No description available.

No description available.

The effects on coliphage T6r of 11 commonly used chemical antibacterial agents have been examined. The effect of the agents on the multiplication of the phage was shown by their effect on the mass lysis of fluid cultures of the host by the phage. Only aminacrine hydrochloride and sodium lauryl sulphate showed significant inhibition of the growth of the phage at concentrations below those inhibiting the host. For the examination of the effect of the agents on free phage particles, a method of assessment giving precise inactivation time estimates has been applied and two methods of interpreting the results have been compared. Large differences have been shown in the effectiveness of different antibacterial agents in inactivating the free phage. The effect on the inactivation time of changes in concentration of the antibacterial agent has been examined for five of the agents and the regression between log inactivation time and log concentration has been shown to be linear. The dilution coefficients for these agents fell into two distinct groups, those for chloramine T and formaldehyde (about 2 and 3 respectively) being very much smaller than those for crystal violet, cetrimide and phenol (about 11,13 and 15 respectively). It has been postulated that this difference in dilution coefficients indicates different mechanisms of inactivation between the two groups and the possible nature of the mechanisms has been discussed. Methods of cultivation of the phage and its stability under different conditions of storage have been investigated. A method of plaque counting by surface drop plating has been developed and its reproducibility examined.

The role of chromatin structure on viral gene expression during HCMV infection of a permissive cell type was investigated. Experiments demonstrated that the MIEP was associated with chromatin throughout infection and was subject to modification consistent with the known temporal transcription of IE genes. However, immediately upon infection, the MIEP was associated with a repressed chromatin structure. This repression was relieved through use of the histone deacetylase (HDAC) inhibitor Trichostatin-A (TSA), confirming that the regulation of IE gene expression at this time was mediated by an inhibitory chromatin structure. Furthermore, down-regulation of the ND10-associated transcriptional repressor, hDaxx, which is known to interact with HDACs, led to similar relief from repression. Further analysis of promoters of prototypic viral early and late genes, UL44 and pp28 respectively, also revealed that all classes of viral genes are subject to chromatin-mediated regulation throughout productive infection. Although differentiation-dependent regulation of MIEP activity underpins viral latency and reactivation, down-regulation of the intrinsic anti-viral repressor hDaxx in <i>in vitro</i> latent model systems did not permit reactivation of IE gene expression. Consequently, repression of IE gene expression by hDaxx does not appear to be involved in regulation of viral latency and reactivation. In conclusion, immediately upon infection of permissive cells, intrinsic repression of HCMV IE gene transcription occurs through the establishment of an inhibitory chromatin structure around the MIEP, mediated by hDaxx and HDACs. This repression is overcome by viral factors before full productive infection can begin. Full lytic infection then involves regulation of viral gene expression through remodelling of chromatin structure at all classes of HCMV promoters.

No description available.

The members of the genus Alphavirus are positive-sense RNA viruses and it is one of two within the family Togaviridae. Most alphaviruses are predominantly transmitted to susceptible vertebrates by a mosquito vector. Alphavirus disease in humans can be severely debilitating, and depending on the particular viral species, infection may result in encephalitis and possibly life threatening symptoms. Chikungunya virus (CHIKV) is the aetiological agent represents a substantial health burden to affected populations, with clinical symptoms that include severe joint and muscle pain, rashes, and fever, as well as prolonged periods of disability in some patients. In recent years, CHIKV has received significant attention from public health authorities as a consequence of the dramatic emergence infections in the Indian Ocean islands and the Caribbean as well as the recent emergence of CHIKV in the Americas. Infections have also been reported around Europe such as in Italy, France and Greece. Currently, no safe, approved or effective vaccine or treatment exists for CHIKV infection. The ubiquitin-proteasome system (UPS), the major intracellular proteolytic pathway, mediates different kinds of cellular processes, which may be targeted by viruses to aid their replication within cells. In recent years it has been well established that both the forward reaction of ubiquitination, and the reverse reaction of deubiquitination are targeted during virus infection to enhance their replication, either by targeting of cellular proteins or encoding viral homologues of key pathway proteins. The reverse reaction is undertaken by a large family of enzymes termed deubiquitylases or DUBs, and many of these have been shown to play a crucial role, not only in virus replication but also in the regulation of the immune system and vesicle trafficking. The DUBs are attractive drug targets and have increasingly been implicated in cellular processes germane to malignancy which makes the continued characterisation of the role of DUBs during virus infection a worthwhile objective. In on-going experiments in the research group a DUB siRNA pools library screen identified 12 DUBs (USP1, USP4, USP5, USP34, USP45, USP46, OTUD6A, UCHL1, JOSD2, BRCC3 and MYSM1). Depletion of these hits in HeLa cells lead to an increase in cell viability following Semiliki Forest Virus (SFV) infection (and predicted to be pro-viral) and thus could potential be candidate antiviral targets. Inroads into understanding the role of the DUB hits during the alphavirus infection, focusing initial on the BSL2 model virus SFV, and extending this to CHIKV (at BSL3). In the present study, further screening focused on the deconvolution siRNA pools for the DUB hits. Investigation of the subsequent follow up experiments with one strong candidate DUB from this list, MYSM1. Two different approaches were taken. Firstly, the effect of depletion of MYSM1 by siRNA treatment was further investigated in HeLa cells. Secondly, the analysis was extended to investigate the role of MYSM1 in fibroblasts utilising MYSM1 genetic knockout murine embryo fibroblasts. Results from this study indicate that depletion of MYSM1 in HeLa cells by siRNAs resulted in a reduction in both SFV and CHIKV replication, as assayed by measuring RNA levels and plaque formation. It was also found that MYSM1 genetic knockout in MEF cells lead to increase in both SFV and CHIKV replication. In addition, depletion of MYSM1 by siRNAs in MRC-5 cells lead to increase in SFV replication. In conclusion, MYSM1 generated interesting data, implying a role during virus infection that appeared to depend on the cell type being infected. Up to now it is unclear what the effector mechanisms are that contribute to these observations, subject to further mechanistic and functional studies, may increase the options available for targeting this vital DUB during Alphavirus infections.

Norovirus has been identified as a common cause of gastroenteritis worldwide, and food as a transmission vehicle has been well documented. Standardised detection methods exist for the detection of norovirus from fresh produce and molluscan bivalves, whilst detection methods for a wider range of food matrices that may be implicated in transmission of norovirus do not currently exist. The detection of norovirus in foods suspected to be implicated in transmission is paramount for appropriate outbreak investigation. The contamination of foods other than shellfish and fresh produce often occurs via food handlers. The proportion of norovirus that is typically transferred from food handlers to food also remains unknown. Understanding this is necessary in order to estimate the risk of infection and the burden of gastroenteritis caused by norovirus that is attributable to food contaminated by food handlers. These questions were addressed by the development of a combined capture, concentration and quantitative detection protocol with the aim to enhanced norovirus recovery from a range of food types. A food surface wash and norovirus capture method that was sensitive, reduced processing time, and increased throughput capacity was applied to a range of ready to eat foods. An automated nucleic acid extraction method which further reduced processing time and increased throughput was validated. Finally the validated method demonstrated that two real time RT-PCR assays currently used for the detection of norovirus in shellfish and fresh produce or in faecal samples were comparable overall, and hence either could be used in combination with the norovirus capture, concentration and extraction protocol described in this thesis. The protocol was applied to a range of food matrices and resulted in < 1% to 55% recovery of norovirus GI and < 1% to 25% recovery of norovirus GII. The optimised protocol was then used to quantify virus transfer between food handlers hands and to food, in simulation experiments where food handlers’ gloved hands were artificially contaminated prior to preparation of a sandwich. This enabled norovirus transfer to food items and to other food handlers to be measured at each stage. Quantitative data demonstrated that 5.9 ± (SD ± 0.1) log10 cDNA copies/μl of norovirus GII inoculum, resulted in a percentage recovery of between 3.0% and 0.02% from Food Handlers and 7.8 ± (SD ± 0.1) log10 cDNA copies/μl of norovirus GI inoculum resulted in a percentage recovery between 9.6% and 0.004% from Food Handlers. The average percentage recovered from sandwich pieces over six replicates was 0.2% for norovirus GII and 1.2% for norovirus GI. The method and protocols developed could be rolled out to official control laboratories and aid foodborne outbreak investigation by allowing testing of food categories that currently are not investigated. Furthermore, this work demonstrated the extent of norovirus transfer from hands to food ingredients and the environment and could be used in risk assessment models. Further work applying these protocols to quantify the transfer from contaminated hands using a range of viral loads will be useful in determining risk more accurately, and to monitor and investigate food premises by introducing this as an additional food and hand hygiene marker.

Human noroviruses (HuNoVs) are distributed globally, affect all age groups and place a significant burden upon health services. The diversity of this RNA virus is thought to play a significant role in the persistence of HuNoVs as the main cause of non-bacterial gastroenteritis globally. Molecular diagnostics have been critical for understanding the epidemiology of outbreaks and sporadic cases, and to design and implement effective intervention strategies and disease control measures. Immunocompromised individuals are widely considered to be a reservoir for epidemic variants of HuNoV and whilst there are studies investigating the emergence of novel strains in an immunocompetent general population, reports at the the individual level are scarce. Three separate methodologies were developed to characterise HuNoV persistence in acute convalescent and chronic infection. First, a standardised quantification method to accurately quantify the most prevalent HuNoV genogroup. Second, a PGM-MB capture method to select HuNoV prior to massively parallel sequencing (MPS). Third, an assay to measure host specific coproantibody responses to three epidemic variants from different epochs. Quantification of longitudinal samples from individuals with acute or chronic HuNoV infection showed the virus distribution was homogenous in stool and an RNA external standard, in contrast to DNA, did not underestimate virus titre. HuNoV PGM-MB capture meant near complete viral genomes could be recovered at variable mean coverage. A bioinformatics pipeline demonstrated over the course of chronic infection allele frequencies were much more variable. In acute infection, minor alleles were present at a much lower frequency, but potential immune escape mutants were present. Immune escape mutants existed as minority variants or conserved mutations in the consensus sequence, and were in the presence of HuNoV specific-coproantibody, which were mapped to the protein surface. In HuNoV chronic infection, immune pressure is variable or non-existent, and therefore epidemic variants could emerge over long periods of infection by random chance. However, under immune pressure exerted by coproantibodies, escape variants may be seen. In three individuals, acute HuNoV symptomatic infection occurred despite the presence of specific secretory Ab responses to the VLP classed as the closest phylogenetic relative. The closest relative (Sydney 2012), differed at two amino acids, one of which has been previously described (A340T) as belonging to an epitope, and another which can be classed as having a potential role in immune escape (A323T). A single individual with acute HuNoV infection established a more prominent response to an earlier strain of HuNoV, rather than two contemporary strains, which proposes a role for Original Antigenic Sin (OAS) or Antigenic Seniority in the secretory Ab immunity. Finally, the use of MPS in outbreak tracking was assessed and compared to the currently used amplicon and Sanger based method. Overall both methods showed significant correlation. However, MPS provided greater depth and the ability to identify variants among samples within an outbreak that represented consensus changes in one or more samples from the same outbreak. This meant that the MPS data would have been able to link all the samples into a single outbreak or transmission network, where the current Sanger sequencing may not have been able to link them all.

No description available.

The envelope glycoproteins of Maedi-Visna virus consist of a surface glycoprotein (gp135) which is responsible for the characteristic spikes on the surface of the virion, and a transmembrane protein (gp41) whose function includes linkage to the surface glycoprotein, anchoring it to the virion envelope. The external glycoprotein is required for attachment to the host cell via a receptor molecule present on the surface of the cell. Cells of the macrophage lineage are the main target cells in MVV infection <I>in vivo. </I>The host humoral response is targeted to the surface glycoprotein resulting in neutralizing antibody production. The relevance of these antibodies is not understood as virus infection persists despite this active immune response. The external glycoprotein has also been shown to be susceptible to antigenic variation. Expression of gp135 as three overlapping fragments in the bacterial pGEX system was undertaken with a view of using the recombinant protein as a source of immunogen to raise monoclonal antibodies. These and the three recombinant fragments could be used for epitope mapping. However, these fragments proved to be toxic to bacterial cells resulting in low yields and high levels of contamination. In depth studies were carried out to improve the yield and attempts were made to raise immune polyclonal sera. Characterization of these sera is described. Recombinant protein studies were extended to express gp135 in the baculovirus expression system. This resulted in a reliable source of recombinant protein that was devoid of contamination and was easily purified. This protein was glycosylated and was recognised by MVV-infected sheep sera. Preliminary studies were carried out to determine its interaction with sheep fibroblasts and hence its use to isolate the host cell receptor. Attempts were made to raise monoclonal antibodies against gp135 purified from virions by lectin affinity chromatography.