Prevalence of hepatitis B and C markers in volunteer blood donors in Crete. A 5-year study.

Greece is a country with an intermediate prevalence of hepatitis B virus (HBV) and hepatitis C virus (HCV) infection. Crete, the third-largest island of the Mediterranean sea, has a different prevalence of viral hepatitis. One-eighth of the total island population, of 550,000, was included in a 5-year study of blood donors from three out of four blood banks, serving three out of four prefectures of the island. Markers for HBV and HCV were studied and evaluated according to geographical area, gender and age of donor. A total of 65219 blood donors were studied. A greater number of males than females were hepatitis B surface antigen (HBsAg) positive (0. 41% vs 0.28%, respectively) with a peak at a younger age for males and older age for females. Males are more frequently exposed to HBV and become carriers more often than females. For HCV, an opposite gender trend was found, females being infected more frequently (0. 49%) than males (0.37%). Statistical differences were found among geographical areas of the island. Hence, Crete is an area of low endemicity for HBsAg in blood donors. The HCV infectivity is more similar to Northern Europe than to other neighbouring countries. Differences in geographical distribution within the island and during different years indicate the need for extended epidemiological surveys for valid results.

Prevalence of hepatitis A, B, and C markers in school children of a rural area of Crete, Greece.

OBJECTIVE: To determine the prevalence of hepatitis A, B, and C markers in children who were attending junior and senior high schools in a high risk area in rural Crete, Greece. METHODS: Three-hundred and thirty-four children who attended the three junior schools and one senior high school in the Agios Vassilios province of Southern Crete were invited to participate in the study. Three hundred and four of them were tested for hepatitis A, B, and C markers. Hepatitis B (HBV) markers (HBsAg and anti-HBc) as well as hepatitis A (anti-HAV) and hepatitis (anti-HCV) antibodies were tested with commercial enzyme-linked immunosorbent assay kits. RESULTS: Six of the 304 children (1.97%) were found to be positive for anti-HAV, 1 (0.33%) to HBsAg, 7 (2.30%) to anti-HBc and none were found positive for anti-HCV. No significant differences were seen between the prevalence of anti-HAV antibodies in males (2%) and females (1.95%), and of anti-HBc antibodies in males (3.33%) and females (1.30%). CONCLUSIONS: The very low prevalence of anti-HAV is obviously due to the improved conditions of hygiene and it raises the question of the possible emergence of this disease at an older age and therefore appropriate preventative strategies should be considered. The low endemicity of hepatitis B in Crete in contrast to other areas of Greece also calls for a vaccination policy probably during adolescence. The absence of hepatitis C markers in the children in contrast to the observed higher prevalence of HCV-infected people in the adult population in the same rural area raises questions regarding possible sources of transmission of hepatitis C during the preceding years.

Current prevalence of hepatitis A, B and C in a well-defined area in rural Crete, Greece.

A seroepidemiological study was carried out in a geographically well-defined area in rural Crete in order to determine the prevalence of A, B and C hepatitis markers in the local population. Serum samples were obtained from 257 subjects (94 males, 163 females), aged 15 years and over, who visited the primary health care services of the Spili Health Centre between July 1993 and March 1994, and from 164 subjects (83 males, 81 females) randomly selected from households in three neighbouring villages of the study area. In samples obtained from the Spili Health Centre, antibodies to hepatitis A virus (anti-HAV) were detected in 234/244 (95.9%) subjects, antibodies to hepatitis B virus core antigen (HBcAb) were detected in 63/257 (24.5%) subjects and antibodies to hepatitis C virus (anti-HCV) were detected in 28/257 (10.9%) subjects. The corresponding figures for those randomly selected from the villages were 135/154 (87.7%), 16/164 (9.8%) and 5/164 (3%) respectively. Hepatitis B surface antigen (HBsAg) was positive in three (1.2%) subjects from the first group, while none of those recruited from the villages were positive for HBsAg. Interestingly, hepatitis markers were closely associated with age. No subjects under the age of 15 years showed evidence of prior hepatitis A infection and approximately 20% of those between 15 and 44 years of age were also negative. By contrast, practically all subjects older than 44 years were anti-HAV positive. Similarly, the majority of all those who were anti-HCV positive were older subjects. Seroepidemiology of hepatitis in this well-defined population seems to be different from other parts of Greece, at least for hepatitis B and C viruses. There is a very low prevalence of HBsAg and a very high incidence of anti-HCV. Low exposure to HAV, as found in other parts of the country, was also found in the younger generation in this rural area of Crete.

Studies on mechanism of action of glycyrrhizin against hepatitis A virus replication in vitro.

Glycyrrhizin (GL) achieved a concentration-dependent inhibition of the replication of hepatitis A virus (HAV) in PLC/PRF/5 cells. GL has been shown to inhibit an early stage of the HAV replication. GL was not virucidal and had no measurable effect on the adsorption of [3H]uridine-labelled virions to cells. GL inhibited HAV penetration of the plasma membrane as measured by the amount of infective virus no longer neutralizable by specific antibody over time.

Inhibition of hepatitis A virus replication in vitro by antiviral compounds.

Forty antiviral compounds were screened for inhibitory effect on hepatitis A virus (HAV) antigen expression in the human hepatoma cell line PLC/PRF/5. Ribavirin, amantadine, glycyrrhizin, and pyrazofurin were selected in this screening test and were studied further. The selectivity indices of these four compounds, calculated as the ratio of 50% cytotoxic dose (determined by the trypan blue exclusion and by inhibition of [3H] leucine incorporation) to the 50% effective dose (determined by the viral antigen expression), were 4.6 and 3.0 with ribavirin, 5.3 and 5.9 with amantadine, 15.2 and 16.9 with glycyrrhizin, and 45.4 and 74.6 with pyrazofurin. All four compounds resulted in concentration-dependent reductions of HAV antigen expression and HAV infectivity. Ribavirin, amantadine, pyrazofurin, and glycyrrhizin emerged, from the present study, as promising candidates for chemotherapy of acute hepatitis A.

Inhibitory effects of atropine, protamine, and their combination on hepatitis A virus replication in PLC/PRF/5 cells.

Atropine, protamine, and the combination of these drugs were tested for their effects on hepatitis A virus (HAV) replication in cell culture. PLC/PRF/5 hepatoma cells were treated simultaneously with nontoxic concentrations of these drugs and inoculated with HAV strain CF 53 at several multiplicities of infection. The yields of infectious HAV after 4 and 15 days were markedly reduced by each drug, especially at the lowest multiplicity of infection. The activities of each drug were irreversible. Atropine was active when it was added as late as 2 h after inoculation with HAV. An anti-HAV effect was also induced by treating cells with atropine prior to inoculation. Protamine was active as late as 6 h postinoculation. The combination of atropine and protamine resulted in an enhanced anti-HAV effect. We concluded that these drugs affect undetermined, but separate, steps in the HAV replication cycle.

Monoclonal antibodies against an immunodominant and neutralizing epitope on hepatitis A virus antigen.

Two monoclonal antibodies (813 and 10.09) were raised against hepatitis A virus (HAV). They recognize an immunodominant epitope and a neutralizing site on HAV.

Long-term survival of hepatitis A virus and poliovirus type 1 in mineral water.

The survival in mineral water of hepatitis A virus (HAV) and poliovirus type 1 was compared, under controlled experimental conditions, at 4 degrees C and room temperature. Viral infectivity titers were determined by cell culture titration, while HAV antigenicity was monitored by radioimmunoassay-endpoint titration. Both viruses persisted longest at 4 degrees C. At this temperature, after 1 year of exposure, the inactivation of either HAV or poliovirus type 1 was not important. At room temperature, poliovirus type 1 was not detected after 300 days, whereas HAV was still infectious. For both temperatures, the computed regression coefficients of best-fit lines for inactivation rates for the two viruses were significantly different. The survival of HAV was also studied at 4 degrees C and room temperature in mineral water with 5- and 50-micrograms/ml protein concentrations (i.e., purity of the virus suspension) for 120 days. As shown by a comparison of the regression coefficients for the inactivation rates, the stability of HAV in mineral water depends on protein concentration and temperature. Radioimmunoassay-endpoint titration results showed inactivation patterns similar to those of cell culture titration, with the most significant reduction in HAV antigenicity at room temperature. At the two temperatures, the infectivity of HAV declined at a faster rate than the antigenicity.

Continuous production of hepatitis A virus in PLC/PRF/5 cell cultures: use of antigen for serology.

The strain CF53 of hepatitis A virus (HAV) previously adapted to growth in PLC/PRF/5 cells was grown in 175 cm2 flasks, at different passages. After infection, cells were incubated at 32 degrees C in RPMI 1640 medium supplemented with 2.5% foetal calf serum (FCS) for 6-12 months. HAV which was released continuously in the culture medium was harvested weekly. Hepatitis A virus antigen (HAAg) and infectious virus production was stable during each passage. The antigen titre, determined by radioimmunoassay, was about 50 for each passage whereas the infectious virus titre increased from 10(3.7) (passage 7) to 10(6.0) TCID50/ml (passage 13). Virus production was not influenced by the FCS concentration (0-2.5%) in the maintenance medium. The cell culture produced HAAg was used for detection of total anti-HAV antibodies, anti-HAV titration and IgM antibody capture assay and the results were identical to those obtained with commercial kits. HAAg produced by this practical and cheap method could easily replace primate derived antigen for the detection of anti-HAV antibodies.

Effect of glutaraldehyde on the antigenicity and infectivity of hepatitis A virus.

The effect of glutaraldehyde on the antigenicity and infectivity of hepatitis A virus (HAV) was examined. The CF 53 strain, adapted to human hepatoma PLC/PRF/5 cells, was treated with glutaraldehyde using three different concentrations, 0.02, 0.10, and 0.50%, for various periods of time, 3, 10, and 30 min, respectively. After the virucidal assays, glutaraldehyde and HAV were separated by gel filtration, then the antigen (radioimmunoassay) titer and the infectivity titer were determined. The greatest antigen titer reduction was about 80% after 30 min using 0.10% glutaraldehyde and within only 3 min using 0.50% glutaraldehyde. Glutaraldehyde is an effective disinfectant against HAV: the infectious virus titer decreased by more than 3 log10 after 30 min using 0.10% glutaraldehyde and within only 3 min using 0.50% glutaraldehyde. Statistical studies showed that the decrease of antigen or infectious virus titer was affected by both glutaraldehyde concentration and exposure time.

Effect of antiviral substances on hepatitis A virus replication in vitro.

The effect of protamine, atropine, selenocystamine, taxifolin, and catechin on the infectivity and antigenicity of the cell culture-adapted hepatitis A virus (HAV) strain CF 53 was studied. The toxicity on uninfected PLC/PRF/5 cells was examined for each antiviral compound by morphological and biochemical methods, in order to determine concentrations without cytotoxic effect. At these concentrations, protamine and taxifolin, added to infected cells for a 15-day period, caused concentration-dependent reductions in the infectivity and antigenicity of HAV. Atropine also caused a concentration-dependent reduction of HAV infectivity but did not affect the antigenicity of the virus. At the highest concentration used, 50 micrograms/ml of protamine, 59 micrograms/ml of taxifolin, and 50 micrograms/ml of atropine, the infectious viral titer reduction was 1.56, 0.77, and 0.68 log10, respectively. Selenocystamine and catechin had no effect on HAV replication.

[Adaptation to the cell line PLC/PRF/5 of hepatitis A virus released in the cell culture medium]. / Adaptation à la lignée cellulaire PLC/PRF/5 du virus de l'hépatite A libéré dans le surnageant de culture.

The propagation of hepatitis A virus (HAV), CF53 strain, released without any cytopathic effect into the PLC/PRF/5 cells supernatant, was studied in the course of six serial passages (6th to 11th). The decrease (from 5 to 1 week) of incubation time required to detect HAV, by RIA, in culture supernatant, the increase in Hepatitis A antigen (from 777 to 10,038 c.p.m./50 microliter) and infectivity titre (from 10(3.0) TCID 50/ml to 10(4.5) TCID 50/ml) were consistent with the adaptation of this virus to the cell line PLC/PRF/5.