Bovine nodular skin disease virus ORF135 gene deletion strain and application thereof

Abstract

The invention relates to a bovine nodular skin disease virus, in particular to a bovine nodular skin disease virus ORF135 gene deletion strain and application thereof. The gene deletion strain is characterized in that the gene deletion strain is constructed by deleting an ORF135 (Open Reading Frame 135) gene from a bovine nodular skin disease virus virulent strain LSDV XJ201901. Compared with a parent strain, the replication level of the constructed deletion strain rLSDVdelta135 in in-vitro cells is equivalent to that of the parent strain, which indicates that gene deletion does not affect the in-vitro multiplication capacity of the virus. In addition, after MDBK cells are infected with the rLSDVdelta135, the mRNA expression level of proinflammatory factors IL-1beta and IL-6 can be remarkably reduced, and meanwhile expression of IFN beta mRNA is not interfered. Further transcriptomics analysis shows that the toxicity and pathogenicity of the deletion strain are obviously reduced on the basis of keeping good immunogenicity; the vaccine not only loses the ability to inhibit natural immune pathways, but also can effectively activate adaptive immune response and obviously reduce inflammatory response and tissue injury risks, and shows significant potential as a safe and efficient attenuated live vaccine.

Description

TECHNICAL FIELD

[0001] The present application relates to bovine tuberous skin disease virus, in particular to bovine tuberous skin disease virus gene deletion strain and its application. BACKGROUND

[0002] Lumpy Skin Disease (LSD) is an acute and subacute infectious disease caused by Lumpy Skin Disease Virus (LSDV) of Capripoxvirus genus in Poxviridae family. The core susceptible population is bovine animals, among which the symptoms of yellow cattle and dairy cattle after infection are the most typical. Although buffaloes and yaks can be infected, the symptoms are usually mild. In terms of harm, infected cattle usually exhibit systemic symptoms such as high fever (body temperature up to 40-41℃), listlessness, and loss of appetite. The skin and mucous membranes (such as the oral cavity, nasal cavity, and reproductive tract mucosa) will develop 1-5 centimeter diameter round or oval firm nodules. Some nodules are prone to bacterial infection after breaking down, causing suppuration and necrosis, which brings huge economic losses to the livestock industry.

[0003] Based on the analysis of the whole genome sequence of LSDV, LSDV can be divided into two main branches, and each main branch can be divided into two sub-branches. LSDV XJ201901 belongs to the vaccine recombinant sub-branch, which may be generated by recombination of wild strong strains and vaccine strains in Europe and Africa. LSDV XJ201901 (GenBank No: OM984485) is a strong strain, and its ORF135 gene is highly homologous to the ORF134 gene of the traditional strong strain Kenya (GenBank No: MN072619).

[0004] Vaccination is an important means that cannot be replaced in the prevention and control system of bovine lumpy skin disease. At present, the vaccine against LSD on the market in China is an inactivated vaccine, but the inactivated vaccine has the disadvantages of large immunization dose and weak cross-protection ability. Internationally, Neethling attenuated vaccine is mainly used for immunization to prevent and control LSD epidemic, and the strain has been isolated for many years. The side effects are serious, and the attenuated vaccine strain can replicate in the vaccinated animals, increasing the risk of recombination and transmission of the strain in non-epidemic areas, which is not suitable for the current domestic bovine lumpy skin disease epidemic.

[0005] Therefore, it is of great significance to construct a safe, effective and suitable LSDV attenuated vaccine for China. By comparing and analyzing the whole genome of wild strong strains and attenuated vaccine strains of LSDV, potential virulence genes of strong strains are screened out, and the virulence genes of domestic LSDV epidemic strains are deleted by using homologous recombination method to reduce their virulence, thereby providing a strong guarantee for the prevention and control of domestic LSD epidemic. SUMMARY

[0006] OBJECTIVE In order to construct a safe, effective and suitable LSDV attenuated vaccine in China, the application provides a bovine dermatophilus virus ORF135 gene deletion strain and a construction method and application thereof.

[0007] Technical scheme The functional domain encoded by the ORF135 gene is crucial for maintaining the virulence of the bovine dermatophilus virus (LSDV), and the functional domain is highly conserved among different virus strains.

[0008] Based on this finding, the application proposes that targeted deletion of the ORF135 gene or the functional domain encoded thereby can be used as a general strategy for constructing an LSDV attenuated vaccine strain. This strategy is expected to be applicable to LSDV strains with different genetic backgrounds. To verify the feasibility of this strategy, the application takes the bovine dermatophilus virus strain LSDV XJ201901 as a specific example. Specifically as follows: An LSDV attenuated strain, characterized in that the ORF135 gene and / or the encoded protein of the attenuated strain are functionally lost or partially lost.

[0009] The LSDV attenuated strain, characterized in that the functional loss or partial loss is achieved by deleting all or part of the sequence of the ORF135 gene.

[0010] A bovine dermatophilus virus gene deletion strain, characterized in that the strain is an LSDV XJ201901 strain with the ORF135 gene deleted.

[0011] The application deletes the ORF135 gene of the LSDV XJ201901 strain by a homologous recombination method and a Cre / loxP system.

[0012] Taking the bovine dermatophilus virus strain LSDV XJ201901 as an example, the application constructs an LSDV gene deletion strain in MDBK cells by using a homologous recombination method and a Cre / loxP system. The method recombines a transfer vector targeting the virulence gene ORF135 with the LSDV XJ201901 strain to obtain rLSDVΔ135eGFP through fluorescent plaque screening, and then transfects a pCMV-Cre plasmid to knock out the fluorescent gene expression cassette, so as to obtain the LSDV gene deletion strain through non-fluorescent plaque purification.

[0013] Specifically: The construction method of the bovine dermatophilus virus gene deletion strain of the application includes the following steps: According to the genome sequence of the bovine dermatophilus strain LSDV XJ201901, the upstream and downstream primers of the homologous arms on the left and right sides of LSDV ORF135 are designed, the genome of the bovine dermatophilus is amplified by PCR, and the sequences of the homologous arms on the left and right sides of ORF135 are obtained respectively, and the homologous arms on the left and right sides of ORF135 are inserted into pBluescript KS(+) by using Xho I, Kpn I, Sac I and Sac II enzyme cutting sites, and a transfer vector pBluhm135-loxPmH5eGFP is obtained. In the previous experiment, it is found that the mH5 promoter can quickly and efficiently start the expression of the fluorescent gene in the MDBK cells infected by the bovine dermatophilus, therefore, the mH5 is selected as the promoter of the screening gene expression cassette.

[0014] The mH5eGFP fragment in the pUC57-mH5eGFP plasmid is amplified by PCR and recovered, connected to pBS302-loxP, and the loxPmH5eGFP is amplified and connected between the left and right homologous arms of pBlu-LSDV135L+R plasmid, and a transfer vector pBluhm135-loxPmH5eGFP is obtained. After the bovine dermatophilus is infected with the MDBK cells, the constructed transfer vector pBluhm135-loxPmH5eGFP is transfected into the MDBK cells, and after purification, the bovine dermatophilus gene deletion fluorescent strain rLSDVΔ135eGFP is obtained. The MDBK cells are infected with the LSDV gene deletion strain rLSDVΔ135eGFP, and the pCMV-Cre plasmid is transfected to knock out the mH5eGFP fluorescent screening expression cassette, and the non-fluorescent plaque is picked up and purified to obtain the LSDV gene deletion strain rLSDVΔ135. A vaccine comprising the LSDV attenuated strain. The use of the attenuated strain or the vaccine in the preparation of a medicine for preventing bovine dermatophilus.

[0015] The use is characterized in that the bovine dermatophilus is caused by the bovine dermatophilus virus. Beneficial effects

[0016] 1、The application analyzes and compares the whole genomes of the traditional virulent strain Kenya strain and the attenuated vaccine strain Neethling strain (GenBank number: KX764645) of bovine nodular dermatopathy virus, finds that there is a significant difference between the genome sequences of the virulent strain and the vaccine strain in ORF134, the mutation of ORF134 of the vaccine strain is truncated into two open reading frames (ORFs), and it is analyzed that ORF134 is a potential virulence gene of the virulent strain of LSDV, and ORF134 of the Kenya virulent strain is highly homologous to ORF135 of the XJ strain. Therefore, the application first proposes to construct a bovine nodular dermatopathy virus gene deletion strain targeting ORF135, i.e., deleting ORF135 of the LSDV XJ201901 strain. Compared with the parent virus, the deletion strain can reduce the mRNA level of intracellular pro-inflammatory factors IL-1beta and IL-6 after infecting MDBK cells, the inflammatory reaction is more moderate, the safety is higher, the immunogenicity is retained while the risk of immunopathology is reduced. In addition, the replication level of the constructed rLSDVΔ135 on the in vitro cells is the same as that of the parent virus, which does not affect the production of the gene deletion virus, and its application is not reported in the literature; the deletion virus strain rLSDVΔ135 is a live attenuated vaccine candidate strain with good application prospect, which retains the immunogenicity while significantly reduces the virulence and inflammatory reaction risk, and can effectively stimulate comprehensive natural immunity and adaptive immune response.

[0017] 2、The deletion virus strain does not have resistance genes, fluorescent markers and other exogenous genes, which meets the current biological safety law of genetically engineered products; at the same time, the deletion of the ORF135 gene can also be used as a molecular marker to distinguish vaccine strains and wild virulent strains, which is beneficial to LSD infection detection and immune monitoring in breeding farms; in addition, the gene deletion strain is constructed from domestic epidemic strains, which will not introduce domestic non-epidemic strains, and has certain biological safety. In addition, since ORF135 is a conserved sequence of the virulent strain of bovine nodular dermatopathy virus, theoretically, deleting any kind of domestic bovine nodular dermatopathy virus strain has similar functions.

[0018] 3、The commonly used methods for obtaining weak viruses include: (1) screening and isolation of weak viruses in nature; (2) passage of heterologous animals or cells to weaken; (3) using genetic engineering methods to recombine the virulent strain. The isolation from nature and the passage of heterologous animals or cells to weaken have the disadvantages of large workload, long time for weakening, etc., such as the swine fever rabbitized attenuated vaccine which is passed in rabbits for 240 generations; And the application can realize the modification of the genome of wild strong strain by genetic engineering method, greatly shortens the vaccine preparation period, and compared with natural mutant strain (most of which are point mutant strains), the gene deletion strain deletes several hundred or even several thousand bases of the target gene, the deletion region is clear, has the advantages of clear mutation characteristics, stability and not easy to return to the original state, and is an important way to study safe and effective new vaccines. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 , Homology analysis of ORF134 protein of LSDV strong strain and vaccine strain; Figure 2 , Detection of transcription level of virulence genes of LSDV infected MDBK cells; Figure 3 , Schematic diagram of transfer vector construction; Figure 4 , Generation of recombinant fluorescent virus; Figure 5 , PCR identification results of recombinant fluorescent virus; Figure 6 , PCR identification results of recombinant deletion LSDV virus; Figure 7 , In vitro growth curve of LSDV parent virus and deletion strain rLSDVΔ135; Figure 8 , Cytokine detection after infection of MDBK by LSDV parent virus and deletion strain rLSDVΔ135, A is IFNβ mRNA level of MDBK cells after infection of virus, B is IL-6 mRNA level of MDBK cells after infection of virus, and C is IL-1β mRNA level of MDBK cells after infection of virus. DETAILED DESCRIPTION

[0020] Example 1 Screening of virulence genes of bovine nodular dermatitis virus Natural immunity is the first line of defense against viral infection in the body, and is also the normal physiological defense barrier of the body. It mainly includes natural physiological barrier (skin and mucosal barrier, placental barrier, blood-brain barrier, etc.) and innate immune response, and immune cells and immune molecules are produced by innate immune response. In order to counteract the immune defense of the host, poxvirus has evolved a variety of immunomodulators, which have a variety of strategies to destroy or evade the host antiviral response triggered by pattern recognition receptors (PRRs), and affect the results of poxvirus infection.

[0021] Promoter is a DNA sequence that controls the initiation of gene transcription. Poxvirus has a unique life cycle, replicating and transcribing in the cytoplasm of host cells without entering the nucleus, so the poxvirus uses the RNA polymerase and related transcription factors encoded by the virus itself, and selecting a suitable promoter is crucial for high-level expression of foreign genes in host cells. The mH5 promoter is a commonly used late promoter in poxvirus vector systems, which has been proven to rapidly and efficiently express foreign genes in MDBK cells.

[0022] Based on the above research progress, the present application compares the main difference proteins of the domestic LSDV epidemic strain LSDV XJ201901 and the Kenya virulent strain and the Neethling vaccine strain, screens potential virulence genes, and verifies the mRNA level expression of the virulence genes in the in vitro infection of LSDV XJ201901.

[0023] 1 Materials and methods 1.1 Cells and viruses MDBK cells were purchased from the China Academy of Sciences Cell Bank; the LSDV XJ201901 strain was preserved in the China Animal Health and Epidemiology Center, and the operation experiments of the live virus of the LSDV involved in the present application were completed in the biosafety level 3 laboratory of the China Animal Health and Epidemiology Center.

[0024] 1.2 Main reagents Fetal bovine serum used for MDBK cells was purchased from PAN Biotech; penicillin and streptomycin were purchased from Biyun Tian Biological Technology Co., Ltd.; 1640 culture medium was purchased from Shanghai Yuancheng Biological Technology Co., Ltd.; the transfection reagent used in the present application was purchased from Promega Company; Opti-MEM was purchased from Gibco; the plasmid extraction kit was purchased from Qiagen; Taq enzyme and DH5a competent cells were purchased from Takara.

[0025] 2 Experimental steps 2.1 Screening of bovine dermal nodular disease virus virulence genes According to the literature research, and by comparing the homology of LSDV XJ201901 and LSDV virulent strain Kenya and LSDV Neethling vaccine strain, the genes that may cause the virulence of LSDV virulent strain to decrease were screened out.

[0026] 2.2 Verification of the transcription level of potential virulence genes in virus-infected cells According to the genome sequence of the LSDV XJ201901 strain, the ORF135 and actin mRNA primers were designed, and the primer sequences are shown in Table 1. The LSDV virus-infected MDBK cells were sampled at 24 h, and the RNA was extracted and reverse transcribed, and then the mRNA level of the potential virulence genes was detected by PCR.

[0027] Table 1 Primers for LSDV mRNA detection

[0028] 3. Experimental Results 3.1 Homology analysis of potential virulence genes between domestic LSDV strains and classic virulent strains and vaccine strains like Figure 1 As shown, gene sequence analysis of LSDV XJ201901, the virulent LSDV Kenya strain, and the LSDV Neethling vaccine strain revealed differences between the virulent strain and the vaccine strain in ORF134 (Kelch-like protein). The vaccine strain's ORF134 mutation truncates it into two proteins. Therefore, ORF135 is a potential virulence gene for the LSDV XJ201901 strain and can serve as a target for gene-deleted attenuated LSDV vaccines.

[0029] 3.2 Detection of transcriptional levels of virulence genes in LSDV-infected MDBK cells 24 hours after LSDV infection of MDBK cells, the culture supernatant was discarded, cells were lysed, RNA was extracted and reverse transcribed, and the LSDVORF135 mRNA level was detected. Results are as follows: Figure 2 The results showed that bands of ORF135 were detected in all cells, indicating that the ORF135 target genes were transcribed 24 hours after LSDV infection of MDBK cells.

[0030] Example 2: Construction and biological characteristics analysis of a gene-deleted strain of bovine nodular dermatitis virus. This invention selects to delete ORF135 of LSDV XJ201901, constructs rLSDVΔ135 gene-deleted strain through homologous recombination, and analyzes its biological characteristics, providing technical support for the development of LSDV gene-deleted vaccines.

[0031] 1. Materials and Methods 1.1 Cells and Viruses MDBK cells were purchased from the Cell Bank of the Chinese Academy of Sciences; the LSDV XJ201901 strain was isolated and preserved by the China Animal Health and Epidemiology Center. All LSDV live virus manipulation experiments involved in this invention were performed in the Biosafety Level 3 laboratory of the China Animal Health and Epidemiology Center.

[0032] 1.2 Main Reagents Fetal bovine serum used in MDBK cells was purchased from PAN Biotech; penicillin and streptomycin were purchased from Beyotime Biotechnology Co., Ltd.; 1640 medium was purchased from Shanghai Yuanpei Biotechnology Co., Ltd.; transfection reagents used in this invention were purchased from Promega; Opti-MEM was purchased from Gibco; plasmid extraction kits were all purchased from Qiagen; qPCR reagents, Taq enzyme, and DH5α competent cells were purchased from Takara.

[0033] 2. Experimental Procedure 2.1 Construction of transfer vector plasmids like Figure 3 As shown, primers for the homologous arms on both sides of LSDVORF135 were designed based on the genome sequence of bovine nodular dermatitis virus strain LSDV XJ201901. The genome of bovine nodular dermatitis virus was amplified by PCR, and the sequences of the homologous arms on both sides of ORF135 were obtained. The homologous arms on both sides of ORF135 were inserted into pBluescript KS(+) using Xho I, Kpn I, Sac I and Sac II restriction sites to obtain the transfer vector pBluhm135-loxPmH5eGFP. Preliminary experimental screening revealed that the mH5 promoter can rapidly and efficiently initiate the expression of fluorescent genes in MDBK cells infected with bovine nodular dermatitis. Therefore, this invention selected mH5 as the promoter for screening gene expression cassettes.

[0034] The mH5eGFP fragment in the pUC57-mH5eGFP plasmid was amplified and recovered by PCR, ligated into pBS302-loxP, and the loxPmH5eGFP was amplified and ligated between the left and right homologous arms of the pBlu-LSDV135L+R plasmid to obtain the transfer vector pBluhm135-loxPmH5eGFP (its nucleotide sequence is shown in SEQ ID NO.23). The transfer vector will achieve complete deletion of the target gene through homologous recombination. The primers used are shown in Table 2.

[0035] Table 2 Primers used to construct the transfer vector

[0036] 2.2 Construction of LSDV gene-deleted strains This invention utilizes homologous recombination to construct a deletion strain, and then transfects the transfer vector into MDBK cells infected with 0.1 MOI LSDV. The specific procedures are as follows: (1) Seed 3 × 10^5 MDBK cells into a six-well plate; (2) After the cell plate is cultured for 16-18h, the cell density is about 60%, the supernatant is discarded, the LSDV virus liquid is diluted with serum-free 1640 culture medium, and the MDBK cells are inoculated at 0.1 MOI infection amount, and are infected at 37 DEG C for 2h; (3) The transfection liquid is prepared: 2ug plasmid + 100ul Opti-MEM + 6ul FuGene transfection reagent, and after being mixed gently, it is placed for 5min; (4) After the MDBK cells are infected for 2h, the virus liquid is discarded, and the 1640 culture medium with 2% FBS and 1% double antibody is replaced, the transfection liquid is added dropwise, and after being mixed, it is placed and cultured at 37 DEG C.

[0037] When the cytopathic effect reaches 80%, the virus is harvested, the virus liquid is inoculated into new MDBK cells after repeated freeze-thawing for 3 times, and the production of the recombinant fluorescent virus is detected by using a fluorescence microscope.

[0038] 2.3 Purification of the LSDV gene deletion fluorescent strain The recombinant virus is purified by the plaque picking purification method, a typical virus plaque with fluorescence is picked in the 1640 culture medium, is inoculated into MDBK cells after freeze-thawing for 3 times at-80 DEG C, is placed and cultured at 37 DEG C after being infected for 2h, and is placed and cultured at 37 DEG C after being infected for 2h. Low-melting-point agarose is laid, and when the virus plaque with fluorescence appears, the operation is repeated until a single virus plaque with fluorescence appears. The single virus plaque is continuously expanded to the third generation, the nucleic acid of the virus is extracted, and the purity of the virus is identified by PCR. The PCR identification primers are listed in Table 3.

[0039] Table 3 PCR primers for identifying the LSDV deletion strain

[0040] 2.4 Knockout and purification of the fluorescent gene of the LSDV gene deletion fluorescent strain The fluorescent strain with good purity is inoculated into MDBK cells at 0.1 MOI dose, is replaced with 2% FBS maintenance liquid after being infected for 2h, and is transfected with the pCMV-Cre plasmid, and is cultured at 37 DEG C until the cytopathic effect is more than 80% to harvest the virus. After repeated freeze-thawing for 3 times, 10ul of the virus liquid is used to infect MDBK cells, and the non-fluorescent plaque is picked to purify, until a single non-fluorescent plaque appears. After the non-fluorescent plaque is continuously expanded for 3 generations, the nucleic acid of the virus is extracted, and the purity of the gene deletion strain is verified by PCR.

[0041] 2.5 In vitro replication kinetics verification of the LSDV gene deletion strain MDBK cells were infected with 0.1 MOI of LSDV-WT and rLSDVΔ135, and viruses were collected at 12 h, 24 h, 48 h, 72 h, 96 h, 120 h and 144 h after infection, respectively. The virus collected at different time points was determined for titer after being frozen and thawed three times at -80°C, and the growth curves of the strains in MDBK cells were plotted.

[0042] 2.6 Detection of cytokine mRNA in MDBK cells infected with LSDV parent virus and deletion virus MDBK cells were infected with 0.1 MOI of LSDV-WT and rLSDVΔ135, and viruses were collected at 4 h, 8 h, 16 h and 24 h after infection, respectively. The cell culture supernatant was discarded, and the cell RNA was extracted. After reverse transcription, the qPCR method was used to detect the mRNA levels of IFNβ, IL-1β and IL-6 in MDBK cells. The detection primers are shown in Table 4.

[0043] Table 4 Primers for detecting cytokine mRNA in MDBK cells

[0044] 2.7 Transcriptome sequencing analysis of LSDV parent virus and deletion virus Nine 100 mm cell culture dishes of MDBK cells were prepared, and the cells were treated when the cell density was greater than 90%. The cells were divided into three groups, each group of three, and the MDBK cells were infected with 0.1 MOI of LSDV-XJ and rLSDVΔ135, respectively, and a blank control group without virus infection. After treatment, the samples were placed in a 37°C cell sample incubator for continuous culture, and the samples were collected at 16 h after infection. The cells were collected in a cryogenic tube with 1 mL Trizol, and then frozen in liquid nitrogen. The transcriptome sequencing analysis was performed by Hangzhou Lianchuan Biological Technology Co., Ltd.

[0045] 3 Experimental results 3.1 Construction of a transfer vector targeting ORF135 In the present application, pBluescript KS(+) is used as the initial plasmid, the homologous arms (1000 bp) flanking the target site are inserted, the same loxp gene sequences are added at both ends of the sequence of the fluorescent screening expression box, and the transfer vector targeting ORF135 pBluhm135-loxPmH5eGFP is obtained by inserting the loxp gene sequences into the homologous arms flanking the target site. Figure 4 ).

[0046] 3.2 Construction of a fluorescent strain with LSDV gene deletion The ORF135-targeting transfer vector was transfected into LSDV-infected MDBK cells. By day 5, 80% cytopathic effect was achieved. The viral load was harvested and subjected to three freeze-thaw cycles at -80°C. 10 μL of the viral load was then inoculated into MDBK cells, and recombinant fluorescent virus production was observed by day 3. Figure 5 ).

[0047] 3.3 Purification of LSDV gene-deleted fluorescent strains The obtained recombinant fluorescent virus was purified by multiple plaque selections. Single fluorescent plaques were then selected and propagated normally three times consecutively. DNA was extracted and identified by PCR. Results are as follows: Figure 6 The results showed that the primers on the left amplified the target eGFP and produced a specific band, while the primers on the right amplified the wild-type target and produced no band. This demonstrates that the rLSDVΔ135eGFP recombinant fluorescent virus is of good purity and free from contamination by the wild-type parent virus.

[0048] 3.4 Knockout and purification of fluorescent genes in LSDV gene-deleted strains After infecting MDBK cells with a highly purified recombinant fluorescent virus, they were transfected with the pCMV-Cre plasmid. By day three, 80% cytopathic effect was achieved. The virus solution was harvested and subjected to three freeze-thaw cycles at -80°C. 10 μL of the virus solution was then inoculated into MDBK cells, resulting in the formation of fluorescent knockout plaques. After repeated plaque removal and purification, a non-fluorescent LSDV deletion strain was obtained. Viral DNA was extracted and identified by PCR. The results are as follows: Figure 7 The results showed that the target gene of the recombinant LSDV was successfully knocked out, and the purity was good.

[0049] 3.5 Validation of in vitro replication kinetics of LSDV gene-deleted strains To verify whether the missing target site affects viral growth and replication at the cellular level, this study measured the viral titer collected at different time points and plotted the growth curve of the LSDV gene-deleted strain. The results are as follows: Figure 8 The results showed that the deletion of the ORF135 gene in the LSDVXJ201901 strain did not affect viral replication in MDBK cells, and ORF135 is a non-essential region for replication of the LSDVXJ201901 strain.

[0050] 3.6 Detection of cytokine mRNA levels after infection with parental LSDV and deletion virus in MDBK To investigate whether infection of cells with a gene-deleted viral strain affects changes in immune levels, this study collected MDBK cells at 4h, 8h, 16h, and 24h after viral infection. The mRNA levels of cytokines IL-1β, IL-6, and IFNβ were detected by qPCR. The structures are shown in the figure below. Figure 8A-C show that rLSDVΔ135 down-regulates IL-1β, IL-6 mRNA levels, while IFNβ mRNA level is not affected after infecting MDBK compared with LSDV WT strain.

[0051] 3.7 Transcriptome sequencing analysis of LSDV parental virus and gene deletion strain Based on the comprehensive analysis of transcriptome data (as shown in Table 5), the gene deletion strain rLSDVΔ135 showed good live attenuated vaccine potential. Compared with the parental virus LSDV XJ201901 infection, rLSDVΔ135 significantly weakened the inflammatory response and improved the safety while maintaining the immunogenicity. Specifically, in terms of natural immunity, rLSDVΔ135 up-regulated genes such as CHUK, IFNE, IRF5, STAT1 and TRAF3, effectively activated NF-κB, interferon and TLR signaling pathways. In terms of inflammatory response, unlike the parental virus that strongly induced IL-1β, IL6, CX3CL1, CXCL3, CXCL5, CXCL8 and GRO1, etc., in the rLSDVΔ135 group, the expression of these genes had no significant change or slightly up-regulated, showing milder inflammation, reduced neutrophil recruitment and reduced risk of tissue damage. At the same time, the strain retained the expression of tissue repair genes such as INHBA and IL20RB, and weakened MMP9, reflecting good safety. In terms of adaptive immunity, rLSDVΔ135 maintained the expression of CCL17, PIK3CD, CD80, IL12RB2, IL7R, TNFRSF4, TNFSF8 and TNFRSF17, etc., and had the ability to recruit Th2 / Treg, promote T / B cell proliferation and differentiation, activate T cells and support antibody secretion. In terms of apoptosis and stress, rLSDVΔ135 retained the activation of pro-apoptotic / stress genes such as CYCS, GADD45A, EIF2S1, BBC3, PMAIP1 and BCL2L11, and down-regulated anti-apoptotic genes such as BIRC3 and XIAP, promoting the clearance of infected cells, which is conducive to virus clearance and body recovery, further ensuring vaccine safety.

[0052] Table 5 Transcriptomic analysis of LSDV parental virus and deletion strain rLSDVΔ135 infected MDBK

[0053] Discussion At present, most of the vaccines used for the prevention and control of LSD in the world are attenuated live vaccines based on LSDV, GTPV or SPPV. Based on the international prevention and control situation, the use of homologous LSDV vaccine can eradicate the LSD epidemic, and the research and use of LSDV vaccine are crucial for the development of the cattle industry. The attenuated LSDV vaccine has the risk of infection caused by recombination with field wild strong virus, while the gene deletion engineering vaccine can effectively distinguish vaccine strains and wild strains. The present application takes domestic LSDV epidemic strain LSDV XJ201901 as the parent virus, and deletes the ORF135 gene to construct the rLSDVΔ135 gene deletion strain, which lays a foundation for the development of a gene engineering vaccine suitable for the prevention and control of domestic LSD epidemic.

[0054] The innate immune system is the first line of defense against the invasion of foreign pathogens, and the inflammation it produces reacts to harmful stimuli and is strictly controlled by the host. Some viruses, such as highly pathogenic coronaviruses, influenza A and Ebola virus, can cause excessive and long-term cytokine response, which is called "cytokine storm", leading to death of the body. Therefore, excessive immune regulation can lead to collapse of the autoimmune system, while insufficient inflammatory factors can lead to persistent infection of pathogens. Persistent or improper regulation can lead to excessive inflammation, causing chronic or systemic inflammatory diseases, and causing immunopathological damage to the body. The present application constructs a LSDV gene deletion strain rLSDVΔ135, which has the same replication capacity as the parent virus, while the levels of inflammatory factors IL-1β and IL-6 induced by the gene deletion strain are significantly lower than those of the parent strain, reducing the high level of inflammatory response induced by the parent virus. Based on the results of further transcriptome analysis, rLSDVΔ135 significantly reduces the expression of pro-inflammatory cytokines and chemokines (such as IL6, CXCL8, GRO1) while effectively activating innate immunity (such as through TRAF3, STAT1, TICAM1) and adaptive immunity (such as through IL7R, TNFSF8, TNFRSF17), indicating that the inflammatory response it induces is more moderate and has higher potential safety. In addition, rLSDVΔ135 retains pro-apoptotic / stress signals and weakens anti-apoptotic signals, suggesting that it may be more easily cleared by the body and be conducive to the recovery of homeostasis after infection. In summary, rLSDVΔ135 significantly reduces the risk of immunopathology while retaining immunogenicity, showing good prospects for attenuated live vaccine application.

[0055] Therefore, rLSDVΔ135 has the potential to develop LSD vaccines.

Claims

1. A bovine tubercle skin disease virus (BVDV) live-attenuated strain, characterized in that, It is constructed by deleting ORF135 gene of virus strain LSDV XJ201901 completely or partially.

2. The attenuated strain according to claim 1, characterized in that, It is constructed by homologous recombination method and Cre / loxP system.

3. The attenuated strain according to claim 2, characterized in that, It is constructed by deleting ORF135 gene of LSDV XJ201901 virus strain in MDBK cells by homologous recombination method and Cre / loxP system.

4. The attenuated strain according to claim 3, characterized in that, It is constructed by the following steps: by constructing a transfer vector targeting the virulence gene ORF135, recombining the transfer vector with the LSDV XJ201901 virus strain, obtaining rLSDVΔ135eGFP by fluorescent plaque screening, then transfecting pCMV-Cre plasmid to knock out the fluorescent gene expression cassette, and obtaining the LSDV gene deletion strain after purification of non-fluorescent plaques.

5. The attenuated strain according to claim 4, wherein, The upstream and downstream primers of the homologous arms on the left and right sides of LSDV ORF135 are designed according to the genomic sequence of the bovine dermatophilus strain LSDV XJ201901, and the genome of the bovine dermatophilus virus is amplified by PCR to obtain the sequences of the homologous arms on the left and right sides of ORF135, and the homologous arms on the left and right sides of ORF135 are inserted into pBluescript KS(+) using Xho I, Kpn I, Sac I and Sac II enzyme cutting sites to form pBlu-LSDV135L+R plasmid; The mH5eGFP fragment in the pUC57-mH5eGFP plasmid is PCR amplified and recovered, connected to pBS302-loxP, and loxPmH5eGFP is amplified and connected between the left and right homologous arms of pBlu-LSDV135L+R plasmid to obtain the transfer vector pBluhm135-loxPmH5eGFP; After infecting MDBK cells with bovine dermatophilus virus, the constructed transfer vector pBluhm135-loxPmH5eGFP is transfected into MDBK cells, and after purification, the bovine dermatophilus virus gene deletion fluorescent strain rLSDVΔ135eGFP is obtained. The LSDV gene deletion strain rLSDVΔ135eGFP is infected into MDBK cells, and pCMV-Cre plasmid is transfected to knock out the mH5eGFP fluorescent screening expression cassette, and the LSDV gene deletion strain rLSDVΔ135 is obtained by picking and purifying non-fluorescent plaques.

6. A vaccine comprising the LSDV attenuated strain of any one of claims 1-5.

7. Use of the attenuated strain of any one of claims 1-5 or the vaccine of claim 6 in the preparation of a medicament for preventing or treating bovine dermatophilus.

8. Use according to claim 7, characterized in that, The bovine dermatophilus is caused by bovine dermatophilus virus. The bovine dermatophilus is caused by bovine dermatophilus virus.

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