Real-time RT-PCR Assay For Detection Of Genotype Ⅳ HEV And The VLP Protein Of HEV Expressed In Pichia Pastoris

Hepatitis, or inflammation of the liver, can be due to one of several causes including viruses, drugs, toxins, autoimmune diseases, etc. Among infectious agents, at least five viruses are known to primarily affect the liver and cause hepatitis—hepatitis A, B, C, and E viruses and the delta agent, abbreviated as HAV, HBV, HCV, HEV, and HDV, respectively. While infection with HAV and HEV causes only an acute, self-limited disease, the other agents are known to persist for long periods in some of those infected, leading to chronic hepatitis. It is estimated that of the 6 billion world population, the numbers of those who have been exposed to these viruses. Hepatitis E is an emerging disease in resource-poor regions of the world. It is estimated that about 2 billion people live in areas endemic for this disease. HEV causes hepatitis E, a disease transmitted predominantly through the fecal-oral route, primarily through contaminated drinking water. Large waterborne outbreaks of hepatitis E have been recorded, besides rampant sporadic infections in endemic areas. Person-to-person transmission of HEV appears to be uncommon. The disease is clinically indistinguishable from hepatitis A. It generally has a low case mortality; however, a peculiar feature of hepatitis E is its high mortality among pregnant women, reaching up to 20~30%.The epidemiology of hepatitis E is more complex than was initially appreciated, and many features remain unexplained. The age-related seroprevalence of anti-HEV antibodies shows three different patterns. In endemic areas such as Asia, the Middle East, and North Africa, anti-HEV seroprevalence increases with age up to 30~40% in adults. However, surprisingly, relatively high rates of anti-HEV positivity were observed in US blood donors in comparison with those expected from the low disease rates. A possible explanation might be exposure to related animal viruses, for example, swine HEV, in these countries.Genotype IV also includes human and animal isolates from China, Japan, Taiwan, and Vietnam. Accumulating evidences indicate that hepatitis E is a zoonotic disease, and domestic pigs, wild boars and maybe other animal species are reservoirs for HEV. The ubiquitous nature of the virus in domestic pigs and wild boars as well as in other animal species raises public health concern for zoonosis and food safety. The clinical symptoms of human hepatitis E could not be distinguished from other types of acute viral hepatitis, and thus accurate diagnosis of hepatitis E must rely on laboratory tests. HEV is a difficult virus to work with since it can not be efficiently propagated in cell culture. Currently, the diagnosis of HEV infection is primarily based on PCR and ELISA. In addition, The inability to reproducibly culture hepatitis E virus makes it impossible to develop traditional live or inactivated vaccines. However, significant progress has been made in developing and testing recombinant subunit vaccines based on the viral capsid protein.The window of detectable HEV viraemia is narrow, which can continue from a few days to weeks after the onset of clinical symptoms. However, the virus continues to be shed in stool for another 2 weeks. In addition, Many studies found the peak viremia and virus shedding into the feces occurs during the incubation period and early acute phase of disease in the course of HEV infection. Thus, that detection of specific IgG and IgM to HEV was not enough to diagnose HEV infection, especially in the early stage. Immunologic methods are not enough to detect specific IgM or IgG against HEV, especially in the early infection stage. To better quantitate and identify genotype IV swine HEV strains that may exist in clinical samples, we have established a rapid, sensitive and stable real-time RT-PCR assay. The primers and probes for the real-time RT-PCR were designed based on the multiple sequence (swine and humans HEV strains) alignments of 11 sequences of the ORF3 region. In study, three genotype IV swine HEV strains were used to standardize the real-time RT-PCR assay. The correlation coefficient (R2) and slope values of the standard curves with plasmid DNA were 0.994 and -3.312, respectively. Although the reaction included 2.8×101 genome equivalent (GE) copies of HEV plasmid DNA, this assay still can efficiently product specific amplification curve. In 3 different repeated tests, ranges of slopes and coefficient of variation values was from 1.35 to 4.46. In the same repeated test, coefficient of variation values ranged from 0.17 to 3.65. Correlation coefficient values of the reactions in the same or different repeated experiments were over 0.99. In comparison of the real-time PCR with nested or conventional RT-PCR, the TaqMan real-time RT-PCR can detected HEV RNA from 4 negative-HEV swine stools (N1, N2, N3 and N4) with 2.34×102, 2.29×102, 1.70×102 and 1.7×101 copies, respectively. However, conventional or nested RT-PCR could not determined the HEV RNA from the negative-HEV swine stools. The results of this study demonstrate that the real-time RT-PCR assay for HEV is broadly reactive, specific, and sensitive for detection of genotype IV swine HEV in clinical specimens.We have studied the antibody response to vaccine-associated epitopes and to epitopes excluded from the vaccine to determine if important epitopes were omitted from the vaccine and if antibody responses to these regions could be used to differentiate between infection and vaccination. Base on the the relevant studies, we analyzed the gene sequences and amino acid sequences of HEV CH-S-1 strain by bioinformatics software, The results showed the highest nucleotide and amino acid homology were 92.5% and 100% between CH-S-1 strain and CH-87 strain, which were genotype IV and subtype IVa. We found three epitopes, which located in the ORF2 protein 235~372aa,424~563aa and 613~674aa, respectively. In this study, we selected the pORF2 382~674aa(p293) to express in the Pichia Pastoris. We attempted to produce a truncated capsid protein, designed p293, in Pichia pastoris. The p293 gene encoded amino acids (aa) 382~674 of HEV ORF2 was designed based on the full length HEV ORF2, cloned into the yeast vector pPIC9K, and expressed in P. pastoris strain GS115. SDS - PAGE and Western blotting demonstrated that the recombinant protein p293 could well expressed in P. pastoris. Under optimized conditions (culture medium pH, 6.0~6.5; methanol concentration added daily, 3.0%; inoculum density, OD600 = 6.0; induction time point, 72~96 h), the yield of purified and concentrated p293 was approximately 80 mg/L. We also observed that p293 secretory expressed in P. pastoris could formed 30 nm viral like particles by using electron microscopy. These results showed that the p293 may has utility in the analysis of cell specific factors in the protein processing and assembly of HEV, and serve as useful antigen for both diagnostic and vaccine purposes.