Seneca virus A mutant strain and application thereof

Abstract

The invention belongs to the technical field of medicines, and provides a Seneca virus A mutant strain and application thereof. The insertion mutation of the screened mutant strain SVA genome at the 1494 site amino acid changes the spatial conformation of the virus 3B protein, enhances the virus replication efficiency and interferes the host immune response; the mutant strain has high titer and hereditary stability, and can solve the problem of virus dosage fluctuation in a traditional challenge experiment; the genome stability and high replication efficiency of the strain provide a standardized tool for vaccine research and development.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to a Senecavirus A mutant strain. BACKGROUND

[0002] The information disclosed in this background section is intended to provide an overview of the general background of the application and is not necessarily a concession that the information was prior art to the present application.

[0003] Senecavirus A (SVA) is the only member of the Senecavirus A genus of the Picornaviridae family. The virus has the ability to spread horizontally or vertically in infected pig populations, and infected animals often show subclinical state. The virus can survive in feed for a long time and can be transmitted by mice, thereby increasing the risk of infection in non-disease farms. The clinical symptoms caused by SVA infection are not significantly different from other vesicular diseases such as foot and mouth disease (FMD), swine vesicular disease (SVD), etc., and all show vesicles containing transparent liquid. Since its outbreak, SVA has been widely prevalent in the world, causing economic losses to the pig industry and hindering the development of the pig industry in various countries around the world.

[0004] The high variability of the SVA genome poses challenges for vaccine development, such as difficulties in selecting strains, risks of immune escape, etc. Traditional virus attenuation or virulent strain screening relies on random passage, which is low in efficiency and poor in targeting. Currently, laboratory diagnosis of SVA mainly relies on etiology and serology methods, but there is no commercial vaccine available for this disease, and once an epidemic occurs, it is difficult to prevent and control.

[0005] SVA is also an oncolytic virus that suppresses the host's innate immune response to facilitate viral replication, and it can multiply in human tumor cells and has been used as a candidate drug for human oncolytic virus therapy.

[0006] It has been confirmed that the serine-alanine mutation of VP2 protein S177 significantly increases the proliferation of SVA, and the mutations of VP1 R88A, S94A and VP2 D166A reduce the proliferation ability of the virus to less than 10% of the WT virus, and the VP1 S98A mutation only retains 50% of the virus proliferation, but it is unknown whether these mutations have an impact on the virulence of the virus. There is little research on SVA 3B, and there is no report on the impact of site mutation on proliferation or virulence. SUMMARY

[0007] In view of the problems in the prior art, the application provides a Senecavirus A mutant strain, which has higher virulence than the wild strain.

[0008] To achieve the above object, the present application adopts the following technical solutions.

[0009] A preparation method of a mutant strain of Senecavirus A (SVA), comprising the following steps: inserting lysine before the 9th amino acid of the 3B protein of a wild strain.

[0010] Preferably, the GenBank number of the Senecavirus A wild strain genome is MZ818785.1.

[0011] The present application also provides a Senecavirus A mutant strain obtained by the above method.

[0012] A mutant strain of Senecavirus A, Porcine senecavirus SVA-1494K, was deposited with the China Center for Type Culture Collection (CCTCC) on June 10, 2025, and the deposit number is CCTCC NO: V202552.

[0013] A mutant strain of Senecavirus A, Porcine senecavirus SVA-CDS, was deposited with the China Center for Type Culture Collection (CCTCC) on June 10, 2025, and the deposit number is CCTCC NO: V202553.

[0014] The above-mentioned Senecavirus A mutant strain is used for preparing an anti-Senecavirus A drug.

[0015] The present application has the following advantages: The insertion mutation of the mutant strain SVA genome at the 1494th amino acid changes the spatial conformation of the 3B protein of the virus, enhances the virus replication efficiency and interferes with the host immune response; the mutant strain has high titer and genetic stability, which can solve the problem of virus dosage fluctuation in traditional challenge experiments; the genetic stability and high replication efficiency of the mutant strain provide a standardized tool for vaccine development.

[0016] Biological preservation information Porcine senecavirus SVA-1494K was deposited with the China Center for Type Culture Collection (CCTCC) on June 10, 2025, and the deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: V202552.

[0017] Porcine senecavirus SVA-CDS was deposited with the China Center for Type Culture Collection (CCTCC) on June 10, 2025, and the deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: V202553.

[0018] Syrian hamster kidney cell BHK-21-Ago2 Δ- , which was preserved in the China Center for Type Culture Collection (CCTCC) on August 13, 2021, and the address of the preservation is Wuhan University, Wuhan, China, and the preservation number is CCTCC NO: C2021206. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 are fluorescence micrographs after pEGFP-5miR and pEGFP-5miRS transfection of BHK cell lines (A) and BHK-Ago2 Δ- cell lines (B); Figure 2 are flow cytometry histograms of BHK cells transfected with different miRNA minics; wherein A-G are miR-21 minic (miR-21), miR-29b minic (miR-29b), miR-30 minic (miR-30), miR-192 minic (miR-192), miR-31 minic (miR-31), miR-93 minic (miR-93), and 5 mixed minics (all 5); Figure 3 is a schematic diagram of the insertion position of 5miRS in the SVA genome; Figure 4 is the growth curve of wild strain SVA-WT and 1494K mutant strain; Figure 5 is the CPE picture of wild strain SVA-WT and 1494K mutant strain; Figure 6 is the growth curve of wild strain SVA-WT and CDS mutant strain; Figure 7 is the 3B protein structure diagram of wild strain (A) and mutant strain (B); Figure 8 is the in vivo immunosuppression of wild strain SVA-WT and mutant strain SVA-1494K; Figure 9 is the HE staining picture of the intestinal tract of mice in different treatment groups; Figure 10 is the effect of different treatment groups on the body weight (A), survival curve (B), and spleen lymphocyte proliferation ability (C) of ICR suckling mice; Figure 11 is the effect of different treatment groups on the CD3 + CD4 + / CD3 + CD8 +Flow cytometry chart; wherein, A-F are CD3 + CD4 + , wild group (SVA-WT) CD3 + CD4 + , CDS group (SVA-CDS) CD3 + CD4 + , control group CD3 + CD8 + , wild group CD3 + CD8 + , CDS group CD3 + CD8 + ; Figure 12 is the load of each tissue organ of different treatment groups; Figure 13 is the survival curve of ICR mice inoculated with different strains after passage. DETAILED DESCRIPTION

[0020] The application provides a preparation method of a Senecavirus A (SVA) mutant strain, comprising the following steps: inserting lysine before the 9th position of the wild strain 3B protein.

[0021] Since SVA belongs to RNA viruses, the RdRp enzyme has low fidelity, and there may be many SNPs or silent mutations that have no effect on protein structure and function in the genome or protein. Preferably, the GenBank number of the Senecavirus A wild strain genome is MZ818785.1.

[0022] A Senecavirus A mutant strain, with the preservation number of CCTCC NO: V202552.

[0023] A Senecavirus A mutant strain, with the preservation number of CCTCC NO: V202553.

[0024] The above-mentioned Senecavirus A is used for preparing an anti-Senecavirus A drug. For example, the virus strain can be used as a strong strain to evaluate the protective efficacy of a vaccine by comparing the cross-protection ability of different vaccines against the high-toxicity strain, to screen antiviral drugs, and to analyze the molecular correlation between virus mutation and host immune suppression, and to study the immune escape mechanism of the virus.

[0025] The application will be further described below in combination with examples and drawings, but the application is not limited by the following examples.

[0026] Example 1: Screening of Senecavirus A mutant strain 1. Construction and verification of in vitro RNAi system Add nucleotides to both ends of the 5miR and 5miRS sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2. Not Following restriction enzyme cleavage at the I restriction site (GCGGCCGC), artificial synthesis was performed. The aforementioned sequences contain the sequences, complementary sequences, and spacer sequences of five endogenous miRNAs highly expressed in mouse cells: miR-21-5p, miR-31-5p, miR-93-5p, miR-192-5p, and miR-29b-3p. Utilizing... Not The two target fragments were cloned into the 3' end of the enhanced green fluorescent protein in the pEGFP-N1 vector by the I restriction site, and named pEGFP-5miR and pEGFP-5miRS, respectively.

[0027] BHK-21-Ago2 Δ- Cell line CCTCC NO:C2021206 (BHK-Ago2) Δ- ) and wild-type BHK-21, according to 4×10 per hole 5 Cells were seeded in 6-well plates and cultured to 70%-80% confluency. Then, 2.5 μg of pEGFP-5miR and pEGFP-5miRS plasmids were transfected, with a negative control group (Mock) without plasmid transfection and a positive control group (pEGFP-N1) transfected with pEGFP-N1 plasmid. After 36 h of culture, green fluorescence expression was observed under a fluorescence microscope.

[0028] The results are as follows Figure 1 As shown: In wild-type BHK-21 cells, the fluorescence intensity of cells transfected with the pEGFP-5miR plasmid was higher than that of cells transfected with the pEGFP-5miRS plasmid. Figure 1 (A) in BHK-Ago2 Δ- In cells, the fluorescence intensity of cells transfected with pEGFP-5miR plasmid was comparable to that of cells transfected with pEGFP-5miRS plasmid. Figure 1 (B); This indicates that gene silencing can also occur in BHK-21 cells in the presence of endogenous miRNAs, while BHK-Ago2 Δ- Cells cannot achieve gene silencing due to the absence of proteins that mediate gene silencing.

[0029] According to the above method, the BHK-21 cell line is transfected with pEGFP-5miR and pEGFP-5miRS plasmids at the same time, and experimental groups of transfection of miR-21 minic (miR-21), miR-31 minic (miR-31), miR-93 minic (miR-93), miR-192 minic (miR-192), miR-29b minic (miR-29b) and all five minics (all 5) are set up respectively, and miR-30 minic (miR-30) is transfected as a control. After transfection for 36 h, the green fluorescence expression is detected by flow cytometry.

[0030] As shown in Figure 2 , the flow cytometry histogram results show that, compared with miR-30 minic, the remaining miRNA mimics alone or the mixture of five kinds can induce miRNA-mediated silencing and inhibit the expression of fluorescent protein. The above results show that the in vitro RNAi system is successfully constructed.

[0031] 2. Mutant strain screening An intermediate containing a 5miRS sequence fragment of about 500 bp is artificially synthesized, which contains a partial SVA 3D motif at the 5' end and a partial SVA 3'-UTR motif at the 3' end. Then, the restriction enzyme sites of Age I and Not I are used for insertion into the wild-type SVA backbone vector pCI-SVA-WT (reference Li C, Wang H, Shi J, et al. Senecavirus-Specific Recombination Assays Reveal the Intimate Link between Polymerase Fidelity and RNA Recombination [J]. Journal of Virology, 2019. 93(13) construction), to construct the pCI-SVA-5miRS plasmid vector, and the insertion position is shown in Figure 3 .

[0032] BHK-21 cells were placed in a 6-well plate for culture, and when the cells grew to 80% density, 2.5 μg of pCI-SVA-5miRS plasmid vector was transfected, and the cell supernatant was harvested after 72 h and named as P0 generation. BHK-21 cells were continuously placed in a 6-well plate for culture, and when the cells grew to 80% density, 100 μL of P0 supernatant was added, and the cells were adsorbed at 37°C, 5% CO2 cell incubator for 1 h; the virus liquid was discarded, and the cells were washed twice with 1×PBS, and 2 mL of DMEM medium containing 2% FBS was added for continuous culture, and the cell supernatant was harvested after 72 h and named as P1 generation. After continuous subculture, the virus titers of P4 and P10 generations were determined, and the P4 and P10 generation virus RNAs were extracted and subjected to deep sequencing using the Illumina NovaSeq6000 platform (sequencing depth ≥ 100×).

[0033] 3. In vitro virulence evaluation According to the screening results, the SNP and mutation sites were analyzed by SnpEff software, the specific mutations were introduced into the pCI-SVA-WT vector by site-directed mutagenesis, the virus was rescued by transfecting BHK-21 cells, and the mutant strains with higher mutation frequency in the CDS region were subjected to virulence evaluation: the RNAs of P4 and P10 harvested viruses were extracted, PCR was performed using high-fidelity enzyme, the products were sequenced, and the strains that still belonged to SVA and had stable mutations were classified and subjected to determination of virus titer (TCID 50 method) and lesion rate (CPE observation), and the high virulence mutant strains 1494K and CDS were screened.

[0034] The specific mutations were introduced into the pCI-SVA-WT vector by site-directed mutagenesis, the virus was rescued by transfecting BHK-21 cells, and the growth curve of the mutant strain was determined. The P4 generation virus growth curve of the 1494K mutant strain is shown in Figure 4 , and the results show that the mutant strain virus titer is always higher than that of the wild type strain after being infected with the same virus amount (MOI=0.01). The P10 generation was taken to determine the virus titer, and the results show that the virus titer of the mutant strain in BHK-21 cells can be as high as 10 10.8 TCID 50 / mL, which is 1.3 log higher than that of the wild type strain (10 9.26 TCID 50 / mL). The CPE appears 6 hours earlier ( Figure 5 ).

[0035] The P4 generation virus titer of the CDS mutant strain is significantly higher than that of the wild type strain ( Figure 6 ).

[0036] The two mutant strains mentioned above have a lysine (Lys, K) inserted at position 1494, i.e., AAA is inserted at position 5417 of the genome. The mutation frequency of the P4 generation virus of the 1494K strain at this site is 99.85%, and the mutation frequency of the P10 generation virus is 100%, indicating stable inheritance. The mutation frequency of the P4 generation virus of the CDS strain at this site is 99.85%, and the mutation frequency of the P10 generation virus is 100%, indicating stable inheritance.

[0037] Table 1 SNPs mutation analysis of P4 generation virus of CDS mutant strain Table 2 SNPs mutation analysis of P10 generation virus of CDS mutant strain Through the WeMol platform, the 3B structure of the insertion site was predicted, and the results showed that the protein structure had changed Figure 7 ).

[0038] The SVA-1494K strain was preserved in the China Center for Type Culture Collection (CCTCC) on June 10, 2025, the preservation address is Wuhan University, Wuhan, China, and the preservation number is CCTCC NO: V202552.

[0039] The SVA-CDS strain was preserved in the China Center for Type Culture Collection (CCTCC) on June 10, 2025, the preservation address is Wuhan University, Wuhan, China, and the preservation number is CCTCC NO: V202553.

[0040] Example 2 In vivo virulence evaluation of Senecavirus A mutant strain 1494K Two-week-old BALB / c mice were randomly divided into control group, mutant strain group and wild strain group (n=10), the control group was injected intraperitoneally with DMEM, the challenge group was inoculated intraperitoneally with 1×10 8 TCID 50 Different viruses, 14 days after infection, serum was collected to detect neutralizing antibodies, spleen was taken to detect lymphocyte proliferation (CCK-8 method) and T cell subsets (flow cytometry), and various organs were taken for HE staining pathological analysis.

[0041] The results show that the body weight gain of the mutant strain infection group of mice decreased significantly Figure 8 Neutralizing antibody titer (1:215) in serum was reduced by 33.85% compared with the wild strain group (1:325) Figure 8 Spleen lymphocyte proliferation ability (95% vs. 113%) and CD3 + CD4 + / CD3 + CD8+ The ratio (0.80 vs. 1.12) was significantly reduced Figure 8 Medium C and Figure 8 Medium E). The viral load in the heart, liver, spleen, lung and intestine of the mutant group mice was significantly higher than that of the wild group mice Figure 8 Medium D), and the pathological results Figure 9 ) showed that the overall structure of the intestinal tissue of the control group was basically normal, the mucosa layer was basically normal, the glands were arranged in order, goblet cells (red arrow) were visible, the interstitial lymphocyte infiltration was large, and the submucosal layer and muscle layer were normal; the overall structure of the intestinal tissue of SVA-WT was abnormal, the mucosal glands were reduced, the gland structure was abnormal (yellow arrow), the goblet cells were reduced (red arrow), the intestinal villus structure was reduced, the interstitial lymphocyte infiltration was large, and the submucosal layer and muscle layer were normal; the overall structure of the intestinal tissue of SVA-1474K was abnormal, the mucosal glands were reduced, the gland structure was slightly abnormal, the surface was eroded (yellow arrow), the goblet cells were reduced (red arrow), the intestinal villus structure was reduced, the interstitial lymphocyte infiltration was large, and the submucosal layer and muscle layer were normal. This shows that the degree of intestinal damage of the mutant strain group is higher than that of the wild strain group.

[0042] Example 3 In vivo virulence evaluation of CDS of Senecavirus A mutant strain 1. ICR mouse model to evaluate virulence 0-day-old ICR mice were randomly divided into control group, mutant strain group and wild strain group (n=10), the control group was injected intraperitoneally with DMEM, the challenge group was inoculated intraperitoneally with 1x10 8.5 TCID 50 Different viruses, observe the clinical condition and virus mortality, weigh the body weight every day after infection, collect the spleen 7 days after infection to detect lymphocyte proliferation (CCK-8 method) and T cell subsets (flow cytometry), and take the same weight of organs to detect viral load. After 7 days of feeding, the ICR mice were sacrificed by the neck, and all organs were ground to supernatant to inoculate the second batch of 0-day-old ICR mice, and observed for 7 days, and the virus mortality was counted.

[0043] The results are shown in Figure 10 The body weight of the mice was weighed every day after challenge, and the body weight of the challenge group and the control group increased smoothly, but from the average body weight, the challenge group mice had no significant difference compared with the control group 1-2 days after challenge, but the body weight change was gradually significant compared with the control group 3-7 days after challenge (p<0.05) Figure 10 Medium A). The mortality rate of the CDS mutant strain infected group of mice within 7 days was 10% (n=10), and there was no death in the wild strain infected group (n=10) Figure 10 Medium B). The spleen lymphocyte proliferation ability (SI=0.85) of the CDS group was higher than that of the wild group Figure 10 Medium C). Flow cytometry showed that the CD3+ CD4 + / CD3 + CD8 + The proportion was higher than that of the wild group, and the CDS group and the wild group were 1.35 and 1.12 times that of the control group, respectively (P < 0.05). Figure 11 The viral load in each tissue and organ is shown in Table 2: the viral load in the liver and intestinal tract of the CDS group was 2-3 times higher than that of the wild group. Figure 12

[0044] After 7 days of feeding, ICR mice were killed by the neck, and all organs were ground to obtain supernatant, which was inoculated into the second batch of 0-day-old ICR mice. The mortality rate of the SVA-CDS group reached 40% (n = 10). Figure 13

[0045] The above only describes the embodiments of the present application and is not intended to limit the present application. The present application can have various modifications and changes for those skilled in the art. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application shall be included in the scope of the claims of the present application.​​​

Claims

1. A method of constructing a mutant strain of Senecavirus A, characterized by, Comprising the following steps: inserting lysine before the 9th amino acid of the wild strain 3B protein; The GenBank number of the genome of the Senecavirus A wild strain is MZ818785.

1.

2. A Senecavirus A mutant strain obtained by the construction method of claim 1.

3. The Seneca Valley virus A mutant of claim 2, wherein, It is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC NO: V202552.

4. The Seneca Valley virus A mutant of claim 2, wherein, It is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC NO: V202553.

5. Use of the Senecavirus A mutant strain of any one of claims 2-4 in the preparation of an anti-Senecavirus A drug.

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