The Molecular Epidemiological Study Of Equine Hepacivirus And Pegivirus Infection In Guangdong

Background:In 2012,equine hepacivirus(EqHV)also known as non-primate hepacivirus(NPHV)is a recently discovered homolog of the hepatitis C virus(HCV)in horses,which was first identified by Kapoor using the serological methods.NPHV infections in horses have some features in common with HCV infections in humans and thus might be interesting as a surrogate model and study the zoonitisis.In 2013,Kapoor and colleagues reported a new pegivirus in horses and provisionally named as equine pegivirus(EPgV).In the meantime,Theiler’s disease associated virus(TDAV)was identified in horses and proposed as the aetiological agent of acute fulminant hepatitis("Theiler’s disease")and was revealed to be a member of the genus Pegivirus by phylogenetic analysis.There is still little information on the frequency and distribution of NPHV and EPgV,the geographic distribution,epidemiology,persistence,genetic diversity and clinical features of NPHV infection are of great importance for study HCV.In this study,we investigate the presence of both NPHV and EPgV,TDAV.Characterization of the NPHV and EPgV natural infection,liver pathogenesis of virus and the tropism in PBMC,giving more information for further investigation.Objective:We screened NPHV,EPgV and TDAV infection to investigate geographical distribution of these three viruses,and also determine viral loads of NPHV/EPgV positive samples.In order to monitor the course of NPHV infection in horses,measured the anti NPHV-NS3 antibodies using the luciferase immunosorption system assay(LISA).Analysis the acute,persistent and resolved infections of NPHV and EPgV,and we studied the tropism in the PBMC and the liver pathogenesis.Method:Horse sera were collected from imported race horses in Guangzhou and Chinese work horses in Foshan of Guangdong province,China during April 2016 to September 2017,the race horse samples were obtained by longitudinal cohort over 17 months.All samples were separated and kept frozen until testing.A nest PCR method was used to detect viral partial gene,positive rate were calculated separately by plasma origin.The positive samples viral loads were determined by qPCR.For amplification of the near full length genome sequence,several fragments were amplified according to the isolate from the database.The NPHV-NS3 gene was amplified and constructed into pNLFl-N vector.Recombinant NPHV-NS3 protein was successfully expressed in Hela cells by cell disruption and collected cell lysates.The activity were determined by LISA.The blood was separated by Filcoll to obtain PBMC,and then the RNA extraction by Trizol,detecting the viral positive and negative strand RNA to determine the tropism in PBMC.Liver transaminase were detected and quantitated using enzyme-linked immunosorbent assay(ELISA).Result:1.NPHV viremia was detected in 8.1%(6/74)of the total horses tested with a prevalence of 11.5%and 0%in race horses and Chinese horses,respectively.EPgV was detected in 10.8%(8/74)of the total horses tested with a prevalence of 5.8%and 22.7%in race horses and Chinese horses,respectively.No TDAV infection found.2.The recombinant plasmid was successfully constructed and transformed into Hela cells,and the result of LISA indicated the activity of the fusion protein.Of the 73 serum samples tested,37.0%(27/73)were positive for anti-NS3 antibody.3.The near full length sequence of two NPHV isolates and one EPgV stain were obtained.4.While EPgV positive and negative strand RNA were all not detected in PBMC,indicating the PBMC may not infected by EPgV.The NPHV positive strand RNA was detected while the negative strand RNA was not,the NPHV infected PBMC or not,either viral replication in it,it warrant further investigation.The liver enzymes result suggested two viruses possibly no liver pathogenesis of viral infection.Conclusion:1.The virame prevalence of NPHV(8.1%)and EPgV(8.7%)were consistent with previously report(NPHV:2.1～11.5%;EPgV:0.5～14%).2.The serogiology test of NPHV(37.0%)were consistent with previous study(22～45%).3.The genetic diversity was shown in NPHV and EPgV.4.The NPHV show chronic infection.5.The NPHV and EPgV did not show tropism in PBMC and possibly no liver pathogenesis.