Analysis Of Complete Genome Of Y2 Strain Of Deer Source And Establishment Of Real-time PCR Assay For Detecting Genotype Of BVDV

Bovine viral diarrhea virus(BVDV)is a pathogen of bovine viral diarrhea-mucosal disease(BVD / MD),which can infect cattle,deer,sheep and other animals,showing a wide range of clinical signs,including minor of the upper respiratory tract signs,short-term reduction of circulating white blood cells and short-term low-grade fever,severe respiratory diseases,gastrointestinal disorders,bleeding syndrome and pneumonia,reproductive disorders and so on.BVDV has caused serious harm and economic losses to the livestock industry in the world.The main research was divided into the following two parts:1.Gene sequencing and genetic variation analysis of Y2 strain of deer sourceBVDV 1b Y2 strain isolated from deer in our laboratory,was studied.Whole gene sequencing of Y2 was performed.According to the conservative area of genome sequence of BVDV 1b type in Gen Bank,primers with 8 pairs were designed.Virus RNA was extracted by Y2 strain proliferation,and 8 fragments of Y2 were amplified by RT-PCR,the fragments were cloned into p LB-simple vector and transformed into Top10 competent cells for sequencing.The whole genome of Y2 was spliced by reference to the published sequence of BVDV 1b type on Genbank.Respectively,the 5'UTR of Y2,the gene and amino acids of whole gene,Npro and E2 of Y2 were compared and their phylogeny trees were established.The whole genome of Y2 was successfully sequenced,and the 12306 bp genome of Y2 was obtained.In the genome: 5'UTR 381 bp,3'UTR 228 bp and CDS region which could encode 3898 amino acids were 11697 bp.Gen Bank accession numbers of Y2 is KY964311.There are 11 potential N-glycosylation sites of capsules of Y2.The Y2 strain was on the same branch as the Osloss,INSP4 ncp,GX4 strain of BVDV-1b,is BVDV 1b subtype.By constructing the phylogenetic tree,it was found that the phylogenetic distance of 5'UTR of Y2 was close to that of VEDEVAC,Osloss and VEDEVAC,and farthest from CP7.The whole gene and amino acid of Y2 were close to IBSP4 ncp,Osloss,GX4 and VEDEVAC,and farthest with 3156.The gene and amino acid of Npro of Y2 was close to Osloss,IBSP4 ncp,VEDEVAC and GX4.The Npro gene was the farthest JL-1,and the amino acid of Npro was the most distant to AU526,AU526 and HP-KY-RK13.The distance of E2 gene and amino acid of Y2 was close to Osloss,BSP4 ncp,GX4,VEDEVAC distance into.But there was farthest distance between E2 gene of Y2 and JL-1 and there was largest difference in amino acids between Y2 and 3156.The results showed the Y2 from deer source was close to BSP4 ncp,Osloss,VEDEVAC and GX4.The whole gene sequencing of Y2 has a great prospect in the research of reverse genetic technology of BVDV,persistent infection,prevention and cure of disease of BVDV.2.Establishment of real-time PCR for BVDV typeStandard positive plasmids and RNA of BVDV-1,BVDV-2,BVDV-3 were constructed respectively.One-step real-time RT-PCR were established by using standard positive RNA.With good specificity for the three methods,no cross-reactivity was observed in the detection of CSFV,PPRV,BVDV-1,BVDV-2,BVDV-3,BDV,bovine rotaviruses and PRV.The sensitivity of the assay was set at 102 RNA copies for BVDV-1 and BVDV-2,at 103 RNA copies for BVDV-3.The repeatability of these methods were better by calculating the intraand interassay CV of 107~105 copies RNA(CV<0.02).45 samples of BVDV-1 and 5 samples of BVDV-3 were detected from 80 known samples,but no samples BVDV-2 were detected.