RESUMO
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.
Assuntos
Animais , Cebidae , Cercopithecidae , Modelos Animais de Doenças , Vírus de Hepatite/patogenicidade , Hepatite Viral Animal/transmissão , Vírus de Hepatite/imunologia , Vírus de Hepatite/fisiologia , Hepatite Viral Animal/virologia , Replicação ViralRESUMO
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
Assuntos
Células Cultivadas , Feto , Hepatovirus/isolamento & purificação , Rim , Imunofluorescência , Técnicas ImunoenzimáticasRESUMO
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
Assuntos
Animais , Hepatovirus/crescimento & desenvolvimento , Linhagem Celular , Hepatovirus/fisiologia , Técnicas Imunoenzimáticas , RNA Viral/biossíntese , Fatores de Tempo , Replicação ViralRESUMO
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