Hepatitis A virus proteins.

The RNA genome of hepatitis A virus (HAV) shares common characteristics of the picornavirus family. However, the nucleotide or amino acid sequences are distantly related with other members of the family. Like other picornaviruses, HAV proteins are cleaved from a large polyprotein (PO), but the processing and some products are quite different. The 3C protein is the sole processing enzyme, and the primary cleavage takes place at the 2A/2B site. Several VP1-2A sites are proposed. In some strains, the intermediate VP1-2A polypeptides are assembled in the virion. The VP4 is very small and not detected in the mature virion. Some mutations in 2B, 2C and 3A proteins are identified to enhance viral replication or to induce cytopathogenic effects in the viruses adapted to cell cultures.

The latest seroepidemiological pattern of hepatitis A in Japan.

Age-specific prevalence of anti-hepatitis A virus antibody (anti-HAV) was surveyed with 2,708 sera collected in 1994 in various areas of Japan. By age-group analyses, we found strong association of anti-HAV antibody with higher age group. The prevalence ratios of antibody in the groups of 30-34, 35-39, 40-44, 45-49, 50-54, 55-59, 60-64 and 65 years or older were 0, 4.2, 22.0, 44.8, 57.6, 76.4, 84.5 and 91.4%, respectively. Geometric mean titers of anti-HAV antibody in the positive age groups were approximately 6,000 mIU/ml. The seropositives among older population were ascribed to the infections more than 40 years ago and the high anti-HAV titers have been maintained since that time. In Japan, people younger than 40 years of age are extremely risky to HAV infection, since 99% have no antibody. Those in forties are also risky since two-thirds of them are seronegative. In Japan, an inactivated vaccine was licensed in 1994. Vaccination may be recommended for such high-risk groups as travelers going to endemic areas, patients who have received blood product medication and child-care staffs.

Quantitation of group-specific a antigen in hepatitis B vaccines by anti-HBs/a monoclonal antibody.

Balb/c mice were immunized with aluminium hydroxide [alum, Al (OH)3]-adjuvanted hepatitis B (HB) vaccines of subtypes adr, ayw or adw. Spleen cells from the immune animals were fused with SP2/O cells. Eight hybridoma clones producing antibodies specific for HB surface antigen (HBsAg) were selected. Monoclonal antibodies (mAbs) of four clones were specific for group-specific antigen/a, and the other of four clones were specific for subtype antigen/d, y, r, or w. The anti-HBs/a mAbs were classified into three non-competitive groups. Quantitation of group-specific determinant a of HBsAg (HBsAg/a) was performed by sandwich enzyme-linked immunosorbent assay (ELISA), in which a solid phase of anti-HBs guinea-pig polyclonal antibodies (pAb), the HBsAg for testing, anti-HBs/a mouse mAb and horseradish peroxidase (HRP)-conjugated anti-mouse IgG were used. The unadsorbed HBsAg was used to establish the standard curve of HBsAg/a. The lower detection limits were 0.5 to 1 ng/ml of HBsAg. Methods of solubilization of alum were investigated to quantify HBsAg/a in adsorbed HB vaccines. The recovery rate was more than 60% if vaccines were prediluted. The recovery of HBsAg/a in HB vaccines produced by the same manufacturer showed the similar recovery rate, and the contents of HBsAg/a in adsorbed HB vaccines could be estimated by the recovery rate determined for adsorbed HB vaccines.

Identification of a surface glycoprotein on African green monkey kidney cells as a receptor for hepatitis A virus.

Very little is known about the mechanism of cell entry of hepatitis A virus (HAV), and the identification of cellular receptors for this picornavirus has been elusive. Here we describe the molecular cloning of a cellular receptor for HAV using protective monoclonal antibodies raised against susceptible African green monkey kidney (AGMK) cells as probes. Monoclonal antibodies 190/4, 235/4 and 263/6, which reacted against similar epitopes, specifically protected AGMK cells against HAV infection by blocking the binding of HAV. Expression cloning and nucleotide sequence analysis of the cDNA coding for epitope 190/4 revealed a novel mucin-like class I integral membrane glycoprotein of 451 amino acids, the HAV cellular receptor 1 (HAVcr-1). Immunofluorescence analysis indicated that mouse Ltk- cells transfected with HAVcr-1 cDNA gained limited susceptibility to HAV infection, which was blocked by treatment with monoclonal antibody 190/4. Our results demonstrate that the HAVcr-1 polypeptide is an attachment receptor for HAV and strongly suggest that it is also a functional receptor which mediates HAV infection. This report constitutes the first identification of a cellular receptor for HAV.

[Molecular epidemiology of hepatitis A virus].

The genome of hepatitis A virus (HAV) is a linear plus-strand RNA molecule of 7,500 nucleotides and it shares common strategies of construction and function of the picornavirus family. Since it has a unique nucleotide sequence homology, HAV has been classified in the genus of hepatovirus, newly added to the family. Nucleotide sequence of the putative VP1/2A junction area was found variable and a 168 nucleotide portion of the region has been compared with many HAV sequences obtained from all over the world. It was found that HAV strains could be identified and classified into 7 genotypes or 9 subgenotypes. Analyses of the nucleotide sequence homology of this particular region is useful, not only in the study of epidemiology of hepatitis A, but also in the study of the molecular epidemiology of HAV.

[Family-acquired hepatitis A--prevalence of hepatitis A among the family in Aichi Prefecture, 1990].

We studied the transmission of hepatitis A virus (HAV) in 45 families, which members were diagnosed as hepatitis A in 8 hospitals in 1990. Feces and sera from 50 patients and their 126 family members were tested for HAV-specific antigen and IgM antibody by ELISA or polymerase chain reaction (PCR) technology. From the interval of the onset of hepatitis or detection of HAV antigen in feces, HAV transmission was recognized in 11 (24.4%) of 45 families. The transmission was found to be concerned with contacts of the children and that from children to parents was found in 4 families and the reverse in 2. HAV antigen was detected from feces of 4 family members before onset of icterus by ELISA and furthermore, 3 by PCR. It was indicated that these methods would be used to prevent the transmission in a family, day-care centers, or institutions for the mentally retarded.

[Sero-diagnosis for human parvovirus B19 infection by IgM and IgG antibody capture method of enzyme-linked immunosorbent assay--study on an epidemic case of erythema infectiosum].

The IgM and IgG antibody capture methods of the enzyme-linked immunosorbent assay (ELISA) for human parvovirus B19 were performed using Horseradish peroxidase (HRPO)-labeled anti B19 monoclonal antibody. Serially obtained serum samples from one erythema infectiosum (E.I.) patient were examined at once by this methods. The dOD values of the IgM and IgG antibodies decreased on the typical curves according to the course of recovery. In the epidemic case of E.I. among students of one nurse school, 1) The first patients was estimated by comparing the change of dOD values of sera obtained at end of the epidemic and 1.5 months later. 2) In the pre-existing antibody positive persons, the dOD values of IgG antibody did not changed during the epidemic. 3) After the E.I. epidemic, and approximately 30% of the students were remained uninfected.

Seroepidemiology of hepatitis A virus in Japan.

A seroepidemiologic study to detect class-specific antibody against hepatitis A virus (HAV) was made with 831 randomly collected sera (415 in 1973 and 416 in 1984) from healthy Japanese. Competitive-inhibition, IgG, IgA, and IgM anti-HAV enzyme-linked immunosorbent assays (ELISA) were used. Both collections showed a low prevalence of IgG anti-HAV in young age groups and it increased rapidly at middle age and plateued at greater than or equal to 94% prevalence in the older age groups. However, two age groups spanning ages 25-34 demonstrated statistically lower IgG anti-HAV age prevalences in 1984 vs 1973 (P less than 0.001), with an average 10-year prevalence shift. These data suggest that there has been no significant level of HAV infection to alter antibody prevalences in Japan from 1973 to 1984. The markedly decreased incidence of HAV infection in Japan has created a presently large and growing population of HAV susceptibles.

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.

Serodiagnosis of type A hepatitis by detection of immunoglobulin M-type antibody to hepatitis A virus.

Serum specimens from 12 patients with type A hepatitis were analyzed for immunoglobulin M-type antibody to hepatitis A virus (IgM anti-HA). A recently developed solid-phase radioimmunoassay kit for IgM anti-HA (HAVAB-M, Abbott Laboratories) and a competitive binding radioimmunoassay kit (HAVAB, Abbott Laboratories) with or without 2-mercaptoethanol treatment, as modified by Yano et al. (Acta Hepatol. Jpn. 21, 704-712, 1980) were used to obtain an M-index. All specimens obtained within 60 days of the onset of illness and specimens from 2 of 4 patients later than 60 days after the onset were positive with the HAVAB-M test. This test gave negative results to sera which were positive for anti-HA by a standard HAVAB test in the following: 3 patients with type B hepatitis; 5 with non-A, non-B hepatitis; 11 healthy adults; and 10 sera strongly positive for rheumatoid factor. The M-index for type A hepatitis in sera within 30 days of the onset (mean value of the M-index, m, = 1.52; standard deviation, SD, = 0.25) was significantly higher than that for non-A hepatitis (m = 1.05; SD = 0.15) and for healthy adults (m = 1.02; SD = 0.10). The simplicity and usefulness of the HAVAB-M test in diagnosis of acute type A hepatitis over those measuring the M-index by HAVAB tests were shown by direct comparison of the results.

Prevalence of antibody to hepatitis A virus in Sri Lanka.

Antibody to hepatitis A virus was studied by immune adherence hemagglutination using 287 serum specimens collected in 1975 by random sampling from healthy individuals living in Colomobo, Sri Lanka. The overall prevalence of antibody to the virus was 76.9% and the prevalence between males and females was approximately the same. The age-specific prevalence of the antibody indicated that hepatitis A in the area is mainly an infection during the early childhood.

A preliminary serologic study of hepatitis A virus infection in Japan.

Ninety-eight acute non-B hepatitis cases recently observed in Japan and household contacts with these cases were subjected to serologic examinations for hepatitis A; 400 serum specimens obtained in 1971 from healthy individuals living in areas near Tokyo and 16 preparations of human immunoglobulin produced in Japan in 1975 and 1976 were examined for antibody to hepatitis A antigen. Hepatitis A virus infection was confirmed in all 25 patients and in 8 of 26 household contacts found in association with non-B hepatitis outbreaks, and also in 11 of 60 sporadic non-B hepatitis patients, but in none of 13 non-B hepatitis patients found in association with blood transfusion. There was no difference between males and females in the prevalence of antibody to hepatitis A antigen among healthy individuals, however, there was a strong relationship to age. Rates of antibody positives were only 2.5% in the groups younger than 20 years of age. An ample amount of antibody to hepatitis A antigen was detected in the preparations of human immunoglobulin. Hepatitis A virus was thus found to be endemic in Japan, but considered not popular during at least these 20 years. Infection with non-A non-B hepatitis virus(es) seems to be common in Japan especially in such cases as sporadic non-B hepatitis or post-transfusion non-B hepatitis.

Hepatitis B e antigen and infectivity of hepatitis B virus.

For confirmation of the difference in the infectivity of hepatitis B surface antigen (HBS Ag)-positive serum according to differences in the e antigen system, four chimpanzees were inoculated with serum positive for hepatitis B e antigen (HBe Ag), and three chimpanzees were inoculated with serum positive for antibody to HBe Ag (anti-HBe). Since the infectivity titrations are not yet completed, the end infectivity titer of each serum is not known. All four chimpanzees given injections of 10(-1), 10(-4), or 10(-8) dilutions of HBe Ag-positive serum developed hepatitis B virus infection, whereas the one chimpanzee injected with undiluted anti-HBe-positive serum became infected, and other chimpanzees injected with diluted anti-HBe-positive sera did not. As judged from the length of the incubation period before appearance of HBS Ag in blood, there seemed to be a remarkable difference in infectivity between the HBe Ag-positive serum and the anti-HBe-positive serum; the former serum was 10(8) times more infectious than the latter.

Hepatitis B antigen-associated deoxyribonucleic acid polymerase activity and e antigen/anti-e system.

Serum samples of 403 asymptomatic blood donors carrying hepatitis B surface antigen (HB5Ag) were concentrated threefold and tested for e antigen and antibody to e antigen (anti-e) by immunodiffusion. Hepatitis B antigen (HBAg)-associated deoxyribonucleic acid (DNA) polymerase activity was specifically determined by the difference in incorporation of [methyl-3H]thymidine 5' -triphosphate into DNA by an aliquot of centrifuged serum samples after it had been treated either with normal rabbit serum or with rabbit antibody to HBSAg. All of 58 serum samples containing e antigen revealed HBAg-associated DNA polymerase activity, whereas none of 96 samples containing anti-e did. In the remaining 249 samples in which neither e antigen nor anti-e was found, 62 showed specific DNA polymerase activity, although at lower levels than the samples containing e antigen.

Purification of hepatitis A antigen from feces and detection of antigen and antibody by immune adherence hemagglutination.

Hepatitis A antigen (HA Ag) was purified from feces collected during acute illness from patients with naturally occurring viral hepatitis, type A. Positive fecal specimens were identified by immune electron microscopy, but for detection of HA Agduring purification immune adherence hemagglutination (IAHA) and microtiter solid-phase radioimmunoassay were used. Isopycnic banding in cesium chloride, rate-zonal separation in sucrose, and preparative zonal electrophoresis were used in various combinations for successive purification, and the purified antigen was successfully used in a test for antibody by IAHA. Seronconversions to HA Ag were demonstrated by IAHA in 20 instances of hepatitis A virus infection, but in none of six cases of type B hepatitis or three cases of post-transfusion hepatitis unrelated to heaptitis A or B viruses, nor in two individuals without hepatitis. In addition, the temporal pattern of antibody development during type A hepatitis was studied in serial sera from an experimentally infected chimpanzee. Antibody titers by IAHA correlated well with antibody ratings determined by immune electron microscopy.