| Hepatitis E is caused by infection with the hepatitis E virus (HEV), is a acute and self-limited viral hepatitis. Highest rates of HEV infections occur in young to middle-aged adults. It often manifests fulminant hepatitis, when HEV infections occur in young to middle-aged adults. A high rate (10–20%) of unexplained fulminant diseases has been reported for pregnant women, especially in the third trimester. HEV is an important pathogen in developing country, and severely harmful to human. In order to develop region-specific diagnostic assays and prevent HEV, it is essential to understand epidemiology and genotypic variation within different hosts. However the epidemiological data of HEV infections in northeastern China is shortage. Since serological assays for HEV have only recently become available, the pathogenesis of HEV is not well understood. To understand the molecular events during early stage of infection, it is important to determine whether a proposed receptor is involved in the attachment of the virus to the cell or in the subsequent fusion reaction between the viral and cellular membrane. However the receptors or the proteins associated with infection of HEV are not well understood. In the study, we conducted a survey to ascertain the present state of HEV epidemiology in northeastern China and some places in Xinjiang, Sichuan, Guangzhou, analyzed the partially conserved nucleotide sequences of HEV strains isolated from patients and animals, and discussed the epidemiological features of the disease. At the same time, we expressed the recombinant swine HEV capsid protein (p293(ORF2 aa382-674)) and the protein encoded by the minor open reading frame (ORF3) of swine HEV (pORF3), explored the interactions between p293(ORF2 aa382-674)/pORF3 and HepG2 cell. In order to establish a culture method and an experimental infection model, experimental infection on swine, Meriones ungaiculatus and chicken embryos were carried out.4530 animal serum samples from Jilin, Inner Mongolia, Heilongjiang, Xinjiang, Sichuan( including 880 cattle serum, 831 sheep serum, 846 swine serum, 798 deer serum, 179 horse serum,432 rhesus serum, 36 dog serum,528 fowl serum) and 5090 human serum samples from Guangzhou and Jilin were collected. Serum samples were tested for HEV-immunoglobulin G (IgG) and HEV-immunoglobulin M (IgM) by using commercial ELISA kits. The seroprevalence of HEV- IgG was 79.67%, 15.73%, 33.07%, 22.02%, 44.11%, 58.33%, 5.11%, 8.80%, 29.98% in swine, horses, cattle, sheep, deer, dogs, fowls, wild Macaca mulatta, respectively. The infection rate in different species had a significant difference. The seroprevalence of HEV- IgG was highest in swine, and lowest in fowl.In 5090 human serum samples, 1526 serum samples were positive to HEV- IgG, and 180 serum samples were positive to HEV-IgM. The mean positive rate of HEV- IgG were 54.36%, 27.70% in Guangzhou and Jilin, respectively. The seroprevalence of HEV- IgG had a significant difference in the two places. In Guangzhou, the people had highest infection rate in development zone. In Jilin province, the mean positive rate was 27.7 percent. The males had higher prevalence than females (37.2% versus 18.4%; P= 0.00). The positive rate of HEV-specific IgG in swine keeper, other rural population, slaughterman, medical personnel, and other urban population was 40.5%(502), 29.1%(361), 57.3%(309), 40.0%(4) and 6.9%(113), respectively. The number of HEV-specific IgG seropositive individuals in other urban population was significant different with the other groups (P < 0.05). The number of HEV-specific IgG seropositive individuals in swine keeper was significant different with other rural population (P < 0.05). There was a significant difference in the number of HEV-specific IgG seropositive individuals when we compared individuals from 40-49 years old with those from 10-19 years old , 20-29 years old, 60-69 years old, and≥70 years old (P<0.05). This difference was not observed when individuals from 40-49 years old were compared with those from 30-39 years old (P=0.31) and 50-59 years old (P=0.59). HEV-specific IgM was detectable in 180 HEV infected individuals, including 143 male and 37 female. There was a significant difference in the number of HEV-specific IgM seropositive individuals when we compared individuals from 40-49 years old with those from 10-19 years old, and≥70 years old (P<0.05). This difference was not observed when individuals from 40-49 years old were compared with those from 10-19 years old (P=0.31), 20-29 years old (P=0.12), 30-39 years old (P=0.18), 50-59 years old (P=0.73), and 60-69 years old (P=0.11). The number of cases was 8 in January, 9 in February, 7 in March, 7 in April, 9 in May, 8 in June, 13 in July, 29 in August, 34 in September, 26 in October, 20 November, 10 in December, and the number of cases with acute HEV infection peaked during August, September and October.In molecular epidemiology study, we detected HEV partial sequences of ORF2 in stool, bile, and serum samples, and acquired 49 sequences, including 16 strains from human, 1 strains from sheep, 32 strains from swine. The homology of all isolated strains was almost above 82.2%-00.0%. When compared with the reference HEV isolates, the homology was 75.6-81.6% homologous to genotype I, 77.7%~80.9% to genotype II, 73.6%~81.0% to genotype III, 80.9%~95.7% to the other strains of genotype IV. It showed that all isolated strains belong to genotype IV. The sequences from human were 86.2%~100.0% homologous to sequences from animal. It indicated that the possibility, which animal HEV might infect humans raises a potential public health problem.In the phylogenetic tree constructed with all isolated strains and 12 strains of genotype IV known subgenotype, it showed that all strains can be divided into eight distinct major branches. The 12 strains of genotype IV known subgenotype can be divided into the branches according to their subgenotype. All strains which belong to the 8th major branche are isolated in this study. We named the strains"IVh" subgenotype. The results showed that IVa, IVb and the new subgenotype IVh are the representative subgenotype in northeastern China.In order to study the action between HEV and hosts cells, we expressed the recombinant swine Hepatitis E Virus capsid protein, and name it"p293(ORF2 aa382-674)". It showed that p293(ORF2 aa382-674) can formed 30~40 nm viral like particles. ELISA indicated that p293(ORF2 aa382-674) can reacted with the HEV positive human and swine serum, and have a good reactionogenicity. Immuno-fluorescens results showed p293(ORF2 aa382-674) can absorbed onto HepG2 cell. This action can be inhibited by the anti-p293(ORF2 aa382-674) antibody. The results indicated that p293(ORF2 aa382-674) maybe act as a analogue HEV, and can be used to study the infection mechanism. We found that p293(ORF2 aa382-674) can act with a 45~60 kDa protein in the HepG2 cell by VOPBA. We found that SLC3A2 4F2 cell-surface antigen heavy chain, TFRC Transferrin receptor protein 1, CD166 antigen maybe the receptor or receptor associated protein by the result of SDS-PAGE, mass spectrometry. Protein encoded by ORF3 (pORF3) of genotype IV swine HEV was expressed. ELISA indicated that p293(ORF2 aa382-674) can reacted with the HEV positive human and swine serum, and have a good reactionogenicity. We put pORF3 onto the NC membrane which transferred the whole protein of HepG2 cell, and found that pORF3 can act with a 45~60 kDa protein. This laid a foundation for study the function of ORF3 and pORF3 in the process of HEV infection.In order to establishment a culture method and experimental infection model, experimental infection on swine, Meriones ungaiculatus and chicken embryos were carried out. After the HEV positive swine feces inoculated into 9~11 days old chicken embryos, the HEV RNA were detectable , and could be lasted on the 3th generation. The HEV RNA were detectable in blood, kidney, spleen, mesenteric lymph node, liver, lung, when the HEV positive swine faeces was inoculated into swine and Meriones ungaiculatus. It was found that HEV can cause pathohistological change and can expess in the liver of the animals. But the clinical symptom was shortage. |
