The pathophysiology of chronic hepatitis in rabbits infected with coxsackievirus B5 (CVB5), (strain Mitchell) was investigated. Three-week-old male New Zealand White rabbits were inoculated intraperitoneally with 1 x 10(5) plaque forming units of virus. Every 3 months for 15 months postinoculation (p.i.) groups of animals were sacrificed for the following tests: interleukin (IL)-6, tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-beta cytokine levels by enzyme-linked immunosorbent assay (ELISA); splenic natural killer (NK) cell function; sequence of a 154-bp section of the 5' noncoding region; antihepatocyte autoantibodies; histologic examination; in situ polymerase chain reaction (ISPCR) of the liver; neutralizing antibody response to CVB5; and viral cultures of liver, spleen, blood, brain, heart, skeletal muscle, and pancreas samples. Histologic evidence of hepatocyte necrosis was evident at each time point, although few inflammatory cells were seen. Liver samples were positive at each time by ISPCR, with viral nucleic acid localized to hepatocyte cytoplasm. Other cells in the liver did not stain. No hepatocyte autoantibodies were detected, and there was no elevation of intrahepatic cytokine levels compared to uninfected controls. There were no mutations in the virus over time. A vigorous neutralizing antibody response to CVB5, Mitchell was generated, but splenic natural killer (NK) function and numbers of splenic NK cells were significantly decreased. Virus culture was positive at 3 months, but negative at further time points. Cultures were negative at 3 months for the other tissues tested. Thus, CVB5, Mitchell causes a chronic hepatitis in rabbits, with virus limited to hepatocyte cytoplasm and no evidence of autoimmunity.