| Hepatitis E virus (HEV), the causative agent of hepatitis E, is an important public health concern in many countries. Accumulating evidences indicate that hepatitis E is a zoonotic disease, which means that the HEV virus exists in natural animal reservoirs. HEV infection is known to be endemic in China. Increasing reports of sporadic hepatitis E cases are likely related to animal origins. Fully understanding the molecular prevalence of HEV in animals is significant important for the control and prevention of hepatitis E. In this study, we collected animal serum samples from different provinces across the country. The prevalence and the genomic diversity of HEV in these animals were investigated and evaluated. To further investigate the molecular mechanism of host range restriction of different HEV genotypes and the pathogenesis of HEV, yeast two-hybrid system was used to screen a human liver cDNA library for identifying proteins interacting with HEV-ORF3. The molecular functions of ORF3 were analyzed and predicted through bioinformatics analysis of the interacting host proteins.1. The prevalence of and genomic diversity of HEV in domestic animalsA total of 6,329 domestic animal sera were collected from 27 provinces and detected for HEV-specific antibodies and antigens by enzyme immunoassays. The average antibody positive rates were 82.2%,28.2%,10.4% and 15.4% in pigs, cattle, goats and rabbits respectively. The positive rate of pigs was statistically higher than those of other animal species.3.58% and 7.1% chicken and ducks were also found to be anti-HEV positive although the positive rates of both of these two animals were significantly lower than those of mammalian animals. The prevalence of anti-HEV antibody in animals varied from province to province, ranging from 10.9% to 100% in pigs,0 to 48% in goats and 0 to 92.9% in cattle. The positive rates for HEV antigen were 1.9% in pigs,1.6% in goats,0.8% in cattle and 3.8% in rabbits tested in the study. HEV antigen positive rates of chicken and ducks were 1.79% and 1.43% respectively.HEV RNA was detected in 15 swine and 22 rabbit samples of various breeds. Phylogenetic analysis of the 304-bp sequences within the viral ORF2 classified all of the 15 swine HEV isolates into HEV Genotype 4, which were further divided into four different subtypes,4a,4b, and two new subtypes. Thus far,4a,4b,4d,4f,4g and the two new subgenotypes were found to be circulating in pigs across the country. All the 22 isolates from rabbit sera were belonged to rabbit HEV-associated genotypes. The rabbit HEV strains were genetically heterogeneous entities, commonly divided into divergent genetic groups.These results indicated that HEV is widely prevalent in domestic animals in China. The highest rate of HEV infection was found in pigs among the tested animals. Genotype 4 HEV as the most common swine HEV is highly diverse, including 4a,4b,4d,4f,4g and two new subgenotypes, which are prevalent in pigs in China. We first demonstrated that rabbit HEV with considerable genetic diversity are prevalent in various breeds of fanned rabbits in China. The view that rabbit HEV belongs to a new genotype was supported by the results of this study. Goats, cattle, chicken and ducks also may be infected with HEV and further studies are needed.2. Analysis of HEV ORF3 protein and host protein interactionsScreenings for host proteins that interact with ORF3 protein derived from genotype 1 HEV W2 strain and genotype 4 W3 strian were performed by using yeast mating hybrid assays. Interactomes were further characterized by co-transforming the ORF3 bait plasmid with each of positive prey plasmids into competent yeast cells. Thirty two genes encoding proteins that interact with the HEV-ORF3 protein were identified. Four of them were reported previously to be the interacting partners of ORF3 protein. The rest of 28 proteins were new HEV-ORF3 binding partners identified in this study. The screening result was further evaluated by a chemiluminescent co-immunoprecipitation assay.We proceeded to map these interactions onto an overall visualized interaction network that comprised a repertoire of connections potentially required for ORF3 protein linked up with the components of the host cellular networks. We then analyzed the biological information of these proteins and tried to understand the context of their connections with ORF3 protein in HEV pathogens. According to "GO DATABASE", the subcellular locations of the 32 proteins include extracellular space, extracellular parts, extracellular region, membrane fraction, mitochondrion, cytoplasmic part, etc. Most of the proteins have multiple locations. These proteins involved in the biological processes of apoptosis, cell adhesion, cell communication, immune system process, ect,. Thus, ORF3 protein may function in multiple ways via interacting with these proteins. The analysis of the interaction network revealed an enrichment of canonical pathways (p<0.05) encompassing blood coagulation, oxidative stress, cellular iron ion homeostasis. Considering the clinical manifestations of hepatitis E and the biological analysis results may suggested that ORF3 likely affect these biological pathways by direct interacting with hub proteins and then trigger the corresponding pathological processes.Tested by yeast two hybrid co-transformation, the binding capacities of ORF3 proteins from 1,3 and rabbit HEV with the 32 host proteins has no significant difference. In comparison, only 13 of the 32 human liver proteins preyed by genotype 1 ORF3 protein were found to interact with the ORF3 protein of avian HEV. Whether the host ranges variation among avian HEV and mammalian HEVs is related to the difference of ORF3 interacting capacity with host proteins is needed further investigation.This is the first report to predict the physiological function of the ORF3 protein. The results provide us a critical clue to further study the HEV pathogenesis.
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