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.

Cellular changes associated with persistent hepatitis A infection in vitro.

The 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.

Molecular cloning of cDNA from hepatitis A virus strain HM-175 after multiple passages in vivo and in vitro.

Hepatitis A virus (HAV) strain HM-175 was passaged six times in marmosets, 59 times in cell culture and purified from infected cell culture supernatant fluid. The viral RNA was extracted, copied into cDNA and the cDNA:RNA hybrids were cloned into the PstI site of plasmid pBR322. The cDNA clones were authenticated by hybridization to RNA extracted from HAV-infected cells and clones representing the 3' end of the genome were identified using a previously authenticated cDNA clone. The clones represented all but 29 bases of the HAV genome. They were compared to HAV strain HM-175 cDNA cloned from viral RNA after three passages in marmosets on the basis of restriction endonuclease mapping and DNA sequencing. No differences were found in either the presence or absence of restriction endonuclease sites using 33 different restriction enzymes. Sequencing of cDNA representing bases 29 to 1002 of the HAV genome revealed eight base changes all of which were within the 5' noncoding region.

Indirect immunofluorescence assay for the detection of hepatitis A virus-specific serum immunoglobulins.

Hepatitis A virus-specific BSC-1 cells were used for the detection of serum immunoglobulins to hepatitis A virus by indirect immunofluorescence. Of 150 serum samples tested, specific immunoglobulin M was detected only in patients with serologically confirmed acute hepatitis A, while specific immunoglobulin G was detected in patients with acute or past clinical hepatitis A as well as many patients with no known history of hepatitis.

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.

Detection of hepatitis A virus and antibody by solid-phase radioimmunoassay and enzyme-linked immunosorbent assay with monoclonal antibodies.

Monoclonal antibodies (K3-2F2 and K3-4C8) raised against hepatitis A virus were used to develop a solid-phase radioimmunoassay and enzyme-linked immunosorbent assay for the detection of hepatitis A virus and antibody. Assays with this pair of monoclonal antibodies were compared in parallel with similarly constructed solid-phase radioimmunoassays and enzyme-linked immunosorbent assays in which human polyclonal serum was used. The monoclonal antibody assay proved to be more sensitive for the detection of hepatitis A virus from fecal specimens as well as for anti-hepatitis A virus immunoglobulin G (IgG) and IgM in sera.

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.

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.

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

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 patient's serum may be of value in differentiating between acute and chronic HBV infection.

Monoclonal antibodies against hepatitis A virus.

Three monoclonal antibodies (K2-4F2, K3-2F2, and K3-4C8) of the immunoglobulin G2a class were raised against hepatitis A virus. The specificity of these antibodies was confirmed by immune electron microscopy, solid-phase radioimmunoassay, and in vitro neutralization in cell culture. Binding studies suggested that they all recognize closely related antigenic determinants. These monoclonal antibodies should prove to be of great value as diagnostic and research reagents.

A cross sectional study of markers of hepatitis B infection in Niue.

To determine whether the island of Niue would be a suitable location to evaluate the efficacy of hepatitis B vaccine, the prevalence of hepatitis B infection in the adult population was studied. Sera were collected from 1147 of 1244 residents above the age of 20 years, and tested for the presence of hepatitis B surface antigen (HBsAg) and specific antibody to the surface and core antigens (anti-HBs and anti-HBc) by solid-phase radioimmunoassay (SPRIA). Hepatitis B was found to be hyperendemic; 11.9% of those tested were found to be carriers of HBsAg and an additional 84% had detectable levels of anti-HBs or anti-HBc indicative of current or past infection. In this population HBV infection appears to occur early in life as the peak prevalence of serological markers was found in young adults. The almost universal infection of the population, their high rate of compliance with the study and the relatively high birth rate indicate that Niue would be a suitable location to evaluate methods of preventing hepatitis B infection.

Characterization of a precipitating antigen detected in the serum of patients with viral hepatitis.

During a search for the aetiological agent of non-A non-B hepatitis, a precipitating antigen was detected in the sera of some patients during the acute phase of their illness. The antigen was detected by agar gel diffusion using antibody from convalescent sera obtained from patients with non-A non-B hepatitis, and from haemophiliac sera. The antigen was usually detected early in the patient's illness, disappearing as liver function tests returned to normal. In some patients specific antibody appeared during the convalescent phase of the disease. The antigen does not appear to be specific for non-A non-B hepatitis, as it could be detected with similar frequency in patients with hepatitis A or hepatitis B and some patients with other liver disorders. Biochemical and biophysical studies suggest that the antigen is probably an abnormal lipoprotein produced as a result of acute liver damage.

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.

Seroepidemiology of infection with hepatitis B virus in Fiji.

A batch of 984 sera obtained from a stratified sample of Melanesians and Indians living in rural and urban areas of Fiji in 1981 were for hepatitis B surface antigen (HBsAg) and antibody to hepatitis B core antigen (anti-HBc) by solid phase radioimmunoassay. The prevalence of hepatitis B infection (as measured by the sum of HBsAg and anti-HBc frequencies of HBsAg negative sera in the two groups) was 81.5% and 17.9%, respectively. No major differences were detected between urban and rural populations. While hepatitis B virus is endemic in Melanesians and Indians, the epidemiology of the infection shows certain differences. Among Melanesians, infection appears to be acquired early in life and peak prevalence of serologic markers of infection occurs during the second decade. Among the Indian population, the prevalence of markers increases steadily with age, presumably as a result of continuous exposure and infection throughout life. the high prevalence of infection and carriers among Melanesians is consistent with previous observations among Pacific populations. The lower prevalence of infection among Indians is remarkable, since they constitute almost half of the total population and live under similar conditions. Since the two populations remain largely separate in terms of housing and schooling, and intermarriage is uncommon, it is no possible to determine whether these differences merely represent different degrees of exposure to the virus or are the reflection of differences in susceptibility or response to infection.

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.

Detection of markers of hepatitis B infection in serum dried on to filter-paper: an application to field studies.

In an attempt to find a cheap, reliable, and convenient method for the transportation and storage of serum specimens during seroepidemiological surveys, a technique in which serum was dried on to pieces of filter paper was developed and evaluated. For the evaluation, a total of 382 sera were selected from the extensive serum collection held by the WHO Collaborating Centre for Virus Reference and Research at Fairfield Hospital, Australia. These sera were dried on to pieces of filter-paper, stored at different temperatures and then tested for the presence of the various markers of infection with hepatitis B virus by solid-phase radioimmunoassay. The results were in complete agreement with those obtained on whole serum specimens. In addition, storage at 4 degrees C, room temperature (22 degrees C), or 37 degrees C for up to 30 days did not alter the sensitivity of the test. This technique may be useful in field surveys, not only for the detection of hepatitis B infection, but also in the study of other diseases and metabolic disorders.

Restricted replication of human hepatitis A virus in cell culture: intracellular biochemical studies.

When hepatitis A virus was inoculated into Vero cells, virus-specified protein and RNA synthesis was detected. Production of viral protein was detected by electrophoretic analysis in polyacrylamide gels by using a double-label coelectrophoresis and subtraction method which eliminated the contribution of host protein components from the profiles of virus-infected cytoplasm. Eleven virus-specified proteins were detected in the net electrophoretic profiles of hepatitis A virus-infected cells. The molecular weights of these proteins were very similar to those detected in cells infected with poliovirus type 1. Virus-specified protein synthesis could be detected at 3 to 6 h and continued for at least 48 h postinfection, but no significant effect on host-cell macromolecular synthesis was observed. Limited viral RNA replication occurred between 2 and 6 h postinfection. The genomic RNA of hepatitis A virus was extracted and shown to be capable of infecting cells and inducing the same set of proteins as intact virus, indicating that the RNA genome is positive stranded. Progeny virus was never detected in the supernatant fluids of infected cell cultures, and the cells showed no observable cytopathology, even though hepatitis A virus-specific proteins and antigens were being produced. The nature of the defect in the replicative cycle of hepatitis A virus in this system remains unknown.