Detection of hepatitis D virus RNA in serum by a reverse transcription, polymerase chain reaction-based assay.

We designed a reverse transcription, polymerase chain reaction-based assay for serum hepatitis D virus RNA. Amplified hepatitis D virus cDNA was revealed by ethidium bromide staining, followed by blotting onto a nylon membrane and hybridization with a 32phosphorus-labelled oligonucleotide, or by a DNA enzyme immunoassay (DEIA) using a double stranded DNA-specific monoclonal antibody. The absolute sensitivity was expressed as number of hepatitis D virus RNA molecules, using a serum of known viral RNA concentration. Three sets of primers were used, encompassing the base positions 66-686 (variable rod-stabilizing region), 701-962 (conserved, viroid-like domain) and 886-1,333 (portion of the open reading frame 5 encoding for the carboxyterminus of the hepatitis D antigen) of the viral genome. The lower detection limits, after amplification of the three RNA portions, as assessed by ethidium bromide staining, were 7.5 x 10(6), 7.5 x 10(4) and 7.5 x 10(2) molecules of hepatitis D virus RNA per assay, respectively. The region encompassing bases 886-1,333 was chosen for blotting and hybridization to a radiolabelled oligonucleotide probe or for a capture-based DNA enzyme immunoassay, where the microplate was coated with this same probe. The two procedures showed comparable sensitivity, i.e., about 10 molecules of viral RNA per assay. The specificity of the assay was further on a panel of both anti-hepatitis D-positive and -negative sera. Amplification of serum hepatitis D virus RNA by reverse transcription/polymerase chain reaction followed by detection of the amplified cDNA by DNA enzyme immunoassay is a promising and feasible routine assay for detecting low amounts of circulating virions.

Chronic infection in woodchucks infected by a cloned hepatitis delta virus.

Two woodchucks (Marmota monax) intrahepatically inoculated with hepatitis delta virus (HDV) complementary DNA clones pSVL-D3 and pSVL-Ag showed virological and pathological signs of acute and chronic HDV infection. HDV-RNA and hepatitis delta antigen (HDAg) were detected in serum by slot-blot hybridization and by western blot five weeks after inoculation. Liver biopsy specimens collected at 8th week post inoculum were positive for HDV-RNA. Anti-HDV antibodies were detected at the 11th and 9th weeks, respectively. Histological finding of hepatocarcinoma and persistence of circulating HDV-RNA and anti-HDV were observed up to the 10th month. Both woodchucks produced "small" and "large" HDAg antigen, although the inoculated cloned DNA bears the coding capability solely for the small antigen. A transient decrease of woodchuck hepatitis virus DNA (WHV-DNA) level was observed during the peak of HDV infection. Successive inoculation of acute-phase serum in three woodchucks resulted in a successful infection in one of the animals.