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A digoxigenin-tailed, synthetic oligodeoxynucleotide was used to detect genomic hepatitis delta virus (HDV) RNA in formalin-fixed, paraffin-embedded liver sections by a nonisotopic in situ hybridization (NISH) procedure. Twenty-three liver samples from chronically HDV-infected patients were studied. Eight liver specimens from humans and chimpanzees without markers of active HDV infection served as negative controls. In three samples, the NISH findings were compared with characteristic nuclear features and with the distribution of the HDV encoded antigen, HDAg, as detected by direct immunofluorescence. All samples from HDV-infected patients were positive for HDV RNA by NISH. The viral genome was exclusively observed within the hepatocytic nuclei. No enzymatic reaction was detected after hybridization with the negative controls. "Sanded" nuclei, a cytopathologic change associated with HDV infection, were HDV RNA-positive, but only a small percentage of infected cells showed that feature. Hepatocytes containing the HDV RNA were sometimes binucleated or exhibited giant nuclei. When HDAg and HDV RNA were sequentially detected within the same sections, the localization of the viral antigen almost completely overlapped with the expression of the HDV transcripts, and vice versa. In conclusion, detection of intrahepatic HDV RNA by NISH is a rapid, sensitive, and specific technique that is easily applicable to routine histopathology and allows correlation of HDV with the morphology of hepatocyte nuclei.

The woodchuck was selected to study the efficacy of liver-targeted antiviral drugs on hepadnavirus replication. Nineteen woodchucks chronically infected with woodchuck hepatitis virus were treated with adenine arabinoside monophosphate or acyclovir monophosphate, either free or conjugated with the liver-targeting molecule lactosaminated human serum albumin. Circulating woodchuck hepatitis virus DNA levels remained unchanged in untreated animals and in those receiving the carrier lactosaminated human serum albumin alone; in contrast, they were consistently lower after 5 days of treatment with the antiviral drugs. Free and conjugated adenine arabinoside monophosphate were active at doses of 10 and 0.75 mg/kg, respectively, and free and coupled ACVMP were active at doses of 20 and 2.6 mg/kg, respectively. These results indicate that the dosages of adenine arabinoside monophosphate and acyclovir monophosphate required to inhibit hepadnavirus growth can be sharply reduced by coupling the drugs to lactosaminated human serum albumin.

Diagnosis of acute hepatitis B virus/hepatitis delta virus (HBV/HDV) coinfection is currently based on detection of anti-HD, however this antibody may be undetectable during the acute phase of hepatitis. To evaluate the entity of misdiagnosis of HBV/HDV coinfection in acute HBsAg-anti-HBc IgM positive hepatitis we examined sera from 245 consecutive patients obtained at admission and day 30, 60, 120, 210 and 400 of their follow-up. Anti-HD was detected in the serum of 26 out of 245 patients (10.6%). In 15% of cases it was present at admission, while in 92% it was found after 30 days. The combined detection of HDV-RNA, HDAg and IgM anti-HD in acute phase sera allowed a correct etiologic diagnosis in 69% of the cases. These findings suggest that the prevalence of HBV/HDV coinfection is underestimated when anti-HD is the only marker to be detected during the acute phase of disease. A correct etiologic diagnosis can only be made by testing acute phase sera for all the available markers of HDV. However, the best cost-effective procedure is to test any patient with HBV markers at presentation for anti-HD, 30-40 days after the onset of symptoms.

HBV-DNA measured by the spot hybridization technique, was found in the sera of 28 of 106 (26.4%) anti-HBe positive carriers of HBsAg. Dane particle-associated HBeAg, HBcAg and HBV-specific DNA-polymerase activity were found in the sera of nine (8.5%), five (4.7%) and two (1.9%) of these patients, respectively. All carriers with serum HBV-DNA had chronic liver disease and 18 had intrahepatic delta-Ag and serum anti-delta at titers higher than 1/5000. Intrahepatic HBcAg was detected in the nuclei of 90% of delta negative individuals; 50% of them also had cytoplasmic fluorescence. Only two of the 18 patients with intrahepatic delta-Ag (11%) had HBcAg in the liver. Viral nucleic acid was not found in the sera of 15 other patients with chronic hepatitis, seven of whom had intrahepatic delta-Ag. Serum HBV-DNA was also negative in the remaining 63 symptomless carriers of HBsAg lacking markers of delta infection. Interestingly, although DNA-polymerase negative, some sera gave autoradiographic spots of high optical density. HBV-DNA was detected in them at concentrations typical of sera which are usually both DNA-polymerase and HBeAg positive. Detection of HBV-DNA in serum represents the most direct and sensitive in vitro assay for assessing HBV infectivity and characterizes HBsAg carriers with HBV-related liver damage and ongoing HBV replication independently from the state of HBeAg/anti-HBe system. In the Mediterranean area, the majority of anti-HBe positive carriers with serum HBV-DNA have chronic liver disease and delta infection.

Nucleic acid hybridization is extremely important in viral hepatitis research and the newly emerging techniques are now becoming an indispensable resource also in the diagnostic laboratory. This paper provides information on methods currently used for detection of viral nucleic acids with special emphasis on the importance of hepatitis B virus DNA in the serum. Herein, we describe the procedures for preparation and labeling of DNA probes and the principles that regulate dot, slot and Southern blot hybridization. Advantages and shortcomings of filter hybridization are discussed together with the alternative approaches to it. Finally, a collection of laboratory protocols is presented in Appendix #1.

The course of chronic hepatitis B was studied in 30 patients who had antibody to hepatitis e antigen and hepatitis B virus DNA in the serum and hepatitis B core antigen in the liver. Over a 2-year period, no patient experienced a sustained spontaneous remission of disease, and follow-up liver histology revealed worsening of the disease in four patients. After 2 years of observation, 24 patients were allocated randomly to one of two groups: 12 patients served as untreated controls and 12 received recombinant human alpha-interferon-2a in a dose of 9 million units intramuscularly three times weekly for 16 weeks. Patients who remained viremic after 16 weeks received 3 million units three times weekly for an additional 8 weeks. Abnormal amino-transferases and serum hepatitis B virus DNA persisted without appreciable changes in all untreated patients. Hepatitis B virus DNA rapidly became undetectable and serum aminotransferases fell to normal in eight treated patients. After the end of treatment, hepatitis B virus DNA became detectable once again in seven patients, in six of whom a peak of aminotransferases (range: 256 to 850 units per liter) ensued; subsequently, hepatitis B virus DNA disappeared, and serum aminotransferases again fell to normal in two of the seven. Overall, hepatitis B virus DNA was no longer detectable in serum and liver histology improved in three treated patients.(ABSTRACT TRUNCATED AT 250 WORDS)

The presence and distribution of hepatitis B core antigen (HBcAg) was studied in the liver of 227 chronic carriers of hepatitis B surface antigen (HBsAg) to investigate its relationship with serum HBV-DNA, the status of hepatitis B 'e' antigen/antibody (HBeAg/anti-HBe) and the underlying liver disease. HBcAg was detected in 144 of the 227 (63%) liver specimens and HBV-DNA in 132 (58%) of the corresponding sera. Serum HBV-DNA showed a constant link with intrahepatic HBcAg. Out of 96 HBeAg-positive patients, 91 (95%) had HBcAg in the liver and 85 (89%) had HBV-DNA in serum. Overall there was a significant link between HBeAg and HBV-DNA in serum, but there was no correlation in 58 out of 227 (26%) cases. In HBeAg/HBV-DNA-positive carriers, HBcAg expression was predominantly nuclear. It was nuclear and cytoplasmic in patients with the highest levels of viremia. Eleven out of 13 (85%) HBV-DNA-positive patients who had only cytoplasmic HBcAg were HBcAg-negative and had low levels of HBV-DNA. Nine of 13 (69%) patients with exclusively cytoplasmic HBcAg had severe chronic liver disease. Neither the presence of HBV-DNA and HBeAg in serum nor the nuclear localization of HBcAg were associated with the severity of liver damage. In the group of HBV-DNA-positive patients (132), the presence of liver disease was significantly connected with the absence of HBeAg in serum (P less than 0.05; C.L. 3-35).(ABSTRACT TRUNCATED AT 250 WORDS)

A biotin-labeled DNA probe specific for hepatitis B virus (HBV) nucleotide sequences was hybridized in situ to liver tissue of 20 patients; 16 were chronic carriers of hepatitis B surface antigen (HBsAg) and 4 had no markers of HBV infection. HBV-DNA was also analyzed in the serum and the liver of these patients by spot and Southern blot hybridization, respectively. Liver specimens from six carriers were positive for HBV-DNA both by in situ and Southern blot hybridization; ten carriers were negative by in situ hybridization, and two of these were positive by Southern blot technique. The staining was granular, mainly cytoplasmic, limited to liver specimens containing replicative forms of HBV-DNA, and associated with detection of HBcAg in hepatocytes by immunofluorescence. The sensitivity of this technique was not sufficient to detect few copies of integrated HBV-DNA. The hybridization procedure was specific, as results were constantly negative in liver specimens of patients without markers of HBV infection, and no reaction was observed using DNA probes lacking HBV-DNA sequences. Detection of HBV-DNA by in situ hybridization, using a biotinylated probe, is a rapid, reproducible, and specific histochemical method. Currently available biotinylated probes are advantageous when absolute sensitivity is not the limiting factor, and they also facilitate studies of the cellular and subcellular distribution of HBV nucleic acids.

A cytoplasmic autoantibody is described which gives a distinct immunofluorescence pattern in tissues of man and of varied animal species. Fluorescence is maximal in human substrates; it is strong in human hepatocytes and nephron cells and weak in thyroid adrenal and pancreatic cells. Complement fixation and fluorescence absorption studies have shown that the homologous antigen is localized in the microsomal membranes of human liver. The autoantibody was found in 13% of 81 carriers of HBsAg with chronic delta infection. It was not detected in patients with acute delta infection or in HBsAg positive and HBsAg negative patients without delta infection. The apparently exclusive occurrence of this antibody in chronic delta infection suggests that its expression is induced by persistence of the viral event.

Vidarabine (ara A) produces severe dose-dependent side-effects. To examine whether its monophosphate ester (ara-AMP) can be effective in the treatment of chronic hepatitis B when given in reduced dosage as a conjugate with lactosaminated human serum albumin (L-HSA), which selectively enters hepatocytes, five patients with chronic type B hepatitis (HBsAg/HBV-DNA positive for at least 2 years) were treated with the conjugate. The daily dose of conjugate given (35 mg/kg) contains 1.5 mg ara-AMP, whereas the usual daily dose of free ara-AMP is 5-10 mg/kg. In three patients HBV-DNA fell to undetectable levels and remained negative in two; in one of them anti-HBe developed. In the other two patients HBV-DNA decreased but was detectable during treatment--one received three cycles of therapy, and became HBV-DNA negative and anti-HBe positive 45 days after the end of treatment; the other remained HBeAg/HBV-DNA positive. No adverse effects were observed, and biochemical variables (including aminotransferases) remained unchanged or decreased with viraemia. No antibodies (IgM and IgG classes) that bound the conjugate were detected. Thus L-HSA-ara-AMP inhibits HBV replication as well as free ara-AMP but at a third to a sixth of the dose.

Seven patients with hepatitis delta virus (HDV) cirrhosis underwent liver transplantation. In every case the HDV infection was florid but accompanied by an inactive hepatitis B virus (HBV) infection. The patients were given anti-HB surface antigen (HBsAg) serum globulins and HBV vaccine. Two patients cleared the HBsAg and the HDV, and are alive and well 14 and 15 months, respectively, after transplantation. HDV infection recurred in the other five patients: hepatitis developed in three, another died, and the fifth was re-transplanted for causes unrelated to viral hepatitis (reinfection was shown by the presence of HD antigen in the graft). Liver transplantation is feasible in patients with HDV disease but involves a high risk of HDV reinfection that cannot be predicted by the virological pattern of the native HBV infection or prevented by conventional HBV prophylaxis.

In recent years molecular biology techniques allowed an extraordinary progress in knowledge of hepatitis viruses. New informations have accumulated on the mechanisms of virus replication and on the pathogenesis of liver damage. In the clinical setting, simple noninvasive methods that use the same technological approach provide a useful diagnostic tool for infections of hepatitis B virus (HBV) and hepatitis delta virus (HDV). Using these tests we may define whether a carrier of hepatitis B surface antigen has a liver damage due to HBV or HDV or to other, non-virological causes. Detection of HBV-DNA and HDV-RNA in serum and liver can also be used as a guide for prognosis and therapy.