Putative neutralization epitopes and broad cross-genotype neutralization of Hepatitis E virus confirmed by a quantitative cell-culture assay

Emerson, Suzanne U.; Clemente-Casares, Pilar; Moiduddin, Nasser; Arankalle, Vidya A.; Torian, Udana; Purcell, Robert H.

doi:10.1099/vir.0.81545-0

Cited by 114 publications

(71 citation statements)

References 19 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…In order to determine if the replication-competent mutants produced infectious virus in S10-3 cells, we applied a newly established in vitro infectivity assay (5). Cell lysates were prepared from wild-type-or mutant-transfected S10-3 cells and tested for the ability to infect naïve cultured cells.…”

Section: Resultsmentioning

confidence: 99%

Mutations within Potential Glycosylation Sites in the Capsid Protein of Hepatitis E Virus Prevent the Formation of Infectious Virus Particles

Graff

Zhou

Torian

et al. 2008

J Virol

Self Cite

101

View full text Add to dashboard Cite

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.Hepatitis E virus (HEV) is transmitted via the fecal-oral route, predominantly through contaminated water. HEV causes acute self-limiting hepatitis and in much of the developing world is responsible for sporadic infections, as well as large epidemics, of acute hepatitis E, especially in Asia and Africa.Four genotypes comprising a single serotype of mammalian HEV have been identified (26,28). HEV was recently classified as the sole member of the genus Hepevirus, family Hepeviridae (3); sequence analysis suggests it is most closely related to rubella virus, an enveloped virus in the family Togaviridae. However, HEV does not contain a lipid envelope (26). The virion is 27 to 30 nm in diameter (1, 26) and has a sedimentation coefficient of 183S (2). The HEV genome is a positivesense RNA, approximately 7.2 kb in length, with a short, capped 5Ј noncoding region and a 3Ј noncoding region preceding a poly(A) tail. Viral genomic RNA is infectious for some cultured cells and nonhuman primates: transfection with capped recombinant genomes results in production of infectious virions in vitro and acute hepatitis and/or seroconversion in vivo (6-8). The coding region of HEV contains three partially overlapping open reading frames (ORFs): ORF1 encodes the nonstructural proteins, ORF2 encodes the capsid protein, and ORF3, which overlaps the N terminus of ORF2, encodes a small protein of 114 amino acids (aa) (13, 15) that might be a regulatory protein (7,19,33,44). The ORF2 and ORF3 proteins are encoded by a bicistronic subgenomic RNA (13,35,45).HEV does not grow sufficiently well either in cell culture or in nonhuman primates to permit direct biochemical analysis. Therefore, various in vitro systems have been utili...

show abstract

Section: Resultsmentioning

confidence: 99%

Mutations within Potential Glycosylation Sites in the Capsid Protein of Hepatitis E Virus Prevent the Formation of Infectious Virus Particles

Graff

Zhou

Torian

et al. 2008

J Virol

Self Cite

101

View full text Add to dashboard Cite

show abstract

“…This tube simplified the elution procedure because the elution and extraction steps were both carried out in the same device. Similarly, the HEV-specific antigen used in the ELISA assay was the same antigen that has been shown to detect all four mammalian genotypes of HEV with a high degree of sensitivity and to stimulate antibodies that neutralize at least three of the four HEV genotypes when incorporated into a vaccine (Emerson et al, 2006;Engle et al, 2002;Purcell et al, 2003;Zhou et al, 2004). Finally, to confirm that the commercial rabbit anti-white-tail deer IgG secondary antibody employed for this study was sufficiently cross-reactive to detect anti-HEV in Sika deer, two Sika deer were immunized with HEV antigen and an ELISA was standardized with pre-immunization and post-immunization sera and filter eluates from these animals.…”

Section: Discussionmentioning

confidence: 99%

Using improved technology for filter paper-based blood collection to survey wild Sika deer for antibodies to hepatitis E virus

Zimmerman

Engle

et al. 2007

Journal of Virological Methods

Self Cite

View full text Add to dashboard Cite

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 disks from freshly killed deer. The disks 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.

show abstract

“…The same study also questioned the linearity of epitopes, as peptides that map to epitopes were not recognized by the neutralizing monoclonal antibodies. More substantial evidence for the nonlinearity of epitopes was presented for the capsid protein of mammalian HEV (Emerson et al, 2006;Schofield et al, 2003;Zhou et al, 2004). Both investigated isolates display several non-silent mutations within this region (Fig.…”

Section: Sequence Comparison and Identification Of Major Genetic Diffmentioning

confidence: 99%

Sequence analysis and comparison of avian hepatitis E viruses from Australia and Europe indicate the existence of different genotypes

Bilić

Jaskulska

Basic

et al. 2009

Journal of General Virology

108

106

View full text Add to dashboard Cite

Avian hepevirus infections were detected in chickens suffering from big liver and spleen disease or hepatitis–splenomegaly syndrome in Australia, the USA and Europe. Available data indicate their genetic relationship to mammalian hepatitis E virus (HEV). In the present study, the near-complete genomic sequences of an Australian and a European isolate of avian hepatitis E virus (avian HEV) are reported for the first time. Furthermore, the phylogenetic relationship to other avian HEVs is determined. Sequence analyses of these isolates identified major genetic differences among avian HEVs. Most of them are located within the open reading frame (ORF)1 region, although only a few lie within conserved motifs of predicted domains. Non-silent mutations in the ORF2 region suggest the presence of potentially different epitopes among avian HEV isolates. Finally, phylogenetic analysis confirmed the distant relationship to mammalian HEV and additionally suggested that the avian HEVs can be separated into three different genotypes: 1 (Australia), 2 (USA) and 3 (Europe), indicating a geographical distribution pattern.

show abstract

Putative neutralization epitopes and broad cross-genotype neutralization of Hepatitis E virus confirmed by a quantitative cell-culture assay

Cited by 114 publications

References 19 publications

Mutations within Potential Glycosylation Sites in the Capsid Protein of Hepatitis E Virus Prevent the Formation of Infectious Virus Particles

Mutations within Potential Glycosylation Sites in the Capsid Protein of Hepatitis E Virus Prevent the Formation of Infectious Virus Particles

Using improved technology for filter paper-based blood collection to survey wild Sika deer for antibodies to hepatitis E virus

Sequence analysis and comparison of avian hepatitis E viruses from Australia and Europe indicate the existence of different genotypes

Contact Info

Product

Resources

About