Hepatitis: Purcell RH

Purcell RH, Emerson SU. · Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD 20892-8009, USA. · J Hepatol. · Pubmed #18192058 No free full text.

Abstract: 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.

Emerson SU, Purcell RH. · Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA. · Pediatr Infect Dis J. · Pubmed #18043454 No free full text.

This publication has no abstract.

Emerson SU, Purcell RH. · National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA. · Rev Med Virol. · Pubmed #12740830 No free full text.

Abstract: Hepatitis E virus (HEV) is a positive-stranded RNA virus with a 7.2 kb genome that is capped and polyadenylated. The virus is currently unclassified: the organisation of the genome resembles that of the Caliciviridae but sequence analyses suggest it is more closely related to the Togaviridae. Hepatitis E virus is an enterically transmitted virus that causes both epidemics and sporadic cases of acute hepatitis in many countries of Asia and Africa but only rarely causes disease in more industrialised countries. Initially the virus was believed to have a limited geographical distribution. However, serological studies suggest that HEV may be endemic also in the United States and Europe even though it infrequently causes overt disease in these countries. Many different animal species worldwide recently have been shown to have antibodies to HEV suggesting that hepatitis E may be zoonotic. Although two related strains have been experimentally transmitted between species, direct transmission from an animal to a human has not been documented. There are four currently recognised genotypes and two of the four contain viruses isolated from swine as well as from humans. Regardless of country of origin or genotype of the virus, most, if not all, strains belong to a single serotype. A promising recombinant vaccine candidate comprised of a truncated capsid protein is currently under evaluation in Nepal.

Forns X, Bukh J, Purcell RH. · Liver Unit, Institut de Malalties Digestives, Hospital Clínic, Villaroel 170, Barcelona 08036, Spain. · J Hepatol. · Pubmed #12399239 No free full text.

This publication has no abstract.

Emerson SU, Purcell RH. · Molecular Hepatitis and Hepatitis Viruses Sections, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA. · Trends Mol Med. · Pubmed #11597521 No free full text.

Abstract: 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.

Bukh J, Forns X, Emerson SU, Purcell RH. · Hepatitis Viruses and Molecular Hepatitis Sections, Laboratory of Infectious Diseases, NIAID, National Institutes of Health, Bethesda, Md 20892-0740, USA. · Intervirology. · Pubmed #11509874 No free full text.

Abstract: 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.

Purcell RH, Emerson SU. · Hepatitis Viruses and Molecular Hepatitis Sections, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA. · ILAR J. · Pubmed #11406718 No free full text.

Abstract: Several useful animal models for both hepatitis A and E have been identified, characterized, and refined. At present, all of the best models utilize nonhuman primates: chimpanzees, tamarin species, and owl monkeys for hepatitis A; and macaque species, chimpanzees, and owl monkeys for hepatitis E. Pigs may prove useful for some studies of hepatitis E, and it is hoped that serological evidence of widespread infection of rats with an HEV-like agent may lead to the development of an animal model based on laboratory rats. As has been the case for each of the hepatitis viruses as they have been discovered, the development of useful and reproducible animal model systems has been critical for moving the field forward as expeditiously as possible.

Farci P, Purcell RH. · Department of Medical Sciences, University of Cagliari, Italy. · Semin Liver Dis. · Pubmed #10895435 No free full text.

Abstract: Hepatitis C virus (HCV) is a major cause of morbidity and mortality worldwide. The infection becomes chronic in about 85% of infected individuals, in the face of a strong humoral and cellular immune response. One of the most important features of HCV is its high degree of genetic variability, which is due to the inherent low fidelity of the viral replication machinery. As a consequence, HCV circulates in vivo as a population of divergent, albeit closely related, genomes exhibiting a distribution that follows the model referred to as a quasispecies. The genetic variability of HCV is complex and has been classified into four hierarchical strata: genotypes, subgenotypes, isolates, and quasispecies. Over the past few years, an extraordinary interest has been focused on the biologic and clinical implications of the genetic variability of HCV. Although there is consensus that the genotypes may influence the out come of antiviral therapy, their clinical significance in the natural history of the disease, as well as in transmission, infectivity, and pathogenesis of HCV infection, remains elusive. Conversely, evidence has accumulated that the quasispecies nature of HCV provides a large reservoir of biologically different viral variants that may have important clinical implications for viral persistence by immune escape mechanisms, for the generation of antiviral drug resistance, and for the development of an effective vaccine. This article reviews the state of the art on the biologic and clinical implications of the genetic variability of HCV.

Forns X, Purcell RH, Bukh J. · Hepatitis Viruses Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA. · Trends Microbiol. · Pubmed #10498948 No free full text.

Abstract: Hepatitis C virus (HCV) is an important cause of chronic liver disease worldwide. This RNA virus circulates as a quasispecies and its genetic heterogeneity has been implicated in the lack of protective immunity against HCV and in its persistence following infection. HCV might escape from immune surveillance by developing mutations in proteins that are subject to immune pressure.

Farci P, Roskams T, Chessa L, Peddis G, Mazzoleni AP, Scioscia R, Serra G, Lai ME, Loy M, Caruso L, Desmet V, Purcell RH, Balestrieri A. · Dipartimento di Scienze Mediche, Università di Cagliari, SS 554 Bivio Sestu, 09042 Cagliari, Italy. · Gastroenterology. · Pubmed #15188169 No free full text.

Abstract: BACKGROUND & AIMS: Little is known about the long-term effects of interferon alpha on clinical outcome and survival of patients with chronic hepatitis D. METHODS: Thirty-six patients with chronic hepatitis D who participated in a randomized controlled trial of a 48-week course of high (9 million units) or low (3 million units) doses of interferon alpha or no treatment were followed for an additional 2 to 14 years. RESULTS: Long-term survival was significantly longer in the high-dose group than in untreated controls (P = 0.003) or in the low-dose group (P = 0.019) but did not differ between patients treated with 3 million units and controls. Among surviving patients at 12 years of follow-up, a biochemical response was present in 7 of 12 treated with 9 million units, in 2 of 4 who received 3 million units, and in none of 3 controls. Long-term alanine aminotransferase (ALT) normalization correlated with improved hepatic function and loss of IgM antibody to hepatitis delta antigen (anti-HD). Patients in the high-dose group had a sustained decrease in HDV replication (P = 0.008), leading to clearance of HDV RNA and, eventually, hepatitis B virus (HBV) in some patients, as well as a dramatic improvement in liver histology with respect to activity grade (P = 0.0004) and fibrosis stage (P = 0.007). Strikingly, we documented an absence of fibrosis in the final biopsy of 4 patients with a long-term biochemical response and an initial diagnosis of active cirrhosis. CONCLUSIONS: High doses of interferon alpha-2a significantly improved the long-term clinical outcome and survival of patients with chronic hepatitis D, even though the majority had active cirrhosis before the onset of therapy.

Christensen PB, Engle RE, Hjort C, Homburg KM, Vach W, Georgsen J, Purcell RH. · Department of Infectious Diseases, Odense University Hospital, Denmark. · Clin Infect Dis. · Pubmed #18781880 No free full text.

Abstract: BACKGROUND: Antibody to hepatitis E virus (anti-HEV) is prevalent in Western countries, where clinical hepatitis E is rarely reported. The aim of this study was to determine the prevalence of anti-HEV among Danish blood donors and Danish farmers. In addition, we compared the prevalence among 2 sets of serum samples obtained from blood donors 20 years apart. METHODS: Samples from 291 Danish farmers and 169 blood donors that were collected in 1983 and samples from 461 blood donors that were collected in 2003 were tested for anti-HEV. Relevant information on HEV exposure was collected by self-administered questionnaire. RESULTS: Anti-HEV testing was performed on samples after 20 years of storage at -20 degrees C. The prevalence of anti-HEV was 50.4% among farmers and 32.9% among donors in 1983 and 20.6% among donors in 2003 (P < .05). Presence of anti-HEV was significantly correlated with increasing age in all 3 groups (P < .05). Among donors who had serum samples obtained in 2003, age, contact with horses, and the presence of antibody to hepatitis A virus were associated with the presence of anti-HEV in multivariate analysis. Among farmers, only age was independently associated with the presence of anti-HEV. CONCLUSION: Anti-HEV was highly prevalent among Danes but has decreased in prevalence over the past 50 years. Our study supports the hypothesis that HEV infection in Denmark may be an asymptomatic zoonotic infection.

Meunier JC, Russell RS, Engle RE, Faulk KN, Purcell RH, Emerson SU. · Viral Envelopes and Retrovirus Engineering, Human Virology Department, INSERM U758, Ecole Normale Supérieure de Lyon, 46 allée d'Italie, 69364 Lyon Cedex 07, France. · J Virol. · Pubmed #18667498 links to  free full text

Abstract: Accumulating evidence suggests that cellular lipoprotein components are involved in hepatitis C virus (HCV) morphogenesis, but the precise contribution of these components remains unclear. We investigated the involvement of apolipoprotein C1 (ApoC1) in HCV infection in the HCV pseudotyped particle system (HCVpp), in the recently developed cell culture infection model (HCVcc), and in authentic HCV isolated from viremic chimpanzees. Viral genomes associated with HCVcc or authentic HCV were efficiently immunoprecipitated by anti-ApoC1, demonstrating that ApoC1 was a normal component of HCV. The infectivities of HCVpp that had been mixed with ApoC1 and, more importantly, untreated HCVcc collected from lysates or media of infected Huh7.5 cells were directly neutralized by anti-ApoC1. Indeed, convalescent anti-HCV immunoglobulin G and anti-ApoC1 each neutralized over 75% of infectious HCVcc particles, indicating that many, if not all, infectious particles were recognized by both antibodies. Moreover, peptides corresponding to the C-terminal region of ApoC1 blocked infectivity of both HCVpp and HCVcc. Altogether, these results suggest that ApoC1 associates intracellularly via its C-terminal region with surface components of virions during viral morphogenesis and may play a major role in the replication cycle of HCV.

Bukh J, Thimme R, Meunier JC, Faulk K, Spangenberg HC, Chang KM, Satterfield W, Chisari FV, Purcell RH. · Hepatitis Viruses Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Building 50, Room 6531, 50 South Dr., MSC 8009, Bethesda, MD 20892-8009, USA. · J Virol. · Pubmed #18550671 links to  free full text

Abstract: Protective immunity after resolved hepatitis C virus (HCV) infection has been reported. However, the breadth of this immunity has remained controversial, and the role of neutralizing antibodies has not been well-defined. In the present study, two chimpanzees (CH96A008 and CH1494) with resolved monoclonal H77C (genotype 1a) infection were rechallenged with low-dose homologous H77C virus about 12 months after viral clearance; CH96A008 became persistently infected, and CH1494 had transient viremia lasting 2 weeks. CH1494 was subsequently either partially or completely protected following five homologous rechallenges with monoclonal H77C or polyclonal H77 and after six heterologous rechallenges with HC-J4 (genotype 1b) or HC-J6 (genotype 2a) viruses. Subsequently, a final challenge with H77C resulted in persistent HCV infection. In both chimpanzees, serum neutralizing antibodies against retroviral pseudoparticles bearing the H77C envelope proteins were not detected during the initial infection or during rechallenge. However, anamnestic cellular immune responses developed during the initial homologous rechallenge, in particular in CH96A008, which developed a persistent infection. Polyprotein sequences of viruses recovered from CH1494 after the two homologous rechallenges that resulted in transient viremia were identical with the H77C virus. In contrast, the polyprotein sequences of viruses recovered from both chimpanzees after homologous rechallenge resulting in persistent infection had numerous changes. These findings have important implications for our understanding of immunity against HCV; even in the best-case scenario with autologous rechallenge, low-level viral persistence was seen in the presence of primed T-cell responses.

Vanwolleghem T, Bukh J, Meuleman P, Desombere I, Meunier JC, Alter H, Purcell RH, Leroux-Roels G. · Center for Vaccinology, Ghent University and Hospital, Ghent, Belgium. · Hepatology. · Pubmed #18452146 No free full text.

Abstract: The role of the humoral immune response in the natural course of hepatitis C virus (HCV) infection is widely debated. Most chronically infected patients have immunoglobulin G (IgG) antibodies capable of neutralizing HCV pseudoparticles (HCVpp) in vitro. It is, however, not clear whether these IgG can prevent a de novo HCV infection in vivo and contribute to the control of viremia in infected individuals. We addressed this question with homologous in vivo protection studies in human liver-urokinase-type plasminogen activator (uPA)(+/+) severe combined immune deficient (SCID) mice. Chimeric mice were loaded with chronic phase polyclonal IgG and challenged 3 days later with a 100% infectious dose of the acute phase H77C virus, both originating from patient H. Passive immunization induced sterilizing immunity in five of eight challenged animals. In the three nonprotected animals, the HCV infection was attenuated, as evidenced by altered viral kinetics in comparison with five control IgG-treated animals. Plasma samples obtained from the mice at viral challenge neutralized H77C-HCVpp at dilutions as high as 1/400. Infection was completely prevented when, before administration to naïve chimeric mice, the inoculum was pre-incubated in vitro at an IgG concentration normally observed in humans. CONCLUSION: Polyclonal IgG from a patient with a long-standing HCV infection not only displays neutralizing activity in vitro using the HCVpp system, but also conveys sterilizing immunity toward the ancestral HCV strain in vivo, using the human liver-chimeric mouse model. Both experimental systems will be useful tools to identify neutralizing antibodies for future clinical use.

Russell RS, Meunier JC, Takikawa S, Faulk K, Engle RE, Bukh J, Purcell RH, Emerson SU. · Hepatitis Viruses and Molecular Hepatitis Sections, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA. · Proc Natl Acad Sci U S A. · Pubmed #18334634 links to  free full text

Abstract: The JFH1 strain of hepatitis C virus (HCV) is unique among HCV isolates, in that the wild-type virus can traverse the entire replication cycle in cultured cells. However, without adaptive mutations, only low levels of infectious virus are produced. In the present study, the effects of five mutations that were selected during serial passage in Huh-7.5 cells were studied. Recombinant genomes containing all five mutations produced 3-4 logs more infectious virions than did wild type. Neither a coding mutation in NS5A nor a silent mutation in E2 was adaptive, whereas coding mutations in E2, p7, and NS2 all increased virus production. A single-cycle replication assay in CD81-deficient cells was developed to study more precisely the effect of the adaptive mutations. The E2 mutation had minimal effect on the amount of infectious virus released but probably enhanced entry into cells. In contrast, both the p7 and NS2 mutations independently increased the amount of virus released.

Engle RE, Russell RS, Purcell RH, Bukh J. · Hepatitis Viruses Section, Laboratory of Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA. · J Med Virol. · Pubmed #18041021 No free full text.

Abstract: A quantitative real-time PCR assay was developed that detects genomic RNA from reference strains representing the six major genotypes of hepatitis C virus (HCV) with equal sensitivity and accurately measured HCV RNA in JFH1 HCV-infected Huh7.5 cells. The method is indirectly calibrated to the first international (WHO 96/790) HCV standard preparation and has a linear dynamic range of 10(2.6)-10(6.5) IU/ml. In addition, the inter- and intra-assay precision were approximately 3% CV and <2% CV, respectively. Comparison with results obtained by commercially available HCV RNA Nucleic Acid Technology kits (Versant HCV RNA 3.0 b-DNA and Amplicor HCV Monitor), that also employ the WHO standard, allowed validation of the TaqMan assay against all major HCV genotypes. Both commercial methods detected HCV RNA over a wide dynamic range, but showed a consistent difference of about 0.3 log10 when evaluating samples of different HCV genotypes. The genome titers obtained with the three methods correlated with the infectivity titers previously determined for the HCV reference strains. TaqMan assays have become an essential tool to follow viral load in clinical samples and cell culture-based experiments and this technology offers significant advantages in linear dynamic range, sensitivity and customization.

Bukh J, Engle RE, Govindarajan S, Purcell RH. · Hepatitis Viruses Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892-8009, USA. · J Med Virol. · Pubmed #18041000 No free full text.

Abstract: GB virus-B (GBV-B) is the virus most closely related to hepatitis C virus (HCV). Thus, we have used GBV-B infection of tamarins, which develop acute hepatitis following experimental infection, as a surrogate model to study protective immunity. As challenge virus, we first produced a GBV-B pool from an infected tamarin, which was not infected with the related GBV-A viruses. Its infectivity titer was 10(6.6) tamarin 50% infectious doses per ml. Next, two tamarins that were convalescent from recombinant GBV-B infection were re-challenged. In the original infection viremia persisted for 8 and 12 weeks, respectively, and both animals developed moderately severe hepatitis. Each tamarin was re-challenged four times with 10(4.3) tamarin 50% infectious doses of the GBV-B challenge virus. In one animal, each re-challenge produced 1-2 weeks of viremia; hepatitis was observed following the first re-challenge. In the other animal, however, only the first re-challenge produced viremia, lasting 1 week. During the primary infection, peak GBV-B titers were about 10(8) genome equivalents/ml in both animals; following re-challenges, peak titers ranged from 10(3) to 10(6) genome equivalents/ml. Analysis of the polyprotein sequence of viruses recovered from both animals following the first re-challenge demonstrated that these did not represent immune escape variants since mutations were not detected. Neutralization studies suggested that the immunity was not humoral in nature. We also demonstrated that the immunity was long-lived: 1 year after the fourth challenge, the animal with sterilizing immunity had low titer viremia for only 1 week following an additional challenge.

Graff J, Zhou YH, Torian U, Nguyen H, St Claire M, Yu C, Purcell RH, Emerson SU. · Molecular Hepatitis Section, LID, NIAID, National Institutes of Health, Room 6537, Building 50, 50 South Drive, MSC 8009, Bethesda, MD 20892-8009, USA. · J Virol. · Pubmed #18032496 links to  free full text

Abstract: Hepatitis E virus is a nonenveloped RNA virus. However, the single capsid protein resembles a typical glycoprotein in that it contains a signal sequence and potential glycosylation sites that are utilized when recombinant capsid protein is overexpressed in cell culture. In order to determine whether these unexpected observations were biologically relevant or were artifacts of overexpression, we analyzed capsid protein produced during a normal viral replication cycle. In vitro transcripts from an infectious cDNA clone mutated to eliminate potential glycosylation sites were transfected into cultured Huh-7 cells and into the livers of rhesus macaques. The mutations did not detectably affect genome replication or capsid protein synthesis in cell culture. However, none of the mutants infected rhesus macaques. Velocity sedimentation analyses of transfected cell lysates revealed that mutation of the first two glycosylation sites prevented virion assembly, whereas mutation of the third site permitted particle formation and RNA encapsidation, but the particles were not infectious. However, conservative mutations that did not destroy glycosylation motifs also prevented infection. Overall, the data suggested that the mutations were lethal because they perturbed protein structure rather than because they eliminated glycosylation.

Meunier JC, Russell RS, Goossens V, Priem S, Walter H, Depla E, Union A, Faulk KN, Bukh J, Emerson SU, Purcell RH. · Molecular Hepatitis Section, LID, NIAID, NIH, Bldg. 50, Rm. 6537, 50 South Dr., MSC 8009, Bethesda, MD 20892-8009, USA. · J Virol. · Pubmed #17977972 links to  free full text

Abstract: The relative importance of humoral and cellular immunity in the prevention or clearance of hepatitis C virus (HCV) infection is poorly understood. However, there is considerable evidence that neutralizing antibodies are involved in disease control. Here we describe the detailed analysis of human monoclonal antibodies (MAbs) directed against HCV glycoprotein E1, which may have the potential to control HCV infection. We have identified two MAbs that can strongly neutralize HCV-pseudotyped particles (HCVpp) bearing the envelope glycoproteins of genotypes 1a, 1b, 4a, 5a, and 6a and less strongly neutralize HCVpp bearing the envelope glycoproteins of genotype 2a. Genotype 3a was not neutralized. The epitopes for both MAbs were mapped to the region encompassing amino acids 313 to 327. In addition, robust neutralization was also observed against cell culture-adapted viruses of genotypes 1a and 2a. Results from this study suggest that these MAbs may have the potential to prevent HCV infection.

Shata MT, Barrett A, Shire NJ, Abdelwahab SF, Sobhy M, Daef E, El-Kamary SS, Hashem M, Engle RE, Purcell RH, Emerson SU, Strickland GT, Sherman KE. · Internal Medicine, Division of Digestive Diseases, University of Cincinnati, Cincinnati, OH 45267-0595, USA. · J Immunol Methods. · Pubmed #17905301 links to  free full text

Abstract: 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.

Goncalvez AP, Engle RE, St Claire M, Purcell RH, Lai CJ. · Molecular Viral Biology Section and Hepatitis Viruses Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA. · Proc Natl Acad Sci U S A. · Pubmed #17517625 links to  free full text

Abstract: Infection with dengue virus (DENV) or any other flavivirus induces cross-reactive, but weakly neutralizing or nonneutralizing, antibodies that recognize epitopes involving the fusion peptide in the envelope glycoprotein. Humanized mAb IgG 1A5, derived from a chimpanzee, shares properties of cross-reactive antibodies. mAb IgG 1A5 up-regulated DENV infection by a mechanism of antibody-dependent enhancement (ADE) in a variety of Fc receptor-bearing cells in vitro. A 10- to 1,000-fold increase of viral yield in K562 cells, dependent on the DENV serotype, was observed over a range of subneutralizing concentrations of IgG 1A5. A significant increase of DENV-4 viremia titers (up to 100-fold) was also demonstrated in juvenile rhesus monkeys immunized with passively transferred dilutions of IgG 1A5. These results, together with earlier findings of ADE of DENV-2 infection by a polyclonal serum, establish the primate model for analysis of ADE. Considering the abundance of these cross-reactive antibodies, our observations confirm that significant viral amplification could occur during DENV infections in humans with prior infection or with maternally transferred immunity, possibly leading to severe dengue. Strategies to eliminate ADE were explored by altering the antibody Fc structures responsible for binding to Fc receptors. IgG 1A5 variants, containing amino acid substitutions from the Fc region of IgG2 or IgG4 antibodies, reduced but did not eliminate DENV-4-enhancing activity in K562 cells. Importantly, a 9-aa deletion at the N terminus of the CH(2) domain in the Fc region abrogated the enhancing activity.

Sakai A, Takikawa S, Thimme R, Meunier JC, Spangenberg HC, Govindarajan S, Farci P, Emerson SU, Chisari FV, Purcell RH, Bukh J. · Hepatitis Viruses Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-8009, USA. · J Virol. · Pubmed #17409145 links to  free full text

Abstract: Both viral and host factors are thought to influence the pathogenesis of hepatitis C virus (HCV) infection. We studied strain HC-TN (genotype 1a), which caused fulminant hepatic failure in a patient and, subsequently, severe hepatitis in a chimpanzee (CH1422), to analyze the relationship between disease severity, host immune response, viral evolution, and outcome. A second chimpanzee (CH1581) was infected from CH1422 plasma, and a third chimpanzee (CH1579) was infected from RNA transcripts of a consensus cDNA of HC-TN (pHC-TN). RNA transcripts of pHC-TN did not replicate in Huh7.5 cells, which were recently found to be susceptible to infection with another fulminant HCV strain (JFH1). The courses of viremia were similar in the three animals. However, CH1581 and CH1579 developed a less severe acute hepatitis than CH1422. CH1579 and CH1422 resolved the infection, whereas CH1581 became persistently infected. CH1579 and CH1581, despite their differing outcomes, both developed significant intrahepatic cellular immune responses, but not antibodies to the envelope glycoproteins or neutralizing antibodies, during the acute infection. We analyzed the polyprotein sequences of virus recovered at five and nine time points from CH1579 and CH1581, respectively, during the first year of follow-up. High mutation rates and high proportions of nonsynonymous mutations suggested immune pressure and positive selection in both animals. Changes were not selected until after the initial decrease in virus titers and after the development of immune responses and hepatitis. Subsequently, however, mutations emerged repeatedly in both animals. Overall, our results indicate that disease severity and outcome of acute HCV infection depend primarily on the host response.

Yu C, Zimmerman C, Stone R, Engle RE, Elkins W, Nardone GA, Emerson SU, Purcell RH. · Laboratory of Infectious Diseases, Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 50 South Drive, Building 50, Bethesda, MD 20892-8009, USA. · J Virol Methods. · Pubmed #17336401 No free full text.

Abstract: Recent reports from Japan implicated wild Sika deer (Cervus nippon) in the zoonotic transmission of hepatitis E to humans. Seroprevalence studies were performed to determine if imported feral populations of Sika deer in Maryland and Virginia posed a similar risk of transmitting hepatitis E virus (HEV). Hunters collected blood on filter paper discs from freshly killed deer. The discs were desiccated and delivered to a collection point. The dried filters were weighed to estimate the amount of blood absorbed and were eluted and collected in one tube via a novel extraction system. The procedure was quantified and validated with negative and positive serum and blood samples obtained from domestic Sika deer before and after immunization with HEV recombinant capsid protein, respectively. None of the 155 tested samples contained antibody to HEV, suggesting that Sika deer in these populations, unlike those in Japan, do not pose a significant zoonotic threat for hepatitis E. However, the new method developed for collecting and eluting the samples should prove useful for field studies of many other pathogens.

Easterbrook JD, Kaplan JB, Vanasco NB, Reeves WK, Purcell RH, Kosoy MY, Glass GE, Watson J, Klein SL. · The W. Harry Feinstone Department of Microbiology and Immunology, The Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205, USA. · Epidemiol Infect. · Pubmed #17224086 No free full text.

Abstract: 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.

Zhou YH, Chen Z, Purcell RH, Emerson SU. · Laboratory of Infectious Diseases, Hepatitis Viruses and Molecular Hepatitis Sections, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA. · Immunol Cell Biol. · Pubmed #17130902 No free full text.

Abstract: Epitope mapping (identification of an antigenic site recognized by an antibody) is an important component of vaccine development and immunological assays. It is widely accepted that in Western blots, antibodies react exclusively with continuous epitopes: discontinuous epitopes are assumed to be irreversibly destroyed by electrophoresis under the denaturing conditions used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Here, we demonstrate that the epitopes recognized by four different monoclonal antibodies were identified as discontinuous epitopes when characterized by radioimmunoprecipitation assays and enzyme-linked immunosorbent assays, yet each of these antibodies reacted with the corresponding antigen on Western blots. Reaction on Western blots may be due to epitope renaturation during or after the transfer of the protein to a membrane. Therefore, positive reactions on Western blots do not necessarily indicate that epitopes are continuous and this caveat should be kept in mind while characterizing them.