Bovine dermoderatosis virus orf145 gene deletion strain and application thereof

Abstract

The present application relates to bovine tuberous skin disease virus, in particular, to bovine tuberous skin disease virus ORF145 gene deletion strain and its application. The gene deletion strain is characterized in that it is constructed by deleting ORF145 gene from bovine tuberous skin disease virus virulent strain LSDV XJ201901. Compared with the parent strain, the replication level of the constructed deletion strain rLSDVΔ145 in in vitro cells is equivalent to that of the parent strain, indicating that the gene deletion does not affect the in vitro proliferation ability of the virus. In addition, after rLSDVΔ145 infects MDBK cells, it can significantly reduce the mRNA expression level of pro-inflammatory factors IL-1beta and IL-6, while not interfering with the expression of IFNbeta mRNA. Further transcriptomic analysis shows that the deletion strain significantly reduces virulence and pathogenicity on the basis of maintaining good immunogenicity; it not only loses the inhibition ability to the innate immune pathway, but also can effectively activate the adaptive immune response, and significantly reduce the risk of inflammation and tissue damage, showing 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 ORF145 gene deletion strain and its application BACKGROUND

[0002] Bovine lumpy skin disease (LSD) is an acute and subacute infectious disease of cattle and buffaloes caused by capripoxvirus. The disease was initially prevalent in Africa, but has rapidly spread to the Middle East, Europe, Asia and other regions in recent decades, becoming a highly concerned cross-border animal epidemic. Its most typical clinical symptoms include high fever, extensive and various sizes of hard nodules on the skin, lymph node enlargement, and significant reduction in milk production. Although the mortality rate is usually not high, it can cause abortion in pregnant cattle, sterility in male cattle, skin damage, and huge economic losses due to reduced milk production and weight gain, causing great harm to livestock production.

[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 ORF145 gene is highly homologous to the ORF144 gene of the traditional strong strain Kenya (GenBank No: MN072619).

[0004] There is no specific treatment for bovine lumpy skin disease, and vaccination can establish an immune barrier to reduce the outbreak and spread of the epidemic, thereby avoiding huge economic losses. Currently, domestic enterprises have been approved to produce LSD inactivated vaccine (NMG strain), which is relatively safe, but the immune period is relatively short and needs to be boosted. Internationally, Neethling attenuated vaccine is mainly used for immunization to prevent and control LSD. This strain has been isolated for many years, and its safety is insufficient. Some cattle show adverse reactions after vaccination, affecting milk production, and the attenuated vaccine strain can replicate in 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 and attenuated vaccine strains of LSDV, potential virulence genes of strong strains are screened, and the virulence genes of domestic LSDV epidemic strains are deleted by homologous recombination, reducing their virulence and providing a strong guarantee for the prevention and control of domestic LSD epidemic. SUMMARY

[0006] In order to construct a safe, effective and suitable LSDV attenuated vaccine in China, the application provides a bovine dermal nodular disease virus ORF145 gene deletion strain and a construction method and application thereof.

[0007] Technical scheme

[0008] The function coded by the ORF145 gene is essential for maintaining the virulence of the bovine dermal nodular disease virus (LSDV), and the function is highly conserved among different virus strains of the wild strong virus. Based on this finding, the application proposes that targeted deletion of the ORF145 gene or the function domain coded 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 of different genetic backgrounds. In order to verify the feasibility of the strategy, the application takes the bovine dermal nodular disease virus strain LSDV XJ201901 as a specific embodiment.

[0009] An LSDV attenuated strain of bovine dermal nodular disease virus, characterized in that the virus strain LSDV XJ201901 is constructed by deleting all or part of the ORF145 gene, and the ORF145 gene is shown as SEQ ID NO. 23.

[0010] The LSDV attenuated strain is constructed by a homologous recombination method and a Cre / loxP system.

[0011] The attenuated strain is constructed by the following steps: constructing a transfer vector targeting the virulence gene ORF145, recombining the transfer vector with the LSDV XJ201901 virus strain, screening rLSDVΔ145eGFP by fluorescent plaque, transfecting a pCMV-Cre plasmid to knock out the fluorescent gene expression box, and purifying the LSDV gene deletion strain by non-fluorescent plaque.

[0012] A vaccine comprising the LSDV attenuated strain.

[0013] The application takes the bovine dermal nodular disease virus strain LSDV XJ201901 as an example, and constructs an LSDV gene deletion strain in MDBK cells by a homologous recombination method and a Cre / loxP system. The method constructs a transfer vector targeting the virulence gene ORF145, recombines the transfer vector with the LSDV XJ201901 virus strain, screens rLSDVΔ145eGFP by fluorescent plaque, transfects a pCMV-Cre plasmid to knock out the fluorescent gene expression box, and purifies the LSDV gene deletion strain by non-fluorescent plaque. The key to the preparation of the attenuated vaccine is the attenuated virus.

[0014] Specifically,

[0015] The method for constructing the bovine papular dermatitis virus gene deletion strain of the application comprises the following steps:

[0016] According to the genome sequence of the bovine papular dermatitis virus strain LSDV XJ201901, upstream and downstream primers of the homologous arms on the left and right sides of LSDV ORF145 are designed, and the genome of the bovine papular dermatitis virus is subjected to PCR amplification, so as to obtain the sequences of the homologous arms on the left and right sides of ORF145.

[0017] The previous experiments of the application have proved that the mH5 promoter can rapidly and efficiently initiate the expression of a fluorescent gene in the MBDK cells infected by the bovine papular dermatitis, and therefore the mH5 is selected as the promoter of the screening gene expression cassette.

[0018] The mH5eGFP fragment in the pUC57-mH5eGFP plasmid is subjected to PCR amplification and recovery, and is connected to pBS302-loxP, and loxPmH5eGFP is amplified and connected between the left and right homologous arms of pBlu-LSDV145L+R plasmid, so as to obtain the transfer vector pBluhm145-loxPmH5eGFP.

[0019] After the MDBK cells are infected by the bovine papular dermatitis virus, the constructed transfer vector pBluhm145-loxPmH5eGFP is transfected into the MDBK cells, and after purification, the bovine papular dermatitis virus gene deletion fluorescent strain rLSDVΔ145eGFP is obtained.

[0020] The MDBK cells are infected by the LSDV gene deletion strain rLSDVΔ145eGFP, and the pCMV-Cre plasmid is transfected to knock out the mH5eGFP fluorescent screening expression cassette, and after the non-fluorescent plaque is picked and purified, the LSDV gene deletion strain rLSDVΔ145 is obtained.

[0021] The bovine papular dermatitis virus gene deletion strain can be used for preparing a vaccine for bovine papular dermatitis. Advantages

[0022] 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: KX764644) 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 ORF144, the ORF144 of the vaccine strain is truncated into two open reading frames (ORFs), and it is found that ORF144 is a potential virulence gene of the LSDV virulent strain, and the ORF144 of the Kenya virulent strain is highly homologous to the ORF145 of the XJ strain. The application first proposes to construct a bovine nodular dermatopathy virus gene deletion strain targeting ORF145, i.e., deleting the ORF145 of the LSDV XJ201901 strain. Compared with the parent virus, the deletion strain can reduce the mRNA level of the pro-inflammatory factors IL-1beta and IL-6 in cells after infecting MDBK cells, the inflammatory reaction is more moderate, the expression of antigen processing and immune related genes is maintained, and good immunogenicity is exhibited. In addition, the replication level of the constructed rLSDVΔ145 on the in-vitro cells is the same as that of the parent virus, the production of the gene deletion virus is not affected, and the application of the rLSDVΔ145 is not reported in the literature; the deletion virus strain rLSDVΔ145 is a live attenuated vaccine candidate strain with good application prospect, which can significantly reduce the virulence and inflammatory reaction risk while maintaining the immunogenicity, and can effectively stimulate comprehensive natural immunity and adaptive immune response.

[0023] 2、The deletion virus strain does not have exogenous genes such as resistance genes and fluorescent markers, and meets the current biological safety law of genetically engineered products; the deletion of the target 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 a domestic epidemic strain, and will not introduce non-epidemic strains in China, and has certain biological safety. In addition, since ORF145 is a conserved sequence of the bovine nodular dermatopathy virus virulent strain, theoretically, deleting any kind of domestic bovine nodular dermatopathy virus strain has similar functions.

[0024] 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 the wild virulent strain by using genetic engineering methods, greatly shortening the vaccine preparation period, and compared with natural mutant strains (most of which are point mutant strains), the gene deletion strain deletes several hundred or even several thousand bases of the target gene, and the deletion region is clear, which has the advantages of clear mutation characteristics, stability and not easy to return to the original state, and is an important way to research safe and effective new vaccines. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 Homology analysis of ORF144 protein of LSDV virulent strain and vaccine strain;

[0026] Figure 2 Detection of transcription level of virulence genes of LSDV infected MDBK cells;

[0027] Figure 3 Schematic diagram of construction of transfer vector;

[0028] Figure 4 Generation of recombinant fluorescent virus;

[0029] Figure 5 PCR identification results of recombinant fluorescent virus;

[0030] Figure 6 PCR identification results of recombinant deletion LSDV virus;

[0031] Figure 7 In vitro growth curve of LSDV parent virus and deletion strain rLSDVΔ145;

[0032] Figure 8 Cytokine detection after infection of MDBK with LSDV parent virus and deletion strain rLSDVΔ145, A is IFNβ mRNA level of MDBK cells after infection with virus, B is IL-6 mRNA level of MDBK cells after infection with virus, C is IL-1β mRNA level of MDBK cells after infection with virus; DETAILED DESCRIPTION

[0033] Example 1 Screening of virulence genes of bovine dermal nodular disease virus

[0034] 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, poxviruses have evolved a variety of immunomodulators, which have a variety of strategies to destroy or evade the host antiviral response triggered by host pattern recognition receptors (PRRs), and affect the results of poxvirus infection.

[0035] Poxviridae is a kind of large double-stranded DNA virus, and its genome structure is complex, which contains multiple promoter elements to regulate the temporal expression of viral genes. The core function of the poxvirus promoter is to drive the transcription of viral genes, and its activity directly affects the efficiency of viral replication and host immune response. The mH5 promoter is a modified vaccinia virus H5 promoter derived from the late promoter H5 of the Tianlun strain of vaccinia virus. The optimized mH5 promoter has high efficiency, stability and host compatibility, and has become a core tool for vaccine opening and gene therapy.

[0036] 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.

[0037] 1.1 Materials and methods

[0038] 1.1.1 Cells and viruses

[0039] MDBK was purchased from the Chinese Academy of Sciences Cell Bank; the LSDV XJ201901 strain was preserved in the China Animal Health and Epidemiology Center; the operation experiment of the live virus of the LSDV involved in the present application was completed in the biosafety level 3 laboratory of the China Animal Health and Epidemiology Center.

[0040] 1.1.2 Main reagents

[0041] The 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.

[0042] 1.2 Experimental steps

[0043] 1.2.1 Screening of bovine dermatophilus virus virulence genes

[0044] According to the literature research, and by comparing the homology of LSDV XJ201901 with 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.

[0045] 1.2.2 Verification of the transcription level of potential virulence genes in virus-infected cells

[0046] According to the genome sequence of the LSDV XJ201901 strain, primers for detecting ORF145 and actin mRNA were designed, and the primer sequences are shown in Table 1. The MDBK cells infected with LSDV were collected 24 h after infection, and the mRNA levels of potential virulence genes were detected by PCR after reverse transcription of the extracted RNA.

[0047] Table 1 LSDV mRNA detection primers

[0048]

[0049] 1.3 Experimental results

[0050] 1.3.1 Homology analysis of potential virulence genes of domestic LSDV strains and classic virulent strains and vaccine strains

[0051] As shown in Figure 1 , by analyzing the gene sequences of LSDV XJ201901, LSDV Kenya virulent strain and LSDV Neethling vaccine strain, it was found that there were differences between the virulent strain and the vaccine strain in ORF144 (Kelch-like protein), and the ORF144 of the vaccine strain Neethling was mutated and truncated to encode two proteins. Therefore, ORF145 is a potential virulence gene of the LSDV XJ201901 strain, and can be used as a target for developing a LSDV gene deletion attenuated vaccine.

[0052] 1.3.2 Detection of transcription levels of virulence genes in LSDV infected MDBK cells

[0053] After the MDBK cells were infected with LSDV for 24 h, the culture supernatant was discarded, and the cells were lysed to extract RNA for reverse transcription, and then the LSDV ORF145 mRNA level was detected. The results are shown in Figure 2 , which shows that ORF145 bands were detected, indicating that the ORF145 target gene was transcribed after the MDBK cells were infected with LSDV for 24 h.

[0054] Example 2 Construction and biological property analysis of bovine dermal nodular disease virus gene deletion strain

[0055] It was found by literature review that LSDV ORF144 is predicted to encode a Kelch-like protein, and F3L and A55R of vaccinia virus also belong to Kelch-like proteins. Studies have shown that A55R and F3L of VACV can inhibit the activation of the NF-κB pathway, and the deletion of A55R does not affect the replication activity of VACV, and VACVΔA55R infected mice can stimulate the increase of phagocytes and CD8+ T cells in the spleen and lymph nodes, enhance CD8+ T cell memory, and provide better protection against infection of VACV-WT, which provides a theoretical basis.

[0056] Therefore, this invention selects to delete ORF145 of LSDV XJ201901, constructs rLSDVΔ145 gene-deleted strain through homologous recombination, and analyzes its biological characteristics to provide technical support for the development of LSDV gene-deleted vaccines.

[0057] 2.1 Materials and Methods

[0058] 2.1.1 Cells and Viruses

[0059] 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.

[0060] 2.1.2 Main Reagents

[0061] 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.; the transfection reagent used in this invention was 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.

[0062] 2.2 Experimental Procedure

[0063] 2.2.1 Construction of transfer vector plasmids

[0064] like Figure 3 As shown, this invention uses pBluescript KS(+) as the initial plasmid. Approximately 1000 bp of homologous arms flanking the target site were amplified by PCR (upstream and downstream primers for the homologous arms flanking LSDV ORF145 were designed based on the genome sequence of the bovine nodular dermatitis virus strain LSDV XJ201901, and the bovine nodular dermatitis virus genome was amplified by PCR). The homologous arms were inserted into pBluescript KS(+) using XhoI, KpnI, SacI, and SacII restriction enzyme sites. Loxp gene sequences were added to both ends of the fluorescent selection expression cassette, and BamHI and EcoRI were inserted between the homologous arms flanking the target site. The transfer vector completely deleted the target gene through homologous recombination, resulting in the transfer vector pBluhm145-loxPmH5eGFP (its nucleotide sequence is shown in SEQ ID NO. 22). The primers used are shown in Table 2.

[0065] Table 2 Primers used to construct the transfer vector

[0066]

[0067] 2.2.2 Construction of LSDV gene deletion strain

[0068] The present application uses the method of homologous recombination to construct the deletion strain, and transfects the transfer vector into the MDBK cells infected with 0.1 MOI LSDV, and the specific operation is as follows:

[0069] 1. 3x10^5 MDBK cells are plated into a six-well plate;

[0070] 2. After the cell plate is cultured for 16-18 h, the cell density is about 60%, the supernatant is discarded, the LSDV virus solution is diluted with serum-free 1640 medium, and the MDBK cells are inoculated at an infection amount of 0.1 MOI, and are incubated at 37°C for 2 h;

[0071] 3. Prepare the transfection solution: 2 ug plasmid + 100 ul Opti-MEM + 6 ul FuGene transfection reagent, mix gently and stand for 5 min;

[0072] 4. After the MDBK cells are infected for 2 h, the virus solution is discarded, and the 1640 medium containing 2% FBS and 1% double antibody is replaced, the transfection solution is added dropwise, and the mixed solution is incubated at 37°C.

[0073] When the cytopathic effect reaches 80%, the virus is harvested, and after repeated freeze-thawing for 3 times, the virus solution is inoculated into new MDBK cells, and the production of recombinant fluorescent virus is detected by fluorescence microscope.

[0074] 2.2.3 Purification of LSDV gene deletion fluorescent strain

[0075] The present application purifies the recombinant virus by the method of picking plaque, picks the typical virus plaque with fluorescence in the 1640 medium, and inoculates the MDBK cells after freeze-thawing for 3 times at-80°C, and is incubated for 2 h, and is plated with low-melting agarose, and 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.

[0076] Table 3 PCR primers for identifying LSDV deletion strain

[0077]

[0078] 2.2.4 Knockout and purification of the fluorescence gene of the LSDV gene deletion fluorescent strain

[0079] The pure fluorescent strain was infected with 0.1 MOI dose of MDBK cells, and after 2 h of infection, the maintenance liquid of 2% FBS was replaced, and the pCMV-Cre plasmid was transfected, and the virus was harvested after 37°C culture until the lesion was more than 80%. After repeated freezing and thawing 3 times, 10ul virus liquid was used to infect MDBK cells, and the non-fluorescent plaque was picked to purify until a single non-fluorescent plaque appeared. After continuous propagation of the non-fluorescent plaque for 3 generations, the viral nucleic acid was extracted, and the purity of the gene deletion strain was verified by PCR.

[0080] 2.2.5 Verification of in vitro replication kinetics of LSDV gene deletion strain

[0081] MDBK cells were infected with 0.1 MOI of LSDV-WT and rLSDVΔ145, respectively, and the virus was collected at 12 h, 24 h, 48 h, 72 h, 96 h, 120 h and 144 h after infection. After freezing and thawing at -80°C for three times, the titers of the virus collected at different time points were determined, and the growth curves of the strains in MDBK cells were drawn.

[0082] 2.2.6 Cytokine mRNA detection after LSDV parent virus and deletion virus infection of MDBK

[0083] MDBK cells were infected with 0.1 MOI of LSDV-WT and rLSDVΔ145, respectively, and the virus was collected at 4 h, 8 h, 16 h and 24 h after infection. The cell culture supernatant was discarded, the cell RNA was extracted, and 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.

[0084] Table 4 Primers for detection of cytokine mRNA in MDBK cells

[0085]

[0086] 2.2.7 Transcriptome sequencing analysis of LSDV parent virus and deletion virus

[0087] Nine 100mm cell culture dishes of MDBK cells were prepared, and when the cell density was greater than 90%, the treatment was performed. The cells were divided into 3 groups, 3 in each group, and the MDBK cells were infected with 0.1 MOI of LSDV-XJ and rLSDVΔ145, respectively, and the blank control group was not infected with virus. 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 after quick freezing in liquid nitrogen, the transcriptome sequencing analysis was performed by Hangzhou Lianchuan Biotechnology Co., Ltd.

[0088] 2.3 Experimental results

[0089] 2.3.1 Construction of the transfer vector targeting ORF145

[0090] The present application uses pBluescript KS(+) as the initial plasmid, inserts the homologous arms (1000 bp) on both sides of the target, adds the same loxp gene sequence at both ends of the sequence of the fluorescent screening expression box, and inserts it between the homologous arms on both sides of the target to obtain the transfer vector pBluhm145-loxPmH5eGFP targeting ORF145. Figure 3 ).

[0091] 2.3.2 Construction of the LSDV gene deletion fluorescent strain

[0092] The transfer vector targeting ORF145 is transfected into MDBK cells infected with LSDV, and the virus liquid is harvested at -80°C after repeated freezing and thawing three times. 10 ul of the virus liquid is inoculated into MDBK cells, and the production of recombinant fluorescent virus is observed on the third day of culture. Figure 4 ).

[0093] 2.3.3 Purification of the LSDV gene deletion fluorescent strain

[0094] The recombinant fluorescent virus obtained is purified by picking plaques several times, and the DNA is extracted for PCR identification. The results are shown in Figure 5 , which shows that the left amplification target eGFP primer can amplify a specific band, and the right amplification wild-type target primer has no band, proving that the purity of the rLSDVΔ145eGFP recombinant fluorescent virus is good, and there is no wild parent virus contamination.

[0095] 2.3.4 Knockout and purification of the fluorescent gene of the LSDV gene deletion strain

[0096] The recombinant fluorescent virus with good purity infects MDBK cells, and the pCMV-Cre plasmid is transfected. The virus liquid is harvested at -80°C after repeated freezing and thawing three times. 10 ul of the virus liquid is inoculated into MDBK, and fluorescent plaques are observed. After several times of picking plaques, the LSDV deletion strain without fluorescence is obtained. The virus DNA is extracted and PCR identified, and the results are shown in Figure 6 , which shows that the target gene knockout of the recombinant LSDV is successful and the purity is good.

[0097] 2.3.5 In vitro replication kinetics verification of the LSDV gene deletion strain

[0098] In order to verify whether the deletion of the target will affect the growth and replication of the virus at the cellular level, the virus titer collected at different time points is determined, and the growth curve of the LSDV gene deletion strain is drawn, and the results are shown inFigure 7 The results showed that the deletion of ORF145 gene of LSDV XJ201901 strain did not affect the replication of the virus in MDBK cells, and ORF145 was a non-essential region for the replication of LSDV XJ201901 strain.

[0099] 2.3.6 Detection of cytokine mRNA levels after infection of MDBK by LSDV parental and deletion viruses

[0100] In order to detect whether the infection of the gene deletion strain can affect the immune level change after the cell is infected, the samples of virus infected MDBK cells for 4h, 8h, 16h and 24h were collected, and the mRNA levels of cytokines IL-1β, IL-6 and IFNβ were detected by qPCR, and the structure was as follows Figure 8 Figures A-C in the figure show that compared with LSDV WT strain, rLSDVΔ145 down-regulates IL-1β and IL-6 mRNA levels after infecting MDBK, and IFNβ mRNA level is not affected.

[0101] 2.3.7 Transcriptome sequencing analysis of LSDV parental and gene deletion strains

[0102] Based on the comprehensive analysis of transcriptome data (Table 5), it was shown that the deletion strain rLSDVA145 exhibited significant potential as an attenuated live vaccine strain. Compared with the virulent strain LSDV XJ201901, rLSDVA145 had significantly reduced virulence and pathogenicity while maintaining good immunogenicity. Specifically, rLSDVA145 failed to suppress the expression of key genes in the innate immune pathway such as ISG15, STING1, STAT2, and NOD2 after infection, indicating that it had lost the immune escape ability possessed by the wild strain; but it could still activate genes such as TRAF3 and STAT1, effectively initiating innate immune responses and laying the foundation for the establishment of subsequent adaptive immune responses. In terms of safety, rLSDVA145 showed obvious advantages. After infection with the wild strain XJ, pro-inflammatory factors such as IL1β and IL6 and chemotactic factors such as CXCL8 and CXCL3 were strongly up-regulated, triggering severe inflammatory responses and tissue damage (such as up-regulation of MMP3 and VEGFC); in the rLSDVA145 infection group, the expression levels of these genes showed no significant change or lower up-regulation, indicating that the inflammatory response induced by rLSDVA145 was more moderate, and the risk of tissue damage was significantly reduced. More importantly, rLSDVA145 could effectively activate a comprehensive adaptive immune response. The strain maintained stable expression of antigen processing-related genes (such as CTSD and CTSF) and costimulatory molecules (such as TNFSF14 and SPP1), which helped to promote antigen presentation and T cell activation; at the same time, it maintained the expression levels of adaptive immunity-related genes (such as IL12RB1, CD70, and TNFSF8), suggesting that it could induce strong and long-lasting cellular and humoral immune responses. In addition, rLSDVA145 also showed safe characteristics in terms of apoptosis regulation: it maintained the expression of pro-apoptotic signals (such as CYCS) while maintaining the expression of anti-apoptotic genes (such as BIRC3), which was beneficial for the clearance of infected cells.

[0103] Table 5 Transcriptomic analysis of MDBK infected with LSDV parent virus and deletion strain rLSDVA145

[0104]

[0105] Discussion

[0106] Currently, 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 recombination with field wild strong viruses to cause infection, while the gene deletion engineering vaccine can effectively distinguish vaccine strains and wild strains. The present application takes the domestic LSDV epidemic strain LSDV XJ201901 as the parent virus, and deletes the ORF145 gene to construct the rLSDVΔ145 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.

[0107] The innate immune system is the first line of defense against the invasion of foreign pathogens, and the inflammation produced by it 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 the death of the body. Therefore, excessive immune regulation can lead to the 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 bovine nodule skin disease virus gene deletion strain rLSDVΔ145, 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, rLSDVΔ145 does not inhibit key genes of natural immunity such as ISG15, STING1 and STAT2 after infection, indicating that its immune escape ability is weakened, but it can still effectively activate genes such as TRAF3 and TRPV2 to start natural immunity and promote subsequent adaptive immunity. Compared with the strong strain LSDV XJ201901, rLSDVΔ145 induced the expression of pro-inflammatory factors such as IL-1β, IL-6 and CXCL8 to be significantly reduced, and the inflammatory response was more moderate, and it could maintain the expression of antigen processing, costimulatory molecules and cell / humoral immunity related genes, showing good immunogenicity and lower risk of inflammatory pathology, further supporting its potential as a candidate strain for attenuated vaccine.

[0108] Therefore, rLSDVΔ145 has the potential to develop a LSD vaccine, and further animal experiments are needed to verify its safety and immune effect in the later stage.

Claims

1. A bovine tubercle skin disease virus (BVDV) live-attenuated strain, characterized in that, The ORF145 gene of the virus strain LSDV XJ201901 is deleted to construct a live attenuated strain, wherein the ORF145 gene of the live attenuated strain is totally or partially deleted; the ORF145 gene is shown as SEQ ID NO.

23.

2. The LSDV attenuated strain according to claim 1, characterised in that, The loss of function is achieved by deleting totally or partially the sequence of the ORF145 gene.

3. The LSDV attenuated strain according to claim 1 or 2, characterised in that, The live attenuated strain is constructed by a homologous recombination method and a Cre / loxP system, and is constructed by the following steps: a transfer vector targeting the ORF145 gene is constructed, the transfer vector is recombined with the virus strain LSDV XJ201901, rLSDVΔ145eGFP is obtained by screening fluorescent plaques, a pCMV-Cre plasmid is transfected to knock out the fluorescent gene expression cassette, and the LSDV live attenuated strain is obtained by purifying non-fluorescent plaques.

4. A vaccine comprising the LSDV live attenuated strain according to any one of claims 1-3.

5. Use of the LSDV live attenuated strain according to any one of claims 1-3 or the vaccine according to claim 4 in the preparation of a drug for preventing bovine dermatophiliasis.

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