WO2016210097A1

WO2016210097A1 - Novel equine parvovirus and uses thereof

Info

Publication number: WO2016210097A1
Application number: PCT/US2016/038968
Authority: WO
Inventors: W. Ian Lipkin; Amit Kapoor; Bud C. Tennant; Thomas J. DIVERS; John M. CULLEN
Original assignee: The Trustees Of Columbia University In The City Of New York
Priority date: 2015-06-23
Filing date: 2016-06-23
Publication date: 2016-12-29

Abstract

The invention is directed to isolated equine parvovirus (EqPV), and isolated nucleic acids sequences and polypeptides thereof. The invention also relates to antibodies against antigens from EqPV. The invention also relates to iRNAs which target nucleic acid sequences of EqPV. The invention is related to methods for detecting the presence of EqPV in an animal. The invention is also related to immunogenic compositions for inducing an immune response against EqPV in an animal.

Description

NOVEL EQUINE PARVOVIRUS AND USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority of United States Provisional Application Serial No., 62/183,546 filed June 23, 2015, which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The invention is in the field of viruses and is related to a new virus found in horses, denoted equine parvovirus (EqPV). The invention includes isolated EqPV, and isolated nucleic acids sequences and polypeptides thereof. The invention also relates to primers and probes. The invention also relates to antibodies against antigens from EqPV. The invention is related to methods for detecting the presence of EqPV in an animal, i.e., a horse, or a serum or plasma product, using primers, probes, and antibodies. The invention also relates to iRNAs which target nucleic acid sequences of EqPV. The invention is also related to immunogenic compositions for inducing an immune response against EqPV in an animal for prevention and treatment of an EqPV infection.

BACKGROUND OF THE INVENTION

Theiler's disease is one of the most commonly described causes of acute hepatic necrosis in adult horses. Theiler's disease, which is limited almost entirely to adult horses, is also referred to as serum hepatitis because most published cases are associated with administration of a biologic of equine origin four to ten weeks prior to the onset of clinical illness. The onset of hepatic failure in Theiler's disease is acute and often rapidly progressive over two to seven days with death in most cases. In horses that survive, recovery is characteristically uncomplicated. The disease typically occurs sporadically and only a small number of horses receiving a biologic product of equine origin develop clinical signs of any liver disease.

In the original report by Theiler (Theiler 1919), one group of 1,154 horses was described that was vaccinated against African Horse Sickness using the simultaneous administration of infectious virus and convalescent equine antiserum. Of these horses, 210 died of acute liver failure. In the same study, the disease also was observed in four horses of 160 that never received inoculations, suggesting the disease could be both infectious and contagious.

Pathologic lesions in horses dying from Theiler's disease are limited primarily to the liver and the brain (Alzheimer type II cells). Characteristically the liver is smaller than normal, and histopathologic findings consistently include widespread centrilobular to midzonal hepatocellular necrosis with hemorrhage. In portal areas, inflammatory infiltrates of monocytes and lymphocytes and moderate bile duct proliferation are found. Liver enzymes in serum, including sorbitol dehydrogenase (SDH) and aspartate transaminase (AST), are increased several fold while gamma-glutamyl transaminase (GGT) is increased but not of the magnitude of the hepatocellular enzymes. Bilirubinemia, bilirubinuria and increased serum bile acids also are observed. In most cases, the association of Theiler's disease with parenteral injection of antiserum or plasma of equine origin suggests an infectious, blood- borne viral cause, and the history, onset, clinical signs, and histopathologic findings of Theiler's disease appear similar to hepatitis B virus infection of human beings.

The first cases reported in the United States occurred during the pandemic of western equine encephalomyelitis (WEE) that began in 1930 in California and spread rapidly throughout the western states. A recommended procedure for vaccination against WEE was the simultaneous administration of virulent WEE virus and equine antiserum against WEE. Two to three months following vaccination, a syndrome was observed that was clinically more severe than that associated with WEE infection and was characterized by jaundice and signs of encephalopathy. Ultimately, the syndrome was shown to be the result of severe hepatitis similar to that reported by Theiler (Marsh 1937a; Marsh 1937b; Cox et al. 1938).

Since these reports, hepatitis following the administration of equine plasma or serum has been described in horses from around the world and cases continue to be admitted to equine referral centers in the United States every year. Acute hepatitis has been reported following treatment with a variety of equine serum products including tetanus antitoxin, anthrax antitoxin, botulinum antitoxin, antiserum against Streptococcus equi, and pregnant mare's serum.

Although the overall incidence of Theiler's disease in adult horses receiving tetanus antitoxin is low, tetanus antitoxin has been the most common blood product of equine origin associated with the disease in the United States for the past 40 years. Thus, there is a need to screen equine serum and plasma products for infectious agents.

Reported herein is the identification of a novel equine virus found in the serum and liver of horses with Theiler' s Disease. Given the impact of the virus on equine health, as well as the similarity of Theiler's Disease with human hepatisis B virus infection, there is a need to detect the novel virus in both the animals and equine plasma and serum products. There is also a need for diagnostic tests, immunogenic compositions, and methods of preventing the infection as well as treating animals (e.g. horses) having the novel equine parvo viral infections. This invention addresses these needs.

SUMMARY OF THE INVENTION

The invention is related to a novel equine parvovirus (EqPV) associated with equine hepatitis. In particular aspects, invention is related to isolated EqPV nucleic acid sequences including cDNA sequences corresponding to sense or anti-sense EqPV RNA sequences and peptides thereof. The invention is also related to antibodies against antigens derived from EqPV. The invention is also related to iRNAs which target nucleic acid sequences of EqPV. The invention is related to methods for detecting the presence of EqPV in an animal (e.g. in horses), and in biologic, serum and plasma products derived from animals. The invention is also related to immunogenic compositions for inducing an immune response against EqPV in an animal (e.g. in horses).

In certain aspects, the invention relates to diagnostic tools and kits useful for screening a sample for equine parvovirus.

In certain aspects, the invention relates to an isolated or non-isolated nucleic acid sequence having the sequence of any of SEQ ID NOs: 1-22 and 25-28, a sequence complementary to the sequence of any of SEQ ID NOs: 1-22 and 25-28, variants and fragments thereof. In certain embodiments, the nucleic acid is a DNA sequence. In certain embodiments, the nucleic acid is an RNA sequence. In certain embodiments, the nucleic acid is a cDNA sequence. In certain embodiments, the nucleic acid is a synthetic nucleic acid.

In certain aspects, the invention relates to oligonucleotide probes for determining the presence or absence of EqPV in a biological sample or biologic, serum and plasma products.

In certain aspects, the invention relates to a primer set for determining the presence or absence of EqPV in a biological sample, or biologic, serum and plasma product wherein the primer set comprises at least one synthetic nucleic acid described herein.

In certain aspects, the invention relates to a method for determining the presence of equine parvovirus (EqPV) in a biological sample or in serum and plasma product, using the nucleic acids, primers and probes of the invention.

In other aspects, the invention is directed to expression constructs, for example but not limited to, plasmids and vectors which comprise the nucleic acid sequence of any of SEQ ID NOs: 1-22 and 25-28, complementary sequences thereof, fragments and/or variants thereof. Such expression constructs can be prepared by any suitable method known in the art. Such expression constructs are suitable for viral nucleic acid and/or protein expression and purification.

In certain aspects, the invention is directed to iRNA molecules which target nucleic acids from EqPV, for example but not limited to any of SEQ ID NOs: 1-3, fragments, and variants thereof, and silence a target gene.

In certain aspects, the invention relates to a method for reducing the levels of an EqPV protein in an animal, viral mRNA in an animal or viral titer in a cell of an animal, the method comprising administering to the animal an iRNA described herein.

In certain aspects, the invention relates to an isolated polypeptide encoded by the nucleic acid of SEQ ID NOs: 1-3, a sequence complementary to the sequence of any of SEQ ID NOs: 1-3, variants and fragments thereof.

In certain aspects, the invention relates to an isolated polypeptide having the sequence of any of SEQ ID NOs: 23-24, variants and fragments thereof.

In certain aspects, the invention relates to an isolated antibody that specifically binds to a polypeptide of the invention (e.g. a polypeptide of SEQ ID NOs: 23-24 or a polypeptide encoded by SEQ ID NOs: 1-3, fragments, variants or sequences complementary thereto).

In certain aspects, the invention relates to a method for determining whether or not a biological sample, or biologic, serum and plasma product contains EqPV, using the antibodies of the present invention.

In certain aspects, the invention relates to kits for determining whether a biological sample, or biologic, serum and plasma product contains EqPV using the nucleic acids, primers, probes, and antibodies of the present invention.

In other aspects, the invention provides methods for identifying and/or generating anti-viral drugs. For example, in one aspect the invention provides methods for identifying drugs that bind to and/or inhibit the function of EqPV-encoded proteins of the invention, or that inhibit the replication or pathogenicity of EqPV of the invention. Methods of identifying drugs that affect or inhibit a particular drug target, such as high throughput drug screening methods, are well known in the art and can readily be applied to the proteins and viruses of the present invention.

In certain aspects, the present invention provides immunogenic compositions capable of inducing an immune response against EqPV including EqPV of the invention comprising SEQ ID NOs: 1-3, or comprising a cDNA sequence complementary to the sense or an anti- sense strand of SEQ ID NOs: 1-3, or comprising a polypeptide encoded by SEQ ID NOs: 1-3, or a cDNA sequence complementary to the sense or an anti-sense strand of SEQ ID NOs: 1- 3, or comprising a polypeptide of any of SEQ ID NOs: 23-24.

In certain aspects, the invention relates to an immunogenic composition comprising an isolated virus described herein.

In certain aspects, the invention relates to a method of inducing an immune response in an animal, the method comprising administering an immunogenic composition described herein.

In certain aspects, the invention relates to an immunogenic composition for inducing an immune response in an animal, wherein the composition comprises a recombinant or isolated polypeptide derived from an equine parvovirus; and a pharmaceutically acceptable vehicle or diluent. In certain embodiments, the composition further comprises an adjuvant. In certain embodiments, the polypeptide is SI -structural protein. In certain embodiments, the polypeptide is NS1 -non-structural protein. In certain embodiments, the composition is for protecting an animal against equine parvovirus. In certain embodiments, the composition protecting an animal against EqPV delays the onset of symptoms associated with EqPV, or reduces the severity of symptoms of EqPV. In certain embodiments, the animal is an equine animal. In certain embodiments, the animal is a horse. In certain embodiments the animal is a human.

In certain aspects, the invention relates to a method of immunizing an animal against EqPV, the method comprising administering to the animal an immunogenic composition for inducing an immune response in an animal, wherein the composition comprises a recombinant or isolated polypeptide derived from an equine parvovirus; and a pharmaceutically acceptable vehicle or diluent. In certain embodiments, the animal is an equine animal. In certain embodiments, the animal is a horse. In certain embodiments, the animal is a human. In certain embodiments, the animal is a domestic farm animal.

In certain aspects, the invention relates to an isolated virus comprising at least 24 consecutive nucleotides from an isolated (or non-isolated) nucleic acid having the sequence of SEQ ID NOs: 1-3; an isolated (or non-isolated) nucleic acid complementary to the sequence of SEQ ID NOs: 1-3; an isolated (or non- isolated) nucleic acid having at least about 60% sequence identity to SEQ ID NOs: 1-3; an isolated (or non-isolated) nucleic acid having at least about 60% sequence identity to a nucleic acid complementary to the sequence of SEQ ID NOs: 1-3; an isolated (or non-isolated) nucleic acid which comprises at least 10 consecutive nucleotides of SEQ ID NOs: 1-3; an isolated (or non-isolated) nucleic acid which comprises at least 10 consecutive nucleotides of a nucleic acid complementary to the sequence of SEQ ID NOs: 1-3; an isolated (or non-isolated) nucleic acid which comprises at least 10 consecutive nucleotides of a sequence having at least about 60% identity to SEQ ID NOs: 1-3; an isolated (or non-isolated) nucleic acid which comprises at least 10 consecutive nucleotides of a sequence having at least about 60% identity to a nucleic acid complementary to the sequence of SEQ ID NOs: 1-3.

In certain aspects, the invention relates to an isolated virus comprising at least 8 consecutive amino acids from the polypeptide encoded by: an isolated (or non-isolated) nucleic acid having the sequence of SEQ ID NOs: 1-3; an isolated (or non-isolated) nucleic acid complementary to the sequence of SEQ ID NOs: 1-3; an isolated (or non-isolated) nucleic acid having at least about 60% sequence identity to SEQ ID NOs: 1-3; an isolated (or non-isolated) nucleic acid having at least about 60% sequence identity to a nucleic acid complementary to the sequence of SEQ ID NOs: 1-3; an isolated (or non-isolated) nucleic acid which comprises at least 10 consecutive nucleotides of SEQ ID NOs: 1-3; an isolated (or non-isolated) nucleic acid which comprises at least 10 consecutive nucleotides of a nucleic acid complementary to the sequence of SEQ ID NOs: 1-3; an isolated (or non-isolated) nucleic acid which comprises at least 10 consecutive nucleotides of a sequence having at least about 60% identity to SEQ ID NOs: 1-3; an isolated (or non- isolated) nucleic acid which comprises at least 10 consecutive nucleotides of a sequence having at least about 60% identity to a nucleic acid complementary to the sequence of SEQ ID NOs: 1-3.

In certain aspects, the invention relates to an isolated virus comprising at least 8 consecutive amino acids from a polypeptide of SEQ ID NOs: 23-24; or a polypeptide having at least about 80% identity to the sequence of the polypeptide of SEQ ID NOs: 23-24.

In certain aspects, the invention relates to an isolated cell comprising an isolated (or non-isolated) nucleic acid of the current invention including nucleic acids having the sequence of SEQ ID NOs: 1-22 and 25-28, a sequence complementary to the sequence of SEQ ID NOs: 1-22 and 25-28, and variants and fragments thereof.

In certain aspects, the invention relates to an isolated cell comprising a polypeptide of SEQ ID NOs: 23-24, and variants and fragments thereof.

In certain aspects, the invention relates to a method for culturing cells of the invention as well as using them to test for EqPV vaccines and drugs. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is the tree showing the results of the genetic and phylogenetic analysis of the NSl-non structural protein of EqPV as described in Example 1.

Figure 2 is the tree the tree showing the results of the genetic and phylogenetic analysis of the SI- structural protein of EqPV as described in Example 1

Figure 3 shows graphs depicting the hepatic biochemistry values and level of viremia in horse 1 experimentally inoculated with EqPV.

DETAILED DESCRIPTION OF THE INVENTION

Identification of a Novel Equine Parvovirus

Recent identification of the three new horse blood borne Flaviviruses, Equine

Hepacivirus (EHCV) a non-Primate Hepacivirus, and Theiler's disease associated virus (TDAV), and Equine Pegivirus (EPgV), of the Pegivirus genus have also been reported (Burbelo et al. 2012; Chandriani et al. 2013). The absence of these viruses in horses with Theiler's disease led to the search for a yet unknown virus as the causative agent of this widespread post- blood inoculation disease.

In collaboration with academic and private equine referral centers in North America, a prospective clinical epidemiological study of acute hepatitis in horses was initiated in January 2014. The purpose of the study was to assess the possible role of these new viruses and other etiologic and environmental factors in the pathogenesis of acute hepatitis. Using unbiased deep sequencing, a new virus in both the serum and liver of a horse that died from Theiler's disease, and DNA of the virus in the tetanus antitoxin product administered to the horse 65 days earlier, was discovered. The new virus belongs to family Parvoviridae and is phylogenetically different and highly divergent from all known parvoviruses of humans and animals. The complete viral genome was obtained. The new virus was named equine parvovirus (EqPV) (Example 1). Subsequent epidemiological studies confirmed EqPV to be a highly prevalent horse infection and the virus was found in several batches of equine serum derived products as well as in fourteen consecutive cases of Theiler's disease (Example 2).

The complete sequence of the circular genomic form of EqPV found in the liver sample of the first case was used to study the genetic relatedness of EqPV with other known animal parvoviruses. Phylogenetic analysis indicated that EqPV represents the prototype of a new parvovirus species and showed only 40% protein identity to its closest genetic relative, the canine parvovirus. It was also determined that EqPV is a common infection of healthy adult horses with serologic evidence of infection in 16% and evidence of viremia in 12% of horses.

An aged mare that was virus and antibody negative for EqPV was inoculated with commercial tetanus antitoxin that was RT PCR positive for EqPV and had earlier been associated with two of the clinical cases of acute hepatitis. Forty-eight days following inoculation, the mare became viremic with a progressive increase in viral load for five weeks at which time clinical and biochemical findings of liver failure were found. Microscopic examination of a liver biopsy sample collected at that time showed acute hepatic necrosis characteristic of Theiler' s disease. One week later, antibody against EqPVcould be detected Early results obtained from a second experimentally inoculated adult horse show a similar course of infection (Example 3).

There was little diversity noted among EqPV found in different animals in the study indicating homogenous spread. Comparison of sequences between Theiler' s disease horses and the specific antitoxin they had been administered found virtually no diversity. It was concluded that EqPV can cause acute hepatitis in horses, is serum transmissible, and is a potential contaminant of biologicals derived from horse products.

Members of the subfamily Parvovirinae are small, nonenveloped icosahedral viruses with single- stranded linear DNA genomes that frequently infect animals, including canines and humans, through the fecal oral route. Canine parvovirus is highly contagious and affects a dog's intestinal tract. Parvovirus B19 affects humans, with Fifth disease being the most common illness (a mild rash illness). An equine parvovirus from a horse fetal liver was described by Wong et al. in 1985. Equine parvovirus is discussed in Harbour in 1987.

The genomes of most parvoviruses are nearly 5,000 nucleotides (nt) in length and comprise two transcriptional units, one encoding the capsid proteins and the other the nonstructural proteins. Protein coding sequences are flanked on each side by noncoding palindromic repeats, also known as inverted terminal repeat (ITR) sequences, which play an important role in viral DNA replication. Based on their replication requirements, parvoviruses can be classified as either autonomous parvoviruses or dependo viruses; the latter require external factors for replication. The most extensively studied dependoviruses are the adeno associated viruses (AAV) that are used as gene therapy vectors. During their replication in the nuclei of infected host cells, the genomes of wild type AAV integrate in a site specific manner (chromosome 19), resulting in latent infection of host cells. Several recent studies detected the presence of AAV genomes in tissues of humans and nonhuman primates as integrated virus in the host genome and/or in episomal closed circular form. As part of their replication cycle, all parvoviruses must enter the nuclei of their host cells and generate a double stranded monomer replicative form.

Without being bound by theory, the new animal virus, termed Equine (Horse) Parvovirus (EqPV), can be a pathogen of domestic, farm, wild horses, and other animals including humans. The EPISOME form of viral DNA was confirmed in liver tissue indicating the acute and persistent nature of the virus infection. The EqPV could potentially cause cross- species transmission and infections in human and other common domestic animal species through therapeutics made using horse serum or derived products. The discovery of this virus and the sequence reported herein can be used to development of a vaccine for EqPV and also provide a useful sample to study the diverging nature of parvoviruses in general.

Also described herein are PCR assays and primers as diagnostic tools to detect the presence of EqPV in infected animals and perhaps more importantly, in biologicals derived from horses and other animals, including but not limited to, equine plasma and serum products to be administered to animals. Isolation of the equine parvovirus, growth of the virus in cell lines, and vaccine development can be performed. Additionally, the development of a means to inactivate virus infectivity and thus interrupt their tranmission to new animals including humans can be performed.

Definitions

The terms used in this specification generally have their ordinary meanings in the art, within the context of this invention and the specific context where each term is used. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner in describing the methods of the invention and how to use them. Moreover, it will be appreciated that the same thing can be said in more than one way. Consequently, alternative language and synonyms may be used for any one or more of the terms discussed herein, nor is any special significance to be placed upon whether or not a term is elaborated or discussed herein. Synonyms for certain terms are provided. A recital of one or more synonyms does not exclude the use of the other synonyms. The use of examples anywhere in the specification, including examples of any terms discussed herein, is illustrative only, and in no way limits the scope and meaning of the invention or any exemplified term. Likewise, the invention is not limited to its preferred embodiments.

In accordance with the present invention, there may be numerous tools and techniques within the skill of the art, such as those commonly used in molecular immunology, cellular immunology, pharmacology, and microbiology. See, e.g., Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y.; Ausubel et al. eds. (2005) Current Protocols in Molecular Biology, John Wiley and Sons, Inc.: Hoboken, N.J.; Bonifacino et al. eds. (2005) Current Protocols in Cell Biology, John Wiley and Sons, Inc.: Hoboken, N.J.; Coligan et al. eds. (2005) Current Protocols in Immunology, John Wiley and Sons, Inc.: Hoboken, N.J.; Coico et al. eds. (2005) Current Protocols in Microbiology, John Wiley and Sons, Inc.: Hoboken, N.J.; Coligan et al. eds. (2005) Current Protocols in Protein Science, John Wiley and Sons, Inc.: Hoboken, N.J.; and Enna et al. eds. (2005) Current Protocols in Pharmacology, John Wiley and Sons, Inc.: Hoboken, N.J.

The singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise.

The term "about" is used herein to mean approximately, in the region of, roughly, or around. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 20%.

As used herein, "EqPV" refers to isolates of the equine parvovirus described herein.

As used herein, a "EqPV gene" refers to any one of the genes identified in the EqPV genome, including but not limited to, SEQ ID NOs: 1-3.

As used herein, the term "animal" refers to a vertebrate, including, but not limited to, equine, (e.g. horses). Thus, the invention can be used in veterinary medicine, e.g., to treat companion animals, farm animals, laboratory animals in zoological parks, and animals in the wild. The invention is can also be used for human medical applications.

As used herein, the term "sample" includes a biological sample or sample from a product such as a biologic derived from an animal, or a plasma or serum product that is to be administered to an animal.

As used herein, the term "biological sample" means a sample of tissue or fluid obtained from an animal, e.g., horse, and includes but is not limited to blood, plasma, and liver tissue.

The terms "treat", "treatment", and the like refer to a means to slow down, relieve, ameliorate or alleviate at least one of the symptoms of the disease, or reverse the disease after its onset. The terms "prevent", "prevention", and the like refer to acting prior to overt disease onset, to prevent the disease from developing or minimize the extent of the disease or slow its course of development.

"Substantially identical," in the context of two nucleic acids or polypeptides, refers to two or more sequences or subsequences that have at least 98%, at least 99% or higher nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.

"Percent identity" in the context of two or more nucleic acids or polypeptide sequences, refers to the percentage of nucleotides or amino acids that two or more sequences or subsequences contain which are the same. A specified percentage of nucleotides can be referred to such as: 60% identity, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity over a specified region, when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.

A "comparison window", as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence can be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2: 482, by the homology alignment algorithm of Needleman and Wunsch (1970) /. Mol. Biol. 48: 443, by the search for similarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. U.S.A. 85: 2444, by computerized implementations of these algorithms (FASTDB (Intelligenetics), BLAST (National Center for Biomedical Information), GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Ausubel).

In the context of nucleic acids base symbols can be used to represent a position on a nucleic acid sequence that can have multiple possible alternative. For example, "W" represents A or T; "S" represents C or G; "M" represents A or C; "K" represents G or T; "R" represents A or G; "Y" represents C or T; "B" represents C, G, or T; "D" represents A, G, or T; "H" represents A, C, or T; "V" represents A, C, or G.

It will be understood that, for the particular EqPV polypeptides described here, natural variations can exist between individual EqPV strains. These variations may be demonstrated by (an) amino acid difference(s) in the overall sequence or by deletions, substitutions, insertions, inversions or additions of (an) amino acid(s) in said sequence. Amino acid substitutions which do not essentially alter biological and immunological activities, have been described, e.g. by Neurath et al in "The Proteins" Academic Press New York (1979). Amino acid replacements between related amino 15 acids or replacements which have occurred frequently in evolution are, inter alia, Ser/Ala, Ser/Gly, Asp/Gly, Asp/Asn, Ile/Val (see Dayhof, M. D., Atlas of protein sequence and structure, Nat. Biomed. Res. Found., Washington D.C., 1978, vol. 5, suppl. 3). Other amino acid substitutions include Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/ Val, Thr/Phe, Ala/Pro, Lys/Arg, Leu/Ile, Leu/Val and Ala/ Glu. Based on this information, Lipman and Pearson developed a method for rapid and sensitive protein comparison (Science (1985) 227:1435) and determining the functional similarity between homologous proteins. Such amino acid substitutions of the exemplary embodiments of this invention, as well as variations having deletions and/or insertions are within the scope of the invention as long as the resulting proteins retain their immune reactivity. It is know that polypeptide sequences having one or more amino acid sequence variations as compared to a reference polypeptide may still be useful for generating antibodies that bind the reference polypeptide.

Nucleic Acids and Uses Thereof

The present invention provides EqPV nucleic acid sequences. These nucleic acid sequences may be useful for, inter alia, expression of EqPV-encoded proteins or fragments, variants, or derivatives thereof, generation of antibodies against EqPV proteins, generation of primers and probes for detecting EqPV and/or for diagnosing EqPV infection, generating immunogenic compositions against EqPV, and screening for drugs effective against EqPV as described herein.

In certain aspects, the invention is directed to a EqPV isolated nucleic acid sequence including cDNA sequences corresponding to EqPV RNA sequences, and mRNA sequences as provided in any of SEQ ID NOs: 1-22 and 25-28.

SEQ ID NO: 1 is the nucleic acid sequence for equine parvovirus complete genome. SEQ ID NO: 2 is the cDNA sequence encoding the NSl-non structural protein of

EqPV.

SEQ ID NO: 3 is the cDNA sequence encoding the SI -structural protein (capsid polypeptide) or virion protein (VP) of EqPV.

SEQ ID NO: 4 is the nucleic acid sequence for TPN-lScrFl :

CAAGGCTACGATTGGCA (SEQ ID NO: 4).

SEQ ID NO: 5 is the nucleic acid sequence for TPN-lScrF2: GGCTACGATTGGCAAGAAGC (SEQ ID NO: 5).

SEQ ID NO: 6 is the nucleic acid sequence for TPN-lScrRl : CCCGGGAATGTCATTGAA (SEQ ID NO: 6).

SEQ ID NO: 7 is the nucleic acid sequence for TPN-lScrR2: CGGGAATGTCATTGAACGGGAA (SEQ ID NO: 7).

SEQ ID NO: 8 is the nucleic acid sequence for TPN-2ScrFl : GTACCAGAGGTAGTGCACAT (SEQ ID NO: 8).

SEQ ID NO: 9 is the nucleic acid sequence for TPN-2ScrF2:

GTAGTGCACATCACGCCATGG (SEQ ID NO: 9).

SEQ ID NO: 10 is the nucleic acid sequence for TPN-2ScrRl : TCAGGGAAGTGGTAGGTATG (SEQ ID NO: 10).

SEQ ID NO: 11 is the nucleic acid sequence for TPN-2ScrR2: GGGAAGTGGTAGGTATGTTCCCA (SEQ ID NO: 11).

Primers for the PCR assay for the EqPV NSl-non structural gene are exemplified by SEQ ID NOs: 12-15.

SEQ ID NO: 12 is the nucleic acid sequence for primer EqPV akl or TPNS-inFl : GGAGAAGAGCGCAACAAATGCA (SEQ ID NO: 12).

SEQ ID NO: 13 is the nucleic acid sequence for primer EqPV ak2 or TPNS-inRl :

AAGACATTTCCGGCCGTGAC (SEQ ID NO: 13).

SEQ ID NO: 14 is the nucleic acid sequence for primer EqPV ak3 or TPNS-inF2: GCGCAACAAATGCAGCGGTTCGA (SEQ ID NO: 14).

SEQ ID NO: 15 is the nucleic acid sequence for primer EqPV ak4 or TPNS-inR2: GGCCGTGACGACGGTGATATC (SEQ ID NO: 15).

Primers for the PCR assay for the EqPV SI -structural (or virion protein (VP)) gene are exemplified by SEQ ID NOs: 16-19. SEQ ID NO: 16 is the nucleic acid sequence for primer EqPV ak5 or TPST-inFl : GTCGCTGCATTCTGAGTCC (SEQ ID NO: 16).

SEQ ID NO: 17 is the nucleic acid sequence for primer EqPV ak6 or TPST-inRl : TGGGATTATACTGTCTACGGGT (SEQ ID NO: 17).

SEQ ID NO: 18 is the nucleic acid sequence for primer EqPV ak7 or TPST-inF2:

CTGCATTCTGAGTCCGTGGCC (SEQ ID NO: 18).

SEQ ID NO: 19 is the nucleic acid sequence for primer EqPV ak8 or EqTPST-inR2: CTGTCTACGGGTATCCCATACGTA (SEQ ID NO: 19).

Primers and the probe for quantitative PCR are exemplified by SEQ ID NOs. 20-22. SEQ ID NO: 20 is the nucleic acid sequence for primer EqPV ak9 or qPCR Forward

Primer: ATGCAGATGCTTTCCGACC (SEQ ID NO:20).

SEQ ID NO: 21 is the nucleic acid sequence for primer EqPV aklO or qPCR Reverse Primer: GCCCCAGAAACATATGGAAA (SEQ ID NO: 21).

SEQ ID NO: 22 is the nucleic acid sequence for probe EqPV akl 1 or qPCR Probe: [6- F AM] ACCGTAGCGGATTCGGGATCTGC [BHQ 1 a- 6F AM] (SEQ ID NO: 22).

Primers for amplifying and cloning the EqPV capsid protein are exemplified by SEQ ID NOs: 25-28.

SEQ ID NO: 25 is the nucleic acid sequence for forward primer EqPV LIPSF1 for amplifying the the VP1 gene of EgPV: AGTAAAGTCAATGGACACCA (SEQ ID NO: 25).

SEQ ID NO: 26 is the nucleic acid sequence for reverse primer EqPV LIPSR1 for amplifying the the VP1 gene of EgPV: GGATCGTGGTATGAGTTC (SEQ ID NO: 26).

SEQ ID NO: 27 is the nucleic acid sequence for a primer EqPV LIPS BamHl for insertion into a plasmid: GAGGGATCCCATGCTTTACCGTATGATC (SEQ ID NO: 27).

SEQ ID NO: 28 is the nucleic acid sequence for a primer EqPV LIPS Xhol for insertion into a plasmid: GAGCTCGAGTCAGAACTGACAGTATTGGTTC (SEQ ID NO: 28).

In certain aspects, the invention relates to variants of EqPV nucleic acid sequence having greater that 60% similarity to the sequence of any of SEQ ID NOs: 1-22 and 25-28.

In certain aspects, the invention is directed to an isolated nucleic acid of any of SEQ ID NOs: 1-22 and 25-28. In certain aspects, the invention is directed to an isolated nucleic acid complementary to any of SEQ ID NOs: 1-22 and 25-28.

In certain aspects, the invention is directed to isolated nucleic acid sequence variants of any of SEQ ID NOs: 1-22 and 25-28 or any sequence complementary to any of SEQ ID NOs: 1-22 and 25-28. Variants of any of SEQ ID NOs: 1-22 and 25-28 include, but are not limited to, nucleic acid sequences having at least from about 50% to about 55% identity to that of any of SEQ ID NOs: 1-22 and 25-28. Variants of any of SEQ ID NOs: 1-22 and 25-28 include, but are not limited to, nucleic acid sequences having at least from about 55.1 % to about 60% identity to that of any of SEQ ID NOs: 1-22 and 25-28. Variants of any of SEQ ID NOs: 1-22 and 25-28 include, but are not limited to, nucleic acid sequences having at least from about 60.1% to about 65% identity to that of any of SEQ ID NOs: 1-22 and 25-28. Variants of any of SEQ ID NOs: 1-22 and 25-28 include, but are not limited to, nucleic acid sequences having at least from about 65.1 % to about 70% identity to that of any of SEQ ID NOs: 1-22 and 25-28. Variants of any of SEQ ID NOs: 1-22 and 25-28 include, but are not limited to, nucleic acid sequences having at least from about 70.1% to about 75% identity to that of any of SEQ ID NOs: 1-22 and 25-28. Variants of any of SEQ ID NOs: 1-22 and 25- 28 include, but are not limited to, nucleic acid sequences having at least from about 75.1% to about 80% identity to that of any of SEQ ID NOs: 1-22 and 25-28. Variants of any of SEQ ID NOs: 1-22 and 25-28 include, but are not limited to, nucleic acid sequences having at least from about 80.1% to about 85% identity to that of any of SEQ ID NOs: 1-22 and 25-28. Variants of any of SEQ ID NOs: 1-22 and 25-28 include, but are not limited to, nucleic acid sequences having at least from about 85.1% to about 90% identity to that of any of SEQ ID NOs: 1-22 and 25-28. Variants of any of SEQ ID NOs: 1-22 and 25-28 include, but are not limited to, nucleic acid sequences having at least from about 90.1% to about 95% identity to that of any of SEQ ID NOs: 1-22 and 25-28. Variants of any of SEQ ID NOs: 1-22 and 25- 28 include, but are not limited to, nucleic acid sequences having at least from about 95.1% to about 97% identity to that of any of SEQ ID NOs: 1-22 and 25-28. Variants of any of SEQ ID NOs: 1-22 and 25-28 include, but are not limited to, nucleic acid sequences having at least from about 97.1% to about 99% identity to that of any of SEQ ID NOs: 1-22 and 25-28. Programs and algorithms for sequence alignment and comparison of % identity and/or homology between nucleic acid sequences, or polypeptides, are well known in the art, and include BLAST, SIM alignment tool, and so forth.

In a further embodiment, the invention provides an isolated nucleic acid having a sequence substantially identical to a nucleic acid of any of SEQ ID NOs: 1-22 and 25-28, or a fragment thereof. In a further embodiment, the invention provides an isolated nucleic acid having a sequence substantially identical to a nucleic acid complementary to any of SEQ ID NOs: 1-22 and 25-28, or a fragment thereof. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 50 consecutive nucleotides from SEQ ID NO: 1 or a sequence complementary to SEQ ID NO: 1. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 100 consecutive nucleotides from SEQ ID NO: 1 or a sequence complementary to SEQ ID NO: 1. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 200 consecutive nucleotides from SEQ ID NO: 1 or a sequence complementary to SEQ ID NO: 1. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 300 consecutive nucleotides from SEQ ID NO: 1 or a sequence complementary to SEQ ID NO: 1. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 400 consecutive nucleotides from SEQ ID NO: 1 or a sequence complementary to SEQ ID NO: 1. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 500 consecutive nucleotides from SEQ ID NO: 1 or a sequence complementary to SEQ ID NO: 1. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 600 consecutive nucleotides from SEQ ID NO: 1 or a sequence complementary to SEQ ID NO: 1. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 700 consecutive nucleotides from SEQ ID NO: 1 or a sequence complementary to SEQ ID NO: 1. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 800 consecutive nucleotides from SEQ ID NO: 1 or a sequence complementary to SEQ ID NO: 1. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 900 or more consecutive nucleotides from SEQ ID NO: 1 or a sequence complementary to SEQ ID NO: 1. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 1000 or more consecutive nucleotides from SEQ ID NO: 1 or a sequence complementary to SEQ ID NO: 1. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 2000 or more consecutive nucleotides from SEQ ID NO: 1 or a sequence complementary to SEQ ID NO: 1. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 3000 or more consecutive nucleotides from SEQ ID NO: 1 or a sequence complementary to SEQ ID NO: 1. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 4000 or more consecutive nucleotides from SEQ ID NO: 1 or a sequence complementary to SEQ ID NO: 1. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 5000 or more consecutive nucleotides from SEQ ID NO: 1 or a sequence complementary to SEQ ID NO: 1. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 5308 or more consecutive nucleotides from SEQ ID NO: 1 or a sequence complementary to SEQ ID NO: 1.

In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 50 consecutive nucleotides from SEQ ID NO: 2 or a sequence complementary to SEQ ID NO: 2. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 100 consecutive nucleotides from SEQ ID NO: 2 or a sequence complementary to SEQ ID NO: 2. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 200 consecutive nucleotides from SEQ ID NO: 2 or a sequence complementary to SEQ ID NO: 2. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 300 consecutive nucleotides from SEQ ID NO: 2 or a sequence complementary to SEQ ID NO: 2. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 400 consecutive nucleotides from SEQ ID NO: 2 or a sequence complementary to SEQ ID NO: 2. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 500 consecutive nucleotides from SEQ ID NO: 2 or a sequence complementary to SEQ ID NO: 2. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 600 consecutive nucleotides from SEQ ID NO: 2 or a sequence complementary to SEQ ID NO: 2. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 700 consecutive nucleotides from SEQ ID NO: 2 or a sequence complementary to SEQ ID NO: 2. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 800 consecutive nucleotides from SEQ ID NO: 2 or a sequence complementary to SEQ ID NO: 2. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 900 or more consecutive nucleotides from SEQ ID NO: 2 or a sequence complementary to SEQ ID NO: 2. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 1000 or more consecutive nucleotides from SEQ ID NO: 2 or a sequence complementary to SEQ ID NO: 2. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 1500 or more consecutive nucleotides from SEQ ID NO: 2 or a sequence complementary to SEQ ID NO: 2. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 1782 or more consecutive nucleotides from SEQ ID NO: 2 or a sequence complementary to SEQ ID NO: 2.

In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 50 consecutive nucleotides from SEQ ID NO: 3 or a sequence complementary to SEQ ID NO: 3. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 100 consecutive nucleotides from SEQ ID NO: 3 or a sequence complementary to SEQ ID NO: 3. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 200 consecutive nucleotides from SEQ ID NO: 3 or a sequence complementary to SEQ ID NO: 3. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 300 consecutive nucleotides from SEQ ID NO: 3 or a sequence complementary to SEQ ID NO: 3. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 400 consecutive nucleotides from SEQ ID NO: 3 or a sequence complementary to SEQ ID NO: 3. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 500 consecutive nucleotides from SEQ ID NO: 3 or a sequence complementary to SEQ ID NO: 3. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 600 consecutive nucleotides from SEQ ID NO: 3 or a sequence complementary to SEQ ID NO: 3. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 700 consecutive nucleotides from SEQ ID NO: 3 or a sequence complementary to SEQ ID NO: 3. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 800 consecutive nucleotides from SEQ ID NO: 3 or a sequence complementary to SEQ ID NO: 3. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 900 or more consecutive nucleotides from SEQ ID NO: 3 or a sequence complementary to SEQ ID NO: 3. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 1000 or more consecutive nucleotides from SEQ ID NO: 3 or a sequence complementary to SEQ ID NO: 3. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 2000 or more consecutive nucleotides from SEQ ID NO: 3 or a sequence complementary to SEQ ID NO: 3. In one embodiment, the invention is directed to an isolated nucleic acid sequence comprising from about 10 to about 2763 or more consecutive nucleotides from SEQ ID NO: 3 or a sequence complementary to SEQ ID NO: 3.

Encompassed by the invention are polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, including any of the nucleic acid sequences disclosed herein, and variants and fragments thereof under various conditions of stringency Polynucleotides homologous to the sequences illustrated in SEQ ID NOs: 1-22 and 25-28, can be identified, e.g., by hybridization to each other under stringent or under highly stringent conditions. The term "nucleic acid hybridization" refers to anti-parallel hydrogen bonding between two single-stranded nucleic acids, in which A pairs with T (or U if an RNA nucleic acid) and C pairs with G. Nucleic acid molecules are "hybridizable" to each other when at least one strand of one nucleic acid molecule can form hydrogen bonds with the complementary bases of another nucleic acid molecule under defined stringency conditions. The stringency of a hybridization reflects the degree of sequence identity of the nucleic acids involved, such that the higher the stringency, the more similar are the two polynucleotide strands. Stringency of hybridization is determined, e.g., by (i) the temperature at which hybridization and/or washing is performed, and (ii) the ionic strength and (iii) concentration of denaturants such as formamide of the hybridization and washing solutions, as well as other parameters. Hybridization requires that the two strands contain substantially complementary sequences. Depending on the stringency of hybridization, however, some degree of mismatches may be tolerated. Under "low stringency" conditions, a greater percentage of mismatches are tolerable (i.e., will not prevent formation of an anti-parallel hybrid). Hybridization conditions for various stringencies are known in the art and are disclosed in detail in at least Sambrook et al.

In certain aspects, the invention relates to a synthetic nucleic acid comprising at least about 10 nucleotides of an isolated (or non-isolated) nucleic acid having the sequence of SEQ ID NOs: 1-22 and 25-28; an isolated (or non-isolated) nucleic acid complementary to the sequence of any of SEQ ID NOs: 1-22 and 25-28; an isolated (or non-isolated) nucleic acid having at least about 60% sequence identity to any of SEQ ID NOs: 1-22 and 25-28; an isolated (or non-isolated) nucleic acid having at least about 60% sequence identity to a nucleic acid complementary to the sequence of any of SEQ ID NOs: 1-22 and 25-28; an isolated (or non-isolated) nucleic acid which comprises at least 10 consecutive nucleotides of any of SEQ ID NOs: 1-22 and 25-28; an isolated (or non-isolated) nucleic acid which comprises at least 10 consecutive nucleotides of a nucleic acid complementary to the sequence of any of SEQ ID NOs: 1-22 and 25-28; an isolated (or non-isolated) nucleic acid which comprises at least 10 consecutive nucleotides of a sequence having at least about 60% identity to any of SEQ ID NOs: 1-22 and 25-28; an isolated (or non-isolated) nucleic acid which comprises at least 10 consecutive nucleotides of a sequence having at least about 60% identity to a nucleic acid complementary to the sequence of any of SEQ ID NOs: 1-22 and 25-28.

In certain aspects, the invention relates to a synthetic nucleic acid comprising the nucleotides of an isolated (or non-isolated) nucleic acid having the sequence of SEQ ID NOs: 1-22 and 25-28; an isolated (or non-isolated) nucleic acid complementary to the sequence of any of SEQ ID NOs: 1-22 and 25-28; an isolated (or non-isolated) nucleic acid having at least about 60% sequence identity to any of SEQ ID NOs: 1-22 and 25-28; an isolated (or nonisolated) nucleic acid having at least about 60% sequence identity to a nucleic acid complementary to the sequence of any of SEQ ID NOs: 1-22 and 25-28; an isolated (or nonisolated) nucleic acid which comprises at least 10 consecutive nucleotides of any of SEQ ID NOs: 1-22 and 25-28; an isolated (or non-isolated) nucleic acid which comprises at least 10 consecutive nucleotides of a nucleic acid complementary to the sequence of any of SEQ ID NOs: 1-22 and 25-28; an isolated (or non-isolated) nucleic acid which comprises at least 10 consecutive nucleotides of a sequence having at least about 60% identity to any of SEQ ID NOs: 1-22 and 25-28; an isolated (or non-isolated) nucleic acid which comprises at least 10 consecutive nucleotides of a sequence having at least about 60% identity to a nucleic acid complementary to the sequence of any of SEQ ID NOs: 1-22 and 25-28.

In yet another aspect, the invention provides a synthetic nucleic acid which has a sequence consisting of from about 10 to about 30 consecutive nucleotides from a nucleic acid sequence selected from the group of sequence consisting of SEQ ID NOs: 1-22 and 25-28.

In yet another aspect, the invention provides a synthetic nucleic acid which has a sequence consisting of from about 10 to about 30 consecutive nucleotides consisting of consecutive nucleotides having a sequence which is a variant of any of SEQ ID NOs: 1-22 and 25-28 having at least about 95.1%, about 95.5%, about 96%, about 96.5%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%, about 99.5% or about 99.9% identity to that of any of SEQ ID NOs: 1-22 and 25-28.

In yet another aspect, the invention provides a synthetic nucleic acid which has a sequence consisting of from about 10 to about 30 consecutive nucleotides from a nucleic acid sequence which is complementary to a nucleic acid sequence selected from the group of sequence consisting of SEQ ID NOs: 1-22 and 25-28.

In yet another aspect, the invention provides a synthetic nucleic acid which has a sequence consisting of from about 10 to about 30 consecutive nucleotides which is complementary to a nucleic acid consisting of consecutive nucleotides having a sequence which is a variant of any of SEQ ID NOs: 1-22 and 25-28 having at least about 95.1%, about 95.5%, about 96%, about 96.5%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%, about 99.5% or about 99.9% identity to that of any of SEQ ID NOs: 1-22 and 25-28.

In yet another aspect, the invention provides a composition comprising one or more nucleic acids, isolated, non-isolated, or synthetic, described herein.

In other aspects the invention is directed to isolated nucleic acid sequences such as primers and probes, comprising nucleic acid sequences of any of SEQ ID NOs: 1-22 and 25- 28. Such primers and/or probes may be useful for detecting the presence of EqPV of the invention, for example in samples of bodily fluids such as blood, saliva, or urine and biological tissue such as liver from an animal, and thus may be useful in the diagnosis of EqPV infection. Such primers and/or probes can also be used to detect EqPV in biologies and plasma and serum products for equine and other origin. Such probes can detect polynucleotides of any of SEQ ID NOs: 1-22 and 25-28 in samples which comprise EqPV represented by any of SEQ ID NOs: 1-22 and 25-28. The isolated nucleic acids which can be used as primer and/probes are of sufficient length to allow hybridization with, i.e. formation of duplex with a corresponding target nucleic acid sequence, a nucleic acid sequences of any of SEQ ID NOs: 1-22 and 25-28, or a variant thereof.

The isolated nucleic acid of the invention which can be used as primers and/or probes can comprise about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 consecutive nucleotides from any of SEQ ID NOs: 1-22 and 25-28, or sequences complementary to any of SEQ ID NOs: 1-22 and 25-28. The isolated nucleic acid of the invention which can be used as primers and/or probes can comprise from about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 and up to about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 and 100 consecutive nucleotides from any of SEQ ID NOs: 1-22 and 25-28, or sequences complementary to any of SEQ ID NOs: 1-22 and 25-28.

The invention is also directed to primer and/or probes which can be labeled by any suitable molecule and/or label known in the art, for example but not limited to fluorescent tags suitable for use in Real Time PCR amplification, for example TaqMan, cybergreen, TAMRA and/or FAM or 6-FAM probes; radiolabels; and so forth. In certain embodiments, the oligonucleotide primers and/or probe further comprises a detectable non-isotopic label selected from the group consisting of: a fluorescent molecule, a chemiluminescent molecule, an enzyme, a cofactor, an enzyme substrate, and a hapten.

In another aspect, the invention provides an oligonucleotide probe which comprises from about 10 to about 50 nucleotides, wherein at least about 10 contiguous nucleotides are at least 95% complementary to a nucleic acid target region within a EqV nucleic acid sequence in any of SEQ ID NOs: 1-3, wherein the oligonucleotide probe hybridizes to the nucleic acid target region under moderate to highly stringent conditions to form a detectable nucleic acid target oligonucleotide probe duplex. In one embodiment, the oligonucleotide probe is at least about 95.5%, about 96%, about 96.5%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%, about 99.5% or about 99.9% complementary to SEQ ID NOs: 1-3. In another embodiment the oligonucleotide probe consists essentially of from about 10 to about 50 nucleotides.

In certain aspects, the invention is directed to primer sets comprising isolated nucleic acids as described herein, which primer set are suitable for amplification of nucleic acids from samples which comprises EqPV represented by SEQ ID NOs: 1-3, or variants thereof.

Primer sets can comprise any suitable combination of primers which would allow amplification of a target nucleic acid sequences in a sample which comprises EqPV represented by SEQ ID NOs: 1-3, or variants thereof. Amplification can be performed by any suitable method known in the art, for example but not limited to PCR, RT-PCR, qPCR, and transcription mediated amplification (TMA).

In certain aspects, the invention relates to a primer set for determining the presence or absence of EqPV in a biological sample, wherein the primer set comprises at least one synthetic nucleic acid sequence selected from the group consisting of the synthetic nucleic acid described herein, including but not limited to a synthetic nucleic acid which has a sequence consisting of from about 10 to about 30 consecutive nucleotides from a nucleic acid sequence selected from the group of sequence consisting of SEQ ID NOs: 1-22 and 25-28 or variants thereof as described herein; or a synthetic nucleic acid which has a sequence consisting of from about 10 to about 30 consecutive nucleotides from a nucleic acid sequence which is complementary to a nucleic acid sequence selected from the group of sequence consisting of SEQ ID NOs: 1-22 and 25-28 or variants thereof as described herein.

Primer sets can be designed by those of skill in the art using the sequences of SEQ ID NOs: 1-3. Examples of primer pairs useful for the detection methods using PCR are:

For amplication of the non-structural gene of EqPV (SEQ ID NO: 2):

GGAGAAGAGCGCAACAAATGCA (SEQ ID NO: 12) and

AAGACATTTCCGGCCGTGAC (SEQ ID NO: 13);

and

GCGCAACAAATGCAGCGGTTCGA (SEQ ID NO: 14) and

GGCCGTGACGACGGTGATATC (SEQ ID NO: 15).

For amplication of the EqPV Sl-structural gene (SEQ ID NO: 3):

GTCGCTGCATTCTGAGTCC (SEQ ID NO: 16) and

TGGGATTATACTGTCTACGGGT (SEQ ID NO: 17);

and

CTGCATTCTGAGTCCGTGGCC (SEQ ID NO: 18) and

CTGTCTACGGGTATCCCATACGTA (SEQ ID NO: 19).

Primers for quantitative PCR (amplication and detection of entire EqPV genome): qPCR Forward Primer: ATGCAGATGCTTTCCGACC (SEQ ID NO: 20) and qPCR Reverse Primer: GCCCCAGAAACATATGGAAA (SEQ ID NO: 21), used in conjunction with qPCR Probe:

[6-FAM] ACCGT AGCGGATTCGGGATCTGC [B HQ 1 a-6F AM] (SEQ ID NO: 22). In certain aspects, the invention relates to a method for determining the presence of equine parvovirus (EqPV) in a biological sample or biologic or a serum or plasma product, the method comprising: a) contacting nucleic acid from a biological sample or biologic or a serum or plasma product with at least one primer which is a synthetic nucleic acid of an isolated (or non-isolated) nucleic acid which comprises at least 10 consecutive nucleotides of SEQ ID NOs: 1-3; an isolated (or non-isolated) nucleic acid which comprises at least 10 consecutive nucleotides of a nucleic acid complementary to the sequence of any of SEQ ID NOs: 1-3; an isolated (or non-isolated) nucleic acid which comprises at least 10 consecutive nucleotides of a sequence having at least about 60% identity to any of SEQ ID NOs: 1-3; an isolated (or non-isolated) nucleic acid which comprises at least 10 consecutive nucleotides of a sequence having at least about 60% identity to a nucleic acid complementary to the sequence of SEQ ID NOs: 1-3, b) subjecting the nucleic acid and the primer to amplification conditions, and, c) determining the presence or absence of amplification product, wherein the presence of amplification product indicates the presence of DNA or RNA associated with of EqPV the sample.

In certain aspects, the invention relates to a method for determining the presence of EqPV in a biological sample or a biologic or a serum or plasma product, the method comprising: a) contacting nucleic acid from a biological sample or a biologic or a serum or plasma product with at least one primer which is a synthetic nucleic acid of an isolated (or non-isolated) nucleic acid having the sequence of any of SEQ ID NOs: 4-21 ; an isolated (or non-isolated) nucleic acid complementary to the sequence of any of SEQ ID NOs: 4-21 ; an isolated (or non-isolated) nucleic acid having at least about 60% sequence identity to any of SEQ ID NOs: 4-21 ; an isolated (or non-isolated) nucleic acid having at least about 60% sequence identity to a nucleic acid complementary to the sequence of any of SEQ ID NOs: 4- 21 ; an isolated (or non-isolated) nucleic acid which comprises at least 10 consecutive nucleotides of any of SEQ ID NOs: 4-21 ; an isolated (or non-isolated) nucleic acid which comprises at least 10 consecutive nucleotides of a nucleic acid complementary to the sequence of any of SEQ ID NOs: 4-21; an isolated (or non- isolated) nucleic acid which comprises at least 10 consecutive nucleotides of a sequence having at least about 60% identity to any of SEQ ID NOs: 4-21 ; or an isolated (or non-isolated) nucleic acid which comprises at least 10 consecutive nucleotides of a sequence having at least about 60% identity to a nucleic acid complementary to the sequence of any of SEQ ID NOs: 4-21, b) subjecting the nucleic acid and the primer to amplification conditions, and, c) determining the presence or absence of amplification product, wherein the presence of amplification product indicates the presence of RNA associated with of EqPV the sample.

The invention also relates to a method for determining the presence of EqPV in a biological sample or a biologic or a serum or plasma product, the method comprising: a) contacting nucleic acid from a biological sample or a biologic or a serum or plasma product with a primer pair chosen from the group consisting of: SEQ ID NO: 12 and SEQ ID NO: 13; SEQ ID NO: 14 and SEQ ID NO: 15; SEQ ID NO: 16 and SEQ ID NO: 17; SEQ ID NO: 18 and SEQ ID NO: 19; and SEQ ID NO: 20 and SEQ ID NO: 21, b) subjecting the nucleic acid and the primer pair to amplification conditions, and, c) determining the presence or absence of amplification product, wherein the presence of amplification product indicates the presence of RNA associated with of EqPV in the sample.

The method further comprises repeating the amplification method with the same or different set of primers.

In a preferred embodiment, the method for determining the presence of EqPV in a biological sample or a biologic or a serum or plasma product, would utilize primer pair SEQ ID NO: 12 and SEQ ID NO: 13 in a first round of amplification and then contacting the amplification product with primers SEQ ID NO: 14 and SEQ ID NO: 15 for a second round of amplification.

In another preferred embodiment, the method for determining the presence of EqPV in a biological sample or biologic or a serum or plasma product, would utilize primer pair SEQ ID NO: 16 and SEQ ID NO: 17 in a first round of amplification and then contacting the amplification product with primers SEQ ID NO: 18 and SEQ ID NO: 19 for a second round of amplification.

In other aspects, the invention is directed to expression constructs, for example but not limited to plasmids and vectors which comprise the nucleic acid sequence of any of SEQ ID NOs: 1-22 and 25-28, complementary sequences thereof, and/or variants thereof. Such expression constructs can be prepared by any suitable method known in the art. Such expression constructs are suitable for viral nucleic acid and/or protein expression and purification.

In certain aspects, the invention is directed to iRNA molecules which target nucleic acids from EqPV, for example but not limited to any of SEQ ID NOs: 1-3, and variants thereof, and silence a target gene.

In certain aspects, the invention relates to an interfering RNA (iRNA) comprising at least 15 contiguous nucleotides of an isolated (or non-isolated) nucleic acid having the sequence of SEQ ID NOs: 1-22 an isolated (or non-isolated) nucleic acid complementary to the sequence of any of SEQ ID NOs: 1-22; an isolated (or non-isolated) nucleic acid having at least about 60% sequence identity to any of SEQ ID NOs: 1-22; an isolated (or nonisolated) nucleic acid having at least about 60% sequence identity to a nucleic acid complementary to the sequence of any of SEQ ID NOs: 1-22; an isolated (or non-isolated) nucleic acid which comprises at least 10 consecutive nucleotides of any of SEQ ID NOs: 1- 22; an isolated (or non-isolated) nucleic acid which comprises at least 10 consecutive nucleotides of a nucleic acid complementary to the sequence of any of SEQ ID NOs: 1-22; an isolated (or non-isolated) nucleic acid which comprises at least 10 consecutive nucleotides of a sequence having at least about 60% identity to any of SEQ ID NOs: 1-22; an isolated (or non-isolated) nucleic acid which comprises at least 10 consecutive nucleotides of a sequence having at least about 60% identity to a nucleic acid complementary to the sequence of any of SEQ ID NOs: 1-22.

In certain aspects, the invention relates to an interfering RNA (iRNA) comprising a sense strand having at least 15 contiguous nucleotides complementary to the anti-sense strand of a gene from a virus comprising a nucleic acid sequence selected from the group of sequences consisting of any of SEQ ID NOs: 1-22.

In certain aspects, the invention relates to a method for reducing the levels of an

EqPV protein in an animal, viral mRNA in an animal or viral titer in a cell of an animal, the method comprising administering to the animal an iRNA described herein.

An "iRNA agent" (abbreviation for "interfering RNA agent") as used herein, is an RNA agent, which can down-regulate the expression of a target gene, e.g. a EqPV gene. An iRNA agent may act by one or more of a number of mechanisms, including post- transcriptional cleavage of a target mRNA sometimes referred to in the art as RNAi, or pre- transcriptional or pre-translational mechanisms. An iRNA agent can be a double stranded (ds) iRNA agent.

A "ds iRNA agent" (abbreviation for "double stranded iRNA agent"), as used herein, is an iRNA agent which includes more than one, and in certain embodiments two, strands in which interchain hybridization can form a region of duplex structure. A "strand" herein refers to a contigouous sequence of nucleotides (including non-naturally occurring or modified nucleotides). The two or more strands may be, or each form a part of, separate molecules, or they may be covalently interconnected, e.g. by a linker, e.g. a polyethyleneglycol linker, to form but one molecule. At least one strand can include a region which is sufficiently complementary to a target RNA. Such strand is termed the "antisense strand". A second strand comprised in the ds iRNA agent which comprises a region complementary to the antisense strand is termed the "sense strand". However, a ds iRNA agent can also be formed from a single RNA molecule which is, at least partly; self- complementary, forming, e.g., a hairpin or panhandle structure, including a duplex region. In such case, the term "strand" refers to one of the regions of the RNA molecule that is complementary to another region of the same RNA molecule. iRNA agents as described herein, including ds iRNA agents and siRNA agents, can mediate silencing of a gene, e.g., by RNA degradation. For convenience, such RNA is also referred to herein as the RNA to be silenced. Such a gene is also referred to as a target gene. In certain embodiments, the RNA to be silenced is a gene product of a EqPV gene.

As used herein, the phrase "mediates RNAi" refers to the ability of an agent to silence, in a sequence specific manner, a target gene. "Silencing a target gene" means the process whereby a cell containing and/or secreting a certain product of the target gene when not in contact with the agent, will contain and/or secret at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% less of such gene product when contacted with the agent, as compared to a similar cell which has not been contacted with the agent. Such product of the target gene can, for example, be a messenger RNA (mRNA), a protein, or a regulatory element.

In the anti viral uses of the present invention, silencing of a target gene can result in a reduction in "viral titer" in the cell or in the animal, wherein "reduction in viral titer" refers to a decrease in the number of viable virus produced by a cell or found in an organism undergoing the silencing of a viral target gene. Reduction in the cellular amount of virus produced can lead to a decrease in the amount of measurable virus produced in the tissues of an animal undergoing treatment and a reduction in the severity of the symptoms of the viral infection. iRNA agents of the present invention are also referred to as "antiviral iRNA agents".

In other aspects, the invention provides methods for reducing viral titer in an animal, by administering to an animal, at least one iRNA which inhibits the expression of a EqPV gene.

Isolated Polypeptides and Uses Thereof

In certain aspects, the invention is directed to an EqPV isolated amino acid sequence as provided in any of SEQ ID NOs: 23-24, variants and fragments thereof.

In certain aspects, the invention is directed to an EqPV isolated amino acid sequence encoded by the nucleic acid of SEQ ID NOs: 1-3, variants and fragments thereof.

The invention is also directed to isolated polypeptides and variants, fragments, and derivatives thereof. These polypeptides may be useful for multiple applications, including, but not limited to, generation of antibodies and generation of immunogenic compositions. For example, the invention is directed to an isolated polypeptides of SEQ ID NOs: 23-24. For example, the invention is also directed to any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1-3. A peptide of at least 8 amino acid residues in length can be recognized by an antibody (MacKenzie et al., (1984) Biochemistry 23:6544-6549). In certain embodiments, the invention is directed to fragments of the polypeptides described herein, that can, for example, be used to generate anytibodies.

In certain aspects, the invention relates to an isolated polypeptide having the sequence of any of SEQ ID NOs: 23-24. In certain aspects, the invention relates to an isolated polypeptide having at least about 80% sequence identity to the polypeptide of SEQ ID NOs: 23-24. In certain aspects, the invention relates to an isolated polypeptide comprising at least 8 consecutive amino acids of the polypeptide of SEQ ID NOs: 23-24. In certain aspects, the invention relates to an isolated polypeptide comprising at least 8 amino acids having at least about 80% identity to the sequence of the polypeptide of SEQ ID NOs: 23-24.

SEQ ID NO: 23 is the amino acid sequence of the NSl-non structural protein, which is an immunogenic polypeptide sequence.

SEQ ID NO: 24 is the amino acid sequence of the SI -structural protein, which is an immunogenic, capsid polypeptide sequence.

In one aspect, the invention is directed to polypeptide variants of an isolated polypeptide of any of SEQ ID NOs: 23-24. Variants of the isolated polypeptides of any of SEQ ID NOs: 23-24 include, but are not limited to, polypeptide sequences having at least from about 50% to about 55% identity to that of an isolated polypeptide of SEQ ID NOs: 23- 24. Variants of an isolated polypeptide of SEQ ID NOs: 23-24 include, but are not limited to, polypeptide sequences having at least from about 55.1 % to about 60% identity to that of an isolated polypeptide of SEQ ID NOs: 23-24. Variants of an isolated polypeptide of SEQ ID NOs: 23-24 include, but are not limited to, polypeptide sequences having at least from about 60.1% to about 65% identity to that of an isolated polypeptide of SEQ ID NOs: 23-24. Variants of an isolated polypeptide of SEQ ID NOs: 23-24 include, but are not limited to, polypeptide sequences having at least from about 65.1 % to about 70% identity to that of an isolated polypeptide of SEQ ID NOs: 23-24. Variants of an isolated polypeptide of SEQ ID NOs: 23-24 include, but are not limited to, polypeptide having at least from about 70.1% to about 75% identity to that of an isolated polypeptide of SEQ ID NOs: 23-24. Variants of an isolated polypeptide of SEQ ID NOs: 23-24 include, but are not limited to, polypeptide sequences having at least from about 75.1% to about 80% identity to that of an isolated polypeptide of SEQ ID NOs: 23-24. Variants of an isolated polypeptide of SEQ ID NOs: 23- 24 include, but are not limited to, polypeptide sequences having at least from about 80.1 % to about 85% identity to that of an isolated polypeptide of SEQ ID NOs: 23-24. Variants of an isolated polypeptide of SEQ ID NOs: 23-24 include, but are not limited to, polypeptide sequences having at least from about 85.1% to about 90% identity to that of an isolated polypeptide of SEQ ID NOs: 23-24. Variants of an isolated polypeptide of SEQ ID NOs: 23- 24 include, but are not limited to, polypeptide sequences having at least from about 90.1 % to about 95% identity to that of an isolated polypeptide of SEQ ID NOs: 23-24. Variants of an isolated polypeptide of SEQ ID NOs: 23-24 include, but are not limited to, polypeptide sequences having at least from about 95.1% to about 97% identity to that of an isolated polypeptide of SEQ ID NOs: 23-24. Variants of an isolated polypeptide of SEQ ID NOs: 23- 24 include, but are not limited to, polypeptide sequences having at least from about 97.1% to about 99% identity to that of an isolated polypeptide of SEQ ID NOs: 23-24.

In certain aspects, the invention relates to an isolated polypeptide having at least about 80% sequence identity to the polypeptide encoded by the nucleic acid of SEQ ID NOs: 1-3. In certain aspects, the invention relates to an isolated polypeptide having at least about 80% sequence identity to the polypeptide encoded by a nucleic acid complementary to the sequence of SEQ ID NOs: 1-3. In certain aspects, the invention relates to an isolated polypeptide comprising at least 8 consecutive amino acids of the polypeptide encoded by the nucleic acid of SEQ ID NOs: 1-3. In certain aspects, the invention relates to an isolated polypeptide comprising at least 8 consecutive amino acids of the polypeptide encoded by a nucleic acid complementary to the sequence of SEQ ID NOs: 1-3. In certain aspects, the invention relates to an isolated polypeptide comprising at least 8 amino acids having at least about 80% identity to the sequence of the polypeptide encoded by the nucleic acid of SEQ ID NOs: 1-3. In certain aspects, the invention relates to an isolated polypeptide comprising at least 8 amino acids having at least about 80% identity to the sequence of the polypeptide encoded by a nucleic acid complementary to the sequence of SEQ ID NOs: 1-3.

In one aspect, the invention is directed to polypeptide variants of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1-3. Variants of any one of the isolated polypeptides encoded by the nucleic sequence acid of SEQ ID NOs: 1-3 include, but are not limited to, polypeptide sequences having at least from about 50% to about 55% identity to that of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1-3. Variants of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1-3 include, but are not limited to, polypeptide sequences having at least from about 55.1 % to about 60% identity to that of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1-3. Variants of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1-3 include, but are not limited to, polypeptide sequences having at least from about 60.1% to about 65% identity to that of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1-3. Variants of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1-3 include, but are not limited to, polypeptide sequences having at least from about 65.1 % to about 70% identity to that of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1-3. Variants of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1-3 include, but are not limited to, polypeptide having at least from about 70.1% to about 75% identity to that of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1-3. Variants of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1-3 include, but are not limited to, polypeptide sequences having at least from about 75.1% to about 80% identity to that of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1-3. Variants of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1-3 include, but are not limited to, polypeptide sequences having at least from about 80.1% to about 85% identity to that of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1-3. Variants of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1-3 include, but are not limited to, polypeptide sequences having at least from about 85.1% to about 90% identity to that of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1-3. Variants of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1-3 include, but are not limited to, polypeptide sequences having at least from about 90.1% to about 95% identity to that of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1-3. Variants of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1-3 include, but are not limited to, polypeptide sequences having at least from about 95.1% to about 97% identity to that of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1-3. Variants of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1-3 include, but are not limited to, polypeptide sequences having at least from about 97.1% to about 99% identity to that of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1-3.

The invention is directed to a polypeptide sequence comprising from about 10 to about 50 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 23-24. The invention is directed to a polypeptide sequence comprising from about 10 to about 100 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 23-24. The invention is directed to a polypeptide sequence comprising from about 10 to about 150 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 23-24. The invention is directed to a polypeptide sequence comprising from about 10 to about 200 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 23-24. The invention is directed to a polypeptide sequence comprising from about 10 to about 250 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 23-24. The invention is directed to a polypeptide sequence comprising from about 10 to about 300 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 23-24. The invention is directed to a polypeptide sequence comprising from about 10 to about 350 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 23-24. The invention is directed to a polypeptide sequence comprising from about 10 to about 400 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 23-24. The invention is directed to a polypeptide sequence comprising from about 10 to about 450 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 23-24. The invention is directed to a polypeptide sequence comprising from about 10 to about 500 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 23-24. The invention is directed to a polypeptide sequence comprising from about 10 to about 600 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 23-24. The invention is directed to a polypeptide sequence comprising from about 10 to about 700 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 23-24. The invention is directed to a polypeptide sequence comprising from about 10 to about 800 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 23-24. The invention is directed to a polypeptide sequence comprising from about 10 to about 900 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 23-24. The invention is directed to a polypeptide sequence comprising from about 10 to about 974 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 23-24.

The invention is further directed to polypeptide sequences having from about 50% to about 99% identity to a polypeptide sequence comprising from about 10 to about 50 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 23-24. The invention is further directed to polypeptide sequences having from about 50% to about 99% identity to a polypeptide sequence comprising from about 10 to about 100 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 23-24. The invention is further directed to polypeptide sequences having from about 50% to about 99% identity to a polypeptide sequence comprising from about 10 to about 150 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 23-24. The invention is further directed to polypeptide sequences having from about 50% to about 99% identity to a polypeptide sequence comprising from about 200 to about 50 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 23-24. The invention is further directed to polypeptide sequences having from about 50% to about 99% identity to a polypeptide sequence comprising from about 10 to about 350 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 23-24. The invention is further directed to polypeptide sequences having from about 50% to about 99% identity to a polypeptide sequence comprising from about 10 to about 400 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 23-24. The invention is further directed to polypeptide sequences having from about 50% to about 99% identity to a polypeptide sequence comprising from about 10 to about 450 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 23-24. The invention is further directed to polypeptide sequences having from about 50% to about 99% identity to a polypeptide sequence comprising from about 10 to about 500 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 23-24. The invention is further directed to polypeptide sequences having from about 50% to about 99% identity to a polypeptide sequence comprising from about 10 to about 600 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 23-24. The invention is further directed to polypeptide sequences having from about 50% to about 99% identity to a polypeptide sequence comprising from about 10 to about 700 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 23-24. The invention is further directed to polypeptide sequences having from about 50% to about 99% identity to a polypeptide sequence comprising from about 10 to about 800 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 23-24. The invention is further directed to polypeptide sequences having from about 50% to about 99% identity to a polypeptide sequence comprising from about 10 to about 900 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 23-24. The invention is further directed to polypeptide sequences having from about 50% to about 99% identity to a polypeptide sequence comprising from about 10 to about 974 consecutive amino acids of an isolated polypeptide of SEQ ID NOs: 23-24. In certain embodiments, the invention is directed to isolated and purified peptides.

The invention is directed to a polypeptide sequence comprising from about 10 to about 50 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1-3. The invention is directed to a polypeptide sequence comprising from about 10 to about 100 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1-3. The invention is directed to a polypeptide sequence comprising from about 10 to about 150 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1- 3. The invention is directed to a polypeptide sequence comprising from about 10 to about 200 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1-3. The invention is directed to a polypeptide sequence comprising from about 10 to about 250 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1-3. The invention is directed to a polypeptide sequence comprising from about 10 to about 300 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1-3. The invention is directed to a polypeptide sequence comprising from about 10 to about 350 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1- 3. The invention is directed to a polypeptide sequence comprising from about 10 to about 400 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1-3. The invention is directed to a polypeptide sequence comprising from about 10 to about 450 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1-3. The invention is directed to a polypeptide sequence comprising from about 10 to about 500 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1-3. The invention is directed to a polypeptide sequence comprising from about 10 to about 600 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1- 3. The invention is directed to a polypeptide sequence comprising from about 10 to about 700 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1-3. The invention is directed to a polypeptide sequence comprising from about 10 to about 800 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1-3. The invention is directed to a polypeptide sequence comprising from about 10 to about 900 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1-3. The invention is directed to a polypeptide sequence comprising from about 10 to about 974 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1- 3. The invention is directed to a polypeptide sequence comprising from about 10 to about 1000 or more consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1-3. The invention is further directed to polypeptide sequences having from about 50% to about 99% identity to a polypeptide sequence comprising from about 10 to about 50 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1-3. The invention is further directed to polypeptide sequences having from about 50% to about 99% identity to a polypeptide sequence comprising from about 10 to about 100 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1-3. The invention is further directed to polypeptide sequences having from about 50% to about 99% identity to a polypeptide sequence comprising from about 10 to about 150 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1-3. The invention is further directed to polypeptide sequences having from about 50% to about 99% identity to a polypeptide sequence comprising from about 200 to about 50 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1-3. The invention is further directed to polypeptide sequences having from about 50% to about 99% identity to a polypeptide sequence comprising from about 10 to about 350 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1- 3. The invention is further directed to polypeptide sequences having from about 50% to about 99% identity to a polypeptide sequence comprising from about 10 to about 400 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1-3. The invention is further directed to polypeptide sequences having from about 50% to about 99% identity to a polypeptide sequence comprising from about 10 to about 450 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1-3. The invention is further directed to polypeptide sequences having from about 50% to about 99% identity to a polypeptide sequence comprising from about 10 to about 500 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1-3. The invention is further directed to polypeptide sequences having from about 50% to about 99% identity to a polypeptide sequence comprising from about 10 to about 600 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1-3. The invention is further directed to polypeptide sequences having from about 50% to about 99% identity to a polypeptide sequence comprising from about 10 to about 700 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1- 3. The invention is further directed to polypeptide sequences having from about 50% to about 99% identity to a polypeptide sequence comprising from about 10 to about 800 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1-3. The invention is further directed to polypeptide sequences having from about 50% to about 99% identity to a polypeptide sequence comprising from about 10 to about 900 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1-3. The invention is further directed to polypeptide sequences having from about 50% to about 99% identity to a polypeptide sequence comprising from about 10 to about 974 consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1-3. The invention is further directed to polypeptide sequences having from about 50% to about 99% identity to a polypeptide sequence comprising from about 10 to about 1000 or more consecutive amino acids from any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1- 3. In certain embodiments, the invention is directed to isolated and purified peptides.

In certain embodiments, the polypeptides of the present invention can be suitable for use as antigens to detect antibodies against SEQ ID NOs: 23-24, and variants thereof. In other embodiments, the polypeptides of the present invention which comprise antigenic determinants can be used in various immunoassays to identify animals exposed to and/or samples which comprise SEQ ID NOs: 23-24, and variants thereof.

Antibodies, Methods of Making, and Methods of Using

In another aspect, the invention is directed to an antibody which specifically binds to amino acids from the polypeptide of an isolated polypeptide of SEQ ID NOs: 23-24. In one embodiment the antibody is purified. The antibodies can be polyclonal or monoclonal. The antibodies can also be chimeric (i.e., a combination of sequences from more than one species, for example, a chimeric mouse-human immunoglobulin), humanized or fully-human. Species specific antibodies avoid certain of the problems associated with antibodies that possess variable and/or constant regions form other species. The presence of such protein sequences form other species can lead to the rapid clearance of the antibodies or can lead to the generation of an immune response against the antibody by an antibody.

In another aspect, the invention is directed to an antibody which specifically binds to amino acids from the polypeptide of any isolated polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1-3. In one embodiment the antibody is purified. The antibodies can be polyclonal or monoclonal. The antibodies can also be chimeric (i.e., a combination of sequences from more than one species, for example, a chimeric mouse-human immunoglobulin), humanized or fully-human. Species specific antibodies avoid certain of the problems associated with antibodies that possess variable and/or constant regions form other species. The presence of such protein sequences form other species can lead to the rapid clearance of the antibodies or can lead to the generation of an immune response against the antibody by an antibody.

An antibody described in this application can include or be derived from any mammal, such as but not limited to, a bird, a dog, a human, a mouse, a rabbit, a rat, a rodent, a primate, or any combination thereof and includes isolated avian, human, primate, rodent, mammalian, chimeric, humanized and/or CDR-grafted or CDR-adapted antibodies, immunoglobulins, cleavage products and other portions and variants thereof.

Any method known in the art for producing antibodies can be used to generate the antibodies described herein. Examplary methods include animal inoculation, phage diplay, transgenic mouse technology and hybridoma techonology.

The antibodies of the present invention can be used to modulate the activity of any polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1-3, variants or fragments thereof. In certain aspects, the invention is directed to a method for treating an animal, the method comprising administering to the animal an antibody which specifically binds to amino acids from the polypeptide of any polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1-3. In certain embodiments, antibody binding to the polypeptide of any polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1-3 may interfere or inhibit the function of the polypeptide, thus providing a method to inhibit virus propagation and spreading.

The antibodies of the present invention can be used to modulate the activity of any polypeptide of any of SEQ ID NOs: 23-24, variants or fragments thereof. In certain aspects, the invention is directed to a method for treating an animal, the method comprising administering to the animal an antibody which specifically binds to amino acids from the polypeptide of any of SEQ ID NOs: 23-24. In certain embodiments, antibody binding to the polypeptide of any of SEQ ID NOs: 23-24 may interfere or inhibit the function of the polypeptide, thus providing a method to inhibit virus propagation and spreading.

In other embodiments, the antibodies of the invention can be used to purify a polypeptide of SEQ ID NOs: 23-24, variants or fragments thereof. In other embodiments, the antibodies of the invention can be used to identify expression and localization of the polypeptide of SEQ ID NOs: 23-24, variants, fragments or domains thereof. Analysis of expression and localization of the polypeptide of SEQ ID NOs: 23-24 can be useful in determining potential role of the polypeptide of SEQ ID NOs: 23-24.

In other embodiments, the antibodies of the invention can be used to purify polypeptides of any polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1-3, variants or fragments thereof. In other embodiments, the antibodies of the invention can be used to identify expression and localization of the polypeptide of any polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1-3, variants, fragments or domains thereof.

Analysis of expression and localization of the polypeptide of any polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1-3 can be useful in determining potential role of the polypeptide of any polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1-3.

In other embodiments, the antibodies of the present invention can be used in various immunoassays to identify animals exposed to and/or samples which comprise antigens from EqPV represented by SEQ ID NOs: 23-24, and variants thereof.

In other embodiments, the antibodies of the present invention can be used in various immunoassays to identify animals exposed to and/or samples which comprise antigens from EqPV represented by any of SEQ ID NOs: 1-22 and 25-28, and variants thereof. Samples include biological samples as defined as a sample of tissue or fluid obtained from an animal, e.g., horse, and includes but is not limited to blood, plasma, and liver tissue, and serum, and biologicas and serum and plasma products derived from an animal such as an equine.

Any suitable immunoassay which can lead to formation of antigen-antibody complex can also be used. Variations and different formats of immunoassays, for example but not limited to ELISA, lateral flow assays for detection of analytes in samples, immunoprecipitation, and Luciferase Immunoprecipitation Systems (LIPS) are known in the art. In various embodiments, the antigen and/or the antibody can be labeled by any suitable label or method known in the art. For example enzymatic immunoassays may use solid supports, or immunoprecipitation. Immnunoassays which amplify the signal from the antigen-antibody immune complex can also be used with the methods described herein.

In certain aspects the invention provides methods for assaying a sample to determine the presence or absence of a EqPV comprising SEQ ID NOs: 23-24, as provided by the invention, and variants thereof. In certain embodiments, methods for assaying a sample, include, but are not limited to, methods which can detect the presence of nucleic acids, methods which can detect the presence of antigens, methods which can detect the presence of antibodies against antigens from a polypeptide of SEQ ID NOs: 23-24, methods which can detect the presence of a polypeptide of SEQ ID NOs: 23-24 as provided by the invention, and fragements and variants thereof.

In certain aspects the invention provides methods for assaying a sample to determine the presence or absence of a EqPV comprising SEQ ID NOs: 1-3, as provided by the invention, and variants thereof. In certain embodiments, methods for assaying a sample, include, but are not limited to, methods which can detect the presence of nucleic acids, methods which can detect the presence of antigens, methods which can detect the presence of antibodies against antigens from polypeptides encoded by SEQ ID NOs: 1-3, or any polypeptide encoded by the nucleic sequence acid of SEQ ID NOs: 1-3, as provided by the invention, and fragments and variants thereof.

In certain aspects, the invention relates to a method for determining whether or not a sample contains EqPV, the method comprising: a) contacting a sample with an antibody that specifically binds to a polypeptide of any of an isolated (or non-isolated) polypeptide of SEQ ID NOs: 23-24; an isolated (or non- isolated) polypeptide having at least about 80% sequence identity to the polypeptide of SEQ ID NOs: 23-24; an isolated (or non-isolated) polypeptide comprising at least 8 consecutive amino acids of the polypeptide of SEQ ID NOs: 23-24; or an isolated (or non-isolated) polypeptide comprising at least 8 amino acids having at least about 80% identity to the sequence of the polypeptide of SEQ ID NOs: 23-24; and b) determining whether or not the antibody binds to an antigen in the biological sample, wherein binding indicates that the biological sample contains EqPV. In certain embodiments, the determining comprises use of a lateral flow assay or ELISA.

In certain aspects, the invention relates to a method for determining whether or not a sample contains EqPV, the method comprising: a) contacting a sample with an antibody that specifically binds to a polypeptide encoded by the nucleic acid of SEQ ID NOs: 1-3; an isolated (or non-isolated) polypeptide having at least about 80% sequence identity to a polypeptide encoded by the nucleic acid of SEQ ID NOs: 1-3; an isolated (or non-isolated) polypeptide comprising at least 8 consecutive amino acids of a polypeptide encoded by the nucleic acid of SEQ ID NOs: 1-3; or an isolated (or non-isolated) polypeptide comprising at least 8 amino acids having at least about 80% identity to the sequence of a polypeptide encoded by the nucleic acid of SEQ ID NOs: 1-3; and b) determining whether or not the antibody binds to an antigen in the biological sample, wherein binding indicates that the biological sample contains EqPV. In certain embodiments, the determining comprises use of a lateral flow assay or ELISA.

In certain aspects the invention provides methods for assaying a sample to determine the presence or absence of a EqPV by detecting the presence of antibodies against antigens from polypeptides encoded by any of SEQ ID NOs: 23-24. In one embodiment the antibody that is detected is against the NS1 (Non-structural protein) gene protein (SEQ ID NO: 23). In one embodiment the antibody that is detected is against the SI (structural protein) gene protein (SEQ ID NO: 24). In one embodiment, antibodies against antigens are detected by immunoassay. In one embodiment, the sample is equine serum. In one embodiment, complex formation between an antibody to the antigen and the antigen is detected.

In certain aspects, the invention relates to a method for determining whether or not a sample has been infected by EqPV, the method comprising: a) determining whether or not a sample contains antibodies that specifically bind to a polypeptide of any one of an isolated (or non-isolated) polypeptide of SEQ ID NOs: 23-24.; an isolated (or non-isolated) polypeptide having at least about 80% sequence identity to the polypeptide of SEQ ID NOs: 23-24; an isolated (or non-isolated) polypeptide comprising at least 8 consecutive amino acids of the polypeptide of SEQ ID NOs: 23-24; or an isolated (or non-isolated) polypeptide comprising at least 8 amino acids having at least about 80% identity to the sequence of the polypeptides of SEQ ID NOs: 23-24.

In certain aspects, the invention relates to a method for determining whether or not a sample has been infected by EqPV, the method comprising: a) determining whether or not a sample contains antibodies that specifically bind to a polypeptide encoded by the nucleic acid of SEQ ID NOs: 1-3; an isolated (or non-isolated) polypeptide having at least about 80% sequence identity to a polypeptide encoded by the nucleic acid of SEQ ID NOs: 1-3; an isolated (or non-isolated) polypeptide comprising at least 8 consecutive amino acids of a polypeptide encoded by the nucleic acid of SEQ ID NOs: 1-3; or an isolated (or non-isolated) polypeptide comprising at least 8 amino acids having at least about 80% identity to the sequence of a polypeptide encoded by the nucleic acid of SEQ ID NOs: 1-3.

Kits

Also provided for are kits for practicing one or more of the above-described methods.

The subject reagents and kits thereof may vary greatly. Reagents of interest include reagents specifically designed for use in determining if an animal has EqPV or biologic or a serum or plasma product contain EqPV. One type of regent that is specifically tailored for the detection of EqPV is at least one oligonucleotide primer specific SEQ ID NOs: 1-3 to amplify nucleic acid obtained from a biological sample, and, optionally, at least one primer suitable to enable sequencing of the amplified nucleic acid and determination of the presence of the mutation.

Examples of primers that can be included as reagents are:

GGAGAAGAGCGCAACAAATGCA (SEQ ID NO: 12) and

AAGACATTTCCGGCCGTGAC (SEQ ID NO: 13);

and

GCGCAACAAATGCAGCGGTTCGA (SEQ ID NO: 14) and

GGCCGTGACGACGGTGATATC (SEQ ID NO: 15);

and

GTCGCTGCATTCTGAGTCC (SEQ ID NO: 16) and

TGGGATTATACTGTCTACGGGT (SEQ ID NO: 17);

and

CTGCATTCTGAGTCCGTGGCC (SEQ ID NO: 18) and

CTGTCTACGGGTATCCCATACGTA (SEQ ID NO: 19);

and

qPCR Forward Primer: ATGCAGATGCTTTCCGACC (SEQ ID NO: 20) and qPCR Reverse Primer: GCCCCAGAAACATATGGAAA (SEQ ID NO: 21), used in conjunction with qPCR Probe:

[6-FAM] ACCGT AGCGGATTCGGGATCTGC [B HQ 1 a-6F AM] (SEQ ID NO: 22).

A further type of reagent is one or more nucleic acid probes comprising or complementary SEQ ID NOs: 1-3. In one embodiment, one or more probes are in an array formation. A variety of different array formats are known in the art with a wide variety of different probe structures, substrate compositions, and attachment technologies. In some embodiments, the arrays include at least 2 nucleic acid probes, in a more preferred embodiment, at least 5 nucleic acid probes, in a more preferred embodiment, at least 10 nucleic acid probes, in a more preferred embodiment, at least 15 nucleic acid probes, in a more preferred embodiment, at least 25 nucleic acid probes, and in a most preferred embodiment, at least 50 nucleic acid probes, said nucleic acid probes comprising or complementary to SEQ ID NOs: 1-3.

A further type of reagent is one or more antibodies as described herein that specifically binds to amino acids from the polypeptide of an isolated polypeptide of SEQ ID NOs: 23 or 24, fragments and variants thereof, or amino acids from the polypeptide of any isolated polypeptide encoded by the nucleic sequence acid of any of SEQ ID NOs: 1-3, fragments and variants thereof.

The kit of the invention may include the above-described primers, probes, arrays, and antibodies as well as additional reagents employed in the various methods, such as: labeling reagents; enzymes such as reverse transcriptase, DNA and RNA polymerases, and the like; various buffers, such as hybridization and washing buffers; signal generation and detection reagents; and reagents for isolation of nucleic acid from a sample. In addition, the kit may include instructions for practicing the methods of the present invention.

The invention also covers systems for practicing one or more of the above-described methods. The subject systems may vary greatly but typically include at least one element to detect EqPV, i.e., one or more reagents described above for detection of EqPV, including primers, probes, arrays, antibodies, and additional reagents for practicing the methods of the invention.

Immunogenic compositions. Methods of Making, and Methods of Using

As used herein, the term immunogenic composition refers to a composition capable of inducing an immunogenic response in an animal or a cell. As used herein, reference to an immunogenic composition can include a vaccine.

In one embodiment, the immunogenic compositions are capable of ameliorating the symptoms of a EqPV infection and/or of reducing the duration of a EqPV associated disease.

In another embodiment, the immunogenic compositions are capable of inducing protective immunity against EqPV associated disease. In a further embodiment, the immunogenic composition can be used to prevent and/or treat an infection caused by EqPV in an animal.

The immunogenic compositions of the invention can be effective against the EqPV viruses disclosed herein, and may also be cross-reactive with, and effective against, multiple different clades and strains of EqPV, and against other parvoviruses.

In certain aspects, the present invention provides immunogenic compositions capable of inducing an immune response against EqPV including EqPV of the invention comprising

SEQ ID NOs: 1-3, or comprising a cDNA sequence complementary to the sense or an anti- sense strand of SEQ ID NOs: 1-3, or comprising a polypeptide encoded by SEQ ID NOs: 1-3, or a cDNA sequence complementary to the sense or an anti-sense strand of SEQ ID NOs: 1-

3, or comprising a polypeptide of any of SEQ ID NOs: 23-24. In certain aspects, the invention relates to an immunogenic composition comprising at least about 24 consecutive nucleotides from an isolated (or non-isolated) nucleic acid having the sequence of SEQ ID NOs: 1-3; an isolated (or non-isolated) nucleic acid complementary to the sequence of SEQ ID NOs: 1-3; an isolated (or non-isolated) nucleic acid having at least about 60% sequence identity to SEQ ID NOs: 1-3; an isolated (or non-isolated) nucleic acid having at least about 60% sequence identity to a nucleic acid complementary to the sequence of SEQ ID NOs: 1-3; an isolated (or non-isolated) nucleic acid which comprises at least 24 consecutive nucleotides of SEQ ID NOs: 1-3; an isolated (or non-isolated) nucleic acid which comprises at least 24 consecutive nucleotides of a nucleic acid complementary to the sequence of SEQ ID NOs: 1-3; an isolated (or non-isolated) nucleic acid which comprises at least 24 consecutive nucleotides of a sequence having at least about 60% identity to SEQ ID NOs: 1-3; an isolated (or non-isolated) nucleic acid which comprises at least 24 consecutive nucleotides of a sequence having at least about 60% identity to a nucleic acid complementary to the sequence of SEQ ID NOs: 1-3, or a polypeptide encoded from any such nucleic acid.

In certain aspects, the invention relates to an immunogenic composition comprising an isolated (or non-isolated) polypeptide of SEQ ID NOs: 23-24; an isolated (or non-isolated) polypeptide having at least about 80% sequence identity to the polypeptide of SEQ ID NOs: 23-24; an isolated (or non-isolated) polypeptide comprising at least 8 consecutive amino acids of the polypeptide of SEQ ID NOs: 23-24; an isolated (or non-isolated) polypeptide comprising at least 8 amino acids having at least about 80% identity to the sequence of the polypeptide of SEQ ID NOs: 23-24. In certain aspects, the invention relates to an immunogenic composition comprising at least 8 consecutive amino acids of a polypeptide described herein.

In other embodiments, the present invention provides immunogenic compositions for inducing an immune response in an animal, wherein the composition includes a recombinant or isolated capsid polypeptide derived from an equine parvovirus; and a pharmaceutically acceptable vehicle or diluent. In one embodiment, this immunogenic composition is a vaccine composition. In one embodiment, the capsid polypeptide employed in the immunogenic compositions is SEQ ID NO: 24.

In other embodiments, the present invention provides immunogenic compositions for inducing an immune response in an animal, wherein the composition includes a recombinant or isolated polypeptide derived from an equine parvovirus; and a pharmaceutically acceptable vehicle or diluent. In one embodiment, this immunogenic composition is a vaccine composition. In one embodiment, the polypeptide employed in the immunogenic compositions is SEQ ID NO: 23.

In certain embodiments, the polypeptide sequence corresponds to the immunogenic protein in both the whole virus and subunit compositions provided by the present invention.

As used herein, the term "capsid polypeptide sequence", and the like, refer to polypeptide sequences and includes derivatives of capsid polypeptide sequences, such as including, but not limited to, capsid polypeptide sequences which include Histidine tags, Xpress™ tags, signal sequences or other epitope tags at the N- and/or C-terminal ends.

The capsid polypeptide composition can be used to protect equine or other animals against EqPV. Thus, the present invention also provides a method of immunizing an equine or other animal against EqPV, wherein the method includes administering to the equine or other animal the capsid polypeptide composition. In certain embodiments, the equine parvovirus from which the capsid polypeptide is derived encodes a polypeptide sequence having at least 90% identity to the amino acid sequence SEQ ID NO: 24. In one embodiment, the capsid polypeptide composition includes an adjuvant.

As used herein, the term "non-capsid polypeptide sequence", and the like, refer to polypeptide sequences and includes derivatives of non-capsid polypeptide sequences, such as including, but not limited to, capsid polypeptide sequences which include Histidine tags, Xpress™ tags, signal sequences or other epitope tags at the N- and/or C-terminal ends.

The non-capsid polypeptide composition can be used to protect equine or other animals against EqPV. Thus, the present invention also provides a method of immunizing an equine or other animal against EqPV, wherein the method includes administering to the equine or other animal the non-capsid polypeptide composition. In certain embodiments, the equine parvovirus from which the non-capsid polypeptide is derived encodes a polypeptide sequence having at least 90% identity to the amino acid sequence SEQ ID NO: 23. In one embodiment, the non-capsid polypeptide composition includes an adjuvant.

The types of immunogenic composition encompassed by the invention include, but are not limited to, attenuated live viral immunogenic compositions, and inactivated (killed) viral immunogenic compositions, where the viruses are no longer capable of replicating or causing disease in animals, but still induce an immune response in an animal.

Inactivated immunogenic composition can be made by methods well known in the art. For example, once EqPV is propagated to high titers, EqPV antigenic mass could be obtained by methods well known in the art. For example, the EqPV viral antigenic mass may be obtained by dilution, concentration, or extraction. All of these methods have been employed to obtain appropriate EqPV antigenic mass to produce immunogenic compositions. EqPV may be inactivated by treatment with formalin (e.g. 0.1-10%), betapropriolactone (BPL) (e.g. 0.01-10%), or with binary ethyleneimine (BEI) (e.g. 1-10 mM), or using other methods known to those skilled in the art.

In addition to killed EqPV production, various means of attenuation are also possible and are well known and described in the art. Attenuation leading to modified live immunogenic compositions can also be used in conjunction with the compositions and methods described herein. Methods of attenuation suitable for use with the viruses described herein include continuous passaging in cell culture, continuous passaging in animals, various methods for generating genetic modifications and ultraviolet or chemical mutagenesis and cold-adaptation.

The EqPV viruses of the invention may be attenuated by removal or disruption of those viral sequences whose products cause or contribute to the disease and symptoms associated with EqPV infection, and leaving intact those sequences required for viral replication. In this way an attenuated EqPV can be produced that replicates in animals, and induces an immune response in animals, but which does not induce the deleterious disease and symptoms usually associated with EqPV infection. One of skill in the art can determine which EqPV sequences can or should be removed or disrupted, and which sequences should be left intact, in order to generate an attenuated EqPV suitable for use as an immunogenic composition.

The immunogenic compositions of the invention may comprise subunit immunogenic compositions. Subunit immunogenic compositions include nucleic acid immunogenic compositions such as DNA immunogenic compositions, which contain nucleic acids that encode one or more viral proteins or subunits, or portions of those proteins or subunits. When using such immunogenic compositions, the nucleic acid is administered to the animal, and the immunogenic proteins or peptides encoded by the nucleic acid are expressed in the animal, such that an immune response against the proteins or peptides is generated in the animal. Subunit immunogenic compositions may also be proteinaceous immunogenic compositions, which contain the viral proteins or subunits themselves, or portions of those proteins or subunits.

To make the nucleic acid and DNA immunogenic compositions of the invention, EqPV sequences disclosed herein may be incorporated into a plasmid or expression vector containing the nucleic acid that encodes the viral protein or peptide. Any suitable plasmid or expression vector capable of driving expression of the protein or peptide in the animal may be used. Such plasmids and expression vectors should include a suitable promoter for directing transcription of the nucleic acid. The nucleic acid sequence(s) that encodes EqPV protein or peptide may also be incorporated into a suitable recombinant virus for administration to the animal. Examples of suitable viruses include, but are not limited to, vaccinia viruses, retroviruses, adenoviruses and adeno-associated viruses. One of skill in the art could readily select a suitable plasmid, expression vector, or recombinant virus for delivery of EqPV nucleic acid sequences of the invention.

To produce the proteinaceous immunogenic compositions of the invention, EqPV nucleic acid sequences of the invention are delivered to cultured cells, for example by transfecting cultured cells with plasmids or expression vectors containing EqPV nucleic acid sequences, or by infecting cultured cells with recombinant viruses containing EqPV nucleic acid sequences. EqPV proteins or peptides may then be expressed in the cultured cells and purified. The purified proteins can then be incorporated into compositions suitable for administration to animals. Methods and techniques for expression and purification of recombinant proteins are well known in the art, and any such suitable methods may be used.

Subunit immunogenic compositions of the present invention may encode or contain any of EqPV proteins or peptides described herein, or any portions, fragments, derivatives or mutants thereof, that are immunogenic in an animal. One of skill in the art can readily test the immunogenicity of EqPV proteins and peptides described herein, and can select suitable proteins or peptides to use in subunit immunogenic compositions.

The immunogenic compositions of the invention comprise at least one EqPV-derived immunogenic component, such as those described herein. The compositions may also comprise one or more additives including, but not limited to, one or more pharmaceutically acceptable carriers, buffers, stabilizers, diluents, preservatives, solubilizers, liposomes or immunomodulatory agents. Suitable immunomodulatory agents include, but are not limited to, adjuvants, cytokines, polynucleotide encoding cytokines, and agents that facilitate cellular uptake of EqPV-derived immunogenic component.

Immunogenic compositions for use in accordance with the present invention thus may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used to induce an immunogenic response. These immunogenic compositions may be manufactured in a manner that is itself known, e.g. by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. Proper formulation is dependent upon the route of administration chosen. When a therapeutically effective amount of protein or other active ingredient of the present invention is administered orally, protein or other active ingredient of the present invention can be in the form of a tablet, capsule, powder, solution or elixr.

When administered in tablet form, the immunogenic composition of the invention may additionally contain a solid carrier such as a gelatin or an adjuvant. The tablet, capsule, and powder contain from about 5 to 95% protein or other active ingredient of the present invention, and from about 25 to 90% protein or other active ingredient of the present invention. When administered in liquid form, a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added. The liquid form of the immunogenic composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol. When administered in liquid form, the immunogenic composition contains from about 0.5 to 90% by weight of protein or other active ingredient of the present invention, and from about 1 to 50% protein or other active ingredient of the present invention.

When a therapeutically effective amount of protein or other active ingredient of the present invention is administered by intravenous, cutaneous or subcutaneous injection, protein or other active ingredient of the present invention will be in the form of a pyrogen- free, parenterally acceptable aqueous solution. The preparation of such parenterally acceptable protein or other active ingredient solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art. One immunogenic composition for intravenous, cutaneous, or subcutaneous injection can contain, in addition to protein or other active ingredient of the present invention, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art. The immunogenic composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art. For injection, the agents of the invention may be formulated in aqueous solutions, physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

For oral administration, the compounds can be formulated readily by combining the active compounds with immunogenicly acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Immunogenic preparations for oral use can be obtained solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol, and cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

Immunogenic preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration. For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

Capsules and cartridges may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampules or in multi- dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

Immunogenic formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Alternatively, the active ingredient maybe in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides. In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intamuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

A carrier for hydrophobic compounds of the invention can be a co-solvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. The co-solvent system may be the VPD co-solvent system. VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol. The VPD co-solvent system (VPD:5W) consists of VPD diluted 1 : 1 with a 5% dextrose in water solution. This co- solvent system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic administration. Naturally, the proportions of a co-solvent system may be varied considerably without destroying its solubility and toxicity characteristics. Furthermore, the identity of the co-solvent components may be varied: for example, other low-toxicity nonpolar surfactants may be used instead of polysorbate 80; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g. polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.

Alternatively, other delivery systems for hydrophobic immunogenic compounds may be employed. Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs. Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity. Additionally, the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent. Various types of sustained-release materials have been established and are well known by those skilled in the art. Sustained- release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days. Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein or other active ingredient stabilization may be employed.

The immunogenic compositions also may comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols. Many of the active ingredients of the invention may be provided as salts with immunogenicly compatible counter ions. Such immunogenicly acceptable base addition salts are those salts which retain the biological effectiveness and properties of the free acids and which are obtained by reaction with inorganic or organic bases such as sodium hydroxide, magnesium hydroxide, ammonia, trialkylamine, dialkylamine, monoalkylamine, dibasic amino acids, sodium acetate, potassium benzoate, triethanol amine and the like.

The immunogenic composition of the invention may be in the form of a complex of the protein(s) or other active ingredient of present invention along with protein or peptide antigens.

The immunogenic composition of the invention may be in the form of a liposome in which protein of the present invention is combined, in addition to other acceptable carriers, with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithins, phospholipids, saponin, bile acids, and the like. Preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S. Pat. Nos. 4,235,871 ; 4,501,728; 4,837,028; and 4,737,323, all of which are incorporated herein by reference.

Other additives that are useful in immunogenic composition formulations are known and will be apparent to those of skill in the art.

An "immunologically effective amount" of the compositions of the invention may be administered to an animal (including a human). As used herein, the term "immunologically effective amount" refers to an amount capable of inducing, or enhancing the induction of, the desired immune response in an animal (including a human). The desired response may include, inter alia, inducing an antibody or cell-mediated immune response, or both. The desired response may also be induction of an immune response sufficient to ameliorate the symptoms of a EqPV associated disease and/or provide protective immunity in an animal (including a human) against subsequent challenge with a EqPV. An immunologically effective amount may be an amount that induces actual "protection" against EqPV associated diseases, meaning the prevention of any of the symptoms or conditions resulting from EqPV associated disease in animals (including humans). An immunologically effective amount may also be an amount sufficient to delay the onset of symptoms and conditions associated with infection, reduce the degree or rate of infection, reduce in the severity of any disease or symptom resulting from infection, and reduce the viral load of an infected animal (including a human).

One of skill in the art can readily determine what is an "immunologically effective amount" of the compositions of the invention without performing any undue experimentation. An effective amount can be determined by conventional means, starting with a low dose of and then increasing the dosage while monitoring the immunological effects. Numerous factors can be taken into consideration when determining an optimal amount to administer, including the size, age, and general condition of the animal, the presence of other drugs in the animal, the virulence of the particular EqPV against which the animal is being vaccinated, and the like. The actual dosage is can be chosen after consideration of the results from various animal studies.

The immunologically effective amount of the immunogenic composition may be administered in a single dose, in divided doses, or using a "prime-boost" regimen. The compositions may be administered by any suitable route, including, but not limited to parenteral, intradermal, transdermal, subcutaneous, intramuscular, intravenous, intraperitoneal, intranasal, oral, or intraocular routes, or by a combination of routes. The compositions may also be administered using a "gun" device which fires particles, such as gold particles, onto which compositions of the present invention have been coated, into the skin of an animal. The skilled artisan will be able to formulate the immunogenic composition according to the route chosen.

Dose sizes of the immunogenic compositions described herein can be in the range of about 2.0 to 0.1 ml depending on the route of administration, but dose sizes are not limited to this range. For inactivated EqPV compositions can contain suitable TCID50 levels of virus prior to inactivation. The antigen content in EqPV preparation can have, but is not limited to, a titer of between 10 to 10,000 units/ml as the amount administered per dose. One of skill in the art will readily be capable of determining a suitable antigen content for the immunogenic compositions described herein.

For immunogenic compositions containing modified live EqPVes or attenuated EqPVes, a therapeutically effective dose can be determined by one of skill in the art. For immunogenic compositions containing EqPV subunit antigens, a therapeutically effective dose can be determined by one of skill in the art. While the amounts and concentrations of adjuvants and additives useful in the context of the present invention can readily be determined by the skilled artisan.

An animal or a human can be inoculated with the immunogenic compositions or formulations described herein to generate an immune response. In certain embodiments, inoculation can be perfomed on horses that are at least 1, 2, 3, 4, 5, 6, or more weeks or older. In certain embodiments, the horses can receive one or more dosages. In certain embodiements, two or more dosages can be administered to the animal 3-4 weeks apart. In certain embodiments, the administration can be by subcutaneous injection. Intramuscular, intradermal, or oral routes of administration can also be used to administer the immunogenic compositions or formulations described herein.

Cells Comprising EqPV and Uses Thereof

EqPV can be used to infect cells. Cells may be cultured in any useful media and any permissive cell or tissues, which may be, or may be derived from any animal or equine cell. As used herein, a cell or a tissue can include, but is not limited to individual cells, tissues, organs, insect cells, rodent cells, avian cells, mammalian cells, hybridoma cells, primary cells, continuous cell lines, and/or genetically engineered cells. Cell lines suitable for propagating, growing, or harboring EqPV nucleic acid sequence or for expressing a polypeptide produced by the EqPV nucleic acid sequence include, animal or equine cells, including but are not limited to dog kidney cells, BSC-1 cells, LLC-MK cells, CV-1 cells, CHO cells, COS cells, murine cells, human cells, HeLa cells, 293 cells, VERO cells, MDBK cells, MDCK cells, MDOK cells, CRFK cells, RAF cells, TCMK cells, LLC-PK cells, PK15 cells, WI-38 cells, MRC-5 cells, T-FLY cells, BHK cells, SP2/0 cells, NSO, PerC6 (human retina cells), chicken embryo cells or derivatives, embryonated egg cells, embryonated chicken eggs or derivatives thereof.

Cell culture media formulations to suitable for culturing cells infected with EqPV viruses described herein include, but are not limted to, Modified Eagle's media MEM, minimum essential media MEM, Dulbecco's modified Eagle's media D-MEM, D-MEM-F12 media, William's E media, RPMI media and analogues and derivative thereof. These can also be specialty cell cultivation and virus growth media as VP-SFM, OptiPro.TM. SFM, AIM V.R media, HyQ SFM4 MegaVir, EX-CELL Vero SFM, EPISERF, ProVero, any 293 or CHO media and analogues and derivatives thereof. The culture media described herein can be supplemented by any additive known from prior art that is applicable for cell and virus cultivation as for example animal sera and fractions or analogues thereof, amino acids, growth factors, hormones, buffers, trace elements, trypsin, sodium pyruvate, vitamins, L- glutamine and biological buffers. One medium is OptiPRO SFM supplemented with L- glutamine and trypsin. In certain embodiments, the cell culture media can be supplemented with 0.1 to 10 units of trypsin. Alternatively, plant derived equivalents of trypsin (e.g. Accutase) ranging from 2-100 units can also be used in cell culture. Cell culture media can be used in the absence or presence of animal-derived components. An example of supplementation with an animal-derived component is gamma-irradiated serum ranging from 0.5-10% final concentration.

An expression vector can be introduced into cells in order to produce proteins (for example, SEQ ID NOs: 23-24) encoded by nucleotide sequences of the invention (for example SEQ ID NOs: 1-3). Cells can harbor an expression vector via introducing the expression vector into an appropriate host cell via methods known in the art.

An expression vector can be introduced into cells in order to produce proteins encoded by nucleotide sequences of the invention (for example SEQ ID NOs: 1-3 or a sequence complementary to SEQ ID NOs: 1-3). Cells can harbor an expression vector via introducing the expression vector into an appropriate host cell via methods known in the art.

A eukaryotic expression vector can be used to transfect cells in order to produce proteins (for example, SEQ ID NOs: 23-24) encoded by nucleotide sequences of the vector. An exogenous nucleic acid (for example any of SEQ ID NOs: 1-22 and 25-28, a cDNA of SEQ ID NOs: 1-3 or a cDNA complementary to SEQ ID NOs: 1-3, fragments, or variants thereof) can be introduced into a cell via a variety of techniques known in the art.

A eukaryotic expression vector can be used to transfect cells in order to produce proteins encoded by nucleotide sequences (for example SEQ ID NOs: 1-3, a cDNA of SEQ ID NOs: 1-3 or a cDNA complementary to SEQ ID NOs: 1-3, fragments, or variants thereof). Mammalian cells can harbor an expression vector via introducing the expression vector into an appropriate host cell via methods known in the art.

An exogenous nucleic acid can be introduced into a cell via a variety of techniques known in the art, such as lipofection, microinjection, calcium phosphate or calcium chloride precipitation, DEAE-dextrin-mediated transfection, or electroporation. Other methods used to transfect cells can also include calcium phosphate precipitation, modified calcium phosphate precipitation, polybrene precipitation, microinjection liposome fusion, and receptor-mediated gene delivery.

Cells to be infected with EqPV or nucleic acids thereof (for example SEQ ID NOs: 1-

3, a cDNA of SEQ ID NOs: 1-3 or a cDNA complementary to SEQ ID NOs: 1-3, fragments, or variants thereof) can be primary and secondary cells, which can be obtained from various tissues and include cell types which can be maintained and propagated in culture.

Various culturing parameters can be used with respect to the host cell being cultured. Appropriate culture conditions for mammalian cells are well known in the art or can be determined by the skilled artisan (see, for example, Animal Cell Culture: A Practical Approach 2^nd Ed., Rickwood, D. and Hames, B.D., eds. (Oxford University Press: New York, 1992)), and vary according to the particular cell selected. Commercially available medium can be utilized. Non-limiting examples of medium include, for example, Dulbecco's Modified Eagle Medium (DMEM, Life Technologies), Minimal Essential Medium (MEM, Sigma, St. Louis, MO); HyClone cell culture medium (HyClone, Logan, Utah); and serum- free basal epithelial medium (CellnTech).

The media described above can be supplemented as necessary with supplementary components or ingredients, including optional components, in appropriate concentrations or amounts, as necessary or desired. Cell medium solutions provide at least one component from one or more of the following categories: (1) an energy source, usually in the form of a carbohydrate such as glucose; (2) all essential amino acids, and usually the basic set of twenty amino acids plus cysteine; (3) vitamins and/or other organic compounds required at low concentrations; (4) free fatty acids or lipids, for example linoleic acid; and (5) trace elements, where trace elements are defined as inorganic compounds or naturally occurring elements that are typically required at very low concentrations, usually in the micromolar range.

The medium also can be supplemented electively with one or more components from any of the following categories: (1) salts, for example, magnesium, calcium, and phosphate; (2) hormones and other growth factors such as, serum, insulin, transferrin, epidermal growth factor and fibroblast growth factor; (3) protein and tissue hydrolysates, for example peptone or peptone mixtures which can be obtained from purified gelatin, plant material, or animal byproducts; (4) nucleosides and bases such as, adenosine, thymidine, and hypoxanthine; (5) buffers, such as HEPES; (6) antibiotics, such as gentamycin or ampicillin; (7) cell protective agents, for example, pluronic polyol; and (8) galactose.

Cells maintained in culture can be passaged by their transfer from a previous culture to a culture with fresh medium. In one embodiment, induced epithelial cells are stably maintained in cell culture for at least 3 passages, at least 4 passages, at least 5 passages, at least 6 passages, at least 7 passages, at least 8 passages, at least 9 passages, at least 10 passages, at least 11 passages, at least 12 passages, at least 13 passages, at least 14 passages, at least 15 passages, at least 20 passages, at least 25 passages, or at least 30 passages.

The cells suitable for culturing according to the methods of the present invention can harbor introduced expression vectors (constructs), such as plasmids and the like. The expression vector constructs can be introduced via transformation, microinjection, transfection, lipofection, electroporation, or infection. The expression vectors can contain coding sequences, or portions thereof, encoding the proteins for expression and production. Expression vectors containing sequences encoding the produced proteins and polypeptides, as well as the appropriate transcriptional and translational control elements, can be generated using methods well known to and practiced by those skilled in the art. These methods include synthetic techniques, in vitro recombinant DNA techniques, and in vivo genetic recombination which are described in Sambrook and Ausubel.

In one embodiment, cells that have been infected with EqPV or contain nucleic acids thereof (for example SEQ ID NOs: 1-3, a cDNA of SEQ ID NOs: 1-3 or a cDNA complementary to a SEQ ID NOs: 1-3, fragments, or variants thereof) can express a variety of markers that distinguish them from uninfected cells. Expression of markers can be evaluated by a variety of methods known in the art. The presence of markers can be determined at the DNA, RNA or polypeptide level.

In one embodiment, the method can comprise detecting the presence of a marker gene polypeptide expression. Polypeptide expression includes the presence of a marker gene polypeptide sequence, or the presence of an elevated quantity of marker gene polypeptide as compared to non-epithelial cells. These can be detected by various techniques known in the art, including by sequencing and/or binding to specific ligands (such as antibodies). For example, polypeptide expression maybe evaluated by methods including, but not limited to, immunostaining, FACS analysis, or Western blot. These methods are well known in the art (for example, US Patent 8,004,661, US Patent 5,367,474, US Patent 4,347,935)

In another embodiment, the method can comprise detecting the presence of nucleic acids (for example any of SEQ ID NOs: 1-22 and 25-28, a cDNA of SEQ ID NOs: 1-3 and 25-28 or a cDNA complementary to SEQ ID NOs: 1-3 and 25-28, fragments, or variants thereof). RNA expression includes the presence of an RNA sequence, the presence of an RNA splicing or processing, or the presence of a quantity of RNA. These can be detected by various techniques known in the art, including by sequencing all or part of the marker gene RNA, or by selective hybridization or selective amplification of all or part of the RNA. In one embodiment, in situ hybridization can be used to detect EqPV nucleic acids.

The EqPV viruses and immunogenic compositions described herein can be be produced in cells. Production of the EqPV viruses and immunogenic compositions described herein may also be accomplished on any useful media and permissive cell or tissues, which may be derived from equine or other animal cell lines. As used herein, a cell or a tissue can include, but is not limited to individual cells, tissues, organs, insect cells, fish cells, mammalian cells, hybridoma cells, primary cells, continuous cell lines, and/or genetically engineered cells, such as recombinant cells expressing a virus. For example, production of the EqPV viruses and immunogenic compositions can be in any cell type, including but not limited to animal or equine cells. Cell lines suitable for producing the EqPV viruses and immunogenic compositions described herein include, but are not limited to animal or equine cells. In certain embodiments, cell lines suitable for producing the EqPV viruses and immunogenic compositions described herein also include dog kidney cells, BSC-1 cells, LLC-MK cells, CV-1 cells, CHO cells, COS cells, murine cells, human cells, HeLa cells, 293 cells, VERO cells, MDBK cells, MDCK cells, MDOK cells, CRFK cells, RAF cells, TCMK cells, LLC-PK cells, PK15 cells, WI-38 cells, MRC-5 cells, T-FLY cells, BHK cells, SP2/0 cells, NSO, PerC6 (human retina cells), chicken embryo cells or derivatives, embryonated egg cells, embryonated chicken eggs or derivatives thereof.

The cell culture system for producing the EqPV viruses and immunogenic compositions described herein can be a traditional adherent monolayer culture. Alternatively, suspension and microcarrier cell culture systems can also be utilized.

Vessels for producing the EqPV viruses and immunogenic compositions described herein include, but are not limted to, roller bottles. For example, alternatively, other useful cell culture formats include flasks, stacked modules and stir tanks.

Production of the EqPV viruses and immunogenic compositions can also be performed using a recombinant expression system that expresses EqPV, a EqPV protein, a fragment of a bocoviral protein or a variant of a EqPV viral protein. The expression system can comprise any suitable plasmid or a linear expression construct known in the art.

Methods of purification of inactivated virus are known in the art and may include one or more of, for instance gradient centrifugation, ultracentrifugation, continuous-flow ultracentrifugation and chromatography, such as ion exchange chromatography, size exclusion chromatography, and liquid affinity chromatography polyethylene glycol or ammonium sulfate precipitation. Additional methods of purification include the use of a nucleic acid degrading agent, a nucleic acid degrading enzyme, such as a nuclease having DNase and RNase activity, or an endonuclease, such as from Serratia marcescens, membrane adsorbers with anionic functional groups or additional chromatographic steps with anionic functional groups (e.g. DEAE or TMAE).

The purified viral preparation of the invention is substantially free of contaminating proteins derived from the cells or cell culture and can comprises less than about 1000, 500, 250, 150, 100, or 50 pg cellular nucleic acid^g virus antigen, and less than about 1000, 500, 250, 150, 100, or 50 pg cellular nucleic acid/dose. The purified viral preparation can also comprises less than about 20 pg or less than about 10 pg. Methods of measuring host cell nucleic acid levels in a viral sample are known in the art. Standardized methods approved or recommended by regulatory authorities such as the WHO or the FDA can be used.

In certain aspects, the invention relates to a method for culturing cells comprising: a) infecting a cell with EqPV, or an isolated (or non-isolated) nucleic acid of the current invention having the sequence of SEQ ID NOs: 1-3, a sequence complementary to the sequence of SEQ ID NOs: 1-22 and 25-28, and variants and fragments thereof, and b) culturing the cells. In certain aspects, the invention relates to a method of testing an EqPV vaccine, comprising: a) contacting cells with an EqPV vaccine; b) contacting cells with EqPV; and c) measuring the number of cells infected with EqPV.

In certain aspects, the invention relates to a method of testing an EqPV drug, comprising: a) contacting cells with an EqPV drug; b) contacting cells with EqPV; and c) measuring the number of cells infected with EqPV.

In certain aspects, the invention relates to a method of testing an EqPV drug, comprising: a) contacting cells with EqPV; b) contacting cells with an EqPV drug; and c) measuring the replication of EqPV.

EXAMPLES

The present invention may be understood by reference to the following non-limiting examples, which are presented in order to more fully illustrate the preferred embodiments of the invention. They should in no way by construed to limit the broad scope of the invention.

Example 1- Identification of a Novel Parvovirus in Horses

Materials and Methods

The first case (Case 1) enrolled had been treated with tetanus antitoxin of equine origin 65 days before the onset of clinical signs of hepatic failure. Serum from Case 1 , liver obtained post mortem, and the tetanus antitoxin the horse received 65 days prior to the development of signs of hepatic failure were PCR negative for the Non-Primate Hepacivirus (NPHV), the Theiler's Disease Associated Virus (TDAV), and Equine Pegivirus (EPgV). Using unbiased, high throughput sequencing, a previously unidentified parvovirus was detected in the serum and the liver of Case 1 (Table 1).

Virus discovery using unbiased high throughput sequencing

Serum and antitoxin samples were centrifuged at 5000 rpm for 10 minutes to remove the sediment. Liver suspension was prepared using approximately 100 mg of liver tissue in 1 ml of PBS and 3mm steal beads using tissue lyser (Qiagen). Serum and liver suspensions were filtered through a 0.45 μπι filter and treated with nucleases to digest free nucleic acids (NAs) for enrichment of viral NA and then extracted in NucliSens buffer using the automated easyMAG system (bio Merieux, United States). Total RNA extracts were reverse transcribed using a Superscript III kit (Invitrogen Life Technologies) with random hexamer primers. The cDNA was RNase H treated prior to second strand DNA synthesis using Klenow fragment (3-5 exonuclease negative) (New England Biolabs). The double stranded cDNA was sheared to a 200 bp average fragment length using a Covaris E210 focused ultrasonicator. Sheared DNA was purified and used for Illumina library construction using a Kapa library preparation kit (KK8234; Kapa Biosystems) and SeqCap EZ Library SR (Nimblegen, Roche). The sequencing libraries were quantified using an Agilent Bioanalyzer 2100. Samples with low concentrations were amplified by increasing PCR cycle numbers from 9 to 14. All sequencing was done on the Illumina HiSeq 2500 platform (Illumina, San Diego, CA, USA), yielding an average of 150 million reads per sequencing lane. Virome analysis included only those sequences that were most closely related to vertebrate viruses and was done using MEGAN version 5.10.5. Sequence data were demultiplexed using Illumina software to generate FastQ files for individual samples. Sequences were filtered using Q30 and mapped against reference genomes from GenBank with Bowtie2 mapper 2.0.6 (http://bowtie bio.sourceforge.net). SAMtools (v 0.1.19) were used to generate the consensus genomes and coverage statistics. Integrative Genomics Viewer (v 2/3/55; Broad Institute) was used to generate the sequence coverage plots. Host derived sequences were identified using Bowtie2 based sequence mappings against the reference host genomes downloaded from the NCBI database. Sequencing data obtained from the clinical samples were preprocessed using PRINSEQ (v 0.20.2) software, and primer trimmed, quality score filtered reads were aligned against the host reference databases to remove the host background. The host subtracted sequence reads were de novo assembled using MIRA (v 4.0) or SOAPdenovo2 (v 2.04) assemblers, and then contigs and unique single sequences were subjected to a homology search using MegaBlast against the GenBank nucleotide database. Sequences that showed poor or no homology at the nucleotide level were subjected to a search with BLASTx against the viral GenBank protein database. Viral sequences from BLASTx analysis were subjected to another round of BLASTx homology search against entire GenBank protein database to correct for biased E values due to the smaller size of the virus only database, and taxonomy was reassigned. Based on the contigs identified for different viral strains, GenBank sequences were downloaded and used for mapping the whole data set to recover partial or complete genomes. The descriptive statistics mean, standard deviation (SD), and median were calculated for viral count and viral rate. The pretransfusion viral rate and the posttransfusion rate were compared by paired Wilcoxon rank sum test.

Complete genome sequencing and genetic analysis of EqPV

Complete genome of the EqPV was acquired using primer walking approach as previously described (Kapoor et al. 2010). In brief, each extension step of PCR used one primer specific for the novel virus sequence and other degenerated to hybridize all known animal parvovirus sequences. After the complete genome was assembled, each base is sequenced in triplicate to confirm the sequence.

To determine the sequence relationship between EqPV and other known parvovirus species, at least one representative virus member, including the reference genome from each species and their translated protein sequences, were used for generating sequence alignments. Sequences and their GenBank accession numbers used for the comparison are shown in Figures 1 and 2. The evolutionary history was inferred by using the Maximum Likelihood Method based on the Le_Gascuel_2008 model (Le and Gascuel 2008). The trees with the highest log likelihood (19539.2965) are shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor Join and BioNJ algorithms to a matrix of pairwise distances estimated using a JTT model, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.9819)). The trees were drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 100 amino acid sequences. There were a total of 295 positions in the final dataset. Evolutionary analyses were conducted in MEGA7 (Kumar et al. 2016). Recombination analysis was performed by Symplot

(http://sray.med.som.jhmi.edu/SCRoftware/simplot/), using reference genomes of all of two closely related porcine parvoviruses. To assess similarity across the genomes, sequence scans were performed using a fragment length of 300 bases and an increment of bases between fragments. DNA secondary structures of genomic termini were predicted by Mfold (Zuker et al. 2003).

RESULTS

Nucleic acids extracted from two vials of tetanus antitoxin and liver sample of the recipient horse that suffered with fatal hepatitis, were enriched for virus derived nucleic acids and subjected to Illumina sequencing. A total of 3.6 and 6.4 million reads were acquired from liver and antitoxin sample, respectively. Bioinformatics analysis revealed presence of a 4.5 kilo base assembled sequence in the horse liver sample that showed distant yet significant protein similarity with known animal parvoviruses. Further analysis indicated the total of approximately 10,398 reads from liver sample and 136 reads from antitoxin sample belonged to the new parvovirus. Thereafter, the in silico assembled sequence data was used to design primers for amplifying complete genome of the new parvovirus, however several attempts to acquire the virus genome termini remained unsuccessful. Considering the presence of high titer virus in liver sample and the precedent of finding virus episome in tissue for related parvoviruses, an inverse PCR based approach was used to acquire the complete virus genome. Inverse PCR assay confirmed that new equine parvovirus exit as episome in the liver tissue. Considering the international committee of virus taxonomy criteria to classify parvoviruses, this new virus represents a new species within the family Parvoviridae, named this virus equine parvovirus (EqPV). The complete genome of EqPV is found in SEQ ID NO: 1.

The complete episome of EqPV comprises of 5308 nucleotides and conceptually codes for two large operon reading frames whose proteins are related to the non structural proteins (NSP) (SEQ ID NO: 2) and the structural proteins (virion protein, VP) (SEQ ID NO: 3) of known animal parvoviruses (Figures 1 and 2).

The genomic termini of parvoviruses plays important role in virus replication and translation. DNA folding programs predicts that the 3' termini of EqPV folds as a 160 nt long hairpin, like found in other members of parvoviruses. The DNA secondary structure at the 5' end of virus genome were not evident but that may be due to the fact that episome form of virus genome often miss some stretches of nucleotide not required for maintain virus persistence.

The NS protein of EqPV contains canonical virus replicase sequence motifs shared by all parvoviruses.

The VP protein region can encode four different structural proteins, predicted based on the presence of methionine codons. Parvoviruses virions are known to contain major and minor VP. In parvoviruses, protein coding sequences are flanked on each side by noncoding palindromic repeats, also known as inverted terminal repeat (ITR) sequences, which play an important role in viral DNA replication. EqPV has perfect palindromic sequence of 478 nucleotides at the 3' end of the VP region. Even the complete genome folding analysis reveals this palindrome to be thermodynamically stable secondary structure. This is the longest palindromic sequence identified in any known parvovirus and it also includes sequence residues that codes for the carboxy terminus of VP. Example 2- Study of Clinical Cases of Acute Hepatitis

Materials and Methods

Prospective Clinical Case Study

Following the recent description of hepatitis in horses associated with the newly described flaviviruses TDAV and NPHV (Burbelo et al. 2012; Chandriani et al. 2013), a prospective clinical case study was initiated in collaboration with clinicians in academic and selected private equine referral centers in North America to determine the prevalence of infection with these viruses in horses with acute severe hepatitis (Theiler' s disease). Case definition included; acute onset of clinical signs of hepatic failure with characteristic laboratory and/or histopathologic findings in the liver that are described in the literature for Theiler' s disease. Serum was obtained and frozen before being shipped from the clinic of origin to Cornell University for virologic study. In some of the horses that died, it also was possible to obtain and freeze specimens of liver that were also shipped for virological study. In horses that received tetanus antitoxin prior to onset of signs of acute liver disease, it was possible to determine the identification number of the lot of commercial tetanus that had been used to treat the horses while in other cases it was possible to narrow the identifying lot number used to two possibilities. Aliqouts of the incriminated lots of commercial tetanus antitoxin also were tested virologically.

PCR and serological assays for EqPV

The NS and VP proteins of EqPV were aligned to all known parvovirus proteins.

Nucleotide and amino acid motifs showing relative conservation among different virus lineages were used to make primers for screening of samples for EqPV and related variants. All PCR mixtures used AmpliTaq Gold 360 master mix (catalog no. 4398881 ; Applied Biosy stems) and 2 μΐ of extracted nucleic acids.

The PCR assay targeting the EqPV NS (non-structural) gene used the following primer pairs: the pair EqPV akl or TPNS-inFl (GGAGAAGAGCGCAACAAATGCA) (SEQ ID NO: 12) and EqPV ak2 or TPNS-inRl (AAGACATTTCCGGCCGTGAC) (SEQ ID NO: 13) in the first round and the pair EqPV ak3 or TPNS-inF2 (GCGCAACAAATGCAGCGGTTCGA) (SEQ ID NO: 14) and EqPV ak4 or TPNS-inR2 (GGCCGTGACGACGGTGATATC) (SEQ ID NO: 15) in the second round of PCR. The amplicon from the first round was used for the second round.

For the first round, the PCR cycle included 8 minutes of denaturation at 95 °C, 10 cycles of 95°C for 40 s, 57°C for 45 s, and 72°C for 45 s, 30 cycles of 95°C for 30 s, 55°C for 45 s, and 72°C for 45 s, and a final extension at 72°C for 5 minutes. In first 10 cycles, the annealing temperature was ramped down by 0.5°C each cycle to allow mutation tolerance during primer hybridization. For the second round, PCR conditions included 8 minutes of denaturation at 95°C, 10 cycles of 95°C for 40 s, 64°C for 45 s, and 72°C for 45 s, 30 cycles of 95°C for 30 s, 58°C for 45 s, and 72°C for 45 s, and a final extension at 72°C for 5 minutes. In first 10 cycles, the annealing temperature was ramped down by 0.5°C each cycle.

The PCR assay targeting the SI (structural) or virion protein (VP) gene used the following primer pairs: EqPV ak5 or TPST-inFl (GTCGCTGCATTCTGAGTCC) (SEQ ID NO: 16) and EqPV ak6 or TPST-inRl (TGGGATTATACTGTCTACGGGT) (SEQ ID NO: 17) in the first round of PCR and the pair EqPV ak7 or TPST-inF2 (CTGCATTCTGAGTCCGTGGCC) (SEQ ID NO: 18) and EqPV ak8 or TPST-inR2 (CTGTCTACGGGTATCCCATACGTA) (SEQ ID NO: 19) in the second round of PCR. The amplicon from the first round was used in the second round.

For the first round, the PCR cycle included 8 minutes of denaturation at 95 °C, 10 cycles of 95°C for 40 s, 56°C for 45 s, and 72°C for 45 s, 30 cycles of 95°C for 30 s, 54°C for 45 s, and 72°C for 45 s, and a final extension at 72°C for 5 min. In first 10 cycles, the annealing temperature was ramped down by 0.5°C each cycle to allow mutation tolerance during primer hybridization. For the second round, PCR conditions included 8 minutes of denaturation at 95°C, 10 cycles of 95°C for 40 s, 63°C for 45 s, and 72°C for 45 s, 30 cycles of 95°C for 30 s, 59°C for 45 s, and 72°C for 45 s, and a final extension at 72°C for 5 minutes. In first 10 cycles, the annealing temperature was ramped down by 0.5°C each cycle.

For quantitative PCR assay, primer pair EqPV ak9 or qPCR Forward Primer (ATGCAGATGCTTTCCGACC) (SEQ ID NO: 20) and EqPV aklO or qPCR Reverse Primer (GCCCCAGAAACATATGGAAA) (SEQ ID NO: 21) was used with probe EqPV akl l or qPCR Probe: 6-F AM] ACCGT AGCGGATTCGGGATCTGC [B HQ 1 a-6F AM] (SEQ ID NO: 22)

Real time PCR conditions included two initial denaturation steps at 50°C for 2 minutes and 95°C for 10 minutes followed by 40 cycles of 95°C for 30 s and 58°C for 60 s. Data was analyzed using StepOne software.

LIPS assay for serology of EqPV

C terminus of EqPV capsid protein was cloned into pREN2 plasmid for making Renilla luciferase fused antigen for LIPS assay. Briefly, the VPl gene of EqPV was amplified using primers EqPV LIPSF1 (5 ' AGTA A AGTC A ATGGAC ACC A) (SEQ ID NO: 25) and EqPV LIPSR1 (5' GGATCGTGGTATGAGTTC 3') (SEQ ID NO: 26). PCR product was sequenced and then used as template to make inserts for LIPS assay using primers with flanking restriction sites, EqPV LIPS BamHl

(5 ' GAGGGATCCC ATGCTTTACCGTATGATC 3') (SEQ ID NO: 27) and EqPV LIPS Xhol (5' GAGCTCGAGTCAGAACTGACAGTATTGGTTC 3'( (SEQ ID NO: 28). Inserts were subsequently sequenced, digested and ligated into pREN 2 expression vector as described previously. Cos cells were transfected and antigen was harvested 60 hrs post transfection.

Infection prevalence, diseases association and molecular epidemiology

To determine the infection prevalence of EqPV, serum samples of 100 horses submitted to Cornell University diagnostic center for equine infectious anemia antibody testing (EDJ) were tested. Horses viremic with EqPV had the same serum tested for evidence of liver disease by measuring serum gamma-glutamyl trans aminopeptidase activity (GGT). RESULTS

As shown in Example 1, a previously unidentified parvovirus was detected in the serum and the liver of Case 1. Subsequent PCR analysis confirmed the presence of DNA from the novel parvovirus (EqPV) in the incriminated lot of tetanus antitoxin with which Case 1 had been treated.

In collaboration with North American private and academic equine referral clinics, a total of 14 cases of acute hepatitis were used in the prospective study (Table 1). Ten of the fourteen horses had received antecedent treatment with commercial tetanus antitoxin prior to onset of signs of liver disease (mean 53.6 days: range 28-63 days), two received allogenic stem cell treatment from donor horses 47 and 53 days earlier as treatment for orthopedic injuries, and two had no history of treatment with a biologic of equine origin.

Serum was available for testing from twelve of the fourteen cases studied. PCR using

SEQ ID NOs: 20 and 21 was performed on the serum and all twelve were PCR positive for EqPV. Four of the twelve serums also were PCR positive for EPgV, one of the 12 also was PCR positive for NPHV, but none of the 12 were positive for TDAV RNA. Eleven of the 14 horses died or were euthanized due to severity of the liver failure. See Table 1.

The livers of eight of these cases were available for PCR analysis. Seven of the eight were test positive for EqPV only, and the other was test positive for EqPV and EPgV. None of the eight liver samples from horses was positive either for NPHV or TDAV. See Table 1.

A total of thirteen separately identified lots (labeled numbers on the vial) of tetanus antitoxin were incriminated in the ten acute hepatitis cases known to have been treated with tetanus antitoxin prior to onset of illness. In six of the ten cases, the lot identification was reasonably certain and in two of these horses the lot number was the same. In four cases, it was reasonably certain that one or the other of two lots had been administered. PCR using SEQ ID NOs: 20 and 21 was performed on the thirteen lots of tetanus antitoxin.

In the six cases where lot identification was reasonably certain, the tetanus antitoxins were PCR positive for EqPV. In the four cases where one or the other of two lots had been administered, seven of the eight lots were PCR positive for EqPV. Seven of the thirteen lots were test positive for both EqPV and for EPgV, five were test positive for EqPV, EPgV, and NPHV and one was test positive for EqPV only. None of the thirteen was positive for TDAV. See Table 1.

Although all thirteen lots of tetanus antitoxin tested were PCR positive for EPgV, and seven were positive for NPHV, only 1 horse inoculated with this product was serum positive for EPgV and 1 different horse positive for NPHV. No horse receiving tetanus antitoxin was positive for either NPHV or EPgV in the liver (N=5). See Table 1.

The serum samples of the 100 additional horses were assayed with PCR targeting the non structural (NS) and virion (VP) or structural (SI) protein regions of EqPV. PCR identified thirteen of 100 horses positive for EqPV viremia. Sequence analysis indicated very low NS and VP sequence heterogeneity (<2% nucleotide differences) among isolates (Figures 1 and 2). These 100 sera were also tested for presence of anti-EqPV IgG using c terminus VPl protein as antigen in LIPS assay. It was determined that all thirteen viremic horses had high titer IgG antibodies. Additionally two non- viremic horses were also seropositive indicating clearance of viremia. The thirteen samples that were virus positive were then biochemically tested for evidence of liver disease using gamma-glutamyltransaminopeptidase (GGT) as a marker and all were within normal range.

Table 1- Results of Prospective Study

Table 1 - Results of Prospective Study continued

Example 3- Experimental Inoculation of Commercial Tetanus Antitoxin

Materials and Methods

Following discovery of EqPV, two clinically normal, aged horses that were PCR negative for EHCV and TDAV each were inoculated two lots of tetanus antitoxin.

Horse 1 , an 18 year old thoroughbred mare from the Cornell University herd was used for the experimental inoculation study. The mare was housed in a biosecurity level 2 facility separated physically from other horses. Prior to inoculation, the mare was PCR negative for EqPV nucleic acids and EqPV antibody as tested by LIPS.

Horse 1 was inoculated with two separately identified lots of tetanus antitoxin. One of the lots has been used to treat two of the horses who developed acute hepatitis as described in Example 2 and the other lot had been used to treat a third horse described in Example 2. Experimental horse 1 was inoculated intravenously with 5.0 ml of each of the two selected lots, and inoculated subcutaneously with 5.0 ml of each of the two selected lots for a total volume of 20.0 ml of tetanus antitoxin inoculated into the horse.

After the completion of the first experimental inoculation study on Horse 1, a second adult horse was inoculated in an identical manner (Horse 2).

Both experimentally inoculated horses were tested for EHCV, TDAV and EqPV prior to inoculation and thereafter at weekly intervals for virological and serological test and biochemical tests for liver disease. Blood samples were collected from the jugular vein before inoculation and at weekly intervals following inoculation. Serum was harvested from clotted blood, frozen and stored at 80°C until used for viral testing. Using fresh plasma, biochemical tests for aspartate aminotransferase (AST), sorbitol dehydrogenase (SDH), gamma glutamyltransferase (GGT), glutamate dehydrogenase (GLDH), bilirubin, and bile acids were performed with an Hitachi Mod P 800 (Roche Diagnostics). Blood sampling and testing was continued until detection and continued clearance of viremia and until development and clearance of evidence of acute hepatitis.

RESULTS

Horse 1 , the first experimentally inoculated horse, remained PCR negative for EqPV nucleic acids on weekly sampling until post-inoculation (PI) day 47 at which time she became PCR positive for EqPV and had increasing viremia on 4 consecutive tested weekly samples with peak viremia occurring on day 81 PI. Antibody tests by LIPS for EqPV remained negative on weekly samples until day 88 PI. LIPS values between PI days 0-70 ranged from 540-2,375 and on day 88 PI the value was 28,420. Weekly blood samples were evaluated for biochemical evidence of liver disease by testing for plasma AST, SDH, GGT, serum bile acids, and serum bilirubin. All values remained normal until day 81 when marked increases in all test except bile acids which increased the 2 days later (Figure 3). The mare became clinically icteric and developed orange colored urine (bilirubinuria) on day 87. There were further increases in all biochemical tests for 6 days after which time values started to return to normal range (Figure 3). A liver biopsy obtained during the period of elevated liver enzyme activity demonstrated generalized and severe single cell hepatic necrosis. Also observed occasionally were confluent groups of necrotic hepatocytes. Occasionally lymphocytes were clustered around a necrotic hepatocyte. Lobular and portal inflammatory cell infiltration was mild. Clinical icterus, and discolored urine were not observed after PI day 94 and biochemical markers of liver disease returned to normal by day 120 although the mare remained viremic.

The second experimentally inoculated horse (Horse 2) developed progressive EqPV viremia beginning at week 7 post inoculation and had marked biochemical evidence of liver disease on week 11 PI, corresponding to peak viremia (data not shown).

Example 4- Experimental Study of Botulinum Antitoxin

Materials and Methods

Frozen (80°C) samples of the Botulinum antitoxin that had been previously sequenced for presence of RNA viruses with discovery of TDAV, along with stored samples from four clinically normal, 2 to 3 year old ponies from that same study that had been experimentally inoculated with the botulism plasma were evaluated for evidence of EqPV infection and dynamics by PCR. Blood samples from before inoculation and at weekly intervals for 10 to 14 weeks following inoculation were available for testing (Chandriani, et ah, 2013).

RESULTS

As shown in Table 2, all of the horses became positive for EqPV at 3-5 week post inoculation (PI).

Table 2 - Time of Horses Testing Positive for EqPV Post Inoculation with Botulinum Antitoxin

REFERENCES

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Claims

What is claimed is:

1. An isolated nucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 1-3.

2. An isolated nucleic acid complementary to a sequence selected from the group

consisting of SEQ ID NOs: 1-3.

3. An isolated nucleic acid which is a variant of any one of SEQ ID NOs: 1-3 and has at least about 60% sequence identity to any of SEQ ID NOs: 1-3.

4. The isolated nucleic acid of claim 3, wherein the variant has at least about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% identity to any of SEQ ID NOs: 1-3.

5. An isolated nucleic acid which is complementary to a variant of any one of SEQ ID

NOs; 1-3 and wherein the variant has at least about 60% sequence identity to SEQ ID NOs: 1-3.

6. The isolated nucleic acid of claim 5, wherein the variant has at least about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% identity to any of SEQ ID NOs: 1-3.

7. An isolated nucleic acid sequence having the sequence of any of SEQ ID NOs: 4-22 and 25-28.

8. An isolated nucleic acid complementary to the sequence of any of SEQ ID NOs: 4-22 and 25-28.

9. An isolated nucleic acid having at least about 60% sequence identity to any of SEQ ID NOs: 4-22 and 25-28.

10. An isolated nucleic acid having at least about 60% sequence identity to a nucleic acid complementary to the sequence of any of SEQ ID NOs: 4-22 and 25-28.

11. The isolated nucleic acid of any of claims 1-10, wherein the nucleic acid comprises at least 10 nucleotides.

12. The isolated nucleic acid of claim 11, wherein the nucleotides are consecutive.

13. The isolated nucleic acid of any of claims 1-12, wherein the nucleic acid is DNA.

14. The isolated nucleic acid of claim 13 wherein the DNA is cDNA.

15. The isolated nucleic acid of any of claims 1-12, wherein the nucleic acid is RNA.

16. An isolated polypeptide encoded by the nucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 1-3.

17. An isolated polypeptide encoded by a nucleic acid complementary to a sequence selected from the group consisting of SEQ ID NOs: 1-3.

18. An isolated polypeptide which is encoded by a variant of any of one of SEQ ID NOs: 1- 3, wherein said variant has at least about 80% sequence identity to SEQ ID NOs: 1-3.

19. The isolated polypeptide of claim 18, wherein the variant has at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% identity to any of SEQ ID NOs: 1-3.

20. An isolated polypeptide which is encoded by a variant of a nucleic acid complementary to a sequence selected from the group consisting of SEQ ID NOs; 1-3, wherein said variant has at least about 80% sequence identity to the polypeptide encoded by a nucleic acid complementary to the sequence of any of SEQ ID NOs: 1-3.

21. The isolated polypeptide of claim 20, wherein the variant has at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% identity to any of SEQ ID NOs: 1-3.

22. An isolated polypeptide comprising the amino acid sequence of SEQ ID NOs: 23 or 24.

23. An isolated polypeptide which is a variant of SEQ ID NOs: 23 or 24 and has at least about 80% sequence identity to SEQ ID NOs: 23 or 24.

24. The isolated polypeptide of claim 23, wherein the variant has at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% identity to SEQ ID NOs: 23 or 24.

25. The isolated polypeptide of any of claims 16-24, wherein the polypeptide comprises at least 8 amino acids.

26. An isolated antibody that specifically binds to a polypeptide encoded by the nucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 1-3, fragments and variants thereof.

27. An isolated antibody that specifically binds to a polypeptide comprising the amino acid sequence of SEQ ID NOs: 23 or 24.

28. The isolated antibody of claims 26 or 27, wherein the antibody is a diagnostic antibody.

29. The isolated antibody of claims 26 or 27, wherein the antibody binds to equine

parvovirus or equine parvovirus polypeptide and inhibits, neutralizes or reduces the function or activity of the equine parvovirus or the equine parvovirus polypeptide.

30. The isolated antibody of claims 26 or 27, wherein the antibody is a polyclonal antibody.

31. The isolated antibody of claims 26 or 27, wherein the antibody is a monoclonal antibody.

32. An immunogenic composition comprising an equine parvovirus nucleic acid.

33. The immunogenic composition of claim 32, wherein the equine parvovirus nucleic acid comprises the nucleic acid of any of SEQ ID NOs: 1-3.

34. The immunogenic composition of claim 33, wherein the equine parvovirus nucleic acid comprises at least 24 consecutive nucleic acids.

35. The immunogenic composition of claim 32, wherein the equine parvovirus nucleic acid is substantially identical to the nucleic acid sequence of any of SEQ ID NOs: 1-3.

36. The immunogenic composition of claim 32, wherein the nucleic acid is a variant of any one of SEQ ID NOs: 1-3 and has at least about 60% sequence identity to any of SEQ ID NOs: 1-3.

37. The immunogenic composition of claim 36, wherein the variant has at least about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% identity to any of SEQ ID NOs: 1-3.

38. The immunogenic composition of any of claims 32-37, wherein the nucleic acid is a cDNA.

39. An immunogenic composition comprising an equine parvovirus polypeptide.

40. The immunogenic composition of claim 39, wherein the equine parvovirus polypeptide is a polypeptide encoded by the nucleic acid of any of SEQ ID NOs: 1-3.

41. The immunogenic composition of claim 40, wherein the nucleic acid comprises at least 24 consecutive nucleic acids.

42. The immunogenic composition of claim 40, wherein the equine parvovirus polypeptide is a polypeptide encoded by the nucleic acid that is substantially identical to the nucleic acid of any of SEQ ID NOs: 1-3.

43. The immunogenic composition of claim 40, wherein the nucleic acid is a variant of any one of SEQ ID NOs: 1-3 and has at least about 60% sequence identity to any of SEQ ID NOs: 1-3.

44. The immunogenic composition of claim 43, wherein the variant has at least about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% identity to any of SEQ ID NOs: 1-3.

45. The immunogenic composition of claim 39, wherein the equine parovirus polypeptide comprises the amino acid sequence of SEQ ID NOs: 23 or 24.

46. The immunogenic compound of claim 45, wherein the polypeptide comprises at least 8 consecutive amino acids.

47. The immunogenic composition of claim 45, wherein the polypeptide is a variant of SEQ ID NOs: 23 or 24 and has at least about 80% sequence identity to SEQ ID NOs: 23 or 24.

48. The immunogenic composition of claim 47, wherein the variant has at least about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% identity to SEQ ID NOs: 23 or 24.

49. An immunogenic composition comprising a killed virus comprising an equine

parvovirus polypeptide.

50. An immunogenic composition comprising an attenuated virus comprising an equine parvovirus polypeptide.

51. The immunogenic composition of claims 32-50 further comprising at least one excipient, additive or adjuvant.

52. A method of inducing an immune response in an animal, the method comprising

administering the immunogenic composition of any one of claim 32-50.

53. A method for preventing and/or treating an equine parvoviral infection in an animal, the method comprising administering the immunogenic composition of any one of claims 32-50.

54. The method of claims 52 or 53, wherein the animal is a horse.

55. Use of the immunogenic composition of any of claims 32-50 in the manufacture of a vaccine for the prevention or treatment of equine parvovirus in an animal.

56. An oligonucleotide probe comprising for about 10 nucleotides to about 50 nucleotides, wherein at least about 10 contiguous nucleotides are at least 95% complementary to a nucleic acid target region within a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-3.

57. The oligonucleotide probe of claim 56, wherein the probe is at least about 95.5%, about 96%, about 96.5%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%, about 99.5% or about 99.9% complementary to SEQ ID NOs: 1-3.

58. The oligonucleotide probe of claim 56, consisting of from about 10 to about 30

nucleotides from a nucleic acid sequence selected from the group consisting of: SEQ ID NOs: 1-3.

59. The oligonucleotide probes of any of claims 56-58, wherein the nucleic acid is cDNA.

60. A synthetic nucleic acid comprising at least about 10 nucleotides of the isolated nucleic acid having the nucleic acid sequence of any of SEQ ID NOs: 1-3.

61. A synthetic nucleic acid comprising at least about 10 nucleotides complementary to the isolated nucleic acid having the nucleic acid sequence of any of SEQ ID NOs: 1-3.

62. A method for determining the presence of equine parvovirus in a sample, the method comprising:

a) contacting nucleic acid from the sample with at least one primer which is a synthetic nucleic acid of claim 60 or claim 61 ;

b) subjecting the nucleic acid and the primer to amplification conditions; and c) determining the presence of amplification product, wherein the presence of amplification product indicates the presence of RNA associated with of equine parvovirus.

63. The method of claim 61, wherein the primer is selected from the group consisting of SEQ ID NOs: 4-21.

64. The method of claim 63, wherein more than one primer is used, and the primers are selected from the group consisting of: SEQ ID NO: 12 and SEQ ID NO: 13; SEQ ID NO: 14 and SEQ ID NO: 15; SEQ ID NO: 16 and SEQ ID NO: 17; SEQ ID NO: 18 and SEQ ID NO: 19; and SEQ ID NO: 20 and SEQ ID NO: 21.

65. A method for determining the presence of NSl-non structural gene of equine parvovirus in a sample, the method comprising:

a) contacting nucleic acid from the sample with primers SEQ ID NOs: 12 and 13; b) subjecting the nucleic acid and the primers to amplification conditions;

c) contacting an amplification product of step b) with primers SEQ ID NOs: 14 and 15; and

d) determining the presence of amplification product in step c), wherein the presence of amplification product indicates the presence of RNA associated with of equine parvovirus.

66. A method for determining the presence of the SI -structural gene of equine parvovirus in a sample, the method comprising:

a) contacting nucleic acid from the sample with primers SEQ ID NOs: 16 and 17; b) subjecting the nucleic acid and the primers to amplification conditions;

c) contacting an amplification product of step b) with primers SEQ ID NOs: 18 and 19; and d) determining the presence of amplification product in step c), wherein the presence of amplification product indicates the presence of RNA associated with of equine parvovirus.

67. A method for determining the presence of equine parvovirus in a sample, the method comprising:

a) contacting nucleic acid from the sample with primers SEQ ID NOs: 20 and 21 ; b) subjecting the nucleic acid and the primer to amplification conditions, and c) determining the presence of amplification product with probe SEQ ID NO: 22, wherein the presence of amplification product indicates the presence of RNA associated with of equine parvovirus.

68. A primer set for determining the presence of equine parvovirus in a sample, wherein the primer set comprises at least one synthetic nucleic acid sequence selected from the group consisting of:

a) a synthetic nucleic acid of claim 60; and

b) a synthetic nucleic acid of claim 61.

69. The primer set of claim 68, wherein the primers are selected from the group consisting of: SEQ ID NO: 12 and SEQ ID NO: 13; SEQ ID NO: 14 and SEQ ID NO: 15; SEQ ID NO: 16 and SEQ ID NO: 17; SEQ ID NO: 18 and SEQ ID NO: 19; and SEQ ID NO: 20 and SEQ ID NO: 21.

70. A method for determining whether a sample contains equine parvovirus the method comprising:

a) contacting the sample with an antibody that specifically binds to a polypeptide encoded by the nucleic acid of any one of SEQ ID NOs: 1-3, fragments and variants thereof; and

b) determining whether or not the antibody binds to an antigen in the biological sample, wherein binding indicates that the biological sample contains equine parvovirus.

71. The method of claim 70, wherein the determining comprises use of a lateral flow assay or ELISA.

72. A method for determining whether a sample has been infected by equine parvovirus, the method comprising: a) determining whether the sample contains antibodies that specifically bind to a polypeptide encoded by the nucleic acid of any one of SEQ ID NOs: 1-3, fragments and variants thereof.

73. The method of claims 62-67 and 70-72 wherein the sample is chosen from the group consisting of a biological sample, a biologic derived from an animal, a plasma product and a serum product.

74. An interfering RNA (iRNA) comprising a sense strand having at least 15 contiguous nucleotides complementary to the anti-sense strand of a gene from a virus comprising the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-3.

75. An interfering RNA (iRNA) comprising an anti-sense strand having at least 15

contiguous nucleotides complementary to the sense strand of a gene from a virus comprising the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-3.

76. A method for reducing the levels of a equine parvoviral protein in an animal, viral

mRNA in an animal or viral titer in a cell of an animal, the method comprising administering to the animal an iRNA of claims 74 and 75.

77. The method of claim 76, further comprising co- administering a second iRNA agent to the animal, wherein the second iRNA agent comprises a sense strand having at least 15 or more contiguous nucleotides complementary to a second gene or gene segment from equine parvovirus comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-3 and an antisense strand having at least 15 or more contiguous nucleotides complementary to the sense strand.

78. An isolated virus comprising at least 24 consecutive nucleotides from the nucleic acid having the nucleic acid sequence of any of SEQ ID NOs: 1-3.

79. An isolated virus comprising at least 8 consecutive amino acids from a polypeptide encoded by the nucleic acid having the nucleic acid sequence of any of SEQ ID NOs: 1 - 3.

80. A method of testing an agent for the prevention and/or treatment of equine parvovirus, comprising:

a) contacting cells with the agent;

b) contacting cells with equine parvovirus; and

c) measuring the number of cells infected with equine parvovirus, wherein if the number of cells infected with the equine parvovirus is decreased or less after contact with the agent, the agent is a preventative and/or therapeutic agent for equine parvovirus.

81. A method of testing an agent for the prevention and/or treatment of equine parvovirus, comprising:

a) contacting cells with equine parvovirus;

b) contacting cells with the agent; and

c) measuring the replication of equine parvovirus,

wherein if the replication of the equine parvovirus is lower after contact with the agent, the agent is a preventative and/or therapeutic agent for equine parvovirus.

82. A kit for screening for equine parvovirus, which comprises (a) at least one primer

comprising an isolated nucleic acid having the nucleic acid sequence of any of SEQ ID NOs: 1-3 to amplify nucleic acids obtained from a sample, and, optionally, (b) primers or adapters suitable to enable sequencing of the amplified nucleic acid and determination of the presence of the equine parvovirus.

83. The kit of claim 82, wherein the primer is chosen from the group consisting of: SEQ ID NOs: 4-21.

84. The kit of claim 82, comprising more than one primer and wherein the primers are

chosen from the group consisting of SEQ ID NO: 12 and SEQ ID NO: 13; SEQ ID NO: 14 and SEQ ID NO: 15; SEQ ID NO: 16 and SEQ ID NO: 17; SEQ ID NO: 18 and SEQ ID NO: 19; and SEQ ID NO: 20 and SEQ ID NO: 21.

85. A kit for screening for equine parvovirus which comprises at least two oligonucleotides complementary to the nucleic acid sequence of any of SEQ ID NOs: 1-3, fragments and variants thereof, in a microarray format for identifying equine parvovirus.