Identification and analysis of a new hepadnavirus in white storks.

We identified, cloned, and functionally characterized a new avian hepadnavirus infecting storks (STHBV). STHBV has the largest DNA genome of all avian hepadnaviruses and, based on sequence and phylogenetic analysis, is most closely related to, but distinct from, heron hepatitis B virus (HHBV). Unique for STHBV among the other avian hepadnaviruses is a potential HNF1 binding site in the preS promoter. In common only with HHBV, STHBV has a myristylation signal on the S and not the preS protein, two C terminally located glycosylation sites on the precore/core proteins and lacks the phosphorylation site essential for the transcriptional transactivation activity of duck-HBV preS protein. The cloned STHBV genomes were competent in gene expression, replication, and viral particle secretion. STHBV infected primary duck hepatocytes very inefficiently suggesting a restricted host range, similar to other hepadnaviruses. This discovery of stork infections unravels novel evolutionary aspects of hepadnaviruses and provides new opportunities for hepadnavirus research.

Frequency of spontaneous mutations in an avian hepadnavirus infection.

In this study, we measured the frequency of revertants of a cytopathic strain of the duck hepatitis B virus that bears a single nucleotide substitution in the pre-S envelope protein open reading frame, resulting in the amino acid substitution G133E. Cytopathic virus mixed with known amounts of a genetically marked wild-type virus was injected into ducklings. Virus outgrowth was accompanied by a coselection of wild-type and spontaneous revertants during recovery of the ducklings from the acute liver injury caused by death of the G133E-infected cells. The frequency of individual revertants in the selected noncytopathic virus population was estimated by determining the ratio of each revertant to the wild-type virus. Spontaneous revertants were found to be present at frequencies of 1 x 10(-5) to 6 x 10(-5) per G133E genome inoculated. A mathematical model was used to estimate that the mutation rate was 0.8 x 10(-5) to 4.5 x 10(-5) per nucleotide per generation.

Reactivation of hepatitis B in a long-term anti-HBs-positive patient with AIDS following lamivudine withdrawal.

BACKGROUND/AIMS: In HIV-infected patients, who have recovered completely from an acute hepatitis B infection and become anti-HBs positive, hepatitis B infection may be reactivated after progression to AIDS. CASE REPORT: We present the case of a homosexual male patient with AIDS who developed clinical and serological reactivation of hepatitis B with detectable HBV-DNA 18 years after complete recovery from acute hepatitis B infection. Prior to reactivation, antiretroviral triple therapy including lamivudine was changed to therapy without lamivudine. After reintroduction of lamivudine in the triple therapy, HBV-DNA became undetectable and the patient lost HBsAg and again developed anti-HBs antibodies. CONCLUSION: The hepatitis B in this patient can be explained best by reactivation of persistent HBV infection, possibly because of transient decline in antibodies against HBs-antigen due to a reduction in CD4+ lymphocyte numbers and B cell dysfunction. This observation points to the clinical relevance of HBV persistence in serum and blood cells of anti-HBs-positive subjects for many years after recovery from acute hepatitis B infection. The possible role of lamivudine withdrawal which immediately preceded HBV breakthrough in our patient is noteworthy. Regular monitoring of markers of HBV infection, including HBV-DNA, in patients with AIDS appears justified after discontinuation of lamivudine.

A hepatitis B virus mutant with a new hepatocyte nuclear factor 1 binding site emerging in transplant-transmitted fulminant hepatitis B.

Hepatitis B virus (HBV) DNA was cloned from serum of a heart transplant recipient who died from fulminant hepatitis B transmitted by the donor. Restriction enzyme analyses of the clones obtained by conventional cloning yielded six HBV variants: a major species (pF-1) representing 88% and five minor species (pF-2 to pF-6), each representing 2% to 4% of the clones. The complete nucleotide sequence of these six variants revealed that five of the six viral genomes, including pF-1, carried a novel 11 base pair (bp) insertion in the core promoter region as well as an 18 bp and an 108 bp in-frame deletion in the pre-S1 region not present in the donor. One genome was identical to the sequence of the donor. Functional analyses of HBV clones generated by in vitro mutagenesis and cassette exchange showed that the 11 bp insertion is a strong binding site for hepatocyte nuclear factor 1 (HNF-1). In transient transfection experiments, the novel HNF-1 sequence motif was shown to result in enhanced viral replication. Immunohistochemical analyses revealed high levels of cytoplasmic and nuclear hepatitis B core antigen (HBcAg) and only scattered hepatitis B surface antigen (HBsAg) expression in the liver. The data in our immunosuppressed patient showed that HBV variants can rapidly accumulate in severe hepatitis B and suggest that the novel HNF-1 binding site may have contributed to the fulminant clinical course, possibly via enhanced viral replication.

Hepatitis B virus infection of tupaia hepatocytes in vitro and in vivo.

For the systematic analysis of various clinical and molecular aspects of hepatitis B virus (HBV) infection, an experimental small animal system of HBV infection would be a great advance. The susceptibility to HBV infection, therefore, of hepatocytes from the tree shrew species tupaia belangeri was studied in vitro and in vivo. Primary hepatocytes isolated from livers of tupaias can be reproducibly infected with HBV. In vitro infection results in viral DNA and RNA synthesis in hepatocytes and secretion hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) into culture medium. Tupaias can also be infected with HBV in vivo, resulting in viral DNA replication and gene expression in tupaia livers. Similar to acute, self-limited hepatitis B in humans HBsAg is rapidly cleared from serum, followed by seroconversion to anti-HBe and anti-HBs. These data clearly tht HBV is infectious to tupaia hepatocytes in vitro and transiently in vivo. Tupaias, therefore, may become a useful model for the experimental analysis of various molecular and clinical aspects of HBV infection, including the significance of HBV quasispecies, the steps involved in hepatocarcinogenesis as well as the evaluation of various antiviral strategies.

Selective transmission of variant genomes from mother to infant in neonatal fulminant hepatitis B.

Hepatitis B virus (HBV) nucleotide sequences isolated from mother/child pairs were analyzed in three cases of neonatal fulminant hepatitis B (FHB). Heterogeneous HBV sequences consistent with both adw2 and ayw subtype were found in all three mothers. In one case, in which the child survived, both subtypes were transmitted. By contrast, only the ayw subtype was present in the two other children with a fatal course of FHB. In one fatal case, studied in greater detail, multiple HBV variants (viral quasi-species) were identified in both mother and child. A direct sequence comparison showed that only a subfraction of the virus pool from the mother was transmitted and that multiple new mutations emerged in the child. These data suggest that a minor HBV subpopulation from the mother may prevail as the dominant species in the child and that neonatal FHB is associated with the selection of mutant strains.

Mitochondrial DNA sequences from Switzerland reveal striking homogeneity of European populations.

Mitochondrial DNA sequences from 74 Swiss individuals were compared to sequences from British and Finish populations. We found that the nucleotide sequence differences between these populations are almost as low as those within the populations. This is in contrast to three African populations, which display substantial differences between each other. The homogeneity of the mitochondrial gene pool in Europe suggests a recent common ancestry for European populations. This may reflect the arrival of anatomically modern humans about 40,000-30,000 years ago or, alternatively, the spread of agriculturalists about 10,000-6,000 years ago. Taking into account the estimated rate of evolution of the mitochondrial control region, the data favor the former explanation.

Polymerase chain reaction detects hepatitis B virus DNA in paraffin-embedded liver tissue from patients sero- and histo-negative for active hepatitis B.

The polymerase chain reaction (PCR) was used to analyse tissues from paraffin blocks of liver needle biopsies retrospectively. Biopsies of 29 patients with proven HBsAg and HBcAg expression in liver tissue and of 8 healthy volunteers served as positive (group 1) and negative tissue controls (group 2), respectively. These were compared with 16 patients with proven HBsAg expression in liver but lack of HBcAg (group 3), with 23 patients with anti-HBc as the only hepatitis B virus (HBV)-related marker (group 4) and with 21 patients with liver disease and without HBV markers in tissue or serum (group 5). PCR detected HBV sequences in all cases of the positive control group and in 94% of group 3, in 65% of group 4, and in 71.4% of group 5, whereas all healthy volunteers were negative. Our data show that PCR is able to detect HBV-DNA sequences in virtually all patients with active viral antigen expression but also in a high proportion of hepatitic patients who are silent for active HB but may or may not show signs of a contact with the HBV. Thus, PCR for HBV-DNA in paraffin sections might become a useful tool for identifying patients carrying HBV-DNA but not expressing HBV antigens.

[Acute myocardial infarct after prophylactic administration of low doses of heparin-ergotamine: coronary artery spasm the cause?]. / Akuter Myokardinfarkt unter "Low-dose-Heparin-Dihydroergotamin": Folge eines Koronararterienspasmus?

There have been several recent publications reporting vasospastic complications under heparin-dihydroergotamine prophylaxis. We report on 2 patients without significant coronary artery stenosis who died of acute myocardial infarction. Both patients had been operated on for a lumbar disc protrusion and were treated with 2500 IU heparin + 0.5 mg dihydroergotamine s.c. twice daily. The possibility of coronary arterial spasm as the cause of the myocardial infarctions is discussed. Publications reporting cases with myocardial infarctions, presumably caused by coronary arterial spasm, and other vasospastic complications under ergotamine therapy are briefly reviewed.