGTCCCAAACACTGTCCTCSTGCAATTCATCATCTCTGGGCCCA 701T R PT I P EES A VAC CTITE ESS GA QTLR 2101 ACAAGGCCTACCATTCCAGAAJAAAGTGCTGTGGCCTGCATAGAGAGTGGTCAACTTCGC 72)L V N G6 G G R C A G RV EI Y HE GS W 2161 TTGGTAAATGGAGGAGGTCGCTATACTGGGAGAGTAGAGATTTATCATGAGGGCTCCTGA 741@ T I C D DS WDUL SDA AUBHVYVVYV CRO QTL 2221 GGCACCATCIGTGATGACAGCTGGGACCTGAGCGATGCCCACGTGGTGTGCAGACAGCTG 761 6G C G E A I N AT G S A HP GR GTG P 2281 GGCTGTGGAGAGGCCATTAATGCCACTGATTCTGCTCATTTIGGAGAAGGAACAGAGCCC 7611 W I. D E M K C N G KE S R I W Q CH § 2341 ATCTGGCTGGATGAGATGAAATGCAATAGAAAAGAATCCCGCATTTGGCAGTGCCATTCA 80H G W G Q Q N C R BE KE DAG GUVTIOC CSE 2401 CATGGCTGAGAGCAGCAAAACTGCAGACACAAGGAGGATGCAGGRGTTATCTGCTCAGAG 821 F M 8 L RL T S BE A S R BR A CAGTZ RTL E 2461 TTCATGTCTCTGAGACTGACCAGTGAAGCCAGCAGAGAGGCCTGTGCAGGGCGTCTAGAA 841 V F Y N 8 A W G S V G R 8 NM S ETT V 2521 GTTTTTTACAACGGAGCTTGGGGCAGTGTTGGCAGGAGTAACATGTCTGAAACCACTGTG 861G V V C R Q L G6 C A DK G K I NS A S 1. 2581 GGTGTAGTGTGCAGGCAGCTGGGCTGTACAGACAAAGGGAAAATCARCTCTGCATCTTTA 88l1D K A M 8 I PM WV DNVAG QTC CTPZKTUGTPTD 2641 GACANGGCCATGTCCATTCCCATGTOGATAGACAATGTTCAGTGTCCAAAAGGACCTGAC 901 T L W Q C P S 8 P Ww BR K RL ARU©PSGSTER

“T/US2005/011502

. 7701 ACGCTGTGOCAGTGCCCATCATCTCCATGOGAGAAGAGACTGAGCCAGACCCTCOGAGGAG 921T W I T C DN KMRL Q EGU PTS CB G 2761 ACCTGGATCACATGTGACAACARGATGAGACTACAAGAAGGACCCACTTCCTGTTCTGGA 941R V E I W H G 6 § WG T V C DD 8 WD L 2821 CGTGTOGAGATCTGGCACGGAGGTTCC TGGGAGACAGTGTGTAATAACTCCTGGGACTTG 9%61N D AQ VVC QQlLGGCGaGEPA ALTE KA MAPK 2881 AACGATGCTCAGGTGGTGTGTCAACAACTIGGCTGTGATCCAGCTTTGAARGCATTCAAA 981E A E FP G Q G T 6G P IT WL NRV K CK G 2941 GAAGCAGAGTTIGGTCAGGGGACTGGACCCATATGGCTCAATGARGTGAAGTGCAAAGGG 1001lN E 8 8 L W D C P A RRWGTUH SE ECG H 3001 AATGAGTCTTCCTTGTGGGATTGTCCTGCCAGACGCTGGGGCCACAGTGAGTGTGGACAC 1021K E D A AVN CTODTI STU RTIKT®PQK A 3061 AAGGAAGACGCTGCAGTGAATTGCACAGATATTTCAACGCGCAAAACCCCACAAAAAGCC 1041 T T 3 Q 8 8 L I AV @ I L GV V L L A I 3121 ACAACAGATCAGTCATCCCTTATTGCAGTCGGAATCCTTGGAGTTGTTCTCTTGGCCATT 1061 V AL FP LT QKRUROQRU QRTLTU VS § 3181 TTCGTCACATTATTCTTGACTCAAAAGCGAAGACAGAGACAGCGGCTTACAGTTTCCTCA 1081R G6 B N L V H Q I Q YR BE MN 8 C L N A 3241 AGAGGAGAGAACTTAGTCCACCAAATTCARTACCGGGAGATGAATTCTTGCCTGAATGCA 110: D D L D L M N 6 8 EN § N E 8 A DF N A 3301 GATGATCTGGACCTAATGAATTCCTCAGARAATTCCAATGAGTCAGCTGATTTCAATGCT 1121A E L I § V § K P L P I 8 G M E K E A I 3361 GCTGAACTAATTTCTGTGTCTAAATTTCTTCCTATTTCTGGARTGGAAAAGGAGGCCATT 1141 L R HT E K ER N G N L 3421 CTGAGGCACACTGAAAAGGAAAATGGGAATTTA 1 MSKLRMVLLE DSGSADVRRH FVNLEEFTIA VVLLLRACFV TSSLGGTTKE LRLVDGENKC SEQ ID 61 SGRVEVKIQR BWGTVCNNGW SMEAVSVICN QLGCPTATKA TGNANSSAGS GRIWMDHVSC 121 RGNRSALWDC KHDGWGKHSN CTHQUDAGVT CSDGSDLEMR LTNGGNMCSG RIEIKFQGQOW NO:36 181 GTVCDDNFNI NHASVVCKQL ECGSAVSFSG SANFGEGSGP IWFDDOLICNG NESALWNCKH 241 QGWGKHNCDH AEDAGVICSK GADLSLRLVD GVTECSGRLE VRFQGEWGTI CDDGWDSHDA 301 AVACKQLGCP TAITAIGRVN ASEGFGHIWL DSVSCQGHEP AVMQCKEHEW GKHYCNHNED 361 AGVTCSDGSD LELRLRGGGS RCAGTVEVEI QRLLGKVCDR GWGLKEADVV CRQLGCGSAL 421 KTSYQVYSKI QATNMNLFLS SCNGNETSLW DCKNWGWGGL TCDHYERAKI TCSAHREPRL 481 VGGDIPCSGR VEVKHGDTNG SVCDSDFSLE AASVLCRELQ CGTVVSILOG AHPGERGNGQI 541 WIRRFQCEGH ESHLSLCFVA PRPBGTCSES RDVGVVCSRY TEIRLVEGKT PCEGRVELKT 601 LNAWGSLCNS HNDIEDAHVI, CQOLEOGVAL STPGGAHPGX GMOQVWRHMF HCTGTROHEMG 661 DCPVTALGAS LCPSGUVASV ICBGNQBQTL SSCNSSSLOP TRPTIPERSA VACIRSGQLR 721 LVNGGGRCAG RVEIYHEGSW GTICDDSWDL SDAHVVCRQL GCGEAINATG EAHPGBGTGP 781 IWLDEMKCNG KESRIWQCHS HGWGQQNCRH KEDAGVICSE PMSLRLTSEA SRRACAGRLE 841 VPYNGANGSV GRSNMSETTV GVVCROLGCA DXGKINSASL DKAMSIPMWV DNVQCPKGPD 901 TLWQCPSSPN EKRLARPSER TWITCDNKMR LQEGPTSCSG RVEIWHGGSW GTVCDDSWDL 961 NDAQVVCQQL GCGPALKAFK EAEFGQGTGP INLNEVKCKG NESSLWDCPA RRWGHSECGH 1021 KEDAAVNCTD ISTRKTPQKA TTGQSSLIAV GILGVVLLAY FVALPLTQKR RQRQRLTVSS 1081 RGENLVHQIQ YREMNSCLNA DDLDLMNSSE NSNESADFNA AELISVSKPL PISGMEKEAI 1141 LRHTEKENGN L 1M 8 K L R M V LL ED 8G GS ADVTVTR RTEKTEH SEQ ID 1 ATGAGCAAACTCAGAATGGTGCTACTTGAAGACTCTGGATCTGCTGACGTCAGAAGACAT NO:37 20/F VN L SPF TTIAUVVUILILULTZBRATCTEV and 38 61 TTTGTCAACTTGAGTCCCTITCACTATTGCTGTGGTCTTACTTCTCCATGCCTGTTTTGTC 41T S$ S$ L G6 G T T KE LRTILVDGTENTZRKC 121 ACCAGTTCTCTTGGAGGAACAACCAAGGAGCTGAGGCTAGTGGRTGGTGARAACAAGTGT 61S G R VE V KI QREWGTJVCNNGW 181 AGTGGGAGAGTGGAAGTGAARATCCAGGAGGAGTGGGGAACGGTGTGTAATAATAGCTGS 81S M E A V S V I CNGQULG GTC CT PTA ATK A 241 AGCATGGAAGCAGTCTCTGTGATTTATAACCAGCTAGGATGTCCAACTGCTATCAAAGCC 100 T G W AN 8 S AGS GR I WMUDUHUYS C 301 ACTGGATGGGCTAATTCCAGTGCAGGTTCTGRACGCATTTGGATGGATCATGTTTCTTGT 121R G NB S A L WD CUKHTDGTWGTI KTH 8S N 361 CGTGGGAATGAGTCAGCTCTTTGGGACTGCAAACATGATGGATGGGGARAGCATAGTAAC 141C T H Q Q D A G VTC SDGSUDULEMR 421 TATACTCACCAACAAGATGCTGGAGTAACTTGCTCAGATGGATCCGATTIGGAAATGAGS 161L T N G 6 N M C § GR IETITZKTPOQGO QW 481 CTGACGAATGGAGGGAATATGTGTTCTGGAAGAATAGAGATCAAATTCCAAGGACAGTGA 181G T V ¢C D DN FNIWNUHASTYTVYVYCGCHKTO QTL 541 GOARCAGTGTGTGATGATAACTTCAACATCARTCATGCATCTGTGGTTTGTAAACAACTT 200E C G § A VS FP 8G SANVPG GEG SG P 601 GAATGTGGAAGTGCTGTCAGTTTCTCTAATTCAGCTAATT TTGGAGARGGCTCTGGACCA 2221 W P DD LI CNGUOYNTETSA ALTWNTGCIKH 661 ATCTGGTTTGATGATCTTATATGCAACGGAAATGAGTCAGCTCTCTGGAACTGCAAACAT

" 7410 CG W 6 KE NCD EHRAETDT AG GUVITGCGSE KX 721 CAAGGATGGGGAAAGCATAACTGTGATCATGCTGAGGATGCTGGAGTGATTTGCTCAARG 2616 ADL S§ L RL VDGVYVTTE BTC CSG GTRTILE 781 GGAGCAGATCTGAGCCTGAGACTGGTAGATGGAGTCACTGAATGTTCAGGARGATTAGAA 2801V R F Q G EWG TTI CDODG WD S HDA 841 GTGAGATTCCAAGGAGAATGGGGGACAATATCTGATGACGGCTGGGACAGTCATGATACT 301A V AC K QL GCUPTA ATILITA AIG GT RUYHN 901 GCTGTGGCATGCAAGCAACTGGGATGTCCAACTGCTATCACCGCCATTGATOGAGTTAAC 321A § EG F 6G H I WLDSVYV SCQGHEP 961 GCCAGTGAGGGATTTGGACACATCTGGCTTGACAGTGTTTCTTGCCAGGGACATGAACCT 341A V W Q C K H HEWG GI KZEKTVYCNEHINTETHD 1021 GCGGTCTGGCAATGTAAACACCATGAATGGGGARAGCATTATTGCAATCACAATGAAGAT 361A G VT CS DOG SDILETLT RTELTZERTGT GG § 1081 GCTGGCGTAACATGTTCTGATGGATCAGATCTGGAGCTAAGACTTAGAGGTGGAGGCAGC 31R C AG T V EVE ETIGQRTLULG GE KV CODR 1141 CGCTGTGCTGAGACAGTTGAGGTGGAGATTCAGAGACTGTTAGGGARGATATATGACAGA 4016 W 6 L XK E ADV V CRAQDL GCG S ATL 1201 GGCTGGGGACTGAAAGAAGCTGATATAGTTTGCAGGCAGCTGGGATGTGGATCTGCACTC 421K T 8 ¥Y Q V Y¥ 8 KI Q AT N MW DL F L 8 1261 AAAACATCCTATCAAGTATACTCCARAATCCAGGCAACAAACATGTGGCTGTTTCTAAGT 4418S ¢ NG N ET S L WDCKUNWO GT WG G L 1321 AGCTGTAACGGAAATGAAACTTCTCTTTGGGACTACAAGAACTGGCARTGGGGTGGRCTT 461T C¢ DH Y E EA KI TCS A HRTEBTZ PRL 1381 ACCTGTGATCACTATGAAGAAGCCARARTTACCTGCTCAGCCCACAGGGAACCCAGACTG 481V @ 6 D I P C § G RV EV KHGUDTWQ 1441 GTTGGAGGAGACATTCCCTGTTCTGGACGCGTIGAAGTAARGCATEGTGACACATGGGGC 5011S V ¢ D 8 DP SLEAXMASV VILCR RTETLQ 1501 TCOGTCTGTGATTCGOATTTCTCTCTGAAAGCTGCCAGCGTTCTATGCAGGGAATTACAG 521C @ TV V § I L G6 6 AHF G BG NG QT 1561 TGTGGCACAGTCGTCTCTATCCTGCGOAGAGCTCACT TTGGAGAGGGAAATAGACAGATC

S541W T E BR P Q C E @ HE S HL STL CP VA 1621 TGGACTGAAGAATTCCAGTGTGAGGAACATGAGTCCCATCTTTCACTCTGCCCAGTAGCA 61 P R P B G T C 8 H 8S RD V 6 VV CSU RY 1681 CCCCGCCCAGAAGGAACTTGTAGCCACAGCAGGGATGTTGGAGTAGTCTGCTCAAGATAC

S81 T E I R L V NG KT P CBE G RV ETL K T 1741 ACAGARATTCGCTTGGTGAATGGCAAGACCCCATATGAGGGCAGAGTGGAGCTCARAACG 601L N A W G6 5S L C N 8 H WD IZ ETDA ATHTUVL 1801 CTTAATACCTGGGAATCCCTCTGCAACTCTCACTGGGACATAGAAGATGCCCACGTTCTT 621C Q QL K C GV AL STUPOGGATHT PG K 1861 TGCCARCAACTTAAATGTGGAGTTCCCCTTTCTACCCCAGGAGGAGCACATTTTGGARAR 641G N ¢G Q V W R HM FP 5 CT G TE Q H M @G 1921 GGAAATGGTCAGATCTGGAGGCATATGTTTCACTGCACTGGGACTGAGCAGCACATAGAA 661D C P V T A L G RAR S L CP 8 GQ V A 8 V 1981 GATTGTCCTGTAACTGCTCTGGITGCTTCACTATGTCCTTCAGAGCAAGTGGCCTCTGTA 6811 C S G N Q 8 Q T LL 8 8 C N S§ S S L G P 2041 ATTTGCTCAGGAAACCAGTCCCAAACACTGTCCTCGTGCAATICATCATCTCTGGECCCA 701T R BT I PEE SAV ACTITESG GS QTLR 2101 ACAAGGCCTACCATTCCAGAAGAAAGTGCTGTGACCTGCATAGAGAGTGGTCAACTTCGC 721L V NG @ GR CAGRVYVETITYTHTE EGS STW 3161 TTGGTAAATGGAGAAGATCACTGTGCTGGGAGAGTAGAGATTTATCATGAGGGCTCCTGE 7416 T I CD DS WDULSDAEKV VVC CRAG QHL 2221 GGCACCATCTGTGATGACAGCTGGGACCTGAGCGATGCCCACGTGGTGTGCAGACAGCTG 7616 C GE A I N ATG S AHTPGTETGTG P 2281 GGCTGTGGAGAGACCATTARTGCCACTGGTTCTGCTCATTTTGGAGAAGGAACAGGGCCC 7811 W L D E M K C N G KES RI NWO QU CHS

T/US2005/011502

To 2341 ATCTGGCTGGATGAGATGAAATGCARTOGARAAGAATCCCGCATITGGCAGTGOCAT TCR 801H G W G Q Q NCR HKZ EDU AGT VTITC CSE 2401 CATGGCTGGGGGCAGCAAAACTGCAGGCACARGGAGGATGCAGGAGTTATCTGCTCAGAG 821F M S L RL T SE ASU RTUEACAGRTLE 2461 TTCATGTCTCTGAGACTGACCAGTGAAGCCAGCAGAGAGGCCTGTGCAGGGOGTCTAGAR 841V FP Y N G A W G3 8 VGRSNMSTETTUV 2521 GTTTTTTACAACGGAGCTTGGGGCAGTGTIGGCAGGAGTAACATGTCTGAAACCACTGTA 8616 Vv Vv C R Q L GG C AD KG XTIUNJGSA ASTI. 2581 GGTGTAGTGTGCAGGCAGCTGGACTATACAGACAAAGGGAAAATCAACTCTGCATCTTTA 881 D K AM 8 I P M WV DNTYGQCTPZEKTGT PD 2641 GACAAGGCCATGTCCATTCCCATGTGGGTGAACAATGTTCAGTGTCCAARAGGACCTGAC 901 T L WQ CP S SP WEJZ KJZRTLATRTEPSTSER 2701 ACGCTGTGGCAGTGCCCATCATCTCCATGGGAGAAGAGACTGACCAGACCCTCGGRGGRG 921 T Ww I T CC D N KM RL QE GPTGSTC CS G 2761 ACCTGGATCACATGTGACAACAAGATGAGACTACARGAAGGACCCACTTCCTGTTCTGGA 941 R V EI W HG G S WG TJVOCDTDTGSWZDL 2821 CGTGTGGAGATCTGGCACGGAGETTCCTGGGGGACAGTGTATGATAACTCCTGGAACTTG 961 N D A Q V V C Q OQ L G €C G PATULTEKATFK 2881 AACGATGCICAGGTGGTGTGTCAACAACTTGACTGTGGTCCAGCTTTGAARGCATTCARA 9812 A EF G Q G T G P I WL NEV KTZG CZK G 2941 GARGCAGAGTTTGGTCAGGGGACTGGACCCATATGGCTCAATGARGTGAAGTGCAAAGAG 1001N BR S S L WD CP A RRTWGOGTEHTSTZ EBC CGH 3001 AATGAGTCTTCCTTGTGGGATTGTCCTGCCAGACHCTEGGACCACAGTGAGTGTGGACAC 1021 X BR D AAV N CT DTI STZ RTEKTTZPGQTZ KA 3061 AAGGAAGACGCTGCGGTGAATTGCACAGATATTTCAACGCGCAAAACCCCACARAAAGCC 1041T T V S S R G EN L V HQIOQYU RTEHMHN 3121 ACAACGGTTTCCTCAAGAGGAGAGAACTTAGTCCACCAAATTCAATACCGGGAGATGAAT 10618 C L N AD DLXTILMNTGSTGSSGGTHTSTEA 3181 TCTTGCCTGARTGCAGATGATCTGAACCTAATGAATTCCTCRAGGAGGCCATTCTGAGGCA 1081 H *+ XK G K W E FP IT T Q 3241 CACTGAAAAGGAARATGGGAATTTATAACCCAG 1 MSKLRMVLLE DSGSADVRRH FVNLSPPTIA VVLLLRACFV TSSLGGTTKE LRLVDGENKC SEQ 1D 61 SGRVEVKIQR EWGTVCNNGW SMEAVSVICN QLGCPTAIKA TGWANSSAGS GRIWMDHVSC 121 RGNESALWDC KHDGWGKHSN CTHQQDAGVT CSDGSDLEMR LTNGGNMCSG RIEIRFQGQW NO:38 181 GTVCDDNPNI NHASVVCKQL ECGSAVSPSG SANPGEGSGP IWFDDLICNG NESALWNCKH 241 QGWGKHNCDH AERDAGVICSK GADLSLRLVD GVTECSGRLR VRFQGEWGTI CDDGWDSHDA 301 AVACKQLGCP TAITAIGRVN ASEGFGHIWL DSVSCQGHEP AVWQCKHHEW GKHYCNHNED 361 AGVTCSDGSD LELRLRGGGS RCAGTVEVEI QRLLGKVCDR GWGLKEADVV CRQLGCGSAL 421 KTSYQVYSKI QATNMWLFLS SCNGNETSLW DCKNWQWGGL TCDHYEEAKI TCSAHREPRL 481 VGGDIPCSGR VEVKHGDTNG SVCDSDPSLE AASVLCRBLQ CGTVVSILGG AHFGRGNGQI

S41 WTEEFQCEGH RSHLSLCPVA PRPRGTCSHS RDVGVVCSRY TEIRLVNGKT PCEGRVELKT 601 LNAWGSLCNS HWDIEDAHVL CQOLKCGVAL STBGGAHFGK GNGQVWRHMF HCTGTEQHMG 661 DCPVTALGAS LCPSGQVASV ICSGNQSQTL SSCNSSSLGP TRPTIPEESA VACIESGQLR 721 LVNGGGRCAG RVEIYHEGSW GTICDDSWDL SDAHVVCRQL GCGEAINATG SAHFGEGTGP 781 IWLDEMKCNG KESRINQCHS HGWGQQNCRH KEDAGVICSE FMSLRLTSEA SREACAGRLE 841 VFYNGAWGSV GRSNMSETTV GVVCRQLGCA DKGKINSASL DKAMSIPMWV DNVQCPKGPD 901 TLWQCPSSPW EKRLARPSEE TWITCDNKMR LQBGPTSCSG RVEIWHGGSW GTVCDDSWDL 961 NDAQVVCQQL GCGPALKAFK EARFGQGTGP IWLNZVKCKG NESSLWDCPA RRWGHSRCGH 1021 KEDAAVNCTD ISTRKTPQKA TTVSSRGENL VHQIQYREMN SCLNADDLNL MNSSGGHSEA 1081 H

IM 8 KL RMV LTLTETDSTGTSA ADVE RTRE SEQ ID 1 ATGAGCARACTCAGAATGGTGCTACTTGARGACTCTGGATCTGCTGACGTCAGAAGACAT NO:39 21P V NL S PF TI AV UV LULULURACTEUV and 40 61 TTTGTCAACTTGAGTCCCTITCACTATTGCTGTGGTCTTACTTCICOGTGCCTGTTTTETC 41T 8 8 L. 6 GG T T XK RL RL VD GENK C 121 ACCAGTTCTCTTGGAGGAACAACCAAGGAGCTGAGGCTAGTGGATGGTGARAACAAGTGT 618 G RV BR V KI Q R EWG GTV VC CNNVNOU GHW 181 AGTGGGAGAGTGGAAGTGAAAATCCAGGAGGAGTAAAGAACGGTGTGTAATAATGGCTGS 818 M E AV 8 VI CNG QTLGTC CT PTA ATITKA 241 AGCATGGAAGCAGTCTCTGTGATTTGTAACCAGCTGGGATGTCCAACTGCTATCAAAGCC

CTT IT GW ANSSAGSGRTINWMDTELUVSEC

301 ACTGGATGOGCTAATTCCAGTGCAGGTTCTGGACGCATTTGGATGGATCATGTTTCTTAT 121 R G N B S A L W D C K HD G@ WA K H 8 N 361 CGTGGGAATGAGTCAGCTCTTTGGGACTGCAAACATGATGGATGGGGAAAGCATAGTAAC 141C T H Q 0 DAGUV TCS DG SDTILEMR 421 TGTACTCACCAACAAGATGCTGGAGTGACTTGCTCAGATGGATCCGATTTGGAAATGAGS 16lL T N G G NN M C 8 G R I B I K FP Q G8 Q WwW 481 CTGACGAATGGAGGGAATATGTGTTCTGGAAGAATAGAGATCAAATTCCAAGGACAGTGR 181G T V €C D DN FN VN HAS VY V CIKOQL 541 GGAACAGTGTGTGATGATAACTTCAACGTCAATCATGCATCTGTGGTTTGTAAACAACTT 200E C 6G 8S A V 8S P S$ G 8S A NP GG EG 8 @¢ Pp 601 GAATGTGGAAGTGCTGTCAGTTTCTCTGGTTCAGCTAATTTTAGAGAAGGCTCTGGACCA 221 I W FP D D L I C N G N BE 8 AL W NCIX H 661 ATCTGGTTTGATGATCTTATATGCAACGGAAATGAGT CAGCTCTCTGGAACTGCAAACAT 241 Q 6 W @ K H N CC DPD H A ED A GG V I C Ss K 721 CARGGATGGGGAAAGCATAACTGTGATCATGCTGAGGATGCTGOAGTAATTTGCTCAMAG 261G A DL 8S L RL V DG V TE EU GCS GURL E 781 GGAGCAGATCTGAGCCTGAGACTGGTAGATGGAGTCACTGAATGTTCAGGAAGATTAGAA 281 V R F Q G BE W GG T I ¢C DPD DG WD BS HD A 841 GTGAGATTCCAAGGAGAATGGGGGACAATATGTGATGACGGCTEGEACAGTCATGATACT 301A VAC K Q LL 6 C P TAIT ATIGT RYUN 901 GCTGTGGCATGCAAGCAACTGGGATATCCAACTGCTATCACCACCATTGGTCGAGTTAAC 321A 8 BE G P GG EI WL DS VV S CQ G HE P 961 GCCAGTGALGGEATTTGGACACATCTGGCTTGACACTG TTT CTTGCCAGGGACATUAACCT 34 A V W Q CC K HE H BE WG K HUY CNUHNUZERD 1021 GOGGTCTGGCAATGTAAACACCATGAATGGGGAAAGCATTATTGCAATCACAATGAAGAT 3A @ VTCSDA@S8DILETLT RTELTRGG GG GS 1081 GCTGGCGTAACATOTTCTGATGGATCAGATCTGGAGCTAAGACTTAGAGGTOGAGACAGC 3831R C A GG T V £E V BE I Q RL L G KV CDR 1141 CGCTCTGCTGGGACAGTTGAGGTGGAGATE CAGAGACTGTTAGGGAAGGTGTGTGACAGA 401G W GL KB ADV VECRQLGC CGS AL 1201 GGCTGGGGACTGAAAGAAGCTGATGTGGTTTGCAGGCAGCTGGGATGTGGATCTGCACTC 421 K T 8 ¥Y¥ Q Vv ¥ S K I Q AT N M ¥ L FP L 8 1261 AAAACATCCTATCAAGTATACTCCAAAATCCAGGCAACAAACATGTGGCTGTTTCTAAGT 441s C N G6 N E T 8 L A D C X N KN Q WG OG IL 1321 AGCTGTAACGGAAATGAAACTTCTCTTTGGGACTGCAAGAACTGECAATGGGGTGGACTT 461 T C D H4 ¥Y BEB BE A K I T C §S A HR RB PRL 1387 ACCTGTGATCACTATGAAGAAGCCAARATTACCTGCTCAGCCCACAGAGAACCCAGACTG 481 V G G6 D I P ¢C 8S G R V BE V K HG DTW G 2441 GTTGGAGGAGACATTCCCIGTTCIGGACGCGTTGAAGTGAAGCATAGTCACACATGGGGC 5018 Vv CC D S$ DP 8S LL BR A A SS VL CUREUL Q 1501 TCCATCTGTGATTCGGATITCTCTCTGGAAGCTGCCAGCGTTCTATGCAGGGAATTACAG $21 ¢C G6 T V V 5s I L G G A H F G EG NG Q 1 1561 TGTGGCACAGTCGTCTCTATCCTIGAGGGAGCTCACTTTGGAGAGGGAAATGGACAGATC 541 W A B E F Q CC BE @ HE S HL SS L CP V Aa 1621 TGGACTGAAGAATTCCAGTGTGAGGGACATGAGTCCCATCTTTCACTCTGCCCAGTAGCA

S61 P R P E G T C S$ H S RD V GG VV C 8 RR Y ! 1681 CCCCGCCCAGAAGGAACTTGTAGCCACAGCAGOGATATTOGAGTAGTCTGCTCAAGATAC se1T B I R L VN G RT P CEGRUYVETLEKT 1741 ACAGARATTCGCTTGGTGAATGGCARGACCCCATGTGAGGGCAGAGTGAAGCTCAAAACG 601 2 N A W @ S L C N § HW DTI EDA AU HUY TL 1801 CTTAATGCCTGGGAGATCCCTCTGCAACTCTCACTOGGACATAGAAGATGCCCACGTTCTT 621C Q Q L XK CG V AL STPGGATEHTEGK 1861 TGCCAACAACTTAAATGTGGAGTTGCCCTTTCTACCCCAGGAGGAGCACATTTTGGAAAA 641G N G Q V W R HM FP HCTGTU RG QU HMG

1921 GGAAATGGTCAGGTCIGGAGGCATATGTTTCACTGCACTAGGACTGAGCAGCACATGGGA 661D C P VT A L GA SLCPS GQ VASEUV 1981 GATTGTCCTGTAACTACTCTGGGTGCTTCACTATGTCCTTCAGGGCAAGTGACCTCTCTA 6811 C S BG N Q S 0 T L § S CN 8 8 8 L G P 2041 ATTTGCTCAGGARACCAGTCCCARACACTGTCCTCGTGCAATTCATCATCTCTGGGCCCA 700T R P T I P ERS AV ACTITESSG GSO QTL R 2101 ACAAGGCCTACCATTCCAGAAGAAAGTGCTGTGGCCTGCATAGAGAGTGGTCAACTTOGE 721L V NG G G R CA GRV EI Y HBG SS W 2161 TTGGTARATGGAGGAGGTCGCTGTGCTGGGAGAGTAGAGATTTATCATGAGGGCTCCTGE 7416 T I ¢ DD BS WDUL SDA AEHUTYTYVC CRO QL 2221 GGCACCATCTGTGATGACAGCTGAGACCTGAGCGATGCCCACGTGGTGTGCAGACAGCTA 7616 C GB A I N ATG SAHTPGETG GTS ap 2281 GGCTOTGGAGAGGCCATTARTGCCACTGGTTCTGCTCATTTTGGAGAAGGAACAGGGCCT 7811 W L D BR M XK C N G KE 8S RI WOQG CEH S 2341 ATCTGGCTGGATGAGATGAAATGCAATGGAARAGAATCCCGTATTTGGCAGTGCCATTCA 80l1H G WG Q Q N CRHIEKETDA AGT VTITC CSE 2401 CATGGCTGGGGGCAGCARARCTGCAGGCACAAGAAGGATGCAGGAGTTATCTGCTCAGAG 821F M S L RL T § EA SR EA ACA AGT RTL E 2461 TTCATGTCTCTGAGACTGACCAGTGARGCCRAGCAGAGAGGCCTGTGCAGAGCGTCTAGAR

B41V P Y N G A W GG 8 V GR S NMSE ETT V 2521 GTTTTTTACAACGAAGCTTGGGGCAGTGTTGGCAGAAGTAACATGTCTGARACCACTATG 8616 V V C R Q L G C A DX GKTIMNTGBA ASL 2581 GGTGTGGTGTGCAGGCAGCTGGACTATGCAGACAAAGGGAAAATCARCTCTGCATCTTTA 881D K AM 8 I P M WV DNTYOQCPTKSGTZ PD 2641 GACAAGGCCATATCCATTCCCATGTGGATGAACAATOTTCAGTITCCARAAGGACCTGAC 901 T L WQCUPS SP WERKURTUELATRTEPTSESTEE 2701 ACGCTETUGCAITACCCATCATCTCCATGAGAGAAGAGACTAGCCAGGCCCTCGGAGAAG 921T W I T CDN KM RLQEGT PTS SCS a 2761 ACCTGGATCACATGTGACAACAAGATGAGACTACAAGRAGGACCCACTTCCTGTTCTOGA 41R V BE I WH GG S WGTVCDODTGSETHWTDL 2821 COTATGAAGATCTGGCACGAAGGTTCCTAGGAGACAGTOTCTGATAACTCCTGAGACTTS 918 D A Q VV CQ QLGC CG GPA ATLTZEKTA ATF EK 2881 ARCGATGCTCAGGTGGTGTGTCAACAACTTOGCTGTGATCCAGCTTTGAAAGCATTCAAR 98. E A E F G Q 6G T 6G P I WL NEJVIKTCK G 2941 GARGCAGAGTTTGGTCAGGGGACTGGACCCATATGGCTCAATGAAGTGAAGTGCAARGGE 1001N E S S L-W D C P ARRUNWGTHTGSETETCG H 3001 ARTGAGTCTTCCTTGTGGGATTGTCCTGCCAGACGCTGGEGCCACAGTGAGTGTGGACAC 10220 K B D A AV NCTA AGOQZXTISTTEHTEKTTP Q 3061 ARGGAAGACGCTGCAGTGAATTGCACAGCACAAAAAATTTCAACGCACAAAACCCCACAR 041K A TT V § § R GE NL V HQTIGQTYTRE 3121 AARGCCACAACAGTTTCCTCAAGAGGAGAGAACTTAGTCCACCAAATTCAATACCGGGAG 061M N S CL NADODTLTODTLUMDNSGSGG GH § 3181 ATGAATTCTTGCCTGAATGCAGATGATCTGGACCTAATGAATTCCTCAGGAGGCCATTCT 1081B A H * K ¢ XK WEP PTIT Q 3241 GAGGCACACTGAAAAGGAAAATGGGAATTTATAACCCAG i 61 SGRVEVKIQE EWGTVCNNGW SMEAVSVICN QLGCPTAIKA TGMANSSAGS GRIWMDHVSC 121 RGNESALWDC KHDGWGKHSN CTHQQDAGVT CSDGSDLEMR LTNGGNMCSG RIEIKFPQGQW 181 GTVCDDNFNV NHASVVCKQL ECGSAVSPSG SANFGRGSGP IWFDDLICNG NESALWNCKH 241 QGWGKHNCDH AEDAGVICSK GADLSLRLVD GVTECSGRLE VRFQGEWGTI CDDGNDSHDA 301 AVACKQLGCP TAITAIGRVN ASEGFGHIML DSVSCQGHBP AVWQCKHHEW GKHYCNHNED 361 AGVTCSDGSD LELRLRGGGS RCAGTVEVEI QRLLGKVCDR GWGLXRADVV CRQLACGSAL 421 KTSYQVYSKI QATMMMLFLS SCNGNETSLN DCENWQWGGL TCDHYREBAKI TCSAHREFRL 481 VGGDIPCSGR VEVKHGDTWG SVCDSDFSLE AASVLCRELQ CGTVVSILGG AHFGEGNGQI 541 WAEEFQCEGH ESHLSLCPVA PRPRGTCSHS RDVGVVCSRY TBIRLVNGKT PCEGRVELKT 601 LNAWGSLCNS HWDIEDAHVL CQQLKCGVAL STPGGAHFGK GNGQVNREMF HCTGTEQHMG 661 DCPVTALGAS LCPSGQVASV ICSGNQSQTL SSCNSSSLGP TRPTIPEESA VACTESGQLR 721 LVNGGGRCAG RVELIYHEGGW GTICDDSWDL SDAHVVCRQL GCGRAINATG SAHPGEGTGP 761 TWLDEMKCHG KESRIWQCHS HGWGQONCRH KEDAGVICSR PMSLRLTSEA SREACAGRLE 841 VPYNGAWGSV GRENMBETTV GVVCRQLGCA DKGKINSASL DKAMSIPMNV DNVQCPKGED

901 TLWQCPSSDW EXRLARPSEE TWITCONKMR LQEGPTSCSG RVEIWHGGSW GIVCDDSWOL 961 NDAQUVCQQL GCGPALKAFK EAEFGQGTGP IWLNEVKCKG NESSLWDCPA RRWGHSECGH

1081 RAH iM 8s KL RM VIL EDGSGGS ADJVRRZRE SEQ ID 1 ATGAGCAAACTCAGAATGGTACTACTTGAAGACTCTGAATCTGCTGACGTCAGAAGACAT NO:41 2lF VNL SPF TTIAVVYLLTILTZERATCTEV and 42 61 TTTGTCAACTTGAGTCCCTTCACTATTGCTGTGGTCTTACTTCTCCATGCCTATTTIGTC 41 T S$ § L 6G 6G T T KEL RLVDG GENK C 121 ACCAGTTCTCTTGGAGAAACAACCARGGAGCTAAGGCTAGTGOATCGTGARAACAAGTGT 618 @G R V E V K I Q E BE W GG T V C N N GCG W 181 AGTOGGAGAGTGGARGTGARAATCCAGGAGGAGTGGGGAACGGTGTGTAATAATGACTGG 81S M EA V S$ VICNGQLO GC CT PTA ATITKRA 241 AGCATGGAAGCAGTCTCTGTGATTTGTAACCAGCTOGGATGTCCARCTGCTATCARAGCC 101T 6G W ANS SS AG SGRTITWMDEHUYS C 301 ACTGAATGAACTAATTCCAGTGCAGGTTCTGRACGCATTTGGATGGATCATGTITCTTGT 121 R 6G N BE 8S A L WD C K H DG W GG K H SH 361 CGTGGGAATGAGTCAGCTCTTTGGGACTGCAMACATGATGGATGGCGARRGCATAGTARC 141C T H Q Q DA GV TCSDGSDTLEHMR R 421 TGTACTCACCAACAAGATGCTGGAGTGACTTGCTCAGATGGATCCGATTTGGAAATGAGS 161L T N G G NM CS GR I BI KTFQGGQ W 481 CTGACGRATGGAGGGAATATGTGTTCTGGAAGAATAGAGATCAAATTCCAAGGACAGTGG 816 T VCDDNTFEGERTINGBHASYVVYVCTEKS QTL 541 GGAACAGTGTGTGATGATAACTTCAACATCARTCATGCATCTGTGGTTTGTAAACAACTT 200E C @ § AVS PSGSANTFEG GETS GTS GP 601 GAATGTGGAAGTGCTGTCAGTTICTCTGGTTCAGCTAATTI TGGAGAAGACTCTGGACCA 2211 W P D DL I CNG NES ATLTWENC CR KH 661 ATCTGGTTTGATGATCTTATATGCAACGOAAATGAGTCAGCTCTCIGGAACTGCOARACAT 2410 G WG KEN CODGHA AETGDA AGU YTITG CS K 721 CAAGGATGGGGAAAGCATAACTGIGATCATGCT! GAGGATGCTGGAGTGATTTACTCAAAG 2616 A DL S LRLVDGT VTTETC CSG GR RTILE 781 GGAGCAGATCTGAGCCTGAGACTGGTAGATGGAGTCACTGAATGTTCAGGAAGATTAGAA 281 V R FP Q G BE W GG T I CD DG WUD S HOD A 841 GTGAGATTCCAAGGAGAATGGGGGACAATATGTGATGACGGCTCGGACAGTCATGATGCT 301A V A CK QL @ C PTATIT ATIU GT RUVYVN 901 GCTGTGGCATGCAAGCAACTGGGATGTCCAACTGCTATCACCGCCATTGGTCGAGTTAAC 321A §S EG F GH I WIL DS V 8S ¢C Q G H = Pp 961 GCCAGTGAGGGATTTGAACACATCTGGCTTGACAGTGTTTCTTGCCAGGGACATGAACCT 341A VW Q C KH HEBEWOGIKGEHTYTGCNTETNED 1021 GCGETCTGGCARTGTAAACACCATGAATGGGGAAAGCATTATTGCAATCACAATGARGAT 361A G V T C 8 D G S DL EL RL R G G G S§ 1081 GCTGGCGTAACATGTTCTGATGGATCAGATCTGGAGCTAACACTTAGAGCTGCAGGCAGE 3831R ¢C A 3G T V BRB V BR I Q RL L GX UV CDR 1141 CGCTGTGCTGGGACAGTTGAGGTGGAGATTCAGAGACTGTTAGGGAAGGTATGLGACAGA 40G W GL KE ADV VCROQQLGT CGSATL 1201 GGCTGGGGACTGAAAGRAGCTGATGTGGTTTGCAGGCAGCTGGGATGTGGATCTGCACTC 42) XK T SS Y Q V Y § XK I Q A T NM W L F L 8 1261 AAARCATCCTATCARGTATACTCCAMAATCCAGGCAACAARCATGTGGCTATTTCTARGT 4418S C N G6 N E T 8 L WDC KNW QWG G L 1321 AGCTGTAACGGARATGAAACTTCTCTTTGGGACTGCARGAACTGGCARTAAGATAGACTT 461T CD H Y ER E A KI T C § A H REP R L 1381 ACCTGTGATCACTATGAAGARGCCAAAATTACCTGCTCAGCCCACAGGGAACCCAGACTS 481V G 6G D I PC S @QRVEVIE KEG GDTUWG 1441 GTTGGAGGAGACATTCCCTGTTCTGAACGCGTTGAAGTGAAGCATGGTGACACATAGEEC 5018 V CDS DPF SLEAASVYVLCR RET Q -93.

" FTTT1501 TCOGICTGTUATTCAGATITCTCT CTGBAAGC TGC CACO TTCTATOCAGGGARTTACAS $21 ¢ 6 T VV 8 I LL. G G A H »» G E G NG QI 1561 TGTAGCACAGTCGTCTCTATCCTGGGGGGAGCTCACT TIGGAGAGGGAAATGGACAGATC

S41 WwW A BE E P Q C E G HE 8 HL 8 L CP V 2a 1621 TGGGCTGAAGARTTCCACTGTGAGGOACATAAGTCCCATCTTTCACTCTCCCCAGTAGCA 561 P R P BE 3 T C SS HR §S RD V G V V C § R Y 1681 CCCOGCCCAGAAGGAACTTGTAGCCACAGCAGGGATGTTGGAGTAGTCTGCTCAAGATAC 581 T BE I R L V N G K T P C BE @ R V EL K T 1741 ACAGAAATTCGCTTGGTGAATGGCAAGACCCCATGTGAGGGCAGAGTGGAGCTCAAAACG 601 L N A W GG 8 LL, CC RNR 8S HEH W DI RD ABH UV L 1801 CTTAATGCCTGGGGATCCCTCTGCAACTCTCACTGGGACATAGAAGATGCCCACGCTICTT 621 C 9 Q L K C G6 V A L 8 T P @ G A H F G K 1861 TGCCAACAACTTAAATGTGGAGTTGCCCTTTCTACCCCAGGAGGAGCACATTTTGGAAAA 641 G N GG Q V W R HM PR CT GTZ RBG QEBMG 1921 GGAAATGGTCAGGTCTGGAGGCATATGTTTCACTGCACTCGGACTGAGCAGCACATGGGA 61D C P VT ALGA ATGSTLTCT PSG QVASV 1981 GATTGTCCTGTAACTGCTICTGGGTGCTTCACTATGTCCTTCAGGRCAAGTAGCCTCTGTA 681 I C 8 G N Q §$ Q TL 8 8 C N § 8S ss L G6 Pp 2041 ATTTGCTCAGGAAACCAGTCCCAAACACTGTCCTCGTGCARTTCATCATCTCIGAGCCCA 701 T R P T I P BE E 8 A V A CI E 8 G Q L R 2101 ACAAGGCCTACCATTCCAGAAGAAAGTGCIGTGGCCTGCATAGAGAGTAATCAACTTCGC 721 L V N GG @ G R C A G R V B I Y BR BE GG 8S W 2161 TTGGTAAATGGAGAAGGTCRCTGTGCTGIGAGAGTAGAGATTTATCATGAGGGCTCCTGE 741 G T I C DD 8 W DUL SD A HV UV CROQL 2221 GGCACCATCTGTGATGACAGCTAGGACCTGAGCGATGCCCACGTOCTGTGCAGACAGCTG 7613 ¢C G BE A I ¥N A T G 8 A HP GB BR G3 TG P 2281 GGCTGTGGAGAGGCCATTAATGCCACTAGTTCTGCTCATTTTOGAGAAGGARCAGGGCCC 7811 W L D B MK C NG KE S RI MW Q C H 8 2341 ATCTGGCTGGATGAGATGAAATGCAATGGAAAAGAATCCOGTATTIGGCAGTGCCATTCA 801H G W GG Q @Q N C R HK E DAG UV I C 8 B 2401 CATGGCTGGGGGCAGCAAAACTGCAGGCACAAGGAGGATGCAGGAGTTATCTGCTCAGAG 821 P M 8S L R L T S E A S R E A C A GR L E 2461 TTCATGTCTCTGAGACTGACCAGTGAAGCCAGCAGAGAGGCCTGCTACAGGGCGTCTAGAA 841 V P Y N G A W G 8S V G R 8S N M 8 B T T V 2521 GTTTTTTACAACGGAGCTTGGGGCAGTGTTGGCAGGAGTAACATATCTGAAACCACTGTG 861 G V V C R Q L G C A DK @ KI NS A S L 2581 GGTGTGGTGTGCAGGCAGCTGAGCTGTGCAGACAAAGGGAAAATCAACTCTGCATCTITA 881 D XK A M 8 I P M W V D N V Q C PP K G PD 2641 GACARGGCCATGTCCATTCCCATGTGGGTGGACAATGTTCAGTGTCCAARAGGACCTAAC 901 T L W Q C P §S 8S P WE X RL A RP SE E 3701 ACGCTGTGGCAGTGCCCATCATCTCCATCGGAGAAGAGACTEGCCAGGCCCTCAGAGGAG 921T W IT T CDN XKMRLOGQTEG GT PTE ECS SG 2761 ACCTGGATCACATGTGACAACARGATGAGACTACAAGAAGGACCCACTTCCTGTTCTAGA 841 R V E I W H G G 8B W G T V C DD S WD L 2821 CGTGTGGAGATCTGGCACGGAGGTTCCTGGEAGACAGT GTGTGATGACTCCTGGGACTTG 961 N D A Q V V CC Q QL GC GP ALI KA ATF K 2881 AACAATGCTCAGGTGGTGTGTCAACAACTTGGC CTGGTCCAGCTTTGAAAGCATTCAAA 381 E A B F 6G Q G T G P I WL NU RVYVIKTUG CT KG 2941 GARGCAGAGTTTGGTCAGGGGACTGAACCCATATGGCTCAATGAAGTGAAGTGCARAGAS 1001 N E 8S 8S L W D C P A RR WG GH SEOCG H 3001 AATGAGTCTTCC TTGTGGGATTGTCCTGCCAGACGCTGUGGCCACAGTGAGTGTGAGACAL 1021 X ED AAV N CTAQIKTIG STU HT KTZ®PQ 3061 AMGGAAGACGCTGCAGTGAATTACACAGCACARAARATTTCAACGCACARAACCCCACAR 1041 K A T T 6 R 8 FL I A P GI L GV VL L 3121 ARAGCCACAACAGGTCGGTCATTCCTTATTGCATTCGGAATCCTTGGAGTTGTICTCTTG

CT" 1o61A I F V A F LTQXKRRQRGQRTELTUV 3181 GCCATTTICGTCGCATTATTCTTGACTCARARGCGAAGACAGAGACAGCGGCTTACAGTT 1081S 8 R @ E NL V H Q I QQ Y R B MN S CL 3241 TCCTCAAGAGGAGAGAACTTAGTCCACCAAATTCAATACCGGGAGATGAATTCTTGCCTG 11010 N A D D L DP L M NBS 8 6 6 H S BR A H 3301 AATGCAGATGATCTGGACCTAATAAATTCCTCAGGAGGCCATTCTGAGGCACAC 1 MSKLRMVLLE DSGSADVRRH FVNLSPFTIA VVLLLRACFV TESLGGTTKE LRLVDGENKC SEQ ID 61 SGRVEVKIQE EWGTVCNNGW SMEAVSVICN QLGCPTAIKA TGWANSSAGS GRIWMDHVSC : 121 RGNESALWDC KHDGWGKHSN CTHQQDAGVT CSDGSDLEMR LTNGGNMCSG RIEIKFQGQW NO: 42 181 GTVCDDNFNI NHASVVCKQL ECGSAVSPSG SANFGEGSGP INFDDLICNG NESALWNCKH 241 QGWGKHNCDH ABDAGVICSK GADLSLRLVD GVTECSGRLE VRFQGEWGTI CDDGWDSHDA 301 AVACKQLGCP TAITAIGRVN ASEGFGHIWL DSVSCQGHEP AVWQCKHHEW GRHYCNHNED 361 AGVTCSDGSD LELRLRGGGS RCAGTVEVEI QRLLGKVCDR GWGLKEADVV CRQLGCGSAL 421 KTSYQVYSKI QATNMWLFLS SCNGNETSLW DCKNWQWGGL TCDHYEEAKI TCSAHREPRL 481 VGGDIPCSGR VBVKHGDTWG SVCDSDPSLE AASVLCRELQ CGTVVSILGG AHPGEGNGQI 541 WAEEFQCEGH BSHLSLCPVA PRPEGTCSHS RDVGVVCSRY TEIRLVNGKT PCEGRVELKT 601 LNAWGSLCNS HWDIEDAHVL CQQLKCGVAL STPGGAHPGR GNGOVWRHMF HCTGTEQHMG 661 DCPVTALGAS LCPSGQVASV ICSGNQSQTL SSCNSSSLGP TRPTIPEESA VACIESGQLR 721 LVNGGGRCAG RVEIYHEGSW GTICDDSWDL SDAHVVCRQL GCGEAINATG SAHFGRGTGP 781 IWLDEMKCNG KEBSRIWQCHS HGWGQONCRH KRDAGVICSE FMSLRLTSEA SREACAGRLE 841 VFYNGAWGSV GRSNMSETTV GVVCRQLGCA DKGKINSASL DXAMSIPMWV DNVQCPKGPD 901 TLWQCPSSPW RKRLARPSEE TWITCDNKMR LQEGPTSCSG RVEIWHGGSW GTVCDDSWDL 961 NDAQVVCQQL GCGPALKAFK EARFGQGTGP INLNEVKCKG NESSLWDCPA RRWGHSBCGH 1021 KEDAAVNCTA QKISTHKTPQ KATTGRSFLI APGILGVVLL AIFVALFLTQ KRRQRQRLTV 1081 SSRGENLVHQ IQYREMNSCL NADDLDLMNS SGGHSEAH 1M §$ KL R M VL L ED SS G S A DVY RU RH SEQ ID 1 ATGAGCAAACTCAGAATGGTGCTACTTGAAGACTCTGGATCTGCTGACGTCAGARGACAT NO:43 21 P V N LL 8 P P T IT A V V L L L R A CUP V and 44 61 TTTGTCAACTTGAGTCCCTTCACTAT TGCTGTGGTCTTACTTCTCCGTGCCTGTTTTGTC 41 T 8 § L GG 3G T T K EL RL V D GG EN KX C 121 ACCAGTTCTCTTGGAGGAACAACCAAGGAGCTGAGGCTAGTGGATAGTGARAACAAGTGT 61 § G R V B V KI Q E E W GG T V CN NG NWN 181 AGTGOGAGAGTGGAAGTGAAAATCCAGGAGGAGTGGGGAACGGTGTGTAATAATGGCTGG 81 Ss M BE A V 8S v I €C N Q L @ C P T A I XX A 241 AGCATGGAAGCAGTCTCTGTGATTTGTAACCAGCTGGGATGTCCAACTACTATCARAGCC 101 T G W A N S 8S A BG 8S @G R I W MD HV ss ¢C 301 ACTGGATGGGCTAATTCCAGTGCAGGTTCTGGACGCATTTGGATGGATCATGTTTCTTGT 122 R @ NWN E S AL WDC KUHDGTWGZ KTH HS N 361 CGTGGGAATGAGTCAGCTCTTTGGGACTGCAAACATGATGGATGGGGAAAGCATAGTAAC 141 C T H Q Q D A G V T C §S DD @ $S DL EMR 421 TGTACTCACCAACAAGATGCTGGAGTAACTTGCTCAGATGGATCCGATTIGGARATGAGG 161L T N G@G G N M C § G R I E I K F Q G Q W 481 CTGACGAATGGAGGGARTATGTGTTCTGGAAGAATAGAGATCAAATTCCAAGAACAGTGS 181 G TT V ¢ DD NF NI NHA SV V CZK QL 541 GGAACAGTGTGTGATGATAACTTCAACATCAATCATGCATCTGTGGTTTGTAAACAACTT 200E C G 8S AV S FS GSA ANTFG GTZ EGS SSG Pp 601 GAATGTGGAAGTGCTGTCAGTTTCTCTGGTTCAGCTAATTTTGGAGAAGGCTCTGGACCA 22] I W F b DL I C N G NE S A L WNC CTIKBH 661 ATCTGGTTTGATGATCTTATATGCAACGGAAATOAGTCAGCTCTCTGGAACTGCARACAT 241Q G W @ K H N CD HAEUDA AGU VTIGC CS SK 721 CAAGGATGGGGAAAGCATAACTGTGATCATGCTGAGGATGCTGGAGTGATTTGCTCAAAG 261G A DL S L RLV DGVTT EBT CSU GTR RTULE 781 GGAGCAGATCTGAGCCTGAGACTGGTAGATAGAGTCACTOAATGTTCAGGAAGATTAGAR 288V RP Q GE NGTTIOCDDTG GU WD S HUD A 841 GTGAGATTCCAAGGAGAATGGGGGACAATATGTGATGACGGCTGGGACAGTCATGATGCT 301A V AC XK ¢L GC PTA ATITA ATI GTR RUVYVN 901 GCTGTGGCATGCAAGCAACTGGGATATCCAACTGCTATCACCGCCATTGGTCGAGTTARC 320A S BR @ P @d H I WILDS VYV SC OQGTHTE FP 961 GCCAGTGAGGGATTTGGACACATCTGGCTTGACAGTGTTTCT TGCCAGGGACATAAACCT

’ 381A V A 0 CK EH EWG GK HTYCNTHYNZED 1021 GCGGTCTGGCAATGTAAACACCATGAATGGGGARRGCATTATTGCAATCACAATGAAGAT 312A 6G V T ¢C S$ DGS DDL BLRILGRGTG GG S 1081 GCTGGCGTAACATGTTCTGATGAATCAGATCTAGAGCTAAGACTTAGAGGTGGAGGCAGT 31R CA GT UV EV ETIOQRTILTELGTE KT YTCTDR 1141 COCTGTACTGGGACAGTTGAGGTGGAGATTCAGAGACTGTTAGGGAAGGTATGTGACAGA 01G W 6G L KE ADV V CRQLGOCAES AD 1201 GGCTGGGGACTGAAAGAAGCTAATATAGTTTGCAGGCAGCTGGAATGTGGATCTGCACTC 421K T § Y Q VY S KI QATNUMGW NLP PULSE 1261 AAARCATCCTATCAAGTATACTCCAAAATCCAGGCAACAAACATGTGACTGTTTCTAAGT 4418 C N G N ET SL WDCZKNUWQWGOG a L 1321 AGCTGTAACGGARATGAAACTTCTCTTTGGOACTGCAAGAACTAGCAATGGGGTGGACTT 461T C D H Y E E AK I TC S A HRTETPR RTE 1381 ACCTGTGATCACTATGAAGAAGCCAARATTACCTGCTCAGCCCACAGGGAACCCAGACTA 481V 6 G D I P C S G R VE V XK HGDTUH G 1441 GTTGGAGGAGACATTCCCTGTTCTGGACGCGTTAAAGTGAAGCATGATOACACATAGEGE 501s VCcDBDPFSLEAAMSTYTLC CRETE Q 1501 TCCGTCTGTGATTCGGATTTCTCTCTGGAAGCTGCCAGCATTCTATGCAGGGAATTACAG 521C 6 T VV S IL GGA ABHTFEFGTETGHNTG GQ 1 1561 TGTGGCACAGTCATCTCTATCCTGGGGGGAGCT CACTTIGGAGAGGOAAATGAACAGATC

S¢1W AR E E F Q CE GHESGSUHTLSTILCTPUV A 1621 TGGGCTGAAGAATTCCAGTGTGAGGGACATGAGTCCCATCTTTCACTCTGCCCAGTAGCA 561 P R P EG T C 8 HS RD V GV VCS SR Y 1681 CCCCGCCCAGAAGGARCTTGTAGCCACAGCAGGGATGTTGGAGTAGTCTGCTCAAGATAC 581T E Z R L VN G KT PCEG GP RTVYVTETELTE KT 1741 ACAGAAATTCGCTTGGTGAATGGCAAGACCCCATGTGAGGGCAGAGTAGAGCTCAAARCS 601L NF A W G 8 L CN 8 HWDTIGETDATEKTVYTEL 1801 CTTAATGCCTGAGGATCCCTCTGCAACTCTCACTGGGACATAGAAGATGCCCACGTTCTT 621C Q Q L EK C 6 VA LST PG GA ATEHTEPG XK 1861 TGCCARCAACTTAARTGTGGAGTTGCCCTTTCTACCCCAGGAGGAGCACATTTTGGAARA 641G N G Q V W R HM F HC TG T B Q H MG 1921 GGAAATGGTCAGGTCTGGAGGCATATGTTTCACTGCACTGGCACTGAGCAGCACATGGGA 661D C P VT AL GAS LCT®PSG QV AGSYV 1981 GATTGTCCTGTAACTGCTCTGGGTGCTTCACTATGTCCTTCAGGGCAAGTGGCCTCTATA 681I C S 6 N Q § Q TL SS CNS GS S11 GP 2041 ATTTGCICAGGAAACCAGTCCCARACACTGTCCTCATGCAATTCATCATCTCTGGGOCCA 701T R P T I P EES A VACTITGESG GG QTELR 2101 ACAAGGCCTACCATTCCAGARGAAAGTGCTGTGECCTGCATAGAGAGTGGTCAACTTCGS 721L V N G 6G 6G R C A G RV BE I Y HE G § W 2161 TTGGTAAATGGAGGAGGTCGCTGTGCTAGAAGAGTAGACATTTATCATGAGEGCTCCTGS 7426 T I ¢ DD 9 ¥ DL §DAEKTVVYVC CRO OTL 2221 GGCACCATCTGTGATGACAGCTGGGACCTGAGCGATGCCCACGTGETGTGCAGACAGCTA 7616 C G BE A I NAT GS AHTPGTESG TGP 2281 GGCTGTGGAGAGGCCATTAATGCCACTGGTTCTACTCATTTTGAAGAAGGARCAGEGCCE 7811 W L D BE M K C NG KE § R I W QC CEH S 2341 ATCTGGCTGGATGAGATGARATGCAATGGARRAGAATCCOGTATTTGGCAGTGCCATTCA ! 01H G W 6 Q Q N Cc RH KBR DA GV I CS RB 2401 CATGGCTGGGGGCAGCAAAACTGCAGGCACAAGGAGCGATGCAGGAGTTATCTGCTCAGAG 821lF M § L R L T S EA S REA ATCA AGT RTELE 2461 TTCATGTCTCTGAGACTGACCAGTGARGCCACCAGAGAGGCCTGTGCAGUGCOTCTAGAR 841V P Y N G A W 6 § V G R 8 N M § ET T V 2521 GTTTTTTACAACGGAGCTTGGGGCAGTATTAGCAGGAGTAACATGTCTGARACCACTGTG 861G V V C R Q L GC A DIXGKTITNSGEA ASL 2581 GGTGTGATGTGCAGGCAGCTGGACTGTGCAGACAAAGGGAAAATCAACTCTACATCTTTA 881D XK A M S I P M W V D NV Q CP KG PD

X 005/011502 ; 2641 GACAAGGCCATATCCATTCCCATGTGGGTGAACARTG TICAGTG TCCARRAGGACCTGAC 901 T L WwW Q C P 8 S P W BR X RL A RP S ER BE 2701 ACGCTGTGGCAGTGCCCATCATCTCCATAGEAGAAGAGACTOGCCAGGCCCTCGGAGGAG 921T W I T C D N KM RL QEGU PTS CSS G 2761 ACCTGGATCACATGTGACAACRAGATGAGACTACAAGAAGGACCCACTTCCTGTTCTGGA 941R V E I WH G G S NGTJUVOCDTDS SUWDL 2821 CGTGTGGAGATCTGGCACGGAGGTTCCTGGGEAACAGTATATAATGACTCCTGGGACTTG 961 N D A Q VVC QQLGG CG GT?PATLTEKA ATF K 2881 AACGATGCTCAGGTGGTCTGTCARCARCTTGACTATGGTCCAGCTTTGAAAGCATTCAAA 981 E A BE F @ Q G T G P I WL NETVTZEKTCTZKG 2941 GAAGCAGAGTTTGGITCAGGGAACTGGACCCATATAGCTCAATAAAGTGAAGTGCAAAGGS 1000 E 8 8 L WD OCZPAIRTPERTWGHSTETGCG GH 3001 AATGAGTCTTCCTTGTGGOATTGTCCTGCCAGACGCTGGGGCCACAGTGAGTGTGGACAC 1022 XK BE D A A VNC CTA AGQIKTIT STU HI KTT?PAQ 3061 AAGGAAGACGCTGCAGTGAATTGCACAGCACAAAAAATTTCAACGCACAAAACCCCACAA 141K A T T 3 Q 8 FL I AF G1I1IUL GY V ULL 3121 AAAGCCACARCAGGTCAGTCATTCCTTATTGCATTCGGAATCCTTGGAGTTGTTCTCTTG 061A I F VA L P LT QKRUPRGOQRUO QRTULTUV 3181 GCCATTTTCGTCGCATTATTCTTGACTCAAAAGCGAAGACAGAGACAGCGGCTTACAGTT 1081S S R G6 BR NL V HQ I QYRGEMENGS CL 3241 TCCTCAAGAGGAGAGAACTTAGTCCACCAAATTCAATACCGGGAGATGAATTCTTGCCTG 2101 N A D D L DL M NS SEBENGSNTES ADP 3301 AATGCAGATGATCTGGACCTAATGAATTCCTCAGAAAATTCCAATGAGTCAGCTGATTTC 1122 KR A A 83 L I S V § K FL PI 8 GMTETZK E 3361 AATGCTGCTGAACTAATTTCTGTGTCTAAATTTCTTCCTATTTCTGGAATGGAAARGGAG 1141 A I L R HT E KB NGN IL 3421 GCCATTCTGAGGCACACTGAAAAGGAAAATGGGAATTTA 1 MSKLRMVLLE DSGSADVRRH FVNLSPFTIA VVLLLRACFV TSSLOGTTKE LRLVDGENKC SEQ ID 61 SGRVBVKIQE EWGTVCNNGW SMEAVSVION QLGCPTAIKA TGMANSSAGS GRIWMDHVSC 121 RGNEBSALWDC KHDGWGKHSN CTHQQUDAGVT CSDGSDLEMR LTNGGEMCSG RIBIKFQGQW NO: 44 181 GTVCDDNPNI NHASVVCKQL ECGSAVSPSG SANPGEGSGP INFDDLICNG NESALWNCKH 241 QGWGKHNCDH AEDAGVICSK GADLSLRLVD GVTECSGRLE VRFQGBWGTI CDDGWDSHDA 301 AVACKQLGCP? TAITAIGRVN ASBGPGHIWL DSVSCQGHEP AVWQCKHHEW GKHYCNHNED 361 AGVTCSDGSD LELRLRGGGS RCAGTVEVEI QRLLGKVCDR GWGLKEADVV CRQLOCGSAL 421 KTSYQVYSKI QATNMWLFLS SCNGNETSLW DCKNWQWGGL TCDHYREAKI TCSAHREPRL 481 VGGDIPCSGR VEVKHGDTWG SVCDSDFSLE AASVLCRELQ CGTVVSILGG AHPGRGNGQI 541 WABRFQCEGH ESHLSLCFVA PRPEGTCSHS RDVGVVCSRY TEIRLVNGKT PCEGRVELKT 601 LNAWGSLCNS HWDIEDAHVL CQQLKCGVAL STPGGAHFGK GNGQVWRHMF HCTGTEQHMG 661 DCPVTALGAS LCPSGQVASV ICSGNQSQTL S5SCNSSSLGP TRPTIPERESA VACIESGQLR 721 LVNGGGRCAG RVEIYHEGSW GTICDDSWDL SDAHVVCRQL GCOEAINATG SAHFGEGTGP 781 IWLDEMKCNG KESRIWQCHS HGWGQONCRH KRDAGVICSE FMBLRLTSEA SREACAGRLE 841 VFYNGANGSV GRSNMSETTV GVVCRQLGCA DKGKINSASL DKAMSIPMWYV DNVQCPKGPD 901 TLWQCPSSPW EXRLARPSEE TWITCDNKMR LQEGPTSCSG RVEIWHGGSW GTVCDDSWDL 961 NDAQVVCQQL GCGPALKAFK EAEFGQGTGP IWLNEVKCKG NESSLWDCPA RRNGHSECGH 1021 KEDAAVNCTA QKISTHRTPQ KATTGQSFLI APGILGVVLL AIFVALFLTQ KRRQRQRLTV 1081 SSRGENLVHQ ZQYREMNSCL NADDLDLMNS SENSNESADF NAAELISVSK FLPISGMEKE 1141 AILRHTEKEN GNL

Example 10 Cloning and characterization of canine CD163 from DHS? cells.

A forward primer 5’simianCD163 (SEQ ID NO: 28) (5’-

CACCGGAATGAGCAAACTCAGAATGG-3’ based on human CD163) and a reverse primer HuCD163-3'Kpn (SEQ ID NO: 29) 5’-

GCTCCGGTACCTAGTCCAGGTCTTCA

TCAAGGTATCTTA-3’) were used to amplify CD163 cDNA from DHS2 cells. Total cellular RNA was prepared from DHS2 cells using the RNeasy kit. RT-PCR .97- q parameters were the same as described in Example 4. RT-PCR products were cloned directionally into the pCDNA3.1D/V5/His/TOPO vector according to the manufacturer’s instruction. Several clones containing large inserts were analyzed.

Several clones with large inserts were analyzed, and these fell into either the v2 or v3 splicing patterns seen in other species. The v2 variant is missing an 81-nucleotide exon (E81) relative to the v3 variant, which results in a reading frame shift and alternative carboxy terminal amino acid sequences. The canine CD163v2 cDNA from DHS82 cells encodes a peptide of 1115 amino acids. When compared to the sequences in Genbank database, it is 83.9% identical to human CD163 (Genbank 722968), 85.1% identical to pig CD163 (Genbank AJ311716), and 74.3% identical to mouse CD 163 (Genbank

AF274883). The nucleotide and amino acid sequences of the two splice variants found in DHB82 cells are provided below (SEQ ID NOS: 45-48).

SEQUERCE LO iM 8 KL R MV P HGW NS GS ADUFPUR RU RTC SEQ ID 1 ATGAGCAAACTCAGAATGGTCCCACATOGAAACTCTGGATCTGCTGACTTTAGAAGATGT NO: 4S 21lF AL L CF 8S AV A VV S IL BSTC COL M and 46 61 TTTGCCCTCTTGTGTCCCTCTGCTGTGGCTGTGGTCTCCATTCTCAGTACCTGTTTGATG 41 T N 8 L GG R A D XK EM RL TUDGZ RTDINSUZC 121 ACCAATTCTCTTGGGAGAGCAGATAAAGAGATGAGGCTAACGGATAGTGAAGACAATTGC 618 G R V E V XK V Q E E W GTVCNUNG W 181 TCCGGGAGAGTGGAAGTGAAAGTCCAGGAGGAGTGGGGAACGGTATATAATAATGGCTGG 81G M D E VS V I CR QL G@G CP TA XI K A 241 GGCATGGATGAAGTCTCTOTGATTTACASGCAGCTGGGATGTCCCACTGCTATCAAAGCC 101A G W A N S R A G 8S GR I WMUDUH HV S C 301 GCTGGATGGGCCAATTCCAGGGCAGGCTCTGGACGAATCTGGATGGATCATGTTTCTTGT 121 R G N E 8 A L W D C K HD G W 3 K H XN C 361 CGAGGGAATGAATCTGCTCTCTGOGACTGCAAACATGATAGATGGGGAAAGCACARCTGC 141s E Q QQ D A G V T €C 8 D G SS SS L EM R L 421 AGTCATCAACAGGATGCTGGAGTAACCTGTTCAGATGGATCCAGTTTGGAGATGAGGTTG 161M N 6 G N Q C § G R I BE V KP Q GG Q W G 481 ATGAACGGCGGAAACCAGTGTTCTGGCAGAATAGAAGTCAAQTTCCAGGGACAGTGGGGA 181 T V € bp D N F N I D ¥ A S V V C K Q L E 541 ACAGTGTGTGATGACAACTTCAACATAGATCATGCTTCTGTGGTITGTAAACAGCTCGAA 201C 6G S AV S F S$ GS ANUP FGEUG GS SG UP I 601 TGTGGAAGTGCTGTCAGTTTCTCTGGTTCAGCTAATT TTOGAGAAGGTTCTGGGCCAATC 221" P D DL V CS 3 NR S AL WNUGCIKH E 661 TGGTTTAATGATCTTGTGTGCAGTGGAARTGAGTCAGCTCTCTGGAACTGCAAGCATGAA 241 G W G X HN C DHA EDV GV I CULL DG 721 GGATGGGGAAAGCATAACTGTGATCACGCTGAGGATGTTGGAGTGATTTGCTTGGATGGA 261A D L 8 L RL V D G3 V TEC S$ GR LE V 781 GCAGATCTGAGCCTGAGACTGGTAGATGGAGTCACTGAATGTTCAGGARGATTAGAAGTA 281 K F Q G E WG T V C DD GWUDSNUDD AOA 841 AAATTCCAAGGGGAATGGGGGACAGTATGTGATAATAGCTGGAATAGTAATGATGCTGCT 301v VC KQLGCUPT AV TATIG GT RUYNA 901 GTGGTATGTAAACARCTGGGATGCCCAACTGCTGTCACCSCCATTGGTCGAGTTAACGCC 321s EG S G HI WL DNULSG CO QO GT DE S A

) 961 AGTGAGAGAAGTGGACACATTTGGCTTAACAATC I TTCCTOCCARGGAGACGARTCTGCT 341 L W Q CC R H HEW GI KU HUY CNU HUNUETDA 1021 CTCTGGCAGTGTAGACACCATGAATGGGGAAAGCATTATTGCARTCATAATGAAGATGCT 316 VT CSD GSD LELZ RLY GS GS GSR 1081 GGTGTGACATITTCTGATGGATCAGACCTGGAGCTGAGACTTGTCGGTGGAGGCAGCCGC 381 C A GG T V E V BI Q KL Lh 8G XK V CDUZRGEG 1141 TGTGCTGGAACAGTGGAGGTTGAAATTCAGAAACTGCTAGGGARAGTATGTAATAGAGAE 401W 6 L KE ADV V CEKODLG GEG CG GSA ATLREK 1201 TGGGGACTGAAAGAAGCCGATGTGGTTTGCAAGCAGTTGGGATGTGGATCTGCTCTCARA 421T 8 Y QR Y § KV RATNTMWILTPETULSR 1261 ACGTCCTATCAGCGTTATTCCAAAGTTAAGGCAACRAACACATGGCTGTTTTIAAGCCGC 441 C 8 8 N E T 8S LL W D C K N WwW Q W G3 GL 8 1321 TGTAGTGGCARTGAARCTTCCCTTTGGGACTACAAGAACTGGCAGTAAGITOGACTGAGT 461C D H Y E BE AKVTC CS SRAHTRTETPT RTDLDUV 1381 TGTGATCACTATGARGAAGCTAAAGTTACCTGCTCAGCCCACAGGGAACCCAGACTAGTT 4816 @ DI P C S G RV EV KH GDTWG T 1441 GGAGGAGATATTCCCTGCTCTGGTCGTGTTAAAGTGARACATGATGACACATGAAACACT 501V ¢ D SDF §LEAA ASU VTL CRTETLR QC 1501 GTCTGTGATTCCGACTTCTCTTTOGAAGCTGCCAGTGTCCTGTGCAGAGAGTTACAGTGT 5213 T V I 8 I L G G A HF GEE GN GOQTIW 1561 GBCACAGTCATCTCCATCCTAGGGGGAGCTCACTTTGGAGAMGGAAATGGACAGATCTIGE

S412 B BR F Q C EG Q BE 8 HL SL CS V A 8 1621 GCTGARGRATTCCAGTGTGAGGGGCAGGAGTCCCATCTTTCACTCTGTTCAGTAGCCTCT

S61R P D GT C 8S H 9 R DV GV VCSTZ RTZYT 1681 CGCCCAGATGAAACCTGTAGCCACAGCAGGGATGTTGGAGTOSTCTGCTCARGATACACS

S818 I R L V N 6G Q S PC EGRVEBELTEKTIL 1741 GAARTCCGCTTOGTGAATGGCCAGTCCCOGTCTAANGGAAGAGTAGAGCTCAAGATACTT 6016 N W G 8 L CN S H N D I EDASEKUYVEP C 1801 GGGAACTGEGGATCCCTCTGCAACTCTCACTGGAACATAGAAGATGCCCATGTTTTCTGT 621 Q Q L XK ¢ GCG V AL SS I P GG G A HP G K a 1861 CAGCAGCTCARATGTGGAGTTGCCCTTTCTATTCCO@GAGAAGCACATTTTGGGAAAGGA 641 8 G Q I W R HM P H CT GTUEGQH HM GD 1921 AGTGGTCAGATCTGGAGGCACATGTTTCACTGCACTOGOACTAAGCAGCACATGGGAGAT 661C P V T A L 6 AT L € S AG QV ASV I 1981 TGCCCTGTAACTGCTCTGGGCGCGACGCTGTGTTCTRCTGEECARGTGGCCTCTATARTC 681C S G N Q S Q T L 68 PC NS T 8 L DP T 2041 TGCTCAGGAAATCAGAGCCAGACGCTATCCCCATGCAATTCAACRTCTCTGGACCCAACA 700R 8 TT S B B S A VACTIA ASG OTLT RL 2101 AGATCTACCACTTCGGAAGAAAGTGCTGTTGCTTGTATTGCGAGTGGGCAACTTCACCTA 721 V N G G G R C AG R I E VY ¥ EG 8 W G 2161 GTAAATGGAGGCGGTCGCTGTGCTCGAAGAATAGAGBTCTACCATGAGGGCTCCTGRGAE 741T I CD J 5 WD LS DAUHVTYVTVC CRO OQTLGa 2221 ACCATCTGTGATGACAGCTGGGACCTGAGTGATGCCCATGTGGTGTGCAGACAGCTAAAE 761C G V A I N ATG S AHPGETGTSG GP I 2281 TGTGGAGTGGCCATTAATGCCACTGICTCTGCTCATT TTGGGGAAGAAACAGGGCCCATC 781 W L D E V N C N G K BE § HI W QQ CR § 2341 TGGCTGGACGAGGTGAACTGTAATOGARAGGAATCTCATATCTGGCAATGCCGCTCACAC 8016 W 6G 0 HR N C RB K ED AG UV ICSE UP 2401 GGCTGGGGACAACACAACTGCAGACATAAGGAGGATGCAGGAGTTATCTACTCAGAGTTC 821M § L R L I D ET S R DTI CAGRTELTEV 2461 ATGTCTCTCAGACTGATTGATGAAACCAGCAGAGACATCTGTRCAGGGCGTCTTGAAGTT

B41 F Y N G A W G S V G K 8 N M 8 AT T V E 2521 TTTTACARTGGAGCTTGGGGCAGCOTTGECARGAGTAATATETCTGCAACCACTGTGGAG 861V V C R QL GCA ADTEKSGSTIDNTPASSZED 2581 GTGGTATGCAGGCAACTGGGTTGIGCAGACAAGGGGAGCATCAACCCTGCATCTTCAGAC

881K FP M 5 RH M N VD NV Q COP KE PDT 2641 AAGCCCATGTCCAGGCACATGTGOGTGGACARTGTCCAGTGTCCAAAAGGACCTGACACE 901L W Q C P 8 S PWEKGQRUVAGSTSS SETET 2701 TTATGGCACTGCCCATCTTICTCCATGGAAACAGAGAGTGGCCAGTTCTTCAGRGGAGACT 921 Ww I T C A NK I RL QB GTS NCGS GR 2761 TGGATCACATGTGCCAACAAGATAAGACTTCARGARGGAACCTCTAATTGTTCTAGACGT 941V EL W H § G § WG TV CDDTGEWDLE 2821 GTGIAGCTCTGGCACGGAGETTCCTGIGAGACAGTGTGCGATGACTCCTGAGACCTTGAA 91D A Q V V CR QL @CGTPATLTEATLTEKE 2881 GATGCACAAGTOGTGTCTCGACAGCTGGECTGTGGCCCAGCATTAGAAGCACTAAAAGAG 981A A P G Q GT GP I WLNJDVZEKTCET KS GN 2941 GCAGCATTTGGTCAGGGGACTGGACCTATATAGCTCARTGACATGAAGTACAAAGGGAAT 1001E 8 8 L W DC P ARGPWGTHSTDTCG GH K 3001 GAGTCTTCCTTGTGGGATTETCCTGCTAGACCCTGGGGGCACAGTGACTCTGGCCACARG 1021E DP A AV RC S EI A MAG QTRTGETSNT PR 3061 GARGATGCTGCTGTGAGGTGCTCAGARATTGCAATGGCCCAAAGATCATCAAATCCTAGA 10416 K S § L VA L GI PG V I LILATPF¥TLTI 3121 GGTCACTCATCCCTTGTTGCATTGGGGATCTTTGGTGTCATTCTTCTGGCCTTTCTCATCS 061A L L L WT QRRURG QGQQRLTJVS SLR 3181 GCTCTCCTCTTGTGGACTCAAAGGCGAAGACAGCAACAGOGGCTTACAGTTTCCTTGAGA 10816 BE N S V HQ I Q YR EMUE SSL KAD 3241 GGAGAGAATTCTGTCCACCAAATTCAATACCGGGARATGAATTCTTCCCTGAARGCAGAT 11000 L D V L T 8S 8 ED H FP XR V H 3301 GATCTGGACGTGCTGACTTOCTCAGAAGACCATTTTGAGGTACAC 1 MSKLRMVPHG NSGSADFRRC FALLCPSAVA VVSILETCIM THSLGRADKE MRLTDOEDNC SEQ ID 61 SGRVEVKVQE EWGTVCNNGW GMDEVSVICR QLGCPTAIKA ACMANSRAGS GRIWMDHVSC 121 RONBSALWDC KHDGWGKENC SHQQDAGVTC SDGSSLEMRL MNGGNQCSGR IEVKPQAQWG | NO:46 181 TVCDDNENID HASVVCKQLE CGSAVSFSGS ANFGRGSGPI WPDDLVCSGN ESALWNCKHE 241 GWGKHNCDHA EDVGVICLDG ADLSLRLVDG VTECSGRLBV KFQGEWGTVC DDGWDSNDAA 301 VVCKQLGCPT AVTAIGRVNA SEGSGHIWLD NLECQGDESA LWQCRHHEWG KHYCNHNEDA 361 GVTCEDGSDL ELRLVGGGSR CAGTVEVEIQ KLLGEVCDRG WGLKEADVVC KQLGCGSALK 421 TSYQRYSKVK ATNTNLFLSR CSGNETSLWD CKNWOWGOLS CDHYEBAKVT CSAHREPRLV 481 GGDIPCSGRV EVKHGDTWGT VCDSDPSLEA ASVLCRELQC GTVISILGGA HPGEGNGQIN 541 ARBPQCEGQE SHLSLCSVAS RPDGTCSHSR DVGVVCSRYT EIRLVNGQSP CEGRVELKIL 601 GNWGSLCNSH NDIEDAHVPC QQLKCGVALS IPGGAHPGRG SGQIWRHMFH CTGTEQHMGD 661 CPVTALGATL CSAGQVASVI CSGNQSQTLS PCNSTSLDPT RSTTSEESAV ACILASGQLRL 721 VNGGGRCAGR IEVYHEGSWG TICDDSWOLS DAHVVCRQLG CGVAINATGS AHFGRGTGPI 781 WLDEVNCNGK ESHINQCRSH GWGQHNCRHX EDAGVICSEF MSLRLIDETS RDICAGRLEV 841 PYNGAWGEVG KSNMSATTVE VVCRQLGCAD KGSINPASSD KPMSREMWVD NVQCPKGPDT 901 LWQCPSSPWK QRVASSSEET WITCANKIRL QEGTSNCSGR VELWHGGSWG TVCDDSWDLE 961 DAQVVCRQLG CGPALEALKE AAPGQGTGPI WLNDVKCKGN ESSLWDCPAR PWGHSDCGHR 1021 EDAAVRCSEI AMAQRSSNPR GHSSLVALGI PGVILLAFLI ALLLWTQRRR QQORLTVELR 1081 GENSVHQIQY REMNSSLKAD DLDVLTSSED HFEVH iM 8 XL RM VP HGTSNGS GE ADPFRTETC SEQ ID 1 ATGAGCAAACTCAGAATGGTCCCACATGGAAACTCTGGATCTGCTGACTTTAGAAGATGT NO: 47 21F A L L C PS AV AVY SS ILS STU CTLHM 61 TTTECCCTCTTATATCCCTCTGCTCTGGCTGTEGTCTCCATTCTCAGTACCTGTTTGATG 41T N 8S L G RAD KEMBRTLTTDGTETDNC 121 ACCARTTCTCTTGGGAGAGCAGATAAAGAGATGAGGCTARCGGATGGTGAAGACAATTGC 61S G R V E V K V Q BEB E WwW GT V CC N NG WwW 181 TCCGAGAGAGTOGAAGTGAARGTCCAGAAGGAGTGGCGAACCGTGTGTAATAATGGCTGS 81@ M DEV §V ICRG QLGT CT PTA ATITKA 241 GGCATGGATGAAGTCTCTGTGATTTGCAGGCAGCTGGGATETCCCACTGCTATCAAAGCC 10LA G W A N 8 R ARGS GRTIWMDUEHTYSC 301 GCTGGATGGACCAATTCCAGGGCAGACTCTGOACGAATCIGGATGGATCATGTTTCTTGT 121R @ N E § A L WD C K HD GWG GI KU HBINSUC 361 CGAGGGAATGAATCTGCTCTCTGGGACTGCARACATGATGGATGGGGAAAGCACAACTGS 1418 H Q Q D AG V TC 8S DGS SLEMTP RL

121 AGTCATCAACAGOATGCTGEAGTAACCTGT TCAGATOGAT CCACTT TGGAGATGAGGTTG 161M N G6 G N Q ¢C S 6G RI EV KF QGOQGHW G 481 ATGAACGGCGGAAACCAGTGTTCTGGCAGAATAGAAGTCAAGTTCCAGGGACAGTGEGGA 181T Vv ¢C DD NP NIDUHASVTYVTVCEKO QTL E 541 ACAGTGTGTGATGACAACTTCAACATAGATCATGCTTCTGTGGTTTGTAARCAGCTCGAR 2000 GS AV SF SGSANTPFGE EG GSG GP I 601 TGTGGAAGTGCTGTCAGTTTCTCTGGTTCAGCTAATTTTGCACAAGGTICTGGACCAATC 220W FP D DL VCSGNTET SA ATLTMWNTC CT KTHE 661 TGGTTTGATGATCTTGTGTGCAGTGAAAATGAGTCAGCTCTCTIGGAACTGCAAGCATGAA 2416 W 8 KH NCD HAETDUTV GV ICTLDG 721 GGATGGGGAAAGCATARCTGTGATCACGCTGAGGATGTTGGAGTGATTTGCTTGGATAGA 261A DL S$ L RL VDGUVTTE ET CS EGR RTILEV 781 GCAGATCTGAGCCTGAGACTGGTAGATGGAGTCACTGAATGTTCAGGARGATTAGAAGTA 261K F Q G B W G T V CDDGWDSENTD A A 841 AAATTCCAAGGGGAATGGGGGACAGTGTGTOATGATGGCTGGGATAGTAATGATGCTGCT 301V V ¢ KQ LGC PTA AVTA ATITG GTR RVYHNA 901 GTGGTATGTAARCAACTGGGATGCCCAACTACTATCACCGCCATTAGTCAAGTTARCGCC 3218 EG 8 3 HI WLDUNTESTC CO QO GDTES A 961 AGTGAGGGAAGTGGACACATTTGGCTTGACAATCTTTCCTGCCARGGAGACGAATCTGCT 341 L W Q CR KH E NG KHYCNTBHTENTETD A 1021 CTCTGGCAGTGTAGACACCATGAATGGGGAAAGCATTATTIGCAATCATAATGAAGATGCT 31G VT C65 DG SDLETELTZRTELVYG GGG S R 1081 GGETGTAACATGTTCTGATGGATCAGACCTGGAGCTGAGACTTGTCAGTOGAGGCAGCCAC 381C A 6 T V B V E I Q KL LGXTV CODTERG 1141 TGTGCTGGIACAGTGGAGGTTGAAATTCAGAAACTGCTAGGGARAGTATCTCATAGAGGC 401W 6 L KE ADV V CKO QLG GT CG GSATL K 1201 TGGGGACTGARAGAAGCCGATGTGATTTOCAAGCAGTTGGGATCTOGATCTGCTCTCARA 421T 8 Y Q R Y S KV KA TNTWLP PLE R 1261 ACGTCCTATCAGCGTTATTCCAAAGTTARGGCARCAAACACATGGCTGTTTTTAAGCCGE 441 C S G N BE T 8 L WD C KN WOQWOaG G TIL S 1321 TGTAGTGGCAATGAAACTTCCCTTTGRAACTACAAGAACTOGCAGTOAGETGEACTGAGE 461C D H Y B 3 A K VTC SAEHBTRTETPZRTELYV 1381 TGTGATCACTATGAAGAAGCTAAAGTTACCTGCTCAGCCCACAGGGAACCCAGACTAGTT 4816 6 D I P C § G RV BR V K HG DT WEG T 1441 GGAGGAGATATTCCCTGCTCTGGTOGTGTTGAAGTAARACATAGTAACACATGGAGCACC 501Vv CD § DP SL EAASV VLC CRTETLTQ QC 1501 GTCYGTGATTCCOACTTCTCTTTAGARGCTGCCAGTGTGCTGTGCAGAGAGTTACAGTGT 5216 T V I § I L G GA HP? GE GUNGOTIMW 1561 GGCACAGTCATCTCCATCCTAGGGGGAGCTCACTTTGGAGAAGGAAATAGACAGATCTAR 541A E E F Q CE G QE S HL SL C8 V A S 1621 GCTGAAGAATTCCAGTGTGAGGGGCABAAGTCCCATCTTTCACTCTGTTCAGTAGCCTCT 561R P D G T CS H S RD V GV V CEU RTUYT 1681 CGCCCAGATGGGACCTGTAGCCACAGCAGGGATGTTRGAGTCETCTGCTCARGATACACG 581E I R L VN GQ S P CEG RV ETLTEKTITHL 1741 GARATCCGCTTGGTGARTGGCCAGTCCCCGTGTGARGGARGAGTGGAGCTCARGATACTT 601G N W 6 § L C N S H WD TIETDATHVFEC 1801 GGGAACTGGGGATCCCTCTGCAACTCTCACTGGGACATAGAAGATGCCCATGTTTTCTGT : 621Q Q L K C G V AL S I PG GA HP G K G 1861 CAGCAGCTCARATGTGGAGTTACCCTTTCTATTCCGGGAGGAGCACATTTTGGGARRGGA 64S G Q I W R HM F EH CT GTGEGQHMGD 1921 AGTGGTCAGATCTGGAGGCACATGTTTCACTGCACTOGGACTGAGCAGCACATAGGAGAT 661C P V T A L G A TL C 8S A GQ UV ASV I 1981 TECCCTGTAACTGCTCTAAACGCAACGCTGTETTCTGCTGGGCARGTGGCCTCTGTAATC 681C S G N Q S Q TL S P CMNE STS L DEP T 2041 TGCTCAGGAAATCAGAGCCAGACGCTATCCCCATGCAATTCAACATCTCTGGACCCAACA

Tr T01R § IT T S EB E88 AV ACI AGS SG QL RT 2101 AGATCTACCACTTCGGAAGAAAGTGCTATTGCTTGTATTGOGAGTOGGCARCTTCGCCTG 7220V N G G GG R C A GR I B V Y H EG S W G 2161 GTAAATGGAGGCGGTCGCTGTGCTGGGAGAATAGAGETCTACCATGAGGGCTCCTARAAC 741T I C DDS WODLGESDAHRTVYVUYCRA QTELG 2221 ACCATCTGTGATGACAGCTGGGACCTGAGTGATGCCCATGTAGTGTGCAGACAGCTGGGC 761C 6 V A I N AT GS AR HPFGETGTG GP I 2281 TGTGGAGTGGCCATTAATGCCACTGGCTCTGCTCATTTTUGGGAAGGAACAGGGCCCATC 781W L D E V N C N G K E 8 H I WQ@CRS H 2341 TGGCTGGACGAGGTGAACTGTAATGGAAAGGARTCTCATATCTGGCAATACCACTCACAC 8016 W G Q H NCR HKTETDA AGT VTIT CSTE? 2401 GGCTGGGAGCAACACAACTGCAGACATAAGGAGGATGCAGGAGTTATCTGCTCAGAGTTC 821M § L R L I DBT 8 RDI CAGT RTILEBYV 2461 ATGTCTCTCAGACTGATTGATGAARCCAGCAGAGACATCTATGCAGGGCATCTTGAAGTT 841 P ¥Y N G A WW G6 8 vV @ K 8 NM 8 A T T V E 2521 TTTTACAATGGAGCTTGGGGCAGCGTTGGCAAGAGTAATATGTCTGCAACCACTGTGGAG

BE1V V ¢ R Q L G C AD X @ 8 I NP A 8 § D 2581 GTGGTATGCAGGCAACTGGGTTGTGCAGACAAGAGGAGCATCAACCCTGCATCTTCAGAC 881K P M S R HM W VD NUVQCT®PTRGTPTDT 2641 AAGCCCATGTCCAGGCACATGTGAATGGACAATATCCAGTGTCCARAAGGACCTGACACC 901 L WwW Q C P 8 8S P WwW X Q R V A 8 8 8 BE B T 2701 TTATGGCAGTGCCCATCTTCTCCATGGAAACAGAGAGTGGCCAGTTCTTCAGAGGAGACC 921W I T C AN KI RL Q EG T 8 NC 8 G R 2761 TGGATCACATGTGCCAACAAGATAAGACTTCAAGAAGGAACCTCTARTTGTTCTGGACGT 941V BE L W H G 6G S WG TV CDTGDGSWNDTLE 2821 GTGGAGCTCTGGCACGGAGGTTCCTGGAGGACAGTATACGATGACTCCTGAGACCTTGAA 961D A Q VV CR QLGC CG ®PATLTEA ATLTEKRE 2881 GATGCACAAGTGGTGTGTCGACAGCTGAGCTATGACCCAGCATTAGAAGCACTAAAAGAG 981A A F G Q " 6 P I WL ND UV KC CIRKGEHN 2941 GCAGCATTTGATCAGGGGEACTGGGCCTATATGGCTCAATGACGTGAAGTGCAAAGGGAAT 101E § S L W DCP ARTPHW¥GTEHTSDGC CGH K 3001 GAGTCTTCCTTGTGGGATTGTCCTGCTAGACCCTAGGGGCACAGTGACTGTAGCCACAAG 1021E D A AV R CS EOI AMAG QT RT ETSNTPR 3061 GAAGATGCTGCTGTGAGGTGCTCAGAAATTGCAATGGCCCARAGATCATCAAATCCTAGA 141 @ BR 8 8S IL V A L G6 I PP G6 V I L L A F L I 3121 GGTCACTCATCCCTTGTTGCATTGGGGATCTTTGGTGTCATTCTTCTGGCCTTTCTCATC 1061 A L L L W T Q R RR Q 0 Q RL TV 8S L R 3181 GCTCTCCTCTTGTAGGACTCAAAGGCAAAGACAGCAACAGCCGCTTACAGTTTCCTTGAGA 10813 BE N S V H Q I Q YR E MNS S L K A D 3241 GGAGAGAATTCTGTCCACCARATTCAATACCGGGARATGAATTCTTCCCTGAAAGCAGAT 1101 D L. OD V L T S S EY P NE S DD F ND A 3301 GATCTGGACGTGCTGACTTCCTCAGAATATCCCAATGAGTCAGATGATTTTAATGATCCT 11216 L I 8S V 8 K S L P I § © 3361 GGGCTAATTTCTGTGTCTAAATCTCITCCTATTTCTGGA 1 MSKLRMVPHG NSGSADFRRC FALLCPSAVA VVGSILSTCLM THSLGRADKE MRLIDGEDNC | SEQ ID 61 SGRVEVKVQR EWGTVCNNGW GMDEVSVICR QLGCPTAIKA AGWANSRAGS GRIWMDHVSC 181 TVCDDNFNID HASVVCKQLE CGSAVSFSGS ANFGEGSGPI WFDDLVCSGN ESALWNCKHE 241 GWGKHNCDHA BDVGVICLDG ADLSLRLVDG VTECSGRLEV KFQGEWGTVC DDGWDSNDAA 301 VVCKQLGCPT AVTAIGRVNA SEGSGHIWLD NLSCQGDESA LWQCRHHEWG KHYCNHNEDA 361 GVICEDGSDL ELRLVOG@SR CAGTVEVEIQ KLLGKVCDRG WGLKEADVVC KQLGCGSALK 421 TSYQRYSKVK ATNTWLFLSR CSGNETSLWD CKNWQWGGLS CDHYEEAKVT CSAHREPRLV 481 GGDIPCSGRV EVKHGDTWGT VCDSDPSLEA ASVLCRELQC GTVISILGGA HFGEGNGQIW

S41 AEEPQCBGQR SHLSLCSVAS RPDGTCSHSR DVGVVCERYT BIRLVNGQSP CERGRVELKIL 601 GNWGSLCNSH WDIEDAHVFC QQLKCGVALS IPGGAHPGKG SGQIWRHMFH CTGTBQHMGD 661 CPVTALGATL CSAGQVASVI CSGNQSQTLS PCNSTSLDPT RSTTSEESAV ACTASGQLRL 721 VNGGGRCAGR IEVYHBGSWG TICDDSWDLS DAHVVCRQLG CGVAINATGS AHFGRGTGPI 781 WLDEVNCNGK BSHINQCRSE GWGQHNCRHK EDAGVICSEP MSLRLIDETS RDICAGRLEV 841 FYNGAWGEVG KSNMSATTVE VVCRQLGCAD KGSINPASSD KPMSRHMWVD NVQCPKGEDT

1081 GENSVHQIQY REMNSSLKAC DLDVLTSSEY PNESDDFNDA GLISVSKSLP ISG A

Example 11. Various cell lines are rendered permissive to North American

PRRSYV infection following transient transfection with pCMV-susCD163v1

Porcine Kidney (PK032495), Norden Labs Swine Testicular (NLST-1), Norden Labs

Dog Kidney (NLDK-1) were obtained from Pfizer Inc. and were grown at 37° C and 5% CO, in growth media consisting of Dulbecco’s Modified Eagle Medium (DMEM,

Invitrogen catalog number 11965) supplemented with 5% fetal bovine serum (FBS), 1mM sodium pyruvate, 2mM L-glutamine and antibiotics. Cell lines Baby Hamster

Kidney (BHK21), Norden Labs Feline Kidney (NLFK-1), and Rabbit Lung (RL) were obtained from Pfizer Inc. and were grown at 37° C and 5% CO, in growth media consisting of Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen catalog number 11965) supplemented with 10% fetal bovine serum (FBS), ImM sodium pyruvate, 2mM L-glutamine and antibiotics. Vero cells were obtained from Pfizer

Inc. and were grown at 37° C and 5% CO; in growth media consisting of Minimum

Essential Medium Alpha (MEM, Pfizer Inc. formulation) supplemented with 10% fetal bovine serum (FBS), 2mM L-glutamine and Gentamicin at 20 micrograms per mL. Cell culture wells (35 mm) containing approximately 1x10° cells were transfected with 2 micrograms per well of plasmid pPCMV-susCD163v1, in DMEM without FBS or antibiotics, using Lipofectamine 2000 (Invitrogen catalog number 11668-027) according to the manufacturer’s instructions. Cell line RL was transfected with 1.0 micrograms per well of plasmid pCMV-susCD163vl. A member of the PAM cell cDNA library without an insert, designated pPAMB (essentially and empty pSport plasmid vector), was used as a negative control plasmid. At 24 hours post transfection wells were aspirated and washed twice with DMEM/5% FBS followed by infection with North American PRRSYV isolate P129. Virus was allowed to adsorb in 0.5 ml growth media for a minimum of two hours, after which additional media was added to a final volume of 2.0ml and incubated overnight. The virus was then removed, wells washed twice with growth media, and fresh growth media added (2.0 ml per well). A time zero sample of culture fluid was immediately taken in order to determine the background level of infectious virus from the inoculum. Ata minimum of 48 hours post infection cultures were screened for permissivity by removing culture fluids in order to assay viable virus, and permissive cells in the monolayer were detected by fluorescent antibody assay (FA). The FA was completed by fixing the monolayer with 80% acetone and stained with FITC-conjugated monoclonal antibody SDOW17 (Rural Technologies Inc), which is specific for the

PRRSV nucleocapsid protein. Viable virus was titrated by inoculating dilutions of culture fluids onto MARC-145 cells. Table 5 shows the results of virus infection by

FA and the presence of progeny virus for each cell line tested.

Failure to detect progeny virus from some cell lines may be the result of low virus titer in the cell culture fluids, below the assay’s limit of detection. Permissivity of Vero cells to PRRSV infection was augmented by the expression of susCD163v1.

Compared to the time zero measurement of background virus, there was nearly a two- log increase in virus titers in Vero cells transfected with pPCMV-susCD163v1, whereas there was less than a one-log in titer increase in cells transfected with negative control plasmid pPAMB. All cell lines except NLDK-1 were positive by FA for permissivity to North American PRRSV isolate P129 infection after transfection with pPCMV- susCD163vl.

Table §

Screening of various cell lines for permissivity to NA PRRSV isolate P129 following transient transfection with pCMV-susCD163v1 or pPAMB

Transfected | Fluorescent Antibody assa cell line pCMV. pPAMB | pCMV-susCD163vl | pPAMB susCD163v1

BHK21 | 4 | aT

PKO32495 | + | 0-4

NKFK1 [| +

NIST [0 + 0 [or ul INE IE I CH

RL + TT +++ = Highly positive ++ = Moderately positive + = Slightly positive - = Not detectible

NT = Not tested

Example 12. BHK21 cells are rendered permissive to European PRRSV infection following transient transfection with pCMV-susCD163v1

Cell line Baby Hamster Kidney (BHK21) was obtained from Pfizer Inc. and grown at 37° C and 5% CO, in growth media consisting of Dulbecco’s Modified Eagle Medium

(DMEM, Invitrogen catalog number 11965) supplemented with 10% fetal bovine serum (FBS), 1mM sodium pyruvate, 2mM L-glutamine and antibiotics. Cell culture wells (35 mm) containing approximately 1x10° cells were transfected with 2 micrograms per well of plasmid pPCMV-susCD163v1, in DMEM without FBS or antibiotics, using Lipofectamine 2000 (Invitrogen catalog number 11668-027) according to the manufacturer’s instructions. At 24 hours post transfection wells were aspirated and washed twice with DMEM/5% FBS followed by infection with

European PRRSV isolate 96V198. Virus was allowed to adsorb for a minimum of 2 hours. The virus was then removed, wells washed twice with growth media, and fresh growth media added (2.0 ml per well). A time zero sample of culture fluid was immediately taken in order to determine the background level of infectious virus from the inoculum. At a minimum of 48 hours post infection cultures were screened for permissivity by removing culture fluids in order to assay viable virus, and permissive cells in the monolayer were detected by fluorescent antibody assay (FA). The FA was completed by fixing the monolayer with 80% acetone and stained with FITC- conjugated monoclonal antibody SDOW17 (Rural Technologies Inc), which is specific for the PRRSV nucleocapsid protein. Viable virus was titrated by inoculating dilutions of culture fluids onto MARC-145 cells. As a result of the transient transfection of BKH21 with pCMV-susCD163v1, cells were rendered permissive to

European PRRSYV isolate 96V198 infection and yielded progeny virus.

Example 13: CD163 genes from multiple animal species render BHK21 cells permissive to PRRS virus infection

BHK21 cells grown in DMEM (Invitrogen catalog number 11965) supplemented with 10% fetal bovine serum, 1mM sodium pyruvate, and antibiotics, were used in transient transfection experiments. Before transfection cells were washed once with

OptiMEM (Invitrogen) without serum or other additives. Lipofectamine 2000 (Invitrogen) was used in all transfection experiments according to the protocol provided by the manufacturer. The transfection mixture consisted of 10 microliters of

Lipofectamine 2000 and 2-3 micrograms of DNA per 35 mm well. After overnight incubation, transfection medium was removed and cells were infected with PRRSV isolate P129. Infection was allowed to progress for 24-48 hours, when cells were fixed with 80% acetone and stained with monoclonal antibody SDOW 17 conjugated with FITC (Rural Technology Inc., Brookings, SD). Staining of the nucleocapsid protein was visualized under a fluorescence microscope.

Table 6. Transient transfection of BHK21 cells with various CD163 genes renders them permissive to PRRS virus infection

Table 6

Plasmid backbone CD163 gene PRRSYV infection (FA)

Swine CDIG3vI pRSV-Script Swine CD163v1 pcDNA3.1D Swine CD163v2 ocDNA3.1D Human CD163v2 pcDNA3.1D Mouse CD163v3 pcDNA3.1D African green monkey H+ (MARC-145 cell)

CD163v2 pcDNA3.1D Vero cells CD163v7 rcDNA3.1D DHS82 cell CD163v2 +++ = Highly positive ++ = Moderately positive + = Slightly positive

Example 14. Generation of PRRSV-permissive BHK21 stable cell lines nsing pCMV-susCD163v1

BHK-21 cells were grown in Dulbecco's Modified Eagle Media (DMEM) supplemented with 10% fetal bovine serum, 1mM sodium pyruvate, and antibiotics.

For transfection, cells were seeded at approximately 90% confluency in 6 well plates and incubated over night at 37°C in 5% CO». Cells were transfected with pCMV- susCD163v1 DNA using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. One day after transfection the cells were trypsinized and re-seeded in 96 well plates in a dilution series. To select for stable transfectants, the media was supplemented with 1 mg/ml Geneticin (G418 sulfate, Invitrogen catalog number 10131-027) from this point forward. Medium was changed every 3-5 days.

Plates were cultured until those wells with colonies derived from single cells reached confluency, at which point the plates were trypsinized and seeded into duplicate 96 well plates. One of the duplicate 96 well plates was infected with PRRSYV isolate

P129 and clones permissive to infection were identified by staining with FITC conjugated monoclonal antibody SDOW17. Positive clones were then expanded from the second duplicate plate. To ensure homogeneity the positive cultures were single- cell cloned by limiting dilution. At each cloning the subclones that displayed robust growth and high PRRSV permissivity were chosen for expansion. Three clones designated BHK/CMV/v1 #3, BHK/CMV/v1 #5, and BHK/CMV/v1 #12 (Figure 6) were selected. These cell lines have maintained the permissive phenotype through 20 passages.

Example 15. Generation of PRRSV-permissive BHK21 stable cell lines using pRSV-susCD163vl

BHK-21 cells were cultured as described in Example 14. BHK-21 cells were transfected with pRSVsusCD163v1 using Lipofectamine 2000 as described in

Example 14. Cloning of transfected cells and screening for permissive clones was performed essentially as described in Example 14. From the original cloning 3 single cell clones were identified as permissive and were subsequently recloned two more times to ensure homogeneity and to attempt to isolate subclones of higher permissivity (see Figure 7). The resulting cell lines were named BHK/RSV/v1, #2, #3, and #4. All of these clones have maintained the permissive phenotype through the highest passage tested (passage 11 for clone #2, passage 7 for clone #3, and passage 5 for clone #4).

Example 16. Generation of PRRSV-permissive feline kidney stable cell lines using pCMV-susCD163v1

Parental Norden Labs Feline Kidney (NLFK) cells were grown at 37 degrees C and 5% CO, in Dulbecco’s Modified Eagle Medium (Invitrogen catalog number 11965-092) supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate, and antibiotics. Several 35 mm wells containing approximately 2x10° cells each were transfected with 4 micrograms per well of pPCMV-susCD163v1, in OptiMEM, using

Lipofectamine 2000 (Invitrogen catalog number 11668-027) according to the manufacturer’s instructions. After overnight incubation, cells were removed from the substrate using Accutase (Innovative Cell Technologies, catalog number AT104) diluted in medium, and seeded into three 96-well plates at three densities (approximately 2 x 107%, 2 x 10%, and 2 x 10 cells per well). The cells were allowed to settle overnight at 37 degrees C before beginning selection of stable transformants.

The next morning medium was replaced with 100 microliters / well fresh medium containing S00 micrograms / ml Geneticin (G418 sulfate, Invitrogen catalog number 10131-027) to select for cells expressing the neomycin resistance gene. Medium was changed every 2 or 3 days to maintain Geneticin potency. After 19 days of selection, the 96-well plate with the lowest initial cell density (approximately 200 cells / well) yielded 70 empty wells and 26 wells with one or more colonies of G418-resistant cells

(calculated number of resistant cells / well is 0.3, using the Poisson distribution).

These 26 wells were split into duplicate wells and allowed to settle overnight. One set of wells was infected with PRRSYV isolate P129, incubated for 24 hours, then fixed with 80% acetone and stained with FITC-conjugated monoclonal antibody SDOW17 (Rural Technologies Inc), which is specific for PRRSV nucleocapsid. Of the 26 clones, 8 contained some cells that were infected by PRRSV. One of these, designated “NLFK-CMV-susCD163v1-G4”, was clearly more permissive than the others with nearly 100% of the cells staining positive for viral antigen.

By cell passage number 5, there was some evidence of phenotypic heterogeneity in the NLFK-CMV-susCD163v1-G4 cell line. Therefore, the cells were single-cell cloned by limiting dilution in G418-containing medium, starting with frozen stocks of NLFK-CMV-susCD163v1-G4 passage 4. Twelve such clones (“A” — “L”) were expanded for study. Of these, clones NLFK-CMV-susCD163v1-G4F and

NLFK-CMV-susCD163v1-G4L were notable for their ability to form discrete plaques (localized areas of CPE) when infected with PRRSYV isolate P129 (see Figure 8).

Example 17. Generation of PRRSV-permissive feline kidney stable cell lines using pRSV-susCD163v1

Norden Labs Feline Kidney (NLFK) cells were grown at 37° C and 5% CO, in

Minimal Essential Medium Alpha Medium (Invitrogen catalog number 12571-071) supplemented with 10% fetal bovine serum and antibiotics. NLFK cells were seeded in 6 well plates at approximately 90% confluency and allowed to attach overnight.

The cells were then transfected with plasmid pRSV-susCD163v1 using Lipofectamine 2000 (Invitrogen) following the manufacturer’s instructions. After 24 hours the cells were cloned as described in Example 14. Screening for PRRSV permissive cell clones was performed as described in Example 14. Four clones were selected from the screening and were single cell cloned by limiting dilution two more times. Four clones named FK/RSV/v1 #1, FK/RSV/vl #2, FK/RSV/v] #3, and FK/RSV/v1 #4 were selected. These cell lines have maintained the PRRSV permissive phenotype through at least 8 passages (see Figure 9).

Example 18. Generation of PRRSV-permissive porcine kidney stable cell lines using pCMV-susCD163v1

Parental Porcine Kidney (PK032495) cells were obtained from Pfizer Inc. and were grown at 37 degrees C and 5% CO, in growth media consisting of Dulbecco's

Modified Eagle Medium (DMEM, Invitrogen catalog number 11965) supplemented with 5% fetal bovine serum (FBS), ImM sodium pyruvate, 2mM L-glutamine and antibiotics. Tissue culture wells (35 mm) containing approximately 1x10° cells each were transfected with 2 micrograms per well of plasmid pCMV-susCD163v1, in

DMEM without FBS or antibiotics, using Lipofectamine 2000 (Invitrogen catalog number 11668-027) according to the manufacturer’s instructions. After overnight incubation, cells were washed with PBS and removed from the substrate using

Accutase (Innovative Cells Technologies, catalog number AT104) and diluted in growth medium containing Geneticin (G418 sulfate, Invitrogen catalog number 10131-027) at 1.0 milligram per ml and seeded into 96-well plates at various densities to ensure recovery of single cell clones after Geneticin selection. Throughout

Geneticin selection, media was changed approximately every 3 to 5 days. After selection, wells containing single cell clones were expanded into duplicate 96-well plates and allowed to incubate until 100% confluency was achieved. One set of wells was screened for PRRSV-permissivity by infecting with PRRSYV isolate P129 for a minimum of 48 hours. Eleven clones were found to be permissive for PRRSV. One of these, designated “PK-CMV-susCD163v1-A10”, clearly retained the permissive phenotype after numerous passages (see Figure 10).

Example 19. Generation of PRRSV-permissive BHK21 stable cell lines using pCMYVScript-susCD163v2

Parental Baby Hamster Kidney (BHK21) cells were obtained from Pfizer Inc. and were grown at 37 degrees C and 5% CO; in growth media consisting of

Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen catalog number 11965) supplemented with 10% fetal bovine serum (FBS), 1mM sodium pyruvate, 2mM L- glutamine and antibiotics. Tissue culture wells (35 mm) containing approximately 1x10° cells each were transfected with 2 micrograms per well of pCMVScript- susCD163v2, in DMEM without FBS or antibiotics, using Lipofectamine 2000 (Invitrogen catalog number 11668-027) according to the manufacturer’s instructions.

After overnight incubation, cells were washed with PBS and removed from the substrate using Accutase (Innovative Cells Technologies, catalog number AT104) and diluted in growth medium containing Geneticin (G418 sulfate, Invitrogen catalog number 10131-027) at 1.0 milligram per ml and seeded into 96-well plates at various densities to ensure recovery of single cell clones after Geneticin selection.

Throughout Geneticin selection, media was changed approximately every 3 to 5 days.

After selection, wells containing single cell clones were expanded into duplicate 96- well plates and incubated until 100% confluency was achieved. One set of wells was screened for permissivity by infecting with PRRSYV isolate P129 and incubating for a minimum of 48 hours. Three clones were found to be PRRSV-permissive, and one of these, designated “BHK-CMVScript-susCD163v2-A9”, was chosen for further study (see Figure 11).

Example 20. Generation of PRRSV-permissive BHK-21 stable cell lines using

PRSV-susCD163v2

BHK-21 cells were cultured as described in Example 14. BHK-21 cells were transfected with the ligated pRSV-susCD163v2 DNA construct described in Example 5 using Lipofectamine 2000 (Invitrogen) following manufacture’s instructions.

Subsequent cloning and selection of PRRSV permissive cell lines was performed as described in Example 14. Of 336 single cell clones screened, 129 were positive.

Several of these cell clones have been passed up to 7 times and they have maintained the PRRSV permissive phenotype (see Figure 12). These cell lines have been named

BHK/RSV/v2, followed by a numerical clone number.

Example 21. Generation of PRRSV-permissive porcine kidney stable cell lines using pCMVScript-susCD163v2

Parental Porcine Kidney (PK032495) cells were obtained from Pfizer Inc. and were grown at 37 degrees C and 5% CO, in growth media consisting of Dulbecco’s

Modified Eagle Medium (DMEM, Invitrogen catalog number 11965) supplemented with 5% fetal bovine serum (FBS), 1mM sodium pyruvate, 2mM L-glutamine and antibiotics. Tissue culture wells (35 mm) containing approximately 1x10° cells each were transfected with 2 micrograms per well of pCMV Script-susCD163v2 in DMEM without FBS or antibiotics, using Lipofectamine 2000 (Invitrogen catalog number 11668-027) according to the manufacturer’s instructions. After overnight incubation, cells were washed with PBS and removed from the substrate using Accutase (Innovative Cells Technologies, catalog number AT104) and diluted in growth medium containing Geneticin (G418 sulfate, Invitrogen catalog number 10131-027) at 1.0 milligram per ml and seeded into 96-well plates at various densitics to ensure recovery of single cell clones after Geneticin selection. Throughout Geneticin selection, media was changed approximately every 3 to 5 days. After selection, wells containing single cell clones were expanded into duplicate 96-well plates and incubated until 100% confluency was achieved. One set of wells was screened for permissivity by infecting with PRRSYV isolate P129 and incubating for a minimum 48 hours. One clone designated “PK-CMVScript-susCD163v2-D1” showed the PRRSV- permissive phenotype.

Example 22. Generation of PRRSV-permissive BHK21 stable cell lines using pcDNA3.1D-humCD163v2

Parental Baby Hamster Kidney (BHK21) cells were obtained from Pfizer Inc. and were grown at 37 degrees C and 5% CO, in growth media consisting of Dulbecco’s

Modified Eagle Medium (DMEM, Invitrogen catalog number 11965) supplemented with 10% fetal bovine serum (FBS), 1mM sodium pyruvate, 2mM L-glutamine and antibiotics. Tissue culture wells (35 mm) containing approximately 1x10° cells each were transfected with 2 micrograms per well of pcDNA3.1D-humCD163v2, in

Geneticin selection, media was changed approximately every 3 to 5 days. After selection, wells containing single cell clones were expanded into duplicate 96-well plates and incubated until 100% confluency was achieved. One set of wells was screened for permissivity by infecting with PRRSV isolate P129, incubated for a minimum 48 hours. Seven candidate clones were found to be PRRSV-permissive.

There was some evidence of phenotypic heterogeneity in each of the seven candidate clones, likely because they were not clonal. Therefore, the candidate clones were single-cell cloned by limiting dilution in G418 containing medium. One single cell clone with clear PRRS-permissivity was obtained and designated BHK-cDNA3.1D- humCD163v2-H9.

Example 23. Generation of PRRSV-permissive feline kidney stable cell lines using pcDNA3.1D-humCD163v2

Parental Norden Labs Feline Kidney (NLFK) cells were grown at 37 degrees C and 5% CO, in growth media consisting of Dulbecco’s Modified Eagle Medium (DMEM,

Invitrogen catalog number 11965) supplemented with 10% fetal bovine serum (FBS), mM sodium pyruvate, 2mM L-glutamine and antibiotics. Tissue culture wells (35 mm) containing approximately 1x10° cells each were transfected with 2 micrograms per well of pcDNA3.1D-humCD163v2 in DMEM without FBS or antibiotics, using

Lipofectamine 2000 (Invitrogen catalog number 11668-027) according to the manufacturer's instructions. After overnight incubation, cells were washed with PBS, removed from the substrate using Accutase (Innovative Cells Technologies, catalog number AT104), diluted in growth medium containing Geneticin (G418 sulfate,

Invitrogen catalog number 10131-027) at 500 micrograms per ml, and seeded into 96- well plates at various densities to ensure recovery of single cell clones after Geneticin selection. Throughout Geneticin selection, media was changed approximately every 3 to 5 days. After selection, wells containing single cell clones were expanded into duplicate 96-well plates and incubated until 100% confluency was achieved. One set of wells was screened for PRRSV-permissivity by infecting with PRRSV isolate P129 for a minimum 48 hours. Five clones were found to be permissive. One of these, designated “FK-cDNA3.1D-humCD163v2-A6”, clearly displayed the permissive phenotype (see Figure 13). NLFK parent cells and one subclone of FK-cDNA3.1D-humCD163v2-A6 were examined for the CD163 expression. Cells were fixed in 80% acetone and reacted with Goat anti-human CD163 (R&D System at 1:200) for one hour following by washing with PBS. For visualization, donkey anti-Goat IgG conjugated with FITC (Biodesign Inc at 1:100) were used. No specific fluorescence was detected in the NLFK parent cells as shown in Figure 21A. The majority of the FK.A6.A2 subclone showed good fluorescent staining indicating the presence of CD163 (Figure 21B).

Example 24. Generation of PRRSV-permissive porcine kidney stable cell lines using pcDNA3.1D-humCD163v2

Modified Eagle Medium (DMEM, Invitrogen catalog number 11965) supplemented with 5% fetal bovine serum (FBS), ImM sodium pyruvate, 2mM L-glutamine and antibiotics. Tissue culture wells (35 mm) containing approximately 1x10° cells each were transfected with 2 micrograms per well of pcDNA3.1D-humCD163v2, in

DMEM without FBS or antibiotics, using Lipofectamine 2000 (Invitrogen catalog number 11668-027) according to the manufacturer’s instructions. After overnight incubation, cells were washed with PBS, removed from the substrate using Accutase (Innovative Cells Technologies, catalog number AT104), diluted in growth medium containing Geneticin (G418 sulfate, Invitrogen catalog number 10131-027) at 1.0 milligram per ml, and seeded into 96-well plates at various densities to ensure recovery of single cell clones after Geneticin selection. Throughout Geneticin selection, media was changed approximately every 3 to 5 days. After selection, wells containing single cell clones were expanded into duplicate 96-well plates and incubated until 100% confluency was achieved. One set of wells was screened for

PRRSV-permissivity by infecting with PRRSV isolate P129 for a minimum 48 hours.

Two clones were found to be permissive. One of these, designated “PK-cDNA3.1D- humCD163v2-B11” clearly showed the PRRSV-permissive phenotype.

Example 25 Generation of PRRSV-permissive feline kidney stable cell line using ligated pRSV-Script MARC CD163v2

A non-cloning based procedure to generate microgram quantities of linear DNA suitable for use in generating stable cell lines expressing CD163 from an RSV promoter was developed (Figure 4). A similar process was adapted to place simian

CD163v2 from MARC-145 cells behind the RSV promoter. The procedure involves the isolation and ligation of two pieces of DNA, one containing the neomycin gene and RSV promoter cassette derived from pRSV-script, and the other containing the

MARC CD163v2 coding sequence from pCDNA3.1D MARC CD163v2. Vector plasmid pRSV-Script was linearized with Hind Ill and Kpn I. Plasmid was first digested with Kpn I and was bunted with the Kienow fragment of E. coli DNA polymerase. This plasmid was then digested with Hind Ill immediately downstream of the RSV promoter. The pCDNA3.1D MARC CD163v2 clone was digested in the vector sequence downstream of the CD163 insert with EcoRV, and Hind II upstream of CD163. The CD163 coding sequence was liberated from the vector. For each plasmid digestion the appropriate fragments were purified from agarose gels. A large- scale ligation reaction was performed as follows. Approximately 20 pig of each DNA fragment was incubated in a volume of 600 pL. with 15 units of T4 DNA ligase. The reaction was incubated at room temperature for 1 hour. Following ligation, a linear piece of DNA containing all of the appropriate elements was purified by agarose gel electrophoresis. Restriction enzyme digestion analysis was performed to confirm the authenticity of each ligated fragment. Ligation of the two DNA fragments via the cohesive Hind HI termini resulted in the placement of the 5° sequences of the MARC

CDI163 gene downstream of the RSV promoter, allowing for directed expression of

CD163 in mammalian cells. Once isolated, the purified DNA was used to transfect selected mammalian cell lines.

Norden Labs Feline Kidney (NLFK) cells were grown at 37° C and 5% CO» in

DMEM supplemented with 5% fetal bovine serum and antibiotics. NLFK cells were seeded in 6 well plates at approximately 90% confluency and allowed to attach overnight. The cells were then transfected with ligated plasmid pRSV-MARC

CD163v2 using Lipofectamine 2000 following the manufacturer’s instructions. After 24 hours the cells were cloned as described in Example 12. Screening for PRRSV permissive cell clones was performed as described in Example 12. One clone was positive for PRRSV infection and designated NLFK-MARC CD163 D4. This D4 clone has maintained the PRRSV permissive phenotype through 9 passages.

Example 26. Growth kinetics of PRRSYV isolate NVSL 94-3 in recombinant

BHK-21 and NLFK cells stably expressing susCD163v1 from the CMV promoter.

The amounts of progeny virus produced by PRRSV-infected BHK-21 or

NLFK cells stably engineered to express susCD163v1 were quantitated. Four cell lines expressing susCD163v1, BHK/CMV/susv1 #3, BHK/CMV/susv1 #5,

BHK/CMV/susv1 #12, and FK/CMV/susvl G4 were seeded at sub confluency in 6 well plates and, after overnight incubation, were infected with the NVSL 94-3 isolate of PRRSV. MARC-145 cells were included in the experiment for comparison. The cells were infected with virus at an m.o.i. of approximately 0.1. Virus was adsorbed for 60-90 minutes and was removed. Cells were washed three times with PBS to remove residual virus. One-milliliter aliquots were harvested from the cultures at 12- hour intervals starting immediately after infection and continued through 96 hrs.

Fresh culture media was added to the cells at various time points to maintain a culture volume sufficient to prevent the cell monolayer from drying out. Culture supernatants were stored at -80° until all samples were collected. The amount of PRRSV present in the culture supernatants was determined by plaque assay on MARC-145 cells. Figure

14 shows that all CD163 expressing recombinant cell lines tested were able to produce progeny PRRSV.

Example 27. Blocking PRRSY infection with anti-CD163 antibody: Transiently transfected cells.

BHK-21 cells, seeded in 24 well plates, were transiently transfected with the plasmid pCDNA3.1D-MARC-CD163v2 described in example 8, using Lipofectamine 2000 as described in example 14. After overnight incubation to allow expression of

CD163, a titration of goat polyclonal antibody specific for human CD163 (R&D

Systems, cat # AF1607) in PBS was added to the cells in a volume of 100pl. Asa control, equivalent amounts of normal goat IgG (R&D Systems, cat # AB-108-C) were used. Following a one-hour incubation at 37°C, the monolayers were infected with approximately 1x10’ pfu of a recombinant P129 strain of PRRSV that expresses

GFP. The cell monolayers, with anti-CD163 antibody and PRRSV, were incubated at 37°C for one hour at which time the virus inoculum/antibody mixture was aspirated, the cell monolayer washed once with PBS, and 1ml of growth medium added to the wells. The cells were incubated for 24 hours at 37°C to allow PRRSV directed GFP expression. For analysis, the cells were trypsinized, resuspended in 500 of PBS and analyzed by flow cytometry to innumerate the PRRSV infected cells via GFP expression. For flow cytometry, uninfected BHK-21 cells were used to set the baseline for fluorescence detection, and approximately 100,000 cells were analyzed from each subsequent sample. The results of this analysis, shown in Figure 15, show that the CD163 specific antibody was able to significantly reduce the number of infected cells when compared to cells incubated with normal goat IgG.

Example 28: Blocking PRRSY infection by anti CD163 antibody: Stably transfected cells.

The NLFK cells that stably express human CD163 (FK-cDNA3.1D- humCD163v2-A6), described in Example 23, were seeded into 24 wells plates. After allowing the cells to attach overnight, a titration of goat polyclonal antibody specific for human CD163 (R&D Systems, cat # AF1607) in PBS was added to the cells in a volume of 100ul. As a control, equivalent amounts of normal goat IgG (R&D

Systems, cat # AB-108-C) were used. Following a one-hour incubation at 37°C, the monolayers were infected with approximately 1x10’ pfu of a recombinant P129 strain of PRRSV that expresses GFP. The cell monolayers, with anti-CD163 antibody and

PRRSYV, were incubated at 37°C for one hour at which time the virus inoculum/antibody mixture was aspirated, the cell monolayer washed once with PBS, and 1mlof growth medium added to the wells. The cells were incubated for 24 hours at 37°C to allow PRRSYV directed GFP expression. For analysis, the cells were trypsinized, resuspended in 500pl of PBS, and analyzed by flow cytometry to innumerate the PRRSV infected cells via GFP expression. Approximately 100,000 cells were analyzed from each sample. The results of this analysis, shown in Figure 16, show that the CD163 specific antibody was able to significantly reduce the number of infected cells when compared to cells incubated with normal goat IgG.

Example 29. Generation of PRRSV-permissive porcine kidney stable cell lines using pRSV-susCD163v2,

Porcine kidney cells (PK032495) were cultured as described in Example 21. For transfection, cells were seeded in a 24 well plate at 80% confluency and allowed to recover overnight. Transfection of ligated pRSV-susCD163v2 DNA described in

Example 5 was performed using Lipofectamine 2000 (Invitrogen) following the manufacturer's instructions. Subsequent cloning and selection of PRRSV permissive cells was performed essentially as described in Example 14. The initial cloning by limiting dilution failed to yield single cell derived clones, so 5 wells with PRRSV permissive cells were recloned by limiting dilution to yield clonal cell lines. 10 clones were selected for further study and one of these clones, PK-RSVScript-susCD163v2 #9 showed the ability to support foci growth of PRRSV early after infection (see

Figure 18).

Example 30. Generation of PRRSV-permissive feline kidney stable cell lines using pRSV-susCD163v2. NLFK feline kidney cells were cultured as described in Example17. For transfection, cells were seeded at approximately 80% of maximal density in 24 well plates. After overnight incubation the monolayers were transfected with ligation derived RSV/susCD163v2 (see example 5) using Lipofectamine following the manufacturer’s instructions. Cloning of the transfected cells and selection of PRRSV permissive cell clones was performed essentially as described in Example 14. Of the 67 cell clones tested for PRRSV permissivity, 20 were found to be positive. An example of the staining observed is shown in Figure 19.

Example 31. Passage of PRRSYV isolate P201 in PK-RSVScript-susCD163v2 cells

Amplification of a PRRSV clinical isolate was performed as follows. Peripheral alveolar macrophage (PAM) cells were seeded at 5.4E6 cells per 10cm2 in a 6 well dish using OptiMEM media supplemented with 2% FBS. After 6 hours the media was aspirated and a 2 ml aliquot of serum harvested from a PRRSV infected pig was added to the cells. Following a 90 minute adsorption, the serum inoculum was removed and replaced with OptiMEM. At approximately 40 post infection the supernatant was harvested and clarified with a 10 minute centrifugation. The supernatant was directly used to infect PK-RSVScript-susCD163v2 clone #9 cells using a 6 hour adsorption. After removal of the inoculum the cells were refed with D-,

MEM. The P201 virus was serially passaged on the PK-RSVScript-susCD163v2 #9 cell line using alternating infected cell and cell free supernatant passes. We observed that for efficient spread of the virus, the cells should be seeded at 50-70 % confluency the day before infection, using flasks of cells that were kept at sub-confluency. To follow the progression of infection, each passage was replicated in multiple wells of identically infected cells and at each day one of the wells was acetone fixed and stained with the FITC labeled monoclonal antibody SDOW17. If the percentage of infected cells was not greater than 50% and significant progression of foci development over the prior days observations were not seen, the cells in an equivalent well were trypsinized and passed to multiple fresh wells. These infected cell passages were typically at a 1:4 split and sometimes included the addition of an equivalent number of cells from an uninfected culture. Alternatively, if the SDOW17 staining revealed that the infected cell foci had spread sufficiently to account for greater than 50% of the total cells, cell free supernatant was harvested and used to infect multiple wells of freshly seeded cells (Figure20). After 11 passages the intervening cell passages were not necessary as the virus was able to grow to sufficient titer to allow consecutive cell free supernatant passaging of the virus.

Example 32. Screening various CD163 cell lines for permissivity to various

European and North American PRRSY isolates

Various CD163 transgenic cell lines were assessed for permissivity to low passage

European and North American PRRSYV isolates (see Table 7). Transgenic CD163 cell lines as described in earlier examples included NLFK-MARC CD163 D4, PK-

RSVScript-susCD163v2clone #9 and PK-CMV-susCD163v1-A10. Each CD163 cell line along with cell lines MARC-145, parental feline kidney, parental porcine kidney cell lines (serving as controls) were planted onto 96-well tissue culture plates. Growth media was removed from monolayers and inoculated with 0.1 mL per well of each

PRRSYV isolate. At day 3 post infection, the plates were fixed with 80% acetone and stained with FITC-conjugated monoclonal antibody SDOW17 (Rural Technologies

Inc.) which is specific for the nucleocapsid. Results of the fluorescent antibody (FA) assay are in Table 7. Table 7

FA results of screening of various CD163 cell lines for permissivity to European and North American

PRRSYV isolates

CD163 Cel Line EOwvize | iz | Pao | u1si | 943 | INDs

FEE EE EE a a I I [PE EE EE EE

MARC145 | wr waar [4 Taree] 4s

Porcine Kidney (parental) | - |. | - | . [ - [ - “Feline Kidney (parent) | - | | - [ - | - [ - * All PRRSV isolates are North American except EU98V226 is a European isolate.

Example 33. Phorbol 12-myristate 13-acetate (PMA) induction of CD163 renders human U937 cells permissive to PRRSYV infection.

Human U937 cells obtained from ATCC (CRL-1593.2) were propagated in

RPMI medium containing serum and additives according to ATCC specifications.

These cells are known to express CD163 when activated by PMA treatment (Gronlund et al., 2000). U937 cells were seeded in duplicate in wells of a 6-well plate. One set of wells was treated with 100 ng/ml of PMA and the other set was left untreated. Three days after PMA stimulation, one well from each set was infected with the P129 isolate of PRRSV. The other well from each set was fixed and stained for expression of CD163 in an indirect immunofluorescent antibody assay using goat- anti human CD163 (R&D System) and donkey anti-goat IgG conjugated with FITC (BioDesign International).

Untreated U937 cells continued propagation to high density 3 days after initial planting. PMA-treated U937 cells stopped propagating, became enlarged, and attached to the surface of the culture wells. A small fraction of untreated U937 were positive for CD163 staining, whereas almost all PMA-treated U937 were positive for

CD163 staining. In untreated U937 no PRRSYV infected cells were observed.

However, hundreds of PMA treated U937 cells became infected by PRRSV. This demonstrates that non-permissive cells can be rendered permissive for PRRSV infection following chemical induction of CD 163 expression.

Additional features and variations of the invention will be apparent to those skilled in the art from the entirety of this application, including the detailed description, and all such features are intended as aspects of the invention. Likewise, features of the invention described herein can be re-combined into additional embodiments that also are intended as aspects of the invention, irrespective of whether the combination of features is specifically mentioned above as an aspect or embodiment of the invention. Also, only such limitations that are described herein as critical to the invention should be viewed as such; variations of the invention lacking limitations that have not been described herein as critical are intended as aspects of the invention.

It will be clear that the invention may be practiced otherwise than as particularly described in the foregoing description and examples.

Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, are within the scope of the invention.

The entire disclosure of all publications cited herein are hereby incorporated by reference to the extent they are not inconsistent to the disclosure herein.

Claims

What is claimed is:

1. A method of facilitating infection of a vertebrate cell by PRRSV that comprises the step of (a) directing increased expression of a CD163 polypeptide by said vertebrate cell.

2. The method of claim 1 that further comprises infecting said cell with PRRSV.

3. The method of claim 1 that further comprises producing a culture of PRRSV.

4. The method of claim 1 in which said cell is mammalian.

5. The method of claim 1 wherein said CD163 polypeptide comprises a transmembrane domain.

6. The method of claim 1 wherein said increased expression is accomplished by introduction of exogenous nucleic acid.

7. The method of claim 1 wheerin said increased expression is accomplished by chemical treatment.

8. The method of claim 1 wherein said cell was PRRSV non-permissive and is rendered PRRSV permissive by step (a).

9. The method of claim 2 in which the PRRSV is of the European genotype.

10. The method of claim 2 in which the PRRSV is of the North American genotype.

11. The method of any of claims 2-10 further comprising the step of producing a PRRSV vaccine.

12. The method of claims 11 wherein the vaccine is killed.

13. The method of claim 11 wherein the vaccine is live attenuated.

14. A method for measuring the propensity of a test cell line to allow infection by PRRSYV comprising:

a) providing a sample containing nucleic acids from the test cell line; b) determining the amount of polynucleotide encoding a CD163 polypeptide or its complement in said sample;

wherein an increased amount of polynucleotide encoding a CD163 polypeptide relative to a control sample derived from a control cell line known not to support the growth of said PRRSV indicates a propensity of the test cell line to support the replication of said PRRSV.

A method for measuring the propensity of a test cell line to allow infection by PRRSV comprising: (a) providing a sample containing polypeptides from the test cell line; (b) determining the amount of CD163 polypeptide in said sample;

15 wherein an increased amount of a CD163 polypeptide relative to a control sample derived from a control cell line known not to support the growth of said PRRSV indicates a propensity of the test cell line to support the replication of said PRRSV.