Epidemiological Investigation Of Swine HEV In Parts Of Shaanxi Province And Analysis Of The Cell Protein Interacting With Avian HEV Capsid Protein

This study covers the epidemiological investigation of swine hepatitis E in part of Shaanxi province and identification and characterization of the host protein interacting with avian hepatitis E virus capsid protein.Hepatitis E,caused by the hepatitis E virus（HEV）,is an important public health concern in many Asian and African developing countries as well in some industrialized countries,where it occurs sporadically.Although HEV only causes subclinical infection in pigs,it has been reported that consumption of raw and undercooked pork can transmit HEV to humans.Therefore,the spread of HEV in pigs is an important public health concern.It is recognized that pig is a major natural reservoir of HEV.Both genotype 3 and 4 HEV are zoonotic and genotype 4 HEV is the the predominant HEV genotype in China,is also the predominant genotype on swine farms in China.To date,six subgenotypes（4a,4b,4d,4g,4h and 4i）have been identified in both humans and pigs in China.Seroepidemiology and genetic characterization of HEV have been performed on pigs from Jilin,Guangdong,Zhejiang,Jiangsu,Shandong provinces and in the city of Shanghai,all regions in eastern China.However,there are only few reports of HEV prevalence on swine farms in north‐west and central regions of China.In this study,the seroprevalence and genetic characterization of HEV infection in swine farms of Shaanxi Province were investigated.The results showed that 13 of 17 farms in five cities are positive（76.47%）for anti‐HEV antibodies.Within positive farms,the proportion of positive pigs ranged from 1.6% to 37.5%.Genetic detection analyses of faecal samples revealed that pigs in four cities and on nine of 17 farms were positive for sequences homologous to a partial ORF2-coding region of HEV（306 bp）,as were 6 of 53 bile and 1 of 26 semen samples.Meanwhile,DNA coding for partial HEV ORF1（1,080 bp）and a longer gene segment coding for partial ORF2（1,594 bp）were successfully amplified fromRNA isolated from pig semen from one HEV‐positive pig.Sequence comparisons of partial ORF2 gene sequences showed that HEV isolates from Shaanxi Province shared the highest identity（81.4%–96.1%）with genotype 4 HEV.Phylogenetic tree analysis grouped these isolates into three subgenotypes（4d,4h and 4i）,with subgenotype 4i the predominant subgenotype.In addition,the HEV isolate from pig semen belonged to subgenotype 4i HEV based on phylogenetic trees constructed both using partial ORF1 and ORF2 gene sequences.In conclusion,HEV infection is endemic on pig farms of Shaanxi Province,China,and 4i is the predominant HEV subgenotype.More important,this is the first study demonstrating detection of HEV RNA in pig semen,suggesting that artificial insemination can transmit HEV in pigs.The second part of this study is the identification and characterization of organic anion transporting polypeptides 1A2（OATP1A2）that interacting with avian HEV ORF2 protein.Avian hepatitis E virus（HEV）is the main causative agent of big liver and spleen disease and hepatitis-splenomegaly syndrome in chickens,which seriously threating the production of chicken industry.Due to the absence of cell culture for avian HEV,the mechanism of virus infection is still not clear.In recent years,the exploration of HEV in vitro culture system is always the focus of HEV research.Avian HEV ORF2,including 606 amino acids（aa）,encodes capsid protein of the virus and contains the major epitope of the viral particle,which is responsible for stimulating a protective humoral immune response.Formerly,studies on avian HEV ORF2 focused on the research of antigenic epitopes and antigenicity of capsid proteins,and few of them explored their functions in viral infection.As a non-enveloped virus,ORF2,the capsid protein of avian HEV may participate in viral attachment and entry through interacting with host factors.ORF2-interacting proteins have been reported in mammalian HEVs.The truncated ORF2 protein p239（aa 368-606）,which can self-assemble into virus-like particles,was used as a bait protein to screen the interacting proteins of host,and confirmed that the target proteins played different roles in virus adsorption and infection.In our study,the truncated avian HEV capsid protein was used as a bait protein to screen the target protein in liver tissue.The interaction was further confirmed and explored the function of the target protein in viral attachment and infection.The results are as follows:1.Identification of CaHEV ORF2-interacting proteins in liver tissueBased on the report of human HEV ORF2 truncated protein,named p239（aa 368–606）,we designed the region of CaHEV ORF2 at aa 313-549 and named it ap237.This protein includes three antigenic domains and a neutralizing epitope.In this study,the recombinant protein GST-ap237 was obtained and incubated with chicken liver tissue lysate to perform GST pull-down.The complexes were subject to mass spectrometry assay.Totals of 236 target proteins interacting with ap237 were analyzed using bioinformatics software.A transmembrane protein OATP1A2 was identified.The interaction between OATP1A2 and CaHEV capsid protein was further verified by immunoprecipitation and confocal assay.The ectodomains of OATP1A2 was tandem and expressed with GST tag in E.coli.The direct binding of OATP1A2 to ORF2 protein was confirmed by GST pull-down and ELISA analysis.2.Impact of OATP1A2 on CaHEV attachment and infectionCell line LMH^1A2-GFP stably expressing OATP1A2 was successfully established and identified.Western blotting and flowcytometry assay showed that the adsorption of ap237 on LMH cells was increased with the high expression of OATP1A2.The attachment and infection of CaHEV to LMH^1A2-GFP,LMH^GFP and LMH cells showed that with the decrease of OATP1A2,the attachment and infection of CaHEV were reduced.To further investigate the function of OATP1A2 on CaHEV attachment and infection to LMH^1A2-GFPcell,some of the substrate（or inhibitor）of the transporter protein OATP1A2 were selected and used to incubate with LMH^1A2-GFP cell before virus incubation.It was found that these substrates（chenodeoxycholic acid and imatinib）or inhibitors（carvedilol）of OATP1A2 significantly blocked the virus attachment and infection to LMH^1A2-GFP cell at dose-dependent manner.Besides,the treatment of anti-1A2^ecto serum and ap237 protein also significantly blocked the virus attachment and infection to LMH^1A2-GFP cell.Immunohistochemistry assay revealed that OATP1A2 protein was present in chicken liver,kidney,testis,brain and spinal cord.Detection of OATP1A2 mRNA and CaHEV RNA in infected chicken tissues showed that CaHEV RNA copies was higher in liver,kidney,testis,brain and spinal cord,in which has a relative higher expression of OATP1A2 mRNA.The copies of CaHEV RNA was consistent with the change of the relative expression level of OATP1A2 mRNA.It was further confirmed that chicken OATP1A2 protein is associated with avian HEV infection.In conclusion,the transmembrane protein OATP1A2 directly binds to avian HEV ORF2 protein and inhance the virus attachment and infection.These effects can be blocked by the substrate or inhibitor of OATP1A2,anti-OATP1A2 serum and ap237 protein.Therefore,it is speculated that OATP1A2 is one of the important proteins involved in the attachment and infection of avian HEV.