- Volume 91, Issue 7, 2010

Analysis of the molecular mechanisms of viral-mediated oncogenesis has contributed enormously to the understanding of the basic principles of normal/malignant cell growth. Transformation by human adenoviruses is a multi-step process involving the modulation of numerous cellular pathways, leading to inhibition of apoptosis and growth arrest. However, the molecular mechanism of how the adenovirus oncogenes facilitate transformation of rodent cells, while concurrently failing to do so for human cells, remains elusive. In this report, we demonstrate for the first time that the transformation capabilities of adenovirus type 5 oncogenes are not restricted to rodent cells, but include cells of the related mammalian order Lagomorpha, inducing considerable morphological alterations, enhanced cell growth and tumour induction in vivo. Furthermore, the established cell lines may represent a suitable tool for further development to generate E4-mutated adenoviruses, which has so far been difficult as mutations within the E4 region often prove to be lethal without a helper-cell system.

Human papillomavirus (HPV) 58 is a high-risk HPV type associated with progression to invasive genital carcinomas. We developed six monoclonal antibodies (mAbs) against HPV58 L1 virus-like particles that bind conformational epitopes on HPV58. The hybridoma cell lines were adapted to serum- and animal component-free conditions and the mAb supernatants were affinity-purified. The six mAbs neutralized HPV58 pseudoviruses (PsVs) and ‘quasivirions’ with different capacities. The mAbs differed in their ability to prevent PsV58 attachment to HaCaT cells, to the extracellular matrix (ECM) deposited by HaCaT cells, to heparin and to purified human laminin 5, a protein in the ECM. These mAbs provide a unique set of tools to study the binding properties of a previously untested, high-risk HPV type and the opportunity to compare these characteristics with the binding of other HPV types.

This study developed and validated a high-throughput human papillomavirus (HPV) serology method based on Luminex technology, using pseudovirions (PsVs) of eight mucosal HPV types (HPV-6, -11, -16, -18, -31, -45, -52 and -58) and two cutaneous HPV types (HPV-5 and -38) bound to heparin-coated beads. Analysis with neutralizing type-specific monoclonal antibodies against the included HPV types indicated the type specificity of the assay. Analysis of negative-control serum samples from 63 children and 71 middle-aged women with up to one lifetime sexual partner indicated high specificity. Positive-control serum samples from subjects with known HPV DNA status or clinical diagnosis found expected sensitivities for most of the HPV types in 219 European serum samples, but lower than expected in 124 samples from Africa. HPV-45 and -52 did not react as expected with the human serum samples. The PsV-Luminex method was used to determine the HPV-seropositivity-associated relative risk for future cervical cancer using 208 serum samples from a prospective study of 18 814 women followed for 23 years, analysed previously with standard HPV-16 ELISA. The PsV-Luminex method gave similar results to ELISA (κ=0.77). As expected, HPV seropositivities assayed using the PsV-Luminex method found an increased risk of cervical cancer for HPV-16 [odds ratio (OR)=7.7, 95 % confidence interval (CI)=2.6–23] and HPV-31 (OR=4.1, 95 % CI=1.6–10.8), non-significant tendencies for increased risk for other mucosal HPV types and no risk for the cutaneous HPV types. In summary, multiplexed HPV serology using mammalian-derived PsVs selected for native conformation by binding to heparin-coated beads was validated as a high-throughput HPV serological method for most of the analysed HPV types.

Serology has been used to indicate past infection by the human polyomaviruses BK virus (BKV) and JC virus (JCV), because the site of primary infection is not established fully. Little is known about BKV and JCV antibody stability over time. We investigated BKV and JCV seroprevalence and antibody stability over time in an Australian population-based study. Serum was collected from 458 adults participating in a longitudinal skin cancer study in Queensland in 1992, 1993 and 1996, and 117 people had a fourth sample collected in 2003. Serum samples were analysed for BKV and JCV VP1 antibodies by multiplex detection using the Luminex platform. The seroprevalence for BKV and JCV over 4.5 years was 97 and 63 %, respectively. The BKV seroprevalence was 99 % in 25–60-year-olds, and 94 % in people older than 60 years. JCV seroprevalence was around 60 % in people younger than 50 years, 68 % in people 50–70 years of age and 64 % in people older than 70 years. BKV seroprevalence was very stable over 11 years, with 96 % of people staying seropositive and 2 % remaining seronegative. JCV antibody status over time was less stable; 57 % of participants remained seropositive and 31 % seronegative. The same proportion of people (4 % each) seroconverted, seroreverted or had fluctuating JCV antibody levels. These results confirm the previously believed stability of polyomavirus antibodies, with BKV antibodies being highly stable and JCV antibodies moderately so. Thus, a single measure can be used as a reasonable indicator of long-term antibody status in epidemiological studies aiming to understand associations between polyomaviruses and disease.

The non-structural X protein, HBx, of hepatitis B virus (HBV) is assumed to play an important role in HBV replication. Woodchuck hepatitis virus X protein is indispensable for virus replication, but the duck hepatitis B virus X protein is not. In this study, we investigated whether the HBx protein is indispensable for HBV replication in vivo using human hepatocyte chimeric mice. HBx-deficient (HBx-def) HBV was generated in HepG2 cells by transfection with an overlength HBV genome. Human hepatocyte chimeric mice were infected with HBx-def HBV with or without hepatic HBx expression by hydrodynamic injection of HBx expression plasmids. Serum virus levels and HBV sequences were determined with mice sera. The generated HBx-def HBV peaked in the sucrose density gradient at points equivalent to the generated HBV wild type and the virus in a patient’s serum. HBx-def HBV-injected mice developed measurable viraemia only in continuously HBx-expressed liver. HBV DNA in the mouse serum increased up to 9 log10 copies ml−1 and the viraemia persisted for more than 2 months. Strikingly, all revertant viruses had nucleotide substitutions that enabled the virus to produce the HBx protein. It was concluded that the HBx protein is indispensable for HBV replication and could be a target for antiviral therapy.

Geminiviruses are often associated with subviral agents called DNA satellites that require proteins encoded by the helper virus for their replication, movement and encapsidation. Hitherto, most of the single-stranded DNA satellites reported to be associated with members of the family Geminiviridae have been associated with monopartite begomoviruses. Cassava mosaic disease is known to be caused by viruses belonging to nine different begomovirus species in the African continent and the Indian subcontinent. In addition to these species, several strains have been recognized that exhibit contrasting phenotypes and infection dynamics. It is established that Sri Lankan cassava mosaic virus can trans-replicate betasatellites and can cross host barriers. To extend these studies further, we carried out an exhaustive investigation of the ability of geminiviruses, selected to represent all cassava-infecting geminivirus species, to trans-replicate betasatellites (DNA-β) and to interact with alphasatellites (nanovirus-like components; previously called DNA-1). Each of the cassava-infecting geminiviruses showed a contrasting and differential interaction with the DNA satellites, not only in the capacity to interact with these molecules but also in the modulation of symptom phenotypes by the satellites. These observations could be extrapolated to field situations in order to hypothesize about the possibility of acquisition of such DNA satellites currently associated with other begomoviruses. These results call for more detailed analyses of these subviral components and an investigation of their possible interaction with the cassava mosaic disease complex.