Studies on the pathogenesis of Hepadnavirus infection

Abstract

Improved methods for the in situ hybridisation detection of messenger RNA ( mRNA ) in sections of liver tissue, were derived by use of an experimental system. This involved the use of tritiated-poly ( dT ) probes to detect poly ( A ) sequences attached to the 3 ' end of mRNA in sections of mouse liver that had been processed in various ways. The improved - methods were applied to the detection of hepatitis B virus ( HBV ) - and hepatitis delta virus ( HDV ) - RNA. In situ hybridisation and immunostaining techniques were then applied to studies of the pathogenesis of HBV and duck hepatitis B virus ( DHBV ) infection. In situ hybridisation studies of liver biopsy tissue from HBV - infected immunosuppressed renal transplant patients demonstrated an anatomical association between piecemeal necrosis and HBV replication at the cellular level in some patients. However, widespread replicative infection of hepatocytes also occurred in some patients in the presence of normal hepatocyte morphology and mild inflammatory changes indicating that at the cellular level virus replication was not necessarily a direct cytopathic process. These findings supported the view that hepatocyte Injury may : ( i ) result from immune - mediated damage directed against cells undergoing replicative, but not restricted infection ; ( ii ) eliminate cells undergoing replicative infection and favour clonal regeneration of cells undergoing restricted infection. Localisation of interferon - alpha ( IFN - alpha ) expression in liver tissue chronically infected with HBV and HDV, identified mononuclear cells and fibroblasts ( but not hepatocytes ) as the main producers of IFN - alpha. IFN - alpha - positive cells were associated with areas of liver tissue containing cells supporting virus replication and exhibiting the greatest degree of liver damage, suggesting that locally produced IFN - alpha may be a natural regulator of virus replication in chronic liver disease. Experimental DHBV infection of Pekin - Aylesbury ducks showed that virus inoculated either intravenously or intraperitoneally, gained access to randomly distributed hepatocytes without first replicating in other cell types in the liver. Virus was seen to disseminate to contiguous cells following anatomical boundaries by the third day post - inoculation. Markers of DHBV infection in liver and serum showed reproducible kinetics, and duck hepatocytes in this system appeared to be highly permissive as large amounts of DHBV DNA and DHBsAg were produced intracellularly without the development of ongoing cytopathology. Hepatocytes were the major cell type responsible for early significant DHBV replication, in contrast to pancreas, kidney, spleen and circulating mononuclear cells where significant levels of infection were detected only after the first week of infection and the onset of viraemia.