Genetic Evolution Of Orf Virus And Duel Transcriptome Study In Interaction With Goat Cells

Contagious ecthyma is a goat-sheep acute and contact-induced aphthous dermatitis caused by orf virus（ORFV）in the genus Parapoxvirus（PPV）.The infected sheep mainly show symptoms such as erythema,pustules,papules,and ulcers formed on the lips and oral mucosa.Seriously,it will affect the feeding of sheep,resulting in death.ORFV is prevalent in all goat and sheep breeding countries in the world,and is a global infectious disease.The incidence of ORFV in adult sheep in our country is between 10%and 86%,and that in lambs is between 30%and 90%.The culling rate of lambs caused by this is about 46%to 93%,or even 100%.As a result,it has caused great economic losses to the goat farming industry.The results of the previous epidemiological investigation revealed that the ORFV generally exists in the goat with healthy appearance,and its virus-carrying rate in the flock can reach more than 95%.ORFV is prevalent in sheep raising countries all over the world,which indicates that ORFV has adapted to different geographical locations,climatic environments,goat and sheep breeds,suggesting that the genome of ORFV has changed under different external factors and pressures,forming variants adapted to different environments and hosts,but the detailed and accurate variation of the genome of each variant of ORFV is still unclear.Secondly,after the ORFV infects the goat and other hosts,does it adapt to the host cell environment through the difference of virulence?What are the transcriptome changes in the natural anti infective immunity of host cells against ORFV infection with different virulence?These issues have not yet been clarified.Based on this,this study collected 17 whole genome sequences and 776 functional gene sequences of parapoxviruses including ORFV isolated in the past 20 years,using phylogenetic analysis,genetic polymorphism analysis,evolution rate analysis,selection pressure analysis,phylogenetic biogeographical analysis and other methods to analyze the genetic evolution of Parapoxvirus isolates,which may reveal the evolutionary characteristics of ORFV in PPV.The pathogenicity of ORFV virulent strain FX0910 and artificial passage attenuated strain aFX0910 derived from this strain were compared in goat lip cells,and the mechanism of virus-host interaction and gene mutation sites related to virus weakening were analyzed.The study obtained the following results:1.The results of the gene evolutionary tree show that since 2010,ORFV accounts for the vast majority of PPV circulating in the world.Computational analysis of the reported PPV gene sequence similarity during 2000-2018 revealed that ORFV eventually evolved into the dominant PPV species during 2010-2018 and spread widely in China and India.2.The nucleotide substitution rates of different genes were inferred by Bayesian method.The results showed that the evolution rates of different gene segments of PPV ranged from 3.56×10^-5 to 4.21×10^-4/nucleotide/year.Among them,the VIR gene with the fastest evolution rate show high variability in the process of virus synthesis and replication,which may be caused by the long-term interaction of the virus with the host immune response.3.FX0910 and aFX0910 infected goat lip cells respectively.The results showed that a large number of cells fell off and died 48 hours after the infection of FX0910,while aFX0910 began to become round and clustered 72 hours after the infection;The proliferation curves of FX0910 and aFX0910 showed that the growth of FX0910 within5 days after infection was significantly higher than that of aFX0910.The above results showed that the infection and replication ability of strong ORFV strain was significantly better than that of weak ORFV strain.It is suggested that the gene mutation or even loss related to virus proliferation and replication may occur in the process of weakening the virulence of ORFV.4.The results of SNP analysis of the gene sequences of strong and weak ORFV strains showed that there were a large number of SNP sites in both virulent and attenuated ORFV strains.The three-dimensional structure map of the encoded proteins（ORF018,ORF019,ORF020 and ORF025）with a confidence level of>90%in the viral genes with the top10 SNPs shows that the sense mutation of ORF018 in the FX0910 strain is expressed in the 425th position of its amino acid sequence（Arg Mutation to His）,the sense mutation in the aFX0910 strain is expressed at 120th position of its amino acid sequence（Thr is mutated to Pro）;ORF019 has two sense mutation sites in the FX0910 strain,184th and 235th position respectively（Ala is mutated to Ser,Ala is mutated to Val）,and the sense mutation in aFX0910 strain occurs at 432th position（Ala is mutated to Thr）;the sense mutation site of ORF020 in FX0910strain is at 101th position（Ala was mutated to Ser）,and no mutated amino acid site was found in the aFX0910 strain;the sense mutation site of ORF025 in the FX0910 strain was at 751th position（Arg was mutated to Cys）,and the sense mutation in the aFX0910strain occurs at 371th position（Asp is mutated to Asn）.5.The differentially expressed genes of FX0910 and aFX0910 infected cells at different time points are not only enriched in"MAPK signaling pathway","Apoptosis"and"Cellular senescence"metabolic pathways,which were related to cell growth and apoptosis,but also enriched in"Notch signaling pathway","FoxO signaling pathway","mTOR signaling pathway"and"TGFβsignaling pathway"and other signaling pathways involved in immune regulation of cells.Weighted gene co-expression network analysis of gene expression profiles of infected cells at different stages showed that FX0910 mainly induces high expression of genes invlolved in innate immune responses,such as IRF9,ISG15,IFI44,IFIT2,IFIT1,OAS1,IFIH1 and IRF7.aFX0910induces high expression of genes involved in immunochemokines in cells,mainly including IL6,CXCL5,CXCL8,CCL2,CCL20 and TGFβ2 genes.Further study of transcription factors showed that STAT2 and STAT1 were the main regulators of these co-expressed genes.The conclusions are as follows:1.In recent years,great changes have taken place in the composition and structure of parapoxvirus strains.ORFV strains continue to increase in the proportion of all isolates and have become the main epidemic strains.2.VIR gene is the virulence gene with the greatest positive selection effect and the fastest evolution rate.The mutation of this gene may be the main reason why sheep mouth sore virus can adapt to different environmental pressures.3.In addition to the difference in gene expression of sheep mouth sore virus,the variation of many structural genes at the amino acid level is also the internal cause of the difference in virulence between different strains of sheep mouth sore virus.4.After infecting the host cells,the natural immune response of the infected cells induced by the weak virus strain is significantly lower than that of the strong virus strain,which is another strategy for the sheep mouth sore virus to adapt to the environment.