Hepatitis E virus: identification of type-common epitopes

Abstract

Large epidemic outbreaks of enterically transmitted non-A, non-B viral hepatitis (ET-NANBH) have been documented in developing countries. A molecular clone derived from the causative agent, the hepatitis E virus (HEV), has recently been described (G.R. Reyes, M.A. Purdy, J.P. Kim, K.-C. Luk, L.M. Young, K.E. Fry, and D. Bradley, Science 247:1335-1339, 1990). We now report the isolation, by serologic screening, of two cDNA clones derived from a fecal sample collected during a 1986 outbreak of ET-NANBH in Telixtac, Mexico. The cDNA clones encode epitopes that specifically reacted with acute- and convalescent-phase sera collected during five different ET-NANBH epidemics and represent the initial cloning of the Mexico strain of HEV. Recombinant fusion proteins expressed from these clones were also recognized by antibodies from cynomolgus macaques experimentally infected with HEV. The cDNA clones were shown to be derived from HEV by their specific hybridization to the previously recognized full-length genomic RNA transcript of approximately 7.5 kb. In addition, however, subgenomic polyadenylated transcripts of approximately 2.0 and approximately 3.7 kb were also identified in HEV-infected cynomolgus monkey liver. Sequences homologous to the epitope clones were isolated from the Burma strain of the virus, and these demonstrated reactivity comparable to that seen with the Mexico strain epitopes. When compared with the available full-length sequence of the Burma strain of HEV, it was discovered that the cDNA clones were encoded in different open reading frames (ORFs). The comparison between Mexico and Burma HEV strains indicated amino acid homologies of 90.5 and 73.5% for these epitope-encoding clones derived from ORF2 and ORF3, respectively. The identification of these clones not only has provided insight into the expression strategy of HEV but has also resulted in a source of recombinant protein useful in the diagnosis of HEV-induced hepatitis.

Hepatitis E virus (HEV): Molecular cloning and sequencing of the full-length viral genome

“…It is of note that ET1 .l did not originally identify these subgenomic messages (Reyes et al, 1990) and other Northern blot studies using probes located 5' to ET1 .l also did not hybridize to these viral-specific transcripts (data not shown). These same subgenomic messages were identified in poly(A)-selected R#A from HEV(M)-infected cyno liver when the epitope-encoding clones were used as probes (Yarbough et al, 1991). The ORF2 epitope is located at the extreme 3' end of that reading frame (Yarbough eZ a/., 1991), therefore indicating that these messages may be co-terminal with the 3' end of the viral genomic transcript.…”

“…The utilization of ORF2 was substantiated by the independent isolation of a cDNA clone by immunoscreening of a hgtl 1 cDNA expression library made from the HEV (Mexico) isolate (Yarbough et a/., 1991). That xgt 11 clone mapped to the 3' end of ORF2.…”

“…The fact that sera from acutely infected humans and animals detected HEV antigens encoded by ORF2 and ORF3 confirmed their expression and established that the virus utilized all three positive-polarity reading frames. The presence of a consensus s&al sequence motif in ORF2, together with the immunodominant seroreactivity of an identified epitupe (Yarbough et a/., 1991) suggested that the viral structural protein(s) were encoded by this region of the genome.…”

Acute sporadic hepatitis E in children: diagnostic relevance of specific immunoglobulin M and immunoglobulin G compared with nested reverse transcriptase PCR

“…At least four major genotypes of HEV have been reported worldwide: genotype 1 (found primarily in Asian countries), genotype 2 (isolated from a single outbreak in Mexico), genotype 3 (identified in swine and humans in the United States and many other countries), and genotype 4 (identified in humans, swine and other animals in Asia) (Jothikumar et al, 2006). Recombinant peptides expressed from ORF2 and ORF3 of the Mexican strain of HEV were shown to react with sera collected from outbreaks of hepatitis E in Pakistan, Burma, Borneo, the USSR, and Somalia (Yarbough et al, 1991). The existence of these so-called type-common epitopes provided the basis for the development of immunoassays that broadly react with antibodies against different HEV strains (Yu et al, 2003).…”

Full coding hepatitis E virus genotype 3 genome amplification method

“…This protein has three linear domains: the S domain, which forms the capsid, and the M and P domains, which are involved in the interaction of the virus with the host cell (Perez-Gracia et al, 2014). It has been described an homologous to alphavirus region (46 AAs long) of unknown function (Yarbough et al, 1991;Purdy et al, 1993;Rodriguez-Frias et al, 2012). The ORF3 protein probably regulates the host cell environment through its interaction with various intracellular pathways.…”