| Hepatitis E has become an important public health problem worldwide,and Hepatitis E Virus(HEV)is the pathogen of Hepatitis E(HE).China is a high-risk area of HE.From 1986 to 1988,a large outbreak of HE occurred in Xinjiang,China.A total of 119,280 cases occurred,with a prevalence rate of 2.96%.The results of the national legal infectious disease report show that in recent years,the number of HE cases in China has continued to rise.In this study,a nested RT-PCR method for detecting HEV was established to investigate the HEV infection in most areas of Sichuan Province.On this basis,the partial region of HEV ORF2 was cloned and analyzed,and its genetic evolution was analyzed to lay a foundation for the study of HEV standard strains in China.In addition,the established HEV-infected rat model was observed in terms of tissue tropism,antibody level,transaminase level and histopathology,and the infection process of the rat model was analyzed according to the observation results,laying a foundation for the study of the pathogenesis of HEV.In this study,two pairs of inner and outer pairs of ORF2 gene conserved regions of multiple genotypes of hepatitis E were designed by comparing the sequences of 20 strains of hepatitis E virus at home and abroad,and the nested PCR primers with a fragment size of 638 bp were amplified.The annealing temperature was optimized,and the HEV nested RT-PCR detection method was established.The obtained fragment was cloned and sequenced.The results showed that the porcine hepatitis E positive disease material was detected by this method,and the target fragment was consistent with the expectation.The annealing temperature of the first round and the second round of amplification was the best at 53℃.The HEV nested RT-PCR established in this study has good specificity and high sensitivity,and can be applied to clinical diagnosis.Using this method,174 pig feces samples and 160 bile samples from various scale farms in Sichuan Province were tested,and the positive rate was 10.5%.The highest positive rate(17.9%)ranged from 5-9 weeks.The highest to lowest pig breeds were Chenghua Pig,Large White Pig,Duroc,Pietrain,Landrace and Hampshire Pig.Nucleotide and amino acid sequence analysis showed that the popular genotype of SHEV in Sichuan Province was gene type IV,which had the highest homology with Beijing strain.The comparison analysis between Sichuan strain and Chinese representative strain showed a large variation,indicating that there may be a new HEV strain.In order to understand the tissue tropism,pathological changes and infection process of SHEV-infected rat model,a pair of primers were designed for the conserved sequence of HEV ORF2 gene,and SYBR Green II real-time quantitative RT-PCR method for detecting HEV was established.The method has good specificity,high sensitivity and good reproducibility.The method is used to quantitatively detect the organ tissues of the SHEV-infected rat model.A SHEV rat model was established by intraperitoneal injection of a genotype IV SHEV-positive pig feces suspension.First,changes in alanine aminotransferase(ALT)and aspartate aminotransferase(AST)in rat serum were examined.Second,HEV RNA in the heart,liver,spleen,lung,kidney,feces and brain of rats at different stages was examined.Third,observe the histopathological changes of rats at different stages.According to the changes of serum HEV IgG,ALT and AST levels in rats,the results of HEV RNA detection can infect SD rats across species,and there is no obvious clinical symptoms after infection.HEV RNA was detected in most tissues and organs after infection,but the viral load was low.The liver has pathological changes in chronic hepatitis,but the lesions are mild. |
