Moleclular Epidemiology Of HEV In Wuhan And The Functions Of ORF3 Protein

Hepatitis E virus (HEV) is one of major causes of epidemically and sporadically, enterically transmitted non-A, non-B hepatitis in many developing countries in Asia, Africa and Latin America. The cases of HEV have been gradually increasing recently. Now the epidemiology of HEV infection is about 10%-20% in China in which most common infections have been found in adolescents and young adults, but it also occurs to a lesser extent in children and elders. Although the overall mortality rate associated with HEV infection is very low, it is reported that the mortality rate is up to 20% in infected pregnant women. In the past few years there were a lot of updated detection against antigen fragments of HEV, but the commercial kit marketed have a poor sensitivity. The sporadical cases of HEV infection were increasing in the past few years in Wuhan, But nowadays only the immuneglobulin M (IgM) against HEV (anti-HEV IgM) and immuneglobulin G (IgG) against HEV (anti-HEV IgG) have mainly been used as diagnostic markers of HEV infection clinically. Many HEV antigens, including synthetic peptides, recombination proteins of ORF3 and many recombination proteins of ORF2, have a better activity on acute sera, but as to chronic sera, it is just another case, or it is very hard to describe the diversity. If those reagents were used as epidemiologic study, the infection rate of HEV would be underestimated obviously, but as diagnostic criterias in clinic it could not tell the difference between the past infection and the current one so as not to satisfy with the need of diagnosis correctly. Therefore, it is an very important question to be solved to find a reliable diagnostic reagent kit on HEV infection including anti-HEV IgM and anti-HEV IgG..There are many different nucleotide sequences of HEV according to the different places in the world. The recent reports of many different derived HEV strains provide a basis for identifying as the same genotypes in HEV with which around 75% nucleotide similarities between two genotypes. according to this criterion, Eight different genotypes have been found in the world, with which at least six subgenotypes had been identifacted. But the HEV strains in the same case are not always just the same one. There are a little difference among those strains with which the difference is about 2-5% of HEV full-length nucleotide sequence. It is termed viral quasispecies. The heterogenicity and Clinic feature of the hepatitis E virus (HEV) are very important to understand its pathopoiesis, to establish specific diagnostic assays and producing vaccine on HEV.The HEV genome contains three open reading frames (ORFs). The ORF1 (about 5kb) is predicted to encode the viral nonstructural polyprotein, the ORF2 (about 2kb) encodes the viral major capsid protein, and the ORF3 encodes a protein of ～13.5kDa, a small protein, which is associated with the cytoskeletal and membrane fractions in expressing cells. Hepatitis-E was regarded a self-limited disease without chronic process for many years. But current research has indicated that there is a cis-reactive element in HEV ORF3. It is found ORF3 enhanced alpha1 microglobulin secretion from Hepatitis E virus ORF3-expressing human hepatoma cells is mediated by the tumor susceptibility gene 101. There is a long way to go before we understand the function of HEV ORF3.Therefore, in our study, we first investigated the epidemiology and the genotypes of HEV among out-and in-patients in the department of infectious diseases of Tongji hospital in Wuhan. Then, we established a method of enzyme-linked immunosorbent assays (ELISA) to detect anti-HEV IgA and reveal its change and significance of anti-HEV IgA in the sera of patients with hepatitis E. Meantime, we constructed a eukaryotic expression vector for expressing hepatitis E virus recombinant pEGFP-ORF3 and pXF2RH-ORF3 fusion protein and obtained a stable transfected HepG2 cell line. At last, we have investigated their effects of on HepG2.Objective1. To investigate the epidemiology and the genotypes of HEV among out-and in-patients in the department of infectious diseases of Tongji hospital in Wuhan and understand the distribution feature of heterogenicity of HEV, diversify of HEV and the relation between genetypes and clinic outcome of the disease.2. To establish a method of enzyme-linked immunosorbent assays (ELISA) to detect anti-HEV IgA and therefore increase the diagnostic specificity and sensitivity of HEV infection. 3. To study the quasispecies groups of hepatitis E virus ORF3 in the patients with hepatitis E infection in Wuhan and find the basis of the function of pORF3 on gene level.4. To construct a eukaryotic expression vector for expressing hepatitis E virus recombinant pEGFP-ORF3 and pXF2RH-ORF3 fusion protein and obtain a stable transfected HepG2 cell line and supple basis to study the function of ORF3.5. To investigate the effect of hepatitis E virus recombinant pEGFP-ORF3 and pXF2RH-ORF3 fusion protein on proliferation and apoptosis of HepG2 cell line.Methods1. Clinical data were elicited from patients' hospital records that are enrolled 916 cases clinically diagnosed as acute hepatitis during the period of August 2003 to August 2004, in which 120 patients were anti-HEV-IgM, IgG positive and of them the conserved genomic sequences of open reading frame 2 (345bp) in the HEV were detected using polymerase chain reaction, 25 of which have been cloned and sequenced. Clustal X and Mega software were used for phylogenetic analysising of genotype Ⅰ, Ⅱ, Ⅲ, Ⅳ in HEV strains of Wuhan.2. In this experiment, instead of Anti-HEV IgG detected in the later phase of HEV infection, a new method was established to assay Anti-HEV IgA, which can be detected in the different phase of the infection. And we compared Anti-HEV IgA assay with anti-HEV IgM and anti-HEV IgG methods in serial sera of 60 patients with HEV-RNA positive.3. We choosed two samples from the sera that were confirmed positive by HEV sequenced. The conserved genomic sequences of open reading frame 2 (346 bp) and the whole ORF3 region in the HEV were detected using RT-nested-polymerase chain reaction. Which was cloned into pMD18-T Vector, and in one sample, 30 clones were sequenced.4. The coding region of ORF3 gene of HEV was amplified by PCR and was digested by Ecol Ⅰ/BamH Ⅰ. This fragment was inserted into eukaryotic expression vector both pEGFP-N2 and pXF2RH with T4 ligase and transformed E. coli DH5α . The positive recombinant plasmid was selected, then the recombinant plasmid was transfected into HepG2 cell by Lipofectamine^TM 2000. Cells containing stable transformants were selected by the ability of resistance to G418 and isolated with a limited dilution. The expression of fusion gene was analyzed by fluorescent microscopy, SDS-PAGE and Western blot, respectively.5. MTT colorimetric assay was used to detect the proliferation activity of HepG2. Flow cytometry was used to observe the cell apoptosis of HepG2.Results1. 25 isolates shared the same genotype IV, with the similarity of nucleotides of 82.61-98.55%. Compared with other genotypes in HEV, They had 76.52-81.74%, 70.43-73.04%, 76.52-81.16%, and 84.35-88.70% homology at the nucleotide sequences in the HEV genotypes I -IV, respectively. Phylogenetic analysis suggested that these 25 isolates represented 3 different subtypes at least but there was no significant difference found in epidemiology feature and liver function among the three subtypes.2. The 60 patients with HEV-RNA positive had both anti-HEV IgA and anti-HEV IgM and 410 patients with HEV-RNA negative were used as control. Periodic serum samples obtained from 60 patients with hepatitis E were tested for HEV RNA, anti-HEV IgM, anti-HEV IgA and anti-HEV IgG. Their HEV-RNAs were detectable in the serum until 20±lld. We used anti-HEV IgM and anti-HEV IgA assays to detect the HEV infection and found that positive results was during 90±15d and 120±23d separately, which demonstrates that the positive rates of anti-HEV IgA was higher than that of anti-HEV IgM and HEV-RNA (P<0.05).3. The homology among the clones from one patient of ORF3 region was 97.97%～99.71%, while there was 89.86% 99.71% at the nucleotide sequences compared with HEV genotype IV. The substitution, deletion and insertion of short sequence were found in 3 open reading frames.4. Recombinant vector pEGFP-ORF3 and pXF2RH-ORF3 were successfully constructed and sequence result indicated that it was identical with reference sequence. The protein on transfected HepG2 cell membrane selected by G418 was confirmed by fluorescent microscopy, and pXF2RH-ORF3 approximately 28.5 kDa was conformed by SDS-PAGE and Western blot. Compared with HepG2 and HepG2/pEGFP-N2 (or pXF2RH) the increase of growth inhibitory rate and apoptosis rate in HepG2/pEGFP-ORF3 (or pXF2RH-ORF3) were not significant (P>0.05).5. Compared with HepG2 and HepG2/pEGFP-N2 (or pXF2RH) cell, proliferative capacity in HepG2/pEGFF-ORF3 (or pXF2RH-ORF3) was slightly increased (P>0.05). The Cell cycle in HepG2/pEGFP-ORF3 (or pXF2RH-ORF3) were Gl= 68.09% and 67.1%, S=19.50% and 19.0%, G2=12.41% and 13.9%, apoptosis rates were 0.83% and 0.86%. The Cell cycle in HepG2 was G1=72.15%, S=14.91%, G2=12.95%, apoptosis rates 1.41%. The changes had not significant.Conclusions1. HEV sequences isolated from patients of Wuhan belong to HEV genotype IV with different subtypes. The infection rate of HEV is gradually increased in Wuhan, in which that of male patients are 3.3 times higher than female patients found in clinical investigation. Age from 30 years to 59 years and seasonality from March to June are susceptible factor for patients to hepatitis E infection.2. The duration of anti-HEV IgA in the serum is longer than that of anti-HEV IgM and anti-HEV IgA assay is a good method to detect HEV infection. It is helpful to diagnose HEV after anti-HEV IgM had disappeared in the serum that demonstrated that Anti-HEV IgA with or without anti-HEV IgM is useful for serological diagnosis of hepatitis E.3. There were HEV quasispecies groups in one patient with HEV infection. The substitution in two C-terminal proline-rich regions of HEV-ORF3 could not influence the translation of the proline and the substitutions in amino-acid sequence of viral protein are worthy of further study.4. The stable transfected HepG2 cell line could express pEGFP-ORF3 and pXF2RH-ORF3 fusion protein.5. Compared with HepG2/pEGFP-N2 (or pXF2RH), We found that there was no significance both in growth inhibitory rate and apoptosis rate in HepG2/ pEGFP-ORF3 (or pXF2RH-ORF3) (P>0.05).