CN113717953A

CN113717953A - Application of attenuated flavivirus in oncolytic

Info

Publication number: CN113717953A
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: 2021-11-30

Abstract

The present invention relates to an oncolytic virus comprising an attenuated flavivirus 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 flavivirus, such that the 3 'untranslated region (3' UTR) of the attenuated flavivirus obtained after the flavivirus is replaced with the part of the nucleotide sequence retains at least the 3 '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 small influence on normal cells; the tumor model of xenograft proves that the oncolytic virus can inhibit the growth of various tumor cells in vivo, and can provide a safe and efficient anticancer therapeutic agent with small toxic and side effects for the clinical tumor treatment.

Description

Application of attenuated flavivirus in oncolytic

Technical Field

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

Background

Tumors are the largest public health problem facing humans and the second leading killer to death. Malignant tumors have become one of the major public health problems that severely threaten the health of people in china and all over the world. The current treatments for tumors mainly comprise two types, one is the traditional treatment method comprising chemotherapy/radiotherapy/surgery treatment, and the other is the immunotherapy mainly based on cytokines/immune cells and antibodies. But both traditional and immunotherapy have therapeutic drawbacks. The biggest defect of the traditional treatment is drug resistance, and the biggest defect of the immunotherapy is low response rate. Therefore, new therapeutic approaches, such as tumor immunotherapy/oncolytic virus, etc., are gradually gaining 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 infect and replicate preferentially in tumor cells compared to normal cells, causing tumor cell lysis. In addition to direct oncolytic, oncolytic viruses can also act as immunogens to activate innate and adaptive anti-tumor immune responses, resulting in effective infiltration of immune cells, transforming a "cold" Tumor Microenvironment (TME) with few immune cells into a "hot" tumor microenvironment with many immune cells, thereby enhancing the ability of the body to recognize, control, and kill tumor cells. In recent years, oncolytic viruses have received increasing attention as the cancer field has moved towards biological and immune based approaches to combat cancer. At present, most researches achieve the oncolytic effect by modifying some virulent strains which exist in the nature and are weak in pathogenicity to enable the virulent strains to be specifically expressed and packaged in tumor cells. Compared with natural virus, the attenuated live vaccine with the oncolytic effect has the advantages of safety, controllability, easy production and the like. There are studies that show that the yellow fever vaccine YFV-17D replicates and kills mouse and human tumor cell lines, but does not kill untransformed human cells. The injection of active YFV-17D in tumor can obviously inhibit the CD8+ T cell dependent transplantation tumor process, thereby generating immune-mediated anti-tumor effect, and finally playing the anti-tumor effect in the form of oncolytic virus. Similarly, studies have shown that the interstitial poliovirus attenuated live vaccine Pvsripo has a significant role in the treatment of gliomas. Pvsripo virus specifically enters glioma cells via the poliovirus receptor CD155 and expands in tumor cells ultimately inducing lytic death of the tumor cells.

West Nile Virus (WNV) belongs to the family of Flaviviridae members of the genus flavivirus, and is also one of the most widely distributed flaviviruses worldwide, but it is only spread in a small area, and does not cause serious disease. WNV infection in humans is mostly manifested as a mild condition such as no symptoms or west nile fever.

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

Disclosure of Invention

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

In one aspect, the present invention provides an oncolytic virus that is an attenuated flavivirus 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 flavivirus, such that the 3 'untranslated region (3' UTR) of the attenuated flavivirus obtained after the flavivirus is replaced with the part of the nucleotide sequence retains at least the 3 'stem loop region (3' SL).

In one aspect, the invention provides a DNA which can be transcribed to produce said 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 cell.

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

The oncolytic virus can obviously infect and crack different cancer cells in an in vitro cell experiment, and does not cause toxic or side effect on normal cells. The results of in vivo animal experiments show 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 anti-cancer therapeutic agent with safety, high efficiency and small toxic and side effects for the clinical tumor treatment.

Drawings

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

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

Figure 3 shows the oncolytic effect of WNV-WT and WNV-poly (a) on tumor cells and normal cells, wherein: FIG. 3A shows the bright field observation of the oncolytic effects of WNV-WT and WNV-poly (A) on tumor cells as well as normal cells; FIG. 3B shows the detection of different doses of WNV-WT and WNV-poly (A) with CCK8 for oncolytic effects on tumor cells as well as normal cells; figures 3C-3F show the differences in replication of WNV-WT and WNV-poly (a) within normal cell MEF (figure 3C) and tumor cells B16F1 (figure 3D), a375 (figure 3E), Huh7 (figure 3F), respectively.

Figure 4 shows that WNV-poly (a) causes apoptosis of tumor cells Huh7, SKOV3, and B16F1, wherein: FIG. 4A shows the apoptotic effect of WNV-poly (A) on tumor cells Huh7, SKOV3, and B16F1 as detected by Annexin-V/PI; FIG. 4B shows WB detection of cleavage of Caspase3 within Huh7, SKOV3, tumor cells after WNV-poly (A) infection.

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

Figure 6 shows an in vivo assay for the oncolytic effect of WNV-poly (a) on melanoma administered once on 3 days; wherein: FIGS. 6A-E show in vivo assays for the oncolytic effect of multiple doses of WNV-poly (A) on mouse melanoma B16F1 tumors; FIGS. 6F-J show the in vivo detection of oncolytic effects of multiple doses of WNV-poly (A) on human melanoma A375 tumors.

Figure 7 shows WNV-poly (a) activates the tumor immune microenvironment; wherein: figure 7A shows the proportion of CD45+ cells in a tumor; figure 7B shows the proportion of individual immune cells in the tumor; FIG. 7C shows the ratio of individual immune cells in the spleen; figures 7D, 7E show activation of CD8+ T cells in tumors; figures 7F-H show the effect of CD8+ T cell clearance on WNV-poly (a) therapy.

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

Figure 9 shows the oncolytic effect of treatment of B16 tumors with a single administration of different doses of WNV-poly (a), wherein: FIG. 9A shows a photograph after a single treatment with different doses of virus; FIG. 9B shows a statistical plot of tumor weight after one treatment with different doses of virus; FIG. 9C is a graph showing the change in tumor volume and mouse body weight after one treatment with different doses of virus; figure 9D shows amplification of WNV-poly (a) within a tumor after one administration; FIGS. 9E-H show the immune response of the tumor and spleen after a single administration.

Figure 10 shows the broad spectrum of WNV-poly (a) oncolytic therapy, wherein: figure 10A shows a graph of a human liver cancer Huh7 model treatment strategy; FIGS. 10B-D show the oncolytic effect of WNV-poly (A) given toxin once in three days on Huh7 tumors detected in vivo; figure 10E shows Huh7 model treatment for mouse viral tissue tropism; figure 10F shows a plot of a human ovarian cancer SKOV3 model treatment strategy; FIGS. 10G-I show the oncolytic effect of WNV-poly (A) given virus once three days on SKOV3 tumors tested in vivo; figure 10J shows treatment of mouse viral tissue tropism by SKOV3 model.

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

Detailed Description

In the present disclosure, unless defined otherwise, scientific and technical terms used herein have the meanings that are commonly understood by those of skill in the art. Also, protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology related terms, and laboratory procedures used herein are all terms and conventional procedures used extensively in the relevant art. Meanwhile, for better understanding of the present disclosure, definitions and explanations of related terms are provided below.

I. Definition of

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

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 exhibits less or substantially no replication in non-cancer cells. In certain embodiments, less replication means at least about 30% greater, or at least about 50% greater, or at least about 80% greater replication in a cancer cell as compared to a corresponding non-cancer cell.

As used herein, the term "oncolytic" refers to a condition in which at least 10% of the proliferating cells are lysed (e.g., at least about 20%, 30% >, or less,40%, 50% or 75% of the cells were lysed). For example, the percentage of cell lysis can be determined by measuring the reduction in size of a neoplasm or the number of proliferating cells in a mammal or by measuring the amount of cell lysis in vitro (e.g., from a biopsy of proliferating cells). The effective amount of 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 aberrantly proliferating cells. For example, for the treatment of humans, about 10 is used depending on the type, size and number of proliferating cells or neoplasms present³To 10¹²Plaque Forming Units (PFU) of virus. An effective amount can be, for example, from about 1.0PFU/kg body weight to about 10¹⁵PFU/kg body weight (e.g., from about 10)²PFU/kg body weight to about 10¹³PFU/kg body weight). Optionally, the effective amount is about 1 × 10⁸To about 1X 10¹²TCID₅₀. Optionally, the effective amount is about 1 × 10¹⁰TCID₅₀。

As used herein, flaviviruses may include, but are not limited to, Japanese Encephalitis Virus (JEV), West Nile Virus (West Nile Virus, WNV), tick-borne encephalitis Virus (TBEV), Yellow Fever Virus (YFV), dengue Virus (dengue Virus, DENV), zika Virus (ZIKV), St Louis Encephalitis Virus (SLEV), Murray Valley Encephalitis Virus (MVEV), Omsk hemorrhagic fever Virus (ompk hemorrhagic fever Virus), and karsanol Forest Disease Virus (Kyasanur Forest Disease Virus) or any related flavivirus thereof.

As used herein, the 5 'stem loop region (SL), dumbbell region (DB), repeat and circularization sequence region 3(RCS3), circularization sequence region 3(CS3), repeat and circularization sequence region 2(RCS2), circularization sequence region 2(CS2), circularization sequence region 1(CS1), and short hairpin-3' stem loop region (sHP-3 'SL) following circularization sequence region 1 of the flavivirus 3' untranslated region are terms within the corresponding art. In other studies, the stem-loop region (SL) is also known 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 within, package, re-infect, or a combination thereof, a host. In general, such attenuation may be in several or all hosts of the virus, or only in one or several hosts of the virus. Thus, attenuation of a live attenuated virus (that is, its ability to infect, replicate within, package, re-infect a host, or a combination thereof, is attenuated) is typically associated with one or more hosts of the virus in which the live attenuated virus is not significantly or measurably attenuated in one or more other hosts of the virus. Live attenuated viruses disclosed herein are typically genetically altered, or may be referred to as mutated, mutant, genetically engineered, recombinant, or a combination.

As used herein, the term "live attenuated vaccine" or "attenuated vaccine" refers to a pharmaceutical composition comprising a live attenuated pathogen, e.g., a virus. The pharmaceutical composition comprises at least one immunologically active component that induces an immune response against the virus in the 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 immunological activity of the active component. The vaccine may additionally include other components typical of pharmaceutical compositions. At least one immunologically active component is one or more 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, resulting in a virus that is infectious or infective. "recombinant virus" refers to a genetically engineered virus produced by recombinant DNA technology, and the virus sequence can be artificially subjected to any genetic manipulation such as deletion, insertion, inversion, substitution, etc., so as to be distinguished from a naturally occurring virus.

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

As used herein, the term "wild-type," in the context of viruses, refers to the type of virus that is prevalent, naturally transmitted and undergoes a typical disease 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. 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 embodiments

In one aspect, the present disclosure provides an oncolytic virus comprising an attenuated flavivirus 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 flavivirus, such that the 3 'untranslated region (3' UTR) of the attenuated flavivirus obtained after the flavivirus is replaced with the part of the nucleotide sequence retains at least the 3 'stem loop region (3' SL).

The attenuated flavivirus of the preceding aspect, wherein said partial nucleotide sequence comprises all or part of a5 'stem-loop region (5' SL) in a3 'noncoding region (3' UTR), all or part of a circularizing sequence region (CS), and/or all or part of a dumbbell region (DB).

In some preferred embodiments of the present disclosure, the 3 ' untranslated region (3 ' UTR) of the attenuated flavivirus retains at least the cyclized sequence region 1(CS1) of the 3 ' untranslated region and the short hairpin-3 ' terminal stem-loop region (sHP-3 ' SL) following the cyclized 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 'terminal 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 (sliii), and stem-loop region iv (SLIV).

In some preferred embodiments of the present disclosure, the cyclized sequence region (CS) is selected from the group consisting of cyclized sequence region 1(CS1), cyclized sequence region 2(CS2), cyclized sequence region 3(CS3), repetitive cyclized sequence region 2(RCS2), and repetitive cyclized sequence region 3(RCS 3).

In some preferred embodiments of the present disclosure, the dumbbell zone (DB) is selected from dumbbell zone 1(DB1) and dumbbell zone 2(DB 2).

An attenuated flavivirus according to any of the preceding aspects, wherein the poly (a) sequence comprises 10-200 adenylates.

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

In some preferred embodiments of the present disclosure, the flavivirus is selected from the group consisting of Japanese Encephalitis Virus (JEV), West Nile Virus (West Nile Virus, WNV), tick-borne encephalitis Virus (TBEV), Yellow Fever Virus (YFV), dengue Virus (DENV), zaka Virus (zika Virus, ZIKV), st.

In some preferred embodiments of the present disclosure, the attenuated flavivirus plaque titer is 1 × 10⁵-1×10⁹PFU/ml; more preferably 5X 10⁴-5×10⁶PFU/ml。

In some preferred embodiments of the present disclosure, the attenuated flavivirus is 10-100 fold less virulent than the parental wild-type virus.

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

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

In some preferred embodiments of the present disclosure, the attenuated flaviviruses 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 set forth 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 flavivirus is an attenuated japanese encephalitis virus. In some preferred embodiments of the present disclosure, the attenuated japanese encephalitis virus comprises a nucleotide sequence having 80% or more identity to the nucleotide sequence shown in SEQ ID No.2, 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 attenuated japanese encephalitis virus has the nucleotide sequence of SEQ ID No. 2.

In some preferred embodiments of the present disclosure, the attenuated flavivirus is an attenuated dengue virus. In some preferred embodiments of the present disclosure, the attenuated dengue virus comprises a nucleotide sequence having 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 flavivirus is an attenuated yellow fever virus. In some preferred embodiments of the present disclosure, the attenuated yellow fever virus comprises a nucleotide sequence that is 80% or more identical to the nucleotide sequence set forth in SEQ ID No.4, preferably a nucleotide sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably a nucleotide sequence that is 98% or 99% or more identical. In a specific embodiment of the 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 Zika virus comprises a nucleotide sequence that is 80% or more identical to the nucleotide sequence set forth in SEQ ID No.5, preferably a nucleotide sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and 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 Zika virus is SEQ ID No. 5.

In some preferred embodiments of the present disclosure, the attenuated flavivirus 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 that is 80% or more identical to the nucleotide sequence set forth in SEQ ID No.6, preferably a nucleotide sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and 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 tick-borne encephalitis virus is SEQ ID No. 6.

In a preferred embodiment of the present disclosure, the partial nucleotide sequence includes a5 'stem-loop region i (sli), a stem-loop region ii (slii), a repeated loop region 3(RCS3), a stem-loop region iii (sliii), a stem-loop region IV (sliv), a loop region 3(CS3), and the 3' untranslated region (3 'UTR) of the attenuated flavivirus retains dumbbell region 1(DB1) of the 3' untranslated region, repeated loop region 2(RCS2), dumbbell region 2(DB2), loop region 2(CS2), loop region 1(CS1), and a short hairpin-3 'stem-loop region (sHP-3' SL) after loop region 1 after the loop region is replaced with a poly (a) sequence.

In a preferred embodiment of the present disclosure, the partial nucleotide sequence includes dumbbell region 1(DB1), repeated circularized sequence region 2(RCS2), dumbbell region 2(DB2), circularized sequence region 2(CS2), circularized sequence region 1(CS1), and the partial nucleotide sequence is replaced with a poly a (poly (a)) sequence, and the 3 ' untranslated region (3 ' UTR) of the attenuated flavivirus retains the 5 ' stem loop region i (sli), stem loop region ii (slii), repeated circularized sequence region 3(RCS3), stem loop region iii sli (ii), stem loop region iv (sliv), circularized sequence region 3(CS3), and short hairpin-3 ' stem loop region (sHP-3 ' SL).

In a preferred embodiment of the present disclosure, the partial nucleotide sequence includes a5 'stem-loop region i (sli), a stem-loop region ii (slii), a repeated-circularized sequence region 3(RCS3), a stem-loop region iii (sliii), a stem-loop region iv (sliv), a circularized sequence region 3(CS3), a dumbbell region 1(DB1), a repeated-circularized sequence region 2(RCS2), a dumbbell region (DB2), a circularized sequence region 2(CS2), and the 3' untranslated region (3 'UTR) of the attenuated flaviviruses retains a circularized sequence region 1(CS1) of the 3' untranslated region and a short hairpin-3 'stem-loop region (sHP-3' SL) after the circularized sequence region 1 after being replaced with a poly (a)) sequence.

In a preferred embodiment of the present disclosure, the partial nucleotide sequence includes a5 'stem-loop region ii (slii), a cyclized region 3(RCS3), a stem-loop region iv (sliv), a cyclized region 3(CS3), a dumbbell region 1(DB1), a cyclized region 2(RCS2), a dumbbell region (DB2), and a cyclized region 2(CS2), and the partial nucleotide sequence is replaced with a poly (a) sequence, and the 3' untranslated region (3 'UTR) of the attenuated flavivirus retains the cyclized region 1(CS1) of the 3' untranslated region and the short hairpin-3 'stem-loop region (sHP-3' SL) after the cyclized region 1.

In a preferred embodiment of the present disclosure, the partial nucleotide sequence includes a5 ' stem loop region ii (slii), a repeated cyclized sequence region 3(RCS3), a dumbbell region (DB), a repeated cyclized sequence region 2(RCS2), a dumbbell region (DB2), and a cyclized sequence region 2(CS2), and the 3 ' untranslated region (3 ' UTR) of the attenuated xanthovirus retains the 5 ' stem loop region (SL) of the 3 ' untranslated region, the cyclized sequence region 1(CS1), and the short hairpin-3 ' stem loop region (sHP-3 ' SL) after the cyclized 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 Factor (TNF), colony stimulating factor, proteins exposed to APC (antigen presenting cell), growth factors, Major Histocompatibility Complex (MHC) class I or II components, inducers or inhibitors of apoptosis, cytostatic agents, immunotoxins and blockers of the immune escape mechanism of 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 CXCL 11.

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

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 the 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 inducer or inhibitor of apoptosis is selected from Bax, Bcl2, and BcIX.

In some preferred embodiments of the present disclosure, the cytostatic agent is selected from 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 present 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 virus.

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 a use of the oncolytic virus, the DNA, the cell and/or the composition for 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 medicament may be administered in one or more doses.

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., agent of the embodiments) to be administered in which the therapeutic effect of the active agent outweighs 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 best therapeutic response.

In one embodiment, the compound (e.g., the live attenuated virus of an embodiment) may 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 can 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 with a needleless system (e.g., a syringe-based syringe-based syringe-based syringe-based syringe-based syringe-syringe

) Or other intradermal administration systems.

In certain embodiments, live attenuated virus vaccines (e.g., west nile virus vaccines can be administered to children or young adults in stable formulations (e.g., albumin and trehalose; FTA, or other formulations for stabilizing live attenuated viruses.) as used herein, "pharmaceutically acceptable carriers" can also include diluents, such as saline and aqueous buffer solutions. To prevent growth of microorganisms or other stabilizing formulations (e.g., for stabilizing live attenuated flaviviruses or other stabilizing formulations).

Pharmaceutical compositions suitable for injectable use may be administered by means known in the art. For example, sterile aqueous solutions (where water soluble) 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 syringeability. The pharmaceutically acceptable carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. For example, proper fluidity can be maintained, for example, by the use of coating materials 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 the addition of various antibacterial or antifungal agents or other agents.

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

In certain embodiments, a single or multiple dose of an attenuated flavivirus (e.g., west nile virus) formulation may also be administered to a subject. In some embodiments, a subject may be treated with a single dose formulation. In other embodiments, the subject may be treated with at least two doses of a live attenuated west nile virus formulation. In certain embodiments, the subject may be administered 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-immunized 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, seronegative subjects may be treated on day 0 and then receive boosters within 6 months, within 5 months, within 4 months, within 3 months, or less of the first dose, in order to generate an enhanced immune response to west nile virus. In certain embodiments, the child may be a child aged from about 2 years to about 17 years. In other embodiments, the child is between 2 and 17 years of age.

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 contain excipients such as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, 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 time 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 materials known.

In certain embodiments, the dose range of a live attenuated flavivirus (e.g., West Nile virus) may be about 10 of that initially administered²To about 10⁶PFU, and optionally, then at least a second administration for up to 12 months within or after 30 days as needed.

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 may 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 of the same day or simultaneously in the same or different anatomical locations. In some embodiments, the pharmaceutical compositions disclosed herein can 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 can be administered to a subject subcutaneously or intradermally. Administration in two or more anatomical sites may include any combination of administrations, including through the same pattern in two or more anatomical sites or through two different patterns including two separate anatomical sites. According to these embodiments, the two or more anatomical sites may comprise different limbs or different regions of the body. In certain embodiments, two doses of the vaccine composition can be continuously introduced into the subject at day 0 in the same or multiple anatomical locations, e.g., to provide protection from all flavivirus (e.g., west nile virus) serotypes (e.g., cross-protection). In other embodiments, the pharmaceutical composition can include a live attenuated west nile virus in combination with other immunogenic agents against other flaviviruses (e.g., zika virus, japanese encephalitis, west nile virus, st. In certain embodiments, the vaccines against west nile virus disclosed herein may be used to reduce other related viral infections, such as zika virus infection.

In some embodiments, the pharmaceutical compositions disclosed herein may 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 potentiates a CD8+ T cell response or other immune response against west nile virus in a child or young adult receiving such pharmaceutical compositions against a flavivirus (e.g., west nile virus) virus.

Topical application is accomplished by topically applying creams, gels, rinses, etc., containing a therapeutically effective amount of a serine protease inhibitor. Transdermal administration is accomplished by applying creams, irrigation solutions, gels, etc., which allow the serpin to penetrate the skin and enter the bloodstream. 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 molecules are 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 proteinaceous 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 a live attenuated virus.

In certain embodiments, the compositions contemplated herein may be partially or fully dehydrated or hydrated. In other embodiments, stable 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 albumins, or members of the albumin gene family. The sugar or polyol agent may include, but is not limited to, monosaccharides, disaccharides, sugar alcohols, trehalose, sucrose, maltose, isomaltose, cellobiose, gentiobiose, laminaribiose, xylobiose, mannobiose, lactose, fructose, sorbitol, mannitol, lactitol, xylitol, erythritol, raffinose, amylases, cyclodextrins, 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 sorbitan monolaurate, octyl glucoside and decyl maltoside), fatty alcohols (cetyl or oleyl alcohol) or cocamides (cocamides) (cocamide MEA, cocamide DEA and cocamide TEA).

In other embodiments, the surfactant can include, but is not limited to, poloxamer 407 (e.g., Pluronic)

) Replacement of poloxamer 407 or poloxamers 335, 338 or 238 other than poloxamer 407, or with poloxamer 407

Other 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 copolymer, poloxamer 407, or more specifically, Pluronic

In other embodiments, a formulation for stabilizing a live virus may comprise one or more live flaviviruses, one or more carbohydrate agents, and one or more amino acids or salts, esters, or amide derivatives thereof. In other embodiments, the formulations used herein stabilize live attenuated flaviviruses for commercial use. In some embodiments, the composition further comprises a buffering agent. 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 sodium chloride (NaCl), monosodium and/or disodium phosphate (Na2HPO4), potassium chloride (KCl) and potassium phosphate (KH)₂PO₄) At least one of (1). 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 MSG or other suitable agents.

In some embodiments, a live attenuated flavivirus, such as west nile virus, may be stabilized in a formulation including, 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 a mannitol 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 22 mM; wherein the methionine concentration is 1mM to 5 mM; wherein the MSG concentration is 8mM to 12 mM; wherein the urea concentration is 0.1% to 0.3% (w/v). Certain formulations for stabilizing live attenuated viruses 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); the sucrose concentration may be from about 4% to about 6% (w/v); alanine concentration can be from about 8.0mM to about 22 mM; 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 can be about 0.1% to 0.2% (w/v); the sucrose concentration may be from about 4.0% to about 6.0% (w/v); the methionine concentration may be about 1.0mM to about 5.0 mM; the urea concentration may be about 0.1% to about 0.3% (w/v). In other embodiments, the stabilizing formulation may comprise recombinant HSA, sucrose, arginine, and urea, wherein the concentration of recombinant HSA may be 0.1% to 0.2% (w/v); the sucrose concentration may be 4% to 6% (w/v); arginine concentration may be 10mM to 50 mM; 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 concentration of recombinant HSA is about 0.1% to 0.2% (w/v); trehalose concentration is about 4% to 6% (w/v); arginine concentration is about 10mM to 50 mM; the urea concentration is about 0.1% to 0.3% (w/v). In other embodiments, the stabilizing composition can include recombinant HSA, trehalose, MSG, and urea. According to these embodiments, the concentration of recombinant HSA may be from about 0.1% to about 0.2% (w/v); the trehalose concentration can be from about 4.0% to about 6.0% (w/v); wherein the MSG concentration is 8mM to 12 mM; 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 shipping 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 domestic animal or a wild animal. In certain embodiments, the immunogenic compositions of the present disclosure can be effective to immunize adolescent subjects, such as human children aged 20 years or less. Although the prior art relating to current dengue virus vaccines/immunogenic compositions for use by children (e.g., dengue/yellow fever chimeras) reports low potency and/or immunogenicity in children 9 years of age or younger, the immunogenic compositions disclosed herein can 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 older. The immunogenic compositions disclosed herein exhibit superior efficacy and immunogenicity as compared to the prior art.

The oncolytic virus can be amplified in BHK (BHK) cells or Vero cells, and the virus titer can reach (1-10) x 10⁷PFU。

Experiments prove that the oncolytic virus disclosed by the invention has cell killing activity on 12 tumor cell lines (including Huh7 (human liver cancer epithelioid cells), A549 (human lung epithelial cell cancer), 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 utilizes a mouse melanoma tumor-bearing model, a human melanoma nude mouse tumor-bearing model and a human hepatoma cell nude mouse tumor-bearing modelTumor model and nude mouse tumor-bearing model of human ovarian cancer, which verifies that the virus dosage of the oncolytic virus is 1 × 10⁷PFU has the function of dissolving tumor in animal body, which proves that the oncolytic virus has good tumor targeting property and anti-tumor effect.

Compared with the classical tumor treatment method, the oncolytic virus can infect and crack various tumor cells in an in vitro cell experiment, has small influence on normal cells, has good tumor tropism and can kill the tumor cells specifically. Can inhibit the in vivo tumor growth of melanoma, liver cancer and ovarian cancer in a xenograft tumor model, delay the tumor progression, and achieve consistent in vitro or in vivo experimental effect. On the other hand, the oncolytic virus of the present invention has better safety and reliability compared to other oncolytic virus therapies because it is an attenuated virus strain. The oncolytic virus can be efficiently replicated in Vero cells, has stable virus passage, and is easy to use and produce and apply in later period. The oncolytic virus is an RNA virus, and the cDNA of the oncolytic virus is easy to operate and is beneficial to later modification and application of a virus genome. The oncolytic virus provided by the invention has a great application prospect in tumor treatment as a novel oncolytic virus.

Examples

The experimental methods of virus infection, immunofluorescence, CCK8 cell survival detection, virus ultraviolet inactivation, virus titer determination, mouse in-vivo tumor-bearing model modeling, mouse in-vivo tumor administration, tumor tissue grinding, virus titration determination and the like, which are related in the part, adopt the conventional methods in the field if no special description exists. The following are merely illustrative of several embodiments of the present invention. It is obvious that the present invention is not limited to the following embodiments, but many variations are possible. Therefore, modifications and improvements based on the disclosure of the present invention should be made by those skilled in the art within the scope of the claims of the present invention.

Example 1: construction of flavivirus attenuated strain clone and virus rescue

1. Cloning construction of attenuated flavivirus strains

A schematic diagram of the construction of the attenuated flavivirus strain is shown in FIG. 1.

With plasmid DNA of infectious clone of Wild Type (WT) West Nile Virus (WNV) strain as backbone, the sequence from SLI to CS2 (including 5 'stem loop region I (SLI), stem loop region II (SLII), repeated cyclized sequence region 3(RCS3), stem loop region III (SLIII), stem loop region IV (SL IV), cyclized sequence region 3(CS3), 35 1(DB 25), repeated cyclized sequence region 2(RCS2), dumbbell region (DB2), cyclized sequence region 2(CS2)) in 3' untranslated region (3 'UTR) was deleted, the cyclized sequence region 1(CS1) of 3' untranslated region and short hairpin-3 'stem loop region (sHP-3' SL) after cyclized sequence region 1 were retained, and poly A sequence (130-A) was inserted into deletion site caused by deletion of sequence from SLI to CS2 to obtain attenuated strain of WNV (130-poly A) plasmid (WNV-A), the nucleotide sequence is shown in SEQ ID NO. 1.

With plasmid DNA of infectious clone of Wild Type (WT) Japanese Encephalitis Virus (JEV) strain as a backbone, sequences from SLI to CS2 (including SLI, SLII, RCS3, SLIII, SLIV, CS3, DB1, RCS2, DB2, CS2) in the 3 ' untranslated region (3 ' UTR) of JEV were deleted, the cyclized sequence region 1(CS1) of the 3 ' untranslated region and the short hairpin-3 ' terminal stem loop region (sHP-3 ' SL) after the cyclized sequence region 1 were retained, and a poly (A) sequence (150 adenylic acids) was inserted at the deletion site due to deletion of the sequence from SLI to CS2 to obtain an attenuated strain plasmid of JEV-poly (A) virus having the nucleotide sequence shown in SEQ ID No. 2.

With plasmid DNA of infectious clone of a wild-type (WT) dengue virus type 2 New Guinei island strain (DENV-2(NGC)) strain as a backbone, sequences from SLII to CS2 (including SLII, RCS3, SL IV, CS3, DB1, RCS2, DB2, CS2) in the 3 ' untranslated region (3 ' UTR) of DENV-2(NGC) were deleted, the cyclized sequence region 1 of the 3 ' untranslated region (CS1) and the short hairpin-3 ' terminal stem loop region after the cyclized sequence region 1 (sHP-3 ' SL) were retained, and a poly (A) sequence (140 adenylic acids) was inserted at the deletion site resulting from deletion of the sequence from SLI to CS2 to give an attenuated strain plasmid of DENV-2(NGC) -poly (A) virus having the nucleotide sequence shown in SEQ ID No. 3.

With plasmid DNA of infectious clone of Wild Type (WT) Yellow Fever Virus (YFV) strain as a backbone, the sequence from SLII to CS2 (including SLII, RCS3, DB) in the 3 ' untranslated region (3 ' UTR) of YFV was deleted, the stem-loop region (SL) of the 3 ' untranslated region, the cyclized sequence region 1(CS1), and the short hairpin-3 ' terminal stem-loop region (sHP-3 ' SL) after the cyclized sequence region 1 were retained, and a poly (A) sequence (130 adenylic acids) was inserted at the deletion site resulting from deletion of the sequence from SLI to CS2, to obtain a V-poly (YF) virus attenuated strain plasmid having the nucleotide sequence shown in SEQ ID NO. 4.

All the strain plasmids replace all or part of 5 ' terminal stem loop region (SL) sequences and all DB sequences in 3 ' UTR regions by poly (A) sequences, at least a cyclized sequence region 1(CS1) of a3 ' untranslated region and a short hairpin-3 ' terminal stem loop region (sHP-3 ' SL) after the cyclized sequence region 1 are reserved, and corresponding poly (A) virus attenuated strain plasmids are obtained respectively.

In vitro transcription of RNA

Linearizing the poly (A) virus attenuated strain plasmid obtained in the step 1, identifying the complete linearization through 0.8% agarose gel electrophoresis, performing phenol chloroform extraction, finally adding 11 mu L of RNAase-free water for dissolving, determining the concentration of DNA by using Thermo Scientific NanoDrop 2000 and detecting the quality of the DNA through electrophoresis. Taking 1 mu g of phenol chloroform extracted DNA as a template, and using an in vitro transcription kit T7 mMESSAGE mMACHINE kit (Ambion), carrying out in vitro transcription according to kit instructions to obtain recombinant poly (A) RNA, namely genomic RNA of WNV-poly (A), JEV-poly (A), DENV-2(NGC) -poly (A) and YFV-poly (A) virus attenuated strains. RNA concentration was determined using Thermo Scientific NanoDrop 2000 and RNA quality was checked using 0.8% freshly prepared agarose gel electrophoresis and stored at-80 ℃ until use.

Poly (A) Virus rescue

The experiments were divided into the following two groups:

(1) experimental groups: 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 lipofection as a positive control.

The specific experimental steps are as follows: the day before transfection, 2X 105 BHK-21 cells were seeded into 35mm cell culture dishes, three 10mm X10 mm coverslips per dish to bring the cells to about 80% on the day of transfection; when transfection is carried out, the medium in the culture dish is discarded, 1ml of Opti-MEM is used for washing once, and then 1ml of Opti-MEM is added (cells are in an infiltration state); adding 1ml of Opti-MEM into a 1.5ml EP tube, adding 4. mu.l of DMRIE-C (mixing DMRIE-C before use) to mix by reversing the top and the bottom, then respectively adding 1. mu.g of recombinant poly (A) RNA obtained by in vitro transcription into an experimental group, adding 1. mu.g of wild type viral RNA into a control group, and reversing the top and the bottom to mix; quickly abandoning the Opti-MEM in the culture dish, and adding the mixture into the dish (the action is light, and the mixture is not blown against the cells); after 4 hours of incubation at 37 ℃ in a CO2 incubator, the cultures were discarded and 2mL of DMEM medium containing 2% FBS was added. Observing the cell states of the experimental group and the control group under a microscope, taking a slide respectively 24, 48 and 72 hours after transfection, fixing the cells by using 5% acetone fixing solution (purchased from national drug group chemical reagent company), fixing for 15 minutes at room temperature, washing for three times by using PBS, storing at 4 ℃, collecting supernatant as poly (A) P0 generation virus of attenuated flavivirus when the IFA detection positive rate is high, and obtaining 4P 0 generation WNV-poly (A), JEV-poly (A), DENV-2(NGC) -poly (A) and YFV-poly (A) attenuated strains, and storing at-80 ℃.

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

The glass slide preserved at 4 ℃ in the step 3 is incubated with a primary antibody, 4G2 monoclonal antibody (flavivirus E protein universal antibody) diluted 1: 500 times of flavivirus attenuated strain antibody at room temperature. Incubating the primary antibody for 1-2 h at room temperature, washing the secondary antibody for 10 times by PBS, and incubating the secondary antibody at room temperature in a dark place, wherein the antibody is a coupled Fluorescein Isothiocyanate (FITC) goat anti-mouse antibody diluted by 1: 125 times. After incubation for 1h with secondary antibody, the slides were washed 10 times with PBS, labeled, and 95% glycerol was spotted at each label, the coverslip was placed cell side down on glycerol drops and observed under a fluorescence microscope at 200 x magnification (figure 1).

FIG. 1 shows that WNV-poly (A), JEV-poly (A), DENV-2(NGC) -poly (A) and YFV-poly (A) attenuated strains have a lower positive rate than WT at each time point, but the number of positive cells gradually increases with the culture time after transfection, which is consistent with the trend of WT, indicating that WNV-poly (A), JEV-poly (A), DENV-2(NGC) -poly (A) and YFV-poly (A) rescue infectious virus particles.

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

Subsequent experiments further investigated the oncolytic effect of attenuated strains of flavivirus on tumor cells using WNV-poly (A) virus.

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

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

control group (MOCK): a tumor cell group cultured in a medium without the addition of a virus;

experimental groups: a group of tumor cells cultured in a medium supplemented with WNV-poly (A).

1. The oncolytic effect of WNV-poly (A) on tumor cells and normal cells is observed in bright field

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 cells are respectively prepared according to the ratio of 8 multiplied by 10⁴The cells/well are plated in 12-well cell culture plates in DMEM or 1640 medium supplemented with 10% serum (FBS) supplemented with 1% penicillin and streptomycin at 37 deg.C and 5% CO₂Culturing in a humidifying incubator. After adherent growth of the cells, the medium was changed to DMEM medium with 2% FBS, and the death of tumor cells was observed in the open field at a dose of WNV-poly (a) with MOI 10 after 72 hours of infection.

The results showed that WNV-poly (A) clearly induced tumor cell death or significant inhibition of growth in all 12 tumor cells tested, but had no significant effect on 3 normal cells tested (FIG. 2A). The above results initially show that WNV-poly (A) has a broad-spectrum oncolytic effect in vitro experiments, and has almost no damage to normal cells.

CCK8 testing the oncolytic Effect of different doses of WNV-poly (A) on tumor cells

In order to further quantitatively verify the in vitro oncolytic effect of WNV-poly (A), a CCK8 detection kit is used for detecting the oncolytic effect of the virus on tumor cells after infection of different MOI doses.

The test method comprises the following steps: respectively inoculating tumor cells Huh7, A549, SKOV3, B16F1, 4T1 and HELA and normal cells MLE-12, MEF and L-02 into a 96-well cell culture plate according to 4000 cells/well, wherein after the cells are grown in an adherent manner, the MOI is respectively as follows: 0. doses of 0.001, 0.01, 0.1, 1, 10 infected with WNV-poly (a), 5 wells per group. After 72 hours, the activity of the cells was measured using the CCK8 test kit. After being added into cells, CCK8 can be reduced by dehydrogenase in mitochondria to generate highly water-soluble orange-yellow formazan products, the number of living cells is indirectly reflected by the OD value read at the detection wavelength 450 of an enzyme-labeling instrument, and the OD value is in direct proportion to the cell proliferation condition and in inverse proportion to cytotoxicity. Each group of data was blank with equal volume of DMEM medium without cells and virus plus CCK8, and control without virus (MOI ═ 0), and the formula ═ plus virus-blank)/(control-blank) × 100 was calculated. Results mean and standard deviation were calculated by GraphPadPrism7 software. Results are plotted in the detection results of the cell activity of WNV-poly (A) with different infection doses on various tumor cells and normal cells.

As shown in FIG. 2B, WNV-poly (A) had significant killing effect on the 6 tumor cells tested, but had no significant effect on the survival of the 3 normal cells tested. And further observing the effect of different MOIs, the WNV-poly (A) shows obvious virus infection dose dependence 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 confirms that WNV-poly (a) has a significant and broad-spectrum oncolytic effect in vitro experiments with little damage to normal cells.

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

This example shows that after tumor cells were infected with WNV-poly (A) virus and WNV-poly (A) virus (WNV-poly (A)) prepared in example 1 and a fixed MOI dose of wild-type West Nile virus (WNV-WT was produced by transfection of WNV infectious clone 3356 strain), their killing effect on normal and tumor cells was observed in the open field (FIG. 3A), and the difference in killing on normal and tumor cells was determined by detecting the killing on normal and tumor cells with different doses of WNV-WT and WNV-poly (A) using CCK8, and further the specificity of killing on tumor cells by WNV-poly (A) was determined by comparing the growth curves on normal and tumor cells of WNV-WT and WNV-poly (A) (FIG. 3B). Wherein:

experimental groups: a group of tumor cells cultured in a medium supplemented with WNV-WT or WNV-poly (A).

1. Bright field observation of the oncolytic effects of WNV-WT and WNV-poly (A) on normal and tumor cells

Respectively mixing 3 tumor cells B16F1, Huh7 and SKOV3 and normal cells MLE-12, MEF and L-02 according to 8 × 10⁴The cells/well were plated in 12-well cell culture plates in 10% serum (FBS) DMEM or 1640 medium supplemented with 1% penicillin and streptomycin at 37 deg.C and 5% CO₂Culturing in a humidifying incubator. After adherent growth of the cells, the culture medium was changed to 2% FBS DMEM, and WNV-WT and WNV-poly (a) were infected with MOI 10, and death of normal cells and tumor cells was observed in the open field 72 hours after infection.

The results show that WNV-WT and WNV-poly (A) were clearly killed against all 3 tumor cells tested, but WNV-WT induced significant cell death in all 3 normal cells, MLE-12, MEF and L-02, while WNV-poly (A) had no killing effect on these 3 cells (FIG. 3A). The above results preliminarily indicate that relative WNV-WT and WNV-poly (A) have oncolytic property to tumor cells. WNV-WT has a killing effect on normal cells, while 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 testing for different doses of WNV-WT and WNV-poly (A) killing of normal and tumor cells:

in order to further quantitatively verify the oncolytic difference of WNV-WT and WNV-poly (A) on tumor cells and normal cells, a CCK8 detection kit is used for detecting the oncolytic effect of the virus on each cell after infection with different MOI doses.

The test method comprises the following steps: respectively inoculating Huh7, SKOV3, B16F1 and normal cells MLE-12, MEF and L-02 into a 96-well cell culture plate according to 4000 cells/well, wherein after the cells grow adherently, according to MOI: 0. doses of 0.001, 0.01, 0.1, 1, 10 infected 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 infection dose-dependent killing of 3 tumor cells, and that WNV-WT had a more pronounced killing effect than WNV-poly (A) (FIG. 3B). From the results of 3 normal cells, WNV-WT had significant killing on all 3 normal cells tested, and the killing was significantly dependent on the virus infection dose, 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.

WNV-WT and WNV-poly (A) replication differential assay in normal MEF and tumor cells Huh7, A375, B16F1

The experiment was carried out by inoculating 2X 10 cells in 35mm cell culture dishes⁵Tumor cells, 5% CO at 37 ℃₂Under the culture condition, when the confluence reaches 60%, adding 500 μ l of diluted WNV-WT and WNV-poly (A) into a small dish according to the MOI of 10, 37 ℃ and 5% CO₂Adsorbing in incubator for 2 hr, discarding virus solution, adding 2ml DMEM medium containing 2% fetal calf serum into each well, and culturing at 37 deg.C with 5% CO₂Culturing under the culture condition, collecting 300 μ l virus supernatant every 24h after infection and supplementing 300 μ l DMEM medium containing 2% fetal calf serum as virus sample at different time points, and storing at-80 deg.C. Performing a plaque experiment on the virus samples at different time points to identify plaque morphology and determine a growth curve of the plaque: in 24-well cell culture platesEach well was inoculated with 1X 10 of the seed⁵For BHK-21 cells, when the confluency of the cells reached 90%, the medium in the wells was discarded, 100. mu.L of the virus collected at the above different time points diluted 10-fold with DMEM medium containing 2% FBS was added, and the mixture was adsorbed at 37 ℃ for 1 hour and sufficiently shaken every 15 minutes. After completion of the adsorption, the virus solution in each well was aspirated off, and DMEM medium containing 2% FBS and a 2% methylcellulose cover were added thereto at 37 ℃ and 5% CO₂After plaque is formed, staining the plaque with staining solution containing 1% crystal violet and 3.7% formaldehyde, treating the plaque at room temperature for 30min, taking out the staining solution in the hole, washing the bottom of the hole with running water, drying the staining solution, counting the staining solution, converting the staining solution into virus titer, and drawing a growth curve. The results indicated that WNV-WT and WNV-poly (A) amplified well on B16F1 (FIG. 3C), A375 (FIG. 3D), Huh7 (FIG. 3E) cells, but WNV-WT amplified significantly better on normal MEF cells than WNV-poly (A) (FIG. 3F).

Example 4: tumor cell apoptosis caused by attenuated strain of West Nile virus

In the embodiment, the killing mode of WNV-poly (A) on tumor cells Huh7, SKOV3 and B16F1 is further determined by Anexin-V/PI staining and WB detection of shearing of intracellular Caspase 3. Wherein:

1. Detection of the apoptotic Effect of WNV-poly (A) on Huh7, SKOV3 and B16F1 by Anexin-V/PI

Tumor cells Huh7, SKOV3 and B16F1 are respectively prepared according to 8 x 10⁴Cells/well were plated in 12-well cell culture plates and cultured overnight in 10% serum (FBS) DMEM or 1640 medium. After adherent growth of the cells, the medium was changed to DMEM medium with 2% FBS, and apoptosis of tumor cells was determined by Anexin-V/PI staining 48 hours after infection at MOI ═ 10 dose.

The experimental method is as follows: infected tumor cells were digested with EDTA-free trypsin and collected into EP tubes. Cells were resuspended in 200. mu.l of annexin V binding buffer. Add 5. mu.l Annexin-V solution to each tube, incubate 15min at 4 ℃ in the dark. Then 5. mu.l of 20. mu.g/ml PI solution was added, mixed gently and incubated at 4 ℃ for 15min in the absence of light. The cells were transferred to a flow tube and tested on the machine. The single Annexin V staining represents that the cell undergoes early apoptosis, the double Annexin V/PI staining represents that the cell undergoes late apoptosis, and the single PI positive represents that the cell undergoes obvious necrosis. The results show that WNV-poly (A) can remarkably 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 and B16F1 are respectively mixed according to the proportion of 2 x 10⁵Cells/well were plated in 12-well cell culture plates, the medium was changed to DMEM with 2% FBS after adherent growth of the cells, and the cells were harvested at a MOI of 10 doses for 24h and 48h of infection, respectively.

The experimental method is as follows: adding 200 μ l of lysis solution containing RIPA to each well, collecting cells by cell scraping, placing in a new EP tube, incubating for 30min on ice, adding 50 μ l of 5 Xloading buffer solution (loadingbuffer), mixing, heating at 100 deg.C for 10min to fully denature, centrifuging at 12000rpm for 2min at 4 deg.C, removing precipitate, subjecting the obtained supernatant to Westem-blot to detect apoptosis-related protein change level, and using beta-actin as control internal reference. The results show that Huh7, SKOV3 showed significant Caspase3 cleavage at both 24h and 48h tested (FIG. 4B). The results of the above experiments demonstrate that WNV-poly (A) is able to cause significant apoptosis of Huh7 and SKOV3, whereas the killer form responsible for B16F1 cell death is not apoptotic. The mechanism by which WNV-poly (A) causes tumor cell oncolysis may be other than apoptosis.

Example 5: inactivation of the oncolytic effects of WNV-poly (A) on tumor cells

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

Tumor cells Huh7 and SKOV3 are arranged according to 2 x 10⁵The cells/well were plated in 12-well cell culture plates, the culture medium was changed to 2% FBS DMEM after adherent growth of the cells, and the MOI was 10UV-WNV-poly (A) and WNV-poly (A) doses were usedStaining with staining solution containing 1% crystal violet and 3.7% formaldehyde after 48 hours of infection, treating at room temperature for 30min, taking out the staining solution in the hole, washing the bottom of the hole with running water, drying, and observing the effect of the UV inactivated virus on tumor cells.

The experimental results show that the results after UV-WNV-poly (A) infection are consistent with those of the MOCK group, the cells grow normally, the crystal violet staining is complete, no virus plaques appear, tumor cells die obviously after WNV-poly (A) infection, and the crystal violet staining shows that the cells are basically free of living cells (figure 5). This suggests that UV-inactivated UV-WNV-poly (A) loses its oncolytic effect on tumor cells compared to WNV-poly (A) infected tumor cells.

Example 6: in vivo detection of oncolytic effect of WNV-poly (A) on melanoma after multiple administration of toxin

In this example, 2 melanoma cell lines, namely mouse melanoma cell B16F1 and human melanoma cell a375, were selected to establish an in vivo transplanted tumor model, and WNV-poly (a) was observed for the therapeutic effect on in vivo transplanted tumors after 3 days of intratumoral toxication. And further detecting the immune response condition in tumors and systems by flow. Wherein:

effect of WNV-poly (A) on tumors in tumor-bearing model mice

Control group (MOCK): 2% FBS DMEM medium without virus was administered intratumorally;

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

The implementation method comprises the following steps: 6-8 week old C57 mice were selected and inoculated subcutaneously with 2X 10⁵Individual mice, melanoma cells B16F1, were divided into two groups, one group was given to each containing 1 x 10 melanoma cells until they were able to palpate the graft tumor⁷PFU WNV-poly (a) 2% FBS DMEM (as experimental group), one group was given an equal volume of 2% FBS DMEM (as control group), treated by direct injection onto tumor bodies, given poisoned once every 3 days, and tumor volume and mouse body weight were measured, mice were sacrificed after four consecutive poisonings, tumor tissue was collected, and weighed. The tumor volume was calculated as (length × width 2)/2. The treatment results of B16F1 mouse melanoma are shown in FIG. 6, and the tumor morphology results visually indicate that WNV-poly (A) has significant treatment effect on B16F1 tumor(FIG. 6A), again, tumor volume reduction (FIG. 6B) and tumor weight loss (FIG. 6C) also indicate that WNV-poly (A) significantly inhibited tumor growth. And as shown in figure 6D, the weight change of mice was not affected after WNV-poly (a) treatment. Further, the tissue virus titer was measured by plaque, and the results showed that no virus was detected in the tumor tissue, brain tissue, and spleen tissue and serum tested (FIG. 6E).

Similar to the establishment of the in vivo transplantation tumor model of the mouse melanoma cell B16F1, the in vivo transplantation tumor model of the human melanoma cell A375 is also constructed. 4-5 weeks old Nude Bab/c mice were selected, subcutaneously inoculated with 1X 10⁷Individual melanoma cells a375, until graft tumors were palpable, mice were divided into two groups, one group administered at 1 x 10 per individual⁷PFU WNV-poly (a), one group was given an equal volume of 2% FBS DMEM, directly injected on tumor bodies for treatment, given once every 3 days, and tumor volume and mouse weight were measured, mice were sacrificed after four consecutive administrations, tumor tissue was collected and weighed. As a result, WNV-poly (A) was found to have a significant inhibitory effect on human melanoma tumor A375, and it did not affect the body weight change of mice, but virus could be detected in tumor tissues (FIGS. 6F-J).

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

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

The effect of WNV-poly (A) on the tumor immune microenvironment was examined by flow assay.

The experiments of FIGS. 7A-7E are divided into the following two groups:

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

control group (MOCK): intratumorally to the virus-free PBS group;

WNV-poly (A) + PBS set: intratumorally administering WNV-poly (A) virome, virus diluted with PBS;

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

WNV-poly (A) + Anti-CD4 set: intratumorally administering antibodies to WNV-poly (A) virus that deplete CD4+ T cells;

WNV-poly (A) + Anti-CD8 set: intratumorally WNV-poly (A) virus was given antibodies that cleared CD8+ T cells.

Tumor tissue and spleen tissue were harvested after sacrifice of mice at the treatment end. For the treatment of mouse spleen tissue, the spleen is picked up, PBS is added, a 200-mesh screen is used for grinding, 1200rpm is adopted, centrifugation is carried out for 5 minutes, the supernatant is discarded, 3ml of erythrocyte lysate is added into the obtained cell sediment, the cell sediment is kept stand at 4 ℃ for 10 minutes, then 20ml of PBS is added for washing, the supernatant is discarded after centrifugation, and the tissue protein after lysis is picked out by a gun tip. Adding 1ml PBS to suspend the cells, dividing the cells into tubes according to 100 mul/tube, adding isotype control antibody to block for 30min, adding various fluorescence labeled antibodies after blocking, incubating for 1h at 4 ℃, washing with PBS, and putting the cells on a machine for detection. For the treatment of mouse tumor tissue, the tumor tissue is cut into small pieces, PBS is added into the small pieces, the small pieces are ground by a 200-mesh screen, the obtained grinding fluid is centrifuged at 700rpm for 1min, the supernatant is collected and centrifuged at 1200rpm for 10min, the obtained cell sediment is added with erythrocyte lysate, the obtained cell sediment is kept stand at 4 ℃ for 10min and then washed once by 20ml of PBS, the supernatant is discarded after the centrifugation, and the cleaved tissue protein is picked out by a gun tip. Adding 1ml PBS to suspend the cells, dividing the cells into tubes according to 100 mul/tube, adding isotype control antibody to block for 30min, adding various fluorescence labeled antibodies after blocking, incubating for 1h at 4 ℃, washing with PBS, and putting the cells on a machine for detection.

Flow-through results showed a significant increase in the proportion of CD45+ immune cells in the tumor microenvironment of the WNV-poly (a) -treated group compared to the control group (MOCK) (fig. 7A), and further analysis of the proportion of each immune cell revealed a significant increase in the proportion of CD8+ T cells and a significant decrease in the proportion of regulatory T cells (Treg cells) (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 revealed a significant increase in both effector CD8 and memory CD8 in the tumor, with no change in the spleen (fig. 7D, E).

To further determine the role of CD8 in WNV-poly (a) oncolytic therapy, CD8+ T cell depletion experiments were performed with Anti-CD8 antibody (Anti-CD 8). Meanwhile, CD4+ T cells and NK cells were cleared with Anti-CD4 antibody (Anti-CD4) and Anti-NK antibody (Anti-NK), respectively, and used as control experiment groups for CD8+ T cell clearing. And simultaneously comparing a control group (MOCK) which is not detoxified only with PBS (as a negative control group) and a detoxified group (as a positive control group) which is diluted with PBS. Experimental results showed that the oncolytic effect of WNV-poly (A) was significantly reduced after CD8+ T cell clearance, but the oncolytic effect of WNV-poly (A) was not completely counteracted by CD8+ T cell clearance, and the control group had no effect on the treatment of WNV-poly (A) by CD4+ T cell clearance and NK cell clearance (FIGS. 7F-H). The results indicate that WNV-poly (A) can obviously induce the activation of CD8+ T cells in tumors and organisms, and further clearing experiments indicate that the therapeutic effect of WNV-poly (A) is partially dependent on CD8+ T cells.

Example 7: effect of bilateral tumor-bearing unilateral WNV-poly (A) administration on oncolytic effects

This example determines whether WNV-poly (A) is effective against distant tumors by a model of bilateral tumor-bearing unilateral toxicity in vivo of mouse melanoma cell B16F1 to further clarify the role of immune response in WNV-poly (A) treatment. Wherein:

control group (MOCK): intratumorally administering a virus-free medium;

experimental groups: WNV-poly (A) -containing medium was administered intratumorally.

The implementation method comprises the following steps: selecting 6-8 week old C57 mouse, inoculating 2X 10 axilla on left side⁵Individual murine melanoma cells, B16F1, were inoculated 5 x 10 to the right underarm⁴Cells, to the left where transplantable tumors could be palpated, mice were divided into two groups, one for treatment on the left, one containing 1X 10 cells each⁷PFU WNV-poly (a) in 2% FBS DMEM medium, one group given an equal volume of 2% FBS DMEM medium, directly injected on tumor bodies, given the virus once every 3 days, and measured tumor volume and mouse body weight, mice were sacrificed after four consecutive doses, tumor tissue was collected and weighed (fig. 8A). The tumor volume was calculated as (length × width 2)/2.

The results of the treatment are shown in fig. 8, and the results of the tumor morphology visually indicate that WNV-poly (a) has significant therapeutic effects on both tumor on the toxic side and tumor on the distal side of B16F1 (fig. 8B). Similarly, tumor volume reduction (fig. 8C) and tumor weight loss (fig. 8D) also suggested that WNV-poly (a) significantly inhibited the growth of distal tumors. And further plaque detection. This result indicates that WNV-poly (A) has significant therapeutic effect on distal tumors and no virus is detected in tumor tissues, suggesting that the therapeutic effect of WNV-poly (A) on distal tissues depends mainly on the immune response it activates.

Example 8: oncolytic effect of virus on tumor cells

Previous experimental results observed that WNV-poly (a) treatment effectively reversed the tumor immunosuppressive microenvironment and significantly stimulated activation of CD8+ T cells, further clearing CD8+ T cells found that their oncolytic effects were indeed diminished, but did not completely counteract the oncolytic effects of WNV-poly (a). This suggests that other oncolytic mechanisms besides immune responses exist in WNV-poly (A) therapy. Research and study literature finds that in the oncolytic treatment of viruses, in addition to the effective tumor suppression of the immune response activated by the virus, the killing of tumor cells by the virus itself also exerts a key oncolytic effect.

To determine the oncolytic effect of WNV-poly (A) itself, this example investigated whether the killing of tumor cells by the virus itself in addition to the immune response during WNV-poly (A) treatment would be effective in oncolytic by comparing the mode of administration of the virus only once. Wherein:

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

control group (MOCK): 2% FBS DMEM medium was given intratumorally;

inactivating the virus group: intratumoral administration of UV-inactivated WNV-poly (A) virus 1X 10⁷ PFU 2% FBS DMEM medium;

WNV-poly(A)-1×10⁷group (2): intratumoral administration of WNV-poly (A) virus 1X 10⁷ PFU 2% FBS DMEM medium;

WNV-poly(A)-1×10⁵group (2): intratumoral administration of WNV-poly (A) virus 1X 10⁵ PFU 2% FBS DMEM medium;

WNV-poly(A)-1×10³group (2): intratumoral administration of WNV-poly (A) virus 1X 10³ PFU 2% FBS DMEM medium;

this example examined the oncolytic effect after a single administration of different doses of WNV-poly (A), and found that WNV-poly (A) is dose-dependent, 1X 10⁷PFU effectively inhibits tumor growth, 1X 10⁵PFU partial tumor effective partial tumor ineffective, 1X 10³PFU virus had no therapeutic effect (FIGS. 9A-C).

Next, this example examined a 1X 10 dose of the drug⁷The results of the viral load in tumors on different days after PFU WNV-poly (A) revealed that the intratumoral viral titer increased 10-fold after three days compared to one day after the administration, and then the tumor viral titers were detected 5 days, 7 days, and 9 days, 11 days, and 14 days after the administration, respectively, and the viral load was gradually decreased until no virus was detected for 11 days (FIG. 9D).

The experiments of FIGS. 9E-9H were divided into the following two groups:

experimental groups: 2% FBS DMEM medium supplemented with WNV-poly (A) intratumorally

To further determine whether the immune system is functioning in a single administration, a single administration of 1X 10 was tested⁷The immune response in the tumor and spleen after PFU WNV-poly (A) resulted in no activation of immune cells in both tumor and spleen, further demonstrating that WNV-poly (A) can exert its own oncolytic effect to effectively inhibit tumor growth without activating immune response (FIG. 9E-H). This result indicates that WNV-poly (A) still has a significant inhibitory effect on melanoma after being administered once, and that the virus replicates in tumor tissues after administration.

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

The present example verifies the oncolytic broad spectrum of WNV-poly (A) by detecting the in vivo oncolytic effect of WNV-poly (A) on human hepatoma cell Huh7 and human ovarian carcinoma cell SKOV 3.

The experiment is divided into the following two groups:

Experimental procedures As shown in FIGS. 10A and 10F, 4-5 week-old Nude Bab/c mice were selected and inoculated subcutaneously with 2X 10⁶Personal hepatoma cell Huh7 or 3X 10⁶Individual ovarian carcinoma cells SKOV3, until the graft tumor was palpable, mice were divided into two groups, one group given to 1 x 10 cells each⁷PFU WNV-poly (a), one group was given an equal volume of 2% FBS DMEM, directly injected on tumor bodies for treatment, given once every 3 days, and tumor volume and mouse weight were measured, mice were sacrificed after four consecutive administrations, tumor tissue was collected and weighed.

The results show that WNV-polv (A) has obvious inhibition effect on human hepatoma cell Huh7 (figure 10B-D) and human ovarian cancer cell SKOV3 (figure 10G-I). And further analysis of tissue tropism of the virus revealed that in the human hepatoma cell Huh7 (fig. 10E) and human ovarian carcinoma cell SKOV3 (fig. 10J) models, the virus could be detected only in tumor tissues in each tissue. The results show that WNV-poly (A) has obvious oncolytic effect on liver cancer and ovarian cancer besides melanoma. This further illustrates the potential broad-spectrum tumor therapeutic potential of WNV-poly (A).

Example 10: detection of therapeutic effects of WNV-poly (A) in different modes of administration

This example demonstrates the efficacy of WNV-poly (A) in a therapeutic regimen by testing the in vivo oncolytic effect of WNV-poly (A) on human hepatoma cell Huh7 in different modes of administration.

The experimental procedure was as follows: 4-5 weeks old Nude Bab/c mice were selected, subcutaneously inoculated with 1X 10⁶When the individual hepatoma cells Huh7 were able to touch the transplanted tumor, the mice were divided into 4 groups, of which three groups were given to each 1X 10 by different administration methods (intramuscular injection (i.m.), intravenous injection (i.v.), intratumoral injection (i.t.))⁷PFU WNV-poly (a), one group was given an equal volume of 2% FBS DMEM, poisoned every 3 days, and tumor volumes were measured, and mice were observed for survival to treatment for 20 days. As a result, it was found that intravenous and intratumoral administration significantly inhibited tumor growth, while intramuscular injection had substantially no therapeutic effect on tumors (fig. 11A). And the venous and intratumoral can be also obviously seen by monitoring the change of the volume of the tumorTherapeutic effect of the administration (fig. 11B). Further observation of the survival rate of mice revealed that intratumoral and intramuscular toxication did not affect the survival of mice, but 40% of mice that were intravenously toxic died (fig. 11C).

In summary, both intravenous and intratumoral administration are effective in tumor treatment, but are safer than intravenous and intratumoral administration.

Sequence listing

<110> Shunhei technology, Inc

Wuhan Institute of Virology, Chinese Academy of Sciences

<120> use of attenuated flavivirus in oncolytic

<130> MTI21152

<160> 6

<170> SIPOSequenceListing 1.0

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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. An oncolytic virus which is an attenuated flavivirus 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 flavivirus, such that the 3 'untranslated region (3' UTR) of the attenuated flavivirus obtained after replacement of the part of the nucleotide sequence by the flavivirus retains at least the 3 'stem loop region (3' SL).

2. The oncolytic virus of claim 1, wherein the partial nucleotide sequence comprises all or part of a5 'stem loop region (5' SL) in a3 'non-coding region (3' UTR), all or part of a circularized sequence region (CS), and/or all or part of a dumbbell region (DB);

preferably, the 3 ' untranslated region (3 ' UTR) of said attenuated flavivirus is replaced by a poly (A) sequence, retaining at least the circularized sequence region 1(CS1) of the 3 ' untranslated region and the short hairpin-3 ' terminal stem-loop region (sHP-3 ' SL) following circularized sequence region 1;

preferably. The 5 'terminal 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 (SLIII), and stem-loop region IV (SLIV):

preferably, the cyclised sequence region (CS) is selected from cyclised sequence region 1(CS1), cyclised sequence region 2(CS2), cyclised sequence region 3(CS3), cyclised sequence region 2(RCS2) and cyclised sequence region 3(RCS 3);

preferably, said dumbbell zone (DB) is selected from dumbbell zone 1(DB1) and dumbbell zone 2(DB 2).

3. The oncolytic virus of claim 1 or 2, wherein the polyadenylic acid (p)_oThe ly (A) sequence comprises 10-200 adenylates;

preferably, the poly (A) sequence comprises 50-180, 100-160 adenylates; more preferably 130-150 adenylates, preferably 130 or 150 adenylates;

preferably, the flaviviruses are selected from Japanese Encephalitis Virus (JEV), West Nile Virus (West Nile Virus, WNv), tick-borne encephalitis Virus (TBEv), yelloW fever Virus (yelloW Virus, YFV), dengue Virus (dengue Virus, DENV), zika Virus (ZIKV), Saint Louis Encephalitis Virus (SLEV), Murray Valley Encephalitis Virus (MVEV), ompak hemorrhagic fever Virus (ompk hemorrhagic fever Virus), and karsanol Forest Disease Virus (Kyasanur Forest Disease Virus);

preferably, the attenuated flavivirus plaque titer is 1 × 10⁵-1×10⁹PFU/ml; more preferably 5X 10⁶-5×10⁸PFU/ml；

Preferably, the attenuated flavivirus is 10-100 fold less virulent than the parental wild-type virus;

preferably, the attenuated flavivirus is rescued from baby hamster kidney cell BHK-21 and/or amplified from Vero cell of Vero cell;

preferably, said dengue virus is selected from the group consisting of dengue virus type 1, type 2, type 3 and type 4;

preferably, the attenuated flaviviruses virus is an attenuated west nile virus; preferably, the attenuated west nile virus comprises a nucleotide sequence that is 80% or more identical to the nucleotide sequence set forth in SEQ ID No.1, 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; more preferably, the nucleotide sequence of the attenuated west nile virus is SEQ ID No. 1;

preferably, the attenuated flavivirus is attenuated Japanese encephalitis virus, the attenuated Japanese encephalitis virus containing and SEQ ID NO.2 nucleotide sequence of 80% or more of the same sex, preferably having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the same sex nucleotide sequence, more preferably with 98% or 99% or more of the same sex nucleotide sequence; more preferably, the attenuated japanese encephalitis virus has the nucleotide sequence of SEQ ID No. 2;

preferably, the attenuated flavivirus is an attenuated dengue virus; preferably, the attenuated dengue virus comprises a nucleotide sequence that is 80% or more identical to the nucleotide sequence set forth in SEQ ID No.3, 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; more preferably, the attenuated dengue virus has the nucleotide sequence of SEQ ID No. 3;

preferably, the attenuated flavivirus is an attenuated yellow fever virus; preferably, the attenuated yellow fever virus comprises a nucleotide sequence that is 80% or more identical to the nucleotide sequence set forth in SEQ ID No.4, 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; more preferably, the nucleotide sequence of the attenuated yellow fever virus is SEQ ID No. 4;

preferably, the attenuated flaviviruses are attenuated zika viruses; preferably, the attenuated Zika virus comprises a nucleotide sequence that is 80% or more identical to the nucleotide sequence set forth in SEQ ID No.5, 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; more preferably, the nucleotide sequence of the attenuated Zika virus is SEQ ID No. 5;

preferably, the attenuated flavivirus is an attenuated tick-borne encephalitis virus; preferably, the attenuated tick-borne encephalitis virus comprises a nucleotide sequence that is 80% or more identical to the nucleotide sequence set forth in SEQ ID No.6, preferably 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably 98% or 99% or more identical; more preferably, the nucleotide sequence of the attenuated tick-borne encephalitis virus is SEQ ID No. 6;

preferably, said partial nucleotide sequence comprises a5 'stem-loop region i (sli), a stem-loop region ii (slii), a repeated circularization sequence region 3(RCS3), a stem-loop region iii (sliii), a stem-loop region iv (sliv), a circularization sequence region 3(CS3), said partial nucleotide sequence being replaced with a poly (a) sequence, said 3' untranslated region (3 'UTR) of said attenuated flaviviruses retaining dumbbell region 1(DB1) of the 3' untranslated region, repeated circularization sequence region 2(RCS2), dumbbell region 2(DB2), circularization sequence region 2(CS2), circularization sequence region 1(CS1), and a short hairpin-3 'stem-loop region (sHP-3' SL) following circularization sequence region 1;

preferably, said partial nucleotide sequence comprises dumbbell region 1(DB1), repeat-circularized sequence region 2(RCS2), dumbbell region 2(DB2), circularized sequence region 2(CS2), circularized sequence region 1(CS1), and said partial nucleotide sequence is replaced with a poly (a) sequence, and the 3 ' untranslated region (3 ' UTR) of said attenuated flavivirus retains the 5 ' stem loop region i (sli), stem loop region ii (slii), repeat-circularized sequence region 3(RCS3), stem loop region iii (sliii), stem loop region 1v (sliv), circularized sequence region 3(CS3), and short hairpin-3 ' terminal stem region (sHp-3 ' SL);

preferably, said partial nucleotide sequence comprises a5 'stem-loop region i (sli), a stem-loop region ii (slii), a repeated circularization sequence region 3(RCS3), a stem-loop region iii (sliii), a stem-loop region iv (sliv), a circularization sequence region 3(CS3), a dumbbell region 1(DB1), a repeated circularization sequence region 2(RCS2), a dumbbell region (DB2), a circularization sequence region 2(CS2), said 3' untranslated region (3 'UTR) of said attenuated xanthovirus retaining a circularization sequence region 1(CS1) of the 3' untranslated region and a short hairpin-3 'stem-loop region (sHp-3' SL) after circularization sequence region 1 after said repeated circularization sequence region 1;

preferably, said partial nucleotide sequence comprises 5 'stem loop region ii (slii), repetitive loop sequence region 3(RCS3), stem loop region iv (SLIV), loop sequence region 3(CS3), dumbbell region 1(DB1), repetitive loop sequence region 2(RCS2), dumbbell region (DB2), loop sequence region 2(CS2), said partial nucleotide sequence being replaced with poly (a) sequence, said 3' untranslated region (3 'UTR) of said attenuated flaviviruses retaining loop sequence region 1(CS1) of the 3' untranslated region and short hairpin-3 'stem loop region (sHp-3' SL) following loop sequence region 1;

preferably, the partial nucleotide sequence comprises a5 ' stem loop region II (SLII), a repeated cyclization sequence region 3(RCS3), a dumbbell region (DB), a repeated cyclization sequence region 2(RCS2), a dumbbell region (DB2) and a cyclization sequence region 2(CS2), wherein after the partial nucleotide sequence is replaced by a poly A (poly A) sequence, the 3 ' untranslated region (3 ' UTR) of the attenuated flavivirus retains the 5 ' stem loop region (SL) of the 3 ' untranslated region, the cyclization sequence region 1(CS1) and the short hairpin-3 ' stem loop region (sHP-3 ' SL) after the cyclization sequence region 1.

4. The oncolytic virus of any one of claims 1-3, wherein the oncolytic virus further comprises at least a sequence encoding at least one exogenous gene;

preferably, the exogenous gene is a therapeutic gene;

preferably, the therapeutic gene is an immunostimulatory gene or a suicide gene;

preferably, the immunostimulatory gene encodes an immunostimulatory protein; preferably, the immunostimulatory protein is selected from the group consisting of cytokines, chemokines, interferons, Tumor Necrosis Factor (TNF), colony stimulating factor, proteins exposed to APC (antigen presenting cell), growth factors, Major Histocompatibility Complex (MHC) class I or II components, inducers or inhibitors of apoptosis, cytostatic agents, immunotoxins and blockers of the immune escape mechanisms of immune checkpoint inhibitors;

preferably, 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;

preferably, the chemokine is selected from CXCL10, CXCL9 and CXCL 11;

preferably, the interferon is selected from IFN7 and IFN α;

preferably, the colony stimulating factor is selected from the group consisting of GM-CSF, G-CSF and M-CSF;

preferably, the protein of the APC (antigen presenting cell) is selected from B7.1 and B7.2;

preferably, the growth factor is selected from the group consisting of transforming growth factor TGF, fibroblast growth factor FGF and vascular endothelial growth factor VEGF;

preferably, the inducer or inhibitor of apoptosis is selected from Bax, Bcl2, and BcIX;

preferably, the cytostatic agent is selected from p21, p16 and Rb;

preferably, the suicide gene is selected from Thymidine Kinase (TK), thymidylate kinase, cytosine deaminase (CDase) and uracil phosphoribosyl transferase (UPRTase).

5. A DNA which can be transcribed to produce an oncolytic virus according to any one of claims 1-3;

preferably, the DNA is an infectious clone of an attenuated strain of a flavivirus virus, preferably the infectious clone is a plasmid.

6. A cell comprising the oncolytic virus of claims 1-3 or the DNA of claim 5.

7. A composition comprising the oncolytic virus of claims 1-3, the DNA of claim 5 and/or the cell of claim 6.

8. Use of the oncolytic virus of claims 1-3, the DNA of claim 5, the cell of claim 6 and/or the composition of claim 7 for the manufacture of a medicament for the treatment of a tumor and/or cancer.

9. The use according to claim 8, wherein the tumor and/or cancer is selected from the group consisting of melanoma, liver and ovarian cancer, lung cancer, breast cancer, cervical cancer and glioma;

preferably, the medicament may be administered in one or more doses.

10. The use according to any one of claims 8 to 10, wherein the medicament is administered locally or systemically.