Application of Hepatitis E Virus-Related Markers on Samples from a Developing Country.

BACKGROUND: Nepal is an endemic area for hepatitis E virus (HEV) epidemics. The research on viral hepatitis in Nepal is limited. METHODS: Serum samples from 170 patients presenting with symptoms of hepatitis were collected from April to May 2014 in Biratnagar, Nepal, and then transported to Xiamen, China, for further evaluation. All samples were tested for HEV RNA, HEV antigen, anti-HEV IgM, anti-HEV IgG and anti-HBc IgM, anti-HCV IgG, and anti-HAV IgM. RESULTS: Sixteen patients were identified as acute hepatitis E with the presence of ≥ 2 HEV acute phase markers (antigen, RNA, and anti-HEV IgM). HEV infection was the major cause of potential active viral hepatitis (59.2%, 16 of 27), followed by HBV (25.9%, 7 of 27, anti-HBc IgM positive), HAV (18.5%, 5 of 27, anti-HAV IgM positive), and HCV (3.7%, 1 of 27, anti-HCV antibodies). All 16 confirmed HE cases were positive for HEV antigen, while 5 cases were HEV RNA positive, as well as 15 anti-HEV IgM positive. The low positive rate of RNA might be related to the collection and/or the transportation of these samples. CONCLUSIONS: This study showed that HEV is a major cause of acute hepatitis in developing countries and regions. Application of immunoassay diagnostic kits, especially the HEV antigen tests, showed great potential for HE detection in these countries and regions.

Classification of human and zoonotic group hepatitis E virus (HEV) using antigen detection.

Hepatitis E virus (HEV) is one of the major pathogens that cause acute viral hepatitis. The human (genotypes 1 and 2) and zoonotic (genotypes 3 and 4) groups of HEV present different epidemiology and clinical features. In this study, we developed a classification method for rapidly classifying HEV into human or zoonotic groups that combines a general antigen test with a zoonotic group-specific antigen test. Evaluation of serial samples from HEV-infected rhesus monkeys indicated that HEV antigen-positive samples can be classified using the antigen-based classification method. The antigen-based classification method was evaluated further on 55 genotyped samples from acute hepatitis E patients, including 9 human and 46 zoonotic groups. The novel method was completely consistent with the sequencing results: 9/9 for the human groups (100%, 95% confidence interval [CI] 66.4-100%) and 46/46 for the zoonotic groups (100%, 95% CI 92.3-100%). This method was also successfully used for the clustering of some samples that could not be clustered by sequencing. Compared with the sequencing-based method, this method is less time-consuming, less expensive, and less technically complex and is therefore ideal for large numbers of samples. In conclusion, this study provides a convenient and sensitive method for classifying different groups of HEV, and it has potentially important public health applications, especially in underdeveloped areas that cannot afford the high cost of nucleic acid testing.

A valuable antigen detection method for diagnosis of acute hepatitis E.

Hepatitis E virus (HEV) is a serious public health problem. The commonly used tests that are specific for current HEV infection diagnosis include the detection of anti-HEV IgM and HEV RNA. Here, we report an improved enzyme-linked immunosorbent assay (ELISA) method for HEV antigen detection with a linear range equivalent to 6.3 × 10(3) to 9.2 × 10(5) RNA copies per ml. The monoclonal antibody (MAb) 12F12, a high-ability MAb that binds HEV virus, was selected as the capture antibody from a panel of 95 MAbs. The positive period of HEV antigenemia in infected monkeys using this test was, on average, 3 weeks longer than previously reported and covered the majority of the acute phase. The positive detection rates of IgM, RNA, and new antigen from the first serum samples collected from 16 confirmed acute hepatitis E patients were 81% (13/16), 81% (13/16), and 100% (16/16), respectively. In three patients, the initial serum specimens that tested negative for IgM, despite the presence of symptoms of acute hepatitis and elevated alanine aminotransferase (ALT) levels, were positive for HEV antigen and HEV RNA. In contrast, the serum samples of the three RNA-negative patients were antigen positive (and IgM positive), possibly due to the degradation of HEV nucleic acids. Our results suggest that this new antigen detection method has acceptable concordance with RNA detection and could serve as an important tool for diagnosing acute hepatitis E.

Long-term HEV carriers without antibody seroconversion among eligible immunocompetent blood donors.

Hepatitis E virus (HEV) is emerging as a potential threat to the safety of blood transfusions. In many countries and regions endemic for HEV, such as China, blood donors are not routinely tested for HEV infection. In this study, 11747 eligible blood donors were screened for anti-HEV immunoglobulin M (IgM)/immunoglobulin G (IgG) and HEV RNA and antigen in China. Twenty-four donors who were positive for both HEV antigen and RNA were followed for ≥ 70 days, and none of these donors reported clinical hepatitis or illness. At least 1 follow-up sample was provided by 17 donors, including 10 with viremia and/or antigenemia for ≥ 70 days and 3 with antigen and RNA positivity for >90 days. Fourteen of the 17 donors did not present with an obvious serologic response during the follow-up period. These results differed from previous reports, in which viremia lasted for 68 days and elicited an antibody response. These donors showed atypical HEV infection progression that differed from that of hepatitis E patients. The presence of these donors presents a challenge for transfusion transmission screening.

Density functional theory methods as powerful tools to elucidate amino acid oxidation mechanisms. A case study on methionine model peptide.

The amino acid oxidation mechanism has been a research focus in recent years. Although various experimental techniques have been employed to address the problem, it is still a great challenge to identify the oxidation intermediates of amino acids. To explore the potential of theoretical methods in helping elucidating amino acid oxidation mechanisms, one-electron oxidation of a methionine model peptide (N-acetylmethionine amide) was investigated by density functional theory (DFT; including TD-DFT) calculations. The theoretical results not only testified the experimentally identified oxidation mechanisms of the peptide to a large extent but also revealed the contribution of protonated species to the peptide oxidation. All of these findings strongly suggest that DFT methodology has great potential in investigating amino acid oxidation mechanisms.

A high-throughput neutralizing assay for antibodies and sera against hepatitis E virus.

Hepatitis E virus (HEV) is the aetiological agent of enterically transmitted hepatitis. The traditional methods for evaluating neutralizing antibody titres against HEV are real-time PCR and the immunofluorescence foci assay (IFA), which are poorly repeatable and operationally complicated, factors that limit their applicability to high-throughput assays. In this study, we developed a novel high-throughput neutralizing assay based on biotin-conjugated p239 (HEV recombinant capsid proteins, a.a. 368-606) and staining with allophycocyanin-conjugated streptavidin (streptavidin APC) to amplify the fluorescence signal. A linear regression analysis indicated that there was a high degree of correlation between IFA and the novel assay. Using this method, we quantitatively evaluated the neutralization of sera from HEV-infected and vaccinated macaques. The anti-HEV IgG level had good concordance with the neutralizing titres of macaque sera. However, the neutralization titres of the sera were also influenced by anti-HEV IgM responses. Further analysis also indicated that, although vaccination with HEV vaccine stimulated higher anti-HEV IgG and neutralization titres than infection with HEV in macaques, the proportions of neutralizing antibodies in the infected macaques' sera were higher than in the vaccinated macaques with the same anti-HEV IgG levels. Thus, the infection more efficiently stimulated neutralizing antibody responses.

The Bama miniature swine is susceptible to experimental HEV infection.

The hepatitis E virus (HEV) is one of the main causes of enterically transmitted hepatitis worldwide. Although the mortality rates associated with HEV are generally low, they can be up to 28% in HEV-infected pregnant women, and the elderly are more susceptible. The reasons for this selective severity are unclear, partially because there is no suitable, easy-to-use model in which to study HEV infection. Non-human primates and standard swine have been identified as being sensitive to infection with HEV and have been used for HEV infection studies. However, studies in these animals have been limited by high housing costs and the difficulty of manipulating these animals. In the current study, we established a model of HEV infection using Bama miniature swine. The model is easy to use and is sensitive to infections with HEV genotypes 3 and 4, which are classified as zoonotic HEVs. In this model, infection of Bama miniature swine with HEV genotypes 3 and 4 caused the typical features. All Bama miniature swine that were infected with HEV genotypes 3 and 4 exhibited significant HEV viremia, shedding, anti-HEV antibody responses and partial liver inflammation. Bama miniature swine may serve as an alternative to standard swine models for the study of zoonotic HEV infection and HEV genotype specificity research.