Molecular Epidemiological Investigation On Swine HEV In Shanghai Suburbs

Hepatitis E virus (HEV) is zoontic disease, caused by HEV, and it does a great harm to human health. Among animals that carry HEV, pigs have the highest infection rate and virus burden.The isolated virus from pigs share the higher homology with human.The research on pigs is high meaningful for prevention and control of the disease. Based on the RT-nPCR method, the study makes molecular epidemiological investigation on swine HEV in Shanghai suburbs by detecting the viral RNA from feces, then we confirm the status of HEV. The result lay a scientific foundation for preventing and controlling the advent of HE.The experiment is divided into two parts:1. Identification of genotype 3 hepatitis E virus in fecal samples in a Shanghai suburbIn the present study, 46 swine fecal specimens were collected from five pig farms located in different Shanghai suburbs. Samples were converted to 10% (w/v) suspensions, viral RNA was extractd with TRIZOL reagent. RT-nPCR and nested-PCR were undertaken using partial nucleotide sequences of Open Reading Frame 2 (ORF2) of HEV to detect HEV RNA. Gel electrophoresis was taken to detect the amplified fragment. The fragment was cloned to pMD19-T vector, then transfered to DH5α, and the positive samples were sequenced. The sequence that we got was aligned and analysised through CLUSTAL-W (version 1.8),Mega 3.1 and Lasergene (version 7.0;DNAstar) .Genetic analysis was based on alignments of an amplified 150-nt ORF2 sequence, the study revealed 12 HEV positive samples among 46-pig fecal specimens examined. Among these, two belong to genotype 3, designate as CSH-3, CSH-4. This is the first time that genotype 3 hepatitis E virus has been identified on the Chinese mainland.2. Epidemiological investigation on swine HEV in Shanghai suburbsHepatitis E virus (HEV) genotype 3 has been identified in swine in Shanghai suburban. To accurately determine the prevalence of HEV infections among Shanghai pig farms, 426 pig fecal samples were collected from 37 pig farms located in all ten Shanghai suburban districts and tested for the presence of HEV RNA using RT-nPCR.Genetic analysis based on an amplified 150-bp ORF2 fragment revealed 111 samples to be HEV positive, and the prevalence of HEV infection within the different districts varied between 0.0% and 41.7%. Choose 1~2 from the positive sample in each farm randomly,and clone the amplified fragment to T vector.Totally, thirty-two samples were sequenced, and phylogenetic analysis indicated that ten isolates belonged to HEV genotype 4. The remaining 22 isolates belonged to genotype 3 and were most closely related to a strain of swine HEV, US-SW, isolated from pigs in the United States.The results before suggest that there have been only genotype 4 exited on mainland China. Our data indicated that genotype 3 HEV was widespread among Shanghai suburbs although further study is required to determine the source and zoonotic nature of the virus.