Bovine nodular skin disease virus ORF145 gene deletion strain and application thereof

Abstract

The invention relates to a bovine nodular dermatosis virus, in particular to a bovine nodular dermatosis virus ORF145 gene deleted strain and application of the bovine nodular dermatosis virus ORF145 gene deleted strain. The gene deletion strain is characterized in that the gene deletion strain is constructed by deleting an ORF145 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 rLSDVdelta145 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 rLSDVdelta145, 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] This invention relates to bovine nodular dermatitis virus, specifically to a bovine nodular dermatitis virus ORF145 gene-deleted strain and its applications. Background Technology

[0002] Lumpy Skin Disease (LSD) is an acute and subacute infectious disease of cattle and buffalo caused by capepoxviruses (capepoxviruses). Originally prevalent in Africa, it has rapidly spread to the Middle East, Europe, and Asia in recent decades, becoming a significant transboundary animal disease. Its most typical clinical symptoms include high fever, widespread appearance of hard nodules of varying sizes on the skin, swollen lymph nodes, and a significant decrease in milk production. Although the mortality rate is usually low, it can lead to abortion in pregnant cows, infertility in bulls, hide damage, and substantial economic losses due to reduced milk production and weight gain, posing a significant threat to livestock production.

[0003] Based on LSDV whole-genome sequence analysis, LSDV can be divided into two main branches, each of which can be further divided into two sub-branches. LSDV XJ201901 belongs to the vaccine recombinant sub-branch, which is likely due to recombination between wild-type virulent strains and vaccine strains from Europe and Africa. LSDV XJ201901 (GenBank ID: OM984485) is a virulent strain, and its ORF145 gene is highly homologous to the ORF144 gene of the conventional virulent Kenya strain (GenBank ID: MN072619).

[0004] There is no specific treatment for bovine nodular dermatitis. Vaccination helps establish an immune barrier, reducing outbreaks and spread, thus avoiding significant economic losses. Currently, domestic companies are approved to produce LSD inactivated vaccines (NMG strain). These vaccines have relatively high safety, but the immunity period is relatively short, requiring booster immunizations. Internationally, the Neethling attenuated live vaccine is mainly used to control LSD. However, this strain has been isolated for many years, and its safety is insufficient. Some cattle have experienced adverse reactions after vaccination, affecting milk production. Furthermore, the attenuated live vaccine strain can replicate in vaccinated animals, increasing the risk of recombination and spread of the strain in non-endemic areas. Therefore, it is not suitable for the current prevalence of bovine nodular dermatitis in China.

[0005] Therefore, developing a safe, effective, and applicable LSDV attenuated vaccine for my country is of great significance. By comparing and analyzing the whole genomes of wild-type virulent LSDV strains and attenuated vaccine strains, potential virulence genes of virulent strains were screened. Homologous recombination was then used to delete virulence genes from prevalent LSDV strains in China, reducing their virulence and providing strong support for the prevention and control of LSD outbreaks in China. Summary of the Invention

[0006] To construct a safe, effective, and applicable LSDV attenuated vaccine for my country, this invention provides a bovine nodular dermatitis virus ORF145 gene-deleted strain, its construction method, and its application.

[0007] Technical solution The function encoded by the ORF145 gene is crucial for maintaining the virulence of bovine nodular dermatitis virus (LSDV), and this function is highly conserved among different virulent wild-type virus strains. Based on this finding, this invention proposes that targeted deletion of the ORF145 gene or its encoded functional domain can serve as a general strategy for constructing attenuated LSDV vaccine strains. This strategy is expected to be applicable to LSDV strains with different genetic backgrounds. To verify the feasibility of this strategy, this invention uses the bovine nodular dermatitis virus strain LSDV XJ201901 as a specific example.

[0008] A live attenuated strain of bovine nodular dermatitis virus (LSDV) is characterized by being constructed by deleting all or part of the ORF145 gene from the LSDV XJ201901 virus strain, as shown in SEQ ID NO.23.

[0009] It was obtained through homologous recombination and the Cre / loxP system.

[0010] The attenuated strain is characterized by being constructed by the following steps: constructing a transfer vector targeting the virulence gene ORF145, recombining the transfer vector with the LSDV XJ201901 virus strain, obtaining rLSDVΔ145eGFP by fluorescent plaque screening, then transfecting the pCMV-Cre plasmid to knock out the fluorescent gene expression cassette, and obtaining the LSDV gene-deleted strain after purification by non-fluorescent plaques.

[0011] A vaccine comprising the said attenuated LSDV strain.

[0012] This invention uses the bovine nodular cutaneous virus strain LSDV XJ201901 as an example. An LSDV gene-deleted strain was constructed in MDBK cells using homologous recombination and the Cre / loxP system. This method involves constructing a transfer vector targeting the virulence gene ORF145, recombining the transfer vector with the LSDV XJ201901 strain, obtaining rLSDVΔ145eGFP through fluorescent plasmid selection, and then transfecting it with the pCMV-Cre plasmid to knock out the fluorescent gene expression cassette. The LSDV gene-deleted strain was then purified using non-fluorescent plasmids. The key to producing a live attenuated vaccine is the attenuated virus.

[0013] Specifically: The method for constructing a bovine nodular dermatitis virus gene-deleted strain of the present invention includes the following steps: Based on the genome sequence of bovine nodular dermatovirus strain LSDV XJ201901, upstream and downstream primers were designed for the homologous arms on both sides of ORF145 of LSDV. The genome of bovine nodular dermatovirus was amplified by PCR, and the sequences of the homologous arms on both sides of ORF145 were obtained. The homologous arms on both sides of ORF145 were inserted into pBluescript KS(+) using Xho I, Kpn I, Sac I and Sac II restriction enzyme sites. Previous experiments have demonstrated that the mH5 promoter can rapidly and efficiently initiate the expression of fluorescent genes in MBDK cells infected with bovine nodular dermatitis. Therefore, this invention selected mH5 as the promoter for screening gene expression cassettes.

[0014] The mH5eGFP fragment in the pUC57-mH5eGFP plasmid was amplified and recovered by PCR, ligated into pBS302-loxP, and loxPmH5eGFP was amplified and ligated into the left and right homologous arms of the pBlu-LSDV145L+R plasmid to obtain the transfer vector pBluhm145-loxPmH5eGFP. After infecting MDBK cells with bovine nodular dermatitis virus, the constructed transfer vector pBluhm145-loxPmH5eGFP was transfected into MDBK cells, and after purification, the bovine nodular dermatitis virus gene-deleted fluorescent strain rLSDVΔ145eGFP was obtained. MDBK cells were infected with the LSDV gene-deleted strain rLSDVΔ145eGFP and transfected with the pCMV-Cre plasmid to knock out the mH5eGFP fluorescent selection expression cassette. The LSDV gene-deleted strain rLSDVΔ145 was obtained by picking and purifying the cells using non-fluorescent plaques. The application of a bovine nodular dermatitis virus gene-deleted strain in the preparation of a bovine nodular dermatitis vaccine. Beneficial effects

[0015] 1. This invention analyzed and compared the whole genomes of the traditional virulent strain Kenya and the attenuated vaccine strain Neethling (GenBank ID: KX764644) of bovine nodular dermatitis virus (BNDV). Significant differences were found in ORF144 between the virulent and vaccine strains. The vaccine strain's ORF144 mutation truncated into two open reading frames (ORFs). Analysis indicated that ORF144 is a potential virulence gene for virulent LSDV strains. The ORF144 of the Kenya virulent strain is highly homologous to the ORF145 of the XJ strain. This invention is the first to propose constructing a bovine nodular dermatitis virus gene deletion strain targeting ORF145, specifically, a strain that deletes ORF145 of the LSDV XJ201901 strain. Compared to the parent strain, infection of MDBK cells with the deletion strain reduced the mRNA levels of intracellular pro-inflammatory factors IL-1β and IL-6, resulting in a milder inflammatory response, while maintaining the expression of antigen processing and immune-related genes, demonstrating good immunogenicity. Furthermore, the constructed rLSDVΔ145 replicates at the same level as the parent virus in vitro, without affecting the production of the gene-deleted virus, and its application has not been reported in the literature. The described gene-deleted virus strain rLSDVΔ145 is a promising candidate strain for a live attenuated vaccine, which significantly reduces virulence and the risk of inflammatory response while maintaining immunogenicity, and can effectively stimulate a full range of innate and adaptive immune responses.

[0016] 2. This deleted virus strain does not carry exogenous genes such as resistance genes or fluorescent markers, complying with current biosafety laws for genetically engineered products. The deletion of the target gene can also serve as a molecular marker to distinguish between vaccine strains and wild-type virulent strains, which is beneficial for LSD infection detection and immune monitoring in farms. Furthermore, the gene-deleted strain was constructed from domestically prevalent strains, avoiding the introduction of non-prevalent domestic strains, thus possessing a certain degree of biosafety. Additionally, since ORF145 is a conserved sequence of virulent bovine nodular dermatitis virus strains, theoretically, deletion of any domestic bovine nodular dermatitis virus strain would have a similar function.

[0017] 3. Common methods for obtaining attenuated virus strains include: (1) screening and isolation of attenuated strains in nature; (2) attenuation by passage in different animals or cells; and (3) recombination of virulent strains using genetic engineering methods. Isolation from nature and attenuation by passage in different animals or cells have disadvantages such as large workload and long attenuation time. For example, the attenuated vaccine for swine fever was obtained after passage in rabbits for 240 generations. However, this invention uses genetic engineering methods to modify the genome of wild virulent strains, which greatly shortens the vaccine preparation cycle. Compared with natural mutant strains (most of which are point mutant strains), gene deletion strains have hundreds or even thousands of bases missing from the target gene. The deletion region is clear, and the mutant traits are clear, stable, and not easy to revert to the ancestral form. It is an important way to study safe and effective new vaccines. Attached Figure Description

[0018] Figure 1 Homology analysis of ORF144 protein between virulent LSDV strain and vaccine strain; Figure 2 Detection of transcriptional levels of virulence genes in LSDV-infected MDBK cells; Figure 3 Schematic diagram of the transfer carrier construction; Figure 4 The generation of recombinant fluorescent viruses; Figure 5 PCR identification results of recombinant fluorescent virus; Figure 6 PCR identification results of recombinant LSDV virus with deletion; Figure 7 In vitro growth curves of parental LSDV virus and the deletion strain rLSDVΔ145; Figure 8 Cytokine detection after MDBK cells were infected with parental LSDV and the deletion strain rLSDVΔ145. A represents the IFNβ mRNA level of MDBK cells after viral infection, B represents the IL-6 mRNA level of MDBK cells after viral infection, and C represents the IL-1β mRNA level of MDBK cells after viral infection. Detailed Implementation

[0019] Example 1: Screening of virulence genes for bovine nodular dermatitis virus Innate immunity is the body's first line of defense against viral infections and a normal physiological defense barrier. It mainly includes natural physiological barriers (skin and mucous membrane barriers, placental barrier, blood-brain barrier, etc.) and the innate immune response, which produces immune cells and molecules. To counteract the host's immune defenses, poxviruses have evolved various immunomodulators with multiple strategies to disrupt or circumvent the host's antiviral response triggered by the pattern recognition receptor (PRR), thus affecting the outcome of poxvirus infection.

[0020] Poxviridae are a class of large, double-stranded DNA viruses with complex genomes containing multiple promoter elements that regulate the temporal expression of viral genes. The core function of poxviral promoters is to drive viral gene transcription, and their activity directly affects viral replication efficiency and the host's immune response. The mH5 promoter, a modified vaccinia virus H5 promoter derived from the late promoter H5 of the Tian Tan strain of vaccinia virus, has become a core tool for vaccine development and gene therapy due to its high efficiency, stability, and host compatibility.

[0021] Based on the above research progress, this invention compared the main differential proteins between the domestically prevalent LSDV strain LSDV XJ201901 and the highly virulent Kenya strain and Neethling vaccine strain, screened potential virulence genes, and verified the expression level of virulence genes at the mRNA level of LSDVXJ201901 in vitro infection.

[0022] 1.1 Materials and Methods 1.1.1 Cells and Viruses MDBK was purchased from the Cell Bank of the Chinese Academy of Sciences; LSDV XJ201901 strain was stored at 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.

[0023] 1.1.2 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; Taq enzyme and DH5α competent cells were purchased from Takara. 1.2 Experimental Procedure 1.2.1 Screening of virulence genes for bovine nodular dermatitis virus Based on literature review and by comparing the homology of LSDV XJ201901 with the virulent LSDV strain Kenya and the LSDV Neethling vaccine strain, genes that may cause a reduction in the virulence of virulent LSDV were screened.

[0024] 1.2.2 Verification of the transcriptional level of potential virulence genes in virus-infected cells Based on the genome sequence of LSDV strain XJ201901, primers for detecting ORF145 and actin mRNA were designed, and the primer sequences are shown in Table 1. MDBK cells were infected with LSDV virus for 24 hours, and RNA was extracted, reverse transcribed, and then PCR was used to detect the mRNA levels of potential virulence genes.

[0025] Table 1 Primers for LSDV mRNA detection

[0026] 1.3 Experimental Results 1.3.1 Homology analysis of potential virulence genes between domestic LSDV strains and classic virulent strains and vaccine strains like Figure 1As shown, gene sequence analysis of LSDV XJ201901, the virulent LSDV Kenya strain, and the LSDV Neethling vaccine strain revealed differences in ORF144 (Kelch-like protein) between the virulent strain and the vaccine strain. The ORF144 mutation in the Neethling vaccine strain truncated two proteins. Therefore, ORF145 is a potential virulence gene for the LSDV XJ201901 strain and can serve as a target for gene-deleted attenuated LSDV vaccines.

[0027] 1.3.2 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 LSDVORF145 mRNA level was detected. Results are as follows: Figure 2 The results showed that bands were detected in all ORF145 targets, indicating that the ORF145 target genes were transcribed 24 hours after LSDV infection of MDBK cells.

[0028] Example 2: Construction and biological characteristics analysis of a gene-deleted strain of bovine nodular dermatitis virus. Literature review revealed that LSDV ORF144 is predicted to encode a Kelch-like protein, and vaccinia virus F3L and A55R also belong to the Kelch-like protein family. Studies have shown that VAV A55R and F3L can inhibit the activation of the NF-κB pathway, and the absence of A55R does not affect VAV replication activity. VAVΔA55R infection in mice can stimulate an increase in phagocytes and CD8+ T cells in the spleen and lymph nodes, enhance CD8+ T cell memory, and provide better protection against VAV-WT infection. The above provides a theoretical basis.

[0029] 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.

[0030] 2.1 Materials and Methods 2.1.1 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.

[0031] 2.1.2 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.; 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. 2.2 Experimental Procedure 2.2.1 Construction of transfer vector plasmids 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.

[0032] Table 2 Primers used to construct the transfer vector

[0033] 2.2.2 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 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. 3. Prepare transfection solution: 2 μg plasmid + 100 μL Opti-MEM + 6 μL FuGene transfection reagent, mix gently and let stand for 5 min; 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.

[0034] 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.

[0035] 2.2.3 Purification of LSDV gene-deleted fluorescent strains 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.

[0036] Table 3. PCR primers for identifying LSDV deletion strains

[0037] 2.2.4 Knockout and purification of fluorescent gene in LSDV gene-deleted fluorescent strains 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.

[0038] 2.2.5 Validation of in vitro replication kinetics of LSDV gene-deleted strains MDBK cells were infected with 0.1 MOI LSDV-WT and rLSDVΔ145. Viruses were collected at 12h, 24h, 48h, 72h, 96h, 120h and 144h post-infection. The viral droplets collected at different time points were measured after three freeze-thaw cycles at -80℃. Growth curves of each strain in MDBK cells were plotted.

[0039] 2.2.6 Detection of cytokine mRNA after infection of MDBK with parental LSDV and deletion virus MDBK cells were infected with 0.1 MOI LSDV-WT and rLSDVΔ145. Viruses were collected at 4 h, 8 h, 16 h and 24 h post-infection. Cell culture supernatant was discarded, and cellular RNA was extracted. After reverse transcription, the levels of IFNβ, IL-1β and IL-6 mRNA in MDBK cells were detected by qPCR. The detection primers are shown in Table 4.

[0040] Table 4 Primers for MDBK cytokine mRNA detection

[0041] 2.2.7 Transcriptome sequencing analysis of parental and deletion viruses of LSDV Nine 100mm cell culture dishes were prepared, and treatment was performed when the cell density was greater than 90%. The cells were divided into three groups of three cells each. The MDBK cells were infected with 0.1 MOI of LSDV-XJ and rLSDVΔ145, respectively, as well as a blank control group without virus infection. After treatment, the samples were placed in a 37℃ cell culture incubator for further culture. The samples were collected 16 hours after infection, and the cells were collected in cryovials with 1 mL of Trizol. After being flash-frozen in liquid nitrogen, the samples were sent to Hangzhou Lianchuan Biotechnology Co., Ltd. for transcriptome sequencing analysis.

[0042] 2.3 Experimental Results 2.3.1 A transfer vector targeting ORF145 was constructed. This invention uses pBluescript KS(+) as the initial plasmid, inserts it into the homologous arms (1000bp) on both sides of the target site, adds the same loxp gene sequence to both ends of the fluorescent selection expression cassette sequence, and inserts it between the homologous arms on both sides of the target site to obtain the ORF145-targeting transfer vector pBluhm145-loxPmH5eGFP. Figure 3 ).

[0043] 2.3.2 Construction of LSDV gene-deleted fluorescent strain The ORF145-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 4 ).

[0044] 2.3.3 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 amplified three times consecutively, and DNA was extracted for PCR identification. The results are as follows: Figure 5The 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 rLSDVΔ145eGFP recombinant fluorescent virus is of good purity and free from contamination by the wild-type parent virus.

[0045] 2.3.4 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 6 The results showed that the target gene of the recombinant LSDV was successfully knocked out, and the purity was good.

[0046] 2.3.5 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 7 The results showed that the deletion of the ORF145 gene in the LSDVXJ201901 strain did not affect viral replication in MDBK cells, and ORF145 is a non-essential region for replication of the LSDVXJ201901 strain.

[0047] 2.3.6 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 8 Figures A through C show that, compared to the LSDV WT strain, rLSDVΔ145 infection of MDBK downregulated IL-1β and IL-6 mRNA levels, while IFNβ mRNA levels were unaffected.

[0048] 2.3.7 Transcriptome sequencing analysis of parental LSDV strains and gene-deleted strains A comprehensive analysis of transcriptome data (Table 5) indicates that the deletion strain rLSDVΔ145 demonstrates significant potential as a live attenuated vaccine strain. Compared to the virulent strain LSDV XJ201901, rLSDVΔ145 maintains good immunogenicity while exhibiting significantly reduced virulence and pathogenicity. Specifically, infection with rLSDVΔ145 failed to inhibit the expression of key genes in the innate immune pathway, such as ISG15, STING1, STAT2, and NOD2, indicating that it has lost the immune evasion ability possessed by the wild-type strain; however, it can still activate genes such as TRAF3 and STAT1, effectively initiating the innate immune response and laying the foundation for the subsequent establishment of adaptive immune responses. In terms of safety, rLSDVΔ145 shows a clear advantage. Infection with the wild-type XJ strain strongly upregulated pro-inflammatory factors such as IL1β and IL6, as well as chemokines such as CXCL8 and CXCL3, triggering a severe inflammatory response and tissue damage (e.g., upregulation of MMP3 and VEGFC). In contrast, the expression levels of these genes in the rLSDVΔ145 infection group showed no significant change or only a low degree of upregulation, indicating a milder inflammatory response and a significantly reduced risk of tissue damage. More importantly, rLSDVΔ145 effectively activated a comprehensive adaptive immune response. This strain maintained stable expression of antigen processing-related genes (such as CTSD and CTSF) and co-stimulatory molecules (such as TNFSF14 and SPP1), contributing to antigen presentation and T cell activation; simultaneously, it maintained the expression levels of adaptive immunity-related genes (such as IL12RB1, CD70, and TNFSF8), suggesting that it may induce a potent and durable cellular and humoral immune response. In addition, rLSDVΔ145 also exhibits a safe characteristic in apoptosis regulation: it maintains the expression of anti-apoptotic genes (such as BIRC3) while maintaining the expression of pro-apoptotic signals (such as CYCS), which is beneficial for the clearance of infected cells.

[0049] Table 5. Transcriptomic analysis of MDBK after infection with parental LSDV and deletion strain rLSDVΔ145.

[0050] discuss Currently, most vaccines used internationally to control LSD are live attenuated vaccines based on LSDV, GTPV, or SPPV. Given the international situation, only homologous LSDV vaccines can eradicate LSD outbreaks. The development and use of LSDV vaccines are crucial for the development of the cattle industry. However, live attenuated LSDV vaccines carry the risk of recombination with virulent wild-type viruses in the field, leading to infection. Gene-deleted engineered vaccines can effectively distinguish between vaccine strains and wild-type strains. This invention uses the prevalent domestic LSDV strain LSDV XJ201901 as the parent virus, deleting the ORF145 gene to construct the rLSDVΔ145 gene-deleted strain, laying the foundation for the development of a genetically engineered vaccine suitable for LSD prevention and control in China.

[0051] The innate immune system is the host's first line of defense against invading pathogens. The inflammation it produces responds 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 prolonged cytokine responses, known as a "cytokine storm," leading to organismal death. Therefore, excessive immune regulation can lead to the collapse of the autoimmune system, while insufficient inflammatory factors can lead to persistent pathogen infection. Persistent or improperly regulated excessive inflammation can cause chronic or systemic inflammatory diseases, resulting in immunopathological damage. This invention constructs a bovine nodular dermatitis virus gene-deleted strain, rLSDVΔ145, whose replication capacity is consistent with the parent strain. However, the gene-deleted strain induces significantly lower levels of the inflammatory factors IL-1β and IL-6 than the parent strain, reducing the high-level inflammatory response induced by the parent strain. Further transcriptomic analysis revealed that rLSDVΔ145 did not suppress key innate immune genes such as ISG15, STING1, and STAT2 after infection, indicating a weakened immune evasion ability. However, it could still effectively activate genes such as TRAF3 and TRPV2, initiating innate immunity and promoting subsequent adaptive immunity. Compared with the virulent strain LSDV XJ201901, rLSDVΔ145 induced significantly reduced expression of pro-inflammatory factors such as IL-1β, IL-6, and CXCL8, resulting in a milder inflammatory response. Furthermore, it maintained the expression of antigen processing, co-stimulatory molecules, and cellular / humoral immune-related genes, demonstrating good immunogenicity and a low risk of inflammatory pathology, further supporting its potential as a candidate strain for attenuated vaccines.

[0052] Therefore, rLSDVΔ145 has the potential to be developed into an LSD vaccine, but further animal experiments are needed to verify its safety and immunogenicity.

Claims

1. An attenuated strain of bovine nodular dermatitis virus (LSDV), characterized in that, It is constructed by deleting all or part of the ORF145 gene from the LSDV XJ201901 virus strain, as shown in SEQ ID NO.

23.

2. The LSDV attenuated strain according to claim 1, characterized in that, It was obtained through homologous recombination and the Cre / loxP system.

3. The attenuated strain according to claim 2, characterized in that, It is constructed by the following steps: a transfer vector targeting the virulence gene ORF145 is constructed, the transfer vector is recombined with the LSDV XJ201901 virus strain, rLSDVΔ145eGFP is obtained by fluorescent plaque screening, and then the pCMV-Cre plasmid is transfected to knock out the fluorescent gene expression cassette. After purification by non-fluorescent plaque, the LSDV gene deletion strain is obtained.

4. A vaccine comprising the attenuated LSDV 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 medicament for the prevention of bovine nodular dermatitis.

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