Daniel W. Bradley

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

n Abstractn n We have recently described the cloning of a portion of the hepatitis E virus (HEV) and confirmed its etiologic association with enterically transmitted (waterborne, epidemic) non-A, non-B hepatitis. The virus consists of a single-stranded, positive-sense RNA genome of approximately 7.5 kb, with a polyadenylated 3 end. We now report on the cloning and nucleotide sequencing of an overlapping, contiguous set of cDNA clones representing the entire genome of the HEV Burma strain [HEV(B)]. The largest open reading frame extends approximately 5 kb from the Fend and contains the RNA-directed RNA polymerase and nucleoside triphosphate binding motifs. The second major open reading frame (ORF2) begins 37 by downstream of the first and extends approximately 2 kb to the termination codon present 65 by from the 3 terminal stretch of poly(A) residues. ORF2 contains a consensus signal peptide sequence at its amino terminus and a capsid-like region with a high content of basic amino acids similar to that seen with other virus capsid proteins. A third open reading frame partially overlaps the first and second and encompasses only 369 bp. In addition to the 7.5-kb full-length genomic transcript, two subgenomic polyadenylated messages of approximately 3.7 and 2.0 kb were detected in infected liver using a probe from the 3 third of the genome. The genomic organization of the virus is consistent with the Fend encoding nonstructural and the 3 end encoding the viral structural gene(s). The expression strategy of the virus involves the use of three different open reading frames and at least three different transcripts. HEV was previously determined to be a nonenveloped particle with a diameter of 27–34 nm. These findings on the genetic organization and expression strategy of HEV suggest that it is the prototype human pathogen for a new class of RNA virus or perhaps a separate genus within the Caliciviridae familyn n

Detection of hepatitis C viral sequences in non-A, non-B hepatitis

The role of hepatitis C virus (HCV) in post-transfusion non-A, non-B hepatitis (NANBH) was investigated by analysing clinical samples for both HCV RNA by cDNA/polymerase chain reaction and antibodies against C100-3 by radioimmunoassay. Of fifteen chronic NANBH patients and one patient with chronic cryptogenic liver disease, ten were positive for anti-C100-3 and seven of the ten had viral sequences in their livers. However, two patients negative for anti-C100-3 also had substantial levels of HCV RNA in their livers. In acute post-transfusion NANBH (one surgical patient and two experimentally infected chimpanzees), HCV RNA was detected in the absence of anti-C100-3. In addition, infectious plasma from a seronegative patient with acute post-transfusion NANBH and a seronegative pool of plasma from a chimpanzee with chronic post-transfusion NANBH had high levels of HCV. These findings show that anti-C100-3-positive patients with chronic post-transfusion NANBH are likely to be viraemic; confirm that antibodies to C100-3 are a marker for infectivity; and suggest that the prevalence of HCV infections may be underestimated from the frequency of antibodies to C100-3 alone.

Molecular cloning and sequencing of the mexico isolate of hepatitis E virus (HEV)

Hepatitis E virus (HEV) is the major causative agent of hepatitis E or what was formerly known as enterically transmitted non-A, non-B hepatitis. The disease has a worldwide distribution but occurs principally in developing countries in any of three forms: large epidemics, smaller outbreaks, or sporadic infections. Genetic variation of different HEV strains was previously noted and it will be important to determine the extent to which this variation may pose problems in the diagnosis and treatment of HEV infection. To analyze differences at the genetic level between HEV(Mexico; M) and the previously characterized HEV(Burma; B) and HEV(Pakistan; P) isolates, overlapping cDNAs were cloned from samples obtained from an infected human and an experimentally inoculated cynomolgus macaque. These cDNA clones, representing the nearly complete (7185-bp) genome of HEV(M), confirmed an expression strategy for the virus that involves the use of 3 forward open reading frames (ORFs). The HEV(M) strain has an overall 76 and 77% nucleic acid identity with the HEV(B) strain and HEV(P) strain, respectively; however, the degree of sequence variation was not uniform throughout the viral genome. A hypervariable region was identified in ORF1 that exhibited a 58 and 54% nucleic acid sequence and 13% amino acid similarity with the Burma strain and the Pakistan strain, respectively. A large number of the nucleotide differences occurred at the third codon position, with the deduced amino acid sequences similarity of 83, 93, and 87% between HEV(M) and HEV(B) isolates in ORF1, ORF2, and ORF3, respectively, and with 84, 93, and 87% amino acid identities between HEV(M) and HEV(P) isolates in ORF1, ORF2, and ORF3, respectively. The nucleotide sequences derived from the highly conserved regions of HEV genome will be useful in developing polymerase chain reaction-based tests to confirm the viral infection. Knowledge of the extent of the sequence variation encountered with HEV will not only aid in the future development of diagnostic and vaccine reagents but also further our understanding of how HEV strain variation might impact the pathological outcome of infection.

Posttransfusion Non-A, Non-B Hepatitis: Physicochemical Properties of Two Distinct Agents

Abstract Two separate and distinct episodes of non-At non-B hepatitis were induced in each of two chimpanzees by two inocula: one containing a chloroform-resistant agent and the other containing a chloroform-sensitive agent. Both agents were recovered from liver tissue and plasma obtained from a single chimpanzee during the acute and chronic phases of infection with a factor VIII concentrate, respectively. The chloroform-resistant agent did not cause unique changes in hepatocytes; in contrast, the chloroform-sensitive agent did induce the formation of cytoplasmic tubules, convoluted endoplasmic reticulum, and dense reticular inclusion bodies. The latter changes are similar in character to those induced in infected cells by some enveloped mammalian RNA viruses.

The sequence of hepatitis E virus isolated directly from a single source during an outbreak in China

In this study an IgM antibody-mediated antigen-capture procedure for direct extraction of hepatitis E virus (HEV) RNA from clinical specimens was developed and used with an efficient method for generating viral cDNA that was subsequently sequenced using the dideoxy chain termination method. This is the first time the complete HEV genome has been isolated directly from a single human clinical specimen obtained during an outbreak of enterically transmitted non-A, non-B hepatitis. When the Chinese-derived sequence was compared with the original isolate of Burmese HEV from an experimentally infected cynomolgus macaque, the homology between the two sequences was 94% and 98.5% at the nucleotide and amino acid levels, respectively. The methods we developed for generating and sequencing genomic HEV cDNA dramatically improved the efficiency of cloning the viral genome and should be helpful for continued analysis of this virus as well as other RNA viruses that have proven to be difficult to clone and sequence directly.

Expression of a hepatitis E virus (HEV)-trpE fusion protein containing epitopes recognized by antibodies in sera from human cases and experimentally infected primates

SummaryA 1700 base cDNA fragment coding for the putative structural gene(s) of hepatitis E virus (HEV) was inserted into the pATH 10 expression vector. The fusion protein (C2) expressed by this plasmid was found to contain epitopes recognized by anti-HEV antibodies. C 2 protein was used in a Western blot format to examine its usefulness in detecting anti-HEV antibodies in well documented human cases of HEV and non-human primates infected with HEV. Both IgM and IgG anti-HEV could be detected in our Western blot assay. This Western blot assay was found not to detect antibodies from acute-phase sera from patients with either HAV or HBV. The C 2 protein contains broadly cross-reactive epitopes, and the Western blot assay was able to detect anti-HEV antibodies in patient sera from Asia, Africa, and North America. The optimum serum dilution for the detection of both IgM and IgG was 1:25.

Application of two RNA extraction methods prior to amplification of hepatitis E virus nucleic acid by the polymerase chain reaction

Amplification of the enterically-transmitted non-A, non-B hepatitis virus (HEV) RNA using conventional reverse transcriptase reactions followed by the polymerase chain reaction (PCR) of the cDNA has not been successful. However, after application of two different RNA capture/extraction methods we were able to amplify HEV nucleic acid from clinical samples and specimens from experimentally infected animals. The first procedure, adapted from an immune electron microscopy (IEM) technique, incorporated an immunocapture step with concentration of the virus-antibody complexes by pelleting in a Beckman airfuge. In the second method, glass powder (or size-fractionated silicon dioxide) was used to capture the RNA from its surrounding milieu by adsorption of the nucleic acid to the silicate particles. Since conventional immunoassays for HEV antigen or antibody are not currently available, the use of these RNA extraction methods, coupled with PCR techniques, will be valuable in screening clinical specimens and in further defining the course of disease using animal infectivity studies.

Parenterally transmitted non-A, non-B hepatitis: Virus-specific antibody response patterns in hepatitis C virus-infected chimpanzees

An established chimpanzee model of parenterally-transmitted non-A, non-B hepatitis was used to define virus-specific immune response patterns in acutely and persistently infected animals. Serial bleedings were obtained from 23 chimpanzees that had been experimentally infected with an isolate of hepatitis C virus, originally recovered from contaminated lots of factor VIII (antihemophilic) materials. Sera were assayed for the presence of antihepatitis C virus by a newly developed radioimmunoassay procedure that incorporated recombinant DNA-expressed viral antigen as a reagent. Twenty-one of 23 hepatitis C virus infected animals were shown to acquire antihepatitis C virus, most within 2-8 weeks after the major peak of alanine aminotransferase activity. All chimpanzees with biochemical, electron microscopic, and histological evidence of chronic disease clearly acquired antibody; 14 of 16 animals observed through the acute phase of disease were also shown to acquire antibody. A booster effect or anamnestic response was noted in two chimpanzees (one of which was negative for antihepatitis C virus following the acute phase of disease) after challenge with hepatitis C virus. Antihepatitis C virus was not neutralizing, because some animals with high levels of antibody were also shown to have high titers of circulating hepatitis C virus. The development and maintenance of anti-hepatitis C virus appears to reflect concomitant virus replication and high potential for infectivity.

The agents of non-A, non-B viral hepatitis

n Abstractn n Recent studies have provided physicochemical and electron microscopic evidence for the existence of two distinct agents of posttransfusion non-A, non-B (NANB) hepatitis. One of these agents is chloroform-resistant and is not associated with the formation of unique ultrastructural structures in infected liver. The other agent is CHCl3-sensitive, induces the formation of characteristic hepatocyte cytoplasmic tubules, and interferes with concurrent HAV or HBV infection in experimentally inoculated chimpanzees. The tubule-forming agent (TFA) has also been shown to pass through an 80 nm capillary pore membrane filter, suggesting that it is a small enveloped (or lipid-containing) virus. The TFA can also be recovered from low titer (⩽ 105 infectious doses/ml) chronic-phase chimpanzee plasma by use of a multi-step purification procedure that assumes the agent is a small enveloped RNA virus with an approximate buoyant density of 1.24 g/cm3 and a sedimentation coefficient of 200–280 S. The apparent lack of nucleic acid homology between the NANB-TFA and HBV further suggests that the NANB-TFA is either Togavirus-like or belongs to another or as yet undefined class of RNA or DNA virus.n n

Vitamin C in plasma: A comparative study of the vitamin stabilized with trichloroacetic acid or metaphosphoric acid and the effects of storage at − 70°, − 20°, 4°, and 25° on the stabilized vitamin

Abstract Vitamin C concentration in human plasma was shown to be affected by storage time and temperature as well as by the kind of stabilizer used. Samples were stored for periods of time up to 21 days at temperatures of 25°, 4°, − 20°, and − 70°. Stabilizers employed in this study included trichloroacetic acid (TCA) and metaphosphoric acid (MPA). Plasma vitamin C demonstrated maximum stability in TCA or MPA when stored at − 70°. Other storage conditions tested yielded less accurate vitamin C values.