Limited hepatitis B virus replication space in the chronically hepatitis C virus-infected liver.

We compared the kinetics and magnitude of hepatitis B virus (HBV) infection in hepatitis C virus (HCV)-naive and chronically HCV-infected chimpanzees in whose livers type I interferon-stimulated gene (ISG) expression is strongly induced. HBV infection was delayed and attenuated in the HCV-infected animals, and the number of HBV-infected hepatocytes was drastically reduced. These results suggest that establishment of HBV infection and its replication space is limited by the antiviral effects of type I interferon in the chronically HCV-infected liver.

Serological evidence for a hepatitis e virus-related agent in goats in the United States.

Hepatitis E virus (HEV) causes an important public health disease in many developing countries and is also endemic in some industrialized countries. In addition to humans, strains of HEV have been genetically identified from pig, chicken, rat, mongoose, deer, rabbit and fish. While the genotypes 1 and 2 HEV are restricted to humans, the genotypes 3 and 4 HEV are zoonotic and infect humans and other animal species. As a part of our ongoing efforts to search for potential animal reservoirs for HEV, we tested goats from Virginia for evidence of HEV infection and showed that 16% (13/80) of goat sera from Virginia herds were positive for IgG anti-HEV. Importantly, we demonstrated that neutralizing antibodies to HEV were present in selected IgG anti-HEV positive goat sera. Subsequently, in an attempt to genetically identify the HEV-related agent from goats, we conducted a prospective study in a closed goat herd with known anti-HEV seropositivity and monitored a total of 11 kids from the time of birth until 14 weeks of age for evidence of HEV infection. Seroconversion to IgG anti-HEV was detected in seven of the 11 kids, although repeated attempts to detect HEV RNA by a broad-spectrum nested RT-PCR from the faecal and serum samples of the goats that had seroconverted were unsuccessful. In addition, we also attempted to experimentally infect laboratory goats with three well-characterized mammalian strains of HEV but with no success. The results indicate that a HEV-related agent is circulating and maintained in the goat population in Virginia and that the goat HEV is likely genetically very divergent from the known HEV strains.

Adaptation of a genotype 3 hepatitis E virus to efficient growth in cell culture depends on an inserted human gene segment acquired by recombination.

An infectious cDNA clone of a genotype 3 strain of hepatitis E virus adapted to growth in HepG2/C3A human hepatoma cells was constructed. This virus was unusual in that the hypervariable region of the adapted virus contained a 171-nucleotide insertion that encoded 58 amino acids of human S17 ribosomal protein. Analyses of virus from six serial passages indicated that genomes with this insert, although initially rare, were selected during the first passage, suggesting it conferred a significant growth advantage. RNA transcripts from this cDNA and the viruses encoded by them were infectious for cells of both human and swine origin, the major host species for this zoonotic virus. Mutagenesis studies demonstrated that the S17 insert was a major factor in cell culture adaptation. Introduction of 54 synonymous mutations into the insert had no detectable effect, thus implicating protein, rather than RNA, as the important component. Truncation of the insert by 50% decreased the levels of successful transfection by ~3-fold. Substitution of the S17 sequence by a different ribosomal protein sequence or by GTPase-activating protein sequence resulted in a partial enhancement of transfection levels, whereas substitution with 58 amino acids of green fluorescent protein had no effect. Therefore, both the sequence length and the amino acid composition of the insert were important. The S17 sequence did not affect transfection of human hepatoma cells when inserted into the hypervariable region of a genotype 1 strain, but this chimeric genome acquired a dramatic ability to replicate in hamster cells.

A naturally occurring human/hepatitis E recombinant virus predominates in serum but not in faeces of a chronic hepatitis E patient and has a growth advantage in cell culture.

Hepatitis E virus is the aetiological agent of acute hepatitis E, a self-limiting disease prevalent in developing countries. Molecular analysis of viral genomic RNA from a chronically infected patient confirmed the recent discovery that chronic infection correlated with extensive diversification of the virus quasispecies: the hypervariable region of some virus genomes in this USA patient contained large continuous deletions and a minor proportion of genomes in faeces and serum had acquired a mammalian sequence that encoded 39 aa of S19 ribosomal protein fused to the virus non-structural protein. Genomes with this insert were selected during virus passage in cultured cells to become the predominant species, suggesting that the inserted sequence promoted virus growth. The results demonstrated that hepatitis E virus can mutate dramatically during a prolonged infection and suggests it may be important to prevent or cure chronic infections before new variants with unpredictable properties arise.

Characterization of hepatitis E-specific cell-mediated immune response using IFN-gamma ELISPOT assay.

In developing countries, hepatitis E (HEV) and hepatitis A (HAV) are the major causes of acute viral hepatitis with similar feco-oral modes of transmission. In contrast to the high seroprevalence of hepatitis A infection, a low seroprevalence of HEV among children in endemic areas has been reported. These data suggest the possibility that silent HEV infection is undiagnosed by the current available methods. Many of the serological tests used for HEV diagnosis have poor specificity and are unable to differentiate among different genotypes of HEV. Moreover, the RT-PCR used for HEV isolation is only valid for a brief period during the acute stage of infection. Cell-mediated immune (CMI) responses are highly sensitive, and long lasting after sub-clinical infections as shown in HCV and HIV. Our objective was to develop a quantitative assay for cell-mediated immune (CMI) responses in HEV infection as a surrogate marker for HEV exposure in silent infection. Quantitative assessment of the CMI responses in HEV will also help us to evaluate the role of CMI in HEV morbidity. In this study, an HEV-specific interferon-gamma (IFN-gamma) ELISPOT assay was optimized to analyze HEV-specific CMI responses. We used peripheral blood mononuclear cells (PBMC) and sera from experimentally infected chimpanzees and from seroconverted and control human subjects to validate the assay. The HEV-specific IFN-gamma ELISPOT responses correlated strongly and significantly with anti-HEV ELISA positive/negative results (rho=0.73, p=0.02). Moreover, fine specificities of HEV-specific T cell responses could be identified using overlapping HEV ORF2 peptides.

Detection of immunoglobulin M antibodies to hepatitis E virus by class capture enzyme immunoassay.

The measurement of antibodies to hepatitis E virus (anti-HEV) has been essential for understanding the epidemiology of hepatitis E. Studies to determine the prevalence of HEV infections require a reliable serologic assay that is sensitive and specific. It is also important to distinguish the acute from the convalescent phase of an infection; this usually requires the detection of the immunoglobulin M (IgM) class of antibody. Few enzyme immunoassays (EIAs) that measure IgM anti-HEV have been described, and most have utilized the sandwich method. The present study describes an EIA that detects IgM anti-HEV by antibody class capture methodology. The assay was validated by using serum and/or plasma panels from experimentally infected nonhuman primates. It was used to demonstrate an anamnestic response and the reappearance of IgM anti-HEV in a chimpanzee experimentally challenged with HEV at two different times 45 months apart. The class capture method was more sensitive than the sandwich EIA when used to test clinical samples from two hepatitis E epidemics in Pakistan; it also had the advantage of distinguishing IgM anti-HEV in the presence of high titers of IgG anti-HEV.

Hepatitis E virus (HEV) capsid antigens derived from viruses of human and swine origin are equally efficient for detecting anti-HEV by enzyme immunoassay.

The recombinant truncated ORF2 (capsid) antigen derived from the Meng strain of swine hepatitis E virus (HEV) differs from that of the Sar-55 strain of human HEV by approximately 5% at the amino acid level. Serial serum samples from two chimpanzees and six rhesus monkeys experimentally infected with HEV were tested with one enzyme immunoassay (EIA) based on the Sar-55 antigen and with a second EIA based on the Meng antigen. We obtained 98% agreement (kappa = 0.952) by direct comparison. The virtually identical results obtained with these antigens in detecting seroconversion following infection with HEV suggests that they were reacting with antibodies that detect the same or very similar epitopes of HEV. We then tested human and swine serum samples for anti-HEV in EIAs that utilized one or the other of the two ORF2 antigens and showed that these results were also virtually identical. The specimens tested included swine sera from the United States, Canada, China, Korea, and Thailand and sera from veterinarians, U.S. and non-U.S. volunteer blood donors, and U.S. and non-U.S. animal handlers. We tested 792 swine sera and obtained 93% agreement (kappa = 0.839). We similarly tested 882 human sera and obtained 99% agreement (kappa = 0.938). Moreover, we found virtually no difference in the levels of prevalence of anti-HEV as measured by the two tests, again suggesting that the antigens derived from human and swine HEV contain the same immunodominant epitopes.

Prevalence of antibodies to hepatitis E virus in veterinarians working with swine and in normal blood donors in the United States and other countries.

Hepatitis E virus (HEV) is endemic in many developing and some industrialized countries. It has been hypothesized that animals may be the source of infection. The recent identification of swine HEV in U.S. pigs and the demonstration of its ability to infect across species have lent credence to this hypothesis. To assess the potential risk of zoonotic HEV infection, we tested a total of 468 veterinarians working with swine (including 389 U.S. swine veterinarians) and 400 normal U.S. blood donors for immunoglobulin G anti-HEV. Recombinant capsid antigens from a U.S. strain of swine HEV and from a human HEV strain (Sar-55) were each used in an enzyme-linked immunosorbent assay. The anti-HEV prevalence assayed with the swine HEV antigen showed 97% concordance with that obtained with the human HEV antigen (kappa = 92%). Among the 295 swine veterinarians tested from the eight U.S. states (Minnesota, Indiana, Nebraska, Iowa, Illinois, Missouri, North Carolina, and Alabama) from which normal blood donor samples were available, 26% were positive with Sar-55 antigen and 23% were positive with swine HEV antigen. In contrast, 18% of the blood donors from the same eight U.S. states were positive with Sar-55 antigen and 17% were positive with swine HEV antigen. Swine veterinarians in the eight states were 1.51 times more likely when tested with swine HEV antigen (95% confidence interval, 1.03 to 2.20) and 1.46 times more likely when tested with Sar-55 antigen (95% confidence interval, 0.99 to 2.17) to be anti-HEV positive than normal blood donors. We did not find a difference in anti-HEV prevalence between veterinarians who reported having had a needle stick or cut and those who had not or between those who spent more time (> or = 80% of the time) and those who spent less time (< or = 20% of the time) working with pigs. Similarly, we did not find a difference in anti-HEV prevalence according to four job categories (academic, practicing, student, and industry veterinarians). There was a difference in anti-HEV prevalence in both swine veterinarians and blood donors among the eight selected states, with subjects from Minnesota six times more likely to be anti-HEV positive than those from Alabama. Age was not a factor in the observed differences from state to state. Anti-HEV prevalence in swine veterinarians and normal blood donors was age specific and paralleled increasing age. The results suggest that swine veterinarians may be at somewhat higher risk of HEV infection than are normal blood donors.

Immunogenicity and protective efficacy of a vaccine prepared from 53 kDa truncated hepatitis E virus capsid protein expressed in insect cells.

Hepatitis E virus (HEV) is an enterically transmitted virus that causes acute hepatitis. Expression of recombinant HEV capsid protein in insect cells results in two major proteolytically-processed products of 56 and 53kDa which consist of amino acids (aa) 112-607 and 112-578, respectively. The only neutralization epitope identified to date is located at least partially between amino acids 578 and 607 meaning it should be present only in the 56 and not in the 53kDa protein. Previously, it was shown that vaccination with the 56kDa protein greatly reduced virus shedding and protected Rhesus monkeys from hepatitis E when challenged with a high intravenous dose of homologous or heterologous HEV. To evaluate the immunogenicity and protective efficacy of the 53kDa protein, we vaccinated Rhesus monkeys with this protein and challenged them with a high or low dose of homologous virus. Vaccination with the 53kDa protein greatly reduced virus shedding but did not protect against hepatitis following the high dose challenge. Virus was not detected in the vaccinated animals following the low dose challenge, suggesting that sterilizing immunity may have been achieved.

A virus discovery method incorporating DNase treatment and its application to the identification of two bovine parvovirus species.

Identification of previously unrecognized viral agents in serum or plasma samples is of great medical interest but remains a major challenge, primarily because of abundant host DNA. The current methods, library screening or representational difference analysis (RDA), are very laborious and require selected sample sets. We have developed a simple and reproducible method for discovering viruses in single serum samples that is based on DNase treatment of the serum followed by restriction enzyme digestion and sequence-independent single primer amplification (SISPA) of the fragments, and have evaluated its performance on known viruses. Both DNA viruses and RNA viruses at a concentration of approximately 10(6) genome equivalents per ml were reproducibly identified in 50 microl of serum. While evaluating the method, two previously unknown parvoviruses were discovered in the bovine sera used as diluent. The near complete genome sequence of each virus was determined; their classification as two species (provisionally named bovine parvoviruses 2 and 3) was confirmed by phylogenetic analysis. Both viruses were found to be frequent contaminants of commercial bovine serum. DNase treatment of serum samples may prove to be a very useful tool for virus discovery. The DNase-SISPA method is suitable for screening of a large number of samples and also enables rapid sequence determination of high-titer viruses.

Prevalence of antibodies to the hepatitis E virus in pigs from countries where hepatitis E is common or is rare in the human population.

Hepatitis E virus (HEV) is a very important public health concern in many developing countries where epidemics of hepatitis E are common. Sporadic cases of clinical hepatitis E not only occur in these countries but also occur uncommonly in patients with no known epidemiological exposure to HEV in industrialized countries. The source of infection in industrialized countries is unknown but it has been suggested that animals might serve as a reservoir for HEV in both settings. We recently identified and characterized an HEV strain (swine HEV) that infects large numbers of pigs in the United States. To assess the potential of pigs to serve as a global reservoir of HEV, we measured the prevalence of HEV antibodies in pigs in two countries where hepatitis E is endemic and two countries where it is not. Swine herds in all four countries contained many pigs that were seropositive for IgG anti-HEV, although the percentage of seropositive pigs varied greatly from herd to herd. A very limited number of pig handlers in the two endemic countries were also tested and most of them were found to be seropositive for HEV. The results from this study suggest that hepatitis E is enzootic in pigs regardless of whether HEV is endemic in the respective human population. J. Med. Virol. 59:297-302, 1999. Published 1999 Wiley-Liss, Inc.

Cloning and characterization of partial cDNAs for woodchuck cytokines and CD3epsilon with applications for the detection of RNA expression in tissues by RT-PCR assay.

Immunologic reagents and methodology are essential to develop further the woodchuck and woodchuck hepatitis virus (WHV) as a model of immune response, inflammation, and immunotherapy in hepatitis B virus (HBV) infection. Partial cDNA clones for the woodchuck CD3epsilon marker of T cells (536 bp) and for selected woodchuck cytokines were developed, including IL-1beta (332 bp), IL-2 (249 bp), IL-4 (205 bp), IL-10 (476 bp), IFN-gamma (476 bp), and TNF-alpha (381 bp). This panel of markers includes sets to measure RNAs for T cells (CD3epsilon), immune response induction (IL-1beta, IL-2), TH subsets (TH1, IL-2/IFN-gamma vs. TH2, IL-4/IL-10), and effector molecules that regulate hepadnavirus replication and liver injury (IFN-gamma, TNF-alpha). Primers representing highly conserved segments of genes from other species were used to derive the partial cDNA clones. Target RNA was obtained from woodchuck peripheral blood mononuclear cells (PBMC) that were stimulated in vitro with ConA, LPS, and human rIL-2. The cDNA clones were validated by 1) comparison with other species for homologies in the nucleotide and predicted amino acid sequences and 2) a first generation assay demonstrating induction of the respective RT-PCR products in stimulated woodchuck PBMC. The corresponding RNAs were also detectable in most cases in the total RNA from the livers of uninfected and WHV-infected woodchucks and differential expression of IFN-gamma and TNF-alpha RNAs was suggested. Second generation, semi-quantitative assays for the RNAs were validated using RT-PCR and dot-blot hybridization with 32P-oligomers derived from the internal sequences of the respective clones. Continued study of the woodchuck immune response to WHV infection using these assays will provide insight into the kinetics and immune mechanisms that initiate and maintain chronic hepadnavirus infection and, hence, enable development of improved immunotherapies for established chronic HBV infection.

Hepatitis B virus (HBV)/hepatitis D virus (HDV) coinfection in outbreaks of acute hepatitis in the Peruvian Amazon basin: the roles of HDV genotype III and HBV genotype F.

Recurring outbreaks of acute hepatitis have been a significant cause of morbidity and mortality among Peruvian military personnel stationed in the Amazon Basin region of Peru. The role of hepatitis B virus (HBV) and hepatitis D virus (HDV) infection was investigated as the possible cause of acute hepatitis among 88 military patients stationed at four different jungle outposts during 1992-1993. Analysis of serum markers indicated that 95% (84/88) had evidence of acute HBV infection; 64% (54/84) were also infected with HDV. Genetic analysis of PCR-amplified HDV and HBV fragments showed exclusively HDV genotype III and HBV genotype F. Furthermore, HDV RNA sequences were similar among patients from the same outpost but different from those at other jungle locations. The data suggested focal sources of HDV infection in the jungle environment of the outposts and, further, confirmed the unique association of HDV genotype III with severe cases of human disease in northern South America.

Hepatocellular carcinoma in Richardson's ground squirrels (Spermophilus richardsonii): evidence for association with hepatitis B-like virus infection.

During studies of seasonal obesity, a high frequency of hepatic neoplasms was observed in Richardson's ground squirrels. Of 12 Richardson's ground squirrels examined thoroughly, 7 had mild or moderate degrees of chronic portal hepatitis and 6 (50%) had hepatocellular carcinoma. Serological tests for hepadnavirus surface antigen, anti-core antibody and virion DNA that recognize the ground squirrel hepatitis virus of California ground squirrels (Spermophilus beecheyi) were uniformly negative. Southern blot analyses of EcoRI digests of liver cell DNA demonstrated 3.2 kb fragments that hybridized with a ground squirrel hepatitis virus-specific probe in nontumorous liver tissue from 6 of 10 ground squirrels and in hepatocellular carcinoma specimens from 2 of 5 squirrels indicating infection with a hepadnavirus related to ground squirrel hepatitis virus. Failure, however, to detect serum antibody to ground squirrel hepatitis core antigen suggested probable antigenic differences between the ground squirrel hepatitis virus of California ground squirrels and the putative Richardson's ground squirrel agent. Further studies are required to fully characterize the hepadnavirus of Richardson's ground squirrels and to determine its relationship to hepatocarcinogenesis in this species.

Protection of chimpanzees from type B hepatitis by immunization with woodchuck hepatitis virus surface antigen.

Two chimpanzees immunized with woodchuck hepatitis virus (WHV) surface antigen (WHsAg) developed antibodies cross-reactive with hepatitis B virus (HBV) surface antigen (HBsAg). After challenge with HBV, one animal was completely protected and the other experienced a subclinical infection, without evidence of liver disease. Three woodchucks immunized with HBsAg developed antibodies to HBsAg which did not cross-react with WHsAg. After challenge with WHV, all three woodchucks developed typical acute infections with associated hepatic lesions. Serological studies with the cross-reactive antibodies raised in chimpanzees suggested that the protective epitopes of WHsAg were related to the group a specificity of HBsAg. These studies indicated that cross-protective epitopes are shared by HBV and WHV; however, the humoral response to these epitopes can vary among species.

Significance of IgM antibody to hepatitis B core antigen in a Greek population with chronic hepatitis B virus infection.

IgM antibody to hepatitis B core antigen (IgM anti-HBc) may indicate an active immune response to persistent infection with hepatitis B virus (HBV). We studied 186 Greek HBsAg carriers for IgM anti-HBc and attempted to correlate it with other HBV and hepatitis delta virus (HDV) markers. Overall, IgM anti-HBc was detected more frequently than HBV DNA in this population (50% vs 34, p less than 0.001); this was also true for the 149 of the 186 HBsAg carriers with antibody to hepatitis B e antigen (anti-HBe) (48% vs 22%, p less than 0.001). The opposite was found in the carriers positive for hepatitis B e antigen (HBeAg): HBV DNA was observed in 93% and IgM anti-HBc in 64% of the cases (p less than 0.05). The detection of these markers was independent of sex. Serum alanine aminotransferase (ALT) levels were significantly more elevated in patients with positive tests for IgM anti-HBc and HBV DNA than in patients positive only for HBV DNA (p less than 0.001) irrespective of their HBeAg or anti-HBe status. Moreover, the detection of elevated ALT was independent of the intensity of the HBV DNA hybridization signal. Antibodies to hepatitis delta antigen (HDAg) were only found in 4 (2.4%) of 167 patients tested.

Antigenic analysis of woodchuck hepatitis virus surface antigen with site-specific radioimmunoassays.

Monoclonal antibodies to five nonoverlapping antigenic domains of woodchuck hepatitis virus surface antigen (WHsAg) were used to develop site-specific radioimmunoassays. The assays were based on the solid-phase sandwich principle in which different combinations of individual domain-specific antibodies were used as immunoadsorbents and radioiodinated probes. Over 85% of the combinations tested were able to detect serum WHsAg, including those using the same antibody as immunoadsorbent and probe. The limits for antigen detection in one site-specific system ranged between 16 and 80 ng of WHsAg per ml. The antigenic similarity of serum WHsAg from 13 colony woodchucks was shown with several combination assay systems. WHsAg was equally immunoreactive in these assay systems whether obtained by immunoaffinity chromatography or standard rate zone centrifugation methods. Further site-specific analysis demonstrated that Formalin treatment of purified antigen did not affect the immunoreactivity of these WHsAg sites.