However, ELISA only measures one analyte at a time and requires a relatively large plasma volume if the complete antibody profile of the sample is to be obtained. Furthermore, assay-to-assay variation and the problem of storage of antigen can influence ELISA results. The bead-based assay described here uses the BioPlexBaculovirus system and purified by metal-affinity chromatography. The antibody reactivity level to the recombinant PfEMP1 proteins in human hyper-immune plasma was measured by ELISA. In parallel, these recombinant PfEMP1 proteins were covalently coupled onto beads each having its own unique detection signal and the human hyper-immune plasma reactivity was detected for each individual protein using a BioPlex100 system. Protein-coupled beads were analysed at two time points seven months apart, before and after lyophilization and the results compared to determine the effect of storage and lyophilization respectively on the beads. Multiplexed protein-coupled beads from twenty eight unique bead populations were evaluated on the BioPlex100 system against pooled human hyper-immune plasma before and after lyophilization.A total of twenty nine PfEMP1 domains were PCR amplified from 3D7 genomic DNA, expressed in the -based assay was sensitive, accurate and reproducible. Four recombinant PfEMP1 proteins C17, D5, D9 and D12, selected on the basis that they showed a spread of median fluorescent intensity (MFI) values from low to high when analysed by the bead-based assay were analysed by ELISA and the results from both analyses were highly correlated. The Spearman's rank correlation coefficients (Rho) were ≥ 0.86, (P < 0.0001) for all comparisons. Bead-based assays gave similar results regardless of whether they were performed on individual beads or on multiplexed beads; lyophilization had no impact on the assay performance. Spearman's rank correlation coefficients (Rho) were ≥ 0.97, (P < 0.0001) for all comparisons. Importantly, the reactivity of protein-coupled non-lyophilized beads decreased with long term storage at 4°C in the dark.The beadUsing this lyophilized multiplex assay, antibody reactivity levels to twenty eight different recombinant PfEMP1 proteins were simultaneously measured using a single microliter of plasma. Thus, the assay reported here provides a useful tool for rapid and efficient quantification of antibody reactivity against PfEMP1 variants in human plasma. Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1)  ethanesulfonic acid (MES), Tween-20, bovine serum albumin (BSA) sodium azide, biotinylated anti-human IgG, biotinylated anti-V5 antibody and phycoerythrin conjugated streptavidin were purchased from Sigma-Aldrich, USA.1-ethyl-3-[3dimethylaminopropyl] carbodiimide hydrochloride (EDC) and The hyper-immune plasma pool was made up of plasma from ten individuals from a malaria endemic area of Liberia. Twenty samples from Danes who have never had malaria were used to make up the naïve pool. Sixty individual plasma samples, twenty each from people living in the three Tanzanian villages Mgome, Ubiri and Magamba with high, moderate and low malaria transmission , respectBaculovirus vector, pAcGP67-A (BD Bioscience), which was designed to contain the V5 epitope upstream of a histidine tag in the C-terminal end of the construct. The identity of the cloned fragments was verified by sequencing. Linearized Bakpak6 Baculovirus DNA (BD Biosciences Clontech) was co-transfected with pAcGP67-A into Sf9 insect cells for generation of recombinant virus particles and histidine-tagged proteins secreted into the supernatant of infected High-Five insect cell were purified on Co2+ metal-chelate agarose columns. Eluted products were dialysed overnight in PBS. The yield, integrity and purity of the recombinant proteins were estimated by analysis on SDS gel, comparing to BSA standards, and by western blotting using the anti-V5 antibody. All of the proteins coupled to the Luminex beads were estimated to be at or above 80% purity. The sizes of the different recombinant proteins ranged from 45 to 60 kDa.Protein expression was as described previously ,10. Brie7 beads/ml) were brought to room temperature, vortexed for one minute and transferred to Eppendorf® tubes. The supernatant was removed after centrifugation for one minute at 16,000 × g. 1 ml of distilled water was added to the beads, vortexed to re-suspend followed by centrifugation for one minute at 16,000 × g. The beads were sonicated in a water bath sonicator into suspension and centrifuged for one minute at 16,000 × g. The supernatant was removed by a pipette and 1 ml of activation buffer  added to the pellet and vortexed to re-suspend. In separate tubes Sulfo_NHS and EDC were reconstituted to 50 mg/ml and 125 μl of each added to the beads, vortexed and incubated at room temperature for twenty minutes with inversions in the dark. The beads were centrifuged for one minute at 16,000 × g, re-suspended in 1 ml of 50 mM MES pH 5.0, centrifuged for one minute at 16,000 × g and the supernatant removed. The MES wash was repeated. The beads were re-suspended in 500 μl of MES. In separate tubes, the different protein samples (100 μg of each) were mixed with MES to a final volume of 500 μl and each was added to a separate bead population and incubated at room temperature for two hours in the dark with inversions. The beads were centrifuged for one minute at 16,000 × g and the supernatant removed. The beads were washed twice in 1 ml of PBS/TBN . In order to determine whether the human IgG detection antibody bound non-specifically to the coated beads, the beads were analysed against naïve plasma sample from Danes who have not been exposed to malaria.The coated beads were diluted 1:333 in Assay Buffer E  and 50 μl aliquots of were dispensed into the wells of a 1.2 μm filter bottom 96-well microtiter plate  pre-wetted with ABE buffer. The beads in 96-well plates were washed three times with ABE using a vacuum manifold . Frozen plasma samples were thawed at room temperature, mixed by vortexing, and spun at 16,000 × g for five minutes to remove particulates. Plasma samples were diluted 1:80 in ABE buffer and 50 μl aliquots of diluted sample was added to the beads and incubated in the dark on a shaking platform at 1100 rpm for thirty seconds followed by 300 rpm for thirty minutes. Excess antibody was removed using a vacuum manifold followed by three washes in ABE. 25 μl of biotinylated human IgG detection antibody diluted 1:500 in ABE was added to the beads, incubated in the dark with shaking at 1100 rpm for thirty seconds followed by 300 rpm for thirty minutes and washed three times in ABE. 50 μl of phycoerythrin-conjugated streptavidin diluted 1:500 in ABE was added to the beads and incubated in the dark with shaking at 1100 rpm for thirty seconds followed by 300 rpm for ten minutes. Excess phycoerythrin conjugated streptavidin was removed followed by three washes in ABE. The beads were then re-suspended in 125 μl of ABE and analysed on the BioPlexEqual volumes of the coated beads were pooled together and mixed by vortexing. This bead mix was divided in half and one half was stored at 4°C in the dark. Sucrose and Tween 20 were added to the other half to 3% and 0.05% respectively, mixed by vortexing and single-use aliquots were lyophilized  in polypropylene vials, sealed under nitrogen gas and stored at -80°C. Immediately prior to use, lyophilized beads were reconstituted with distilled water and used for analysis as described above.et al [o-phenylenediamine substrate (Dako) activated with H2O2 was added to each well. The plates were incubated in the dark at room temperature before adding 100 μl of 2.5 M H2SO4. Optical densities were measured at 492 nm (OD492). All samples were analysed in duplicate. On each assay microtiter plate, a reference positive control plasma pool was included in addition to negative control wells without plasma (background levels). Results were calculated as arbitrary ELISA units (EU) to account for plate-to-plate variation as described by Jensen et al [Antibody reactivity levels to the recombinant PfEMP1 proteins were measured in an ELISA system as described by Jensen et al . The welen et al :These same samples were analysed by the bead-based assay and the results compared to those from ELISA.100 system. Comparing the MFI obtained for each coupled domain analysed against both plasma pools showed a significantly higher MFI value for the pooled hyper-immune plasma in all cases  by the bead-based assay were compared to those obtained by ELISA  = 0.97, (P < 0.0001)Figure , indicatTo determine the stability of the coupled beads after storage in the dark at 4°C, five protein-specific beads were analysed against the pooled hyper-immune plasma immediately after coupling (white bars) and after seven months of storage (black bars) and the MFI values from the two time points were compared  = 0.99, (P < 0.0001) Figure . The intInter-assay and intra-assay variation expressed as coefficient of variation (CV) were determined using the pooled hyper-immune plasma. The inter-assay CV was 13.4% when measured in a single well on ten consecutive plates. The intra-assay CV was 8.0% when measured in eight wells on a single plate.et al [P. falciparum malaria proteins. The multiplex assay described here measures antibody recognition to twenty eight recombinant P. falciparum erythrocyte membrane protein 1 (PfEMP1) proteins simultaneously in 1 μl of plasma sample. To demonstrate effective coupling, the coated beads were analysed individually against pooled hyper-immune plasma  and pooled naïve plasma (Danes) and were able to show a significantly higher recognition of the pooled hyper-immune plasma compared to the pooled naïve plasma for each individual coated bead (Figure One of the major challenges faced by researchers dealing with malaria in young children is the limited amount of plasma volume easily obtainable from infants and toddlers. Immunoassays such as ELISA require approximately 1 μl of sample for each analyte. In contrast, the multiplex assay has a major advantage of being able to theoretically analyse one hundred antigens simultaneously in 1 μl of sample. Fouda et al  reportedJacobsen  suggesteStability of reagents is especially important if results are to be compared over an extended period. It has been shown that coupled beads remained stable for nine months with storage at 4°C . There wThe multiplex assay described here can measure simultaneously antibodies against twenty eight recombinant PfEMP1 proteins in human plasma. The assay also uses less sample volume, is faster and less labour intensive compared to ELISA. The assay shows a novel approach for maintaining the stability and extending the shelf life of coated beads thus reducing batch-to-batch assay variation. Thus, the assay described here provides a useful tool for studying the immunology of malaria especially in young children where the plasma volume available is usually limited.The authors declare that they have no competing interests.GKKC carried out the bead assay, did the data analysis and writing of the manuscript, JK participated in the study design, TGT, ATRJ, and LT participated in the study, data analysis and writing of the manuscript, JL collected the human plasma samples used. All authors read and approved the final manuscript.Specific PfEMP1 domain primers used for PCR. Primers used to specifically amplify the PfEMP1 encoded fragments, the restriction enzyme sites are underlined and the extra bases precede the restriction sites.Click here for file


Culicoides vector to survive and overwinter. Another chapter is about Hendra and Nipah viruses, which are newly emerging in Southeast Asia and Australia. There are also informative chapters on arteriviruses, coronaviruses, and pestiviruses. Finally, in 1 chapter, Hans-Dieter Klenk and colleagues write about viruses of birds, including avian influenza. They discuss the molecular mechanism of pathogenesis and host range for the virus everyone fears may give rise to the next influenza pandemic.In this multi-author work, Mettenleiter and Sobrino have compiled 10 chapters that describe what is currently known about the molecular biology of some of the most interesting viruses of veterinary importance, from the tiny circovirus of pigs  to the highly complex African swine fever virus . It is fitting that the first chapter describes foot-and-mouth disease virus, which was the first animal virus to be described by Loeffler and Frosch, who worked in Griefswald-Insel Riems, where Mettenleiter is currently the president of the Friedrich-Loeffler Institut. All 10 chapters are written by experts in their respective fields. Mettenleiter is a coauthor for a chapter about herpesviruses, whereas Sobrino is a coauthor for one on foot-and-mouth disease virus. Polly Roy wrote a chapter about bluetongue virus, one of the major threats to the livestock industry worldwide, which recently emerged in Europe, perhaps because global warming has allowed the The book would have been improved by including a chapter on paramyxoviruses, of which rinderpest virus of cattle and Newcastle disease virus of birds are 2 important examples. But, overall, this compilation is excellent and is rounded off by a scholarly and provocative epilogue about animal virology by Esteban Domingo and Marian C. Horzinek. It is almost worth buying the book for these 10 pages alone.


The diagnosis of rabies is routinely based on clinical and epidemiological information, especially when exposures are reported in rabies-endemic countries. Diagnostic tests using conventional assays that appear to be negative, even when undertaken late in the disease and despite the clinical diagnosis, have a tendency, at times, to be unreliable. These tests are rarely optimal and entirely dependent on the nature and quality of the sample supplied. In the course of the past three decades, the application of molecular biology has aided in the development of tests that result in a more rapid detection of rabies virus. These tests enable viral strain identification from clinical specimens. Currently, there are a number of molecular tests that can be used to complement conventional tests in rabies diagnosis. Indeed the challenges in the 21st century for the development of rabies diagnostics are not of a technical nature; these tests are available now. The challenges in the 21st century for diagnostic test developers are two-fold: firstly, to achieve internationally accepted validation of a test that will then lead to its acceptance by organisations globally. Secondly, the areas of the world where such tests are needed are mainly in developing regions where financial and logistical barriers prevent their implementation. Although developing countries with a poor healthcare infrastructure recognise that molecular-based diagnostic assays will be unaffordable for routine use, the cost/benefit ratio should still be measured. Adoption of rapid and affordable rabies diagnostic tests for use in developing countries highlights the importance of sharing and transferring technology through laboratory twinning between the developed and the developing countries. Importantly for developing countries, the benefit of molecular methods as tools is the capability for a differential diagnosis of human diseases that present with similar clinical symptoms. Antemortem testing for human rabies is now possible using molecular techniques. These barriers are not insurmountable and it is our expectation that if such tests are accepted and implemented where they are most needed, they will provide substantial improvements for rabies diagnosis and surveillance. The advent of molecular biology and new technological initiatives that combine advances in biology with other disciplines will support the development of techniques capable of high throughput testing with a low turnaround time for rabies diagnosis. Validated diagnostic tests that confirm the presence of rabies virus or a lyssavirus variant have been the foundation of rabies control strategies in many countries. Historically, histopathological techniques such as the Sellers Stain technique The fluorescent antibody virus neutralisation (FAVN) test These internationally approved methods have provided accurate and timely information of animal and human rabies cases thereby supporting surveillance for rabies and providing a greater understanding of the epidemiology of this disease . For numValidated diagnostic tests capable of confirming the presence of rabies virus in clinical samples have improved the quality, accuracy and speed of rabies diagnosis in many national reference laboratories thereby supporting rabies control strategies with the global vision of dog rabies elimination in developing countries. Ante-mortem testing for human rabies is now possible using molecular techniques.Adoption of rapid and affordable rabies diagnostic tests for use in developing countries highlights the importance of participation in projects that link laboratories from the developed and the developing countries.The advent of molecular biology and new technological initiatives that combine advances in biology with other disciplines will support the development of microchip, biosensor and robotics-based techniques capable of high throughput testing with a low turnaround time for rabies diagnosis.st century, it is expected that diagnostic virology techniques for high throughput rabies virus detection will progress rapidly towards the use of molecular diagnostics replacing more conventional testing techniques such as virus isolation and histopathology. It is also possible that immunological tests, measuring ‘surrogate’ markers such as cytokines and electrolytes, will augment the standard diagnostic approach; nevertheless they will continue to remain oddities outside the realms of the routine diagnostic laboratory and be confined to a few reference laboratories. Semi-automated or automated instruments and robotics-based techniques are useful when large numbers of the same test are undertaken and these tests will continue to increase in popularity and use, especially in central reference laboratories rather than in each local or regional facility. New technological advances will undoubtedly be faster, more accurate and may, in time, offer a cost-effective alternative to traditional rabies diagnostic tests. These paradigm shifts including modern advances in technology will lead to the effective control of rabies in animals and wildlife Molecular tools based on the detection and manipulation of the genetic information of the virus are becoming more widely accepted and accessible for the diagnosis of rabies. The advent of molecular biology is changing the face of diagnostic virology generally enabling high throughput and short turnaround-time analysis of samples. In the 21Barrat J (1996) Simple technique for the collection and shipment of brain specimens for rabies diagnosis. In: Meslin FX, Kaplan MM, Koprowski H, editors. Laboratory techniques in rabies. Geneva: World Health Organisation. pp 425–432.Bourhy H, Kissi B, Tordo N (1993) Molecular Diversity of the Lyssavirus Genus. Virology 194: 70–81.Rupprecht CE, Hanlon CA, Slate D (2006) Control and prevention of rabies in animals: paradigm shifts. Developments in Biologicals 125: 103–111.Sacramento D, Bourhy H, Tordo N (1991) PCR technique as an alternative method for diagnosis and molecular epidemiology of rabies virus. Molecular Cellular Probes 5: 229–240.Willoughby RE Jr, Tieves KS, Hoffman GM, Ghanayem NS, Amlie-Lefond CM, Schwabe MJ, Chusid MJ, Rupprecht CE (2005). Survival after treatment of rabies with induction of coma. N. Engl. J. Med. 16: 2508–1514.The principal focus of this review is to highlight the new developments in virology related to techniques for the diagnosis and surveillance of rabies. Literature reviews were identified through Web of Science, PubMed and Scopus using various search phrases. This review also drew on information provided to international organisations, mainly WHO and OIE, funded by the UK Department for Environment, Food and Rural Affairs (Defra) in an advisory context on diagnostic and surveillance strategies for rabies. This review however, does not reflect the views of Defra, WHO or OIE. This review provides information on some of the latest developments and diagnostic techniques for determining the presence of rabies virus in diagnostic samples. Our aim is to provide a viewpoint on the current thinking in diagnostic virology for rabies, reflecting the ‘neglected’ nature of this tropical disease and the contrasting needs of diagnostic laboratories in developed and developing countries.A direct Rapid Immunohistochemical Test (dRIT) for the postmortem detection of rabies virus antigen in brain smears has been developed Another method for the detection of rabies virus antigen from postmortem samples is a recently developed rapid immunodiagnostic test (RIDT) based on the principles of immunochromatography Various conventional RT-PCR protocols for the diagnostic amplification of lyssavirus genome fragments have been published –3. SincClassical RT-PCR assays proved to be a sensitive and specific tool for routine diagnostic purposes With the introduction of fluorogenic probes, detection of sequence specific templates can be achieved in real-time. Specificity is ensured by an inherent hybridization reaction, and cross-contamination is avoided due to the closed tube nature of the test in situ, whereby the relatively lower isothermal temperatures of NASBA compared to in-situ RT-PCR ensure that cell integrity is maintained The use of a rapid automated NASBA technique was successfully applied to the ante-mortem saliva and cerebrospinal fluid (CSF) of four rabies patients in Thailand and shown to have a ten-fold increase in sensitivity compared to RT-PCR Bst 1 DNA polymerase. The technique is rapid, generating large quantities of target sequence within minutes. For the amplification of RNA viruses, a reverse transcription step is undertaken prior to the LAMP reaction. Primer sets have been successfully developed to detect a range of pathogenic viruses including West Nile virus LAMP offers an alternative DNA amplification method to the polymerase chain reaction for applications to the ante-mortem saliva and CSF testing. The originators of the technique suggest that it amplifies with high specificity, efficiency and without the need for thermal cycling Development of RT-LAMP assays for use in diagnosis and surveillance is challenged by the considerable sequence variation observed within the rabies virus genome Microarray linked to sequence independent PCR amplification offers the ability to rapidly identify pathogenic viruses from post-mortem samples A stable potentiometric immunosensor for the detection of various analytes of interest from complex real world samples such as blood, serum and milk has been described 5 infectious units/ml) were produced that proved 100% specific and highly sensitive compared with neutralisation titres achieved using the FAVN r = 0.89). Using pseudotypes expressing EBLV-1 (genotype 5) and EBLV-2 (genotype 6) G-proteins, neutralising antibody titres broadly correlated with the degree of G-protein diversity. A vaccine study in Tanzania compared the two assays with pseudotypes showing 100% specificity and 94.4% sensitivity to the FAVN with a high correlation of antibody titres (r = 0.92). Incorporation of Lagos bat virus (genotype 2), Mokola virus (genotype 3) and Duvenhage virus (genotype 4) G-proteins, as well as lacZ as a reporter gene, makes the pseudotype assay an attractive option for serosurveillance in Africa and other resource limited countries. In addition, as the pseudotype assay uses substantially less input serum (10 µl) compared to FAVN and RFFIT, multiple tests can be undertaken on samples where collection volumes are limited or valuable e.g. bat sera.Serological assays are not suitable as diagnostic tools for routine rabies testing as the host response to infection varies substantially between individuals. However, serology is still useful, particularly to monitor the development of the immune response. We would suggest that detection of rabies antibodies in serum and CSF, early after presentation and in the absence of a history of vaccination may be a positive indicator for a therapeutic intervention. Viral pseudotypes, the core of one virus coated with envelope protein derived from a second virus, offers a safe alternative to the use of pathogenic viruses in neutralisation assays. Using pseudotypes expressing genotype 1 CVS-11 glycoprotein, high titre stocks (1.3–3.2×10ante mortem diagnosis of human rabies Due to the neurotropic nature of rabies virus, infection results in enormous viral replication in the CNS in the final stage of the disease that leads to massive antigen and viral genome concentrations. This makes detection of viral antigen in brain tissue by tests such as the FAT or the dRIT intra-vitam in suspect human cases, when conventional diagnostic methods have failed and post-mortem material is not available (The development of ‘real-time’ RT-PCR techniques allows the quantification of this RNA in ‘real-time,’ giving a relatively quick and reliable method for the measuring levels of viral RNA. PCR based techniques are not currently recommended by the WHO for routine post-mortem diagnosis of rabies. However, in laboratories with strict quality control procedures in place and demonstrable experience and expertise, these molecular techniques have been successfully applied for confirmatory diagnosis and epidemiological surveys. For these reasons, it is likely that international bodies will accept their use in the future for routine rabies diagnosis. Reverse transcription PCR has been reported to confirm rabies diagnosis vailable  [57]. Rast century. However, we should not discount alternatives that have the benefit of isothermal amplification that will enable implementation in laboratories where access to thermal cyclers is an obstacle. A NASBA technique was successfully applied to the saliva and CSF of four living patients with rabies and detected rabies viral RNA 2-days after the onset of symptoms. This technique has also been adapted to investigate rabies virus replication in-situ. LAMP also falls into this category and can be adapted for use with lateral flow devices thus making its application very simple.It is evident that the RT-PCR dominates genetic detection of rabies virus and it seems probable that this technique will dominate rabies diagnosis in the 21Existing assays for rabies virus antibody prevalence studies either require high containment facilities or do not distinguish between neutralising and non-neutralising antibodies st century, it is possible that these techniques will replace conventional tests (st century, these technologies will have a demonstrable impact on people living in developing countries, especially where rabies is still considered a ‘neglected’ disease. By contrast in the developed world, these new technological advances will undoubtedly be faster, more accurate and cost-effective leading to a ‘Theragnostics Approach’ that combines therapeutics with diagnostics for the human treatment of rabies. Interest in treating human rabies aggressively is gaining momentum, largely due to the reported success in treating a 15-year-old girl, in whom clinical rabies developed one month after she was bitten by a bat, using a combination of therapeutic coma with antiviral drugs whilst allowing for the host immune system to confer immunity – The ‘Milwaukee Protocol’ (Currently, high-throughput rabies virus molecular detection methods augment standard diagnostic tests or are in the process of development and refinement for use alone. As we progress through the 21al tests . Obstaclrotocol’  [62],[63


Leucine rich repeats (LRRs) are present in over 60,000 proteins that have been identified in viruses, bacteria, archae, and eukaryotes. All known structures of repeated LRRs adopt an arc shape. Most LRRs are 20-30 residues long. All LRRs contain LxxLxLxxNxL, in which "L" is Leu, Ile, Val, or Phe and "N" is Asn, Thr, Ser, or Cys and "x" is any amino acid. Seven classes of LRRs have been identified. However, other LRR classes remains to be characterized. The evolution of LRRs is not well understood.Here we describe a novel LRR domain, or nested repeat observed in 134 proteins from 54 bacterial species. This novel LRR domain has 21 residues with the consensus sequence of LxxLxLxxNxLxxLDLxx(N/L/Q/x)xx or LxxLxCxxNxLxxLDLxx(N/L/x)xx. This LRR domain is characterized by a nested periodicity; it consists of alternating 10- and 11- residues units of LxxLxLxxNx(x/-). We call it "IRREKO" LRR, since the Japanese word for "nested" is "IRREKO". The first unit of the "IRREKO" LRR domain is frequently occupied by an "SDS22-like" LRR with the consensus of LxxLxLxxNxLxxLxxLxxLxx or a "Bacterial" LRR with the consensus of LxxLxLxxNxLxxLPxLPxx. In some proteins an "SDS22-like" LRR intervenes between "IRREKO" LRRs.Proteins having "IRREKO" LRR domain are almost exclusively found in bacteria. It is suggested that IRREKO@LRR evolved from a common ancestor with "SDS22-like" and "Bacterial" classes and that the ancestor of IRREKO@LRR is 10 or 11 residues of LxxLxLxxNx(x/-). The "IRREKO" LRR is predicted to adopt an arc shape with smaller curvature in which β-strands are formed on both concave and convex surfaces. LRR (leucine rich repeat) domains are present in over 60, 000 proteins listed in PFAM, PRINTS, SMART, InterPro and PANTHER databases . LRR-con10-helix, polyproline II helix, and an extended structure or a tandem arrangement of β-turns. In most LRR proteins the β-strands on the concave surface and (mostly) helical elements on the convex surface are connected by short loops or β-turns. Seven classes of LRRs have been recognized, characterized by different lengths and consensus sequences of the VS part of the repeats  also have some IRREKO@LRR domains.The present observations indicate that IRREKO@LRR is a new class of LRR. This is supported by several additional observations. The identification of LRRs by PFAM or SMART occurs in a large number of IRREKO@LRR proteins including The IRREKO@LRRs show a nested periodicity consisting of alternating 10- and 11- residue units with the consensus of Lxx(L/C)xLxxNx(x/-). The IRREKO@LRR domains in many proteins contain a mixture of both subtypes. The first LRR of the LRR domains is frequently "SDS22-like" or "Bacterial" classes. In addition, among the IRREKO@LRR domain "SDS22-like" class occurs in some proteins. The two subtypes of IRREKO@LRR appear to have evolved from a common precursor. Further, the "IRREKO" domain evolved from a precursor common to "SDS22-like" and "Bacterial" classes. The precursor of IRREKO@LRR is shorter sequence - LxxLxLxxNx(x/-) -. This parsimonious evolutionary scenario for three LRR classes, "IRREKO", "SDS22-like", and "Bacterial" LRRs is shown in Figure STT, where "T" is "typical" LRR and "S" is "Bacterial" LRR; they include the SLRP subfamily , the TLR7 family , the FLRT family , and OMGP  is an LRR protein. It is identified in the data bases of InterPro, PFAM, PRINTS and SMART. The InterPro data base indicates that the LRR domain contains nine repeats. The PFAM program predicts that yddK contain one significant LRR (residues 216-238) and seven insignificant LRRs .The putative uncharacterized protein yddK from We recently developed a new method that utilizes known LRR structures to recognize and align new LRR domains and incorporate multiple sequence alignments and secondary structure predictions . This meThis new method was used for multiple sequence alignments of LRRs in the yddK protein. This analysis predicted not nine repeats of the LRRs but 13 repeats and also revealed that their "phasing" differ significantly. We noticed that LRRs, 1, 5 7, 8, 9, and 10 contain a unique domain whose consensus is LxxLxLxxNxLxxLxLxxxxx with 21 residues. The variable segment offers a characteristic hydrophobic pattern unidentified previously Figure . Each LRLRR proteins having the IRREKO@LRR domains were identified in three steps:Step 1: Detection of LRR proteins containing the six, novel LRRs in E-coli yddk by using FASTAStep 2: Identification of the IRREKO@LRRs in individual LRR proteins by a newmethod.Step 3: Iteration of these two steps using novel LRRs in newly identified LRR proteinsstep 1, we performed similarity search using the six, novel LRRs as probes by FASTA at the Bioinformatic Center, Institute for Chemical Research, Kyoto University on April 27, 2009 http://www.genome.ad.jp/. This procedure detected many yddK homologs from Escherichia coli strains and Shigella flexneri [Q0T447 and Q83R94] with significant similarity . In addition, two other proteins were detected with significant similarity . One is SSON_1653 that is 387 residues long [Q3Z1L5]. The other is SD1012_2081 with 163 residues [B3WXZ7]. In step 2, we performed multiple sequence alignment among their LRR domains of SSON_1653 and Sd1012_2081. SSON_1653 contains 14 LRRs and 9 of the 12 repeats consist of LxxLxLxxNxLxxL(D/N)(L/F)xxxxx where "L" is Leu, Val, or Ile. Sd1012_2081 contains 4.5 LRRs; 3.5 of these repeats consist of LxxLxLxxNxLxxIx(I/A/F)xxaxx In step 3, the above procedures were iterated to identify other LRR proteins having this IRREKO@LRR domain.In http://emboss.bioinformatics.nl/cgi-bin/emboss/dotmatcher. A radar chart is a graphical method displaying multivariate data in the form of a two-dimensional chart of three or more quantitative variables represented on axes starting from the same point http://en.wikipedia.org/wiki/Radar_chart. For a given observation, the length of each ray is the occurrence frequency of each amino acid at two positions of "IRREKO" LRR with 21 residues. Multiple sequence alignments were performed by CLUSTALW at the Bioinformatic Center. The protein secondary structure prediction was performed by SSpro4.0 http://contact.ics.uci.edu/sspro4.html[http://129.128.185.184/proteus/#[The dot-matrix comparisons were performed using the BLOSUM62 scoring matrix and a window size of 21 residues pro4.html and Protproteus/#. Signal proteus/#.NM (corresponding author) carried out the molecular genetic studies, participated in the sequence alignment and drafted the manuscript. HM performed dot plot analysis and radar chart analysis. TM contributed to the data analysis including the sequence alignment. KY conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.Table 1: IRREKO@LRR proteins. Database; Protein accession number or identification number in EMBL or NCBI. Consensus; The consensus sequences of complete IRREKO@LRRs with 21 residues are shown. Bold uppercase letters indicate more than 60%, normal uppercase letters indicate more than 50% and less than 60%, and normal lowercase letters indicate less than more than 30% and less than 50%. "L" in the consensus sequence denotes Leu, Val, or Ile. "x" denotes any residues. Length; The length of complete amino acid sequences of proteins. LRR repeat; The repeat number of LRR domain. Number is the repeat number of complete IRREKO@LRRs with 21 residues. The numeral in the parenthesis is total repeat number of LRRs. 1st LRR; The LRR class of the first repeat of LRR domain. SIGNAL; The Occurrence (○) and no-occurrence (-) of signal peptide sequence. LRRNT; The pattern of cysteine clusters of the N-terminal side of LRR domain.Click here for fileFigure S1: Sequence alignments of the LRR domain in seventeen IRREKO@ LRR proteins. (A) Escherichia coli yddk; (B) Bifidobacterium animalis BIFLAC_05879; (C) Vibrio harveyi HY01 A1Q_3393; (D) Shewanella woodyi ATCC 51908 SwooDRAFT_0647; (E) Unidentified eubacterium SCB49 SCB49_09905; (F) Colwellia psychrerythraea CPS_3882; (G) Listeria monocytogenes lmo0331 protein; (H) Treponema denticola TDE_0593; (I) Polaromonas naphthalenivorans Pnap_3264; (J) Ddelta proteobacterium MLMS-1 MldDRAFT_4836; (K) Kordia algicida OT-1 KAOT1_04155; (L) Coprococcus eutactus ATCC 27759 COPEUT_03021; (M) Clostridiales bacterium 1_7_47_FAA Cbac1_010100006401; (N) Listeria lin1204/LMOf6854_0364; (O) Escherichia coli SMS-3-5 EcSMS35_1703; (P) Escherichia coli O157:H7 ECS2075/Z2240; (Q) Trichomonas vaginalis G3 TVAG_084780. Overall consensus sequences of IRREKO@LRRs - LxxLxLxxNxLxxLDLxx(N/L/Q/x)xx or LxxLxLxxNxLxxLDLxx(N/L/Q/x)xx - are shown. The consensus amino acids are highlighted with reverse-contrast. Also the consensus amino acids of "SDS22-like" LRR with the consensus of LxxLxLxxNxLxxLxxLxxLxx and of "Bacterial" LRR with the consensus of LxxLxxNxLxxLPxLPxx are highlighted with reverse-contrast. Cysteines of the cysteine clusters at the N-terminal side of LRR domain are shown by underlined bold letter. Cons., the overall consensus sequences of IRREKO@LRRs; SIGNAL, signal peptide sequence; LRR; leucine rich repeat (LRR); IRREKO, IRREKO LRR; SDS22; "SDS22-like" LRR; BAC; "Bacterial" LRR; ISLAND, Island region interrupting LRRs; N-TERM, the N-terminal region of proteins; C-TERM, the C-terminal region of proteins; LRRNT; the region of cysteine clusters at the N-terminal side of LRR domain.Click here for fileFigure S2: Self-dot matrices for four IRREKO@LRR proteins. (A) Bifidobacterium animalis BIFLAC_05879; (B) Vibrio harveyi HY01A1Q_3393, (C) Listeria monocytogenes lmo0331 protein; (D) Treponema denticola TDE_0593. A window size of 21 residues was used. The threshold is 30 in the upper panel and 10 or 15 in the lower panel. Residues used are full lengths for the self-dot matrices; residue 1-186, 1-278, 1-633, and 1-631 of BIFLAC_05879, HY01A1Q_3393, lmo0331 protein, TDE_0593, respectively, were used. The abscissa and the ordinate are residues number.Click here for fileFigure S3: Protein secondary structure prediction in five IRREKO@LRR proteins by the Proteus and SSpro4.0 programs. (A) Escherichia coli yddk; (B) Bifidobacterium animalis BIFLAC_05879; (C) Vibrio harveyi HY01 A1Q_3393; (D) Listeria monocytogenes lmo0331 protein; (E) Shewanella woodyi ATCC 51908 SwooDRAFT_0647; (F) Treponema denticola TDE_0593. The highly conserved segment of individual LRRs is highlighted by a shadow. For comparison, its consensus sequence is shown in bold letters. Abbreviations: h/H, helix; c/C, coil; e/E, β-strand.Click here for file


These findings suggest that testing cloacal or oral swabs might be a low-resource approach to detect WNV in dead birds.We evaluated if postmortem cloacal and oral swabs could replace brain tissue as a specimen for West Nile virus (WNV) detection. WNV was detected in all three specimen types from 20 dead crows and jays with an average of >10 West Nile virus [WNV]) transmission throughout the eastern United States Since 1999, surveillance of bird deaths has become a standard epidemiologic method for detecting the spread and continued presence of West Nile virus  on cloacal and oral  swabs of corvidGiven that birds with acute WNV infection frequently shed the virus in cloacal or oral cavities –6 and thCorvus brachyrhynchos), 4 Fish Crows (Corvus ossifragus), and 4 Blue Jays (Cyanocitta cristata), that had died (or in one case had been euthanized after being moribund for 24 hours) after experimental infection with the New York 1999 strain of WNV. , especially corvids, for detection of infectious virus particles or RNA in brain or other viscera. We have shown that postmortem oral and cloacal swabs, in addition to brain, are effective samples to collect for WNV detection in experimentally infected corvids. A potential implication of these findings, pending field trials using corvids and other species routinely collected as part of avian mortality surveillance, is that WNV may be detected by simply collecting swabs from carcasses and forwarding the swabs (frozen) to a virology laboratory for testing. Eliminating multiple steps currently necessary for WNV testing of bird carcasses may conserve valuable public health resources and reduce the risk of exposure for laboratory personnel.


Mycobacterium tuberculosiscomplex genome. This region consists of 36 bp repetitive sequences separated bynon-repetitive unique spacer sequences. Around this region there are several genescoding for proteins of unknown function. To determine whether the M. smegmatis, M.avium, M. marinum and M. leprae genomes contain sequences and ORFs similar tothose of the DR locus of the M. tuberculosis complex, we analysed the correspondingregions in these species. As a first step, some conserved genes that flank the DR genes were used as markersto locate the corresponding orthologues in M. smegmatis, M. avium, M. marinum andM. leprae in silico. Most of these M. tuberculosis marker genes have highly similarorthologues located in the same order and orientation in the other mycobacteria. Incontrast, no orthologues were found for ORFs Rv2801–Rv2824, suggesting that thesegenes are unique to M. tuberculosis within the genus Mycobacterium.We observed thatin M. smegmatis and M. avium, Rv2800 and Rv2825 are adjacent.This observation was experimentally confirmed by PCR. In conclusion, as the DR locus and the ORFsaround it are absent in M. smegmatis and M. avium and, as it is possible that thesespecies are older than M. tuberculosis, we postulated that the DR locus was acquiredby the M. tuberculosis complex species or by an ancestor bacterium.The direct repeat (DR) region is a singular locus of the


Predator-prey models for virus-host interactions predict that viruses will cause oscillations of microbial host densities due to an arms race between resistance and virulence. A new form of microbial resistance, CRISPRs  are a rapidly evolving, sequence-specific immunity mechanism in which a short piece of invading viral DNA is inserted into the host's chromosome, thereby rendering the host resistant to further infection. Few studies have linked this form of resistance to population dynamics in natural microbial populations.Sulfolobus islandicus from a single, isolated hot spring from Kamchatka, Russia to determine the effects of CRISPR immunity on microbial population dynamics. First, multiple housekeeping genetic markers identify a large clonal group of identical genotypes coexisting with a diverse set of rare genotypes. Second, the sequence-specific CRISPR spacer arrays split the large group of isolates into two very different groups and reveal extensive diversity and no evidence for dominance of a single clone within the population.We examined sequence diversity in 39 strains of the archeaon S. islandicus is indicative of a lack of strain dominance, in contrast to the prediction for a resistant strain in a simple predator-prey interaction. Based on evidence for the independent acquisition of resistant sequences, we hypothesize that CRISPR mediated clonal interference between resistant strains promotes and maintains diversity in this natural population.The evenness of resistance genotypes found within this population of  Virus-host interactions are a prominent driver of microbial diversity in natural environments. The simplest models describe these interactions through predator-prey dynamics which result in temporal fluctuations in strain dominance similar to ecological Lotka-Volterra models Cellulophaga baltica infected with two virulent bacteriophages Flavobacterium diversified physiologically and in phage susceptibility to a panel of phages. In addition, Lennon et al. demonstrated variation in the cost of resistance that could result in a stable level of diversity within a population that is higher than would be predicted if populations were evolving through clonal competition for resources in the absence of viral predation Oscillations of different species' abundance are theorized to be damped by fitness trade-offs associated with the physiological costs of viral resistance, resulting in a stable level of diversity of coexisting strains within a population at any one time Streptococcus thermophilusStaphylococcus epidermidisPredictions from theoretical and experimental studies of virus-host interactions have been challenging to study in wild populations because establishing linkage between genotype and resistance phenotype is difficult using culture independent molecular tools Leptospirilum group II from acid mine drainage To examine the effects of CRISPR immunity on population dynamics, Tyson and Banfield reconstructed CRISPR loci from two different populations of one species of S. islandicus from a hot spring in the Mutnovsky Volcano region of Kamchatka, Russia. We use multi-locus sequence analysis (MLSA) from a set of core housekeeping genes present in S. islandicus to determine overall host diversity and compare this to the diversity identified in CRISPR spacer sequences from each isolate.In order to understand the ways in which virus-host interactions mediated by CRISPRs affect population dynamics, it is necessary to link signatures of resistance among coexisting strains to genotypic variation within a population. Analysis of natural population dynamics at a strain-specific level is needed to test predictions of current models about the way that virus-host interactions affect population dynamics Sulfolobus islandicus from a single hot spring in the Mutnovsky Volcano region of Kamchatka, Russia. As shown in S. islandicus in this spring has been well sampled with the 39 strains described here. Chao1 richness is estimated to be 20 OTUs when each individual is unique (one OTU at 0.01% divergence) S. islandicus from the Mutnovsky population have up to three CRISPR loci , new spacer sequences are added at the leader end in response to invasion of mobile elements Sulfolobus as it does in Streptococcus, the variability observed at the leader end is likely to have resulted from recent interactions of S. islandicus with viruses or plasmids and indicates that these loci are actively acquiring resistance in this population. For several pairs of isolates, the only remaining evidence of shared spacer sequences in the same position are the very first conserved spacer at the trailer end of the locus  BLAST matches to a database of Sulfolobus genomes, viruses, and plasmids. The majority (87%) of these match viruses; 22% of total matches are to viruses integrated into S. islandicus genomes and 51% of total matches are to SSV (Sulfolobus spindle-shaped virus) sequences, a non-lytic virus that has been isolated from around the world, with many sequenced isolates. The rest of the matches (less than 7% each) are to plasmids and other Sulfolobus genomes or other integrated elements within them. As we have shown previously, we do not see spacers that match 100% to a portion of the same genome in which the spacers are located, as is evident in the fully sequenced subset of isolates from this population Of the 756 unique spacers, only 50 have significant  of the independently acquired matching spacer pairs are located on the leader ends of loci in both strains, which indicates that both spacers were recent acquisitions of an element that was present in the spring at that time. Pairs with both trailer end spacers make up 27% of the matches and mixed leader and trailer matches make up the remaining 29%. We did not observe a locus preference for spacers as has been suggested previously Sulfolobus viruses, plasmids, and S. islandicus genomes, there are two viruses (SSV) that are matched 100% by different spacers from different isolates. One virus has two spacers that match it while the other has three spacers. Just like the overlapping spacer matches, the spacers that match the same virus represent independent acquisitions, by different isolates, of the same virus. It is unlikely that the high frequency of spacer matches is due to a particular, rare sequence on the genome that is especially effective in resistance because we and others have shown that spacers are derived from throughout viral genomes SulfolobusMatches between independently acquired spacers from isolates with different CRISPR arrays demonstrates that isolates share a common viral pool from which they are independently infected. When spacers are compared to S. islandicus from a single hot spring. This diversity is underestimated by MLSA sequences, but is revealed by CRISPR spacers present in these strains. Relationships described by MLSA are quite different from those observed using CRISPR locus sequences supporting the rapid evolution of CRISPR sequences relative to the rest of the genome. In addition, the population structure revealed by the individual-level of resolution provided by CRISPRs shows no evidence of a selective sweep or an epidemic structure in which one set of sequences is at high abundance relative to the rest of the population. Without a dominant clone, this snapshot of diversity within a single population is unlikely to follow a simple predator-prey model in which there are oscillations in strain abundance dependent upon resistance.Our results show a significant amount of diversity in a population of Leptospirillum sp, in which evidence of selective sweeps were identified in shared spacer sequences at the trailer end of the repeat spacer region of the locus as well as surrounding genes Sulfolobus population include: 1) the addition of spacers in the bacterial population are slower, leaving time for a selective sweep to occur, 2) viruses in the Leptospirillum population are more virulent, causing a stronger selection, 3) the two studies have observed dynamics at different times during oscillations within populations, 4) there are differences in the number of interactions between strains in the highly structured biofilm and well mixed hot spring environments, and 5) there is a difference in recombination frequency between Leptospirillum and Sulfolobus that preserves the diversity of both genotype and CRISPR arrays in the S. islandicus population. In addition, in the metagenome study, as opposed to this study of isolates, it is difficult to link individual spacers within a CRISPR array and to link these arrays to very similar specific genotypes.This is in contrast to the metagenome study of the clonal S. islandicus isolates from this pool are recombining suggests that they are not completely isolated from one another Spatial substructure would physically isolate hosts and/or virus populations from one another, allowing aggregate diversity to persist Theoretical and experimental studies have demonstrated that diversity of coexisting strains can arise in a population due to tradeoffs and variation in resistance phenotypes, and their costs are often associated with the efficiency of nutrient uptake S. islandicus population due to clonal interference among individuals that have independently acquired resistance to viruses in their CRISPR loci. Different clones, each with a different CRISPR spacer to the same virus, compete with one another and therefore prevent a sweep that would purge all diversity from the environment We propose that CRISPR diversity may be maintained within this S. islandicus that may play an additional role in the maintenance of diversity within this population.This is conceptually similar to the theoretical model described by Rodriguez-Valera et al. in which diversity is maintained due to the rapid evolution of virus receptors in genomic islands Streptococcus sp. In that study, when the host is challenged by one virus, multiple resistant hosts are found, each with different spacers that give immunity to the same virus The rapid acquisition of independent CRISPR spacers consistent with our model has been shown by Barrangou et al. in laboratory infections of Sulfolobus species Our proposal that diversity is maintained through clonal interference among independently acquired CRISPR variants depends upon there being a fitness advantage to resistance and consequent cost of viral infection. However, both lytic and non-lytic viruses infect Sulfolobus islandicus strains from hot spring M16, located in the Mutnovsky Volcano region of Kamchatka, Russia were isolated and DNA was extracted as in Whitaker et al. S. islandicus strains were previously isolated and sequenced S. islandicus core genes 2, 0.14 uL 0.2 mM dNTP, 0.5 uL of each 10 uM primer, and 0.14 uL 5 u/uL GoTaq DNA Polymerase (Promega). PCR conditions for all loci were as follows: 94°C for 5 min, 30 cycles of 94°C 30 sec, annealing temperature , AB1r (5′CCATACGGCTTCCCTAGATTTAGATT), A1.2r (5′CATCAACAGTTAGCGGAAGTGAGG), A1.2f (5′GGGAGGTAGGGTGTTGTCCTAAA), ABrU (5′TCCCACCCTCATGCTGGAATTCTT), and 16.43.AB1r (5′GGAATGGGAATTGCTGAAATAGCG) to amplify the AB1 locus. Primers AB2f (5 CTAGTTGCTTCCATTAAGTCGCTC), AB2r (5′TCCCGGGTTTAGTAGGGAGTGAAA), A2.2f (5′TGCCTTGTCTCATTAATGCGCGG), and A2.2r (5′GGGAGGTAGGGTGTTGTCCTAAA) were designed to amplify the AB2 locus. CDr (5′CGGTCACATGAGGAGTAAAGGA), CDf (5′CGTCCCATCACTTGCTTTGAGCAT), CDf3 (5′TTGAATGAGGCTTACCGGAAGGGA), and CDr3 (5′TTAGGCCCAGAAGGGAACCATCAA) were designed to amplify the CD locus.Primers to amplify the CRISPR loci were designed by genomic comparison of the CRISPR region of strains of All loci were amplified by PCR in 20 ul reactions with 4 ul 5x Phusion HF Buffer, 200 uM dNTP, 0.5 uM primer, and 0.02 U/ul Phusion DNA Polymerase (Finnzymes). PCR conditions for all loci were as follows: 98°C for 30 sec, 30 cycles of 98°C 30 sec, annealing temperature (depending on primer set) 10 sec, 72°C 2 min 30 sec, and a final incubation at 72°C for 5 min. The various primer sets amplified at the following annealing temperatures: AB1f/AB1r at 57°C, AB1f/16.43.AB1r at 57.5°C, AB1f/ABrU at 64°C, A1.2f/A1.2r at 59.2°C, AB2f/AB2r at 53–57°C, A2.2f/A2.2r at 58.5°C, CDf/CDr at 51–57°C, CDf3/CDr at 55°C, and CDf3/CDr3 at 54°C. PCR products were sequenced with both forward and reverse primers at the Core DNA Sequencing Laboratory . Sequences were deposited in GenBank, and accession numbers (HQ198372-HQ198558) are listed in GATAATCTACTATAGAATTGAAAG and C repeat is GATTAATCCTAAAAGGAATTGAAAG. Spacers were grouped according to the ends of the loci they came from, and BLASTn <0.001 was used to find 100% spacer matches. Strains were grouped within each locus as being ancestrally related if spacers in the array matched each other in the same spacer context . The results are interpreted manually and shown in CRISPR PCR products were sequenced as with MLSA amplicons and manually trimmed and checked for sequencing errors using Sequencher 4.9 . Individual spacers were removed from the sequences by manually extracting the sequence between the repeats sequences: A repeat is Sulfolobus genome, virus, and plasmid sequences. This database included all Sulfolobus genome, virus, and plasmid sequences found on the Sulfolobus Database (http://dac.molbio.ku.dk/dbs/Sulfolobus/cbin/mutagen.pl –01/01/10), plus the Sulfolobus islandicus genomes of L.S.2.15, L.D.8.5, M.14.25, M.16.4, M.16.27, Y.G.57.14, Y.N.15.51 http://www.jgi.doe.gov). Spacers were blasted against this database using BLASTn with parameters r = 1, q = −1, G = −4 and significant matches were E<0.001.Spacers were compared to a database of Rarefaction curves were constructed using Mothur Figure S1S. islandicus isolates from a single hot spring. The number of isolates (X-axis) is plotted against the number of OTUs (Y-axis) determined by the level of divergence for each line .Rarefaction curves of MLSA and CRISPR sequences. Rarefaction curves of (A) the concatenated nucleotide alignment of 12 MLSA loci and (B) the concatenated coded CRISPR spacer arrays from 39 (0.62 MB TIF)Click here for additional data file.Figure S2Primer design and implementation. Primer design schematic (A) and table of primer sets and temperatures used for each strain at each CRISPR locus (B). In (A), primers are shown by their position on the reference genome M.16.27, with the head of the arrow matching the 5′ end of the primer. Arrows above the schematic indicate the approximate location of primers on M.16.27 while arrows below the schematic indicate primers designed on other fully sequenced genomes or PCR products that do not match sequence in M.16.27. In (B), the primer pair used for each locus is listed, with annealing temperature used if multiple temperatures are used for that primer pair. ‘X’ and ‘NA’ as in (0.57 MB TIF)Click here for additional data file.Table S1Pairs of independently acquired spacers match the same virus or plasmid. Each spacer pair is numbered and spacer names are given as an isolate number followed by a locus position number as in (0.30 MB TIF)Click here for additional data file.Table S2S. islandicus isolates. Isolates are listed with their isolation method. There are seven different isolation methods that yielded colonies: DT (dextrin and tryptone) spread plate as described in Isolation methods for (0.33 MB TIF)Click here for additional data file.Table S3MLSA primers. MLSA loci and primers listed with annealing temperature (T) and length of amplicon. * indicates loci used in (0.52 MB TIF)Click here for additional data file.Table S4MLSA sequence allele accession numbers. Allele numbers for each of the 12 MLSA loci for each strain are shown in the table on the left. Locus marker codes correspond to (0.67 MB TIF)Click here for additional data file.Table S5CRISPR loci accession numbers. Accession numbers for CRISPRs are listed by strain and locus. The first number at each locus corresponds to the leader end sequence and the second number corresponds to the trailer end. ‘NA’ and ‘X’ as in (0.82 MB TIF)Click here for additional data file.


Apis mellifera L. are known to reside at low levels in colonies, with typically no apparent signs of infection observed in the honeybees. Reverse transcription-PCR (RT-PCR) of regions of the RNA-dependent RNA polymerase (RdRp) is often used to diagnose their presence in apiaries and also to classify the type of virus detected.Single-stranded RNA viruses, infectious to the European honeybee, Varroa destructor virus (VDV); and the dicistroviruses, acute bee paralysis virus (ABPV), Israeli paralysis virus (IAPV) and Kashmir bee virus (KBV), shared greater than 98% and 92% homology across the RdRp conserved domains, respectively.Analysis of RdRp conserved domains was undertaken on members of the newly defined order, the Picornavirales; focusing in particular on the amino acid residues and motifs known to be conserved. Consensus sequences were compiled using partial and complete honeybee virus sequences published to date. Certain members within the iflaviruses, deformed wing virus (DWV), Kakugo virus (KV) and RdRp was validated as a suitable taxonomic marker for the assignment of members of the order Picornavirales, with the potential for use independent of other genetic or phenotypic markers. Despite the current use of the RdRp as a genetic marker for the detection of specific honeybee viruses, we provide overwhelming evidence that care should be taken with the primer set design. We demonstrated that DWV, VDV and KV, or ABPV, IAPV and KBV, respectively are all recent descendents or variants of each other, meaning caution should be applied when assigning presence or absence to any of these viruses when using current RdRp primer sets. Moreover, it is more likely that some primer sets (regardless of what gene is used) are too specific and thus are underestimating the diversity of honeybee viruses. Varroa destructor, a parasite commonly detected in apiaries [V. destructor, virus-associated symptoms are observed, including deformed wings and paralysis [Apis mellifera L [Iflavirus, Cheravirus, and Sadwavirus [Iflavirus and BQCV, KBV and ABPV have been assigned to the family Dicistroviridae [Iflavirus, however have not yet been formally classified [V. destructor mites, VDV, has now been identified [Iflavirus genus [Honeybee populations are known to be infected by numerous viruses that reside in colonies yet show no apparent signs of infection . These vapiaries . Evidencaralysis . Over 18lifera L -5. They lifera L . The newdwavirus . Honeybeoviridae ,8. DWV aassified . In addientified . The VDVus genus .The use of RT-PCR to detect the RNA viruses in honeybees is a routinely implemented technique and is often coupled with phylogenetic analyses to investigate similarities or differences between virus isolates. Typically, sequences encoding capsid genes ,12 and sIn this study, we assessed the suitability of the RdRp to not only detect, but to differentiate between the different picorna-like viruses found within the order Picornavirales. This is considered especially important in light of the ever increasing entries in sequence databases of viruses belonging to the order Picornavirales and the tentative assignments of viruses to particular families/genera, often based on partial sequences ,20. We ath and 5th positions are conserved across all members; the family Dicistroviridae and the genus Iflavirus are the most variable in this domain, with only 3 and 2 conserved amino acids respectively, and these two members were the only two not to have the conserved motif KDE. Domain II was highly variable, where only one amino acid, Arginine (R), was conserved for 7 out of the 8 members, the exception being the family Dicistroviridae, which had a potential Lysine (K) substitution at this position for BQCV, Triatoma virus (TRV) and Himetobi P virus (HiPV), yet both have basic amino acid properties  residues and 2 conserved sites of amino acids with aromatic side chains in domain IV. The genus Iflavirus had a substitution of either Glycine (G) or Serine (S) at the 2nd conserved aspartate site , the honeybee viruses analysed in this study have been assigned or tentatively assigned (these will be discussed as assigned viruses for the purpose of this paper) to 2 separate groups within the order Picornavirales, the family Dicistroviridae and the genus sa Table . CBPV, wIn general, BQCV shared more conserved motifs with other members within the family Dicistroviridae, but it also had the most amino acid substitutions across all domains Table . The amiSBV shows the most amino acid differences in this group, with DWV, VDV and KV showing a high level of similarity. These 3 viruses are identical at the amino acid level in domains I, II, III, VI and VII Table . Only 2 Iflavirus was less clear. Domains III to VIII revealed clearer separation between these two members , which shares the property of being an aromatic amino acid. Hence, the comparison of the consensus amino acid sequence for each group supports the current classification of these viruses together within this order and suggests that their RdRp share similar properties or activities . One potential subdivision could be within the family Dicistroviridae, with KBV, ABPV, CrPV, TSV and DCV forming a genus due to their high similarity within this family. Future analyses could address whether these viruses differ in any other way to the other members of the family Dicistroviridae in their RdRp enzymology or with respect to their epidemiology, transmission or persistence. Much more information is being brought to light regarding the importance of the motifs in the structure and functioning of RdRp [The analysis of the RdRp of the order Picornavirales shows that there is enough sequence variability for the subdivision of this order into the 8 families and genera, as previously assigned based on features described by Christian et al.  Table 22. BrieflNosema apis [With the RdRp being confirmed as a good marker for resolving hierarchical structures within the order Picornavirales, sequences of honeybee viruses deposited in GenBank were investigated further to assess the application of RdRp for differentiating between these viruses. Within the family Dicistroviridae, BQCV shows consistent amino acid differences with KBV, IAPV and ABPV across all 8 domains, yet is more closely related to these viruses than any other honeybee virus Table . KBV, IAema apis . ABPV, IThe RdRp lacks a proof reading function and hence is more prone to errors, leading to frequent nucleotide changes and subsequently, amino acid substitutions ,24. The At this point it is also important to re-evaluate the data obtained from the particular primer sets employed in RT-PCR for the routine detection of the viruses in colonies. Analysis of primers employed by Tentcheva et al.  and BakeIflavirus with VDV, KV and DWV sharing a greater than 98 % homology across the 8 domains and only 2 amino acid substitutions  [A similar scenario was detected in the genus s Tables  &5. Agaii et al. , none ofi et al. . No cleai et al. , DWV was tested) , also le tested) , being l tested) . The pat tested) , VDV has tested) .In addition, the detection of these iflaviruses through RT-PCR can be unreliable, depending on the purpose of the study, as the likelihood of detecting all the known variants is high. DWV-specific primers used by Tentcheva et al.  and BakeTo date, only a region of the RdRp of CBPV has been sequenced and based on traditional classification requirements, it is difficult to assign a family/genus for this virus. Based on our analysis CBPV is clearly a member of the order Picornavirales, however, it appears that it is very divergent from the other characterised honeybee viruses and thus should be assigned as the type strain for a new genus and/or family.We have validated the use of the RdRp as a taxonomic marker for the classification of the order Picornavirales and, to an extent, for the viruses infecting the honeybee. The evidence supports the assignment of DWV, VDV and KV as variants of the same virus, with it also being proposed that ABPV, IAPV and KBV, are also variants of the same virus. We suggest that care should be taken when using molecular tools to ascertain whether certain viruses are present in any given sample and thus will affect the prediction of cause and effect. The data presented here provides further foundations for understanding the ecology of these viruses and the interactions they have with their hosts, therefore being useful for beekeeping practises. The results potentially also provide further information on the evolution of these honeybee viruses in the context of the order Picornavirales.Multiple amino acid and nucleotide sequences of the RNA-dependent RNA polymerase (RdRp) protein for the single-stranded RNA viruses were selected from NCBI Tables  &3 and wThe author(s) declare that they have no competing interests.ACB performed all the experimental work, carried out the genetic analysis and wrote the manuscript. DCS co-ordinated the development of the project, performed the multiple sequence alignments and oversaw the research.


The HSV-1 UL20 protein (UL20p) and glycoprotein K (gK) are both important determinants of cytoplasmic virion morphogenesis and virus-induced cell fusion. In this manuscript, we examined the effect of UL20 mutations on the coordinate transport and Trans Golgi Network (TGN) localization of UL20p and gK, virus-induced cell fusion and infectious virus production. Deletion of 18 amino acids from the UL20p carboxyl terminus (UL20 mutant 204t) inhibited intracellular transport and cell-surface expression of both gK and UL20, resulting in accumulation of UL20p and gK in the endoplasmic reticulum (ER) in agreement with the inability of 204t to complement UL20-null virus replication and virus-induced cell fusion. In contrast, less severe carboxyl terminal deletions of either 11 or six amino acids  allowed efficient UL20p and gK intracellular transport, cell-surface expression and TGN colocalization. However, while both 211t and 216t failed to complement for infectious virus production, 216t complemented for virus-induced cell fusion, but 211t did not. These results indicated that the carboxyl terminal six amino acids of UL20p were crucial for infectious virus production, but not involved in intracellular localization of UL20p/gK and concomitant virus-induced cell fusion. In the amino terminus of UL20, UL20p mutants were produced changing one or both of the Y38 and Y49 residues found within putative phosphorylation sites. UL20p tyrosine-modified mutants with both tyrosine residues changed enabled efficient intracellular transport and TGN localization of UL20p and gK, but failed to complement for either infectious virus production, or virus-induced cell fusion. These results show that UL20p functions in cytoplasmic envelopment are separable from UL20 functions in UL20p intracellular transport, cell surface expression and virus-induced cell fusion. Herpes simplex viruses (HSV) specify at least eleven virus-specified glycoproteins, as well as several non-glycosylated membrane associated proteins, most of which play important roles in multiple membrane fusion events during virus entry and intracellular virion morphogenesis and egress -8. SpreaThe most prevalent model for morphogenesis and egress of infectious herpes virions includes sequential de-envelopment and re-envelopment steps in transit to extracellular spaces: a) primary envelopment by budding of capsids assembled in the nuclei through the inner nuclear leaflet leading to the production of enveloped virions within perinuclear spaces; b) de-envelopment by fusion of viral envelopes with the outer nuclear leaflet leading to the accumulation of unenveloped capsids in the cytoplasm; c) assembly of sets of tegument proteins on the cytoplasmic capsids, as well as potentially on vesicle sites to be used for cytoplasmic envelopment; d) re-envelopment of cytoplasmic tegumented capsids into TGN-derived vesicles. This final event in cytoplasmic virion envelopment is thought to be largely mediated by interactions between tegument proteins and cytoplasmic portions of viral glycoproteins embedded within the TGN-derived membranes. Cytoplasmically enveloped viruses are thought to be transported to extracellular spaces within Golgi or TGN-derived vesicles .The UL20 gene encodes a 222 amino acid non-glycosylated transmembrane protein that is conserved by all alphaherpesviruses. The UL20p is a structural component of extracellular enveloped virions and it is expressed in infected cells assuming a predominantly perinuclear and cytoplasmic distribution . An initIn this manuscript, we show that the amino and carboxyl termini of UL20p contain distinct domains that function in infectious virion production and intracellular gK/UL20 transport.Previously, we reported on the construction and characterization of a panel of 31 mutations within the UL20 gene . These mIt was previously shown that deletion of the HSV-1 UL20 and the PRV UL20 genes resulted in up to two logs reduction in infectious virus production relative to their parental wild type strains. The targeted set of single or double UL20 mutants and UL20p truncations were tested for their ability to complement the HSV-1(KOS) UL20-null virus. Complementation experiments involved transfection of Vero cells with plasmids encoding wild-type or mutant UL20 genes, followed by infection with the UL20-null virus as reported previously ,30 and dWe previously showed that syncytial mutations in either gB or gK failed to cause virus-induced cell fusion in the absence of the UL20 gene . FurtherTransport and localization of UL20p and gK was further assessed by transient coexpression of gK and UL20p and simultaneous detection of the TGN compartment. We showed previously that in the absence of UL20p, gK was localized exclusively to reticular-like compartments and was absent from the Golgi and TGN. A similar pattern was detected for UL20p in the absence of gK . In contWe reported previously that UL20 and gK are co-expressed on infected cell surfaces and co-internalize to TGN cytoplasmic membranes. Similar findings were produced in transient co-transfection experiments with both UL20 and gK genes . SimilarWe showed previously that UL20 and gK are functionally interdependent for their intracellular transport, cell-surface expression and TGN localization  and thatPreviously, we characterized a series of carboxyl terminal truncations including the 204t and 211t encoding carboxyl terminal truncations of 18 and 11 aa respectively. These two UL20p truncations failed to complement for infectious virus production and virus-induced cell fusion, while the 216t coding for a 6 aa truncation enabled virus-induced cell fusion, but failed to complement for infectious virus production . We showDomain I is the largest domain 63 aa) and it includes stretches of acidic amino acid  clusters, which could form electrostatic interactions with other proteins . Further aa and iWe reported previously that the Y49A, CL49 and 216t mutant viruses produced syncytial plaques, although their ultrastructural phenotypes seemed to be similar to that of the UL20-null virus . We showThese results show that UL20p domains required for UL20p and gK intracellular transport and TGN localization can be functionally segregated from domains involved in infectious virus production and virus-induced cell fusion. The results suggest that virus-induced cell fusion mechanisms are not required for cytoplasmic virion envelopment.African green monkey kidney (Vero) cells were obtained from ATCC . The Vero-based UL20 complementing cell line, G5, was a gift of Dr. P. Desai,  . Cells wpCR2.1-UL20, which was used as the parental vector for UL20 mutagenesis, was generated by cloning a 773 bp DNA fragment containing the UL20 gene, obtained by PCR amplification of HSV-1(KOS) viral DNA, into pCR2.1/TOPO (Invitrogen) as described in detail previously . The genConfluent Vero monolayers in six well plates were transfected with 2 μg of wild-type or mutant UL20 plasmid with Lipofectamine 2000 as described by the manufacturer (Invitrogen). Six hours post-transfection, the monolayers were infected with a UL20-null virus at an MOI of 1. Infections were placed on a rocker for 1 hour at 4°C, and then transferred to 37°C for 2 hours. Residual virus was inactivated using an acid wash  for 2 min, and monolayers were subsequently washed 3 times with DMEM to restore the pH to a normal level. Infections were incubated at 37°C for 24 hours. After repeated freeze/thaw cycles, virus stocks were titered in triplicate on Fd20-1 cells, which effectively complement the UL20-null defect . The comThe complementation assay was performed essentially as we described previously for addressing the role of the HSV-1 UL11 protein in virion morphogenesis . BrieflyComplementation of the UL20-null virus by transient expression of the wild-type UL20 gene caused the production of up to 40% of total viral plaques appearing to have similar morphology and size to the HSV-1(F) parental virus.z axis, averaged six times, were collected at the indicated zoom in series in the different channels at 1,024- by 1,024-pixel resolution as described previously [Cell monolayers were grown on coverslips in six-well plates. Cell monolayers were transfected with the indicated UL20 and/or gK plasmid combinations by using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions and prepared for confocal microscopy approximately 30 h posttransfection. Cells were washed with TBS and fixed with electron microscopy-grade 3% paraformaldehyde  for 15 min, washed twice with phosphate-buffered saline-50 mMglycine, and permeabilized with 1.0% Triton X-100. Monolayers were subsequently blocked for 1 h with 7% normal goat serum and 7% bovine serum albumin in TBS (TBS blocking buffer) before incubation for 2 h with either anti-V5 , for recognition of gK, or anti-FLAG , for recognition of UL20p, diluted 1:500 in TBS blocking buffer. Alternatively, simultaneous detection of gK and UL20p in cotransfected cells was accomplished by concurrent incubation with murine anti-V5 and rabbit anti-FLAG  diluted 1:500 in TBS blocking buffer. Cells were then washed extensively and incubated for 30 min with Alexa Fluor 594 and/or Alexa Fluor 647-conjugated anti-immunoglobulin G diluted 1:500 in TBS blocking buffer. After incubation, excess antibody was removed by washing five times with TBS. TGN were identified with a donkey anti-TGN46 primary antibody and an Alexa Fluor 488-conjugated sheep anti-donkey secondary antibody . Specifieviously ,29,42. Ieviously .Internalization assays were modified from similar assays performed previously ,44,45. BThe author(s) declare that they have no competing interests.J. Melancon performed most of the experiments. K. G. Kousoulas wrote the manuscript.


Daphnia magna and its bacterial parasite Pasteuria ramosa.A central hypothesis in the evolutionary ecology of parasitism is that trade-offs exist between resistance to parasites and other fitness components such as fecundity, growth, survival, and predator avoidance, or resistance to other parasites. These trade-offs are called costs of resistance. These costs fall into two broad categories: constitutive costs of resistance, which arise from a negative genetic covariance between immunity and other fitness-related traits, and inducible costs of resistance, which are the physiological costs incurred by hosts when mounting an immune response. We sought to study inducible costs in depth using the crustacean D. magna-P. ramosa system.We designed specific experiments to study the costs induced by exposure to this parasite, and we re-analysed previously published data in an effort to determine the generality of such costs. However, despite the variety of genetic backgrounds of both hosts and parasites, and the different exposure protocols and environmental conditions used in these experiment, this work showed that costs of exposure can only rarely be detected in the We discuss possible reasons for this lack of detectable costs, including scenarios where costs of resistance to parasites might not play a major role in the co-evolution of hosts and parasites. Parasites are thought to be a major cause of evolutionary change due to the deleterious fitness effects they impose on their hosts -3. CoevoThese costs fall into two broad categories. Constitutive costs of resistance arise from a negative genetic covariance between immunity and other fitness-related traits (a genetic-based trade-off) ,7,9,10. Microphallus sp, an experimental study found that Potamopyrgus snails are still resistant, which would not be expected if resistance bore fitness costs ,29,308,2D. magna-P. ramosa interaction. The first experiment investigated inducible costs of resistance with different P. ramosa strains and doses on 4 Daphnia clones , andulations . A thirdulations ]), whichulations . While iulations ], the evulations ,41,42,45ulations ,12,36,37ulations  and backulations ,24, and D. magna-P. ramosa system, coupled with a lack of apparent costs, supports a "matching-allele" coevolution scenario in this system [Costs can potentially contribute to the maintenance of resistance polymorphism in host-parasite interactions ,30,47,48s system .D. magna-P. ramosa does not appear to suggest a crucial role of immunity costs in their coevolution [Moving beyond simple genetic models, complex immune systems may incorporate substantial redundancy ,43 to favolution ,31,33,44volution ,41,53,54volution ), suggesPL designed, carried out and analyzed experiments 1 and 2, and drafted the manuscript. PV designed, carried out and analyzed experiment 3, and helped to draft the manuscript. TL participated in the design of the study, analyzed the data for experiments 4 and 5, and helped to draft the manuscript. All authors read and approved the final manuscript.Experimental details and infection costs. A1: Detailed experiments protocols. A2: Analyses of infection costs. Table A3: Infection levels for the different host-parasite combinations. Fig. A4: Experiment 2 spore loads. Fig. A5: Experiment 3 spore loads.Click here for file


Arboviruses are transmitted in nature exclusively or to a major extend by arthropods. They belong to the most important viruses invading new areas in the world and their occurrence is strongly influenced by climatic changes due to the life cycle of the transmitting vectors. Several arboviruses have emerged in new regions of the world during the last years, like West Nile virus (WNV) in the Americas, Usutu virus (USUV) in Central Europe, or Rift Valley fever virus (RVFV) in the Arabian Peninsula. In most instances the ways of introduction of arboviruses into new regions are not known. Infections acquired during stays in the tropics and subtropics are diagnosed with increasing frequency in travellers returning from tropical countries, but interestingly no attention is paid on accompanying pet animals or the hematophagous ectoparasites that may still be attached to them. Here we outline the known ecology of the mosquito-borne equine encephalitis viruses , WNV, USUV, RVFV, and Japanese Encephalitis virus, as well as Tick-Borne Encephalitis virus and its North American counterpart Powassan virus, and will discuss the most likely mode that these viruses could expand their respective geographical range. All these viruses have a different epidemiology as different vector species, reservoir hosts and virus types have adapted to promiscuous and robust or rather very fine-balanced transmission cycles. Consequently, these viruses will behave differently with regard to the requirements needed to establish new endemic foci outside their original geographical ranges. Hence, emphasis is given on animal trade and suitable ecologic conditions, including competent vectors and vertebrate hosts. During the last decades the appearance of new infectious diseases and an increasing invasion of diseases into new areas created a new category of pathogens: emerging and re-emerging pathogens. Most of the emerging viruses are zoonotic which means they can infect both animals and humans . As outlAedes albopictus, Aedes japonicus), is well-known and, in several countries, is under close observation, the risk and the importance of animal trade for the importation of arboviruses has not been studied extensively . More than 150 of these are known to cause human and/or animal diseases. For many of those viruses, only limited information is available regarding their vector and host spectrum. Hence, we have chosen some prominent examples of important arboviruses causing human and animal diseases, which belong to the genera alphaviruses (family Togaviridae), flaviviruses (family Flaviviridae), and phleboviruses (family Bunyaviridae) to discuss the animal aspect in virus dispersal.The last International Catalogue of Arboviruses listed more than 500 arboviruses and related viruses [; http://Culex tarsalis [[Aedes and six species of the genus Culex [Aedes and vertebrate hosts including rice rats (Oryzomys spp.), rabbits and introduced European hares (Lepus europaeus) [Western Equine Encephalomyelitis (WEE) is caused by the Western equine encephalomyelitis virus (WEEV) which belongs to the genus Alphavirus in the family Togaviridae . The virarsalis [, Figure arsalis [. Howeverus Culex . In Soutropaeus) -26. Humaropaeus) . Therefohttp://diseasemaps.usgs.gov/; as of December 8th 2009). Less land irritation and consequently less breading opportunities for vector mosquito species have been claimed for the fading of the virus. To some extent the use of vaccines, which are available for equines but not for humans, might have attributed to this situation. Nevertheless, WEEV has been used to develop chimeric vaccines in combination with other alphaviruses such as Sindbis or eastern equine encephalitis viruses [[In humans, WEEV causes severe encephalitis with higher manifestation rates in children and in elderly persons. Fatality rates may be up to 5% . WEEV isviruses [; see belAedes eggs  may be possible means of importation [Culex pipiens and from Aedes vexans. Both mosquito species form a major part of the Central European mosquito fauna. For a natural transmission cycle, WEEV is dependent on passerine birds and possibly also on small wild mammals. Both groups of animals are abundant in Europe and although no data are available on the potential of European species to serve as natural maintenance or amplifying hosts, there are no obvious reasons to argue against a potential for transmission in European species. Hence, the risk of the introduction of WEEV into Europe seems to be low, although the required components for a natural transmission cycle of WEEV seem to be available  is caused by eastern equine encephalomyelitis virus (EEEV) which is also a member of the genus Alphavirus in the family Togaviridae. EEEV causes severe disease in humans, in horses and in some game animals . In humaCuliseta melanura and passerine and wading birds of different species . The results of studies of transovarial transmission of EEEV in mosquitoes are conflicting. Probably EEEV is not transmitted via infection of eggs to the next mosquito generation while for Coquilletidia perturbans transovarial transmission could be proved [EEEV occurs in North and South America. While the natural transmission cycle(s) in South America are not well understood, transmission in Eastern North America is mainly dependent on ornithophilic mosquitoes of the species s Figure . The cycAedes vexans and Culex pipens are among the most abundant mosquito species in Europe. However, at least in North America, Culiseta melanura seems to be the main vector for EEEV. The genus Culiseta is a rather species poor genus (five species worldwide), which has been claimed to be the reason for higher levels of genetic identity in viruses transmitted by Culiseta mosquitoes than in viruses that mainly use Culex or Aedes vector species [The risk of an importation of EEEV into Europe or other areas outside of the American continent seems to be low. Basically, an importation seems possible via infected mosquitoes, infected birds  and also via infected reptiles and amphibians. As already mentioned for WEEV, no frequent migration of birds between the Americas and Europe exists. Therefore an introduction seems only possible as a result of human activities . Although no studies on the vector competence of European mosquito species for EEEV are available,  species . In contCulex (Melanoconion) and rodents  [[Venezuelan equine encephalomyelitis is caused by a complex of viruses  which belongs to the genus Alphavirus in the family Togaviridae. The complex includes seven different virus species and a number of subtypes and varieties . VEEV ocntomys) [, Figure ntomys) [. Howeverntomys) [. Other wntomys) [,39.Aedes and Psorophora which have a peridomestic/peri-agricultural behaviour and may transmit VEEV to equines, donkeys and mules. Equids develop high virus titres and therefore may serve as amplifying hosts for VEEV. An equine-mosquito-cycle may induce an extensive virus circulation with a spill-over to humans and cause epidemic VEE  and replicate to higher titres in this mosquito species than in mosquitoes involved in endemic transmission (Melanoconion) [Culex and Aedes in Europe are competent for VEEV has not been studied so far. However, American strains of Aedes albopictus were found to be capable of transmitting VEEV [The epidemic occurrence of VEEV during the last two decades shows that it is highly variable in nature and that single amino acid changes in the viral genome may cause major changes in vector competence of mosquitoes or in the pathogenicity in equids -43. Studoconion) . The inting VEEV ,46. TherWest Nile virus (WNV) is a member of the Japanese encephalitis group of the genus Flavivirus in the family Flaviviridae. The evolutionary origin of WNV seems to be in Central Africa, from where it spread over various parts of the world and locally new genotypes emerged . ActuallCulex [Aedes, Anopheles, Aedomyia, Mansonia, Coquilletidia) and of hard and soft ticks  for the endemic and epidemic transmission cycles remains to be determined [Rana ridibunda) was found to be viremic and was able to transmit the virus to blood-sucking Culex pipiens [Like other members of the Japanese encephalitis serogroup, WNV in nature is maintained in a bird-mosquito cycle Figure . WNV wasCulex . The imptermined . Varioustermined ,55. WNV  pipiens . TherefoWNV is an often cited example of a dispersing arbovirus since it invaded into North America in 1999 . From thCulex pipiens), climate warming, and perhaps also the decline of infections with the closely related St. Louis encephalitis virus, an indigenous virus of the Japanese encephalitis serogroup in the Americas [The exact way of introduction of WNV into North America is still unclear. Several additional factors are discussed which improved the establishment and transmission of WNV in this new environment Table . Among tAmericas . HoweverAmericas ,59. In aAmericas . The intAmericas ,61,62.Flavivirus of the family Flaviviridae. JEV is transmitted in a natural transmission cycle involving mosquitoes of the genus Culex and water birds (mainly egrets and herons) [Japanese encephalitis virus (JEV) is a member of the similarly named serogroup in the genus  herons) . Actuall herons) . Current herons) . Up to 3 herons) . JEV inf herons) .Culex. The primary vector is Culex tritaeniorhynchus, which is associated with rice paddies and irrigated crop fields in whole Southeast Asia. Culex tritaeniorhynchus feeds on water birds and on larger mammals, also on pigs and therefore transmits JEV to this important amplifying host, and also to equids and to humans. Other Culex species, like Culex pipens, Culex vishnui and Culex bitaeniorhynchus may play a local role for the transmission of JEV  and the Asian cattle egret (Bubulcus ibis coromandus) [Culex tritaeniorhynchus. Therefore, an enzootic or an epizootic cycle may be initiated from mosquitoes directly after diapause. The invasion of JEV in new areas in Southeast Asia during the last decades has been mainly associated with the increase of human populations and, consequently, in increasing areas of rice paddies and pig farming [The natural transmission cycle mainly involves mosquitoes of the genus V Figure . The natomandus) . There i farming . JEV rec farming ,70.Japanese encephalitis virus shows a clear tendency of expansion. One mechanism of spread involves the air transport of infected mosquitoes. This method of spread was shown by the introduction of JEV into Pacific islands like Guam or Saipan ,72. A reRift Valley fever (RVF) is a disease which was first described as an entity during an epizootic outbreak in 1930 - 1931 in Kenya . There, Phlebovirus of the family Bunyaviridae. It is transmitted in an enzootic cycle among wildlife and mosquitoes [Aedes, Ochlerotatus, Stegomyia, Anopheles, Culex, Neomelaniconion, Eretmapodites and others) as vectors [[Aedes. Aedes macintoshi seems to play a major role in Eastern Africa [Aedes macintoshi lays infected eggs into the ground and these eggs need one or more severe flooding to hatch. Therefore an inter-epidemic period  and an epidemic period  can be distinguished. The occurrence of epidemic periods is clearly associated with heavy rains which are closely linked to warming of the Indian Ocean during the El Nino Southern Oscillations  can be infected and develop systemic infection. However, only Aedes vexans and Culex erraticus developed virus titres which were high enough to transmit the virus to laboratory animals [These examples show that RVFV, without any doubt, is one of the most aggressive migrating arbovirus. The routes of dispersal detected so far seem to be in parallel with the great migration routes of camels. Therefore, there is some good evidence that viremic, but non-symptomatic infected camels transported the virus to Egypt and possibly also to the Arabian Peninsula . As also animals ,90. TherFlavivirus in the family Flaviviridae [Culex in South Africa in 1959. Since that time the virus was isolated several times from mosquitoes, rodents and birds throughout Sub-Saharan Africa [Turdus merula) and some other bird species. USUV could be detected the following years and its area of distribution extended into south-east (Hungary), south , west (Switzerland), and north  of the original location of emergence where it also caused mortality in birds [Usutu virus (USUV) belongs to the Japanese encephalitis serogroup within the mosquito-borne cluster of the genus iviridae . It was n Africa . There hn Africa . In 2001in birds ,94,95. Iin birds ,97. USUVCulex, Coquillettidia and Mansonia are thought to be the main vectors. In Austria, Culex spp. may play a major role, although USUV so far has not been isolated from mosquitoes but has been detected in overwintering Culex pipiens pools by real time RT-PCR (our own unpublished results). There seems to be a mode of adaptation of the virus to the new bird species and/or to the new mosquito species in Europe. After high mortality rates in blackbirds during the first two years of emergence of USUV, in the following years increasing rates of seropositive birds were detected in Austria which gave evidence for a continuing circulation of USUV with a somewhat lower pathogenicity, inducing an herd immunity in the bird populations [USUV is thought to be maintained in nature in a mosquito-bird transmission cycle. In Africa ornithophilic mosquitoes of the genera ulations .USUV appears as an impressive example for the introduction and permanent establishment of a so-called "tropical" arbovirus in moderate climates. In a recent study, it was argued that USUV is mainly maintained in a natural cycle in areas of Austria with a minimum of at least ten hot days (> 30°C) . In thisFlavivirus in the family Flaviviridae [Ixodes ricinus and Ixodes persulcatus). The natural vertebrate hosts of TBE virus are small rodents of the genera Myodes and Apodemus, although other Rodentia or Eulipotyphla (formerly: Insectivora) may contribute to the natural transmission cycle [[Ixodes ricinus in Europe, larvae become active above 10°C while nymphs start searching for suitable hosts at 7°C. This means, among many other factors, that a fast warming in spring will be beneficial for co-feeding and in turn will result in higher numbers of TBEV-positive ticks [So far, only the invasive potential of mosquito-borne arboviruses has been discussed. The example of tick-borne encephalitis virus (TBEV) shows that also tick-transmitted arboviruses may be able to invade new areas. TBEV is a flavivirus of the tick-borne group of the genus iviridae . It is dn cycle [; see Fign cycle [. In contn cycle [. The skin cycle [. Adults ve ticks ,103.Ixodes ricinus) on every 30th bird which migrated in autumn from Northern Europe towards the South. About one out of 2200 migrating birds carried a TBEV-infected tick [TBEV is the most important tick-transmitted arbovirus of human pathogenicity in Europe and Asia ,105. An ted tick . These dted tick . At leasted tick . The antth [Ixodes cookie and woodchucks and mustelids; Ixodes marxi and squirrels; Ixodes scapularis and white-footed mice [Dermacentor ticks, namely D. andersoni and D. variablis but the corresponding enzootic cycle has not ben explored in further detail. Vertical transmission of POWV was observed in Ixodes scapularis [Powassan virus (POWV) is the sole member of the tick-borne encephalitis serological complex of flaviviruses in North America. It received its name after the town Powassan in Ontario, Canada, were it was isolated from the brain of a child deceased after encephalitis in the late 1950th  and a coth . The latth -118. Morted mice . POWV haapularis . The curapularis . One wayapularis . So thisIt is interesting that for other tick-borne arboviruses, similar results on the importance of human activities for the spread into new, non-endemic areas are evolving. Kyasanur Forest virus, a virus related to TBEV is limited to some regions (Karnataka) in India . HoweverArboviruses are maintained in nature in complex transmission cycles between arthropods and vertebrates. They have developed strategies of adaptation and evolution to spread into new areas and eventually become established. Several recent examples show, that tropical arboviruses are capable to spread to countries with moderate climates. While bird-associated mosquito-borne viruses seem to be transported mainly by migrating birds, human activities  play a major role for arboviruses where humans play a role as natural vertebrate hosts. Also for tick-borne arboviruses, mainly human activities seem to contribute to the spread over long distances and the establishment in new ecosystems changed by human activities. In most cases of newly emerging zoonotic arboviruses, the ways of introduction remain obscure. Future research should aim at exploring the circumstances of these events. A better understanding of how arboviruses travel and why they become established in other geographic areas will be of great benefit for human and veterinary public health, because it may help to prevent devastating outbreaks of arboviral diseases in humans and animals.The authors declare that they have no competing interests.Both authors contributed equally to this work.