Anthony G. Coulepis

Immunoglobulin M antibodies against hepatitis B core antigen in patients with chronic hepatitis B infection

Summary A batch of sera obtained from subjects with acute hepatitis B virus (HBV) infection, chronic carriers of hepatitis B surface antigen (HBsAg) who were either asymptomatic or who had chronic active hepatitis, and 32 sera from patients with HBsAg negative chronic active hepatitis were examined for the presence of antibodies against hepatitis B core antigen (anti‐HBc) by radioimmunoassay (RIA). Sera containing anti‐HBc were fractionated on sucrose density gradients to separate immunoglobulin M (IgM) and the titre of anti‐HBc IgM was determined. In patients with acute HBV infection, anti‐HBc IgM was detected during the acute phase of the illness with titres ranging from 1:128 to 1:4,096 (geometric mean titre 1:709). The titre of anti‐HBc IgM fell rapidly over the following months and in most patients persisted at low levels for several years. Anti‐HBc IgM was also detected in subjects with chronic HBV infection but with significantly lower titres. In asymptomatic carriers, anti‐HBc IgM titres ranged from 1:4 to 1:32 (geometric mean titre 1:12), whilst carriers with chronic active hepatitis had titres ranging from 1:4 to 1:128 (geometric mean titre 1:35). By using a standardized assay procedure, the titre of anti‐HBc IgM in a patients serum may be of value in differentiating between acute and chronic HBV infection.

Taxonomic Classification of Hepatitis A Virus

Sufficient data have accumulated to permit the ICTV Ad Hoc Study Group on the Taxonomy of Hepatitis Viruses to recognize hepatitis A virus as a picornavirus. Within the family Picornaviridae, hepatitis A virus closely resembles members of the genus Enterovirus.

Taxonomic classification of human hepatitis B virus

Sufficient data have accumulated to permit the ICTV Study Group on the Nomenclature of Hepatitis Viruses to recognize human hepatitis B virus as a member of a unique group of viruses and to classify it, together with a number of related animal viruses, into a new family called the Hepadnaviridae. Over the past decade, the International Committee on Taxonomy of Viruses (ICTV) has been active in the development of a classification system for viruses. The majority of viruses infecting vertebrate hosts have been classified into families and genera on the recommendations of the Vertebrate Virus Subcommittee (VVSC). In June 1980, the VVSC authorized the formation of an ad hoc Study Group on the Nomenclature of Hepatitis Viruses under the Chairmanship of Dr. Ian D. Gust. This paper represents the first report of the Study Group on the Taxonomic Classification of Human Hepatitis B Virus.

Biophysical and biochemical characterization of hepatitis A virus.

Biophysical and biochemical analysis of hepatitis A virus has shown it to be a 27- to 32-nm icosahedral particle with 32 capsomers. The mature virion has a buoyant density of 1.33-1.34 g/cm3, a sedimentation coefficient of 156-160S, and is composed of four polypeptides with molecular weights of 30,000-33,000 (VP1), 24,000-27,000 (VP2), 21,000-23,000 (VP3), and 7,000-14,000 (VP4). The genome of hepatitis A virus consists of a single piece of single-stranded RNA which sediments at 32-35S and has a buoyant density of 1.64 g/cm3. The molecular weight of RNA is 2.25 x 10(6) when measured under nondenaturing conditions and 2.8 x 10(6) when measured under fully denaturing conditions. The genome contains a 40-80 nucleotide sequence of polyadenylic and is capable of infecting cell cultures. These findings, together with the observation that the virion is stable at pH 3.0 and resistant to ether and a temperature of 60 degrees for 1 h, indicate that hepatitis A virus should now be classified as an Enterovirus within the family Picornaviridae.

The polypeptides of hepatitis A virus.

Hepatitis A virus was purified from fecal specimens obtained from 3 patients with naturally acquired hepatitis A, by a process of differential centrifugation, chloroform extraction, column chromatography, and isopycnic ultracentrifugation. Analysis of purified virus by discontinuous SDS-PAGE revealed three major polypeptides with molecular weights of 34,000, 25,500, and 23,000 daltons. These polypeptides appear to be specific for hepatitis A virus and have similar molecular weights to three of the four major polypeptides reported for members of the genus Enterovirus within the family Picornaviridae.

Cellular changes associated with persistent hepatitis A infectionin vitro

SummaryThe rate of division, morphology and ultrastructure of BSC-1 cells, persistently infected with hepatitis A virus (HAV), were compared with uninfected cells for 60 days after splitting of the cells. Both control and infected cells showed a biphasic growth pattern marked firstly by increasing cell density and high mitotic rate (exponential phase) and then high constant cell density and little mitosis (stationary phase). Immunoperoxidase studies showed that hepatitis A antigen (HAAg) appeared as cytoplasmic granules approximately one third of the way through the exponential phase in infected cells. The percentage of cells with HAAg rose until the early stationary phase when virtually all cells contained antigen. Radioimmunoassay demonstrated an increase in HAAg per cell in the stationary phase. Radioimmunofocus assay and immune electron microscopy confirmed the presence of HAV in infected cells in the stationary phase. Thin sectioning electron microscopy showed cytoplasmic annulate lamellae in infected cells of both phases but not in control cells.

Purification of hepatitis A virus from human feces.

Hepatitis A virus was purified fecal specimens obtained from 2 patients with naturally acquired hepatitis A. The purification procedure involved differential centrifugation, organic solvent extraction, agarose gel filtration, ion-exchange chromatography, and isopycnic ultracentrifugation in cesium chloride. Using immune electron microscopy and discontinuous SDS-PAGE, this procedure was found to be effective in removing extraneous material from hepatitis A virus. There was significant recovery of virus as judged by immune electron microscopy and solid-phase radioimmunoassay. Using this protocol, it has been possible to obtain virus preparations of sufficient purity and high enough titer to enable biochemical studies to proceed.

Annulate lamellae and lytic HAV infection in vitro

The aim of this study was to examine the relationship between viral infection and annulate lamellae (AL) production by using quantitative and qualitative electron microscopy to document the size and numbers of AL in BS-C-1 cells infected with a lytic strain of hepatitis A virus (HAV). The progress of the HAV infection was found to occur in two phases. In phase 1, cell proliferation and cell death were roughly the same as that of the mock infected control, but there was an increase with time in the amount of hepatitis A antigen in the infected cells. In phase 2 cell division was minimal and cell death became manifest. AL were detected in both infected and control cells. Quantitative analysis indicated that the average number of AL was greater in infected cells compared to that in control cells in phase 1; in infected cells there were greater numbers of AL in phase 1 than in phase 2; the average number of membraneous leaves/AL was greater in infected cells than in control cells. Quantitative analysis also indicated that AL were very rare, with only about three AL per entire control cell and eight AL per entire infected cell. The study clearly establishes that viral infection can stimulate AL production. The data suggest stimulation of AL production in the virus infected cells was linked to the synthesis of viral antigen. Ultrastructural observations indicated that AL could be derived from either the rough endoplasmic reticulum or the nuclear membrane.

Detection of delta infection using reagents obtained from the serum of patients infected with HBV

A microtitre solid-phase radioimmunoassay (SPRIA) was developed for the detection of delta antigen (delta Ag) and antibody (anti-delta) using sera from subjects who had been infected with this agent as the source of antigen and antibody. The assay was compared with reference tests, which use delta antigen extracted from liver tissue obtained at autopsy, and found to be equally sensitive and specific.

Restrictive events in the replication of hepatitis A virus in vitro.

Hepatitis A virus was purified from the feces of 2 patients with unrelated, naturally acquired infections and was inoculated into FRhK-4 cells. Analysis of protein synthesis by double-label coelectrophoresis and subtraction allowed the resolution of virus-specific proteins synthesized during infection. In FRhK-4 cells the two strains of virus studied produced markedly different profiles of virus-specified proteins, with an accumulation of high-molecular-weight proteins for strain HM790 relative to strain HM175, suggesting a level of restriction in the processing of the viral polyprotein.