Many plant and animal viruses are spread by insect vectors. Cauliflower mosaic virus (CaMV) is aphid-transmitted, with the virus being taken up from specialized transmission bodies (TB) formed within infected plant cells. However, the precise events during TB-mediated virus acquisition by aphids are unknown. Here, we show that TBs react instantly to the presence of the vector by ultra-rapid and reversible redistribution of their key components onto microtubules throughout the cell. Enhancing or inhibiting this TB reaction pharmacologically or by using a mutant virus enhanced or inhibited transmission, respectively, confirming its requirement for efficient virus-acquisition. Our results suggest that CaMV can perceive aphid vectors, either directly or indirectly by sharing the host perception. This novel concept in virology, where viruses respond directly or via the host to the outside world, opens new research horizons, that is, investigating the impact of ‘perceptive behaviors’ on other steps of the infection cycle.DOI:http://dx.doi.org/10.7554/eLife.00183.001 Viruses are infectious agents that can replicate only inside a living host cell. When a virus infects an animal or plant, it introduces its own genetic material and tricks the host cells into producing viral proteins that can be used to assemble new viruses. An essential step in the life cycle of any virus is transmission to a new host: understanding this process can be crucial in the fight against viral epidemics.Many viruses use living organisms, or vectors, to move between hosts. In the case of plant viruses such as cauliflower mosaic virus, the vectors are often aphids. When an aphid sucks sap out of a leaf, virus particles already present in the leaf become attached to its mouth, and these viruses can be transferred to the next plant that the insect feeds on. However, in order for cauliflower mosaic virus particles to become attached to the aphid, structures called transmission bodies must form beforehand in the infected plant cells. These structures are known to contain helper proteins that bind the viruses to the mouth of the aphid, but the precise role of the transmission body has remained obscure.Now Martinière et al. show that the transmission body is in fact a dynamic structure that reacts to the presence of aphids and, in so doing, boosts the efficiency of viral transmission. In particular, they show that the action of an aphid feeding on an infected leaf triggers a rapid and massive influx of a protein called tubulin into the transmission body. The transmission body then bursts open, dispersing helper protein-virus particle complexes throughout the cell, where they become more accessible to aphids. This series of events increases viral transmission rates twofold to threefold.The results show that a virus can detect insect vectors, likely by using the sensory system of its host, and trigger a response that boosts viral uptake and thus transmission. This is a novel concept in virology. It will be important to discover whether similar mechanisms are used by other viruses, including those that infect animals and humans.DOI:http://dx.doi.org/10.7554/eLife.00183.002 Potato virus Y transmission  and ticks are especially significant for vertebrate viruses, and likewise aphids, white flies and other sap-feeding bugs are consequential for plant viruses. These vectors are ideal, as their variety of mouth parts can puncture cells, blood vessels, and plant sap vessels with great precision, thus enabling efficient uptake and injection of pathogens without killing the host. Vector transmission can be classified into two main transmission modes. In circulative transmission, the virus is taken up by the vector together with the nutrients , where it actively crosses from the intestine into the vector interior. Then it cycles through the hemocoel  to the salivary glands, where the virus can be secreted together with the saliva into a new host. The second transmission mode is alternatively referred to as mechanical transmission  or non-circulative transmission (for plant viruses). In this transmission mode, the arthropod vector only briefly comes into contact with the virus, in which it transiently attaches to the vector mouth parts and is subsequently released; an internalization step does not occur. The viral proteins involved in this seemingly simple process have been well-described in the literature, often down to the molecular level (for review see Cauliflower mosaic virus (CaMV), the virus studied here, is a non-circulative virus transmitted by aphids. CaMV binds to a receptor protein located at the tip of the aphid's needle-like mouth parts, the stylets  and mixed-network phenotypes were induced by unidentified stresses during leaf handling, whereas the tubulin-less phenotype (Tub−|TBs) corresponded to unstressed ‘standby’ TBs found under normal conditions. As the only known role for TBs is in transmission, this further raised the question of whether and how the presence of tubulin within TBs impacts transmission by aphids. To investigate this phenomenon, we first aimed to identify artificial stresses that could trigger specific transformation of standby TBs into Tub+|TBs and mixed-networks. Heat-shock, wounding and CO2 exposure all induced TB transformation  were significantly increased only in wounded or CO2-exposed leaves, and were rarely observed in heat-shocked or untreated leaves. This indicates that the different stresses had different and specific effects on TBs.At the beginning of this study was the observation that several different TB phenotypes could be discerned in infected tissues, as viewed by double-labeling experiments using antibodies against the TB marker P2 and the microtubule protein α-tubulin. Thus, typical TBs were detected, and these were rather large (2–5 μm in diameter) and mostly ovoid single cytoplasmic inclusions, having a cortex heavily labeled by P2 antibody and a less intensely labeled interior. Most importantly, little if any tubulin was detected in this regular form of the TB . Interesormation . Quantiformation  establisHaving access to an experimental system to specifically induce TB transformation, we next examined what role(s) the different TB morphs might play in CaMV aphid-transmission. Here, we pursued three complementary objectives: i) to characterize the dynamics of TB transformation; ii) to investigate whether aphid feeding activity can trigger this transformation; and iii) to test whether TB transformation is required for successful CaMV transmission.+|TBs and mixed-networks were detected readily at the wounding sites; by contrast, the cells of control tissue, or tissue wounded after fixation, predominantly harbored standby TBs . Photo-bleached GFP-tubulin in TBs was rapidly replaced by fresh cytoplasmic GFP-tubulin  and the TB phenotype analyzed by immunofluorescence. Tubndby TBs . We next-tubulin , suggest2) induced TB transformation. As in leaves, heat treatment of protoplasts only induced Tub+|TBs (data not shown), whereas CO2 and azide stimulated Tub+|TBs as well as mixed-networks (−|TBs. After 5 min (CO2 treatment) or 40 min (azide treatment) most cells displayed mixed-networks , this tubulin entry can be followed by a total disruption of the TB. This includes an even dispersion of the components of the CaMV transmissible complex  onto microtubules throughout the cortical cytoplasm. This redistribution might render the CaMV transmissible complexes more readily accessible to aphid vectors.Taken together, these results demonstrate that mixed-network formation is always preceded by a massive entry of tubulin into the standby TB, which in itself does not induce a major ultrastructure change. However, under specific stress conditions such as azide or CO+|TBs, and then again into mixed-networks, on a time scale that is fully compatible with aphid intracellular probing (+|TBs (2 treatment (The results in  probing . However probing . In addi probing . In contg (+|TBs . Mixed-ng (+|TBs . Quantifg (+|TBs . In parag (+|TBs . This rereatment . Taken t+|TBs and/or mixed-networks) should display a significant impact on CaMV transmission. We therefore investigated whether TB transformation is required for aphid-transmission of CaMV. Assessing the effect of TB transformation on transmission was however not straightforward, since aphids themselves provoke TB transformation. As a possible means to circumvent this problem, we observed while quantifying TB morphs  and successful aphid transmission, and presents direct biological evidence in support of our transmission hypothesis.To further confirm the role of TB changes in aphid-transmission of CaMV, we pursued an independent approach and examined a CaMV mutant impaired in transmission. This mutant, CaMV P2-TC, harbors a 7-amino acid insertion, including a tetracysteine tag  at posit2 and azide were correlated strongly with increased transmission rates. These results additionally indicate that the TB reaction is required for transmission.Combined, our results establish that the TB is a dynamic structure, which in the presence of an aphid vector can react immediately to promote CaMV transmission . Transmi2—could trigger TB transformation. This reveals a certain level of specificity in TB activation, although it is more broad than the classical pattern recognition receptor-mediated defense responses of plants against pathogens (including insects) that are often species or even isolate-specific (One enduring question that deserves further attention is precisely where the virus particles come from that are recruited onto the microtubules. Whereas the origin of microtubule-associated P2 is clearly the TB (since the TB is the only source of P2), the origin of the virus particles aligning on the microtubules is less clear. They could derive either from the TB, which contains some virus particles, or from the many virus factories dispersed throughout the cytoplasm . We werespecific . This brThe significance of this remarkable phenomenon described here extends beyond CaMV transmission to broader fields of research. First, this opens up a fascinating new direction within virology, to explore whether other viruses form transmission morphs in response to vector-sensing by the host. Second, the transient accumulation of tubulin in TBs, followed by redistribution of TB contents on microtubules uncovers unforeseen capacities of tubulin/microtubule dynamics and raises further questions pertinent to cell biology. For example: How can apparently soluble tubulin concentrate in the TB and what is its function there? Does this serve as the source for the mixed-networks? Most strikingly, our work reveals that a virus can detect external stresses (probably by using its host's perception system) and respond in a way that is somewhat independent of the host's response. We propose naming this phenomenon: ‘virus perceptive behavior’. This concept is nicely illustrated by three compelling observations made in this study. First, TB transformation occurs while the host plant is still in the process of transducing the triggering signal. This shows that the virus appropriates the host's perception machinery itself, rather than relying upon downstream reactions that take tens of minutes (or hours) to manifest and establish local and systemic defense responses . Second,Whether such viral perceptive behaviors play a role in the vector-transmission of other viruses is entirely unknown, and will thus be a question of great priority in the field of research on virus transmission. Viruses tightly regulate all the different steps of their life cycle, from intracellular replication and short- and long-distance intra-host movement, to inter-host spread. In this sense, the ability to specifically trigger the ‘transmission-mode’ at the right time and the right place seems like a valuable adaptation for avoiding the deleterious interference between these various functions.On a more broad scope, our results highlight many unexpected research horizons to explore in the biology of these fascinating pathogens. The possible instances in which viruses could react directly to cues from the host environment, the diversity of sensorial pathways that could be exploited in both animal and plant hosts, and the number of key life cycle steps that could be optimized accordingly all inspire questions that will shape future research directions in this field. Finally, aside from being an academic challenge, this phenomenon also represents a potential Achilles heel in viral transmission that could lead to novel virus control strategies.Brassica rapa cv. ‘Just Right’) and transgenic Arabidopsis thaliana Col0 plants with a gl1 marker expressing GFP-TUA6 under control of the 35S promoter  on eggplant and cultivated by G. Labonne . The population was started from a single virginiparous female.A non-viruliferous clonal Sf9/baculovirus system, the tetracysteine sequence and an additional serine were introduced into the unique XhoI site in the P2 coding region of plasmid p119-P2, using the same strategy as for cloning CaMV-P2TC described above. Recombinant baculovirus was obtained by homologous recombination as described in Sf9 cells were harvested 48 h after inoculation and total cell extracts were prepared in SES buffer and stored at −20°C until use.To produce recombinant P2-TC using the http://www.unilever.com) for 3 min. The leaves were then washed three times with water, prior to overnight incubation in protoplast medium M  containing freshly added 0.5% cellulose ‘Onozuka’ R10 and 0.05% macerozyme R10 . Protoplasts were separated from undigested tissue by filtration through Miracloth (http://www.merckmillipore.com), and washed three times with buffer M by centrifugation at 80×g for 5 min in a swing-out rotor. Prior to treatments, protoplasts were maintained fourfold diluted in buffer M at room temperature with slow agitation (5 rpm) for 2 h.Protoplasts were prepared from healthy or infected (14 dpi) leaves of turnip plants as described in 2 atmosphere (15 min). Azide (100× concentration) or oryzalin (1000× concentration) stock solutions were added to water or DMSO, respectively. Pure solvent was used as a control. For CO2 treatment, leaves or protoplasts were placed in a plastic box filled with CO2 that was generated by sublimation of dry ice in water, contained in a small beaker in the box. We visually confirmed the displacement of the water cloud initially created by the subliming CO2 to assure that the heavier CO2 had replaced the air (no more water vapor visible); only then was the plant material placed in the box. We also verified that the dry ice did not lower the temperature of the atmosphere in the box. For TB reversion, protoplasts were cycled every 15 min between the plastic box and standard bench-top conditions for the CO2 treatment or the azide was removed by replacing the protoplast medium with fresh medium after centrifugation of the protoplasts for 5 min at 80×g in a swing-out rotor. Protoplast viability was verified by the fluorescein diacetate test , 0.02% azide (40 min), or COate test . For heaLeaf segments (5- to 7-mm-long) of turnip or Arabidopsis were fixed in 1% glutaraldehyde prepared in stabilizing buffer . The tissue was then embedded in either Steedman's wax, as described in 4, and embedded in Epon resin (http://www.emsdiasum.com). For immunoelectron microscopy, protoplasts were fixed with 0.5% glutaraldehyde and 2% paraformaldehyde and embedded in LR Gold resin (http://www.emsdiasum.com). All primary antisera and secondary antibodies were used at 1:25 dilution. The grids were observed in a Jeol JEM 100CX II electron microscope (http://www.jeol.com) operated at 60–80 kV.Samples for electron microscopy and immunoelectron microscopy were processed as described in http://www.sigmaaldrich.com; http://plant.neogeneurope.com). For secondary antibodies, we used Alexa 488 and Alexa 594 conjugates (http://www.lifetechnologies.com) or 10 nm colloidal gold conjugates (http://www.bbigold.com).The following antibodies or antisera were used: rabbit anti-P2 , anti-P3g), aliquots were separated by SDS/PAGE using 12% gels. Proteins were transferred onto nitrocellulose membranes and antigens were revealed by the NBT-BCIP reaction as described in Plant tissue was ground in liquid nitrogen, and the powder was resuspended in 2× Laemmli buffer  and boil4, washed with TS and blocked with TS containing 5% dry milk powder (TS-M) for 30 min. The slides were incubated with primary antisera  for at least 2 h. After two rinses with TS, slides were incubated for at least 2 h with secondary antibodies diluted 1:300. After two rinses with TS, slides were mounted in antifading medium, which optionally included DAPI (50 ng/ml).After treatments, protoplasts were fixed for 20 min at room temperature with 1% glutaraldehyde in 0.5 M mannitol and 50 mM HEPES pH 8, and washed with TS buffer . Protoplasts were immobilized on polylysine-coated slides, incubated for 15 min with 0.2% NaBH4, washed with TS and incubated for 90 min in an enzyme solution  and 2% driselase . The 50-μm vibratome sections were incubated for 1 h with 0.2% NaBH4 in a 24-well plate. All sections were then blocked with 3% BSA or 5% BSA in TS supplemented with 0.01% Tween20 for 30 min and incubated with primary antisera in 1% BSA/0.01% Tween20 in TS-M for at least 12 h at the following dilutions: 1:200 for rabbit anti-P2 and rabbit anti-P4 and 1:100 for mouse anti-α-tubulin. After two rinses with TS or PBS, slides were incubated for at least 12 h with Alexa Fluor conjugates at a 1:200 dilution. After two rinses with TS or PBS, slides were mounted as described above.The 14-μm microtome sections were immobilized on polylysine-coated slides, incubated for 1 h with 0.2% NaBHhttp://www.zeiss.com) or Leica SP2 (http://www.leica.com) confocal microscopes operated in sequential mode to avoid crosstalk. Raw images were processed using LSM, ZEN or LAS software and final figures were prepared using GIMP 2.6.11 (http://www.gimp.org) and OpenOffice 3.4 (http://www.openoffice.org). Quantification of TB phenotype was performed by counting 200–300 cells in 10 different, randomly chosen microscopy fields per treatment.Slides were observed with Zeiss LSM510 or LSM700 (http://www.parafilm.com) for a 1 h pre-acquisition period in a humid chamber. Then, either protoplasts or suspensions containing purified virus particles, P2 and P3 in SES buffer (http://www.certiseurope.fr) as described in Groups of about 500 aphids were placed inside copper rings covered with stretched Parafilm M membranes , which included a TB, was photobleached. Three iterations of the 488-nm laser at 100% intensity were used for the bleaching. For recovery of the fluorescence in TBs, images were recorded for 110 s, with a 512 × 512 px picture size, a scan speed of 167 ms/frame and a delay between frames of 0.2 s. For controls, to account for fast tubulin diffusion in the cytosol, recovery was recorded in identical ROIs for only 5.9 s, with a 100 × 50 px picture size, a scan speed of 16 ms/frame and a delay between frames of 23 ms. We verified that the energy of the 488-nm laser used for the post-bleach added no bleaching effect by recording a control region outside the bleaching ROI. The experiment was repeated 21 times for wild type TB bleaching, 23 times for TB-TC bleaching and 18 times in the case of cytoplasmic soluble tubulin. Average intensities in all ROIs including the background signal were measured using ImageJ 1.44p software (http://imagej.nih.gov/ij), before exporting data into Microsoft Excel 2007 (http://www.microsoft.com).FRAP experiments were performed according to n is normalized intensity, It is intensity at any time t, Imin is the minimum intensity post bleach and Imax is the mean intensity pre-bleach.Fluorescence recovery data was normalized as follows:Non-linear regression was used to model FRAP data. In this case, a one-phase exponential curve was used:(1/2) = 0.69/k. Finally, t(1/2) was used to calculate the diffusion rate as D = (0.88 R2)/(4 t(1/2)), where D is the diffusion rate and R is the radius of the bleaching area.From this curve, the half time of recovery was calculated as thttp://www.prioruk.com) and placed either on the stage of a Leica confocal LSI macroscope equipped with a 0.56–16× zoom and a 5× objective or a Zeiss LSM700 confocal microscope with a 10× objective. Either a whole potted plant  or a detached leaf taped to a slide and with a water-soaked paper wrapped around its stalk  was placed under the objective. The microelectrode was carefully brought up to the leaf, and the epidermis was touched or pierced. The approach, the mechanical stress and the reaction of the plant leaf epidermis cell were all recorded by time lapse fluorescence microscopy using acquisition settings as described above for the LSM700 microscope, or excitation with a 488-nm diode laser and an emission bandwidth from 505–550 nm for the LSI macroscope. The pinholes were opened to record sections approximately with 20-μm thickness, and the microscope settings were selected for minimal acquisition times at the expense of image quality.A pulled glass microelectrode was fitted on a micromanipulator (http://www.jmp.com), R 2.9.2 (http://www.r-project.org) and Vassarstats (http://vassarstats.net/) software. The p values <0.05 were regarded as statistically significant.For FRAP, D values were compared with a two-tailed t-test. TB activation states close to and distant from salivary sheaths, as well as transmission rates, were analyzed using GLM and hierarchical GLM models with a binomial distribution. For P2-TC transmission experiments, the Mann–Whitney test was used. To test for differences in TB states between wild type-infected and P2-TC-infected tissue, a nominal logistic model was used since three parameters were analyzed. Statistical analyses were carried out using JMP 10 ( review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent ; Georg Jander (peer reviewer); Saskia Hogenhout (peer reviewer).Thank you for choosing to send your work entitled “Perceiving the outside world: a virus uses the host sensory system for instantaneous transmission by an insect vector” for consideration at The reviewers discussed their comments before we reached this decision. Our goal is to provide the essential revision requirements as a single set of instructions, so that you have a clear view of the revisions that are necessary for us to publish your work.eLife. However, eLife is a broad audience journal and it is critical that the manuscript is written in a manner that will be accessible to its readership. Accordingly:The discovery that aphid feeding and other stresses can trigger a tubulin-dependent reorganization of transmission bodies in plant cells infected with CaMV, that this reorganization depends on viral protein P2, and that the “mixed networks” mediate viral transmission are original and significant contributions and highly appropriate for 1. The manuscript should be re-written to provide background information and to improve the writing. Special attention should be given to the Introduction and Results section ‘TB transformation enables efficient transmission’. Consider obtaining detailed feedback from a colleague who is not in the plant virus field.2. The confocal work should include line scans to show the position of tubulin and EM images are needed for tubulin minus and plus transmission bodies.3. Label the figures in a manner that is more intuitive with each micrograph separately labeled and provide clear explanations for some of the figures. Specifically, the graphs in Figure 3B need to be explained, Figures 2, 3, 4, and 5K need improved labeling, and Figure 6C was difficult to interpret.4. Figure 1 should be expanded to provide an overall model for the paper with a timeline of key events. Consider moving it to the end.5. We would ask you to re-phrase the title and abstract of your study. In our view, this is mandatory as it indicates that viruses indeed make use of the host machinery for vector-mediated transmission. While this hypothesis seems plausible, it was not investigated in your study to an extent that would justify this rather strong statement. 1. The manuscript should be re-written to provide background information and to improve the writing. Special attention should be given to the Introduction and Results section ‘TB transformation enables efficient transmission’. Consider obtaining detailed feedback from a colleague who is not in the plant virus field.We took great care to present our results in a way appropriate for a broad audience. Many parts of the manuscript were rewritten . The text was edited for language and readability by a native English-speaking biologist not from the plant virus field. We hope that the text is now easier to understand.2. The confocal work should include line scans to show the position of tubulin and EM images are needed for tubulin minus and plus transmission bodies.Line scans were added to Figure 2A–C and the new Figures 5A,B and 6A–C. This was a cool idea because the profiles show clearly the differential distribution of P2 within a TB (TB cortex with strong P2 label and center region with weaker P2 label and the opposite for the a-tubulin label). The text was adapted accordingly.3. Label the figures in a manner that is more intuitive with each micrograph separately labeled and provide clear explanations for some of the figures. Specifically, the graphs in Figure 3B need to be explained, Figures 2, 3, 4, and 5K need improved labeling, and Figure 6C was difficult to interpret.The original 6 figures were split up into 11 newly designed figures. We took care to improve the labeling: where possible, each panel has now its proper character label.4. Figure 1 should be expanded to provide an overall model for the paper with a timeline of key events. Consider moving it to the end.To leave some “suspense” we explain the biological model in Figure 1 as before, and present the different steps of TB activation in the new Figure 11 that summarizes the results.5. We would ask you to re-phrase the title and abstract of your study. In our view, this is mandatory as it indicates that viruses indeed make use of the host machinery for vector-mediated transmission. While this hypothesis seems plausible, it was not investigated in your study to an extent that would justify this rather strong statement.To take account of your remarks on the title, we changed it to “Looking outside: A virus responds instantly to the presence of the vector on the host and forms transmission morphs”. We believe that this title better reflects our work and is less speculative. If you have any suggestions for alternative titles, we will be happy to consider them. The Abstract, the Digest and the impact statement were also changed to respect the fact that, although the results suggest it, we do not directly prove viral use of the host sensory system.


Foamy viruses synthesize the Pol precursor protein from a specific transcript. Thus, in contrast to orthoretroviruses, e.g. human immunodeficiency virus, no Gag-Pol precursor protein is synthesized. Foamy viral Pol is processed into a mature protease-reverse transcriptase fusion protein (PR-RT) and the integrase. To avoid premature processing of the Gag and Pol precursors the activity of the protease domain needs to be inhibited before virus assembly. We have demonstrated recently that an independent foamy virus protease domain as well as the PR-RT fusion protein are monomers lacking protease activity. We thus postulated protease activation through induction of dimerization by an additional factor. Here, we identify a specific protease activating RNA motif (PARM) located in the pol region of viral RNA which stimulates foamy virus protease activity in vitro and in vivo, revealing a novel and unique mechanism of retroviral protease activation. This mechanism is strikingly different to that of orthoretroviruses, where the protease can be activated even in the absence of viral RNA during the assembly of virus-like particles. Although it has been shown that the integrase domain is important for Pol uptake, activation of the foamy virus protease is integrase independent. We show that only RNAs containing the PARM result in significant activation of the protease. DNA harboring the PARM is not capable of protease activation. Structure determination of the PARM by selective 2’ hydroxyl acylation analyzed by primer extension (SHAPE) revealed a distinct RNA folding, important for protease activation and thus virus maturation.


AbstractTorrenticolidae Piersig, 1902 (Acari: Hydrachnidia) from streams in South Korea and the Russian Far East are presented. Detailed descriptions or redescrptions are provided for eight species of the genera Torrenticola Piersig, 1896 and Monatractides K. Viets 1926. Two species are described as new to science: Torrenticola kimichungisp. n. and Monatractides abeisp. n. Five species are reported as first records from Korea: Torrenticola brevirostris ; Torrenticola dentifera Wiles, 1991; Torrenticola recentis Tuzovskij, 2003; Torrenticola ussuriensis ; and Torrenticola turkestanica . Torrenticola nipponica  is reported for the first time from Russia.New records of water mites of the family  Torrenticolidae are presently known from all continents except Antarctica, with more than 400 species described so far  is known from South Korea , Torrenticola amplexa , Torrenticola elliptica Maglio, 1909, Torrenticola recentis Tuzovskij, 2003 and Torrenticola ussuriensis , are known from the Russian Far East http://species-id.net/wiki/Torrenticola_brevirostrisAtractides brevirostris Halbert, 1911: 16.35°38'25.623"N, 126°34'53.1438"E, 10.x.2012, Pešić & Choi 1/0/0 .SOUTH KOREA: CR9 Ne myeon Mt, Naebyeansan NP, stream near Naebyeansan Info Center, General features. Idiosoma roundish; Cxgl-4 subapical; posterior suture line of Cx-IV starting at right angle from genital field; excretory pore and Vgl-2 away from the line of primary sclerotization .SOUTH KOREA: CR9 Ne myeon Mt, Naebyeansan NP, stream near Naebyeansan Info Center, General features. Idiosoma elongated; frontal platelets anteriorly bulging ; RUSSİA, Primory Territory, Partizansky District, Partizanskay River basin, Tigrovaya River, 43°11.401'N, 133°12.660'E; depth 30 cm below the sediment surface; substrate: cobbles, pebbles, sand; 12.vi.2010, Semenchenko & Sidorov, three males (490-492-kas–IBSS), two females (493-494-kas–IBSS), dissected and slide mounted.Holotype male (NIBRIV0000268846), dissected and slide mounted, SOUTH KOREA: CR4 Seoraksan NP, stream near Temple, PageBreakIdiosoma elongated ; medial suture line of Cx-II+III in male short (L 74-85 μm); suture line of Cx-IV extended posterior to the genital field; excretory pore posterior to the line of primary sclerotization, Vgl-2 posterior to excretory pore; male genital field with maximum width at the anterior margin; gnathosoma deep with a short rostrum; P-4 with ventral setae on flat hump.General features. Idiosoma elongated; Cxgl-4 subapical; suture line of Cx-IV evident and curved, starting posterior from genital field, laterally curved anteriorly : Idiosoma . Idiosoma  , Torrenticola nanshihensis Pešić PageBreaket al., 2011 (Taiwan), Torrenticola projectura Pešić et al., 2012 (Taiwan), Torrenticola retractipora Lundblad, 1969 (Burma), Torrenticola siamis Pešić & Smit, 2009 (Thailand), and Torrenticola subterranea Imamura, 1957 (Japan). Males of Torrenticola brevirostris and Torrenticola nanshihensis differ in a prominent suture line of Cx-IV starting at a right angle from the genital field, excretory pore on the same level as Vgl-2 and more away from the line of primary sclerotization and P-4 stockier with well developed ventral tubercles . Torrenticola projectura clearly separates in having P-3 with a long tapering ventral protrusion which curves distally .http://species-id.net/wiki/Torrenticola_nipponicaAtractides nipponicus Enami, 1940: 221.37°41.262'N, 127°01.706'E, 19 m asl., 7.x.2012, Pešić & Choi 2/0/0 . RUSSIA: Primory Territory, Partizansky District, Partizanskay River basin, Tigrovaya River, 43°11.401'N, 133°12.660'E; depth 30 cm below the sediment surface; substrate: cobbles, pebbles, sand; 12.vi.2010, Semenchenko & Sidorov 2/2/0 .SOUTH KOREA: CR1 Seoul, Dobong stream, General features. Idiosoma roundish; dorsal shield with colour pattern as illustrated in PageBreakMeasurements. Male  Idiosoma . Gnathosomal bay L 148-153 (132-141), Cx-I total L 278-294 (284-286), Cx-I mL 129-141 (145-165), Cx-II+III mL 69-91 (79-92); ratio Cx-I L/Cx-II+III mL 3.2-4.0 (3.1-3.6); Cx-I mL/Cx-II+III mL 1.55-1.87 (1.57-2.08). Genital field L/W 156-181/117-133 (168-172/125-132), L/W ratio 1.33-1.36 (1.26-1.38); ejaculatory complex L 231 (224-251); distance genital field-excretory pore 131-178 (138-145), genital field-caudal idiosoma margin 183-231 (172-205). Gnathosoma vL 273-303 (277-297); chelicera total L 319-347 (323-343); palp total L 279-316 (284-291), dL: P-1, 34-37 (33-36); P-2, 92-101 (92-95); P-3, 52-63 (51-59); P-4, 82-93 (82-92); P-5, 19-22 (17-18); P-2/P-4 ratio 1.09-1.13 (1.03-1.12).PageBreakPageBreakGnathosomal bay L 168-178, Cx-I total L 303-310, Cx-I mL 132-145, Cx-II+III mL 30-40; ratio Cx-I L/Cx-II+III mL 7.75-10.1; Cx-I mL/Cx-II+III mL 3.63-4.4. Genital field L/W 185-195/172-178, L/W ratio 1.04-1.13; distance genital field-excretory pore 208-210, genital field-caudal idiosoma margin 297-310. Gnathosoma vL 330-336; chelicera total L 420; palp total L 341-345, dL: P-1, 41-43; P-2, 115-116; P-3, 65-67; P-4, 100-102; P-5, 18-19; P-2/P-4 ratio 1.13-1.16.Female . Idiosoma  which was based on one male and seven females from River Inôzava, Uzi region in Japan , a species which differs in the gnathosomal rostrum not distinctly set off from the gnathosomal base, P-2 shorter than P-4, the suture line of Cx-IV starting at right angle from the genital field, the excretory pore and Vgl-2 more distanced from the line of primary sclerotization and the genital field in male is less elongated.The male specimens from South Korea and Russia fit the description of in Japan . HoweverA permanent shaded sandy/bouldary stream at low elevations ; the speJapan , South Korea . New for the fauna of Russia.Tuzovskij, 2003http://species-id.net/wiki/Torrenticola_recentisTorrenticola recentis Tuzovskij, 2003: 45.437°41.262'N, 127°01.706'E, 19 m asl., 7.x.2012, Pešić & Choi 6/6/0 ; CR2 Seoul, Ui-dong stream 37°39.554'N, 127°00.249'E, 114 m asl., 7.x.2012, Pešić & Choi 3[1 juvenile]/0/1; CR4 Seoraksan NP, stream near Temple, 38°10.399'N, 128°29.050'E, 196 m asl., 8.x.2012 Pešić & Karanović 7/2/5; CR7 Odesean NP, stream, 37°49.642'N, 128°42.170'E, 215 m asl., 9.x.2012, Pešić & Karanović 6/6/0 ;; CR11 Mudeung Mt., stream, 35°8'50.2584"N, 126°59'18.942"E, 11.x.2012, Pešić & Choi 2/2/1; CR12 JiriSan NP, stream near waterfall, 35°22'47''N, 127°29'10''E, 11.x.2012, Pešić & Choi 4/12[1 juvenile]/0 (1/0/0 mounted); CR14 Duckyu San NP, stream, 35°53'50"N, 127°46'35"E, 11.x.2012, Pešić & Choi 0/2/0. RUSSIA, Primory Territory, Khasansky District, “Kedrovaya Pad National Nature Biosphere Reserve”, Sea of Japan basin, PageBreakPageBreakKedrovaya River, 43°06.056'N, 131°33.310'E; depth 12–50 cm; substrate: boulders, cobbles, pebbles; 8.xi.1993, Tiunova 2/2/0 (IBSS).SOUTH KOREA: CR1 Seoul, Dobong stream, General features. Idiosoma roundish; dorsal shield with colour pattern as illustrated in Measurements. Male . Idiosoma . Idiosoma http://species-id.net/wiki/Torrenticola_ussuriensisAtractides ussuriensis Sokolow, 1940: 347.38°03.961'N, 128°10.516'E, 225 m asl., 8.x.2012 Pešić & Karanović 0/1/0 .SOUTH KOREA: CR3 River Inje, General features. Shoulder platelets fused to the large dorsal plate http://species-id.net/wiki/Torrenticola_turkestanicaAtractides turkestanicus Sokolow, 1926: 74.38°03.961'N, 128°10.516'E, 225 m asl., 8.x.2012 Pešić & Karanović 1/1/0 .SOUTH KOREA: CR3 River Inje, General features. Idiosoma elongated ; dorsal shield with colour pattern as illustrated in Measurements. Male: Idiosoma . The only difference with the original description is found in a broader genital field in the type specimen. Later on, populations provisionally assigned to Torrenticola turkestanica, mainly based on the approved non-identity with the alternative species, were reported from Indian Himalayas .The specimens examined from River Inje fit the description of imalayas  and Thaiimalayas . The popimalayas , a much PageBreakTorrenticola japonica Imamura, 1953, a species described based on a single female from a stream in Shinjô-mura in Japan . From the latter species, Torrenticola japonica differs in a broader idiosoma, a dorsal shield with broader shoulder platelets and posterior suture line of Cx-PageBreakIV not extending far beyond genital field , additional material and selection of a neotype from the locus typicus is necessary to clarify its taxonomy.in Japan , resembleld see: . HoweverTorrenticola elliptica Maglio, 1909, from a stream in Shinjô-mura in Japan. However due to the presence of subrectangular ventrodistal projections on P-2 and P-3 and a convex ventral margin of P-2, his illustrations show a general conformity with Torrenticola japonica and Torrenticola turkestanica. It is very likely that the specimen attributed by Torrenticola elliptica is conspecific with Torrenticola japonica, especially as both species were collected from the same location and on the same day.A permanent sandy/bouldery river with considerable exposure to sunlight .Tadjikistan (Sokolow 1926), Indian Himalayas , Thailanurn:lsid:zoobank.org:act:F9C4384E-4673-4FC9-B098-A002FCDD1CDAhttp://species-id.net/wiki/Monatractides_abei37°39.554'N, 127°00.249'E, 114 m asl., 7.x.2012, Pešić & Choi. Paratypes: SOUTH KOREA, CR1 Seoul, Dobong stream, 37°41.262'N, 127°01.706'E, 19 m asl., 7.x.2012, Pešić & Choi, one female (NIBRIV0000268854), dissected and slide mounted; RUSSIA, Primory Territory, Partizansky District, Partizanskay River basin, Tigrovaya River, 43°11.401'N, 133°12.660'E; depth 30 cm below the sediment surface; substrate: cobbles, pebbles, sand; 12.vi.2010, Semenchenko & Sidorov, one female (497-kas–IBSS), dissected and slide mounted.Holotype male (NIBRIV0000268853), dissected and slide mounted, SOUTH KOREA, CR2 Seoul, Ui-dong stream, Lateral margins of dorsal shield nowhere subparallel; distal margins of P-3 with several pointed extensions; genital field in male with slightly protruding anteriolateral angles; medial suture line of Cx-II+III in female relatively long (L 90–105 μm).PageBreakGeneral features. Lateral margins of dorsal shield nowhere subparallel ; medial suture line of Cx-II+III relatively long.parallel , 11I; thparallel : proximaparallel . Male. MPageBreakMeasurements. Male: Idiosoma . Idiosoma , known from the Western Palaearctic, due to the presence of an elongated ventral seta on P-4 and the similar shape of the ejaculatory complex. Males of Monatractides madritensis differ in a more slender idiosoma with subparallel lateral margins of the dorsal shield, and a slender genital field with more protruding anterior margins of the genital flaps forming a more acute angle  as Torrenticola stadleri (syn. to Monatractides stadleri), but he noted that his specimens differ from the later species in P-4 bearing an elongated ventral seta. Given the latter character and the shape of genital field in Enami’s specimens, with slightly protruding anteriolateral angles in male, resembling the female, suggest that these specimens are probably conspecific with Monatractides abei sp. n.Monatractides abei sp. n. was collected in two sandy/bouldary streams, shaded by riparian vegetation .


Seroprevalence of antibodies to influenza A(H1N1)pdm09 virus among 193 emergency department health care personnel was similar among 147 non–health care personnel . Working in an acute care setting did not substantially increase risk for virus infection above risk conferred by community-based exposures. Transmission of infectious disease from acutely ill patients to health care personnel (HCP) is a critical concern during disease outbreaks. During the initial months after the emergence of influenza A(H1N1)pdm09 virus, comparisons to prior pandemic viruses pdm09. These early outbreaks and the concomitant surge in patient volumes in our emergency department (ED) provided the opportunity to evaluate and compare risk for A(H1N1)pdm09 virus infection among frontline HCP and non-HCP from the same community in a virus-naive population before availability of the A(H1N1)pdm09 monovalent vaccine.Written informed consent was obtained and the study approved by the Human Subjects Review Board of the Feinstein Institute for Medical Research of the North Shore–Long Island Jewish Health System. Long Island Jewish Medical Center and the adjoining Cohens Children’s Hospital are tertiary care teaching hospitals in Queens, New York. During April 24–June 11, 2009, the volume of all-cause ED visits to these 2 institutions increased by 62% compared with the same period during 2008. There were 5,100 visits with influenza-like illness (ILI) as the primary manifestation, which coincided with a surge of ILI visits to EDs throughout New York, New York pdm09 monovalent vaccine before enrollment. Assuming a 20% seroprevalence of antibodies to A(H1N1)pdm09 among the general population and a type I error probability of 5% and type II error probability of 20% (power 80%), a sample size of 140 HCP and 140 non-HCP would be sufficient to show a 15% difference in seroprevalence between HCP and non-HCP.Serum samples were tested by using hemagglutination inhibition and microneutralization assays with A/Mexico/4108/2009, an A/California/07/2009 (H1N1)–like virus  or hypothesized to be exposure risk factors were included. Analyses were performed by using SAS version 9.2 software .Separate analyses comparing seropositive and seronegative persons were performed for HCP and non-HCP by using either a χWe enrolled 193 HCP and 147 non-HCP in the study. Non-HCP were older  than HCP  and less likely to recall symptoms of an ILI . A similAmong 193 HCP, 41 (21.2%) were seropositive for antibodies to A(H1N1)pdm09 virus; of these, 12 (29.3%) reported no influenza-like symptoms during the study period. Age, sex, and HCP role were not associated with seropositivity. However, a higher proportion of attending physicians who took care of children were seropositive than those who took care of adults . Seropositive HCP did not work more ED shifts than seronegative HCP  or temporary influenza treatment center shifts  during April 24–June 11, 2009. The proportion of seropositive HCP who reported contact with a patient with suspected or confirmed A(H1N1)pdm09 was similar .Among 147 non-HCP, 24 (16.3%) were seropositive for antibodies to A(H1N1)pdm09 virus. A higher proportion of persons living with children <18 years of age were seropositive  than those not living with children <18 years of age. However, this finding did not reach statistical significance.Among the 340 study participants, 65 (19%) were seropositive for antibodies to A(H1N1)pdm09 virus. HCP were no more likely to be seropositive than were non-HCP . In a multivariate model that included age, sex, receipt of seasonal influenza vaccine, having children <18 years of age in the household, and occupation, only living in a household with children <18 years of age was associated with being seropositive .We found that 21% of frontline HCP working in the ED or specially designated influenza areas during the first wave of A(H1N1)pdm09 virus circulation in New York, New York, were seropositive for antibodies to A(H1N1)pdm09 virus, similar to non-HCP. Overall, our estimated seroprevalence among HCP and non-HCP was 19%, which was similar to estimates after the first wave of A(H1N1)pdm09 virus circulation from other studies pdm09 virus in other countries ranges from 7% to 30% pdm09 virus. However, we found that that our criteria for seropositivity were highly specific for detection of A(H1N1)pdm09 virus infection.


Graminella nigrifrons Forbes) transmits two viruses that use different strategies for transmission: Maize chlorotic dwarf virus (MCDV) which is semi-persistently transmitted and Maize fine streak virus (MFSV) which is persistently and propagatively transmitted. To date, little is known regarding the molecular and cellular mechanisms in insects that regulate the process and efficiency of transmission, or how these mechanisms differ based on virus transmission strategy.Insects are the most important epidemiological factors for plant virus disease spread, with >75% of viruses being dependent on insects for transmission to new hosts. The black-faced leafhopper  that also infects the leafhopper, including induction of immune responses in the hemocoel and at the cell membrane. The significant induction of the innate immune system in G. nigrifrons fed on a foregut-borne virus (MCDV) that does not infect leafhoppers was less expected. The changes in transcript accumulation that occur independent of the mode of pathogen transmission could be key for identifying insect factors that disrupt vector-mediated plant virus transmission.We expected, and found, changes in transcript expression in  Among the most difficult plant diseases to control are those transmitted by insect vectors. Pathogens and their vectors have co-evolutionary histories that are intricately intertwined with their ecologies, environments and genetic interactions. While much progress has been made towards understanding the interactions among vectors and pathogens required for transmission of human and animal diseases ,2, much Modes of plant virus transmission by insects fall into four categories: non-persistent, semi-persistent, persistent-circulative, and persistent-propagative. Non-persistently-transmitted and semi-persistently-transmitted viruses do not breach the gut barrier in the insect vector; rather they are retained in the insect stylet or foregut prior to transmission. Because of their location outside of insect cells and the few barriers they must cross prior to introduction into a new plant host, these viruses can be transmitted within seconds to hours after acquisition. Most viruses transmitted in a non-persistent manner are transmitted by aphids; aphids, leafhoppers and whiteflies transmit semi-persistently-transmitted viruses ,4. PersiGraminella nigrifrons (Forbes) is found in the eastern U.S., feeding on a wide range of grasses and cereal crops  to the NCBI sequence read archive under the accession [SRP022856]. The Transcriptome Shotgun Assembly project has been deposited at DDBJ/EMBL/GenBank under the accession GAQX00000000.1. The version described in this paper is the first version, GAQX01000000. All the supporting data are included as additional files.The authors declare that they have no competing interests.BJC, MGR, AM conceived and designed the study. BJC and YP conducted the sampling and did the molecular work . BJC and SW carried out the read filtering, trimming and assembly. BJC and SW performed the differential expression analysis. BJC, LRS, MGR, AM wrote and revised the manuscript. All authors read and approved the final manuscript.Ortholog amino acid sequence similarity for assembled transcripts.Click here for fileGene and enzyme ontologies assigned to assembled transcripts using nr database. Click here for fileG. nigrifons fed on virus-infected maize for 4 h and 7 d.Transcripts differentially expressed in Click here for fileThe 25 top-ranked transcripts with the greatest up-or down-regulation at each time point. Click here for fileThe four differentially expressed transcripts from next generation sequencing validated by RT-qPCR. Click here for filede novo assembly. Preprocessing parameters of Click here for file


Female genital tract mucosae are bathed in acidic secretions, which, in the case of HIV-1 infected women, likely contain virus coated with antibody. Thus, uninfected male sexual partners are often exposed to an acidic milieu containing virus in the form of immune complexes. We investigated the impact of low pH and antibody on transcytosis, a potentially critical mechanism by which HIV-1 passes through genital tract epithelia to infect susceptible target cells.HIV-1 was incubated with Env-specific monoclonal and polyclonal antibodies at pH 6.0 or pH 7.4 and exposed to the apical surface of tight junction-forming human endometrial carcinoma (HEC-1) cells in transwell plates. The quantity and infectivity of transcytosed virus was measured by RT-PCR and infection of TZMbl cells, respectively.We found that the combination of acidic pH and Env-specific antibody augmented transcytosis as much as 30-fold compared with Env-specific antibody at neutral pH or compared with non-specific antibody or no antibody at neutral or acidic pH. The pH and antibody dependence of enhanced transcytosis was blocked by antibody specific for the Fc neonatal receptor (FcRn) or by treatment with bafilomycin A1 (which inhibits acidification of endosomes). Non-neutralizing antibodies resulted in a lower quantity of total transcytosed virus, measured by RT-PCR, than did neutralizing antibodies. However, the ratio of total to infectious virus was much higher for neutralizing antibodies, indicating that neutralizing antibodies efficiently allow transcytosis while blocking infectivity of the transcytosed virus; the non-neutralizing antibodies facilitate transcytosis  without blocking infectivity.These results demonstrate that acidity and Env-specific antibody greatly enhance transcytosis of virus across mucosal epithelial cells via FcRn. Since male penile and urethral tissues express FcRn, our results suggest a novel mechanism by which antibody, and in particular, non-neutralizing antibody, might facilitate female-to-male transmission following sexual exposure.


TDT) to one that accounts for locus heterogeneity.Locus heterogeneity is one of the most documented phenomena in genetics. To date, relatively little work had been done on the development of methods to address locus heterogeneity in genetic association analysis. Motivated by Zhou and Pan's work, we present a mixture model of linked and unlinked trios and develop a statistical method to estimate the probability that a heterozygous parent transmits the disease allele at a di-allelic locus, and the probability that any trio is in the linked group. The purpose here is the development of a test that extends the classic transmission disequilibrium test  is approximately 0 for all MOI, there are parameter settings for which the power difference can be substantial. Our multi-locus simulations suggest that our method has good power to detect association as long as the markers are reasonably well-correlated and the genotype relative risk are larger. Results of both single-locus and multi-locus simulations suggest our method maintains the correct type I error rate.Our simulations suggest that, for sufficiently large sample size (1000 trios) our method has good power to detect association even the proportion of unlinked trios is high (75%). While the median difference  that allows for locus heterogeneity among coded trios. Benefits of our method include: estimates of parameters in the presence of heterogeneity, and reasonable power even when the proportion of linked trios is small. Also, we have extended multi-locus methods to TDT-HET and have demonstrated that the empirical power may be high to detect linkage. Last, given that we obtain PPBs, we conjecture that the TDT-HET may be a useful method for correctly identifying linked trios. We anticipate that researchers will find this property increasingly useful as they apply next-generation sequencing data in family based studies.We have developed an extension of the  In genetics, heterogeneity is a major feature of human traits. Genetic heterogeneity occurs when the same or clinically indistinguishable phenotypes are caused by different genetic factors. This can be due to multiple variants located in the same locus  or to mutations located in different loci (locus heterogeneity).The focus of this work is locus heterogeneity, specifically heterogeneity caused by having an unknown subset of pedigrees in a sample being unlinked to a disease locus while the rest are linked ,2.There are many reported examples of locus heterogeneity, including breast cancer -6, matur(θ) for each pedigree in a sample; the B-test ) are determined using the disease allele frequency p  for formulas determining mating type frequencies for the first locus. For each consecutive locus l, 2 ≤ l ≤ 4, the mating type frequencies for the ith mating type is determined in the following fashion.Here, we consider 4 correlated SNPs in each simulation. Mating types for the first locus (labeled MT [i]) arel][i] = ρ × MT[l - 1][i] + (1 - ρ) × X, where X~U.1. Define τ^1,abc to one that accounts for locus heterogeneity. Our results suggest that use of permutation p-values enable us to correctly maintain correct type I error rates at the 5% and 1% significance levels. Power simulations using disease MOIs suggest that power can be disease model dependent, with the TDT being slightly more powerful for dominant MOIs, and the TDT-HET being more power for recessive MOIs. Also, we find that our statistic can have high power, even in the presence of locus heterogeneity, when the GRR is larger.In this work, we present a mixture model of linked and unlinked trios and develop a statistical method to estimate the probability TDT-HET statistic and the corresponding permutation p-value appears to be about the same as that of ordinary TDT for the Idiopathic scoliosis Candidate Loci data set even though results of the TDT-HET analysis suggest that there is locus heterogeneity for several loci. Based on our simulations, we might conjecture that the single-locus MOI for each SNP is multiplicative.It is interesting to note that the value of the rth iteration step estimates, we did not investigate this form of the TDT-HET statistic further. Given the extensive amount of work already present, we consider this work to be beyond scope of the present manuscript. We plan to follow up this research and report our findings in another manuscript.We computed parameters for the situation where linked and unlinked trio types come from populations with different sets of parental mating type frequencies, but apart from determining the xabc is linked to a locus is seeing whether the inequality:As noted in Results, Idiopathic Scoliosis Candidate Loci section, Ott documents that a decision rule for determining whether a particular trio type α does not even measure the proportion of linked families within the sample, as is commonly supposed.is satisfied. Having said that, Terwilliger and Ott  report tnd Logue  documentTDT-HET statistic is that it provides estimates that each of the 10 types of trios  all trios that satisfy inequality (2); and (ii) all trios for which at least one parent is heterozygous and that also satisfy inequality (2).This decision rule potentially reduces the number of trios that we need consider when looking for linked trios. We can further reduce the number of trios considered by adding the condition that we only consider trios in which at least one parent is heterozygous. Thus, the two decision rules we consider here for selecting linked trios using the TDT statistic, our analogous decision rules are: (i) all trios; and (ii) all trios for which at least parent is heterozygous.For the We plan to perform an extensive analysis to evaluate the empirical probabilities that each statistic can correctly identify linked trios. We can simulate linked and unlinked trios using the method implemented in the FASTSLINK software ,80. We cGiven that next generation sequencing data applied to families is bound to identify large amounts of locus heterogeneity, any methods that increase the probability of identifying true disease variants should be welcome. We realize that, though, the probabilities of correctly identifying linked trios may be dependent upon the true proportion of linked trios. One way we can reduce heterogeneity is to look at larger family sizes. We plan to apply our statistic to such families and investigate its performance.TDT statistic, TDT-HET, that allows for locus heterogeneity among coded trios. This method is an extension of TDT, in that our simulation results suggest it has approximately the same power as the original TDT. Results of our simulations suggest that our method maintains correct type I error for the null hypothesis (R1 = 1.0). Benefits of our method include: estimates of parameters in the presence of heterogeneity , and reasonable power even when the proportion of linked trios is lower. Also, we have extended Hoh, Ott, and colleagues' SumStat method to TDT-HET. The parameter estimation above, particular, estimation of the probability that a trio is linked will be useful as we enter the age of next-generation sequencing, where one can expect extensive levels of locus heterogeneity given the rare disease frequencies.Motivated by the recent work of Zhou and Pan , we haveTDT-HET method and provided advice on application of the Expectation-Maximization Algorithm. SS performed the genotyping for the Idiopathic Scoliosis candidate loci samples and, together with CAW, wrote the section describing the Idiopathic Scoliosis candidate loci sample collection. CAW graciously provided samples for the Idiopathic Scoliosis candidate loci example. DG co-developed the TDT-HET statistic, wrote software code to compute the statistic, performed a number of simulations and wrote the majority of the manuscript. All authors read and approved the final manuscript.DL co-developed the TDT-het statistic, performed a number of analyses and simulations and wrote a significant portion of the manuscript. SB and SJF both helped develop the Appendix. Full details of the derivation of the TDT-HET statistic, including notation.Click here for file


New viruses pathogenic to plants continue to emerge due to mutation, recombination, or reassortment among genomic segments among individual viruses. Tospoviruses cause significant economic damage to a wide range of crops in many parts of the world. The genetic or molecular basis of the continued emergence of new tospoviruses and new hosts is not well understood though it is generally accepted that reassortment and/or genetic complementation among the three genomic segments of individual viruses could be contributing to this variability since plants infected with more than one tospovirus are not uncommon in nature.Iris yellow spot virus (IYSV) and Tomato spotted wilt virus (TSWV), were investigated for inter-virus interactions at the molecular level in dually-infected plants. Datura (Datura stramonium) is a permissive host for TSWV, while it restricts the movement of IYSV to inoculated leaves. In plants infected with both viruses, however, TSWV facilitated the selective movement of the viral gene silencing suppressor (NSs) gene of IYSV to the younger, uninoculated leaves. The small RNA expression profiles of IYSV and TSWV in single- and dually-infected datura plants showed that systemic leaves of dually-infected plants had reduced levels of TSWV N gene-specific small interfering RNAs (siRNAs). No TSWV NSs-specific siRNAs were detected either in the inoculated or systemic leaves of dually-infected datura plants indicating a more efficient suppression of host silencing machinery in the presence of NSs from both viruses as compared to the presence of only TSWV NSs.Two distinct and economically important tospoviruses, Bunyaviridae family.Our study identifies a new role for the viral gene silencing suppressor in potentially modulating the biology and host range of viruses and underscores the importance of virally-coded suppressors of gene silencing in virus infection of plants. This is the first experimental evidence of functional complementation between two distinct tospoviruses in the  Bunyaviridae consists of five genera and viruses with negative-stranded RNA genomes. All but one genera cause serious diseases in animals and humans Tospovirus, in this family consists of viruses that infect plants. Tospoviruses cause some of the most destructive diseases in important crop plants and world-wide annual losses could be as high as U.S. $1 billion Tomato spotted wilt virus (TSWV) was the first tospovirus described and is one of the most-studied tospoviruses The virus family Tospovirus was thought to be a monotypic genus consisting of only TSWV until a second tospovirus, Impatiens necrotic spot virus (INSV), was described Iris yellow spot virus (IYSV) was the third tospovirus to be reported Groundnut ring spot virus (GRSV) in tomato in Florida For many years, genus i.e. the exchange of genomic segments among different parents. This is facilitated by the presence of mixed infections in plants under natural conditions.The increasing diversity of tospoviruses has been attributed to shifts in crop production, differing specificity of thrips vectors, and the segmented nature of the viral genome which could result in genetic reassortants. Moreover, it is generally accepted that tospoviruses, like other RNA viruses, show high mutation rates because of the lack of proofreading ability of their replicases N/GC) in antisense orientation. The S RNA codes for a non-structural protein (NSs) in sense direction and the nucleocapsid protein (N) in antisense direction. The NSm and GN/GC proteins coded by the M RNA play important roles in cell to cell virus movement in plant host, and in vector transmission, respectively The genome of tospoviruses consists of three RNAs, large (L), medium (M) and small (S). The L RNA is organized in negative sense orientation, whereas the M and S RNAs are in ambisense The segmented genome of tospoviruses facilitated genetic reassortment studies to map genetic determinants for symptomatology Citrullus lanatus) plants by Groundnut bud necrosis virus (GBNV) and Watermelon bud necrosis virus (WBNV) under field conditions in central India. Similarly, TSWV and IYSV were shown to infect and co-exist in onion plants under field conditions in Georgia, where 7% of the onion (Allium cepa) plants tested were dually infected with these two distinct tospoviruses Datura stramonium) is a differential host for IYSV and TSWV. It is considered a permissive host for TSWV and a restrictive host for IYSV with respect to virus movement following infection  (N/GC and RdRp) of these two RNAs could be amplified from inoculated leaves but not from uninoculated systemic leaves (data not shown); further confirming that IYSV was restricted to only inoculated leaves  . By 20–2on (DPI) . The viron (DPI) . This waD. stramonium plants, when mechanically inoculated with TSWV, first show a few small concentric ring symptoms, 10–12 DPI on inoculated leaves (N/GC) and L (RdRp) RNA were amplified using gene-specific primers. Both inoculated  were analyzed for IYSV and TSWV N and NSs gene expression using gene-specific cDNA probes  in the USA due to genome reassortment of two viruses, GRSV and Tomato chlorotic spot virus (TCSV), with the large (L) and small (S) RNAs coming from GRSV and the medium (M) RNA from TCSV . This exchange of genetic material and interaction between co-infecting viruses resulting in synergism or complementation could lead to changes in biological properties and increased diversity of viruses, especially those with segmented genomes due to genetic reassortment Mixed infections of plants with viruses is a natural phenomenon, and the outcome of this inter-virus interaction is influenced by several factors including the host plant's genetic background and growth stage, genetic relatedness of the viruses, and environmental conditions. The outcomes include elimination of one virus from the mixed infection by the second virus, co-existence and continued replication of both viruses for the remainder of the plant's life, or increased titer of one virus resulting in more severe symptoms (synergism). Mixed infections could also result in genetic recombination or genetic reassortment between the co-infecting viruses that could lead to generation of new viruses or strains with a subsequent expansion of the host range Tospovirus.Synergistic interactions between viruses leading to more severe disease have been described for positive-stranded RNA viruses and DNA viruses We utilized a model system wherein the experimental host, datura, responds differentially to two distinct tospoviruses, IYSV and TSWV. While this host is susceptible to infection by both viruses, it is a permissive host to TSWV (where the virus invades the plant and causes systemic infection), whereas IYSV infection is confined to inoculated leaves and fails to move to younger, uninoculated leaves Datura plants inoculated with IYSV alone showed symptoms of infection only on the inoculated leaves, with younger systemic leaves remaining symptomless. On the other hand, TWSV inoculation resulted in symptoms on the inoculated as well as systemic leaves . This waD. stramonium. NSs have been reported as the silencing suppressor in tospoviruses Dual infection of datura plants with IYSV and TSWV resulted in an increase in the symptom severity of the inoculated as well as the systemic leaves with plants succumbing to the virus 40 days post-inoculation (dpi) as compared to 80 dpi in the case of single infection with TSWV. In addition, the IYSV NSs was selectively expressed in the younger, uninoculated leaves of dually infected Availability of antisera specific to each of the TSWV and IYSV N proteins and the IYSV NSs protein enabled us to determine the expression of these genes in individually and dually infected plants. While the relative levels of these proteins as reflected by the absorbance values varied among individual plants within a treatment, we were able to consistently detect these proteins. TSWV N could be detected in inoculated as well as systemic leaves as expected. IYSV N and NSs could be found only in inoculated leaves in singly infected plants (since the virus is confined to inoculated leaves in datura). In case of dually infected plants, the IYSV NSs protein was expressed in uninoculated leaves, albeit to a lower level.Tospovirus spp. including TSWV and a tomato-infecting strain of Tomato yellow ring virus (TYRV-t) using partial N gene sequences. They showed that transgenic resistance against TYRV-t does not hold against the soybean strain (TYRV-s) and is broken by TYRV-t when co-inoculated with TYRV-s. TYRV-t could be detected in the systemic leaves of a resistant line in co-inoculated plants implying a transcomplementation event involving a protein of TYRV-s. They further showed that the presence of TYRV-s encoded silencing suppressor (NSs) was crucial for rescuing of TYRV-t. The severe symptoms in datura plants dually infected with TSWV and IYSV, compared to those individually infected either with TSWV or IYSV, could be explained by the fact that the level of the silencing suppressor, NSs, of both viruses was much higher in dually infected than in singly infected Datura  (Tomato chlorotic spot virus (TCSV) has recently been described Tobacco mosaic virus and Cucumber mosaic virus produce a synergistic disease with severe malformations Tospovirus spp. such as TYRV and IYSV with N gene variation of only 10 to 15%.Genetic reassortment has long been accepted as one of the means by which new viruses or strains are generated among viruses with segmented genomes. Such a naturally occurring genetic reassortant between GRSV and The model system we developed facilitates further studies on the nature of protein-protein interactions between the silencing suppressors of distinct tospoviruses in dually infected plants, the nature of the plant factors that interact with the viral proteins, and the effect of dual infection on the profile of virus-specific small RNAs produced as a result of host-induced gene silencing.Arachis hypogaea) plants, were maintained on experimental hosts, Datura stramonium and Nicotiana benthamiana under controlled conditions in a greenhouse.IYSV and TSWV, originally isolated from naturally infected onion and peanut (D. stramonium and N. benthamiana were dusted with silicon carbide (600 mesh) and the homogenate was manually applied using cotton buds. Inoculated plants were maintained at 25/18°C (day/night), 14 h day light and observed for development of symptoms. For establishing mixed infections, leaves from D. stramonium plants individually infected with TSWV or IYSV were homogenized together in equal amount of tissue and mechanically inoculated on to healthy D.stramonium and N. benthamiana plants as stated earlier. All experiments were repeated at least three times. The inoculated as well as systemic leaves of both hosts were harvested 25–30 days after inoculation, and stored at 4°C for further analysis.Virus-infected tissue was homogenized by grinding in 0.01 M sodium phosphate buffer (pH 7.0) containing 0.4% β-Mercaptoethanol and the homogenate was used as the inoculum for mechanical inoculation to experimental hosts. Fully expanded leaves (25–30 days old) of E. coli-expressed NSs protein which was used in a direct antigen-coated ELISA format Virus infection of inoculated plants was confirmed by ELISA using commercially available kits for TSWV and IYSV  following manufacturer's instructions. IYSV NSs-specific polyclonal antiserum was made to Total plant RNA was extracted separately from the symptomatic and asymptomatic IYSV and TSWV-infected plant tissue using the Plant RNeasy mini kit . RT-PCR was performed to detect the presence of various genomic RNAs  of TSWV and IYSV using gene-specific primers .8000/NaCl as described previously Symptomatic tissues were collected from inoculated and systemic leaves from co-infected datura plants and snap frozen immediately in liquid nitrogen. Frozen leaf tissue was homogenized in liquid nitrogen using a mortar and pestle. Total RNA was extracted using TRIZOL reagent  following the manufacturer's instructions. Low molecular weight RNAs were selectively precipitated by PEG32P dATP. Small RNA was separated on a 17% polyacrylamide- 7M urea gel and transferred to positively charged nylon membrane  by the semi-dry blot method 32P-radiolabeled probes by end-labelling with T4 polynucleotide kinase enzyme for oligo probes and random-priming method with Megaprime kit  for larger double-stranded DNA probes following manufacturer's instructions. The radiolabeled probes were column-purified. The blots were washed twice in 2× SSC, 0.1% SDS at 40°C for 10 minutes, exposed to X-ray film , and the film was developed. Blots were stripped in boiling 2× SSC, 0.1% SDS for 10–15 minutes, and verified for residual radiation by overnight exposure before re-probing with a different probe.Total RNA (20 ug) from plant samples was subjected to Northern blot analysis


Nicotiana benthamiana under co-infection in a plant host. The accumulation of viral RNA was quantified using qPCR at 1, 2 and 8 days post infection (dpi), and two different methods were employed to predict the dominating virus. TMV was predicted to dominate the game in the long run and this prediction was confirmed by both qRT-PCR at 8 dpi and the death of co-infected plants after 15 dpi. In addition, we validated our model by using data reported in the literature. Ten out of fourteen reported co-infection outcomes agreed with our predictions. Explanations were given for the four interactions that did not agree with our model. Hence, it serves as a valuable tool in making long term predictions using short term data obtained in virus co-infections.Mixed virus infections in plants are common in nature and their interactions affecting host plants would depend mainly on plant species, virus strains, the order of infection and initial amount of inoculum. Hence, the prediction of outcome of virus competition in plants is not easy. In this study, we applied evolutionary game theory to model the interactions between Hibiscus latent Singapore virus (HLSV) and Tobacco mosaic virus (TMV) in  Viral infections are common in plants and it is not unusual for more than one virus species to infect the same host. These mixed infections usually generate effects that are not observed in single infections as plants are often found to be co-infected with multiple viruses Nicotiana benthamiana Domin which leads to plant death. Hibiscus latent Singapore virus (HLSV) is a new member of Tobamovirus and unlike TMV, it does not cause necrosis in its host. Instead, it induces mild symptoms such as slight crinkling of leaves in N. benthamianaN. benthamiana can be cross-protected against TMV by HLSV (unpublished data). However, co-inoculation of both viruses onto the same plant has not been carried out. Here we applied evolutionary game theory to examine the interactions between these two viruses in co-infection.TMV is a tobamovirus with a broad host range Evolutionary game theory was formulated to analyze a population of interacting players, whose fitness are frequency dependent In a game involving two players and two strategies, a pay-off matrix is used to model their interactions .a if playing against Player A, a fitness of b against Player B. On the other hand, Player B has a fitness of c against Player A and a fitness of d against Player B. Under the assumption of a well-mixed population where players encounter each other randomly, there are four outcomes which depend on the relative fitness between the two players resulting from their interactions. Player A dominates the game if a>c and b>d. Similarly, player B dominates the game if a<c and b<d. In any mixed population, it will converge towards a homogenous population containing the dominant player. The fitness of the dominant player is a result of the interactions between both players and it is relatively higher than the opponent. However, when there is no clear dominance, i.e. when a>c and b<d, player A plays better against player A, but not against player B and vice versa. Both strategies are in strict Nash equilibrium a<c and b>d, both strategies are equally competitive. The well mixed population will remain heterogeneous as it is more advantageous for both players to interact with opponents, as compared to a homogenous population.Each player has a different fitness depending on which other individual it has interacted (played) with. Player A has a fitness of In the context of a co-infection, the viruses are both the players and the strategies. By virtue of being a virus itself is a strategy because each species of viruses possesses a set of behaviours such as replication, expression of coat protein, virion assembly, virus movement, etc, which differentiates it from other virus species/players. Application of game theory to the interactions between plant viruses during mixed infection has yielded interesting insights into the synergism between different species of viruses N. benthamiana plants infected with HLSV and/or TMV under co-infection. The collected short term data was then used to model their interactions and to predict their long term interaction outcome.In this study, we investigated the virus accumulation in To compare the fitness of the two viruses under single and mixed infections, we computed the relative virus accumulation. It was computed as the ratio of the amount of viral RNA accumulated in each infection compared with the values obtained for HLSV in single infections. The relative means ± SEM of viral RNA accumulation for each virus at 1 and 2 dpi, respectively, with single and mixed infections, were compared , using tAt 2 dpi, HLSV accumulated at 0.983±0.237, a 1.7% decrease but the decrease was not statistically significant . HLSV takes time to replicate and as such the difference in viral RNA accumulation between 1 and 2 dpi was relatively small. On the other hand, TMV increased by 241% to 3.299±0.176 . In mixed infections, HLSV accumulation in 2 dpi decreased to 0.146±0.045, an 85.1% decrease . Similarly, TMV level in mixed infections experienced a 1.85% decrease, to 3.238±0.664 . The presence of HLSV lowers the relative amount of TMV, as observed on the first day.The relative accumulation of the two viruses allows us to examine their relative fitness. We have constructed the pay-off matrix by arranging the relative accumulation of virus in single and mixed infections for both days .d = 0.967±0.354>c = 0.730±0.335) but a lower fitness when competing against HLSV (a = 1.000±0.113>b = 0.629±0.162). Modelling with one of the four scenarios described above, the two viruses were in Nash equilibrium; both viruses had equally competitive strategies and were able to overcome each other, depending on which strategy was established first. One of the prediction methods is to calculate the expected pay-off values, or fitness of each player TMV had a higher fitness when competing against its own population . The x-value (0.556) of the intersection between two lines represents the proportion of HLSV in an ESS. It is estimated that in a population consisting of 55.6% HLSV and 44.4% TMV, both viruses will have the same fitness value. However, such condition is difficult to achieve in actual experiments, because viruses cannot be perfectly mixed in a biological system. Also, any fluctuations of initial virus amount could lead to oscillations and chaotic behaviours d = 3.299±0.176>c = 3.238±0.664) or against HLSV (c = 3.238±0.664> a = 0.983±0.237). The expected pay-off for TMV was calculated to be 3.3135±0.3435, being significantly higher than that of HLSV (0.5645±0.1206). Using Straffin chart as an alternative method . Hence, these two methods proved to be useful in making long term predictions using short term data.To investigate the validity of our proposed model, we conducted a literature search of scientific papers published on plant virus co-infection and characterization of interactions of two viruses in a single host plant. A total of 14 virus-virus interactions out of 9 papers that contained data on the short term and long term quantifications of the viruses were investigated N. benthamianaN. benthamiana, we were unable to determine the upper line in the Straffin chart because of the relatively large error bars . Chenopodium amaranticolor plants at 12-leaf stage were inoculated with the diluted HLSV or TMV (in 10 µl) in a Latin square design, followed by 8 h post-inoculation of dark treatment. Plants were grown under 16 h light and 8 h dark at 23°C. The number of local lesions on each inoculated leaves was counted at 6 dpi as an indication of the number of virus infectious unit. Ten ng each of HLSV and TMV provide comparable amount of viral infectious units.The concentrations of purified HLSV and TMV virons were measured spectrophotometrically using a Nanodrop spectrophotometer (Nanodrop Technologies) in triplicates, after which serial dilutions were performed . Both single and mixed infections were carried out on two new fully expanded leaves per plant. A mock control group at 6–8 leaf stage was treated with the same amount of inoculation buffer. A total of 23 plants were used for each HLSV, TMV, mixed and mock inoculations. The plants were kept at 16 h light and 8 h dark at 23°C until sample collection at 1, 2 and 8 dpi. Half of the infected leaf was cut and stored at −80°C for total RNA extraction. The remaining half of the leaf was left on the plant for observation of symptom expression in order to confirm the infection.Ten ng of HLSV or TMV were inoculated on each chosen leaf of N. benthamiana leaves as described 5′-AGAGGTCCAAACCAAACCAG-3′) or HLSV (5′- AGCCCAGGATAAACCTGAAG-3′) primer; for mixed infection, both primers were added (1 pmol each). Actin gene was used as an internal control and was amplified using 1 pmol of oligo-dT primer.Total RNAs were extracted from collected 5′-TAGAGTAGACGACGCAACGG-3′) and qTMV-R (5′-AGAGGTCCAAACCAAACCAG-3′) or qHLSV-F (5′-GAGACTTTGAATGCAACGCA-3′) and qHLSV-R (5′-AGCCCAGGATAAACCTGAAG-3′). All four primers were included for cDNA obtained from mixed infection samples. The actin gene in each sample, which was used as the reference gene, was amplified in triplicates using primers qActin-F (5′-CTTGAAACAGCAAAGACCAGC-3′) and qActin-R (5′-GGAATCTCTCAGCACCAATGG-3′). Relative viral RNA accumulation was determined using 2−ΔΔCt method All the cDNA samples were subjected to 50 × dilutions for qPCR analysis using BioRad CFX384 Real Time System. Each cDNA was amplified in triplicates each containing 0.5 µl cDNA, 2.5 µl 2 × SYBR Green PCR Master Mix (KAPA), 20 nM of the primers qTMV-F  and Wheat streak mosaic rymovirus (WSMV) in N84Ht corn .(TIF)Click here for additional data file.Figure S2Straffin chart for the interactions between Zucchini yellow mosaic virus (ZYMV) and Cucumber mosaic virus (CMV) in zucchini cv. Escandarani .(TIF)Click here for additional data file.Figure S3Straffin chart for the interactions between Sweet potato chlorotic stunt virus (SPCSV) and Sweet potato feathery mottle virus common strain (SPFMV-C) in sweet potato .(TIF)Click here for additional data file.Figure S4Straffin chart for the interactions between Sweet potato chlorotic stunt virus (SPCSV) and Sweet potato virus G (SPVG) in sweet potato .(TIF)Click here for additional data file.Figure S5Capsicum annuum cv. Early Calwonder .Straffin chart for the interactions between Cucumber mosaic virus (CMV) and Pepper mottle virus (PepMoV) in (TIF)Click here for additional data file.Figure S6Straffin chart for the interactions between Zucchini yellow mosaic virus (ZYMV-SD) and Cucumber mosaic virus (CMV-Fny) in cucumber .(TIF)Click here for additional data file.Figure S7Straffin chart for the interactions between Zucchini yellow mosaic virus (ZYMV-SD) and Cucumber mosaic virus (CMV-Fny) in bottle gourd .(TIF)Click here for additional data file.Figure S8Nicotiana benthamiana .Straffin chart for the interactions between Tomato chlorosis virus (ToCV) and Tomato infectious chlorosis virus (TICV) in (TIF)Click here for additional data file.Figure S9Physalis wrightii .Straffin chart for the interactions between Tomato chlorosis virus (ToCV) and Tomato infectious chlorosis virus (TICV) in (TIF)Click here for additional data file.Figure S10Arabidopsis thaliana .Straffin chart for the interactions between Turnip mosaic virus (TuMV) and Cauliflower mosaic caulimovirus (CaMV) in (TIF)Click here for additional data file.Figure S11Nicotiana benthamiana .Straffin chart for the interactions between Tomato rugose mosaic virus (ToRMV) and Tomato yellow spot virus (ToYSV) in (TIF)Click here for additional data file.Figure S12Straffin chart for the interactions between Tomato rugose mosaic virus (ToRMV) and Tomato yellow spot virus (ToYSV) in tomato .(TIF)Click here for additional data file.Figure S13Straffin chart for the interactions between Triticum mosaic virus (TriMV) and Wheat streak mosaic virus (WSMV) in wheat cv. Tomahawk .(TIF)Click here for additional data file.Figure S14Straffin chart for the interactions between Triticum mosaic virus (TriMV) and Wheat streak mosaic virus (WSMV) in wheat cv. Arapahoe .(TIF)Click here for additional data file.


The control of foot-and-mouth disease virus (FMDV) outbreaks in non-endemic countries relies on the rapid detection and removal of infected animals. In this paper we use the observed relationship between the onset of clinical signs and direct contact transmission of FMDV to identify predictors for the onset of clinical signs and identify possible approaches to preclinical screening in the field. Threshold levels for various virological and immunological variables were determined using Receiver Operating Characteristic (ROC) curve analysis and then tested using generalized linear mixed models to determine their ability to predict the onset of clinical signs. In addition, concordance statistics between qualitative real time PCR test results and virus isolation results were evaluated. For the majority of animals (71%), the onset of clinical signs occurred 3–4 days post infection. The onset of clinical signs was associated with high levels of virus in the blood, oropharyngeal fluid and nasal fluid. Virus is first detectable in the oropharyngeal fluid, but detection of virus in the blood and nasal fluid may also be good candidates for preclinical indicators. Detection of virus in the air was also significantly associated with transmission. This study is the first to identify statistically significant indicators of infectiousness for FMDV at defined time periods during disease progression in a natural host species. Identifying factors associated with infectiousness will advance our understanding of transmission mechanisms and refine intra-herd and inter-herd disease transmission models. Aphthovirus genus within the Picornaviridae family, is the causative agent of foot-and-mouth disease (FMD), one of the world’s most important infectious animal diseases, responsible for huge global losses of livestock production and trade, as well as frequent and highly disruptive large-scale epidemics [Foot-and-mouth disease virus (FMDV), a member of the pidemics ,2. The dpidemics ,4.There are seven immunologically distinct serotypes and more than 60 antigenic variations ,6 and maA recent publication  reportedThe aim of the present study was to utilize the relationship between the onset of clinical signs and direct contact transmission of FMDV to identify possible predictors of the onset of clinical signs as well as identify candidates for preclinical screening in the field. Such information will advance our knowledge of the transmission mechanisms and improve the model predictions that are used in disease control. The assumption that the likelihood of transmission is decreased if control can be implemented just 24 h earlier provides strong support for investment in the development of practical tools for preclinical diagnosis. If we can identify infected cattle before they show signs of disease using tests in the laboratory then perhaps these can be used in the field during an outbreak. Measures of concordance between qualitative real time (qRT)-PCR results and virus isolation results were also determined in each experiment. These measures of concordance are useful in evaluating the performance of both methods of virus detection.Details of the methods used in this paper have been published elsewhere  but are 5.7 TCID50 of cattle adapted FMDV O UKG 34/2001. Forty eight hours after challenge naïve animals  were introduced to the inoculates and were challenged by direct contact exposure for 24 h. The inoculates were removed from the study and the animals exposed to infection (n = 8) were used to attempt transmission to further naive cattle (“recipients”) at two, four, six and eight (in experiments 3 and 4 only) days post infection for a period of 8 h each time. A total of 28 recipients were used in this study design.Four individual animal experiments using 100–150 kg Holstein Friesian calves were performed. For each experiment, two animals (referred to later as inoculates) were selected at random, and were needle challenged intradermolingually (1) with 1 × 10Individual donors were examined daily for the presence of clinical signs . Rectal temperature was also recorded. Blood and nasal fluid samples were taken daily for the first 8 days following challenge and then every other day for up to 14 days after challenge. The samples were transferred immediately to the laboratory for processing; nasal fluid was stored at −80°C and heparinised blood aliquoted and stored for subsequent virus detection in bovine thyroid cells (BTY) culture as described earlier . Aliquot50) of airborne FMDV per animal per challenge period (8 hrs).Several air samples using multiple devices were collected simultaneously, each hour, during all but 2 of the 28 challenge periods. Air samples were collected using an all-glass Cyclone sampler (operated for 5 min at a flow rate of approximately 390l/min) and an all glass Porton impinger sampler (operated for 5 min at a flow rate of 11l/min). These sampling periods were determined as the optimal sampling configurations for the instruments -22. The Live virus was detected in the biological samples collected  and in the collecting media from the air samples using BTY primary cell cultures ,23,24. GRNA  was extracted using the MagNA Pure LC total nucleic acid isolation kit  and an automated nucleic acid robotic workstation according to the manufacturer’s instruction . The samples were eluted in a volume of 50 μL and stored at −80°C for later analysis.As these experiments were performed with the O UKG 34/01 isolate, a homologous RNA standard was used  of FMDV O UKG 34/01) as described by Quan et al. . The revTo determine the amount of FMDV RNA in extracts of the total nucleic acid from blood, OPF, and nasal fluid a qRT-PCR was performed according to the methodology previously described ,32. In tSerum samples were tested for the presence of antibodies to FMDV using a liquid phase blocking ELISA (LPBE) ,36.Type-I interferon (IFN) biological activity was measured in serum samples from donor animals by using an Mx/chloramphenicol acetyltransferase (Mx/CAT) reporter gene assay .10 TCID50 per mL of nasal fluid was set because values below this threshold may be misleading and may represent low viral titres or insufficient sample collection due to limited nasal secretions. The baseline for Type 1 IFN of >1 IU per ml serum had been established previously [Table eviously . For allIdentification of the predictors of the onset of clinical signs was performed using a Generalised Linear Mixed Model (GLMM). The onset of clinical signs (as opposed to transmission) was chosen as the response variable as it was previously shown that there is a significant association between transmission and the onset of clinical signs  see Fig. Onset oOnly data up to and including day 8 were used in the statistical analyses as after day 8 data was not collected daily. In addition, all donor animals had exhibited clinical signs by day 8. To account for the repeated measures structure in the data (i.e. daily sampling) analyses using multiple explanatory variables were performed using a GLMM with covariance pattern model . The GLMM was fitted with a binomial distribution and a logit link function. The response variable was the presence or absence of any clinical signs. Donor was fitted as the sole random effect, other variables were fitted as fixed effects. Given the small sample size (n=8 donors) only single factor models were generated. In total 63 variables were analysed.χ2/DF) was used to compare the fit of different models. Unfortunately no significance test of model fit is available, however, the χ2/DF can identify models that given their fixed and random effect specifications more (or less) closely meet the specified distribution . The closer the χ2/DF is to unity the better the model and data meet the assumed residual distribution.Diagnostics were performed and plots of residuals were examined, confirming the goodness-of-fit of each model. Odds ratios (OR) and their associated 95% confidence intervals were estimated in the final models for factors statistically significantly associated with the onset of clinical signs. The generalised chi-square/DF (Virus in the blood and OPF was measured using both the virus isolation method (from heparinised blood samples) and the qRT-PCR method (from serum samples). The relationship between the two methods was examined by looking at the temporal trends using PROC LOESS (SAS version 9.3). LOESS is a nonparametric method for estimating local regression surfaces. In addition, agreement between the two methods was examined using Cohen’s kappa .Preclinical predictors were identified in the data by examining the virological and immunological variables in the time frame surrounding the onset of clinical signs. Data from ±4 days from the onset of clinical signs for each donor animal were included.p <0.05 would be reported as exhibiting formal statistical significance.Prior to analysis, it was specified that results with As previously described  there wep = 0.021) with the onset of clinical signs  of airborne FMDV per animal per challenge period (8 h). Data were available for only 26 transmission days, 2 days were missing. The total amount of virus in the air ranged from 3–4.5 TCID50, therefore values for airborne virus were treated as either detected or not detected. A positive air sample was statistically significantly associated with transmission of FMDV  curve analysis was used to determine the best cut-off for the virological variables measured in this study Figure . The cutTable The pattern of virus detection with time (days) was similar for both the qRT-PCR and the virus isolation (VI) methods Figure . InteresFigure In summary, virus is first detectable in the OPF, but detection of virus in the blood and nasal fluid may also be good candidates for preclinical indicators. Interestingly, the donor that did not transmit in this study never had any measurable amount of virus in the blood. However, virus was detectable in the OPF and nasal fluid.p < 0.05). On the day that clinical signs appear temperature increases by an average of 1°C  were positively associated with onset of clinical signs. This result is not surprising given that they were chosen a priori as useful measures of the transmission of FMDV . It doesAir sample results were not included in previous analysis of this data  because Temperature was a good indicator of the onset of clinical signs and of transmission . HoweverFor OPF there was no difference between the two methods of virus detection. The levels recorded using qRT-PCR tended to be higher at the peak but for both methods there was perfect agreement with respect to detection of virus. Measurement of virus in the blood using the VI method resulted (in some cases) in low levels of virus being detected earlier. Virus was detectable by qRT-PCR (in some cases) even when it was not using the VI method, but this always occurred later in the course of infection. It is unknown whether the virus detected by qRT-PCR at these stages is inactivated or at such a low concentration that it is unable to be detected by virus isolation.This analysis has confirmed the close association between the onset of clinical signs and the transmission of FMDV from an infected bovine reported previously . In addiIt has been argued that early detection of FMDV infection was critical to effective control of outbreaks and could help remove the need for pre-emptive culling . As clinThe authors declare that they have no competing interests.Conceived and designed the experiments: BC, MEJW, BMB, NDJ, PVB, SJC. Performed the experiments: BMB, NDJ, DG. Analyzed the data: MCT, IH. Contributed reagents/materials/analysis tools: BMB, NDJ, DG, MW, ER, SJC. Wrote the paper: MCT, MEJW. Revised the draft for important intellectual content: PB, BC, MEJW. Approved the final version for publication: BMB, NDJ, MCT, DG, SJC, MW, ER, PVB, RH, IH, MEJW, BC. All authors read and approved the final manuscript.Paul V Barnett and Bryan Charleston are Jenner Institute investigators.


To the Editor: Sandfly fever Naples virus, Sandfly fever Sicilian virus, and Toscana virus  have been recognized as etiologic agents of human illnesses in European countries bordering the Mediterranean Sea. These viruses are responsible for rapidly resolving diseases with nonspecific symptoms such as fever and myalgia. However, infection with Toscana virus may involve the central nervous system; severity may range from aseptic meningitis to meningoencephalitis  and elevated γ-glutamyltransferase (104 IU/L) and C-reactive protein levels (57 mg/L). Cerebrospinal fluid (CSF) analysis showed 3,500 leukocytes/μL , and glucose and protein levels of 2.5 mmol/L and 2.749 mg/L, respectively. Blood and CSF cultures were bacteriologically sterile. Polymerase chain reaction (PCR) assays of CSF for herpes simplex virus were also negative. The patient received intravenous amoxicillin and acyclovir for 3 days. The patient recovered after 6 days without sequelae.Serum and CSF samples collected during the acute phase were tested for immunoglobulin (Ig)M and IgG antibodies to a battery of arboviruses. These samples contained no antibodies  to flaviviruses, dengue virus, West Nile virus, and tickborne encephalitis, Tahyna virus, or Sandfly fever Sicilian virus . HoweverAedes albopictus) and Vero (E6 clone) monolayers. Toscana virus was found only on Vero cells by indirect immunofluorescence by using mouse hyperimmune ascitic fluid against Toscana virus. In contrast, no fluorescence was found by using mouse hyperimmune ascitic fluids against Rift Valley fever and Sandfly fever Sicilian virus.Virus isolation was attempted by incubation of peripheral blood mononuclear cells and CSF collected on the day of onset with C6/36 (S segment of Toscana virus genome was partly amplified from culture supernatants by reverse-transcription PCR and sequenced . NucleotPhlebotomus vectors, has been recognized as a major cause of aseptic meningitis in Italy and Spain. P. perniciosus, proven to be a vector of Toscana virus (Toscana virus, transmitted to humans by na virus , is abunna virus . Thus, TFinally, this work suggests that, in addition to West Nile virus, Toscana virus should now be considered as a potential etiologic agent of acute meningitis in the southeastern part of France. Entomologic and epidemiologic surveys, however, will have to be conducted in the near future to determine the risk for the people living in that area.