Our label-free optical imaging method does require neither elaborated sample preparation nor complex optical systems, offering a good platform for rapid and sensitive counting of viral particles.Here we report label-free optical imaging of single particles of the influenza virus attached on a glass surface with a simple objective-type total internal reflection dark-field microscopy (TIRDFM). The capability of TIRDFM for the imaging of single viral particles was confirmed from fine correlation of the TIRDFM images with fluorescent immunostaining image and scanning electron microscopy image. The density of scattering spots in the TIRDFM images showed a good linearity against the virus concentration, giving the limit of detection as 1.2×10 The rapid detection of viruses, especially the influenza virus, in the early stage of infection is important for preventing pandemics. Conventional methods of virus detection include the plaque-formation assay of infective viruses on cultured mammalian cells, the enzyme-linked immunosorbent assay (ELISA) of viral proteins, and the amplification of viral genomes by the polymerase chain reaction. Another approach that is useful for virus detection is the optical imaging of single viral particles under an optical microscope. Single viral particles can be imaged with fluorescent-labeled antibodies by using a fluorescence microscope. Although fluorescence imaging enables quantitative estimation of the virus concentration in a sample by counting single particles, it is time consuming and is hampered by the photobleaching of fluorescent dyes.The optical imaging of a single influenza viral particle without labeling may provide great benefits for rapid diagnosis. However, label-free imaging of single viral particles with conventional optical microscopy is difficult because of their small size (∼100 nm) and small difference in refractive index between viral particles and surrounding medium. Thus far, several methods of label-free optical imaging for single viral particles have been reported, including interferometric measurement 4 plaque-forming units per milliliter (pfu/mL). Our label-free method will offer a good platform for rapid and sensitive counting of viral particles.Here, we report a method for label-free imaging of single influenza virus particles by using a simple dark-field microscopy. In the present work, we employed objective-type total internal reflection dark-field microscopy (TIRDFM), which has been previously developed and used for high-speed single-particle imaging of colloidal gold attached to single motor proteins This study was approved by Review Board of the institute of scientific and industrial research at Osaka University and Review Board of the graduate school of engineering at University of Tokyo.g for 20 min at 4°C. The supernatant was passed through a membrane filter with a pore size of 0.45 µm. The solution was then subjected to 10–60% sucrose density gradient centrifugation for 2 h at 120,000 g at 4°C in a swing-bucket rotor. After the centrifugation was completed, 2-mL aliquots of the gradient starting from the top of the tube were collected manually. Each fraction was analyzed using sodium dodecyl sulfate (SDS) polyacrylamide gels. The solutions from selected fractions were ultracentrifuged for 2 h at 120,000 g at 4°C in a fixed-angle rotor, and the pellet was suspended in phosphate-buffered saline (PBS). The concentration of infective influenza A/Puerto Rico/8/34 (H1N1) virus was determined by a plaque-formation assay 2 incubator, the cell sheets were stained with methylene blue solution and the plaques formed in each well were counted.Influenza A/Puerto Rico/8/34 (H1N1) was inoculated (50 pfu/egg) into the allantoic fluid of 11-day-old chicken embryonated eggs for 3 days at 37°C. The allantoic fluid solution was centrifuged at 2,500 N-hydroxysuccinimide ester (Cy3-NHS). After incubation for 90 min, non-reacted Cy3-NHS was removed using a desalting column .A goat anti-influenza A (H1N1) polyclonal antibody  was reacted with Cy3 conjugated with 2 in front of the objective lens. For fluorescence imaging of the same field of view, the perforated mirror was replaced with a dichroic mirror  and an emission filter , and objective-type TIRFM was performed. The laser power was set at 6.3 µW/µm2 in front of the objective lens. Fluorescence images were obtained with same CMOS camera at 60 frames/s.l-lysine  was infused into the sample chamber such that the surface of the coverslips was covered with it. After 20 min, the excess poly-l-lysine was washed away with PBS without Mg2+and Ca2+(PBS(-)). Then, purified viral particles diluted with PBS(-) were infused into the sample chamber and incubated at room temperature so that the viruses gradually and nonspecifically bound to the glass surface. After 1 h, the chamber was washed with PBS(-). Light scattered by the viruses on the bottom coverslip was then observed with TIRDFM.The sample chamber was constructed with 2 glass coverslips , cleaned using 10 M KOH, and separated by 2 spacers that were approximately 50 µm thick. Poly-6 pfu/mL viral particles were mixed with 400 nM Cy3-labeled antibody at a volume ratio of 1∶1 for 4 h at room temperature; the mixture was then stored on ice for 30 h. Subsequently, it was infused into the sample chamber. After 30 min, the sample chamber was washed using PBS(-). First, Cy3-labeled antibody was observed with TIRFM. After photobleaching of Cy3, the emission filter was removed and the dichroic mirror was replaced with a perforated mirror. Light scattered by the viral particles was then observed with TIRDFM. The total observed area was 2592 µm2. The scattering and fluorescence images were both analyzed using the ImageJ software . The scattering spots were defined as spots with an area composed of consecutive pixels in which each pixel had an intensity higher than the threshold intensity of 19 arbitrary units (AUs) in 8-bit (0–255 AU) images. This intensity threshold was determined to distinguish the scattering spots from the weak background scatter and to determine the XY coordinate of the spot. Similarly, fluorescence spots were defined as a spots with an area composed of consecutive pixels in which each pixel had an intensity above the threshold intensity of 25 AU in 8-bit (0–255 AU) images. This intensity threshold was used to distinguish the fluorescent spots from the background and to determine the XY coordinate of the spot.For observation of the same field of view with TIRDFM and TIRFM, 1.0×102. To count the scattering spots, the images were analyzed using the ImageJ software. The scattering intensity of each spot was defined as the average intensity over an 8×8-pixel area.The dependence of density of the scattering spot observed with TIRDFM on virus concentration was measured by changing the virus concentration in sample infused into the chamber. Total observed area for each concentration was set at 6480 µm5 pfu/mL) was infused into the sample chamber consisting of 2 Poly-l-lysine-coated glass coverslips. After 1 h, the excess virus suspension was washed away with PBS(-), and 2.5% glutaraldehyde was infused into the chamber so that the amino groups in the viruses were cross-linked and the viral particles were firmly immobilized on the surface of the glass coverslip. After observation with TIRDFM, the bottom coverslip was dried and coated with osmium. The sample was observed at 3,000–5,000×magnification using a scanning electron microscope with a tungsten filament as an electron source . The locations of the areas observed with TIRDFM and SEM were identified by an etched pattern on the glass coverslip. The ImageJ sofware was used to analyze both TIRDFM and SEM images.The virus suspension   at 8,000–15,000×magnification.The correlation between images obtained by different methods was assessed quantitatively as follows: For images obtained by TIRDFM and TIRFM, the XY coordinate of each spot in each image was calculated. Then, a single fluorescence (scattering) spot located at the nearest neighbor for a single scattering (fluorescence) spot was selected and the distance between these 2 spots was measured. To correlate the images obtained by TIRDFM and SEM, the SEM image was scaled and rotated to adjust the etched pattern markers on the glass coverslip because the magnification and orientation of images obtained by TIRDFM and SEM were different. After the adjustment, the spots in each image were analyzed as described above.6 pfu/mL) was infused into the sample chamber, which consisted of 2 Poly-l-lysine-coated glass coverslips. After 1 h, the excess virus was washed away with PBS(-), and 2.5% glutaraldehyde in PBS(-) was infused into the chamber so that the amino groups in the viruses were cross-linked and viral particles were firmly immobilized on the surface of the glass coverslip. The sample chamber was then disassembled, and the bottom coverslip was cut into 5 mm×5 mm-pieces and washed with distilled water. The coverslip pieces were rapidly frozen by contact with a pure copper block cooled in liquid helium , freeze-dried at −80°C for 15 min, and rotary-shadowed with platinum/carbon and carbon . The replicated virus immobilized on the surface was observed at 30,000×magnification with a conventional transmission electron microscope (TEM)  equipped with a side-mounted CCD camera  2.The virus solution  on the virus surface . The aver of the scattering object and is proportional to r6Consistent with the results of TEM observation, the distribution of the scattering intensity of each spot obtained with TIRDFM showed a single peak with a small population with a higher intensity . The sca−3 spots/µm2) plus 3 times the standard deviation (SD) of the background (4 pfu/mL, which is comparable to the LOD of the influenza virus determined using ELISA The density of the scattering spots at different virus concentrations was analyzed . The spockground . The LOD5 pfu/mL, the spot density of bound viruses was 0.065 spots/µm2 (5 pfu/mL would correspond to the density of only 3.1×10−5 viruses/µm2, if we assume that all virus particles have infectious ability and are bound to the glass surface. Therefore, the density of scattering spots was approximately ∼2000 times higher than that of viruses with infectious ability. It is known that most of influenza virus particles cannot accomplish infection because of defective infectious process including 8-genome packing. To understand the mechanism of infection and efficiency of infection, it is important to investigate the ratio of particles that have infectious ability by counting the influenza virus particle. In the previous studies using electron microscopy, the ratio of the observed particles to the particles with infectious ability was reported to be 10−100 In our experiment, concentration of viruses with infectious ability was determined in advance by plaque formation assay. When virus concentration was 1.0×10pots/µm2 . Conside4 pfu/mL, which is comparable to that for ELISA. The LOD can be further improved by selective immobilization of the virus on the glass coverslip by using an antibody. Specific binding of the virus to the glass surface may also permit virus detection in saliva and nasal mucus with TIRDFM. In addition, other viruses, typically those that are larger than the influenza virus, may also be detected with TIRDFM.In this study, we performed label-free imaging of single particles of the influenza virus with TIRDFM. The optical system for TIRDFM is simpler than other systems reported previously


The lack of effective antiviral drugs restricts the control of the dangerousRNA-containing influenza A (H1N1) virus. Extracellular ribonuclease of Bacilli(binase) was shown to manifest antiviral activity during single- andmulti-cycle viral replication in the range of concentrations non-toxic toepithelial cells and 0.01-0.1 multiplicity of infection. During antiviraltreatment for 15-30 min, the concentration of 1 μg/ml binase reduced theamount of focus-forming units of viruses by a factor of 3-10 and suppressed thevirus-induced cytopathic effect in A549 human lung cells. The possiblemechanisms of interaction between the virus and enzyme are discussed. Positivecharges in both binase and viral hemagglutinin cause electrostatic interactionwith negatively charged sialic acid on the host cell’s surface followedby its penetration into the cell. Capsid elimination and release of viral RNAfrom endosome to the cytoplasm allows catalytic RNA cleavage by internalizedbinase. The data obtained confirm that binase is an effective antiviral agentagainst the pandemic influenza A (H1N1) virus. Certain progress in this fieldis associated with clarifying the detailed mechanism underlying the antiviralaction of binase and development of the most effective way for its practicaluse. Ranapipiens, BS-RN ase from bovine testicles, and microbial RN ases fromBacillus amyloliquefaciens and B. intermedius aspotential therapeutic agents. Some cytotoxic RN ases are cancer-selective-3 and an pumilus ), barnas pumilus , thusre pumilus . Binase, pumilus , 8. An iresponse. Binase response.The search for effective antivirus products is an urgent task justified by thewide variability and global distribution of viruses. The aim of this study wasto examine the action of binase on the pandemic influenza A/Hamburg/04/09(H1N1) virus, the causative agent of the 2009 influenza epidemics. We foundthat a short-term (15-30 min) treatment of viruses with increasingconcentrations of binase caused a proportional 3- to 10-fold decrease in thevirus’ ability to infect A549 lung adenocarcinome cells. A binaseconcentration of 1 μg/mL was the most efficient and caused no inhibitionof epithelial cells viability; this fact suggests that binase could be apromising antiviral agent.Bacterial RNaseB. pumilus 7P, binase,  was homogenouslyisolated from the culture fluid of Escherichia coli BL21carrying the pGEMGX1/ent/Bi plasmid, according to A. Schulga etal. . The binase effecton the virus infectivity was analyzed with the infection of A549 cells with RNase preincubated with the virus for 15-60 min. The cells were dark-incubated atroom temperature for 1 h to adsorb the virus; the unadsorbed virus was removed,and the infected cells were incubated in DMEM supplemented with 0.36% of bovineserum albumin and 1 μg/mL of TPCK trypsin at 5% COStatistical analysisThe statistical analysis of the results from four runs of each experiment wasperformed by standard methods in Microsoft Excel 2010 and the SigmaPlot 10software.Binase cytotoxity against A549 adenocarcinoma cellsBinase at a concentration approaching 1 mg/mL of the medium (corresponding to82 μM) exerts a concentration- and time-dependent inhibiting  effect on the viability of A549 human lung adenocarcinomacells. The cytotoxic binase concentration that caused 50% cell lethality (CC50)was 420-490 μg/mL after a one- or two-day exposure, respectively A, B.A in vitro infectivity of virusparticles by penetrating into a virus’ capsid and destroying its viralgenomic RN A [Rana catesbeiana not only inhibitsthe replication of the Japanese encephalitis virus, but also stimulatesapoptosis in virus-infected cells [Most RN ases involved in antiviral cell protection are synthesized by the hostcells, and these enzymes direct the cells towards apoptotic death. In animals,the antiviral effect is exerted by ribonucleases from the RN Ase A family, 22, incmic RN A . Examinamic RN A . Onconasmic RN A ; howevered cells .We founed cells . This faBinase reduces the infectious titer of the influenza A (HIN1) virus6 FFU/mL.The hemagglutination assay showed that the initial virus fluid contained theA/Hamburg/04/09 virus with a hemagglutination titer of 32 HA units/mL.This va-4-101 μg/mL) for 30 or 60 min; the A549 cellswere infected with the resulting suspension, and the degree of their infectionat 0.1 MOI was estimated.To determine the antiviral effect of binase, the virus was co-preincubated withthe enzyme at concentrations of less than cytotoxic,Table 1Independently of the cell infectivity level, the maximum antiviral effect wasobserved after 30 min of binase- virus co-preincubation; a longer period (60min) of co-preincubation caused a threefold decrease in the antiviral effect.With a Thus, binase concentrations not toxic for A549 cells (1-10 μg/mL) inhibitthe reproduction of the influenza A (H1N1) virus when pretreated with binasefor 15-30 min.Immunofluorescent methods demonstrated that binase had penetrated into the A549cells even within the first hours of incubation .The catBinase demonstrates antiviral activity against the viruses of rabies, thehoof-and-mouth disease, some plant viruses, and the seasonal influenza virus, 10, 32.B. intermedius (binase) e antiviral toxicityhave no toxic effect on human epithelial cells. The pretreatment of viralparticles with binase at approximately a 1 μg/mL concentration caused asignificant (up to tenfold) decrease in virus infectivity and suppressed thedevelopment of the virus-induced cytopathic effect in a A549 human lung cellline at various multiplicity of infections both during single-cycle andmulti-cycle virus reproduction. Fine-tuned mechanisms of antiviral activityneed further examination, although it is conceivable that they include both thecharge-charge interaction between cation binase and the negatively chargedhemagglutinin receptors on the surface of a host cell and catalytic cleavage ofthe viral RN A inside cells. The data confirm the applicability of binase as aneffective antiviral agent against the pandemic influenza A (H1N1).The high death rates that accompany influenza impose a serious social andeconomic burden on society; therefore, containing pandemics of influenza Atype/H1N1 viruses is one of the urgent tasks facing the scientific community.Rapid spreading of a viral infection can be checked by using broad-specificitydrugs whose effectiveness is independent of particular mutations in thevirus’ genome. We have shown that concentrations of secretableribonuclease from


Anopheles mosquitoes collected in Xinjiang, China, in 1990. Recombinant plasmid pBR-XJ160 is an infectious full-length cDNA clone of XJ-160 virus, from which rescued virus BR-XJ160 can be obtained by transcription in vitro and transfection. The BR-XJ160 virus raised in BHK-21 cells was indistinguishable from the XJ-160 virus in its biological properties, including its plaque morphology, growth kinetics and suckling mouse neurovirulence. On basis of pBR-XJ160, the effects of substitutions within nonstructural protein 1 (nsP1) or nsP2 on the infectivity and pathogenesis of Sindbis virus (SINV) have been investigated. We have also confirmed the essential role of E2 glycoprotein, especially the domain of 145-150 (amino acid) aa, in SINV infection through the interaction with cellular heparan sulfate (HS). In addition, we have developed XJ-160 virus-based vector system, including replicon vector, defective helper (DH) plasmids and the packaging cell lines (PCLs). Here we provide an update of main development in the field concerned with XJ-160 virus.As a Sindbis-like virus (SINLV), XJ-160 virus was isolated from a pooled sample of  Alphavirus in the family Togaviridae that has more than 30 members . The detection method is simple, rapid and specific, being expected to be used in clinical examination and epidemiological surveillance as well as rapid screening of viral biological warfare agents.Anopheles mosquitoes collected in Xinjiang in 1990. Then its reverse system was constructed, on which we have performed a series of research work, including the effects of mutagenesis of nsPs on biological characteristics of SINV, the role of E2 glycoprotein in the nonreciprocal interaction between SINLV and SINV as well as the function of E2 glycoprotein interacting with HS in the cellular infection. We have constructed RNA- or DNA-based vector derived from XJ-160, and the PCLs for XJ-160 virus was also selected. Currently, we have labeled XJ-160 virus by inserting the reporter gene (EGFP or GLUC) into the genome. The labeled viruses were used to research on the cell tropism of SINV in detail. We first found that SINV could infection renal carcinoma cell persistently in large range, and this discovery indicate that SINV have the potential to be used in kidney cancer treatment.As a SINLV, XJ-160 virus was isolated from a pooled sample of The authors declare that they have no competing interests.WZ collected the references concerned with this review and drafted the manuscript, and GL also contributed to edit and revise the manuscript. Both authors read and approved the final manuscript.


Trichosanthes comprises 90–100 species that occur from India to Japan and southeast to Australia and Fiji. Most species have large white or pale yellow petals with conspicuously fringed margins, the fringes sometimes several cm long. Pollination is usually by hawkmoths. Previous molecular data for a small number of species suggested that a monophyletic Trichosanthes might include the Asian genera Gymnopetalum  and Hodgsonia . Here we test these groups’ relationships using a species sampling of c. 60% and 4759 nucleotides of nuclear and plastid DNA. To infer the time and direction of the geographic expansion of the Trichosanthes clade we employ molecular clock dating and statistical biogeographic reconstruction, and we also address the gain or loss of petal fringes.The Cucurbitaceae genus Trichosanthes is monophyletic as long as it includes Gymnopetalum, which itself is polyphyletic. The closest relative of Trichosanthes appears to be the sponge gourds, Luffa, while Hodgsonia is more distantly related. Of six morphology-based sections in Trichosanthes with more than one species, three are supported by the molecular results; two new sections appear warranted. Molecular dating and biogeographic analyses suggest an Oligocene origin of Trichosanthes in Eurasia or East Asia, followed by diversification and spread throughout the Malesian biogeographic region and into the Australian continent.Hodgsonia and Trichosanthes, followed by two losses in the latter coincident with shifts to other pollinators but not with long-distance dispersal events. Together with the Caribbean Linnaeosicyos, the Madagascan Ampelosicyos and the tropical African Telfairia, these cucurbit lineages represent an ideal system for more detailed studies of the evolution and function of petal fringes in plant-pollinator mutualisms.Long-fringed corollas evolved independently in  Trichosanthes[Deeply divided or fringed petal lobes are known from a range of angiosperm families, including Caryophyllaceae, Celastraceae, Cucurbitaceae, Myrtaceae, Orchidaceae, Saxifragaceae, and Tropaeolaceae . While thosanthes, Figure hosanthes,4. Experhosanthes,6. A preTrichosanthes with currently 90–100 species of mainly perennial, 3 to 30 m long climbers that are usually dioecious and have medium-sized fleshy fruits. Referring to the petal fringes, Linnaeus formed the genus name from the Greek words for 'hair' (genitive τριχός) and 'flower' (Άνθoς). Trichosanthes has its center of diversity in Southeast Asia, but ranges from India throughout tropical and subtropical Asia east to Japan, and southeast to New Guinea, Australia, and Fiji [T. cucumerina L., is a widely cultivated vegetable in tropical and subtropical regions around the globe, and another 15 species are commonly used in Asian traditional medicine [Trichosanthes is lacking  and unusual seeds. The petal margins in Gymnopetalum are entire  [lar data . Based ospecies; ) and Hodspecies; ) possiblspecies; . Both sh, Japan) . Trichosanthes occurring in the Flora Malesiana region have been grouped into six sections, the typical sect. Trichosanthes and sections Cucumeroides (Gaertn.) Kitam., Edulis Rugayah, Foliobracteola C.Y.Cheng & Yueh, Involucraria (Ser.) Wight, and Asterosperma W.J.de Wilde & Duyfjes [T. truncata C.B.Clarke, is in its own section, Truncata C.Y.Cheng & C.H.Yueh [Gymnopetalum have been allocated to two sections that differ in flower morphology, the typical sect. Gymnopetalum with just one species from southern India and Sri Lanka and sect. Tripodanthera (M.Roem.) Cogn. with three southeast Asian and Malesian species [Based on mainly fruit and seed characters, the 43 species of  Duyfjes ,22. The C.H.Yueh . The fou species .Trichosanthes using a broad sampling of some 60% of its species, including the type species of each section name, plus representatives of Gymnopetalum, Hodgsonia, and other Sicyoeae as well as more distant outgroups. The well-resolved phylogeny, combined with field observations on flower shape and color, allows us to test whether petal fringes in Old World Sicyoeae evolved just once as would be the case if Gymnopetalum and Hodgsonia were nested inside it [Trichosanthes clade.Here we test the monophyly and phylogenetic placement of nside it  or multiTrichosanthes is paraphyletic because Gymnopetalum is embedded in it, while Gymnopetalum is polyphyletic because its four species do not group together. Instead, G. tubiflorum (Wight & Arn.) Cogn. groups with species from sections Trichosanthes and Cucumeroides (1.00 PP/84 ML support), while G. orientale W.J.de Wilde & Duyfjes, G. chinense (Lour.) Merr., and G. scabrum (Lour.) W.J.de Wilde & Duyfjes are sister to section Edulis (1.00 PP/86 ML). The Trichosanthes/Gymnopetalum clade  is sister to Luffa, a genus of seven or eight species of which we included five. This sister group relationship, however, is only weakly supported  is only distantly related to the Trichosanthes/Gymnopetalum clade.Phylogenies obtained under Bayesian or Maximum Likelihood (ML) optimization revealed no statistically supported incongruences, defined as nodes with Bayesian posterior probabilities (PP) >0.95 or ML bootstrap support >75. A Bayesian consensus tree is shown in Figure Trichosanthes (see Background), three are supported by the molecular results, namely sections Asterosperma , Cucumeroides , and Edulis . Three other sections with more than one species  are not monophyletic in their current circumscriptions. To achieve a more natural classification, a revised infrageneric classification has been proposed including two new sections [Of the seven sections previously proposed in sections .Trichosanthes originated during the Oligocene , were approximately 120 m below those of today, resulting in the complete exposure of the Sunda Shelf; even sea level reduction by just 40 m already connected Indochina, Sumatra, Java, and Borneo [Collision between the Eurasian and Australian tectonic plates started in the Late Oligocene, about 25 Ma ago, and the Sahul Shelf (carrying New Guinea) and Sunda Shelf  reached their present proximity only by the Late Miocene, some 10 Ma ,34. Mid-d Borneo ,36. No ld Borneo ,38.Trichosanthes are numbered in Figure T. subvelutina F.Muell. ex Cogn. and its sister clade on the Asian mainland and areas of the Sunda Shelf, dated to 23.8 (29.4-18.4) Ma; (ii) the disjunction between T. edulis Rugayah, T. dentifera Rugayah, T. laeoica C.Y.Cheng & L.Q.Huang, T. schlechteri Harms from New Guinea, and T. odontosperma W.E.Cooper & A.J.Ford from Australia on the one hand, and Gymnopetalum chinense, widespread in Asia as far East as Flores, and G. orientale in Sulawesi, the Lesser Sunda Islands, and the Moluccas on the other (this is dated to 16.7 (22.1-11.2) Ma, but the position of G. scabrum relative to G. chinense and G. orientale remains unclear; compare Figures T. wawrae Cogn. from Thailand, peninsular Malaysia, Sumatra, and Borneo, and its sister clade T. papuana F.M.Bailey/T. pentaphylla F. Muell. ex Benth. from New Guinea and Australia, which dates to 7.1 (11.2-3.3) Ma.The most striking transoceanic disjunctions in  23.8 29.-18.4 Ma;Trichosanthes range expansion between New Guinea and Australia occurred during the Pliocene/Pleistocene, when these two regions were repeatedly connected due to the above-mentioned sea level changes [T. odontosperma  Ma, while that of the sister species pair T. papuana from the Aru Islands and New Guinea, and T. pentaphylla from Australia  also would require population-level sampling. Both species have fleshy red fruits and small seeds, probably dispersed by birds.The geographic history of F.Muell. ), a wideHodgsonia and Trichosanthes and were lost in three of the four species formerly placed in the genus Gymnopetalum . The two inferred losses , and it therefore seems likely that there is a shift from predominantly nocturnal sphingid pollinators to diurnal bee or butterfly pollinators. The loss of fringes does not coincide with long-distance dispersal events to insular habitats (where hawkmoths might be absent), and the trigger for the pollinator shifts so far is unknown.The phylogeny obtained here implies that long-fringed corollas evolved independently in the Asian genera Viola species, more complex corolla outlines correlate with higher fruit set [Trichosanthes-hawkmoth system. Another untested possibility is that the fringes with their highly increased surface area and exposed position might be involved in scent production  or produce a waving motion, which has been shown to increase pollinator attraction in other systems [Trichosanthes, wind tunnel experiments with naive hawkmoths, and detailed field observations are required to test these possibilities.The adaptive function of the corolla fringes in pollinator attraction requires experimental study. An innate preference for radial patterns  and highruit set  but it r systems . AnatomiGymnopetalum into a then monophyletic Trichosanthes[. Our molecular phylogenies reveal that long-fringed petals evolved independently in Hodgsonia and Trichosanthes/Gymnopetalum, followed by two losses of corolla fringes in the latter clade, most likely associated with pollinator shifts. Molecular dating and a biogeographic analysis indicate an Oligocene initial diversification of Trichosanthes in mainland Asia. The lineage then diversified and spread in Malaysia  during the late Miocene and Pliocene, reaching the Australian continent several times.Molecular evidence supports the inclusion of hosanthes. Our molTrichosanthes have corolla fringes, and these are absent in three of the four Gymnopetalum species, except G. orientale, which can have short-fimbriate petal margins (fringes up to 5 mm length).Herbarium specimens from A, BRI, CNS, E, GH, K, KUN, KYO, L, LE, M, MO, P, S, UC, UPS and US were obtained on loan or studied during herbarium visits. Determination of herbarium material was verified using identification keys -16,19,42rbcL and matK, the trnL and trnLtrnF intron and spacer, and rpl20-rps12 spacer. Data for rbcL and the trnL region were taken from previous studies [Hodgsonia, all four of Gymnopetalum, and 52 of Trichosanthes, representing approximately 60% of the accepted species in the latter genus. Type species of all sections were included: Gymnopetalum tubiflorum (Wight & Arn.) Cogn. , Gymnopetalum chinense (Lour.) Merr. (G. sect. Tripodanthera), Trichosanthes postarii W.J.de Wilde & Duyfjes (T. sect. Asterosperma), Trichosanthes pilosa Lour. (T. sect. Cucumeroides), Trichosanthes edulis Rugayah (T. sect. Edulis), Trichosanthes kirilowii Maxim. (T. sect. Foliobracteola), Trichosanthes wallichiana (Ser.) Wight (T. sect. Involucraria), Trichosanthes villosa Blume (T. sect. Pseudovariifera), Trichosanthes cucumerina L. (T. sect. Trichosanthes), Trichosanthes truncata C.B.Clarke (T. sect. Truncata), Trichosanthes subvelutina F.Muell. ex Cogn. (T. sect. Villosae). Species names and their authors, specimen voucher information, and GenBank accession numbers for all sequenced markers (including 262 new sequences) are summarized in Table We included six DNA regions, namely the nuclear ribosomal ITS region (ITS1-5.8S-ITS2), the chloroplast genes  studies ,20,43,44Total DNA was extracted using the Carlson/Yoon DNA isolation procedure  and a MimatK with primers matK-2.1a [matK-1440R [rpl20rps12 using the primers rpl20 and rps12[Polymerase chain reaction (PCR) amplification of purified total DNA was performed in 200 μl reaction tubes with a total volume of 50 μl. Each tube contained a mixture of 5 μl reaction buffer , 3 μl MgCl2 (25 mM), 1 μl dNTP’s (10 μM), 0.25 μl Taq-polymerase , 0.25 μl BSA (Roche Diagnostics), 12.5 μl of each primer (2 mM), 14.5 μl Milli-Q water and 1 μl template DNA. The ITS region was amplified using the primer pair ITS-P17 and ITS-26 S-82R  with theatK-2.1a  and matKtK-1440R , 95° 5 mand rps12, 95° 5 mrpl20-rps12 (100% of taxa), ITS (96%), matK (84%), trnL-F spacer (31%), trnL intron (28%), and rbcL (20%). The three latter regions increased statistical support values at early-branching clades. Sequences were concatenated, and gap-coded using the Simmons and Ochoterena simple method [Sequence trace files were compiled into contigs with the program Gap4 and edited using Pregap4 , both pae method  implemene method .Selection of best-fit models of nucleotide substitution for the nuclear and plastid data partitions relied on the Akaike Information Criterion (AIC and AICc) as implemented in JModelTest version 0.1.1 ,56. LikeBayesian tree searching used MrBayes  on the Chttp://www.treebase.org; study number 12339).The data matrix and trees have been deposited in TreeBASE . Absolute ages were taken from the geologic time scale of Walker and Geissman [Calibration relied on Cucurbitaceae fossils assigned to particular nodes  TheBulgaria  dated toBulgaria  dated toBulgaria . (C) TheGeissman . We alsoBiogeographic reconstruction relied on statistical dispersal-vicariance analysis using S-DIVA version 2.0  as impleHB conceived the study, carried out the molecular genetic analyses, and drafted the manuscript. HS participated in the design of the study and data analysis, and also contributed field observations. SR and MT participated in the design and coordination of the study, and SR also helped with clock calibration and writing. All authors read and approved the final manuscript.


Bunyaviridae) are small mammal-associated zoonotic and emerging pathogens that can cause hemorrhagic fever with renal syndrome (HFRS). Puumala virus, the main etiological agent carried by the bank vole Myodes glareolus is responsible for a mild form of HFRS while Dobrava virus induces less frequent but more severe cases of HFRS. Since 2000 in Europe, more than 3000 cases of HFRS have been recorded, in average, each year, which is nearly double compared to the previous decade. In addition to this upside long-term trend, significant oscillations occur. Epidemic years appear, usually every 2–4 years, with an increased incidence, generally in localized hot spots. Moreover, the virus has been identified in new areas in the recent years. A great number of surveys have been carried out in order to assess the prevalence of the infection in the reservoir host and to identify links with different biotic and abiotic factors. The factors that drive the infections are related to the density and diversity of bank vole populations, prevalence of infection in the reservoir host, viral excretion in the environment, survival of the virus outside its host, and human behavior, which affect the main transmission virus route through inhalation of infected rodent excreta. At the scale of a rodent population, the prevalence of the infection increases with the age of the individuals but also other parameters, such as sex and genetic variability, interfere. The contamination of the environment may be correlated to the number of newly infected rodents, which heavily excrete the virus. The interactions between these different parameters add to the complexity of the situation and explain the absence of reliable tools to predict epidemics. In this review, the factors that drive the epidemics of hantaviruses in Middle Europe are discussed through a panorama of the epidemiological situation in Belgium, France, and Germany.In Europe, hantaviruses ( Bunyaviridae) are carried by rodents, insectivores, and – as recently confirmed – by bats  is located in this region. The Ardennes extend into N-France and in W-Germany.Although scattered data concerning clinically apparent human hantavirus infections are available from 1976 on in Belgium, this was only reliably and on a national level recorded from 1996 on by the Reference Laboratory for Vector-Borne Diseases (RLVBD) in Brussels, that was appointed National Reference Laboratory for Hantavirus Infections; the data were regularly reported to the Scientific Institute of Public Health (SIPH), the reporting organism to the public that is usually – but erroneously – cited as the source of the data  is the only hantaviral serotype that is known to infect humans in Belgium, it induces a mild form of HFRS called nephropathia epidemica (NE); on average 150 cases occur yearly. From 2005 on there is a definite increase in the yearly number of NE cases in Belgium , carried by the bank vole  act as dead-end hosts. So far, only infected humans can become ill although no sufficient evidence exists to exclude other mammals as competent hosts  or performing work that disturbs carrier nests  or other activities that put the host in prolonged contact with the carrier’s habitat  outside the carrier if conditions are favorable  and foxes (Vulpes vulpes) are on the rise since the 1980s in accordance with bank vole population fluctuations  that predate on Microtus arvalis, M. glareolus, A. sylvaticus, Apodemus flavicollis, Rattus norvegicus) and two insectivore species  are readily captured in live traps. In the study Ugglan Special No2 traps  that are suitable for trapping the vast majority of Apodemus, Myodes, Microtus, Sorex, and Crocidura species were used. Talpa europaea is also common but requires a specific trapping strategy due to its subterranean life. In Belgium, M. arvalis is the carrier for Tula virus  but this virus is so far not found in Belgium, R. norvegicus carries Seoul virus (SEOV) which is readily present in Belgian rural brown rats  in Belgium just by climate change is difficult because the key years for changes in hantavirus activity were 1999 and 2005; if milder climatic conditions caused the increase in cases, these events should have been visible much earlier as the key years for climatic changes in Belgium occurred long before Figure . If – ev–2011 in “Mast” is a term used to describe the seeds of shrubs and trees that are eaten by wildlife. “Hard mast” refers to nuts of beech and oak, whereas “soft mast” refers to leaves, flowers, and berries of a variety of species. A number of different levels of mast are used such as full, half, dispersed, or deficient but these criteria are not always quantitatively measured. The mast theory – although recently claimed by some in Belgium – is by no means new. Charles Elton (1900–1991) was the first to systematically study animals in their natural habitats and their interactions with their surroundings Elton, .Hundreds of animal species use mast as staple food. No less than 171 species  are known to depend on it in beech or oak stand habitats  and 95–1,292 (March to October) flowering plant speciesSus scrofa (wild boar) or Capreolus capreolus (roe deer) and various bird species, it seems obvious that rodents need to rely on other food sources for most – if not the whole – of the year. Besides evidence for an animal-based food preference in spring  represents more than 36% of the total tree stand composition  remains possible  were detected during the peak of prevalence in bank voles  captured in the Jura region during 2002, 2003, and 2004  trapped in the Department of Jura, close to the Swiss border  is in charge of hantavirus infection surveillance in France. The serological diagnosis relies on indirect immunofluorescence since 1982. In 1993, a confirmation with the research of specific IgM antibodies using an enzyme-linked immunosorbent assay (ELISA) has been added  see a summer peak in human PUUV cases. Figure Apodemus agrarius-associated strain DOBV-Aa (“Saarema”) was found in A. flavicollis in the Federal States of Mecklenburg-Western Pomerania, Lower Saxony, Brandenburg, and also in Schleswig-Holstein , and other parameters are still at an early stage. This also includes factors that might have a direct impact on the virus stability in the environment. In general, Germany consists of a broad variety of landscapes formed by former glaciers. In the North these include the Islands of the North- and Baltic Sea, the North German Plain. In Central Germany uplands and numerous river valleys, e.g., Rhine are present. Southern Germany is characterized by several linear hills and high mountain ranges such as Swabian and Franconian Alb, Bohemian Forest, and the Alps. There are three main biogeographic regions, the Atlantic, the continental-middle European, and the alpine . Here, increased species diversity was correlated with higher PUUV estimates in the host rodent, which is in line with the “Amplification Effect.” With regard to this phenomenon several hypotheses may be formulated: (i) species other than the primary host could have the ability of functioning as a further reservoir for PUUV and thus lead to an increased transmission of the virus and overall prevalence; (ii) the presence of a species with a low reservoir capability could lead to higher contact rates between the primary hosts and subsequent higher transmission of the virus . In conclusion, despite the important aspects of deadwood for a healthy ecosystem and the tendency to launch such traditional and untouched forests enhanced deadwood may be one factor to increase a risk for PUUV infections.Landscape attributes might have a beneficiary effect on rodent host population dynamics. There exists excellent data on the influence of topography and soil properties on dynamics of common voles from almost 40 years collected in eastern Germany. The analyses showed for example that mean elevation, area-related percentage of Chernozem soils, and soil air capacity are variables that have an influence on the common vole outbreak risks at different sites  bank voles may be more dependent on tree seeds such as beechnut. Data from Lower Saxony from 16 years on beech fructification and the abundance of bank voles showed that the fruits have a positive effect on the population in the next year. For negative trends the context between fruit and vole was true in 75% of the cases  has an influence on population dynamics of bank voles. In contrast to other rodent species . Interestingly, as compared to other years between 2001 and 2007, in 2005 there also seemed to be a beech crop failure (Piechotowski et al., Mild winters and springs may positively influence rodent survival rates and early food supply and thus affect rodent population dynamics. The density of the rodent population may well drive the distribution and prevalence of disease/associated agents in the rodent host. For As described earlier the viability of a hantavirus outside the host may be dependent on climatic factors such as temperature and moisture. Yet, additional factors such as UV radiation may also have an impact on virus tenacity. In experimental studies for appropriate inactivation of Hantaan virus the virus was effectively inactivated through UV irradiation in cell cultures and supernatants (Kraus et al., There is good evidence that in the wild PUUV-prevalences in bank voles are age-dependent (Bernshtein et al., R. norvegicus and Calomys musculinus show a similar pattern regarding transmission of SEOV and Junin virus, respectively (Mills and Childs, Male bank voles are believed to be of greater risk of acquiring and spreading PUUV and thus play an important role in the dynamics of PUUV epidemics (Bernshtein et al., Our findings indicate that the occurrence of clinically apparent human hantavirus infections in Belgium, France, and Germany are highly variable in time and space. Comparing the epidemiological features in the three forenamed countries also indicated that hantavirus outbreaks in our three countries are also not always triggered by the same factors.We defined six major groups that could act as triggers or regulators of hantavirus outbreaks in Western Europe; behavioral factors of reservoir and host, landscape and changes in land-use, climate, food availability, biodiversity, and physiological features of the reservoir population. Care should however be taken not to generalize the relationships as some have already been proven wrong in the past (Brown and Ernest, The ever-present pitfall however, is relating the occurrence of infectious diseases in general and hantavirus infections in particular to one particular trigger, e.g., food availability. Climate change, for instance, is a likely facilitator but climate change is an ancient and ongoing process (Ruddiman, The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


It has been shown that visual geometrical shape categories (rectangle and triangle) are graded structures organized around a prototype as demonstrated by perception and production tasks in adults as well as in children. The visual prototypical shapes are better recognized than other exemplars of the categories. Their existence could emerge from early exposure to these prototypical shapes that are present in our visual environment. The present study examined the role of visual experience in the existence of prototypical shapes by comparing the haptic recognition of geometrical shapes in congenitally blind and blindfolded adolescents.To determine whether the existence of a prototype effect  depended on visual experience, congenitally blind and blindfolded sighted adolescents were asked to recognize in the haptic modality three categories of correct shapes  varying in orientation  among a set of other shapes. A haptic prototype effect was found in the blindfolded sighted whereas no difference between prototypical and non prototypical correct shapes was observed in the congenitally blind. A control experiment using a similar visual recognition task confirmed the existence of a visual prototype effect in a group of sighted adolescents.These findings show that the prototype effect is not intrinsic to the haptic modality but depends on visual experience. This suggests that the occurrence of visual and haptic prototypical shapes in the recognition of geometrical shape seems to depend on visual exposure to these prototypical shapes existing in our environment. Geometrical shapes  can be considered as categories including an infinite number of particular shapes that share common properties “these subjective shape distributions originate not in simple empirical observation but rather in a nexus of subtle mental stereotypes about regularity of form and pattern”. The findings of Gentaz and his colleagues However, the origins of the bias toward prototypical shape are still in debate. Feldman,  Consequently, the question was to determine whether the presence of prototypical shapes holds true for objects encoded in the haptic modality. This question was not trivial because of several specificities of haptic perception Therefore, the comparison between congenitally blind and blindfolded sighted adolescents could allow us an evaluation of the role of visual experience in the manifestation of the prototype effect. In the latter populations, the haptic modality is coordinated with the visual modality whereas in the former one, it is not. Lederman et al. (1990) In the present research, we investigated therefore with the same haptic recognition task whether the “prototype effect” exists in haptics by testing a group of congenitally blind adolescents and a group of blindfolded sighted adolescents. The “prototype effect” corresponds to the better recognition of prototypical shapes than non prototypical shapes and occurs independently of the rate of global performances. This means that it may be present in relatively accurate performances as well as in poor ones. Each participant was asked to recognize with their haptic modality the category of correct shapes  varying in orientation  among a set of other geometrical shapes varying in angle between sides and in number of side. Our aim was to determine whether the performances and manual exploratory strategies observed in the congenitally blind would be different from those found in blindfolded sighted adolescents. To analyze the performances, the recognition time (seconds) and the number of correct responses were measured. In order to ensure that the differences on performances observed between the two groups were not due to different haptic strategies, the manual exploratory procedures used by participants during the recognition of each shape stimuli were analyzed. If visual experience was not involved in the existence of prototypical shape, the same pattern of results (an intrinsic haptic prototype effect or no haptic prototype effect) should be observed in both populations. By contrast, if the prototype effect was in some way dependent on visual experience, no haptic prototype effect should be observed in the congenitally blind adolescents, whereas this effect would be present in the same tasks and the same conditions in blindfolded sighted adolescents.Fourteen congenitally blind (three girls and eleven boys) without associated disorders, aged 15.5 years on average (SD = 3.16) integrated in ordinary schools in France but receiving special support from teachers for visually impaired participated in this study. None of the blind subjects having light perception could discriminate shape or hand movements. Three categories of geometric shapes were presented to subjects: square, rectangle and triangle. For each category of shape, there was a correct shape and two distorted shapes, each varying from the correct shape according to a criterion: the angle between sides (changing the degree of the angle between sides of the correct shape) or the distortion of the base (distort the base in order to change the shape into a polygon with an interior angle of 140°). Concerning the change in the degree of the angle, the four right angles (90°) of squares and rectangles have been modified in two obtuse angle of 105° (the top right and the left bottom angle of the shape) and in two acute angle of 75° (the top left and the right bottom angle). For triangles the three angles of 60° have been modified in three angles of 20°(left bottom angle), 30° (right bottom angle) and 130°(top angle). Stimuli consisted of embossed geometric shapes cut in foam of 4 mm thick. As some authors had observed that picture size influences recognition rate For each category of geometric shapes, correct and distorted shapes  were presented in a canonical way  or in an oriented way . The correct shapes presented on the canonical way were called «prototypical shape» and those presented in an oriented way were called “non prototypical shape”. For each category of geometric shapes , there were twelve shapes: one correct shape and two distorted shapes, each presented in two size format  and in two presentation format  . In sum,To examine the relevance of the set of stimuli used in this main haptic experiment, we carried a control experiment  in orderParticipants performed a haptic recognition task of geometrical shapes. The experimenter presented one by one the different shapes  to the participant whose task was to correctly identify (with no time limit) by touch embossed geometric shapes . The non-dominant hand held the support of the shape on the table and the other dominant hand explored the shape. The sighted participants worked blindfolded by a mask. The shapes were randomly presented. The instruction was: “I'm going to show you embossed geometric shapes and you will have to touch them. Then, you have to tell me whether you think the shape that you touched is a square, rectangle, triangle or you'll say “none” if you think it is none of these three shapes”. No feedback on responses and no instructions on how to explore were given to participants.The whole experiment was videotaped in order to measure the recognition time of each stimulus and the manual exploratory procedure used by each participant. To analyze the performances, the nature of responses  and the recognition time (seconds) were measured. Regarding the nature of responses, we considered as correct recognition when a subject identified a square for the shapes 1-2-7-8, a rectangle for the shapes 3-4-9-10 and a triangle for the shapes 5-6-11-12-29-30-35-36 cf.  and as fPreliminary ANOVAs on performances with visual status as intersubject factor, shape and size as intrasubject factor and age as continuous predictor revealed that the age factor and size factor were not significant and did not interact with other factors [all F<1]. Consequently, 2 ×2 (shape) ANOVAs were carried out on recognition rates and recognition times for correct responses considering the visual status factor  as intersubject and the shape factor  as intrasubject.2 = .45]: Correct shapes were better identified by the congenitally blind with a recognition rate of 96.43% (SD = 8.31) than by the sighted . The analysis did not reveal a main effect of shape : The prototypical correct shapes  were not better recognized than the non prototypical one . Finally, the interaction between shape and visual status was not significant . It should be noted that the change in the orientation of square did not lead the subjects to perceive it as a diamond in haptics (even if a square rotated 45 degrees is not a diamond since it still has 4 right angles). Indeed, the subjects equally recognized “diamonds” and “square on their bases” as correct square (74.11% and 76.79% respectively).Regarding the recognition rates (in %), results showed a main effect of visual status . Results showed a significant interaction between the visual status and shape factors . A HSD Tukey test revealed that while no difference was observed between prototypical correct shapes (M = 5.06 and SD = 2.32) and non prototypical correct shapes (M = 5.24 and SD = 1.96) in the congenitally blind (p = .96) the blindfolded sighted recognized the prototypical correct shapes (M = 5.40 and SD = 1.26) faster than the non prototypical one (M = 6.88 and SD = 1.74) (p<.01).Regarding the recognition times (in sec.), results showed that the main effect of visual status was not significant . The analysis also revealed that the prototypical correct shapes  were faster recognized than the non prototypical ones  . This means that the two groups did not use in the same manner the three exploratory procedures: blindfolded sighted used almost exclusively metric procedure whereas congenitally blind used also the other exploratory procedures. The main effect of shape was not significant  and the interaction between the visual status factor and the shape factor was not significant . Finally the accuracy of the main exploratory procedure (metric) was examined as a function of group. In the congenitally blind, 7.03% of metric procedures were associated to an incorrect response versus 20.94% in the blindfolded sighted.The objective of this study was to investigate the role of visual experience on the haptic recognition of geometrical shapes by congenitally blind and blindfolded sighted adolescents and in particular the occurrence of prototypical shapes. First, the recognition time analysis showed that the blindfolded sighted recognized prototypical shapes faster than non prototypical shapes while no difference was observed in the congenitally blind. Moreover, results showed that in the congenitally blind as in the blindfolded sighted, exploration procedures did not vary depending on the shape orientation. The differences observed between these two groups were thus not related to the characteristics of the exploratory movements of the participants. Nevertheless, the analysis of haptic recognition rates showed that both congenitally blind and blindfolded sighted recognized the prototypical shapes and the non prototypical ones in the same way. Yet, the measure of this recognition rate probably did not allow us to highlight such fine distinctions since the proposed task was not limited in time and was succeeded by participants at a very high level. More particularly, recognition rates of the congenitally blind were at ceiling. It could then be argued that the task was too simple for blind subjects whatever the shapes and that this factor could have masked the occurrence of the prototype effect on recognition time in congenitally blind. The fact that congenitally blind appeared more skilled than blindfolded sighted on this task contrasts with the results of old studies on the recognition of geometric shapes In our study, the prototype effect was observed in the haptic modality only on recognition times and when participants benefited from visual experience. This result contrasts the findings of Woods et al. (2008) Our findings suggest that the prototype effect is not intrinsic to the haptic modality but depends on visual experience. Since prototypical shape and non prototypical shape differed mainly in their orientation, our results were in line with those of Gori et al. (2010) Finally, though our findings provide new support for understanding how objects are represented in memory and subsequently recognized by the sighted, results do not allow us to determine whether the internal representations of geometric categories of congenitally blind differ from those of the sighted. Indeed, Woods et al. (2008) Experiment S1The visual recognition of geometrical shapes in adolescents.(DOC)Click here for additional data file.


Juvenile Arthritis Disease Activity Score (JADAS) is a recently developed composite tool for scoring disease activity in juvenile idiopathic arthritis (JIA). JADAS consists of four items; the joint count, the physician and the patient’s/parent’s global assessment and the erythrocyte sedimentation rate (ESR) as an inflammatory marker. C-reactive protein (CRP) has been suggested as an alternative inflammatory marker .The aim of the study was to validate and compare the CRP versus the ESR as an inflammatory marker and to validate in JADAS in a Nordic population-based setting.We included newly diagnosed cases of JIA from defined geographical areas of Denmark, Finland, Sweden and Norway with disease onset in 1997 to 2000 followed longitudinally during the first eight years of the disease. The construct validity, predictive and discriminative ability, and sensitivity to change of JADAS were assessed by comparing with other measures of disease activity and outcome.At the first study visit with all JADAS items available in 389 children, the correlation between JADAS27-CRP and JADAS27-ESR was 0.99. Children with higher JADAS scores had increased risk of concomitant pain, physical disability and use of disease modifying anti-rheumatic drugs (DMARD). Higher JADAS score at the first study visit also significantly predicted physical disability, damage, and DMARD during the disease course and no remission off medication at the final study visit. Median JADAS of the oligoarticular persistent category differed significantly compared to median score of all other categories . Sensitivity to change was demonstrated as change in JADAS score compared to the American College of Rheumatology paediatric measures of improvement criteria, showed mostly excellent classification ability.The JADAS-CRP and JADAS-ESR correlate closely, show similar test characteristics, and are demonstrated to be feasible and valid tools to assess disease activity in all categories of JIA.


This virus was named after the town of Lassa at the southern end of Lake Chad in northeastern Nigeria, where the first known patient, a nurse in a mission hospital, had lived and worked when she contracted this infection in 1969. The virus was discovered as part of a plan to identify unknown viruses from Africa by collecting serum specimens from patients with fevers of unknown origin. Lassa virus, transmitted by field rats, is endemic in West Africa, where it causes up to 300,000 infections and 5,000 deaths each year.


An encephalitis outbreak among horses was caused by a pathogenic variant of Kunjin virus. NSW2011, that is most closely related to WNV Kunjin (WNVKUN), the indigenous WNV strain in Australia. Studies in mouse models for WNV pathogenesis showed that WNVNSW2011 is substantially more neuroinvasive than the prototype WNVKUN strain. In WNVNSW2011, this apparent increase in virulence over that of the prototype strain correlated with at least 2 known markers of WNV virulence that are not found in WNVKUN. Additional studies are needed to determine the relationship of the WNVNSW2011 strain to currently and previously circulating WNVKUN strains and to confirm the cause of the increased virulence of this emerging WNV strain.To determine the cause of an unprecedented outbreak of encephalitis among horses in New South Wales, Australia, in 2011, we performed genomic sequencing of viruses isolated from affected horses and mosquitoes. Results showed that most of the cases were caused by a variant West Nile virus (WNV) strain, WNV KUN infections are infrequent and less severe ; infection is confined to inland areas where flooding supports large mosquito populations and water birds are a reservoir and amplifying host. Even in years when WNVKUN has caused disease in humans, disease has rarely been observed or confirmed in horses  and West Nile virus (WNV) Kunjin (KUN) strain are the main etiologic agents of arboviral encephalitis in humans, which usually occurs as isolated sporadic cases or occasional small outbreaks, mainly in northwestern Australia and rarely in southern regions , panel AKUN, we conducted genomic sequencing, antigenic profiling, in vitro growth kinetics, and mouse virulence studies on virus isolates from diseased animals and mosquitoes.In 2011, an outbreak of encephalitis occurred among horses in NSW. To analyze this strain of WNVIn late February 2011, neurologic disease was reported in several horses in northwestern and southwestern NSW. The number of cases and geographic distribution gradually increased. By mid-June 2011, specimens from ≈300 horses were submitted to the virology laboratory at Elizabeth Macarthur Agriculture Institute . Many more horses probably were affected. Diseased horses were located throughout most of NSW, west of the Great Dividing Range, but also extending through the Hunter River Valley region, Sydney Basin, and Illawarra coastal region immediately south of Sydney , panel BWhole brains were removed from 12 horses at postmortem examination. Half of each brain was fixed in 10% neutral buffered formalin; the other half was held fresh at 4°C. Upon receipt, we collected small pieces of fresh and formalin-fixed tissue from several locations in the cerebrum and cerebellum and along the brain stem and cervical spinal cord. If virus isolation could not be performed on fresh samples within 24 h after receipt, we held the samples at −80°C until tested. Before testing, we prepared 10% tissue homogenates in RPMI medium  containing antimicrobial drugs.Mosquitoes were collected throughout NSW, as part of the NSW Arbovirus Surveillance and Mosquito Monitoring Program, by using dry ice–baited light traps. The mosquitoes were submitted live to the Medical Entomology Laboratory at Westmead Hospital  for species identification, arbovirus isolation, and virus identification . We used prototype WNVKUN MRM61C (NY99) 4132 strains (NSW2011. Stocks of WNVKUN MRM61C and WNVNY99 4132 that most closely resembled the low-passage level WNVNSW2011 were prepared by electroporation of BHK21 cells with WNVKUN or WNVNY99 RNA (prepared from corresponding infectious cDNA clones) and passaging them 1× in C6/36 cells. Viral supernatants were harvested 5 days later. Viral titers for each viral stock were determined by plaque assay on Vero 76 cells.We propagated Vero 76 cells in Dulbecco modified minimum essential medium  supplemented with 10% fetal bovine serum (FBS). C6/36 NSW2011, WNVKUN, and WNVNY99; we used 2-fold dilutions of serum from an initial dilution of 1:20. Results were scored as 80% reduction in virus growth or 100% inhibition of virus growth. Reduction in virus growth was determined by assessing the extent of cytopathic effect in each well. Inhibition of virus growth was determined by the absence of viral antigen in the cells of each well when tested with a WNV-reactive monoclonal antibody (mAb) in ELISA.We conducted microneutralization tests  on a magnetic particle handling system  to extract total nucleic acid from clarified 10% brain homogenate (50 μL) or tissue culture fluid. Purified nucleic acids were eluted in 50 μL of kit elution buffer and used immediately for PCR amplification or stored frozen at ≈−20°CWe used a published WNV real-time reverse transcription PCR (rRT-PCR)  containing antimicrobial drugs and 2% FBS. We regularly examined cultures by light microscopy for cytopathic effects. We used rRT-PCR to confirm the identity of virus isolates in culture supernatants or to confirm that there was no virus replication in the absence of cytopathology. Viral RNA recovered from culture fluid at the first or second passage in BHK21 cells was sequenced as described below. A virus isolate obtained from the first brain examined was designated WNVNSW2011. Virus isolation was conducted on homogenates of mosquitoes by similar methods but with passage onto BHK and PSEK cells after initially being placed on C6/36 cells. We identified virus isolates by using immunoassays with generic and specific mAbs.Supernatant from the 10% brain homogenate was placed on monolayers of NSW2011 virus harvested from the first passage in C6/36 cells was used to examine plaque morphology and virulence in mice. The virus was passaged 1× in Vero76 cells for 4 d and 1× in C6/36 cells for 5 d. Virus supernatant was centrifuged at 500 × g at 4°C for 5 min before being stored at −80°CWNVKUN and WNVNY99 by using a fixed-cell ELISA (KUN); mAb 2B2, anti-E (reactive with WNV); mAb 3.1112G, anti-NS1 (reactive with WNV); mAb 5H1, anti-NS5 ; mAb 17D7, anti-E (specific for strains of WNV with glycosylated E); mAb 3.101C, anti-E (specific for strains of WNV with unglycosylated E); and the pan–flavivirus-reactive mAbs   by using ClustalW .For sequencing of the whole genome, we used total nucleic acid purified from virus-infected cell culture supernatant as template in 5 RT-PCRs with primers designed to cover the coding regions of any WNV genome . RT and 1 genome  to ampliNSW2011 sequence as described (www.mbio.ncsu.edu/BioEdit/bioedit.html) for manual editing before construction of phylogenetic trees. Maximum-likelihood trees were constructed by using PhyML  or young adult (4 weeks old) Swiss outbred CD1 mice were injected intraperitoneally with 10-fold dilutions of virus. The mice were monitored for 21 days after injection and euthanized when signs of encephalitis were evident. All animal procedures had received prior approval from The University of Queensland Animal Ethics Committee.NSW2011. Eight isolates of WNVNSW2011 were isolated from Culex annulirostris Skuse mosquitoes during the 2011 vector season. Of the 8 isolates, 5 were from mosquitoes collected in the Riverina region of southwestern NSW ; 2 were from the Murray region in southern NSW ; and 1 was collected at Lower Portland in the outer western Sydney region of NSW. No other isolates of WNV were obtained.Viral RNA was detected by WNV-specific rRT-PCR in fresh brain tissue from 6 of 12 horses showing signs of encephalitis. Viruses were isolated from 4 of these samples; each showed distinct cytopathology in BHK21 and Vero cells. rRT-PCR of the culture fluids and immunoperoxidase staining of the cells with pan–flavivirus-reactive and WNV-specific mAbs confirmed the isolation of a West Nile–like virus. The first isolate was named NSW2011 and designated WNVNSW2011 in a fixed-cell ELISA  and of WNVKUN and WNVNY99  of virus in the assay  between WNVNSW2011 and WNVKUN and an 89-aa difference (38 nonconserved changes) between WNVNSW2011 and WNVNY99  at residue 49 that is not contained in WNVKUN, confirming the critical role of this residue for 5H1 binding. Together these data suggest that WNVNSW2011 represents a virulent WNV strain that has emerged in Australia. However, the amino acid substitution in NS3, which is believed to be associated with increased virulence of WNVNY99 in birds in North America (NSW2011. Sequencing of the key regions of the other WNV isolates obtained during this outbreak (3 from horses and 8 from mosquitoes) showed that each was indistinguishable from WNVNSW2011.A comparison of the nucleotide sequence of the complete coding region of the first isolate of WNVisolates . A detai WNVNY99 . At leasVNSW2011 . The glyNSW2011 (NY99 (4.7 mm ± 0.8 mm) than to the prototype WNVKUN (2.8 mm ± 0.4 mm). To confirm the presence of an N-linked glycan on the E protein of WNVNSW2011, we used Western blot to analyze endoglycosidase-digested viral protein. Analysis showed that the E protein of WNVNSW2011 and WNVNY99 migrated slightly faster than the undigested control protein. This result is consistent with N-linked glycosylation , compared with WNVKUN (LD50 13.4 PFU), induced neurologic signs (50 for WNVNY99 (0.1 PFU) was lower than that for WNVNSW2011, and neurologic signs developed more rapidly  mice. Compared with WNVNSW2011, WNVNY99 exhibited a lower LD50  and a shorter time to death  (Injection of 18- to 19-day-old (weanling) mice with 10-fold dilutions of virus showed that substantially lower doses of WNVic signs ; and theic signs , panel C rapidly , panel C000 PFU) , panel CKUN now has been detected on the eastern seaboard of NSW, close to major urban areas, including the largest 3 cities . This detection occurred despite relatively small mosquito populations in many of these areas, suggesting that the virus is more virulent and probably transmitted more efficiently than other strains between mosquito vectors and mammalian hosts. Characterization of virus isolated from the brain of an animal that died showed a variant strain of WNV most closely related to WNVKUN. Typing of WNVNSW2011 by reactivity with a panel of mAbs indicated the virus was antigenically more similar to the native Australian WNVKUN strains than to exotic WNV strains. However, for WNVNSW2011, the reaction profile of mAbs 17D7 and 3.101C differed from that of the prototype WNVKUN. Similar to WNVNY99 and other virulent strains of WNV, WNVNSW2011 E protein was glycosylated at residue 154. This finding was further confirmed by gene sequencing and endoglycosidase F digestion analysis. Glycosylation of WNV E protein at this site is thought to enhance virus dissemination in the infected host by increasing the efficiency of assembly and release of virus particles from infected cells . In contrast, increased mortality among birds of any species was not reported during the 2011 outbreak in southeastern Australia. The lack of disease in birds in Australia supports the hypothesis that the amino acid substitution observed in WNVNY99 (Ala→Pro at aa 249 in NS3)  was experimentally infected with WNVNY99 , the new virus has several amino acid substitutions that are likely to be the reason for enhanced virulence in horses. More extensive epidemiologic studies in the field and experimental studies in the laboratory are required to determine the relation of WNVNSW2011 to other currently and previously circulating WNVKUN strains and to confirm which viral proteins and amino acid residues are associated with increased virulence of WNVNSW2011 in horses.Taken together, our results show that the WNV


Begomoviruses are single-stranded DNA viruses that cause economically important diseases of many crops throughout the world and induce symptoms in plants, including enations, leaf curling and stunting, that resemble developmental abnormalities. MicroRNAs (miRNAs) are small endogenous RNAs that are involved in a variety of activities, including plant development, signal transduction and protein degradation, as well as response to environmental stress, and pathogen invasion.African cassava mosaic virus (ACMV), Cabbage leaf curl virus (CbLCuV), Tomato yellow leaf curl virus (TYLCV) and Cotton leaf curl Multan virus/Cotton leaf curl betasatellite (CLCuV/CLCuMB), were used in this study. Ten developmental miRNA were studied. N. benthamiana plants were inoculated with begomoviruses and their miRNA profiles were analysed by northern blotting using specific miRNA probes. The levels of most developmental miRNA were increased in N. benthamiana by TYLCV, CLCuMV/CLCuMB and CbLCuV infection with a common pattern despite their diverse genomic components. However, the increased levels of individual miRNAs differed for distinct begomoviruses, reflecting differences in severity of symptom phenotypes. Some of these miRNA were also common to ACMV infection.The present study was aimed at understanding the deregulation of miRNAs upon begomovirus infection. Four distinct begomoviruses Our results have shown a common pattern of miRNAs accumulation upon begomovirus infection. It was found that begomoviruses generally increase the accumulation of miRNA and thus result in the decreased translation of genes involved in the development of plants. Identification of common miRNAs that are deregulated upon begomovirus infection may provide novel targets for control strategies aimed at developing broad-spectrum resistance. After heating that at 65°C for 5 min, sample was placed on ice for 2 min before loading it to TBE gel . Gel was run in 1× TBE buffer at 150-180 volts for 2 to 2.5 hours. After removing gel from the cassette it was photographed under UV trans-illumination and details of the samples were documented.RNA was transferred to Hybond N+ (Amersham) by a semi-dry blotting system (Bio-Rad) at 10-12 volts for 45-60 min. The membrane was air dried, UV cross-linked and stored at 4°C between two sheets of wet Whatman filter paper.Arabidopsis miRNAs  were end-labeled using a DIG olig-labeling kit according to manufacturer instructions . The sequences of oligo-nucleotides used for end labeling are listed in Table Oligonucleotide primers complementary to N. benthamiana plants were set at 1 and other data calculated relative to this value. The data shown in Figure The intensity of bands was quantified by using software ImageJ. Data from these analyses were used to normalize the intensity of each band, based on rRNA loaded in each well. For virus infection the values for the miRNA species in non-infected N. benthamiana plants were agro-infiltrated with infectious clones of ACMV, CLCuMV/CLCuMB, CbLCuV and TYLCV in two independent experiments. Potato virus X (PVX) was used as reference for virus infection with an RNA genome. All plants were kept in the same green house for duration of each experiment.A group of 15 The authors declare that they have no competing interests.IA, BP performed the experiments. IA, RWB, SM and CMF conceived the study and wrote the manuscript. All authors read and approved the final manuscript.


Since 1975, Lyme disease has become the most common vectorborne inflammatory disease in the United States. To assess the economic impact of Lyme disease (LD), the most common vectorborne inflammatory disease in the United States, cost data were collected in 5 counties of the Maryland Eastern Shore from 1997 to 2000. Patients were divided into 5 diagnosis groups, clinically defined early-stage LD, clinically defined late-stage LD, suspected LD, tick bite, and other related complaints. From 1997 to 2000, the mean per patient direct medical cost of early-stage LD decreased from $1,609 to $464 (p<0.05), and the mean per patient direct medical cost of late-stage LD decreased from $4,240 to $1,380 (p<0.05). The expected median of all costs , aggregated across all diagnosis groups of patients, was ≈$281 per patient. These findings will help assess the economics of current and future prevention and control efforts. Borrelia burgdorferi. LD usually begins with an initial expanding skin lesion, erythema migrans (EM), which may be followed by musculoskeletal, neurologic, and cardiac manifestations in later stages of the disease (Lyme disease (LD) is a multisystem, multistage, inflammatory tickborne disorder caused by the spirochete Since the first case reported in 1975 . In 2002Assessing the economic impact of LD will help assess the economics of current and future prevention and control efforts. Although several studies of cost estimates of LD have been published  on the Maryland Eastern Shore, an area where LD is endemic . The stuB. burgdorferi infection  direct medical costs of LD diagnosis and treatment, 2) indirect medical costs, 3) nonmedical costs, and 4) productivity losses. Intangible costs  were not incorporated. Consumer price index (CPI) for medical care was used to adjust all medical payments into year 2000 dollars  of physician visits, consultation, serology, procedure, therapy, hospitalization/emergency room (ER), and other related costs (Appendix 2).Indirect medical costs, nonmedical costs, and productivity losses were all acquired from a patient questionnaire used in 1997 and 1998. The questionnaire was sent to LD patients with informed consent forms. Collection of these data was restricted to those 2 years. In this study, indirect medical costs refer to extra prescription and nonprescription drug costs that patients paid out of pocket.The patient's questionnaire also collected information on nonmedical payments made for home or health aides and miscellaneous services, such as travel (transportation) and babysitting. Each patient's transportation costs to a physician's office were estimated by using the US federal government reimbursement rate, multiplying the reported total travel miles per patient by $0.365/mile. Total travel mileage per patient was calculated by counting the number of physician visits and multiplying total visits by the distance of a round trip to the physician's office.We used patient-reported time lost from work to estimate productivity losses due to LD on the basis of the human capital method and valued the time lost by using age- and sex-weighted productivity valuation tables  aggregated across all diagnosis groups of patients:Expected mean cost of a LD outcome = Σ direct medical costs, indirect medical costs, nonmedical costs, and productivity losses .Because the distribution of cost data is often not normal, we also calculated the medians of these costs and used both mean and median to estimate the most likely per capita cost of LD on the Maryland Eastern Shore. The median cost of an LD outcome was calculated by using the following formula:Expected median cost of a LD outcome = Σ direct medical costs, indirect medical costs, nonmedical costs, and productivity losses Differences between annual mean direct medical costs were analyzed by using 1-way analysis of variance followed by a Bonferroni test. Differences were considered significant for p values <0.05. Additionally, we used a multivariate linear regression model to estimate the relative impact of a number of factors on the direct medical costs of LD. The ordinary linear regression (OLS) method was applied by using SAS 8.2  and Stata SE . The dependent variable was total direct medical cost per LD patient. We transformed total direct medical costs by using natural logarithms because the data were highly skewed. Independent variables of the equation included cohort year, LD diagnosis groups, diagnostic and treatment procedures, and patient characteristics . All independent variables, except age, were binomial . Baseline costs  referred to those costs accrued by a woman who had tick bite only (without EM symptoms) diagnosed in 1997 during an office visit. Such a patient had no hospital or ER stay, no serologic tests, no consultation from other physicians, no antimicrobial drug therapy, and no other procedures outside a physician office and hospital/ER. Additional direct medical costs were added or subtracted to the baseline costs for each independent variable of interest if significant (Appendix 3). We tested heteroscedasticity in Stata and corrected mild heteroscedasticity by using "robust" and "hc3" procedures. We also tested both linearity and multicollinearity in SAS and Stata.From 1997 to 2000, we identified 3,415 LD-relevant patients in the 5 counties studied on Maryland Eastern Shore . Among tFrom 1997 to 2000, the mean cost of therapy of all diagnosis groups decreased 75%, from $189 to $47, and the mean cost of hospitalization/ER decreased 61%, from $41 to $16 . During A patient with clinically defined early-stage LD paid an average of $164 in 1997 and $307 in 1998 (in 2000 dollars) for extra prescription and nonprescription drugs . Those wUsing multivariate linear regression analysis, we found that patients with clinically defined early- and late-stage LD had direct medical costs that were ≈50% and 100%, respectively, higher (p<0.001) relative to patients who only had tick bite, if the impact from other factors was not considered . MoreoveIn year 2000 dollars, the expected mean total cost attributable to LD was $1,965 per patient, and the expected median total cost attributable to LD was estimated at $281 per patient . For LD Previous studies of the economic impact of LD were often based on numerous assumptions and experts' suggestions  costs $2,970 in direct medical costs plus $5,202 in indirect medical costs, nonmedical costs, and productivity losses. In 2002, 23,763 LD cases were reported to CDC. Hence, the estimated nationwide annual economic impact of LD and relevant complaints was ≈$203 million (in 2002 dollars). However, since LD cases reported on the basis of the surveillance case definition are believed to be underreported  instead of surveillance case definition of LD because of limited data. Thus, we may have overestimated the number of LD cases. As a result of case definition, our estimation of cost not only included the cost of LD  but also the costs of LD-relevant complaints . Second, medical charges used in our study may not reflect the true cost. Third, our results are likely to underestimate the costs per case because some of the costs were not included. Costs that were omitted included any costs incurred by a patient beyond the study period. Likewise, Steer et al. reported that ≈7% of LD cases remained asymptomatic within the 20-month study  and relevant complaints were calculated as follows:YY is the total direct medical costs for patient i and itC is the direct medical cost of type t for patient i. Different types (t) of direct medical costs involved in this study included physician visits, consultation, serology, procedure, therapy, hospitalization/emergency room, and other relevant costs.Where Cost of physician visits: refers to total visit charges per patient with primary provider or physician managing LD. It includes charges for procedures only performed in the office at the time of the visit. All other procedures were covered in the cost of procedure category.Cost of consultation: refers to total charges per patient accrued at the time of consultation including only those procedures  done in the office at that time. All other procedures ordered by the consult but done off site  were covered in the cost of procedure category.Cost of serology: refers to total charges of LD serology tests  for each patient.Cost of procedure: refers to total charges per patient of all other procedures not covered in office visits, consultation, or hospital and emergency room.Cost of therapy: refers to total charges per patient of therapy including antibiotics and additional costs associated with intravenous therapy Cost of hospitalization/emergency room: refers to total hospital and/or emergency room charges per patient. It includes physician fees and ambulance fees.Other relevant costs: refer to other appropriate charges for each patient such as charges for additional laboratory tests.Yi = Xi β + εiWe used a multivariate linear regression model to estimate the relative impact of a number of factors on the direct medical costs of Lyme disease (LD).Yi, the dependent variable, is the total direct medical costs for patient i and Xi is a vector of covariates. εi is a mean-zero random error. Baseline costs came from the intercept term, which refers to a female patient who had tick bite only (without erythema migrans symptoms) diagnosed in 1997 during an office visit. Such a patient had no hospital or emergency room stay, no serologic tests, no consultation from other physicians, no antimicrobial drug therapy, and no other procedures outside physician office and hospital/emergency room. Additional direct medical costs were added or subtracted to the baseline costs for each independent variable of interest if statistically significant. Therefore, total direct medical costs per LD patient = baseline costs + cost difference of different patient diagnosis group  + costs of serologic test + costs of other procedure outside physician office and hospital/emergency room + costs of hospital and/or emergency room stay + costs of consultation from other physicians + costs of therapy + miscellaneous costs + cost difference with or without erythema migrans + cost difference of gender + cost difference of each additional year of age + cost difference of cohort year, if statistically significant.where


Flavivirus, are emerging in Italy as important human and animal pathogens. Migratory birds are involved in the spread of Flaviviruses over long distances, particularly from Africa to Europe. Once introduced, these viruses can be further be dispersed by short-distance migratory and resident bird species. Thus far, there is still a considerable knowledge gap on the role played by different bird species in the ecology and transmission mechanisms of these viruses. The Region of Trentino-Alto Adige  is located on the migratory route of many of the short- and long-distance migratory birds that cross the Alps, connecting northern Europe and western Asia with southern Europe and Africa. Until now, only a silent circulation of WNV and USUV within the territory of the Province of Trento has been confirmed by serological screening, whilst no cases of infected humans or animals have so far been reported. However, continuous spillover events of both viruses have been reported in neighbouring Regions. The aim of this study was to monitor the circulation of WNV and USUV in Trentino-Alto Adige, in order to detect if active virus shedding occurs in migratory birds captured during their seasonal movements and to evaluate the role that different bird species could play in the spreading of these viruses.West Nile virus (WNV) and Usutu virus (USUV), both belonging to the genus We carried out a biomolecular survey on oral and cloacal swabs collected from migratory birds during seasonal migrations. Birds belonging to 18 transaharian and 21 intrapaleartic species were examined during spring (n = 176) and autumn (n = 146), and were tested using a generic nested-PCR.All samples tested negative for Flaviviruses. The possible causes of unapparent shedding, along with ecological and epidemiological implications are discussed.The lack of detection of active virus shedding in these bird species does not exclude the circulation of these viruses within the Trentino-Alto Adige region, as reported in previous studies. The possible ecological implications are discussed. Flavivirus, family Flaviviridae, contains more than 70 viruses subdivided into three groups, according to their route of transmission: 1) arthropod-borne, infecting a range of vertebrate hosts through mosquito or tick bites; 2) those spread by an unknown vector, presumed to be limited to infecting vertebrates only, or; 3) those spread by insects only, called ‘insect-specific flaviviruses’ or ‘mosquitoes-only flaviviruses’, because they replicate only in mosquito-derived cells. When considering their observed pathogenicity for humans, those with highest impact on human health in Europe belong to the first group, and include West Nile Virus (WNV), Usutu Virus (USUV), and Tick-borne Encephalitis Virus [The genus th century and has since then radiated into Europe, India, Asia, Australia and America. During the last decade, new strains with various pathogenic characteristics have been discovered [Culex spp., and birds that are the amplifying hosts. It infects a broad range of avian and mammalian species, but has also been reported to infect reptiles and amphibians. Other mechanisms of transmission include mites and ticks, organ transplant, blood transfusion, breastfeeding, intrauterine infection, and the fecal-oral route [WNV is a zoonotic agent that has been reported in Africa since the beginning of the 20scovered -5. WNV ial route -11 and ral route . Surveilal route -15 and ral route . In 2011al route ,18. Baseal route . WNV infal route ,20,21.Flavivirus isolated for the first time from Culex neavei (Cx. neavei) mosquitoes in South Africa in 1959. It is maintained in nature by a mosquito-bird transmission cycle, with the genus Culex as the main vector, and for several years it has been considered a virus with very low pathogenicity for humans and animals [Turdus merula), Great Gray Owls (Strix nebulosa) and Barn Swallows (Hirundo rustica) [USUV is another  animals . It was  animals  and then animals , resultirustica) ,24. In trustica) ,25, Switrustica) , the UK rustica)  and Germrustica) . Moreoverustica) ,26. In 2Flaviviruses, as reviewed by some authors e.g.: [Wild birds are believed to have the potential to maintain, transport, and disperse several rs e.g.: . Wild birs e.g.: , much rers e.g.: ,26,29,31rs e.g.: ,33 and rAmong the non-vectorial transmission routes of WNV between birds, oral and fecal viral shedding plays a central epidemiological role for many reasons. The fecal-oral secretions and excretions can contaminate the environment, leading to a high number of individuals coming into contact with the virus. In addition, this transmission route can take place in several ways, such as direct and indirect contact , intra- and inter-species socialization, feeding of the nestlings, cannibalism and scavenging of infected carcasses. In fact, the viremia in orally-infected animals is similar to the one reached after mosquito bites ,10,11,34Very little is currently known about USUV, mainly because it was historically confined to Africa, and because its pathogenicity to humans and animals has only recently been recognised. Moreover, these studies have focused in detecting the virus in dead birds e.g.: ,28, throFlaviviruses, we carried out a biomolecular survey to detect if active virus shedding occurs in migratory birds captured during their seasonal migrations, and to evaluate the role of different species in spreading these viruses.Trentino Alto-Adige, a mountainous Region in northern-eastern Italy, is located on many of the short- and long-distance routes of migratory birds that, from northern Europe, cross the Alps on their way to western Asia or Africa and viceversa ,42. So fFlaviviruses. The positive control tested always positive, and the negative one resulted always negative.A total of 43 birds were captured during the autumn of 2011, 176 during spring 2012, and 103 during autumn 2012  previously tested negative in study also carried out in Italy [Garrulus glandarius), domestic chicken, European Nightjar (Caprimulgus europaeus), European Bee-eater (Merops apiaster), Barn Swallow, Cetti’s Warbler (Cettia cetti), Blue Tit (Parus ceruleus) [Anseriformes tested in Finland [In previous studies, the oro-fecal shedding of USUV was detected in domestic goose (domestica) and domomesticus). Alternin Italy . Moreoveeruleus) , and for Finland .Anseriformes, screened by [Corvidae of British Colombia tested by [Cuculidae, Motacillidae, Silvidae, Turdidae (order Passeriformes) and Strigidae (order Strigiformes) were analysed and all tested negative [Threskiornithidae and Accipitridae[Charadriiformes , some to genus Calidris and Tringa, some to the orders Anseriformes (Anas spp.), Gruiformes  and Passeriformes, family Motacillidae  and others to the family Corvidae (Hooded Crow Corvus corone cornix).With respect to WNV, the species tested by , the 11 eened by , and theested by  resultednegative . This fuipitridae. The traipitridae. The tesCorvidae, such as American Crows (Corvus brachyrhynchos), Fish Crows (Corvus ossifragus), Blue Jays (Cyanocitta cristata), Common Ravens (Corvus corax), Black-billed Magpies (Pica pica), Little Raven (Corvus mellori) for e.g.: [Anatidae (order Anseriformes) such as Canada Goose (Branta canadensis), Wild Mallard (Anas platyrhynchos) and Domestic Goose [Galliformes, such as Northern Bobwhite (Colinus virginianus), Turkey , domestic chicken, Red-legged Partridge (Alectoris rufa) [Gruiformes, such as American Coot (Fulica americana) [Charadriiformes, such us: Killdeer (Charadrius vociferus), Ring-billed Gull (Larus delawarensis) [Columbiformes, such as Mourning Dove (Zenaida macroura) and Rock Dove (Columba livia) [Psittaciformes, such as Monk Parakeet (Myiopsitta monachus) and Budgerigar (Melopsittacus undulatus) [Passeriformes, such as American Robin (Turdus migratorius), Common Grackle , House Finch (Carpodacus mexicanus), House Sparrow (Passer domesticus), Great-tailed Grackles , Cedar Waxwing (Bombycilla cedrorum), Northern Mockingbird (Mimus polyglottus), Barn Swallow (Hirundo rustica), Cliff Swallow (Petrochelidon pyrrhonota) [Buteo swainsoni), Ferruginous Hawk , Peregrine Falcon , Golden Eagle (Aquila chrysaetos), American Kestrel , and some species of North American owls (family Strigidae) like Great Horned Owl, (Bubo virginianus) [On the other hand, additional studies have detected oro-fecal shedding of WNV in bird species of different families and orders. These include or e.g.: ,48-52; Aic Goose ,20,53; Gis rufa) ,54,55 anericana) ; Charadrarensis) ; Columbia livia) ; Psittacdulatus) ; Passerirhonota) ,56,57; sinianus) ,49,58,59Fringillidae, Lanidae, Paridae, Muscicapidae, Silvidae, Turdidae, Hirundinidae and Picidae[Motacillidae, Prunellidae, Emberizidae, Cuculidae, Egitalidae, Strigidae, previously never screened for USUV, may not be important shedders of this virus.The results of our study further corroborate the results of a previous study also carried out in Italy, which found there was no evident oro-faecal shedding of USUV in the families d Picidae. Our resCuculidae, Motacillidae, Silvidae, Turdidae (order Passeriformes) and Strigidae (order Strigiformes) [Motacillidae[Galliformes that was not included in the current study; and secondly, the birds were experimentally infected with the virus, and so the results may not reflect those seen in natural conditions in the wild [Columbiformes, Psittaciformes, Charadriiformes, Gruiformes, Galliformes, Anseriformes, Falconiformes). Studies that have been carried out on Strigiformes and Passeriformes, also investigated different species to the ones included in our study .Considering the migratory birds tested in Italy so far, what has been said for USUV is also valid for WNV. Moreover, this has also been confirmed in India in birds belonging to the families iformes)  and in Gacillidae. Of the the wild . The othCuculiformes and Piciformes is not intense or it lasts only few days. Regarding Strigiformes and Passeriformes, their shedding seems low also for USUV, but for WNV, various families or species could have an important role, such as Corvidae, Hirundinidae, Icteridae, Turdidae, Fringillidae, Passeridae, Bombycillidae, Mimidae and Tytonidae. There are several factors that could explain these different results, for example, the limited number of subjects that were tested and the taxonomical differences between the birds screened. Also, an additional reason could be the period of the year during which the study was carried out in relation to the bird’s physiology: migration requires morphological and physiological changes [Accordingly, it seems that the oro-fecal shedding of USUV and WNV in  changes  that cou changes , thus be changes ,54 and rFlaviviruses have been recorded in this region of Italy. Their circulation is then apparently very limited, in contrast to the high number of cases and the pathogenicity observed in animals, mosquitoes and humans in the neighbouring regions  and spring 2012 (March to May). Intrapaleartic and transaharian migratory birds were captured by ornithologists using net labyrinths authorized by ISPRA  within the European Union for Bird Ringing (EURING) which includes ethical approval. The research protocol was also approved by the Wildlife Management Committee of the Autonomous Province of Trento . These activities are carried out to provide data on migration patterns, demography and ecological processes. The sampling sites included: Faedo (Trento) and Tiarno di Sopra (Trento) during the 2011 autumnal ringing campaing; Cloz (Trento), Campi al lago , Campodenno (Trento), Calavino (Trento) and Sarche (Trento) during the 2012 spring ringing campaing; Faedo (Trento), Tiarno di Sopra (Trento) and San Michele all’Adige (Trento) during the 2012 autumnal ringing campaing . Samples were kept refrigerated during transport to the laboratory, where they were stored at -80°C until analysis. Each bird was manipulated only for few minutes and prior to its release, each one was marked by standard procedures using metal leg rings, according to EURING procedures. Date of capture, species, ring number, age, weight and other morphobiometric parameters were recorded for each individual.Flaviviruses, we used a generic nested RT-PCR that amplifies a region of the NS5 gene that is well-conserved within this genus, according to [Molecular analyses were performed in the laboratory of Veterinary Sciences Department of University of Torino . For RNA extraction, each swab was dissolved in 200 μl of phosphate-buffered saline (PBS) buffer  and the suspension obtained was centrifuged for 5 minute at 8000 rpm. 140 μl of the supernatant was added to 560 μl of Buffer AVL and carrier RNA, prepared according to QIAamp® Viral RNA Mini Handbook . The samples were then processed following this protocol. In the final step, RNA was eluted in 60 μl of Buffer AVE. After quantification with Thermo Scientific Nanodrop 2000 , up to 1 μg of RNA was reverse-transcribed according to QiagenQuantiTect® Reverse Transcription Kit Handbook . For the screening of rding to , with moEURING: European Union for Bird Ringing; ISPRA: Istituto Superiore per la Protezione e la Ricerca Ambientale; PBS-buffer: Phosphate-buffered saline; PCRs: Polymerase chain reactions; USUV: Usutu virus; WNV: West Nile virus.The authors declare that they have no competing interests.MG and AR conceived and directed the study. MG, DA and FR carried out the bird netting and swabs collection. MG carried out the molecular analysis and drafted the manuscript. LB participated in the molecular analysis. MaG was involved in the interpretation of the data. All authors participated in the revision of the manuscript and approve the submitted version. All authors read and approved the final manuscript.