Co-circulation of genotypes IA and IB of hepatitis A virus in Northeast Brazil

The Northeast region is the location of most cases of acute hepatitis A virus (HAV) in Brazil. In the present study, the genotypes of HAV strains from Pernambuco State, one of most populous states in the Northeast region, were characterized. Blood samples positive for anti-HAV IgM from 145 individuals (mean age = 29.1 years), collected during 2002 and 2003, were submitted to nested RT-PCR for amplification of the 5'non-translated region (5'NTR) and VP1/2A regions of the HAV genome. The VP1/2A and 5'NTR regions were amplified in 39 and 21 percent of the samples, respectively. Nucleotide sequencing was carried out in 46 percent of VP1/2A and in 53 percent of 5'NTR isolates. The identity in nucleotide sequence of the VP1/2A region ranged from 93.6 to 100.0 percent. Phylogenetic analysis of the VP1/2A sequences showed that 65 percent belong to sub-genotype IA and 35 percent to sub-genotype IB. Co-circulation of both sub-genotypes was observed in the two years studied. Distinct clusters of highly related sequences were observed in both sub-genotypes, suggesting endemic circulation of HAV strains in this area. In the 5'NTR isolates, 92.7-99.2 percent identity was observed and two isolates presented one deletion at position 413. Phylogenetic analysis showed that genotype IA strains cluster in the tree in the same way as genotype IB strains, but one IIIA isolate from Spain clusters with genotype IB strains. These results do not allow us to state that 5'NTR could be used to genotype HAV sequences. This is the first report of co-circulation of sub-genotypes IA and IB in this region, providing additional information about the molecular epidemiology of HAV strains in Brazil.

Genetic variability of hepatitis A virus isolates in Rio de Janeiro: implications for the vaccination of school children

The epidemiology of hepatitis A virus (HAV) infection is shifting from high to intermediate endemicity in Brazil, resulting in increased numbers of susceptible individuals and a greater potential for the emergence of outbreaks. Universal vaccination against HAV has been recommended for children, but updated sero-epidemiological data are necessary to analyze the level of natural immunity and to identify candidates for preventive measures. In addition, more molecular studies are necessary to characterize the genotypes involved in HAV infections and outbreaks. Sera from 299 school children (5-15 years old) and 25 school staff members, collected during an outbreak of HAV at a rural public school in June 2000, were tested for IgM and total anti-HAV antibodies (ELISA). Viral RNA was amplified by RT-PCR from anti-HAV IgM-positive sera and from 19 fecal samples. Direct nucleotide sequencing of the VP1/2A region was carried out on 18 PCR-positive samples. Acute HAV infection was detected by anti-HAV IgM in 93/299 children and in 3/25 adult staff members. The prevalence of total anti-HAV antibodies in IgM-negative children under 5 years of age was only 10.5 percent. HAV-RNA was detected in 46 percent IgM-positive serum samples and in 16 percent stool samples. Sequence analysis showed that half the isolates belonged to subgenotype IA and the other half to IB. On the basis of these data, mass vaccination against HAV is recommended without prevaccination screening, especially for children before they enter school, since nearly 90 percent of the children under 5 years were susceptible. Molecular characterization indicated the endemic circulation of specific HAV strains belonging to subgenotypes IA and IB.

In situ enzyme immunoassay for titration of a Brazilian hepatitis A virus strain (HAF-203)

Hepatitis A virus (HAV) replicates relatively slowly in cell culture without a cytopathic effect, a fact that limits the use of tissue culture assays. The radioimmunofocus assay is the standard method for HAV titration, although it is labor intensive and requires the use of radioisotopes. A simple, rapid and objective infectivity assay based on an in situ enzyme immunoassay (EIA) is described here for a Brazilian cell culture-adapted HAV strain (HAF-203). The assay uses a peroxidase-labeled polyclonal antibody to fixed monolayers as an indicator of infection. EIA may be completed within 7 days using serial 5-fold dilutions of the virus, yielding a titer of 5.024 log 50 percent tissue culture infective dose (TCID50)/ml for HAF-203. This technique had a detection limit of 1.1 log TCID50/ml and the specificity was demonstrated by detecting no reaction on the columns of uninfected wells. The reproducibility (with intra- and inter-assay coefficients of variation ranging from 1.9 to 3.8 percent and from 3.5 to 9.9 percent, respectively) and quantitation of the assay were demonstrated by close agreement in virus infectivity titers among different assays of the same amount of virus and between assays of different amounts of virus. Furthermore, this assay does not require the use of radiolabeled antibodies. We describe here an efficient EIA that is highly reproducible and that could be used to monitor HAV growth in cell culture and to determine the quantity of HAV antigen needed for diagnostic assays. This is the first report of the infectious titer of the Brazilian cell culture-adapted HAV strain (HAF-203).

The use of non-human primates as animal models for the study of hepatitis viruses

Hepatitis viruses belong to different families and have in common a striking hepatotropsin and restrictions for propagation in cell culture. The transmissibility of hepatitis is in great part limited to non-human primates. Enterically transmitted hepatitis viruses (hepatitis A virus and hepatitis E virus) can induce hepatitis in a number of OLD World and New Worls monkey species, while the host range of non-human primates susceptible to hepatitis viruses transmitted by the parenteral route (hepatitis B virus, hepatitis C virus and hepatitis delta virus) is restricted to few species of Old World monkeys, especially the chimpanzee. Experimental studies on non-human primates have provided an invaluable source of information regarding the biology and pathogenesis of these viruses, and represent a still indispensable tool for vaccine and drug testing.

Intragastric infection induced in marmosets (Callithrix jacchus) by a Brazilian hepatitis A virus (HAF-203)

Several specied of non-human primates have been used in studies on experimental infection with hepatitis A virus (HAV). Attempts to infect a South-American marmoset (Callithrix jacchus) with a Brazilian HAV isolate (HAF-203) are described here. Four seronegative animals were inoculated intragastrically and one was sacrificed on day 11,20,47 and 62 after infection. One uninfected animal was included as control. Liver, small intestine, lymph node, spleen and kidney samples were collected for histological diagnosis and immunocytochemistry studies. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) serum enzymes and anti-HAV antibodies were monitored by a colorimetric procedure (Abbott) and an enzyme immunoassay (ELISA), respectively. Feces were collected daily for HAV antigen (HAVAg) detection by ELISA. Increased levels of HAVAg were detected in hepatocytes 11 days after infection, with a gradual decrease during the course of infection. Shedding of HAVAg in feces was observed from the late incubation to the early acute phase (20th day to 47th day after infection). The end of the incubation period was indicated by the initial increases in serum ALT and AST. Severe hepatic lesions such as piecemeal necrosis and bridging necrosis were detected during the acute phase, coinciding with the maximum transaminase levels and the appearance of anti-HAV antibodies. On the 62nd day (convalescent phase), the hepatic tissue showed evidence of regeneration and the transaminase values had returned to baselines. The serological, biochemical, antigenic and histological evidence of hepatitis A was similar to that observed in several primate models inoculated with other HAV isolates. The data suggest that C. jacchus can be a valuable model for the study of hepatitis A and for the evaluation of HAV vaccines

Fast growth of a Brazilian hepatitis A virus (HAF-203) in a primate cell line

A hepatitis A virus (HAV, HAF-203) isolated in Brazil was submitted to 8 serial passages through fetal Rhesus kidney cells (FRhK-4). The kinetics of replication were monitored by enzyme immunoassay (EIA-HAVAg) and cDNA-RNA dot blot hybridization. The maximum level of RNA, which was observed 21 days post-infection (p.i.) during the 3rd passage, when HAVAg was still undetectable by EIA, served as a basis to establish subsequent passages every 21 days p.i. This schedule of passage resulted in a progressive reduction of time between culture infection and HAVAg and RNA production, together with an enhancement in antigen titer content of cell lysates. During the 7th passage, maximum HAVAg and RNA levels were detected at 7 days. Fourteen days after the 8th passage, clear morphological modifications appeared, suggesting a good adaptation of HAF-203 to FRhK-4 cells. Obtaining a fast-growing Brazilian HAV is very important for the development of vaccines

Primary isolation of a brazilian strai9n of hepatitis A virus (HAF-203) and growth in a primate cell line (FRhK-4)

Although hepatitis A is endemic in Brazil, this is the first report describing the isolation of a Brazilian strain of hepatitis A virus (HAV). Fecal specimens obtained from patients in the acute phase of hepatitis A were iunoculated into fetal Rhesus kidney cell cultures (FRhK-4). Only one inoculum, denoted HAF-203, could be propagated serially. Both cell lysates and tissue culture fluids of infected cells were used as inocula and evaluated for viral antigen and RNA content by enzyme immunoassay and cDNA-RNA hybridization, respectively. Cell lysates gave better yields when used as viral inocula. After three passages, viral RNA and antigen were detected in cell lysates 4 and 14 days post-infection, respectively. Using tissue culture fluid as inoculum, the incubation period was decreased from 49 to 7 days after 4 serial passages, reflecting the adaptation of HAF-203 to growth in FRhK-4 cells. FRhK-4 cells can now be used for HAV antigen production for diagnostic assays and molecular characterization

Resposta imune à vacina da hepatite B (Hevac B, França) em unidade de diálise / Immuneresponse to B hepatitis vaccine (Hevac B, France) in a dialysis service

Foi feita uma avaliaçäo da resposta imunológica a vacina da hepatite B (Hevac B, Instituto Pasteur, França) nos pacientes e pessoal de uma unidade de hemodiálise (HD). 88,4% (23/26) desenvolveram anticorpos contra o antígeno de superfície da hepatite B (anti-HBs) entre os elementos do pessoal da HD; 75% /12) dos pacientes adultos e 87,5% (7/8) dos pacientes infantis também desenvolveram o anticorpo, todos após seis meses da primeira dose de vacina. Nenhum dos recipientes da vacina apresentou o antígeno de superficie da hepatite B (AgABs) no sangue em qualquer momento. Ficou demonstrado alto índice de resposta a vacinaçäo contra a hepatite B no pessoal e nos pacientes da HD, particularmente nas crianças, com bons títulos de anti-HBs após quatro injeçöes da vacina Hevac B