Mutant strain of yellow fever virus and use thereof

Abstract

The application relates to the field of virus and vaccine technology, and discloses a yellow fever virus mutant strain and application thereof. The yellow fever virus mutant strain provided by the application is obtained based on a new yellow fever virus neutralization site, has obvious attenuation characteristics, and after animals are immunized by using the mutant strain, it is found that compared with a wild type yellow fever virus, the mutant strain can induce higher neutralization activity against the yellow fever virus carrying the site of an epidemic strain. The mutant strain provided by the application provides an important target for development of a new generation of live attenuated yellow fever virus vaccine, not only provides the possibility of updating the existing live attenuated yellow fever virus vaccine, but also lays a foundation for further research on viruses of the genus flavivirus.

Description

Technical Field

[0001] This invention relates to the field of virus and vaccine technology, specifically to a yellow fever virus mutant strain and its application. Background Technology

[0002] Yellow fever virus (YFV) belongs to the genus Flaviviridae in the family Flaviviridae. It is an important mosquito-borne virus that causes acute, infectious yellow fever. Currently, there are no specific antiviral drugs for yellow fever virus; vaccination with a live attenuated yellow fever vaccine is the most effective measure for preventing and controlling yellow fever.

[0003] The genome of yellow fever virus is a single-stranded positive-sense RNA that is translated into a polyprotein. It is cleaved by viral proteases and host signal peptidases during and after translation to form three structural proteins (capsid protein C, pre-membrane protein prM, and envelope protein E) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 proteins).

[0004] Although yellow fever virus vaccines are currently available and in use, research has found that existing live attenuated yellow fever vaccines offer reduced protection against current circulating strains, and that neutralizing antibodies can escape from the yellow fever virus, posing new challenges to the prevention and control of yellow fever. Therefore, there is an urgent need to develop a more effective next-generation yellow fever vaccine targeting circulating strains of the yellow fever virus. Summary of the Invention

[0005] The purpose of this invention is to overcome the problems existing in the prior art, such as the lack of attenuated live vaccine strains with more effective protective effects against currently circulating yellow fever virus strains, and to provide a yellow fever virus mutant strain and its application. The mutant strain provided by this invention is obtained based on a novel yellow fever virus neutralizing site, and its virulence is significantly reduced compared to wild-type yellow fever virus strains. Using this mutant strain can induce higher neutralizing activity against yellow fever virus carrying circulating strain sites, indicating that the mutant strain of this invention has the potential for clinical application in updating existing yellow fever virus attenuated live vaccines.

[0006] To achieve the above objectives, the first aspect of the present invention provides a yellow fever virus mutant strain, which, compared to the wild-type virus strain, includes a mutation at amino acid position 83 of the E protein.

[0007] A second aspect of the present invention provides a DNA molecule comprising an open reading frame in the genome of a yellow fever virus, wherein, compared to the open reading frame and nucleotide sequence in the genome of a wild-type virus strain, the DNA molecule comprises a mutation in the nucleotide encoding the 83rd amino acid of the E protein.

[0008] A third aspect of the present invention provides a construct comprising the DNA molecule or fragment thereof described in the second aspect.

[0009] The fourth aspect of this invention provides the use of the mutant strain or a portion thereof described in the first aspect, and / or the DNA molecule or a fragment thereof described in the second aspect, and / or the construct described in the third aspect, the use comprising:

[0010] (1) Applications in yellow fever virus research; and / or,

[0011] (2) Use in the preparation of medicines for the prevention and / or treatment of diseases caused by yellow fever virus infection.

[0012] The fifth aspect of the present invention provides a pharmaceutical composition comprising the mutant strain or a portion thereof described in the first aspect, and / or the DNA molecule or a fragment thereof described in the second aspect, and / or the construct described in the third aspect.

[0013] The sixth aspect of the present invention provides the application of a mutated gene site in altering the virulence of yellow fever virus, wherein the gene site is a nucleotide in the yellow fever virus genome encoding the 83rd amino acid of the envelope protein;

[0014] The alteration of yellow fever virus virulence includes either increasing or decreasing virulence.

[0015] Through the above technical solution, the present invention can achieve at least the following beneficial effects:

[0016] (1) This invention has discovered an important neutralizing site for yellow fever virus for the first time, providing an important target for the development and application of a new generation of live attenuated yellow fever virus vaccines, and laying the foundation for further research on various viruses in the Flaviviridae genus.

[0017] (2) The yellow fever virus mutant strain provided by the present invention has obvious attenuation characteristics and can effectively induce the production of neutralizing antibodies that protect against flaviviruses carrying epidemic strain sites, and has the potential to be used to prepare a new generation of live attenuated flavivirus vaccines. Attached Figure Description

[0018] Figure 1 This is a schematic diagram showing the location of the mutation site of the yellow fever recombinant mutant virus A83E in the genomic cDNA sequence.

[0019] Figure 2 This is a comparison diagram of the plaque features of the parent virus 17D and the mutant virus A83E in Example 3.

[0020] Figure 3 This is a comparison diagram of the proliferation characteristics of parental virus 17D and mutant virus A83E in BHK-21 cells in Example 3.

[0021] Figure 4 This is a comparison of the neutralization sensitivity of parental virus 17D and mutant virus A83E to serum from mice immunized with 17D in Example 3.

[0022] Figure 5 This is a comparison of survival curves of mice after intracranial inoculation with parental virus 17D and mutant virus A83E in Example 3.

[0023] Figure 6 This is a comparison diagram of the neutralizing antibodies induced by parental virus 17D and mutant virus A83E in Example 3. Detailed Implementation

[0024] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0025] In this invention, unless otherwise specified, "YF-17D" refers to the yellow fever virus strain YF-17D (also known as "yellow fever virus vaccine strain 17D"), which is one of the vaccine strains used in the currently commercially available live attenuated yellow fever virus vaccines. For specific nucleic acid and protein sequence information related to YF-17D, please refer to NC_002031.

[0026] The inventors of this invention discovered a novel yellow fever virus neutralizing site (amino acid 83 of the E protein) during their research. Mutation targeting this site yielded a mutant strain with significantly reduced virulence and the ability to induce immune protection. Compared to existing vaccine strains, this mutant strain exhibits further reduced virulence but higher neutralizing activity against yellow fever viruses carrying circulating strains, demonstrating its potential to update existing live attenuated yellow fever vaccines.

[0027] Based at least on the above findings, the first aspect of the present invention provides a yellow fever virus mutant strain, which, compared to the wild-type virus strain, includes a mutation at amino acid position 83 of the E protein.

[0028] According to a preferred embodiment of the present invention, the wild-type virus strain is YF-17D.

[0029] In this invention, there are no particular restrictions on the specific mutation method of the 83rd amino acid of the E protein in the mutant strain; for example, it can be a substitution mutation. There are no particular restrictions on the specific substitution mutation method, as long as the virulence of the mutated virus strain changes compared to the wild-type virus (e.g., decreases or increases).

[0030] According to a preferred embodiment of the present invention, in the mutant strain, the 83rd amino acid of the E protein is mutated from alanine (A) to glutamic acid (E).

[0031] The yellow fever virus mutant strain provided by this invention may contain only a mutation at amino acid position 83 of the E protein, or it may contain other mutations simultaneously. These other mutations may be mutations in the E protein, mutations in other proteins of the yellow fever virus, or mutations in non-coding regions of the genome.

[0032] According to a particularly preferred embodiment of the present invention, the amino acid sequence of the E protein in the mutant strain is as shown in SEQ ID NO:1.

[0033] AHCIGITDRFIEGVHGGTWVSATLEQDKCVTVMAPDKPSLDISLETVAIDRPAEVRKVCYNAVLT

[0034] HVKINDKCPSTGEAHLEEENEGDNACKRTYSDRGWGNGCGLFGKGSIVACAKFTCAKSMSLFEVD

[0035] QTKIQYVIRAQLHVGAKQENWNTDIKTLKFDALSGSQEVEFIGYGKATLECQVQTAVDFGNSYIAE

[0036] METESWIVDRQWAQDLTLPWQSGSGGVWREMHHLVEFEPPHAATIRVLALGNQEGSLKTALTGA

[0037] MRVTKDTNDNNLYKLHGGHVSCRVKLSALTLKGTSYKICTDKMFFVKNPTDTGHGTVVMQVKVS

[0038] KGAPCRIPVIVADDLTAAINKGILVTVNPIASTNDDEVLIEVNPPFGDSYIIVGRGDSRLTYQWHKEG

[0039] SSIGKLFTQTMKGVERLAVMGDTAWDFSSAGGFFTSVGKGIHTVFGSAFQGLFGGLNWITKVIMGAVLIWVGINTRNMTMSMSMILVGVIMMFLSLGVGA(SEQ ID NO:1)

[0040] Preferably, in the mutant strain, no amino acid sequence mutations have occurred in any of the proteins other than the E protein (including structural and non-structural proteins).

[0041] A second aspect of the present invention provides a DNA molecule comprising an open reading frame in the genome of a yellow fever virus, wherein, compared to the nucleotide sequence of the open reading frame in the genome of a wild-type virus strain, the DNA molecule comprises a mutation in the nucleotide encoding the 83rd amino acid of the E protein.

[0042] According to a preferred embodiment of the present invention, the wild-type virus strain is YF-17D.

[0043] Preferably, the mutation includes changing the 83rd amino acid of the E protein to glutamic acid.

[0044] Any method that can induce the above-mentioned mutations in the yellow fever virus E protein is applicable to the DNA molecule provided by this invention. Those skilled in the art can design and adjust it themselves based on the codon table and the genomic nucleotide sequence of the wild-type yellow fever virus strain.

[0045]

[0046] In the DNA molecule provided by this invention, the nucleotide sequences encoding proteins other than E protein may or may not be mutated.

[0047] Preferably, the nucleotide sequences encoding proteins other than protein E are free from mutations (i.e., these nucleotide sequences encode proteins identical to YF-17D). For example, the nucleotide sequences encoding proteins other than protein E may be free from any mutations (including sense and nonsense mutations) or may contain nonsense mutations.

[0048] A third aspect of the present invention provides a construct comprising the DNA molecule or fragment thereof described in the second aspect.

[0049] In this invention, "construction" refers to synthetic DNA comprising a vector and a (exogenous) DNA fragment carried within the vector. Typically, the vector can be a plasmid, which can be a naturally isolated plasmid or a plasmid artificially constructed or modified as needed.

[0050] According to some preferred embodiments of the present invention, the construct may also contain gene elements such as promoters and enhancers.

[0051] The construct provided by the present invention may contain the complete DNA molecule described in the second aspect (i.e., the nucleotide sequence of the open reading frame of the yellow fever virus mutant strain provided by the present invention), or may contain a portion thereof, such as the E protein encoding gene of the yellow fever virus mutant strain provided by the present invention.

[0052] The fourth aspect of this invention provides the use of the mutant strain or a portion thereof described in the first aspect, and / or the DNA molecule or a fragment thereof described in the second aspect, and / or the construct described in the third aspect, the use comprising:

[0053] (1) Applications in yellow fever virus research; and / or,

[0054] (2) Use in the preparation of medicines for the prevention and / or treatment of diseases caused by yellow fever virus infection;

[0055] The drug preferably includes antibodies and / or vaccines, and the vaccine is preferably a live attenuated vaccine.

[0056] In this invention, "yellow fever virus research" can refer to any research targeting yellow fever virus or the disease caused by yellow fever virus. For example, it can be research on the functional sites of yellow fever virus itself or its function and mechanism of action, research on the effects of yellow fever virus infection on the body, or drug development for anti-yellow fever virus infection or to alleviate the symptoms caused by yellow fever virus infection.

[0057] The fifth aspect of the present invention provides a pharmaceutical composition comprising the mutant strain or a portion thereof described in the first aspect, and / or, the DNA molecule or a fragment thereof described in the second aspect, and / or, the construct described in the third aspect.

[0058] The pharmaceutical composition provided by this invention can be used as an active ingredient in the preparation of a medicament for the prevention and / or treatment of diseases caused by yellow fever virus infection. It should be noted that, in this medicament, the pharmaceutical composition provided by this invention can be the sole active ingredient, or it can be used in conjunction with other ingredients that have the effect of preventing and / or treating diseases caused by yellow fever virus infection as active ingredients.

[0059] The sixth aspect of the present invention provides the application of a mutated gene site in altering the virulence of Flavivirviruses, wherein the gene site is a nucleotide corresponding to the 83rd amino acid encoding the envelope protein in the yellow fever virus genome;

[0060] The alteration of viral virulence in the Flavivir genus includes either increasing or decreasing viral virulence.

[0061] Existing research has shown that the nucleotide sequence homology of the E protein encoding genes of Flavivir viruses is high, all cysteine ​​residues in the E protein are highly conserved, and the E proteins of different Flavivir viruses share similar structural and functional characteristics. Therefore, those skilled in the art can, based on the novel neutralizing site of yellow fever virus discovered in this invention, combine it with the amino acid corresponding to the 83rd amino acid of the E protein of yellow fever virus YF-17D in other Flavivir viruses and the nucleotide position of its encoding gene to mutate other viruses and obtain mutant strains with altered virulence.

[0062] According to a preferred embodiment of the present invention, the virus of the Flavivir genus includes at least one of yellow fever virus, Japanese encephalitis virus, tick-borne encephalitis virus, dengue virus, Zika virus, and West Nile virus.

[0063] Preferably, when the yellow fever virus is a yellow fever virus, the mutation causes the amino acid at position 83 of the envelope protein to change from alanine to glutamic acid.

[0064] More preferably, the yellow fever virus is YF-17D.

[0065] The present invention will be described in detail below through embodiments. It should be understood that the following embodiments are only used to further explain and illustrate the content of the present invention by way of example, and are not intended to limit the present invention.

[0066] Unless otherwise specified, all reagents / materials used in the following examples are commercially available products purchased from reputable chemical / biological reagent or material suppliers, and all reagents are of analytical grade.

[0067] Example 1

[0068] This example illustrates the construction of a full-length genome clone of the yellow fever recombinant mutant strain.

[0069] This embodiment is based on the full-length infectious clone pACNR-FLYF17Dx of the yellow fever virus vaccine strain (containing the full-length cDNA of the yellow fever virus YF-17D genome, constructed according to the method described by BREDENBEEK, et al. 2003. A stable full-length yellow fever virus cDNA clone and the role of conserved RNA elements inflavivirus replication). Using reverse genetics, a full-length infectious clone of the yellow fever recombinant mutant virus A83E was constructed. The mutation site of A83E is located in the genome as follows: Figure 1 As shown.

[0070] Site-directed mutagenesis primers were designed according to the NEB Q5 site-directed mutagenesis kit instructions. Using the full-length infectious clone pACNR-FLYF17Dx as a template, site-directed mutagenesis PCR was performed to obtain a DNA fragment containing the A83E mutation. The circularized full-length clonal plasmid of the yellow fever recombinant mutant strain was finally obtained using NEB's KLD enzyme. The primer sequences used are detailed in Table 1, the PCR reaction system in Table 2, and the PCR reaction procedure in Table 3.

[0071] Table 1:

[0072] Primer name Sequence (5′-3′) SEQ ID NO: A83E-F GGCCCACCTAGAAGAAGAGAACGAAGG 3 A83E-R TCTCCAGTGCTGGGGCAC 4

[0073] Table 2:

[0074] Components Volume (μL) Q5 Hot Start High-Fidelity 2×Master Mix 12.5 Forward primer (10 μM) 1.25 Reverse primer (10 μM) 1.25 plasmid template 1 (approximately 20 ng) Nuclease-free water 9

[0075] Table 3:

[0076]

[0077] According to the reaction system in Table 4, the PCR products were treated with KLD enzyme.

[0078] Table 4:

[0079] Components Volume (μL) PCR products 1 2×KLD reaction buffer 5 10×KLD enzyme Mix 1 Nuclease-free water 3

[0080] The culture was incubated at room temperature for 5 min, then transformed into HB101 competent cells (TaKaRa), plated on solid LB medium (ampicillin-resistant), and incubated at 37°C for 14 h. Single clones were picked for colony PCR. Positive bacteria were added to liquid LB medium (ampicillin-resistant) for amplification culture. Plasmids were extracted and identified by enzyme digestion. Correctly identified plasmids were sent to Sangon Biotech (Shanghai) Co., Ltd. for further sequencing verification. Correct enzyme digestion and sequencing indicated successful construction of the mutant plasmid. Based on the characteristics of the mutation site, the obtained full-length infectious clone was named pACNR-YF17D-A83E.

[0081] Example 2

[0082] This example illustrates the rescue of yellow fever recombinant mutant virus.

[0083] The positive bacteria containing the pACNR-YF17D-A83E plasmid obtained in Example 1 were expanded and cultured. A large number of plasmids were prepared using a plasmid large-scale extraction kit (Invitrogen), linearized by XhoI restriction enzyme digestion, and extracted with phenol-chloroform. Using the linearized plasmids as templates, in vitro transcription was performed using SP6 RiboMAX Express Large Scale RNA Production Systems (Promega). The reaction system is shown in Table 5.

[0084] Table 5:

[0085] Components Volume (μL) SP6 transcription 5×buffer 6 rNTPs (25mM ATP, CTP, UTP, GTP) 6 Linear DNA template 10 (2-3 μg) SP6 enzyme 3 <![CDATA[Rino m 7 G Cap Analog]]> 1 Nuclease-free water 4

[0086] After preparing the reaction system according to Table 5, react at 37℃ for 4 hours. Then add 2 μL of DNase and incubate at 37℃ for 30 minutes. Purify the transcriptomic RNA according to the RNeasy mini kit (Invitrogen) instructions, quantify it, aliquot it, and store it at -80℃ for later use.

[0087] Transcriptomic RNA was transfected into monolayers of BHK-21 cells (purchased from ATCC, catalog number CCL-10) using Lipofectamine 3000 (Invitrogen) transfection reagent according to the manufacturer's instructions. After transfection, the cells were incubated at 37°C in a 5% CO2 incubator. Once cell pathogenesis was observed, the supernatant was collected by centrifugation. The supernatant was reseeded into BHK-21 cells, and after cell pathogenesis was observed, the supernatant was collected by centrifugation. The supernatant was then frozen at -80°C as seed culture for recombinant mutant viruses.

[0088] Example 3

[0089] This example illustrates the biological characteristics of the yellow fever recombinant mutant virus A83E.

[0090] (1) Plaque characteristics of yellow fever recombinant mutant virus A83E

[0091] The yellow fever recombinant mutant virus A83E seed culture obtained in Example 2 and the parental virus 17D (rescued using reverse genetics technology based on the full-length infectious clone pACNR-FLYF17Dx of the yellow fever virus vaccine strain) were serially diluted 10-fold to 10-fold. -1 10 -2 10 -3 10 -4 10 -5 10 -6 and 10 -7 A concentration gradient of 300 μL / well was used to inoculate different concentration gradients of seed culture into a monolayer of BHK-21 cells in 12-well plates. After incubation for 1 h, the virus culture was discarded, and 1% agar containing DMEM medium (2% FBS) was added to cap the cells. The plates were then incubated at 37°C with 5% CO2 for 4 days. After fixation with 4% formaldehyde at room temperature for 1 h, the agar caps were discarded, and the cells were stained with crystal violet at room temperature for 10 min. The morphology of the plaques was observed, and the plaque-forming units (PFU) were calculated.

[0092] The results are as follows Figure 2 As shown in the figure, both the yellow fever recombinant mutant virus A83E and the parental virus 17D can form relatively uniform vacuoles with clear edges, and the vacuoles of the mutant virus are similar to those of the parental strain.

[0093] (2) Proliferation characteristics of yellow fever recombinant mutant virus A83E on BHK-21 cells

[0094] BHK-21 cells in 24-well plates were infected with yellow fever recombinant mutant virus A83E and parental virus 17D (both seeded at MOI = 0.01). After adsorption for 1 h in a 37°C, 5% CO2 incubator, the virus solution was discarded, and DMEM medium containing 2% FBS was added. The cells were then incubated at 37°C in a 5% CO2 incubator. Cell supernatants were collected at 0 h, 24 h, 48 h, and 72 h post-inoculation. Infectious virus particles in the supernatant were quantified using the plaque assay, and proliferation curves were plotted.

[0095] The results are as follows Figure 3 As shown in the figure, both the yellow fever recombinant mutant virus A83E and the parent virus 17D can effectively replicate and proliferate in the BHK-21 cell line, and the A83E mutation does not affect the survival of the virus.

[0096] (3) Neutralization sensitivity characteristics of yellow fever recombinant mutant virus A83E to serum from 17D-immunized mice

[0097] The plaque reduction neutralization assay was used to determine the neutralizing antibody titer in serum. First, serum from mice immunized with parental 17D virus was prepared into suspensions diluted 1:70, 1:210, 1:630, and 1:1890. These suspensions were then mixed separately with equal volumes of recombinant mutant virus A83E containing 100 PFU and parental 17D virus solution, and incubated at 37°C for 1 h. The mixture was then used to infect monolayers of BHK-21 cells in 12-well plates. After 1 h of incubation, the virus solution was discarded, and a 1% agar cap containing DMEM medium (2% FBS) was added. The plates were then incubated at 37°C with 5% CO2 for 4 days. After fixation with 4% formaldehyde at room temperature for 1 h, the agar cap was discarded, and the plates were stained with crystal violet at room temperature for 10 min. Plaque morphology was observed, and the serum dilution that reduced the number of plaques by 50% was calculated as the serum neutralizing antibody titer (PRNT). 50 ).

[0098] The results are as follows Figure 4 As shown in the figure, the neutralizing antibody titer of yellow fever recombinant mutant virus A83E against serum from mice immunized with parental virus 17D is significantly lower than that of parental virus 17D against serum from mice immunized with parental virus 17D. This indicates that the neutralizing sensitivity of yellow fever recombinant mutant virus A83E against serum from mice immunized with 17D is significantly reduced, suggesting that the neutralizing ability of serum from mice immunized with 17D is decreased against mutant viruses carrying mutation sites of the circulating strain, resulting in the inability of existing 17D vaccines to effectively protect against infection by the circulating yellow fever strain.

[0099] (4) Neurovirulence characteristics of yellow fever recombinant mutant virus A83E

[0100] Yellow fever recombinant mutant virus A83E and parental virus 17D were intracranially inoculated into 3-4 week old BALB / c mice (Vitalliwa). Death within 24 hours post-inoculation was considered nonspecific death. The morbidity and mortality of the mice were observed over 21 days, and survival curves were plotted.

[0101] The results are as follows Figure 5 As shown in the figure, at the same dose, the yellow fever recombinant mutant virus A83E caused the death of 50% of mice, while the lethality of the parent virus 17D was 75%, significantly higher than that of the mutant virus of this invention. These results indicate that the mouse neurotoxicity of the yellow fever recombinant mutant virus A83E is weaker than that of the parent virus 17D, exhibiting a clear attenuation characteristic.

[0102] (5) Immunogenicity of yellow fever recombinant mutant virus A83E

[0103] 2×10 4Six-week-old BALB / c mice were immunized intraperitoneally with PFU yellow fever recombinant mutant virus A83E and parental virus 17D, respectively (Vitolver), with PBS used as a negative control. Blood was collected from the retroorbital venous plexus on day 28 post-immunization, centrifuged at 4°C for 3 hours, inactivated at 56°C for 30 minutes, and then frozen at -20°C for later use.

[0104] First, the two sera were prepared into suspensions diluted 1:70, 1:210, 1:630, and 1:1890. These suspensions were then mixed separately with an equal volume of viral suspension containing 100 PFU of the recombinant mutant virus A83E. The suspensions were incubated at 37°C for 1 hour, and then used to infect monolayers of BHK-21 cells coated in 12-well plates. After 1 hour of incubation, the viral suspension was discarded, and a 1% agar cap containing DMEM medium (2% FBS) was added. The plates were then incubated at 37°C with 5% CO2 for 4 days. After fixation with 4% formaldehyde at room temperature for 1 hour, the agar cap was discarded, and the plates were stained with crystal violet at room temperature for 10 minutes. The morphology of the empty plaques was observed, and the serum dilution that reduced the number of empty plaques by 50% was calculated as the serum neutralizing antibody titer (PRNT). 50 ).

[0105] The results are as follows Figure 6 As shown in the figure, the yellow fever recombinant mutant virus A83E can induce high levels of neutralizing antibodies against the A83E carrying the circulating strain site, with antibody titers significantly higher than those produced by immunization with the parent virus 17D. These data indicate that the yellow fever recombinant mutant virus A83E can effectively induce protective neutralizing antibodies against yellow fever virus carrying the circulating strain site, suggesting its potential for application in next-generation live attenuated yellow fever vaccines.

[0106] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A yellow fever virus mutant strain, characterized in that, Compared to the wild-type virus strain, this mutant strain includes a mutation at amino acid position 83 of the E protein.

2. The yellow fever virus mutant strain according to claim 1, wherein, The wild-type virus strain is YF-17D; Preferably, in the mutant strain, the 83rd amino acid of the E protein is mutated from alanine to glutamic acid; More preferably, in the mutant strain, the amino acid sequence of the E protein is as shown in SEQ ID NO:

1.

3. A DNA molecule, characterized in that, The DNA molecule contains an open reading frame from the yellow fever virus genome, wherein, compared to the nucleotide sequence of the open reading frame in the wild-type virus strain genome, the DNA molecule contains a mutation in the nucleotide encoding amino acid 83 of the E protein.

4. The DNA molecule according to claim 3, wherein, The wild-type virus strain is YF-17D; Preferably, the mutation includes changing the 83rd amino acid of the E protein to glutamic acid.

5. The DNA molecule according to claim 3 or 4, wherein, The DNA molecule includes the nucleotide sequence shown in SEQ ID NO:

2.

6. A construct, characterized in that, The construct comprises the DNA molecule or a fragment thereof as described in any one of claims 3-5.

7. The mutant strain or a portion thereof as claimed in claim 1 or 2, and / or, the DNA molecule or fragment thereof as claimed in any one of claims 3-5, and / or, the construct of claim 7, wherein the application includes: (1) Application in yellow fever virus research; And / or, (2) Use in the preparation of medicines for the prevention and / or treatment of diseases caused by yellow fever virus infection; The drug preferably includes antibodies and / or vaccines, and the vaccine is preferably a live attenuated vaccine.

8. A pharmaceutical composition, characterized in that, The composition comprises the mutant strain or a portion thereof as described in claim 1 or 2, and / or, a DNA molecule or a fragment thereof as described in any one of claims 3-5, and / or, the construct as described in claim 7.

9. The application of mutant gene sites in altering the virulence of Flavivir viruses, among which, The gene locus is a nucleotide corresponding to the 83rd amino acid encoding the envelope protein in the yellow fever virus genome; The alteration of viral virulence in the Flavivir genus includes either increasing or decreasing viral virulence.

10. The application according to claim 9, wherein, The viruses of the Flavivir genus include at least one of yellow fever virus, Japanese encephalitis virus, tick-borne encephalitis virus, dengue virus, Zika virus, and West Nile virus; Preferably, when the yellow fever virus is a yellow fever virus, the mutation causes the amino acid at position 83 of the envelope protein to change from alanine to glutamic acid; More preferably, the yellow fever virus is YF-17D.

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