Molecular Epidemiology Of Swine Hepatitis E Virus In Shanghai Suburbs And Reverse Genetics Of HEV

Hepatitis E is a fecal-oral transmission, zoonotic disease, which is caused by hepatitis E virus (HEV). Pig is the big reservoir of HEV. Therefore, finding out the existence status of the disease in pig herds is important for preventing the HEV.Molecular epidemiology of HEV:458samples deruved from22pig farms in shanghai suburbs. We used RT-nPCR to detect the HEV RAN in the samples. HEV RNA positive rate was26.2%. Genotype4HEV was the major genotype in shanghai, the subtypes were4i、4d and4b. Genotype3HEV is the secondary, just having3b subtype. Virus rescue for the clone of SAAS-JDY5strain:This study was aimed to test the infectivity of the full-length cDNA clone of SAAS-JDY5in vitro. Capped RNAs transcribed in vitro with T7polymerase were transfeced with Huh-7cells and injected into SD rat (clean grade) liver intrahepatically to reseuce the virus. IFA result showed ORF2was expressed in Huh-7cell, indicating that the transfected viral RNA was replication-competent in the Huh-7cells.. Animal experimental result showed that not all SD rat could be infected clone of HEV3SAAS-JDY5strain (3/5).Genetic mark detected from HEV RNA positive fecal samples confirmed that the RNA was derived from clone of HEV3SAAS-JDY5strain.To establish HEV-Gluc reporter replicon construction and the Gluc development The full-length HEV cDNA clone SAAS-JDY5was modified to introduce the Gluc reporter gene as an in-frame fusion with the ORF2and ORF3genes. After transcription, the capped RNA were transfected with Huh-7and HepG2cells, respectively. The Gluc activity were detected in the culture supernatant of Huh-7and HepG2cells.The result indicated Gluc express increased markedly at the third day of the post-transfection. At the fifth day the Gluc quantity in the experimental group and control group expressed was different significantly. The day can be the detecting day.Intergenotypic chimeric HEVs and its replicon with the genotype1,3and4HEV JR in the backbone of genotype3and4swine HEV construction:The conserved nucleotides on the stem-loop (SL) structure in the intergenic region (JR) of the HEV genome in virus replication is important. The JR of genotype3HEV of SAAS-JDY5strain was replaced with corresponding JR of genotype1and4HEV, respectively; The JR of genotype4HEV of SAAS-FX17strain was replaced with corresponding JR of genotype1and4HEV, respectively. Four chimeric virus with intergenotypic JR and its Gluc repicon were obtained. The Gluc activity could be detected on the5th day of postransfection, which will assess the effect of JR replacement on HEV replication efficiency.