Review

The importance of hepatitis B viral DNA in serum and liver*

The polymerase chain reaction was used to investigate the presence of hepatitis B virus DNA in sera of 61 children with chronic hepatitis B and negative results on dot biot hybridization tests. Our results demonstrate that most chronic carriers of hepatitis B surface antigen in childhood have hepatitis B virus DNA detectable by polymerase chain reaction in their serum and must be considered infectious.

The sensitivity of four different non-radiolabelled HBV-DNA probes (digoxigenin-, biotin-, acetylaminoflorene-, chemiprobe-labelled) in detecting HBV-DNA were compared with a ³²P-labelled HBV-DNA probe using a spot hybridisation assay. Of the non-radiolabelled probes, the probe using digoxigenin labelling was the most sensitive with reliable and reproducible detection to between 1–3 pg HBV-DNA. The use of digoxigenin-labelled HBV-DNA hybridisation assay deserves wider application for the detection of HBV-DNA because of a number of advantages including ease, speed, safety and cost.

In hepatitis B virus carriers who are anti-HBe positive despite ongoing viral replication (HBcAg in liver and HBV-DNA in serum) the natural course of hepatitis is severe and the response to interferon is low. We investigated whether a new hepatitis B virus (HBV) strain could be involved. A translational termination codon at the carboxyterminal end of the pre-C region responsible for the lack of HBeAg secretion was found in 18 of 19 HBV clones isolated from seven pedigreed patients with this clinical syndrome. The same findings were confirmed by direct sequencing. One of these patients underwent a liver transplant and HBV infection of the new liver resulted in high titered viremia and intrahepatic expression of HBcAg, without detectable HBeAg in serum. Another patient was superinfected by hepatitis delta virus (HDV) and developed high titres of total and IgM anti-HD. In spite of this, chronic hepatitis remained unchanged during 7 years of follow-up. These data strongly suggest that a viable precore minus mutant of hepatitis B virus is responsible for the lack of HBeAg in the serum of these patients. The HBV variant may explain the peculiar geographic distribution of anti-HBe positive hepatitis. The variations in the virus genome sequence may cause the more severe form of liver disease and modify the pathogenicity in the case of HDV superinfection.

In recent years, there has been increased recognition of the importance of viral infections. In addition, new antiviral agents have become available. These factors have led to a marked increase in utilization of viral diagnositc services. In this review, both conventional and rapid methods for viral diagnosis are presented, with emphasis on recent advances. The antiviral agents currently available and the major drugs under investigation are also briefly discussed. It is hoped that this review will serve as a useful adjunct for the management of patients with virus infections.

This chapter discusses biochemical methods for the detection of hepatitis B virus (HBV) constituents. Hepatitis B virus is the smallest of the DNA viruses that infect man. Only one other animal DNA virus that infects pigs is smaller. The four viruses—HBV, woodchuck hepatitis virus, ground squirrel hepatitis virus, and duck hepatitis virus—form a distinct group of reverse transcribing viruses that show strong similarities in virion size, in the length of the genome, and in other properties. Two biochemical techniques are applicable to the detection of HBV DNA: dot hybridization that provides a semiquantitative test for its presence or absence, and southern blot analysis, by means of which HBV DNA sequences are located in nucleic acid fragments that are separated according to electrophoretic mobility. The principal tool for studying the molecular forms of HBV DNA is the southern blot technique. DNA forms are separated according to size and secondary and tertiary structure by means of gel electrophoresis and are transferred from the gel to a membrane either by blotting or by electrophoresis.