CN113717953B

CN113717953B - Application of attenuated flaviviruses in oncolysis

Info

Publication number: CN113717953B
Application number: CN202110899303.1A
Authority: CN
Inventors: 张波; 袁绍鹏; 刘静; 占顺利; 高磊
Original assignee: Beijing Shunlei Technology Co ltd; Wuhan Institute of Virology of CAS
Current assignee: Beijing Shunlei Technology Co ltd; Wuhan Institute of Virology of CAS
Priority date: 2021-08-05
Filing date: 2021-08-05
Publication date: 2024-07-26
Anticipated expiration: 2041-08-05
Also published as: CN113717953A

Abstract

The present invention relates to an oncolytic virus comprising an attenuated flaviviridae virus comprising a poly (a) sequence, wherein the poly (a) sequence is used to replace a part of the nucleotide sequence of the 3 'untranslated region (3' utr) of the flaviviridae virus such that the 3 'untranslated region (3' utr) of the attenuated flaviviridae virus obtained after the flaviviridae virus is replaced with a part of the nucleotide sequence retains at least the 3 'terminal stem loop region (3' sl), and uses thereof. In vitro cell experiments prove that the oncolytic virus can infect and kill various tumor cells, but has less influence on normal cells; the tumor-dissolving virus can inhibit the growth of various tumor cells in vivo in a xenograft tumor model, and can provide an anticancer therapeutic agent with safety, high efficiency and small toxic and side effects for the treatment of clinical tumors.

Description

Application of attenuated flaviviruses in oncolysis

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a broad-spectrum oncolytic application of an attenuated west nile virus.

Background

Tumors are the biggest public health problem facing humans and are also the second leading killers of death. Malignant tumors have become one of the major public health problems severely threatening the health of people in China and worldwide. Currently, treatments for tumors mainly include two types, one type is a traditional treatment method including chemotherapy/radiotherapy/surgery treatment, and the other type is an immunotherapy based on cytokines/immune cells and antibodies. But there are therapeutic drawbacks to either conventional or immunotherapy. The biggest disadvantage of traditional therapies is drug resistance, while the biggest disadvantage of immunotherapy is low response rate. Therefore, new therapeutic approaches, such as tumor immunotherapy/oncolytic virus, are increasingly receiving attention.

Oncolytic viruses are a class of natural or recombinant viruses that have the effect of directly killing tumor cells and/or activating tumor immune responses. Oncolytic viruses preferentially infect and replicate in tumor cells compared to normal cells, thereby causing tumor cell lysis. In addition to direct oncolysis, oncolytic viruses can also act as immunogens to activate innate and adaptive anti-tumor immune responses, resulting in efficient infiltration of immune cells, converting a "cold" Tumor Microenvironment (TME) with few immune cells into a "hot" tumor microenvironment with more immune cells, thereby enhancing the ability of the body to recognize, control and kill tumor cells. In recent years, as the cancer field progresses toward biological and immune-based methods for combating cancer, oncolytic viruses have received increasing attention. At present, most researches modify some naturally existing virulent seeds to make the seeds specifically express and package in tumor cells so as to realize an oncolytic effect. Compared with natural viruses, the attenuated live vaccine with the oncolytic effect has the advantages of safety, controllability, easy production and the like. There are studies showing that yellow fever vaccine YFV-17D replicates and kills mouse and human tumor cell lines, but does not kill untransformed human cells. The YFV-17D with activity can obviously inhibit the CD 8+T cell dependent transplanted tumor process by intratumoral injection, thereby generating immune mediated anti-tumor effect and finally playing the anti-tumor effect in an oncolytic virus mode. Similarly, studies have shown that the live attenuated vaccine Pvsripo of the interstitial poliovirus has a significant role in the treatment of gliomas. Pvsripo virus specifically enters glioma cells through the poliovirus receptor CD155 and expands in tumor cells ultimately inducing lytic death of the tumor cells.

West Nile Virus (WNV), a member of the flaviviridae family of flaviviruses, is also one of the most widely distributed flaviviruses worldwide, but it spreads only in a small range without causing serious disease. Most of the human infections WNV are asymptomatic or develop mild conditions such as West Nile fever.

At present, oncolytic virus therapy has the characteristics of high efficiency, good killing effect, small toxic and side effects and the like, and the development of new safe and effective oncolytic viruses has become a new hotspot in the field of tumor treatment research.

Disclosure of Invention

The invention aims to provide a novel anticancer therapeutic agent which is safe, efficient and has small toxic and side effects for the treatment of clinical tumors.

In one aspect, the invention provides an oncolytic virus that is an attenuated flaviviridae virus comprising a poly (a) sequence, wherein the poly (a) sequence is used to replace a portion of the nucleotide sequence of the 3 'untranslated region (3' utr) of the flaviviridae virus such that the 3 'untranslated region (3' utr) of the attenuated flaviviridae virus obtained after the flaviviridae virus is replaced with a portion of the nucleotide sequence retains at least the 3 'terminal stem loop region (3' sl).

In one aspect, the invention provides a DNA that can be transcribed to produce the oncolytic virus.

In one aspect, the invention provides a cell comprising said oncolytic virus or DNA.

In one aspect, the invention provides a composition comprising said oncolytic virus, DNA and/or said cells.

In one aspect, the invention provides the use of said oncolytic virus, said DNA, said cells and/or said composition in the manufacture of a medicament for the treatment of a tumor and/or cancer.

The oncolytic virus can obviously infect and lyse different cancer cells in an in vitro cell experiment, and can not cause toxic or side effect on normal cells. The in vivo animal experiment result shows that the oncolytic virus can obviously inhibit the growth of melanoma, liver cancer and ovarian cancer. Therefore, the oncolytic virus has wide application prospect, and provides an anticancer therapeutic agent with safety, high efficiency and small toxic and side effects for the treatment of clinical tumors.

Drawings

FIG. 1 shows a schematic of the construction of a clone of a flavivirus attenuated strain with a poly (A) sequence replacing the 3' UTR and virus rescue, wherein: a shows WNV-poly (A) clone construction and virus rescue IFA validation; b shows JEV-poly (A) clone construction and virus rescue IFA validation; c shows DENV-2 (NGC) -poly (A) clone construction and virus rescue IFA validation; d shows the construction of YFV-poly (A) clones and virus rescue IFA validation.

FIG. 2 shows the oncolytic effect of WNV-poly (A) on tumor cells and normal cells, wherein: FIG. 2A shows the oncolytic effect of WNV-poly (A) on tumor cells and normal cells in bright field observation; FIG. 2B shows the oncolytic effect of different doses of WNV-poly (A) on tumor cells and normal cells using CCK 8.

FIG. 3 shows the oncolytic effect of WNV-WT and WNV-poly (A) on tumor cells and normal cells, wherein: FIG. 3A shows the oncolytic effect of WNV-WT and WNV-poly (A) on tumor cells and normal cells in bright field; FIG. 3B shows the oncolytic effect of different doses of WNV-WT and WNV-poly (A) on tumor cells and normal cells using CCK 8; FIGS. 3C-3F show the replication differences of WNV-WT and WNV-poly (A) in normal cells MEF (FIG. 3C) and tumor cells B16F1 (FIG. 3D), A375 (FIG. 3E), huh7 (FIG. 3F), respectively.

FIG. 4 shows WNV-poly (A) causes apoptosis of tumor cells Huh7, SKOV3 and B16F1, wherein: FIG. 4A shows the apoptotic effect of WNV-poly (A) detection with Annexin-V/PI on tumor cells Huh7, SKOV3 and B16F 1; FIG. 4B shows the WB detection of cleavage of Caspase3 in tumor cells Huh7, SKOV3 after WNV-poly (A) infection.

FIG. 5 shows the oncolytic effect of inactivated WNV-poly (A) on tumor cells in vitro.

FIG. 6 shows the oncolytic effect of WNV-poly (A) on melanoma tested in vivo once a3 day dosing; wherein: FIGS. 6A-E show in vivo detection of oncolytic effects of multiple-dose WNV-poly (A) on murine melanoma B16F1 tumors; FIGS. 6F-J show in vivo detection of oncolytic effects of multiple-administered WNV-poly (A) on human melanoma A375 tumors.

FIG. 7 shows WNV-poly (A) activating tumor immune microenvironment; wherein: FIG. 7A shows the proportion of CD45+ cells in the tumor; FIG. 7B shows the proportion of individual immune cells in a tumor; FIG. 7C shows the proportion of individual immune cells in the spleen; FIGS. 7D, 7E show activation of CD8+ T cells in tumors; FIGS. 7F-H show the effect of CD8+ T cell depletion on WNV-poly (A) treatment.

FIG. 8 shows oncolytic effect on murine melanoma following double sided tumor-bearing unilateral administration of WNV-poly (A), wherein: FIG. 8A shows a schematic photograph of a double sided tumor bearing single sided viral therapy; FIG. 8B shows a statistical plot of tumor weights after treatment with a bilateral tumor-bearing unilateral virus; FIG. 8C is a graph showing tumor volume and mouse body weight change after bilateral tumor-bearing unilateral viral therapy; figure 8D shows viral load of bilateral tumors.

FIG. 9 shows oncolytic effects after one treatment of B16 tumors with different doses of WNV-poly (A), wherein: FIG. 9A shows a schematic photograph of a single dose of different doses of virus therapy; FIG. 9B shows a statistical plot of tumor weights after one treatment with different doses of virus; FIG. 9C is a graph showing tumor volume and mouse body weight changes after one viral treatment at different doses; FIG. 9D shows the amplification of WNV-poly (A) in tumors after one dose; FIGS. 9E-H show the tumor and spleen immune response after one dose.

FIG. 10 shows the broad spectrum of WNV-poly (A) oncolytic treatment, wherein: FIG. 10A shows a human liver cancer Huh7 model therapeutic strategy diagram; FIGS. 10B-D show oncolytic effects on Huh7 tumors in vivo with WNV-poly (A) administered once a three day period; FIG. 10E shows Huh7 model treatment of viral tissue tropism in mice; FIG. 10F shows a schematic of a human ovarian cancer SKOV3 model treatment strategy; FIGS. 10G-I show oncolytic effects on SKOV3 tumors in vivo with WNV-poly (A) administered once a three day period; figure 10J shows SKOV3 model treatment of mouse viral tissue tropism.

FIG. 11 shows the therapeutic effect detection of different modes of administration of WNV-poly (A) in the Huh7 model, wherein: fig. 11A shows the therapeutic effect on human liver cancer Huh7 model after intramuscular (i.m.), intravenous (i.v), intratumoral (i.t.) administration; FIG. 11B is a graph showing tumor volume change after treatment by different modes of administration; figure 11C shows the survival rate of mice after treatment with different modes of administration.

Detailed Description

In this disclosure, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Also, protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology-related terms and laboratory procedures as used herein are terms and conventional procedures that are widely used in the corresponding arts. Meanwhile, in order to better understand the present disclosure, definitions and explanations of related terms are provided below.

I. Definition of the definition

As used herein and unless otherwise indicated, the term "about" or "approximately" means within plus or minus 10% of a given value or range. Where integers are required, the term refers to rounding up or down to the nearest integer within plus or minus 10% of a given value or range.

As used herein, the term "oncolytic virus" is a virus that exhibits enhanced replication and lysis in cancer cells compared to non-cancer cells. In certain embodiments, the oncolytic virus exhibits selective replication in cancer cells and less or substantially no replication in non-cancer cells. In certain embodiments, less replication means that replication in a cancer cell is at least about 30% greater, or at least about 50% greater, or at least about 80% greater than that of a corresponding non-cancer cell.

As used herein, the term "oncolytic" refers to at least 10% of the proliferating cells being lysed (e.g., at least about 20%, 30%, 40%, 50% or 75% of the cells being lysed). For example, the percentage of cytolysis may be determined by measuring the decrease in the size of a neoplasm or the number of proliferating cells in a mammal or by measuring the amount of cytolysis in vitro (e.g., from a biopsy of proliferating cells). An effective amount of the virus will be determined on an individual basis and may be based (at least in part) on the particular virus used; size, age, sex of the individual; and the size and other characteristics of the abnormally proliferating cells. For example, for the treatment of humans, viruses of about 10 ³ to 10 ¹² Plaque Forming Units (PFU) are used, depending on the type, size and number of proliferating cells or neoplasms present. An effective amount may be, for example, from about 1.0PFU/kg body weight to about 10 ¹⁵ PFU/kg body weight (e.g., from about 10 ² PFU/kg body weight to about 10 ¹³ PFU/kg body weight). Optionally, the effective amount is about 1x10 ⁸ to about 1x10 ¹²TCID₅₀. Optionally, the effective amount is about 1x10 ¹⁰TCID₅₀.

As used herein, flaviviridae may include, but are not limited to, japanese encephalitis virus (Japanese encephalitis virus, JEV), west Nile Virus (WNV), tick-borne encephalitis virus (tick-bome encephalitis, TBEV), yellow fever virus (yellow fever virus, YFV), dengue virus (dengue virus, DENV), zika virus (zika virus, ZIKV), st.louis encephalitis virus (st.louis ENCEPHALITIS VIRUS, SLEV), murray valley encephalitis virus (Murray VALLEY ENCEPHALITIS virus, MVEV), jawski hemorrhagic fever virus (Omsk hemorrhagic fever virus), and casinosen virus (Kyasanur Forest Disease Virus), or any related flaviviruses thereof.

As used herein, the 5' end stem loop region (SL), dumbbell region (DB), repetitive cyclization sequence region 3 (RCS 3), cyclization sequence region 3 (CS 3), repetitive cyclization sequence region 2 (RCS 2), cyclization sequence region 2 (CS 2), cyclization sequence region 1 (CS 1), and short hairpin-3 ' end stem loop region (sHP-3 ' SL) following cyclization sequence region 1 are all terms of art. In other studies, the stem loop region (SL) was also referred to as the Variable Region (VR).

As used herein, the term "live attenuated virus" or "attenuated virus" refers to a virus that has been altered from the original parent virus or wild-type virus in such a way that its ability to infect, replicate in, package in, re-infect the host, or a combination thereof, is diminished. In general, such attenuation may be in several or all of the hosts of the virus, or only in one or several of the hosts of the virus. Thus, attenuation of a live attenuated virus (i.e., its ability to infect a host, replicate within a host, package, re-infect a host, or a combination thereof is attenuated) is generally associated with one or more hosts of the virus, wherein the live attenuated virus is not significantly or measurably attenuated in one or more other hosts of the virus. The live attenuated viruses disclosed herein are typically genetically altered, or may be referred to as mutated, mutant, genetically engineered, recombinant, or combinations.

As used herein, the term "live attenuated vaccine" or "attenuated vaccine" refers to a pharmaceutical composition comprising a live attenuated pathogen, such as a virus. The pharmaceutical composition comprises at least one immunologically active component that induces an immune response against a virus in a subject, protects the subject from death or possible death due to the virus, or both, and optionally may include one or more additional components that enhance the immune activity of the active component. The vaccine may additionally comprise other components typical for pharmaceutical compositions. At least one immunologically active component is one or more of the live attenuated viruses described herein.

As used herein, the term "viral rescue" is the process of introducing an in vitro construct comprising viral sequences into a suitable cell to produce an infectious or infectious virus. "recombinant virus" refers to genetically engineered viruses produced by recombinant DNA techniques, and any genetic manipulation of the viral sequences such as deletion, insertion, inversion, substitution, etc., may be performed artificially to distinguish from naturally occurring viruses.

As used herein, the term "recombinant RNA virus" refers to a virus described herein that comprises heterologous RNA.

As used herein, the term "wild-type" in the context of a virus refers to the type of virus that is prevalent, naturally transmitted, and develops a typical outbreak. In other embodiments, in the case of viruses, the term "wild-type" refers to the parent virus.

As used herein, the term "suicide gene" refers to a gene encoding a protein capable of converting a prodrug into a cytotoxic drug. Representative examples of suitable suicide genes include, but are not limited to, thymidine Kinase (TK), thymidylate kinase, cytosine deaminase (CDase), and uracil phosphoribosyl transferase (UPRTase).

As used herein, the term "multiplicity of infection" or "MOI" is the average number of infectious viral particles per infected cell. The MOI was calculated by dividing the number of infectious viral particles added (ml added x PFU/ml) by the number of cells added (ml added x cells/ml).

Detailed description of the preferred embodiments

In one aspect, the present disclosure provides an oncolytic virus comprising an attenuated flaviviridae virus comprising a poly (a) sequence, wherein the poly (a) sequence is used to replace a portion of the nucleotide sequence of the 3 'untranslated region (3' utr) of the flaviviridae virus such that the 3 'untranslated region (3' utr) of the attenuated flaviviridae virus obtained after the flaviviridae virus is replaced with a portion of the nucleotide sequence retains at least the 3 'terminal stem loop region (3' sl).

The attenuated flaviviruses according to the previous aspect, said partial nucleotide sequence comprising all or part of the 5 'terminal stem loop region (5' sl) in the 3 'non-coding region (3' utr), all or part of the circularized sequence region (CS), and/or all or part of the dumbbell region (DB).

In some preferred embodiments of the present disclosure, the 3' untranslated region (3 ' UTR) of the attenuated flaviviridae retains at least the circularized sequence region 1 (CS 1) of the 3' untranslated region and the short hairpin-3 ' end stem loop region (sHP-3 ' SL) following the circularized sequence region 1 after the partial nucleotide sequence is replaced with a poly (A) sequence.

In some preferred embodiments of the present disclosure, the 5 'end stem-loop region (5' sl) in the 3 'non-coding region (3' utr) is selected from the group consisting of stem-loop region I (SLI), stem-loop region II (SLII), stem-loop region III (SLII), and stem-loop region iv (SLIV).

In some preferred embodiments of the present disclosure, the circularized sequence region (CS) is selected from the group consisting of circularized sequence region 1 (CS 1), circularized sequence region 2 (CS 2), circularized sequence region 3 (CS 3), repetitive circularized sequence region 2 (RCS 2) and repetitive circularized sequence region 3 (RCS 3).

In some preferred embodiments of the present disclosure, the dumbbell region (DB) is selected from dumbbell region 1 (DB 1) and dumbbell region 2 (DB 2).

The attenuated flaviviridae virus according to any of the preceding claims, wherein the poly (a) sequence comprises 10-200 adenylates.

In some preferred embodiments of the present disclosure, the poly (a) sequence comprises 50-180, 100-160 adenylates; more preferably 130 to 150 adenylates, preferably 130 or 150 adenylates.

In some preferred embodiments of the present disclosure, the flaviviridae virus is selected from the group consisting of japanese encephalitis virus (Japanese encephalitis virus, JEV), west Nile Virus (WNV), tick-borne encephalitis virus (tick-borne encephalitis, TBEV), yellow fever virus (yellow fever virus, YFV), dengue virus (dengue virus, DENV), zika virus (zika virus, ZIKV), st.louis ENCEPHALITIS VIRUS, SLEV), murray valley encephalitis virus (Murray VALLEY ENCEPHALITIS virus, MVEV), jak hemorrhagic fever virus (Omsk hemorrhagic fever virus), and cassinol virus (Kyasanur Forest Disease Virus).

In some preferred embodiments of the present disclosure, the attenuated flaviviridae virus plaque titer is 1 x 10 ⁵-1×10⁹ PFU/ml; more preferably 5X 10 ⁴-5×10⁶ PFU/ml.

In some preferred embodiments of the present disclosure, the attenuated flaviviruses have a 10-100 fold reduced virulence compared to the parental wild-type virus.

In some preferred embodiments of the present disclosure, the attenuated flaviviruses are rescued on baby hamster kidney cells BHK-21 and/or the attenuated flaviviruses are amplified on Vero cells.

In some preferred embodiments of the present disclosure, the dengue virus is selected from dengue virus type 1, type 2, type 3, and type 4.

In some preferred embodiments of the present disclosure, the attenuated flaviviridae virus is an attenuated west nile virus. In some preferred embodiments of the present disclosure, the attenuated west nile virus comprises a nucleotide sequence having 80% or more identity to the nucleotide sequence depicted in SEQ ID No.1, preferably a nucleotide sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably a nucleotide sequence having 98% or 99% or more identity. In a specific embodiment of the present disclosure, the nucleotide sequence of the attenuated west nile virus is SEQ ID No.1.

In some preferred embodiments of the present disclosure, the attenuated flaviviridae is an attenuated japanese encephalitis virus. In some preferred embodiments of the present disclosure, the attenuated Japanese encephalitis virus comprises a nucleotide sequence that is 80% or more identical to the nucleotide sequence shown in SEQ ID NO.2, preferably a nucleotide sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, more preferably a nucleotide sequence that is 98% or 99% or more identical. In a specific embodiment of the present disclosure, the nucleotide sequence of the attenuated Japanese encephalitis virus is SEQ ID NO.2.

In some preferred embodiments of the present disclosure, the attenuated flaviviruses are attenuated dengue viruses. In some preferred embodiments of the present disclosure, the attenuated dengue virus comprises a nucleotide sequence that has 80% or more identity to the nucleotide sequence set forth in SEQ ID NO.3, preferably a nucleotide sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably a nucleotide sequence having 98% or 99% or more identity. In a specific embodiment of the present disclosure, the nucleotide sequence of the attenuated dengue virus is SEQ ID No.3.

In some preferred embodiments of the present disclosure, the attenuated yellow fever virus is an attenuated yellow fever virus. In some preferred embodiments of the present disclosure, the attenuated yellow fever virus comprises a nucleotide sequence having 80% or more identity to the nucleotide sequence set forth in SEQ ID No.4, preferably a nucleotide sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably a nucleotide sequence having 98% or 99% or more identity. In a specific embodiment of the present disclosure, the nucleotide sequence of the attenuated yellow fever virus is SEQ ID No.4.

In some preferred embodiments of the present disclosure, the attenuated flaviviruses are attenuated zika viruses. In some preferred embodiments of the present disclosure, the attenuated card virus comprises a nucleotide sequence having 80% or more identity to the nucleotide sequence set forth in SEQ ID NO.5, preferably a nucleotide sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably a nucleotide sequence having 98% or 99% or more identity. In a specific embodiment of the present disclosure, the nucleotide sequence of the attenuated zika virus is SEQ ID No.5.

In some preferred embodiments of the present disclosure, the attenuated flaviviridae is an attenuated tick-borne encephalitis virus. In some preferred embodiments of the present disclosure, the attenuated tick-borne encephalitis virus comprises a nucleotide sequence with 80% or more identity to the nucleotide sequence shown in SEQ ID NO.6, preferably a nucleotide sequence with 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably a nucleotide sequence with 98% or 99% or more identity. In a specific embodiment of the present disclosure, the nucleotide sequence of the attenuated tick-borne encephalitis virus is SEQ ID NO.6.

In a preferred embodiment of the present disclosure, the partial nucleotide sequence comprises 5' terminal stem loop region I (SLI), stem loop region II (SLII), repetitive cyclization sequence region 3 (RCS 3), stem loop region III (SLIII), stem loop region IV (SL IV), cyclization sequence region 3 (CS 3), and the 3' untranslated region (3 ' UTR) of the attenuated flaviviruses retains dumbbell region 1 (DB 1), repetitive cyclization sequence region 2 (RCS 2), dumbbell region 2 (DB 2), cyclization sequence region 2 (CS 2), cyclization sequence region 1 (CS 1), and short hairpin-3 ' terminal stem loop region (sHP-3 ' SL) following cyclization sequence region 1 after the partial nucleotide sequence is replaced with a poly (A) sequence.

In a preferred embodiment of the present disclosure, the partial nucleotide sequence comprises dumbbell region 1 (DB 1), repetitive cyclizing sequence region 2 (RCS 2), dumbbell region 2 (DB 2), cyclizing sequence region 2 (CS 2), cyclizing sequence region 1 (CS 1), and the 3 'untranslated region (3' utr) of the attenuated flaviviridae retains 5 'end stem loop region I (SLI), stem loop region II (SLI), repetitive cyclizing sequence region 3 (RCS 3), stem loop region III (SLI), stem loop region IV (SLIV), cyclizing sequence region 3 (CS 3), and short hairpin-3' end stem loop region (sHP-3 'sl) in the 3' untranslated region after the partial nucleotide sequence is replaced with a polyadenylation (poly (a)) sequence.

In a preferred embodiment of the present disclosure, the partial nucleotide sequence comprises 5 'terminal stem loop region I (SLI), stem loop region II (SLII), repetitive cyclization sequence region 3 (RCS 3), stem loop region III (SLIII), stem loop region IV (SLIV), cyclization sequence region 3 (CS 3), dumbbell region 1 (DB 1), repetitive cyclization sequence region 2 (RCS 2), dumbbell region (DB 2), cyclization sequence region 2 (CS 2), the partial nucleotide sequence being replaced with a poly (A) sequence, the 3' untranslated region (3 'UTR) of the attenuated flaviviridae virus retaining cyclization sequence region 1 (CS 1) of the 3' untranslated region and short hairpin-3 'terminal stem loop region (sHP-3' SL) following cyclization sequence region 1.

In a preferred embodiment of the present disclosure, the partial nucleotide sequence comprises 5 'terminal stem loop region II (SLII), repetitive cyclizing sequence region 3 (RCS 3), stem loop region IV (SL IV), cyclizing sequence region 3 (CS 3), dumbbell region 1 (DB 1), repetitive cyclizing sequence region 2 (RCS 2), dumbbell region (DB 2), cyclizing sequence region 2 (CS 2), the 3' untranslated region (3 'UTR) of the attenuated yellow virus retains cyclizing sequence region 1 (CS 1) of the 3' untranslated region and short hairpin-3 'terminal stem loop region (sHP-3' SL) following cyclizing sequence region 1 after the partial nucleotide sequence is replaced with a poly (A) sequence.

In a preferred embodiment of the present disclosure, the partial nucleotide sequence comprises 5' terminal stem-loop region II (SLII), repetitive cyclizing sequence region 3 (RCS 3), dumbbell region (DB), repetitive cyclizing sequence region 2 (RCS 2), dumbbell region (DB 2), cyclizing sequence region 2 (CS 2), and the 3' untranslated region (3 ' UTR) of the attenuated flaviviridae virus retains 5' terminal stem-loop region (SL) of the 3' untranslated region, cyclizing sequence region 1 (CS 1), and short hairpin-3 ' stem-loop region (sHP-3 ' SL) following cyclizing sequence region 1 after the partial nucleotide sequence is replaced with a poly (A) sequence.

In some preferred embodiments of the present disclosure, the oncolytic virus further comprises at least a sequence encoding at least one exogenous gene.

In some preferred embodiments of the present disclosure, the exogenous gene is a therapeutic gene.

In some preferred embodiments of the present disclosure, the therapeutic gene is an immunostimulatory gene or a suicide gene.

In some preferred embodiments of the present disclosure, the immunostimulatory gene encodes an immunostimulatory protein; preferably, the immunostimulatory protein is selected from the group consisting of cytokines, chemokines, interferons, tumor Necrosis Factors (TNF), colony stimulating factors, proteins exposed to APC (antigen presenting cells), growth factors, major histocompatibility complex class I or II (MHC) components, apoptosis inducers or inhibitors, cytostatics, blockers of immune escape mechanisms of immunotoxins and immune checkpoint inhibitors.

In some preferred embodiments of the present disclosure, the cytokine is an interleukin, more preferably, the cytokine is selected from the group consisting of IL-2, IL-6, IL-12, IL-15, and IL-24.

In some preferred embodiments of the present disclosure, the chemokine is selected from CXCL10, CXCL9, and CXCL11.

In some preferred embodiments of the present disclosure, the interferon is selected from IFN7 and ifnα.

In some preferred embodiments of the present disclosure, the colony stimulating factor is selected from the group consisting of GM-CSF, G-CSF, and M-CSF.

In some preferred embodiments of the present disclosure, the protein of APC (antigen presenting cell) is selected from B7.1 and B7.2.

In some preferred embodiments of the present disclosure, the growth factor is selected from the group consisting of transforming growth factor TGF, fibroblast growth factor FGF, and vascular endothelial growth factor VEGF.

In some preferred embodiments of the present disclosure, the apoptosis inducer or inhibitor is selected from the group consisting of Bax, bcl2 and BcIX.

In some preferred embodiments of the present disclosure, the cytostatic agent is selected from the group consisting of p21, p16, and Rb.

In some preferred embodiments of the present disclosure the suicide gene is selected from the group consisting of Thymidine Kinase (TK), thymidylate kinase, cytosine deaminase (CDase) and uracil phosphoribosyl transferase (UPRTase).

In one aspect, the disclosure provides a DNA that can be transcribed to produce the oncolytic virus.

In some preferred embodiments of the present disclosure, the DNA is an infectious clone of an attenuated strain of a flavivirus.

In some preferred embodiments of the present disclosure, the infectious clone is a plasmid.

In one aspect, the present disclosure provides a cell comprising the oncolytic virus or the DNA.

In one aspect, the present disclosure provides a composition comprising the oncolytic virus, the DNA, and/or the cell.

In one aspect, the present disclosure provides the use of the oncolytic virus, the DNA, the cells and/or the composition in the manufacture of a medicament for the treatment of a tumor and/or cancer.

In some preferred embodiments of the present disclosure, the tumor and/or cancer is selected from melanoma, liver and ovarian cancer, lung cancer, breast cancer, cervical cancer, and glioma.

In some preferred embodiments of the present disclosure, the drug may be administered one or more times.

In some preferred embodiments of the present disclosure, the drug may be administered locally or systemically.

Embodiments herein provide for administering a composition to a subject in a biocompatible form suitable for in vivo drug administration. By "biocompatible form suitable for in vivo administration" is meant a form of the active agent (e.g., a pharmaceutical agent of an embodiment) to be administered, wherein the therapeutic effect of the active agent exceeds any toxic effect. Administration of a therapeutically effective amount of a therapeutic composition is defined as an amount effective over the dosage and period of time necessary to achieve the desired result. For example, the therapeutically active amount of a compound may vary with factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody to elicit a desired response in the individual. The dosage regimen may be adjusted to provide the optimal therapeutic response.

In one embodiment, the compound (e.g., a live attenuated virus of an embodiment) can be administered in a convenient manner, e.g., subcutaneously, intravenously, by oral administration, inhalation, intradermally, transdermally, intravaginally, topically, intranasally, or rectally. Depending on the route of administration, the active compound may be contained in a protective buffer, such as albumin and trehalose, poloxamer 407/trehalose/albumin (FTA). In one embodiment, the composition may be administered orally. In another embodiment, the composition may be administered intravenously. In one embodiment, the composition may be administered intranasally, e.g., by inhalation. In another embodiment, the composition may be used in a needleless system (e.g.) Or other intradermal administration system.

As used herein, "pharmaceutically acceptable carrier" may also include diluents such as saline and aqueous buffer solutions.

Pharmaceutical compositions suitable for injectable use may be administered by means known in the art. For example, sterile aqueous solutions (in the case of water solubility) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions may be employed. In some embodiments, the composition may be sterile and may be fluid for ease of injectability. The pharmaceutically acceptable carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycols, and the like), and suitable mixtures thereof. For example, proper fluidity can be maintained by the use of a coating material such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. Prevention of microorganisms can be achieved by adding various antibacterial or antifungal agents or other agents.

In some embodiments, the solution may be administered in a manner compatible with the dosage formulation and in a therapeutically effective amount at the time of formulation. According to these embodiments, the formulation may be readily administered in a variety of dosage forms, such as the types of injectable solutions described above.

In certain embodiments, single or multiple doses of an attenuated flavivirus (e.g., west nile virus) formulation may also be administered to a subject. In some embodiments, the subject may be treated with a single dose formulation. In other embodiments, the subject may be treated with at least two doses of the live attenuated west nile virus formulation. In certain embodiments, the subject may administer the attenuated west nile virus composition on day 0 and a booster dose within about 3 months of the first dose. In certain embodiments, the subject is a non-immune subject (seronegative) that has never been exposed to west nile virus. In other embodiments, the subject may have been previously exposed to west nile virus and/or west nile virus infection (seropositive). According to these embodiments, a seronegative subject may be treated on day 0 and then receive a booster within 6 months, 5 months, 4 months, 3 months or less of the first dose in order to generate an enhanced immune response to the west nile virus. In certain embodiments, the child may be a child from about 2 years old to about 17 years old. In other embodiments, the child is 2 years to 17 years old.

In another embodiment, a nasal solution or spray, aerosol or inhalant may be used to deliver a live attenuated flavivirus (e.g., west nile virus) virus formulation to a subject. Certain formulations may comprise excipients, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like.

Pharmaceutical compositions may be prepared with carriers that protect the active ingredient from rapid elimination from the body, such as timed release formulations or coating materials. Such carriers may include controlled release formulations such as, but not limited to, microencapsulated delivery systems and biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters (polyorthoesters), polylactic acid, and other substances known.

In certain embodiments, the dose range of live attenuated flaviviruses (e.g., west nile virus) may be about 10 ² to about 10 ⁶ PFU administered initially, and optionally followed by at least a second administration within 30 days or up to 12 months after as desired.

In certain embodiments, the pharmaceutical compositions disclosed herein may be administered to a subject in one or more doses. In some embodiments, the immunogenic compositions disclosed herein can be administered to a subject in a single dose or in two or more doses over a predetermined period of time, including but not limited to, within about 6 months, within about 120 days, within about 90 days, within about 80 days, within about 70 days, within about 60 days, within about 50 days, within about 40 days, within about 30 days, within about 20 days, within about 10 days, within about 5 days, or less, or within hours or minutes on the same day or simultaneously in the same or different anatomical locations. In some embodiments, the pharmaceutical compositions disclosed herein may be administered within about 90 days of each other, within about 60 days of each other, within about 30 days of each other, and within less than about 30 days of each other. In some embodiments, the compositions disclosed herein may be administered to a subject subcutaneously or intradermally. Administration in two or more anatomical sites may include any combination of administration, including by the same pattern in two or more anatomical sites or by two different patterns including two separate anatomical sites. According to these embodiments, the two or more anatomical locations may comprise different limbs or different regions of the body. In certain embodiments, two doses of the vaccine composition may be introduced into the subject serially at the same or multiple anatomical locations on day 0, e.g., to provide protection from all flaviviridae (e.g., west nile virus) serotypes (e.g., cross-protection). In other embodiments, the pharmaceutical composition may include a combination of live attenuated west nile virus with other immunogenic agents against other flaviviruses (e.g., zika virus, japanese encephalitis, west nile virus, st.Louis encephalitis virus, yellow fever, or other viruses). In certain embodiments, the vaccines against west nile virus disclosed herein can be used to reduce other related viral infections, such as, for example, pick up virus infections.

In some embodiments, the pharmaceutical compositions disclosed herein can be used to increase the immune response of a target formulation in a subject by combining an immunogenic composition against a flavivirus (e.g., west nile virus) virus with an agent that boosts a cd8+ T cell response or other immune response against west nile virus in a child or young human receiving such a pharmaceutical composition against a flavivirus (e.g., west nile virus) virus.

Topical application is accomplished by topical application of creams, gels, lotions, and the like containing a therapeutically effective amount of serine protease inhibitors. Transdermal administration is accomplished by application of creams, lotions, gels, etc. that enable the serine protease inhibitors to penetrate the skin and enter the blood stream. In addition, osmotic pumps may be used for administration. The necessary dosage will vary with the particular condition being treated, the method of administration, and the rate at which the molecule is cleared from the body.

Other embodiments relate to methods of reducing inactivation of a live attenuated virus, including, but not limited to, combining one or more live attenuated viruses with a composition capable of reducing inactivation of a live attenuated virus (e.g., a flavivirus). These compositions may include, but are not limited to, one or more protein agents; one or more sugar or polyol agents; and optionally one or more EO-PO block copolymers, wherein the composition can reduce inactivation or stabilize the live attenuated virus.

In certain embodiments, the compositions contemplated herein may be partially or fully dehydrated or hydrated. In other embodiments, stabilized protein agents contemplated for use in pharmaceutical or non-pharmaceutical compositions herein may include, but are not limited to, whey protein, human serum albumin, recombinant human serum albumin (rHSA), bovine Serum Albumin (BSA), other serum albumin, or albumin gene family members. Sugar or polyol agents may include, but are not limited to, monosaccharides, disaccharides, sugar alcohols, trehalose, sucrose, maltose, isomaltose, cellobiose, gentiobiose, laminaribose, xylobiose, mannobiose, lactose, fructose, sorbitol, mannitol, lactitol, xylitol, erythritol, raffinose, amylase, cyclodextrin, chitosan, or cellulose. In certain embodiments, surfactants may include, but are not limited to, nonionic surfactants such as alkyl poly (ethylene oxide), copolymers of poly (ethylene oxide) and poly (propylene oxide) (EO-PO block copolymers), poly (vinyl pyrrolidone), alkyl polyglucosides (e.g., sucrose monostearate, lauryl diglucoside or sorbitol monolaurate (sorbitanmonolaureate), octyl glucoside and decyl maltoside), fatty alcohols (cetyl or oleyl alcohol (olelyl alcohol)) or cocoamide (cocamides) (cocoamide MEA, cocoamide DEA and cocoamide TEA).

In other embodiments, the surfactant may include, but is not limited to, poloxamer 407 (e.g., pluronic) Either in place of poloxamer 407 or in addition to poloxamer 407, poloxamer 335, 338 or 238, or in combination withOther EO-PO block copolymers having similar properties.

In some embodiments, the vaccine composition may include, but is not limited to, one or more protein agents that are serum albumin; one or more trehalose saccharides; and one or more surfactant polymer agents, such as EO-PO block copolymers, poloxamer 407, or more specifically Pluronic

In other embodiments, the formulation for stabilizing a live virus may comprise one or more live flaviviruses, one or more carbohydrate reagents, and one or more amino acids or salts, esters, or amide derivatives thereof. In other embodiments, the formulation used herein stabilizes the live attenuated flavivirus for commercial use. In some embodiments, the composition further comprises a buffer. According to these embodiments, the buffer may include, but is not limited to, phosphate Buffered Saline (PBS). According to these embodiments, the buffer may include at least one of sodium chloride (NaCl), monosodium and/or disodium phosphate (Na 2HPO 4), potassium chloride (KCl), and potassium phosphate (KH ₂PO₄). In some embodiments, the buffer of the composition may comprise sodium chloride at a concentration of 25mM to 200 mM. In other embodiments, the compositions disclosed herein may comprise urea and/or other suitable agents for MSG.

In some embodiments, live attenuated flaviviruses such as west nile virus may be stabilized in a formulation comprising, but not limited to, recombinant HSA at a concentration of 0.1% to 0.2% (w/v); and/or sucrose at a concentration of about 4.0% to about 6.0% (w/v); and/or mannitol at a concentration of about 2% to 4% (w/v); and/or alanine at a concentration of about 8.0mM to about 22.0 mM; and/or methionine at a concentration of about 1.0mM to about 5.0 mM; and/or MSG at a concentration of about 8.0mM to 12.0 mM; and/or urea at a concentration of about 0.1% to about 0.3% (w/v). In certain embodiments, the composition may comprise recombinant HSA, trehalose, mannitol, alanine, methionine, MSG, and urea. In other embodiments, the stabilizing composition may comprise HSA at a concentration of about 0.1% to about 0.2% (w/v); trehalose at a concentration of about 4% to about 6% (w/v); mannitol at a concentration of about 2% to about 4% (w/v); wherein the alanine concentration is 8mM to 22mM; wherein methionine concentration is 1mM to 5mM; wherein the MSG concentration is 8mM to 12mM; wherein the urea concentration is 0.1% to 0.3% (w/v). Certain formulations for stabilizing a live attenuated virus may include, but are not limited to, recombinant HSA, sucrose, alanine, and urea. According to these embodiments, the HSA concentration may be about 0.1% to about 0.2% (w/v); sucrose concentration may be about 4% to about 6% (w/v); the alanine concentration can be about 8.0mM to about 22mM; and the urea concentration may be about 0.1% to about 0.3% (w/v). Other stabilizing formulations may include recombinant HSA, sucrose, methionine, and urea. Recombinant HSA concentration may be about 0.1% to 0.2% (w/v); sucrose concentration may be about 4.0% to about 6.0% (w/v); methionine concentration may be about 1.0mM to about 5.0mM; the urea concentration may be about 0.1% to about 0.3% (w/v). In other embodiments, the stabilization formulation may comprise recombinant HSA, sucrose, arginine, and urea, wherein the recombinant HSA concentration may be 0.1% to 0.2% (w/v); sucrose concentration may be 4% to 6% (w/v); arginine concentration may be 10mM to 50mM; the urea concentration may be 0.1% to 0.3% (w/v). Other stabilizing formulations may include recombinant HSA, trehalose, arginine, and urea, wherein the recombinant HSA concentration is about 0.1% to 0.2% (w/v); trehalose concentration from about 4% to 6% (w/v); Arginine concentration is about 10mM to 50mM; urea concentration is about 0.1% to 0.3% (w/v). In other embodiments, the stabilizing composition may include recombinant HSA, trehalose, MSG, and urea. According to these embodiments, the recombinant HSA concentration may be about 0.1% to about 0.2% (w/v); trehalose concentration may be about 4.0% to about 6.0% (w/v); wherein the MSG concentration is 8mM to 12mM; and wherein the urea concentration is 0.1% to 0.3% (w/v).

Some embodiments herein relate to a partially or fully dehydrated live attenuated virus composition for transport or other reasons. According to these embodiments, the composition may be 20% or more; 30% or more; 40% or more; 50% or more; 60% or more; 70% or more; 80% or more; or 90% or more dehydrated. According to these embodiments, the viral vaccine composition may be dehydrated and rehydrated in any known stabilizing composition prior to administration of the pharmaceutically acceptable composition to a subject.

In certain embodiments, the subject may be a mammal, such as a human or veterinary and/or a domestic animal or livestock or wild animal. In certain embodiments, the immunogenic compositions of the present disclosure may be effective in immunizing adolescent subjects, such as human children 20 years of age or less. Although the prior art relating to current dengue virus vaccines/immunogenic compositions (e.g., dengue/yellow fever chimeras) for use by children reports low potency and/or immunogenicity in children 9 years of age or less, the immunogenic compositions disclosed herein may produce an effective immune response in children from about 1 year of age to about 17 years of age or from about 1 year of age to about 9 years of age or more. The immunogenic compositions disclosed herein exhibit superior efficacy and immunogenicity compared to the prior art.

The oncolytic virus can be amplified in BHK cells or Vero cells, and the virus titer can reach (1-10) multiplied by 10 ⁷ PFU.

Experiments prove that the oncolytic virus has cell killing activity on 12 tumor cell lines (including Huh7 (human liver cancer epithelial-like cells), A549 (human lung epithelial cell carcinoma), PC9 (non-small cell lung cancer cells), H1975 (non-small cell lung cancer cells), SKOV3 (human ovarian epithelial adenocarcinoma cells), U87MG (human brain epithelial glioblastoma cells), U118MG (human brain glioma cells), B16F1 (mouse melanoma cells), 4T1 (mouse mammary epithelial carcinoma cells), HELA (human cervical epithelial adenocarcinoma cells), A375 (human skin epithelial melanoma, A2058 (human skin/lymph node melanoma))). In addition, the invention verifies that the oncolytic virus has oncolytic effect in animals when the viral dose of the oncolytic virus is 1 multiplied by 10 ⁷ PFU by utilizing a mouse melanoma-bearing model, a human melanoma nude mouse tumor-bearing model, a human liver cancer cell nude mouse tumor-bearing model and a human ovarian cancer nude mouse tumor-bearing model, and proves that the oncolytic virus has good tumor targeting and anti-tumor effects.

Compared with classical tumor treatment methods, the oncolytic virus can infect and lyse various tumor cells in vitro cell experiments, has small influence on normal cells, has good tumor tropism, and can specifically kill the tumor cells. Can inhibit the in vivo tumor growth of melanoma, liver cancer and ovarian cancer in the xenograft tumor model and delay the tumor progress, i.e. the in vitro or in vivo experimental effect is consistent. On the other hand, compared with other oncolytic virus therapies, the oncolytic virus of the invention has better safety and reliability because of being an attenuated virus strain. The oncolytic virus can be efficiently replicated in Vero cells, and the virus passage is stable, so that the oncolytic virus is easy to use for later production and application. The oncolytic virus is RNA virus, and the cDNA is easy to operate, thereby being beneficial to the later reconstruction and application of viral genome. The oncolytic virus has great application prospect in tumor treatment as a novel oncolytic virus.

Examples

The experimental methods of virus infection, immunofluorescence, CCK8 detection cell survival, virus ultraviolet inactivation, virus titer determination, modeling of tumor-bearing models in mice, intratumoral administration in mice, grinding of tumor tissues, virus titration determination and the like are all conventional methods in the art unless otherwise specified. The following list is merely a few specific embodiments of the present invention. Obviously, the invention is not limited to the following embodiments, but many variations are possible. Modifications and improvements will readily occur to those skilled in the art based upon this disclosure, and are therefore intended to be within the scope of the invention as claimed.

Example 1: construction of attenuated flavivirus clones and virus rescue

1. Construction of attenuated strain clone of flavivirus

The construction of the attenuated flavivirus clones is schematically shown in FIG. 1.

The plasmid DNA of infectious clone of West Nile Virus (WNV) strain of Wild Type (WT) is taken as a skeleton, sequences from SLI to CS2 (comprising 5 'end stem loop region I (SLI), stem loop region II (SLII), repetitive cyclization sequence region 3 (RCS 3), stem loop region III (SLIII), stem loop region IV (SL IV), cyclization sequence region 3 (CS 3), dumbbell region 1 (DB 1), repetitive cyclization sequence region 2 (RCS 2), dumbbell region (DB 2) and cyclization sequence region 2 (CS 2)) in the 3' untranslated region (CS 1) and short hairpin-3 'end stem loop region (sHP-3' SL) after cyclization sequence region 1 are reserved, and poly (A) sequences (130 adenosines) are inserted at deletion sites caused by deletion of sequences from SLI to CS2, so that WNV-A virus attenuated strain is obtained, and nucleotide sequence of which is shown as SEQ ID No. 1.

The sequence from SLI to CS2 (including SLI, SLII, RCS, SLIII, SLIV, CS, DB1, RCS2, DB2, CS 2) in the 3' untranslated region (3 ' UTR) of JEV was deleted, the circularized sequence region 1 (CS 1) of the 3' untranslated region and the short hairpin-3 ' terminal stem-loop region (sHP-3 ' SL) after the circularized sequence region 1 were retained, and poly (A) sequence (150 adenylates) was inserted at the deletion site due to deletion of the sequence from SLI to CS2, to obtain a JEV-poly (A) virus attenuated strain plasmid, the nucleotide sequence of which is shown in SEQ ID NO. 2.

The nucleotide sequence of the DENV-2 (NGC) -poly (A) virus attenuated strain is shown as SEQ ID NO.3 by deleting the sequence (including SLII, RCS3, SL IV, CS3, DB1, RCS2, DB2, CS 2) from SLII to CS2 in the 3' untranslated region (3 ' UTR) of DENV-2 (NGC) with the plasmid DNA of the infectious clone of the dengue virus type 2 New Egizzard strain (DENV-2 (NGC)) of wild-type (WT) as a backbone, retaining the cyclized sequence region 1 (CS 1) of the 3' untranslated region and the short hairpin-3 ' stem loop region (sHP-3 ' SL) after the cyclized sequence region 1, and inserting the poly (A) sequence (140 adenylates) at the deletion site due to deletion of the sequence from SLI to CS 2.

The plasmid DNA of infectious clone of Yellow Fever Virus (YFV) strain of Wild Type (WT) is taken as a skeleton, sequences (including SLII, RCS3 and DB) from SLII to CS2 in a 3' untranslated region (3 ' UTR) of YFV are deleted, a stem-loop region (SL) of the 3' untranslated region, a cyclized sequence region 1 (CS 1) and a short hairpin-3 ' end stem-loop region (sHP-3 ' SL) after the cyclized sequence region 1 are reserved, and a poly (A) sequence (130 adenylates) is inserted at a deletion site caused by deletion of the sequences from SLI to CS2, so that a plasmid of YFV-poly (A) virus attenuated strain is obtained, the nucleotide sequence of which is shown as SEQ ID NO. 4.

The strain plasmids are all obtained by replacing all or part of 5' end stem loop region (SL) sequences and all DB sequences in a 3' UTR region with poly (A) sequences, and retaining at least cyclized sequence region 1 (CS 1) of a 3' untranslated region and short hairpin-3 ' end stem loop region (sHP-3 ' SL) after cyclized sequence region 1, thereby respectively obtaining corresponding poly (A) virus attenuated strain plasmids.

Poly (A) in vitro transcription of RNA

Linearizing the poly (A) virus attenuated strain plasmid obtained in the step 1, carrying out phenol-chloroform extraction after 0.8% agarose gel electrophoresis to identify that linearization is complete, finally adding 11 mu L of RNAase-free water for dissolution, measuring the concentration of DNA by using Thermo Scientific NanoDrop 2000,2000 and detecting the quality of the DNA by electrophoresis. 1. Mu.g of phenol-chloroform extracted DNA was used as a template, and the recombinant poly (A) RNA, i.e., genomic RNA of the attenuated strains of WNV-poly (A), JEV-poly (A), DENV-2 (NGC) -poly (A) and YFV-poly (A), was obtained by in vitro transcription using an in vitro transcription kit T7 MMESSAGE MMACHINE KIT (Ambion) according to the kit instructions. The concentration of RNA was determined by Thermo Scientific NanoDrop 2000, and the quality of RNA was checked by electrophoresis on a freshly prepared agarose gel at 0.8% and stored at-80℃for further use.

Poly (A) virus rescue

The experiments were divided into the following two groups:

(1) Experimental group: transfecting the recombinant poly (A) RNA obtained by in vitro transcription in the step 2 into BHK-21 cells by using a liposome transfection method;

(2) Control group: wild-type viral RNA was transfected into BHK-21 cells using a liposome transfection method as a positive control.

The specific experimental steps are as follows: on the day before transfection, 2X 105 BHK-21 cells were seeded in 35mm cell culture dishes, each dish was placed with three 10mm X10 mm coverslips to bring the cells to about 80% on the day of transfection; during transfection, the culture medium in the culture dish is firstly discarded, and 1ml of Opti-MEM is used for washing once, and then 1ml of Opti-MEM is added (so that cells are in an infiltration state); 1ml of Opti-MEM and 4 mu l of DMRIE-C (which are mixed before use) are added into a 1.5ml EP tube, and are mixed up and down, 1 mu g of recombinant poly (A) RNA obtained by in vitro transcription is respectively added into an experimental group, and 1 mu g of wild-type viral RNA is added into a control group, and are mixed up and down; the Opti-MEM in the dish was rapidly discarded and the mix was added to the dish (action was light, not blown against the cells); after culturing in a CO2 incubator at 37℃for 4 hours, the cultures were discarded, and 2mL of DMEM medium containing 2% FBS was added to each. The cell status of the experimental group and the control group was observed under a microscope, and after 24, 48 and 72 hours of transfection, one slide was taken, the cells were fixed with 5% acetone fixing solution (purchased from national drug group chemical reagent company), room temperature was fixed for 15 minutes, PBS was washed three times, and stored at 4℃and when the IFA detection positive rate was high, the supernatants were collected as poly (A) P0 generation viruses of attenuated flaviviruses, namely, 4P 0 generation WNV-poly (A), JEV-poly (A), DENV-2 (NGC) -poly (A) and YFV-poly (A) attenuated strains were obtained, respectively, and stored at-80 ℃.

4. Indirect Immunofluorescence (IFA) detection of viral protein expression of recombinant poly (A)

The slide preserved at 4 ℃ in the step 3 is incubated with primary antibody at room temperature, and the attenuated strain of the flavivirus is a 1:500-fold dilution of the 4G2 monoclonal antibody (the general antibody of the flavivirus E protein). After primary antibody is incubated for 1-2 h at room temperature and PBS is washed for 10 times, secondary antibody is incubated at room temperature in dark, and the antibody is a coupled Fluorescein Isothiocyanate (FITC) goat anti-mouse antibody diluted 1:125 times. After incubation for 1h with secondary antibody, the slides were removed and labeled with 95% glycerol at each site, the coverslips were placed with the cell side down on glycerol drops and observed under a fluorescence microscope using 200 x magnification (fig. 1).

The results in FIG. 1 show that WNV-poly (A), JEV-poly (A), DENV-2 (NGC) -poly (A) and YFV-poly (A) attenuated strains were each less positive than WT at each time point, but positive cell numbers gradually increased with increasing incubation time after transfection, consistent with the WT trend, indicating that WNV-poly (A), JEV-poly (A), DENV-2 (NGC) -poly (A) and YFV-poly (A) each rescue infectious viral particles.

In addition, WNV-poly (A), JEV-poly (A), DENV-2 (NGC) -poly (A) and YFV-poly (A) were serially passaged for 10 passages, and the polyA insertion position and number remained substantially unchanged by about 130-150A, indicating that the above poly (A) viruses have excellent genetic stability.

Subsequent experiments used WNV-poly (A) virus to further investigate oncolytic effect of attenuated strains of Flaviviridae on tumor cells.

Example 2: oncolytic effect of WNV-poly (A) on tumor cells in vitro

This example demonstrates the in vitro oncolytic effect of the virus by infecting tumor cells with a fixed MOI dose of WNV-poly (A) virus attenuated virus (WNV-poly (A)) prepared in example 1 (FIG. 2A), and examining the killing of tumor cells by different doses of WNV-poly (A) with CCK8 (FIG. 2B). Wherein:

Control group (MOCK): a group of tumor cells cultured in a medium without virus;

experimental group: tumor cell groups cultured with medium supplemented with WNV-poly (A).

1. Open field observation of oncolytic effect of WNV-poly (A) on tumor cells and Normal cells

12 Tumor cells Huh7, A549, PC9, H1975, SKOV3, U87MG, U118MG, B16F1, 4T1, HELA, A375 and A2058 and 3 normal cells MLE-12, MEF and L-02 were inoculated into a 12-well cell culture plate according to 8X 10 ⁴ cells/well, and a DMEM or 1640 medium with 10% serum (FBS) was used, and 1% penicillin and streptomycin were added to the medium, and cultured in a humidified incubator at 37℃and 5% CO ₂. After cell attachment growth, the medium was changed to DMEM medium with 2% fbs, and tumor cell death was observed in the bright field 72 hours after infection at a dose of WNV-poly (a) with moi=10.

The results showed that WNV-poly (a) significantly induced death or significant inhibition of growth of tumor cells in all 12 tumor cells examined, but it had no significant effect on the 3 normal cells examined (fig. 2A). The above results primarily indicate that WNV-poly (A) has a broad spectrum of oncolytic effects in vitro experiments and has little damage to normal cells.

CCK8 assay for oncolytic Effect of different doses of WNV-poly (A) on tumor cells

To further quantitatively verify the in vitro oncolytic effect of WNV-poly (A), the oncolytic effect of virus on tumor cells after infection at different MOI doses was detected using CCK8 detection kit.

The testing method comprises the following steps: tumor cells Huh7, A549, SKOV3, B16F1, 4T1, HELA and normal cells MLE-12, MEF and L-02 are respectively inoculated into 96-hole cell culture plates according to 4000 cells/hole, and after cell wall-attached growth, the tumor cells are respectively grown according to MOI: 0. doses of 0.001, 0.01, 0.1, 1, 10 infected WNV-poly (A), 5 multiple wells per group. After 72 hours, cell activity was detected using CCK8 detection kit. After CCK8 is added into cells, the CCK8 can be reduced by dehydrogenase in mitochondria to generate a orange yellow formazan product with high water solubility, the OD value read at the detection wavelength 450 of an enzyme label instrument indirectly reflects the number of living cells, and the OD value is directly proportional to the proliferation condition of the cells and inversely proportional to cytotoxicity. For each group of data, the same volume of DMEM medium without cells and virus was added with CCK8 as a blank group, and no virus was added (moi=0) as a control group, and the formula = (added virus group-blank group)/(control group-blank group) ×100 was calculated. The results were averaged and standard deviation calculated by GraphPadPrism7 software. Results the results of the cell activity assays of different doses of WNV-poly (A) on each tumor cell and normal cell were plotted.

As shown in FIG. 2B, WNV-poly (A) had significant killing effect on 6 tumor cells examined, but it had no significant effect on survival of 3 normal cells examined. And further observing the effect of different MOIs, the WNV-poly (A) has obvious virus infection dose dependency on the killing of tumor cells, and the death of the tumor cells is obviously increased along with the increase of the virus infection dose. This result further established that WNV-poly (a) has a remarkable and broad-spectrum oncolytic effect in vitro experiments and has little damage to normal cells.

Example 3: in vitro differences in killing of WNV-wT and WNV-poly (A) on normal and tumor cells

This example further determined the specificity of WNV-poly (A) killing on tumor cells by comparing the growth curves of WNV-WT and WNV-poly (A) on normal cells and tumor cells by brightly observing their killing on normal cells and tumor cells after infection of tumor cells with wild-type West Nile Virus (WNV-WT produced by transfection of WNV-infectious clone 3356 strain) at a fixed MOI dose (FIG. 3A) and detecting killing on normal cells and tumor cells by WNV-WT and WNV-poly (A) at different doses with CCK8 (FIG. 3B). Wherein:

experimental group: tumor cell groups cultured with WNV-WT or WNV-poly (A) medium.

1. Open field observations of oncolytic effects of WNV-WT and WNV-poly (A) on normal and tumor cells

3 Kinds of tumor cells B16F1, huh7, SKOV3 and normal cells MLE-12, MEF, L-02 were inoculated into a 12-well cell culture plate according to 8X 10 ⁴ cells/well, and 10% serum (FBS) medium of DMEM or 1640 was added with 1% penicillin and streptomycin, and cultured in a 5% CO ₂ humidified incubator at 37 ℃. After cell attachment growth, the medium was changed to DMEM medium with 2% fbs, and WNV-WT and WNV-poly (a) were infected at moi=10, and death of normal cells and tumor cells was observed in the open field 72 hours after infection.

The results showed that WNV-WT and WNV-poly (A) were clearly visible in the 3 tumor cells tested, but in the 3 normal cells MLE-12, MEF and L-02, WNV-WT induced significant cell death, whereas WNV-poly (A) had no killing effect on these 3 cells (FIG. 3A). The above results preliminarily indicate that WNV-WT and WNV-poly (A) have oncolytic properties against tumor cells. WNV-WT has killing effect on normal cells, whereas WNV-poly (A) has no killing effect on normal cells. The above results indicate that WNV-poly (A) has oncolytic specificity for tumor cells.

Cck8 assay for killing of normal and tumor cells at different doses WNV-WT and WNV-poly (a):

To further quantitatively verify the oncolytic variability of WNV-WT and WNV-poly (A) to tumor cells and normal cells, the oncolytic effect of the virus on each cell after infection at different MOI doses was tested using the CCK8 assay kit.

The testing method comprises the following steps: huh7, SKOV3, B16F1 and normal cells MLE-12, MEF, L-02 were seeded at 4000 cells/well in 96-well cell culture plates, and after cell attachment growth, the MOI was: 0. doses of 0.001, 0.01, 0.1, 1, 10 were infected with WNV-WT and WNV-poly (A), respectively, 5 multiple wells per group. Cell survival after 72 hours of infection was determined according to the CCK8 assay described in example 2.

The results showed that WNV-WT and WNV-poly (A) had dose-dependent killing of all 3 tumor cells, and WNV-WT was more pronounced than WNV-poly (A) (FIG. 3B). From the results of 3 normal cells, WNV-WT had significant killing of all 3 normal cells examined, and the killing was significantly dose dependent on viral infection, but WNV-poly (A) had no significant killing effect on normal cells. The above results further indicate that WNV-poly (A) has oncolytic specificity for tumor cells.

Differential detection of replication of WNV-WT and WNV-poly (A) in normal cells MEF and tumor cells Huh7, A375, B16F1

In the experiment, 2X 10 ⁵ tumor cells are inoculated in a 35mm cell culture dish, when the confluence reaches 60% under the culture condition of 5% CO ₂ at 37 ℃, 500 mu l of diluted WNV-WT and WNV-poly (A) are added into the small dish according to the MOI of the infection complex of 10, after the diluted WNV-WT and WNV-poly (A) are adsorbed for 2 hours in a culture box with 5% CO ₂ at 37 ℃, 2ml of DMEM culture medium containing 2% bovine serum is added into each hole, the culture is carried out under the culture condition with 5% CO ₂ at 37 ℃, 300 mu l of virus supernatant is respectively collected every 24 hours after the infection, 300 mu l of DMEM culture medium containing 2% bovine serum is added as a virus sample at different time points, and the virus sample is stored at-80 ℃. Virus samples at different time points were subjected to plaque experiments to identify plaque morphology and their growth curves were determined: 1X 10 ⁵ BHK-21 cells were inoculated into each well of a 24-well cell culture plate, and when the cell confluency reached 90%, the medium in the well was discarded, 100. Mu.L of virus collected at the above different time points diluted 10-fold with DMEM medium containing 2% FBS was added, and adsorbed at 37℃for 1 hour, and shaking was performed sufficiently every 15 minutes. After the adsorption, the virus solution in each well is sucked and removed, a DMEM culture medium containing 2% FBS and a cover of 2% methylcellulose are added, the culture is carried out in an incubator containing 5% CO ₂ at 37 ℃ for 4 days, after plaque formation, the plaque is stained with a staining solution containing 1% crystal violet and 3.7% formaldehyde, after the room temperature treatment is carried out for 30min, the staining solution in the well is taken out, the bottom of the well is washed by running water, and after the drying, the virus titer is counted and converted, and a growth curve is drawn. The results indicate that WNV-WT and WNV-poly (A) can be well amplified on B16F1 (FIG. 3C), A375 (FIG. 3D), huh7 (FIG. 3E) cells, but that WNV-WT can be amplified significantly better on normal cells MEF than WNV-poly (A) (FIG. 3F).

Example 4: west Nile Virus attenuated strain causes tumor cell apoptosis

In this example, the killing pattern of WNV-poly (A) on tumor cells Huh7, SKOV3, B16F1 was further determined by detecting intracellular Caspase3 cleavage by Anexin-V/PI staining and WB detection. Wherein:

1. Apoptosis of WNV-poly (A) on Huh7, SKOV3 and B16F1 was detected with Anexin-V/PI

Tumor cells Huh7, SKOV3, B16F1 were inoculated in 12-well cell culture plates at 8×10 ⁴ cells/well, respectively, and cultured overnight in DMEM or 1640 medium with 10% serum (FBS). After cell attachment growth, the medium was changed to DMEM medium with 2% fbs, and apoptosis of tumor cells was determined by Anexin-V/PI staining after 48 hours of infection with moi=10 dose.

The experimental method is as follows: tumor cells after infection were digested with EDTA-free trypsin and collected into EP tubes. Cells were resuspended with 200 μl annexin v binding buffer. Mu.l of Annexin-V solution was added to each tube and incubated at 4℃for 15min in the absence of light. Mu.l of 20. Mu.g/ml PI solution was added, gently mixed and incubated at 4℃for 15min in the absence of light. Cells were transferred into flow tubes and tested on-machine. Annexin V single-stain indicates early apoptosis of cells, annexin V/PI double-stain indicates late apoptosis of cells, PI single-positive indicates obvious necrosis of cells. The results show that WNV-poly (A) can significantly induce apoptosis of tumor cells Huh7 and SKOV3, and has obvious necrosis promoting effect on B16F1 (FIG. 4A).

Detection of cleavage of Caspase3 in Huh7, SKOV3 and B16F1 cells following WNV-poly (A) infection

Tumor cells Huh7, SKOV3, B16F1 were seeded in 12-well cell culture plates at 2×10 ⁵ cells/well, respectively, medium was changed to DMEM with 2% fbs after cell wall-attachment growth, and cell samples were collected at moi=10 doses at 24h and 48h of infection, respectively.

The experimental method is as follows: 200 μl of RIPA-containing lysate is added to each well, cells are scraped with cells and collected into a new EP tube, incubated on ice for 30min, 50 μl of 5 Xloading buffer (loadingbuffer) is added, mixed and heated at 100deg.C for 10min to allow sufficient denaturation, 4℃and 12000rpm, centrifuged for 2min, the precipitate is removed, and the obtained supernatant is subjected to Westem-blot to detect the level of apoptosis-related protein change, with β -actin as a control reference. The results showed that Huh7, SKOV3 showed significant Caspase3 cleavage at both 24h and 48h of detection (FIG. 4B). The above experimental results demonstrate that WNV-poly (A) is capable of causing significant apoptosis of Huh7 and SKOV3, whereas the killing form responsible for B16F1 cell death is not apoptosis. The mechanism by which WNV-poly (A) causes tumor cell oncolysis may exist in addition to apoptosis.

Example 5: inactivating oncolytic effect of WNV-poly (A) on tumor cells

In this example, a known titer of WNV-poly (A) virus was inactivated by UV to obtain UV-inactivated WNV-poly (A) (UV-WNV-poly (A)) and the virus was determined to have been completely inactivated by the plaque assay described above.

Tumor cells Huh7 and SKOV3 were inoculated in a 12-well cell culture plate at2×10 ⁵ cells/well, the culture medium was changed to DMEM of 2% fbs after cell wall-attachment growth, and the cells were stained with a staining solution containing 1% crystal violet and 3.7% formaldehyde at moi=10 UV-WNV-poly (a) and WNV-poly (a) doses for 48 hours, after treatment at room temperature for 30min, the staining solution in the wells was removed, the bottoms of the wells were rinsed with running water, and the effect of UV-inactivated virus on tumor cells was observed after drying.

Experimental results show that the results after UV-WNV-poly (A) infection are consistent with the MOCK group, the cells grow normally, crystal violet staining is complete, no viral plaques appear, and tumor cells die significantly after WNV-poly (A) infection, and crystal violet staining shows substantially no living cells (FIG. 5). This suggests that UV-inactivated UV-WNV-poly (A) loses oncolytic effect on tumor cells compared to WNV-poly (A) infected tumor cells.

Example 6: in vivo detection of oncolytic effects on melanoma following multiple dosing of WNV-poly (A)

In this example, 2 melanoma cell lines, namely, mouse melanoma cell B16F1 and human melanoma cell A375, were selected, an in vivo transplantation tumor model was established, and the therapeutic effect of WNV-poly (A) on in vivo transplantation tumors was observed once 3 days of intratumoral administration. And further detecting the immune response conditions of the tumor and the system through flow. Wherein:

effect of wnv-poly (a) on tumor in tumor bearing model mice

Control group (MOCK): intratumorally administering 2% fbs DMEM medium without virus;

experimental group: the 2% FBS DMEM medium supplemented with WNV-poly (A) was intratumorally administered.

The implementation method comprises the following steps: c57 mice of 6-8 weeks old were selected, inoculated subcutaneously with 2×10 ⁵ murine melanoma cells B16F1, and until the transplanted tumors could be reached, the mice were divided into two groups, one group was given 2% fbs DMEM (as an experimental group) each containing 1×10 ⁷ PFU WNV-poly (a), one group was given an equal volume of 2% fbs DMEM (as a control group), treated by direct injection onto the tumor body, once every 3 days, and tumor volume and mouse weight were measured, and after four consecutive administrations, the mice were sacrificed, tumor tissues were collected, and weighed. The tumor volume calculation formula is (length×width 2)/2. The results of treatment of melanoma in B16F1 mice are shown in fig. 6, where the tumor morphology results visually indicate that WNV-poly (a) has significant therapeutic effects on B16F1 tumors (fig. 6A), and similarly, reduced tumor volume (fig. 6B) and reduced tumor weight (fig. 6C) indicate that WNV-poly (a) significantly inhibited tumor growth. And as shown in fig. 6D, there was no effect on the change in mouse body weight after WNV-poly (a) treatment. Further detection of tissue virus titer by plaque revealed that no virus was detected in the tumor tissue, brain tissue, and spleen tissue and serum detected (fig. 6E).

Similar to the establishment of an in vivo transplantation tumor model of a mouse melanoma cell B16F1, an in vivo transplantation tumor model of a human melanoma cell A375 is also constructed. Nude Bab/c mice of 4-5 weeks old were selected, subcutaneously vaccinated with 1 x 10 ⁷ human melanoma cells a375, and until the transplanted tumors could be reached, the mice were divided into two groups, one group given each 1 x 10 ⁷ PFU WNV-poly (a), one group given an equal volume of 2% fbs DMEM, directly injected on the tumor for treatment, dosed once every 3 days, and tumor volume and mouse weight were measured, mice were sacrificed after four consecutive doses, tumor tissues were collected, and weighed. As a result, WNV-poly (A) was found to have a significant inhibitory effect on human melanoma tumor A375, and did not affect weight changes in mice, but was able to detect virus in tumor tissue (FIGS. 6F-J).

The in vivo tumor-bearing model result shows that WNV-poly (A) has remarkable inhibiting effect on human melanoma cells and mouse melanoma.

Effect of WNV-poly (A) on tumor immune microenvironment

The effect of WNV-poly (A) on tumor immune microenvironment was examined by flow-through this experiment.

The experiments of figures 7A-7E were divided into two groups:

The experiment of fig. 7F was divided into the following five groups:

control group (MOCK): intratumoral administration of PBS group without virus;

WNV-poly (a) +pbs group: intratumorally administering WNV-poly (A) virus, the virus diluted with PBS;

WNV-poly (A) +anti-NK group: intratumorally administering antibodies to WNV-poly (A) virus that clear NK cells;

WNV-poly (a) +anti-CD4 group: intratumorally administering antibodies to WNV-poly (A) virus that clear CD4+ T cells;

WNV-poly (a) +anti-CD8 group: antibodies that clear CD8+ T cells were added intratumorally to WNV-poly (A) virus.

Tumor tissue and spleen tissue were harvested after mice were sacrificed at the treatment endpoint. For the treatment of mouse spleen tissue, spleen is taken, PBS is added to the spleen, the spleen is ground by a 200-mesh screen, the centrifugation is carried out at 1200rpm for 5 minutes, the supernatant is discarded after the centrifugation, 3ml of erythrocyte lysate is added to the obtained cell sediment, the mixture is kept stand at 4 ℃ for 10 minutes, 20ml of PBS is added to wash the mixture for one time, the centrifugation is completed, the supernatant is discarded, and the lysed tissue protein is picked out by a gun tip. Cells were suspended by adding 1ml PBS, and the cells were pipetted at 100. Mu.l/tube, blocked by adding isotype control antibody for 30min, after blocking, various fluorescent-labeled antibodies were added, incubated at 4℃for 1h, washed with PBS, and then detected on a machine. For the treatment of mouse tumor tissues, the tumor tissues are sheared into small blocks, then PBS is added into the small blocks and is ground by a 200-mesh screen, the obtained grinding liquid is centrifuged at 700rpm for 1min, the supernatant is collected and centrifuged at 1200rpm for 10min, the obtained cell sediment is added into erythrocyte lysate, the erythrocyte lysate is placed at 4 ℃ for 10min, then 20ml PBS is added for washing, the supernatant is removed after centrifugation, and the cracked tissue protein is picked out by a gun tip. Cells were suspended by adding 1ml PBS, and the cells were pipetted at 100. Mu.l/tube, blocked by adding isotype control antibody for 30min, after blocking, various fluorescent-labeled antibodies were added, incubated at 4℃for 1h, washed with PBS, and then detected on a machine.

Flow results showed that the proportion of cd45+ immune cells in the tumor microenvironment of WNV-poly (a) treated group was significantly increased compared to control group (MOCK) (fig. 7A), and that the proportion of cd8+ T cells was found to be significantly increased and the proportion of regulatory T cells (Treg cells) was significantly decreased when the proportion of individual immune cells was further analyzed (fig. 7B). A significant reduction in the proportion of Treg cells was also observed in the spleen (fig. 7C). And further analysis of the phenotype of cd8+t found a significant increase in both effector CD8 and memory CD8 in the tumor, whereas there was no change in the spleen (fig. 7d, e).

To further determine the role of CD8 in WNV-poly (A) oncolytic therapy, a CD8+ T cell depletion experiment was performed with an Anti-CD8 antibody (Anti-CD 8). Meanwhile, CD4+ T cells and NK cells were cleared with Anti-CD4 antibody (Anti-CD 4) and Anti-NK antibody (Anti-NK), respectively, and used as a control experiment group for CD8+ T cell clearance. And at the same time, a control group (MOCK) which was not detoxified by PBS alone (as a negative control group) and a detoxified group diluted with PBS (as a positive control group) were compared. Experimental results show that the oncolytic effect of WNV-poly (A) is significantly reduced after CD8+ T cell depletion, but the removal of CD8+ T cells does not completely counteract the oncolytic effect of WNV-poly (A), and the control group has no effect on the treatment of WNV-poly (A) by CD4+ T cell depletion and NK cell depletion (FIG. 7F-H). The results show that WNV-poly (A) can obviously induce the activation of CD8+ T cells in tumors and organisms, and further clearing experiment results show that the therapeutic effect of WNV-poly (A) is partially dependent on CD8+ T cells.

Example 7: effect of double sided tumor-bearing unilateral administration of WNV-poly (A) on oncolytic Effect

This example demonstrates whether WNV-poly (A) is effective against distant tumors by a model of double sided tumor-bearing unilateral dosing in mouse melanoma cells B16F1 to further clarify the role of immune responses in WNV-poly (A) therapy. Wherein:

control group (MOCK): intratumoral administration of medium without virus;

experimental group: culture medium to which WNV-poly (A) was added was intratumorally administered.

The implementation method comprises the following steps: the 6-8 week old C57 mice were selected, left side underarm was inoculated with 2X 10 ⁵ murine melanoma cells B16F1, right side underarm was inoculated with 5X 10 ⁴ cells, and when the transplanted tumors could be touched to the left side, the mice were divided into two groups, left side was given treatment, one group was given 2% FBS DMEM medium each containing 1X 10 ⁷ PFU WNV-poly (A), one group was given an equal volume of 2% FBS DMEM medium, directly injected onto the tumor mass, and the tumor volume and the weight of the mice were measured once every 3 days, and after four consecutive administrations, the tumor tissues were collected and weighed (FIG. 8A). The tumor volume calculation formula is (length×width 2)/2.

The treatment results are shown in FIG. 8, and the tumor morphology results visually indicate that WNV-poly (A) has significant therapeutic effects on both B16F 1-administered and distal tumors (FIG. 8B). Likewise, tumor volume reduction (fig. 8C) and tumor weight reduction (fig. 8D) also demonstrated that WNV-poly (a) significantly inhibited growth of distant tumors. And further plaque detection. This result indicates that WNV-poly (A) has a significant therapeutic effect on the distal tumor and that no virus is detected in the tumor tissue, indicating that the therapeutic effect of WNV-poly (A) on the distal tissue is primarily dependent on its activated immune response.

Example 8: oncolytic effect of virus itself on tumor cells

Previous experimental results observed that WNV-poly (A) treatment was effective in reversing the tumor immunosuppressive microenvironment and significantly stimulated activation of CD8+ T cells, and further clearing CD8+ T cells found that their oncolytic effect was indeed reduced, but did not completely counteract the oncolytic effect of WNV-poly (A). This suggests that there are other oncolytic mechanisms in addition to immune responses in WNV-poly (a) treatment. The research literature has found that in oncolytic treatment of viruses, in addition to the effective inhibition of tumor by virus-activated immune responses, the killing of tumor cells by the viruses themselves also plays a key oncolytic effect.

To determine the oncolytic effect of WNV-poly (A) itself, this example explores whether killing of tumor cells by the virus itself, except for immune response, was responsible for oncolysis during WNV-poly (A) treatment by comparing the modes of administration to which it was administered only once. Wherein:

the experiments of fig. 9A-C were divided into the following five groups:

control group (MOCK): intratumoral administration of 2% fbs DMEM medium;

inactivated virus group: intratumorally administered 2% FBS DMEM medium supplemented with 1X 10 ⁷ PFU of ultraviolet inactivated WNV-poly (A) virus;

WNV-poly (a) -1 x 10 ⁷ group: intratumorally was given 2% FBS DMEM medium supplemented with WNV-poly (A) virus 1X 10 ⁷ PFU;

WNV-poly (a) -1 x 10 ⁵ group: intratumorally was given 2% FBS DMEM medium supplemented with WNV-poly (A) virus 1X 10 ⁵ PFU;

WNV-poly (a) -1 x 10 ³ group: intratumorally was given 2% FBS DMEM medium supplemented with WNV-poly (A) virus 1X 10 ³ PFU;

This example examined oncolytic effects following one treatment with different doses of WNV-poly (A) and found that one treatment with WNV-poly (A) was dose dependent, 1X 10 ⁷ PFU was effective in inhibiting tumor growth, 1X 10 ⁵ PFU was not effective in part of the tumor, and 1X 10 ³ PFU was not effective (FIGS. 9A-C).

Next, the present example examined the intratumoral viral load at different days after one administration of 1X 10 ⁷ PFU WNV-poly (A), and as a result, it was found that the intratumoral viral titer was increased 10-fold after three days compared to one day after administration, and then the oncological viral titers were examined 5 days, 7 days, and 9 days, 11 days, and 14 days after administration, respectively, and the viral load was found to be gradually decreased until no virus was detected for 11 days (FIG. 9D).

The experiments of fig. 9E-9H were divided into two groups:

Experimental group: intratumoral administration of WNV-poly (A) in 2% FBS DMEM medium

To further determine whether the immune system was functioning in one administration, the tumor and spleen immune responses were examined after 1X 10 ⁷ PFU WNV-poly (A) administration, and as a result, it was found that no immune cell activation was found in either tumor or spleen, further demonstrating that WNV-poly (A) could exert its oncolytic effect by itself to effectively inhibit tumor growth without activating immune responses (FIGS. 9E-H). This result shows that WNV-poly (A) has a significant inhibitory effect on melanoma once administered and that the virus replicates in tumor tissue after administration.

Example 9: broad-spectrum oncolytic effects of WNV-poly (A)

In this example, the broad spectrum of WNV-poly (A) oncolysis was demonstrated by examining the in vivo oncolysis of WNV-poly (A) against human hepatoma cells Huh7 and human ovarian carcinoma cells SKOV 3.

The experiment was divided into the following two groups:

Experimental procedure As shown in FIGS. 10A and 10F, 4-5 week old Nude Bab/c mice were selected, subcutaneously inoculated with 2X 10 ⁶ human hepatoma cells Huh7 or 3X 10 ⁶ human ovarian carcinoma cells SKOV3, and when the transplanted tumors could be touched, the mice were divided into two groups, one group was given to each 1X 10 ⁷ PFU WNV-poly (A), one group was given with an equal volume of 2% FBS DMEM, directly injected on the tumor body for treatment, administered once every 3 days, and the tumor volume and the weight of the mice were measured, and after four consecutive administrations, the tumor tissues were collected and weighed.

As a result, WNV-polv (A) was found to have a significant inhibitory effect on both human hepatoma cell Huh7 (FIGS. 10B-D) and human ovarian carcinoma cell SKOV3 (FIGS. 10G-I). And further analysis of tissue tropism of the virus found that in the human hepatoma cell Huh7 (FIG. 10E) and human ovarian carcinoma cell SKOV3 (FIG. 10J) models, only the virus could be detected in tumor tissue in each tissue. The result shows that WNV-poly (A) has obvious oncolytic effect on liver cancer and ovarian cancer besides obvious oncolytic effect on melanoma. This further illustrates the potential broad spectrum tumor treatment potential of WNV-poly (A).

Example 10: therapeutic efficacy detection of different modes of WNV-poly (A)

This example demonstrates the effectiveness of WNV-poly (A) therapy by detecting its oncolytic effect in human hepatoma cell Huh7 in vivo with different modes of administration.

The experimental procedure was as follows: nude Bab/c mice of 4-5 weeks old were selected, subcutaneously vaccinated with 1×10 ⁶ human hepatoma cells Huh7 until they could be touched to the transplanted tumors, the mice were divided into 4 groups, three of which were given each 1×10 ⁷ PFU WNV-poly (a) by different modes of administration (intramuscular injection (i.m.), intravenous injection (i.v), intratumoral injection (i.t.), one group was given an equal volume of 2% fbs DMEM, administered once every 3 days, and tumor volumes were measured, and survival of the mice was observed until treatment for 20 days. As a result, it was found that the growth of tumor was significantly inhibited after intravenous and intratumoral administration, whereas intramuscular injection had substantially no therapeutic effect on tumor (FIG. 11A). And the therapeutic effect of intravenous and intratumoral administration can also be seen significantly by monitoring the volume change of the tumor (fig. 11B). Further observations of the survival rate of mice revealed that intratumoral and intramuscular administration did not affect survival of mice, but 40% of mice with intravenous administration died (fig. 11C).

In conclusion, intravenous and intratumoral administration are both effective for tumor treatment, but safer than intravenous intratumoral administration.

Sequence listing

<110> Beijing Shun Deg science and technology Co., ltd

Chinese academy of sciences' Wuhan virus institute

<120> Use of an attenuated flaviviridae virus in oncolysis

<130> MTI21152

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

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<213> West Nile virus

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ataacattca caacgacatc aaacgtggtt gttccgctgc tagccctgct aacacccggg 4020

ctgagatgct tgaatctgga tgtgtacagg atactgctgt tgatggtcgg aataggcagc 4080

ttgatcaggg agaagaggag tgcagctgca aaaaagaaag gagcaagtct gctatgcttg 4140

gctctagcct caacaggact tttcaacccc atgatccttg ctgctggact gattacatgt 4200

gatcccaacc gtaaacgcgg atggcccgca actgaagtga tgacagctgt cggcctgatg 4260

tttgccatcg tcggagggct ggcagagctt gacattgact ccatggccat tccaatgact 4320

atcgcggggc tcatgtttgc tgctttcgtg atttctggga aatcaacaga tatgtggatt 4380

gagagaacgg cggacatttc ctgggaaagt gatgcagaaa ttacaggctc gagcgaaaga 4440

gttgatgtgc ggcttgatga tgatggaaac ttccagctca tgaatgatcc aggagcacct 4500

tggaagatat ggatgctcag aatggtctgt ctcgcgatta gtgcgtacac cccctgggca 4560

atcttgccct cagtagttgg attttggata actctccaat acacaaagag aggaggcgtg 4620

ttgtgggaca ctccctcacc aaaggagtac aaaaaggggg acacgaccac cggcgtctac 4680

aggatcatga ctcgtgggct gctcggcagt tatcaagcag gagcgggcgt gatggttgaa 4740

ggtgttttcc acaccctttg gcatacaaca aaaggagccg ctttgatgag cggagagggc 4800

cgcctggacc catactgggg cagtgtcaag gaggatcgac tttgttacgg aggaccctgg 4860

aaattgcagc acaagtggaa cgggcaggat gaggtgcaga tgattgtggt ggaacctggc 4920

aagaacgtta agaacgtcca gacgaaacca ggggtgttca aaacacctga aggagaaatc 4980

ggggccgtga ctttggactt ccccactgga acatcaggct caccaatagt ggacaaaaac 5040

ggtgatgtga ttgggcttta tggcaatgga gtcataatgc ccaacggctc atacataagc 5100

gcgatagtgc agggtgaaag gatggatgag ccaatcccag ccggattcga acctgagatg 5160

ctgaggaaaa aacagatcac tgtactggat ctccatcccg gcgccggtaa aacaaggagg 5220

attctgccac agatcatcaa agaggccata aacagaagac tgagaacagc cgtgctagca 5280

ccaaccaggg ttgtggctgc tgagatggct gaagcactga gaggactgcc catccggtac 5340

cagacatccg cagtgcccag agaacataat ggaaatgaga ttgttgatgt catgtgtcat 5400

gctaccctca cccacaggct gatgtctcct cacagggtgc cgaactacaa cctgttcgtg 5460

atggatgagg ctcatttcac cgacccagct agcattgcag caagaggtta catttccaca 5520

aaggtcgagc taggggaggc ggcggcaata ttcatgacag ccaccccacc aggcacttca 5580

gatccattcc cagagtccaa ttcaccaatt tccgacttac agactgagat cccggatcga 5640

gcttggaact ctggatacga atggatcaca gaatacaccg ggaagacggt ttggtttgtg 5700

cctagtgtca agatggggaa tgagattgcc ctttgcctac aacgtgctgg aaagaaagta 5760

gtccaattga acagaaagtc gtacgagacg gagtacccaa aatgtaagaa cgatgattgg 5820

gactttgtta tcacaacaga catatctgaa atgggggcta actttaaggc gagcagggtg 5880

attgacagcc ggaagagtgt gaaaccaacc atcataacag aaggagaagg gagagtgatc 5940

ctgggagaac catctgcagt gacagcagct agtgccgccc agagacgtgg acgtatcggt 6000

agaaatccgt cgcaagttgg tgatgagtac tgttatgggg ggcacacgaa tgaagacgac 6060

tcgaacttcg cccattggac tgaggcacga atcatgctgg acaacatcaa catgccaaac 6120

ggactgatcg ctcaattcta ccaaccagag cgtgagaagg tatataccat ggatggggaa 6180

taccggctca gaggagaaga gagaaaaaac tttctggaac tgttgaggac tgcagatctg 6240

ccagtttggc tggcttacaa ggttgcagcg gctggagtgt cataccacga ccggaggtgg 6300

tgctttgatg gtcctaggac aaacacaatt ttagaagaca acaacgaagt ggaagtcatc 6360

acgaagcttg gtgaaaggaa gattctgagg ccgcgctgga ttgacgccag ggtgtactcg 6420

gatcaccagg cactaaaggc gttcaaggac ttcgcctcgg gaaaacgttc tcagataggg 6480

ctcattgagg ttctgggaaa gatgcctgag cacttcatgg ggaagacatg ggaagcactt 6540

gacaccatgt acgttgtggc cactgcagag aaaggaggaa gagctcacag aatggccctg 6600

gaggaactgc cagatgctct tcagacaatt gccttgattg ccttattgag tgtgatgacc 6660

atgggagtat tcttcctcct catgcagcgg aagggcattg gaaagatagg tttgggaggc 6720

gctgtcttgg gagtcgcgac ctttttctgt tggatggctg aagttccagg aacgaagatc 6780

gccggaatgt tgctgctctc ccttctcttg atgattgtgc taattcctga gccagagaag 6840

caacgttcgc agacagacaa ccagctagcc gtgttcctga tttgtgtcat gacccttgtg 6900

agcgcagtgg cagccaacga gatgggttgg ctagataaga ccaagagtga cataagcagt 6960

ttgtttgggc aaagaattga ggtcaaggag aatttcagca tgggagagtt tcttttggac 7020

ttgaggccgg caacagcctg gtcactgtac gctgtgacaa cagcggtcct cactccactg 7080

ctaaagcatt tgatcacgtc agattacatc aacacctcat tgacctcaat aaacgttcag 7140

gcaagtgcac tattcacact cgcgcgaggc ttccccttcg tcgatgttgg agtgtcggct 7200

ctcctgctag cagccggatg ctggggacaa gtcaccctca ccgttacggt aacagcggca 7260

acactccttt tttgccacta tgcctacatg gttcccggtt ggcaagctga ggcaatgcgc 7320

tcagcccagc ggcggacagc ggccggaatc atgaagaacg ctgtagtgga tggcatcgtg 7380

gccacggacg tcccagaatt agagcgcacc acacccatca tgcagaagaa agttggacag 7440

atcatgctga tcttggtgtc tctagctgca gtagtagtga acccgtctgt gaagacagta 7500

cgagaagccg gaattttgat cacggccgca gcggtgacgc tttgggagaa tggagcaagc 7560

tctgtttgga acgcaacaac tgccatcgga ctctgccaca tcatgcgtgg gggttggttg 7620

tcatgtctat ccataacatg gacactcata aagaacatgg aaaaaccagg actaaaaaga 7680

ggtggggcaa aaggacgcac cttgggagag gtttggaaag aaagactcaa ccagatgaca 7740

aaagaagagt tcactaggta ccgcaaagag gccatcatcg aagtcgatcg ctcagcagca 7800

aaacacgcca ggaaagaagg caatgccact ggagggcatc cagtctctag gggcacagca 7860

aaactgagat ggctggtcga acggaggttt ctcgaaccgg tcggaaaagt gattgacctt 7920

ggatgtggaa gaggcggttg gtgttactat atggcaaccc aaaaaagagt ccaagaagtc 7980

agagggtaca caaagggcgg tcccggacat gaagagcccc aactagtgca aagttatgga 8040

tggaacattg tcaccatgaa gagtggagtg gatgtgttct acagaccttc tgagtgttgt 8100

gacaccctcc tttgtgacat cggagagtcc tcgtcaagtg ctgaggttga agagcatagg 8160

acgattcggg tccttgaaat ggttgaggac tggctgcacc gagggccaag ggaattttgc 8220

gtgaaggtgc tctgccccta catgccgaaa gtcatagaga agatggagct gctccaacgc 8280

cggtatgggg ggggactggt cagaaaccca ctctcacgga attccacgca cgagatgtat 8340

tgggtgagtc gagcttcagg caatgtggta cattcagtga atatgaccag ccaggtgctc 8400

ctaggaagaa tggaaaaaag gacctggaag ggaccccaat acgaggaaga tgtaaacttg 8460

ggaagtggaa ccagggcggt gggaaaaccc ctgctcaact cagacaccag taaaatcaag 8520

aacaggattg aacgactcag gcgtgagtac agttcgacgt ggcaccacga tgagaaccac 8580

ccatatagaa cctggaacta tcacggcagt tatgatgtga agcccacagg ctccgccagt 8640

tcgctggtca atggagtggt caggctcctc tcaaaaccat gggacaccat cacgaatgtt 8700

accaccatgg ccatgactga cactactccc ttcgggcagc agcgagtgtt caaagagaag 8760

gtggacacga aagctcctga accgccagaa ggagtgaagt acgtgctcaa cgagaccacc 8820

aactggttgt gggcgttttt ggccagagaa aaacgtccaa ggatgtgctc tcgagaggag 8880

ttcataagaa aggtcaacag caatgcagct ttgggtgcca tgtttgaaga gcagaatcaa 8940

tggaggagcg ccagagaggc agttgaagat ccaaaatttt gggagatggt ggatgaggag 9000

cgcgaggcac atctgcgggg ggaatgtcac acttgcattt acaacatgat gggaaagaga 9060

gagaaaaaac ccggagagtt cggaaaggcc aagggaagca gagccatttg gttcatgtgg 9120

cttggagctc gctttctgga gttcgaggct ctgggttttc tcaatgaaga ccactggctt 9180

ggaagaaaga actcaggagg aggtgtcgag ggcttgggcc tccaaaaact gggttacatc 9240

ctgcgtgaag ttggcacccg gcctgggggc aagatctatg ctgatgacac agctggctgg 9300

gacacccgca tcacgagagc tgacttggaa aatgaagcta aggtgcttga gctgcttgat 9360

ggggaacatc ggcgtcttgc cagggccatc attgagctca cctatcgtca caaagttgtg 9420

aaagtgatgc gcccggctgc tgatggaaga accgtcatgg atgttatctc cagagaagat 9480

cagaggggga gtggacaagt tgtcacctac gccctaaaca ctttcaccaa cctggccgtc 9540

cagctggtga ggatgatgga aggggaagga gtgattggcc cagatgatgt ggagaaactc 9600

acaaaaggga aaggacccaa agtcaggacc tggctgtttg agaatgggga agaaagactc 9660

agccgcatgg ctgtcagtgg agatgactgt gtggtaaagc ccctggacga tcgctttgcc 9720

acctcgctcc acttcctcaa tgctatgtca aaggttcgca aagacatcca agagtggaaa 9780

ccgtcaactg gatggtatga ttggcagcag gttccatttt gctcaaacca tttcactgaa 9840

ttgatcatga aagatggaag aacactggtg gttccatgcc gaggacagga tgaattggta 9900

ggcagagctc gcatatctcc aggggccgga tggaacgtcc gcgacactgc ttgtctggct 9960

aagtcttatg cccagatgtg gctgcttctg tacttccaca gaagagacct gcggctcatg 10020

gccaacgcca tttgctccgc tgtccctgtg aattgggtcc ctaccggaag aaccacgtgg 10080

tccatccatg caggaggaga gtggatgaca acagaggaca tgttggaggt ctggaaccgt 10140

gtttggatag aggagaatga atggatggaa gacaaaaccc cagtggagaa atggagtgac 10200

gtcccatatt caggaaaacg agaggacatc tggtgtggca gcctgattgg cacaagagcc 10260

cgagccacgt gggcagaaaa catccaggtg gctatcaacc aagtcagagc aatcatcgga 10320

gatgagaagt atgtggatta catgagttca ctaaagagat atgaagacac aactttggtt 10380

gaggacacag tactgtagat atttaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 10440

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 10500

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaacagcat attgacacct gggatagact 10560

aggagatctt ctgctctgca caaccagcca cacggcacag tgcgccgaca atggtggctg 10620

gtggtgcgag aacacaggat ct 10642

<210> 2

<211> 10663

<212> DNA

<213> Japanese encephalitis virus

<400> 2

agaagtttat ctgtgtgaac ttcttggctt agtatcgttg agaagaatcg agagattagt 60

gcagtttaaa cagtttttta gaacggaaga taaccatgac taaaaaacca ggagggcccg 120

gtaaaaaccg ggctatcaat atgctgaaac gcggcctacc ccgcgtattc ccactagtgg 180

gagtgaagag ggtagtaatg agcttgttgg acggcagagg gccagtacgt ttcgtgctgg 240

ctcttatcac gttcttcaag tttacagcat tagccccgac caaggcgctt ttaggccgat 300

ggaaagcagt ggaaaagagt gtggcaatga aacatcttac tagtttcaaa cgagaacttg 360

gaacactcat tgacgccgtg aacaagcggg gcagaaagca aaacaaaaga ggaggaaatg 420

aaggctcaat catgtggctc gcgagcttgg cagttgtcat agcttgtgca ggagccatga 480

agttgtcgaa tttccagggg aagcttttga tgaccatcaa caacacggac attgcagacg 540

ttatcgtgat tcccacctca aaaggagaga acagatgctg ggtccgggca atcgacgtcg 600

gctacatgtg tgaggacact atcacgtacg aatgtcctaa gcttaccatg ggcaatgatc 660

cagaggatgt ggattgctgg tgtgacaacc aagaagtcta cgtccaatat ggacggtgca 720

cgcggaccag gcattccaag cgaagcagga gatccgtgtc ggtccaaaca catggggaga 780

gttcactagt gaataaaaaa gaggcttggc tggattcaac gaaagccaca cgatatctca 840

tgaaaactga gaactggatc ataaggaatc ctggctatgc tttcctggcg gcggtacttg 900

gctggatgct tggcagtaac aacggtcaac gcgtggtatt taccatcctc ctgctgttgg 960

tcgctccggc ttacagtttt aattgtctgg gaatgggcaa tcgcgacttc atagaaggag 1020

ccagtggagc cacttgggtg gacttggtgc tggaaggaga tagctgcttg acaatcatgg 1080

caaacgacaa accaacattg gacgtccgca tgattaacat cgaagctagc caacttgctg 1140

aggtcagaag ttactgctat catgcttcag tcactgacat ctcgacggtg gctcggtgcc 1200

ccacgactgg agaagcccac aacgagaagc gagctgatag tagctatgtg tgcaaacaag 1260

gcttcactga ccgtgggtgg ggcaacggat gtggactttt cgggaaggga agcattgaca 1320

catgtgcaaa attctcctgc accagtaaag cgattgggag aacaatccag ccagaaaaca 1380

tcaaatacga agttggcatt tttgtgcatg gaaccaccac ttcggaaaac catgggaatt 1440

attcagcgca agttggggcg tcccaggcgg caaagtttac agtaacaccc aatgctcctt 1500

cgataaccct caaacttggt gactacggag aagtcacact ggactgtgag ccaaggagtg 1560

gactgaacac tgaagcgttt tacgtcatga ccgtggggtc aaagtcattt ctggtccata 1620

gggagtggtt tcatgacctc gctctcccct ggacgtcccc ttcgagcaca gcgtggagaa 1680

acagagaact cctcatggaa tttgaagagg cgcacgccac aaaacagtcc gttgttgctc 1740

ttgggtcaca ggaaggaggc ctccatcagg cgttggcagg agccatcgtg gtggagtact 1800

caagctcagt gaagttaaca tcaggccacc tgaaatgtag gctgaaaatg gacaaactgg 1860

ctctgaaagg cacaacctat ggcatgtgta cagaaaaatt ctcgttcgcg aaaaatccgg 1920

cggacactgg tcacggaaca gttgtcattg aactctccta ctctgggagt gatggcccct 1980

gcaaaattcc gattgtttcc gttgcgagcc tcaatgacat gacccccgtt gggcggctgg 2040

tgacagtgaa ccccttcgtc gcgacttcca gtgccaactc aaaggtgctg gtcgagatgg 2100

aacccccctt cggagactcc tacatcgtag ttggaagggg agacaagcag atcaaccacc 2160

attggcacaa agctggaagc acgctgggca aggccttttc aacaactttg aagggagctc 2220

aaagactggc agcgttgggc gacacagcct gggactttgg ctctattgga ggggtcttca 2280

actccatagg aaaagccgtt caccaagtgt ttggtggtgc cttcagaaca ctctttgggg 2340

gaatgtcttg gatcacacaa gggctaatgg gtgccctact gctctggatg ggcgtcaacg 2400

cacgagaccg atcaattgct ttggccttct tagccacagg gggtgtgctc gtgttcttag 2460

cgaccaatgt gcatgctgac actggatgtg ccattgacat cacaagaaaa gagatgagat 2520

gtggaagtgg catcttcgtg cacaacgacg tggaagcctg ggtggatagg tataaatatt 2580

tgccagaaac gcccagatcc ctagcgaaga tcgtccacaa agcgcacaag gaaggcgtgt 2640

gcggagtcag atctgtcact agactggagc accaaatgtg ggaagccgta agggacgaat 2700

tgaacgtcct gctcaaagag aatgcagtgg acctcagtgt ggttgtgaac aagcccgtgg 2760

gaagatatcg ctcagcccct aaacgcctat ccatgacgca agagaagttt gaaatgggct 2820

ggaaagcatg gggaaaaagc atcctctttg ccccggaatt ggctaactcc acatttgtcg 2880

tagatggacc tgagacaaag gaatgccctg atgagcacag agcttggaac agcatgcaaa 2940

tcgaagactt cggctttggc atcacatcaa cccgtgtgtg gctgaaaatt agagaggaga 3000

gcactgacga gtgtgatgga gcgatcatag gcacggctgt caaaggacat gtggcagtcc 3060

atagtgactt gtcgtactgg attgagagtc gctacaacga cacatggaaa cttgagaggg 3120

cagtctttgg agaggtcaaa tcttgcactt ggccagagac acacaccctt tggggagatg 3180

atgttgagga aagtgaactc atcattccgc acaccatagc cggaccaaaa agcaagcaca 3240

atcggaggga agggtataag acacaaaacc agggaccttg ggatgagaat ggcatagtct 3300

tggactttga ttattgccca gggacaaaag tcaccattac agaggattgt agcaagagag 3360

gcccttcggt cagaaccact actgacagtg gaaagttgat cactgactgg tgctgtcgca 3420

gttgctccct tccgccccta cgattccgga cagaaaatgg ctgctggtac ggaatggaaa 3480

tcagacctgt taggcatgat gaaacaacac tcgtcagatc acaggttgat gctttcaatg 3540

gtgaaatggt tgaccctttt cagctgggcc ttctggtgat gtttctggcc acccaggagg 3600

tccttcgcaa gaggtggacg gccagattga ccattcctgc ggttttgggg gccctacttg 3660

tgctgatgct tgggggcatc acttacactg atttggcgag gtatgtggtg ctagtcgctg 3720

ctgctttcgc agaggccaac agtggaggag acgtcctgca ccttgctttg attgccgttt 3780

ttaagatcca accagcattt ctagtgatga acatgcttag cacgagatgg acgaaccaag 3840

aaaacgtggt tctggtccta ggggctgcct ttttccaatt ggcctcagta gatctgcaaa 3900

taggagtcca cggaatcctg aatgccgccg ctatagcatg gatgattgtc cgagcgatca 3960

ccttccccac aacctcctcc gtcaccatgc cagtcttagc gcttctaact ccggggatga 4020

gggctctata cctagacact tacagaatca tcctcctcgt catagggatt tgctccctgc 4080

tgcacgagag gaaaaagacc atggcaaaaa agaaaggagc tgtactcttg ggcttagcgc 4140

tcacatccac tggatggttc tcgcccacca ctatagctgc cggactaatg gtctgcaacc 4200

caaacaagaa gagagggtgg ccagctactg agtttttgtc ggcagttgga ttgatgtttg 4260

ccatcgtagg tggtttggcc gagttggata ttgaatccat gtcaataccc ttcatgctgg 4320

caggtctcat ggcagtgtcc tacgtggtgt caggaaaagc aacagatatg tggcttgaac 4380

gggccgccga catcagctgg gagatggatg ctgcaatcac aggaagcagt cggaggctgg 4440

atgtgaaact ggatgatgac ggagattttc acttgattga tgatcccggt gttccatgga 4500

aggtctgggt cctgcgcatg tcttgcattg gcttagccgc cctcacgcct tgggccatcg 4560

ttcccgccgc tttcggttat tggctcactt taaaaacaac aaaaagaggg ggcgtgtttt 4620

gggacacgcc atccccaaaa ccttgctcaa aaggagacac cactacagga gtctaccgaa 4680

ttatggctag agggattctt ggcacttacc aggccggcgt cggagtcatg tacgagaatg 4740

ttttccacac actatggcac acaactagag gagcagccat tatgagtgga gaaggaaaat 4800

tgacgccata ctggggtagt gtgagagaag accgcatagc ttacggaggc ccatggaggt 4860

ttgaccgaaa atggaatgga acagatgacg tgcaagtgat cgtggtagaa ccggggaagg 4920

ctgcagtaaa catccagaca aaaccaggag tgtttcggac tcccttcggg gaggttgggg 4980

ctgttagtct ggattacccg cgaggaacat ccggctcacc cattctggat tccaatggag 5040

acattatagg cctatacggc aatggagttg agcttggcga tggctcatac gtcagcgcca 5100

tcgtgcaggg tgaccgtcag gaggaaccag tcccagaagc ttacacccca aacatgttga 5160

gaaagagaca gatgactgtg ctagatttgc accctggttc agggaaaacc aggaaaattc 5220

tgccacaaat aattaaggac gccatccagc agcgcctaag aacagctgtg ttggcaccga 5280

cgcgggtggt agcagcagaa atggcagaag ctttgagagg gctcccagta cgatatcaaa 5340

cttcagcagt gcagagagag caccaaggga atgaaatagt ggatgtgatg tgccacgcca 5400

ctctgaccca tagactgatg tcaccgaaca gagtgcccaa ctacaaccta tttgtcatgg 5460

atgaagctca tttcaccgac ccagccagta tagccgcacg aggatacatt gctaccaagg 5520

tggaattagg ggaggcagca gccatcttta tgacagcgac cccgcctgga accacggatc 5580

cttttcctga ctcaaatgcc ccaatccatg atttgcaaga tgagatacca gacagggcat 5640

ggagcagtgg atacgaatgg atcacagaat atgcgggtaa aaccgtgtgg tttgtggcga 5700

gcgtaaaaat ggggaatgag attgcaatgt gcctccaaag agcggggaaa aaggtcatcc 5760

aactcaaccg caagtcctat gacacagaat acccaaaatg taagaatgga gactgggatt 5820

ttgtcattac caccgacatc tctgaaatgg gggccaactt cggtgcgagc agggtcatcg 5880

actgtagaaa gagcgtgaaa cccaccatct tagaagaggg agaaggcaga gtcatcctcg 5940

gaaacccatc tcccataacc agtgcaagcg cagctcaacg gaggggcaga gtaggcagaa 6000

accccaacca agttggagat gaataccact atgggggggc taccagtgaa gatgacagta 6060

acctagccca ttggacagag gcaaagatca tgttagacaa catacacatg cccaatggac 6120

tggtggccca gctctatgga ccagagaggg aaaaggcttt cacaatggat ggcgaatacc 6180

gtctcagagg tgaagaaaag aaaaacttct tagagctgct taggacggct gacctcccgg 6240

tgtggctggc ctacaaggtg gcgtccaatg gcattcagta caccgacaga aagtggtgtt 6300

ttgatgggcc gcgtacgaat gccatactgg aggacaacac cgaggtagag atagtcaccc 6360

ggatgggtga gaggaaaatc ctcaagccga gatggcttga tgcaagagtt tatgcagatc 6420

accaagccct caagtggttc aaagactttg cagcagggaa gagatcagcc gttagcttca 6480

tagaggtgct cggtcgcatg cctgagcatt tcatgggaaa gacgcgggaa gctttagaca 6540

ccatgtactt ggttgcaacg gctgagaaag gtgggaaagc acaccgaatg gctctcgaag 6600

agctgccaga tgcactggaa accatcacac ttattgtcgc cattactgtg atgacaggag 6660

gattcttcct actaatgatg cagcgaaagg gtatagggaa gatgggtctt ggagctctag 6720

tgctcacgct agctaccttc ttcctgtggg cggcagaggt tcctggaacc aaaatagcag 6780

ggaccctgct gatcgccctg ctgctgatgg tggttctcat cccagaaccg gaaaaacaga 6840

ggtcacagac agataaccaa ctggcggtgt ttctcatctg tgtcttgacc gtggttggag 6900

tggtggcagc aaacgagtac gggatgctag aaaaaaccaa agcagatctc aagagcatgt 6960

ttggcggaaa gacgcaggca tcaggactga ctggattgcc aagcatggca ctggacctgc 7020

gtccagccac agcctgggca ctgtatgggg ggagcacagt cgtgctaacc cctcttctga 7080

agcacctgat cacgtcggaa tacgtcacca catcgctagc ctcaattaac tcacaagctg 7140

gctcattatt cgtcttgcca cgaggcgtgc cttttaccga cctagacttg accgttggcc 7200

tcgtcttcct tggctgttgg ggtcaaatca ccctcacaac gtttctgaca gccatggttc 7260

tggcgacact tcactatggg tacatgctcc ctggatggca agcagaagca ctcagggctg 7320

cccagagaag gacagcggct ggaataatga agaatgccgt tgttgacgga atggtcgcca 7380

ctgatgtgcc tgaactggaa aggactactc ctctgatgca aaagaaagtc ggacaggtgc 7440

tcctcatagg ggtaagcgtg gcagcgttcc tcgtcaaccc taatgtcacc actgtgagag 7500

aagcaggggt gttggtgacg gcggctacgc ttactttgtg ggacaatgga gccagtgccg 7560

tttggaattc caccacagcc acgggactct gccatgtcat gcgaggtagc tacctggctg 7620

gaggctccat tgcttggact ctcatcaaga acgctgataa gccctccttg aaaaggggaa 7680

ggcctggggg caggacgcta ggggagcagt ggaaggaaaa actaaatgcc atgagcagag 7740

aagagttttt taaataccgg agagaggcca taatcgaggt ggaccgcact gaagcacgca 7800

gggccagacg tgaaaataac atagtgggag gacatccggt ttcgcgaggc tcagcaaaac 7860

tccgttggct cgtggagaaa ggatttgtct cgccaatagg aaaagtcatt gatctagggt 7920

gtgggcgtgg aggatggagc tactacgcag caaccctgaa gaaggtccag gaagtcagag 7980

gatacacgaa aggtggggcg ggacatgaag aaccgatgct catgcagagc tacggctgga 8040

acctggtctc cctgaagagt ggagtggacg tgttttacaa accttcagag cccagtgaca 8100

ccctgttctg tgacataggg gaatcctccc caagtccaga agtagaagaa caacgcacac 8160

tacgcgtcct agagatgaca tctgactggt tgcaccgagg acctagagag ttctgcatta 8220

aagttctctg cccttacatg cccaaggtta tagaaaaaat ggaagttctg cagcgccgct 8280

tcggaggtgg gctagtgcgt ctccccctgt cccgaaactc caatcacgag atgtattggg 8340

ttagtggagc cgctggcaat gtggtgcacg ctgtgaacat gaccagccag gtactactgg 8400

ggcgaatgga tcgcacagtg tggagagggc caaagtatga ggaagatgtc aacctaggga 8460

gcggaacaag agccgtggga aagggagaag tccatagcaa tcaggagaaa atcaagaaga 8520

gaatccagaa gcttaaagaa gaattcgcca caacgtggca caaagaccct gagcatccat 8580

accgcacttg gacataccac ggaagctatg aagtgaaggc tactggctca gccagctctc 8640

tcgtcaacgg agtggtgaag ctcatgagca aaccttggga cgccattgcc aacgtcacca 8700

ccatggccat gactgacacc accccttttg gacagcaaag agttttcaag gagaaagttg 8760

acacgaaggc tcctgagcca ccagctggag ccaaggaagt gctcaacgag accaccaact 8820

ggctgtgggc ccacttgtca cgggaaaaaa gaccccgctt gtgcaccaag gaagaattca 8880

taaagaaagt caacagcaac gcggctcttg gagcagtgtt cgctgaacag aatcaatgga 8940

gcacggcgcg tgaggctgtg gatgacccgc ggttttggga gatggttgat gaagagaggg 9000

aaaaccatct gcgaggagag tgtcacacat gtatctacaa catgatggga aaaagagaga 9060

agaagcctgg agagtttgga aaagctaaag gaagcagggc catttggttc atgtggcttg 9120

gagcacggta tctagagttt gaagctttgg ggttcctgaa tgaagaccat tggctgagcc 9180

gagagaattc aggaggtgga gtggaaggct caggcgtcca aaagctggga tacatcctcc 9240

gtgacatagc aggaaagcaa ggagggaaaa tgtacgctga tgataccgcc gggtgggaca 9300

ctagaattac cagaactgat ttagaaaatg aagctaaggt actggagctc ctagacggtg 9360

aacaccgcat gctcgcccga gccataattg aactgactta caggcacaaa gtggtcaagg 9420

tcatgagacc tgcagcagaa ggaaagaccg tgatggacgt gatatcaaga gaagatcaaa 9480

gggggagtgg acaggtggtc acttatgctc ttaacacttt cacgaacatc gctgtccagc 9540

tcgtcaggct gatggaggct gagggggtca ttggaccaca acacttggaa cagctaccta 9600

ggaaaaacaa gatagctgtc aggacctggc tctttgagaa tggagaggag agagtgacca 9660

ggatggcgat cagcggagac gactgtgtcg tcaagccgct ggacgacaga ttcgccacag 9720

ccctccactt cctcaacgca atgtcaaagg tcagaaaaga catccaggaa tggaagcctt 9780

cgcatggctg gcacgattgg cagcaagttc ccttctgctc taaccatttt caggagattg 9840

tgatgaaaga tggaaggagt atagttgtcc cgtgcagagg acaggatgag ctgataggca 9900

gggctcgcat ctctccagga gctggatgga atgtgaagga cacagcttgc ctggccaaag 9960

catatgcaca gatgtggcta ctcctatact tccatcgcag ggacttgcgt ctcatggcaa 10020

atgcgatttg ctcagcagtg ccagtggatt gggtgcccac aggcaggaca tcctggtcaa 10080

tacactcgaa aggagagtgg atgaccacgg aagacatgct gcaggtctgg aacagagtct 10140

ggattgaaga aaatgaatgg atgatggaca agactccaat cacaagctgg acagacgttc 10200

cgtatgtggg aaagcgtgag gacatctggt gtggcagcct catcggaacg cgatccagag 10260

caacctgggc tgagaacatc tatgcggcga taaaccaggt tagagctgtc attgggaaag 10320

aaaattatgt tgactacatg acctcactca ggagatacga agacgtcttg atccaggaag 10380

acagggtcat ctagtgtgat aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 10440

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 10500

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa agcatattga 10560

cacctgggaa tagactggga gatcttctgc tctatctcaa catcagctac taggcacaga 10620

gcgccgaagt atgtagctgg tagtgaggaa gaacacagga tct 10663

<210> 3

<211> 10523

<212> DNA

<213> Dengue virus type 2

<400> 3

agttgttagt ctacgtggac cgacaaagac agattctttg agggagctaa gctcaacgta 60

gttctaacag ttttttaatt agagagcaga tctctgatga ataaccaacg aaaaaaggcg 120

agaaatacgc ctttcaatat gctgaaacgc gagagaaacc gcgtgtcgac tgtacaacag 180

ctgacaaaga gattctcact tggaatgctg cagggacgag gaccattaaa actgttcatg 240

gccctggtgg cgttccttcg tttcctaaca atcccaccaa cagcagggat actgaagaga 300

tggggaacaa ttaaaaaatc aaaagccatt aatgttttga gagggttcag gaaagagatt 360

ggaaggatgc tgaacatctt gaacaggaga cgcagaactg caggcatgat cattatgctg 420

attccaacag tgatggcgtt ccatttaacc acacgtaacg gagaaccaca catgatcgtc 480

agtagacaag agaaagggaa aagtcttctg tttaaaacag aggatggtgt gaacatgtgt 540

accctcatgg ccatggacct tggtgaattg tgtgaagata caatcacgta caagtgtcct 600

tttctcaggc agaatgaacc agaagacata gattgttggt gcaactctac gtccacatgg 660

gtaacttatg ggacgtgtac caccacagga gaacacagaa gagaaaaaag atcagtggca 720

ctcgttccac atgtgggaat gggactggag acacgaactg aaacatggat gtcatcagaa 780

ggggcctgga aacatgccca gagaattgaa acttggatct tgagacatcc aggctttacc 840

ataatggcag caatcctggc atacaccata ggaacgacac atttccaaag agccctgatt 900

ttcatcttac tgacagctgt cgctccttca atgacaatgc gttgcatagg aatatcaaat 960

agagactttg tagaaggggt ttcaggagga agctgggttg acatagtctt agaacatgga 1020

agctgtgtga cgacgatggc aaaaaacaaa ccaacattgg attttgaact gataaaaaca 1080

gaagccaaac aacctgccac tctaaggaag tactgtatag aggcaaagct gaccaacaca 1140

acaacagatt ctcgctgccc aacacaagga gaacccagcc taaatgaaga gcaggacaaa 1200

aggttcgtct gcaaacactc catggtggac agaggatggg gaaatggatg tggattattt 1260

ggaaaaggag gcattgtgac ctgtgctatg ttcacatgca aaaagaacat gaaaggaaaa 1320

gtcgtgcaac cagaaaactt ggaatacacc attgtgataa cacctcactc aggggaagag 1380

catgcagtcg gaaatgacac aggaaaacat ggcaaggaaa tcaaaataac accacagagt 1440

tccatcacag aagcagagtt gacaggctat ggcactgtca cgatggagtg ctctccgaga 1500

acgggcctcg acttcaatga gatggtgttg ctgcaaatgg aaaataaagc ttggctggtg 1560

cacaggcaat ggttcctaga cctgccgttg ccatggctgc ccggagcgga cacacaagga 1620

tcaaattgga tacagaaaga gacattggtc actttcaaaa atccccatgc gaagaaacag 1680

gatgttgttg ttttgggatc ccaagaaggg gccatgcaca cagcactcac aggggccaca 1740

gaaatccaga tgtcatcagg aaacttactg ttcacaggac atctcaagtg caggctgagg 1800

atggacaaac tacagctcaa aggaatgtca tactctatgt gcacaggaaa gtttaaagtt 1860

gtgaaggaaa tagcagaaac acaacatgga acaatagtta tcagagtaca atatgaaggg 1920

gacggttctc catgtaagat cccttttgag ataatggatt tggaaaaaag acatgtttta 1980

ggtcgcctga ttacagtcaa cccaatcgta acagaaaaag atagcccagt caacatagaa 2040

gcagaacctc cattcggaga cagctacatc atcataggag tagagccggg acaattgaag 2100

ctcaactggt ttaagaaagg aagttctatc ggccaaatga ttgagacaac aatgagggga 2160

gcgaagagaa tggccatttt aggtgacaca gcttgggatt ttggatccct gggaggagtg 2220

tttacatcta taggaaaggc tctccaccaa gttttcggag caatctatgg ggctgccttc 2280

agtggggtct catggactat gaaaatactc ataggagtca ttatcacatg gataggaatg 2340

aattcacgca gcacctcact gtctgtgtca ctagtattgg tgggagtcgt gacgctgtat 2400

ttgggagtta tggtgcaggc cgatagtggt tgcgttgtga gctggaaaaa caaagaactg 2460

aagtgtggca gtgggatttt catcacagac aacgtgcaca catggacaga acaatacaag 2520

ttccaaccag aatccccttc aaagctagct tcagctatcc agaaagctca tgaagagggc 2580

atttgtggaa tccgctcagt aacaagactg gaaaatctga tgtggaaaca aataacacca 2640

gaattgaatc acattctatc agaaaatgag gtgaagttga ctattatgac aggagacatc 2700

aaaggaatca tgcaggcagg aaaacgatct ctgcagcccc agcccactga gctgaagtat 2760

tcatggaaaa catggggcaa agcgaaaatg ctctctacag agtctcataa ccagaccttt 2820

ctcattgatg gccccgaaac agcagaatgc cccaacacaa acagagcttg gaattcgctg 2880

gaagttgaag actatggctt tggagtattc accaccaata tatggctaaa gttgagagaa 2940

aagcaggatg tattctgcga ctcaaaactc atgtcagcgg ccataaaaga caacagagcc 3000

gtccatgccg atatgggtta ttggatagaa agtgcactca atgacacatg gaagatagag 3060

aaagcctctt tcatcgaagt taaaagctgc cactggccaa agtcacacac cctctggagt 3120

aatggagtgt tagaaagtga gatgataatt ccaaagaatt tcgctggacc agtgtcacaa 3180

cacaactaca gaccaggcta ccatacacaa acagcaggac catggcatct aggtaagctt 3240

gagatggact ttgatttctg cgaaggaacc acagtggtgg tgactgagga ctgtggaaat 3300

agaggaccct ctttaagaac aactactgcc tctggaaaac tcataacaga atggtgctgc 3360

cgatcttgca cattaccacc gctaagatac agaggtgagg acggatgctg gtacgggatg 3420

gaaatcagac cattgaaaga gaaagaagag aatttggtca actccttggt cacagccgga 3480

catgggcaga ttgacaactt ttcactagga gtcttgggaa tggcattgtt cctggaagaa 3540

atgctcagga cccgagtagg aacgaaacat gcaatactac tagttgcagt ttcttttgtg 3600

acattgatca cagggaacat gtcctttaga gacctgggaa gagtgatggt tatggtgggc 3660

gctactatga cggatgacat aggtatgggc gtgacttatc ttgccctact agcagccttc 3720

aaagtcagac caacttttgc agctggacta ctcttgagaa agttgacctc caaggaattg 3780

atgatgacta ccataggaat cgtactcctc tcccagagca ccataccaga gaccattctt 3840

gaactgactg atgcgttagc cttgggcatg atggtcctta aaatggtgag aaaaatggaa 3900

aagtatcaat tggcagtgac tatcatggct atcttgtgcg tcccaaatgc agtgatatta 3960

caaaacgcat ggaaagtgag ttgcacaata ttggcagtgg tgtccgtttc cccactgttc 4020

ttaacatcct cacagcagaa agcggattgg ataccattag cattgacgat caagggtctc 4080

aatccaacag ctatttttct aacaaccctt tcaagaacca acaagaaaag gagctggcca 4140

ctaaatgagg ctatcatggc agtcgggatg gtgagcattt tggccagttc actcctaaag 4200

aatgacattc ccatgacagg accattagtg gctggagggc tcctcactgt gtgctacgtg 4260

ctcactggac gatcggccga tttggaactg gagagagccg ccgatgtcaa atgggaagat 4320

caggcagaga tatcagggag ctctccaatc ctgtcaataa caatatcaga agatggtagc 4380

atgtcgataa aaaacgaaga ggaagaacaa acactgacca tactcattag aacaggattg 4440

ctggtgatct caggactttt tcctgtatca ataccaatca cggcagcagc atggtacctg 4500

tgggaagtga agaaacaacg ggctggagta ttgtgggatg tcccttcacc cccacccgtg 4560

ggaaaggctg aactggaaga tggagcctat agaatcaagc aaaaagggat tcttggatat 4620

tcccagatcg gagccggagt ttacaaagaa ggaacattcc atacaatgtg gcatgtcaca 4680

cgcggcgctg ttctaatgca taaaggaaag aggattgaac catcatgggc ggacgttaag 4740

aaagacctaa tatcatatgg aggaggctgg aagctagaag gagaatggaa ggaaggagaa 4800

gaagtccagg tcttggcatt ggagcctgga aaaaatccaa gagccgtcca aacaaaacct 4860

ggtcttttca aaaccaacgc cggaaccata ggtgccgtat ctctggactt ttctcctgga 4920

acctcaggat ctccaatcat cgacaaaaaa ggaaaagttg tgggtcttta tggtaatggt 4980

gttgttacaa ggagtggagc atatgtgagt gctatagccc agactgaaaa aagtattgaa 5040

gacaatccag agatcgaaga tgatattttt cgaaagagaa aattgaccat catggacctc 5100

cacccaggag cgggaaagac gaagagatac cttccggcca tagtcagaga ggctataaaa 5160

cggggcctga ggacattaat cctggccccc actagagtcg tggcagctga aatggaggaa 5220

gccctaagag gacttccaat aagataccaa accccagcca tcagagctga gcacaccggg 5280

cgggagattg tggacctaat gtgtcatgcc acattcacta tgaggctgct atcaccagtt 5340

agagtgccaa attacaacct gatcatcatg gacgaagccc atttcacaga cccagcaagt 5400

atagcggcta gaggatacat ctcaactcga gtagagatgg gtgaggcagc tgggattttc 5460

atgacagcca ctcctccggg aagcagagac ccattccctc agagcaatgc accaatcatg 5520

gatgaagaaa gagaaatccc tgaacgttcg tggagttctg gacatgagtg ggtcacggat 5580

tttaaaggga agactgtttg gttcgttcca agtataaaag caggaaatga tatagcagct 5640

tgcctgagaa aaaatggaaa gaaagtgata caactcagta ggaagacctt tgattctgag 5700

tatgtcaaga ctagaaccaa tgattgggac ttcgtggtca caactgacat ttcagaaatg 5760

ggtgccaact tcaaggctga gagggttata gaccccagac gctgcatgaa accagttata 5820

ctaacagatg gtgaagagcg ggtgatcctg gcaggaccta tgccagtgac ccactctagt 5880

gcagcacaaa gaagagggag aataggaaga aatccaaaaa atgaaaatga ccagtacata 5940

tacatggggg aacctctgga aaatgatgaa gactgtgcac actggaaaga agctaaaatg 6000

ctcctagata acatcaacac acctgaagga atcattccta gcatgttcga accagagcgt 6060

gaaaaggtgg atgccattga tggtgaatac cgcttgagag gagaagcaag gaaaaccttt 6120

gtggacctaa tgagaagagg agacctacca gtctggttgg cctacagagt ggcagctgaa 6180

ggcatcaact acgcagacag aaggtggtgt tttgatggaa ttaagaacaa ccaaatcttg 6240

gaagaaaatg tggaggtgga aatctggaca aaagaagggg aaaggaagaa attaaaaccc 6300

agatggttgg atgccaggat ctactctgac ccactgacgc taaaggaatt caaggagttt 6360

gcagctggaa gaaagtccct gaccctgaac ctaatcacag aaatgggtag gcttccaact 6420

ttcatgactc agaaggcaag agacgcactg gacaacttag cagtgctgca cacggctgaa 6480

gcaggtggaa gggcgtacaa tcatgctctc agtgaactgc cggagaccct ggagacattg 6540

cttttactga cacttctggc tacagtcaca ggaggaatct ttttattctt gatgtccgga 6600

aggggtatag ggaagatgac cctgggaatg tgctgcataa tcacggccag tattctccta 6660

tggtacgcac aaatacagcc acactggata gcagcttcaa taatactgga gttttttctc 6720

atagttttgc ttattccaga accagaaaag cagagaacac cccaagataa ccaattgacc 6780

tacgttgtca tagccatcct cacagtggtg gccgcaacca tggcaaacga gatgggtttc 6840

ctggaaaaaa cgaagaaaga tctcggattg ggaagcatta caacccagca acccgagagc 6900

aacatcctgg acatagatct acgtcccgca tcagcatgga cgctgtatgc tgtggccaca 6960

acatttgtca caccaatgtt gagacacagc attgaaaatt cctcagtgaa cgtgtcccta 7020

acagctattg ccaaccaagc cacagtgtta atgggtcttg ggaaaggatg gccattgtca 7080

aagatggaca tcggagttcc ccttctcgcc attggatgct actcacaagt caaccccata 7140

actctcacag cagctctttt cttactggta gcacattatg ccatcatagg gccaggactc 7200

caagcaaaag caaccaggga agctcagaaa agagcagcag cgggcatcat gaaaaaccca 7260

actgtcgatg gaataacagt gattgaccta gatccaatac cctatgatcc aaagtttgaa 7320

aagcagttgg gacaagtaat gctcctagtc ctctgcgtga ctcaagtgtt gatgatgagg 7380

actacatggg ctctgtgtga ggcgttaacc ttagcgaccg ggcctatctc cacattgtgg 7440

gaaggaaatc cagggaggtt ttggaacact accattgcag tgtcaatggc taacattttt 7500

agagggagtt acttggccgg agctggactt ctcttttcca tcatgaagaa cacaaccaac 7560

acgagaaggg gaaccggtaa cataggagag acgcttggag agaaatggaa aagccgattg 7620

aacgcattgg ggaaaagtga attccagatc tacaagaaaa gtggaatcca ggaagtggat 7680

agaaccttag caaaagaagg cattaaaaga ggagaaacgg accatcacgc tgtgtcgcga 7740

ggctcagcaa aactgagatg gttcgtcgag agaaatatgg tcacaccaga agggaaagta 7800

gtggacctcg gttgcggcag aggaggctgg tcatactatt gtgggggact aaagaatgta 7860

agagaagtca aaggcctgac aaaaggagga ccaggacatg aagaacccat ccccatgtca 7920

acatatgggt ggaatctagt acgtcttcaa agtggagttg acgttttctt cactccgcca 7980

gaaaagtgtg acacattgtt gtgtgacata ggggagtcgt caccaaatcc cacggtagaa 8040

gcaggacgaa cactcagagt ccttaactta gtggaaaatt ggttgaacaa caacacccaa 8100

ttttgcataa aggttctcaa cccatacatg ccctcagtca tagaaaaaat ggaagcacta 8160

caaaggaaat atggaggagc cttagtgagg aatccactct cacgaaactc cacacatgag 8220

atgtactggg tatccaatgc ctccgggaac atagtgtcat cagtgaacat gatttcaagg 8280

atgttgatca acagattcac aatgagacac aagaaagcca cttacgagcc agatgtagac 8340

ctcggaagcg gaacccgcaa catcggaatt gaaagtgaga taccaaacct agacataatc 8400

gggaaaagaa tagaaaaaat aaaacaagag catgaaacat catggcacta tgaccaagac 8460

cacccataca aaacgtgggc ttaccatggc agctatgaaa caaaacaaac tggatcagca 8520

tcatccatgg tgaacggagt ggtcagactg ctgacaaaac cttgggacgt cgtccccatg 8580

gtgacacaga tggcaatgac agacacgact ccatttggac aacagcgcgt ttttaaagaa 8640

aaagtggaca cgagaaccca agaaccgaaa gaaggcacaa agaaactaat gaaaatcacg 8700

gcagagtggc tttggaaaga actagggaag aaaaagacac ctaggatgtg cactagagaa 8760

gaattcacaa gaaaggtgag aagcaatgca gccttggggg ccatattcac tgatgagaac 8820

aagtggaagt cggcacgtga ggctgttgaa gatagtaggt tttgggagct ggttgacaag 8880

gaaaggaatc tccatcttga aggaaagtgt gaaacatgtg tgtataacat gatgggaaaa 8940

agagagaaga agctagggga gttcggcaag gcaaaaggca gcagagccat atggtacatg 9000

tggcttggag cacgcttctt agagtttgaa gccctaggat tcttgaatga agatcactgg 9060

ttctccagag agaactcctt gagtggagtg gaaggagaag ggctgcacaa gctaggttac 9120

attttaagag acgtgagcaa gaaagaggga ggagcaatgt atgccgatga caccgcagga 9180

tgggacacaa gaatcacact agaagaccta aaaaatgaag aaatggtaac aaaccacatg 9240

gaaggagaac acaagaaact agccgaggcc attttcaaat taacgtacca aaacaaggtg 9300

gtgcgtgtgc aaagaccaac accaagaggc acagtaatgg atatcatatc gagaagagac 9360

caaagaggta gtggacaagt tggtacctat ggactcaata ctttcaccaa tatggaagcc 9420

caactaatca gacagatgga gggagaagga gtcttcaaaa gcattcagca cctgacagtc 9480

acagaagaaa tcgccgtgca aaactggtta gcaagagtag ggcgcgaaag gttatcaaga 9540

atggccatca gtggagatga ttgtgttgtg aaacctttag atgacaggtt cgcaagcgct 9600

ttaacagctc taaatgacat gggaaaggtt aggaaagaca tacaacaatg ggaaccttca 9660

agaggatgga acgattggac acaagtgccc ttctgttcac accatttcca tgagttaatc 9720

atgaaagacg gacgcgtact tgtagttcca tgcagaaacc aagatgaact gattggtaga 9780

gcccgaattt cccaaggagc tgggtggtct ttgcgagaga cggcctgttt ggggaagtcc 9840

tacgcccaaa tgtggagctt gatgtacttc cacagacgtg acctcaggct ggcggctaat 9900

gctatttgct cggcagtccc atcacattgg gttccaacaa gtagaacaac ctggtccata 9960

cacgccaaac atgaatggat gacaacggaa gacatgctga cagtctggaa cagggtgtgg 10020

attcaagaaa acccatggat ggaagacaaa actccagtgg aatcatggga ggaaatccca 10080

tacttgggga aaagagaaga ccaatggtgc ggctcattga ttgggctaac aagcagggcc 10140

acctgggcaa agaacatcca aacagcaata aatcaagtta gatcccttat aggcaatgag 10200

gaatacacag attacatgcc atccatgaaa agattcagaa gagaagagga agaggcagga 10260

gtcctgtggt agaaggcaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 10320

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 10380

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaacag catattgacg ctgggaaaga 10440

ccagagatcc tgctgtctcc tcagcatcat tccaggcaca gaacgccaga aaatggaatg 10500

gtgctgttga atcaacaggt tct 10523

<210> 4

<211> 10775

<212> DNA

<213> Yellow fever virus

<400> 4

agtaaatcct gtgtgctaat tgaggtgcat tggtctgcaa atcgagttgc taggcaataa 60

acacatttgg attaatttta atcgttcgtt gagcgattag cagagaactg accagaacat 120

gtctggtcgt aaagctcagg gaaaaaccct gggcgtcaat atggtacgac gaggagttcg 180

ctccttgtca aacaaaataa aacaaaaaac aaaacaaatt ggaaacagac ctggaccttc 240

aagaggtgtt caaggattta tctttttctt tttgttcaac attttgactg gaaaaaagat 300

cacagcccac ctaaagaggt tgtggaaaat gctggaccca agacaaggct tggctgttct 360

aaggaaagtc aagagagtgg tggccagttt gatgagagga ttgtcctcaa ggaaacgccg 420

ttcccatgat gttctgactg tgcaattcct aattttggga atgctgttga tgacgggtgg 480

agtgaccttg gtgcggaaaa acagatggtt gctcctaaat gtgacatctg aggacctcgg 540

gaaaacattc tctgtgggca caggcaactg cacaacaaac attttggaag ccaagtactg 600

gtgcccagac tcaatggaat acaactgtcc caatctcagt ccaagagagg agccagatga 660

cattgattgc tggtgctatg gggtggaaaa cgttagagtc gcatatggta agtgtgactc 720

agcaggcagg tctaggaggt caagaagggc cattgacttg cctacgcatg aaaaccatgg 780

tttgaagacc cggcaagaaa aatggatgac tggaagaatg ggtgaaaggc aactccaaaa 840

gattgagaga tggttcgtga ggaacccctt ttttgcagtg acggctctga ccattgccta 900

ccttgtggga agcaacatga cgcaacgagt cgtgattgcc ctactggtct tggctgttgg 960

tccggcctac tcagctcact gcattggaat tactgacagg gatttcattg agggggtgca 1020

tggaggaact tgggtttcag ctaccctgga gcaagacaag tgtgtcactg ttatggcccc 1080

tgacaagcct tcattggaca tctcactaga gacagtagcc attgatagac ctgctgaggt 1140

gaggaaagtg tgttacaatg cagttctcac tcatgtgaag attaatgaca agtgccccag 1200

cactggagag gcccacctag ctgaagagaa cgaaggggac aatgcgtgca agcgcactta 1260

ttctgataga ggctggggca atggctgtgg cctatttggg aaagggagca ttgtggcatg 1320

cgccaaattc acttgtgcca aatccatgag tttgtttgag gttgatcaga ccaaaattca 1380

gtatgtcatc agagcacaat tgcatgtagg ggccaagcag gaaaattgga ataccgacat 1440

taagactctc aagtttgatg ccctgtcagg ctcccaggaa gtcgagttca ttgggtatgg 1500

aaaagctaca ctggaatgcc aggtgcaaac tgcggtggac tttggtaaca gttacatcgc 1560

tgagatggaa acagagagct ggatagtgga cagacagtgg gcccaggact tgaccctgcc 1620

atggcagagt ggaagtggcg gggtgtggag agagatgcat catcttgtcg aatttgaacc 1680

tccgcatgcc gccactatca gagtactggc cctgggaaac caggaaggct ccttgaaaac 1740

agctcttact ggcgcaatga gggttacaaa ggacacaaat gacaacaacc tttacaaact 1800

acatggtgga catgtttctt gcagagtgaa attgtcagct ttgacactca aggggacatc 1860

ctacaaaata tgcactgaca aaatgttttt tgtcaagaac ccaactgaca ctggccatgg 1920

cactgttgtg atgcaggtga aagtgtcaaa aggagccccc tgcaggattc cagtgatagt 1980

agctgatgat cttacagcgg caatcaataa aggcattttg gttacagtta accccatcgc 2040

ctcaaccaat gatgatgaag tgctgattga ggtgaaccca ccttttggag acagctacat 2100

tatcgttggg agaggagatt cacgtctcac ttaccagtgg cacaaagagg gaagctcaat 2160

aggaaagttg ttcactcaga ccatgaaagg cgtggaacgc ctggccgtca tgggagacac 2220

cgcctgggat ttcagctccg ctggagggtt cttcacttcg gttgggaaag gaattcatac 2280

ggtgtttggc tctgcctttc aggggctatt tggcggcttg aactggataa caaaggtcat 2340

catgggggcg gtacttatat gggttggcat caacacaaga aacatgacaa tgtccatgag 2400

catgatcttg gtaggagtga tcatgatgtt tttgtctcta ggagttgggg cggatcaagg 2460

atgcgccatc aactttggca agagagagct caagtgcgga gatggtatct tcatatttag 2520

agactctgat gactggctga acaagtactc atactatcca gaagatcctg tgaagcttgc 2580

atcaatagtg aaagcctctt ttgaagaagg gaagtgtggc ctaaattcag ttgactccct 2640

tgagcatgag atgtggagaa gcagggcaga tgagatcaat gccatttttg aggaaaacga 2700

ggtggacatt tctgttgtcg tgcaggatcc aaagaatgtt taccagagag gaactcatcc 2760

attttccaga attcgggatg gtctgcagta tggttggaag acttggggta agaaccttgt 2820

gttctcccca gggaggaaga atggaagctt catcatagat ggaaagtcca ggaaagaatg 2880

cccgttttca aaccgggtct ggaattcttt ccagatagag gagtttggga cgggagtgtt 2940

caccacacgc gtgtacatgg acgcagtctt tgaatacacc atagactgcg atggatctat 3000

cttgggtgca gcggtgaacg gaaaaaagag tgcccatggc tctccaacat tttggatggg 3060

aagtcatgaa gtaaatggga catggatgat ccacaccttg gaggcattag attacaagga 3120

gtgtgagtgg ccactgacac atacgattgg aacatcagtt gaagagagtg aaatgttcat 3180

gccgagatca atcggaggcc cagttagctc tcacaatcat atccctggat acaaggttca 3240

gacgaacgga ccttggatgc aggtaccact agaagtgaag agagaagctt gcccagggac 3300

tagcgtgatc attgatggca actgtgatgg acggggaaaa tcaaccagat ccaccacgga 3360

tagcgggaaa gttattcctg aatggtgttg ccgctcctgc acaatgccgc ctgtgagctt 3420

ccatggtagt gatgggtgtt ggtatcccat ggaaattagg ccaaggaaaa cgcatgaaag 3480

ccatctggtg cgctcctggg ttacagctgg agaaatacat gctgtccctt ttggtttggt 3540

gagcatgatg atagcaatgg aagtggtcct aaggaaaaga cagggaccaa agcaaatgtt 3600

ggttggagga gtagtgctct tgggagcaat gctggtcggg caagtaactc tccttgattt 3660

gctgaaactc acagtggctg tgggattgca tttccatgag atgaacaatg gaggagacgc 3720

catgtatatg gcgttgattg ctgccttttc aatcagacca gggctgctca tcggctttgg 3780

gctcaggacc ctatggagcc ctcgggaacg ccttgtgctg accctaggag cagccatggt 3840

ggagattgcc ttgggtggcg tgatgggcgg cctgtggaag tatctaaatg cagtttctct 3900

ctgcatcctg acaataaatg ctgttgcttc taggaaagca tcaaatacca tcttgcccct 3960

catggctctg ttgacacctg tcactatggc tgaggtgaga cttgccgcaa tgttcttttg 4020

tgccgtggtt atcatagggg tccttcacca gaatttcaag gacacctcca tgcagaagac 4080

tatacctctg gtggccctca cactcacatc ttacctgggc ttgacacaac cttttttggg 4140

cctgtgtgca tttctggcaa cccgcatatt tgggcgaagg agtatcccag tgaatgaggc 4200

actcgcagca gctggtctag tgggagtgct ggcaggactg gcttttcagg agatggagaa 4260

cttccttggt ccgattgcag ttggaggact cctgatgatg ctggttagcg tggctgggag 4320

ggtggatggg ctagagctca agaagcttgg tgaagtttca tgggaagagg aggcggagat 4380

cagcgggagt tccgcccgct atgatgtggc actcagtgaa caaggggagt tcaagctgct 4440

ttctgaagag aaagtgccat gggaccaggt tgtgatgacc tcgctggcct tggttggggc 4500

tgccctccat ccatttgctc ttctgctggt ccttgctggg tggctgtttc atgtcagggg 4560

agctaggaga agtggggatg tcttgtggga tattcccact cctaagatca tcgaggaatg 4620

tgaacatctg gaggatggga tttatggcat attccagtca accttcttgg gggcctccca 4680

gcgaggagtg ggagtggcac agggaggggt gttccacaca atgtggcatg tcacaagagg 4740

agctttcctt gtcaggaatg gcaagaagtt gattccatct tgggcttcag taaaggaaga 4800

ccttgtcgcc tatggtggct catggaagtt ggaaggcaga tgggatggag aggaagaggt 4860

ccagttgatc gcggctgttc caggaaagaa cgtggtcaac gtccagacaa aaccgagctt 4920

gttcaaagtg aggaatgggg gagaaatcgg ggctgtcgct cttgactatc cgagtggcac 4980

ttcaggatct cctattgtta acaggaacgg agaggtgatt gggctgtacg gcaatggcat 5040

ccttgtcggt gacaactcct tcgtgtccgc catatcccag actgaggtga aggaagaagg 5100

aaaggaggag ctccaagaga tcccgacaat gctaaagaaa ggaatgacaa ctgtccttga 5160

ttttcatcct ggagctggga agacaagacg tttcctccca cagatcttgg ccgagtgcgc 5220

acggagacgc ttgcgcactc ttgtgttggc ccccaccagg gttgttcttt ctgaaatgaa 5280

ggaggctttt cacggcctgg acgtgaaatt ccacacacag gctttttccg ctcacggcag 5340

cgggagagaa gtcattgatg ccatgtgcca tgccacccta acttacagga tgttggaacc 5400

aactagggtt gttaactggg aagtgatcat tatggatgaa gcccattttt tggatccagc 5460

tagcatagcc gctagaggtt gggcagcgca cagagctagg gcaaatgaaa gtgcaacaat 5520

cttgatgaca gccacaccgc ctgggactag tgatgaattt ccacattcaa atggtgaaat 5580

agaagatgtt caaacggaca tacccagtga gccctggaac acagggcatg actggatcct 5640

agctgacaaa aggcccacgg catggttcct tccatccatc agagctgcaa atgtcatggc 5700

tgcctctttg cgtaaggctg gaaagagtgt ggtggtcctg aacaggaaaa cctttgagag 5760

agaatacccc acgataaagc agaagaaacc tgactttata ttggccactg acatagctga 5820

aatgggagcc aacctttgcg tggagcgagt gctggattgc aggacggctt ttaagcctgt 5880

gcttgtggat gaagggagga aggtggcaat aaaagggcca cttcgtatct ccgcatcctc 5940

tgctgctcaa aggagggggc gcattgggag aaatcccaac agagatggag actcatacta 6000

ctattctgag cctacaagtg aaaataatgc ccaccacgtc tgctggttgg aggcctcaat 6060

gctcttggac aacatggagg tgaggggtgg aatggtcgcc ccactctatg gcgttgaagg 6120

aactaaaaca ccagtttccc ctggtgaaat gagactgagg gatgaccaga ggaaagtctt 6180

cagagaacta gtgaggaatt gtgacctgcc cgtttggctt tcgtggcaag tggccaaggc 6240

tggtttgaag acgaatgatc gtaagtggtg ttttgaaggc cctgaggaac atgagatctt 6300

gaatgacagc ggtgaaacag tgaagtgcag ggctcctgga ggagcaaaga agcctctgcg 6360

cccaaggtgg tgtgatgaaa gggtgtcatc tgaccagagt gcgctgtctg aatttattaa 6420

gtttgctgaa ggtaggaggg gagctgctga agtgctagtt gtgctgagtg aactccctga 6480

tttcctggct aaaaaaggtg gagaggcaat ggataccatc agtgtgttcc tccactctga 6540

ggaaggctct agggcttacc gcaatgcact atcaatgatg cctgaggcaa tgacaatagt 6600

catgctgttt atactggctg gactactgac atcgggaatg gtcatctttt tcatgtctcc 6660

caaaggcatc agtagaatgt ctatggcgat gggcacaatg gccggctgtg gatatctcat 6720

gttccttgga ggcgtcaaac ccactcacat ctcctatgtc atgctcatat tctttgtcct 6780

gatggtggtt gtgatccccg agccagggca acaaaggtcc atccaagaca accaagtggc 6840

atacctcatt attggcatcc tgacgctggt ttcagcggtg gcagccaacg agctaggcat 6900

gctggagaaa accaaagagg acctctttgg gaagaagaac ttaattccat ctagtgcttc 6960

accctggagt tggccggatc ttgacctgaa gccaggagct gcctggacag tgtacgttgg 7020

cattgttaca atgctctctc caatgttgca ccactggatc aaagtcgaat atggcaacct 7080

gtctctgtct ggaatagccc agtcagcctc agtcctttct ttcatggaca aggggatacc 7140

attcatgaag atgaatatct cggtcataat gctgctggtc agtggctgga attcaataac 7200

agtgatgcct ctgctctgtg gcatagggtg cgccatgctc cactggtctc tcattttacc 7260

tggaatcaaa gcgcagcagt caaagcttgc acagagaagg gtgttccatg gcgttgccaa 7320

gaaccctgtg gttgatggga atccaacagt tgacattgag gaagctcctg aaatgcctgc 7380

cctttatgag aagaaactgg ctctatatct ccttcttgct ctcagcctag cttctgttgc 7440

catgtgcaga acgccctttt cattggctga aggcattgtc ctagcatcag ctgccttagg 7500

gccgctcata gagggaaaca ccagccttct ttggaatgga cccatggctg tctccatgac 7560

aggagtcatg agggggaatc actatgcttt tgtgggagtc atgtacaatc tatggaagat 7620

gaaaactgga cgccggggga gcgcgaatgg aaaaactttg ggtgaagtct ggaagaggga 7680

actgaatctg ttggacaagc gacagtttga gttgtataaa aggaccgaca ttgtggaggt 7740

ggatcgtgat acggcacgca ggcatttggc cgaagggaag gtggacaccg gggtggcggt 7800

ctccaggggg accgcaaagt taaggtggtt ccatgagcgt ggctatgtca agctggaagg 7860

tagggtgatt gacctggggt gtggccgcgg aggctggtgt tactacgctg ctgcgcaaaa 7920

ggaagtgagt ggggtcaaag gatttactct tggaagagac ggccatgaga aacccatgaa 7980

tgtgcaaagt ctgggatgga acatcatcac cttcaaggac aaaactgata tccaccgcct 8040

agaaccagtg aaatgtgaca cccttttgtg tgacattgga gagtcatcat cgtcatcggt 8100

cacagagggg gaaaggaccg tgagagttct tgatactgta gaaaaatggc tggcttgtgg 8160

ggttgacaac ttctgtgtga aggtgttagc tccatacatg ccagatgttc tcgagaaact 8220

ggaattgctc caaaggaggt ttggcggaac agtgatcagg aaccctctct ccaggaattc 8280

cactcatgaa atgtactacg tgtctggagc ccgcagcaat gtcacattta ctgtgaacca 8340

aacatcccgc ctcctgatga ggagaatgag gcgtccaact ggaaaagtga ccctggaggc 8400

tgacgtcatc ctcccaattg ggacacgcag tgttgagaca gacaagggac ccctggacaa 8460

agaggccata gaagaaaggg ttgagaggat aaaatctgag tacatgacct cttggtttta 8520

tgacaatgac aacccctaca ggacctggca ctactgtggc tcctatgtca caaaaacctc 8580

aggaagtgcg gcgagcatgg taaatggtgt tattaaaatt ctgacatatc catgggacag 8640

gatagaggag gtcacaagaa tggcaatgac tgacacaacc ccttttggac agcaaagagt 8700

gtttaaagaa aaagttgaca ccagagcaaa ggatccacca gcgggaacta ggaagatcat 8760

gaaagttgtc aacaggtggc tgttccgcca cctggccaga gaaaagaacc ccagactgtg 8820

cacaaaggaa gaatttattg caaaagtccg aagtcatgca gccattggag cttacctgga 8880

agaacaagaa cagtggaaga ctgccaatga ggctgtccaa gacccaaagt tctgggaact 8940

ggtggatgaa gaaaggaagc tgcaccaaca aggcaggtgt cggacttgtg tgtacaacat 9000

gatggggaaa agagagaaga agctgtcaga gtttgggaaa gcaaagggaa gccgtgccat 9060

atggtatatg tggctgggag cgcggtatct tgagtttgag gccctgggat tcctgaatga 9120

ggaccattgg gcttccaggg aaaactcagg aggaggagtg gaaggcattg gcttacaata 9180

cctaggatat gtgatcagag acctggctgc aatggatggt ggtggattct acgcggatga 9240

caccgctgga tgggacacgc gcatcacaga ggcagacctt gatgatgaac aggagatctt 9300

gaactacatg agcccacatc acaaaaaact ggcacaagca gtgatggaaa tgacatacaa 9360

gaacaaagtg gtgaaagtgt tgagaccagc cccaggaggg aaagcctaca tggatgtcat 9420

aagtcgacga gaccagagag gatccgggca ggtagtgact tatgctctga acaccatcac 9480

caacttgaaa gtccaattga tcagaatggc agaagcagag atggtgatac atcaccaaca 9540

tgttcaagat tgtgatgaat cagttctgac caggctggag gcatggctca ctgagcacgg 9600

atgtgacaga ctgaagagga tggcggtgag tggagacgac tgtgtggtcc ggcccatcga 9660

tgacaggttc ggcctggccc tgtcccatct caacgccatg tccaaggtta gaaaggacat 9720

atctgaatgg cagccatcaa aagggtggaa tgattgggag aatgtgccct tctgttccca 9780

ccacttccat gaactacagc tgaaggatgg caggaggatt gtggtgcctt gccgagaaca 9840

ggacgagctc attgggagag gaagggtgtc tccaggaaac ggctggatga tcaaggaaac 9900

agcttgcctc agcaaagcct atgccaacat gtggtcactg atgtattttc acaaaaggga 9960

catgaggcta ctgtcattgg ctgtttcctc agctgttccc acctcatggg ttccacaagg 10020

acgcacaaca tggtcgattc atgggaaagg ggagtggatg accacggaag acatgcttga 10080

ggtgtggaac agagtatgga taaccaacaa cccacacatg caggacaaga caatggtgaa 10140

aaaatggaga gatgtccctt atctaaccaa gagacaagac aagctgtgcg gatcactgat 10200

tggaatgacc aatagggcca cctgggcctc ccacatccat ttagtcatcc atcgtatccg 10260

aacgctgatt ggacaggaga aatacactga ctacctaaca gtcatggaca ggtattctgt 10320

ggatgctgac ctgcaactgg gtgagcttat ctgaaacacc atctaacagg aataaccggg 10380

atacaaacca cgggtggaga accggactcc ccacaacctg aaaccgggat ataaaccacg 10440

gctggagaac cggactccgc acttaaaatg aaacagaaac cgggataaaa actacggatg 10500

gagaaccgga ctccacacat tgagacagaa gaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 10560

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 10620

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaccatatt gacgccaggg 10680

aaagaccgga gtggttctct gcttttcctc cagaggtctg tgagcacagt ttgctcaaga 10740

ataagcagac ctttggatga caaacacaaa accac 10775

<210> 5

<211> 10625

<212> DNA

<213> zika virus

<400> 5

agttgttgat ctgtgtgagt cagactgcga cagttcgagt ttgaagcgaa agctagcaac 60

agtatcaaca ggttttattt tggatttgga aacgagagtt tctggtcatg aaaaacccaa 120

aaaagaaatc cggaggattc cggattgtca atatgctaaa acgcggagta gcccgtgtga 180

gcccctttgg gggcttgaag aggctgccag ccggacttct gctgggtcat gggcccatca 240

ggatggtctt ggcgattcta gccttcttga gattcacggc aatcaagcca tcactgggtc 300

tcatcaatag atggggttca gtggggaaaa aagaggctat ggaaataata aagaagttca 360

agaaagatct ggctgccatg ctgagaataa tcaatgctag gaaggagaag aagagacgag 420

gcgcagatac taatgtcgga attgttggcc tcctgctgac cacagctatg gcagcggagg 480

tcactagacg tgggagtgca tactatatgt acttggacag aaacgatgct ggggaggcca 540

tatcttttcc aaccacattg gggatgaata agtgttatat acagatcatg gatcttggac 600

acatgtgtga tgccaccatg agctatgaat gccctatgct ggatgagggg gtggaaccag 660

atgacgtcga ttgttggtgc aacacgacgt caacttgggt tgtgtacgga acctgccatc 720

acaaaaaagg tgaagcacgg agatctagaa gagctgtgac gctcccctcc cattccacta 780

ggaagctgca aacgcggtcg caaacttggt tggaatcaag agaatacaca aagcacttga 840

ttagagtcga aaattggata ttcaggaacc ctggcttcgc gttagcagca gctgccatcg 900

cttggctttt gggaagctca acgagccaaa aagtcatata cttggtcatg atactgctga 960

ttgccccggc atacagcatc aggtgcatag gagtcagcaa tagggacttt gtggaaggta 1020

tgtcaggtgg gacttgggtt gatgttgtct tggaacatgg aggttgtgtc accgtaatgg 1080

cacaggacaa accgactgtc gacatagagc tggttacaac aacagtcagc aacatggcgg 1140

aggtaagatc ctactgctat gaggcatcaa tatcggacat ggcttcggac agccgctgcc 1200

caacacaagg tgaagcctac cttgacaagc aatcagacac tcaatatgtc tgcaaaagaa 1260

cgttagtgga cagaggctgg ggaaatggat gtggactttt tggcaaaggg agcctggtga 1320

catgcgctaa gtttgcatgc tccaagaaaa tgaccgggaa gagcatccag ccagagaatc 1380

tggagtaccg gataatgctg tcagttcatg gctcccagca cagtgggatg atcgttaatg 1440

acacaggaca tgaaactgat gagaatagag cgaaggttga gataacgccc aattcaccaa 1500

gagccgaagc caccctgggg ggttttggaa gcctaggact tgattgtgaa ccgaggacag 1560

gccttgactt ttcagatttg tattacttga ctatgaataa caagcactgg ttggttcaca 1620

aggagtggtt ccacgacatt ccattacctt ggcacgctgg ggcagacacc ggaactccac 1680

actggaacaa caaagaagca ctggtagagt tcaaggacgc acatgccaaa aggcaaactg 1740

tcgtggttct agggagtcaa gaaggagcag ttcacacggc ccttgctgga gctctggagg 1800

ctgagatgga tggtgcaaag ggaaggctgt cctctggcca cttgaaatgt cgcctgaaaa 1860

tggataaact tagattgaag ggcgtgtcat actccttgtg taccgcagcg ttcacattca 1920

ccaagatccc ggctgaaaca ctgcacggga cagtcacagt ggaggtacag tacgcaggga 1980

cagatggacc ttgcaaggtt ccagctcaga tggcggtgga catgcaaact ctgaccccag 2040

ttgggaggct gataaccgct aaccccgtaa tcactgaaag cactgagaac tccaagatga 2100

tgctggaact tgatccacca tttggggact cttacattgt cataggagtc ggggagaaga 2160

agatcaccca ccactggcac aggagtggca gcaccattgg aaaagcattt gaagccactg 2220

tgagaggtgc caggagaatg gcagtcttgg gagacacagc ctgggacttt ggatcagttg 2280

gaggcgctct caactcattg ggcaagggca tccatcaaat ttttggagca gctttcaaat 2340

cattgtttgg aggaatgtcc tggttctcac aaattctcat tggaacgttg ctgatgtggt 2400

tgggtctgaa cacaaagaat ggatctattt cccttatgtg cttggcctta gggggagtgt 2460

tgatcttctt atccacagcc gtctctgctg atgtggggtg ctcggtggac ttctcaaaga 2520

aggagacgag atgcggtaca ggggtgttcg tctataacga cgttgaagcc tggagggaca 2580

ggtacaagta ccatcctgac tccccccgta gattggcagc agcagtcaag caagcctggg 2640

aagatggtat ctgtgggatc tcctctgttt caagaatgga aaacatcatg tggagatcag 2700

tagaagggga gctcaacgca atcctggaag agaatggagt tcaactgacg gtcgttgtgg 2760

gatctgtaaa aaaccccatg tggagaggtc cacagagatt gcccgtgcct gtgaacgagc 2820

tgccccacgg ctggaaggct tgggggaaat cgtacttcgt cagagcagca aagacaaata 2880

acagctttgt cgtggatggt gacacactga aggaatgccc actcaaacat agagcatgga 2940

acagctttct tgtggaggat catgggttcg gggtatttca cactagtgtc tggctcaagg 3000

ttagagaaga ttattcatta gagtgtgatc cagccgttat tggaacagct gttaagggaa 3060

aggaggctgt acacagtgat ctaggctact ggattgagag tgagaagaat gacacatgga 3120

ggctgaagag ggcccatctg atcgagataa aaacatgtga atggccaaag tcccacacat 3180

tgtggacaga tggaatagaa gagagtgatc tgatcatacc caagtcttta gctgggccac 3240

tcagccatca caataccaga gagggctaca ggacccaaat gaaagggcca tggcacagtg 3300

aagagcttga aattcggttt gaggaatgcc caggcaccaa ggtccacgtg gaggaaacat 3360

gtggaacaag aggaccatct ctgagatcaa ccactgcaag cggaagggtg atcgaggaat 3420

ggtgctgcag ggagtgcaca atgcccccac tgtcgttcca ggctaaagat ggctgttggt 3480

atggaatgga gataaggccc aggaaagaac cagaaagtaa cttagtaagg tcaatggtga 3540

ctgcaggatc aactgatcac atggatcact tctcccttgg agtgcttgtg attctgctca 3600

tggtgcagga agggctgaag aagagaatga ccacaaagat catcataagc acatcaatgg 3660

cagtgctggt agctatgatc ctgggaggat tttcaatgag tgacctggct aagcttgcaa 3720

ttttgatggg tgccaccttc gcggaaatga acactggagg agatgtagct catctggcgc 3780

tgatagcggc attcaaagtc agaccagcgt tgctggtatc tttcatcttc agagctaatt 3840

ggacaccccg tgaaagcatg ctgctggcct tggcctcgtg tcttttgcaa actgcgatct 3900

ccgccttgga aggcgacctg atggttctca tcaatggttt tgctttggcc tggttggcaa 3960

tacgagcgat ggttgttcca cgcactgata acatcacctt ggcaatcctg gctgctctga 4020

caccactggc ccggggcaca ctgcttgtgg cgtggagagc aggccttgct acttgcgggg 4080

ggtttatgct cctctctctg aagggaaaag gcagtgtgaa gaagaactta ccatttgtca 4140

tggccctggg actaaccgct gtgaggctgg tcgaccccat caacgtggtg ggactgctgt 4200

tgctcacaag gagtgggaag cggagctggc cccctagcga agtactcaca gctgttggcc 4260

tgatatgcgc attggctgga gggttcgcca aggcagatat agagatggct gggcccattg 4320

ccgcggtcgg tctgctaatt gtcagttacg tggtctcagg aaagagtgtg gacatgtaca 4380

ttgaaagagc aggtgacatc acatgggaaa aagatgcgga agtcactgga aacagtcccc 4440

ggcttgatgt ggcgctagat gagagtggtg atttctccct ggtggaggat gacggtcccc 4500

ccatgagaga gatcatactc aaggtggtcc tgatgaccat ctgtggcatg aacccaatag 4560

ccataccctt tgcagctgga gcgtggtacg tatacgtgaa gactggaaaa aggagtggag 4620

ctctatggga tgtgcctgct cccaaggaag taaaaaaggg ggagaccaca gatggagtgt 4680

acagagtgat gactcgtaga ctgctaggtt caacacaagt tggagtggga gttatgcaag 4740

agggggtctt tcacaccatg tggcacgtca caaaaggatc cgcgctgaga agcggtgaag 4800

ggagacttga tccatactgg ggagatgtca agcaggatct ggtgtcatac tgtggtccat 4860

ggaagctaga tgccgcctgg gacgggcaca gcgaggtgca gctcttggcc gtgccccccg 4920

gagagagagc gaggaacatc cagactctgc ccggaatatt taagacaaag gatggggaca 4980

ttggagcggt tgcgctggat tacccagcag gaacttcagg atctccaatc ctagacaagt 5040

gtgggagagt gataggactt tatggcaatg gggtcgtgat caaaaatggg agttatgtta 5100

gtgccatcac ccaagggagg agggaggaag agactcctgt tgagtgcttc gagccttcga 5160

tgctgaagaa gaagcagcta actgtcttag acttgcatcc tggagctggg aaaaccagga 5220

gagttcttcc tgaaatagtc cgtgaagcca taaaaacaag actccgtact gtgatcttag 5280

ctccaaccag ggttgtcgct gccgaaatgg aggaagccct tagagggctt ccagtgcgtt 5340

atatgacaac agcagtcaat gtcacccact ctggaacaga aatcgtcgac ttaatgtgcc 5400

atgccacctt cacttcacgt ctactacagc caatcagagt ccccaactat aatctgtata 5460

ttatggatga ggcccacttc acagatccct caagtatagc agcaagagga tacatttcaa 5520

caagggttga gatgggcgag gcggctgcca tcttcatgac cgccacgcca ccaggaaccc 5580

gtgacgcatt tccggactcc aactcaccaa ttatggacac cgaagtggaa gtcccagaga 5640

gagcctggag ctcaggcttt gattgggtga cggatcattc tggaaaaaca gtctggtttg 5700

ttccaagcgt gaggaacggc aatgagatcg cagcttgtct gacaaaggct ggaaaacggg 5760

tcatacagct cagcagaaag acttttgaga cagagttcca gaaaacaaaa catcaagagt 5820

gggactttgt cgtgacaact gacatttcag agatgggcgc caactttaaa gctgaccgtg 5880

tcatagattc caggagatgc ctaaagccgg tcatacttga tggcgagaga gtcattctgg 5940

ctggacccat gcctgtcaca catgccagcg ctgcccagag gagggggcgc ataggcagga 6000

atcccaacaa acctggagat gagtatctgt atggaggtgg gtgcgcagag actgacgaag 6060

accatgcaca ctggcttgaa gcaagaatgc tccttgacaa tatttacctc caagatggcc 6120

tcatagcctc gctctatcga cctgaggccg acaaagtagc agccattgag ggagagttca 6180

agcttaggac ggagcaaagg aagacctttg tggaactcat gaaaagagga gatcttcctg 6240

tttggctggc ctatcaggtt gcatctgccg gaataaccta cacagataga agatggtgct 6300

ttgatggcac gaccaacaac accataatgg aagacagtgt gccggcagag gtgtggacca 6360

gacacggaga gaaaagagtg ctcaaaccga ggtggatgga cgccagagtt tgttcagatc 6420

acgcggccct gaagtcattc aaggagtttg ccgctgggaa aagaggagcg gcttttggag 6480

tgatggaagc cttgggaaca ctgccaggac acatgacaga gagattccag gaagccattg 6540

acaacctcgc tgtgctcatg cgggcagaga ctggaagcag gccttacaaa gccgcggcgg 6600

cccaattgcc ggagacccta gagaccatta tgcttttggg gttgctggga acagtctcgc 6660

tgggaatctt tttcgtcttg atgaggaaca agggcatagg gaagatgggc tttggaatgg 6720

tgactcttgg ggccagcgca tggctcatgt ggctctcgga aattgagcca gccagaattg 6780

catgtgtcct cattgttgtg ttcctattgc tggtggtgct catacctgag ccagaaaagc 6840

aaagatctcc ccaggacaac caaatggcaa tcatcatcat ggtagcagta ggtcttctgg 6900

gcttgattac cgccaatgaa ctcggatggt tggagagaac aaagagtgac ctaagccatc 6960

taatgggaag gagagaggag ggggcaacca taggattctc aatggacatt gacctgcggc 7020

cagcctcagc ttgggccatc tacgctgcct tgacaacttt cattacccca gccgtccaac 7080

atgcagtgac cacttcatac aacaactact ccttaatggc gatggccacg caagctggag 7140

tgttgtttgg tatgggcaaa gggatgccat tctacgcatg ggactttgga gtcccgctgc 7200

taatgatagg ttgctactca caattaacac ccctgaccct aatagtagcc atcattttgc 7260

tcgtggcgca ctacatgtac ttgatcccag ggctgcaggc agcagctgcg cgtgctgccc 7320

agaagagaac ggcagctggc atcatgaaga accctgttgt ggatggaata gtggtgactg 7380

acattgacac aatgacaatt gacccccaag tggagaaaaa gatgggacag gtgctactca 7440

tagcagtagc cgtctccagc gccatactgt cgcggaccgc ctgggggtgg ggggaggctg 7500

gggccctgat cacagctgca acttccactt tgtgggaagg ctctccgaac aagtactgga 7560

actcctctac agccacttca ctgtgtaaca tttttagggg aagttacttg gctggagctt 7620

ctctaatcta cacagtaaca agaaacgctg gcttggtcaa gagacgtggg ggtggaacag 7680

gagagaccct gggagagaaa tggaaggccc gcttgaacca gatgtcggcc ctggagttct 7740

actcctacaa aaagtcaggc atcaccgagg tgtgcagaga agaggcccgc cgcgccctca 7800

aggacggtgt ggcaacggga ggccatgctg tgtcccgagg aagtgcaaag ctgagatggt 7860

tggtggagcg gggatacctg cagccctatg gaaaggtcat tgatcttgga tgtggcagag 7920

ggggctggag ttactacgcc gccaccatcc gcaaagttca agaagtgaaa ggatacacaa 7980

aaggaggccc tggtcatgaa gaacccatgt tggtgcaaag ctatgggtgg aacatagtcc 8040

gtcttaagag tggggtggac gtctttcata tggcggctga gccgtgtgac acgttgctgt 8100

gtgacatagg tgagtcatca tctagtcctg aagtggaaga agcacggacg ctcagagtcc 8160

tttccatggt gggggattgg cttgaaaaaa gaccaggagc cttttgtata aaagtgttgt 8220

gcccatacac cagcactatg atggaaaccc tggagcgact gcagcgtagg tatgggggag 8280

gactggtcag agtgccactc tcccgcaact ctacacatga gatgtactgg gtctctggag 8340

cgaaaagcaa caccataaaa agtgtgtcca ccacgagcca gctcctcttg gggcgcatgg 8400

acgggcccag gaggccagtg aaatatgagg aggatgtgaa tctcggctct ggcacgcggg 8460

ctgtggtaag ctgcgctgaa gctcccaaca tgaagatcat tggtaaccgc attgaaagga 8520

tccgcagtga gcacgcggaa acgtggttct ttgacgagaa ccacccatat aggacatggg 8580

cttaccatgg aagctatgag gcccccacac aagggtcagc gtcctctcta ataaacgggg 8640

ttgtcaggct cctgtcaaaa ccctgggatg tggtgactgg agtcacagga atagccatga 8700

ccgacaccac accgtatggt cagcaaagag ttttcaagga aaaagtggac actagggtgc 8760

cagatcccca agaaggcact cgtcaggtta tgagcatggt ctcttcctgg ttgtggaaag 8820

agctaggcaa acacaaacgg ccacgagtct gtaccaaaga agagttcatc aacaaggttc 8880

gtagcaatgc agcattaggg gcaatatttg aagaggaaaa agagtggaag actgcagtgg 8940

aagctgtgaa cgatccaagg ttctgggctc tagtggacaa ggaaagagag caccacctga 9000

gaggagagtg ccagagttgt gtgtacaaca tgatgggaaa aagagaaaag aaacaagggg 9060

aatttggaaa ggccaagggc agccgcgcca tctggtatat gtggctaggg gctagatttc 9120

tagagttcga agcccttgga ttcttgaacg aggatcactg gatggggaga gagaactcag 9180

gaggtggtgt tgaagggctg ggattacaaa gactcggata tgtcctagaa gagatgagtc 9240

gcataccagg aggaaggatg tatgcagatg acactgctgg ctgggacacc cgcatcagca 9300

ggtttgatct ggagaatgaa gctctaatca ccaaccaaat ggagaaaggg cacagggcct 9360

tggcattggc cataatcaag tacacatacc aaaacaaagt ggtaaaggtc cttagaccag 9420

ctgaaaaagg gaagacagtt atggacatta tttcgagaca agaccaaagg gggagcggac 9480

aagttgtcac ttacgctctt aacacattta ccaacctagt ggtgcaactc attcggagta 9540

tggaggctga ggaagttcta gagatgcaag acttgtggct gctgcggagg tcagagaaag 9600

tgaccaactg gctgcagagc aacggatggg ataggctcaa acgaatggca gtcagtggag 9660

atgattgcgt tgtgaggcca attgatgata ggtttgcaca tgccctcagg ttcttgaatg 9720

atatggggaa agttaggaag gacacacaag agtggaaacc ctcaactgga tgggacaact 9780

gggaggaagt tccgttttgc tcccaccact tcaacaagct ccatctcaag gacgggaggt 9840

ccattgtggt tccctgccgc caccaagatg aactgattgg ccgggcccgc gtctctccag 9900

gggcgggatg gagcatccgg gagactgctt gcctagcaaa atcatatgcg caaatgtggc 9960

agctccttta tttccacaga agggacctcc gactgatggc caatgccatt tgttcatctg 10020

tgccagttga ctgggttcca actgggagaa ctacctggtc aatccatgga aagggagaat 10080

ggatgaccac tgaagacatg cttgtggtgt ggaacagagt gtggattgag gagaacgacc 10140

acatggaaga caagacccca gttacgaaat ggacagacat tccctatttg ggaaaaaggg 10200

aagacttgtg gtgtggatct ctcatagggc acagaccgcg caccacctgg gctgagaaca 10260

ttaaaaacac agtcaacatg gtgcgcagga tcataggtga tgaagaaaag tacatggact 10320

acctatccac ccaagttcgc tacttgggtg aagaagggtc tacacctgga gtgctgtaag 10380

caccaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 10440

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 10500

aaaaaaaaaa aaaacagcat attgacgctg ggaaagacca gagactccat gagtttccac 10560

cacgctggcc gccaggcaca gatcgccgaa tagcggcggc cggtgtgggg aaatccatgg 10620

tttct 10625

<210> 6

<211> 10903

<212> DNA

<213> Tick-borne encephalitis virus

<400> 6

agattttctt gcacgtgcat gcgtttgctt cggacagcat tagcagcggt tggtttgaaa 60

gagatattct tttgtttcta ccagtcgtga acgtgttgag aaaaagacag cttaggagaa 120

caagagctgg ggatggtcaa gaaggccatc ctgaaaggta aggggggcgg tccccctcga 180

cgagtgtcga aagagaccgc aacgaagacg cgtcaaccca gagtccaaat gccaaatggg 240

cttgtgttga tgcgcatgat ggggatcttg tggcatgccg tagccggcac cgcgagaaac 300

cccgtattga aggcgttctg gaactcagtc cctctgaaac aggcaacagc agcactgcgg 360

aagatcaaaa ggacggtgag tgccctaatg gttggcttgc aaaaacgtgg gaaaaggagg 420

tcagcgacgg actggatgag ctggttgctg gtcatcactc tgttggggat gacgcttgct 480

gcaacggtga ggaaagaaag ggatggctca actgtgatca gagctgaagg aaaggatgca 540

gcaactcagg tgcgtgtgga gaatggcacc tgtgtgatcc tggctactga catggggtca 600

tggtgtgatg attcactgtc ctatgagtgt gtgaccatag atcaaggaga agagcctgtt 660

gacgtggatt gtttttgccg gaacgttgat ggagtctatc tggagtacgg acgctgtggg 720

aaacaggaag gctcacggac aaggcgctca gtgctgatcc catcccatgc tcagggagag 780

ctgacgggaa ggggacacaa atggctagaa ggagactcgc tgcgaacaca ccttactaga 840

gttgagggat gggtctggaa gaacaagcta cttgccttgg caatggttac cgttgtgtgg 900

ttgaccctgg agagtgtggt gaccagggtt gccgttcttg ttgtgctcct gtgtttggca 960

ccggtttacg cttcgcgttg cacacacttg gaaaacaggg actttgtgac tggtactcag 1020

gggactacga gggtcacctt ggtgctggaa ctgggtggat gtgttactat aacagctgag 1080

gggaagcctt caatggatgt gtggcttgac gccatttacc aggagaaccc tgctaagaca 1140

cgtgagtact gtttacacgc caagttgtcg gacactaagg ttgcagccag atgcccaaca 1200

atgggaccag ccactttggc tgaagaacac cagggtggca cagtgtgtaa gagagatcag 1260

agtgatcgag gctggggcaa ccactgtgga ctgtttggaa agggtagcat tgtggcctgt 1320

gtcaaggcgg cttgtgaggc aaaaaagaaa gccacaggac atgtgtacga cgccaacaaa 1380

atagtgtaca cggtcaaagt cgaaccacac acgggagact atgttgccgc aaacgagaca 1440

catagtggga ggaagacggc atccttcaca atttcttcag agaaaaccat tttgactatg 1500

ggtgagtatg gagatgtgtc tttgttgtgc agggtcgcta gtggcgttga cttggcccag 1560

accgtcatcc ttgagcttga caagacagtg gaacaccttc caacggcttg gcaggtccat 1620

agggactggt tcaatgatct ggctctgcca tggaaacatg agggagcgca aaactggaac 1680

aacgcagaaa gactggttga atttggggct cctcacgctg tcaagatgga cgtgtacaac 1740

ctcggagacc agactggagt gttactgaag gctctcgctg gggttcctgt ggcacacatt 1800

gagggaacca agtaccacct gaagagtggc cacgtgacct gcgaagtggg actggaaaaa 1860

ctgaagatga aaggtcttac gtacacaatg tgtgacaaaa caaagttcac atggaagaga 1920

gctccaacag acagtgggca tgatacagtg gtcatggaag tcacattctc tggaacaaag 1980

ccctgtagga tcccagtcag ggcagtggca catggatctc cagatgtgaa cgtggccatg 2040

ctgataacgc caaacccaac aattgaaaac aatggaggtg gcttcataga gatgcagctg 2100

cccccagggg ataacatcat ctatgttggg gaactgagtc atcaatggtt ccaaaaaggg 2160

agcagcatcg gaagggtttt ccaaaagacc aagaaaggca tagaaagact gacagtgata 2220

ggagagcacg cctgggactt cggttctgct ggaggctttc tgagttcaat tgggaaggcg 2280

gtacatacgg tccttggtgg cgctttcaac agcatcttcg ggggagtggg gtttctacca 2340

aaacttttat taggagtggc attggcttgg ttgggcctga acatgagaaa ccctacaatg 2400

tccatgagct ttctcttggc tggaggtctg gtcttggcca tgacccttgg agtgggggcg 2460

gatgttggtt gcgctgtgga cacggaacga atggagctcc gctgtggcga gggcctggtc 2520

gtgtggagag aggtctcaga atggtatgac aattatgcct actacccgga gacaccgggg 2580

gcccttgcat cagccataaa ggagacattt gaggagggaa gctgtggtgt agtcccccag 2640

aacaggctcg agatggccat gtggagaagc tcggtcacag agctgaattt ggctctggcg 2700

gaaggggagg caaatctcac agtggtggtg gacaagtttg accccactga ctaccgaggt 2760

ggtgtccctg gtttactgaa aaaaggaaag gacataaaag tctcctggaa aagctggggc 2820

cattcaatga tctggagcat tcctgaggcc ccccgtcgct tcatggtggg cacggaagga 2880

caaagtgagt gtcccctaga gagacggaag acaggtgttt tcacggtggc agaattcggg 2940

gttggcctga gaacaaaggt cttcttggat ttcagacagg aaccaacaca tgagtgtgac 3000

acaggagtga tgggagctgc agtcaagaac ggcatggcaa tccacacaga tcaaagtctc 3060

tggatgagat caatgaaaaa tgacacaggc acttacatag ttgaactttt ggtcactgac 3120

ctgaggaact gctcatggcc tgctagccac actatcgata atgctgacgt ggtggactca 3180

gagttattcc ttccggcgag cctggcagga cccagatcct ggtacaacag gatacctggc 3240

tattcagaac aggtgaaagg gccatggaag tacacgccta tccgagttat cagagaggag 3300

tgtcccggca cgaccgttac catcaacgcc aagtgtgaca aaagaggagc atctgtgagg 3360

agtaccacag agagtggcaa ggttatccca gaatggtgct gccgagcgtg cacaatgcca 3420

ccagtgacgt tccggactgg aactgattgc tggtatgcca tggaaatacg gccagtccat 3480

gaccaggggg ggcttgttcg ctcaatggtg gttgcggaca acggtgaatt acttagtgag 3540

ggaggagtcc ccggaatagt ggcattgttt gtggtccttg aatacatcat ccgtaggagg 3600

ccctccacgg gaacgacggt tgtgtggggg ggtatcgtcg ttctcgctct gcttgtcacc 3660

gggatggtca ggatagagag cctggtgcgc tatgtggtgg cagtggggat cacattccac 3720

cttgagctgg ggccagagat cgtggccttg atgctactcc aggctgtgtt tgagctgagg 3780

gtgggtttgc tcagcgcatt tgcattgcgc agaagcctca ccgtccgaga gatggtgacc 3840

acctactttc ttttgctggt cctggaattg gggctgccgg gtgcgagcct tgaggagttc 3900

tggaaatggg gtgatgcact ggccatgggg gcgctgatat tcagggcttg cacggcagaa 3960

ggaaagactg gagcggggct tttgctcatg gctctcatga cacagcagga tgtggtgact 4020

gtgcaccatg gactggtgtg cttcctaagt gtagcttcgg catgctcggt ctggaggctg 4080

ctcaagggac acagagagca gaagggattg acctgggttg tccccctggc tgggttgctt 4140

gggggagagg gctctggaat cagactgctg gcgttttggg agttgtcagc gcacagagga 4200

agacgatctt tcagtgaacc actaactgtg gtaggagtca tgctaacact ggccagcggc 4260

atgatgcgac acacttccca ggaggctctc tgtgcactcg cagtggcctc gtttctcttg 4320

ctgatgctgg tgctggggac aagaaagatg cagctggttg ccgaatggag tggctgcgtt 4380

gaatggtatc cggaactagt gaatgagggt ggagaggtta gcctgcgggt ccggcaggac 4440

gcgatgggta actttcactt gactgagctc gagaaagaag agagaatgat ggctttttgg 4500

ctgattgccg gcttggcagc ttcggccatt cactggtcag gcattcttgg tgtgatggga 4560

ctgtggacgc tcacggaaat gctgaggtca tcccgaaggt ctgacctggt tttctctgga 4620

caggggggtc gagagcgtgg tgacagacct ttcgaggtta aggacggtgt ctacaggatt 4680

tttagccccg gcttgttctg gggtcagaac caggtgggag ttggctacgg ttccaagggt 4740

gtcttgcaca cgatgtggca cgtgacgaga ggagcggcgc tgtctattga tgacgctgtg 4800

gccggtccct actgggctga tgtgagggaa gatgtcgtgt gttacggagg agcctggagc 4860

ctggaggaaa aatggaaagg tgaaacagta caggtccatg ccttcccacc ggggagggcg 4920

catgaggtgc atcagtgcca gcctggggag ttgatccttg acaccggaag aaagcttggg 4980

gcaataccaa ttgatttggt gaaaggaaca tcaggcagcc ccattctcaa cgcccaggga 5040

gtggttgtgg ggctatacgg aaatggccta aaaactaatg agacctacgt cagcagcatt 5100

gctcaagggg aagcggagaa gagtcgcccc aacctcccac aggctgttgt gggtacgggc 5160

tggacatcaa agggtcagat cacagtgctg gacatgcacc caggctcggg gaagacccac 5220

agagtcctcc cggagctcat tcgccaatgc attgacaggc gcctgagaac gttggtgttg 5280

gctccaactc gtgtggtact caaagaaatg gagcgtgctt tgaatgggaa acgggtcagg 5340

ttccactcac cagcagtcag tgaccaacag gctggagggg caattgtcga tgtgatgtgt 5400

cacgcaacct atgtcaacag aaggctactc ccacagggaa gacaaaattg ggaggtggca 5460

atcatggatg aggcccactg gacggacccc cacagcatag ctgccagagg tcatttgtat 5520

actctggcaa aagaaaacaa gtgtgcactg gtcttgatga cagcgacacc tcctggtaag 5580

agtgaaccct ttccggagtc caatggagcc attactagtg aggaaagaca gattcctgat 5640

ggggagtggc gtgacgggtt tgactggatc actgagtatg aagggcgcac cgcctggttt 5700

gtcccttcga ttgcaaaagg tggtgctata gctcgcacct tgagacagaa ggggaaaagt 5760

gtgatttgtt tgaacagcaa aacctttgaa aaggactact ccagagtgag ggatgagaag 5820

cctgactttg tggtgacgac tgatatctcg gagatgggag ctaaccttga cgtgagccgc 5880

gtcatagatg ggaggacaaa catcaagccc gaggaggttg atgggaaagt cgagctcacc 5940

gggaccaggc gagtgaccac ggcttccgct gcccaacggc gcggaagagt tggtcggcaa 6000

gacggacgaa cagatgaata catatactct ggacagtgtg atgatgatga cagtggatta 6060

gtgcaatgga aagaggcgca aatacttctt gacaacataa caaccttgcg ggggcccgtg 6120

gccaccttct atggaccaga acaggacaag atgccggagg tggccggtca ctttcgactc 6180

actgaagaga aaagaaagca cttccgacat cttctcaccc attgtgactt cacaccgtgg 6240

ctggcatggc acgtcgcagc gaatgtatcc agcgtcacgg atcgaagctg gacatgggaa 6300

gggccggagg caaatgccgt ggatgaggcc agtggtgact tggtcacctt taggagcccg 6360

aatggggcgg agagaactct caggccggtg tggaaggacg cacgtatgtt caaagaggga 6420

cgtgacatca aagagttcgt ggcgtacgcg tctgggcgtc gcagcttcgg agatgttctg 6480

acaggaatgt cgggagttcc tgagctcctg cggcacagat gcgtcagtgc cctggatgtc 6540

ttctacacgc ttatgcatga ggaacctggc agcagggcaa tgagaatggc ggagagagat 6600

gccccagagg cctttctgac tatggttgag atgatggtgc tgggtttggc aaccctgggt 6660

gtcatctggt gcttcgtcgt ccggacttca atcagccgca tgatgctggg cacgctggtc 6720

ctgctggcct ccttgctact cttgtgggca ggtggcgtcg gctatgggaa catggctgga 6780

gtggctctca tcttttacac gttgctgacg gtgctgcagc ctgaggcggg aaaacagaga 6840

agcagtgacg acaacaaact ggcatatttc ttgctgacgc tctgcagcct tgctggactg 6900

gttgcagcca atgagatggg ctttctggag aagaccaagg cagacttgtc cacggcgctg 6960

tggagtgaac gggaggaacc ccggccatgg agtgaatgga cgaatgtgga catccagcca 7020

gcgaggtcct gggggaccta tgtgctggtg gtgtctctgt tcacacctta catcatccac 7080

caactgcaga ccaaaatcca acaacttgtc aacagtgccg tggcatctgg tgcacaggcc 7140

atgagagacc ttgggggagg tgcccccttc tttggtgtgg cgggacatgt catgaccctc 7200

ggggtggtgt cactgattgg ggctactccc acctcactga tggtgggcgt tggcttggcg 7260

gcactccatc tggccattgt ggtgtctggt ctggaggctg aattaacaca gagagctcat 7320

aaggtctttt tctctgcaat ggtgcgcaac cccatggtgg atggggatgt catcaaccca 7380

ttcggggagg gggaggcaaa acctgctcta tatgaaagga aaatgagtct ggtgttggcc 7440

acagtgttgt gcctcatgtc ggtggtcatg aaccgaacgg tggcctccat aacagaggct 7500

tcagcagtgg gactggcagc agcgggacag ctgcttagac cggaggctga cacgttgtgg 7560

acgatgccgg ttgcttgtgg catgagtggt gtggtcaggg gtagcctgtg ggggtttttg 7620

cctcttgggc atagactctg gcttcgagcc tctgggggta ggcgtggtgg ttctgaggga 7680

gacacgcttg gagatctctg gaagcggagg ctgaacaact gcacgaggga ggaattcttt 7740

gtgtacaggc gcaccggcat cctggagacg gaacgtgaca aggctagaga gttgctcaga 7800

agaggagaga ccaatgtggg attggctgtc tctcggggca cggcaaagct tgcctggctt 7860

gaggaacgcg gatatgccac cctcaaggga gaggtggtag atcttggatg tggaaggggc 7920

ggctggtcct attatgcggc atcccgaccg gcagtcatga gtgtcagggc atataccatt 7980

ggtggaaaag ggcacgaggc tccaaagatg gtaacaagcc tgggttggaa cttgattaaa 8040

ttcagatcag gaatggacgt gttcagcatg cagccacacc gggctgacac tgtcatgtgt 8100

gacatcggag agagcagccc agatgccgct gtggagggtg agaggacaag gaaagtgata 8160

ctgctcatgg agcaatggaa aaacaggaac cccacggctg cctgtgtgtt caaggtgctg 8220

gccccatatc gcccagaagt gatagaggca ctgcacagat tccaactgca atgggggggg 8280

ggtctggtga ggaccccttt ttcaaggaac tccacccatg agatgtatta ctcaacagcc 8340

gtcactggga acatagtgaa ctccgtcaat gtacagtcga ggaaactttt ggctcggttt 8400

ggagaccaga gagggccaac caaggtgcct gaactcgacc tgggagttgg aacgaggtgt 8460

gtggtcttag ctgaggacaa ggtgaaagaa caagacgtac aagagaggat cagagcgttg 8520

cgggagcaat acagcgaaac ctggcatatg gacgaggaac acccgtaccg gacatggcag 8580

tactggggca gctaccgcac ggcaccaacc ggctcggcgg cgtcactgat caatggggtt 8640

gtgaaacttc tcagctggcc atggaacgca cgggaagatg tggtgcgcat ggctatgact 8700

gacacaacgg ctttcggaca gcagagagtg ttcaaagata aagttgacac aaaggcacag 8760

gagcctcagc ccggtacaag agtcatcatg agagctgtaa atgattggat tttggaacga 8820

ctggcgcaga aaagcaaacc gcgcatgtgc agcagggaag aattcatagc aaaagtgaaa 8880

tcaaatgcag ccttgggagc ttggtcagat gagcaaaaca gatgggcaag tgcaagagag 8940

gctgtagagg atcctgcatt ctggcgcctc gtggatgaag agagagaaag gcacctcatg 9000

gggagatgtg cgcactgcgt gtacaacatg atgggcaaga gagaaaagaa actgggagag 9060

ttcggagtgg cgaaaggaag tcgggccatt tggtacatgt ggctggggag tcgctttttg 9120

gagttcgagg ctcttggatt cttgaatgaa gaccattggg cctctagaga gtccagtgga 9180

gctggagttg agggaataag cttgaactac ctgggctggc acctcaagaa gttgtcaacc 9240

ctgaatggag gactcttcta tgcagatgac acagctggct gggacacgaa agttaccaat 9300

gcagacttag aggatgaaga acagatccta cggtacatgg agggtgagca caaacaattg 9360

gcaaccacaa taatgcaaaa agcataccat gccaaagtcg tgaaggtcgc gaggccttcc 9420

cgtgatggag gctgcatcat ggatgtcatc acaagaagag accaaagagg ttcgggtcag 9480

gttgtgacct atgcccttaa caccctcacc aacataaagg tgcaattaat ccgaatgatg 9540

gaaggggaag gggtcataga ggcagcggat gcacacaacc cgagactgct tcgagtggag 9600

cgctggctga aagaacacgg agaagagcgt cttggaagaa tgctcgtcag tggtgacgat 9660

tgtgtggtga ggcccttgga tgacagattt ggcaaagcac tttactttct gaatgacatg 9720

gccaagacca ggaaggacat tggggaatgg gagcactcag ccggcttttc aagctgggag 9780

gaggtaccct tttgttcaca ccatttccac gagctagtga tgaaggatgg acgcaccctg 9840

gtggtgccgt gccgagacca agatgaactc gttgggaggg cgcgcatctc accggggtgc 9900

ggctggagtg tccgcgagac ggcctgcctt tcaaaagcct acgggcagat gtggctgctg 9960

agctacttcc accgacgaga cctgaggacg ctcgggcttg ccattaactc agcagtgcct 10020

gccgattggg ttcctaccgg ccgcacgacg tggagcattc atgccagtgg ggcctggatg 10080

accacagaag acatgctgga cgtttggaac cgggtgtgga ttctggacaa ccctttcatg 10140

cagaacaagg aaagggtcat ggagtggagg gatgttccgt acctccctaa agctcaggac 10200

atgttatgtt cctcccttgt tgggaggaga gaaagagcag aatgggccaa gaacatctgg 10260

ggagcggtgg aaaaggtgag gaagatgata ggtcctgaaa agttcaagga ctatctctcc 10320

tgtatggacc gccatgacct gcactgggag ctcagactgg agagctcaat aatctaaacc 10380

cagaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 10440

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 10500

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaccccctg accagcaaag ggggcagacc 10560

ggtcaggggt gaggaatgcc cccagagtgc attacggcag cacgccagtg agagtggcga 10620

cgggaaaatg gtcgatcccg acgtagggca ctctgaaaaa ttttgtgaga ccccctgcat 10680

catgataagg ccgaacatgg tgcatgaaag gggaggcccc cggaagcacg cttccgggag 10740

gagggaagag agaaattggc agctctcttc aggatttttc ctcctcctat acaaaattcc 10800

ccctcggtag agggggggcg gttcttgttc tccctgagcc accatcaccc agacacaggt 10860

agtctgacaa ggaggtgatg tgtgactcgg aaaaacaccc gct 10903

Claims

1. Use of an oncolytic virus in the manufacture of a medicament for the treatment of a tumor, wherein,

The oncolytic virus is an attenuated West Nile virus, and the nucleotide sequence of the attenuated West Nile virus is shown as SEQ ID NO.1 by deleting the sequence from SLI to CS2 in the 3' untranslated region of the wild type West Nile virus, retaining the cyclized sequence region 1 of the 3' untranslated region and the short hairpin-3 ' end stem loop region after the cyclized sequence region 1, and inserting a poly (A) sequence into the deletion position caused by deleting the sequence from SLI to CS 2;

the sequence from SLI to CS2 in the 3 'untranslated region comprises a 5' end stem-loop region I, a stem-loop region II, a repeated cyclization sequence region 3, a stem-loop region III, a stem-loop region IV, a cyclization sequence region 3, a dumbbell region 1, a repeated cyclization sequence region 2, a dumbbell region 2 and a cyclization sequence region 2;

The poly (A) sequence has 130 adenylates;

the tumor is selected from melanoma, liver cancer, ovarian cancer, lung cancer, breast cancer, cervical cancer and glioma.

Use of DNA in the manufacture of a medicament for the treatment of a tumor, wherein said DNA is transcribed to produce an oncolytic virus,

The poly (A) sequence has 130 adenylates;

3. Use of a cell in the manufacture of a medicament for the treatment of a tumor, wherein the cell comprises an oncolytic virus or DNA,

The poly (A) sequence has 130 adenylates;

Said DNA is transcribed to produce said oncolytic virus;

4. Use of a composition in the manufacture of a medicament for treating a tumor, wherein the composition comprises an oncolytic virus, DNA and/or cells;

The poly (A) sequence has 130 adenylates;

Said DNA is transcribed to produce said oncolytic virus;

The cell comprises the oncolytic virus or the DNA;

the tumor comprises melanoma, liver cancer, ovarian cancer, lung cancer, breast cancer, cervical cancer and glioma.