The practical and methodological challenges inherent in online behaviour change studies are both novel and complex. We relate our experiences of estimating and managing information technology (IT) research and intervention costs in an ongoing internet trial in the hope that others will find this information useful.Actual IT costs were approximately twice those that had been originally estimated by external contractors. These original estimates for IT costs allowed little scope for the identification of new needs, which was intrinsic to the iterative nature of the research enterprise.Making greater provision for the uncertain nature of these costs in future studies is a key practical lesson for the planning of future online behaviour change studies. Estimating research and intervention costs in the planning of any large study is a considerable practical challenge facing researchers. Overestimation may threaten the granting of research funding and underestimation can threaten the viability, quality and completion of the study. For most trials costs can be divided between those required to develop the intervention materials (both active and control) and the research evaluation. There is a particular need to take account of both the practical and methodological challenges inherent in online studies as the internet is increasingly used to both deliver healthcare and public health interventions, and to undertake research. Here thThe Down Your Drink randomised controlled trial was fundOur chosen modus operandi involved a technical consultant liaising between the research team and the programmers who were based in a small private sector IT company. A competitive tendering process had occurred for the original establishment of the website some years previously and it would have been difficult for technical reasons to involve new personnel. As it was essential to have current expertise on web design and the user interface, we opted to work with the private sector rather than with an academic IT department. For this case study the use of a private company had the advantage that resources used in the trial were more specifically monitored than may have been the case in a large academic department. Communications between the various parties were effective, though constrained in ways to be described. We examined the literature for guidance on IT costs and had informal discussions with other e-health researchers, but at that time (during 2006) there was little collective experience of on-line trials. Some of the costs we present are specific to our model and our circumstances, though the lessons drawn from this experience are more clearly generalisable.In a total research grant awarded of approximately £350,000* over three and a half years, we were awarded £21,400 for website development and maintenance costs, exactly as requested . We received £15,400 for website development and maintenance and £6,000 for the technical consultant who also fulfilled a project management function. These costings were based on estimates we received from both external contractors in the course of the research grant application, and before we started the work on redeveloping the existing website. Our funding award involved a pilot study being undertaken online 3 months after the initiation of the project, leading into a main trial after approximately one year.After the project was initiated, a work plan was put together to provide a more detailed specification of how the awarded funds were to be initially spent -- see Table These tasks were duly implemented. In so doing, it quickly became apparent that additional work would be required. This stemmed from the fundamentally iterative nature of the enterprise. Once particular tasks were completed, only then could it be established whether additional work was or was not needed. For example, testing new material with users was judged essential to the successful conduct of the study. It is difficult to know in advance, however, precisely what need for further work will emerge from this feedback and thus what the costs will be.It should be emphasised that we were not striving to include the latest graphics and other technical innovations. All along we were mindful of the need to provide easy access to those with slow download speeds, and to make the entire process of trial participation and engagement with the interventions as straightforward as possible. Formative evaluations of progress made led in a series of further iterations to the identification of new needs for further work to be done. Information on these additional costs is presented in detail in Table Some developments were also initiated or required by other parties. For example, one participant who wished to withdraw from the study contacted the ethical committee because they no longer wished to be bothered by automated e-mail messages. The ethical committee required that this be done, and we were very happy to do it, though this did involve unforeseen programming costs. We sought and obtained additional funding support for the newly identified extra work (see acknowledgements). If we had not been able to obtain additional funding, the conduct of the study would have been jeopardised.There are two other types of research costs described in Table Towards the end of the study, further work was identified to fulfil our initial commitment to make the website freely available after the end of the trial. This work was thus neither specifically an intervention nor a research cost and totalled £4,140. These were added to the total programming costs of £10,986 for research purposes, £19,448 on the development of the intervention website, £1,645 on the control website, and £8,460 on consultancy costs to give a final total for IT costs of £44, 679. Research-specific IT costs were much lower than were intervention IT costs, and costs for our Technical Consultant (which embraced both research and intervention activities) were far from trivial. Research staff costs have not been included here.In total the actual costs of the IT work were more than twice what had originally been estimated . It was almost as if the elephant in the room was twice as big as we thought it would be, notwithstanding our efforts to obtain well informed estimates. With the benefit of hindsight our original estimates allowed little scope for the identification of new IT needs, beyond the need for maintenance costs, and thus were grossly inadequate. Perhaps we could have given more scrutiny to the estimates we received from contractors. Even so, the fundamental problem that we faced was that many of the additional costs could not have been specifically anticipated in advance as the need for them only became apparent during the course of the methodological and intervention design work that was the substance of the research itself. And we were reliant on the estimates provided by external expert IT contractors, precisely because they had expertise which we did not.Current research funding systems rely on academic departments taking the risk for any underestimation of resources, while most research funding bodies have mechanisms to claw back those resources not used. This risk is managed by most universities through some pooling of risks across different projects and different funding streams to support research activities. However, these systems do not usually extend to flexibility in external contracts for specialist services such as IT support.There is a limited pool of knowledge about how to do studies of this type effectively and efficiently and we certainly do not suggest that our particular model, involving an external technical consultant, should be taken up more widely. Particularly noteworthy for internet intervention developmental work is the fact that we deliberately eschewed advanced interactive features and videos on grounds of cost and user engagement. We were also aware of the need to limit not just the direct IT costs but also the hidden costs of research project staff time devoted to IT issues. Although these are difficult to separate from wider research costs, they may well exceed the IT specific costs and it was our experience that such staff time was extended by the inevitable communication problems encountered in bridging the worlds of academic research and the internet in addressing ongoing issues. Making greater provision for the essentially unknowable nature of these costs in future studies is thus a key practical lesson for the planning of future internet behaviour change studies.*The value of financial costs may be approximately converted to U.S. dollars or Euros by multiplying by 1.5 or 1.2 respectively, though exchange rates obviously do vary.The authors declare that they have no competing interests.JM wrote the first draft of the paper and led the writing process. OO gathered the costs data. OO, CG, ZK, SL, EM and PW provided input into revisions. All authors are involved in the parent trial from which this short report is derived and read and approved the final manuscript.


Viruses that have spent most of their evolutionary time associated with a single host lineage should have sequences that reflect codivergence of virus and host. Several examples for RNA viruses of host-virus tree congruence are being challenged. DNA viruses, such as mastreviruses, are more likely than RNA viruses to have maintained a record of host lineage association.Wheat dwarf virus (WDV), a member of the Mastrevirus genus, from different regions of China were sequenced. The analysis of these 28 entire genomes and 18 entire genome sequences of cereal mastreviruses from other countries support the designation of wheat, barley and oat mastrevirus isolates as separate species. They revealed that relative divergence times for the viruses WDV, Barley dwarf virus (BDV), Oat dwarf virus (ODV) and Maize streak virus (MSV) are proportional to divergence times of their hosts, suggesting codivergence. Considerable diversity among Chinese isolates was found and was concentrated in hot spots in the Rep A, SIR, LIR, and intron regions in WDV genomes. Two probable recombination events were detected in Chinese WDV isolates. Analysis including further Mastrevirus genomes concentrated on coding regions to avoid difficulties due to recombination and hyperdiversity. The analysis demonstrated congruence of trees in two branches of the genus, but not in the third. Assuming codivergence, an evolutionary rate of 10-8 substitutions per site per year was calculated. The low rate implies stronger constraints against change than are obtained by other methods of estimating the rate.The full genomes of 28 isolates of We report tests of the hypothesis that mastreviruses have codiverged with their monocotyledonous hosts over 50 million years of evolution. The tests support the hypothesis for WDV, BDV and ODV, but not for MSV and other African streak viruses. Viruses are a class of genetic elements dependent on suitable host cells for their propagation. Viruses belonging to diverse viral groups have been proposed to have codiverged with their hosts based on congruence of phylogenetic trees for the viruses with those for their hosts. In codivergence, congruence results from long association of the viral and host lineages. The term codivergence is preferred to describe this situation since, unlike the term "coevolution", it does not imply that the association necessarily provides mutual benefits to the partners .Potyvirus divergence suggested that this genus emerged shortly after the beginning of agriculture [Tobamovirus tree has supported the congruence of host and virus trees [-5 substitutions per site in the shorter term [The best studied examples of codivergence  include iculture , much laiculture  supportsus trees . Furtherus trees  and Greeus trees , virusester term .The viruses in these major examples of putative codivergence of viruses and hosts have RNA genomes that replicate using error prone RNA-dependent RNA polymerases encoded by the viral genomes. Such viruses are expected to evolve more rapidly than viruses with DNA genomes which use host DNA-dependent DNA polymerases with proof-reading ability for replication of their genomes and could be subject to the action of DNA repair systems on replication errors or spontaneous mutations . Thus, fGeminiviridae replicate their DNA using a host DNA polymerase and encapsidate circular single-stranded DNAs [Mastrevirus, Curtovirus, Topocuvirus and Begomovirus, classified depending on their vectors, host range and genomic characteristics [Members of the ded DNAs . This pleristics -20. Durieristics .Wheat dwarf virus (WDV) from the Mastrevirus genus of Geminiviridae, we observed patterns that suggested that viruses in this genus have substitution frequencies consistent with their replication by host DNA polymerases. The genus Mastrevirus consists of viruses with circular single-stranded (ss) DNA genomes in geminate (twinned) virions [Psammotetix striatus L. to barley, wheat, oats, rye and many wild grasses [Barley dwarf virus (BDV), and Oat dwarf virus (ODV), designations used in this paper.In the process of studying populations from China of  virions , and has grasses ,26. It w grasses  in the w grasses ,28. Its  grasses , Tunisia grasses , Turkey  grasses , Zambia  grasses . The com grasses . Compari grasses ,28. Schu grasses  suggesteMaize streak virus as an outgroup. The topologies of the two types of trees were identical at all branch points that were well supported by bootstrap analysis (> 70%) but differed at some branch points with low statistical support  as proposed by the ICTV [Bootstrap analysis supported an unambiguous host-dependent clustering of wheat-, barley-, and oat-derived samples. The isolates also fell into subpopulations seemingly structured according to geographic origin. Wheat isolates from China were > 94.8% identical in nucleotide sequence, whereas > 92% identities were seen between the wheat isolates of China and those of other countries. Greater than 94% nucleotide sequence identities were found among barley isolates. Nucleotide sequence identities were < 69% between wheat and barley isolates. The oat-derived isolate exhibited 59.7%–69.3% identities to those of barley, and 68.3%–69.4% to those of wheat. The values for wheat-barley, barley-oat and oat-wheat comparisons were below the threshold for the ICTV ,25.Unscaled evolutionary distances from the common ancestors to their sequence progeny were deduced from the NJ tree. The distances indicated that the barley isolates diverged from wheat isolates much more recently than the oat isolate did. The most basal barley isolate was TR2 from Turkey, followed by those from Germany and the CSR. Wheat isolates were separated into two groups, the first one from Europe and the second one from France, the CSR, and China. The grouping of all of the Chinese isolates in one clade with the CSR and French isolates suggested that they evolved from the same ancestor. The Chinese wheat isolates were divided into two clades, but were not clustered by geographical source or collection time Figure . For exaTriticeae. Together with the Aveneae, which includes oats, they belong to the BEP clade of the Poaceae, while maize represents the PACCAD clade of the Poaceae [Aveneae (oat) and Triticeae (barley and wheat) has been placed at 25 Mya [2 values were greater than 0.99 regardless of whether middle, high, or low values for host divergence times were used, with the middle values giving the best fit. Thus, we cannot reject the hypothesis that the mastreviruses codiverged with their hosts. Relative divergence times for viral evolution were converted to Mya times using the slope of the relationship of the middle host divergence times with virus distance, using the assumption that variation occurs according to a linear molecular clock. The results /year.Barley and wheat are members of the tribe  Poaceae . Maize a Poaceae  and betw Poaceae . The est Poaceae . The divt 25 Mya . In the s Figure  suggest Mastrevirus family in the phylogenetic analysis. To align these additional sequences with the previously analyzed sequences in an unambiguous and reliable fashion, we considered elimination of regions of high diversity and regions prone to be different due to recombination. Regions with high nucleotide diversity were difficult to align reliably. To identify regions that could be reliably aligned, the level of genetic diversity (θw and π) [To explore further the possibility of the codivergence of virus and host, we included additional members of the w and π) ,39 of WDw and π) ,28. ConsA plot of the diversity values as a function of position in the genome revealed that determinations of diversity over large genomic regions obscure regions of high diversity embedded in a background of low diversity Figure . The diva/Ks ratios of all coding regions were lower than 0.22, which suggested negative or purifying selection (Ka/Ks ratios < 1 indicating purifying selection) acting on the sequences, with no genes under positive selection (Ka/Ks ratio > 1 indicating positive selection). The CP gene had the lowest ratio, whereas that of the Rep A gene was highest , Sugarcane streak virus (ScSV), and Urochloa streak virus (USV)). Single available sequences of Chloris striate virus (ChlStrV), Digitaria streak virus (DigSV), Miscanthus streak virus (MisSV), Eragrostis streak virus (ESV), Tobacco yellow dwarf virus (TYDV), Bean yellow dwarf virus (BeYDV) and Chickpea chlorotic dwarf virus (ChPCDV) also were included in the alignment [see Additional file rbcL gene sequences from the plastids of respective host plants for which the virus was named, recognizing that the plant named in the virus name is not necessarily the one from which the virus is most commonly isolated. The rbcL tree is shown in each Figure rbcL trees of the host plants. Viruses named for hosts in the PACCAD clade were not monophyletic since MisSV was basal to the clade of BEP clade viruses in both Rep-RepA and MP-CP trees. Among the remaining PACCAD clade viruses, ChlStrV was in basal position and ESV and ScSV branched together in both viral trees. In the MP-CP tree, insufficient bootstrap support among the other PACCAD clade viruses prevented further comparison. Relationships between viruses with PACCAD host names and the hosts are not straightforward as noted by the off-vertical lines in Figure rbcL trees suggests that a uniformly ticking molecular clock should not be supported. The dicotyledonous plants were on a relatively longer branch than the viruses bearing their names. MisSV had diversified more than the BEP clade viruses in the MP-CP region, but the opposite was true in the Rep-RepA region. ChlStrMV also showed anomalous branch lengths. Such anomalies are signs of recombination events after which adjustment of the recombined segments to one another is needed [To facilitate alignment, consensus MP-CP and Rep-RepA sequences were generated for mastreviruses with multiple sequenced representatives , compared to that for the majority of geminiviruses (89%) . In our Mastrevirus trees to one another . It is also noteworthy that adding the third datable branch (PACCAD clade from BEP clade) led to a near perfect correlation of host divergence times with relative viral divergence times  which have been isolated from native grasses of the genera Setaria, Cenchrus, Paspalum and Eragrostis, indicating a wide host range [Non-congruence of PACCAD clade host branching patterns with branching patterns of viruses named for those hosts seems to argue against the codivergence hypothesis. However, it must be remembered in this regard that virus names are based on the host plant from which the virus was first isolated. Thus, the names do not necessarily reflect the plant lineage in which the virus spent most of its time evolving. Indeed, non-congruence is expected for viruses that can efficiently infect many species of plants. MSV isolates have been found in many grass genera including Triticum . FurtherTriticum . Similarst range . In contst range .Begomovirus. Inoculation of plants with infectious cloned DNA of Tomato yellow leaf curl China virus (TYLCCV) resulted in the subsequent recovery of viral sequences with substitutions at a frequency of about 10-4 substitutions per site during a 60 day growth period in plants [-4 per site per year for the related Tomato yellow leaf curl virus [Begomovirus investigations, while the deeper phylogenetic tree construction employed in our Mastrevirus work focuses on the areas of the genome with low nucleotide diversity. These are also the areas subjected to strong purifying selection. The apparent discrepancy between very long-term and short or long-term evolution rates in the Geminiviridae is reminiscent of similar findings in the Tobamovirus analysis. Understanding this apparent difference awaits further analysis.Estimates of short term and longer term evolution rates are available for viruses in the sister genus, n plants . Consistrl virus . Thus onrl virus ,49. Thesrl virus , four nuTriticum aestivum L.) cultivar Fengkang No. 8 by vector leafhoppers  to increase virus concentration and to allow serological typing or sequencing of polymerase chain reaction (PCR) products. The wheat plants were later tested for WDV with ELISA using an antiserum . Leaves were collected from WDV-positive plants displaying typical symptoms of WDV infection, and stored at -80°C. Details of the isolates, their names, provinces of collection, original host plant, and years of collection are shown in additional file [see Additional file WDV was collected throughout China during field surveys in the growing seasons 2004 to 2006. The 28 isolates described here originated from wheat planted in different agro-ecological areas in China, including the northwestern (Shaanxi and Gansu provinces), northern (Shanxi and Hebei provinces), central (Henan province), and southwestern (Yunan province) areas. All the field isolates were inoculated to the susceptible wheat . Sequence data were assembled using DNASIS version 3.5 (Hitachi) or BIOEDIT version 5.0.9 [EF536859 through EF536886.Nucleotide sequences of the entire genome of each isolate were determined using the above PCR fragments. The purified fragments were cloned into the pMD18-T vector . The plasmids were transformed into on 5.0.9 . The nucMaize streak virus (MSV) (NC_001346) [θw) [Complete genomes of the 28 WDV isolates sequenced in this study and 18 entire sequences of other WDV, BDV and ODV isolates obtained from the NCBI database  were analyzed. The coding and intergenic regions were annotated by reading frame or following NCBI's annotations. The complete genome sequences of the WDV, BDV and ODV genomes were aligned with CLUSTAL W V.1.8. MEGA V.4.0  determin_001346)  were use_001346)  was used46) [θw)  and the 46) [θw) , were es46) [θw) . Also, trbcL sequences were retrieved from GenBank/DDBJ/EMBL. They are identified in the legend of Figure s) and non-synonymous (Ka) positions of genes were calculated by DnaSP version 4.10.2 [To evaluate the sequence relationships among mastrevirus genomes, a selection of mastrevirus sequences available at the time was obtained [see Additional file n 4.10.2 .-4/site during a 60 day growing period for Tomato yellow leaf curl China virus (TYLCCNV). That value is clearly an overestimate of the average frequency.The average frequency with which mastrevirus sequence sites mutate in evolution is unknown. For highly mutable sites the substitution frequency has been estimated  at 3 × 1Nevertheless, since our interest was in determining the relative ratios of divergence times of WDV, BDV and ODV from MSV, of ODV from WDV and BDV, and of WDV from BDV, the number was used to obtain divergence time estimates with MEGA software . Host dihttp://darwin.uvigo.es/rdp/heath2006.zip). The transversion and transition differences of all pairs of sequences were calculated using the discalc program  and these were compared in diplomo scatter plots [To investigate the extent of recombination within the data set, the aligned sequences were examined using the Recombination Detection Program (RDP3) , GENECONer plots .BLW performed molecular methods, gathered sequence information from GenBank and research, conducted bioinformatics analysis, and wrote the manuscript. UM carried out evolution time analysis and the codivergence analysis between virus and host, and edited the manuscript. XFW designed the project, provided tools/reagents and edited the manuscript. XYG designed and conducted bioinformatics analysis. LJF conducted bioinformatics analysis. GHZ aided the study design and provided tools/reagents. All authors read and approved the final manuscript.WDV sequences of wheat isolates from China obtained in this research. Isolate names, collected times, regions and GenBank accession numbers used in this study.Click here for fileSequences from GenBank for sequence comparision. The sequences obtained from the NCBI database used in phylogenetic comparison and codivergence analysis.Click here for fileThe primers used for PCR and corresponding annealing temperature. Primer's name, sequences and corresponding annealing temperature used in this study.Click here for file


P < 0.012). Significant and suggestive two-locus effects were also obtained for region 6p21 in combination with loci 18q21, 8p23, 1q41, and 6p22, while the highest 2D peaks excluding region 6p21 were observed at locus pairs 8p23-18q21 and 1p21-18q21. The 2D peaks were further examined using combined microsatellite and single-nucleotide polymorphism (SNP) marker genotypes in 744 families. The two-locus evidence for linkage increased for region pairs 6p21-18q12, 6p21-16p12, 6p21-8p23, 1q41-6p21, and 6p21-6p22, but decreased for pairs of regions that did not include locus 6p21. In conclusion, we obtained evidence for multi-locus interactions in rheumatoid arthritis that are mediated by the major susceptibility locus at 6p21.We performed a genome-wide search for pairs of susceptibility loci that jointly contribute to rheumatoid arthritis in families recruited by the North American Rheumatoid Arthritis Consortium. A complete two-dimensional (2D) non-parametric linkage scan was carried out using 380 autosomal microsatellite markers in 511 families. At each 2D peak we obtained the most likely underlying genetic model explaining the two-locus effects, defining epistasis as a departure from an additive or a multiplicative two-locus penetrance function. The highest peak in the surface identified an epistatic interaction between loci 6p21 and 16p12 (two-locus lod score = 18.02, epistasis  Multiple loci are likely to influence susceptibility to rheumatoid arthritis (RA). Genome-wide scans for multiple interacting loci have been performed in model organisms  and morer2 > 0.1.The genotyped sample of families provided by NARAC to the Genetic Analysis Workshop 15 consisted of 757 families , in which at least one individual per family was genotyped . We initA1 and VD1) and 2 (VA2 and VD2), and the four epistatic variances . Different genetic models can be fitted to the data by restricting the number of free variance components, for example, two-locus additive (ADD) or multiplicative (MUL) models and single-locus (SL) models are nested within the general epistatic model.Non-parametric linkage analysis was carried out initially using the single-locus maximum lod score (MLS) test statistic . Two-locε, captures the degree of epistasis) and the maximum-likelihood estimate of ε can also be used to indicate the degree of epistasis .In the variance-component framework, epistasis can be defined as a departure from an additive or a multiplicative two-locus penetrance function. To assess the evidence for epistasis as a departure from additivity, the two-locus MLS under the general model may be compared to the MLS under the nested additive model with no interaction terms (GEN-ADD). Alternatively, a test for epistasis would compare the MLS under the general model with that under the multiplicative model (GEN-MUL). The MLS under a two-locus epsilon-epistatic model  (where aWe used previously published  significSingle-locus (SL) linkage analysis of the microsatellite genome-scan data in 511 NARAC families Figure  indicateWe performed a 2D linkage scan by computing the two-locus general model MLS at each marker-pair grid coordinate in the genome Figure . For eacP < 0.001) and the most likely model describing this interaction was a model of extreme epistasis . Because the 2D peaks involving 6p21 always surpassed the genome-wide significance threshold of 5.85 (which assumes a null effect at either locus), we also assessed the significance of two-locus results involving 6p21 using simulations of chromosome pairs in which the null model included a single-locus effect at 6p21 alone. The results from these analyses indicated significant (chromosome-wide) effects at regions 16p12 (P = 0.012), 18q21 (P = 0.045), and 8p23 (P = 0.05) and a suggestive effect at 1q42 (P = 0.12), which was independent of 6p21. The two-locus effects observed at locus pairs 6p21-18q21, 6p21-8p23, 6p21-1q42, and 6p21-6p22 were best explained by the additive or multiplicative two-locus models (see Table P = 0.34) and the most likely underlying genetic model describing these effects was an additive two-locus model , and is lowest in the analyses of the combined data under sex-specific maps, 0.93 (P = 0.048).We also examined the evidence for two linked loci on chromosome 6 in more detail, using sex-averaged and sex-specific maps Figure . AssuminWe performed a genome-wide search for pair-wise interactions that contribute to RA susceptibility in the NARAC family collection. The highest peak on the 2D surface involved an epistatic interaction between two loci on 6p21 and 16p12. We also detected pairs of loci that jointly contribute to RA under two-locus additive and multiplicative models, 6p21-18q21, 6p21-8p23, and 6p21-1q41. Suggestive evidence for a secondary gene on 6p22, independent of the major locus on 6p21, was also obtained, but addition of SNP genotypes and use of more precise sex-specific maps in the two-locus analyses of chromosome 6 reduced the evidence for linkage at 6p22, indicating that these results should be interpreted with caution. Our findings are consistent with previous interaction analyses of genetic interactions in RA [Multi-locus linkage analysis methods have been developed and applied to complex human traits. Such methods are useful in detecting novel loci that contribute to the trait susceptibility only through their genetic interactions, and in establishing the type of interaction among susceptibility loci. The approach used in this study can examine entire genomes for potential pair-wise or higher-order interactions, however, interpreting the genome-wide significance of the findings is challenging, in particular when there is strong single-locus evidence for linkage. All of the regions involved in the 2D peaks in our analyses have at least suggestive single-locus effects. Therefore, the results of this study are more useful for elucidating the nature of the interactions between previously identified RA susceptibility loci, rather than in identifying novel loci for RA susceptibility. The two-locus findings are also useful in potentially refining the susceptibility regions, by yielding narrower 1-lod unit support intervals.Our primary results were based on analyses of the microsatellite genotype data alone, while SNP genotypes were added to confirm the effects observed at the two-locus peaks. The inclusion of SNP genotypes in the 2D scan of the peaks allowed for more informative analyses, with more confidence in the two-locus effects observed at the 2D peaks. A follow-up to this study would involve a complete 2D genome-wide analysis of the combined SNP and microsatellite data in the NARAC families. These analyses prove computationally prohibitive at present.The analyses of the NARAC data indicated that if genetic interactions contribute to RA, they are most likely mediated by the major locus at 6p21. This study was aimed at searching for pair-wise interactions in RA, but higher dimension interactions may also exist and should be examined in future analyses.The author(s) declare that they have no competing interests.


Cryptococcus gattii, an emerging fungal pathogen of humans and animals, is found on a variety of trees in tropical and temperate regions. The ecological niche and virulence of this yeast remain poorly defined. We used Arabidopsis thaliana plants and plant-derived substrates to model C. gattii in its natural habitat. Yeast cells readily colonized scratch-wounded plant leaves and formed distinctive extracellular fibrils (40–100 nm diameter ×500–3000 nm length). Extracellular fibrils were observed on live plants and plant-derived substrates by scanning electron microscopy (SEM) and by high voltage- EM (HVEM). Only encapsulated yeast cells formed extracellular fibrils as a capsule-deficient C. gattii mutant completely lacked fibrils. Cells deficient in environmental sensing only formed disorganized extracellular fibrils as apparent from experiments with a C. gattii STE12α mutant. C. gattii cells with extracellular fibrils were more virulent in murine model of pulmonary and systemic cryptococcosis than cells lacking fibrils. C. gattii cells with extracellular fibrils were also significantly more resistant to killing by human polymorphonuclear neutrophils (PMN) in vitro even though these PMN produced elaborate neutrophil extracellular traps (NETs). These observations suggest that extracellular fibril formation could be a structural adaptation of C. gattii for cell-to-cell, cell-to-substrate and/or cell-to- phagocyte communications. Such ecological adaptation of C. gattii could play roles in enhanced virulence in mammalian hosts at least initially via inhibition of host PMN– mediated killing. Arabidopsis thaliana has proven to be a tractable host for the study of bacterial pathogenesis, especially in efforts to unravel virulence factors that are shared between plant pathogens and animal pathogens ‘Primary’ pathogenic fungi that cause serious diseases in healthy humans occur in relatively specialized niches in nature. This trait differentiates them from ‘opportunistic’ fungal pathogens that afflict immunodeficient individuals, since ‘opportunistic’ pathogenic fungi are more widely distributed in soil and plant vegetable matter. During the last decade, more than 400 species of ‘opportunistic’ fungi have been recognized as pathogens for humans and animals C. gattii is an encapsulated yeast that causes pulmonary and cerebromeningeal cryptococcosis. C. gattii is an emerging pathogen that has triggered serious public health concerns due to (i) its appearance in previously unknown geographic areas, (ii) its outbreaks among healthy humans, pets, and wildlife, (iii) the intractable nature of cryptococcal disease, and (iv) the difficulty of diagnosis in clinical laboratories. Reliable estimates of cryptococcosis due to C. gattii are currently lacking, but one estimate suggests that one-third to one-tenth of cryptococcosis cases worldwide are caused by C. gattiiC. gattii from the closely related pathogen C. neoformans, since the reagents required are not readily available. Current information on C. gattii comes either from environmental surveys, which are patchy and far between or from retrospective evaluation of C. neoformans/C. gattii clinical isolates.C. gattii is readily distinguished from closely related pathogen C. neoformans by occurrence on trees, rather than the pigeon droppings colonized by the latter. Other diagnostic characteristics of C. gattii include the presence of distinctive cigar-shaped yeast morphology in the host cerebrospinal fluid, agglutinating serotypes, creatinine assimilation and smooth, elongate, rod shaped basidiospores of the teleomorphic form Filobasidiella bacillisporaC. gattii, but other questions have arisen. Why do rural to semi-urban forested areas pose higher risks for C. gattii infection? How do trees figure in the infectious cycle? What is the nature of the infectious propagules, and how are they spread in the environment? Why are apparently healthy humans and animals so prone to infection by this pathogen?C. gattii has been associated with decayed hollows, in the trunks and/or branches of over 50 species of angiosperms and gymnosperms. The growing body of sampling data supports the idea that C. gattii is long established among the fungal flora of native trees in many parts of the world C. gattii–plant associations. Huerfano et al. C. gattii can survive on and be recovered from experimentally infected stems of almond seedlings 100 days post-inoculation, demonstrative of this pathogen's ability to colonize and thrive on live plants. Previously, we reported that C. gattii will grow profusely on wood and wood extracts from a variety of trees C. gattii ecological fitness and virulence. Xue et al. C. neoformans and C. gattii can colonize, cause leaf chlorosis, and produce basidiospores on young seedlings of A. thaliana and Eucalyptus camaldulensis in the laboratory; by extensions, fungal infectious propagules are to be produced on plants in nature C. gattii–plant interactions could elucidate unknown facets of C. gattii biology and virulence.To date, C. gattii; use of enriched culture media to grow laboratory strains could mask the appearance of certain attributes, especially those that appear in nature early in the establishment of infection in the humans and animals. Further experiments focused on interactions between fungal cells and human phagocytes (PMN), to elucidate the factors that determine if the pathogen persists or is eliminated from the host.We set out to develop an experimental system that captures native habitat and growth of wild-type Blood collection procedures from human volunteers complied with the New York State Department of Health Institutional Review Board (IRB) guidelines. Informed consent was obtained from the volunteers prior to the collection of specimens. All animal studies were conducted under full compliance with the guidelines of the Wadsworth Center's Institutional Animal Care and Use Committee (IACUC) in facilities accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). The protocol approval numbers were 06-333 and 09-333. A daily animal welfare chart was maintained to monitor any overt signs of pain and illness and all animals were promptly euthanized according to recommended institution procedures.C. gattii NIH444 , the wild-type strain isolated from cerebrospinal fluid (CSF), was a gift from Dr. K.J. Kwon-Chung . We have previously described this strain as optimum for C. gattii molecular pathogenesis studies C. gattii ste12αΔ, a mutant strain with targeted knockout of the transcription factor STE12α, was included as it exhibits reduced colonization of woody substrates, and reduced pathogenicity in mice as a result of defective environmental sensing C. gattii , because the capsule of C. neoformans has critical roles in fungal biology and virulence C. gattii cap59Δ mutant was constructed by homologous recombination of the cap59::NAT disruption cassette based on sequences from C. neoformans CAP59C. gattii cells were cultured in YPD broth at 30°C with shaking (180 rpm), and were maintained in YPD agar and preserved at −70°C in 15% sterile glycerol or in liquid N2. Un- treated black cherry (Prunus serotina) wood chips were obtained from Roger Dziengeleski, Finch, Pruyn & Co., Inc. . Leaf agar was prepared by mincing of 10 g of A. thaliana leaves into small pieces with scissors, and addition of 2% agar with 0.1% glucose followed by autoclaving for 15 min at 121°C A. thaliana ecotype Columbia (Col-0) and Landsberg erecta (Ler-0), and various mutant lines eds1 , nahG , npr1 , sid1 (SA-induction deficient), rpm1 (resistance to Pseudomonas syringae pv maculicola 1), were grown in a greenhouse at the Biology Department, Syracuse University, Syracuse, NY C. gattii cells were subcultured twice in YPD broth at 30°C with 180 rpm shaking and were then collected by centrifugation , washed twice in deionized sterile water (DSW) and re-suspended to a final concentration of 1.0×106 cells/ml, by counting in a hemacytometer. A. thaliana plants from the growth chamber were placed in a sealed carrier case and moved to a BSL-2 safety cabinet. Four to six leaves on each A. thaliana plant were marked with a glass marker. Leaves were lightly wounded on the adaxial leaf surface with a 2–3 mm shallow scratch (without complete puncture) on either side of the mid-vein with a 27-gauge syringe needle 6C. gattii cells/ml were placed at the wound site, and allowed to air dry (5–10 min). Plants were replaced in modified growth incubator maintained at 20–23°C, 12 hr light/dark cycle, and 50–70% humidity. Macro-photographic images of A. thaliana plants and leaves were obtained with a consumer digital camera.A. thaliana leaves were chosen at random and cut from the plant 7 days post-inoculation and homogenized with a tissue grinder in 1 ml of DSW, and the homogenate was serially diluted. 100-µL of each dilution was plated onto YPD agar and incubated for 3–5 days at 25°C, and colonies were counted for determination of fungal viability.Infected For light microscopy, leaves were cut from plants 7 or 14 days post-inoculation, fixed in 2% glutaraldehyde (EM grade) in phosphate-buffered saline (PBS) or in 0.2 M sodium cacodylate buffer, pH 7.4, alcohol dehydrated by graded series , and stored for further processing in 100% ethanol at 4°C C. gattii. The natural substrates were intended to recapitulate growth of C. gattii on plants in nature, and for comparisons to growth on organic or synthetic substrates. Wild-type and C. gattii cap59Δ mutant strains were grown to mid-logarithmic phase in YPD broth. Cells were collected by centrifugation, washed two times in minimal asparagine medium, with 1% glucose, and re-suspended in the same medium to a final concentration of 1×106 cells/ml. Wild-type C. gattii cap59Δ mutant cells were treated with sodium azide (2 mM), followed by incubation at 65°C for 30 minutes to render them non-viable; these cells were used in experiments that compared effects of viable and non-viable cells. The plant-based and artificial substrates used were: autoclaved black cherry wood shavings , consumer brown paper bag, Whatman #1 qualitative filter paper with 98% cellulose , Thermanox® plastic coverslips surface treated for optimal cell adhesion , and glass coverslip  and dialysis membrane. The substrate was placed at the bottom of 18-mm round wells of a 24-well flat-bottom polystyrene cell culture plate. One milliliter of 1×106 cells in minimal asparagine medium, was added to each well in one row of the 24 wells in duplicate. The culture plates were covered and incubated for 24- or 48-hr at 25°C. The cell suspension was removed, and wells were washed 3 times with 0.05% Tween-80 in Tris-buffered saline (TBS), to remove non-adherent cells. Substrates were fixed with 2% glutaraldehyde in 0.2 M sodium cacodylate buffer, pH 7.4 for 24 hr at 4°C, and dehydrated by exposure to a graded ethanol-water series, and stored in 100% ethanol in plates. Average numbers of attached cells were determined by counting cells in four SEM fields per sample.A number of natural and artificial substrates were used to study growth characteristics of A. thaliana leaves inoculated with C. gattii were harvested at indicated intervals, fixed in 2% glutaraldehyde (EM grade) in phosphate-buffered saline (PBS) or 0.2 M sodium cacodylate buffer, pH 7.4, alcohol-dehydrated by graded series , and stored in 100% ethanol. The dehydrated specimens were critical point dried with liquid CO2 in a tousimis Samdri 795 drier , gold-sputter-coated, observed and imaged with a LEO 1550 Variable Pressure field emission gun SEM , according to procedures followed in our laboratory C. gattii attachment to the natural or artificial substrates was obtained similarly to our quantification of mating in SEM images described previously C. gattii cells. Mean average counts were obtained from determining the number of C. gattii cells in at least four or more SEM images.A. thaliana leaves that have been scratch-wounded and inoculated with C. gattii cells were harvested and fixed in 2% glutaraldehyde (EM grade) in 0.2 M sodium cacodylate buffer, pH 7.4 overnight. Leaves were washed three times with 0.2 M sodium cacodylate buffer and stained with 1% osmium tetroxide (OsO4) in 0.1 M sodium cacodylate for 1-hr at 4°C, rinsed two times with DSW, and dehydrated through a graded ethanol series A. thaliana leaves were cut and trimmed with a hot razor blade, and mounted on pre-formed TEM pegs. Sections were cut approximately 1 µm thick, on a microtome and were mounted on formvar-coated grids. Grid-mounted sections were then stained with 2% uranyl acetate for 60 min, followed by Reynold's lead stain for 20 min; they were then allowed to air dry, and were stored in an EM grid box. Sections were observed and imaged with the 1.2 MeV AEI EM7 MK II high voltage electron microscope (HVEM) at the Wadsworth Center.C. gattii microtubes, since these proteins are important determinats of cell shape C. gattii wild-type and cap59Δ mutant strain were grown in YPD broth, 30°C, 180 rpm for 12–16 hr and treated with thiabendazole, cytochalasin B, mebendazol, or latrunculin B  or rapid onset of cerebromeningeal cryptococcosis  as described previously C. gattii cells were grown with two successive passages on YPD agar and A. thaliana leaf-agar at 25°C for 4 days. Four- to six-week old male BALB/c mice were obtained from Charles River Laboratories, Inc. Groups of 8 BALB/c mice were inoculated either via IN or IV route with 30 µl or 100 µl DSW containing 105 cells or 104 cells, respectively. Mice were given food and water ad libitum, and were observed daily according to a welfare score sheet that addresses appearance of disease symptoms, general malaise, and any apparent pain and discomfort in the infected animals. After signs of progressive poor health or discomfort were noted, the infected animals were euthanized by CO2 inhalation followed by cervical dislocation. Data from five infected mice (IN or IV) were used to determine Kaplan-Meyer survival curve using SAS software . An assessment of colonization and multiplication within lungs and brain of infected mice was obtained by using three mice from each group, which were euthanized at day 7 (IV) or day 14 (IN) post infection. None of the animals displayed any signs of overt illness at these two time periods. One-half of lung or brain harvested from infected animals was preserved for histopathological analyses, in Bouin's fixative (brain section) or buffered formalin (lungs). The other halves of target organs were used to calculate relative fungal loads. Tissues were weighed and homogenized in 1 ml DSW, serial dilutions were plated on YPD agar, and incubated at 30°C for determination of CFU. Histopathological examinations were done as described previously Virulence of in vitro interactions between human PMN and C. gattii can be used for a complimentary evaluation of virulence outcome in mouse models, and to assess the likely outcome of initial interactions between C. gattii and phagocyte cells C. gattii cells grown on leaf agar to C. gattii cells grown on YPD agar; a clinical isolate of Candida glabrata was used as control as was C. gattii cap59Δ mutant. Blood was obtained from human volunteers per protocol approved by the New York State Department of Health Institutional Review Board. PMN were isolated by Ficol-Paque (Pharmacia LKB Biotechnology) centrifugation 2, followed by plating on YPD agar for CFU determination C. gattii-PMN interactions were carried using Thermanox® plastic cover slips in 24-well flat-bottom tissue culture plates. PMNs and yeast cells were mixed at a 1∶10 ratio, added to each well, and incubated for 4 hr at 37°C under 5% CO2. Following incubation, cells were fixed with 2% glutaraldehyde and processed as described earlier.We have previously shown that A. thaliana ecotype Col-0 leaves inoculated with C. gattii wild-type strain showed noticeable chlorotic lesions around the initial wound site. Leaves inoculated with C. gattii cap59Δ mutant strain had smaller chlorotic lesions  extracellular fibrils of variable lengths on the cells  extehe cells , and c) ii cells . The dimii cells .C. gattii extracellular fibrils was carried out in HVEM using thick-section microscopy of inoculated leaves  than did mice infected with C. gattii cells grown on traditional YPD agar (C. gattii cells grown on leaf agar and cells grown on YPD agar was even more significant (p<0.0001).Kaplan-Meyer survival curves revealed that mice infected IV with YPD agar . For IN C. gattii cells grown on leaf agar had higher total fungal counts than lungs and brains of mice infected with C. gattii cells grown on YPD agar . This observation was also consistent with more proliferation of C. gattii cells with extracellular fibrils. Unfortunately, it was not practicable to examine these infected tissues by electron microscopy. Therefore, we are unable to provide any evidence of the presence or absence of the C. gattii extracellular fibrils in the infected murine tissues.Lungs and brains of mice infected with YPD agar . This ra damaged . NotablyYPD agar  than waseaf agar . C. gattC. gattii cells grown on leaf agar were significantly more resistant to in vitro killing by human PMN than were C. gattii cells grown on YPD agar (p<0.05). Controls cells in this assay including Candida glabrata and C. gattii cap59Δ mutant were highly susceptibility to PMN killings as expected. PMN assay results were consistent with earlier findings in this study from mouse survival experiments, and these findings raised the possibility that at least one mechanism behind the increased virulence of C. gattii cells with extracellular fibrils is their greater ability to escape from PMN-mediated host defenses.YPD agar ; p<0.05.C. gattii extracellular fibrils in PMN interactions. Neutrophils extracellular traps (NETs) were prominent against control organism Candida glabrata. NETs from one neutrophil entrapped many Candida glabrata cells  leading to extracellular fibrils formation.A. thaliana scratch wound model over other inoculation methods, because it is more consistent with natural wounding of plant tissues as a result of herbivory, insect attack, animal damage or abiotic damaging events C. neoformans. For example, in landmark studies, Staib and colleagues established that pigeon dropping could provide adequate nutrition for C. neoformans asexual and sexual growth, notable because these droppings are a relatively nutrient-poor source for microbial growth C. neoformans undergoes filamentation and sexual mating on pigeon manure agar C. gattii. Extracellular fibrils formation could be an adaptation for colonization on plant leaves, which offer an inhospitable surface for microbial colonization due to limited nutrients, extreme exposure to temperature changes, dehydration and radiation, and unique inter-specific competition Cryptococcus species from the Tremellales lineage that includes C. gattiiWe preferred our C. gattii infections are most likely acquired from host exposure to environments where the fungus grows on or in association with leaves and trees especially tree hollows C. gattii on plant substrates, and then examining its virulence in a murine model of cryptococcosis. We utilized growth on A. thaliana leaf agar, which induced C. gattii to produce extracellular fibrils. Mice infected using C. gattii cells with extracellular fibrils had higher rates of lung and brain colonization by the fungus and significantly lower survival. Histopathological examination of diseased tissues also showed extensive fungal proliferation and destruction of pulmonary tissue in infected mice; the host response appeared rather limited, in terms of PMN infiltration. The ease with which we were able to produce a hypervirulent variant of the parent C. gattii strain, most notably for IN infection, by simply changing the growth substrate, underscored the crucial effect that natural habitat could exert as a modulator of innate virulence of a pathogen. Alternately, C. gattii grown on YPD agar did not express all putative virulence factors. Further experiments are warranted to clarify the roles that growth substrates exert on the virulence of C. gattii.C. gattii cells grown on plant-based substrates showed expected correlation with the increased virulence seen in mice. Thus, two independent experimental approaches indicated that C. gattii with extracellular fibrils display hypervirulence trait. An insight into possible mechanism was achieved when fungus–neutrophils preparations were examined by SEM. We discovered extensive NETs in the presence of C. gattii cells with extracellular fibrils. This observation was significant given that NETs have not been described previously in any interactions with either C. neoformans or C. gattii. More surprisingly, the extent of NET formation did not correlate with fungal killing: more traps were seen in preparations with extracellular fibrils-bearing C. gattii cells that were less susceptible to neutrophils killing. This observation was surprising in view of previous publications on the pathogenic yeast Candida albicans, and several bacterial pathogens; NETs were found to be efficient weapons in PMN-mediated microbial killing C. gattii extracellular fibrils associated virulence is the result of either inhibition of, or escape from, PMN-mediated killing C. gattii. Our findings on neutrophils impairment are significant in view of early landmark studies by Murphy and colleagues C. gattii and C. neoformans; the former, but not the latter, could inhibit PMN chemotaxis and chemokinesis. Our studies also support a role for the microbial capsule in inhibition of NET-mediated killing, as shown earlier for bacteria; encapsulated Streptococcus pneumoniae can escape from the microbicidal action of NETs and from confinement in the lungs, and then cause systemic disease To examine cause-effect relationship between extracellular fibrils formation and hypervirulence by a complementary approach, we utilized in vitro fungus–PMN assay since this assay is rapid and results correlate well with virulence properties seen in animal model C. gattii–plant interactions, and relevance of such interactions to fungal virulence. For example, we know little about the nature of the host plant responses to the presence of the fungus; are plant defense mechanisms critical in permitting colonization? It would also be important to examine C. gattii mutant strains deficient in actin, tubulin and extra-cellular transport for characterization of extracellular fibrils formation. Immune effector cells other than neutrophils are also likely critical in allowing eventual survival and dissemination of C. gattii cells from lungs; do C. gattii extracellular fibrils interact with these cell types? In summary, we believe that examination of pathogenic fungi in conditions that simulate their specialized niches would be a fruitful approach to discern what distinguishes these pathogens from myriad of non-pathogenic fungi from similar environment. Increased knowledge of factors that modulate fungal virulence traits will eventually allow better design of antifungal drugs and vaccines.Additional studies are needed to expand our understanding of Figure S1C. gattii colonized A. thaliana mutant plant leaves. A. thaliana ecotype Columbia (Col-0) and various mutant ecotypes such as eds1 , nahG , npr1 , sid2 (SA-induction deficient), rpm1 (resistance to Pseudomonas syringae pv maculicola 1), and pad4  were grown in a greenhouse at the Biology Department, Syracuse University, Syracuse, NY . Four-to six-week old plants were transferred to the Mycology Laboratory of the Wadsworth Center where they were maintained at 20–23°C with a 12 hr light/dark cycle under 50–70% humidity, in a modified incubator with HEPA filtration. C. gattii cells were subcultured twice in YPD broth at 30°C with 180 rpm shaking and were then collected by centrifugation, washed twice in deionized sterile water (DSW) and re-suspended to a concentration of 1.0×106 cells/mL. Four to six leaves on each A. thaliana plant were lightly wounded on the adaxial surface on either side of the mid-vein with a 27-gauge syringe needle . Two 5-µl drops of 106C. gattii cells/mL were placed at the wound site, and allowed to air dry (5–10 min). Plants were replaced in modified growth incubator maintained at 20–23°C, 12 hr light/dark cycle, and 50–70% humidity. After 7 days, inoculated plants were transferred to a BSL 2 cabinet, leaves excised and photographed with a digital camera. Whole plants and close up of inoculated leaves showed varying levels of scars. .(8.56 MB DOC)Click here for additional data file.Figure S2C. gattii showed enhanced colonization of A. thaliana mutant plant leaves. A number of mutant plants with genotypes derived from A. thaliana Col-0 ecotype were inoculated as described in supplementary C. gattii wild-type cells. The colonization was highest in eds1 and nahG mutants. Overall, colonization was eds1>nahG>sid2>npr1>rpm1. The experiment was repeated once.(0.14 MB DOC)Click here for additional data file.Figure S3C. gattii extracellular projections were formed on agar with plant substrates. Dialysis tubing was cut to small squares approximately 1 cm×1 cm and sterilized by boiling in water for 20–30 min. Three squares of sterilized tubing were then laid flat over YPD, A. thaliana leaf, black cherry wood chip or Niger seed agar plates. Each square was inoculated with 100 µl of 107 cells/ml C. gattii wild-type cells. Plates were allowed to dry and incubated at 25°C for 4 days. Small blocks of agar (∼1 cm×1 cm) were removed and fixed in 2% glutaraldehyde 0.2 M sodium cacodylate buffer, and dehydrated by graded alcohol series, critical point dried, gold sputter coated and imaged by. (A) YPD agar, (B) A. thaliana leaf agar, (C) Niger seed agar, and (D) Black cherry wood chip agar. SEM micrographs of one representative C. gattii cell imaged from (E) YPD agar, (F) A. thaliana leaf agar, (G) Niger seed agar, and (H) Black cherry wood chip agar, scale bar 1 µm. Notably, C. gattii extracellular fibrils were absent on YPD agar in contrast to abundant formation on agar supplemented with plant substrates.(3.15 MB DOC)Click here for additional data file.Figure S4C. gattii extracellular fibrils are altered by cytoskeletal protein inhibitors. We examined the roles of actin, tubulins and other cytoskeletal proteins on the formation of C. gattii extracellular fibrils since these proteins are important determinants of cell shape . C. gattii wild-type and cap59Δ mutant strain were grown in YPD broth for 12–16 hr. Thiabendazole (25–150 µg/ml), cytochalasin B (25–150 µg/ml), mebandazol (20–80 µg/ml), or latrunculin B (100 µ/ml–400 µM/ml), were added to individual cultures and incubated for an additional 6 hr (2). An aliquot of yeast cells were also treated with 2 M sodium azide at 65°C for 30 min to render them non-viable. Cells were collected by centrifugation, washed twice with SDW, and re-suspended to a concentration of 108 cells/ml. Cells were serially diluted in SDW and plated on YPD agar to determine any loss in viability. None of the drug treatments caused significant loss of viability. Six to eight leaves each from at least three different A. thaliana plants were scratch wounded and inoculated with two 5-µl drops of 1×106C. gattii cells obtained from each treatment described above. Controls included leaves from wounded plants inoculated with SDW, and non-wounded, non-inoculated A. thaliana plants. Leaves were harvested 7-days post inoculation and prepared for light microscopy and SEM as described in the previous section. Microfilament (Actin) and microtubule (Tubulin) inhibitors cytochalasin B (Top left), latrunculin B (Top right), thiabendazole (Bottom left) or mebandazole (Bottom right) alter C. gattii cell attachment and extracellular fibril formation in a concentration dependent manner. Lower magnification (left panel) and higher magnification images (right panel) for each treatment are shown with red borders demarcating original inoculation sites. The concentrations used were cytochalasin B ; latrunculin B ; thiabendazole ; mebandazole . A concentration dependent effect on extracellular fibril formation is most pronounced with latrunculin and mebendazole. Click here for additional data file.Figure S5C. gattii cells, utilizing the Thermonox® plastic cover slip assay. Thiabendazole (25–150 µg/ml), cytochalasin B (25–150 µg/ml), mebandazol (20–80 µg/ml), or latrunculin B (100 µM/ml–400 µM/ml), were added to individual cultures and incubated for an additional 6 hr Click here for additional data file.Figure S6C. gattii cells grown on leaf agar. BALB/c mice were inoculated either via IV  or IN  route with either 30 µl or 100 µl containing 104 (IN) or 105 (IV) C. gattii cells passage two times on YPD, Niger seed (NGS), A. thaliana leaf (LEAF), or black cherry wood chip agar (WOOD). Mice were sacrificed 7 days (IV) or 14 days (IN) post infections and CFUs per gram brain or lung tissue was determined. Notably, enhanced colonization of brain and lung tissues by C. gattii grown on A. thaliana leaf agar is independent of the route of infection.Enhanced colonization of brain and lung tissues by (0.21 MB DOC)Click here for additional data file.


Streptococcus have not been clarified. We previously reported that mutations of csrS/csrR genes, a two-component negative regulator system for multiple virulence genes of Streptococcus pyogenes, are found among the isolates from STSS patients. In the present study, mutations of another negative regulator, rgg, were also found in clinical isolates of STSS patients. The rgg mutants from STSS clinical isolates enhanced lethality and impaired various organs in the mouse models, similar to the csrS mutants, and precluded their being killed by human neutrophils, mainly due to an overproduction of SLO. When we assessed the mutation frequency of csrS, csrR, and rgg genes among S. pyogenes isolates from STSS (164 isolates) and non-invasive infections (59 isolates), 57.3% of the STSS isolates had mutations of one or more genes among three genes, while isolates from patients with non-invasive disease had significantly fewer mutations in these genes (1.7%). The results of the present study suggest that mutations in the negative regulators csrS/csrR and rgg of S. pyogenes are crucial factors in the pathogenesis of STSS, as they lead to the overproduction of multiple virulence factors.Streptococcal toxic shock syndrome (STSS) is a severe invasive infection characterized by the sudden onset of shock and multiorgan failure; it has a high mortality rate. Although a number of studies have attempted to determine the crucial factors behind the onset of STSS, the responsible genes in group A  streptococcus (GAS) causes life-threatening severe invasive diseases, including necrotizing fasciitis and streptococcal toxic shock-like syndrome. Although many studies have attempted to determine factors that are crucial for the onset of streptococcal toxic shock syndrome (STSS), bacterial factors responsible for it have not been clarified. By comparing genome sequences of clinical GAS isolates from STSS with those of non-invasive infections, we showed that mutations of negative regulator genes  were detected at a high frequency of more than 50% in STSS isolates, but at a low frequency of less than 2% in non-invasive isolates. These mutations of negative regulators were found in various emm-genotyped STSS isolates but not in a particular emm genotype. These mutants enhanced virulence in mouse models. Such results indicated that mutations of bacterial negative regulators are crucial for the pathogenesis of STSS due to the overproduction of multiple virulence factors under the de-repressed conditions.Group A  Streptococcus pyogenes  is one of the most common human pathogens. It causes a wide variety of infections, ranging from uncomplicated pharyngitis and skin infections to severe and even life-threatening manifestations, such as necrotizing fasciitis (NF) and bacteremia. Several streptococcal virulence factors, including pyrogenic exotoxins, streptokinase, and streptolysins, are reportedly involved in these diseases. Streptococcal toxic shock syndrome (STSS) is a severe invasive infection that has been recently characterized by the sudden onset of shock and multiorgan failure; it has a high mortality rate, ranging from 30% to 70% csrS (covS) gene—a sensor gene of a two-component regulatory system—were detected in a panel of clinical isolates from severe invasive streptococcal infections, but not in non-STSS isolates csrS mutated strains from being killed by neutrophils, resulting in increased virulence in lethality in the mouse model csrS gene into csrS-mutated STSS isolates dramatically decreased their virulence in lethality csrR (covR) mutations were found in the clinical isolates of STSS patients csrS/csrR mutations are closely associated with the onset of STSS.Recently, we and others have reported that mutations in the csrS/csrR gene sequence in each STSS isolate csrS/csrR gene of STSS isolates csrS/csrR genes in a mass of isolates, and what mutations other than csrS/csrR genes may be responsible for the onset of STSS.However, several study groups that investigated the csrS/csrR genes of 164 GAS strains that have been isolated from STSS patients in Japan since 1992. Almost one-half of the STSS isolates had a mutation in the csrS/csrR genes. In addition, we found a mutation in the rgg (ropB) gene, instead of the csrS/csrR genes, in the clinical isolates of patients with STSS. Since the rgg gene negatively regulates various virulence genes in a manner similar to that of the csrS gene, a mutation of the rgg gene in STSS clinical isolates increased the expression of several virulence genes and virulence in lethality in the mouse model. Such mutations were detected at a high frequency in more than 50% of STSS isolates. These findings suggest that mutations in the negative regulators such as csrS/csrR and rgg of S. pyogenes bring about overproduction of a number of virulence factors, such as SLO, and play a crucial role in the onset of STSS.In this study, we sequenced the csrS gene of emm49 clinical isolates from STSS patients csrR gene in emm3 clinical isolates from STSS patients csrS/csrR mutations play important roles in the pathogenesis of STSS. To evaluate the frequency of these csrS/csrR mutations in isolates from clinical cases of STSS csrS and csrR genes in STSS clinical isolates from sterile sites (164 isolates) and non-STSS clinical isolates from non-sterile sites (59 isolates). The diagnoses, sites of bacteria isolation, and characteristics of S. pyogenes isolates are described in S. pyogenes isolates, 55 isolates  (33.5%) had mutations in the csrS gene, 19 isolates  (11.6%) had mutations in the csrR gene, and 2 isolates (1.2%) had mutations in both genes  had mutations in neither csrS nor csrR ; csrR mutation, 0% (0/59); total, 1.69% (1/59); p = 0.00000000062 by χ2 analysis]. Although csrS/csrR mutations were more common among STSS isolates examined than among non-STSS isolates, they were not present in all STSS isolates. This may suggest that mutations in other regulatory genes may also be found among STSS isolates.In our previous study, we reported that there were various types of mutations in the th genes  and 2. Tnor csrR  and 2. OS. pyogenes isolates. We determined the sequence of the csrS/csrR genes from a panel of emm-matched GAS isolated from STSS patients; NIH1 , NIH3, NIH8, NIH34, NIH152-3, NIH249, NIH327, and NIH352 were clinical isolates of the emm3 genotype from STSS and C500, OT22, and K33 were emm3 non-STSS isolates . To determine whether other emm3 STSS strains have possible mutations in their genomes, we used comparative genome sequencing (CGS) emm3 STSS and non-STSS isolates. We found several genes with SNPs in the NIH1 genome in comparison with that of non-invasive isolates K33. Three  of them had non-synonymous amino acid change in NIH1 but not in K33 and C500  than non-STSS isolates, due to a mutation in the csrS gene. Therefore, we examined whether a panel of emm3-genotyped STSS isolates possessing mutations in the csrS or rgg gene and emm3 non-STSS isolates lacking mutations could produce SLO . The comparison of the supernatants at the same growth condition (data not shown) showed that larger amounts of SLO were secreted by STSS isolates possessing mutations in the csrS gene  or rgg gene  than by non-STSS isolates  , the SLO secretion was decreased to the level of that in non-STSS isolates (rgg gene was responsible for increased SLO production in the culture supernatant as that of csrS gene was.We and K33) . These disolates . In contisolates . Taken tS. pyogenesrgg gene, we performed real-time polymerase chain reaction (RT-PCR) with specific primers for each virulence-associated gene. The amounts of mRNA of protein G-related alpha2-macroglobulin-binding protein (grab), nicotine adenine dinucleotide glycohydrolase (nga), streptodornase (phage-associated) (sdn), streptokinase (ska), and slo in the STSS isolate of NIH34 with the rgg mutation were larger than those of the pharyngitis isolate of K33 with the intact rgg gene (speB) and streptolysin S (sagA) genes in the STSS isolate of NIH34 were less than a half of those in the non-STSS isolate of K33 (mac), C5a peptidase (scpA), IL-8 protease (scpC), superantigen (speA), and DNA gyrase (gyrA) genes in NIH34 were almost the same as those in K33  (rgg mutation in the K33 strain (K33rgg) resulted in higher lethality among infected mice than the K33 strain (p = 0.00067) and exerted a level of lethality similar to NIH34. The NIH34 strain into which an intact rgg gene (NIH34+rgg) had been introduced exhibited less lethality than the NIH34 strain (p = 0.0000097) and possessed the same level of lethality as the K33 strain. We confirmed that bacteria isolated from kidney or liver of infected mice at day 6 retained the mutation (data not shown). Therefore, the mutation of the rgg gene in the STSS isolates contributed to enhanced lethality in the mouse model. Histopathological examination of mice infected with NIH34 or K33rgg strains was carried out. Scattered multiple inflammatory foci containing bacterial colonies were observed in the kidney. The foci were accompanied with neutrophil infiltration, cell debris and hyalinization (rgg+ strains (rgg resulted in significantly larger lesions as compared to the infection with NIH34rgg+ or K33 (p<0.01) (rgg mutants but not the intact rgg strains. We confirmed that bacteria isolated from lesions retained the mutation (data not shown).This showed that subcutaneous inoculation of mice led to the systemic spreading in the rgg mutant. These results suggest that the rgg-mutated strains isolated from STSS patients are more virulent in vivo than strains from patients with non-invasive infections, and that the increase in virulence in vivo is canceled by introducing an intact rgg gene.To elucidate the role of 0.00027) . Introdunization . In cont strains . In anot(p<0.01) . Bacterirgg mutation on resistance to killing by neutrophils. As shown in rgg or between NIH34 and NIH34rgg+. Furthermore, the scpC mutation in the NIH34 strain did not have a significant influence on the migration of human neutrophils, compared to the csrS mutation, as previously reported (rgg mutation (rgg gene had little influence on the migration of human neutrophils in response to IL-8. As previously reported rgg-mutated strains (NIH34 and K33rgg), whereas strains with the intact rgg gene (K33 and NIH34rgg+) did not cause obvious impairment of neutrophils  is a cytotoxic protein secreted through the SLO complex nga expression was negatively regulated by the rgg gene (nga mutant against neutrophils. The neutrophil-killing activity was significantly decreased in an nga-deficient mutant (NIH34nga), but to a lesser extent as compared to the activity of NIH34slo. Therefore, these findings strongly suggest that SLO is a factor essential for neutrophil-killing activity in rgg-mutated emm3 STSS isolates, and that Nga partially influences the neutrophil-killing activity.In our previous study, using the Transwell system, we showed that SLO, which causes necrosis in neutrophils, and an IL-8 protease of ScpC are important for bacterial resistance to killing by neutrophils reported . This fimutation . Collecttrophils . In the rgg gene , we examcsrS mutation in the emm49-genotyped strains was a key to the onset of severe invasive streptococcal infections csrS mutant showed higher lethality in a mouse model and more efficiently killed human neutrophils than the non-mutated strain csrS gene with that in the rgg gene, in terms of in vivo virulence in lethality and impairment of neutrophil function in vitro. Intraperitoneal infection of mice with the csrS mutant (K33csrS) caused earlier death and higher lethality than did infection with the rgg mutant (K33rgg) (p = 0.017)  . Further mutants . These a as ScpC  and 3A,Brgg gene or the csrS/csrR genes in STSS clinical isolates. We sequenced the rgg gene in strains isolated from sterile sites of STSS patients and found that 42 of 164 (25.6%) isolates carried some mutations  in the rgg gene. To determine whether these mutations contributed to a loss of Rgg function, we examined the level of SLO and SpeB rgg mutation rgg mutants (strains with mutation only in rgg) showing no increase of SLO expression were emm12-genotyped strains and had a mutation at the same position in comparison with other non-invasive strains. This mutation was synonymous in the level of amino acid, so we defined the mutants are functional as shown in rgg, csrS, csrR, both csrS and csrR, both csrS and rgg, and both csrR and rgg genes, respectively, so that a total of 57.3% of the STSS isolates carried mutations in one or more of these negative regulator genes . This finding suggests that mutations in the csrS/csrR genes or the rgg gene are crucial factors causing severe invasive infections, such as STSS.In this study, we found that there are mutations in the or genes  and 2. Oor genes  and 2. TS. pyogenes has become a serious health problem in both developed and developing countries. In this study, we found a high frequency of mutations of negative regulators in STSS clinical isolates. The rgg mutant killed human neutrophils, impaired multiple organs, and enhanced lethality in the mouse model, similar to the csrS mutant. These findings suggest that the impairment of negative regulators of S. pyogenes virulence genes induces neutrophil incompetence and subsequent STSS infection. This study is the first to show that clinical S. pyogenes isolates from STSS patients have mutations in one or more of genes––rgg, csrS, and csrR––which are involved in the negative regulation of multiple virulence genes.Since the late 1980s, STSS caused by csrS/csrR genes of 5 emm49 strains isolated from patients with severe invasive infections emm49 genotype possess mutations in the csrS and csrR genes: 46.3% of the STSS isolates including various emm genotypes had non-functional mutations in one or more of the csrS/csrR genes. This finding suggests that mutations in the csrS/csrR genes are commonly recognized in STSS clinical isolates with various emm genotypes.In our previous study, we found mutations in the rgg and csrS genes of the isolates. The loss of function incurred by the mutation in the rgg gene in emm3-genotyped S. pyogenes affected the regulatory network of the virulence-associated genes; hence, the mutated strains could resist killing by neutrophils and caused damage to various organs in the mouse models. Therefore, the mutated emm3-genotyped S. pyogenes strains may potentially cause severe infections such as STSS in humans. Hollands et al. rgg (ropB) gene reduces M1T1 group A streptococcal virulence. We examined the contribution of Rgg to the pathogenesis of systemic infections by using a clinical emm1-genotyped STSS isolate, NIH186, and an emm1-genotyped pharyngitis isolate, Se235. NIH186 and Se235rgg, both of which had a mutation in the rgg gene, showed higher lethality than NIH186rgg+ and Se235, in both of which the rgg gene is intact (data not shown). The rgg mutants impaired neutrophils to a greater extent than the rgg-intact strains did  was much higher than that in non-invasive isolates (1.7%). These results suggest that mutations in the negative regulators of various virulence genes are important to the STSS onset. However, 42.7% of the STSS isolates did not have mutations in the csrS mutant (K33csrS) showed higher lethality than the rgg mutant. However, in the present study, the mortality rate of STSS patients infected with the rgg mutant was 60.9%, while that of patients infected with the csrS mutant was 47.2% (data not shown). These findings suggest that the rgg mutant also causes high lethality in humans, which may indicate differences in disease severity between humans and mice. Streptokinase is highly specific for human plasminogen, exhibiting little or no activity to those of other animal species rgg mutant.In the mouse model, the Staphylococcus aureus (CA-MRSA) causes severe invasive infections, resulting in NF or even death agragr regulator results in increased expression of virulence factors and increased virulence. Severe invasive infections are caused not only by S. pyogenes but also by other bacteria such as other Streptococcus, Staphylococcus aureus, Vibrio vulnificus, and Aeromonas spp. Such severe invasive infections may be caused by the coordinated overexpression of multiple virulence factors that are affected by the global regulatory network.Collectively, we showed that mutations of negative regulators that result in the overproduction of multiple virulence factors are important to the onset of severe invasive infections such as STSS. Recently, it has been reported that community-associated methicillin-resistant This study complies with the guidelines of the declaration of Helsinki. This study protocol was approved by the institutional individual ethics committees for the use of human subjects  and the animal experiments . Written informed consent was obtained from all study participants or their legal guardians for the patients who died. All clinical samples and healthy human neutrophils were stripped of personal identifiers not necessary for this study. All animal experiments were performed according to the Guide for animal experiments performed at National Institute of Infectious Diseases, Japan.S. pyogenes strains and plasmids used in this study are described in Escherichia coli DH5α was used as a host for plasmid construction and was grown in a Luria-Bertani liquid medium with shaking or on agar plates at 37°C. S. pyogenes was cultured in Todd-Hewitt broth supplemented with 0.5% yeast extract (THY medium) without agitation or on tryptic soy agar supplemented with 5% sheep blood. Cultures were grown at 37°C in a 5% CO2 atmosphere. When required, antibiotics were added to the medium at the following final concentrations: erythromycin, 300 µg/mL for E. coli and 1 µg/mL for S. pyogenes; and spectinomycin (Sp), 25 µg/mL for each of E. coli and S. pyogenes. The growth of S. pyogenes was turbidimetrically monitored at 600 nm, using a MiniPhoto 518R .The csrS, csrR, and rgg genes were determined by automated sequencers, i.e., an Applied Biosystems 3130xl Genetic Analyzer and an Applied Biosystems 3130 Genetic Analyzer . Sequencing data were deposited in the DNA Data Bank of Japan (DDBJ).The nucleotide sequences of the Male five to six-week-old outbred ddY and hairless mice were purchased from SLC  and maintained in specific pathogen-free (SPF) conditions. All animal experiments were performed according to the guidelines of the Ethics Review Committee of Animal Experiments of the National Institute of Infectious Diseases, Japan.rgg was amplified from the NIH34 (for emm3) and NIH186 (for emm1) chromosomal DNA, using the primers of rgg-del1 and rgg-del2  were then selected by using spectinomycin-containing agar plates. Deficiency of the native rgg gene was verified by PCR.A 692-bp DNA fragment containing the internal region of rgg-del2 . The PCRcsrS was amplified from the K33 chromosomal DNA, using the primers of csrS-def1 and csrS-def2  and F482 (for emm1) was purified and used as a template for the PCR amplification of the intact rgg gene. The primers used were 5′-GGGGATCCTTATGGCTATATCATAGCTG-3′ (sense) and 5′-GGGAATTCTGTTGAGATAAACTACACC-3′ (antisense). The PCR fragment was ligated into the plasmid pSF152, and the resultant plasmids pSFrgg3+ (for emm3) and pSFrgg1+ (for emm1) were used for chromosomal integration into the mutated rgg gene of isolates from STSS patients, as described previously r) were then selected by using spectinomycin (Sp)-containing agar plates. Integration of the intact rgg gene was confirmed by PCR.The replacement of a mutated 600 = 1.0) was passed through a 0.45-mm pore size membrane filter , to remove the remaining cells. Proteins in the resulting cell-free supernatant were precipitated with 10% trichloroacetic acid and resuspended in a sample loading buffer containing a saturated Tris base. Samples were heated at 100°C for 3 min and separated on sodium dodecyl sulfate (SDS)–12.5% polyacrylamide gels. To detect SLO, the proteins on the gels were electrophoretically transferred onto a PVDF membrane. The membrane was blocked with 5% nonfat milk +0.2% Tween-20 and reacted with primary anti-SLO polyclonal antibody , secondary antibody peroxidase-conjugated anti-rabbit Ig , and an ECL Plus Western blotting Detection System .A total of 1 mL of the supernatant of an overnight bacterial culture (ODS. pyogenes SSI-1 (GenBank accession No. BA000034).Complete-genome comparisons were performed with an array-based service (CGS) provided by NimbleGen Systems Inc.  Total RNA was extracted from bacterial cells using the RNeasy Protect Bacteria Mini Kit , according to the manufacturer's instructions. Complementary DNA synthesis was performed with the PrimeScript RT reagent kit  , also following the manufacturer's instructions. Transcript levels were determined using the ABI PRISM Sequence Detection System 7000 (Applied Biosystems) and Premix Ex Taq  (Takara). For real-time amplification, the template was equivalent to 5 ng of total RNA. Measurements were performed in triplicate; a reverse-transcription-negative blank of each sample and a no-template blank served as negative controls. The primers and probes used are listed in 2 atmosphere, pelleted by centrifugation, washed twice with sterile phosphate-buffered saline (PBS), suspended in sterile PBS. A total of 1×107 CFU of GAS suspended in 0.5 mL of PBS was injected intraperitoneally into five to six-week-old ddY outbred male mice (10–16 mice/GAS isolate). The number of surviving mice was compared statistically, using the Kaplan-Meier log-rank test. For the subcutaneous infection model, male hairless mice Hos:Hr-1 were injected with 1×107 CFU of GAS in a 100-µl suspension of GAS in PBS. The lesion area was measured daily and analyzed. Dissemination in kidney and spleen of GAS was evaluated by colony counting at day 7 post-infection.GAS was grown to late-log phase (OD600 = 0.6−0.8) at 37°C in a 5% COFor histopathological analysis, the tissues from GAS-infected mice were directly fixed in 10% neutral-buffered formalin, embedded in paraffin, sectioned and stained with hematoxylin and eosin (H&E).Human neutrophils were isolated from the venous blood of five healthy volunteers, in accordance with a protocol approved by the Institutional Review Board for Human Subjects, National Institute of Infectious Diseases 5 neutrophils in Roswell Park Memorial Institute (RPMI) medium containing 25 mM HEPES and 1% FCS in Transwell inserts  were placed in 24-well plates containing 600 µl medium or 100 nM interleukin (IL)-8 solution ; the plates were then incubated with or without 5×106 bacteria for 1 h at 37°C, in advance of the assay. After 1 h of incubation, cells in the lower wells were collected and 104 10-µm microsphere beads  were added. Cells were stained with propidium iodine  for flow cytometry to quantify the viable neutrophils; analysis was performed, using the FACS Calibur .Chemotaxis assays were performed as previously described http://www.ddbj.nig.ac.jp/index-e.html) accession numbers for the genes and gene products discussed in this paper are: TK283 csrR locus - AB517797; TK929 csrR locus - AB517804; NIH43 csrR locus - AB517807; NIH75 csrR locus - AB517814; NIH136 csrR locus - AB517819; NIH157 csrR locus - AB517822; NIH212 csrR locus - AB517826; NIH216 csrR locus -AB517827; NIH252-2 csrR locus - AB517838; NIH259 csrR locus - AB517839; NIH273 csrR locus - AB517842; NIH300 csrR locus - AB517850; NIH301 csrR locus - AB517851; NIH323-1 csrR locus - AB517853; NIH381-1 csrR locus - AB517863; NIH404 csrR locus - AB517867; NIH406 csrR locus - AB517868; NIH447 csrR locus - AB517877; NIH5 csrS locus - AB517796; TK76 csrS locus - AB517800; NIH18 csrS locus - AB517801; TK280 csrS locus - AB517803; NIH35 csrS locus - AB517805; NIH44 csrS locus - AB517809; NIH49 csrS locus - AB517810; NIH55 csrS locus - AB517812; NIH75 csrS locus - AB517815; NIH102 csrS locus - AB517817; NIH152-3 csrS locus - AB517820; NIH156-1 csrS locus - AB517821; NIH205 csrS locus - AB517823; NIH200-4 csrS locus - AB517825; NIH220-1 csrS locus - AB517828; NIH222 csrS locus – AB517829; NIH230 csrS locus - AB517830; NIH236 csrS locus - AB517831; NIH238 csrS locus - AB517833; NIH243 csrS locus - AB517834; NIH253-1 csrS locus - AB517835; NIH250-2 csrS locus - AB517836; NIH263-2 csrS locus - AB517840; NIH268 csrS locus - AB517841; NIH283-1 csrS locus - AB517844; NIH286 csrS locus - AB517845; NIH287-1 csrS locus - AB517846; NIH296 csrS locus - AB517847; NIH297 csrS locus - AB517849; NIH317 csrS locus - AB517852; NIH325-1 csrS locus - AB517854; NIH345 csrS locus - AB517855; NIH372 csrS locus - AB517859; NIH437 csrS locus - AB517862; NIH403 csrS locus - AB517866; NIH421 csrS locus - AB517871; NIH424-1 csrS locus - AB517873; NIH433 csrS locus - AB517874; NIH453 csrS locus - AB517875; Se202 csrS locus - AB517643; NIH3 rgg locus - AB517795; NIH8 rgg locus - AB517798; TK65 rgg locus - AB517799; NIH18 rgg locus - AB517802; TK1097 rgg locus - AB517806; NIH43 rgg locus - AB517808; NIH50 rgg locus - AB517811; NIH60 rgg locus - AB517813; NIH91 rgg locus - AB517816; NIH118 rgg locus - AB517818; NIH186 rgg locus - AB517824; NIH236 rgg locus - AB517832; NIH250.2 rgg locus - AB517837; NIH273 rgg locus - AB517843; NIH293 rgg locus - AB517848; NIH357 rgg locus - AB517856; NIH366 rgg locus - AB517857; NIH371 rgg locus - AB517858; NIH372 rgg locus - AB517860; NIH374-2 rgg locus - AB517861; NIH381-1 rgg locus - AB517864; NIH390 rgg locus - AB517865; NIH406 rgg locus - AB517869; NIH409 rgg locus - AB517870; NIH422 rgg locus - AB517872; NIH445 rgg locus - AB517876.The DNA Data Bank of Japan (DDBJ) (Table S1emm3 and emm1 genotype S. pyogenes and plasmids used in this studyStrains of (0.06 MB DOC)Click here for additional data file.Table S2Amino acid difference in comparison with K33(0.04 MB DOC)Click here for additional data file.Table S3Primers used for the construction of deletion mutants(0.03 MB DOC)Click here for additional data file.Table S4Primers used for RT-PCR(0.06 MB DOC)Click here for additional data file.Figure S1rgg mutation of emm1-genotyped S. pyogenes on survival of human neutrophils. Human neutrophils migrated in the lower wells of a Transwell system in response to IL-8. The migrated human neutrophils were brought into contact with various emm1 GAS strains   , and the(0.05 MB TIF)Click here for additional data file.


Lycaon pictus) in Tanzania, killing 49 of 52 animals within 2 months. The causative agent was identified as Canine distemper virus (CDV) by means of histologic examination, virus isolation, reverse transcriptase-polymerase chain reaction analysis, and nucleotide sequencing. This report emphasizes the importance of adequate protection against infectious diseases for the successful outcome of captive breeding programs of endangered species.In December 2000, an infectious disease spread through a captive breeding group of African wild dogs ( Lycaon pictus) is a highly endangered carnivore found in Africa south of the Sahara. Its population, estimated at <5,500, has declined dramatically in recent decades. Suggested causes for this decline include habitat loss, killing by humans, reduced prey availability, competition with other carnivores, and infectious diseases, including rabies and canine distemper The African wild dog , determined by methods similar to those used in a study of large felids Neutralizing antibody levels to Morbillivirus-specific primers P1: 5´ATGTTTATGATCACAGCGGT 3´ and P2: 5´ATTGGGTTGCACCACTTGTC 3´, which have been used before for phylogenetic analysis of morbilliviruses Morbillivirus P-gene fragment. Phylogenetic analysis of the nucleotide sequences from the resulting PCR fragments demonstrated that the causative virus was most closely related to CDV (Canis familiaris), lions (Panthera leo), and bat-eared foxes  from East Africa in the 1990s , virus isolation (n=2), and reverse transcriptase-polymerase chain reaction (RT-PCR) with d to CDV  and clushe 1990s . P-gene These results show that the primary cause of death of these African wild dogs was CDV infection. Canine distemper is highly infectious for many species of carnivores and causes high death rates in immunologically naïve populations Potential routes of transmission of this virus to the captive breeding groups are by direct contact with infected domestic dogs or wild carnivores or indirectly by contact with humans or their equipment. Domestic dog populations in some parts of Tanzania are endemically infected with CDV and were considered to be the source of infection for canine distemper in Serengeti lions in 1994 Phoca vitulina) and dogs against phocine distemper virus infection Because vaccination with live attenuated virus had been suspected of causing past deaths of African wild dogs (Conservation of endangered species, both free-living and captive, has been jeopardized by infectious disease outbreaks in the past


During 1999 and 2000, a disease outbreak of West Nile (WN) virus occurred in humans, horses, and wild and zoological birds in the northeastern USA. In our experiments, WN virus infection of young domestic geese (Anser anser domesticus) caused depression, weight loss, torticollis, opisthotonus, and death with accompanying encephalitis and myocarditis. Based on this experimental study and a field outbreak in Israel, WN virus is a disease threat to young goslings and viremia levels are potentially sufficient to infect mosquitoes and transmit WN virus to other animal species.