| Sheep pox is currently the most harmful livestock pox virus disease which can infect goats,sheep and other livestock.Animals infected with goat pox will develop fever,systemic acne,respiratory and digestive tract damage and lymphadenopathy,resulting in high mortality in sheep and abortion of pregnant sheep,which has brought huge economic losses to the global sheep industry.Therefore,in order to solve the above-mentioned problems,it is urgent to strengthen the study on the pathogenic mechanism of sheep pox virus infection.In this study,the vaccinia virus Tiantan strain(VTT)was used as a vector to study the biological function of the goat pox virus N1 protein and explore the pathogenic mechanism of goat pox virus.The specific test content is as follows:1.Using plaque purification method,the three recombinant virus VTT?N1L(recombinant virus deleting vN1L gene),VTTN1Lrev(recombinant virus rescued after N1L gene deletion,vN1L gene),and VTTg N1L(Recombinant virus with GTPV N1L gene replacing vN1L gene)was screened and purified.And the target gene and purity of the recombinant virus were verified by ordinary PCR and fluorescent quantitative PCR.2.The cloned and purified recombinant virus and the parental strain were inoculated into BHK-21,MDCK,Hela and PK-15 cells,and analyzed the differences in virus genetic stability,host range,replication ability and virulence.3.The mouse was inoculated with the recombinant virus and the parental strains intracranially.The clinical manifestations and deaths of the mouse were observed,and pathological examination of brain tissue was performed to analyze the distribution of the virus in different tissues and organs,as well as inflammatory factors,Th1 and Th2 factor transcription levels were used to evaluate the differences in pathogenicity of the three recombinant viruses.4.Sequencing transcriptomes of moice brain tissues and bioinformatics analysis of the sequencing data were carried out to screen differentially expressed genes,and then functional annotation of these differentially expressed genes was performed to explore the potential functional mechanism of vN1L and gN1L genes.5.The recombinant plasmid pEGFP-g N1L was constructed and transfected into BHK-21 cells to explore the subcellular localization of gN1 protein.The results: 1.The purified recombinant viruses VTTgN1L,VTTN1Lrev and VTT?N1L were obtained after selection of 6-8 generation plaques.2.The three recombinant viruses can be stably inherited in BHK-21 cells for more than30 generations,and there is no significant difference in host range.VTTgN1L has the fastest replication speed in BHK-21,MDCK,Hela,and PK-15 cells,followed by VTT?N1L,VTTN1 Lrev and VTT,which was further confirmed that the vN1L gene is a non-essential gene for virus replication.3.After inoculation of the parental strains and the recombinant in mice,the other three groups of mice except the VTT?N1L group showed loss of appetite,rough hair,shivering etc.The mortality and half lethal showed that VTTgN1L virus have the most power toxicity,followed by VTTN1 Lrev,VTT,and VTT?N1L have the lowest virulence.From the perspective of pathological damage,VTTgN1L brain tissue is the most serious,and the smallest is VTT?N1L.All four viruses can penetrate the blood-brain barrier,but VTTgN1L can infect organs such as brain,heart,liver,spleen or lungr.VTTN1 Lrev and VTT can infect organs such as brain,liver,and lung or spleen,and VTT?N1L can only infect organs such as brain and lung.The above results indicate that gN1 can replace the role of vN1 protein and its pathogenicity is higher than vN1 protein.The deletion of the v N1L gene can significantly reduce the invasion of the virus.Fluorescent quantitative detection of various cytokines showed that VTT?N1L induced higher expressions of pro-inflammatory factors TNF-? and IL-1? in mice than VTTgN1L and VTTN1 Lrev.The expression of anti-inflammatory factor IL-10 induced by VTT,VTTg N1L and VTTN1 Lrev viruses in mice was significantly higher than that of VTT?N1L virus group,indicating that the gN1 and vN1 protien have anti-inflammatory effects.4.Transcriptome analysis showed that VTTg N1L,VTT?N1L,and VTT N1Lrev had 421 differentially expressed genes compared with the control group VTT,including 282 up-regulated genes and139 down-regulated genes.174 differentially expressed genes were identified when VTTgN1L virus group mice compared with VTT virus group mice,of which 59 down-regulated expression genes,115 up-regulated expression genes;VTTN1Lrev virus group compared with VTT virus group of mice 86 differentially expressed genes were identified,of which 30 were down-regulated and 56 were up-regulated;161 differentially expressed genes were identified when VTT?N1L virus group mice compared with VTT virus group mice,of which 50 genes were down-regulated and 111 genes were up-regulated.VTT?N1L vs VTT: 0 inflammation-related genes,5 immune-related genes,and3 apoptosis-related genes;VTTgN1L vs VTT: 1 inflammation-related genes,8immune-related genes,and 6 apoptosis-related genes were identified;VTTN1Lrev vs VTT: 1 inflammation-related gene,2 immune-related genes,and4 apoptosis-related genes were identified;VTTgN1L vs VTTN1Lrev: 0inflammation-related genes,2 immune-related genes,and 5 apoptosis-related genes were identified.5.The results of subcellular localization showed that gN1 protein is expressed in the cytoplasm.Conclusion: The vN1L gene is both a non-essential gene for virus replication and a virulence gene.gN1 can replace vN1 protein in terms of pathogenicity and the pathogenicity is stronger than vN1 protien.VTT virus lacking the vN1L gene is significantly weakened;gN1L and vN1L genes have anti-inflammatory and anti-apoptotic effects which mechanism of action is similar;gN1 and vN1 proteins also have anti-inflammatory effects;the gN1 protein was expressed in the cytoplasm,indicating that it did not affect the host function as genetic material. |
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