Melon necrotic spot virus (MNSV) and Prunus necrotic ringspot virus (PNRSV).In plants, RNA silencing plays a fundamental role as defence mechanism against viruses. During last years deep-sequencing technology has allowed to analyze the sRNA profile of a large variety of virus-infected tissues. Nevertheless, the majority of these studies have been restricted to a unique tissue and no comparative analysis between phloem and source/sink tissues has been conducted. In the present work, we compared the sRNA populations of source, sink and conductive (phloem) tissues in two different plant virus pathosystems. We chose two cucurbit species infected with two viruses very different in genome organization and replication strategy; Our findings showed, in both systems, an increase of the 21-nt total sRNAs together with a decrease of those with a size of 24-nt in all the infected tissues, except for the phloem where the ratio of 21/24-nt sRNA species remained constant. Comparing the vsRNAs, both PNRSV- and MNSV-infected plants share the same vsRNA size distribution in all the analyzed tissues. Similar accumulation levels of sense and antisense vsRNAs were observed in both systems except for roots that showed a prevalence of (+) vsRNAs in both pathosystems. Additionally, the presence of overrepresented discrete sites along the viral genome, hot spots, were identified and validated by stem-loop RT-PCR. Despite that in PNRSV-infected plants the presence of vsRNAs was scarce both viruses modulated the host sRNA profile.We compare for the first time the sRNA profile of four different tissues, including source, sink and conductive (phloem) tissues, in two plant-virus pathosystems. Our results indicate that antiviral silencing machinery in melon and cucumber acts mainly through DCL4. Upon infection, the total sRNA pattern in phloem remains unchanged in contrast to the rest of the analyzed tissues indicating a certain tissue-tropism to this polulation. Independently of the accumulation level of the vsRNAs both viruses were able to modulate the host sRNA pattern.The online version of this article (doi:10.1186/s12864-015-1327-5) contains supplementary material, which is available to authorized users. Arabidopsis thaliana, four classes of DCL enzymes (DCL1 to DCL4) have been reported, DCL1 mainly contributes to the production of miRNAs [In most eukaryotes RNA silencing is an essential mechanism which controls gene expression in a wide range of biological processes. In addition, RNA silencing plays an important role as defence against viruses in plants, invertebrates and possibly also in mammals -4. Doublf miRNAs  and DCL4f miRNAs -11. In if miRNAs -16. Subsf miRNAs ,18. In pf miRNAs .In plants, a series of grafting experiments demonstrated that RNA silencing has a systemic nature and that the long-distance transmission of the putative sequence-specific signaling molecule must be transported through the phloem .Little is known about the nature of the systemic signal that mediates the process and how it is transmitted from the site of initiation to the rest of the plant. However, the detection of sRNAs in the phloem of different plant species -24 as weMelon necrotic spot virus (MNSV) and Prunus necrotic ringspot virus (PNRSV), respectively, were analyzed.During last years deep-sequencing analyses in different plant/virus systems have allowed to study the vsRNA profile of a high variety of infected tissues ,31-42. NMelon necrotic ringspot virus (MNSV) is a plant virus belonging to the genus Carmovirus within the family Tombusviridae [Prunus necrotic ringspot virus (PNRSV) is a positive single-stranded RNA virus, member of the genus Ilarvirus in the family Bromoviridae and with a tripartite genome. RNAs 1 and 2 encode the replicase subunits P1 and P2. RNA 3 is bicistronic and contains the coding sequences of the putative movement protein (MP) gene and the CP gene. CP synthesis occurs via a subgenomic monocistronic mRNA (RNA 4) [sviridae ,44 whichsviridae . The opesviridae -49. The sviridae . By cont (RNA 4) ,52.Our findings showed, in both systems, an increase of the 21-nt total sRNAs together with a decrease of those with a size of 24-nt in all the infected tissues, except for the phloem where the ratio of 21/24-nt sRNAs remained constant. Comparing the vsRNAs, both PNRSV and MNSV infected plants share the same vsRNA size distribution in all the analyzed tissues. Although in PNRSV-infected plants the percentage of vsRNAs was one hundred times less than that observed for the MNSV- infected plants, both viruses were able to modulate the host sRNA profile. In addition, similar accumulation levels of sense and antisense vsRNAs were observed in both systems except for roots that showed a prevalence of (+) vsRNAs in both pathosystems. The biological significance of these results is discussed.To determine virus-induced changes in the sRNA profiles of cotyledon, leaf, root and phloem samples of MNSV- and PNRSV-infected or mock-inoculated melon and cucumber plants, we conducted Solexa sRNA profiling (HiSeq2000) and bioinformatic analysis of the sequence. The results were further confirmed by polyacrylamide gels stained with silver nitrate (PAGE-SN). A total of 16 sRNA libraries were constructed generating between 6 to 15 million reads each  of the 21-nt sRNAs was observed whereas the percentage of the population with a length of 24 nucleotides diminished considerably (around 5% and below). Compared to the PAGE-SN  together with a lower amount of 24-nt (from 25% to 12% in cotyledon and 35% to 14% in leaf) species , 23 21%) and 21 (15%) nucleotides  [Size distribution analysis showed that the vsRNAs were predominately 21-nt in length for both MNSV and PNRSV in leaves, cotyledons, roots and phloem Figure . In melous (TCV) . ValidatIn cucumber the results were similar, approximately 50% of the vsRNAs from PNRSV infected leaves, cotyledons, roots and phloem had a length of 21 nucleotides, however, they were hardly detected in infected cotyledon, leaf and root tissues  identified the four homologues of A. thaliana DCL1 to 4 in both C. sativus and C. melo genome  from each library were plotted against the corresponding virus sequence considering both polarity and number of reads. As it is shown in Figures A. thaliana, the 5′ terminal nucleotide of the sRNAs influences their selective loading into specific AGOs. Thus, investigating the 5′-terminal nucleotide of vsRNAs can give insight into vsRNA function. Figures In As described above, both MNSV and PNRSV vsRNAs mapped along the whole corresponding virus genome. However, PNRSV vsRNA distribution was more heterogeneous with a higher accumulation along the RNA 3 Figure . AdditioAs these “hot-spots” may have biological relevance, we validated their presence by stem-loop RT-PCR . As posiRNA silencing controls countless biological processes including, in the case of plants, defence against biotic and abiotic stresses. When a plant is infected by a virus, the silencing machinery has to combine efforts to guarantee the correct development of the plant on the one hand and an appropriate defence against the pathogen on the other. This involves, among other aspects, a silencing signal with nucleotide-sequence specificity that spreads over long distances through the phloem tissue. Next generation sequencing has allowed a considerable progress in identifying and characterizing the hallmark molecules in this process, the vsRNAs. However, in most of these studies, the characterization of the vsRNAs has been usually restricted to a particular tissue. In addition, when phloem tissue was analyzed, its vsRNA population has not been compared, with very few exceptions, with sink and/or source tissues. In this work we deep-sequenced the sRNA population of four tissues; cotyledon as source, symptomatic-primary leaf and root as sink tissues and phloem as conductive tissue. In addition, we compared two different plant-virus systems, MNSV and PNRSV, infecting melon and cucumber plants, respectively.Cucumber mosaic virus (CMV), a member belonging to the same family than PNRSV, showed also a lower percentage of virus-derived sRNAs (around 14%) in Arabidopsis thaliana.Sequence analysis of the data revealed a remarkable difference between the total number of vsRNA reads in both systems. Consistent with our data, Donaire et al.  found hiCauliflower mosaic virus (CMV), for instance, uses decoy RNAs to evade silencing [Red clover mosaic virus (RCNMV) suppresses RNA silencing recruiting DCL1 proteins or their homologs to the replication complexes [Magnaporthe oryzae virus 2 (MoV2), a dsRNA mycovirus, accumulates very low amounts of vsRNAs (0.5%). None of the viral proteins exhibit RNA silencing suppressor activity but MoV2 seems to evade silencing by replicating within viral particles [Alfalfa mosaic virus (AMV), require a specific and efficient interaction of their CPs with the viral genome for replication and translation [Our study did not reveal a significant correlation between virus accumulation and number of vsRNAs. Thus, other factors like differences in virus counter defense strategies in the respective host plants likely accounts for the differences in vsRNA accumulation in the plant-virus systems tested. Little is known about the MNSV and PNRSV VSRs. For MNSV, the two viral proteins, p42 and p7B have been described to have VSR activity . They weilencing , Red cloomplexes  and highomplexes . Like PNarticles . Similarnslation . It woulImportantly, despite the fact that PNRSV infection hardly produced any vsRNAs, total sRNA profiles MNSV and PNRSV infected plants showed a clear increase of the 21-nt species in all the tissues being less evident in phloem. A sharp decline of 24-nt sRNA relative abundance was also observed in all the tissues except for the phloem sap where the amount remains similar and even higher than the 21-nt species. This indicates that virus infection modulates dicer activities independent of whether the viral RNA is targeted by silencing or not.Citrus tristeza virus (CTV)-infected Mexican lime and sweet orange but not in sour orange [Sweet potato chlorotic stunt virus, SPCSV) increases 600-fold the titer of an unrelated potyvirus  in non-phloem tissue resulting in highly symptomatic plants. It was hypothesized that SPCSV interferes with the systemic phloem dependent signaling required for SPFMV resistance [Similar to our findings, a 2.5-3 fold reduction of 24-nt sRNAs relative abundance compared to healthy plants was detected in r orange . Interesr orange . A similr orange . In SPVDsistance . Only mosistance . WhetherCucumber yellows closterovirus (CuYV) infected pumpkin plants. In contrast to what we report here, the authors observed a distribution pattern clearly different from the healthy one. However, this apparent contradiction needs to be carefully considered since CuYV vsRNAs, mostly of 20 and 21-nt, represented 57% of total sRNAs. In our study, due to the low percentage of vsRNAs in infected phloem (10% in melon and close to 0% in cucumber), the bulk of the sRNA population in phloem belongs to the endogenous sRNA group. Consequently the size distribution analysis of the host genome sRNAs showed the same pattern than for the total sRNAs  tissues, in two different pathosystems for the first time. Our results indicate that the antiviral silencing machinery in melon and cucumber acts mainly through DCL4. One of the most interesting results to have emerged from our sRNA analysis is that the total sRNA pattern in phloem remains unchanged upon infection, unlike the other analyzed tissues. In addition, and independently of the accumulation level of vsRNAs, both viruses were able to modulate the host sRNA pattern.Cucumis melo cv. Galia and Cucumis sativus cv. Supermarketer plants were inoculated with purified virions and crude virus-containing extracts from infected plants respectively, 9 days after germination. As a control, mock-inoculated plants were grown in parallel and under the same greenhouse conditions (16 hours at 24°C and 8 hours at 18°C in daylight conditions). Inoculated-cotyledons, root, phloem and primary leaf from mock-inoculated and symptomatic infected plants were harvested 15 days after inoculation (15 dpi).Cotyledons of both To collect the exudates, the first petiole and the stem internode between the first and second leaf were cut with a sterile razor blade and with the help of a pipet the phloem was collected in an Eppendorf-tube containing 1 ml of TRIzol.Total RNA extraction was identical for all the samples and carried out by using conventional TRIzol (Sigma-Aldrich) protocol according to the manufacturer’s instructions. The quality of the samples was verified by Northern-blot analysis using specific digoxigenin-labelled riboprobes for each virus and evaluating both A260/230 and A260/280 absorption ratios (Nanodrop- Thermo Scientific).To enrich the samples in sRNAs, 3 volumes of polyethylene glycol (MW 8000) and NaCl to final concentrations of 5% and 500 mM respectively were added. After incubation on ice for 30 minutes, the samples were centrifuged at 15000 rpm for 15 min at 4°C. The upper phase was transferred to a new tube and 4 volumes of phenol were added. Then the tubes were vortexed thoroughly and centrifuged during 2 minutes at maximum speed. Afterwards the upper phase was precipitated and the pellet containing sRNAs was resuspended in 50 μl of water.2O/acetic acid (ratio 50:40:1) followed by a second 45 minutes incubation in the same solution with a ratio of 10:89:1. Thereafter the gel was dipped in 150 ml of H2O with 0.3 g AgNO3 for 45 minutes. Then, the gel was rinsed 3 times with sterile water and developed in a formaldehyde solution . The reaction was stopped by washing with sterile water.1 μg of each fractionated sample was separated in a 17% polyacrylamide/7 M urea gel for the subsequent silver staining. For this purpose, the gel was soaked for 45 minutes in ethanol/H2). Detection of the vsRNAs was carried out using specific riboprobes labeled with (α-32P) ATP. After overnight hybridization, the membranes were washed twice with 2X SSC plus 0.1% SDS for 10 min at room temperature, and once with 0.1X SSC plus 0.1% SDS at 55°C for 15 min, and examined with a bioimage analyzer (Fujifilm FLA-5100).In order to detect vsRNAs, melon (2 μg) and cucumber (4 μg) fractionated RNA samples were separated in a 17% polyacrylamide/ 7 M urea gel in 0.5X TBE, transferred onto a positively-charged nylon membrane using the Bio-Rad Trans-Blot Cell and covalently UV cross-linked to the membrane (700 × 100 μJ/cmhttp://www.genoscreen.com). Small RNAs were fractionated from total RNA by acrylamide gel purification. Single strand ligation of 3′ and 5′ adaptors was done before a second acrylamide gel purification. Reverse transcription and PCR amplification were performed to generate the cDNA colonies template library. To verify quality and ensure that there was no contamination, libraries were then titrated by a 1x50bp run on HiSeq2000.Production and sequencing of the libraries were carried out by the biotechnology company GenoScreen  and known virus genomes (MNSV Al: DQ339157 or PNRSV NcM1.NctSp.mur1: AJ306818) by the Bioinformatics Service at the IBMCP (http://www.ibmcp.upv.es). Count of matching sequence reads were normalized to the total number of reads after last filtering step and given in parts per millionEach sRNA library was aligned with the corresponding plant (Melonomics: melon_genome_pseudomolecules_V3.5.fasta http://www.thermoscientific.com/en/home.html) and 40 U RNase Inhibitor were added in a final volume of 20 μl and pulsed RT was performed as follow: 30 min at 16°C, pulsed RT of 60 cycles at 30°C for 30 s, 42°C for 30 s and 50°C for 1 s. Then samples were incubated at 85°C for 5 min. PCR was carried out with the following components: 0.2 μM of each primer . The PCR reaction was performed as follows: 2 min at 94°C, 25 cycles 15 s at 94°C and 1 min at 60°C.We used the protocol described by Varkonyi-Gasic, et al.  with somArabidopsis thaliana DCL proteins  were blasted against the corresponding database (http://www.phytozome.net and http://www.melonomics.net) to identify the four homologues in C. sativus and C. melo. Accession numbers: Cucsa.260100, Cucsa.356600, Cucsa.055950, Cucsa.350750 (C. sativus) and MELO3C005929P1/30P1, MELO3C010042P1, MELO3C011495P1, MELO3C010254P1 (C. melo).To analyze the differential expression of DCL transcripts, qRT-PCR was performed with 100 ng of total RNA using one step SYBR PrimeScript RT-PCR Kit II (Takara) following the manufacturer’s recommendations in an Applied Biosystems 7500 Real-Time PCR System. Each biological replicate was assayed in triplicate. Gene-specific oligonucleotide primers were designed using Primer Express® version 3.0 software (Applied Biosystems). Primer information is shown in Additional file


The aim of this study was biological and molecular characterization of classical swine fever (CSF) challenge virus from India.CSF challenge virus maintained at Division of Biological standardization was experimentally infected to two seronegative piglets. The biological characterization was done by clinical sign and symptoms along with postmortem findings. For molecular characterization 5’-nontranslated region, E2 and NS5B regions were amplified by reverse transcription polymerase chain reaction and sequenced. The sequences were compared with that of reference strains and the local field isolates to establish a phylogenetic relation.The virus produced symptoms of acute disease in the piglets with typical post-mortem lesions. Phylogenetic analysis of the three regions showed that the current Indian CSF Challenge virus is having maximum similarity with the BresciaX strain (USA) and Madhya Pradesh isolate (India) and is belonging to subgroup 1.2 under Group 1.Based on biological and molecular characterization of CSF challenge virus from India is described as a highly virulent virus belonging to subgroup 1.2 under Group 1 along with some field isolates from India and Brescia strain. Classical swine fever (CSF) is most important contagious viral disease of pigs and wild boar and causes high mortality . The disThe genome of CSFV is a single strand RNA of the positive sense, approximately 12,300 nucleotides in length. It has non-translated regions at either end (5’NTR and 3’NTR) of its genome, encompassing a single open reading frame encoding a large protein that is cleaved into smaller fragments. These cleaved proteins include a N-terminal protease, four structural proteins  toward the 5’ end of the genome and seven non-structural proteins  in the 3’ two-thirds of the genome . Three rIn the present study, 5`NTR and NS5B region for typing and molecular characterization of the CSF challenge virus, while E2 region is used for molecular characterization. For biological characterization, two seronegative piglets were experimentally infected with CSF challenge virus.The aim of the present study was biological and molecular characterization of CSF challenge virus from India.Animal experiments were approved by Committee for the Purpose of Control and Supervision of Experiments on Animals, Ministry of Environment and Forests, New Delhi and bio-security measures were taken during the whole study.The CSFV challenge virus, used in this study was kindly provided by Veterinary Biological Product Institute, Mhow, M.P., in 2005 and the same has been maintained in the Division of Biological Standardization by pig passages every year. The virus is being used for potency testing of CSF vaccine and also supplied to various State Biological Units.th day post infection (dpi). The piglets were sacrificed after 11th days for post mortem studies after moribund condition.In the present experiment, two seronegative piglets, aged around 2 months were used. Piglets were monitored for a period of 2 weeks prior to experiments for general clinical sign and rectal temperature. For the experimental infection, both piglets were injected subcutaneously into neck region with 10 ml 20% spleen suspension, containing challenge virus. Piglets were examined twice a day for the clinical signs and change in rectal temperature. Blood was collected (heparinized) at the peak rectal temperature for isolation of viral RNA. Total blood leukocyte count was calculated using Newberg’s hemocytometer using the standard method before infection and 11Total RNAs were extracted from the CSFV infected blood or spleen by TRIzol reagent (Life Technologies) as per the manufacturer’s protocol. The total RNAs were reverse transcribed to generate cDNA by MMLV-Reverse Transcriptase enzyme (Promega) using random hexamers. The cDNA obtained was further subjected to PCR for the three regions of 5’ NTR, E2 and NS5B using primers shown in PCR reactions were performed in 25 μl of reaction mixture, which contained 2.5 μl of ×10 buffer, 200 μM of each dNTP, 10 pmol of each primer, 50 ng of each DNA sample, and 1.5 units of Taq polymerase (Thermo scientific). Amplification was carried out with initial denaturation step of 94°C for 4 min, followed by 35 cycles of 94°C for 1 min, annealing temperature (Ta) for 1 min, 72°C for 1 min, and a final extension of 72°C for 10 min. Annealing temperature (Ta) was standardized at 50°C, 55°C and 56°C for PCR of 5’ NTR, E2 and NS5B regions respectively . The PCRThe PCR products were gel purified using QIAquick Gel Extraction Kit  as per the manufacturer’s protocol. The purified PCR products were sequenced using Sanger`s sequencing method and the partial genomic sequences of 5’ NTR, E2 and NS5B gene of Indian CSF challenge virus were submitted to GenBank and their accession numbers are JF903852 (5`NTR), JF903851 (E2), and JF903853 (NS5B).et al. (1996) and Paton et al. (2000) was used. Phylogenetic analysis was performed for the 150 bp fragment for 5’ NTR, 308 bp fragment for E2, and 409 bp sequence for NS5B region of the CSF viral genome. The sequences from different sources were obtained and manually edited to obtain the desired sequence length for uniform alignment.Sequences for the phylogenetic analyses were obtained from already published ,10,14,15et al. (1996) and Paton et al. (2000) were used as the starting point. Phylogenetic and molecular evolutionary analyses were conducted using MEGA version 6 [Alignment of nucleotides sequences were analyzed by the Clustal W method. Sequences were assembled into multiple sequences alignment. The phylogenetic tree derived from sequences was constructed by neighbor-joining method of MEGA version 6 . To acceersion 6 .3-4800 cells/mm3 and 19000 cells/mm3-2650 cells/mm3)  .rd to 4th days onwards, animal had reduced feed intake, and after 4 dpi, animals were completely fed-off. After 5 days, dpi animals became recumbent with vigorous belly movement. After 6-8 dpi animals were recumbent, with dyspnea, eyes closed and inability to stand, and nervous signs were observed. Posterior paralysis was observed in both the animals 8 dpi  were enlarged and hemorrhagic , and most closely related to the Madhya Pradesh isolate, while most of the vaccine viruses  are clustered together in the different clade . PhylogeOn phylogenetic analysis of 150 bp region of 5`NTR region, virus can be grouped into various groups and subgroups ,17. UsinOn phylogenetic analysis of 409 bp region of NS5B region, CSF challenge virus was grouped into Group 1 and subgroup 1.2 as already described -10. On pOn the basis of clinical signs, postmortem legion and genetic characterization of 5`NTR, E2 and NS5B regions the CSF challenge virus have been found to be a highly virulent virus belonging to Group 1.2 which is more closely related to Indian field outbreak isolate of Madhya Pradesh and BresciaX strain of USA, followed by Brescia strain of Switzerland. This is the first report of characterization of Indian CSF challenge virus which describes it to be a highly virulent and designated as 1.2 on the basis of partial genome.PK, PD implemented the study design. PK and VU carried out the work part and PK, VU and PD did analysis part and wrote the manuscript. All authors have read and approved the final manuscript.


Equid herpesvirus type 1 (EHV-1) causes outbreaks of abortion and neurological disease in horses. One of the main causes of these clinical syndromes is thrombosis in placental and spinal cord vessels, however the mechanism for thrombus formation is unknown. Platelets form part of the thrombus and amplify and propagate thrombin generation. Here, we tested the hypothesis that EHV-1 activates platelets. We found that two EHV-1 strains, RacL11 and Ab4 at 0.5 or higher plaque forming unit/cell, activate platelets within 10 minutes, causing α-granule secretion (surface P-selectin expression) and platelet microvesiculation . Microvesiculation was more pronounced with the RacL11 strain. Virus-induced P-selectin expression required plasma and 1.0 mM exogenous calcium. P-selectin expression was abolished and microvesiculation was significantly reduced in factor VII- or X-deficient human plasma. Both P-selectin expression and microvesiculation were re-established in factor VII-deficient human plasma with added purified human factor VIIa (1 nM). A glycoprotein C-deficient mutant of the Ab4 strain activated platelets as effectively as non-mutated Ab4. P-selectin expression was abolished and microvesiculation was significantly reduced by preincubation of virus with a goat polyclonal anti-rabbit tissue factor antibody. Infectious virus could be retrieved from washed EHV-1-exposed platelets, suggesting a direct platelet-virus interaction. Our results indicate that EHV-1 activates equine platelets and that α-granule secretion is a consequence of virus-associated tissue factor triggering factor X activation and thrombin generation. Microvesiculation was only partly tissue factor and thrombin-dependent, suggesting the virus causes microvesiculation through other mechanisms, potentially through direct binding. These findings suggest that EHV-1-induced platelet activation could contribute to the thrombosis that occurs in clinically infected horses and provides a new mechanism by which viruses activate hemostasis. The min vivo ,9. Varioin vivo –14 and pin vivo ,16. ThisEquid herpesvirus type 1 (EHV-1) is a double stranded DNA virus and a member of the Alphaherpesviridae subfamily. EHV-1 is highly contagious causing outbreaks of respiratory and neurologic disease, abortion, and neonatal mortality . SimilarThe role of platelets in the pathogenesis of EHV-1 infection is unknown. Since platelets are integral to thrombosis, we hypothesized that EHV-1 would associate with and activate equine platelets. We found that platelets were activated within 10 minutes of exposure to two EHV-1 strains, RacL11 and Ab4, and released α-granule contents and underwent microvesiculation. The α-granule secretion was mediated by thrombin, generated by virus-associated TF-triggered activation of factor X (FX). In contrast platelet microvesiculation was partly TF and thrombin-independent.Preparation of virus and mock controlsAll reagents were from Sigma-Aldrich  unless otherwise stated. ORF16 gene, which encodes envelope glycprotein C, en passant mutagenesis was performed using an infectious bacterial artificial chromosome (BAC) clone of Ab4 (pAb4) as previously described [KanR) as a template, a PCR fragment was amplified, digested with DpnI to remove residual template DNA, and then the purified product was electroporated into E. coli GS1783 -competent cells containing pAb4. The first round of Red recombination produced kanamycin resistant (KanR) colonies, which were screened for correctness by restriction digest. The KanR gene was excised using growth in LB medium that was supplemented with 1% L-(+)-Arabinose . After a second step of Red recombination, KanR-deficient colonies were isolated and analyzed by restriction enzyme digestion, PCR verification, and DNA sequencing (The RacL11 and Ab4 strains of EHV-1 plaque purified strains (<3 passages) were used in the study. RacL11 was isolated from an aborted fetus , whereasescribed . Two priquencing . The rec2 flasks in Dulbecco’s Modification of Earl’s Medium  supplemented with L-glutamine, glucose, sodium pyruvate and 10% fetal bovine serum  at 37°C with 5% carbon dioxide in a tissue culture incubator. One-day-old confluent monolayers of RK cells were infected with virus at 0.1 plaque-forming units (PFU)/cell until a cytopathic effect of approximately 90% was achieved. The flasks were then subjected to two freeze-thaw cycles. The culture media were centrifuged at 1800 x g for 15 minutes at 4°C then were ultracentrifuged at 175,000 x g for 1 hour at 4°C (with no brake) on a 60, 30 and 10% sucrose gradient. The band (containing virus) between the 30% and 60% sucrose layers was removed, diluted in phosphate-buffered saline  and ultracentrifuged again. The supernatant was discarded and the pellet was reconstituted in PBS, separated into aliquots and stored at -20°C until use (multiple freeze-thaw cycles were avoided). Virus titers were determined using a standard plaque assay. Freeze-thaw lysates from uninfected RK cells were processed similarly and served as mock-infected negative controls.Virus was propagated in RK cells  and purified on a discontinuous sucrose gradient using ultracentrifugation. The RK cells were cultured in 75 cmBlood was collected from the jugular vein of clinically healthy horses using an 18-G needle  and 6 ml syringe (Covidien) that was prefilled with 3.2% citrate or acid-citrate dextrose (ACD), maintaining an anticoagulant to blood ratio of 1:9 and 1:5, respectively. The needle was inserted first and blood was allowed to drip through the needle for a few seconds before syringe attachment. Blood was then slowly drawn into the syringe. In select experiments, blood was also collected into citrate vacutainer tubes, with or without added corn trypsin inhibitor . The samples were immediately transported to the laboratory for preparation of platelet-rich plasma (PRP) or washed platelets, which occurred within 15–30 minutes of sample collection. The sample collection protocol was approved by the Institutional Animal Care and Use Committee at Cornell University (#2007–0086), which follows several federal and state guidelines, including the U.S. Department of Agriculture Animal Welfare Act (1966), Regulation  and the Guide for the Care and Use of Agricultural Animals in Research and Teaching (2010).g at 21°C for 10 minutes. The supernatant or PRP was removed and a platelet count was measured on the PRP using an automated hematology analyzer . For preparation of washed platelets, ACD-anticoagulated blood was diluted in a 1:1 ratio with platelet buffer I , then centrifuged at 450 x g at 21°C for 5 minutes to obtain the platelet-rich fraction. This fraction was removed and centrifuged at 1250 x g at 21°C for 10 minutes. The platelet pellet was then washed three times with platelet buffer I and gently resuspended in platelet buffer II . A platelet count was measured as above. Platelet-rich plasma or washed platelets were rested for 30 minutes at room temperature before exposure to treatments or virus.For preparation of PRP, 3.2% citrate-anticoagulated blood from clinically healthy horses was transferred to a 15 ml polypropylene tube and the red blood cells were allowed to settle using gravity sedimentation for 20 minutes at room temperature. The resulting leukocyte-platelet-rich plasma was removed and centrifuged at 250 x 5 final concentration) in flow buffer  with supplemental glycine-proline-arginine-proline . Calcium chloride (2.5 mM) was added to the buffer for most experiments. The platelets were exposed to virus at various PFUs/cell for 10 minutes at 37°C, using PBS and RK lysate as negative controls. As positive platelet activation controls, platelets were stimulated for 10 minutes at 37°C with bovine thrombin  for P-selectin expression or a mixture of bovine thrombin and convulxin  for PDMP quantification . In select experiments, platelets in PRP were exposed to the thrombin inhibitor hirudin (10 units), a goat polyclonal anti-rabbit TF antibody  with a goat IgG control , or an inhibitory murine monoclonal antibody against equine MHC-I [g for 20 minutes at 4°C, then centrifugation twice at 22,000 x g for 20 minutes at 4°C. This is an accepted method for obtaining PDMP-depleted plasma [Platelets were diluted  and FX  in HEPES buffer  for 15 minutes at 37°C. Then a chromogenic substrate  was added and the color change was measured kinetically every 15 seconds at an optical density of 405 nm at 37°C with a plate spectrophotometer . The rate of change in optical density (Vmax) was recorded for the first 60 seconds (in the linear portion of the curve) and converted to amount of FXa generated (nM) based on a standard curve created from serial dilutions of human FXa (Haematologic Technologies Inc). The standard curve was linear between FXa concentrations of 0.17 and 5.43 nM. Negative controls included virus without added FVIIa, FX or substrate. Assays were run in duplicate and results were averaged.5 platelets as PRP (from the same horse as the CTI plasma), with virus or RK lysate alone, platelets, and CTI-MDP alone as negative controls. After a 10 minute incubation at 37°C, a fluorogenic substrate with calcium was added, then fluorescence intensity was measured at one minute intervals for 120 minutes using excitation and emission wavelengths of 360 and 460 nm, respectively, with a plate spectrofluorometer (Spectromax M3). Thrombograms represent a mean of three experiments. Reported results of lag time (minutes) and total thrombin generated (derived from the area under the curve and expressed as nM*minute) were obtained from the manufacturer’s software (SoftMax Pro 6.2.1).Virus-induced thrombin generation was measured in equine MDP using calibrated automated thrombography and a single lot of a commercially available kit, with calibrators . Equine citrate-anticoagulated plasma can generate thrombin via contact activation in this system. To inhibit factor XIIa and subsequent contact activation, blood was collected from the jugular vein of a single horse into a CTI-containing citrate vacutainer and CTI-MDP was prepared as described above. Virus or RK lysate  were added to CTI-MDP, with or without 1 x 10Data is expressed as means with standard deviation (SD). Means of 2 groups were compared with a paired T test or student T test, as appropriate. Means of 3 or more groups were compared using an Analysis of Variance (ANOVA) or Repeated ANOVA, as appropriate, with a Tukey’s multiple comparison post-hoc test . A p value (two tailed) was set at < 0.05.Both strains of EHV-1 induced surface P-selectin expression and shedding of PDMPs at 0.5–1 PFU/cell, with little to no activation occurring at lower infectious doses . We alsoTo determine if EHV-1-induced activation of platelets required plasma, we washed ACD-anticoagulated platelets free of plasma and exposed them to RacL11 and positive and negative controls in the presence or absence of equine plasma. Using P-selectin as the marker for platelet activation and thrombin alone as the positive control, we found that expression was abolished in washed platelets and re-established by the addition of 5 ul equine MDP . We thenThe prototypical Alphaherpesvirus HSV can initiate thrombin generation through extrinsic or intrinsic/contact pathways ,30. We uSince factor-deficient equine plasma is not available, we added factor-deficient human MDP to washed equine platelets to examine the role of the extrinsic pathway in EHV-1-induced platelet activation. Addition of 5 ul of equine or human MDP to washed equine platelets was sufficient to re-establish platelet P-selectin expression after exposure to RacL11 . Unlike Herpes simplex virus can also generate thrombin through the contact and intrinsic pathways . Also, tUsing a combination of functional clotting assays with FVII- and FX-deficient plasma, inhibitory anti-TF antibodies and flow cytometry and electron microscopy techniques, Sutherland et al have shown that HSV envelopes constitutively express TF and phospholipid derived from the host cell . We perfTo more directly test the role of TF in thrombin generation and platelet activation, we pre-incubated a goat polyclonal anti-rabbit TF antibody (152 ug/mL) with an equivalent concentration of goat IgG as a negative control, with virus or RK lysate for 10–15 minutes at room temperature before adding the virus or controls to equine PRP. The anti-TF antibody abolished P-selectin expression on EHV-1-exposed platelets  but onlyPrevious studies with HSV have shown that the virus-encoded envelope protein gC is a weak direct activator of FX and co-operates with TF to generate FXa and promote virus replication within human umbilical vein endothelial cells ,32. We eg for 5 minutes at 22°C, then performed plaque assays on RK cells. Cells not exposed to virus and virus without cells treated to similar washes served as negative controls, whereas virus not subjected to the centrifugation washes was used as a positive control. We found that infectious virions could be retrieved from washed platelets , suggesting that the virus binds to platelets without any requirement for plasma and calcium.To examine if EHV-1 binds to platelets, we exposed washed platelets  to EHV-1 for 10 minutes at 37°C, washed the platelets three times in PBS by centrifugation at 850 x To determine if EHV-1 required platelets to generate thrombin or could generate thrombin in a plasma milieu alone, virus  was incubated with equine CTI-MDP in the presence or absence of platelets and thrombin generation was measured using a fluorescent substrate. At 1 PFU/cell, the Ab4 strain generated small amounts of thrombin in CTI plasma alone without autologous platelets. Autologous platelets shortened the lag time and boosted thrombin generation with both virus strains . Ab4 genEquine platelets express major histocompatibility complex class I molecules (MHC-I) , which iWe show here for the first time that EHV-1 associates with and activates platelets, inducing α-granule secretion and microvesiculation. Platelet activation is largely mediated by thrombin generated through TF-FVIIa-triggered activation of FX, although there is a thrombin-independent component to microvesiculation. Our working model of EHV-1-induced platelet activation is that the virus rapidly binds to the outer platelet membrane. Once sufficient virus particles have attached (0.5 PFU/cell or higher), microvesiculation is induced and thrombin generation is triggered via TF incorporated within the virus envelope and propagated via virus- or platelet-associated phospholipid. This yields a platelet membrane-localized thrombin burst, which activates platelets via protease-activated receptors. Although free virus with both strains (depending on virus load) can generate thrombin in the absence of platelets, platelets amplify thrombin generation . Our datStrain-dependent differences were observed in procoagulant activity, thrombin generation and platelet activation responses. The reason for these observed differences is unknown. It is possible that these differences are due to the inherent imprecision of plaque assays for virus quantification or a higher ratio of virions to PFU for RacL11 versus Ab4. However, the observed differential response was maintained across several virus preparations leading us to hypothesize that there are structural differences between the strains that affect their procoagulant ability. For instance, the higher procoagulant activity with RacL11 suggests this strain contains more TF in its envelope or TF is in a “decrypted”, more procoagulant conformational state. Surprisingly, the higher procoagulant activity with the purified system did not translate into more robust thrombin generation in a plasma milieu with RacL11, even though this strain induced stronger microvesiculation. This could be due to strain-related differences in envelope phospholipid content or glycoproteins, which may affect assembly or activity of coagulation factors on the virus or platelet surface. There are minor strain-associated differences in envelope glycoprotein sequences, but this does not translate into differences in virus attachment on other cells . RacL11 Higher infectious viral doses, particularly with the RacL11 strain, yielded strong microvesiculation, usually to a greater degree than the thrombin-convulxin control. When marked, this microvesiculation was accompanied by a decrease in P-selectin expression, which was not seen with thrombin or thrombin-convulxin stimulation. This was attributed to loss of membrane-anchored P-selectin as a consequence of microvesiculation, as observed previously with calcium ionophore-treated equine platelets . The losHow the virus associates with platelets is unknown. Equine platelets express MHC-I , a knownin vitro or in vivo contain high concentrations of interleukin-1β (IL-1β) and increase vascular permeability in an IL-1β-dependent manner [Virus-induced platelet activation can have several consequences for the host. Activation (or apoptosis) could be part of a host protective or innate immune response to virus infection and may facilitate viral clearance. This immune response can improve survival in virus-infected hosts, despite an ensuing thrombocytopenia that can manifest as hemorrhage if sufficiently severe ,42,43. Pt manner . Potentit manner –20. Finat manner .In conclusion, our studies highlight a new mechanism by which an enveloped virus, EHV-1, can rapidly activate platelets, namely through generation of thrombin by virus-associated TF. The virus-associated TF is likely derived from the host cell membrane and expressed on the virus envelope. We hypothesize that other enveloped viruses, such as cytomegalovirus and HSV, which incorporate TF within their envelope , could pS1 FigA) or PDMPs (B) was then quantified (n = 4). At the higher PFUs/cell of 0.5, 1 and 5, both strains induced P-selectin expression and microvesiculation. RacL11 induced significantly stronger microvesiculation than Ab4, with an associated decrease in P-selectin expression. * p ≤ 0.001 versus RK, 0.01 and 0.05 PFU/cell for each virus strain. ** p < 0.001 versus Ab4 at 0.5, 1, and 5 PFU/cell.Platelets were exposed for 10 minutes to RacL11 and Ab4 EHV-1 strains at increasing PFU/cell  with rabbit kidney (RK) lysate as a negative control. The mean ± SD percentage of platelets positive for P-selectin ((EPS)Click here for additional data file.S2 FigA) and release of platelet-derived microparticles  in response to Ab4 at 1 plaque forming unit/cell or thrombin  (n = 5). No P-selectin expression or PDMP release occurred in PBS-treated negative control platelets. * p < 0.05 versus untreated platelets. P-selectin expression was abolished in washed platelets exposed to Ab4, but re-established with addition of platelet-derived microparticle-depleted citrate-anticoagulated equine (E) or human plasma containing all coagulation factors (Full) or human plasma deficient in factors IX, XI or XII. In contrast, addition of human FVII- or FX-deficient plasma did not re-establish P-selectin expression, unless supplemental purified FVIIa (1 nM) was added to FVII-deficient plasma (FVII- + FVIIa) . * p < 0.05 versus washed platelets with no added plasma, ** p < 0.05 versus Full plasma. In contrast to P selectin, PDMPs were still present in washed platelets with or without microparticle-depleted equine or human plasma, indicating a plasma-independent component to EHV-1-induced microvesiculation. The degree of microvesiculation trended lower when FVII- or FX-deficient plasma was added to Ab4-exposed washed platelets and supplemental purified human FVIIa significantly boosted PDMP percentages in FVII-deficient plasma . Data shown are mean ± SD.** p < 0.05 versus Full plasma.Addition of hirudin (10 units) to equine citrate-anticoagulated platelet-rich plasma reduced P-selectin expression ((EPS)Click here for additional data file.S3 FigA) or platelet-derived microparticles  was quantified by flow cytometry (n = 3). CTI did not significantly inhibit these markers of platelet activation.Platelet-rich plasma prepared from blood collected into citrate anticoagulant with or without CTI (50 ug/mL) was exposed to the RacL11 and Ab4 strains of EHV-1 at 1 plaque forming unit/cell or rabbit kidney (RK) cell lysate for 10 minutes at 37°C, then the mean ± SD percentage of platelets expressing P-selectin ((EPS)Click here for additional data file.


Leishmania infantum, the causative agent of zoonotic visceral leishmaniasis in the Western Mediterranean basin is present affecting both humans and dogs. We investigated the capacity of dogs to be used as sentinels for sandfly-borne phleboviruses as previously shown for leishmaniasis.Sandfly-borne phleboviruses are present in North Africa where they can infect humans in regions where A total of 312 sera were collected from guard dogs in two different bioclimatic regions (governorates of Kairouan and Bizerte) of Tunisia where zoonotic visceral leishmaniasis has been reported. These sera were tested for the presence of neutralising antibodies against 3 phleboviruses: Toscana virus, Punique virus and Sicilian virus. In the governorate of Kairouan, seroprevalence rates of 7.5%, 43.5%, and 38.1% were observed for Toscana, Punique and Sicilian virus, respectively. A high proportion of sera from the governorate of Bizerte were hemolyzed and showed high cytotoxicity for the cells and subsequently precluded detailed interpretation of this batch. However, validated results for 27 sera were in agreement with data observed in the governorate of Kairouan.Toscana virus is present in the governorate of Kairouan but at a lower rate compared to Punique and Sicilian viruses. These three sandfly-borne phleboviruses can infect dogs. Direct detection and isolation of the viruses are now to be attempted in animals as well as in humans. Our findings showed that guard dogs are good sentinels for virus transmitted by sandflies and strongly suggested that the high seroprevalence rates observed in dogs merit further attention. Phlebovirus, family Bunyaviridae) were not only geographically restricted to southern Europe, but were also present in North Africa [Phlebotomus pernicious and Phlebotomus longicuspis, which are considered as possible vectors of phleboviruses [Leishmania infantum, etiologic agent of zoonotic visceral leishmaniasis (ZVL) in Tunisia [Recent studies have indicated that sandfly-borne phleboviruses  two viruses belonging to the Sandfly fever Naples species (Toscana virus and Punique virus), (ii) and one virus belonging to the Sandfly fever Sicilian species (Sicilian virus) as described previously . NeutralFrom the governorates of Kairouan and Bizerte, 194 and 118 sera were collected from guard dogs, respectively. The virus microneutralisation (VNT) assay was performed in 96-well microtitre plates using Vero cells as previously described  with sli4 cells in 5% foetal bovine serum was added to each well, and incubated at 37°C in presence of 5% CO2. After 5 days, the microplates were read under an inverted microscope, and the presence or absence of cytopathic effect was noted. The titre  was recorded. The threshold for positivity was defined as 1:20 [For this study the 3 virus strains were (i) Toscana virus strain MRS2010-4319501 , (ii) PuThe results are presented in Table The results obtained from the analysis of 147 sera from Kairouan showed that the 3 viruses (or very closely related viruses) were circulating in the region. Seroprevalence rates of 43.5%, and 38.1% were observed for Punique virus and Sicilian virus, respectively. In contrast, only 7.5% of dog sera possessed TOSV-NT-Ab. Of the 11 sera that showed positive results with Toscana virus, 7 did not contain PUNV-NT-Ab demonstrating that these dogs had been infected with Toscana virus only and not with Punique virus. For the 4 remaining sera, the respective titres for TOSV-NT-Ab/PUNV-NT-Ab were 40/40 for one serum, 40/160 for one serum, and 20/160 for 2 sera. Three sera showed a difference ≥ two-fold dilutions in favour of Toscana virus, which is undisputable evidence of Toscana virus past infection. Therefore, past infection with Toscana virus is unambiguous for 10 of the 11 reactive sera. Since neutralisation is the most specific and discriminative serological technique; we can exclude that cross-reactivity is a valid explanation for the finding of NT Ab against two related viruses. Thus, it is most likely that these dogs have been successively infected by Toscana virus and Punique virus. Toscana virus is present in the governorate of Kairouan although it is much less frequently infecting dogs than Punique and Sicilian viruses.P. perniciosus is the predominant sandfly species in the governorate of Bizerte, P. longicuspis is the most abundant sandfly species in the governorate of Kairouan [P. perniciosus is more anthropophilic compared to P. longicuspis which is more zoophilic leading to a difference in human versus dog biting rates and subsequently to a difference in the infection status with Toscana virus and Punique virus in humans compared to dogs within the governorates of Bizerte and Kairouan. To address this hypothesis, the forage ration of the two sandfly species needs to be determined in both governorates.In the governorate of Kairouan, the presence for TOSV-NT-Ab in only 7% of dogs versus 43% of PUNV-NT-Ab was unexpected; indeed, it is in contrast with the high rate of TOSV-NT-Ab and low rate of PUNV-NT-Ab recently reported in humans from the governorate of Bizerte . A possiKairouan ,10,12. SThe high rate of SFSV-NT-Ab observed in the governorate of Kairouan demonstrates that Sicilian virus or a very closely virus related to Sicilian virus is present and circulates at high levels in the region. Such findings are in agreement with the identification of sequences corresponding to a Sicilian-like virus, provisionally named Utique virus in the village of El-Felta located within the governorate of Sidi Bouzid adjacent to the governorate of Kairouan .The results obtained from the 27 sera collected from dogs in the governorate of Bizerte for which the VNT was interpretable showed that 16 sera contained neutralising antibodies against Sicilian virus which is coherent with the detection of Utique virus in 7 pools of sandflies collected from the same region in 2010  but alsoThe results of this study provided further evidence that Toscana, Punique and Sicilian viruses are present in Tunisia and showed that guard dogs may represent excellent sentinels for virus transmitted by sandflies. The existence and nature of vertebrate reservoir of sandfly-borne phleboviruses is unknown; however, the high seroprevalence rates observed in dogs in this study leads to further investigations concerning the possible role of dogs in the transmission dynamic of theses arboviruses.Whether Punique virus and Sicilian virus represent a threat for humans in Tunisia, as previously shown for Toscana virus , needs tIn conclusion, the results of this study showed that Toscana, Punique and Sicilian viruses are circulating in several regions of Tunisia, and dogs are frequently infected with these viruses for which they could serve as sentinels.


Nanopore sequencing, a novel genomics technology, has potential applications for routine biosurveillance, clinical diagnosis, and outbreak investigation of virus infections. Using rapid sequencing of unamplified RNA/cDNA hybrids, we identified Venezuelan equine encephalitis virus and Ebola virus in 3 hours from sample receipt to data acquisition, demonstrating a fieldable technique for RNA virus characterization. Portable and reliable molecular epidemiology techniques and field approaches for assessing virus genomes are desired to inform clinical diagnostics and public health operations. Need for such methods has been highlighted by the recent Middle East respiratory syndrome and Ebola virus disease (EVD) epidemics, during which it became necessary to characterize novel viruses and to evaluate genetic drift, transmission chains, and zoonotic introductions.www.ncbi.nlm.nih.gov/RefSeq/]). From the results of these analyses, we determined that the current and future versions of nanopore sequencing technology can be used to rapidly identify and characterize pathogens. .To determine if nanopore sequencing can be used as an accelerated viral genome sequencing tool, we utilized a rapid cDNA/RNA–hybrid library preparation procedure to sequence cell cultures of Venezuelan equine encephalitis virus vaccine (VEEV) strain TC-83 or Ebola virus (EBOV) isolate Makona-C05 stock IRF0137. To evaluate nanopore sequencing for rapid, field-deployable pathogen characterization, we collected raw read data and statistics for VEEV and EBOV sequence runs on the MinION sequencing device . To determine the level of identification and accuracy of genome characterization over sequencing runtime, these reads were then mapped to VEEV and EBOV genomes and to reference databases . The coverage increased from 15–60 min from the 3′ end of the VEEV genome with reads aligning directionally from the 3′ to 5′ end of the VEEV genome -tailed RNA genome. Thus, poly-dT primers can be used for cDNA production without further genomic RNA manipulation. The workflow to isolate the RNA and prepare it for sequencing  took ≈3 V genome , panel Bhttp://www.ncbi.nlm.nih.gov/genomes/GenomesGroup.cgi?taxid=10239&opt=Virus), plus the VEEV TC-83 genome database (GenBank accession no. L01443). We then used LAST to align nanopore reads against this set of references (www.onecodex.com), and found that the sample contained VEEV ,,http://wned VEEV .http://www.thermofisher.com/us/en/home/brands/product-brand/trizol.html) was poly(A)-tailed, a single complementary strand of cDNA synthesized, and RNA/cDNA hybrids sequenced. The EBOV samples sequenced on MinION rapidly provided usable, accurate data, despite less raw data than the VEEV TC-83 dataset (137kbp for EBOV versus 2.4Mbp for VEEV at 60 min). Using 15-and 60-min time points and an identical alignment strategy to VEEV TC-83 above, we detected EBOV as the top hit within the sequencing dataset when compared to all virus RefSeq sequences  and EBOV (a negative-stranded RNA virus) prepared from cell-culture supernatants. This method definitively identified VEEV TC-83 and differentiated it from wild-type VEEV in ≈3 hours, including only 15 min of data acquisition on MinION. VEEV TC-83 was also differentiated from other alphavirus genomes, facilitating strain-level identification of TC-83. EBOV was also identified rapidly by this approach, differentiating the virus in the sample analyzed here from available virus reference genomes. However, variant/isolate level characterization was not possible due to limited data generated from the RNA/cDNA–hybrid approach.The method applied here is greatly accelerated compared to traditional next-generation sequencing library preparation, and was used with reagents and equipment suitable for austere conditions . This study confirmed the possibility of accurate RNA virus genome characterization from RNA/cDNA hybrids by using limited sample manipulation, albeit from relatively pure samples. If samples derived directly from clinical matrices  were used, this method would probably not support the necessary depth to characterize virus genomes unless the pathogen titer within these samples was high. As the depth of sequence data obtained from nanopore sequencing approaches continues to improve (Utilization of nanopore sequencing in Western Africa (Description of determining the ability of using nanopore sequencing to provide rapid genomic data for RNA virus pathogens, supported by data collection and analysis techniques.


The recent Ebola virus (EBOV) epidemic highlights the need for efficacious virucidal products to help prevent infection and limit the spread of Ebola virus disease. However, there is limited data on the efficacy of virucidal products against EBOV, because the virus has a high biosafety level and is only available in a few laboratories worldwide.The virucidal efficacy of antiseptics and disinfectants can be determined using the European Standard EN14476:2013/FprA1:2015. Modified vaccinia virus Ankara (MVA) was introduced in 2014 as a reference virus for the claim ‘virucidal active against enveloped viruses for hygienic hand rub and hand wash’. For EBOV, also an enveloped virus, the suitability of MVA as a surrogate needs to be proven.in vitro efficacy of four povidone iodine (PVP-I) formulations against EBOV: 4 % PVP-I skin cleanser; 7.5 % PVP-I surgical scrub; 10 % PVP-I solution; and 3.2 % PVP-I and 78 % alcohol solution. The formulations were tested with MVA to define the test conditions, and as a secondary objective the suitability of MVA as a surrogate for enveloped viruses like EBOV was assessed.The aim of this study was to test the According to EN14476, a standard suspension test was used for MVA. Large-volume plating was used for EBOV to increase test sensitivity and exclude potential after-effects. All products were tested under clean (0.3 g/L BSA) and dirty (3.0 g/L BSA + 3.0 mL/L erythrocytes) conditions with MVA for 15, 30, and 60 s. The concentration-contact time values obtained with MVA were verified for EBOV.Viral titres of MVA and EBOV were reduced by >99.99 % to >99.999 % under clean and dirty conditions after application of the test products for 15 seconds.All products showed excellent virucidal efficacy against EBOV, demonstrating the important role PVP-I can play in helping to prevent and limit the spread of Ebola virus disease. The efficacy against both test viruses after 15 s is helpful information for the implementation of guidance for people potentially exposed to EBOV, and confirms the excellent virucidal efficacy of PVP-I against enveloped viruses. MVA was found to be a suitable surrogate for enveloped viruses like EBOV. In December 2013, the Ebola virus (EBOV) epidemic began in Guinea, and on March 23, 2014, the Ebola virus outbreak was officially communicated by the World Health Organization (WHO). This outbreak in West Africa  has been the largest and most complex outbreak since the virus was discovered.EBOV is spread mainly through contact with body fluids of symptomatic patients or contaminated surfaces , so healConsidering that EBOV is a deadly threat, it is clear that only chemical disinfectants with proven virucidal efficacy can be used. This can be achieved by ensuring that disinfectants pass a virucidal activity test performed in compliance with good laboratory practice and country-specific standards. In Europe, EN14476  has inclAlthough enveloped viruses are deemed to be more susceptible to disinfectants, they may react differently than non-enveloped viruses with regard to the concentration and required application time of the active ingredient. Therefore, it is necessary to test an enveloped model virus for the claim ‘virucidal active against enveloped viruses’. Despite the usual role of enveloped viruses as blood-borne pathogens, e.g. human immunodeficiency, hepatitis B and hepatitis C viruses, they are also responsible for severe outbreaks including severe acute respiratory syndrome (SARS), Middle East respiratory syndrome coronavirus (MERS-Co), influenza pandemic, and hemorrhagic fever viruses such as EBOV.The CEN/TC 216/WG1 committee, which establishes standardised European testing methods and requirements for the antimicrobial efficacy of chemical disinfectants and antiseptics, intended to implement a new test virus to assess antimicrobial efficacy against all enveloped test viruses. The choice of model viruses is of great importance when establishing such a standard test method. The requirements for model viruses include high resistance to disinfectants and drying, combined with simple virus propagation in cell culture (e.g. growing in high titres). The experts in WG1 suggested the enveloped virus modified vaccinia virus Ankara (MVA) as the reference virus for the claim ‘virucidal active against enveloped viruses for hygienic hand rub and hand wash’. This virus has been shown to produce similar results to vaccinia virus strain Elstree in virucidal testing , and wasPovidone iodine (PVP-I) has been known for a long time as a broad spectrum microbicide against bacteria, fungi, protozoans and viruses . In a wain vitro data for the general virucidal activity of PVP-I is available, demonstrating efficacy against enveloped viruses such as mumps, herpes simplex, rubella, measles, influenza, human immunodeficiency and corona viruses, and non-enveloped viruses including adeno-, rota-, polio-, coxsackie- and rhinovirus  and eight parts by volume of the test product. Aliquots of 100 μL from each test product at each dilution were then inoculated into six wells of a 96-well microtitre plate containing 200 μL cell suspension BHK-21 cells. The cell cultures were observed for cytotoxic effects for the same incubation time as was later used for the suspension tests.When using cell culture to evaluate disinfectant activity on viruses, the target cells are often very sensitive to the active ingredient. However this cytotoxic effect seen 7–108 tissue culture infectious dose 50 % [TCID50]/mL) and one part by volume of the organic load were mixed with eight parts by volume of the PVP-I hand wash or hand rub product. The test products were examined undiluted and as 1:10 and 1:100 solutions. After the specified contact time , the virucidal activity was immediately suppressed by dilution with nine volumes of ice-cold medium (MEM + 2.0 % FCS) and without delay the assay was serially diluted 10-fold. Due to the immediate titration, no after-effect of the product could occur. Infectivity was determined by means of end point dilution titration in microtitre plates. Aliquots of 100 μL from each dilution were placed in six wells of a sterile polystyrene flat-bottomed 96-well microtitre plate containing 200 μL BHK-21 cells. Cultures were observed for cytopathic effects after eight days of inoculation. For the virus control, doubly distilled water was applied instead of the test product. All tests were performed under clean conditions (0.3 g/L BSA) and dirty conditions (3.0 g/L BSA + 3.0 mL/L erythrocytes) as interfering substance.Tests were carried out once, in accordance with EN14476:2013/FprA1:2015 at 20 °C ± 1 °C [50/mL, including standard deviation. Titre reduction is presented as the difference between the virus titre after contact with the test product and the control virus titre. This difference is given as a reduction factor including its 95 % confidence interval. A reduction in virus titre of ≥4 log10 (corresponding to an inactivation of ≥99.99 %) was regarded as evidence of sufficient virucidal activity. The calculation was performed according to EN14476 [The virus titres were determined using the Spearman-Kärber method , 25 and 10 TCID50/mL. To demonstrate at least a 4 log10 reduction in virus titre, it is necessary for test mixtures containing low virus titres to undergo detoxification by molecular sieving, or to use a more sensitive assay such as LVP [10. In LVP, a high volume of the lowest apparently non-cytotoxic dilution of the inactivation assay test mixture is added to the detector cell line and the cultures are monitored for virus-specific effects. Using this method, larger reductions in virus titre can be shown even at lower viral loads.Usually, only low titres of EBOV can be harvested in cell culture, resulting in a range of 5.00 to 7.70 logh as LVP . MoleculThe inactivation tests were conducted once at 20 °C ± 1 °C. One part EBOV suspension (100 μL) was mixed with 100 μL of either 0.3 g/L BSA (clean conditions) or 3.0 g/L BSA + 3.0 mL/L erythrocytes (dirty conditions) as the interfering substance. The virus-protein mixture was added to 8 parts (800 μL) of the 1:10 diluted test product, with the exception of 4 % PVP-I skin cleanser which was diluted to 1:100. After a contact time of 15 s, 600 μL of the test mixture for the 10 % PVP-I solution, 7.5 % PVP-I surgical scrub, and 3.2 % PVP-I/alcohol solution were added to 57.0 mL ice-cold medium resulting in a 1:96 dilution of the test product. For the 4 % PVP-I skin cleanser, 120 μL of the test mixture was added to 57.6 mL which corresponds to a 1:481 dilution. After dilution, the samples were added in 200 μL aliquots to microtitre plates (288 wells) containing the indicator cells in 100 μL cell culture medium. The cells were cultivated for an incubation period of 6 days, and inspected microscopically for virus foci (virus-induced changes in cell morphology) ).50 can be calculated by the use of the following formula, which is derived from the Taylor series:The most probable average number of TCIDw” is the plated volume per well, “n” number of inoculated wells, and “np” is the number of successfully infected wells). is 0.69 or ln(0.5) = 0.69. This implies that at least 69 trials are necessary to successfully infect 50 % of 100 wells. Therefore this factor is needed to calculate the TCID50. In addition, if the Poisson formula is used, the dilution factor of the prediluted sample should be considered.However, one TCIDUsing LVP, the lowest apparently non-cytotoxic dilution of the test mixture is added to ice-cold medium after the specified contact time, immediately suppressing the virucidal effects of the test product. This makes it a more precise method for very short incubation times because after-effects of the test product on the sample can be excluded, compared to the endpoint titration Spearman-Kärber method. With this method, it might sometimes be difficult to carry out the necessary pipetting quickly enough, particularly while wearing a BSL-4 protective suit when working with EBOV. In addition, the detection of residual virus can be improved by the testing of a large sample volume. In cases where there are small numbers of virus particles present, the Poisson formula used to calculate virus titre is more precise than the Spearman-Kärber formula.The test concentrations and contact periods were chosen in order to observe the point at which each test preparation produced efficient virus inactivation.10 reduction in virus titre of at least 4 [10 reduction factors produced by each test product under clean and dirty conditions at each time point are shown in Table To demonstrate virucidal efficacy, disinfectant and antiseptic products are required to produce a log least 4 . The logViral titres of MVA were reduced by >99.99 % to >99.999 % under clean and dirty conditions after application of undiluted and 1:10 dilutions of 10 % PVP-I solution, 7.5 % PVP-I surgical scrub and 4 % PVP-I skin cleanser for as little as 15 s. The 3.2 % PVP-I/alcohol solution had a similar effect, except under dirty conditions at the 1:10 dilution for 15 s, when the reduction factor was 3 (99.9 % reduction).10 reduction of MVA was 5.3 times higher at a 30 s contact time (0.4 g/L versus 0.075 g/L) and 4 times higher at a 15 s contact time (0.4 g/L versus 0.1 g/L), than under clean conditions  may have an influence on the activity of disinfectants, including PVP-I products , 29. In The results of suspension tests only allow the virucidal efficacy of a product to be predicted, and do not give information about its effectiveness in practice. In order to further improve our knowledge of the time/concentration relationships of hand rubs and hand wash products which reflect daily needs in clinical surroundings, more practical testing would be necessary. EN1500  describeHand hygiene is an infection control procedure with clearly demonstrated efficacy, and remains the cornerstone of efforts to reduce the spread of infection. According to WHO guidelines, hand hygiene is the most important infection prevention and control measure against EBOV . GeneralSince the skin is a routine source of infection transmission, the use of PVP-I for hand disinfection may be an alternative to WHO recommended alcohol-based hand rubs. Medicated soaps containing PVP-I are available. Thus, the hygiene measure of hand washing is supported by the virucidal effect of PVP-I. This could be extended beyond surgical use. Healthcare professionals, patients, family members and professionals at risk of infection could use these products to protect themselves after exposure to patients.PVP-I soap has shown similar efficacy to alcohol-based hand sanitisers against norovirus in a hand wash model . ChlorheIn another study in a neonatal ICU setting, a PVP-I based hand wash showed antimicrobial efficacy comparable to an alcohol-based hand rub, with both being superior to use of soap and water alone . In relaThe results of our study support these findings, and suggest that hand washing with PVP-I based products can help to prevent infection with EBOV. In addition to hand washing, PVP-I preparations may have other applications in reducing the spread of EBOV, as they are not limited to use on intact skin but can also be administered to the mucosa of the mouth, nose, eye, urinary and genital tract , 37. TreAll of the PVP-I products tested showed excellent efficacy and fast virucidal activity against both MVA and EBOV after as little as 15 s. The European test virus MVA was even more resistant to PVP-I under dirty conditions than EBOV. Therefore, we consider virucidal tests with MVA a suitable alternative for the claim ‘virucidal active against enveloped viruses for hygienic hand rub and hand wash’.These results suggest that PVP-I represents an effective measure to help prevent infection and limit the spread of Ebola virus disease. This is helpful information for the implementation of appropriate guidance for people exposed to EBOV, and confirms the excellent virucidal efficacy of PVP-I against enveloped viruses.


This is the first Canadian case of Chikungunya virus (CHIKV) infection reported in a traveller returning from the Caribbean. Following multiple mosquito bites in Martinique Island in January 2014, the patient presented with high fever, headaches, arthralgia on both hands and feet, and a rash on the trunk upon his return to Canada. Initial serological testing for dengue virus infection was negative. Support therapy with nonsteroidal anti-inflammatory drugs was administered. The symptoms gradually improved 4 weeks after onset with residual arthralgia and morning joint stiffness. This clinical feature prompted the clinician to request CHIKV virus serology which was found to be positive for the presence of IgM and neutralizing antibodies. In 2014, over four hundred confirmed CHIKV infection cases were diagnosed in Canadian travellers returning from the Caribbean and Central America. Clinical suspicion of CHIKV or dengue virus infections should be considered in febrile patients with arthralgia returning from the recently CHIKV endemic countries of the Americas. Leptospira spp., Plasmodium spp., and other viruses should also be considered in the differential diagnosis.The present report is the first published case of a Canadian patient acquiring a Chikungunya virus (CHIKV) infection after travelling to a newly endemic country in the Americas. The CHIKV was introduced in the island of Saint-Martin in December 2013 and spread rapidly to neighboring countries in the Caribbean, South, Central, and North America (Florida) through local transmission cycles. There is no treatment to cure the disease; supportive treatment with nonsteroidal anti-inflammatory drugs is available to relieve symptoms. The clinical picture of CHIKV and dengue virus infection shares several similar signs and symptoms. Therefore, a prompt request of laboratory tests for dengue and Chikungunya viruses in travellers returning from America endemic regions is recommended to confirm an arboviral disease and to adjust the treatment options. Other pathogens which mimic Chikungunya infection such as Zika virus,The patient is a 57-year-old Caucasian male with no past relevant medical history. He travelled to Martinique from mid-January to February 2, 2014, where he stayed in a villa, in the suburb of the city, close to the mountains. The villa was well maintained, with no evidence of mould. The patient recalled multiple mosquito bites, mostly during the first few days of his stay. No other particular infectious exposition was noted during his activities, and he did not experience episodes of fever, gastrointestinal or respiratory illness during his trip.9 cells/L) and a discrete neutropenia and lymphopenia . No eosinophilia was noted, and the platelets levels remained normal. Further analysis revealed a negative urine culture and a normal sedimentation rate.About three days upon his return to the province of Québec, Canada, he presented with a viral syndrome including myalgia, reduced general condition, fever, and increasingly progressive headaches over two to three days. On February 7, he sought medical advice for arthralgia in both hands and feet, joint swelling/synovitis, and a nonpetechial rash on the thorax. The workup was negative for renal and liver anomalies but did reveal leucopenia  was initiated. Upon treatment, the fever stopped but the musculoskeletal symptoms persisted for another two weeks.9 cells/L).Follow-up testing from blood drawn on February 17 showed normal liver and renal function, haemoglobin, platelets, and sedimentation rates. Leucocytes had returned to normal levels  screening test [A battery of serological tests foring test  was obtaing test  with a n envelope E1 gene from both the acute and the first convalescent sample was attempted by conventional RT-PCR [Amplification of the CHIKVl RT-PCR . Successl RT-PCR  and showl RT-PCR , 5 aboutEight weeks after the patient first visited the doctor, the patient reported a return to normal health . Aedes aegypti and Aedes albopictus mosquitoes in tropical and subtropical climates. Of importance, dengue virus is also transmitted by these arthropod vectors and coinfections with both viruses have been described [Chikungunya virus (CHIKV) is an alphavirus of the Togaviridae family and is a member of the arbovirus group. The virus is transmitted to humans mostly by the bites of the urban and periurbanescribed , 7. Follescribed . Despiteescribed , 10.Differential diagnosis should be carried out according to epidemiological information such as place of residence, travel history, and mosquito exposure . Other zThe first case of CHIKV infection was reported in 1953 in Tanzania . SubsequCHIKV was introduced for the first time in the Americas in December 2013 on the island of Saint-Martin in the Caribbean . Since tHistorically, Canadian patients acquire CHIKV infections following travels to African and Asian countries where the virus is endemic and case numbers have averaged 10–15 a year . As descRetrospective analysis of laboratory findings for 386 Québec patients with suspected Chikungunya infection in 2014 revealed that 133 (34%) patients were positive by the EIA IgM screening test and 105 (27%) were confirmed by PRNT testing. The detection of CHIKV in acute sera by a specific RT-PCR test identified an additional 17 confirmed cases. These cases were found to be reactive by EIA but not confirmed by PRNT indicating that samples from these cases were too acute to harbor neutralizing antibodies to the virus.According to a recent report, the number of CHIKV infection cases could rise significantly since Canada has the fourth highest number of travellers to the Caribbean (64 736 Canadian travellers between May and July 2012) . In 2015 Ae. albopictus which is a vector better adapted to temperate climates [ Ae. albopictus and Ae. aegypti populations throughout the Americas are highly competent for transmitting a variety of CHIKV genotypes. This finding is consistent with the rapid spread of the virus currently observed and the accompanying outbreak of CHIKV disease throughout the Caribbean. Seven months following the first case in America, eleven locally acquired CHIKV infection cases were reported in Florida in June 2014 [The envelope gene of the CHIK strain identified in this report did not exhibit the A226V substitution. This mutation was found to enable CHIKV variants to be efficiently transmitted by the Asian tiger mosquitoclimates . Based uclimates . Howeverclimates  indicateune 2014 . PresentSeveral thousands of people are travelling to the islands of the Caribbean each year and consequently are at risk of being infected by dengue, CHIKV, or the recently introduced Zika virus (ZIKAV). Similarly to the dengue virus, ZIKAV is a flavivirus which is found in some African and Asian countries. ZIKAV was detected for the first time in Brazil in March 2015 and has spread rapidly to Central America and the Caribbean . Prompt


Bombus terrestris and its tissue tropism. B. terrestris is a common pollinator of wild flowers in Europe and is used for biological pollination in agriculture. Infection experiments demonstrated a similar pathology and tissue tropism in bumblebees as reported for honeybees. The effect of oral administration of virus-specific dsRNA was examined and resulted in an effective silencing of the virus, irrespective of the length. Interestingly, we observed that non-specific dsRNA was also efficient against IAPV. However further study is needed to clarify the precise mechanism behind this effect. Finally we believe that our data are indicative of the possibility to use dsRNA for a broad range viral protection in bumblebees.Israeli acute paralysis virus (IAPV), a single-stranded RNA virus, has a worldwide distribution and affects honeybees as well as other important pollinators. IAPV infection in honeybees has been successfully repressed by exploiting the RNA interference (RNAi) pathway of the insect’s innate immune response with virus-specific double stranded RNA (dsRNA). Here we investigated the effect of IAPV infection in the bumblebee  Nosema ceranae [Managed bees are used worldwide as pollinators to aid food production in open field as well as in greenhouses . Honeybe ceranae ,17,18,19 ceranae  responsi ceranae  and also ceranae . This RN ceranae ,24,25. SIn contrast to honeybees, bumblebees are reared in a closed environment, therefore it should be feasible to produce pathogen-free bumblebees via a combination of screening and preventative measures . dsRNA cet al. [ad libitum access to 50% sugar water  [g). Supernatant was collected and centrifuged at 40,000× g for 4 h at 4 °C. The pellet was suspended in nuclease free water and stored at −80 °C. We performed a quality control on both the viral stock and the control inoculum. Only the virus stock contained IAPV, checked with primers described by Cox-Foster et al. [8 virus particles/µL. We screened for possible contaminating viruses in both viral stock and control inoculum, we tested for slow bee paralysis virus, Kashmir bee virus, acute bee paralysis virus and DWV with PCR and all were negative. Finally, the infectivity of the prepared viral stock and control inoculum solution was tested by injection of 2 µL control inoculum and viral stock. Four days after injection the infection status was determined with PCR [For the production of the viral stock we obtained 240 adult bumblebees from virus-free colonies . One hundred twenty bumblebees were injected with 2 µL of IAPV solution containing 200 virus particles. The starting virus solution originated from honeybees and was kindly provided by Joachim de Miranda , and quantified by transmission electron microscopy  as described by Meeus et al. . The othBiobest) . Four dar et al. . The virwith PCR . The virad libitum access to 50% sugar water  [n = 17), or with 1 × 107 (n = 16), 2 × 107 (n = 16) and 1 × 108 (n = 18) virus particles in a 20 µL droplet of 50% sugar water . Feeding was done under red light, to minimize disturbance of the bees. After inoculation behavior and mortality were followed for 20 days. After 20 days, the infection status of the bees was checked by PCR as described by Cox-Foster et al. [7 or 1 × 108 were sacrificed for tissue tropism analysis, these bumblebees were not included in the survival analysis.Bumblebees of the same age were kept in micro-colonies per treatment, in an incubator  at 30 °C, 60% RH and constant darkness. Bees had Belgium) . Prior t®, Biobest). On the third day the bees also received 1 × 107 virus particles suspended in 20 µL of 50% sugar water  [To test the effect of dsRNA on IAPV infectivity and mortality, we fed dsRNA on a daily basis for 6 consecutive days to bumblebees of the same age. Each day a 4h-starved bee received 2 µg of dsRNA in 20 µL of 50% sugar water (BioglucBiobest) . Bees we® II Reverse Transcriptase, Life Technologies, Carlsbad, CA, USA) using 500 ng of RNA and oligo-dT primers, according to manufacturer’s protocol. The cDNA was 10 times diluted and IAPV was quantified using a CFX96™ Real-Time PCR Detection system , performing each reaction in duplicate. The total reaction volume of 20 µL contained 10 µL GoTaq® qPCR Master Mix, , 1 µL (10 µM) Forward and 1 µL (10 µM) Reverse primer . Further RNA extraction proceeded according manufacturer’s protocol  and were stored at −80 °C until further use. RNA concentrations and purity were determined using a NanoDrop ND-1000 spectrophotometer. A two-step real time RT-PCR was used for quantification of IAPV. First cDNA was made , except in the elution step where nuclease free water was used to elute the dsRNA instead of elution buffer. Eluted dsRNA was subjected to a DNAse treatment. dsRNA for GPF  was produced in the same way, starting from a plasmid containing the nucleotide sequence for GFP. The sequences of all dsRNA fragments were compared to the genome of B. terrestris and did not contain any similarity longer than 20 bp [The RNA extract from the IAPV stock solution was subjected to a DNase treatment and subsequently to RT-PCR with oligo-dT primers, as described earlier. The fragments corresponding to the structural protein-coding region, dsVP586  were PCR amplified with primers  carrying7 IAPV particles is near the minimum amount required for oral infection in bumblebees, and reported no significant effect on mortality with this dose [i.e., 1 × 107, 2 × 107 and 1 × 108) and tested the infection status  in different body parts  after four and seven days. For all treatments we also followed mortality and bee behavior.Previous studies showed that 0.5 × 10his dose . Here we8 virus particles, we were able to detect IAPV in the head, gut and fat body both four and seven days post infection (p.i.). Feeding of 1 × 107 virus particles had a slower infection progression as IAPV was only detectable in the gut at 4 days p.i., while it took until day seven for virus to appear in the other tested body parts. This reduced infection progression was also apparent in the survival scoring of the bumblebees. 8 IAPV virus particles resulted in significant increased mortality after 20 days  compared to the no virus control. Feeding of 2 × 107 virus particles resulted in reduced survival, although not significant . When 1 × 107 virus particles were fed, there was nearly no increase in mortality, which is consistent with previous reports showing only sub-lethal effects [After a single oral administration of 1 × 10 effects . IAPV th effects . Experim8 or 2 × 107) of virus particles were fed and shortly before dying. In honeybees infected with IAPV similar symptoms of spasms have been reported shortly before dying [Looking at the behavior of the bumblebees we noticed disorientated movements: the bumblebees moved around lifting their paws high in the air and tipping over frequently. These observations were not recorded before, and were only seen when high amounts  and dsGFP (n = 10) three days before and three days after inoculation could not prevent infection. After eight days all tested body parts   were infected.The aim of this experiment was to evaluate if oral administration of dsRNA specific for IAPV could prevent infection in bumblebees. We opted to administer 1 × 10p < 0.001), the gut (p = 0.015) and the fat body (p = 0.014) in the dsGFP-treated samples compared to the dsVP586 treatment. This result was somewhat unexpected, but actually the impact of non-specific dsRNA  and the IAPV-specific dsRNA  on mortality followed the same pattern. The dsGFP control had a significantly higher survival compared to the virus-specific treatment  (In = 0.009) a. The dst p = 0.0 and the et al. [Additionally, the effect of dsGFP in bumblebees on the relative viral titer seems much higher than reported in honeybees. Maori et al.  reportedet al. . In shriet al. .p < 0.05). The length of the dsRNA molecule did not have a significant effect on the virus silencing with the IAPV-specific fragment, as there was no significant difference in virus titer between all three virus-specific fragments (p > 0.10). The viral titer of the dsGFP control was significantly different from the only-virus control (p < 0.05) but not from the virus-specific treatments (p > 0.10). These findings confirm the positive effect of dsGFP found in the first experiment.The lower effect observed for the virus-specific dsRNA (dsVP586) could be due to the differences in length of the dsRNA. The used dsVP586 is much longer than the dsGFP fragment. Indeed size can be a limiting factor in uptake of the dsRNA molecules . To elucBombus impatiens by detection of the negative strand [Apis pollinators [Apis pollinators. We reported here on the pathology and tissue distribution of IAPV infection of B. terrestris. Infection with IAPV seems to have similar effects on mortality as reported for honeybees when high doses are fed [9 RNA copies have been detected in bee feces for chronic bee paralysis virus (CBPV) [IAPV and other dicistroviruses have been reported in several key pollinators as well as in other insects belonging to different orders . Active e strand . Howeverlinators . As theslinators , the imp are fed ,33. The  are fed . At colo are fed . This is are fed . The bios (CBPV) , giving s (CBPV) . Indeed s (CBPV) . This cas (CBPV) . The imps (CBPV) ,42.The risk of pathogen spillover associated with the use of commercial bumblebees is well described ,40,43,44B. terrestris [The effect of IAPV-specific dsRNA molecules on the virus titer suggests the presence of a functional specific antiviral RNAi immune response in bumblebees, as all the genes required for the RNAi machinery are present in the genome of rrestris .Our results, done in bumblebees, demonstrate that a non-specific dsRNA molecule has the same or even higher potency to silence IAPV. In honeybees, virus-specific dsRNA proofed to be more effective to silence viruses compared to non-specific dsRNA ,17,18. TThe effect of non-specific dsRNA molecules on virus titer and survival has been reported in several invertebrates including honeybees ,24, thusN-terminal kinase immune pathways are more or equally important and triggered by non-specific dsRNA molecular patterns.Although the exact immune response pathway is unknown, it seems that non-specific dsRNA acts as a PAMP triggering the anti-viral defense. It could be that PAMP recognition strengthens the conventional RNAi pathway, making it more efficient when the actual virus infects and triggers the virus-specific RNAi machinery. But it could also be that in bumblebees other viral defense pathways like the JAK-STAT, Imd, Toll or Jun-Feeding experiments in honeybees, all show a clear effect of virus-specific dsRNAs ,16,18; i7 virus particles, it could significantly lower the virus titer. This treatment could possibly protect bumblebees from high infections when placed in a natural environment and prevent possible spillback.Aside from which pathways is triggered, the fact that non-specific dsRNA can silence IAPV opens avenues to prevent replication of multiple viruses in bumblebees. However, the effect of random dsRNA on other viruses in bumblebees first need to be tested. Although our preventive treatment did not lower the infection success of 1 × 10


Scleractinia. While hybrid embryos can be producedin vitro, it has been difficult to identify adult hybrids in the wild. Here, we tested the potential for hybridization between two closely related species in the family Fungiidae. We mixed approximately 5000 eggs ofCtenactis echinata with sperm fromC. crassa. No hybrid embryos were produced. This observation adds to a growing body of evidence for pre-zygotic barriers to hybridization in corals and challenges the claim that hybridization is a major source of evolutionary novelty in the order.Hybridization is often cited as a potential source of evolutionary novelty in the order While small numbers of hybrid embryos can be produced in a few speciesin vitro3, the evidence for hybrids in the field is often equivocal because the genetic techniques used for corals cannot distinguish between hybridization and incomplete lineage sorting4. In fact, only one of the over 1300 species in the order is generally accepted to be unequivocally of hybrid origin:Acropora prolifera5. Nonetheless, hybridization is often invoked as a source of evolutionary novelty in the orderScleractinia7.Hybridization is a controversial topic in coral reef ecologyCtenactis echinata andC. crassa. These species are sympatric, often dominating large multi-specific assemblages of fungiid corals throughout the central Indo-Pacific8. These species can generally be distinguished on the basis of the density of septa and the shape of septal dentitions, however, in Okinawa, these features are very similar and the most useful diagnostic character is a strong arch in the corallum ofC. crassa  we predict that hybridization between these species is unlikely. This observation adds to a growing body of evidence indicating strong pre-zygotic barriers to hybridization in many scleractinian corals13.While the species are relatively easy to identify, determining the sex of each individual prior to spawning is impossible without destructive sampling to expose the gametes. Consequently, we placed each individual in a separate 20 L bucket containing sea water in the open air at approximately 20:00 h in order to sex each individual once gametes had been released. On the night of 27 July between 22:30 and 23:30 h three I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Fungia fungites were taken? I assume the photos were taken from a GBR specimen, since this species is a brooder and not a broadcaster in Okinawa.I note that the authors preferred to retain Figures 1C and 1D in spite of my recommendation to delete them, since they have nothing to do with the contents of the article. I do not insist that they do it, but it may confuse the readers. I wonder though where did the photos ofI have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Ctenactis echinata andCtenactis crassa, closely related fungiid corals that naturally release gametes at the same time. The authors recognise the limitations of this observation - only small numbers of eggs (100's) were observed and only eggs fromC. echinata were available to be fertilized  - but rightly point out that this study provides further evidence that hybridization is not as widespread amongst scleractinian corals as often portrayed in the literature. However, the analysis of reciprocal crosses and a larger numbers of eggs is necessary before it can be said with some confidence that these congeners can not hybridize.This Observation Article reports the lack of cross-fertilization betweenI have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Dear Bernie,Thank you for your comments. We would just like to point out that while we only examined 100 eggs every few hours under the microscope, we used approximately 5000 eggs in the fertilization experiment, all of which had broken down after 24 h suggesting none had fertilized. We have added a sentence to the revised text to draw attention to this. crass to crassa, in the 3rd line of the Abstract.The title is appropriate for the content of the article. The abstract represents a suitable summary of the work. Please correct:  'spawning occurs following the full moons from July to August' (reference 8 in the manuscript) is:Proc. Roy. Soc. Biol. B 275: 2335-2343Loya Y. & K. Sakai (2008). Bidirectional sex change in mushroom corals..Article content: The design, methods and analysis of the results been clearly explained and are appropriate for the topic being studied. Figures 1C and 1D appear to be irrelevant to the article since they show different species. I suggest deleting them. A proper reference to the statement C. echinata males withC. crassa females; however they also did not test the positive control of crossingC. echinata males withC. echinata females . Nevertheless, this does not diminish their prediction that hybridization between these species is unlikely. The paper contributes further information to the controversial topic of potential hybridization and breeding incompatibilities within the mating systems of broadcast spawning reef corals.Data and Conclusions: The authors note that they did not crossI have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Dear Yossi,Thank you for your comments. We have corrected the typos you identified, changed the reference as requested and added a sentence to clarify the controls that were used to test for gamete viability. Images 1 C & D are presented as an example of the spawning behavior of fungiids.