Sequence Analysis Of Full-length Genome Of Canine Parvovirus In Some Regions Of Anhui Province From 2018 To 2019 And The Establelishment Of SYBR Green ? Detection Method

Canine parvovirus disease is a highly contagious and deadly disease caused by canine parvovirus(canine parvovirus,CPV).Since CPV-2 was first discovered in the United States in 1978,its rapid rate of genetic variation and continuous host range changes have significantly increased its difficulties in detection,prevention and treatment.In China,studies on genetic variation of canine parvovirus have been carried out in many regions,except Anhui.Therefore,it is necessary and urgent to investigate the prevalence of canine parvovirus in Anhui province,analyze the whole genome sequence and establelish a fluorescence quantitative detection method for the current pandemic strain.In this study,feces or anal swabs suspected of canine parvovirus infection were collected from several animal hospitals in parts of Anhui from 2018 to 2019.A pair of primers were designed according to the conserved region of CPV VP2 gene on NCBI to identify fecal samples and classify CPV strains from positive samples.Ten strains were selected from the positive samples for genome-wide cloning and their sequences were analyzed for genetic variation.Design based on the current epidemic strain of SYBR green?fluorescence quantitative PCR detection primer,thus establelish a detection method.The results showed that a total of 31 positive samples were detected,including 3strains of New CPV-2a,accounting for 9.68%of the total.New CPV-2b was 1 strain,accounting for 3.23%.CPV-2c was 27 strains and the proportion was 87.1%.The results of whole genome sequence analysis of 10 CPV isolates showed that,on the whole,the CPV isolates from Anhui province were closely related to each other and formed a monophyletic group,which was far from the isolates from abroad.In addition,CPV-2a and CPV-2b subtypes form independent branches,while CPV-2c subtypes form another independent branch.NS1 gene and VP2 gene are out of sync in the branch of genetic evolution,which provides a basis for gene recombination.CPV Anhui isolates showed three previously unknown mutations in NS1:D358N,A587T and V596A.Two new right hairpin secondary structures have been discovered that may affect the replication of the virus.The NS1 gene identified two potential positive selection sites.This study establelished a against canine parvovirus Anhui isolates SYBR green?fluorescence quantitative PCR detection method,and it can detect 5.407×10~1copy/?L virus nucleic acid.It has sharp specificity,high sensitivity and good repeatability.In summary,the main epidemic strain of CPV in some regions of Anhui is CPV-2c,and New CPV-2a and New CPV-2b participate in the co-circulation.This study enriched the epidemiological data of CPV in Anhui region,carried out an in-depth study on the genetic variation of CPV Anhui strain,and provided a reliable diagnosis method of CPV by fluorescence quantitative PCR.