Attenuated virus of flavivirus virus and use thereof

1 . An attenuated flavivirus virus, comprising a polyadenylic acid (poly(A)) sequence, wherein the polyadenylic acid (poly(A)) sequence is used for replacing a part of nucleotide sequence of a 3′ untranslated region (3′UTR) of the flavivirus virus, so that the 3′ untranslated region (3′UTR) of the attenuated flavivirus virus obtained in response to the part of the nucleotide sequence of the flavivirus virus being replaced at least retains a 3′-end stem loop region (3′SL), the flavivirus virus is West Nile virus (WNV) or dengue virus (DENV).

2 . The attenuated flavivirus virus according to claim 1 , wherein the part of nucleotide sequence comprises all or a part of a nucleotide sequence of a 5′-end stem loop region (5′SL), all or a part of a nucleotide sequence of a cyclization sequence region (CS), and/or all or a part of a nucleotide sequence of a dumbbell region (DB) in a 3′ untranslated region (3′UTR);

optionally, after the part of nucleotide sequence is replaced by the polyadenylic acid (poly(A)) sequence, the 3′ untranslated region (3′UTR) of the attenuated flavivirus virus retains at least a cyclization sequence region 1 (CS1) and a short hairpin 3′ stem loop region (sHP-3′SL) behind the cyclization sequence region 1 in the 3′ untranslated region;

optionally, the 5′-end stem loop region (5′SL) in the 3′ untranslated region (3′UTR) is selected from a stem loop region I (SLI), a stem loop region II (SLII), a stem loop region III (SLIII) and a stem loop region IV (SLIV);

optionally, the cyclization sequence region (CS) is selected from a cyclization sequence region 2 (CS2), a cyclization sequence region 3 (CS3), a repetitive cyclization sequence region 2 (RCS2) and a repetitive cyclization sequence region 3 (RCS3);

optionally, the dumbbell region (DB) is selected from a dumbbell region 1 (DB1) and a dumbbell region 2 (DB2).

3 . The attenuated flavivirus virus according to claim 1 , wherein the polyadenylic acid (poly(A)) sequence comprises 10-200 adenylates;

optionally, the polyadenylic acid (poly(A)) sequence comprises 50-180, 100-160 adenylates;

optionally, the polyadenylic acid (poly(A)) sequence comprises 130-150 adenylates,

optionally, the polyadenylic acid (poly(Ab) sequence comprises 130 or 150 adenylates.

4 . A DNA, which can be transcribed to produce an RNA genome of the attenuated flavivirus virus according to claim 1

optionally, the DNA is an infectious clone of an attenuated strain of the flavivirus virus;

optionally, the infectious clone is a plasmid.

5 . A cell, comprising the attenuated flavivirus virus according to claim 1 or a DNA that can be transcribed to produce an RNA genome of the attenuated flavivirus virus.

6 . A vaccine, comprising the attenuated flavivirus virus according to claim 1 , a DNA that can be transcribed to produce an RNA genome of the attenuated flavivirus virus, and/or a cell comprising the attenuated flavivirus virus or the DNA.

7 . A pharmaceutical composition, comprising the attenuated flavivirus virus according to claim 1 , a DNA that can be transcribed to produce an RNA genome of the attenuated flavivirus virus, a cell comprising the attenuated flavivirus virus or the DNA and/or a vaccine comprising the attenuated flavivirus virus, the DNA, and/or the cell, and a pharmaceutically acceptable vector.

8 . A method for preparing the attenuated flavivirus virus according to claim 1 , the method comprising substituting a part of nucleotide sequence of a 3′ untranslated region (3′UTR) of a wild-type virus with a polyadenylic acid (poly(A)) sequence, wherein

optionally, the attenuated flavivirus virus is obtained by means of rescue on baby hamster kidney cells BHK-21 and/or amplification on African green monkey kidney Vero cells.

9 . A method of stimulating an immune response to a flavivirus virus, comprising immunologically administering to the individual an effective mount of an attenuated flavivirus virus vaccine, wherein the attenuated flavivirus virus vaccine comprises: the attenuated flavivirus virus according to claim 1 , a DNA that can be transcribed to produce an RNA genome of the attenuated flavivirus virus and a cell comprising the attenuated flavivirus virus or the DNA.

10 . A method of treating or preventing diseases caused by flavivirus virus, comprising administering to an individual an effective mount of a medicament, wherein the medicament comprises: the attenuated flavivirus virus according to claim 1 , a DNA that can be transcribed to produce an RNA genome of the attenuated flavivirus virus, a cell comprising the attenuated flavivirus virus or the DNA, a vaccine comprising the attenuated flavivirus virus, the DNA, and/or the cell and/or the pharmaceutical composition comprising the attenuated flavivirus virus, the DNA, the cell and/or the vaccine.

11 . The attenuated flavivirus virus according to claim 1 , the attenuated flavivirus virus has a plaque titer of 5×10 4 −5×10 6 PFU/ml.

12 . The attenuated flavivirus virus according to claim 1 , the attenuated flavivirus virus is 10-100 times less virulent than a parent wild-type virus.

13 . The attenuated flavivirus virus according to claim 1 , the attenuated flavivirus virus is obtained by means of rescue on baby hamster kidney cells BHK-21 and/or amplification on African green monkey kidney Vero cells.

14 . The attenuated flavivirus virus according to claim 1 , the dengue virus is selected from 1-type, 2-type, 3-type and 4-type dengue viruses.

15 . The attenuated flavivirus virus according to claim 1 , the attenuated West Nile virus comprises a nucleotide sequence having 80% or more identity with a nucleotide sequence shown in SEQ ID NO. 1;

optionally, the attenuated West Nile virus comprises a nucleotide sequence having more than 85% identity with the nucleotide sequence shown in SEQ ID NO. 1;

optionally, the attenuated West Nile virus comprises a nucleotide sequence having more than 90% identity with the nucleotide sequence shown in SEQ ID NO. 1;

optionally, the attenuated West Nile virus comprises a nucleotide sequence having more than 95% identity with the nucleotide sequence shown in SEQ ID NO. 1;

optionally, the attenuated West Nile virus comprises a nucleotide sequence having more than 96% identity with the nucleotide sequence shown in SEQ ID NO. 1;

optionally, the attenuated West Nile virus comprises a nucleotide sequence having more than 97% identity with the nucleotide sequence shown in SEQ ID NO. 1;

optionally, the attenuated West Nile virus comprises a nucleotide sequence having more than 98% identity with the nucleotide sequence shown in SEQ ID NO. 1;

optionally, the attenuated West Nile virus comprises a nucleotide sequence having more than 99% identity with the nucleotide sequence shown in SEQ ID NO. 1;

optionally, the nucleotide sequence of the attenuated West Nile virus is SEQ ID NO. 1.

16 . The attenuated flavivirus virus according to claim 1 , the attenuated dengue virus comprises a nucleotide sequence having 80% or more identity with a nucleotide sequence shown in SEQ ID NO. 3;

optionally, the attenuated dengue virus comprises a nucleotide sequence having more than 85% identity with the nucleotide sequence shown in SEQ ID NO. 3;

optionally, the attenuated dengue virus comprises a nucleotide sequence having more than 90% identity with the nucleotide sequence shown in SEQ ID NO. 3;

optionally, the attenuated dengue virus comprises a nucleotide sequence having more than 95% identity with the nucleotide sequence shown in SEQ ID NO. 3;

optionally, the attenuated dengue virus comprises a nucleotide sequence having more than 96% identity with the nucleotide sequence shown in SEQ ID NO. 3;

optionally, the attenuated dengue virus comprises a nucleotide sequence having more than 97% identity with the nucleotide sequence shown in SEQ ID NO. 3;

optionally, the attenuated dengue virus comprises a nucleotide sequence having more than 98% identity with the nucleotide sequence shown in SEQ ID NO. 3;

optionally, the attenuated dengue virus comprises a nucleotide sequence having more than 99% identity with the nucleotide sequence shown in SEQ ID NO. 3;

optionally, the nucleotide sequence of the attenuated dengue virus is SEQ ID NO. 3.

17 . The attenuated flavivirus virus according to claim 1 , the part of nucleotide sequence comprises a 5′-end stem loop region I (SLI), a stem loop region II (SLII), a repetitive cyclization sequence region 3 (RCS3), a stem loop region III (SLIII), a stem loop region IV (SLIV), a cyclization sequence region 3 (CS3), a dumbbell region 1 (DB1), a repetitive cyclization sequence region 2 (RCS2), a dumbbell region 2 (DB2), and a cyclization sequence region 2 (CS2), and after the part of nucleotide sequence is replaced by the polyadenylic acid (poly(A)) sequence, the 3′ untranslated region (3′UTR) of the attenuated flavivirus virus retains a cyclization sequence region 1 (CS1) and a short hairpin 3′ stem loop region (sHP-3′SL) behind the cyclization sequence region 1 in the 3′ untranslated region.

18 . The attenuated flavivirus virus according to claim 1 , the part of nucleotide sequence comprises a 5′-end stem loop region II (SLII), a repetitive cyclization sequence region 3 (RCS3), a stem loop region IV (SLIV), a cyclization sequence region 3 (CS3), a dumbbell region 1 (DB1), a repetitive cyclization sequence region 2 (RCS2), a dumbbell region 2 (DB2), and a cyclization sequence region 2 (CS2), and after the part of nucleotide sequence is replaced by the polyadenylic acid (poly(A)) sequence, the 3′ untranslated region (3′UTR) of the attenuated flavivirus virus retains a cyclization sequence region 1 (CS1) and a short hairpin 3′ stem loop region (sHP-3′SL) behind the cyclization sequence region 1 in the 3′ untranslated region.

19 . The method according to claim 9 , the attenuated flavivirus virus vaccine is an attenuated West Nile virus vaccine;

optionally, the attenuated flavivirus virus vaccine is an attenuated dengue virus vaccine.

20 . The method according to claim 10 , the method is used for treating or preventing diseases caused by the West Nile virus;

optionally, the method is used for treating or preventing diseases caused by the dengue virus.