ABSTRACT
Common marmosets (Callithrixjacchus) were orally inoculated with a Brazilian strain (HAF-203) of hepatitis A virus (HAy). Three monkeys were euthanized at postinoculation hours 6, 12 and 24 to investigate the early events of HAV infection. Following others three inoculated and one control marmosets remained throughout the 46 day to evaluation of viral excretion. Different samples were collected to detect sequential presence of HAV RNA by nested reverse transcription-polymerase chain reaction (RT-PCR) in liver, saliva, bile and stools at 6 hours to 461h days postinoculation. Liver tissues were examined by immunofluorescence assay in a confocal laser-scanning microscope for the presence of HAV antigen. HAV RNA was detected in saliva during the course of the study, in bile from 24 hours to 46 days. in stools from 7 to 46 days and liver at 12 hours postinfection. In immunofluorescence of liver stained preparations, viral antigen was present at six hours after inoculation throughout the remainder of the 46-day study. The animals developed histological and biochemical acute hepatitis after second week postinoculation. Spleen, duodenum, and mesenteric lymph nodes specimens were negative for HAV antigens. This study supports the possibility that in Callithrixjacchus orally inoculated with hepatitis A virus the saliva route may be additional way of viral elimination. The viral replication in the liver was responsible for biliary HAV presence and latter HAV detection in fecal samples.
Subject(s)
Antigens, Viral/analysis , Callithrix , Hepatitis A virus/immunology , Hepatitis A/immunology , Monkey Diseases/immunology , Virus Replication/immunology , Animals , Disease Models, Animal , Hepatitis A/pathology , Hepatitis A/transmission , Hepatitis A Antigens , Hepatitis A virus/growth & development , Hepatitis A virus/isolation & purification , Liver/immunology , Liver/pathology , Liver/virology , Monkey Diseases/transmission , RNA, Viral/analysis , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Callithrix jacchus is considered a reliable animal model for hepatitis A virus (HAV) infection. All three HAV orally inoculated marmosets developed hepatitis - the infection was monitored by continuous virus shedding, high levels of serum enzyme alanine aminotransferase, specific antibody and seroconversion 3-6 weeks after HAV inoculation. HAV antigen was detected in liver by immunofluorescence 4 days post inoculation (PI) and onwards. To gain insight into the biological role of inducible nitric oxide synthase (iNOS) during immune-related acute liver injury the enzyme was searched in frozen biopsies: immunofluorescent labeling was found in the cytoplasm of liver cells mainly Kupffer's cells and spleen macrophages (CD68+) starting 11 days PI with maximum intensity on the fifth to sixth week PI. Necroinflammatory liver lesions characteristic of viral hepatitis were also observed at 10 days PI with maximum severity at 4 to 6 weeks PI. Furthermore, T lymphocytes (CD2+) were raised at this time point. No difference was evident in the frequency of B lymphocytes (CD20+). Therefore, iNOS expression preceded necroinflammatory liver lesion and maximal immunofluorescence reaction was coincident with tissue injury, supporting the hypothesis that NO contributes to hepatic cytotoxic mechanism but also to virus clearance. The concomitant rise in T-lymphocyte population may suggest a role for these cells in this and/or other independent HAV-induced pathological changes.
Subject(s)
Hepatitis A/enzymology , Hepatovirus , Liver/pathology , Nitric Oxide Synthase/biosynthesis , T-Lymphocytes/immunology , Animals , Callithrix , Disease Models, Animal , Enzyme Induction , Fluorescent Antibody Technique , Hepatitis A/pathology , Immunophenotyping , Liver/enzymology , Liver/virology , Necrosis , Nitric Oxide Synthase Type II , Spleen/virology , T-Lymphocytes/virologyABSTRACT
A thorough phenotypic characterization of yellow fever (YF) virus generated from cDNA is a necessary prerequisite for mapping virulence/attenuation determinants and exploring the potential of YF attenuated virus 17D as a carrier for heterologous protective epitopes. In this study, YF virus was produced from 17D cDNA clones after lipofectin-mediated RNA transfection of certified primary cultures of chicken embryo fibroblasts (YFiv5.2/SL). This virus was passaged once in embryonated chicken eggs according to current YF vaccine manufacture methodology to produce the experimental virus (YFiv5.2/VL). These viruses were characterized in established monkey neurovirulence safety tests and quantitative clinical and histologic scores were derived for each virus. The experimental vaccine viruses (YFiv5.2/SL and VL) compared favorably with another well-known YF vaccine strain (17DD) used as control virus for the histologic score. Although slightly higher clinical neurovirulence was observed for YFiv5.2 as compared with the 17DD virus, it should not preclude the use of YFiv5.2 for mapping YF virus virulence determinants.
