- Volume 84, Issue 7, 2003

The incidence of human cytomegalovirus (CMV) genotype gB2 (UL55) is high in patients with human immunodeficiency virus (HIV) infection in the San Francisco Bay area of California. Virus neutralizing antibody (NAb) to human CMV strain Ad169, a gB2 laboratory strain, was measured prospectively in HIV-infected patients, with CD4 T-lymphocyte counts <200, who were at risk for CMV-associated disease. Patients were grouped according to CMV DNA copy number, as quantified by PCR, and presence or absence of CMV-induced retinitis. Mean NAb titres were similar in all patient groups and unrelated to either virus load or outcome of CMV infection. Both gB2 and mixtures of gB2 with other gB genotypes were represented in isolates from blood and/or urine, even in the presence of high titres of antibody to the gB2 genotype challenge virus.

Attachment of, and cell–cell fusion induced by, human cytomegalovirus were studied in the presence of neutralizing monospecific antibodies against antigenic domains 1 (AD-1) or 2 (AD-2) of glycoprotein B (gB, gpUL55). Efficient inhibition of the virion-mediated fusion event was consistently observed for the human AD-2-specific antibody as determined by a reporter gene activation assay based on permissive astrocytoma cells. In contrast, antibodies directed against the major neutralizing gB epitope AD-1 reduced fusion only by 20–60 %. Virus attachment via heparan sulfate was unaffected by the antibodies under the conditions used. Virus receptor binding as examined by heparin treatment of adsorbed virus was significantly reduced only if the virus had been coated with the AD-2-specific antibody. Neutralization of virus infectivity by the AD-2-specific antibody thus seems most likely to result from interference with a receptor-binding event during initial virus–host cell interaction.

Epstein–Barr virus (EBV) has been suggested to play a role in hepatocellular carcinoma (HCC). However, reports on detailed EBV transcript analyses in HCCs are limited. It was shown recently that expression of the transforming BARF1 (BamHI A rightward open reading frame 1) gene of EBV is restricted to latently EBV-infected epithelial malignancies, i.e. nasopharyngeal carcinoma and gastric carcinoma. The aim of this study was to test the presence of EBV in Dutch HCCs. A semiquantitative DNA PCR-enzyme immunoassay (PCR-EIA) for the BamHI W fragment of EBV was used to assess the presence of EBV in frozen and paraffin-embedded tissues of 16 HCCs. In addition, several RNA detection techniques, i.e. nucleic acid sequence-based amplification (NASBA), RT-PCR, RNA in situ hybridization (RISH) and immunohistochemistry (IHC), were applied. Five of 16 HCCs and two of four hepatitis C virus hepatitis samples were weakly positive for EBV DNA by PCR-EIA. Using sensitive RNA transcription techniques, no transcripts were found for BARF1, EBNA-1 and BARTs (BamHI A rightward transcripts) in any of the liver tissues tested. In addition, RISH for EBER1/2 and BARTs and IHC for EBNA-1, LMP-1 and ZEBRA, performed on the paraffin-embedded tissue of the PCR-EIA-positive cases and on adjacent non-neoplastic liver tissues, were negative. The absence of epithelial-specific BARF1 transcripts and other EBV transcripts and proteins in the EBV DNA PCR-positive cases argues strongly against a role for EBV in HCC.

The Epstein–Barr virus latent membrane protein (LMP 1) functions as a constitutively active signalling molecule and associates in lipid rafts clustered with other signalling molecules. Using immunofluorescent confocal microscopy, LMP 1 was shown to have an heterogeneous distribution among individual cells which was not related to the cell cycle stage. LMP 1 was shown to localize to intracellular compartments in cells other than the plasma membrane. Co-labelling of cells with both an LMP 1 antibody and an antibody to the Golgi protein GS15 revealed that the intracellular LMP 1 partly co-localized with the Golgi apparatus. Further confirmation of intracellular LMP 1 localization was obtained by immunoelectron microscopy with rabbit polyclonal LMP 1 antibodies and cryosectioning. As well as being present in intracellular foci, LMP 1 co-localized in part with MHC-II and was present on exosomes derived from a lymphoblastoid cell line. Preparations of LMP 1 containing exosomes were shown to inhibit the proliferation of peripheral blood mononuclear cells, suggesting that LMP 1 could be involved in immune regulation. This may be of particular relevance in EBV-associated tumours such as nasopharyngeal carcinoma and Hodgkin's disease, as LMP 1-containing exosomes may be taken up by infiltrating T-lymphocytes, where LMP 1 could exert an anti-proliferative effect, allowing the tumour cells to evade the immune system.

In order to investigate whether previous findings of ubiquitous skin papillomavirus infection in Caucasians apply to populations from other parts of the world, skin swab samples from Bangladesh, Japan, Ethiopia and Zambia were analysed in parallel with Swedish samples. The prevalence of HPV DNA in the material from Bangladesh was 68 %, Japan 54 %, Ethiopia 52 %, Zambia 42 % and Sweden 70 %. A great multiplicity of genotypes was demonstrated by the finding of 88 HPV types or putative types in 142 HPV DNA-positive samples in total. Double or multiple genotypes were frequently found in the same sample. The most prevalent HPV type was HPV-5, with an overall prevalence of 6·5 %. This was also the only type that was found in samples from all of the countries in the study. The results presented show that commensal skin HPV infections have a worldwide distribution with a very broad spectrum of genotypes.

The small t antigen (st-ag) of simian virus 40 can exert pleiotropic effects on biological processes such as DNA replication, cell cycle progression and gene expression. One possible mode of achieving these effects is through stimulation of NFκB-responsive genes encoding growth factors, cytokines, transcription factors and cell cycle regulatory proteins. Indeed, a previous study has shown that st-ag enhanced NFκB-mediated transcription. This study demonstrates that promoters possessing a consensus TATA box (i.e. TATAAAAG) in the context of either NFκB- or Sp1-binding sites are trans-activated by st-ag. Overexpressing the general transcription factor hTAFII130/135, but not hTAFII28 or hTAFII80, stimulated the activity of promoters in a consensus TATA box-dependent mode. Converting the consensus TATA motif into a non-consensus TATA box strongly impaired activation by st-ag and hTAFII130/135. Conversely, mutating a non-consensus TATA motif into the consensus TATA box rendered the mutated promoter inducible by st-ag and hTAFII130/135. Mutation of the TATA box had no effect on TNFα- or RelA/p65-mediated induction of NFκB-responsive promoters, indicating a specific st-ag effect on hTAFII130/135. St-ag stimulated the intrinsic transcriptional activity of hTAFII130/135. Substitutions in the conserved HPDKGG motif in the N-terminal region or a mutation that impaired the interaction with protein phosphatase 2A abrogated the ability of st-ag to activate hTAFII130/135-mediated transcription. These results indicate that trans-activation of promoters by st-ag may depend on a consensus TATA motif and suggest that such promoters recruit the general transcription factor hTAFII130/135.

The simian virus 40 (SV40) capsid is composed of 72 pentamers of VP1, the major protein of SV40. These pentamers are arranged in a T=7d icosahedral surface lattice, which is maintained by three types of appropriately arranged, non-equivalent interactions between the pentamers. However, it remains unclear how these interactions are achieved. In this study, the in vitro assembly of recombinant VP1 was analysed. Electron microscopy observations revealed that these recombinant VP1 proteins assembled into structurally polymorphic particles depending on environmental conditions. VP1 pentamers assembled efficiently into virus-like particles (VLPs) when high concentrations of ammonium sulfate were present. However, in the presence of 1 M NaCl and 2 mM CaCl2 at neutral pH, VP1 pentamers formed not only VLPs but also produced tiny T=1 icosahedral particles and tubular structures. The exclusion of CaCl2 resulted in the exclusive formation of tiny particles. In contrast, in the presence of 150 mM NaCl at pH 5, the VP1 pentamers produced only extraordinarily long tubular structures. VP1 is thus quite unique in that it can assemble into such diverse structures. These observations provide clues that will help elucidate the mechanisms underlying SV40 capsid formation.

The bidirectional activity of the precore/core promoter of hepatitis B virus (HBV) has been demonstrated in cultured cell lines. However, HBV antisense transcripts (asRNAs) have not been demonstrated in vivo. In the present study using liver tissue from patients with chronic hepatitis, an anchored 5′RACE mapping the 5′ ends at position 1680/1681, 1655 or 1609/1602 was carried out. In limited cases, RLM-3′RACE detected asRNAs to terminate at four or five consecutive dT residues in the 0·7 kb downstream region. PCR of oligo(dT)-primed cDNA did not amplify a typical polyadenylated asRNA. RT-PCR using various primers did not detect any spliced forms. Competitive RT-PCR estimated the copy numbers of the asRNAs to be 0·05–0·4 % of total sense RNAs. All sequenced asRNAs had ORF6 but, in one patient, the asRNA initiating at position 1680/1681 had additional initiation and termination codons in front of ORF6. Therefore, asRNAs are transcribed by RNA polymerase III at a low level, encompass a dispensable ORF6 gene and might be retained in the nucleus. The endogenous asRNAs complementary to the common ends of all sense RNAs suggest antisense-mediated self-regulation of hepadnavirus.

To understand the possible origin of hepatitis B virus (HBV), three of the four hyperendemic, primitive accessible tribes of the Andaman and Nicobar islands, India, were investigated. The Nicobarese tribe was investigated in 1989 and 1999. The S gene from 65 HBV isolates was amplified by PCR and sequenced. Genotyping and serotyping were carried out on the basis of phylogenetic and amino acid analyses of S gene. All 20 Nicobarese-89 isolates, nine Onges-99 isolates and the single Andamanese-99 HBV isolate were classified as genotype D. Of the Nicobarese-99 isolates, 32 (91·4 %) and three (8·6 %) were genotypes D and A, respectively. Per cent nucleotide identity between the S sequences representing different tribes varied from 98·06 to 98·59 % and varied from mainland isolates by 1·6–2·0 %. Although southeast Asian origin is postulated for the Nicobarese tribe, the presence of different genotypes suggests introduction of HBV after migration to these islands, probably from mainland India, 200 years back, when these islands became inhabited as a part of penal settlement during the British regimen.

A brain homogenate prepared from a terminally ill hamster infected with scrapie strain 263K was serially diluted and administered orally to groups of hamsters. The undiluted brain homogenate led to clinical scrapie in all animals inoculated. The attack rate decreased with dilutions of the homogenate, and subclinical infections were identified among the healthy survivors at 520 days post-infection by Western blotting. The number of animals succumbing to disease and the combined number of Western blot-positive survivors plus diseased hamsters were used to calculate the LD50 and ID50 of the inoculum. The model system represents an approximation to the transmission of TSEs such as new variant Creutzfeldt–Jakob disease (vCJD) via dietary exposure to the infectious agent and suggests that, due to the rather small difference between the calculated LD50 and ID50, the number of clinical cases will not be vastly exceeded by the number of subclinical carriers of the disease.