Animals ; China ; epidemiology ; Disease Reservoirs ; Hepatitis E ; epidemiology ; transmission ; Hepatitis E virus ; genetics ; isolation & purification ; Humans ; Rodentia ; virology ; Swine ; virology

OBJECTIVETo establish a specific TaqMan-based Real-time PCR assay for the detection of hepatitis E virus (HEV).

METHODSAccording to the references, primers-probe sets which were located in ORF2, the conservative part of HEV genome were designed and therefore we established a HEV TaqMan real-time RT-PCR assay with great performance of specificity, sensitivity and reproducibility. And then it was used in the detection of HEV RNA in clinical samples.

RESULTSThe HEV Real-time RT-PCR assay established in this study were able to detect HEV RNA with a detection limit of 10 copies/reaction. When the detection of a same sample was repeated for several times, coefficients of variation (CV) was all less than 1.53%. Our data also suggested that there were 1.87 x 10(6)-8.12 x 10(9) RNA copies in 1 ml of the clinical samples.

CONCLUSIONThe TaqMan-based Real-time PCR assay established in this study was specific and precise for the rapid detection of HEV RNA. It was applied successfully in the pathogen detection of clinical samples.

DNA Primers ; genetics ; Hepatitis E ; virology ; Hepatitis E virus ; genetics ; isolation & purification ; Humans ; RNA, Viral ; genetics ; metabolism ; Real-Time Polymerase Chain Reaction ; methods ; Taq Polymerase ; metabolism

Hepatitis E, caused by hepatitis E virus (HEV) infection, usually leads to an acute clinical course, and is the most common diagnosis among cases of acute viral hepatitis. From 2008, there have been increasing reports of chronic HEV infection in immunocompromised patients such as organ transplant recipients. Without intervention with antiviral treatment, approximately 60% of HEV infections in organ transplant recipients evolve into chronic HEV infections. Of these chronic hepatitis E patients, 10% may develop liver fibrosis and progress to liver cirrhosis. This article reviews chronic HEV infection and treatment in organ transplant recipients.
Animals ; Antiviral Agents ; therapeutic use ; Hepatitis E ; drug therapy ; virology ; Hepatitis E virus ; genetics ; isolation & purification ; physiology ; Hepatitis, Chronic ; drug therapy ; virology ; Humans ; Transplant Recipients ; Transplants ; virology

OBJECTIVETo investigate the presentation of a neutralization epitope-containing peptide antigen of hepatitis E virus (HEV) on chimeric virus-like particles (VLPs) of hepatitis B surface antigen (HBsAg).

METHODSThe gene fragment corresponding to amino acids (aa) 551-607 (HEnAg) of HEV capsid protein, which contains the only neutralization epitope identified to date, was fused via a synthetic glycine linker in frame with the gene of HBsAg. The resulted fusion gene was then integrated through transformation into the genome of Pichia pastoris under the control of a methanol-induced alcohol oxidase 1 (AOX1) promoter and expressed intracellularly. The expression products in the soluble cell extracts were characterized by Western blot, ELISA, CsCl density gradient analysis, and electron microscopic visualization.

RESULTSThe novel fusion protein incorporating HBsAg and the neutralization epitope-containing HEnAg was expressed successfully in Pichia pastoris with an expected molecular weight of approximately 32 kD. It was found to possess the ability to assemble into chimeric HBV/HEV VLPs with immunological physical and morphological characteristics akin to HBsAg particles. Not only did the chimeric VLPs show high activity levels in a HBsAg particle-specific ELISA but they were also strongly immunoreactive with hepatitis E (HE) positive human serum in a HEV specific ELISA, indicating that HEnAg peptide fragments were exposed on VLP surfaces and would be expected to be readily accessible by cells and molecules of the immune system. Similarity between chimeric VLPs to highly immunogenic HBsAg particles may confer good immunogenicity on surface-displayed HEnAg.

CONCLUSIONThe chimeric HBV/HEV VLPs produced in this study may have potential to be a recombinant HBV/HEV bivalent vaccine candidate.

Epitopes ; Genetic Engineering ; Hepatitis Antigens ; biosynthesis ; genetics ; immunology ; Hepatitis B Surface Antigens ; biosynthesis ; genetics ; immunology ; Hepatitis B virus ; genetics ; immunology ; Hepatitis E virus ; genetics ; immunology ; Pichia ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification ; Vaccines, Synthetic

OBJECTIVETo investigate the seroprevalence of HEV infection and genotype.

METHODSELISA were used for detecting anti-HEV IgG of the serum samples, the nested reverse transcriptase PCR (RT-nPCR) was used for detecting HEV RNA in patient serum and swine bile samples. All samples were collected in 2005-2007 in some districts in Sichuan province. The primers used for genotyping were the ORF2 region of HEV genome.

RESULTSThe anti-HEV IgG was detected positive in childrens 6.10% (41/672), adults 42.26% (280/ 661), swines 88.89% (32/36), chickens negative (0/59). 1 case of 15 serum samples of anti-HEV IgM positive and 3 of 54 swine bile samples were positive for HEV RNA by RT-PCR.Sequence analysis of 4 isolates has 92.1% to 98.6% nucleotide sequence homology. These isolates from human and swine were identified closely related to Ch-T21 strain 90.1%-96.9% sequence homology, which belonged to HEV genotype 4B.

CONCLUSIONSThe swine were the risk factors in the spread of hepatitis E virus.

Animals ; Chickens ; Child ; Child, Preschool ; China ; Enzyme-Linked Immunosorbent Assay ; Genotype ; Hepatitis Antibodies ; immunology ; Hepatitis E ; classification ; epidemiology ; genetics ; Hepatitis E virus ; genetics ; immunology ; isolation & purification ; Humans ; Phylogeny ; Seroepidemiologic Studies ; Swine

BACKGROUNDTo establish a new assay for detecting the quantity of HBV DNA with PCR and enzyme-linked immunosorbent assay(ELISA).

METHODSThe products of PCR using primers pre-labeled with biotin were hybridized with the capture probes that were immobilized on the microtiter strips and then bond with Sav-Ap. The quantity of DNA was detected by measuring the yellow color at 405 nm wave length.

RESULTSTotally 125 sera from patients with hepatitis B were tested for HBV DNA by this method,the sera were also tested for HBV immunological markers by solid phase radio immuno-assay (SPRIA). The HBV positive rate with PCR-ELISA was 64.9% (24/37) in samples which were positive for HBsAg, HBeAg and HBcAb; and 34.2% (13/38) in sera which were positive for HBsAg, HBeAb and HBcAb; in sera positive for HBsAg and HBcAb or only HBcAb, the positive rate was 6.7% (1/15) and 5.9% (2/34) respectively.

CONCLUSIONSThe PCR ELISA assay is simple and suitable for clinical laboratory in quantitative determination of HBV DNA.

DNA Probes ; DNA, Viral ; analysis ; biosynthesis ; genetics ; Enzyme-Linked Immunosorbent Assay ; Hepatitis B ; virology ; Hepatitis B Antibodies ; blood ; Hepatitis B Surface Antigens ; blood ; Hepatitis B e Antigens ; blood ; Hepatitis B virus ; genetics ; isolation & purification ; Humans ; Polymerase Chain Reaction ; Sensitivity and Specificity

OBJECTIVETo explore the relationship between HBV core promoter mutations and liver damage or HBeAg status.

METHODSNested polymerase chain reaction (nPCR) was used for amplification of HBV DNA core promoter in 59 sera from patients with chronic hepatitis B in Guangxi, then the HBV DNA positive products were sequenced by direct sequencing.

RESULTSThe HBV DNA positive rate of was 59.3%(35/59). All the patients were infected by mutants. The commonest mutation was the double mutation (A --> T at nt1762 and G --> A at nt1764), counting for 57.1% (20/35). The next was C --> G at nt1799, counting for 54.4% (19/35), but this was no function. A --> G at nt1752 (resulting in isoleucine to valine) was seen in 37.1% (13/35) of the HBV DNA positive patients, and T --> C at nt1753 was seen in 20% (7/35). The significant difference in the frequency of T1762A1764 mutant was found between HBeAg positive patients (31.3%) and negative patients (79.0%).

CONCLUSIONSHBV core promoter mutations are common among patients with chronic hepatitis B in Guangxi. T1762A1764 mutant is associated with HBeAg status and chronic hepatitis.

Adolescent ; Adult ; Female ; Hepatitis B Core Antigens ; genetics ; Hepatitis B e Antigens ; blood ; Hepatitis B virus ; genetics ; isolation & purification ; Hepatitis B, Chronic ; virology ; Humans ; Liver ; pathology ; Male ; Middle Aged ; Point Mutation ; Promoter Regions, Genetic ; genetics

OBJECTIVETo investigate the southern region of Zhejiang hepatitis E virus (HEV) infection.

METHODSA cluster sampling strategy was used to sample all blood donors from February to October in 2008 in Wenzhou blood center. Their blood was tested for IgG and IgM antibody against HEV. Reverse transcriptase-polymerase chain reaction(RT-PCR) and sequencing were applied to detect its genotype and sequence homology in HEV IgM-positive specimen.

RESULTSThe prevalence of anti-HEV IgG in 3044 cases of blood donors was 33.28%. IgG increased with age. There are certain increase in positive rates between the 20-year-old group and over 40 years of age group from 21.16% to 50.36%. The positive rate of IgM was 0.92%. The ratio of infection among different age group was the highest in the age range from 31 to 40 years and up to 1.90%. IgG and IgM through their negative and positive analysis of samples found in their group with donors age, sex and blood type does not significantly related to each other. Nucleic acids were found in three cases through PCR amplification in all 28 cases of HEV IgM positive samples. The total positive rate was one-thousandth, of which two cases for gene 4, 1 cases of infection for gene 1.

CONCLUSIONThe results indicate that there was a certain percentage of HEV virus in voluntary blood donors in south Zhejiang.

Adolescent ; Adult ; Blood Donors ; China ; epidemiology ; Female ; Genotype ; Hepatitis Antibodies ; blood ; Hepatitis E ; blood ; epidemiology ; virology ; Hepatitis E virus ; classification ; genetics ; immunology ; isolation & purification ; Humans ; Immunoglobulin G ; blood ; Immunoglobulin M ; blood ; Male ; Middle Aged ; Phylogeny ; Young Adult

The primers and probes for the Real-time RT-PCR were designed based on the multiple sequence (swine and humans HEV strains) alignments of the ORF3 region of genotype 4 HEV. A rapid, sensitive and stable TaqMan Real-time RT-PCR assay was established, and its specificity and sensitivity were assessed, and comparison of the Real-time RT-PCR with conventional and nested RT-PCR was performed. The results found that the crossing points showed linearly proportional to the logarithm of the input copy number. The correlation coefficient (R2) and the slope value of the standard curves with plasmid DNA were 0.994 and -3.312, respectively. The efficiency (E) of the PCR was 100%. Coefficients of variation values of the different diluted plasmid DNA were low in the same or different repeated experimental group. In addition, the assay was able to correctly detect genotype 4 HEV RNA from swine fecal samples. The sensitivity of established assay was 100-fold higher than that of conventional RT-PCR and 10-fold higher than nested RT-PCR.
Animals ; DNA Primers ; genetics ; Disease Reservoirs ; virology ; Feces ; virology ; Fluorescence ; Genotype ; Hepatitis E ; virology ; Hepatitis E virus ; classification ; genetics ; isolation & purification ; Humans ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Sensitivity and Specificity ; Swine ; virology

OBJECTIVETo understand phylogenetic characteristics of sporadic hepatitis E virus (HEV) in eastern China.

METHODSFour hundred and thirteen sera were collected from sporadic hepatitis E cases in 14 second- or first-class hospitals in Eastern China from 2005 to 2008 and detected with a nested RT-PCR assay. Partial nucleotide sequences of the HEV isolates were determined for phylogenetic analysis with prototype sequences in the GenBank.

RESULTSThe male-to-female sex ratio of the patients was 1.75:1 with 61.5% of them aged 40 - 69 years old. HEV RNA was detected in 140 out of 413(34%)serum samples. Phylogenetic analysis revealed that all the 140 HEV isolates belonged to genotype IV, sharing 77.9% - 88.3%, 80.8% - 90.6%, 73.4% - 85.2% and 91.0% - 95.4% nucleotide sequence identities with prototype I, II, III and IV HEV isolates respectively.

CONCLUSIONIt was evident that genotype IV HEV served as the main causative agent of sporadic HEV infection in Eastern China. However the viral origin and evolution needs further clarification.

Adult ; Aged ; Base Sequence ; China ; epidemiology ; Female ; Genotype ; Hepatitis E ; epidemiology ; virology ; Hepatitis E virus ; classification ; genetics ; isolation & purification ; Humans ; Male ; Middle Aged ; Molecular Sequence Data ; Phylogeny ; RNA, Viral ; genetics