John Monjardino

Hypervariable region of hepatitis C virus envelope glycoprotein (E2/NS1) in an agammaglobulinemic patient

In an agammaglobulinemic patient with chronic hepatitis C, a previously identified hypervariable region of the major envelope glycoprotein remained unchanged for 2.5 years. Serum-derived RNA amplified by reverse transcription-polymerase chain reaction was cloned in a bacterial vector, and a minimum of three independent clones were sequenced by dideoxy chain termination reaction. Comparison of consensus sequences from three different time points during the chronic phase of infection showed absolute homology at both amino acid and nucleotide levels. This finding provides support for the role of antibody selection in generating genetic variation and viral persistence; also, it is consistent with the hypothesis that an epitope within this region is the site of virus neutralization. The observations show that the hepatitis seen in hepatitis C virus infection is not dependent on the humoral immune response.

Cloning and sequencing of RNA of hepatitis delta virus isolated from human serum

The RNA of hepatitis delta virus (HDV) 1682 nucleotides long, has been cloned from a human serum isolate. Comparison with the three complete published sequences shows that a region of the HDV genome, between positions 620 and 1350, which contains sequences involved in replication and possibly pathogenicity, is highly conserved.

Cloning and expression of an immunodominant region of the hepatitis delta antigen.

A cDNA clone prepared from hepatitis delta virus (HDV) RNA extracted from human serum was subcloned in the bacterial expression vector pPL31 to produce a fusion protein consisting of the first 98 amino acids of MS2 polymerase and of 64 amino acids from near the N-terminal region of hepatitis delta antigen (HDAg). The fusion protein was shown to be related to HDAg by a commercial sandwich immunoassay (Abbott) and immunoblotting with human anti-HDAg serum. Antiserum against the fusion protein was raised in rabbits and used to identify HDAg extracted from the serum and liver of an HDV-infected woodchuck and chimpanzee and from the serum of an HDV-infected human, by immunoblotting and immunohistology. A single, major polypeptide of 24K was detected in both serum and liver extracts, with a minor polypeptide of 26K sometimes present. Liver extracts also contained lower Mr polypeptides thought to be degradation products, the major species being 22.5K. The same pattern of staining was obtained with human anti-HDAg serum. Absorption experiments with the expressed protein and cross-competition experiments with the rabbit antiserum suggest that a major immunodominant region of HDAg is present near the N-terminal end of the antigen, between positions 1561 and 1368 on the genome. Both the expressed protein and rabbit antiserum were shown to be good diagnostic reagents.

Sequence, expression and reconstitution of an HCV genome from a British isolate derived from a single blood donation

Morphological analysis of hepatitis C virus and development of antiviral drugs to eradicate this agent have been seriously hampered by the low viraemias observed during natural infection and the unavailability of a cell culture system for virus propagation. Recently a low‐grade hepatitis has been reported in chimpanzees after intrahepatic transfection of full‐length synthetic HCV RNA and successful infections shown to be critically dependent on the integrity and genetic homogeneity of the reconstituted clone. In this study we describe and characterize a full HCV RNA sequence derived from a case of chronic sporadic hepatitis. The genotype was shown to be 1a with a low level of intraclonal sequence heterogeneity, and processing of both structural and nonstructural proteins has been documented. The assembly of the full genome has also been achieved.

Purification of woodchuck hepatitis surface antigen using a monoclonal antibody raised against the antigen.

Woodchuck hepatitis virus (WHV) is closely related to the human hepatitis B virus (HBV) and infection of woodchucks with WHV creates a useful model for studies of immunity, pathogenesis and therapy of HBV infection. To increase the usefulness of this model, monoclonal antibodies were raised to woodchuck hepatitis surface antigen (WHsAg) and one of these antibodies was used to purify the antigen by affinity chromatography from serum, a simpler and quicker method of purification than the current ultracentrifugation methods. The bands found by SDS-polyacrylamide gel electophoresis of WHsAg were the major 25 and 29 kilodalton (kDa) bands and a triplet of 45, 51 and 55 kDa which are thought to be the glycosylated and unglycosylated middle and large WHsAg. Both the antibody and the antigen are valuable reagents for the study of WHV infection.