Bovine dermoderatosis virus orf 19 / 20 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 ORF19 / 20 gene deletion strain and its application. The gene deletion strain is characterized in that it is constructed by deleting ORF019 and ORF020 genes from bovine tuberous skin disease virus virulent strain LSDV XJ201901. Compared with the parent strain, the replication level of the constructed deletion strain rLSDVΔ19 / 20 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 rLSDVΔ19 / 20 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 tubercle skin disease virus, in particular to bovine tubercle skin disease virus ORF19 / 20 gene deletion strain and its application BACKGROUND

[0002] Lumpy skin disease (LSD) is an acute and subacute infectious disease caused by lumpyskin disease virus (LSDV). LSDV belongs to Poxviridae, Capripoxviras (CPV), has high host specificity, mainly infects cattle and buffaloes. The morbidity of LSD epidemic is 5%~45%, and the mortality is about 5%, and can reach 20% in severe cases. The body temperature of infected cattle rises, and skin nodules appear on the body surface. With the development of the disease, the skin nodules will slowly break down and even necrosis, and if accompanied by secondary bacterial infection, it is easy to lead to the death of cattle. After infection, the cow shows infertility, pregnant cows have miscarriage, and lactating cows have a sharp decline in milk production, which seriously affects the production performance of cattle.

[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 produced by recombination of wild strong strains and vaccine strains in Europe and Africa. LSDV XJ201901 (GenBank No: OM984485) is a strong strain, and compared with the traditional strong strain Kenya strain (GenBank No: MN072619), ORF019 is truncated into two open reading frames ORF019 and ORF020, and the truncation does not change the core function of the encoded protein, and the K-like beta helix and the BTB / POZ domain are independently encoded by two ORFs, which together maintain the structural and functional integrity consistent with the original protein.

[0004] Vaccination is an important means to prevent and control lumpy skin disease. At present, the main prevention and control in China is through vaccination of goatpox attenuated vaccine and inactivated vaccine, but the 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, and the strain has been isolated for many years, has serious side effects, and some vaccine strains 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 lumpy skin disease epidemic situation.

[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 genomes of the wild strong LSDV strain and the attenuated vaccine strain, potential virulence genes of the strong strain are screened, and the virulence genes of the domestic LSDV epidemic strain are deleted by using homologous recombination method to reduce the virulence, thereby providing a powerful guarantee for the prevention and control of domestic LSD epidemic. SUMMARY

[0006] In order to construct a safe, effective and suitable LSDV attenuated vaccine for China, the present application provides a bovine nodule skin disease virus ORF019 and ORF020 gene deletion strain and a construction method and application thereof.

[0007] TECHNICAL SCHEME

[0008] The function coded by ORF019 / ORF020 gene is essential for maintaining the virulence of bovine nodule skin disease virus (LSDV), and the function is highly conserved among different strains of viruses. Based on this finding, the present application proposes that targeted deletion of ORF019 / ORF020 gene or the function domain coded thereby can be used as a general strategy for constructing LSDV attenuated vaccine strain. This strategy is expected to be applicable to LSDV strains with different genetic backgrounds. In order to verify the feasibility of the strategy, the present application takes bovine nodule skin disease virus strain LSDV XJ201901 as a specific embodiment.

[0009] The bovine nodule skin disease virus gene deletion strain is characterized in that the strain is obtained by deleting ORF019 and ORF020 genes from the LSDV XJ201901 strain, wherein the nucleotide sequence of ORF019 is SEQ ID NO. 25, and the nucleotide sequence of ORF020 is SEQ ID NO. 26.

[0010] The present application deletes ORF019 and ORF020 genes from the LSDV XJ201901 strain by using a homologous recombination method and a Cre / loxP system.

[0011] The present application takes bovine nodule skin disease virus strain LSDV XJ201901 as an example, and constructs a LSDV gene deletion strain in MDBK cells by using a homologous recombination method and a Cre / loxP system. The method is to construct a transfer vector targeting virulence genes ORF019 and ORF020, recombine the transfer vector with the LSDV XJ201901 strain, screen rLSDVΔ19 / 20eGFP by fluorescent plaque, transfect pCMV-Cre plasmid to knock out the fluorescent gene expression box, and purify the non-fluorescent plaque to obtain the LSDV gene deletion strain. The key to the preparation of the attenuated vaccine is the attenuated virus.

[0012] Specifically:

[0013] The method for constructing the bovine nodular dermatopathy virus gene deletion strain of the application comprises the following steps:

[0014] According to the genome sequence of the bovine nodular dermatopathy virus strain LSDV XJ201901, left and right upstream primers of the homologous arms of LSDV ORF019 and ORF020 are designed, and the genome of the bovine nodular dermatopathy virus is subjected to PCR amplification, so as to obtain the sequences of the left and right homologous arms of ORF19 / 20, respectively.

[0015] The laboratory found in the early stage that the poxvirus promoter mH5 can start the expression of an exogenous gene in MDBK cells, so the mH5eGFP fragment in the pUC57-mH5eGFP plasmid is recovered by PCR amplification, connected to pBS302-loxP, and the loxPmH5eGFP is amplified and connected between the left and right homologous arms of the pBlu-LSDV1920L+R plasmid, so as to obtain the transfer vector pBluhm1920-loxPmH5eGFP.

[0016] After the bovine nodular dermatopathy virus infects the MDBK cells, the constructed transfer vector pBluhm1920-loxPmH5eGFP is transfected into the MDBK cells, and after purification, the bovine nodular dermatopathy virus gene deletion fluorescent strain rLSDVΔ19 / 20eGFP is obtained.

[0017] The MDBK cells are infected with the LSDV gene deletion strain rLSDVΔ19 / 20eGFP, and the pCMV-Cre plasmid is transfected to knock out the mH5eGFP fluorescence screening cassette, and the LSDV gene deletion strain rLSDVΔ19 / 20 is obtained by picking and purifying the non-fluorescent plaque.

[0018] A vaccine comprising the LSDV attenuated strain.

[0019] The LSDV attenuated strain or the vaccine is used for preparing a drug for preventing bovine nodular dermatopathy.

[0020] Beneficial effects

[0021] 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 ORF019, wherein the ORF019 of the vaccine strain is truncated into two open reading frames (ORFs), and the ORF019 of the virulent strain remains complete, indicating that the complete ORF019 is a potential virulence determinant. Although the ORF019 of the virulent strain XJ201901 also exists similar truncation (forms ORF019 and ORF020), but it still shows a strong toxic phenotype, it is speculated that the truncation does not completely destroy the protein function. Based on this, the application first proposes to construct a gene deletion vaccine with ORF019 as a target, by deleting the complete ORF019 of the virulent strain (i.e. deleting the ORF019 and ORF020 of XJ201901), the genetic characteristics of the natural vaccine strain are simulated to obtain a safe and effective vaccine candidate strain. Compared with the parent virus, the mRNA levels of intracellular proinflammatory factors IL-1beta and IL-6 of the deletion virus strain after infecting MDBK cells can be reduced. In addition, the replication level of the constructed rLSDVΔ19 / 20 on the in vitro cells is the same as that of the parent virus, and does not affect the production of the gene deletion virus, and its application is not reported in the literature; the deletion virus strain rLSDVΔ19 / 20 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 and adaptive immune responses.

[0022] 2、The deletion virus strain does not carry exogenous genes such as resistance genes and fluorescent markers, and meets the current biological safety law of genetically engineered products; the deletion of ORF019 and ORF020 can 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 farms; in addition, the gene deletion virus is constructed from domestic prevalent strains, and will not introduce domestic non-prevalent strains, and has certain biological safety. In addition, since the functions of the proteins encoded by ORF019 and ORF020 are relatively conservative, theoretically, deleting any one of the domestic bovine nodular dermatopathy virus strains has similar functions.

[0023] 3、The commonly used methods for obtaining weak virus strains include: (1) screening and isolation of weak virus strains in nature; (2) passage of heterologous animals or cells to weaken; (3) using genetic engineering methods to recombine strong virus strains. 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 passage of the rabbitized attenuated porcine pestivirus vaccine in rabbits to 240 generations; and the present application can realize the modification of the genome of the wild strong virus strain by using genetic engineering methods, greatly shortening the vaccine preparation cycle, and compared with natural mutant strains (most of which are point mutant strains), the gene deletion strain 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

[0024] Figure 1 , Homology analysis of ORF019 protein of LSDV strong strain and vaccine strain;

[0025] Figure 2 , Detection of transcription level of virulence genes of LSDV infected MDBK cells;

[0026] Figure 3 , Schematic diagram of transfer vector construction;

[0027] Figure 4 , Generation of recombinant fluorescent virus;

[0028] Figure 5 , PCR identification results of recombinant fluorescent virus;

[0029] Figure 6 , PCR identification results of recombinant deletion LSDV virus;

[0030] Figure 7 , In vitro growth curve of LSDV parent virus and deletion strain rLSDVΔ19 / 20;

[0031] Figure 8 , Cytokine detection after infection of MDBK with LSDV parent virus and deletion strain rLSDVΔ19 / 20, A is the IFNβ mRNA level of MDBK cells after infection with virus, B is the IL-6 mRNA level of MDBK cells after infection with virus, and C is the IL-1β mRNA level of MDBK cells after infection with virus. DETAILED DESCRIPTION

[0032] Example 1: Screening of virulence genes of bovine nodular dermatopathy virus

[0033] Natural immunity is the first line of defense against viral infection and the normal physiological defense barrier of the body. It mainly includes natural physiological barriers (skin and mucosal barrier, placental barrier, blood-brain barrier, etc.) and innate immune response, which produces immune cells and immune molecules. In order to counteract the host's immune defense, poxviruses have evolved a variety of immunomodulators, which have a variety of strategies to destroy or evade the host's antiviral response triggered by pattern recognition receptors (PRRs), affecting the outcome of poxvirus infection.

[0034] Poxviridae is a kind of large double-stranded DNA virus, which has a complex genome structure and 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 is a core tool for vaccine opening and gene therapy due to its high efficiency, stability and host compatibility.

[0035] 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 (Neethling vaccine strain is a live attenuated vaccine virus strain for preventing lumpy skin disease (LSD), i.e. bovine lumpy skin disease), screens potential virulence genes, and verifies the mRNA level expression of the virulence genes in the in vitro infection of LSDV XJ201901.

[0036] 1.1 Materials and methods

[0037] 1.1.1 Cells, viruses and plasmids

[0038] MDBK was purchased from the Chinese Academy of Sciences Cell Bank; the LSDV XJ201901 strain was preserved in the Chinese Animal Health and Epidemiology Center; the operation experiment of the live virus of the present application was completed in the biosafety level 3 laboratory of the Chinese Animal Health and Epidemiology Center.

[0039] 1.1.2 Main reagents

[0040] 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 DH5α competent cells were purchased from Takara.

[0041] 1.2 Experimental steps

[0042] 1.2.1 Screening of virulence genes of bovine contagious pustular dermatitis virus

[0043] According to literature research, and by homology alignment 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.

[0044] 1.2.2 Verification of transcription level of potential virulence genes in virus-infected cells

[0045] According to the genome sequence of the LSDV XJ201901 strain, the ORF019, ORF020 and actin mRNA primers were designed, and the primer sequences are shown in Table 1. The MDBK cells infected with LSDV 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.

[0046] Table 1 LSDV mRNA detection primers

[0047]

[0048] 1.3 Experimental results

[0049] 1.3.1 Homology analysis of potential virulence genes of domestic LSDV strains and classic virulent strains and vaccine strains

[0050] By analyzing the gene sequences of LSDV XJ201901, Kenya virulent strain and Neethling vaccine strain, Figure 1 it was found that there was a significant difference between the virulent strain and the vaccine strain in the ORF019 (Kelch-like protein) region. The ORF019 of the vaccine strain Neethling was truncated to form two open reading frames, ORF019a and ORF019a1. However, the ORF019 of the virulent strain XJ201901 was also truncated (named ORF019 and ORF020), but it still showed a strong virulent phenotype, and it was speculated that the truncation did not completely destroy the protein function. Based on this, we proposed a hypothesis: ORF019 and its truncated product are key virulence factors of LSDV. Therefore, simultaneous deletion of ORF019 and ORF020 in XJ201901 is expected to completely destroy the function of the gene, thereby constructing an attenuated vaccine strain. This study first proposed a strategy of developing a LSDV gene deletion vaccine targeting ORF019 / ORF020.

[0051] 1.3.2 Detection of transcription level of virulence genes in LSDV-infected MDBK cells

[0052] The culture supernatant was discarded after the LSDV infected MDBK cells for 24 hours, and the RNA was extracted from the lysed cells, and the mRNA levels of LSDV ORF019 and ORF020 were detected after reverse transcription. Figure 2 It is shown that the bands of ORF019 and ORF020 are detected, which indicates that the transcription of the target genes of ORF019 and ORF020 occurs after the LSDV infected MDBK cells for 24 hours.

[0053] Example 2 Construction and biological property analysis of bovine dermal nodular disease virus gene deletion strain

[0054] It is found by referring to the literature that the LSDV ORF019 encodes a Kelch-like protein, and the F3L and A55R (F3L and A55R are two very important genes in the genome of vaccinia virus) of vaccinia virus (VACV) also encode a Kelch-like protein, and researches show that A55R and F3L 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 for the infection of VACV-WT; the ORF019 of SPPV encodes a Kelch protein, and the deletion of 0RF019 does not affect the replication of SPPV, and the clinical symptoms of sheep infected by SPPVΔ019 are obviously reduced.

[0055] Therefore, the ORF019 and ORF020 of the LSDV XJ201901 are selected to be deleted together, the rLSDVΔ19 / 20 gene deletion strain is constructed by homologous recombination, and the biological properties are analyzed, so as to provide technical support for the development of the LSDV gene deletion vaccine.

[0056] 2.1 Materials and methods

[0057] 2.1.1 Cells and viruses

[0058] The MDBK cells are purchased from the cell bank of the Chinese Academy of Sciences; the LSDV XJ201901 strain is isolated and preserved by the China Animal Health and Epidemiology Center; the operation experiment of the live virus of the LSDV involved in the present application is completed in the biosafety level 3 laboratory of the China Animal Health and Epidemiology Center.

[0059] 2.1.2 Main reagents

[0060] Fetal bovine serum used by MDBK cells is purchased from PAN Biotech; penicillin, streptomycin are purchased from Biyun Tian Biotechnology Co., Ltd.; 1640 culture medium is purchased from Shanghai Yuancheng Biotechnology Co., Ltd.; the transfection reagent used in the application is purchased from Promega Company; Opti-MEM is purchased from Gibco; the plasmid extraction kit is purchased from Qiagen; qPCR reagent, Taq enzyme and DH5α competence are purchased from Takara;

[0061] 2.2 Experimental steps

[0062] 2.2.1 Construction of transfer vector plasmid

[0063] According to the genomic sequence of the bovine nodular dermatitis virus strain LSDV XJ201901, the upstream and downstream primers of the homologous arms on the left and right sides of LSDV ORF019 and ORF020 are designed, and the genome of the bovine nodular dermatitis virus is PCR amplified, and the sequences of the homologous arms on the left and right sides of ORF19 / 20 are obtained, and the Xho I, Kpn I, Sac I and Sac II enzyme cutting sites are used to insert the homologous arms on the left and right sides of ORF19 / 20 into pBluescript KS(+);

[0064] 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-LSDV1920L+R plasmid, to obtain the transfer vector pBluhm1920-loxPmH5eGFP, that is, the same loxp gene sequence is added at both ends of the sequence of the fluorescent screening expression cassette, and is inserted between the homologous arms on both sides of the target point by using BamH I and EcoR I, and the transfer vector will realize the complete deletion of the target gene by homologous recombination (as shown in Figure 3 ).

[0065] The primers used are shown in Table 2.

[0066] Table 2 Primers used for constructing transfer vector

[0067]

[0068] 2.2.2 Construction of LSDV gene deletion strain

[0069] The application uses the method of homologous recombination to construct the deletion strain, and the transfer vector is transfected into the MDBK cells infected with 0.1 MOI LSDV, and the specific operation is as follows:

[0070] 1、 3×10^5MDBK cells are plated into a six-well plate;

[0071] 2. After culturing the cell plates for 16-18 hours, the cell density is about 60%. Discard the supernatant, dilute the LSDV virus solution with serum-free 1640 medium, and inoculate MDBK cells with an infection dose of 0.1 MOI. Incubate at 37°C for 2 hours.

[0072] 3. Prepare transfection buffer: 2 μg plasmid + 100 μL Opti-MEM + 6 μL FuGene transfection reagent, mix gently and let stand for 5 min;

[0073] 4. Two hours after infecting MDBK cells, discard the virus solution and replace it with 1640 medium containing 2% FBS and 1% penicillin antibody. Add transfection solution, mix well, and incubate at 37°C.

[0074] When the cytopathic effect reached 80%, the virus was harvested. After three freeze-thaw cycles, the virus solution was inoculated into new MDBK cells, and the production of recombinant fluorescent virus was detected using a fluorescence microscope.

[0075] 2.2.3 Purification of LSDV gene-deleted fluorescent strains

[0076] This invention employs a plaque-picking purification method to purify recombinant viruses. Typical fluorescent viral plaques are picked and placed in 1640 medium, subjected to three freeze-thaw cycles at -80°C, and then inoculated into MDBK cells. After 2 hours of incubation, low-melting-point agarose is layered. The process is repeated until fluorescent viral plaques appear, until a single fluorescent viral plaque is observed. The single viral plaque is continuously propagated to the third generation, and viral nucleic acid is extracted. Viral purity is identified by PCR. The PCR primers are listed in Table 3.

[0077] Table 3. PCR primers for identifying LSDV deletion strains

[0078]

[0079] 2.2.4 Knockout and purification of fluorescent gene in LSDV gene-deleted fluorescent strains

[0080] MDBK cells were infected with a highly purified fluorescent virus strain at a dose of 0.1 MOI. After 2 hours of infection, the medium was replaced with 2% FBS and transfected with pCMV-Cre plasmid. The cells were cultured at 37°C until more than 80% of the cells showed lesions, at which point the virus was harvested. After three freeze-thaw cycles, 10 μL of the virus solution was used to infect MDBK cells. Non-fluorescent plaques were removed for purification until a single non-fluorescent plaque appeared. The non-fluorescent plaques were continuously propagated for three generations, and viral nucleic acid was extracted. The purity of the gene-deleted strain was verified by PCR.

[0081] 2.2.5 Validation of in vitro replication kinetics of LSDV gene-deleted strains

[0082] MDBK cells were infected with 0.1 MOI of LSDV-WT and rLSDVΔ19 / 20, and viruses were collected at 12 h, 24 h, 48 h, 72 h, 96 h, 120 h and 144 h post-infection, respectively. The virus collected at different time points was determined for titer after being frozen and thawed three times at -80 DEG C, and the growth curves of the strains in MDBK cells were plotted.

[0083] 2.2.6 Detection of cytokine mRNA in MDBK cells infected with LSDV parent virus and deletion virus

[0084] MDBK cells were infected with 0.1 MOI of LSDV-WT and rLSDVΔ19 / 20, and viruses were collected at 4 h, 8 h, 16 h and 24 h post-infection, respectively. The cell culture supernatant was discarded, and cell RNA was extracted. After reverse transcription, the IFN beta, IL-1 beta and IL-6 mRNA levels in the MDBK cells were detected by qPCR method. The detection primers are shown in Table 4.

[0085] Table 4 Primers for detection of cytokine mRNA in MDBK cells

[0086]

[0087] 2.2.7 Transcriptome sequencing analysis of LSDV parent virus and deletion virus

[0088] 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, and each group had three MDBK cells which were infected with 0.1 MOI of LSDV-XJ and rLSDVΔ19 / 20, respectively, and a blank control group without virus infection. After treatment, the samples were placed in a 37 DEG C cell sample incubator for continuous culture, and the samples were collected at 16 h post-infection. The cells were collected in a cryogenic tube with 1 mL Trizol, and were quickly frozen in liquid nitrogen. The transcriptome sequencing analysis was performed by Hangzhou Lianchuan Biotechnology Co., Ltd.

[0089] 2.3 Experimental results

[0090] 2.3.1 Construction of a transfer vector targeting ORF019 / 20

[0091] In the present application, pBluescript KS(+) is used as an initial plasmid, the homologous arms (1000 bp) on both sides of the target point are inserted, the same loxp gene sequences are added at both ends of the sequence of the fluorescent screening expression box, and the loxp gene sequences are inserted between the homologous arms on both sides of the target point, so that the transfer vector targeting ORF019 / 20 pBluhm1920-loxPmH5eGFP is obtained (the nucleotide sequence is shown as SEQ ID NO. 24).

[0092] 2.3.2 Construction of a LSDV gene deletion fluorescent strain

[0093] The transfer vector targeting ORF19 / 20 was transfected into MDBK cells infected with LSDV, and cultured to the fifth day when the lesion reached 80%. After repeated freezing and thawing of the virus liquid at -80°C three times, 10ul of the virus liquid was inoculated into MDBK cells, and the production of recombinant fluorescent virus was observed on the third day of culture. Figure 4 ).

[0094] 2.3.3 Purification of LSDV gene deletion fluorescent strain

[0095] The obtained recombinant fluorescent virus was purified by picking plaques several times, and a single fluorescent plaque was picked and continuously expanded three times, and DNA was extracted for PCR identification. The results are shown in Figure 5 The left amplification target eGFP primer can amplify a specific band, and the right amplification wild virus target primer has no band, proving that the purity of the rLSDVΔ19 / 20eGFP recombinant fluorescent virus is good, and there is no wild parent virus contamination.

[0096] 2.3.4 Knockout and purification of fluorescent gene of LSDV gene deletion strain

[0097] After the recombinant fluorescent virus with good purity infected MDBK cells, the pCMV-Cre plasmid was transfected, and the lesion reached 80% on the third day of culture. After repeated freezing and thawing of the virus liquid at -80°C three times, 10ul of the virus liquid was inoculated into MDBK, and fluorescent plaques were observed. After several times of picking and purifying plaques, LSDV deletion strain without fluorescence was obtained. Virus DNA was extracted and PCR identified, and the results are shown in Figure 6 The target gene knockout of the recombinant LSDV was successful, and the purity was good.

[0098] 2.3.5 In vitro replication kinetics verification of LSDV gene deletion strain

[0099] In order to verify whether the deletion of the target point will affect the growth and replication of the virus at the cellular level, the virus titer collected at different time points was determined, and the growth curve of the LSDV gene deletion strain was drawn, and the results are shown in Figure 7 The deletion of ORF019 and ORF020 genes of LSDV XJ201901 strain does not affect the replication of the virus in MDBK cells, and ORF019 and ORF020 are non-essential regions for the replication of LSDV XJ201901 strain.

[0100] 2.3.6 Detection of cytokine mRNA levels after infection of MDBK by LSDV parent virus and deletion virus

[0101] To detect whether the infection of gene deletion strain can affect the immune level changes, 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 show that, compared with the LSDV WT strain, rLSDVΔ19 / 20 down-regulates the mRNA levels of IL-1β and IL-6 after infecting MDBK, while the mRNA level of IFNβ is not affected.

[0102] 2.3.7 Transcriptome sequencing analysis of LSDV parent virus and gene deletion strain

[0103] Based on the comprehensive analysis of transcriptome data (Table 5), the deletion strain rLSDVΔ19 / 20 showed significant potential as a live attenuated vaccine strain. Compared with the virulent strain LSDV XJ201901, rLSDVΔ19 / 20 significantly reduced virulence and pathogenicity while maintaining good immunogenicity. Specifically, rLSDVΔ19 / 20 failed to suppress the expression of key genes in the natural immune pathway such as ISG15, STING1, and STAT2 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 TRPV2, effectively initiating a natural immune response and laying the foundation for the establishment of subsequent adaptive immune responses. In terms of safety, rLSDVΔ19 / 20 showed a clear advantage. 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 a severe inflammatory response and tissue damage (such as up-regulation of MMP3 and VEGFC); in the rLSDVΔ19 / 20 infection group, the expression levels of these genes showed no significant change or lower up-regulation, indicating that the inflammatory response induced by rLSDVΔ19 / 20 was more moderate, and the risk of tissue damage was significantly reduced. More importantly, rLSDVΔ19 / 20 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 cell immunity-related genes (such as IL15RA, CTSC, and IL15RA) and humoral immunity-related genes (such as CD40 and TNFRSF17), suggesting that it could induce a strong and long-lasting cellular and humoral immune response. In addition, rLSDVΔ19 / 20 also showed safe characteristics in terms of apoptosis regulation: it maintained the expression of pro-apoptotic signals (such as CYCS) while down-regulating anti-apoptotic genes (such as XIAP), which was conducive to the clearance of infected cells.

[0104] Table 5 Transcriptomic analysis of LSDV parent virus and deletion strain rLSDVΔ19 / 20 infected MDBK

[0105]

[0106]

[0107] Discussion

[0108] 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, only 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 viruses, 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 respectively deletes ORF019 and ORF020 genes, constructs rLSDVΔ19 / 20 gene deletion strain, and lays a foundation for the development of gene engineering vaccine suitable for domestic LSD epidemic prevention and control.

[0109] 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 coronavirus, influenza A and Ebola virus, etc. can cause excessive and long-term cytokine response, which is called "inflammatory factor storm", leading to the death of the body. Therefore, excessive immune regulation can lead to the collapse of the autoimmune system, and insufficient inflammatory factors can lead to persistent infection of pathogens. Persistent or improper regulation can cause excessive inflammation, causing chronic or systemic inflammatory diseases, and causing immunopathological damage to the body. The present application constructs a LSDV gene deletion strain rLSDVΔ19 / 20, 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 further transcriptome analysis results, rLSDVΔ19 / 20 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, CXCL16 and IL15RA, start natural immunity and promote subsequent adaptive immunity. Compared with the strong strain LSDV XJ201901, rLSDVΔ19 / 20 induces significantly reduced expression of pro-inflammatory factors such as IL-1β, IL-6 and CXCL8, and the inflammatory response is more moderate, and it can 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.

[0110] Therefore, rLSDVΔ19 / 20 has the potential to develop LSD vaccine, and further animal experiments are needed to verify its safety and immune effect.

Claims

1. A bovine tubercle skin disease virus (BVDV) live-attenuated strain, characterized in that, The ORF019 gene and the ORF020 gene of the virus strain LSDV XJ201901 are deleted to construct the attenuated strain, and the ORF019 gene and the ORF020 gene of the attenuated strain lose functions; the nucleotide sequence of the ORF019 gene is SEQ ID NO. 25, and the nucleotide sequence of the ORF020 gene is SEQ ID NO.

26.

2. The LSDV attenuated strain according to claim 1, characterised in that, The functions are lost by deleting all or part of the sequences of the ORF019 gene and the ORF020 gene.

3. The LSDV attenuated strain according to claim 1 or 2, characterised in that, The attenuated strain is obtained by a homologous recombination method and a Cre / loxP system, and is constructed by the following steps: a transfer vector targeting the ORF019 gene and the ORF020 gene is constructed, the transfer vector is recombined with the virus strain LSDV XJ201901, rLSDVΔ19 / 20eGFP is obtained by screening fluorescent plaques, a pCMV-Cre plasmid is transfected to knock out the fluorescent gene expression box, and the LSDV attenuated strain is obtained by purifying non-fluorescent plaques.

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

5. Use of the LSDV 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 nodular dermatosis.

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