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VIRUSES
Exploiting Marine and Freshwater Microbial Biodiversity Research for Biotechnology |
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3rd VEG Workshop 18 - 19 February 2004
AbstractsWednesday 18th FebruaryRING-FISH – Detecting single genes in situK. Zwirglmaier1*, W. Ludwig1 & K.-H Schleifer1. 1 Technical University, Munich, Germany. * Present address: Dept. of Bio. Sci., University of Warwick, Coventry. Fluorescence in situ hybridisation (FISH) using rRNA targeted oligonucleotide probes has become a standard method in environmental microbiology. Apart from its value as a phylogenetic marker ribosomal RNA has always been the favoured molecule for FISH due to its high abundance in all cells. Plasmids, chromosomal DNA and other nucleic acid molecules have previously been regarded as unsuitable because of their low copy number. Here, we present an improved FISH technique based on polynucleotide probes, which goes beyond the detection of high copy intracellular nucleic acids such as rRNA. It allows for the first time the in situ detection of low copy number targets such as genes or gene fragments encoded on plasmids, chromosomal DNA or other nucleic acid molecules and therefore has the potential to become a new valuable tool for environmental microbiologists. The method was successfully applied to detect an antibiotic resistance gene (plasmid-encoded beta-lactamase), a standard housekeeping gene (GAPDH, chromosomal encoded), as well as some putative virulence factors in the plant pathogen Xanthomonas campestris. Because of the characteristic hybridisation signal obtained with this method – a halo-like ring shaped concentration of fluorescence in the cell periphery – the term RING-FISH (Recognition of INdividual Genes) was created to differentiate it from conventional FISH. Zwirglmaier, K., et al., (2004). Mol. Microbiol. 51 : 89-96
Genomic Analysis of ØJL001: Insights into its Interaction with a Sponge-Associated Alpha-ProteobacteriumJayme Lohr Center of Marine Biotechnology, University of Maryland, Baltimore, USA. Current Address: Marine Biological Association, Plymouth. Bacteriophage ØJL001 infects a novel marine bacterium in the a subclass of the Proteobacteria isolated from the marine sponge Ircinia stobilina. ØJL001 is a siphovirus and forms turbid plaques on its host. The genome sequence of ØJL001 was determined in order to better understand the interaction between the marine phage and its sponge-associated host bacterium. The complete genome sequence of ØJL001 comprised 63,469 bp with an overall G+C content of 62%. The genome has 91 predicted open reading frames (ORFs) and 17 ORFs have been assigned putative functions. Although ØJL001 appears to be a temperate phage, the integrase gene was not identified in the genome. DNA hybridization analysis showed that the ØJL001 genome does not integrate into the host chromosome. The genome of ØJL001 contains many putative genes involved in phage DNA replication. (e.g. helicase, DNA polymerase, thymidylate synthase). Genomic analysis and DNA hybridization experiments therefore suggest that ØJL001 could be a pseudolysogenic phage. Phylogeny based on DNA polymerase gene sequences indicates that ØJL001 is related to a group of Siphoviruses that infect mycobacteria. Designation of ØJL001 as a siphovirus is consistent with the morphology of the phage visualized by electron microscopy. The unique marine phage-host system presented here provides a model system to study the role of phage in sponge microbial communities.
Persistent baculovirus infections in a laboratory-reared culture of Trichoplusia ni and field populations of Pieris brassicaeRobert D. Possee1, Richard Hitchman1,2, Robert Graham1, John P. Burden1, Linda A. King2 and Rosie S. Hails1. CEH Oxford, Institute of Virology and Environmental Microbiology1, Mansfield Road, Oxford OX1 3SR UK. School of Biological and Molecular Sciences2, Oxford Brookes University, Gipsy Lane, Headington, Oxford OX3 0BP UK Baculoviruses are best known for the lethal infections they induce in susceptible insect larvae. The virus-infected host typically liquefies after death and releases large quantities of occlusion bodies with the potential to continue the replication cycle in a new insect. If a host is not immediately available, dogma has it that the virus can survive in the environment for extended periods. However, persistent virus infections have also been identified in populations of Mamestra brassicae reared in the laboratory and those recently collected in the field. In contrast with sublethal baculovirus infections of insects, the host appears to incur little cost by carrying these persistent infections. Recent studies have shown that the results with M. brassicae are not unique. Trichoplusia ni is a host commonly used to propagate a number of baculoviruses, including Autographa californica nucleopolyhedrovirus (AcNPV). We used it to analyse replication of Panolis flammea (Pf) NPV in an alternative host. Subsequent purification of virus DNA from polyhedra derived from PfNPV-challenged T. ni revealed the progeny virus was unrelated to the inoculum. Further analysis using restriction enzymes suggested that the virus amplified in T. ni was most closely related to AcNPV or TnNPV, although significant differences in fragment sizes were evident. Direct evidence for the presence of a persistent baculovirus infection in T. ni was provided by using reverse transcription coupled with the polymerase chain reaction to detect polyhedrin-specific transcripts present in mRNA prepared from healthy larvae. These data suggest that a laboratory-maintained culture of T. ni harbours a persistent baculovirus closely related to AcNPV or TnNPV. In related studies, populations of Pieris brassicae from the field were also shown to contain baculovirus-specific sequences, suggesting that these insects also have a persistent virus infection.
Microsatellite sequences in large DNA viruses and their use in studying virus diversityBurden., J.P., Thurston., M., Field, D., Hails., R.S., Possee, R.D. Increasing numbers of viral genomes are appearing in the public sequence databases. The huge amount of information contained in these genomes can provide insights into the genes present in the virus and how these may interact, as well as indicating genotypic relatedness between virus types. Attention is now focussing on how such information can be exploited to answer questions in viral ecology. Microsatellite loci, short direct repeats of 1-6 bp, are among the most common and the most mutable DNA sequences found in nature. Since their discovery, the ability to use hypermutable microsatellites as molecular markers has revolutionised a range of disciplines from ecological and evolutionary genetics to forensics and genome mapping. Msatminer is a package of perl scripts, designed for the detection and investigation of microsatellite repeats in genetic sequences developed by the Molecular Evolution and Bioinformatics group at CEH Oxford, which has been used to identify putative microsatellite sequences in baculovirus genomes.Baculoviruses are large DNA viruses which infect invertebrate species and to date 23 baculovirus genomes have been sequenced. Recent research at CEH Oxford has demonstrated that natural populations of the Cabbage Moth (Mamestra brassicae) carry a persistent, symptomless baculovirus infection1. Sequencing of PCR amplified viral polyhedrin and ie1 genes from persistently infected M. brassicae populations has shown that the majority of these persistent infections are by the M. brassicae nucleopolyhedrovirus (MbNPV). However, one population was persistently infected with the closely related Panolis flammea nucleopolyhedrovirus (PfNPV). Using Msatminer fourteen putative microsatellite sequences were identified in the genomes of the baculoviruses Mamestra configurata nucleopolyhedrovirus A (McNPV-A) and Mamestra configurata nucleopolyhedrovirus B (McNPV-B), both closely related to MbNPV and PfNPV. These sequences are currently being amplified and sequenced from the persistently infected populations of M. brassicae to see whether they can provide a greater resolution of the relationships between these persistent infections than can be achieved using viral gene sequences, which may be highly conserved. 1 Covert infections as a mechanism for long term persistence of baculoviruses. Burden, J.P., Nixon, C.P., Hodgkinson, A.S., King, L.A., Sait, S.M. & Hails R.S. Ecology Letters, 2003 (6) 524-531.
Umbravirus-luteovirus mutualism in wild carrotM.Naylor (1), D.W.Pallett (1), M-L.Edwards (1), H.C.Godfray (2), J.I.Cooper (1) and H. Wang (1)
Genetic and biological diversity of Turnip mosaic virus in wild cabbage (Brassica oleracea subsp. oleracea) populations in the U.K.P. J. HUNTER*, C. OBERMEIER*, K. TOMIMURA†, R. HIROTA†, K. OHSHIMA†, M. NAYLOR‡ and J. A. WALSH* *Horticulture Research International, Wellesbourne, Warwick, CV35 9EF, UK †Laboratory of Plant Virology, Saga University 840-8502, Japan ‡CEH Oxford, OX1 3SR, UK The relative incidence, pathotype, coat protein gene nucleotide and amino acid sequences of Turnip mosaic virus (TuMV) isolates from populations of wild cabbage (Brassica oleracea ssp. oleracea) at three clustered sites in southern England, one site in northern England and one site in north Wales were compared. Incidences of infection ranged from 4% at one of the sites in southern England to 25.4% at another of the sites in southern England. All isolates from the 3 clustered sites in southern England and one distant site in northern England analysed belonged to pathotype 1, whereas all the isolates analysed from Wales belonged to pathotype 3. Phylogenetic analysis of nucleotide sequences of the coat protein-coding region of seven isolates from each site showed little variation. There was more variation in isolates from the two geographically separated sites; two distinct coat protein genotypes of TuMV were identified within each of these sites. All the isolates were associated with the ‘world-B’ group most of which have been isolated from brassicas supporting the hypothesis of host-specific adaptation in TuMV.
The Molecular Characterisation of Turnip Rosette Virus.Jonathan Reeves CEH Oxford The previously undetermined Turnip Rosette Sobemovirus (TrosV), isolated from Ringstead in Dorset, was propagated in tendergreen mustard plants. The RNA was extracted and poly (A) tailed from which cDNA was made and then cloned. The 5’ race system was used to clone the extreme 5’ end of the genome. The clones were then sequenced and the sequences assembled. The genome of the virus consists of 66 bp of 5’ untranslated region, 134 bp of 3’ untranslated region and 4 open reading frames (ORF); ORF 1, 2a, 2b and the coat protein. ORF’s 2a and 2b are overlapping and the polyprotein is expressed using a frameshift mechanism. The coat protein sequence was then used as a focus of analysis when TrosV was passaged through a number of generations of differing hosts and mutations in the coat protein recorded. Some host specific and non-host specific non-random substitution mutations were observed. More interestingly a frameshift causing, non-random insertion mutation at position 649 in the coat protein was found. From the crystal structure of four characterised Sobemovirus coat proteins, it was predicted that the coat protein sub-unit of the 649 mutant would lose two calcium binding sites. Whether or not the entire binding site would be lost, or whether a water molecule acting as ligand will be enough to keep the site, is unknown.
MVX DISEASE AND dsRNA ELEMENTS IN AGARICUS BISPORUSAdriana Soares, Sarah Holcroft, Inyoung Choi, Helen Grogan, Mike Challen, Peter Mills. Horticulture Research International, Wellesbourne, Warwick, CV35 9EF, UK Double-stranded RNA (dsRNA) elements are routinely associated with the MVX disease of Agaricus bisporus. MVX comprises a wide range of symptoms including; bare cropping areas on commercial beds (primordia disruption), crop delay, premature veil opening, off- or brown-coloured mushrooms, sporophore malformations and loss of crop yield and/or product quality. The 26 MVX dsRNA elements range between 640 bp and 20.2 kbp; three of which (16.2, 9.4 and 2.4 kbp) are routinely found in mushrooms asymptomatic for MVX. Hitherto, sequence information generated from dsRNAs shows novelty. Work is in progress to characterise the MVX dsRNA elements, identify the type and number of viruses and, to develop PCR diagnostics and control strategies for the industry. Progress in molecular characterisation and studies of in vitro transmission will be reported. Thursday 19th FebruaryCharacterisation of cyanophages associated with Baltic Sea Nodularia and Synechococcus populationsCaroline Jenkins and Paul Hayes. School of Biological Sciences, University of Bristol, BS8 1UG Cyanophages, bacteriophages that infect cyanobacteria, are found in association with their hosts in both marine and freshwater environments. We have isolated a collection of 30 lytic cyanophages from the brackish Baltic Sea that infect either the filamentous cyanobacterium Nodularia or the picoplanktonic cyanobacterium Synechococcus. We have also isolated, from plaques that appear spontaneously in lawns of stressed host cells, a collection of thirteen cyanophages that could be temperate. Transmission electron microscopy has revealed that both the lytic and the potentially temperate cyanophages in our collection are members of the Cyanomyoviridae, Cyanopodoviridae or Cyanosiphoviridae. The genetic diversity of these cyanophages was investigated through the amplification and partial sequencing of a T4 g20 homologue (the gene that encodes the portal vertex protein): this portal vertex protein-encoding gene is conserved among the Baltic Sea isolates and cyanophages from various aquatic environments.
Photosynthesis genes in Synechococcus phagesMartha Clokie, Andy Millard, Shaun Bailey, Nick Mann Department of Biological Sciences, University of Warwick, Gibbet Hill Road Coventry, CV4 7AL, U. K. We have found photosynthesis genes in a large fraction of phage that infect the cyanobacteria Synechococcus. We are interested in determining whether the acquisition of these genes is a rare or a common and we are attempting to establish their ecological significance. Plants and cyanobacteria have two photosynthetic reaction centres, PSI and PSII. The PSII is where an electron is removed from a water molecule and water is converted to molecular oxygen. Two large proteins are at the core of this reaction centre. They are called D1 and D2 and are encoded by genes psbA and psbB. We have found both of these genes in cyanophage genomes. We have complete sequence information for the D1 region and the genes either side of it for five phages which were collected from a variety of geographical locations. A phylogenetic analysis of the D1 genes is presented, and possible relationships discussed. Genetic organisation of the D1 region is highly variable in the different phages. Futhermore, some phage D1 genes have a small group I intron at the 3’end. The ecological significance of these genes is being investigated by looking at the expression of mRNA and protein throughout an infection cycle. Protein labelling experiments have shown markedly different profiles expression throughout infection. Real-time PCR is being used to establish at which point expression of D1 from the host is terminated and if phage expression occurs.
An insight into the virus-host dynamic during an Emiliana huxleyi bloom. Schroeder, D. C. 1, Biggi, G. F.1, Hall, M.1, Davy, J. E.1, Malin, G. 2 & Wilson, W. H. 1 1Marine Biological Association, The Laboratory, Citadel Hill, Plymouth, PL1 2PB 2School of Environmental Sciences, University of East Anglia, Norwich, NR4 7TJ Emiliania huxleyi is a marine coccolithophorid, which has a global distribution. E. huxleyi is an important species with respect to past and present marine primary productivity, sediment formation and climate change. E. huxleyi-specific viruses (EhV) were isolated from E. huxleyi blooms off the coast of Plymouth, UK and from an E. huxleyi bloom induced during a mesocosm experiment in a fjord near Bergen, Norway. Denaturing gradient gel electrophoresis was used as a tool to elucidate both the viral and host community structures at different stages during the progression of a viral induced collapse of an E. huxleyi bloom.
Coral viruses: electron microscope artefacts or something to take seriously?Willie Wilson1,2, Simon K. Davy3, Joanne E. Davy4, Isobel Francis5, Cornelia Muncke5, Sarah Burchett5, Amy Dale1,6 & Piers Davies1 & Jayme Lohr2,6 1Marine Biological Association of the UK, Citadel Hill, Plymouth, Devon, PL1 2PB. 2Plymouth Marine Laboratory, Prospect Place, The Hoe, Plymouth, PL1 2PB 3School of Biological Sciences, Victoria University of Wellington, PO Box 600, Wellington, New Zealand. 4Centre for Marine Studies, University of Queensland, Brisbane, QLD 4072, Australia. 5Institute of Marine Studies, University of Plymouth, Devon, PL4 8AA. 6Department of Biological Sciences, University of Plymouth, Devon, PL4 8AA. Previously, we have demonstrated that symbiotic dinoflagellates (zooxanthellae) in the temperate sea anemone Anemonia viridis harbour a latent virus, which enters into a lytic cycle at elevated temperatures and contributes to the degeneration of its zooxanthella host. However, evidence for viruses in tropical zooxanthellae and their hosts, such as reef corals, has been lacking. Here, we screened for viruses in the reef corals Pavona sp. and Acropora sp., as well as in the tropical zoanthid Zoanthus sociatus. These anthozoans were stressed (i.e. bleached) at elevated temperatures (³32 oC) and thin sections prepared for transmission electron microscopy (TEM). TEM revealed numerous hexagonal virus-like particles (VLPs) both in the zooxanthellae and in the animal host’s tissues. Subsequently, the seawater from around bleaching Acropora sp. was sampled and analysed by Analytical Flow Cytometry (AFC) for the presence of VLPs. AFC showed a distinct group of virus sized particles that did not appear in seawater from around non-bleaching Acropora sp. TEM, however, showed these VLPs to be ‘droplet’ shaped rather than hexagonal. Considerable work is still required though to fully characterise these viruses and to determine their infection mechanism, but the potential for viruses to infect reef corals is clear and such infections may play an important role in determining the health of reef systems in the face of global climate change.
Host-phage interactions in the marine environmentNick Mann University of Warwick. The interactions of lytic bacteriophages with their hosts in the oceans is likely to be far more subtle than a simple infection-lysis cycle. Even the most basic questions relating to contact rates between phages and their hosts have not been satisfactorily answered yet. However, it is these contact rates that are likely to have been one of the most potent selection pressures on phage evolution. Furthermore, hosts in the oligotrophic central gyres of the oceans are subject to chronic starvation and the physiological status of the host can have profound effects on the course and outcome of phage-host interactions, beginning with adsorption and ending with lysis. In this context control of lysis timing in general and the pseudolysogenic response in particular are likely be key strategies for parasites of the starved. These ideas are discussed in both a theoretical and experimental context.
Lambda integrase gene family: markers for phage diversity, environmental regulation, and gene transfer in freshwater bacteria.C. Balding, S.A. Bromley, R.W. Pickup and J.R. Saunders Temperate bacteriophages play an important role in generating microbial variation and diversity. The integrase gene family will be used to detect temperate bacteriophages in a population of freshwater bacteria, and to monitor their activity and diversity. The integrase family comprises a wide range of diverse genes. The integrase genes encoded by enterobacterial phages were aligned, and divided into nine sub-groups. Degenerate PCR primers have been designed to each sub-group, to detect all known integrase sequences from temperate enterobacterial phages. Priest Pot is being used as the main field site for the detection of temperate bacteriophages in the environment. Lysogenic Escherichia coli colonies have been isolated from Priest Pot, and the primer sets are being used to detect the bacteriophage integrase genes in these lysogens.
The VT-phage infection process in Escherichia coli.Darren Smith, Paul Loughnane, Paul Fogg, Jon Saunders, Alan McCarthy & Heather Allison. School of Biological Sciences, University of Liverpool. Lysogenic bacteriophages are a major vector for the horizontal transfer of genetic information, including virulence factors. Verocytotoxigenic bacteriophages (VT-phages) encode toxins (VT1 or VT2) within the late gene region that are important virulence factors for enteroheamorrhagic E. coli (EHEC/VTEC). VTEC are a serious global health concern because infection can lead to haemorrhagic colitis (HC) and haemolytic uremic syndrome (HUS), which may be fatal. We have shown that at least some VT-phages, although categorised as lambdoid-like, do not conform to the lambda bacteriophage model. Firstly, they use a different receptor for infection which is located on the outer membrane and highly conserved across the Enterobacteriacae, but analogues with differing degrees of homology can be found in all Gram negative bacteria. Furthermore, we recently demonstrated that a single VT-phage can multiply infect an E. coli host cell to produce a multiple lysogen, which is a direct contradiction to the established lambda bacteriophage infection model. These features would appear to be relevant to the rapid evolution and spread of VT-genes amongst E.coli populations.
Genetics of the phage growth limitation system in Streptomyces coelicolor A3(2).Jo Leafe, Paul Sumby and Maggie Smith1 Institute of Genetics, University of Nottingham, Queens Medical Centre, Nottingham NG7 2UH, UK. 1Present address; Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD. The phage growth limitation (Pgl) system, encoded by Streptomyces coelicolor A3(2), confers protection against the temperate bacteriophage fC31 and its homoimmune relatives. The Pgl phenotype is characterised by the ability of Pgl+ hosts to support a phage burst on initial infection but subsequent cycles are severely attenuated. Previously two adjacent genes pglY and pglZ were shown to be required for Pgl1,2. We have described two novel genes, pglW and pglX, that are also part of this system3. pglW encodes a 169 kDa protein that includes putative motifs for both serine/threonine protein kinase activity and DNA binding. pglX encodes a 136 kDa protein with putative adenine-specific DNA methyltransferase activity. S1 mapping of transcripts initiating upstream of pglW indicated that, like pglYZ, pglWX are expressed in uninfected cultures. A plasmid encoding pglWXYZ was able to confer the Pgl+ phenotype to S. lividans implying that these four genes constitute the whole system. Recent work addresses the mechanism of phase variation in which the switch from Pgl+ to Pgl- and vice versa occurs at a high frequency (approx. 10-3 to 10-4). We also describe attempts to identify the target of Pgl. 1 Bedford, D.J. et al. (1995) Two genes involved in the phase-variable phi C31 resistance mechanism of Streptomyces coelicolor A3(2). J Bacteriol 177 (16), 4681-4689 2 Laity, C. et al. (1993) Genetic analysis of the phic31-specific phage growth limitation (Pgl) system of Streptomyces coelicolor A3(2). Molecular Microbiology 7, 329-336 3 Sumby, P. and Smith, M.C. (2002) Genetics of the phage growth limitation (Pgl) system of Streptomyces coelicolor A3(2). Mol Microbiol 44 (2), 489-500
Tools for genetic and functional genomic analysis of the plant and animal pathogens, Erwinia and Serratia: fun with phages – new and old.Ian Foulds, Jinghua Guo, and George Salmond. Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QW, UK We are interested in diverse biological features of specific strains of the Gram-negative phytopathogen, Erwinia and the opportunistic animal and insect pathogen, Serratia. In addition to exhibiting virulence, our strains also make bioactive secondary metabolites such as the b-lactam antibiotic (1-carbapen-2-em-3-carboxylic acid; carbapenem [Car]) and the red tripyrolle antibiotic, prodigiosin (Pig). Genome sequence information is now available for some of the strains we are investigating and so we require facile and productive systems for functional genomic analysis in these strains. In addition, we have been investigating other strains of Erwinia and Serratia for which there is still no genomic sequence information. Therefore we have developed a series of genetical tools for the analysis of multiple features in the biology and physiology of these pathogens. We have isolated a spectrum of bacteriophages that infect our chosen strains and have used these phages for the dissection of various processes, including bacterial quorum sensing, antibiotic and pigment synthesis and regulation, protein secretion and virulence. In this short presentation, we will describe the use of our phages in making and analysing Erwinia and Serratia mutants and we will outline a new project area on the interactions between the pathogen, an extrachromosonal replicon and the world of phages.
Migration of enteric viruses in deep aquifers; intergranular processes, sorption and survivalK.E. Collins*, E.M. Joyce, A..A. Cronin, J. Rueedi, S. Pedley, Robens Centre for Public and Environmental Health, Building AW Floor 2, University of Surrey, Guildford GU2 7XH, UK. Tel. (0044) 01483 684581, Fax. (0044) 01483 689971, Email k.brown@surrey.ac.uk J.H. Tellam, K. Parker, R. Greswell, M. Riley, S.H. Rahman, Earth Sciences, School of Geography, Earth and Environmental Sciences, University of Birmingham, Birmingham, B15 2TT UK, *Corresponding Author: Katie Collins Recent findings show that deep intergranular flow-dominated systems cannot be assumed to be virus-free. This has major implications for public health, especially with regard to land use policies relating to sewage disposal, landfill, other waste disposal facilities, abattoirs, and even agricultural burials of infected animals. If the risk is to be assessed it is necessary to gain a quantitative description of virus migration. Ultimately, this needs to be based on field scale experiments that can take into account large-scale heterogeneities and the presence of fractures. In order to allow interpretation of such tests, laboratory investigations of intergranular migration processes are required. In addition, pathogenic viruses cannot be used in field tests, and it is therefore necessary to establish which non-pathogenic viruses (e.g. bacteriophages) could be used as surrogates for specific groups of pathogenic organisms (e.g. enteric viruses). Collaboration between the Robens Centre for Public and Environmental Health (RCPEH) at the University of Surrey and the University of Birmingham aims to determine the transport and survival of enteric viruses through UK Triassic sandstone aquifer cores. The Birmingham team is concerned with the construction of sandstone columns, silica colloid interactions and modelling of virus transport. In parallel to this, the RCPEH are working on laboratory-scale investigations to examine the possible use of bacteriophages as surrogates for enteric viruses and the specific survival and transport of both pathogenic viruses and bacteriophage within the sandstone columns. To date little information is available regarding the transport, attenuation, and survival of pathogenic viruses in groundwater systems. This inhibits attempts to model virus behaviour. This collaboration aims to resolve these unknown issues by developing a quantitative description of virus-matrix sorption, virus-colloid interactions and the effects of these on virus transport and infectivity. Three different bacteriophages (MS2, fX174 and PRD1) are being investigated for their similarities to enteric viruses (poliovirus, rotavirus, enteric adenovirus and norovirus) for use as surrogates. Laboratory scale column and batch experiments are being carried out to identify specific characteristics that will help to predict enteric virus movements and persistence within sandstone aquifers. Batch experiments have been set up to measure the survival and adsorption characteristics of enteric viruses and bacteriophage. Adsorption measurements are to include the presence and absence of silica colloids. Column work is to be carried out in a controlled environment to mimic field conditions. This work is being carried out with research funding from the NERC.
Fate and transport of phage as surrogates for pathogenic viruses in UK aquifersE.M. Joyce*, K. Collins, A.A. Cronin, J. Rueedi, S. Pedley, Robens Centre for Public and Environmental Health, Building AW Floor 2, University of Surrey, Guildford GU2 7XH, UK. Tel. (0044) 01483 684581, Fax. (0044) 01483 689971, Email e.joyce@surrey.ac.uk A.J. Hart, Environment Agency, Olton Court, 10 Warwick Road, Solihull, West Midlands, B92 7HX, UK. *Corresponding Author: Eadaoin Joyce Recent challenges with microbial contaminants entering the subsurface have highlighted the lack of reliable information in assessing the fate and transport of pathogens within groundwater systems. Additionally, recent enteric virus monitoring in urban areas of the UK have demonstrated deep porous systems cannot be assumed to be virus free (Environment Agency 2002, Microorganisms in groundwater: tracers and troublemakers. R&D Technical Report P2-290/TR, Bristol, UK - ISBN 1857059441). These findings have major implications for public health, with regard to groundwater protection and land use management policies, especially those relating to sewage leakage and disposal as well as possible contamination from landfills and cemeteries. There is also a need to be able to understand the survival and transport characteristics of pathogenic viruses in the environment in preparation for future emergency situations, such as a repeat of the 2001 Foot and Mouth epidemic. As pathogenic viruses cannot be legally released into aquifers, this work will incorporate the use of conservative tracers and bacteriophage as surrogates for pathogenic viruses to determine their fate and transport characteristics in a typical UK aquifer (Triassic sandstone). The selection of the most appropriate surrogate bacteriophage for particular viruses has been identified by both literature reviews and associated laboratory work. The latter will quantify the processes at the micro scale (cm’s), including issues associated with colloidal transport of viruses while the majority of this work involves tracer testing at a field-scale (meters). After testing the field site, bacteriophage will be introduced directly into the groundwater so that the fate and transport responses can be monitored over specific time periods. This will allow relative phage transport breakthrough to be compared and evaluated. The laboratory and field data will supplement and validate the information collected in the background literature review. This information will be used to develop a database of viral properties providing a matrix to compare the physical properties of the virus against those of the phage surrogate tested in the subsurface. This information can be then used in environmental management policies and future emergency situations or epidemics by allowing quick hypothesis-testing of phage properties and behaviour in the subsurface to allow inferences be drawn with the virus of interest. The project is currently ongoing and due to report in August 2005.
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