Sequence Analysis Of The Complete Genome Of Swine Hepatitis E Virus And Generation Of Infectious Clone

Hepatitis E is a self-limited acute illness which caused by Hepatitis E virus (HEV). The illnessmay be particularly severe among pregnant women, with mortality rates reaching as high as 20ï¼….Human healthy will be severe harmed. Hepatitis E virus (HEV) was previously considered anenterically transmitted non-A, non-B hepatitis virus. Using an immunoelectronmieroscopictechnique. Balagan et al observed a type of particles 27 to 30 nm in diameter in the stool sample ofa volunteer subject. They further proved this virus was a new non-A, non-B type that represents aserious threat to human health. Reyes et al cloned the genome of this virus. The virus and thehepatitis caused by the virus were named as HEV and hepatitis E, respectively, at an internationalconference held in Tokyo Japan in 1989. The HEV genome is a single-stranded, positive-senseRNA molecule of approximately 7.5 kb. The viral genome contains three open reading frames(ORFs) flanked by two non-coding regions (NCRs) at the 5'- and 3'-ends. The order of thesequence is 5'-NCR, ORF1, ORF2, ORF3, 3'-NCR and a polyadenylated tail.Studies have shown that HEV genomic sequences from different regions present specificdifferences, while sequences from the same region exhibit relative stability. The main HEVgenotypes found in China are genotypeâ… andâ…£and sporadic hepatitis E is dominated by HEVâ…£infection. In this study, we analyzed the complete genome sequence of HEV swDQ isolated fromstool samples of swine in an area of epidemic swine HEV in Heilongjiang Province to identify thegenotype of HEV that prevailed in that region and further investigate the relationship betweenswine and human HEV. The overlapping fragments of the swDQ genome were amplified and thefull-length genome was obtained by rapid amplification of the cDNA ends (RACE).HEV swDQ was divided into 15 fragments for RT-PCR amplification. The obtained sequenceswere assembled to a complete genomic sequence and submitted to the GenBank database(accession number DQ279091). The full-length genome of swDQ strain is 7,234 bp (excluding thepolyA tail) with 26 bp of 5'-NCR and 69 bp of 3'-NCR. There are 30RFs between the 5'- and3'-NCRs. ORF1, between nt 27 and 5,144, has 5,118 bp encoding 1,705 amino acids. ORF2,between nt 5,141 and 7,165, has 2,025 bp encoding 674 amino acids, and ORF3, between nt 5,169and 5,513, has 345 bp encoding 114 amino acids. Homology of the nucleotides and amino acidswere compared and a phylogenetic analysis was performed using DNAstar and Clustal software. Inthe ORF-1 region, the swDQ strain shared 81.9ï¼…to 83.5ï¼…and 93.5ï¼…to 94ï¼…sequence identitieswith HEV genotypeâ…£at the nucleotide and amino acid levels, respectively. Sequence similarities in the ORF2 region were 87.1ï¼…to 88.1ï¼…and 96.4ï¼…to 97.6ï¼…, respectively, and sequencesimilarities in the ORF3 region were 94.4ï¼…to 96.5ï¼…and 90.3ï¼…to 96.5ï¼…, respectively. Combinedwith the phylogenetic tree analysis, these results indicated the swDQ stain was HEV genotypeâ…£.Here, we present the first description of the complete genome sequence of HEVâ…£isolated fromswine in Heilongjiang, China.Many HEV sequences have been documented; however, lack of an appropriate in vitro culturesystem has limited further study of HEV. If infectious clone of swine HEV is constructed, we canstudy replication and expression of HEV, self-recombination and induced recombination, and RNAvirus interacting with hosts by insertion, deletion, mutation in DNA level. We can also developantiviral strategies and novel viral vectors. In China, rescue of swine HEV infectious clone remainsvacant. In this study, on the basic of swDQ. We divided HEV genome into 6 fragments, and carriedout RT-PCR. Production of RT-PCR was cloned. Recombinant plasmid of pilE, pCHE wereconstructed including of the complete genome cDNA.In pHE, T7promoter was inserted in5'-upstream of HEV genome. NruI as molecular label was inserted in 3'-downstream. Aftersequencing, it was revealed that 7 nucleotides were deleted and 1 nucleotide was inserted.Mutations were restored by PCR, and we obtained recombinant pHEa. The RNA was transcribed invitro followed by transfection into A549 cells and the HepG2 cells. Positive reaction is received byindirect immunofluorescence and RT-PCR. The T7 promoter and hammerhead ribozyme areintroduced immediately upstream of 5'-end, and HDV ribozymes is introduced 3'-end. Twodetections were carried out the same as pHEa when pCHE was transfected into A549 cells and theHepG2 cells. These results showed that the recombinant plasmids of pHEa and pCHE containedfull-length genomic cDNA of HEV swDQ are infectious. This is the frist time in our country thatinfectious clone vector of swine HEV is successfully constructed.In summary, prevalence and distribution of hepatitis E occurred in swine herds in heilongjianprovince is censused. Complete genome sequence was analyzed about swDQ HEV from swinestool, and decide HEV genotype in order to study on relation between swine HEV and humam HEV.In addition two infectious clone vectors contained CMV, 13-actin promoter and T7 promoterrespectively are constructed based on obtaining genome sequence. We hope to make transcript withinfection and restore swine HEV. This research work will lay fundamentals for molecular biologystudy of HEV and development of genetic engineering vaccines against HEV, and fills in thedomestic gaps.