Here, we investigated the effects of asymmetry in replichore organisation in Escherichia coli. We show that large chromosome inversions from the terminal junction of the replichores disturb the ongoing post-replicative events, resulting in inhibition of both cell division and cell elongation. This is accompanied by alterations of the segregation pattern of loci located at the inversion endpoints, particularly of the new replichore junction. None of these defects is suppressed by restoration of termination of replication opposite oriC, indicating that they are more likely due to the asymmetry of replichore polarity than to asymmetric replication. Strikingly, DNA translocation by FtsK, which processes the terminal junction of the replichores during cell division, becomes essential in inversion-carrying strains. Inactivation of the FtsK translocation activity leads to aberrant cell morphology, strongly suggesting that it controls membrane synthesis at the division septum. Our results reveal that FtsK mediates a reciprocal control between processing of the replichore polarity junction and cell division.Bacterial chromosomes are organised as two replichores of opposite polarity that coincide with the replication arms from the  ori and terminates in the diametrically opposite zone, ter, defining two replication arms. These are also called replichores to account for base composition and sequence motif polarity biases from ori to ter. This organisation as symmetrical replichores is conserved in bacteria. Here, we have investigated the effect of replichore asymmetry in E. coli. We show that it blocks the cell cycle at a step posterior to chromosome replication. This phenotype is not corrected by restoration of the termination of replication opposite ori, strongly suggesting that it is due to the asymmetry of replichore polarity. FtsK, a DNA-translocase associated with the division septum that processes the terminal junction of replichore polarity, is essential for growth and for the controlled blockage of cell growth in cells with asymmetric replichores. These results reveal a reciprocal control between the processing of the terminal junction of the replichores by FtsK and the progression of the cell cycle.In most bacteria, chromosomes consist of a single replication unit. Replication initiates at a single  Cell proliferation involves coordination between replication of the genome, segregation of sister chromosomes to daughter cells and cell division. In bacteria, these processes are not necessarily sequential but can overlap: segregation begins during the replicative C period (S phase), continues during the post-replication D period (G2/M) and may last until constriction of the division septum.Escherichia coli, chromosome replication initiates at a single origin, oriC, and progresses bidirectionally to the termination region, ter. The ter region is flanked by multiple replication terminators  and a second endpoint outside the Ter macrodomain provoke a specific cell-cycle defect in E. colioriC does not suppress the D period blockage. Chromosome loci at the edge of the inversion show defects in their intracellular localisation, particularly the displaced PJ. Most interestingly, translocation by FtsK becomes essential in the inversion-carrying strain and its inactivation leads to defects in peptidoglycan synthesis.We have previously reported that large chromosome inversions with a fixed endpoint at the dif-sp5) inversion, was chosen as a representative strain carrying a large inversion from the terminal junction of replichore polarity (PJ)  . It carre new PJ  and is te new PJ  and riche new PJ . This gre new PJ . Cells we new PJ  are overe new PJ . These dAfter a shift to non-permissive conditions, cells rapidly stop growing and the fraction of double-cells increased  inversion since other strains carrying interdomain inversions from the terminal PJ display the same behaviour (for example the Inv(dif-sp39) strain in dif position do not show over-representation of double-cells (the Inv(dif-ydfE) strain in These observations were confirmed by analysis of cell size distribution of XL151 by flow cytometry . Non-grodif-sp39); To gain insight into the cell cycle of XL151, we first analysed the timing of replication initiation using flow cytometry of cells in which initiation of replication and cell division had been blocked with rifampicin and cephalexin . In XL15dif-sp5) displaces replication terminators and extends one replication arms (ori/ter ratio was measured by southern blot hybridisation (ydeQ and TerA loci when compared to the TerE locus in XL151 in southern blot hybridisations (not shown).The C period is expected to be longer in XL151 than in wt since Inv and replichore 1 locus (crl) separate after a moderate period of colocalisation time  and migrate from mid-cell to the quarter-cell positions while sister ter loci (ydeQ and ydeV) stay colocalised for a longer time , remaining at mid-cell until division (ydeV and ydeQ further shows that the immediate vicinity of dif is the last separated zone of sister chromosomes In the conditions used (64 min doubling time for the wt), sister origin region   does not displace the ydeV locus on the chromosome but changes both its distance from the PJ and the timing of its replication with respect to termination. This is accompanied by a dramatic modification of its segregation pattern and a shorter colocalisation time (compare crl locus segregates in a manner similar to ydeV in accord with its new position (ydeQ locus remains at the PJ (dif-sp5) inversion provokes a looser positioning of all tested loci and an early separation of the ydeQ locus with respect to cell division which may be responsible for or linked to the prolonged D period in XL151.In XL151, we observed that the positioning of loci is generally looser than in the wt; localisation between the mid- and quarter-cell positions is more frequently observed (ity (PJ) . Segregae origin . The fre compare . The crlposition . The ydet the PJ  and segrt the PJ . Most fotus gene was mutated to inactivate Ter sites. The resulting strain grows with a 86 min doubling time, intermediate between wt and XL151 : double-cells are over-represented in permissive and non-permissive conditions , which keeps some features of a terminal locus, particularly a high Ci, independently of its replication timing. The replication timing rather influences the home positioning of loci after their segregation  allele which translocates with no orientation control ftsKwt gene , which lead to a major imbalance of the two replichores. In such strains, the replication period (C) increases as a consequence of the lengthening of one replication arm. The post-replicative period (D) is lengthened or blocked depending on growth conditions, accounting for the accumulation of cells arrested at a late step of the cell cycle. These cells harbour apparently segregated nucleoïds and most often a constricting division septum, indicating that the blocked step is in late D period near the time of cell separation. Importantly, restoring termination of replication to the region opposite A striking feature of strains carrying large inversions from the terminal PJ is that cell elongation is delayed or blocked at the same time as cell division. This phenotype is in contrasts to cell division phenotypes reported previously. When inhibition directly targets divisome components, with conditional mutants mukB- strains in which the structure and/or segregation of the chromosome is affected dif site to resolve chromosome dimers The KOPS-directed DNA translocase activity of the FtsK protein becomes essential for growth after inversion, whereas it is mainly required when a chromosome dimer has formed in the wt strain We assume that FtsK action is required and/or slowed by the displacement of the PJ and that this, in turn, slows or blocks the closure of the division septum. It follows that septum closure depends on a correct processing of the replichore junction by FtsK, independently of the presence of chromosome dimers. In this view, non-permissive conditions, which accelerate the C and D periods, may not allow sufficient time for FtsK to reach the PJ before the end of the next replication cycle that may counteract FtsK translocation to the PJ. We propose that cell division is controlled by FtsK translocation to the PJ in strains harbouring asymmetric replichores. Whether or not this control operates in wt strains remains to be shown.ATPftsK mutant of strain XL151 provokes the appearance of aberrant cell morphologies reminiscent of those due to defects in peptidoglycan synthesis ftsK, 44ftsK, is suppressed by inactivation of DacA (PBP5) E. coliB.subtilisOur data strongly suggest that the translocase activity of FtsK is involved in the control of membrane synthesis in inversion-carrying strains. Depletion of FtsK in an Analysis of the intracellular positioning of chosen loci in wt and XL151 reveals different roles for the proximity with the replichore polarity junction (PJ) and the termination zone in their segregation pattern. Loci neighbouring the PJ in the wt strain remain colocalised (cohesive) long after their replication. This does not depend on the timing of their replication since the PJ keeps a high colocalisation index (Ci) after inversion independently of its replication timing. In contrast, loci far from the PJ in both the wt and inverted strain displays a low Ci, even when present close to the termination zone. Taken together, these data strongly suggest that sister-chromatid cohesion at a given locus is not controlled by its replication timing but more likely due to its proximity with the PJ. Termination of replication nevertheless has a strong influence on segregation of neighbouring loci but affects the positioning of loci after their migration rather than their colocalisation time. Loci close to the termination zone tend to remain in the middle third of the cell whereas those located along the replichore migrate towards quarter positions. This is consistent with the proposal that the timing of replication of a locus determine its home position after migration MlulacI ΔattB::Sp/Sm zdd347::lacZ::attB rpsL (StR); dif-sp5), Inv(dif-ydfE) and Inv(dif-sp39) inversions keeping dif at the replichore polarity junction were previously described ATP-ftsKCm xerC::Gm alleles slmA::Tc and tus::Tc mutations were constructed using standard techniques, inserted on the chromosome using integration-excision vectors and transferred by P1 transduction. Plasmid pFX465 carrying an arabinose-inducible ftsK allele was kindly given by FX Barre. The parS-Cm fragment from pGBKD3-parS was inserted on the chromosome at positions 4,413,507 (aidB), 257,144 (crl), 1,584,764 (ydeQ) and 1,596,522 (ydeV) using standard in vitro constructs and the “lambda red” technique Bacterial strains were grown in L broth or M9 broth . Ampicilin (25g/ml), kanamycin (50g/ml), streptomycin (200g/ml), gentamycin (7.5g/ml) and chloramphenicol (15g/ml) were added when required. The wild type strain (wt) is derived from XL13 (MG1655 Δwww.flowjo.com) for plotting and analysis.Flow cytometry was performed as described in parS insertion and plasmid pALA2705 were grown at 30°C in M9 broth  without IPTG  to OD600 = 0,3, concentrated 50 fold by centrifugation and placed on a polylysine-coated glass slide. Microscopy was carried out on a Zeiss upright axioplan II microscope. Images were analysed using the Metamorph software.Cells carrying a The co-localisation index is the mean copy number of a locus calculated from flow cytometry data divided by its mean number of foci per cell. Detail are in Sup.Table 3.Figure S1Duration of the C period in wild type and XL151 strains.(1.12 MB TIF)Click here for additional data file.Figure S2Cell cycle of the wild type (A) and the XL151 (B) strains.(0.69 MB TIF)Click here for additional data file.Figure S3The oriented translocation activity of FtsK is essential in XL151.(4.80 MB TIF)Click here for additional data file.Table S1Cell size distribution of the different strains in different growth conditions analysed by flow cytometry.(0.03 MB DOC)Click here for additional data file.Table S2Quantification of the number of replication origin per cell.(0.04 MB DOC)Click here for additional data file.Table S3Calculation of the Colocalisation index (Ci).(0.04 MB DOC)Click here for additional data file.Video S1Depletion of FtsK in XL151. XL151 ftsK(ATP-)/pFtsKwt cells were plated on minimal medium containing glucose after 3 hours growth under depletion conditions in liquid medium and photographed every 5 mn over a 5 hour period.(4.32 MB MOV)Click here for additional data file.Video S2Depletion of FtsK in XL151. XL151 ftsK(ATP-)/pFtsKwt cells were plated on minimal medium containing glucose after 3 hours growth under depletion conditions in liquid medium and photographed every 5 mn over a 5 hour period.(3.20 MB MOV)Click here for additional data file.


Salmonella control policies for broiler production were described and compared. The control options were the Zoonosis Directive 92/117/EC and the more intense strategy, the Finnish Salmonella Control Programme (FSCP).Costs and benefits of two Salmonella control costs in primary and secondary production and the direct and indirect losses due to Salmonella infections in humans in 2000.The comparison included the The total annual costs of the FSCP were calculated to be 990 400 EUR (0.02 €/kg broiler meat). The average control costs in the broiler production chain were seven times higher with the FSCP than with the Zoonosis Directive alone. However, the public health costs were 33 times higher with the Zoonosis Directive alone. The value of one prevented loss of life per year exceeded the annual control costs of the FSCP.Due to significant savings in public health costs compared to costs of FSCP, the FSCP was found to be economically feasible. Salmonella infections are compulsory notifiable in Finland. In 2000, a total of 2624 human Salmonella cases were reported in Finland. The incidence was thus 51/100 000 inhabitants. Of these cases, about 80% were of foreign origin 20]].Salmonella control policies available in Finland, namely the minimum requirements set by Zoonosis Directive 92/117/EEC and the FSCP. The comparison was based on data from 2000.The aim of this study was to compare the costs and benefits of the two Salmonella Control Programme (FSCP). The difference between these systems was that the Zoonosis Directive set measures only for breeding flocks of poultry and FSCP for all levels of live animal production and also for cutting plants. Salmonella Typhimurium and Salmonella Enteritidis were the only serovars causing preventive measures by the Zoonosis Directive whereas in the FSCP all Salmonella serovars are controlled. In the FSCP meat from positive flocks is heat treated and delivered only in the domestic market. The Zoonosis Directive was chosen as the lower level of salmonella control instead of no control at all since politically it would be the level of control if the FSCP was discontinued. A situation with no salmonella control at all was thus considered an unrealistic alternative. To analyse the costs and benefits of these control policies, the costs were divided into seven subcategories: 1) official Salmonella control costs, 2) additional control of primary and secondary production, 3) market disturbances, 4) feed control, 5) additional (not FSCP) control costs to society, 6) public health losses and 7) losses due to premature death. The net benefits of both of the policies were calculated, and the policies were then ranked according to the net benefits. The benefits were at least partly underestimated, as the number of disabilities and the economic consequences of chronic health effects were not included in the analysis. The control costs of the FSCP in primary and secondary production were estimated based on information gathered from broiler-producing companies and FSCP statistics. The main input values are presented in the Additional file In this analysis, costs of two control options were compared. The first option was the Zoonosis Directive and the second a more intense strategy, the Finnish Salmonella in poultry breeding flocks. According to this directive, faecal specimens from parent-rearing houses are analysed three times per flock. When Salmonella Typhimurium or Salmonella Enteritidis is suspected, internal organs are also analysed. In hatcheries, chickens are studied for Salmonella every two weeks by sampling bottom papers from boxes or meconium. Of the over 2500 serovars of Salmonella, only the detection of the two most important ones (S. Typhimurium and S. Enteritidis) launch eradication measures in breeding flocks. No requirements are set for broilers in meat production.The main objective of this directive is to stipulate a reporting system on zoonoses. It also orders monitoring, control and eradication of invasive serovars of Salmonella from cage bottom paper or meconium samples when the birds are one day old and from faecal samples at four weeks of age and at two weeks prior to entering the laying house. In the laying period, faecal samples are analysed every eighth week. Chickens from every breeding flock are investigated at hatcheries for Salmonella every two weeks. Since 2001, official sampling supervised by a veterinarian has been compulsory in hatcheries every eighth week. In 2000, the frequency of official sampling was only once a year. Surface swabs from hatchery structures are also analysed. In the FSCP, any serovar of Salmonella is sufficient to launch an intervention.The FSCP covers all the requirements of the Zoonosis Directive and also includes some additional requirements, therefore setting higher costs than the Zoonosis Directive to society and the broiler industry of Finland. The FSCP for broilers encompasses breeding and commercial flocks, hatcheries and, unlike the Zoonosis Directive, also poultry meat cutting plants. Breeding flocks are investigated for Salmonella is detected in a breeding flock, official restrictions are imposed on the farm. A positive result is confirmed by another sampling. The official restrictions include prohibition of egg and animal delivery. In addition an epidemiological investigation is done to identify the source and possible spread of the infection. Hatching eggs originating from the flock are destroyed. The official restrictions result in slaughter or killing of positive breeding flocks. Restrictions are lifted only after the premises have been emptied and disinfected, and surface swab samples yield test negative /([p(lose)*ad(lose)]. The sum p(win)*ad(win)+p(lose)*ad(lose) equals E(E(distance | outcome)) = E(distance), where "outcome" is the indicator function for the type of event, which is either win (outcome = one) or lose (outcome = zero), and where we denote ad(win) = E(distance | win) and ad(lose) = E(distance | lose), respectively. By distance, we denote the absolute value of the difference BC-1.Salmonella cases between control options 1 (Zoonosis Directive) and 2 (FSCP), a quantitative microbiological risk assessment model was built. The two key parameters defining the costs (number of human cases and number of infected flocks) were obtained from the risk assessment model SalmonellThe median BC ratio for the FSCP was 4.00 (90% range 0.04–21.25) , although they can be the most critical input values used in the calculations. However, in our study, we benefited from the results achieved by the risk assessment model developed to study the public health effects of the FSCP on broiler production [ and 23]23]]. TheSalmonella as a cause of health problems may have been underestimated in our study. Helms et al. [Salmonella, Campylobacter and Yersinia enterocolitica) had an increased long-term risk of death even after effects of pre-existing illnesses were taken into account. One-year relative mortality among Salmonella patients was 2.85 times higher than among matched controls. In addition, Salmonella infections may lead to sequelae, including joint and heart problems  [The role of s et al.  reportedyelitis)  and uveiyelitis) . Had theSalmonella cases was 554 EUR when mortality costs were not included. The median cost of reported cases when deaths were included was 589 EUR. The median cost of all cases was 222 EUR. This difference arises from the number of unreported cases clearly being high and the health costs in this group being lower than in reported cases. When the proportion of deaths is high, the costs per case are higher too. Roberts et al. [Salmonella case to be 606 GBP (857 EUR). Mortality costs were not included in that research, but the impact of illness on the ability to carry out normal activities was estimated. Persson & Jendteg [Salmonella case is 2126 USD (1734 EUR) including costs of mortality. ERS uses "willingness-to-pay" (WTP) estimates as the cost of premature death, which was 6.6 million USD (5 384 280 EUR) in 2001.The median cost of reported human s et al.  calculat Jendteg  estimate Jendteg , the aveSalmonella control costs in a broiler production chain in Denmark [Salmonella in the broiler production pyramid.Based on our calculations, the cost per kilogram of broiler meat, 0.02 EUR/kg, 98% of which was paid by the industry, was relatively low. This result was close to USD 0.02/kg for  Denmark . AlthougSalmonella-positive flocks has a minor impact since of the amount of exported poultry meat is very small. This study was also based on the assumption that the origin of consumed broiler meat would be the same, i.e. domestic, even if the FSCP were be abandoned and control was based only on the Zoonosis Directive. However, in real life this might not be the case since with removal of additional guarantees agreed by the EC, import from EU and third world countries would probably increase significantly. If this resulted in the replacement of half of the current retail broiler meat by meat with 20–40% contamination, 33–93 times more human cases would be detected compared with the expected value under the current situation [In the FSCP, the greatest cost to the industry was caused by the heat treatment of meat from positive flocks, whereas for society the greatest cost was due to premature deaths. The significance of heat treatment is affected by the variety of products. In Finland, most broiler meat is sold as fresh, and therefore, the opportunities to use heat-treated broiler meat are limited. For the Finnish broiler industry, the consequences of prohibition of export of meat originating from ituation .Cost-benefit analysis has been criticized as being mechanistic and unable to account for preferences other than through market prices. Dorfman  listed tSalmonella control in the broiler production chain compared with the old Zoonosis Directive. Thus, the results presented here also apply to the current situation. EU legislation was renewed in 2003 to improve the prevention and control of zoonoses. Salmonella has been identified as a priority target, especially in poultry production. Based on our study, this is reasonable considering the long term public health and economic impact of the societal level. However, the economic efficiency of the FSCP is also based on Salmonella control measures, such as feed control, which has been used for decades in Finland. Therefore, the BC ratios would probably be different if these measures were applied in a situation with a higher prevalence of Salmonella in broiler production.This study shows that the FSCP control policy has been successful. One prevented loss of life covers the control costs in the broiler production chain. The new zoonosis legislation in the EU only slightly augments In conclusion, the FSCP for broilers is an economically feasible programme for society compared with the lower level of control provided by the Zoonosis Directive. The FSCP is viable from the poultry meat producer', consumers' and tax payers' points of view.BC = benefit-costCBA = cost-benefit analysisSalmonella Control ProgrammeFSCP = Finnish The author(s) declare that they have no competing interests.SK, JP and RM were involved in the study design. SK, TL and RM constructed the calculation model in Excel. TL analyzed statistically the data and implemented @risk simulation model. JP was responsible for the economic model. JR and RM were responsible for the risk assessment used as input to this simulation model. SK and RM were responsible for the manuscript preparation. All authors have participated in the manuscript revision and have read and accepted the final manuscript.The input variables and values/distributions used in the model.


Worldwide, approximately 1.8 million children die from diarrhea annually, and millions more suffer multiple episodes of nonfatal diarrhea. On average, in up to 40% of cases, no etiologic agent can be identified. The advent of metagenomic sequencing has enabled systematic and unbiased characterization of microbial populations; thus, metagenomic approaches have the potential to define the spectrum of viruses, including novel viruses, present in stool during episodes of acute diarrhea. The detection of novel or unexpected viruses would then enable investigations to assess whether these agents play a causal role in human diarrhea. In this study, we characterized the eukaryotic viral communities present in diarrhea specimens from 12 children by employing a strategy of “micro-mass sequencing” that entails minimal starting sample quantity (<100 mg stool), minimal sample purification, and limited sequencing (384 reads per sample). Using this methodology we detected known enteric viruses as well as multiple sequences from putatively novel viruses with only limited sequence similarity to viruses in GenBank. Diarrhea is one of the leading infectious causes of death worldwide with an estimated 1.8 million deaths annually, primarily in young children in developing countries. There are many known causes of diarrhea; however, the causes of ∼40% of the cases are still unknown. One possibility is that viruses that we currently do not know about are responsible for these cases. Thus, we used an experimental strategy termed “micro-mass sequencing” to systematically identify viruses present in stool from a number of patients suffering from diarrhea. Sequences from a number of novel viruses were detected, some which differed quite significantly from any previously described virus. These new viruses may or may not be responsible for causing diarrhea. Future studies will specifically address the potential of these viruses to cause human disease. One implication of this study is that there are likely to be many more unknown viruses that can be identified in this fashion. Furthermore, by studying these viruses, we will come to a more complete understanding of the role viruses play in diarrhea. Ultimately, this may lead to the development of therapeutics and/or vaccines that decrease the disease burden of diarrhea. While traditional sequencing approaches are designed to characterize genomes of a single species of interest, metagenomic approaches, such as mass sequencing, transcend species boundaries allowing one to explore the makeup of microbial communities. Such methods provide a holistic look at microbial diversity within a given sample, completely bypassing the need for culturing In this study, mass sequencing was applied to explore specifically the viral communities present in pediatric patients suffering from diarrhea. We anticipated that the viral communities would vary significantly from specimen to specimen and that it would be desirable to sample broadly from multiple patients to obtain an overall perspective on the diversity of viruses that might be present. Toward this end, a simple yet robust experimental strategy was developed that circumvented certain technical and economic limitations of conventional mass sequencing. In both previous viral metagenomic studies of the human gut, large quantities of fecal matter (∼500 g) were collected from adults and then extensively purified to enrich for viral particles −5) alignments. The remaining 556 (28%) sequences had no significant similarity to any sequences in the nr database and were therefore categorized as being of ‘unknown’ origin. The 1,457 identifiable sequences were further classified into categories based on their proposed origin  by enzyme immunoassay. It was our expectation that this sample would yield sequences derived from the infecting rotavirus. In this library, 107 non-redundant sequence reads were identified as viral in origin, almost all of which possessed ≥90% amino acid (aa) BLAST identity to known rotavirus sequences in Genbank. The sequence data included cloned fragments from all 11 RNA segments of the rotavirus genome.An additional 11 stool specimens were then selected that had tested negative in conventional PCR and enzyme immunoassays for the known diarrhea viruses . Despite such screening, sequences derived from the canonical enteric viruses were detected in a number of samples. For example, calicivirus sequences were detected in D02 and D06, astrovirus sequences in D04, and adenoviruses were detected in D05 and D12. Almost all individual sequence reads in these cases possessed >90% aa identity to existing viral sequences in Genbank.Parvoviridae family, was detected in two samples, D11 and D12. These viruses are known to infect the gastrointestinal tract, but are not thought to be enteric pathogens. For productive infections or reactivation from a latent state, AAV requires co-infection with a helper virus that is most commonly an adenovirus or less typically, a herpesvirus Adeno-associated virus (AAV), a member of the In many of the libraries, individual sequence reads were detected that possessed ≤ 90% aa identity to their highest scoring BLAST hit . Furthermore, we wished to sample broadly from multiple patients because of the large number of viruses known or suspected to be associated with diarrhea. Therefore, rather than sequence few specimens in great depth as has been done previously  Our analysis detected viruses, bacteria, host, phage and other sequences . The preViral sequences were detected in all but one sample. Interestingly, a number of DNA viruses  were detected in our analysis, despite our use of a methodology focused on purification of RNA. While it is possible that RNA transcripts from these viruses were purified Analysis of this initial cohort of 12 specimens yielded a wealth of original findings. In contrast to previous metagenomic studies of stool Sequences were detected in this study from at least 9 putatively novel viruses. For 7 of these sequences, the degree of divergence observed based on phylogenetic analysis suggested that they might represent novel virus subtypes or genotypes of picobirnavirus, enterovirus, TT virus and norovirus , and S5.Sequences representing a divergent norovirus were detected in sample D06 . PhylogeIn two samples, much more highly divergent sequences were detected. In D08, phylogenetic analysis of 7 unique sequence reads strongly suggested that a novel astrovirus species was present . The obsNodaviridae. Nodaviruses are small single-stranded, positive sense, bipartite RNA viruses, divided into two genera, the alphanodaviruses (insect viruses) and the betanodaviruses (fish viruses). Currently, none of the established family members are known to naturally infect mammals although experimental manipulation of the viral genome has enabled viral replication in a wide array of organisms including mammals Another novel sequence detected appeared by phylogenetic analysis to belong to the family The micro-mass sequencing approach, like any other experimental methodology capable of detecting novel viruses (such as culture or degenerate PCR), cannot of course by itself determine whether the newly detected agent is pathogenic. However, this strategy can generate novel, testable hypotheses such as “Are these novel viruses involved in the etiology of human diarrhea?” and “What is the true host of these viruses?” that could not be asked in the absence of the knowledge that these viruses existed.556 out of the 2013 (28%) unique high quality sequences were binned as unassigned by the BLAST criteria. Of these, 23 were identified as containing repetitive elements or low-complexity sequence by RepeatMasker Our data suggest that micro-mass sequencing might be of great diagnostic utility for a number of reasons. First, viruses escaping detection in conventional assays were detected by micro-mass sequencing. In theory, the sensitivity of this strategy is limited only by the depth of sequencing. As demonstrated here, even shallow sequencing performed better than conventional diagnostics in some instances. In addition, the unbiased nature of the method enabled detection of viruses not conventionally tested for. Moreover, co-infections were detected in multiple samples. Furthermore, for multi-segmented viruses such as rotaviruses, reassortment of segments between species is a major mechanism of viral evolution that can lead to the emergence of more virulent strains In summary, we have shown that micro-mass sequencing can define the diversity of viral communities found in fecal samples from diarrhea patients. Both known viruses and novel viruses were detected by sequencing only a few hundred colonies from each sample library. These studies will serve as the springboard for further interrogations of the roles of these diverse viruses in the gastrointestinal tract. Finally, our detection of multiple novel viruses in this initial, limited exploration of a dozen samples suggests that broader sampling of patient specimens is likely to be highly fruitful in terms of identification of additional novel viruses.Stool samples were collected from children under the age of 5 who were admitted to the Royal Children's Hospital, Melbourne, Victoria, Australia with acute diarrhea between 1978 and 1999.Stool samples were collected between 2003–2005 at the Emergency Department of the Children's Hospital and Regional Medical Center in Seattle, Washington, USA as part of a prospective study attempting to discern the cause of unexplained pediatric diarrhea.Specimens were tested by routine enzyme immunoassays (EIA) and culture assays for rotaviruses, adenoviruses, and common bacterial and parasitic pathogens as previously described Campylobacter jejuni, Escherichia coli O157∶H7 and non-O157∶H7 Shiga toxin-producing E. coli, Salmonella, Shigella, and Yersinia) following standard culture assays, Clostridium difficile toxin by a cytotoxicity assay, parasites by microscopy and antigen testing Specimens were tested for the presence of a number of bacterial species  were resuspended in 6 volumes of PBS. A subset of the archived specimens had been previously diluted and were further diluted 1∶1 in PBS. The stool suspensions were centrifuged  and supernatants were harvested and then passed through 0.45 µm filters. RNA was extracted from 100 µL of the filtrates using RNA-Bee  according to manufacturers instructions. Approximately, 100–300 nanograms of RNA from each sample was randomly amplified following the Round AB protocol as previously described Sequence traces were subjected to quality assessment and base-calling using Phred ClustalX (1.83) was used to perform multiple sequence alignments of the protein sequences associated with select sequence reads. Available nucleotide or protein sequences from known viruses were obtained from Genbank for inclusion in the phylogenetic trees. Selected sequences from Genbank included those with the greatest similarity to the sequence read in question based on the BLAST alignments as well as representative sequences from all major taxa within the relevant virus family. The protein alignments created by ClustalX were input into PAUP Astrovirus trees: Human astrovirus 1 (NC_001943); Human astrovirus 2 (L13745); Human astrovirus 3 (AAD17224); Human astrovirus 4 (DQ070852); Human astrovirus 5 (DQ028633); Human astrovirus 6 (CAA86616); Human astrovirus 7 (AAK31913); Human astrovirus 8 (AF260508); Turkey astrovirus 1 (Y15936); Turkey astrovirus 2 (NC_005790); Turkey astrovirus 3 (AY769616); Chicken astrovirus (NC_003790); Ovine astrovirus (NC_002469); and Mink astrovirus (NC_004579).Nodavirus tree: Striped Jack Nervous Necrosis virus (Q9QAZ8); Macrobrachium rosenbergii nodavirus (Q6XNL5); Black Beetle virus (YP_053043.1); Flockhouse virus (NP_689444.1); Epinephelus tauvina nervous necrosis virus (NC_004136.1); Nodamura virus (NC_002691.1); Boolarra virus (NC_004145.1); Pariacoto virus (NC_003692.1); and Redspotted grouper nervous necrosis virus (NC_008041.1).Picornavirus trees: Human coxsackievirus A1 (AAQ02675.1), Human coxsackievirus A18 (AAQ04836.1), Human coxsackievirus A19 (AAQ02681.1), Human coxsackievirus A21 (AAQ04838.1), Human coxsackievirus A24 (ABD97876.1), Human poliovirus 1 (CAD23059.1), Human coxsackievirus A2 (AAR38840.1), Human coxsackievirus A4 (AAR38842.1), Human coxsackievirus A5 (AAR38843.1), Human coxsackievirus A16 (AAV70120.1), Human enterovirus 89 (AAW30683.1), Human enterovirus 91 (AAW30700.1), Human enterovirus 90 (BAD95475.1), Human enterovirus 71 (CAL36654.1), Echovirus 1 strain Farouk (AAC63944.2), Human coxsackievirus B2 (AAD19874.1), Human enterovirus 86 (AAX47040.1), Human coxsackievirus B5 (AAF21971.1), Human echovirus 29 (AAQ73089.1), Human enterovirus 68 (AAR98503.1), Human enterovirus 70 (BAA18891.1), Bovine enterovirus (NP_045756.1), Porcine enterovirus A (NP_653145.1), Porcine enterovirus B (NP_758520.1), Simian enterovirus A (NP_653149.1), Human rhinovirus A (ABF51203.1), Human rhinovirus B (NP_041009.1).Picobirnavirus trees: Human picobirnavirus strain 1-CHN-97 (AF246939.1), Human picobirnavirus strain 4-GA-91 (AF246940.1), Human picobirnavirus strain Hy005102 (NC_007027.1), Human picobirnavirus strain 2-GA-91 (AF245701.1), Human picobirnavirus strain 1-GA-91 (AF246612.1), Porcine picobirnavirus 2 (EU104360.1).Anellovirus trees: TGP96 Torque teno virus (AB041962), Pt-TTV8-II Torque teno virus (AB041963), CBD231 TTV-like mini virus (AB026930), Mf-TTV9 Torque teno virus (AB041959), Mf-TTV3 Torque teno virus (AB041958), KC009/G4 Torque teno virus (AB038621), TA278/G1 Torque teno virus (AB008394), Pt-TTV6 Torque teno virus (AB041957), TUS01/G3 Torque teno virus (AB017613), PMV/G2 Torque teno virus (AF261761), JT33F/G5 Torque teno virus (AB064606), MD1-073 Torque teno midi virus (AB290918), MD2-013Torque teno midi virus (AB290919), Tbc-TTV14 Torque teno virus (AB057358), Sd-TTV31 Torque teno virus (AB076001), Fc-TTV4 Torque teno virus (AB076003), Cf-TTV10 Torque teno virus (AB076002), So-TTV2 Torque teno virus (AB041960), At-TTV3 Torque teno virus (AB041961).Calicivirus trees: Camberwell (AAD33960.1), MD-2004 (ABG49508.1), Carlow(ABD73935.1), Snow Mountain virus (AAN08111.1), Mc37 (AAS47823.1), Hawaii(AAB97767.2), Norwalk(AAB50465.1), Southampton (AAA92983.1), Chiba(BAB18266.1), Hesse(AAC64602.1), BoJena-DEU-98 (CAA09480.1), Murine (AAO63098.2), SU17(BAC11827.1), Dumfries (AAM95184.2), SU25-JPN(BAC11830.1), SU1-JPN(BAC11815.1), Desert Shield (AAA16284.1), Melksham (CAA57461.1), Toronto-24 (AAA18929.1), Sw918 (BAB83515.1), OH-QW101 (AAX32876.1).Bacteroides caccae (ZP_01959575.1), Acanthamoeba mimivirus (YP_142599.1), Eubacterium dolichum (ZP_02077753.1), Staphylococcus phage K (YP_024462.1), Lactobacillus phage LP65 (YP_164778.1), Lactococcus phage bIL170 (NP_047162.1), Lactococcus phage r1t (NP_695069.1), Burkholderia vietnamiensis G4 (YP_001119011.1), Streptococcus pyogenes (NP_607538.1), Tetrahymena thermophila (XP_001029162.1), Bacteroides vulgatus (YP_001300673.1)Endonuclease-like sequences for D03 tree (mimvirus-like sequences): Figure S1Composite analysis of all sequences. Sequences from all 12 libraries were categorized based on the best tBLASTX scores  as viral, phage, bacterial, human, fungal, other, or unassigned. Numbers in parenthesis represent the number of sequences in each category.(0.21 MB TIF)Click here for additional data file.Figure S2Phylogenetic analysis of picobirnavirus-like sequence reads. Phylogenetic trees were generated by comparing the translated amino acid sequence of individual sequence reads to members of the unclassified taxa picobirnavirus. The trees were created using the maximum parsimony method with 1,000 replicates. Bootstrap values over 700 are shown.(0.43 MB TIF)Click here for additional data file.Figure S3Phylogenetic analysis of Picornaviridae-like sequence reads. Phylogenetic trees were generated by comparing the translated amino acid sequence of individual sequence reads to members of the Picornaviridae family. The trees were created using the maximum parsimony method with 1,000 replicates. Bootstrap values over 700 are shown. CVA = Coxsakievirus A, CVB = Coxsackievirus B, BEV = Bovine Enterovirus, EV = Enterovirus, HRVA = Human Rhinovirus A, HRVB = Human Rhinovirus B, PEV = Porcine Enterovirus, PV = Poliovirus, SEVA = Simian Enterovirus A.(0.66 MB TIF)Click here for additional data file.Figure S4Phylogenetic analysis of anellovirus-like sequence reads. Phylogenetic trees were generated by comparing the translated amino acid sequence of individual sequence reads to anelloviruses. The trees were created using the maximum parsimony method with 1,000 replicates. Bootstrap values over 700 are shown.(0.37 MB TIF)Click here for additional data file.Figure S5Phylogenetic analysis of Caliciviridae-like sequence reads. Phylogenetic trees were generated by comparing the translated amino acid sequence of individual sequence reads to the A) NS4 (3A-like) protein or B) NS7 (RNAP) protein of caliciviruses. The trees were created using the maximum parsimony method with 1,000 replicates. Bootstrap values over 700 are shown.(0.58 MB TIF)Click here for additional data file.Figure S6Phylogenetic analysis of endonuclease-like sequence reads. Phylogenetic trees were generated by comparing the translated amino acid sequence of two individual sequence reads to endonuclease sequences derived from mimivirus, phage, and bacterial species representing some of the top scoring BLAST hits. The trees were created using the maximum parsimony method with 1,000 replicates. Bootstrap values over 700 are shown.(0.54 MB TIF)Click here for additional data file.


We evaluated a personal protective equipment removal protocol designed to minimize wearer contamination with pathogens. Following this protocol often resulted in virus transfer to hands and clothing. An altered protocol or other measures are needed to prevent healthcare worker contamination. Caring for patients with communicable diseases places healthcare workers (HCWs) at risk. Infected HCWs may not only incur serious illness or death themselves but may spread infection to others. Methods to prevent HCW infections include vaccination  to protect HCWs from pathogen exposure during patient care. PPE includes use of barriers  and respiratory protection  to protect mucous membranes, airways, skin, and clothing from contact with infectious agents  addressed this concern by designing a protocol to minimize contamination to the wearer during PPE removal  (6). How4 PFU of MS2 in 5 drops of 5 μL each. Participants performed a healthcare task (measuring blood pressure on a mannequin) and then removed PPE according to CDC protocol. Hands, items of PPE, and scrubs worn underneath were sampled for virus. Hands were sampled by using the glove juice method  and massaged for 60 seconds to cover all hand surfaces with solution. PPE items were immersed in 1.5% beef extract, pH 7.5, and agitated on a shaker for 20 minutes. Eluent from hands and PPE was assayed by the most probable number (MPN) enrichment infectivity assay  donned by volunteers was contaminated with bacteriophage MS2, a nonenveloped, nonpathogenic RNA virus suspended in 0.01 mol/L phosphate-buffered saline and GloGerm , synthetic beads that fluoresce under UV light . Sites of contamination were as follows: front shoulder of gown, back shoulder of gown, right side of N95 respirator, upper right front of goggles, and palm of dominant hand. Each site was contaminated with a total of 10When an a priori value of 25% was used for the 95% upper confidence limit when p (transfer) = 0, the sample size was N = 10. Protocols were approved by the University of North Carolina (UNC) Biomedical Institutional Review Board, and written informed consent was obtained. Enrolled participants met the following inclusion criteria: >18 years of age, nonpregnant, nonallergic to latex, no active skin disorders, and medical evaluation approval for N95 respirator fit testing and use  and giveTen study participants were enrolled in this study: 9 women and 1 man. Nine participants were right-handed, and 1 was left-handed. Transfer of virus to both hands, the initially uncontaminated glove on the nondominant hand, and the scrub shirt and pants worn underneath the PPE was observed in most volunteers . Because10 MPN for hands and 1–4 log10 MPN for scrubs. The mean amount of virus recovered from the right hand (the dominant hand of 90% of volunteers) was greater than that recovered from the left hand. While removal of gloves and gowns required 2 hands, mask and goggle removal was one-handed, which could have resulted in larger quantities of virus being transferred to the dominant hand during removal. In the single left-handed study participant, recovery of virus was greater from the left hand than the right (1.82 log10 vs. 0.98 log10 MPN). The mean amount of virus recovered from scrub shirts was significantly greater than that recovered from pants (p = 0.01), possibly because of contact with hands when the gown is pulled away from the shoulder during removal.The amount of virus recovered was 1–3 logPPE is vital for protecting HCWs from occupationally acquired infection during patient care, particularly from droplet- or airborne-transmitted diseases. However, removing PPE after patient care without contaminating skin or clothes is important. Although PPE is usually worn only for short periods, viruses such as influenza (Developing and validating an algorithm for removing PPE that prevents contamination of the skin and clothes of HCWs are key to interrupting nosocomial transmission of infectious agents. These experiments demonstrate that the current CDC algorithm is insufficient to protect HCWs from contamination during PPE removal. However, options that might prevent such contamination do exist, including double gloving, use of surgical protocols for PPE removal, and PPE impregnated with an antimicrobial agent.A double-glove removal sequence would begin with removal of the outer glove, followed by removal of goggles or face shield, gown, and respirator/mask, and finishing with removal of the inner glove followed by hand hygiene; handling of PPE with ungloved hands is avoided. Borrowing PPE protocols from surgery, in which the ends of gown sleeves are tucked underneath gloves during wear, might also reduce contamination. When the HCW is finished, goggles and respirator are removed first, and gown and gloves are then removed together by peeling off both at the same time, again avoiding handling PPE with ungloved hands. Finally, the use of PPE impregnated with antimicrobial agents might also reduce or eliminate contamination of skin and clothes.This study also indicates the need for continued emphasis on hand hygiene. A barrier to improving hand hygiene compliance rates is the belief that gloves make hand hygiene unnecessary (


Homalodisca coagulata (Say) (Hemiptera: Cicadellidae). The virus, named Homalodisca coagulata virus -1, HoCV-1, was associated with increased mortality of cultured 5th instar H. coagulata. To identify the presence of H. coagulata viral pathogens, cDNA expression libraries were made from adult and nymphs. Analysis using reverse transcriptase PCR demonstrated that the virus was present in midgut tissues. As the viral capsid proteins are commonly used in classification of newly discovered viruses, the capsid proteins (CP) of the virus discovered in H. coagulata was examined. The order of the polyprotein subunits of HoCV-1 capsid proteins was determined to be CP2, CP4, CP3, and CP1. The CP4/CP3 (AFGL/GKPK) cleavage boundary site was clearly identified when the sequences were aligned. The putative CP3/CP1 (ADVQ/SAFA) cleavage site and the putative CP2/CP4 (VTMQ/EQSA) cleavage site of HoCV-1, respectively, were located in the same region as that of the other viruses. After alignment, the CP3/CP1 cleavage sites and CP2/CP4 cleavage sites of the viruses analyzed fell within 50 amino acids of one another. As with the cricket paralysis virus, HoCV-1 was found to be mainly comprised of β-sandwiches in CP1-3 with a jelly roll topological motif. CP4 of HoCV-1 appeared to be mainly α-helical in structure. CP1-4 domains are most homologous to insect picorna-like virus coat proteins as was demonstrated by the results of the BLASTP and PSI-BLAST tests, and is strongly supported by the structural modeling. While sequence homology between the cricket paralysis virus and HoCV-1 was low, the global structure of the proteins was conserved. Sequence identities were analyzed by in silico comparison to known genes in the public database, NCBI. Phylogenetic analysis performed using the optimized protein alignment generated a phylogram containing 5 clades. Clade 1 consisted of Drosophila C virus, Clade 2 consisted of cricket paralysis virus, Clade 3 of Triatoma virus, Plautia stali intestine virus, Himetobi P virus, black queen cell virus, and HoCV-1. Clade 4 encompassed acute bee paralysis virus and Kashmir bee virus, and Clade 5 consisted of Rhopalosiphum padi virus. Analysis of the capsid protein of this new leafhopper virus provided significant evidence that it is related to other ssRNA insect viruses within the Family, Dicistroviridae. The HoCV-1, capsid protein sequence has been deposited in GenBank, Accession number: DQ308403.A new virus that infects and causes increased mortality in leafhoppers was isolated from the glassy-winged sharpshooter,  Homalodisca coagulata (Say) (Hemiptera: Cicadellidae) is the primary vector of Pierce’s disease of grapes, which is caused by the bacterial plant pathogen, Xylella fastidiosa. There are numerous plant diseases caused by the Xylella bacteria which are known as ‘Scorch’ diseases due to the dry leaf appearance in susceptible plants. In grapes, susceptible plants have reduced yields and may die after infection thus causing severe economic losses. Research to examine the interactions between H. coagulata and Xylella requires mass rearing of large numbers of H. coagulata for use in transmission experiments. Attempts to mass rear H. coagulata, resulted in an observable increase in mortality during the 5th instar stage  making it difficult to produce adult H. coagulata for research needs. Mortality in cultured insects is often due to the presence of undetected insect pathogens. Insect cell cultures have been used to detect insect pathogens . Our approach was to identify the presence of H. coagulata viral pathogens, using cDNA expression libraries, which provide a overview of the transcripts in adults and nymphs. As viral capsid protein (CP) sequences, are commonly used in classification of newly discovered viruses we chose to elucidate the CP of the virus discovered in H. coagulata. Sequence identities were analyzed by comparison to known genes in the public database, NCBI. Viral sequences which were identified were then validated by amplification and bidirectional sequencing. A new viral pathogen of the H. coagulata was identified and shown to be significantly related to insect viruses within the Family, Dicistroviridae. The virus herein was named: Homalodisca coagulata virus–1, HoCV-1.Few viral pathogens are known from leafhoppers. The glassy-winged sharpshooter, H. coagulata, and a 5th instar cDNA library was made from 140 nymphs collected from citrus groves near Riverside, California. Insects were collected into liquid nitrogen and total RNA subsequently extracted using the guanidinium salt-phenol-chloroform procedure as described by www.promega.com) and poly(A)+ RNA was purified using a MicroPoly(A)Pure™ kit  following the manufacturer’s instructions. A directional cDNA library was constructed in Lambda Uni-ZAP® XR Vector using Stratagene’s ZAP-cDNA Synthesis kit . The resulting DNA was packaged into lambda particles using Gigapack® III Gold Packaging Extract (Stratagene). Mass excision of the amplified library was carried out using Ex-Assist® helper phage (Stratagene). An aliquot of the excised, amplified library was used for infecting XL1-Blue MRF’ cells and subsequently plated on LB agar containing 100 μg/ml ampicillin. Bacterial clones containing excised pBluescript SK(+) phagemids were recovered by random colony selection. pBluescript SK(+) phagemids were grown overnight at 37°C and 240 rpm in 96-deep well culture plates containing 1.7 ml of LB broth, supplemented with 100 μg/ml ampicillin. Archived stocks were prepared from the cell cultures using 75 μl of a LB-amp, glycerol mixture and 75 μl of cells. These archived stocks are held at the U.S. Horticultural Research Laboratory where they are kept in an ultra low temperature freezer (−80° C). Plasmid DNA was extracted using the Qiagen 9600 liquid handling robot and the QIAprep 96 Turbo miniprep kit according to the recommended protocol . Bidirectional sequencing of HoCV-1 clones were completed using T3 and T7 primers, sequencing reactions were performed using the ABI PRISM® BigDyeTM Primer Cycle Sequencing Kit  along with a universal T3 primer. Sequencing reaction products were precipitated with 70% isopropanol, resuspended in 15 μl of sterile water and loaded onto an ABI 3730xl DNA Analyzer (Applied Biosystems). Two other H. coagulata cDNA libraries  were also examined for virus sequences.A whole body, adult cDNA library was made from 160 adult H. coagulata adults, collected near Riverside, California, were tested for presence of HoCV-1, using reverse transcriptase PCR (rtPCR), with the amplicons sequenced and compared using Blast analyses, NCBI database to identify homologous viral sequences. Upon arrival at the laboratory, H. coagulata individuals were removed from RNA later® and washed in 1X phosphate buffered saline (PBS), pH 7.0 at 4°C. Tissues from whole insects were dissected in cold 1X PBS, pH 7.0. Samples were ground directly in Buffer RLT (Qiagen) using sterile plastic pestles. RNA extractions were performed using the RNeasy® Mini Kit (Qiagen) following the manufacturer’s instructions. Total RNA was eluted in RNase-free water and quantified by UV spectrophotometry. An equal concentration (500 ng) of total RNA was retrotranscribed for each sample reaction by first combining RNA, 1 μl dNTPs (10mM), and 1 μl oligo(dT)17 primer (2.0 μg/μl) for a reaction volume of 13 μl. The mixture was incubated at 65°C for 5 min after which, 4 μl 5x buffer, 2 μl dithiothreitol (DTT), 1 μl RNasin® ribonuclease inhibitor (40 U/μl) (Promega), and 1 μl SuperScriptTM III (200 U/μl) (Invitrogen) were added. The reaction mixture was then incubated at 55°C for 1 hr and subsequently terminated at 65°C for 10 min. Primers were designed using Primer3  GenBank® accession number DQ308403 . Amplification was performed using 22.5 μl Platinum® PCR Supermix (Invitrogen), 1 μl of the forward (5′-TCC GAG TTC TCA GCC AAA CT-3′) and reverse (5′-CGG CAT ATC GAA ATG AGG TT-3′) primers combined (10 μM) each, and 1.5 μl cDNA. The reaction mixture was subjected to an initial denaturation at 95°C for 2 min followed by 35 cycles of 95°C for 30 sec, 60°C for 30 sec, and 72°C for 1 min, and concluded with a final DNA extension at 72°C for 5 min. Validation of five amplicons by sequencing were completed, after which samples were considered positive when a visible amplicon (443 nucleotides) was present after separation on a 1 % agarose (TAE) gel stained with ethidium-bromide, EtBr (0.5 μg/ml).Midgut and salivary gland tissues from approximately 40 ® . Low-quality bases (confidence score <20) were trimmed from both ends of sequences. All quality trimming, vector trimming and sequence fragment alignments were executed using Sequencher® software . Amplicons were sequenced and compared by Blast analyses for adults and midgut and salivary gland tissues that tested positive for HoCV-1 by rtPCR to identify homologous viral sequences.Base confidence scores were designated using TraceTunerhttp://www.ncbi.nlm.nih.gov) with the sequence comparisons made to protein databases . Contig assembly parameters were set using a minimum overlap of 50 bases and 90% identity match. Multiple alignments were performed with CLUSTAL X, version 1.83  using the following sequences (with their respective GenBank® accession numbers): ABPV (NC_002548), BQCV (NC_003784), CrPV (NC_003924), DCV (NC_001834), HiPV (NC_003782), KBV (NC_004807), PSIV (NC_003779), RhPV (NC_001874), TrV (NC_003783). Protein molecular weights were approximated using suite 2 of the Stothard method for sequence manipulation (Stothard 2000). Phylogenetic trees were constructed using the capsid protein, CP, sequences via the neighbor-joining method using PAUP* version 4.0 (Swofford 2003). For each tree, confidence levels were estimated using the bootstrap resampling procedure (2000 replications).The potential status of the of the HoCV-1 capsid protein was determined based on Blast homology searches using the National Center for Biotechnology Information Blast server  and sequence fragment alignments were executed using Sequencher® software . Sequence corresponding to vector contaminants was removed from the dataset. To estimate the number of genes represented in the library and the redundancy of specific genes, ESTs were assembled into contigs using Sequencher®. Contig assembly parameters that were set using a minimum overlap of 50 bases and 95% identity match.Vector and quality trimming, base quality scores generated with TraceTunerhttp://www.ncbi.nlm.nih.gov/gquery/gquery.fcgi). The theoretical molecular weight and pI value of the predicted protein was calculated by Compute PI/MW on the ExPASy server (http://au.expasy.org). Compute pI/MW calculates the molecular weight of an input sequence by adding the average isotopic mass of each amino acid plus one water molecule.Standard DNA and protein sequence analyses performed used BLASTn and BLASTp, respectively  1997. Thhttp://robetta.bakerlab.org/). Robetta is a full-chain protein structure prediction server and permits both initial and comparative models of protein domains. In addition, Robetta performs domain parsing and 3-D modeling included fragment library generation. The programs utilize the Ginzu domain parsing and fold detection method and the Rosetta fragment insertion method .Structural modeling was performed using the Robetta automated server  were performed using CLUSTAL X version 1.81 algorithm . Guide tH. coagulata. Viral sequences were initially discovered through analysis of the cDNA library generated from whole bodies of adults. Approximately 5.8% (500) of the clones were homologous to viral sequences as indicated by BLASTX results. Analysis using rtPCR showed that the virus was in the midgut tissues, but not in salivary glands (Dicistroviridae. The capsid protein sequence of HoCV-1 was submitted into GenBank (accession number: DQ308403), and was used to perform TBLASTX search of the NCBI database. Sequences with significant amino acid identity were downloaded for comparison to the HoCV-1 sequence. Comparisons were made to capsid protein sequences of: cricket paralysis virus (CrPV) [NC_003924], Drosophila C virus (DCV) [NC_001834], Triatoma virus (TrV) [NC_003783], Himetobi P virus (HiPV) [NC_003782], Plautia stali intestine virus (PSIV) [NC_003779], black queen cell virus (NC_003784), acute bee paralysis virus (ABPV) (NC_002548), Kashmir bee virus (KBV) [NC_004807], and Rhopalosiphum padi virus (RhPV) [NC_001874] . The comparison of the predicted amino acid sequence of putative HoCV-1 capsid protein identified several clusters of conserved amino acids  . There were few secondary structures found in CP4 of both CrPV and HoCV-1. CP4 of HoCV-1 appeared to be mainly α-helical in structure. CP1-4 domains are most homologous to insect picorna-like virus capsid proteins as demonstrated by the BLASTP and PSI-BLAST results, as well as by the structural modeling. While sequence homology between CrPV and HoCV-1 was low  . Other species within this genus are: Drosophila C virus (DCV), Plautia stali intestine virus (PSIV), Himetobi P virus (HPV), Triatoma virus (TrV), black queen cell virus (BQCV), and Rhopalosiphum padi virus (RhPV). Tentative species within this genus include: acute bee paralysis virus (ABPV), and four other candidate species: aphid lethal paralysis virus (ALPV), Kashmir bee virus (KBV), cloudy wing virus (CWV) and Taura syndrome virus (TSV) . Planthoppers are a taxonomically closely related insect group to leafhoppers within the Hemiptera. The virus was detected primarily in midgut tissues of H. coagulata, and this may be the primary site of infection and replication at least in leafhoppers.Analysis of the HoCV-1 capsid protein demonstrated that the virus is taxonomically related to members within the Family us (TSV) . The useus (TSV) . HoCV-1 us (TSV) . with HiH. coagulata. Future research will need to focus on whether HoCV-1 can be used to reduce H. coagulata populations as a biological control agent, either by augmentation and release, or through other means such as bioengineering. Insect pathogens reduce populations naturally in the wild and if more individuals can be exposed to viral infections then the virus may ultimately be useful as a biological control measure . Many crops of economic importance such as grapes, almonds, pecans, and other woody tree crops, especially in California, and throughout the Southeastern part of the United States would benefit if leafhopper populations could be safely reduced. Further studies need to evaluate the mode(s) of virus transmission and stability in H. coagulata populations under field conditions. The largest obstacle with all viral pathogens is the development of a mass propagation system. Although cell cultures of H. coagulata have been established  the ability for mass production of leafhopper viruses for commercial applications still remain unknown. The potential usefulness of HoCV-1 as a biological control agent against the H. coagulata and in reducing the spread of Pierce’s Disease, awaits the answers to these questions.Invading insects are constantly exposed to native pathogens as they move into new environmental niches. In this case, a leafhopper-infecting virus, HoCV-1, was identified that infects


Although the risk of a herpes B virus (Cercopithecine herpes virus 1) infection is low, the clinical course of the infectious disease is generally unfavourable. A high safety standard can be achieved if people with professional contact to primates apply proper organisational, technical and personal safety precautions. The risk can be considerably reduced if animal keepers, laboratory assistants and scientists receive adequate information about the pathology of herpes B virus and are well trained in the necessary procedures and the precautions. For this reason, comprehensive and regular training, information and instruction must be provided to all primate workers and to laboratory workers who come into contact with potentially infectious material. After potential contamination, the risk for the affected worker must be assessed immediately and post-exposure chemoprophylaxis performed if necessary. This necessitates internal risk assessment. An interdisciplinary group of experts has developed an action plan for Germany. Infection with the herpes B virus is rare, but nevertheless important, as transmission of the pathogen from macaques to human has led to sporadic deaths. Since 1930, over 43 infections with clinical symptoms have been diagnosed in different countries [In humans, herpes B virus infection normally develops into encephalomyelitis, with an unfavourable prognosis ,3. As anA first draft of this recommendation was presented at the symposium on herpes B infections on 13 November 2008 in the German Primate Centre in Goettingen and obtained explicit consent. Suggestions from the participants have been incorporated into the present version.Herpes B virus  is a DNA virus. The official nomenclature is "Cercopithecine herpesvirus 1". Another synonym is "Herpesvirus simiae". The virus is closely related to herpes simplex virus  of humans  in its sMacaca (M.) mulatta (rhesus monkeys), M. fascicularis (long-tailed macaques), M. radiata (bonnet macaques), M. fuscata (Japanese macaques), M. cyclopis (rock macaques), M. arctoides (stump-tailed macaques) and M. nemestrina (pigtail macaques). Experimental or accidental infections, which mostly lead to death, have been reported for Erythrocebus pata (patas monkeys), Colobus guereza (guereza), Cebus apella (brown capuchin monkeys), Callithrix jacchus (common marmoset) and Cercopthecus neglectus (De Brazza's monkeys). Moreover, fatal clinical outcomes are known for African macaques . According to current knowledge, long-tailed macaques  in the Mauritius Islands are free of the agent.B virus is an enzootic agent of Asian macaque monkeys (primary host). Natural infections, which are usually latent, have been described for monkeys, including B virus may occur in natural reservoirs for macaques and in primate centres which keep macaques . B virus is classified in risk group 3, according to the German guideline "TRBA 462" . AccordiB virus is generally transmitted to humans percutaneously or transcutaneously by infected tissue or body fluids  of macaques, especially after bites or scratches. Transmissions can also happen through wound or mucous contact with macaque saliva, injuries while handling animal enclosures accidents with cell cultures, needlesticks and with sharps during autopsy. Approximately half of the affected persons are animal keepers with direct contact to the animals. Another half is laboratory staff, e.g. while handling monkey cell cultures or blood components . The lasWith the exception of Asian macaques, the primary hosts, non-human primates develop severe, mostly fatal clinical pictures with various changes in the organs, as well as severe vesicular exanthemas and pneumonia with giant cells and nuclear inclusion bodies, which are detectable by light microscopy .After infections with B virus, human patients develop various symptoms, which normally develop within four weeks after exposure [Additional file If human exposure is suspected, the material must be collected from the suspect animal and tested in special institutions. The common detection methods are:- Serological detection of B virus antibodies in serum via ELISA, IgG antibodies can only be detected quantitatively two to six weeks after the first infection with B viruses. For this reason, serological detection is only useful and necessary for retrospective analysis. However, this detection method is not necessary for considering and deciding about initiating therapeutic measures, as treatment must be started quickly if a patient may have been infected. Furthermore, it should be remembered that seronegative animals can be infected as well and may eliminate the virus before seroconversion, leading to the infection of other animals.- Virological detection of B virus DNA through PCR in fluids from herpes blisters and CSF, otherwise post-mortem in spinal ganglia/trigeminal ganglia. DNA detection depends on the localisation and quality of the sampling and the clinical picture which the animal presents. The virus is persistent in ganglia. The location of the infected ganglia apparently depends on the site of entry ,12. Duri- Detection through culture of B virus in cell monolayers . Virus culture is the most reliable method but only possible in special laboratories under L4 conditions (see box).A serum sample must be taken from the affected person for medical diagnosis after possible exposure. During international transport, guidelines must be considered when handling infectious material (UN 3373).At the moment, there is no approved institution for handling human samples in Germany. For this reason, it would be desirable to establish a national reference laboratory [Additional file In institutions keeping primates, the personnel must be regularly instructed about special risks and dangers of infection. The experience has been that this should be repeated every six months. Generally it must be assumed that, in doubtful cases, the macaque is excreting virus, so that a range of safety measures must be considered when handling the animals [Additional file Measures to be taken directly by affected persons (and first aid workers):a) In the case of an injury to skin:➢ Wound must be immediately and thoroughly cleaned for 15 minutes under running water and then disinfected (e.g. with 10% povidone iodine solution).➢ Wound must be dressed superficially.b) In the case of contamination of eye or oral cavity:➢ Rinse under running water for approximately 15 minutes.c) Further measures:➢ Identification of the animal causing the infection.➢ Documentation of the procedure.➢ Consultation of the physician named in the accident kit [Additional file Measures to be taken by the employer:a) Examination and testing must be performed as soon as possible after the accident by a suitable physician . This includes virological and serological diagnosis and prophylactic antiviral therapy.b) Testing and sampling  by a veterinarian of the animal causing the infection.c) Documentation of the course of the accident.d) Notification to the responsible insurer of a suspected occupational disease, either by the involved doctor or by the employer.Measures to be taken by the physician:a) Immediately after the accident, it must be checked if the wound has been cleaned and disinfected or if the eye or oral cavity have been rinsed sufficiently. If necessary, repeat this and carry out wound care.b) Clinical  examination of the affected persons must be carried out directly after the accident. Risk assessment must be done for the specification of a post-exposure chemoprophylaxis. Regular follow-ups must be performed at short intervals for approximately six weeks.c) Serological diagnosis must be carried out directly after the accident (blank), as well as after three and six weeks. Later tests (e.g. three months after exposure) may sometimes be useful for patients receiving post exposure prophylaxis.d) The patient must be informed about the clinical and prodromal symptoms and the agreement as to which measures should be taken in the case of occurrence (e.g. immediate consultation of the physician).e) Information about chemoprophylaxis and, if required, initiation of treatment, as soon as possible after exposure. The recommended drug is valacyclovir  three times daily, 1 g for 14 days. An alternative drug is acyclovir  five times daily, 800 mg for 14 days [f) Comprehensive medical documentation.The type of wound, the type of exposure, the depth of the wound and the localisation of the wound must be specified and a risk assessment must be carried out.In the case of pathogen transmission through a wound on the head, the upper body or the neck, possible CNS changes may occur before other symptoms [Additional file Deep wounds, especially those caused by bites, are more difficult to clean than superficial wounds. Therefore, decontamination of deep wounds may be relatively ineffective. For this reason, these wounds present a greater risk. Studies on rabies virus have shown that superficial wounds and scratches on the extremities less frequently lead to fatal disease than do deeper bites. Therefore, it is recommended  to perfoSuperficial wounds and scratches can be cleaned easily and are therefore normally assessed as low risk exposures. [Additional files B virus exists in a latent form in the CNS of infected macaques and will be excreted intermittently from the mucous membrane of the infected animals. Therefore, injuries with needles and objects which may contain material from the CNS, the eyes, the eye lids or the mucous membranes, should be assessed as high risk exposure. There is no reliable information about the tenacity of B virus.Although a case of viremia in a sick monkey has been described in the literature, it can be stated that viremia rarely occurs in clinically normal animals. Therefore, injuries with needles contaminated with the peripheral blood of clinically normal monkeys are assessed as low risk exposures. Sharp injuries have been associated with two cases of human B virus infection so far. One of the injuries occurred with a needle which was contaminated with the tissue from the eye of a monkey. The other injury was caused by a needle which had possibly been used for injecting a monkey ,13 Addi, 5 and 6There has been no case of herpes B disease in Germany in macaque keepers as of yet. However, injuries such as bites or needlesticks are frequent in animal workers and in the diagnostic laboratory. To avoid contamination with herpes B, pre-exposure preventive measures must be specified and implemented at the workplace. If contamination is suspected, post-exposure measures must also be precisely specified, so that the correct steps will be taken at once to ensure the necessary diagnosis and post-exposure chemoprophylaxis against herpes B viruses (herpes B-PEP).These recommendations for the prevention and the post-exposure prophylaxis of herpes B have been developed in collaboration by occupational physicians, virologists, veterinarians and laboratory specialists. The initial draft has already been introduced and discussed during the symposium on "Herpes B virus infections" of the German Primate Centre (DPZ) in Goettingen on November 13, 2009. Suggestions made during this symposium have been incorporated into these recommendations.This publication presents the first expert recommendations on pre- and post-exposure prevention after potential contamination with herpes B virus when working with macaques in Germany. The parties - the injured person, the employer and the  physician - now have a sustainable plan of action for the timing of the diagnostic and therapeutic procedure after a macaque bite and other similar accidents. This should provide them with confidence in their actions in the case of emergency.The authors declare that they have no competing interests.TR prepared and drafted the manuscript. All authors provided scientific information of their special fields and helped to draft the manuscript. All authors read and approved the manuscript.Symptoms suggestive of herpes B infection. Symptoms suggestive of herpes B infection.Click here for fileAddresses for B virus diagnosis. Addresses for B virus diagnosis in Germany.Click here for fileSafety measures for keeping macaques. Safety measures for keeping macaques.Click here for fileSituations indicating post-exposure chemoprophylaxis. Situations indicating post-exposure chemoprophylaxis.Click here for fileSituations with the possible indication of post-exposure chemoprophylaxis. Situations with the possible indication of post-exposure chemoprophylaxis.Click here for fileSituations that do not warrant post-exposure chemoprophylaxis. Situations that do not warrant post-exposure chemoprophylaxis.Click here for file


Chlamydiaceae cause a broad spectrum of diseases in a wide range of hosts, including man, other mammals, and birds. Upper respiratory and genital diseases are common clinical problems caused by Chlamydiaceae. Very little is known about chlamydial infections in dogs. Few clinical reports on natural disease in dogs describe mainly conjunctival and upper respiratory signs, and the role of Chlamydiaceae in genital disease is unclear. The present study aimed at studying the prevalence of Chlamydiaceae in healthy dogs and in dogs with genital or upper respiratory disease, including conjunctivitis.Bacteria belonging to the family Chlamydiaceae was used to detect any chlamydial species within this family. Swab samples from the conjunctiva and the mucosal membranes of the oropharynx, rectum and genital tract were taken from 79 dogs: 27 clinically healthy dogs, 25 dogs with clinical signs from the genital tract and 28 dogs with conjunctivitis. There were 52 female and 27 male dogs. From 7 of the male dogs, additional semen samples were analysed.A real-time polymerase chain reaction (PCR) for Chlamydiaceae were detected from any dog.No Chlamydiaceae in dogs probably are related to infection from other species, and that dogs in general do not harbour Chlamydiaceae. Bacteria belonging to the family Chlamydiaceae do not seem to be of major importance for genital or ocular disease in Swedish dogs.Although the number of dogs that was included is limited, the results suggest that cases of  Chlamydiales. The Chlamydiales comprises four families: Chlamydiaceae, Parachlamydiaceae, Simkaniaceae, and Waddliaceae. The family Chlamydiaceae contains two genera: Chlamydia and Chlamydophila [Chlamydiaceae cause a broad spectrum of diseases in a wide range of hosts, including man, other mammals, and birds. Reproductive disturbances are common, among them salpingitis in man (caused by Chlamydia trachomatis), genitourinary disease in koalas (caused mainly by Chlamydophila pecorum) and abortions in sheep and cattle (caused mainly by Chlamydophila abortus) [Chlamydiaceae in reproductive disturbances, and chlamydiae have been isolated from the vagina of cats, e.g. [Chlamydiaceae are also common ocular pathogens, for instance in humans (C. trachomatis), koalas (Cp. pecorum) and cats (Chlamydophila felis). Avian chlamydiosis, caused by Chlamydophila psittaci, occurs worldwide and Cp. psittaci has been detected from a wide variety of both wild and domesticated birds. Avian chlamydiosis is the most important animal chlamydiosis transmissible to man , but other chlamydiae, such as Cp. abortus and Cp. felis, also have a zoonotic potential [Cp. felis infection in humans hitherto has been verified in only one, HIV-positive, case [Chlamydiae are obligately intracellular bacteria belonging to the bacterial order ydophila . Bacteriabortus) ,3. In cats, e.g. . Bacterive, case .Chlamydiaceae to cause genital or upper respiratory disease including ocular disease in dogs. Detection of Chlamydiaceae requires special cultivation techniques, or analysis with polymerase chain reaction (PCR), and therefore these bacteria are seldom searched for in clinical cases of genital disease or reproductive disturbances. Other aetiological agents, such as canine herpes virus or extracellular aerobic bacteria, that are known to have the capacity of causing genital disease and reproductive problems in dogs, are searched for more frequently. Many cases of conjunctivitis are also treated on an empirical basis, without attempts to make an etiological diagnosis, and if a causative agent is searched for, this is rarely chlamydiae.Very little is known regarding the potential of Chlamydiaceae can be detected on the mucosa of healthy dogs, from dogs with reproductive disturbances or genital disease, or from dogs with upper respiratory signs including conjunctivitis. A real-time PCR that detects all members of the family Chlamydiaceae was used. No Chlamydiaceae was detected from any of the 4-5 sampling sites from the 79 dogs in the present study.The aim of the present study was to study if members of the family The study was approved by the Uppsala ethical committee for animal research (C 310/05) and by the Swedish animal welfare agency (2006-0047). All dog owners gave their informed written consent.79 privately own dogs of 41 different breeds, five mixed breeds and 5 with unknown breed, were included in the study. There were 52 females and 27 males. They were categorized as clinically healthy (N = 27), with clinical symptoms from the genital tract (N = 25), or from the upper respiratory tract, including ocular signs (N = 28). One bitch was sampled twice, first with cystic endometrial hyperplasia (CEH)/endometritis and 2 years later with conjunctivitis.Signs from the genital tract included CEH/endometritis or vaginal discharge (N = 12), pyometra (N = 6), vulvar lesions (N = 1), poor semen quality (N = 1), failure to conceive (N = 1), prostatic disease (N = 1), aberrant oestrous cycles (N = 2) and balanoposthitis (N = 1). Upper respiratory signs included conjunctivitis (N = 20), conjunctivitis and cough or sneezing (N = 4), and only cough or sneezing (N = 4). The dogs were sampled with cotton tipped swabs without any transport or culture medium. From each dog, four samples were taken: from the conjunctiva, oropharynx, rectum and genital tract (vagina or preputium) by rolling swabs against the mucosa. From male dogs, an additional fifth swab was dipped in semen samples, when such a sample was available (N = 7).Chlamydiaceae. We have modified this real-time PCR and developed an internal control to monitor any inhibition from the sample material [At the laboratory, DNA was extracted from the swabs for PCR analysis according to the protocol by Sachse and Hotzel . The sammaterial .Chlamydiaceae were detected in any sample.Seventeen of the bitches (22%) were in heat and seventeen were in metoestrus. Of the 79 dogs, 27 (34%) were stud dogs or breeding bitches. For 24 dogs (30%), previous reproductive disturbances or genital disease was reported, and 26 dogs (33%) had previously had conjunctivitis. No Chlamydiaceae were not detected from any of the samples.The present study aimed at studying if healthy dogs or dogs with genital or upper respiratory including ocular disease harboured chlamydiae on their mucosa. In chronically infected ewes, the periovulatory period has been described to be optimal for detecting chlamydiae . To incrCp. psittaci, which was an even higher number than for the cats in the same study [Cp. psittaci: 19.8% in a population of healthy dogs (N = 1028) and 26% of dogs with various clinical signs (N = 99) [Cp. abortus has been described [Cp. psittaci have been described: two out of three dogs in a household were infected, probably from a cockatiel [Cp. psittaci was also detected in a sample from a dog from a colony of dogs experiencing recurrent periods of dyspnoea, keratoconjunctivitis and small litter sizes and that had been kept together with a parrot and two canary birds [Cp. caviae has been detected, a common chlamydial species in rabbits and guinea pigs, and from samples from 4 dogs with conjunctivitis, Cp. felis was detected [Few seroepidemiological studies on chlamydiae in dogs have been performed. In a Japanese study, using a complement fixation test, 9.5% of dogs were seropositive for dogs N = 28 and 26escribed . The souescribed . Coughinockatiel . The humockatiel . Cp. psiry birds . Humans ry birds . From ondetected .Cp. psittaci was detected in a sample from one of the dogs [C. trachomatis directly into the prostatic gland [Chlamydiaceae were detected from the dog with prostatic disease in the present study. Although it has been suggested that the dog might be a model for chlamydial prostatitis [A causative relationship between chlamydial infection and genital tract disease has not been reported in dogs, although small litter sizes were reported in a colony of dogs in which the dogs . In an eic gland . The dogstatitis  it does Cp. psittaci, and it was speculated that the dog had been infected by an avian host [Other clinical signs experienced in dogs infected with chlamydiae include hepatitis after experimental infection , and a sian host .Contact with another animal species infected with chlamydiae is thus almost always present when clinical disease caused by chlamydiae is present in dogs. Infection with chlamydiae is a potential differential etiological diagnosis in cases of especially upper respiratory disease in dogs with bird contact, and in cases of conjunctivitis after contact with cats (and maybe guinea pigs) with clinical chlamydiosis.Cp. pneumoniae by PCR and immunohistochemistry from atherosclerotic lesions of aortas, coronary and splenic arteries of dogs [Cp. pneumoniae may play an important role in the development of canine atherosclerotic lesions [Cp. pneumoniae was neither detected in samples from dogs with idiopathic pericardial effusions [Cp. psittaci, due to the persistent nature of the infection in a colony of dogs [No case of suspected transmission of chlamydial infection between dogs has been reported. Chronic infection in dogs has been suggested, due to the identification of  of dogs . As no c lesions . Such chffusions , nor in ffusions . It has  of dogs .http://www.smittskyddsinstitutet.se. The major source of infection is thought to be psittacine and other birds. Psittacosis in birds is also notifiable in Sweden, the number of cases being less than ten per year http://www.sjv.se. In Swedish dairy cows, Cp. abortus is absent or the prevalence is very low whereas Cp. pecorum was identified by PCR from vaginal swabs from 2/12 animals (17%) [Cp. psittaci in faeces of passerine birds in Sweden is relatively low, around 3%  . The pre . On thectivitis . The risChlamydiaceae were detected from dogs in the present study. Although the number of dogs was limited, the results suggest that single cases of Chlamydiaceae in dogs are probably related to infection from other species, and that dogs in general do not harbour Chlamydiaceae. The frequency with which chlamydial infections can be diagnosed in dogs within a certain area can thus be expected to be dependent on the prevalence in other animal species, and the frequency of contact between dogs and these infected animals. Bacteria belonging to the family Chlamydiaceae do not seem to be of major importance for ocular or genital disease in Swedish dogs. Chlamydial infection could however be considered a potential differential diagnosis in dogs with upper respiratory disease and bird contacts, or with conjunctivitis and contact with cats.No The authors declare that they have no competing interests.BSH designed and coordinated the study and had the major responsibility for writing and finalising the manuscript. SH, CLF and BSH collected the samples and GB was responsible for the PCR analysis. All authors participated actively in the writing of the manuscript, and all read and approved the final manuscript.


Ciona intestinalis , a major model system for evolutionary and developmental genetics of chordates, harbours two cryptic species. To assess the degree of intra- and inter-specific genetic variability, we report the identification and analysis of C. intestinalis SNP (Single Nucleotide Polymorphism) markers. A SNP subset was used to determine the genetic distance between Hox-5 and -10 genes.The tunicate C. intestinalis sp. A. In total, 128 SNPs and 32 one bp indels have been identified within 8 Kb DNA. SNPs in coding regions cause 4 synonymous and 12 non-synonymous substitutions. The highest SNP frequency was detected in the Hox5 and Hox10 intragenic regions. In C. intestinalis, these two genes have lost their archetypal topology within the cluster, such that Hox10 is located between Hox4 and Hox5. A subset of the above primers was used to perform successful amplification in C. intestinalis sp. B. In this cryptic species, 62 SNPs were identified within 3614 bp: 41 in non-coding and 21 in coding regions. The genetic distance of the Hox-5 and -10 loci, computed combining a classical backcross approach with the application of SNP markers, was found to be 8.4 cM . Based on the physical distance, 1 cM corresponds to 39.5 Kb. Linkage disequilibrium between the aforementioned loci was calculated in the backcross generation.DNA fragments were amplified from 12 regions of C. intestinalis cryptic species. We provide the first reliable computation of genetic distance in this important model chordate. This latter result represents an important platform for future studies on Hox genes showing deviations from the archetypal topology.SNPs here described allow analysis and comparisons within and between  C. intestinalis L., the tunicate species most widely used for research purposes, was recently resolved with the discovery of two cryptic taxa, named C. intestinalis spp. A and B .Among SNPs in non-coding regions, the TC. intestinalis sp. A, perfectly amplify homologous loci in C. intestinalis sp. B. Opposite to coding regions, frequency and Ts/Tv ratio in non-coding regions are very similar. Altogether, data are suggestive of genome behavior in the two cryptic species with shared and divergent traits.In conclusion, all EPIC and non-EPIC primers that were designed taking advantage of the genome sequence of C. intestinalis  and the correlation coefficient (rw v.3.32  with a pXP v.3.0  with a tXP v.3.0 . The genXP v.3.0 .LC (corresponding author) performed acquisition and analysis of data and drafted the manuscript; MB and EB developed the SNP detection procedure; NA gave a fundamental help in revisiting and improving the quality of the manuscript; PS is the PI who conceived and coordinated the project, and made important contributions to the text. All authors read and approved the final manuscript.


We describe the third known case of hantavirus pulmonary syndrome (HPS) due to Bayou virus, from Jefferson County, Texas. By using molecular epidemiologic methods, we show that rice rats  are frequently infected with Bayou virus and that viral RNA sequences from HPS patients are similar to those from nearby rice rats. Bayou virus is associated with O. palustris; this rodent appears to be its predominant reservoir host.


Interspecies transmission of pathogens may result in the emergence of new infectious diseases in humans as well as in domestic and wild animals. Genomics tools such as high-throughput sequencing, mRNA expression profiling, and microarray-based analysis of single nucleotide polymorphisms are providing unprecedented ways to analyze the diversity of the genomes of emerging pathogens as well as the molecular basis of the host response to them. By comparing and contrasting the outcomes of an emerging infection with those of closely related pathogens in different but related host species, we can further delineate the various host pathways determining the outcome of zoonotic transmission and adaptation to the newly invaded species. The ultimate challenge is to link pathogen and host genomics data with biological outcomes of zoonotic transmission and to translate the integrated data into novel intervention strategies that eventually will allow the effective control of newly emerging infectious diseases. Most of the well-known human viruses persist in the population for a relatively long time, and coevolution of the virus and its human host has resulted in an equilibrium characterized by coexistence, often in the absence of a measurable disease burden.Aedes aegypti, for example, has led to massive outbreaks of dengue fever in South America and Southeast Asia. Intense pig farming in areas where frugivorous bats are common is probably the direct cause of the introduction of Nipah virus into pig populations in Malaysia, with subsequent transmission to humans. Bats are an important reservoir for a plethora of zoonotic pathogens: two closely related paramyxoviruses—Hendra virus and Nipah virus—cause persistent infections in frugivorous bats and have spread to horses and pigs, respectively When pathogens cross a species barrier, however, the infection can be devastating, causing a high disease burden and mortality. In recent years, several outbreaks of infectious diseases in humans linked to such an initial zoonotic transmission  have highlighted this problem. Factors related to our increasingly globalized society have contributed to the apparently increased transmission of pathogens from animals to humans over the past decades; these include changes in human factors such as increased mobility, demographic changes, and exploitation of the environment (for a review see Osterhaus The similarity between human and nonhuman primates permits many viruses to cross the species barrier between different primate species. The introduction into humans of HIV-1 and HIV-2 (the lentiviruses that cause AIDS), as well as other primate viruses, such as monkeypox virus and Herpesvirus simiae, provide dramatic examples of this type of transmission. Other viruses, such as influenza A viruses and severe acute respiratory syndrome coronavirus (SARS-CoV), may need multiple genetic changes to adapt successfully to humans as a new host species; these changes might include differential receptor usage, enhanced replication, evasion of innate and adaptive host immune defenses, and/or increased efficiency of transmission. Understanding the complex interactions between the invading pathogen on the one hand and the new host on the other as they progress toward a new host–pathogen equilibrium is a major challenge that differs substantially for each successful interspecies transmission and subsequent spread of the virus.New molecular techniques such as high-throughput sequencing, mRNA expression profiling, and array-based single nucleotide polymorphism (SNP) analysis provide ways to rapidly identify emerging pathogens  and to analyze the diversity of their genomes as well as the host responses against them. Essential to the process of identification and characterization of genome sequences is the exploitation of extensive databases that allow the alignment of viral genome sequences and the linkage of these genomics data to those obtained by classical viral culture and serological techniques, and epidemiological, clinical, and pathological studies Because zoonotic pathogens typically may cause variable clinical outcomes in human hosts that differ in age, nutritional status, genetic background, and immunological condition, deciphering the complex interactions between evolving pathogens and their hosts is a great challenge. The genome sequences of many host species have become available the last decade, and with them a range of novel tools are available to study virus–host interactions at the molecular level. This progress, together with advances in high-throughput sequencing technology and, not least, in (bio)informatics and statistics, allows us to analyze the “genome-wide” networks of gene interactions that control the host response to pathogens. By comparing and contrasting the outcomes of infection with closely related pathogens in different but related host species, we can further delineate the various host pathways involved in the different outcomes. The power of this approach was nicely demonstrated for SIV infection of various primate host species. Natural reservoir hosts of SIV do not develop AIDS upon infection, whereas non-natural hosts, such as rhesus macaques and pig-tailed macaques, when infected experimentally with SIV, develop AIDS in a similar manner to HIV-infected humans. Transcriptional profiling indicates that SIV infection of these species produces a distinctive host response Influenza is caused by RNA viruses of the Orthomyxoviridae family. Whereas fever and coughs are the most frequent symptoms, in more serious cases a fatal pneumonia can develop, particularly in the young and the elderly. Typically, influenza is transmitted through the air by coughs or sneezes, creating aerosols containing the virus; but influenza can also be transmitted by bird droppings, saliva, feces, and blood. Birds and pigs play an important role in the emergence of new influenza viruses in humans. Fecal sampling of migratory birds has revealed that they harbor a large range of different subtypes of influenza A viruses After introduction of a new influenza A virus from an avian or porcine reservoir into the human species, viral genomics studies are essential to identify critical mutations that enable the circulating virus to spread efficiently, interact with different receptors, and cause disease in the new host. For example, the importance of residue 627 of the PB2 protein of the viral polymerase in determining species restriction has been demonstrated through these kinds of approaches Microarray-assisted mRNA expression profiling of emerging zoonotic viral infections, including influenza A virus, is used to phenotype the host response in great detail. By comparing mRNA expression in individuals infected with an emerging virus to expression in individuals infected with a related established virus, researchers can generate a “molecular fingerprint” of the host response genes or pathways specifically involved in the often-exuberant host responses to the emerging virus. By using genetically engineered influenza A viruses, a role for the nonstructural NS1 viral protein in evasion of the innate host response has been demonstrated Paguma larvata), a wild cat-like animal hunted for its meat, and subsequently to humans at live animal markets in southern China Coronaviruses (CoVs) primarily infect the upper respiratory and gastrointestinal tract of mammals and birds. Five different currently known CoVs infect humans and are believed to cause a significant percentage of all common colds in human adults. Surprisingly, recent studies revealed that approximately 6% of bats sampled in China were positive for CoVs angiotensin-converting enzyme 2 (ACE2), which encodes the SARS-CoV receptor, also affects the efficiency by which the virus can enter cells Genome analyses have provided evidence that genetic variation in the spike gene of these viruses from civets is associated with increased transmission of the virus Rapid identification of newly emerging viruses through the use of genomics tools is one of the major challenges for the near future. In addition, the identification of critical mutations that enable viruses to spread efficiently, interact with different receptors, and cause disease in diverse hosts through, for instance, enhanced viral replication or circumvention of the innate and adaptive immune responses, needs to be further expanded. Although microarray-assisted transcriptional profiling can provide us with a wealth of information regarding host genes and gene-interacting networks in virus–host interactions, future research should focus on combining data obtained in different experimental settings. Therefore, the careful design of complementary sets of experiments using different formats of virus–host interactions is absolutely needed for successful genomics studies