Investigation Of Molecular Epidemiology And Genetic Evolution Of EqHV And EqPV-H In Guangdong Province

Equine Hepacivirus(EqHV),one of the newest members of the Flaviviridae family,can transmit vertically and horizontally in infected horses.Since the first detection of EqHV in 2011,more than 10 countries in equine groups detected the presence of the virus,EqHV nucleic acid positive rate is 0.9%～34.1%.The virus may be associated with liver damage in equine animals.But so far,the information about EqHV in the domestic prevalence is still limited.Equine Parvovirus(Eq PV)is a single-stranded DNA virus belonging to Parvoviridae family.It can cause equine serum hepatitis,abortion,arthritis,and neurological damage and other symptoms.Equine parvovirus-hepatitis(EqPV-H)was first reported in 2018 and was associated with Equine serum hepatitis.Animal studies have shown that EqPV-H can cause viremia in equine acute hepatitis.China has a thriving horse racing industry with a large number of horses,but there is still a lack of genetic evolution and pathogenicity of EqPV-H.In view of the fact that EqHV and EqPV-H are newly discovered equine viruses,the epidemiological investigation and genetic evolution analysis of these two viruses have not been carried out in China.Therefore,from 2017 to 2019,216 equine serum samples were collected from several racetracks in Guangdong Province.The total prevalence rates of EqHV and EqPV-H were 34.25% and 10.84%,respectively.In order to obtain the genomic information and the genetic evolution of EqHV and EqPV-H in China,4 strains of EqHV and 11 strains of EqPV-H were whole genome sequencing.Through bioinformatics software and genetic analysis,it was found that the strain WSU-2013 in EqHV was a recombinant strain,and its NS5 A and NS5 B genes were recombined.The major parent-like strain of WSU-2013 was NPHV-H3-011 and the minor parent-like strain of WSU-2013 was NPHV-F8-068.These EqHV strains all belonged to EqHV subtype 1.In EqPV-H analysis,two Chinese strains(C11 and C14)were found to be recombined,the major parent-like strain was BCT-01 from the United States and the minor parent-like strain was H29 from China.Subsequently,based on the nucleotide and Amino acid homology analysis and genotyping of HCV genotypes and subtypes,EqHV is divided into 1 genotype and 3 subtypes for the first time.Further analysis showed that the EqHV strains in China belonged to subtypes 1 and 3,which provided important information for the control of EqHV in China.At the same time,in order to establish the detection method of EqHV,we also expressed the EqHV NS3 protein in prokaryotic expression,and prepared the polyclonal antibody of EqHV NS3 protein.The NS3 gene was amplified by RT-PCR from the virus RNA extracted from EqHV nucleic acid positive equine serum samples and cloned into p MAL-c5 x to construct the prokaryotic expression plasmid p MAL-c5x-NS3.The correctly sequenced plasmid was transformed into the expression strain Rosetta(DE3)and then the recombinant NS3 protein was induced by IPTG and purified.The antigenic properties of NS3 protein were analyzed by Western-blot.The anti EqHV NS3 polyclonal antibody was obtained by immunizing New Zealand white rabbits with the purified recombinant NS3 protein as antigen and mixed with water soluble adjuvant Gel 01 st for several times,the titer of polyclonal antibody against EqHV NS3 protein was more than 1:1,000,000.This study examined the prevalence of EqHV and EqPV-H in equine of Guangdong Province,the complete genome sequences of EqHV and EqPV-H strains were obtained.The recombinant strains were found in EqHV and EqPV-H.EqHV was genotyped for the first time and the genetic evolution characteristics of EqHV in guangdong province were determined.Polyclonal antibodies against EqHV NS3 protein were prepared,which provided technical reserve for the subsequent laboratory detection of EqHV in Chinese horses.