Aanalysis Of Genotype And ORF3 Gene Mutation Of Hepatitis E Virus In Wuhan

BackgroundHepatitis E virus(HEV) is a major cause of acute, self-limiting disease. It is primarily transmitted by the fecal–oral route and frequently results in sporadic,as well as epidemic, hepatitis. The clinical manifestations of acute hepatitis E is similar to those of hepatitis A, but the mortality rate of hepatitis E is higher than that of other hepatitides, especially up to 30%–100% in infected pregnant women. China is one of the high incidence areas of hepatitis E.So far, the world's largest hepatasit E outbreak had occurred in Xinjiang Uighur Autonomous Region between1986 and1988 with about 12,000 individuals infected and 707 people died,which did great harm to society and economic. In recent years, the incidence of acute sporadic hepatitis E has been rising in our country.HEV is a a positive sense, single-stranded RNA virus without envelope.The genome of is approximately 7.3 kb and contains three open reading frames (ORFs).ORF1 locates in the 5 'end of the genome and encodes non-structural protein which is essential for HEV replication.ORF2 locates in the 3 'end of the genome and encodes the viral capsid protein.ORF3 also locates in the 3' end of the genome and encodes a small phosphoprotein whose function(s) has not been fully defined. ORF3 of genotypeⅠ,ⅡandⅢwas 372 bp and encodes a protein of 123 amino acids.Compared to ORF3 of genotypesⅠ,ⅡandⅢ, ORF3 of genotype 4 contains a nucleotide insertion(U) which changes the downstream reading frames and this frame shift is predicted to shorten the ORF3 protein by 9 amino acids,including 114 amino acids.For these reasons,in our study,we firstiy investigate general situation(such as gender and age) and performed genotyping of HEV isolates among out-and in-patients with acute hepatitis E in the department of Infectious Diseases of Tongji Hospital in Wuhan.Then we detect and analyze the mutation of typeⅣHEV ORF3 and the encoded protein.Objective1. To investigate the epidemiological characteristics and genotype of hepatitis E virus (HEV) in Wuhan,which will provide some clues to determine genotype distribution of HEV in Wuhan.2. To detect and analyze the gene mutation of GenotypeⅣHEV ORF3 in Wuhan,which will further lay the foundation forinvestigating clinical significance of GenotypeⅣHEV ORF3 gene mutation.Methods1. 162 serum samples were collected from out-and in-patients with acute hepatitis E in the department of Infectious Diseases of our hospital between April 2009 and June 2010 and they were saved in -80℃after new package to test for HEV RNA. General situations(such as gender and age) of patients were surveyed.2. 162 serum samples collected from patients with acute hepatitis E were tested for HEV RNA by reverse transcription-nested polymerase chain reaction (RT-nPCR). The PCR products were sequenced. Then the sequencing results were used for phylogenetic analysis of HEV3. Two gene fragments of 41 GenotypeⅣHEV RNA(5020～5392nt and 5347～5956nt,EF570133) were amplified by reverse transcription-nested polymerase chain reaction (RT-nPCR).The PCR products were sequenced.Then these sequences were stitched(containing complete HEV ORF3) with ContigExpress and HEV ORF3s were aligned with MegAlign(DNAStar software package).The mutations of HEV ORF3 gene and ORF3 protein were analysed.Results1. The ratio of male and female patients with acute hepatitis E was 4.40:1 .The patients aged from 30 to 59 years accounted for 71.60%.41 out of 162 serum samples were positive for HEV RNA.The positive rate was 25.31%.Phylogenetic analysis showed that the 41 isolates belonged to genotypeⅣHEV. They shared 84.7.0%～100% identity with each other at the nucleotide level.The genotypeⅣisolates could be further divided into 5 subtypes, designating as A-E. The nucleotide sequence homologies within the five subtypes were 91.3%～100%,98.0%,92.0%～100%,98.0%～100% and 100%,respectively.SubtypeⅣ-A had 91.3%～96.0%,92.0%～97.3% and 94.7%～99.3% nucleotide sequence homology with the HEV isolates from Guangxi,Shanghai and Xian.SubtypeⅣ-D had 95.3%～96.7% nucleotide sequence homology with the HEV isolate from the Beijing.2. Both of HEV RNA gene fragments were amplified in 18 HEV isolates.There were 47 nucleotide substitutions of ORF3 gene in 18 HEV isolates.Among them ,21 nucleotide substitutions were found in the first or second nucleotide of codon and 26 nucleotide substitutions were found in the third nucleotide of codon.There were 19 amino acid substitutions of ORF3 protein in 18 HEV isolates.Conclusion1. AHE mostly occurs in male.People aged from 30 to 59 years are more susceptible to infection.The pathogen of acute hepatitis E is genotypeⅣHEV in Wuhan.The genotypeⅣisolates could be further divided into 5 subtype, designating asⅣ-A toⅣ-E. There is a close relationship between subtypeⅣ-A and Guangxi,Shanghai and Xian strains.There is a close relationship between subtypeⅣ-D and Beijing strain.2. There are a large number of nucleotide substitutions in GenotypeⅣHEV ORF3 gene in Wuhan. The significance of these gene mutations further needs to be studied in the progression of acute hepatitis E.