The two regimens have different tolerability profiles; therefore, costs for managing adverse events may be an important factor in selecting therapy.Bevacizumab plus interferon-Costs of managing adverse events affecting patients with metastatic RCC eligible for treatment with bevacizumab plus IFN or sunitinib were evaluated using a linear decision analytical model. Management costs were calculated from the published incidence of adverse events and health-care costs for treating adverse events in the United Kingdom, Germany, France and Italy.vs €804), Germany (€1785 vs €1367), France (€2590 vs €1618) and Italy (€891 vs €402). The main cost drivers were lymphopaenia, neutropaenia, thrombocytopaenia, leucopaenia and fatigue/asthaenia for sunitinib; and proteinuria, fatigue/asthaenia, bleeding, anaemia and gastrointestinal perforation for bevacizumab plus IFN.Adverse event management costs were higher for sunitinib than for bevacizumab plus IFN. The average cost per patient for the management of grade 3–4 adverse events was markedly lower with bevacizumab plus IFN compared with sunitinib in the United Kingdom (€1475 The costs of managing adverse events are lower for bevacizumab plus IFN than for sunitinib. The potential for cost savings should be considered when selecting treatments for RCC. Renal cell carcinoma (RCC) is the most common cancer of the kidney, with an incidence of 40 000  annually and accounting for 26 000 deaths  in Europe  significantly improved median progression-free survival =0.63, P=0.0001) and objective response rate  compared with IFN plus placebo  and ORR  (Bevacizumab (Avastin) is a humanised monoclonal antibody that precisely inhibits vascular endothelial growth factor (VEGF), the key mediator of tumour angiogenesis. In combination with cytotoxic chemotherapy, bevacizumab has demonstrated significant clinical benefits in several tumour types including metastatic colorectal  and median PFS  when compared with IFN , an orally active inhibitor of multiple receptor tyrosine kinases including VEGF receptors 1–3 and platelet-derived growth factor receptor-The results of these recent phase III trials in RCC suggest that the efficacy of bevacizumab plus IFN is comparable with that of sunitinib in the first-line treatment setting . These dvs 44%), while efficacy was maintained.The most frequently reported grade 3–4 adverse events in phase III trials of bevacizumab plus IFN include fatigue and asthaenia, hypertension, anorexia, bleeding, pyrexia and proteinuria; the majority of these are mild to moderate and manageable, and only a low incidence of grade 3–4 events is observed . The primary perspective of this analysis was the same as that of the UK National Health Service.βstab (EBM) catalogue 2008  funding system catalogue (2008) and the from Einheitlicher BewertungsmaIn France, the cost of drugs was obtained from the Banque Claude Bernard database  and fromThe assessment for Italy used information on the cost of treating adverse events from the report of a Delphi panel of experts from five clinical practices  and dataThe base-case analysis conducted using the decision analytical model included all grades of adverse events, using as a threshold the cumulative total of events responsible for ⩾80% of total management costs. An additional scenario analysis was also conducted based only on the costs of managing grade 3–4 adverse events. As modelling studies are associated with uncertainty associated with input parameters, the robustness of the model was tested using sensitivity analyses based on varying hospitalisation costs within plus or minus 10% and by excluding the two most costly adverse events for bevacizumab plus IFN and with sunitinib. In Germany, medication costs are not included in the EBM cost estimates. Therefore, in Germany only, additional sensitivity analyses were conducted: first, 5% was added to the ambulatory medication costs to capture the costs of medications used to manage adverse events, and second, the effect of assigning a cost of €0.03 or €0.06 per EBM physician point was investigated.The average cost per patient of managing all-grade and grade 3–4 adverse events varied across the countries assessed . The linA similar trend of higher management costs per patient with sunitinib compared with bevacizumab plus IFN was observed for grade 3–4 adverse events . In the The main drivers of adverse event management costs for sunitinib and bevacizumab plus IFN were generally consistent across the countries examined . NeutropSensitivity analyses based on a 10% difference in hospitalisation costs and excluding the costs of treating the principal adverse events associated with treatment were consistent with the overall analyses and demonstrated that bevacizumab plus IFN remained the least expensive treatment with respect to costs for managing adverse events compared with sunitinib . AdditioThe results of the original analysis for the United Kingdom, Germany and France indicated that the vast majority of management costs for adverse events was associated with the development of grade 3–4 events. On this basis, only grade 3–4 adverse events were assessed for management costs in Italy (The average cost per patient of managing grade 3–4 adverse events for sunitinib (€891) was higher than that for bevacizumab plus IFN (€402); this difference represents an average cost saving of €489 (55%) per patient . ConsistSensitivity analyses were consistent with the main results and confirmed the cost savings of bevacizumab plus IFN in Italy. A 10% difference in hospitalisation costs and excluding the costs of treating the principal adverse events associated with sunitinib and bevacizumab plus IFN showed cost savings that were consistent with those of other countries .The treatment of metastatic RCC has been transformed by the recent introduction of molecularly targeted agents. Sunitinib and bevacizumab, in combination with IFN, are approved for first-line treatment of patients with metastatic RCC and recent clinical studies demonstrate that they provide comparable levels of efficacy  showed that the model remained stable over the entire range of plausible values for a given parameter, and was therefore robust. The main cost drivers for sunitinib were lymphopaenia, neutropaenia, thrombocytopaenia, leucopaenia and fatigue/asthaenia. In contrast, the main cost drivers for the management of adverse events associated with bevacizumab plus IFN were fatigue/asthaenia, proteinuria, bleeding, anaemia and GI perforation. The majority of the increased cost associated with sunitinib was related to the management of haematological toxicities, which accounted for little or none of the cost of managing adverse events associated with bevacizumab plus IFN; chemotherapy-related haematological adverse events are associated with an economic burden due to costly hospitalisation/treatment costs and negatively affect patient quality of life (The management strategies available for dealing with adverse events due to sunitinib and bevacizumab plus IFN may also be relevant when choosing first-line treatment for RCC. Reducing the dose of IFN used in combination with bevacizumab substantially improves the tolerability and management of IFN-related adverse events, enabling patients to remain on therapy while maintaining efficacy , suggestThis study considered the cost of adverse event management for bevacizumab plus IFN and sunitinib in the United Kingdom, Germany, France and Italy. The average adverse event management costs varied across these countries but showed an overall trend of consistently lower costs for bevacizumab plus IFN versus sunitinib. Management costs varied between the countries, but country-specific cost calculations/tariff lists provide a probable explanation. In addition, direct costs from a prospective study may be needed to confirm the cost savings observed in this study. The linear decision analytical model used in this study used health-care costs according to standard clinical practice from a variety of sources and relied on adverse event data from individual clinical trials that may not be fully comparable or reflective of adverse events in daily clinical practice. In addition, the linear decision analytical model did not permit a statistical analysis of the cost differences between sunitinib and bevacizumab plus IFN nor did it permit an analysis of the effect of adverse events on treatment efficacy; additional studies may be needed to confirm these data. The study also highlights that there is no standardisation of treatment methods or costs across different countries, meaning that the potential effect of adverse event management costs should be assessed on an individual country basis. The poorly defined pathophysiology and management strategies of many sunitinib-associated adverse events may not have been captured by this analysis, that is, the potential of having to try different management approaches to identify the most effective may represent increased ‘hidden’ costs. Moreover, the analysis used costs for the management of haematological adverse events and hand-foot syndrome based on historical chemotherapy-associated costs. This could have underestimated the costs of managing sunitinib-associated adverse events. However, the utilisation of chemotherapy-associated costs will remain the standard approach until specific data for targeted therapies are available. Finally, this study did not consider drug administration costs or initial drug acquisition costs because country-specific initiatives may result in significant cost differences, making standardisation across countries difficult.In conclusion, this study demonstrated that the different tolerability profiles of bevacizumab plus IFN and of sunitinib result in markedly lower adverse event management costs for bevacizumab plus IFN. As these regimens have comparable efficacy in the first-line treatment of metastatic RCC, the predicted cost savings in relation to the management of bevacizumab plus IFN could provide benefits to physicians and payers and may be an important consideration when making therapeutic choices. These findings raise important points and potential resource benefits in the current cost-conscious oncology environment, in which there is a demand for novel agents that provide the greatest benefit.


S10-spc-α is a 17.5 kb cluster of 32 genes encoding ribosomal proteins. This locus has an unusual composition and organization in Leptospira interrogans. We demonstrate the highly conserved nature of this region among diverse Leptospira and show its utility as a phylogenetically informative region. Comparative analyses were performed by PCR using primer sets covering the whole locus. Correctly sized fragments were obtained by PCR from all L. interrogans strains tested for each primer set indicating that this locus is well conserved in this species. Few differences were detected in amplification profiles between different pathogenic species, indicating that the S10-spc-α locus is conserved among pathogenic Leptospira. In contrast, PCR analysis of this locus using DNA from saprophytic Leptospira species and species with an intermediate pathogenic capacity generated varied results. Sequence alignment of the S10-spc-α locus from two pathogenic species, L. interrogans and L. borgpetersenii, with the corresponding locus from the saprophyte L. biflexa serovar Patoc showed that genetic organization of this locus is well conserved within Leptospira. Multilocus sequence typing (MLST) of four conserved regions resulted in the construction of well-defined phylogenetic trees that help resolve questions about the interrelationships of pathogenic Leptospira. Based on the results of secY sequence analysis, we found that reliable species identification of pathogenic Leptospira is possible by comparative analysis of a 245 bp region commonly used as a target for diagnostic PCR for leptospirosis. Comparative analysis of Leptospira strains revealed that strain H6 previously classified as L. inadai actually belongs to the pathogenic species L. interrogans and that L. meyeri strain ICF phylogenetically co-localized with the pathogenic clusters. These findings demonstrate that the S10-spc-α locus is highly conserved throughout the genus and may be more useful in comparing evolution of the genus than loci studied previously. Leptospira. Spirochetes belong to an ancient branch of eubacteria, with Leptospira representing its deepest division Leptospira are genetically diverse bacteria. Genetic classification of this genus is based on DNA homology and divides pathogenic Leptospira into seven main species: L. interrogans, L. borgpetersenii, L. weilii, L. noguchii, L. santarosai, L. kirschneri and L. alexanderiL. biflexa and L. meyeri, and L. fainei and L. inadai exemplifying species with an intermediate status L. interrogans serovars Lai and Copenhageni and two strains of L. borgpetersenii serovar Hardjo has revealed the occurrence of frequent gene rearrangements and fragmentation, perhaps indicating a rapid adaptation to new environments by pathogenic LeptospiraL. borgpetersenii reflects lower environmental survivability corresponding to limited potential for indirect transmission L. interrogans, a species that frequently passes through surface water between mammalian hosts Leptospirosis is one of the most widespread zoonotic diseases in the world and is caused by pathogenic spirochetes within the genus S10-spc-α ribosomal protein cluster of L. interrogans serovar Lai fus, tuf, secY, adk and infA, code for ribosomal proteins. The secY gene codes for preprotein translocase, a gene that has diagnostic value and potential for resolving taxonomic questions in LeptospiraS10-spc-α locus differentiates Gram-positive from Gram-negative bacteria L. interrogans S10-spc-α locus is unique, as it contains all genes found in the Escherichia coli locus, and all genes except map that are found in the Bacillus subtilis locus L. interrogans S10-spc-α locus is not typical of other spirochetes; several genes found in the S10-spc-α locus of L. interrogans are translocated to different portions of the Borrelia burgdorferi and Treponema pallidum genomes Leptospira genome S10-spc-α locus is conserved amongst Leptospira, or if the genetic organization shared among Borrelia and Treponema may occur among some Leptospira species, and predate divergence of Leptospira from other spirochete genera.We previously characterized the S10-spc-α locus in Leptospira, and report that this locus is highly conserved throughout the genus. These data suggest that maintenance of the S10-spc-α operon structure is essential regardless of the extent of other rearrangements that have occurred during Leptospira evolution. Comparative sequence analysis of four segments of the S10-spc-α locus provides new information on phylogenetic relationships between pathogenic Leptospira.In this study, we examined genetic organization and content of the L. interrogans strains  for each of the 40 primer pairs tested. These data indicate that the S10-spc-α locus is well conserved in L. interrogans . However, marked strain differences were found within the species L. santarosai (8), L. noguchii (9), L. weilii (15) and L. alexanderi (14). Predictably, because genetic relatedness is used to differentiate Leptospira species, the amplification profiles varied depending on the species from which the template DNA was isolated  pathogenic status frequently failed to generate products or yielded anomalous sized amplicons. These data imply a marked divergence in the S10-spc-α sequence content from pathogenic Leptospira  were aligned with BLAST and the results visualized by ACT  a basal clade, with bootstrapping value of 81%, that includes L. fainei and L. inadai, two species with intermediate pathogenic status, and the saprophytic L. meyeri strain Veldrat Semarang 173; and 2) a sister clade, supported with a 68% bootstrap value, that contains pathogenic Leptospira species . DNA amplification of target sequences from the intermediate strains L. fainei strain BUT 6, L. inadai strain 10, and L. meyeri strain Veldrat Semarang 173 was not successful. Therefore, these strains were not included in the analysis. The sequences for the saprophytic strain Patoc I were deduced from its genome sequence P>0.1). The shortest G1–G2 fragment (245 bp) showed the highest nucleotide diversity, π value of 0.14, whereas in the 300–301 fragment π was 0.09. Congruently, the mean divergence values (D) for pairwise comparisons ranged from 0.103 to 0.171 for the 300–301 and G1–G2 fragment, respectively. The lowest phylogenetic signal was obtained for 300–301 sequences. In contrast, the 621–625 and 624–650 fragments showed a phylogenetic signal slightly higher than the combined data set  occurred in nodes showing the lowest bootstrap support in distance topologies.L. interrogans and L. noguchii, with L. kirschneri located in a basal position. This clade is consistently recovered in all topologies . The phylogenetic tree produced from secY sequence data was compared to a tree derived from the extracted sequences of the 245 bp fragment flanked by primers G1 and G2  that flank the G1 and G2 annealing sites. These primers amplify secY sequences from all pathogenic strains (data not shown).The 20-mer primers G1 and G2 amplify a 285 bp fragment of 1 and G2 . These tLeptospira species reveal extensive plasticity, including rearrangements, duplications, and disruptions of otherwise conserved segments of the genome L. interrogans strain Lai contained a large ribosomal protein locus spanning the S10, spc, and α loci identified in widely divergent eubacterial genera fus, the first gene in the operon, and comprises one of the longest known prokaryotic transcripts S10-spc-α locus is well conserved across the genus Leptospira, a finding that is somewhat remarkable given the extent of rearrangements that have disrupted synteny during Leptospira evolution. The conserved S10-spc-α organization includes the presence of the 5′ fus gene coding for elongation factor EF-G and the genes adk (adenylate kinase), infA (IF1), and rpsD (S4) located at the 3′ end of the locus, genes that are dispersed in the B. burgdorferi and T. pallidum genomes Leptospira S10-spc-α locus is unique among spirochetes Leptospira are one of the oldest branches in spirochete evolution. Conservation of the Leptospira S10-spc-α locus is in stark contrast to the unique organization of rRNA genes, where the rrs, rrl, and rrn genes are not closely linked to each other, but are dispersed throughout the larger of two chromosomes comprising the Leptospira genome Whole genome sequence analyses of different S10-spc-α locus showed a number of regions that were more consistently amplified than other regions . It should be noted that genes contained in the S10-spc-α locus are duplicated in L. borgpetersenii strain L550, but are found as unique copy genes in all other sequenced Leptospira genomes, including L. borgpetersenii strain JB197 secY genes.Three anomalies were found during comparison of the binary and sequence-based phylogenetic trees. First, the two strains of species sed tree . HoweverS10-spc-α locus includes the secY gene encoding preprotein translocase. Primer pair G1–G2 is positioned within this gene and directs amplification of a 285 bp fragment from all pathogenic species except L. kirschneriLeptospira spp. discrimination. Data generated in the present study is more comprehensive than previous reports; phylogenetic trees based on the G1–G2 segment are in accordance with rrs based trees, showing that analysis of this small fragment can be used to identify species.The S10-spc-α locus contributes to a better understanding of Leptospira evolution. Trees generated from analysis of sequence data generated here provide analysis of more conserved loci than those studied previously Leptospira from other spirochetes. Finally, from a practical standpoint, we demonstrate that analysis of a 245 bp segment of secY is suitable for rapid identification of Leptospira species.Genetic analysis of the Leptospira strains used in this study were from the reference collections of the WHO/FAO/OIE Collaborating Center for Reference and Research on Leptospirosis at KIT Biomedical Research, Amsterdam, The Netherlands, and the USDA Leptospirosis Reference Center at the National Centers for Animal Health, Ames, USA  following the manufacturer's instructions. DNA concentration was determined using a Nano-Drop-1000 spectrophotometer  and by visual comparison with Smart Ladder SF  after agarose gel electrophoresis in 1.5% agarose gels, stained with ethidium bromide according to standard procedures S10-spc-α locus were amplified by PCR from various Leptospira strains using primers listed in L. interrogans and L. borgpetersenii genome sequences L. biflexa Patoc I genome sequence before publication  using the following program: denaturation for 5 min. at 94°C, followed by 34 cycles consisting of annealing, 1 min at 52°C, primer extension, 2 min at 72°C, denaturation, 1 min at 94°C. PCR products were separated by agarose gel electrophoresis and visualized as described above.PCR amplification products were purified by QIAquick PCR purification kits (Qiagen Corp.) prior to DNA sequencing. Nucleotide sequences were determined by dye termination reactions separated on ABI Prism 310 and ABI 3700  DNA sequencers. Sequencing was done on both complementary and forward strands and repeated at least twice to obtain reliable sequence data. Sequence data were edited using Sequencher .S10-spc-α locus, for each of the analyzed species, was codified in a discrete binary 40-character matrix covering a complete set of 24 taxa, representing eleven Leptospira species . The negative skew of the distribution of three lengths, under parsimony criterion, is originated from trees with low scores based in highly informative data The presence (1) or absence (0) of correctly amplified fragments within the  species . The chaS10-spc-α locus were obtained to conduct a distance and parsimony-based phylogenetic analysis of pathogenic Leptospira using MEGA4 and PAUP* v. 4.0b10, respectively. Nucleotide diversity and diverse sequence parameters were obtained with MEGA4 D (two-tailed test) was obtained assuming that D follows the beta distribution and the confidence limits given in equation 47 and D by computer simulations using the coalescent algorithm. In distance analysis, midpoint rooted trees were obtained by the neighbor-joining method with Tamura-Nei distances rplE, rpsN, rpsH (primer pair 301–300), rplB, rpsS (primer pair 621–625), rplC, rplD (primer pair 624–650) and secY (primer pairs G1–G2)  were used for the reconstruction of phylogenetic relationships between 131 strains, using L. biflexa strain Patoc I as an outgroup. The secY sequences were stripped to a standard size of 1289 bp, whereas the sequences derived from G1–G2 were significantly shorter: 245 bp. Using this latter fragment, a comparison of monophyletic clustering and resolution of Leptospira species respect to the standard 16S rDNA data was done.Sequence data from four loci within the s G1–G2) . This daS10-spc-α locus sequences from L. interrogans, L. borgpetersenii, and L. biflexa was done using BLASTN Alignment of Figure S1Alignment of the L. biflexa, L. interrogans and L. borgpetersenii S10-spc-a genome sequences. Regions of greater than 80% sequence identity are shown as blue (between L. interrogans and L. borgpetersenii) and red (L. biflexa and L. interrogans). White regions indicate segments where sequence identity drops below 80%. Regions of similarity were determined using Blastn under default settings except the -m 8 output option was used. The display was generated using ACT. Note that the orientation of these sequences shown in the figure is consistent with the genomic sequence data in GenBank and are inverted relative to the direction of transcription. GenBank accession numbers for the genomes of L. interrogans, L. borgpetersenii and L. biflexa are AE016823, CP000348, CP000786, respectively.(2.24 MB DOC)Click here for additional data file.Table S1Leptospira strains used for the S10-spc-α locus study.(0.25 MB DOC)Click here for additional data file.Table S2All primers used for the S10-spc-α locus analysis.(0.09 MB DOC)Click here for additional data file.Table S3Amplification through the S10-spc-α operon of Leptospira spp. Positive and negative PCR scores for amplification reactions along the locus from various strains.(0.05 MB DOC)Click here for additional data file.


Neisseria meningitidis. Our analysis demonstrated that all but one of the hyperinvasive isolates established by multilocus enzyme electrophoresis and MLST were grouped into one of six genealogical lineages, each of which contained substantial variation. Due to the confounding effect of recombination, evolutionary relationships among these lineages remained unclear, even using 20 loci. Analyses of the seven loci in the standard MLST scheme using the same methods reproduced this classification, but were unable to support finer inferences concerning the relationships between the members within each complex.Despite the increasing popularity of multilocus sequence typing (MLST), the most appropriate method for characterizing bacterial variation and facilitating epidemiological investigations remains a matter of debate. Here, we propose that different typing schemes should be compared on the basis of their power to infer clonal relationships and investigate the utility of sequence data for genealogical reconstruction by exploiting new statistical tools and data from 20 housekeeping loci for 93 isolates of the bacterial pathogen  Neisseria meningitidis, the meningococcus, which is a commensal inhabitant of the human nasopharynx that occasionally invades the host, causing devastating septicaemia and meningitis ; carB (carbamoyl phosphate synthase large subunit); dhpS (dihydropteroate synthase); glnA (glutamine synthetase); gpm (phosphoglycerate mutase); pyk (pyruvate kinase); rpiA (ribose-5-phosphate isomerase A); and tal  . Missing data, namely the sequences of the eight novel gene fragments for 14 isolates as well as that of gpm for isolate BZ 147, were represented by a string of Ns in the ClonalFrame input file. The values of r/m (the ratio of rates at which a given nucleotide becomes substituted through recombination and mutation) and ρ/θ (the ratio of rates at which recombination and mutation events occur), as well as the relative age of the groupings, were estimated using ClonalFrame.The principal analysis tool was Csion 1.1 , a statikelihood , using tkelihood , and usison tool . The conN. meningitidis) was assessed by comparison with the reconstruction based on the nucleotide sequences from all 20 loci (∼4.3 % of the entire genome). Assuming the genealogy reconstructed using the 20 loci to be predominantly correct, this gave a measure of inferential power of the seven-locus dataset. We also considered the support given by the 20-locus analysis to the clusters found by the seven-locus analysis. Under the same assumption, this gave a measure of the accuracy of the inference based on the seven-locus (i.e. MLST) dataset. Taken together, these two measures, power and accuracy, revealed the quality of the genealogical reconstruction based on the seven MLST gene fragments.The quality of the genealogy reconstructed on the basis of the seven MLST loci  to 561 bp (rpi) in length with between 11 (aspA and glnA) and 33 (dhps) alleles per locus in the 93 isolates examined (Table 1gpm (5 % polymorphic sites) and the most diverse was dhps (21.2 % polymorphic sites); alternations in this gene confer resistance to sulphonamides  to 0.273 . Allele numbers were assigned to each unique gene sequence and the data are available at http://pubmlst.org/neisseria  and N45/96 (ST-41/44 complex) failed at all loci for technical reasons and no sequence was obtained for the lonalFrame using 20 gene fragments clustered 78 of the 107 isolates into six lineages  that they predominantly contained, which replicated MLST clonal complex designations. All of the isolates previously thought to belong to hyperinvasive lineages were located in one of these lineages, with the exception of isolate 322/85 (ST-2), which was characterized as lineage VI by MLEE  was the monophyly of ST-4. A less conservative cut-off for the interpretation of the seven-locus results generated several misassignments. Furthermore, many relationships found with strong confidence by the 20-locus analysis were not inferred based on only seven loci, with 16 phylogenetic relationships not found at all (support of 0/10) and five found with weak statistical support (between 1/10 and 4/10). The seven-locus analysis therefore had low power to infer relationships within the six hyperinvasive lineages represented in this dataset.The number of clusters found by the 20-locus analysis approximately followed the coalescent expectation , b, exceThe utility of pathogen typing methods principally depends on the genealogical information they provide. Investigation of both disease outbreaks and longer-term epidemiological phenomena relies on distinguishing isolates that share a common ancestor from those that do not. For short-range epidemiology, typing methods are ideally highly discriminatory , so thatdhps and gln were also found in ST-66, and together provided strong evidence that the ST-8 strain above branch A was more closely related to ST-66 than it is to any of the other ST-8 strains in the sample   : membersly 2009) .


Stenella coeruleoalba, were found dead along the coast of the Spanish Mediterranean. Of 10 dolphins tested, 7 were positive for a virus strain closely related to the dolphin morbillivirus that was isolated during a previous epizootic in 1990.In July 2007, >100 striped dolphins,  Morbillivirus, the dolphin morbillivirus (DMV), killed thousands of striped dolphins  in the Mediterranean Sea during 1990–1992  in 2001–2003 was again close to the maximum reported for this species in the western Mediterranean . Stranding rate was also similar during each episode, with an initial low rate at the beginning of July, ≈2 weeks with no stranded dolphins reported, and then a sharp increase of stranding in mid-August (panel B). The most apparent difference between each episode was the size of animals collected: the mean size ± SD of dolphins collected in 1990 and 2007 was 180.9 ± 28.6 cm (n = 34) and 159.9 ± 40.9 cm (n = 17), respectively. This difference is statistically significant .In the Gulf of Valencia, the number of stranded animals during July through August 2007 was similar to that recorded in 1990 during the same months , panel Ad-August , panel BWe examined 10 dolphins (5 adults and 5 juveniles) immediately after their death and collected samples of brain, lung, spleen, liver, and lymph nodes for histologic and molecular studies. Immunohistochemical examination for DMV antigen was performed. A monoclonal antibody to canine distemper virus , known to react with DMV, was used as primary antiserum at a dilution of 1:200. The secondary antibody, a biotinylated goat anti-mouse immunoglobulin serum, was used at the same dilution. Finally, the avidin-biotin peroxidase complex was incubated at a dilution of 1:100. Sections were counterstained with hematoxylin.panel A), lymphoid cells, neurons, and/or bile duct epithelium. The staining pattern was similar to that described for animals affected by the 1990 epizootic , was found concomitantly with DMV infection. For 5 dolphins, immunostaining was observed in epithelial cells and syncytia in the lungs , panel Actgcactraayttgttytgrayngagt-3′ [position 1044–1071] confirmed the other samples as positive.Tissues from the 3 dolphins analyzed by immunohistochemistry were also examined for morbillivirus nucleic acid by reverse transcription–PCR (RT-PCR). We used nested sets of universal morbillivirus primers  based on the conserved N terminus of the morbillivirus N gene. Tissue samples from 1 dolphin tested positive by N1/N2, and the product was sequenced in its entirety. PCR with nested primers  (the primer sequences having been removed); phylogenetic studies were performed with the MEGA (version 4) package (www.megasoftware.net). This alignment indicated that this strain was very closely related to the virus that caused the epizootic in 1990 (panel B). Overall, 7 of the 10 dolphins  examined during this study were positive for DMV by immunohistochemistry or PCR. PCR products spanning other viral genes are currently being generated and sequenced to develop a more detailed phylogenetic analysis of the virus currently circulating.The N1/N2 sequence (European Molecular Biology Laboratory accession no. EU124652) was aligned to the same region for each of the morbilliviruses with ClustalW , have followed a similar course of infection. However, in the current epizootic, younger animals were apparently more severely affected by the disease. Although these results are based on stranded dolphins and stranding rates do not necessarily correlate directly with death rates (These results show that DMV is again circulating in the Mediterranean population of striped dolphins. The new epizootic resembles the previous die-offs in 1990: both epizootics began in approximately the same region and at the same time and, according to the stranding patterns , panel BThese observations further indicate that DMV did not persist as an enzootic infection in the Mediterranean striped dolphins after the 1990–1992 epizootic, that adult dolphins that had survived the first epizootic still had some immunity against the virus, and that unpredictable epizootics may recur. The relatively high density of striped dolphins, their gregarious behavior, and the decreasing level of specific immunity will likely favor the propagation of the virus among the entire Mediterranean population. For this reason, abnormally high die-offs might be expected in other areas of the Mediterranean Sea in the coming months, and measures should be taken to recover and analyze the carcasses.Phoca vitulina, from northern Europe (Tursiops truncatus, from the western Atlantic and Gulf of Mexico (S. coeruleoalba.Recurrent morbillivirus epizootics in marine mammals were described in harbor seals,


Objective: The current Centers for Disease Control and Prevention(CDC) guidelines include 2 drugs, doxycycline and ofloxacin, fortreatment of the chlamydial component of outpatient pelvicinflammatory disease (PID). Although ofloxacin costs about $90more than doxycycline, doxycycline is frequently associated withside effects and patient compliance with this drug is probablypoor. Because clinicians have little information by which to judgethe tradeoffs between price and compliance for these 2antibiotics, we examined the impact of patient compliance in theevaluation of the costs and benefits of using each drug. Methods: The incidence and direct costs of PID sequelae  resulting afterpartially treated chlamydial PID were taken from previousestimates. For differing levels of antibiotic compliance, theprobability of cure, probability of the occurrence of sequelae,and the associated cost of each were calculated. Because therelationship between partial antibiotic compliance and PID cure isunknown, we included 3 plausible relationships in our analyses.The sensitivity analysis was performed by varying key assumptionsand examining the effect of each on future costs.Results: The average probability of future PID sequelae attributable tochlamydia is slightly less than 2%, with an associated cost of $1,272. With an average compliance for doxycycline of 50%, animprovement in compliance of as little as 1.8–3.5 percentage points (51.8–53.5%), depending on theassumption used regarding partial compliance and cure, would makethe use of ofloxacin less costly than doxycycline in the long run.Even with a cost difference of $90 between the 2 drugs, a10-percentage-point increase in compliance (to 60% compliance) with the more expensive drug would save $2.63 for each $1.00 spent.Conclusions: Since the long-term costs of PID are likely to overshadow the immediate cost of providing treatment, physicians should carefully consider thelikelihood of patient compliance in selecting an antibiotic.


Cryptococcus gattii emerged on Vancouver Island, British Columbia (BC), Canada, in 1999, causing human and animal illness. Environmental sampling for C. gattii in southwestern BC has isolated the fungal organism from native vegetation, soil, air, and water.C. gattii is currently established and forecast areas that could support C. gattii in the future. We also examined the utility of ecological niche modeling (ENM) based on human and animal C. gattii disease surveillance data.Our aim was to help public health officials in BC delineate where C. gattii in BC. Human and animal surveillance and environmental sampling data were used to build and test the models based on 15 predictor environmental data layers.We performed ENM using the Genetic Algorithm for Rule-set Prediction (GARP) to predict the optimal and potential ecological niche areas of p-value < 0.001) for C. gattii in BC. The models identified optimal C. gattii ecological niche areas along the central and south eastern coast of Vancouver Island and within the Vancouver Lower Mainland. Elevation, biogeoclimatic zone, and January temperature were good predictors for identifying the ecological niche of C. gattii in BC.ENM provided very accurate predictions (> 98% accuracy, C. gattii in BC. These results are shared with public health to increase public and physician awareness of cryptococcal disease in regions at risk of environmental colonization of C. gattii.The use of human and animal case data for ENM proved useful and effective in identifying the ecological niche of  Cryptococcus gattii appeared on Vancouver Island, British Columbia (BC), Canada, in 1999, causing human and animal illness  to predict the spatial distribution of C. gattii by relating the environmental characteristics of field observations to predictor variables. We used the Genetic Algorithm for Rule-set Prediction (GARP) to model the ecological niche of C. gattii in BC. We also examined the utility of ENM based on human and animal C. gattii disease surveillance data.The epidemiology, genetic characterization, geographic distribution, and environmental sampling for lsewhere . The purC. gattii have varied with the region under study. General characterizations from studies in Australia  may not yet reflect its full potential geographic range in BC .The concept of the ecological niche was proposed by A common challenge in determining the geographic distribution and range of plant and animal species is the lack of data, especially in remote areas. Extensive sampling is typically required, but in most cases, field sampling is prohibitively expensive and limited by terrain. Absence data are even more scarce because it is difficult to determine when a species does not (transiently) exist in an area . HoweverC. gattii is an invasive species, one that is expanding into ecological niches that it has not previously been known to occupy. As such, information derived from its distribution in previously known habitats cannot be used to inform the development of our ENM (contra M contra . FurtherM contra .We selected the Genetic Algorithm for Rule-set Prediction (GARP) software package for our study because it has a proven record of accuracy and performance for predicting the ecological niches of a variety of species .GARP, developed by GARP employs four types of rules to identify the ecological niche of a species: atomic, range, negated range, and logistic regression . The rulPredictive accuracy of the model—defined as the sum of all correct predictions (both presence and absence) divided by the sum of all predictions—is a function of the number of correct predictions and of errors of commission and omission . A commiWe used ArcGIS 9.1 and ArcView 3.2  to perform geographic information system (GIS) mapping. The Geostatistical and Spatial Analyst extensions were used to interpolate continuous surface layers and manipulate raster data sets for ENM in Desktop GARP 1.1.6 .C. gattii surveillance and environmental sampling data as input data to build and test the ecological niche models . A recreational grade  Global Positioning System receiver  was used to record the latitude and longitude coordinates from where the samples were collected, which was then mapped using ArcGIS.Environmental sampling for al parks . LongituC. gattii biogeography in the ENM: elevation, aspect, and slope  and in We used 15 environmental data layers believed to be relevant to C. gattii human and animal cases and environmental positive samples were used, in separate models, to train and test the respective models. GARP is able to use a variety of different data types in the ENM process because it employs a number of different rule types . The C. gattii, aspect direction, biogeoclimatic zone, and soil data sets were nominal data. The elevation, slope, temperature, and precipitation data sets were ratio data.Locations of C. gattii  were used as the ENM input data points. These locations, mapped by address of residence, do not definitively represent the presence of C. gattiiin the local area or where exposure to C. gattii occurred, but instead represent proxies for where exposure may have likely occurred because people, and especially companion animals, tend to spend most of their time and activities around their area of residence. Travel-related cases from the BC mainland and from areas currently nonendemic on northern Vancouver Island were excluded from the ENM process. In all, 122 human and 135 animal cases from endemic areas on Vancouver Island and the Gulf Islands were used in the ENM of C. gattii.Human and animal cases within the known endemic areas for C. gattii presence.We also used (384) positive environmental sampling locations from permanently established sites as ENM input data points. These locations represent confirmed sites of C. gattii ENM because the empirical data from Vancouver Island were used to forecast the ecological niche of C. gattii on the BC mainland.A small number of mainland cases , with precise location information and without travel to Vancouver Island or other endemic sites, were identified on the BC mainland. Fifty-five transiently positive environmental sampling locations, including seven samples from four sites from the BC mainland, were also identified. The locations of these mainland cases and transient positive environmental samples were used to verify the predictive accuracy of the Desktop GARP can identify which environmental factors are more important than others for predicting the ecological niche of a species by “jackknifing” environmental data layers, which involves running multiple ENM tasks with only a subset of the layers . The jacThe human and animal cases and positive environmental sampling locations were divided into training (70%) and testing (30%) sub-data sets. All 15 environmental data layers were jackknifed individually through 30 model runs, for a total of 450 model runs for each scenario. Each model run consisted of a maximum of 1,000 iterations or until the convergence limit of 0.01 was reached.C. gattii was performed using only the significant environmental data layers identified in the jackknifing procedure. For these final runs, the input C. gattii data sets were again divided into training (70%) and testing (30%) sub-data sets, and 20 model runs were performed. A separate binary predictive layer was created from each model run with training and testing accuracy > 95%; cell values of 1 indicate locations with appropriate ecological conditions to support C. gattii, whereas values of 0 indicate unsuitable locations. “Optimal” C. gattii ecological niche areas had 11–20 model output grid data layer agreement, and “potential” C. gattii ecological niche areas had 1–10 model output grid data layer agreement.A final ENM of p < 0.001) and small commission and omission errors, based on jackknifing the environmental data layers with the distribution of C. gattii cases and positive environmental sampling locations as the dependent variables. July minimum temperature and July total precipitation also had training and testing accuracy > 95% (p < 0.001) based on the jackknifing of environmental data layers with the distribution of animal cases.The elevation, biogeoclimatic zone, and January average, maximum, and minimum temperature data layers had training and testing accuracy > 95%  and Conversely, across all scenarios, aspect, slope, July maximum temperature, annual total precipitation, soil drainage, and soil development were poor predictor variables for modeling the ecological niche of C. gattii ecological niche prediction maps for each ENM scenario: human cases, animal cases, and positive environmental sampling locations , and Vancouver Lower Mainland (N49°15′ W123°00′). The C. gattii ecological niche predictions based on the environmental sampling locations produced the most conservative results. For example, potential C. gattii ecological niche areas on the Queen Charlotte Islands (N53°00′ W132°00′) and central BC coast are reduced or absent in the positive environmental sampling locations results . We overlaid the 11 human and 13 animal C. gattii cases and 4 positive environmental sampling sites on the BC mainland with the forecasted C. gattii ecological niche models.We used the nontravel  human and animal C. gattii ecological niche areas based on the ENM scenario using the distribution of human C. gattii cases to construct the model. The remaining four animal cases and one positive environmental sampling site were located within 2.5 km of the nearest forecasted C. gattii ecological niche areas.All 11 human cases, 9 of 13 animal cases, and 3 of 4 positive environmental sampling sites on the BC mainland were located in the forecasted C. gattii cases to construct the model, all 11 human cases, all 13 animal cases, and all 4 positive environmental sampling sites on the BC mainland were located in the forecasted C. gattii ecological niche areas , daily January average temperatures > 0°C, and presence within the coastal Douglas fir and the very dry regions of the coastal western hemlock biogeoclimatic zones are strongly associated with the ecological niche of C. gattii. Of particular note is the association of daily January average temperatures > 0°C within the forecasted ecological niche areas of C. gattii, and the general absence (or transiently positive nature) of C. gattii in geographic areas where the daily January average temperatures are < 0°C. This finding suggests that C. gattii may not colonize regions with prolonged, below-freezing temperatures.C. gattii based on the distribution of animal cases to construct the model also identifies mild daily July minimum temperatures (11.5°C) and moderate July precipitation (41 mm) in areas associated with C. gattii in the environment.The ENM of C. gattii is found in the environment. We mapped cases by their address of residence, so these case locations were proxies to where exposure to C. gattii may have occurred. Unfortunately, from an ENM and epidemiologic perspective, the high mobility of people in developed countries such as Canada, and the duration of time between exposure to C. gattii and onset of symptoms , makes identifying the actual place of exposure difficult to ascertain. Nonetheless, ENM of C. gattii based on the distribution of human cases is appropriate, but caveats pertaining to the limitations of the data, and possible resulting model errors due to the “uncertainty” of these data (whether each case truly represents the presence of C. gattii in the area), should be noted and understood.The validity of any ecological niche model is dependent on the modeling methods, quality, and completeness of the data on species occurrence and on the quality and completeness of the predictor environmental data layers used in the model . TherefoC. gattii in an area because companion animals travel less and because their contact with soil and woody material (environs where C. gattii are most likely to be encountered) are more direct than those to humans—thereby providing more reliable location data for the ENM of C. gattii than human case data. Currently, the number of reported animal cases is approximately 1.5 times greater than the number of reported human cases. New reports of cryptococcosis in previously nonendemic C. gattii geographic areas in BC have been identified first in animal cases and then in human cases. However, although a greater number of animal records have been reported to veterinary and public health, the detail of disease surveillance and other relevant information relating to travel history is generally less complete than those of the human cases. Also, a rigorous system for reporting and tracking animal C. gattii cases is not in place at the present time.Animal cryptococcosis surveillance data, especially the feline cases, may be more appropriate for identifying the true presence of C. gattii, which was performed during the initial stages of the environmental sampling strategy before C. gattii had been isolated from the BC environment. For example, random samples were obtained from the west coast of Vancouver Island, Gulf Islands, Sunshine Coast, and Vancouver Lower Mainland  in the Vancouver Lower Mainland alone. Finally, based on the characteristics of optimal ecological niche areas for C. gattii identified in this study, we hypothesize that the San Juan Islands and Puget Trough of Washington State and the Willamette Valley of Oregon State will also eventually become endemic areas for C. gattii as anthropogenic, wind, and mechanical dispersal of C. gattii into these areas continue.Only a small percentage of the province (< 2%) is geographically located within the forecasted ecological niche area of . gattii . HoweverC. gattii in BC are limited to the central and south eastern coast of Vancouver Island, the Gulf Islands, Sunshine Coast, and Vancouver Lower Mainland. Small, isolated geographical areas on the Queen Charlotte Islands, the BC central coast, west coast of Vancouver Island, and southern BC interior have environmental conditions that could potentially support the establishment of C. gattii. These areas are characterized by low-lying elevations, daily January average temperatures > 0°C, and presence within the coastal Douglas fir and very dry regions of the coastal western hemlock biogeoclimatic zones. In particular, freezing temperatures experienced in areas throughout the rest of BC appear to be the primary environmental limiting factor for C. gattii.In summary, our study found that the “optimal” ecological niche areas of C. gattii ecologic niche models was extremely high: training and testing accuracy was > 98%, and fewer than two commission and omission errors were committed per model run. The use of human and animal cryptococcosis surveillance data to model the ecological niche of C. gattii was effective and corresponded well to the forecasted ecological niche of C. gattii based on the distribution of positive environmental sampling locations. In particular, animal surveillance data proved to be good indicators for C. gattii presence in an area. This finding suggests that surveillance for animal cryptococcosis may be useful as an early alert system for human disease in BC. Therefore, we recommend the creation of a comprehensive, provincewide animal cryptococcosis surveillance system to track laboratory confirmed and clinically diagnosed cases of cryptococcosis due to C. gattii.The predictive accuracy of the


A homeostatic relationship with the intestinal microflora is increasingly appreciated as essential for human health and wellbeing. Mutations in the leucine-rich repeat (LRR) domain of Nod2, a bacterial recognition protein, are associated with development of the inflammatory bowel disorder, Crohn's disease. We investigated the molecular mechanisms underlying disruption of intestinal symbiosis in patients carrying Nod2 mutations.in vitro, altered their metabolism and disrupted the integrity of the plasma membrane. Antibiotic activity was also expressed by the LRR domains of Nod1 and other pattern recognition receptors suggesting that the LRR domain is a conserved anti-microbial motif supporting innate cellular immunity.In this study, using purified recombinant LRR domains, we demonstrate that Nod2 is a direct antimicrobial agent and this activity is generally deficient in proteins carrying Crohn's-associated mutations. Wild-type, but not Crohn's-associated, Nod2 LRR domains directly interacted with bacteria in vitro, supports the hypothesis that a deficiency in direct bacterial killing contributes to the association of Nod2 polymorphisms with the disease.The lack of anti-bacterial activity demonstrated with Crohn's-associated Nod2 mutations  Crohn's disease is a chronic relapsing inflammatory bowel disorder characterised by transmural and discontinuous lesions of the intestinal tract H. pylori's part in the pathogenesis of stomach ulcers, a single pathogen associated with development of Crohn's disease has not been demonstrated although several have been proposed Escherichia coli strain  is a biosafety level 1 bacterium that, to our knowledge, has not demonstrated any previous evidence of pathogenic potential. We next generated GFP-expressing E.coli and repeated the experiment to confirm that Nod2 was recruited to bacteria-containing structures within the cell . Examination of the pellet by microscopy revealed aggregation of LRR-treated bacteria suggesting Nod2 LRR domains were directly influencing the phenotype of a broad range of bacteria . Therefotabolism . These iE.coli were incubated with increasing doses of the recombinant LRR domains of either Nod2 or Nod2 3020insC and the integrity of the bacterial membrane evaluated using a flow cytometric assay previously used to study the activity of anti-bacterial defensins E.coli were only partially susceptible to Nod2-induced membrane depolarisation, gram positive B. subtilis were completely depolarised by less than 10 µg/ml Nod2 .We further extended our analysis of Nod2 anti-bacterial activity using a variety of established anti-bacterial assays to confirm our initial observations. In an agar-diffusion assay anti-bacterial activity of Nod2 could be observed with a clear zone of growth exclusion of  domains . We did l number . CompariP. anaerobius) to 4 µM (B. thetaiotamicron). This establishes the Nod proteins as among the most potent endogenous anti-microbial peptides yet identified The most pronounced observation was the clear sensitivity of the aerobic gram-positive bacteria tested in this assay to the Nod2 LRR domain. This was not observed when anaerobic bacteria were tested for LRR sensitivity in a standard minimal inhibitory concentration (MIC) assay. This assay assesses the potency of antibiotics by determining the minimal concentration at which no visible bacterial growth can be observed. The sensitivity of 9 anaerobic bacterial strains to Nod2 and Nod1 LRR domains using this assessment are presented in These results have major implications for the pathogenesis of Crohn's disease. The compromised anti-bacterial activity demonstrated by the Crohn's-associated LRR domains should be considered alongside the signalling properties of the protein in any model of the molecular mechanisms associated with Nod2's role in Crohn's disease. Normal physiological events such as stress are associated with the onset of relapse in IBD patients and have been demonstrated to increase epithelial barrier permeability resulting in elevated levels of intracellular bacteria in normal animals Mycobacteria, Listeria and Salmonella infection B. subtilis as a model organism, we observed anti-bacterial activity using LRR domains derived from Nalp3, NAIP and TLR2 indicating that the LRR domain is a common anti-bacterial motif. Significant effects on Listeria monocytogenes were also observed with Nalp3 and TLR2 LRR domains using an ATP-based assay supporting this conclusion , nt2165 to nt3286 of Nod1 (NM_006092), nt2856 to nt3857 of Nalp3 (NM_004895), nt3692 to nt4930 of NAIP (NM_004536), nt2204 to nt3526 of CIITA (NM_000246) and nt58 to nt1983 of TLR2 (NM_003264). The integrity of all inserted fragments were confirmed by DNA sequencing.Escherichia coli Rossetta (DE3) cells (Novagen) and purified from inclusion bodies. The bacteria pellet were solubilized using Guanidine-HCl 6M and affinity purified by chromatography on a Ni–NTA column. Protein refolding was performed by a fast 10 fold dilution in PBS. A final purification step was performed using a HiLoad 16/60 Superdex 200 size-exclusion column. Purified proteins were visualized by Coomassie blue staining. The LRR domains were generally unstable, aggregated and demonstrated significant degradation upon storage. Nevertheless, comparable antibacterial activity was consistently demonstrated with over 20 individual protein preps.The LRR domains of Nod1, Nod2 and Nod2 3020insC, Nalp3, NAIP, CIITA and TLR2 were cloned in pDEST17 (Invitrogen) expressed in Anti-Nod2 rabbit antisera recognising recombinant human LRR domains purified from bacteria were generated by Eurogentec. Antibodies recognising the antigen were purified from the serum using a Nod2 LRR domain protein column and passed through a bacterial affinity column (Pierce) to remove contaminating anti-bacterial antibodies.Bacterial viability was evaluated by standard MIC assay protocol in accordance with NCCLS standards 1H-NMR spectra were acquired using standard pulse sequences at 300±1 K using a Bruker-600 Avance spectrometer (1H frequency of 600.26 MHz). 1H-NMR spectra were acquired with 256 FIDs, 65,536 complex data points, a spectral width of 7.2 kHz, and a relaxation delay of 1 s. Metabolite assignments were made on the basis of previous reported data and in certain cases confirmed by spiking Cells were extracted with methanol/chloroform/water (1∶1∶0.9) as previously described Figure S1E.coli (ATCC 25922) at an MOI of 1000∶1 as indicated and incubated for 2 hours. Cells were examined by immunofluorescence with αNod2 polyclonal antibody (generated and affinity purified as described in E.coli affinity column to remove E.coli interacting antibodies), FITC-conjugated phalloidin to detect actin and stained with DAPI to detect DNA . Nod2 is distributed at low levels throughout the cytoplasm of unstimulated cells and colocalizes with intracellular bacteria following incubation with E.coli.Nod2 localisation of endogenous protein in SW480 intestinal epithelial cells in response to bacteria. SW480 cells were inoculated with (0.20 MB PDF)Click here for additional data file.Figure S2E.faecalis. Bacteria were incubated with either BSA (left panel) or Nod2 LRR domains . Bacteria were processed and analysed using either anti-Nod2 antibody (left and centre panels) or without primary antibody (right panel) as controls. Insets demonstrate the presence of bacteria following staining with fluorescent membrane dye in each of the images. Bar = 50 µm.Immunofluorescent detection of recombinant Nod2 LRR domains with (0.31 MB PDF)Click here for additional data file.Figure S3Staphylococcus aureus, c,d, Streptococcus pneumoniae, e,f, Enterococcus faecalis. g–h, E.coli were incubated as described above and visualised by phase microscopy.Bacterial aggregation following incubation with recombinant Nod2 LRR domains. Bacteria were incubated overnight with 1 mg/ml  or 20 µg/ml (g) of BSA or equal concentrations of purified Nod2 LRR domains . a–f, Treated bacteria were incubated with membrane dye to help visualisation. Each sample was mixed by vortexing, a sample placed on a coverslip and observed by fluorescent microscopy. a,b, (0.16 MB PDF)Click here for additional data file.Figure S4L.monocytogenes viability as demonstrated by ATP-coupled luminescent assay. L.monocytogenes (5X105 bacteria/100 µl) were incubated with increasing concentrations of the indicated recombinant LRR domains for 6 hours at 37°C and ATP levels assessed by luminescent assay (BacTiter-glo: Promega). Values shown are relative to controls incubated in the absence of LRR domains (100%). Results are representative of two experiments for TLR2 and Nalp3. Recombinant LRR domains derived from CIITA and NAIP showed no activity in a single assay up to 50 µg/ml.TLR2 and Nalp3 LRR domains inhibit (0.05 MB PDF)Click here for additional data file.Table S1E. coli (µmol/mg dry wt).Metabolite levels in treated (0.03 MB DOC)Click here for additional data file.Table S2LRR anti-bacterial activity against aerobic bacteria.(0.04 MB DOC)Click here for additional data file.


Trauma represents an important public health concern in the United Kingdom, yet the acute costs of blunt trauma injury have not been documented and analysed in detail. Knowledge of the overall costs of trauma care, and the drivers of these costs, is a prerequisite for a cost-conscious approach to improvement in standards of trauma care, including evaluation of the cost-effectiveness of new healthcare technologies.Using the Trauma Audit Research Network database, we examined patient records for persons aged 18 years and older hospitalised for blunt trauma between January 2000 and December 2005. Patients were stratified by the Injury Severity Score (ISS).A total of 35,564 patients were identified; 60% with an ISS of 0 to 9, 17% with an ISS of 10 to 16, 12% with an ISS of 17 to 25, and 11% with an ISS of 26 to 75. The median age was 46 years and 63% of patients were men. Falls were the most common cause of injury (50%), followed by road traffic collisions (33%). Twenty-nine percent of patients were admitted to critical care for a median length of stay of 4 days. The median total hospital length of stay was 9 days, and 69% of patients underwent at least one surgical procedure. Seven percent of the patients died before discharge, with the highest proportion of deaths among those in the ISS 26–75 group (32%). The mean hospital cost per person was £9,530 . Costs varied significantly by Glasgow Coma Score, ISS, age, cause of injury, type of injury, hospital mortality, grade and specialty of doctor seen in the accident and emergency department, and year of admission.The acute treatment costs of blunt trauma in England and Wales vary significantly by injury severity and survival, and public health initiatives that aim to reduce both the incidence and severity of blunt trauma are likely to produce significant savings in acute trauma care. The largest component of acute hospital cost is determined by the length of stay, and measures designed to reduce length of admissions are likely to be the most effective in reducing the costs of blunt trauma care. Severe injury remains a common cause of death and permanent disability across all ages in the United Kingdom, and implies substantial costs to society in medical care, premature death, disability and use of social services ,2. InjurThe incidence of polytrauma (defined as an injury severity score (ISS) > 15) is currently 10,500 per year, equivalent to one patient per 1,000 presenting in A&E departments . While tIn light of the clinical and economic consequences of severe injury, the Royal College of Surgeons and the British Orthopaedic Association recommend developing a national system of trauma services based on the geographic trauma systems for England, Wales and Northern Ireland . In addiRecommendations for improving standards of care are likely to require additional funding and resources, raising questions about the cost-effectiveness of such healthcare improvements. While the outcomes of severe injury have been well documented over the past two decades and some information exists on the standards of trauma care, there are little data on the medical costs of severe injury in the United Kingdom. In particular, there is no information on the major determinants of hospital costs; for example, types of injuries and patient characteristics that drive the total costs of trauma care. Such information would usefully contribute to policy decision-making by targeting high-cost components in trauma care. It would also be a useful resource for evaluating the cost-effectiveness of trauma care interventions.The aim of the present paper is to analyse the determinants of the costs of trauma care in England and Wales. We examine recent data on acute hospital treatment for blunt trauma and we estimate the acute hospital costs from the perspective of the NHS. We also identify the most important predictors of acute care costs in terms of patient and injury characteristics.Data for this study were obtained from the Trauma Audit Research Network (TARN). Data from TARN have been used to examine a range of topics in trauma and trauma care, including trends in trauma care , the effThe TARN registry includes 50% of all hospitals receiving trauma patients in England and Wales . The regData for this study came exclusively from the TARN database without author access to patient records. The TARN database is supported by the Healthcare Commission. Specific informed patient consent or ethical approval is not required because no patient identifiers are retained by TARN electronically or on paper. The TARN has Patient Information Advisory Group approval.We included all patients hospitalised for blunt trauma in TARN hospitals between 1 January 2000 and 31 December 2005. Each hospital completes for every patient a data entry sheet with information on age, gender, cause and type of injuries, severity of injuries, treatment provided at the scene of incident, treatment en route to hospital, treatment in the A&E department, and any other care received at the hospital, including diagnostic tests, surgical procedures, and length of stay. Records of patients transferred between hospitals are linked so that full data for these patients are available.In our study, injury severity is measured using the Injury Severity Score (ISS) . The ISSWe also used data from the TARN registry on the characteristics of the treatment received. These data included the mode of arrival at the hospital, the time from the emergency call to arrival at the A&E department (when by ambulance), the highest grade and specialty of the doctor attending the patient in the A&E department, the time spent in the A&E department, surgery, admission to critical care, and the length of stay. We also used data on whether or not the treating hospital had a neurosurgical centre and/or a spinal injury unit as a rough indicator of specialisation in trauma care. We excluded from the analysis patients younger than 18 years of age at the time of injury.To estimate costs we took the perspective of the NHS in England and Wales; that is, only costs to the NHS in these regions were considered. We focused on the costs of the treatment received during the initial acute hospitalisation. We calculated treatment costs for each patient based on the following cost components: cost of transportation , cost of hospital stay , and cost of all surgical procedures performed. Resource use for every component was recorded for each patient in the TARN database using the data entry sheet. For all resource use we assigned unit costs obtained from external sources to each item  in the study period Table . Sixty pAcross all patients the most common cause of injury was falls (50%), followed by RTC (33%); RTC was the most common cause of injury among patients with an ISS of 10 to 75. The median Glasgow Coma Score was 15 (interquartile range 15 to 15) across the whole sample, but was only 10 (interquartile range 4 to 15) in the ISS 26–75 group. The most common serious injuries (with AIS ≥ 3) were injuries to the lower limbs (40%), the head (20%), the upper limbs (18%) and the chest (13%). The average patient sustained 1.5 injuries with an AIS score ≥ 3, ranging from 0.8 in the ISS 0–9 group to 4.4 in the ISS 26–75 group. Seven percent of patients died in hospital, with the highest proportion of deaths among those in the ISS 26–75 group (32%). Of the hospital deaths observed, the mean (standard deviation (SD)) time to death was 9.3 days (20.3 days) from the date of arrival at the hospital. The time to death was 7.5 days (21.7 days) for RTC, 7.6 days (17.1 days) for falls more than 2 metres, 14.4 days (19.7 days) for falls less than 2 meters, and 5.6 days (9.3 days) for blows.Eighty percent of the patients arrived at the hospital by ambulance, 11% by car, and 6% by helicopter Table . The meaSixty nine percent of patients underwent at least one surgical procedure; the most common were bone fixation (47%), wound debridement (6%) and intracranial procedures (6%). Open reduction and internal fixation was the most common procedure in the ISS 0–9 group, the ISS 10–16 group and the ISS 17–25 group . Evacuation of extradural haematoma/subdural haematoma was the most common procedure in the ISS 26–75 group (9%).Twenty-nine percent of patients were admitted to critical care. The percentage ranged from 10% in the ISS 0–9 group to 79% in the ISS 26–75 group. It is noteworthy that the percentage of patients in the ISS 26–75 group admitted to critical care was less than 100%. This finding may be explained by an insufficient number of critical care beds available to meet the demand. The median length of stay in critical care among those who were admitted was 4 days (interquartile range 2 to 10 days). As expected, the median length of stay in critical care was shortest in the ISS 0–9 group  and longest in the ISS 26–75 group . The median total length of stay, including the length of stay on a general ward and in critical care, was 9 days (interquartile range 5 to 19 days) across the whole sample – ranging from 8 days (interquartile range 5 to 14 days) in the ISS 0–9 group to 17 days (interquartile range 6 to 39 days) in the ISS 26–75 group.Fifty-two percent of patients were treated in a hospital with a neurosurgical centre, and 6% of patients were treated in a hospital with a spinal injuries unit.The mean (SD) total hospital cost per patient was £9,530  Table . Across There was little impact of gender on costs, but costs increased with age in the ISS 0–9 group and the ISS 10–16 group, and decreased in the elderly in the other ISS groups; this age effect is possibly associated with a higher mortality in the elderly. Treatment costs were highest among patients injured in a RTC, and among patients in coma upon arrival at the hospital (Glasgow Coma Score ≤ 8). Every type of serious injury was associated with higher treatment costs; limb injuries had the smallest additional impact on cost, and abdominal injuries had the largest additional impact. Treatment costs increased with the number of serious injuries sustained by the patient.In the ISS 0–9 group and the ISS 10–16 group, mortality was associated with higher treatment costs compared with being alive at discharge. Conversely, mortality was associated with lower treatment costs in the ISS 17–25 group and the ISS 26–75 group. Across all patients, mortality implied a slight increase in treatment costs.The impact of treatment patterns on treatment costs are presented in Table Surgery for bone fixation was associated with higher treatment costs among patients in some, but not all, ISS groups. Wound debridement and intracranial procedures were associated with higher mean costs in all ISS groups.As expected, treatment costs were positively correlated with the length of stay – both for critical care length of stay and for total length of stay. Mean treatment costs did not vary by whether or not the hospital in which the patient was treated had either a neurosurgical centre or a spinal injuries unit.The results of the multivariate regression analysis of costs are presented in Table This study provides the first detailed description of the demographic and clinical characteristics of blunt trauma patients in England and Wales, their causes of injury, the acute treatment received and outcomes. The present study is the first to provide a detailed assessment of the NHS hospital costs associated with blunt trauma in England and Wales. The average blunt trauma patient was male, 46 years old, injured by a fall, with an ISS in the range 0 to 9. The most frequent serious injuries were to the upper and lower limbs, head and chest. Around 30% of all blunt trauma patients were admitted to critical care for a median of 4 days. The total median hospital length of stay was 9 days; 7% of the patients died before hospital discharge. The average cost of acute treatment to the NHS was £9,500 per patient.Few studies have been published on the cost of acute treatment for blunt trauma in the United Kingdom; hence it is difficult to assess the robustness of our cost estimates. In one study, Sikand and colleagues examined the cost implications of treating 69 polytrauma patients (defined as ISS > 15) admitted to an English university hospital in 2000 [Our results suggest that influential determinants of cost were the cause of injury  and the age of the patient. This influence emphasises the importance of continued public health education and accident prevention measures. From the hospital perspective, the most important cost drivers are length of stay in critical care and total hospital length of stay, accounting for 75% of total costs and far exceeding other components such as transport to hospital, immediate resuscitation in the A&E department, and trauma surgery. In the hospital setting, this suggests that steps to reduce the hospital length of stay  are likely to be more effective, all else equal, than other measures aimed at reducing the costs of blunt trauma care.The year of admission was a significant predictor of costs, with lower mean treatment costs in the latter part of our study. This may be due to changes in the NHS costs over the period 2000 to 2005, or to improvement in the hospital care pathways introduced to enhance the cost-effectiveness of trauma care; however, the time series is too short to be able to draw any firm conclusions.We chose to examine the costs of blunt trauma because, in the United Kingdom, this is the largest group of trauma patients – representing 98% of patients in the TARN registry. Any interventions directed at blunt trauma victims or improvements in the care of blunt trauma victims are therefore likely to have the largest public health benefit.Since 1994 the Department for Transport in England has published annual costs estimates for road casualties for use in cost–benefit analyses of road safety schemes. The latest estimates of the medical and ambulance costs associated with slight, serious and fatal injuries from road traffic collisions in 2005 were £850, £11,460 and £840, respectively [The mortality rate observed in the current study (7%) suggests that no improvement in survival after blunt trauma has occurred over the past decade in England and Wales. In the most recent assessment among 129,979 blunt trauma patients admitted to hospital between 1989 and 2000, the in-hospital mortality rate was 5.6% among patients with a complete revised trauma score  and 7.4% among those with an incomplete revised trauma score  [We acknowledge a number of limitations to our study. First, we did not include treatment cost occurring after the initial hospitalisation period, including the costs of rehabilitation, home care support and any subsequent hospitalisations related to blunt trauma.Second, the retrospective nature of the study implies reliance on the quality and completeness of the data reported to the TARN registry. We observed incomplete data on a number of important treatment parameters, particularly the time from emergency call to arrival at the A&E department and surgical procedures. If these data, particularly those relating to surgical procedures, are not missing at random across the recorded patient characteristics, then our results could be biased.Third, we excluded patients who were admitted for fewer than 3 days and who died before they reached the hospital. Those patients who were not admitted and those who died will have incurred lower treatment costs than those who were admitted, and therefore our estimates are likely to have overestimated the costs of blunt trauma care. Since those who were not admitted are likely to be less severely injured than those patients who were admitted, the extent of the overestimation is likely to be higher for those in the less severely injured ISS groups.Fourth, the indirect costs related to lost productivity and time spent away from other activities, as well as the costs associated with the pain and suffering by victims and relatives, were not included. The cost of follow-up care and the indirect costs can be substantial, and are likely to represent the majority of the lifetime societal costs of blunt trauma.Finally, the unit costs used in the analysis are, by necessity, crude. In particular, the unit costs of hospital stays are not specific to blunt trauma patients. Clearly it would have been more appropriate to use the actual costs incurred by each trauma patient included in the analysis, yet these data were not available for our study.In this study we provide the first detailed information on treatment patterns, mortality and costs of blunt trauma in the acute care setting in England and Wales. Our findings indicate that the initial hospital costs associated with blunt trauma vary significantly by patient characteristics, by injury characteristics and by treatment characteristics. The largest component of treatment cost, however, is determined by the total hospital length of stay. Measures to reduce the hospital length of stay – either directly in terms of organisation and staffing, or indirectly in terms of better health outcomes – are likely to be the most effective in reducing the costs of blunt trauma care.• The mean acute hospital cost per blunt trauma patient in England during 2000 to 2005 was £9,530.• The length of stay on a general ward accounted for the largest percentage of these costs (46%), followed by the length of stay in critical care (29%).• Acute hospital costs vary significantly by Glasgow Coma Score, ISS, age, cause of injury, body region of injury, hospital mortality, grade and specialty of doctor seen in the A&E department, and year of admission.A&E = accident and emergency; AIS = Abbreviated Injury Scale; ISS = Injury Severity Score; NHS = National Health Service; RTC = road traffic collision; SD = standard deviation; TARN = Trauma Audit Research Network.The present study was funded by Novo Nordisk A/S. MCC is an employee of Novo Nordisk A/S, and VM is an employee of Novo Nordisk Ltd. SM has received consultancy fees and SR has received honoraria from Novo Nordisk Ltd. TARN received an unrestricted grant for making data available for the present study.MCC conceived of the study, participated in study design and coordination, interpreted data, and helped to draft the manuscript. SM participated in the design of the study, performed the statistical analyses, and helped to draft the manuscript. SR and VM participated in the study design and interpretation of data, and helped to draft the manuscript. FEL participated in the study design, the coordination of data collection, and helped to draft the manuscript. All authors read and approved the final manuscript.


Penaeus japonicus (RV-PJ) infection in Kuruma shrimps. When schizophyllan was administered orally at a dose of 50 or 100 mg/kg body weight per day, the survival rates after virus challenge were significantly higher than those of the control groups. High phagocytic activities were observed in the haemocytes of the schizophyllan-fed shrimps.These results suggest that schizophyllan confers effective protection against viral infection by increasing antiviral immune responses, and that it could be used to boost immunity to virus infection in animals or in invertebrates.An oral administration of fungal polysaccharide schizophyllan has augmented protective immune responses to Sendai virus infection in mice and the rodshaped DNA virus of


Rabies virus exists in dogs on Sri Lanka as a single, minimally divergent lineage only distantly related to other rabies virus lineages in Asia. Stable, geographically isolated virus populations are susceptible to local extinction. A fully implemented rabies-control campaign could make Sri Lanka the first Asian country in >30 years to become free of rabies virus. A dog rabies virus collected in 1986 from Colombo served as a reference sample for mutational change in Sri Lankan rabies virus over time. Sri Lankan samples were submitted to virus typing by both antigenic and genetic methods.The antigenic profile of 31 Sri Lankan samples, displaying 3+ to 4+ antigen distribution, was determined by indirect immunofluorescence methods with a panel of 20 nucleoprotein-specific MAbs obtained from the Centers for Disease Control and Prevention and the Wistar Institute and compared with published reaction patterns for these MAbs . Only 10 nucleotide substitutions  were found when the entire protein encoding sequence of the N gene (1350 bp) was compared for isolates from 1986 and 2001 , and the two samples were identical over an additional 56 bp of nontranslated sequence between the stop codon and polyadenylation signal (bp 1421–1476).No evidence was found for a wildlife reservoir for rabies independent of rabies in domestic dogs. Mutations at nucleotide position 1231 and 1408 separated the virus samples into two clusters , but theIn contrast to the limited sequence diversity among samples from Sri Lanka, extensive sequence divergence was found when samples from Sri Lanka were compared with samples from other Asian countries, and genetic change was much more evident in the nontranslated sequence (17.2% to 31.2% divergence) as compared with the protein-encoding sequence . Additionally, samples from Vietnam, Indonesia, Laos, the Philippines, and Thailand contained an extra nucleotide in the nontranslated region as compared with the Pasteur vaccine strain of rabies virus. The exact position of this extra nucleotide could not be determined because it varied in relation to different sample groups, and the single extra nucleotide likely represents multiple insertion and deletion events.Molecular aspects of the epidemiology of rabies within Sri Lanka and between Sri Lanka and other countries were determined by constructing phylogenetic trees . BecauseThe phylogenetic analysis showed that all samples positive for rabies in diagnostic tests from Sri Lanka were rabies virus. Samples from Sri Lanka clustered with the Pasteur vaccine strain of rabies virus and other samples identified as genotype 1 lyssavirus . Three lIn contrast to the genetic diversity of Asian rabies virus samples, samples of African 1 rabies virus  no evidence for outside introduction of the virus to Sri Lanka in the recent past. Historically, rabies of this type has been susceptible to control with a fully implemented vaccination campaign and an animal-control effort that addresses local cultural and religious attitudes toward management of stray animals. The economic impact of rabies on Sri Lanka argues for national and international cooperation in a strong rabies-control program for this island nation. The prospects are good that such an effort could eliminate rabies virus from the island and create the first rabies virus–free country of the millennium.


Transmissible gastroenteritis virus [TGEV]), a virus that our laboratory has been studying for 16 years Coronaviridae family Endemic Balkan nephropathy (EBN), a disease restricted to three Balkan countries , is characterized by a progressive shrinking of the kidneys and, in some cases, tumors in the proximal regions of the urinary tract The EBN-associated virus was isolated from primary kidney cells cultures, grown from fresh renal biopsy specimens of clinically confirmed cases of EBN 6 to 107 PFU/mL. No specific neutralization was observed when polyvalent or monoclonal antibodies that neutralized TGEV or the human coronaviruses OC43 or 229E were used in a standard neutralization assay. Furthermore, we observed no reactivity by immunofluorescence microscopy with the same antisera and specific monoclonal antibodies A titration method was set up for the EBN-associated virus in Vero cells, as described for coronaviruses Since coronavirus morphology is easily recognized by electron microscopy, Vero cells infected with the EBN-associated virus were embedded in resin for electron microscopy, and ultrathin sections were examined. Coronaviruses interacting with the cell membrane or inside the cell cytoplasm were easily seen when grown in swine testicle cells , A and CSupernatants of Vero cells infected with the EBN-associated virus were concentrated 100-fold by ultracentrifugation and visualized by negative staining with 2% uranyl acetate. Using purified TGEV as a standard, we observed only one type of spherical virion with a homogeneous mean virion size 28.4 nm ± 2 nm in diameter  , F. In cThe virus source used in these experiments is the same as that previously analyzed


Archaea. Less than 100 have been identified and described and very few of these have had their genomic sequences determined. Here we report the genomic sequence of a previously undescribed archaeal virus.We are profoundly ignorant about the diversity of viruses that infect the domain Halorubrum saccharovorum were isolated from a hypersaline lake in Inner Mongolia. Two lytic viruses infecting these were isolated from the lake water. The BJ1 virus is described in this paper. It has an icosahedral head and tail morphology and most likely a linear double stranded DNA genome exhibiting terminal redundancy. Its genome sequence has 42,271 base pairs with a GC content of ~65 mol%. The genome of BJ1 is predicted to encode 70 ORFs, including one for a tRNA. Fifty of the seventy ORFs had no identity to data base entries; twenty showed sequence identity matches to archaeal viruses and to haloarchaea. ORFs possibly coding for an origin of replication complex, integrase, helicase and structural capsid proteins were identified. Evidence for viral integration was obtained.Haloarchaeal strains with 16S rRNA gene sequences 98% identical to The virus described here has a very low sequence identity to any previously described virus. Fifty of the seventy ORFs could not be annotated in any way based on amino acid identities with sequences already present in the databases. Determining functions for ORFs such as these is probably easier using a simple virus as a model system. Eukarya, Bacteria and Archaea is a firmly established biological tenet . The lake was sampled in September 2003. It had substantially evaporated over the summer, exposing expanses of [pink salt – encrusted] mud flats and had been reduced to small pools and lagoons of salt – saturated colourless water, pH 8.5. The pink colouration of the salt crystals indicated the presence of haloarchaea. The chemical composition of lake water was determined using laser inductively coupled plasma optical emission spectrometry by the Department of Geology, University of Leicester. Carbonate/bicarbonate concentrations were determined by titration with HObviously this is a seasonal chemical analysis of the lake water, the composition of which continually varies, more dilute in spring following the winter thaw and then gradually becoming concentrated by the hot summer winds. We used trial and error techniques to find an appropriate medium where we could pour both top and bottom agars. Medium composition was influenced by very high salt concentrations interfering with agar solidification and causing "salting out" of some of the components. The eventual salt composition of this medium was identical to that determined for the lake above with the following exceptions; Na was at 2.85 M, Cl was at 2.6 M, S was at 0.642 M, Ca and Li were omitted completely.Halorubrum saccharovorum with 1289 identical nucleotides. It is also closely related to Hrr. lacusprofundi  and the recently described Hrr. aidingense . All three species were originally isolated from hypersaline environments, a salt pan in San Francisco, Deep lake Antarctica and Xin-Jiang in China respectively . Strain BJ1 B11is most closely related, at 98% identity, to ectively ,20. Fig Plaques for BJ1 required one to two weeks to appear on plates because the host is slow growing. Plaque size for BJ1 was variable between experiments ranging from 1–5 mm in diameter, probably due to slight changes in growth conditions; they were also irregularly shaped and turbid. No attempt was made to optimise plaque formation by modifying temperature, salt concentrations or host strain.Siphoviridae family, (see the Discussion).Virus BJ1 has an icosahedral head, collar and tail, Fig. . The icoBamHI, HaeIII, SstI and XhoI, confirmed that the DNA was double stranded e.g. . PFGE also suggested a genomic size greater than 23 kb but less than 48 kb  followed by melting at 80°C failed to show any change in the number of bands compared to un-melted digests (data not shown).See Figure In the absence of an obvious end for the genome from our sequencing experiments we analysed the cumulative GC skew of the sequence Fig. . Skew miHalobacterium  saccharovorum 16S rRNA gene sequence (Accession HSU17364), which is the closest phylogenetic match to the phage host was complemented (AGGAGGUGA) and used to search 5–15 bp upstream of each putative start site for the presence of putative ribosome binding sites (RBS). 51 of the 70 ORFs had sequences suggestive of a RBS,  had a low calculated isoelectric point (pI < 5), which is similar to the acidic proteins of halophilic organisms ,25. Just-9 to 10-4) to small segments of several haloarchaeal sequences i.e. Natronomonas pharaonis, Halobacterium sp. NRC-1 and Har. marismortui. BlastX analysis identified four regions of the genome having significant matches to data-base proteins either from haloviruses or haloarchaea, discussed below. The putative ORFs were individually analysed using BlastX and BlastP. InterPro was also used to search for functional domains. Using these approaches we were unable to ascribe any match or function to 50 of the 70 ORFs i.e. E values were greater than 0.05. Of the 20 we could match i.e. E value less than 0.05, most were to haloarchaeal virus entries or to haloarchaea. These results are summarised in Table BlastN analysis of the whole virus genome showed significant matches  is 42048ACTATCCGACtggGTCGGATAGT42070; again both are present in putative ORFs and their significance is unclear although the last palindrome is located 209 nucleotides from the 3' end of the genome. The BJ1 genome has a low incidence of CTAG and GATC sequences, just three of each of these palindromes being present. This incidence is low, both compared to the statistically expected incidence, (every 256 base pairs) and compared to the related tetramers CGAG and GCTC which were both found 36 times. CTAG and GATC sequences appear to be selected against by many haloviruses e.g. these palindromes are absent from the genomes of HF1, HF2, His2 and SH1  was added to each plate and the virus harvested by scraping off the soft top agar and homogenising by vortexing for 30 s. Agar and cell debris was pelleted by centrifugation at 10 000 rpm for 20 mins. The supernatant was transferred to a fresh clean tube. To increase the yield of virus particles, the pellet was resuspended in 2 ml halovirus diluent and the previous steps of homogenisation and centrifugation were repeated. Combined supernatants were passed through a 0.45 μm filter and then a 0.22 μm filter to further remove agar and cell debris. To remove any exogenous non-virus nucleic acids DNase I and RNase A were each added to a final concentration of 1 μg/ml and the sample left at room temperature for 30 min.Attempts to purify virus nucleic acid from infected liquid cultures were unsuccessful. Accordingly 30 μl of virus stock (~102HPO4 and 0.034% w/v KH2PO4). To extract genomic nucleic acid from the virus, the pellet was mixed with an equal volume of phenol chloroform and centrifuged for 30 s. The top nucleic acid containing aqueous layer was transferred to a fresh tube. Excess phenol chloroform was removed by ether extraction. The nucleic acid was ethanol precipitated, redissolved in 20 μl Tris-EDTA, pH 8.0 and left to rehydrate at 4°C overnight. An extraction from 20 plates typically yielded 1–2 μg nucleic acid.Virus particles were precipitated by the addition of 1/8 volume polyethylene glycol (PEG) 6000 solution  PEG 6000) and left to incubate for 15 min at room temperature. Virus particles were pelleted by centrifugation at 13 000 × g for 5 min. The supernatant was carefully removed and the pellet resuspended in 100 μl phosphate buffered saline , RNase A (Sigma) or Exonuclease III (NEB) in the manufacturers reaction buffer and incubated at 37°C for 10 min, 60 min or 30 min respectively. Reactions were electrophoresed on Tris-Acetate-EDTA (TAE) agarose gels and stained with SYBR green. Viral nucleic acids were ran on a 1% agarose pulse field gel (BioRad) in 0.5× TBE buffer at 14°C in a CHEF DR-II apparatus (BioRad). The run time was 22 h with a voltage gradient of 6 V/cm and a linearly ramped pulse time of 50 to 90 s at an angle of 120°.1 BamHI . Final coverage of the genome was 4-fold with the majority sequenced on both of the strands or, where bidirectional sequencing was impractical, with multiple sequence runs on the same strand.BJ1 genomic DNA was digested with I vector . ResultiPotential ORFs were assigned using the programs FGENESB  and GeneGC skew was calculated using the online base composition tools at . BLAST (Eulyn Pagaling collected samples on the field trip, did all of the culture and virus isolation work and analysed and interpreted data and revised the manuscript. Richard Haigh determined and analysed much of the genomic sequence data and revised the manuscript. W Grant, D Cowan, B Jones, Y Ma, and A Ventosa, participated in the experimental design, collected samples on the field trip analysed and interpreted data and revised the manuscript. Shaun Heaphy participated in the experimental design, collected samples on the field trip, supervised laboratory work, analysed and interpreted data and wrote the manuscript. All authors read and approved the final manuscript.