Study Of The Molecular Epidemiology Hepatitis E And Inferaction Mechanism Between Hev Its Host

Hepatitis E (HE) is an important public health problem in developing countries. The hepatitis E virus (HEV), a member of the genus Hepevirus, is the causative agent of HE. HEV is a non-enveloped virus with a positives-stranded RNA genome approximately 7.2 kb in length and contains three open reading frames (ORFs). HEV isolates can be divided into four distinct genotypes according to the phylogenetic analysis based on nucleotide sequences.HEV is believed to be transmitted by the faecal-oral route, and outbreaks of hepatitis E are attributed to water contaminated with HEV. HEV and antibodies to HEV have been reportedly found in a wide variety of animals, especially swine. Animals may act as an important role in the cross-species transmission of HEV. China owns large number of wild-life animal sources, but the infection status of HEV in this group is still unclear. Since 2000, genotype 4 HEV has become the dominant cause of hepatitis E disease in China. A recent report showed that genotype 4 HEV is freely transmitted between humans and swine in eastern ChinaAlthough the great progress have been made in the research field of HEV, there are still some unclear points, including (1) The interaction mechanism between HEV and its host cell; (2) The replication mechanism of HEV in host cell; (3) The translation of HEV in host cells;(4) Pathogenesis of HEV.In order to elucidate some points mentioned above, we performed the following research:1. Study of the molecular epidemiology of HE.(1) Detection of HEV in the wild-life animals 191 fecal samples were collected from two wild-life zoo lie in Shanghai city and Anhui Province, respectively. The HEV RNA was detected by using RT-nPCR method. The results indicated that 11 of 39 fecal samples collected from Anhui Province were positive for HEV RNA. Sequence analysis showed that all the 11 HEV strains belonged to genotype 4, sharing more than 96-100% sequence identities with each other, which suggested that cross-species infection of HEV happened in the wild-life zoo. Moreover, 5of 7 serum samples collected from the workers in the zoo were positive for anti-HEV antibodies, suggesting that human in the zoo were also infected by this HEV strain, though we had not isolate HEV RNA from these sera.(2) Prevalence of HEV in the human and swine populations in the north part of Anhui ProvinceThe prevalence of HEV is related with season, geography environment, and the human living habit. More and more cases of HEV cross-species infection have been reported all over the world. The north part of Anhui province is a farming area where swine farms rich distribute. We collected 1476 human sera from general human population and 554 fecal samples from general swine groups from this area. The serum samples were detected for the prevalence of anti-HEV antibodies and the antibody-positive sera were further detected for HEV RNA. The fecal samples were tested for the HEV RNA. Our results indicated that 7.93% (117/1476) of the sera were positive for anti-HEV IgG or IgM, 0.95% (14/1476) of the sera were positive for HEV RNA. The HEV positive rate of the fecal samples was 7.0% (39/554). Sequence analysis of the 53 strains showed that these strains clustered into 3 main groups, the strains from pigs were divided into 2 groups, and all the strains isolated from human population clustered together, forming a group without swine HEV strains, which suggested that no cross-human-swine infection of HEV were involved into this area. The full-length ORF2 gene of 3 representative HEV strains in the 3 main groups were determined and sequence analysis indicated all of them contain 2025 bp, encoding a putative 674 aa proteins. Phylogenetic analysis showed that these 3 strains clustered into 3 genetically distinct groups.(3) Isolation and characterization of several HEV strains in eastern ChinaThis study analyzed the HEV strains isolated from an infant, horse, shanghai swine and other 67 HEV strains we cloned or referenced from GenBank. The results suggested the following contents: (1) This is the first time that HEV strain was isolated from an infant less than one-year-old, the full-length ORF2 gene of this strain was determined and shared 97% sequence identity with a Japanese HEV strain, AB197673; (2) A genotype 3 HEV strain was detected in one horse serum, and this HEV strain clustered closely with a genotype 3 HEV strain isolated from a pig in Zhejiang province; (3) Analysis of the complete genome of Shanghai swine HEV strain indicated that the genome organization of it was identical to genotype 1-3, but inconsistent with previous genotype 4 HEV strains, that is, ORF2 of this shanghai swine HEV strain codes 660aa not 674 aa. This result was confirmed by western blot assay.2. The mechanism of HEV interation with the host cell(1) Identification of a 55kDa protein as a candidate receptor of HEV on liver cell of pig.In the present study, we try to find the candidate receptor of HEV on cell surface of different tissues, including stomach, dodecadactylon, lung, liver, mesenteric lymph node, kidney, and spleen of pig, using virus overlap proteins binding assay (VOPBA). The results indicated that a 55kDa aimed protein band was identified on the liver and stamoch cell surface, and this protein band was purified and could significantly reduce the CPE of A549 caused by HEV infection. The protein band was subjected to Mass spectrum assay, and the results showed the components of this protein band were very complicated and radixin, integral membrane protein 2B, ATP synthase subunit alpha liver isoform, and cytochrome P450 2C49 may be the candidate receptor of HEV on the pig liver cell surface.(2) Screening of proteins that interact with HEV pORF3 in the human liver cDNA expression library using CytoTrap yeast two-hybrid system.So far, there has no good cell system for HEV culture, which greatly hinders the study of the mechanism of HEV interaction with its host cell. The true function of pORF3 of HEV is still unclear. In the present study, using CytoTrap yeast two-hybrid system, we screened 10 proteins which interacted with HEV pORF3, which will be good base for elucidating the function of HEV pORF3 in the process of HEV infection its host cells. (3) Te comparative proteomic study of A549 cell infected with HEV.The molecular mechanism of interaction between HEV and host cell is still unclear. In the present study, combining 2-dismension electrophoresis (2-DE) and MALDI-TOF, we compared the protein expression of the HEV infected A549 cell and control cell. After the A549 cells were incubated with HEV, cytopathic effect assay, dot-blot assay, RT-PCR and immune electronic microscope (IEM) were used to confirm that the A549 cells were infected by HEV. The total proteins of the experimental and control cell were extracted and subjected to 2-DE and the protein dots which showed different between experimental and control cell were sliced from the gel and were analyzed by MALDI-TOF. The results indicated that 31 protein dots showed difference between experimental and control cells. Nine of them were related to metabolism of big molecular in cell, 6 of them were components of cytoskeleton, 6 of them join in molecular modification in cell, 4 of them join in the process of membrane bubble transmission, 2 of them joined in the process of signal transduction, and one of them was the capsid protein of HEV. The mRNA expression of 15 of them was identified using RT-PCR method, the protein expression of two protein dots were confirmed by western blot assay. This suggested that the compared proteomic results were convinced. From the results of compared proteome, we speculated that HEV infection reduced the speed of metabolism and the assembly of cytoskeleton so as to provide suitable environment for HEV replication.