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.

Mutational analysis of the hepatitis C virus E1 glycoprotein in retroviral pseudoparticles and cell-culture-derived H77/JFH1 chimeric infectious virus particles.

Cell entry by enveloped viruses is mediated by viral glycoproteins, and generally involves a short hydrophobic peptide (fusion peptide) that inserts into the cellular membrane. An internal hydrophobic domain within E1 (aa262-290) of hepatitis C virus (HCV) may function as a fusion peptide. Retrovirus-based HCV-pseudotyped viruses (HCVpp; genotype 1a) containing Ala or Pro substitutions at conserved amino acid positions within this putative fusion peptide were generated. Mutation of conserved residues significantly reduced efficiency of HCVpp entry into Huh-7 cells. The majority of amino acid substitutions appeared to disrupt necessary interactions between E1 and E2. For some mutants, reductions in HCVpp-associated E1 were associated with the incorporation of a high molecular weight, hyperglycosylated E2 that displayed decreased CD81-binding. Other entry-deficient mutants displayed normal E1E2 incorporation into pseudoparticles and normal CD81-binding, and therefore might affect viral fusion. One mutant (S283P) consistently displayed two- to threefold higher infectivity than did wild-type. Three mutations that decreased HCVpp infectivity also reduced levels of HCVcc infectious virus production. However, the S283P mutation had a different effect in the two systems as it did not increase production of infectious HCVcc. This comprehensive mutational analysis of the putative HCV fusion peptide provides insight into the role of E1 in its interaction with E2 and in HCV entry.

Hepatitis E: an emerging awareness of an old disease.

Although hepatitis E was recognized as a new disease in 1980, the virus was first visualized in 1983 and its genome was cloned and characterized in 1991, the disease is probably ancient but not recognized until modern times. Hepatitis E is the most important or the second most important cause of acute clinical hepatitis in adults throughout Asia, the Middle East and Africa. In contrast, hepatitis E is rare in industrialized countries, but antibody (anti-HEV) is found worldwide. HEV is a small round RNA-containing virus that is the only member of the genus Hepevirus in the family Hepeviridae. Although similar to hepatitis A virus in appearance, there are significant differences between the two viruses. Hepatitis E is principally the result of a water-borne infection in developing countries and is thought to be spread zoonotically (principally from swine) in industrialized countries. Because diagnostic tests vary greatly in specificity, sensitivity and availability, hepatitis E is probably underdiagnosed. At present, control depends upon improved hygiene; a highly efficacious vaccine has been developed and tested, but it is not presently available.

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.

A survey of zoonotic pathogens carried by Norway rats in Baltimore, Maryland, USA.

Norway rats (Rattus norvegicus) carry several zoonotic pathogens and because rats and humans live in close proximity in urban environments, there exists potential for transmission. To identify zoonotic agents carried by rats in Baltimore, Maryland, USA, we live-trapped 201 rats during 2005-2006 and screened them for a panel of viruses, bacteria, and parasites. Antibodies against Seoul virus (57.7%), hepatitis E virus (HEV, 73.5%), Leptospira interrogans (65.3%), Bartonella elizabethae (34.1%), and Rickettsia typhi (7.0%) were detected in Norway rats. Endoparasites, including Calodium hepatica (87.9%) and Hymenolepis sp. (34.4%), and ectoparasites (13.9%, primarily Laelaps echidninus) also were present. The risk of human exposure to these pathogens is a significant public health concern. Because these pathogens cause non-specific and often self-limiting symptoms in humans, infection in human populations is probably underdiagnosed.

Determination and analysis of the complete genomic sequence of avian hepatitis E virus (avian HEV) and attempts to infect rhesus monkeys with avian HEV.

Avian hepatitis E virus (avian HEV), recently identified from a chicken with hepatitis-splenomegaly syndrome in the United States, is genetically and antigenically related to human and swine HEVs. In this study, sequencing of the genome was completed and an attempt was made to infect rhesus monkeys with avian HEV. The full-length genome of avian HEV, excluding the poly(A) tail, is 6654 bp in length, which is about 600 bp shorter than that of human and swine HEVs. Similar to human and swine HEV genomes, the avian HEV genome consists of a short 5' non-coding region (NCR) followed by three partially overlapping open reading frames (ORFs) and a 3'NCR. Avian HEV shares about 50 % nucleotide sequence identity over the complete genome, 48-51 % identity in ORF1, 46-48 % identity in ORF2 and only 29-34 % identity in ORF3 with human and swine HEV strains. Significant genetic variations such as deletions and insertions, particularly in ORF1 of avian HEV, were observed. However, motifs in the putative functional domains of ORF1, such as the helicase and methyltransferase, were relatively conserved between avian HEV and mammalian HEVs, supporting the conclusion that avian HEV is a member of the genus Hepevirus. Phylogenetic analysis revealed that avian HEV represents a branch distinct from human and swine HEVs. Swine HEV infects non-human primates and possibly humans and thus may be zoonotic. An attempt was made to determine whether avian HEV also infects across species by experimentally inoculating two rhesus monkeys with avian HEV. Evidence of virus infection was not observed in the inoculated monkeys as there was no seroconversion, viraemia, faecal virus shedding or serum liver enzyme elevation. The results from this study confirmed that avian HEV is related to, but distinct from, human and swine HEVs; however, unlike swine HEV, avian HEV is probably not transmissible to non-human primates.

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.

Four chimpanzee monoclonal antibodies isolated by phage display neutralize hepatitis a virus.

Chimpanzee immunoglobulins are virtually identical to human immunoglobulins and may have clinically useful applications. Four chimpanzee monoclonal antibodies (MAbs) to the hepatitis A virus (HAV) capsid were isolated from a combinatorial cDNA library of gamma1/kappa antibody genes using phage display. Competition assays indicated that three of the MAbs recognized the same or overlapping epitopes, whereas the fourth recognized a different, nonoverlapping epitope on the HAV capsid. All four MAbs neutralized the homologous HAV strain, HM-175, in a radioimmunofocus assay and two of the four MAbs neutralized a heterologous simian HAV strain, AGM-27. From these data, we conclude that the MAbs must recognize at least three epitopes on the HAV capsid. Furthermore, competition assays performed with neutralizing murine MAbs suggested that three of the chimpanzee MAbs recognized epitopes on the HAV capsid which have not been defined previously.

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.

Host range studies of GB virus-B hepatitis agent, the closest relative of hepatitis C virus, in New World monkeys and chimpanzees.

GB virus-B (GBV-B) is a member of the Flaviviridae family of viruses. This RNA virus causes acute resolving hepatitis in experimentally infected tamarins, but its natural host remains unknown. GBV-B and a related virus, GBV-A, were recovered from serum containing the "GB agent," which was believed to have originated from a surgeon (initials: GB) with acute hepatitis. GBV-B has special interest because it is the virus related most closely to hepatitis C virus, which is an important cause of acute and chronic liver disease in humans. In the present study, we found that the host range of GBV-B includes owl monkeys. Tamarins and owl monkeys belong to two different families of New World monkeys. The natural history of GBV-B in the two owl monkeys studied was similar to that previously found for tamarins and was characterized by early appearance of viremia and viral clearance. However, the peak viral titers of GBV-B observed in owl monkeys (10(5) genome equivalents [GE] /ml) were lower than those observed in experimentally infected tamarins (10(7)-10(8) GE/ml) and acute hepatitis was observed in only one animal. If GBV-B were indeed a virus of humans, it would be expected to infect chimpanzees, a surrogate of humans, because all recognized human hepatitis viruses are transmissible to chimpanzees and cause hepatitis. However, in the present study, we failed to transmit GBV-B to a naive chimpanzee. In addition, a second naive chimpanzee transfected intrahepatically with RNA transcripts from an infectious clone of GBV-B did not become infected. Thus, chimpanzees are apparently not susceptible to GBV-B. Finally, we failed to detect GBV-B in acute-phase serum from surgeon GB. Our data suggest that GBV-B is not a human virus and that GBV-B, like GBV-A, is a virus of New World monkeys.

Recombinant hepatitis E virus genomes infectious for primates: importance of capping and discovery of a cis-reactive element.

Hepatitis E virus recombinant genomes transcribed in vitro from two cDNA clones differing by two nucleotides were infectious for chimpanzees. However, one cDNA clone encoded a virus that was attenuated for chimpanzees and unable to infect rhesus monkeys. The second cDNA clone encoded a virus that infected both chimpanzees and rhesus monkeys and caused acute hepatitis in both. One mutation differentiating the two clones identified a cis-reactive element that appeared to overlap the 3' end of the capsid gene and part of the 3' noncoding region. Capping of the RNA transcripts was essential for infectivity.

Epidemiology of acute hepatitis in the Stann Creek District of Belize, Central America.

Hepatitis is common in the Stann Creek District of southern Belize. To determine the etiologies, incidence, and potential risk factors for acute jaundice, we conducted active surveillance for cases. Cases of jaundice diagnosed by a physician within the previous 6 weeks were enrolled. Evaluation included a questionnaire and laboratory tests for hepatitis A, B, C, D, and E, a blood film for malaria, and a serologic test for syphilis. Etiologies of jaundice among 62 evaluable patients included acute hepatitis A, 6 (9.7%), acute hepatitis B, 49 (79.0%), hepatitis non-A-E, 2 (3.2%), and malaria, 5 (8.1%). There were no cases of acute hepatitis E. One patient each with antibody to hepatitis C and D were detected. The annualized incidence of hepatitis A was 0.26 per 1,000. All cases of hepatitis A were in children 4-16 years of age. The annualized incidence of hepatitis B, 2.17 per 1,000, was highest in adults aged 15-44 years (4.4 per 1,000) and was higher in men (36 cases; 3.09 per 1,000) than women (13 cases; 1.19 per 1,000). Four (31%) of the women with hepatitis B were pregnant. The annualized incidence was significantly higher in Mestizo (6.18 per 1000) and Maya (6.79 per 1,000) than Garifuna (0.38 per 1,000) or Creole (0.36 per 1,000). Persons with hepatitis B were significantly more likely to be born outside of Belize (82%), had been in Belize < 5 years (73%), and lived and worked in rural areas (96%) than was the general population. Of those > or = 14 years of age with hepatitis B, only 36% were married. Few persons admitted to transfusions, tattoos, IV drug use, multiple sexual partners, visiting prostitutes, or sexually transmitted diseases. Only 1 of 49 had a reactive test for syphilis. Six patients were hospitalized (including 3 with acute hepatitis B and one with hepatitis A), and none to our knowledge died. Acute hepatitis B is the most common cause of viral hepatitis in the Stann Creek District, but the modes of transmission remain obscure. Infants, women attending prenatal clinics, and new workers are potential targets for immunization with hepatitis B vaccine.

Recombinant vaccines for hepatitis E.

Hepatitis E virus causes epidemics of acute hepatitis in many developing countries. It infrequently causes disease in developed countries, but avirulent strains might circulate. Some evidence suggests that hepatitis E might be a zoonosis. There is probably only a single serotype. A candidate vaccine consisting of baculovirus-expressed recombinant capsid protein protected macaques from hepatitis E--it passed phase I clinical trials and is currently scheduled for phase II/III clinical trials.

Identification of the 5' terminal sequence of the SAR-55 and MEX-14 strains of hepatitis E virus and confirmation that the genome is capped.

Hepatitis E virus (HEV) is a nonenveloped virus with a genome of single-stranded, positive-sense RNA. The 5' terminal sequence of two HEV strains (SAR-55 and MEX-14) was determined by a 5' RNA ligase-mediated rapid amplification of cDNA ends (RACE) method designed to select capped RNAs. The 5' noncoding region of the SAR-55 and MEX-14 strains were amplified, confirming that the genomic RNA of HEV is capped. The 5' noncoding region of the SAR-55 strain had 25 nucleotides, which is two less than reported for the Burmese strain, and that of the MEX-14 strain had 24 nucleotides, which is 21 more than reported previously [Huang et al., 1992].

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.

High-level expression of hepatitis C virus (HCV) structural proteins by a chimeric HCV/BVDV genome propagated as a BVDV pseudotype.

A chimeric cDNA genome was constructed in which the core, E1 and E2 genes of hepatitis C virus (HCV) replaced the core, E(rns), E1 and E2 genes of bovine viral diarrhea virus (BVDV). High levels of HCV structural proteins were expressed in a small number of human or bovine cells following transfection with chimeric RNA. However, in one cell line, bovine embryonic trachea cells [EBTr(A)], the number of cells expressing HCV proteins increased to greater than 70% following serial passage of culture medium. These cells were persistently infected with a non-cytopathogenic BVDV helper virus. In these cells, the chimeric genome was packaged into infectious particles that accumulated in the culture medium at a titer as high as 10(7)-10(9) genome equivalents per ml. The virus particles were pseudotypes, because they were neutralized by anti-BVDV but not by anti-HCV.

Studies of hepatitis C virus in chimpanzees and their importance for vaccine development.

Persistent infection with hepatitis C virus (HCV) is an important cause of chronic liver disease worldwide. Therefore, the development of vaccines to prevent HCV infection, or at least to prevent progression to chronicity, is a major goal. Potential HCV vaccine candidates include recombinant proteins, recombinant viruses, DNA constructs, synthetic peptides and virus-like particles. Various vaccine candidates have been shown to generate humoral and cellular immune responses in animals, primarily in mice. However, the efficacy of most vaccine candidates in protecting against HCV has not been tested because the chimpanzee, the only animal other than humans that is susceptible to HCV, is not readily available, requires special facilities, and is very expensive. The course of infection in chimpanzees is similar in its diversity to that in humans and detailed studies in this model are beginning to define the immune responses that can terminate HCV infection. Of relevance for vaccine evaluation was the titration in chimpanzees of different HCV variants to provide well-characterized challenge pools. In addition, monoclonal virus pools generated from chimpanzees infected with cloned viruses make it possible now to examine immunity to HCV without the confounding factor of antigenic diversity of the challenge virus (quasispecies). The vaccine trials performed in chimpanzees to date all have tested the efficacy of immunizations with various forms of the envelope proteins of HCV.