The development and application of quantitative PCR-based assays for the detection of hepatitis C and related viruses

PCR has previously been used to measure hepatitis C virus (HCV) levels during antiviral chemotherapy, in viral cultures and in diverse clinical samples. The use of this technique led to a rapid expansion of our understanding of this clinically important virus. However, the majority of methods previously used to quantify HCV were laborious and relatively insensitive. This thesis describes the development of PCR-based quantitative methods for HCV RNA and their application in a number of research settings. Significant advances in assay speed and throughput are demonstrated, as well as important improvements in sensitivity. The aim of the project was to develop and optimise quantitative PCR methods which could conveniently be applied to serum or plasma and also to cells and cell culture supernatants from in vitro culture experiments. The PCR method developed was shown to be effective in providing quantitative viral monitoring data from several trials of antiviral chemotherapy. Viral RNA levels were also monitored in the serum of a patient infected during pregnancy, in a cultured hepatocyte line and the method was also applied to the study of a potential small primate model. The quantity of virus in cultured hepatocytes and in plasma pools prior to fractionation was demonstrated to be very low. In addition a novel single tube 'hot-start' RT-PCR method (RT-HS-PCR) is described. This method was shown to increase the sensitivity of RT-PCR and to provide quantitative data. The discovery of a new flavivirus (GBV-C), closely related to HCV, provided an opportunity to demonstrate the flexibility of this quantitative PCR method. The technique has now been applied to a wide range of DNA and RNA viruses in several laboratories within the UK

The Parasexual Cycle in Candida albicans Provides an Alternative Pathway to Meiosis for the Formation of Recombinant Strains

Candida albicans has an elaborate, yet efficient, mating system that promotes conjugation between diploid a and α strains. The product of mating is a tetraploid a/α cell that must undergo a reductional division to return to the diploid state. Despite the presence of several “meiosis-specific” genes in the C. albicans genome, a meiotic program has not been observed. Instead, tetraploid products of mating can be induced to undergo efficient, random chromosome loss, often producing strains that are diploid, or close to diploid, in ploidy. Using SNP and comparative genome hybridization arrays we have now analyzed the genotypes of products from the C. albicans parasexual cycle. We show that the parasexual cycle generates progeny strains with shuffled combinations of the eight C. albicans chromosomes. In addition, several isolates had undergone extensive genetic recombination between homologous chromosomes, including multiple gene conversion events. Progeny strains exhibited altered colony morphologies on laboratory media, demonstrating that the parasexual cycle generates phenotypic variants of C. albicans. In several fungi, including Saccharomyces cerevisiae and Schizosaccharomyces pombe, the conserved Spo11 protein is integral to meiotic recombination, where it is required for the formation of DNA double-strand breaks. We show that deletion of SPO11 prevented genetic recombination between homologous chromosomes during the C. albicans parasexual cycle. These findings suggest that at least one meiosis-specific gene has been re-programmed to mediate genetic recombination during the alternative parasexual life cycle of C. albicans. We discuss, in light of the long association of C. albicans with warm-blooded animals, the potential advantages of a parasexual cycle over a conventional sexual cycle

HIV Capsid is a Tractable Target for Small Molecule Therapeutic Intervention

Despite a high current standard of care in antiretroviral therapy for HIV, multidrug-resistant strains continue to emerge, underscoring the need for additional novel mechanism inhibitors that will offer expanded therapeutic options in the clinic. We report a new class of small molecule antiretroviral compounds that directly target HIV-1 capsid (CA) via a novel mechanism of action. The compounds exhibit potent antiviral activity against HIV-1 laboratory strains, clinical isolates, and HIV-2, and inhibit both early and late events in the viral replication cycle. We present mechanistic studies indicating that these early and late activities result from the compound affecting viral uncoating and assembly, respectively. We show that amino acid substitutions in the N-terminal domain of HIV-1 CA are sufficient to confer resistance to this class of compounds, identifying CA as the target in infected cells. A high-resolution co-crystal structure of the compound bound to HIV-1 CA reveals a novel binding pocket in the N-terminal domain of the protein. Our data demonstrate that broad-spectrum antiviral activity can be achieved by targeting this new binding site and reveal HIV CA as a tractable drug target for HIV therapy

Trends in Mortality Rates for Infectious and Parasitic Diseases in Australia: 1907-1997

AIMS: To characterize long-term mortality trends for infectious and parasitic diseases in Australia during the twentieth century, explore influencing factors and provide suggestions to health policy-makers. METHODS: A descriptive study was conducted. Deaths due to communicable diseases from 1907 to 1997 were tallied, according to the International Classifi­cation of Diseases version 9 (ICD-9). Trends in infectious disease mortality in overall population and in the 0−4 years age group were examined and standardized by sex. Death rates were also studied for: (i) diarrhoea/enteritis, (ii) pneumonia and all respir­atory diseases and (iii) tuberculosis. RESULTS: There has been a substantial decline in ­mortality from communicable diseases over the study period. The death rate dropped from 258.9 per 100 000 population in 1907 to 7.2 per 100 000 pop­ulation in 1997. Six phases of the decline were observed. CONCLUSIONS: A combination of improved living conditions and access to readily available treatments over the twentieth century played an important role in the reduction of infectious disease mortality in Australia.P. Bi, M. Whitby, S. Walker and K. A. Parto

Co-existing conditions for deaths from infectious and parasitic diseases in Australia

© ElsevierObjective: To examine the frequency distribution of co-existing conditions for deaths where the underlying cause was infectious and parasitic diseases. Materials and methods: Besides the underlying cause of death, the distributions of co-existing conditions for deaths from infectious and parasitic diseases were examined in total and by various age and sex groups, at individual and chapter levels, using 1998 Australian mortality data. Results: In addition to the underlying cause of death, the average number of reported co-existing conditions for a single infectious and parasitic death was 1.62. The most common co-existing conditions were respiratory failure, acute renal failure – non-specific causes, ischaemic heart disease, pneumonia and diabetes. When studying the distribution of co-existing conditions at the ICD-9 chapter level, it was found that the circulatory system diseases were the most important. There was an increasing trend in the number of reported co-existing conditions from 60 years of age upwards. Gender differences existed in the frequency of some reported co-existing conditions. The most common organism types of co-existing conditions were other bacterial infection and other viruses. Conclusions: The study indicated that the quality of death certificates is less than satisfactory for the 1998 Australian mortality data. The findings may be helpful in clarifying the ICD coding rules and the development of disease prevention strategies.Peng Bi, Kevin A. Parton and Michael Whitbyhttp://www.elsevier.com/wps/find/journaldescription.cws_home/701730/description#descriptio

Small-Molecule Inhibition of Human Immunodeficiency Virus Type 1 Infection by Virus Capsid Destabilization▿

Human immunodeficiency virus type 1 (HIV-1) infection is dependent on the proper disassembly of the viral capsid, or “uncoating,” in target cells. The HIV-1 capsid consists of a conical multimeric complex of the viral capsid protein (CA) arranged in a hexagonal lattice. Mutations in CA that destabilize the viral capsid result in impaired infection owing to defects in reverse transcription in target cells. We describe here the mechanism of action of a small molecule HIV-1 inhibitor, PF-3450074 (PF74), which targets CA. PF74 acts at an early stage of HIV-1 infection and inhibits reverse transcription in target cells. We show that PF74 binds specifically to HIV-1 particles, and substitutions in CA that confer resistance to the compound prevent binding. A single point mutation in CA that stabilizes the HIV-1 core also conferred strong resistance to the virus without inhibiting compound binding. Treatment of HIV-1 particles or purified cores with PF74 destabilized the viral capsid in vitro. Furthermore, the compound induced the rapid dissolution of the HIV-1 capsid in target cells. PF74 antiviral activity was promoted by binding of the host protein cyclophilin A to the HIV-1 capsid, and PF74 and cyclosporine exhibited mutual antagonism. Our data suggest that PF74 triggers premature HIV-1 uncoating in target cells, thereby mimicking the activity of the retrovirus restriction factor TRIM5α. This study highlights uncoating as a step in the HIV-1 life cycle that is susceptible to small molecule intervention

Genome-wide analysis in a murine Dnmt1 knockdown model identifies epigenetically silenced genes in primary human pituitary tumors

DNA methylation at promoter CpG islands (CGI) is an epigenetic modification associated with inappropriate gene silencing in multiple tumor types. In the absence of a human pituitary tumor cell line, small interfering RNA-mediated knockdown of the maintenance methyltransferase DNA methyltransferase (cytosine 5)-1 (Dnmt1) was used in the murine pituitary adenoma cell line AtT-20. Sustained knockdown induced reexpression of the fully methylated and normally imprinted gene neuronatin (Nnat) in a time-dependent manner. Combined bisulfite restriction analysis (COBRA) revealed that reexpression of Nnat was associated with partial CGI demethylation, which was also observed at the H19 differentially methylated region. Subsequent genome-wide microarray analysis identified 91 genes that were significantly differentially expressed in Dnmt1 knockdown cells (10% false discovery rate). The analysis showed that genes associated with the induction of apoptosis, signal transduction, and developmental processes were significantly overrepresented in this list (P < 0.05). Following validation by reverse transcription-PCR and detection of inappropriate CGI methylation by COBRA, four genes (ICAM1, NNAT, RUNX1, and S100A10) were analyzed in primary human pituitary tumors, each displaying significantly reduced mRNA levels relative to normal pituitary (P < 0.05). For two of these genes, NNAT and S100A10, decreased expression was associated with increased promoter CGI methylation. Induced expression of Nnat in stable transfected AtT-20 cells inhibited cell proliferation. To our knowledge, this is the first report of array-based "epigenetic unmasking" in combination with Dnmt1 knockdown and reveals the potential of this strategy toward identifying genes silenced by epigenetic mechanisms across species boundaries

A pilot study of combination therapy with ribavirin plus interferon alfa for interferon alfa-resistant chronic hepatitis C

Background/Aims: In chronic hepatitis C, interferon alfa (IFN-α) therapy fails to achieve a sustained response in approximately 75% of patients. Similarly, ribavirin induces only a transient response. The aim of this study was to evaluate whether ribavirin and IFN-α in combination could be effective in IFN-α-resistant chronic hepatitis C. Methods: Twenty patients with chronic hepatitis C resistant to a previous course of IFN-α were randomly assigned to receive either ribavirin combined with IFN-α or IFN-α alone for 6 months. Results: Serum alanine aminotransferase levels decreased significantly during therapy in both treatment groups, but after therapy, the levels remained significantly decreased only in the combination therapy group. Nine months after treatment, sustained normalization of aminotransferase levels, associated with sustained loss of serum hepatitis C virus RNA, was observed in 40% of the patients in the combination therapy group but in none of the patients treated with IFN-α alone (P < 0.05). The sustained response was accompanied by reduced hepatic necroinflammatory activity on biopsy. Conclusions: These findings suggest that ribavirin plus IFN-α combination therapy is able to induce a sustained biochemical and virological response in a significant proportion of patients with IFN-α-resistant chronic hepatitis C. © 1994