No abstract

To study possible extrahepatic sites for the replication of hepatitis C virus (HCV), we examined fresh and cultured peripheral blood mononuclear lenkocytes (PBML), as well as different subpopulations of PBML of HCVinfected patients, for the presence of viral genomic and antigenomic RNA. Sense and antisense oligonucleotide primers derived from HCV sequences were used for reverse transcription (RT) followed by an amplification with the polymerase chain reaction assay (PCR). Using antisense primers for RT, genomic viral RNA could be detected in serum, liver, total PBML and B lymphocytes of chronically infected patients. However, only liver tissue and PBML specimens were positive when a sense primer was used. To demonstrate further the specificity of these findings, total PBML were stimulated using pokeweed mitogen and synthesis of HCV RNA was determined by incorporation of [3H]uridine into nascent viral RNA molecules using a hybrid release assay.

A major antibody combining site on foot and mouth disease virus (FMDV) serotype O1K has been identified in a predicted surface helix of viral protein 1 (VP1) between amino acid residues 144 and 159. A hexadecapeptide covering this sequence elicits high titers of antibodies that specifically recognize and neutralize FMDV. The high quality of the immune response is attributed to a particularly stable conformation of the antigenic amino acid sequence, which is most likely an alpha‐helix.

To study the replication strategy of the human hepatitis B virus, the 5' end of the RNA pregenome and the initiation sites of DNA plus and minus strands have been mapped. The RNA pregenome was found to be terminally redundant by 120 nucleotides; it is initiated within the pre-C region and may also function as mRNA for synthesis of the major core protein and the hepatitis B virus reverse transcriptase. The hepatitis B virus DNA minus strand is initiated within the direct repeat sequence DR1, it contains a terminal redundancy of up to eight nucleotides, and its synthesis does not require any template switch. The DNA plus strand is primed by a short oligoribonucleotide probably derived from the 5' end of the RNA pregenome, and its synthesis is initiated close to the direct repeat sequence DR2. For its elongation to pass the discontinuity in the DNA minus strand an intramolecular template switch occurs using the terminal redundancy of this template. Thus, the route of reverse transcription and DNA replication of hepatitis B viruses is fundamentally different from that of retroviruses.

Transmissible spongiform encephalopathies (TSEs) are neurological diseases that are associated with the conversion of the normal host-encoded prion protein (PrP-sen) to an abnormal protease-resistant form, PrP-res. Transmission of the TSE agent from one species to another is usually inefficient and accompanied by a prolonged incubation time. Species barriers to infection by the TSE agent are of particular importance given the apparent transmission of bovine spongiform encephalopathy to humans. Among the few animal species that appear to be resistant to infection by the TSE agent are rabbits. They survive challenge with the human kuru and Creutzfeldt-Jakob agents as well as with scrapie agent isolated from sheep or mice. Species barriers to the TSE agent are strongly influenced by the PrP amino acid sequence of both the donor and recipient animals. Here we show that rabbit PrP-sen does not form PrP-res in murine tissue culture cells persistently infected with the mouse-adapted scrapie agent. Unlike other TSE species barriers that have been studied, critical amino acid residues that inhibit PrP-res formation are located throughout the rabbit PrP sequence. Our results suggest that the resistance of rabbits to infection by the TSE agent is due to multiple rabbit PrP-specific amino acid residues that result in a PrP structure that is unable to refold to the abnormal isoform associated with disease.Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative disorders that include kuru and variant Creutzfeldt-Jakob disease (vCJD) in humans, chronic wasting disease (CWD) in deer and elk, scrapie in sheep and goats, and bovine spongiform encephalopathy (BSE). A critical event in TSE pathogenesis is the conversion of the normal, proteasesensitive host prion protein (PrP-sen) to an abnormal, partially protease-resistant form (PrP-res) that is closely associated with TSE. PrP-sen is a glycoprotein with an apparent molecular mass of 33 to 37 kDa, which is anchored to the cell membrane by a glycosylphosphatidylinositol anchor (3). No characteristic sequence differences or chemical modifications between PrPsen and PrP-res have been identified (13), suggesting that the two isoforms differ mainly in their conformation (8,23,31,33).Initial transmission of the TSE agent from one species to another is usually associated with prolonged incubation times, which decrease as the TSE agent adapts over multiple passages through the new species. Understanding this so-called "species barrier" is of particular importance, because the BSE agent has crossed the human species barrier to cause vCJD and there are concerns that the CWD agent in North America could cross species barriers and pose a threat to human health. Transmission of TSE agents to laboratory animals such as mice, hamsters, and rats has greatly enhanced our understanding of the TSE species barrier. These animals are susceptible to a variety of TSE agents, including those from sheep, humans, and cattle. Rabbits are the only mammalian species reported to be resist...