Primary Studies On Immunological Charaterization And Transcriptomics Analysis Of CD2v Protein Of African Swine Fever Virus

African swine fever(ASF)is an acute,febrile,and highly contagious swine disease caused by African swine fever virus(ASFV).ASFV is the only arthropod-borne virus in the DNA virus family,mostly infects domestic pigs,wild boars and soft ticks(Amblyops tick)in nature.At present,there is no effective vaccine for ASF,the prevention and control measures are mainly strengthened quarantine detection and strict prevention of virus intrusion.In August 2018,the disease invaded China and spread rapidly throughout the country,made a huge economic loss to Chinese pig breeding industry.Therefore,the development of suitable vaccine strains and detection methods is of great significance for the prevention and treatment of ASF.1.Preparation and immunological detection of CD2v antibodiesThe ASFV genome contains more than 180 reading frames,which can encode about 150 to 200 proteins.Among them,the CD2v protein is encoded by the ASFV EP402R gene.The CD2v extracellular domain has a high homology with the CD2 of mammals(such as humans).It is found that it can mediate the adhesion of ASFV-infected cells or virons to red blood cells,a phenomenon named hemadsorption.CD2v is expressed in ASFV late infection and three different-sized proteins can be detected in ASFV-infected cells,they are 89kDa full-length glycosylated protein,26kDa C-terminal non-glycosylated fragment,and 63kDa N-terminal glycosylation fragment produced by proteolysis.In this part,we made preliminary research of the EP402R gene,constructed the recombinant prokaryotic expression plasmid to express CD2v truncated proteins.Meanwhile,we prepared the CD2v polyclonal antibodies successfully in order to carry out serological diagnostic technology and biological functions of CD2v protein in the future.2.Transcriptional expression profiles of BM cells infected with ASFV with CD2v gene deletionUsing high throughput sequencing technology,this study is aim to analyze transcriptome changes at different time points in porcine bone marrow macrophage cells infected with ASFV SY18 and SY18ΔCD2v strains.The results showed that,referring to the currently known pig genome sequence database,a total of 6060458700 bps were detected.The numbers of mRNAs in SY18 or SY18ΔCD2v infected BMDM were 23067 and 22341 respectively.In comparion with uninfected groups,the numbers of differentially expressed mRNAs in BMDM after SY18 and SY18ΔCD2v infected 8 h were 1322 and 823 respectively,infected 16 h were 2015 and 1295 respectively.Meanwhile,GO function annotation and KEGG biological pathway enrichment analysis were performed on mRNAs with significant differences.The results showed that genes such as DDX58/RIG-I,MX1,UBE2L6,STAT2 involved in interferon pathway were obviously increased,which proved that interferon pathway was activated during virus infection.In addition,chemotactic factors including CCL5,CXCL11,CCR5 and inflammatory factors TIMP1,CD47,MMP3,THBS1 were significantly increased,while CX3CR1,PLAU,F8,SERPIND1 were down-regulated compared to uninfected group.Fluorescence quantitative PCR confirmed the significant changes of various signaling molecules of the interferon pathway during SY18 and SY18ΔCD2v infection.