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Analysis of Molecular Epidemiology of Swine Hepatitis E Virus (Hev) in Shanghai and Construction of Infectious Cdna Clone of Swine Genotype3Hev

Author: SiFuSheng
Tutor: YangZuo; LiZhen
School: Nanjing Agricultural College
Course: Preventive Veterinary Medicine
Keywords: swine hepatitis E virus genotype3 epidemiology completesequence cloning reverse genetics
CLC: S858.28
Type: PhD thesis
Year: 2011
Downloads: 23
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Abstract


Hepatitis E (HE), formerly known as non A, non B hepatitis, is a fecal-oral transmission disease caused by hepatitis E virus (HEV) and showing the global distribution. It is a zoonotic disease that has significant harm to human health, and has caused the outbreak of acute hepatitis in many developing countries. Studies have confirmed that HEV can infect pigs, cattle, goats, sheep, deer, primates, chickens, dogs, shellfish, rodents and other animals. Pigs have the highest infection rate and maximum viral load in these animal hosts, and the viruses isolated from pigs have high homology with human strains. Therefore, the existence status of the disease in pig herds has important public health significance on preventing and controlling the incidence of human hepatitis E.1Molecular epidemiological study of Hepatitis E Virus in ShanghaiIn order to accurately understand the prevalence of hepatitis E in shanghai,1487stool samples were collected from39pig farms in the suburbs of Shanghai. The partial sequence of HEV ORF2region was amplified by RT-nPCR method, and then sequenced and phylogenetic analyzed for studying the existing state of the virus. The results showed that297of1487samples were positive for HEV RNA (positive rate was20%). In the297samples,158samples were positive for the genotype3HEV,139were positive for the genotype4HEV, the total positive rate of each was10.6%and9.3%, respectively. Genotype3HEV accounted for the dominance in the infection. The HEV positive rate of all tested farms was from12.1%to36%, and the classification results showed that the genotypes of the HEV infection in the suburbs of Shanghai was genotype3and genotype4, among the12selected representative strains,8strains belonged to3b sub-genotype,4strains belonged to4b,4d and a new sub-genotype.We found that there was a middle scale negative relevance between positivity rates of genotype3and4HEVs in the different farms, which suggested a competition for niches between the two genotypes. In addition, the2-6months pigs had the highest infection rate in the three concerned groups (29.7%), the infection rate of1-8weeks pigs took second place in the survey, i.e.6.9%, the sows were only3.1%, which was the lowest among the three groups.Phylogenetic analysis showed that two new strains of subtype4HEV were identified. In addition, the genotype3HEV had the highest homology with a Japanese strain in the phylogenetic tree. The results showed that genotype3HEV was more widely distributed in Shanghai. However, previous studies had shown that there was only genotype4HEV in mainland China, thus its origin required further study.2The complete genome sequence determination and phylogenetic analysis of the SAAS-JDY5genotype3HEVTo further analyze the genotype3HEV infection in Shanghai suburb, the complete sequence of the genotype3HEV strain (SAAS-JDY5) was amplified by RT-nPCR and Overlap PCR method, and the both ends of5’and3’end sequences were amplified by the RACE (rapid amplification of cDNA ends) method.The sequencing results showed that the full-length genome of SAAS-JDY5strain was7225bp (excluding the3’poly (A) tail) with26bp of5’-NCR and73bp of3’-NCR. There were3ORFs between the5’-NCR and3’-NCR, ORF1, from nt27to5135, has5109bp by encoding1702amino acids; ORF2, from nt5170to7152, has1983bp by encoding660amino acids; and ORF3, from nt5159to5500, has342bp by encoding113amino acids. The obtained sequence was submitted to the GenBank database and the accession number was FJ527832. The obtained sequence of SAAS-JDY5was compared with other88reported HEV sequences at nucleotide and amino acid level, and we found that the SAAS-JDY5complete sequences had74-87%nucleotide homology with the genotype Ⅰ, Ⅱ, Ⅳ HEV; However, it had the highest homology (90.8%) with genotype3HEV. In the ORF1region, the SAAS-JDY5strain shared86.1%to88.7%and96.3%to96.9%sequence identities with other genotype3HEV at the nucleotide and amino acid levels, respectively. The sequence and amino acid similarities in the ORF2region were88.7%to90.8%and97.6%to98.9%, respectively. In the ORF3region, they were93.8%to98.1%and92.6%to98.4%, respectively.Phylogenetic analysis showed that the nucleotide of SAAS-JDY5was in the same branch with genotype3HEV. Our study firstly report the complete genome sequence of genotype3HEV isolated from swine in Shanghai, China.3The expression, identification of the recombinant HEV capsid protein and preparation of polyclonal antibodiesSince there is a lack of an efficient in vitro cell culture system and a practical animal model for HEV study, HEV-specific antibodies are detected by the ORF2antigen at present.In this study, the ORF2region (a.a381-614) of SAAS-JDY5was expressed by constructing a expression vector pET-32a-p234, which was designated p234protein. It was confirmed that the recombinant protein (p234) had a good immunogenicity and the ability to response to swine anti-HEV positive serum. High titer antiserum was obtained by immunization of rabbits by the purified protein, which laid a solid foundation for our further research.4Construction the swine genotype3HEV infectious cDNA clone of a Shanghai strain and the research on the in vitro transcription activity of the cloneDue to the lack of a suitable in vitro cell culture system, it was diffcult to proceed the futher study of HEV, thus greatly hampered the progress of HEV research. Reverse genetic system can be a valuable tool for studying the molecular biology of HEV. In this study, based on the obtained complete sequence of SAAS-JDY5strain, we cloned9overlapping DNA fragments through a series of DNA cloning and subcloning, and then constructed the recombinant plasmid designated pGEM4z-HEV which containing the full-length genome. In the pGEM4z-HEV plasmid, we introduced a unique XbaI restriction site and a T7RNA polymerase core promoter at the extreme5’terminus, and short stretch of15adenosines (A) and HindⅢ site at the3’end of the HEV cDNA clone. The plasmid pGEM4z-HEV was subjected to complete sequencing and14nucleotides mutations were identified. Silent mutations were kept as genetic markers and non-silent mutations were corrected by megaprimer PCR mutagenesis.In order to verify the infectivity of the constructed infectious cDNA clone, we firstly carried out cell transfection test with huh7cells. Swine genotype3HEV complete genomic RNA was synthesized by in vitro transcription with pGEM4z-HEV as template. Transfected huh7cells were cultured for6days and tested by RT-nPCR and fluorescence assay (IFA). Results proved that the viral protein synthesis was successfully expressed in the infected cells, which indicated that the infectious HEV cDNA clone was successfully constructed.5The preliminary study of artifical infect SD rats by genotype3HEVFollowing the successful test on Huh7cells, we conducted animal experiments to check infection nature of the HEV cDNA clone. The recombinant plasmid pGEM4z-HEV were linearized and used as a template for in vitro RNA transcription, and SD rats (SPF) was injected with the in vitro synthesized RNA intrahepaticly. Feces and serum samples were collected from the animals at0,3,7,14,21,28,35,45,50,55days post the injection, then the fecal samples were conducted RT-nPCR detection of HEV RNA. The serum samples were conducted double antigen sandwich ELISA and RT-nPCR for the detection of anti-HEV specific antibody and HEV RNA. Our results showed that HEV RNA was detected in the stool samples at7days post infection; The viremia began at about14days post infection and existed till28days post infection; Rats had their sero conversion at about14days post inoculation, however, the animals in the negative group remained sero negative throughout the study. In order to detect the possibility of natural infection, two sets of primers were used for the detection of the genetic markers. The results identified the genetic markers in the extracted HEV RNA. Our research confirmed that the genotype3swine HEV can infect SD rats, which suggested that the SD rat can be used as an animal model for the research of HEV replication and pathogenesis.In summary, a detailed epidemiological investigation of hepatitis E prevalence in Shanghai revealed a middle scale negative relationship between genotype3and4HEV. The complete sequence of the SAAS-JDY5strain which was isolated from our previous investigation was obtained and a complete sequence analysis was carried out. In addition, an infectious HEV cDNA clone was constructed and confirmed to be able to infect cultured cells and SD rats. The research laid the foundation for more advanced research on HEV molecular biology. The research also suggested the possibility that SD rats be used as an animal model of HEV replication and pathogenesis study.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Livestock, poultry, wildlife diseases > Livestock > Pig
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