Spliced RNA of woodchuck hepatitis virus.

Polymerase chain reaction was used to investigate RNA splicing in liver of woodchucks infected with woodchuck hepatitis virus (WHV). Two spliced species were detected, and the splice junctions were sequenced. The larger spliced RNA has an intron of 1300 nucleotides, and the smaller spliced sequence shows an additional downstream intron of 1104 nucleotides. We did not detect singly spliced sequences from which the smaller intron alone was removed. Control experiments showed that spliced sequences are present in both RNA and DNA in infected liver, showing that the viral reverse transcriptase can use spliced RNA as template. Spliced sequences were detected also in virion DNA prepared from serum. The upstream intron produces a reading frame that fuses the core to the polymerase polypeptide, while the downstream intron causes an inframe deletion in the polymerase open reading frame. Whereas the splicing patterns in WHV are superficially similar to those reported recently in hepatitis B virus, we detected no obvious homology in the coding capacity of spliced RNAs from these two viruses.

Structure and expression of the woodchuck herpesvirus genome.

This report describes the genome structure and location from which immediate-early transcription originates in the recently characterized woodchuck herpesvirus (herpesvirus marmota: HVM). Cross-hybridization of restriction fragments indicates that the HVM genome contains a tandem array of 1.5-kb repeat units. Additionally, terminal labeling and exonuclease experiments demonstrate that the repeated sequences lie at the termini of the genome. Hybridization of probes representing immediate-early transcription indicates that only a single predominant species of immediate-early RNA originates from a region near one end of unique sequences in the HVM genome. These results show remarkable similarity with group 2 of the gammaherpesvirinae. However, no homology was detected by conventional Southern blot hybridization between HVM and the gamma-2 prototype, herpesvirus saimiri. Therefore, we propose HVM to be a new member of the gammaherpesvirinae subfamily of herpesviruses.

Extrahepatic replication of woodchuck hepatitis virus in chronic infection.

We describe studies of woodchuck hepatitis virus nucleic acids in liver and other tissues of chronically infected woodchucks, using Southern and Northern blot hybridization techniques. Single-stranded and covalently closed circular replicative DNA molecules were distinguished from partly double-stranded virus genomes. In most animals the liver contained more virus than any other organ, but all extrahepatic organs studied (spleen, thymus, pancreas, and kidney) contained viral DNA and significant amounts of viral RNA. In the spleen, partly double-stranded virus genomes were present at higher levels than in any other organ but liver, but nonetheless replicative intermediates were not detected. The observation of RNA transcripts in the absence of detectable covalently closed circular DNA template suggests that a small proportion of cells in extrahepatic tissues are infected with WHV.

Characterization of a herpesvirus isolated from woodchuck hepatocytes.

A DNA virus with the characteristics of a herpesvirus has been isolated from woodchuck hepatocytes cultured in vitro. We refer to this virus as herpesvirus of marmots (HVM). Electron microscopy of thin sections of HVM-infected cells showed nucleocapsids with a hexagonal outline and a diameter of 80 nm. Enveloped virions were seen in cytoplasmic vacuoles and outside the cell. Negatively stained virus particles purified from cell supernatants were enveloped with the characteristic appearance of herpesviruses. The DNA was double-stranded with a molecular size of approximately 140 kb and a G + C content of 73%. The virus replicated with a lytic effect in kidney cells of owl monkeys and African green monkeys, baby hamster kidney cells, feline kidney cells and WCH-17, a cell line derived from a woodchuck hepatoma. An indirect immunofluorescence assay has shown the presence of antibody to HVM in seven out of 37 animals tested. An important reason for studying HVM lies in its possible role in infection or the disease produced by woodchuck hepatitis virus, an animal model for human hepatitis B virus.

Instability of v-src sequences in nonhuman primate tumors cultured in vitro.

We have analyzed the DNA of marmoset tumors induced and marmoset cells transformed by Rous sarcoma virus (RSV) and derivative viruses of various types. Southern blot hybridization was used to determine the presence of v-src gene sequences. We failed to detect v-src DNA in high-passage cells derived from marmoset tumors induced in vivo or from marmoset cell lines transformed in vitro. The inability to detect src sequences was not related to selection of revertants in culture, since all cell lines retained transformed morphology and cells transformed in vitro retained the ability to induce sarcomas after transplantation into adult allogeneic marmosets. By contrast, we detected integrated proviruses in cells analyzed 32 to 60 days after in vitro transformation. The proviral sequences appeared to be identical to the transforming virus but were apparently unstable and continued to transpose.

Cloning and structural analysis of integrated woodchuck hepatitis virus sequences from hepatocellular carcinomas of woodchucks.

Woodchuck hepatitis virus (WHV), like the related hepatitis B virus, induces in its natural host hepatocellular carcinomas that contain integrated viral sequences. As a first step in determining whether and how the integrated sequences contribute to formation of the tumors in which they are found, we have cloned two such integrations of WHV and have determined their structure by restriction mapping and heteroduplex electron microscopy. The identity of the cloned sequences was confirmed by comparison of restriction sites in the clones with those located by Southern blot analysis of tumor DNA. Viral sequences in both integrations are extensively rearranged, and in neither were all parts of the viral genome represented. In this respect, the behavior of WHV in vivo is similar to that of other DNA tumor viruses that have been studied in vitro. We discuss the implications of these results in relation to possible mechanisms for tumor induction by WHV.

Persistence of hepatitis B surface antigen in the bedbug Cimex hemipterus (Fabr.).

The fate of hepatitis B virus in the bedbug was investigated to assess this insect's potential as a vector. Colony-reared Cimex hemipterus (Fabr.) were fed once on blood positive for hepatitis B surface antigen (HBsAg). The insects were sampled at intervals thereafter and tested for HBsAg by radioimmunoassay. HBsAg persisted for up to six weeks in the bedbug's body after a single HBsAg-positive meal, during which time several further HBsAg-negative blood meals were taken. This result explains the high rates of field infection in bedbugs and further supports the hypothesis that bedbugs may play a role in transmission of hepatitis B virus.