In July 2018, recurrent hepatitis E cases were reported from a factory in Qingdao City, China. The aim of this study was to identify additional cases, and help prevent future incidents by identifying possible risk factors for infection.Participants were asked to provide blood samples for hepatitis E virus (HEV) IgM and IgG antibodies screening, as well as liver function test. A questionnaire that assessed demographics, potential risk factors, and clinical symptoms was completed by participants. HEV RNA genotyping was performed using a nested Reverse Transcriptional Polymerase Chain Reaction (RT-PCR) method. Adjusted Poisson regression model for participant characteristics and risk factors was constructed for multivariate analysis.Overall, 41(14.5%, 41/283) participants had recent acute infection (21 of these were symptomatic). The result of multivariate analysis demonstrated a significant association of acute HEV infection with consumption of pig liver within the past two months (Relative Risk 2.61, 95% confidence interval (CI) 1.10-6.17, p=0.0294). Sequencing of HEV RNA from seventeen acute cases indicated three HEV isolates of genotype 4 induced this outbreak.This was probably a common-source foodborne hepatitis E outbreak, related to the consumption of undercooked pig liver.
Hepatitis A virus (HAV) is the most common cause of infectious hepatitis throughout the world, spread largely by the fecal-oral route. To characterize the genetic diversity of the virus circulating in China where HAV in endemic, we selected the outbreak cases with identical sequences in VP1-2A junction region and compiled a panel of 42 isolates. The VP3-VP1-2A regions of the HAV capsid-coding genes were further sequenced and analyzed. The quasispecies distribution was evaluated by cloning the VP3 and VP1-2A genes in three clinical samples. Phylogenetic analysis demonstrated that the same genotyping results could be obtained whether using the complete VP3, VP1, or partial VP1-2A genes for analysis in this study, although some differences did exist. Most isolates clustered in sub-genotype IA, and fewer in sub-genotype IB. No amino acid mutations were found at the published neutralizing epitope sites, however, several unique amino acid substitutions in the VP3 or VP1 region were identified, with two amino acid variants closely located to the immunodominant site. Quasispecies analysis showed the mutation frequencies were in the range of 7.22x10-4 -2.33x10-3 substitutions per nucleotide for VP3, VP1, or VP1-2A. When compared with the consensus sequences, mutated nucleotide sites represented the minority of all the analyzed sequences sites. HAV replicated as a complex distribution of closely genetically related variants referred to as quasispecies, and were under negative selection. The results indicate that diverse HAV strains and quasispecies inside the viral populations are presented in China, with unique amino acid substitutions detected close to the immunodominant site, and that the possibility of antigenic escaping mutants cannot be ruled out and needs to be further analyzed.
Production of high-value recombinant proteins in transgenic seeds is an attractive and economically feasible alternative to conventional systems based on mammalian cells and bacteria. In contrast to leaves, seeds allow high-level accumulation of recombinant proteins in a relatively small volume and a stable environment. We demonstrate that single-chain variable fragment (scFv)-Fc antibodies, with N-terminal signal sequence and C-terminal KDEL tag, can accumulate to very high levels as bivalent IgG-like antibodies in Arabidopsis thaliana seeds and illustrate that a plant-produced anti-hepatitis A virus scFv-Fc has similar antigen-binding and in vitro neutralizing activities as the corresponding full-length IgG. As expected, most scFv-Fc produced in seeds contained only oligomannose-type N-glycans, but, unexpectedly, 35-40% was never glycosylated. A portion of the scFv-Fc was found in endoplasmic reticulum (ER)-derived compartments delimited by ribosome-associated membranes. Additionally, consistent with the glycosylation data, large amounts of the recombinant protein were deposited in the periplasmic space, implying a direct transport from the ER to the periplasmic space between the plasma membrane and the cell wall. Aberrant localization of the ER chaperones calreticulin and binding protein (BiP) and the endogenous seed storage protein cruciferin in the periplasmic space suggests that overproduction of recombinant scFv-Fc disturbs normal ER retention and protein-sorting mechanisms in the secretory pathway.
To analyse the genetic characteristics of the capsid protein VP3-VP1 region of hepatitis A virus strains circulated in China.The nucleotide sequences of VP3-VP1-2A region of 42 HAV IgM positive serum samples were sequenced and analysed for nucleotide and amino acid identities and genetic characteristics of the VP3-VP1 region.The nucleotide and amino acid identities in the VP1-2A junction region among the 42 strains were 89.1% - 100% and 97.3% - 100%; while in the complete VP3-VP1 region, the identities were 87.6%-100% and 98.8%-100%. Strains with identical nucleotide sequences in the VP1-2A junction region had 98.4%-100% nucleotide identity in the complete VP3-VP1 region and 0-2 amino acid differences in this region. There were no amino acid changes at neutralizing antigenetic sites of VP3-VP1 region within the 42 HAV strains.All the 42 HAV strains belonged to genotype I, with 40 strains clustering to sub-genotype IA and 2 to sub-genotype IB. Different HAV strains analysed in this paper differed in the nucleotide sequences of the VP3-VP1 region, but the amino acid sequences were highly conserved with no changes at neutralizing antigenetic sites. Both the nucleotide and amino acid sequences of the strains with the same VP1-2A junction region were identical or closely related when compared in the complete VP3-VP1 region.
Detection of hepatitis viral infections has traditionally relied on the circulating antibody test using the enzyme-linked immunosorbent assay. However, multiplex real-time PCR has been increasingly used for a variety of viral nucleic acid detections and has proven to be superior to traditional methods. Hepatitis A virus (HAV) and hepatitis E virus (HEV) are the major causes of acute hepatitis worldwide; both HAV and HEV infection are a main public health problem. In the present study, a one-step multiplex reverse transcriptase quantitative polymerase chain reaction assay using hydrolysis probes was developed for simultaneously detecting HAV and HEV. This novel detection system proved specific to the target viruses, to be highly sensitive and to be applicable to clinical sera samples, making it useful for rapid, accurate and feasible identification of HAV and HEV.
In China, hepatitis E virus (HEV) is prevalent and causes disease, but its epidemiological profile is not well understood. We used a commercial enzyme-linked immunosorbent assay to detect total antibodies to hepatitis E virus in 15,862 serum samples collected during the Third National Viral Hepatitis Prevalence Survey. The results were analyzed to calculate estimates of HEV seroprevalence and to examine the effects of some putative risk factors. The seroprevalence of HEV in the general Chinese population during the period from 2005 through 2006 was 23.46% (95% confidence interval [CI], 18.41%–28.50%). The farming population, the age group of 15–60 year olds, and those living in the Midwest or Mideast region and in Xinjiang province had the highest seroprevalence estimates. The prevalence of HEV is high in China. The seroprevalence rate of HEV shows an unbalanced distribution among areas with different geographic location and economic development levels. The characteristics of the distribution associated may be due to the route of HEV transmission (via contaminated water or animal reservoirs). Within the same region, the seroprevalence of HEV is generally increased with age.
With advances in viral surveillance and next-generation sequencing, highly diverse novel astroviruses (AstVs) and different animal hosts had been discovered in recent years. However, the existence of AstVs in marmots had yet to be shown. Here, we identified two highly divergent strains of AstVs (tentatively named Qinghai Himalayanmarmot AstVs, HHMAstV1 and HHMAstV2), by viral metagenomic analysis in liver tissues isolated from wild Marmota himalayana in China. Overall, 12 of 99 (12.1 %) M. himalayana faecal samples were positive for the presence of genetically diverse AstVs, while only HHMAstV1 and HHMAstV2 were identified in 300 liver samples. The complete genomic sequences of HHMAstV1 and HHMAstV2 were 6681 and 6610 nt in length, respectively, with the typical genomic organization of AstVs. Analysis of the complete ORF 2 sequence showed that these novel AstVs are most closely related to the rabbit AstV, mamastrovirus 23 (with 31.0 and 48.0 % shared amino acid identity, respectively). Phylogenetic analysis of the amino acid sequences of ORF1a, ORF1b and ORF2 indicated that HHMAstV1 and HHMAstV2 form two distinct clusters among the mamastroviruses, and may share a common ancestor with the rabbit-specific mamastrovirus 23. These results suggest that HHMAstV1 and HHMAstV2 are two novel species of the genus Mamastrovirus in the Astroviridae. The remarkable diversity of these novel AstVs will contribute to a greater understanding of the evolution and ecology of AstVs, although additional studies will be needed to understand the clinical significance of these novel AstVs in marmots, as well as in humans.
To establish a specific TaqMan-based Real-time PCR assay for the detection of hepatitis A virus in serum samples.According to the references, primers-probe sets which were located in 5'-NCR, the most conservative part of HAV genome were designed and therefore we established a TaqMan real-time RT-PCR assay with great performance of specificity, sensitivity and reproducibility. And then it was used in the detection of HAV RNA in serum from HAV patients.The HAV Real-time RT-PCR assay established in this study were able to detect HAV RNA and its detection limit ranged from 0.1CCID50/reaction to 0.01CCID50/reaction. When the detection of a same sample was repeated for three times, coefficients of varistion (CV) of intra- and inter-assay were calculated and they were all less than 2.0% and 2.6% respectively. Our data suggested that there were 5.18 x 10(2) - 4.93 x 10(7) RNA copies in 1 ml of the serum from acute HAV patients.The TaqMan-based Real-time PCR assay established in this study was specific and precise for the rapid detection of HAV RNA. It was applied successfully in the pathogen detection of clinical samples.
[Objective] To detect Genotype of Hepatitis A virus,so as to provide molecular epidemiologic basis for outbreak of Hepatitis A.[Methods] Serum samples were collected from Hepatitis A patients in several epidemic places.The primers used for genotyping were from the VPI-2A region of HAV RNA genome.HAV RNA from different patient serum samples were amplified,sequenced,and used for phylogenetic anaysis by Neighbor Joining(NJ) method.[Results] Among 27 serum samples,there was 25 samples showed the same exposure,and the same nucleotide sequence.For the two serum samples without the same exposure,the HAV gene of them showed significant difference for samples with the same exposure,and the homology was only 94%.But all of them belonged to IA subgroup(in the same sub-genotype,nucleotide sequence variation ﹤ 7.5%).[Conclusion] The genotyping analysis indicated that there were different HAV strains existing in Guiyang,but the epidemic was induced by the same exposure,the result showed it was consistent with the field epidemiology.
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