Blueberry shoestring virus (BSSV), a member of the genus Sobemovirus, which has not so far been reported. dsRNAs were isolated from BSSV-infected blueberry plants using the dsRNA-binding protein, reverse-transcribed, amplified, and sequenced using NGS. A contig of 4,020 nucleotides (nt) that shared similarities with sequences from other Sobemovirus species was obtained as a candidate of the BSSV genomic sequence. Reverse transcription (RT)-PCR primer sets based on sequences from this contig enabled the detection of BSSV in all BSSV-infected plants tested but not in healthy controls. A recombinant protein encoded by the putative coat protein gene was bound by the BSSV-antibody, indicating that the candidate sequence was that of BSSV itself. Our results suggest that a combination of DECS analysis and NGS, designated here as “DECS-C,” is a powerful method for detecting novel plant viruses.The presence of high molecular weight double-stranded RNA (dsRNA) within plant cells is an indicator of infection with RNA viruses as these possess genomic or replicative dsRNA. DECS  analysis has been shown to be capable of detecting unknown viruses. We postulated that a combination of DECS analysis and next-generation sequencing (NGS) would improve detection efficiency and usability of the technique. Here, we describe a model case in which we efficiently detected the presumed genome sequence of  Arabidopsis thaliana, converted to complementary DNAs (cDNAs), amplified, cloned into plasmid vectors, and randomly selected clones are sequenced. These sequences can be searched against a database  search) to determine the species of the virus. The DECS method can also be used to detect RNA viruses in a small volume of plant tissues and to collect dsRNAs within a short time [When plants exhibiting virus-like symptoms are found on a farm or during a routine plant inspection, the rapid identification of the causal agents is important for taking adequate and timely measures for their control. Conventionally, serodiagnostic tests and/or PCR assays are widely used to detect plant viruses. These methods are effective for detection of viruses commonly known to infect particular plant species. However, it is difficult and time-consuming to detect pathogenic viruses using these methods for new virus strains or infection of new host plants; in these circumstances, different assays need to be performed. For example, the pathogenic causal agents of many graft-transmissible diseases affecting woody crops remain to be elucidated; this is largely because of the difficulty in detecting and isolating candidate viruses from woody crops, and of artificially infecting new host plants to reproduce the disease symptoms. The majority of identified plant viruses are RNA viruses, which possess double-stranded RNA (dsRNA) as the genome or as a replicative form. Therefore, isolating dsRNA is an effective approach for detection of viruses. To date, CF-11 cellulose powder chromatography, digestion with adequate nuclease and/or lithium chloride precipitation have been used to isolate dsRNAs ,3. The mort time . DECS anort time ,7,8.DECS analysis involves a cloning step to determine the sequences of dsRNAs. When a large amount of trapped viral dsRNA is obtained, even a small number of sequencing operations  may suffice as a detection procedure because the clones are likely to contain some viral sequences. However, if a small amount of trapped dsRNA is available, then sequencing of a larger number of clones  might be needed to ensure the detection of viral sequences. The amount of viral dsRNA that accumulates in plant cells is influenced by the host plant species, the time of sampling, and the plant tissue types sampled. Furthermore, it takes considerable time to connect fragments and/or to perform rapid amplification of cDNA ends (RACE) to determine the whole genomic sequence of the virus. For this latter purpose, it is necessary to increase the number of clones that are sequenced, which makes the procedure more laborious and time consuming. Therefore, the use of a more efficient sequencing technology would significantly improve the detection sensitivity of DECS analysis and speed up the determination of whole genome sequences. One option would be to use next-generation sequencing NGS, which allows the generation of considerably larger amount of sequence data in a given time. In recent years, many new plant viruses have been detected from diseased plants using NGS technology ,13,14,15Blueberry shoestring virus (BSSV)-infected blueberry plants. BSSV has been reported to cause extensive damage to blueberry plants in the U.S.A. and Canada, and has caused huge economic losses through decreased yields [Sobemovirus. At present, its nucleotide sequence remains to be determined. Hence, we selected this virus to evaluate the combination of DECS and NGS for the detection of a virus whose sequence of which is unknown.In this study, we used a combination of NGS and DECS analysis in an attempt to improve the sensitivity and rapidity of detection of virus-like sequences in d yields ,17,18,19Four BSSV infected scions were provided by Annemick Schilder, Michigan State University, USA, under a special permit (permit number: 22Y307) issued by the Ministry of Agriculture, Forestry and Fisheries, Japan. BSSV infection of stems and leaves of these scions was confirmed by double antibody sandwich enzyme-linked immunosorbant assay (DAS-ELISA) using a kit specific for BSSV . Each of the four scions were grafted onto four individual healthy blueberry seedlings as rootstocks, labeled as No. 1 to No. 4, and cultivated under greenhouse conditions. After confirming that the disease symptoms were present in all BSSV samples, tissues were collected for subsequent experiments.Newly emerged branches weighing about four grams were collected from the blueberry seedlings grafted with BSSV-infected scions, and total nucleic acids were extracted as previously described . Next, 1Using TransPlex Whole Transcriptome Amplification (WTA) Kit , extracted dsRNA was reverse-transcribed into cDNA with the supplied random primer following the manufacturer’s protocol, and the cDNA was then amplified. Amplification was carried out using ExTaq DNA polymerase  and 20 cycles of PCR. Next, the 50 μL WTA products were reacted with 30 μL AMPure XP , and rinsed twice with 1 mL of 70% ethanol. Thereafter, the products were eluted with 30 μL RNase-free water. In total, 100 ng WTA products were used to construct the NGS library using the Ion Xpress Plus Fragment Library Kit and sequenced on the Ion PGM system . WTA products including various sizes of DNA fragments were digested to 200 bp using Ion shear Plus Reagents Kit (Thermo Fisher Scientific), according to the manufacturer’s instructions.http://www.ncbi.nlm.nih.gov/genome/viruses/) comprising 5660 sequence data. Second, de novo assembly analysis was performed to obtain contigs. We performed BLAST search analyses for the obtained contigs to identify their similarities to known virus sequences. A phylogenetic analysis was conducted by the neighbor-joining method using Mega 6.0 software with a bootstrap resampling analysis (1000 replications), and the sobemovirus-like nucleotide sequences were compared with those of other known species of the genus Sobemovirus, using the information available in the NCBI database. The known sobemoviruses include Cocksfoot mottle virus , Rice yellow mottle virus-CI , Imperata yellow mottle virus , Velvet tobacco mottle virus , Lucerne transient streak virus , Subterranean clover mottle virus , Southern bean mosaic virus , Sesbania mosaic virus , Sowbane mosaic virus , Southern cowpea mosaic virus , Ryegrass mottle virus , and Turnip rosette virus . In addition, Cocksfoot mild mosaic virus , a tentative species of sobemovirus, was included with Potato leaf roll virus  as an outgroup, which is a member of the genus Polerovirus.Analysis of sequence data was performed using Genomics Workbench ver.7.0 . First, the mapping analysis was performed using the complete genomes of all viruses and viroids registered in the NCBI database   . The anaPepper mild mottle virus (PMMoV) CP gene was amplified from pBI-PIW-CP [Escherichia coli strain Rosetta (DE3)  harboring each expression vector for 3 h at 30 °C in the presence of 1 mM isopropyl-β-d(–)-thiogalactopyranoside. His-tagged proteins were purified using TALON® Metal Affinity Resin (Takara Bio) according to the manufacturer’s protocol. His-tagged CPs were separated by SDS-polyacrylamide gel electrophoresis (PAGE) with NuPAGE Novex 12% Bis-tris Gels (Thermo Fisher Scientific). Both purified recombinant CPs were detected by immunoblotting using a previously described method [The putative coat protein (CP) gene of BSSV was amplified from cDNAs of BSSV-infected blueberry plants by PCR using pET-BSSV-CP-F and pET-BSSV-CP-R primers . The PepI-PIW-CP , using pd method  with a mBSSV infection in the stems and leaves of the infected blueberry scions was examined using ELISA. The stem samples tested positive for BSSV infection whereas the leaf samples tested negative. Although the grafted blueberry seedlings showed no symptoms of BSSV for one year, the leaves were red and narrow, or had turned red in the main vein  after thSobemovirus, as revealed by the mapping tools of Genomics Workbench ver.7 . The putative BSSV genomic sequence shares up to 52% identity with other sobemoviruses  predicted. ORF1 (nt No. 85-492), ORF2a (nt No. 462-2204), ORF2b (nt No. 1796-3400), and ORF3 (nt No. 3186-4001) were predicted to encode a 15.4 kDa movement protein, a 65.55 kDa polyprotein protein (Protease-VPg), a 62.22 kDa RNA-dependent RNA polymerase protein, and a 27.85 kDa coat protein, respectively. Each sequence of ORFs was compared with those of other sobemoviruses (Next, using the oviruses ; 75% ideemovirus . Comparaoviruses ; there wThe entire region of the candidate BSSV genome sequence was amplified from the cDNA obtained from the BSSV-infected plant using new primer sets that were based on sequences obtained in the NGS analysis . In addiTo further confirm that the candidate sequence equates to the BSSV genome, we tested whether the putative CP was recognized with a commercial BSSV antibody. A recombinant CP expressed from the putative BSSV CP gene was successfully detected by an immunoblot assay using the BSSV antibody . A recomde novo assembly of virus-like sequence candidates, and a BLAST search of the virus sequence database [de novo assembly of NGS data, confirming the utility of NGS in characterization of virus genomes. The original DECS protocol includes a cloning process, which is time consuming and laborious and requires facilities for recombinant DNA experiments. Using NGS technology, the cloning process before sequencing can be eliminated in the DECS analysis. We would like to propose a name for the “clone-free” DECS analysis combined with NGS protocol as “DECS-C”. Recently, NGS has become more convenient than before, because its cost is decreasing every year. We believe that DECS-C can be a powerful and convenient method to search for plant RNA viruses.In this study, we have successfully and efficiently detected the BSSV sequence using DECS analysis combined with NGS. The procedure for screening NGS data for virus-like sequences includes the filtering out of plant genome sequences, database ,28,29. HFor diagnostic purposes, only part of the virus genome is required for proving the presence of a virus in the plant tissue. Therefore, we may need fewer reads for diagnosis, which would enable us to mix and analyze multiple different libraries in a single NGS run to reduce operational costs. Nevertheless, we cannot rule out the possibility that other viruses might exist in plant materials of interest in quite low amounts and contribute to disease development. It will be necessary to concentrate viral sequences during NGS library preparation in order to enhance detection sensitivity. Although our present protocol used a recombinant dsRNA-binding protein alone to enrich dsRNA, additional treatments, such as nuclease digestion in the presence of higher concentrations of salt, could improve the quality of dsRNA preparation and the efficiency of virus detection with a smaller number of NGS reads. In addition, the operational cost could also be reduced if the exhaustive amplification step before library preparation is omitted. Our current protocol included two amplification steps: first, the amplification of the dsRNA-derived cDNA using a WTA kit; and second, emulsion PCR or bridge amplification depending on the type of NGS. Different manufacturers have released library construction kits for small amounts of RNA for NGS analysis, and these likely make it possible to omit the first amplification step. Collectively, our next challenges should include the improvement of dsRNA quality, the simplification of the NGS library construction, and the reevaluation of the number of sequence reads required to detect plant viruses. We anticipate that DECS-C will evolve through these efforts to become a more powerful, convenient, and cost-effective method, which could be used not only for research purposes but also for routine diagnosis of plant virus diseases .The protocol developed in this study, involves a BSSV detection method with quantitative RT-PCR based on the sequence of only one BSSV strain. It would be preferable to design primers based on highly conserved regions of the genomic sequences of different strains for a more reliable diagnostic method, because RNA viruses are known to evolve rapidly . Therefo


Sabethes intermedius) in Sena Madureira, Acre state, northern Brazil. All genes showed similarities with those belonging to members of the family Rhabdoviridae.We report here the first complete open reading frame (ORF) genome sequence of Xiburema virus (XIBV), that of strain BE AR362159, isolated from mosquitoes ( Rhabdoviridae is a large family of viruses comprising >46 viruses distributed in six recognized genera  (−) possessing five main polycistronic genes: those for nucleocapsid (N), phosphoprotein (P), matrix (M), glycoprotein (G), and polymerase (L) (The family dovirus) , five spdovirus) . They arrase (L) . Some rhrase (L) . Intergerase (L) , 4.Vesiculovirus, Lyssavirus, and Ephemerovirus cause diseases in mammals. An exception is the sigma virus (type species Drosophila melanogaster sigma virus [DmelSV]), which is transmitted vertically among parent flies   . The vecd sperm) , while fc health –9.Sabethes intermedius) in the municipality of Sena Madureira, Acre state, northern Brazil  in 1974. Complement fixation and neutralization tests were used to antigenically characterize and define Xiburema virus as an ungrouped virus. Transmission electronic microscopy (TEM) was also used to confirm the bullet-shaped nature of the virions, as previously described, placing it as a member of the Rhabdoviridae (Xiburema virus (XIBV) (strain BE AR362159) was isolated from mosquitoes (oviridae .The XIBV particles were precipitated using polyethylene glycol (PEG) centrifugation, as previously described , and thede novo hybrid assembly strategy using both Ion Torrent and GS FLX 454 reads simultaneously with the software Mira 4.0. Visual inspection was performed with the software Geneious version 6.1.4. The total genome recovered was 12,240 nucleotides (nt) in length, with a mean coverage of 383-fold. The five main genes 3′-N-P-M-G-L-5' were recognized, as well as two putative accessory genes (pAG1 and pAG2) between the G and L genes.The genome was obtained by employing a This is the first report of the complete genome sequence for Xiburema virus, an ungrouped Brazilian rhabdovirus.KJ636781.The complete genome sequence has been deposited in GenBank under the accession no.


Acarapis woodi). In addition, 108 samples of brood were screened and yielded 4 honey bee viruses. Virus prevalence varied from common  to complete absence . When colonies were forced into one of two classes, strong or weak, the weak colonies contained more pathogens in adult bees. Among observed pathogens, only deformed wing virus was able to predict colony strength. The effect was negative such that colonies testing positive for deformed wing virus were likely to have fewer combs of bees or brood. This study constitutes the first record for Nosema ceranae in Great Britain. These results contribute to the growing body of evidence linking pathogens to poor honey bee health.Inspectors with the UK National Bee Unit were asked for 2007-2008 to target problem apiaries in England and Wales for pathogen screening and colony strength measures. Healthy colonies were included in the sampling to provide a continuum of health conditions. A total of 406 adult bee samples was screened and yielded 7 viral, 1 bacterial, and 2 microsporidial pathogens and 1 ectoparasite ( Varroa destructor is a common thread wherever high bee morbidity is reported [Since the middle of the last decade honey bee decline has galvanized a multi-national effort to understand the extent of the problem, characterize its causative or associated factors, and develop mitigating practices and policies. The problem is concentrated in the Northern Hemisphere where winter losses are reported from around 30% in the USA , 10–85% reported ,12,13,14V. destructor mites or acute pesticide kill, leaving pathogens as the risk factors of interest. Our broad goal was to characterize the pathogen profiles that best predict colony decline in this region.This paper is part of a growing body of evidence that associates specific risk factors with colony death or decline. These papers vary in the case definitions used to categorize colonies that are healthy or unhealthy, the risk categories considered, and the geographic ranges included ,16,17,18http://www.nationalbeeunit.com). NBU inspectors routinely record the number of colonies found dead during each inspection. The annual percentage of colonies found dead during the inspection season (Apr-Oct) for 2002–2010 are shown in The monitoring of honey bee health is performed by a government funded apiary inspection programme in England and Wales, operated by the National Bee Unit  and tested using PCR or real-time RT PCR as appropriate. This sample size disparity is explained by improved analytical capacity in 2008; it is also the reason why subsequent statistical analyses were run by year. DNA extracts were prepared from adult bees by grinding to homogeneity in a ‘long special universal’ bag using a Lenze grinder (Bioreba). DNA was extracted from cleared lysate using a Wizard Magnetic DNA Purification System for Food  in conjunction with a Kingfisher ML magnetic particle processor (Thermo Electron Corporation) as described by Ward et al. [Apis iridescent virus (AIV), black queen cell virus (BQCV), chronic bee paralysis virus (CPBV), deformed wing virus (DWV), Israeli acute paralysis virus (IAPV), Kashmir bee virus (KBV), and sacbrood virus (SBV). Adult bee samples were screened for Nosema apis , N. ceranae  and Melissococcus plutonius [AJ307465-955F/1016R/975T, designed to Apis mellifera 18S rRNA [The nucleic acid was recovered from bulk samples of adult bees and brood samples . Samples were deemed positive when both duplicate reactions had a CT value less than 40 [All TaqMan assays were set up using PCR Core Reagent Kits (PE Biosystems Cat. No. 4304441) consisting of 1 × buffer A, 0·025-U μL–1 AmpliTaq Gold, 0·2 mM dNTPs and 5·5 mM MgCl2. All sets of primers were used at 300 nM and probes at 100 nM with 10 μL of template, giving a final volume of 25 μL per reaction. Reactions were carried out within the ABI Prism 7900HT Sequence Detector System (PE Biosystems) beginning with 50°C for 2 min, 95°C for 10 min, and 40 cycles of 95°C for 15 s and 60°C for 1 min. RT PCR reactions were identical to those for PCR except for the addition of 0.2U MMLV (Fermentas) and the addition of a 30 min RT step at 48°C . The  than 40 .Response variables (number of combs of bees or brood) were analyzed by year 2007 or 2008) and by material sampled (adults bees or brood). First, we classified each colony as “strong” if it had >5 combs of bees or “weak” if it had ≤5 combs of bees . In exploratory epidemiological work, including honey bee disorders, it is common to assign subjects into such “case definitions” as strong vs. weak, well vs. unwell, then work retrospectively to find predictors of the condition  or 2008 ,24,25. WDuring 2002–2010, between 19,044–39,463 colonies were inspected annually. The highest number of colony losses reported in any one year was 3,253. The two years of this study, 2007–2008, constituted a period of high colony mortality immediately preceding an improving trend in 2009–2010 .V. destructor and “failed queens.” Relatively few colonies were found with symptoms of acute pesticide poisoning, and subsequent chemical analysis suggested significant chemical residues in only 2 colonies from 2007 (actives implicated: bendiocarb and dieldrin) and 5 colonies from 2008  [The triage procedure yielded data on known or suspected causes of colony death for the two years of intense sampling, 2007 and 2008, and are shown in methrin) . The mosAcarapis woodi). In addition, 181 samples of brood  were screened and yielded 4 honey bee viruses. Virus prevalence varied from common  to complete absence (IAPV). Adult bees tended to contain more viruses than brood; indeed ABPV and KBV were only found in adults.The number of positive adult bee and brood samples for each tested pathogen is given in P = 0.061) for brood samples in 2008  or weak (≤5 combs of bees), the weak colonies contained significantly more pathogens in adults . Among t in 2008 . When saCompared to strong colonies, weak colonies were more frequently associated with higher numbers of pathogens in adult bees , a trendIt is important to note that these significant differences were detectable in a data cloud with a large amount of random variation. These large and spurious effects include (1) a wide geography from over 400 apiaries across England and Wales, (2) from 52 participating beekeepers, (3) 30 NBU inspectors making subjective strength determinations across a range of (4) non-standardized comb dimensions: beekeepers in the UK use no fewer than 7 hive dimensions . In spitV. destructor [Varroa levels [Varroa. More experiments are required to test both hypotheses.Our results are consistent with overriding conclusions drawn by other teams searching for pathogens that predict honey bee decline. Foremost, our study substantiates the importance of DWV. This pathogen has been among the most implicated predictors of bee decline from surveys in Germany , Greece structor ,29 the pa levels , it is pa levels  for coloa levels . PerhapsIAPV and KBV have been identified as significant risk factors in the development of CCD in the USA ,7. InterN. ceranae has been implicated in colony losses in Spain [N. ceranae in Great Britain; however its congener N. apis was more frequently detected than N. ceranae in adult bees. Neither microsporidian was significantly associated with poor colony strength, supporting a premise that the virulence of these pathogens is variable and region-specific [Nosema spp. or the ectoparasite Acarapis woodi, these data suggest adult bees may be particularly useful in future surveys interested in determining pathogen prevalence.The microsporidian in Spain ,32, Greein Spain , and Belin Spain . The prespecific . Viral pVarroa control, queen maintenance, and timely feeding to reduce colony losses. A clear link was demonstrated between poor colony strength and presence of DWV. Owing to the tight link between Varroa and DWV, and until effective anti-viral products are available to combat this virus, the only recourse available to beekeepers is to control Varroa as a proxy for controlling DWV. Pathogens are a significant fraction of the causes contributing to bee decline, not just from known diseases like Nosemosis, but also from emerging pests and pathogens outside of our normal consideration, as was shown with the trypanosomatid bee parasite Crithidia mellificae in Belgium [Our results suggest that beekeepers in England and Wales should concentrate on  Belgium . Long te


Sweet potato feathery mottle virus, Sweet potato virus G, Sweet potato virus C, and Sweet potato virus 2) was associated with 51% reduction in adventitious root number among plants grown without nitrogen. The effect of virus treatments on first order LR development depended on the presence or absence of nitrogen. In the presence of nitrogen, only plants infected with Sweet potato chlorotic stunt virus showed reductions in first order LR length, number, and density, which were decreased by 33%, 12%, and 11%, respectively, when compared to the controls. In the absence of nitrogen, virus tested and infected plants manifested significant reductions for all first order LR attributes. These results provide evidence that virus infection directly influences sweetpotato yield potential by reducing both the number of adventitious roots and LR development. These findings provide a framework for understanding how virus infection reduces sweetpotato yield and could lead to the development of novel strategies to mitigate virus effects on sweetpotato productivity.It has been shown that virus infections, often symptomless, significantly limit sweetpotato productivity, especially in regions characterized by low input agricultural systems. In sweetpotatoes, the successful emergence and development of lateral roots (LRs), the main determinant of root architecture, determines the competency of adventitious roots to undergo storage root initiation. This study aimed to investigate the effect of some plant viruses on root architecture attributes during the onset of storage root initiation in ‘Beauregard’ sweetpotatoes that were grown with or without the presence of nitrogen. In two replicate experiments, virus-tested plants consistently failed to show visible symptoms at 20 days regardless of nitrogen treatment. In both experiments, the severity of symptom development among infected plants ranged from 25 to 118% when compared to the controls (virus tested plants grown in the presence of nitrogen). The presence of a complex of viruses ( Ipomoea batatas) is propagated vegetatively and in the process, propagating material gradually accumulates pathogens, especially viruses that cause decline in yield and quality. At least 30 different viruses have been isolated from sweetpotato Sweet potato chlorotic stunt virus (SPCSV) is a phloem-restricted, whitefly-transmitted virus that is widespread in Africa and South America. SPCSV by itself causes significant yield reduction, but also represses resistance in sweetpotato to other viruses leading to synergistic interactions that reduce yields 80–90% Sweet potato feathery mottle virus (SPFMV), Sweet potato virus C (SPVC), Sweet potato virus G (SPVG), and Sweet potato virus 2 (SPV2) Sweetpotato  3−) It has been shown in model systems that intrinsic and environmental variables influence LR development. Internal cues of LR formation include auxin, ethylene, abscisic acid, cytokinin, and strigolactones Ipomoea setosa, by PCR for geminivirus detection as described by Li et al. I. setosa and testing the symptomatic I. setosa by ELISA on nitrocellulose membranes (NCM-ELISA) using antisera provided by S. Fuentes , multiplex PCR for potyviruses Sweet potato mild mottle virus, Sweet potato latent virus, Sweet potato chlorotic fleck virus, Sweet potato mild speckling virus, Sweet potato leaf curl virus and other related geminiviruses, Sweet potato chlorotic stunt virus (SPCSV), Sweet potato collusive virus, and Cucumber mosaic virus (CMV). However, the possibility that it was infected by viruses not yet recognized in sweetpotato cannot be eliminated. ‘Beauregard’ plants infected with the U.S. strain of SPCSV, isolate BWFT-3 which was initially obtained by single-whitefly transmission Virus-tested (V0) plants of sweetpotato cv. ‘Beauregard’, mericlone B-14 (V0 B-14), were produced originally by meristem-tip culture −1 soil mix. Source plants were grown in 32-cell Speedling trays  until 25–30 cm long terminal vine cuttings could be taken.Source plants for cuttings for experiments were produced in a greenhouse at the Louisiana State University Agricultural Center, Baton Rouge . A rigorous program of insecticide application, sticky card trapping, and sanitation was routinely employed to manage potential insect vectors of viruses. Plants were propagated in 1∶1∶1 river silt: sand: Jiffy Mix Plus  amended with Osmocote 14-14-14  at 3.5 gm Kg3 at the rate of 50 kg·ha−1 while each of P2O5 and K2O was supplied at the rate equivalent to 134 kg·ha−1. Similar P2O5 and K2O rates were used for plants grown without nitrogen (-N). The greenhouse temperature regime was 29°C for 14 hr (day) and 18°C for 10 hr (night). Photosynthetic photon flux (PPF) for plant production and subsequent experiments ranged from 300 to 800 m−2 s−1. High intensity mercury vapor lamps were used to extend daylength to 14 h per day. PPF was measured at the canopy level with a quantum sensor . The relative humidity averaged 60%. Temperature and relative humidity were monitored at the canopy level using an integrated temperature and relative humidity sensor . The moisture of the growing substrate was maintained ≈65 to 75% of field capacity (≈12% volumetric water content). Growth substrate moisture was measured with ECH2O soil moisture sensors  inserted vertically at the 2–7 cm depth.Experiments were conducted in the same greenhouse. There were two replicate experiments, and the planting dates were 13 November and 19 December 2013. In all experiments, vegetative terminal vine cuttings were selected that were 25–30 cm long, with five to six fully opened leaves, approximately 5 mm diameter at the basal cut, and with uniform distribution of nodes. Cuttings were planted (2–3 nodes under the growth substrate surface) in 10 cm-diameter polyvinyl chloride pots (height = 30 cm) with detachable plastic bottoms. The growth substrate and experimental conditions used in this study were based on previously developed methodology for measuring the effect of growth substrate moisture and nitrogen content on root architecture development at the onset of storage root initiation in ‘Beauregard’ During the experiments, plants infected with the potyvirus complex developed typical symptoms of chlorotic spotting followed by development of purple borders around the spots on leaves. Plants infected with SPCSV developed interveinal purple blotches. In both cases, symptoms developed primarily, but not exclusively on the older leaves. Virus symptom severity was assessed the day before each experiment was terminated by rating each leaf by visual estimation of the proportion of the leaf showing symptoms using a 0 to 3 scale in which: 0 =  no symptoms, 1 =  <1/3 of the leaf involved, 2 = 1/3–2/3 of leaf area involved, and 3 = >2/3 of the leaf involved. The mean rating for all leaves on a plant was used for statistical analysis and comparison of treatments.All experiments were arranged in a randomized complete block design where a pot (1 plant per pot) was considered a replicate. There were four replicates in the first experiment but one replicate was lost prior to data collection. There were five replicates in the second experiment. All experiments were terminated after 20 days by removing the detachable plastic bottoms tilting the pots and removing the growth substrate gradually using a stream of water.Data collection followed the procedures described in previous work Root attribute data from each experiment were pooled after verifying the lack of planting date effects. Root length and number were transformed using log 10 and square root transformation, respectively. Statistical analyses were performed using SAS Proc Mixed .). Fisher's LSD test at the 0.05 probability level was used to test for statistical significance. The data presented were means and standard error of the means from non-transformed data.No virus symptoms were observed on the non-inoculated, virus-tested plants at any time during propagation of source plants or during the experiments. Virus symptoms first appeared on the infected plants at about 2 wk after transplanting and became gradually more pronounced until the experiments were terminated at 20 days after transplanting ,2. VirusRepresentative images of LR development from each combination of nitrogen and virus treatment levels are shown in Other than demonstrating that certain strains of SPFMV can cause russet crack and determining effects of some viruses on storage root yield, little is known about how sweetpotato viruses affect the developing adventitious root system, especially during the critical storage root initiation period. Previous work has provided evidence that virus infected sweetpotato cuttings of the cultivar ‘Kokei No. 14′ had more adventitious roots but with lower weight when compared to virus-tested plants Arabidopsis plants that were transformed to express the Beet necrotic yellow vein virus p25 protein showed abnormal root branching, accompanied by significant changes in the levels of auxin, jasmonic acid and ethylene precursor, ACC Cucumber mosaic virus-infected Arabidopsis root growth patterns were accompanied by significant changes in indole-3-acetic acid (IAA), trans-zeatin riboside and dihydrozeatin riboside Solanum lycopersicon), infection by Tomato aspermy virus resulted in reduced LR development, which was accompanied by increased miR164 levels Tomato spotted wilt virus showed organ-specific responses although the virus was present in similar concentrations It has been documented that auxin is the integrator of intrinsic and environmental signals that affect LR development It has been suggested that root architecture may hold the key to the next green revolution


IFITM3 was linked to outcomes during the 2009 H1N1 pandemic. To identify variant host genes associated with increased virus replication and severe disease, we performed a quantitative trait locus analysis on pro-inflammatory cytokine production 48 hours after intranasal infection with highly pathogenic H5N1 influenza virus.Genetic variation in the human population is a key determinant of influenza disease severity. A single nucleotide polymorphism in the antiviral gene Samd9l, Ica1, and Slc25a13. To study the role of Slc25a13, we obtained Slc25a13 knockout line, but upon challenge with H5N1 influenza virus observed no effect on CCL2 production, or morbidity and mortality.Pro-inflammatory cytokines CCL2, TNFα and IFN-α, were measured by ELISA in lung homogenates of DBA/2J (D2), C57BL/6J (B6) and 44 different BXD recombinant inbred mouse strains. Virus titer was also assessed in a subset of these animals. CCL2 (8-fold), TNFα (24-fold) and IFN-α (8-fold) concentrations varied significantly among the different BXD RI strains. Importantly, cytokine concentration correlated very well  with virus titer suggesting that early cytokine production is due to increased virus infection and replication. Linkage analysis of cytokine concentration revealed a significant locus on chromosome 6 associated with differences in TNFα, IFN-α and CCL2 cytokine concentration (LRS =26). This locus accounted for nearly 20% of the observed phenotypic variation in the BXD population studied. Sequence and RNA expression analysis identified several candidate host genes containing missense mutations or deletions; Samd9l and Ica1, may be important for the control of influenza virus infection and pathogenesis.A novel genetic locus on chromosome 6 modulates early pro-inflammatory cytokine production and virus replication after highly pathogenic influenza virus infection. Candidate genes, The online version of this article (doi:10.1186/1471-2164-15-1017) contains supplementary material, which is available to authorized users. IFITM3 gene was associated with hospitalization and more severe disease after the pandemic H1N1 influenza infection . The. The5]. Qivr6.1 that were detected by microarray in the context of uninfected or H5N1 IAV infected lung tissue. These included the three polymorphic host genes Samd9l, Ica1 and Slc25a13, but not Col28a1. A comparison of baseline RNA expression identified 5 host genes whose expression levels were significantly different (P <0.01), albeit the difference was less than 2-fold. These included Ica1, Col1a2, Calcr, Tfpi2 and Bet1. One probe (1431380_at), detecting a transcript located within an intron of Ica1, was detected only in B6 animals. Within the locus we also identified host genes whose expression increased  or decreased  2-fold (P < 0.05) 72 hours after infection with H5N1 IAV in D2, B6 or both mouse strains. Importantly we did not identify a transcript that was differentially expressed only in D2 or B6 mice suggesting that there are no sequence polymorphisms in important transcription factor binding sites.Using previously published RNA expression data  from lunIca1, Samd9l, Tfpi2 and Calcr correlated well  with TNF-α cytokine concentration in these mice.Finally, we evaluated available lung micro array data from the QTL gene-network website, correlating TNF-α cytokine levels 48 hours after H5N1 IAV infection with basal gene expression in 37 BXD mouse strains . InteresSlc25a13) is a member of the mitochondrial carrier family of proteins and catalyzes the calcium-dependent exchange of cytoplasmic glutamate with mitochondrial aspartate across the mitochondrial inner membrane [Slc25a13 controls the level of cytokine production. Therefore we predicted that the absence of this gene would increase cytokine production upon HK213 infection. To control for the appropriate genetic background we used BALB/cAnCrl as controls for the Slc25a13-/- knockout mice congenic on the BALB/cAnN background. Compared to HK213 infected control animals, the Slc25a13-/- animals produced equal amounts of CCL2, suggesting that the lack of Slc25a13 expression did not affect H5N1 induced inflammation  that harbors three candidate genes associated with increased inflammation early after H5N1 IAV infection. Samd9l is a Sam domain containing protein with similarities to SAMD9 which has been shown to be important during virus infection and innate immunity [Samd9l is expressed in many different tissues including lung [Samd9l expression increases West-Nile virus replication [Samd9l knockout mice are characterized by a delay in homotypic endosome fusion resulting in persistence of ligand-bound cytokine receptors [Samd9l is important for the trafficking, maturation and acidification of the endosome, it may act as an antiviral host gene against IAV infection. Interferon induced transmembrane protein 3 (IFITM3) is a host protein with potent antiviral activity. IFITM3 blocks entry of many difference viruses, including influenza, at the stage of membrane fusion in the endosome. Deletion of Ifitm3 in C57BL/6 mice resulted in increased virus load in the lungs and increased morbidity and mortality after influenza virus infection [Ifitm3 protein between the D2 and B6 mice, the increase in viral load and associated disease in Ifitm3-/- mice is similar to the phenotype observed in D2 mice. An alternative hypothesis is that Samd9l affects the persistence of ligand-bound cytokine receptors which may increase cytokine signaling and inflammation which in turn results in more recruitment of inflammatory cells and virus infection [Samd9l against another RNA virus [Samd9l our priority candidate gene.Here we have identified a highly significant and novel locus on the extreme proximal portion of chromosome 6  and is involved in the regulation of secretory vesicle trafficking [Ica1 is highly expressed in nervous, glandular and muscle tissue and to a lesser extend in other tissues such as the respiratory tract. Differences in Ica1 protein structure or expression can affect the sympathetic nervous system activity and neuropeptide secretion altering the host response to infection [Ica1 could potentially affect the virus-encoded matrix 2 (M2) ion channel activity in the TGN to neutralize the acidic pH of the TGN in order to prevent activation of HA to its fusogenic form. This activity activates NLRP3 resulting in the formation of the inflammasome, cleavage of pro-IL-1β [Ica1 may alter the replication kinetics of IAV by changing intra-cellular trafficking of TGN-associated vesicles required for trafficking of the viral glycoproteins HA and NA to the surface of the cells. Interestingly the amino acid residue at position 312 in the C57BL/6J strain was predicted to be damaging , suggesting that the Ica1 allele in DBA/2J mice may enhance virus replication.syndrome . ICA1 waglaucoma . Ica1 tofficking , 31. Icanfection –34. Chanro-IL-1β , 36 and Finally, Col28a1 is a von Willebrand factor A domain-containing protein with many imperfections in the collagenous domain . The 69 Morbidity and mortality after influenza virus infection is affected by genetic variation of important host genes. Identification of the polymorphism and affected host genes will provide significant insight into the pathogenesis after influenza virus infection. In order to identify host genes with important effects on influenza pathogenesis, we performed a quantitative trait locus analysis on genetically diverse mouse strains comparing the early inflammatory response after H5N1 virus infection. This analysis identified a highly significant H5N1 control locus (LRS 26) on the extreme proximal part of mouse Chr. 6 that is homologous to a 5 megabase interval on human Chr. 7p22 and 7q21 respectively.Severe disease after influenza virus infection is mediated by both host and viral factors and many studies suggest that viral load and rapid replication are the determining factor for the outcome of the disease. These findings are supported by potent effects of neutralizing antibodies at early time points and the relatively short period of time (48 hours) in which treatment with antiviral therapeutics is considered most effective. In contrast to these studies, several others suggest that inflammation is independent of viral load and caused by underlying illnesses, or differences in viral protein function, essentially changing the host response to infection from protective (disease prevention) to pathogenic (disease promoting). Interferon antagonist functions of NS1 or the endonuclease function of PA-X are important examples of this process . This viA major advantage of using inflammatory cytokines as a biomarker for viral load is availability of commercially available and validated assays and the increased dynamic range between the different mouse strains. Although we observed a 50-fold difference in viral titers between D2 and B6 parental strains, on a logarithmic scale this difference is much smaller (1.4-fold). In contrast, the cytokine data is presented on a linear scale greatly increasing our dynamic range between the B6 and D2 animals while maintaining large fold differences in cytokine concentrations between D2 and B6. Thus a QTL analysis with cytokine concentration as the phenotype is more sensitive and likely to yield loci containing genetic polymorphisms involved in virus replication or antiviral activity. Although we provide evidence that inflammatory cytokine concentration in lung homogenates can be used as a surrogate biomarker for viral load between different mouse strains, extensive validation in nasal washes or serum from human patients infected naturally or experimentally with influenza virus needs to be done before this can be used in clinical specimens.Qivr or quantitative trait for influenza virus resistance) on chromosomes 2, 5, 7, 16, 17 and 19 [HrI) located on chromosomes 1, 7, 15, and 16 [Qivr1), associated with increased levels of TNFα, contains a well-known host antiviral gene ribonuclease L (Rnasel). The presence of at least sixteen different missense mutations between B6 and D2 (including three that are predicted damaging) make this gene a likely candidate. Qivr13 contains four host genes harboring non-synonymous polymorphisms including Erbb2ip, a regulator of Nod2-dependent NF-kappaB signaling, and a member of the nuclear importin family (Ipo11). Finally Qivr6.2 contains several polymorphic host genes including Mxd1 and Anxa4.Several loci and polymorphisms have been associated with resistance to influenza virus disease using a variety of different genetic mouse models. BXD RI strains were used to identify genetic loci  in baseline expression of host genes nor did we identify lack of induction of host genes in the susceptible D2 strain. Therefore we consider it unlikely that sequence variation in regulatory or non-coding regions of the genes present in this locus is causing the difference in phenotype.To identify candidate genes we focused primarily on sequence variation that affects the protein (non-synonymous polymorphisms). However sequence variation in regulatory regions of the gene, such as transcription factor binding sites or micro-RNA target sites, can change the level of protein expression and as such impact virus infection and inflammation. At least for Slc25a13 was one of four candidate genes within the Qivr6.1 locus associated with increased cytokine production following H5N1 IAV infection. However, mice lacking this gene did not demonstrate a change in cytokine production, suggesting that Slc25a13 is not the host gene whose polymorphism affected the early innate immune response. Previously, Slc25a13 was found to associate with the PA gene of a related highly pathogenic H5N1 influenza virus (A/Vietnam/1203/04) [Slc25a13-/- had no discernable effect on the host response or pathogenesis in vivo. Alternatively the effects of Slc25a13 gene deletion vary between mouse strains. We tested the effect of Slc25a13 on a Balb/cAn genetic background that is different from both parental strains used to generate the BXD family. It is feasible that the deletion of Slc25a13 in C57BL/6J or DBA/2J mice would change the cytokine profile after H5N1 virus infection. Deletion of the Slc25a13 gene was previously reported to have variable effects on metabolism dependent on the genetic background of the mouse strains [Slc25a13 may not mimic the effects of the genetic changes found in the D2 strain. The amino-acid changes may have altered gene function that is not captured in the Slc25a13 knockout mouse strain.1203/04) . The abs strains . AlternaIn conclusion, gene polymorphisms in the genome of the infected host can have a profound impact on the course of an H5N1 influenza virus infection. Although severe disease outcomes are often attributed to inflammation with excessive production of proinflammatory cytokines, the pathogenicity of influenza infection is rooted in increased virus titers in the lungs of the susceptible hosts.Severe disease after influenza virus infection is associated with excessive production of inflammatory cytokines and recruitment of innate immune cells into the infected tissue. An increase in dose or enhanced virus replication contributes to the excessive production of inflammatory cytokines. Identification of host-factors that facilitate virus replication and pathogenesis is important for the discovery of host targets for the development of novel antiviral therapies.Samd9l and Ica1 as strong candidate genes in this locus.In this report we provide further evidence that excessive production of inflammatory cytokines is due to higher virus loads. We also show that pro-inflammatory cytokine production following infection with highly pathogenic H5N1 influenza virus is dependent on the genetic background of the mouse strain. Quantitative trait locus analysis identified a locus on chromosome 6 that is associated with early production of pro-inflammatory cytokines and increased virus replication in BXD recombinant inbred mouse strains. Expression and coding sequence analysis identified Slc25a13tm1Lct), congenic on a BALB/cAnN genetic background, were obtained from the Jackson Laboratory [Female DBA/2J  and C57BL/6J  mice were purchased from the Jackson Laboratory  and housed at St Jude Children’s Research Hospital, Memphis, TN, USA. Female BXD recombinant inbred (RI) mouse lines derived from crosses between B6 and D2 stock were acquired from Oak Ridge National Laboratory . Mice deficient in citrin . To assess morbidity and mortality after HK213 virus infection, animals were weighed every second day until 21 days post-infection. Animals that lost more than 30% of their initial body weight were sacrificed per approved animal protocol.4 EID50 were obtained 2 days after infection and immediately homogenized in 2.0 ml tubes containing 1.0 ml PBS and a stainless steel ball for two 30-sec periods at 30 Hz . After a 30-sec spin at 16,000 × g, the supernatant was collected, aliquoted, and stored at –80°C. Virus titers were determined on MDCK as described previously [Lungs from B6, D2, and BXD strains infected with 10eviously . The conhttp://www.genenetwork.org). Interval mapping evaluates a potential QTL at regular intervals and estimates the significance at each location by using 2000 or more permutation tests [http://www.genenetwork.org (ID numbers 12971–12973). Loci associated with increased production of pro-inflammatory cytokines were identified and are referred to here as Qivr (QTL for influenza virus resistance). The QTL boundaries were defined as a drop in peak LRS of 7 as this is considered the best approximation of a 95% confidence interval in QTL mapping [Quantitative trait loci (QTL) mapping was performed using the WebQTL module of GeneNetwork , Pon2 (NM_183308.2), Pon3 (NM_ 173006.1), Samd9l (NM_010156.3), Ica1 (NM_010492.3), Hepacam2 (NM_178899.5), Slc25a13 (NM_015829.3), Asb4 (NM_023048.5), Ccdc132 (NM_024260.5), C1galt1 (NM_052993.3). Col28a1 (NM_001037865.1) was partially amplified and sequenced around exon 16–18 to confirm an in-frame deletion.Candidate host genes were amplified by PCR using cDNA generated from RNA extracted from HK213 virus-infected lung tissue obtained from B6 and D2 strains. The cDNA was generated using gene-specific primers and Superscript III First-Strand Synthesis System (Invitrogen) according to the manufacturer’s protocol. For a list of primers see Additional file Slc25a13-/- animals were analyzed for statistical significant using Student’s t-test. The Student’s t-test was also used to analyze differences in cytokine concentration in lung homogenates between the different mouse strains or between HK213-infected and mock-infected samples. The Pearson product-moment correlation coefficient was determined for cytokine concentration and virus titer using GraphPad Prism 6 software on untransformed data.Statistical analyses of differences in mortality were determined by using the log-rank test. Differences in morbidity and CCL2 production between control and 4 EID50 of HK213 virus in 30 μl PBS. Twenty-four, 48 and 72 hours post inoculation the lungs of the inoculated animals were collected, homogenized in sterile PBS, and stored at -80°C. The concentration of IFN-α in these homogenates was quantified by ELISA. (PDF 19 KB)Additional file 1: Figure S1: Kinetics of IFN-α production following H5N1 influenza virus infection in DBA/2J and C57BL/6 mice. DBA/2J and C57BL/6J were inoculated with 104 EID50 of HK213 virus in 30 μl PBS. Forty-eight hours post inoculation the lungs of the inoculated animals were collected, homogenized in sterile PBS, and stored at -80°C. The concentration of TNF-α, IFN-α and CCL2 in these homogenates was quantified by ELISA. (PDF 83 KB)Additional file 2: Table S1: TNF-α, IFN-α and CCL2 concentrations in lung homogenates of the recombinant inbred BXD animals. BXD mice were inoculated with 10Qivr6.1 locus. (PDF 80 KB)Additional file 3: Table S2: Primer sequences used to amplify the coding region of various candidate genes in


Background: Schizophrenia is a disabling mental disorder with high prevalence and that usually  requires long-term follow-up and expensive lifelong treatment. The cost of schizophrenia treatment consumes a significant amount of the health services' budget in western countries.Objective: The aim of the study was to find out about the costs related to schizophrenia across different european countries and compare them.Results: Schizophrenia treatment costs an estimated 18 billion euros annually worldwide. The direct costs associated with medical help are only part of the total expenditure. The indirect costs are an equally (or even more)important part of the total cost. These expenses are related to the lack of productivity of schizophrenic patients and the cost that relatives have to bear as a result of taking care of their affected relatives.Conclusions: Although data on the cost of schizophrenia may vary slightly between different european countries, the general conclusion that can be drawn is that schizophrenia is a very costly disorder. Not only because of direct costs related to medical procedures, but also due to the non-medical (indirect) costs. Together this suggests the need to investigate cost-efficient strategies that could provide a better outcome for schizophrenic patients, as well as the people who care for them. It is one of the most stigmatizing diseases of all time5. Schizophrenic disorders present with a wide range of symptoms, both positive and negative, leading to cognitive, social and functional impairment. Therefore most individuals with schizophrenia are considered disabled and claim benefits6. The debilitating nature of the disorder means that patients receive lifelong treatment, and a large proportion of them need to be admitted to a hospital inpatient unit on multiple occasions throughout their lifetime3.Schizophrenia is a chronic psychiatric condition that affects around 1% of the population worldwide7. By these we mean the inability to work and also the way that schizophrenia affects the patients´ environment. The need to take care of and support schizophrenic relatives is a major reason for members of their families to take sick leave or even sacrifice their own career8. All of this, plus the high expense associated with the newest antipsychotic drugs, makes the costs of schizophrenia management as high as 3% of the total healthcare budget of western countries1.All of the above factors lead to the high costs associated not only with the treatment of schizophrenia but also related to social impairmentThe aim of this paper is to give a global view on the problem and to emphasize certain cost-inducing aspects of schizophrenia management by reviewing past research on the costs of schizophrenia management.9. The direct costs of treating schizophrenia include cost of hospitalization (short- and long-term), outpatient follow-up, residential and day care, pharmaceutical interventions, laboratory testing and social security payments, whereas the indirect costs are mainly related to the loss of productivity11. The age of onset of the disorder, usually in the late teens or early 20s, can preclude patients from even starting to work12. Later on, most of the patients receive benefits for incapacity for work due to disability6. Nowadays, most schizophrenic patients receive a disability certificate and eventually do not work. Up to 80% of schizophrenic patients in the UK do not have paid employment3. In Italy and Spain, three out of four patients with schizophrenia are excluded from the job market13. Thus, some authors consider the loss of productivity as accounting for the majority of the indirect costs4.Regardless of the authors’ origin, they all agree unanimously that the costs associated with the treatment and care of patients with schizophrenia can be divided into two important groups: direct and indirect costs14, whereas others suggest that indirect costs can outnumber the direct ones up to three or four times15.Speaking of indirect costs, it is also important to consider the indirect cost associated with caregivers to schizophrenic patients, who contribute with their time and in-kind services . Therein3. These are expenses of a non-financial nature. They try to accomplish the hard task of reflecting the patients´ quality of life, including side-effects of pharmaceutical interventions and stress and anxiety, both caused by the disease itself and also the treatment process. Although this group might have no direct financial impact, it is worth considering these factors as they probably affect the cooperation between patient and health providers17. Furthermore, we can also take into account the intangible costs of the caregivers of schizophrenic patients3. Daily care of a schizophrenic relative can be a very challenging and exhausting experience18. Social stigma and the lack of sympathy and understanding may lead to anxiety and depression in caregivers as well as sufferers5. This could damage the relationship between the caregiver and the caretaker, which may lead to an increased rate of patient deterioration and worse prognosis in the long-term18.Some authors also distinguish a third group of costs, called “intangible costs”The main part of data used in this article come from psychiatric wards in Spain, France, Sweden, Poland, United Kingdom and Ukraine . Data frschizophrenia costs in Europe”, “(indirect and direct costs) schizophrenia”, “schizophrenia costs worldwide”, “schizophrenia costs United States”, “schizophrenia and disability”, “antipsychotic treatment in Europe” as a part of the abstract, title or included anywhere in the whole paper. We analyzed 41 articles that we managed to find according to the criteria we adopted.The research data for this article was collected by the use of the PubMed database in April 2015, having used key words: “We have taken into account comparable data, e.g. annual expenses on schizophrenia treatment, expenses per capita on schizophrenia treatment. In addition, we have taken into account these possible differences concerning data collecting methods used by the particular researchers, as well as different years in which the researchers conducted their studies, the comparison can only estimate the true cost of schizophrenia treatment.We wanted to include only the latest data coming from research conducted after year 2000. Due to the fact that there has been very little research done in this field in general we decided to analyze papers from the whole PubMed dataset. We did however exclude the earliest data  since we found them irrelevant .The nature of data varies between different authors. Some articles give total amounts of money in relation to the direct and indirect schizophrenia treatment costs in a particular country. The others give numbers per patient. The percentage approximations of pharmacological costs are also present, directly obtained from papers.We included only those articles showing general information about costs of schizophrenia across different countries, and excluded those ones related only to specific services, like acute inpatient units, where stated costs did not include rehabilitation expenses or full treatment options, what could lead to important bias when comparing pharmacological expenses or indirect costs.16.This paper points at the magnitude of the problem of schizophrenia treatment costs and estimates the huge impact that this mental disorder has on patients´ environment and society at a financial level across several different countriesWhat definitely strikes attention when it comes to available data is that the estimated indirect cost represents a significant part of the total cost of schizophrenia. It is particularly important to bear that in mind in order to manage schizophrenia efficiently.16. This statement is equally consistent for both Western  may occur early in life, before young people begin their career14.In some countries, like France and UK, the indirect costs outnumber the direct ones 4,15. OTherefore, it could be possible that the indirect costs are even higher in the countries who register its workforce in this way.19. Although good adherence to treatment means no reduction in the budget for pharmaceutical interventions, this cost does not seem to represent a significant percentage when compared to total cost of the illness (drugs costs vs. total costs – see3.Many authors raise the issue of patients´ adherence to prescribed therapy. An optimal control of schizophrenic symptoms is proven to lead to fewer numbers of hospitalizations and less need to use other approaches of formally organized patient care20. So it is of crucial importance to investigate whether better control of symptoms could allow patients to get a chance to attain and keep a job, and how this fact could affect their quality of life. Moreover, we should consider how this could be used to lighten the burden of care that relatives and caregivers experience and thus reduce the indirect costs which make up a significant part of the costs of this illness.When the symptoms of schizophrenia remain under control, patients experience a smaller risk of mental impairment and social exclusion which extends to participation in work opportunitiesTherefore, it is our suggestion that the future of schizophrenia treatment should address more carefully important elements of the financial aspects of the disease, such as cost-efficacy of treatment, including psychological therapies and psychoeducational approaches for both patients and their families. A wider view on the matter is needed. I think the article may have those tables proposed by the authors.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. Was not fully addressed, some phrases still contain the term "schizophrenic." The text should be more thoroughly reviewed.Frey 2014 in Germany was not included.Was not addressed. It's a bit confusing whether the paper aims to describe findings from economic studies in Europe vs worldwide. Not clearly stated where the 18 billion figure comes from. Not sure then why the study byA few concerns that I have: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. Changing the terminology "schizophrenic patients" to "patients with schizophrenia" or "individuals with schizophrenia".In the abstract the objective states the aim is to report on the costs of schizophrenia across different European countries, yet the results states "18 billion euros worldwide." Given that the manuscript reviews economic studies in Europe, it should be modified to focus on European findings.The last few sentences in paragraph 3 of the introduction describe limitations of this review; these sentences should be described in the methods section. It is not completely clear to me what the authors are referring to when they mention "other data" that could not be compared.A paragraph following the introduction or within the introduction detailing the types of costs that are typically measured in these type of studies could replace table 1.Detailing the types of costs that were measured in the different studies would be a valuable contribution to Table 2.The article offers a concise, brief summary of an important topic. The following are minor revisions that will improve the manuscriptI have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Change the terminology "schizophrenic patients" to "patients with schizophrenia" or "individuals with schizophrenia".The last few sentences in paragraph 3 have been moved to the methods section.A brief paragraph following the introduction of table 1 have been included. A brief paragraph following the introduction of table 2 have been included.Best regards and thanks for your quick and good review of our paperThanks for your review, we have made the following changes in our latest version Brief and nice review of an important topic related to economic cost of schizophrenia. Results are presented clear. However, I think that tables with examples are not necessary and should be considered to be deleted. The text does not need any change in my consideration.We have read this submission. We believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however we have significant reservations, as outlined above. Thanks for your quick review and your comments. According to the reviewer comments, we have deleted table 4, and we have maintained table 1, 2 and 3. I hope that this changes could contribute to the final acceptance of the paper.


Influenza viruses cause annual seasonal epidemics and occasional pandemics. It is important to elucidate the stringency of bottlenecks during transmission to shed light on mechanisms that underlie the evolution and propagation of antigenic drift, host range switching or drug resistance. The virus spreads between people by different routes, including through the air in droplets and aerosols, and by direct contact. By housing ferrets under different conditions, it is possible to mimic various routes of transmission. Here, we inoculated donor animals with a mixture of two viruses whose genomes differed by one or two reverse engineered synonymous mutations, and measured the transmission of the mixture to exposed sentinel animals. Transmission through the air imposed a tight bottleneck since most recipient animals became infected by only one virus. In contrast, a direct contact transmission chain propagated a mixture of viruses suggesting the dose transferred by this route was higher. From animals with a mixed infection of viruses that were resistant and sensitive to the antiviral drug oseltamivir, resistance was propagated through contact transmission but not by air. These data imply that transmission events with a looser bottleneck can propagate minority variants and may be an important route for influenza evolution. Influenza viruses cause respiratory infections in annual seasonal outbreaks and intermittent pandemics. The spread of virus is difficult to control because transmission is efficient and may occur from asymptomatic or presymptomatic individuals135http://www.cdc.gov/flu/pandemic-resources/tools/risk-assessment.htm)101217The ferret is the favoured animal model for studying influenza transmission. This is because ferrets and humans show a similar distribution of sialic acid (SA) receptors that enable incoming virus to enter target cells, and ferrets display clinical signs after infection reminiscent of influenza-like symptoms in humansThese observations suggest that RD transmission may exert a more stringent bottleneck than DC transmission. Implications for virus evolution are that whilst genetic diversity might expand in an infected host, only a fraction of that diversity might be transmitted. Comprehending the circumstances under which minority populations might be transmitted onwards is crucial for understanding the emergence of new traits such as drug resistance, antigenic drift or extended host range1920et al. also described a tight bottleneck that restricted the RD transmission of H7N9 avian influenza viruses to mammalset al. found that the number of viruses that passed between hosts during RD transmission between animals was small, indeed as few as two barcoded viruses initiated infectionTo address the stringency of the influenza transmission bottleneck, several groups have analysed sequence variation between viruses in donor and recipient animals during transmission experiments. During the rather inefficient RD transmission of avian-like H5N1 or H1N1 viruses between ferrets, next generation sequencing (NGS) demonstrated that HA variants that had been minority genotypes in donor animals were uniquely transmitted, suggesting a tight bottleneck22Here, we also used a genetic tagging approach to monitor the dose of virus transmitted between ferrets by either contact or RD routes. Donor ferrets were infected with a mixed population of two viruses whose genomes differed only by reverse engineered synonymous mutations that could be easily differentiated. We then quantified the mixtures in sentinel animals that acquired infection by the RD route or by DC during co-housing. We found that mixtures were more readily transmitted during contact transmission events, whereas a bottleneck was imposed at RD transmission. Moreover, this was also the case during infections with a mixture of drug-sensitive and drug-resistant variants: drug resistance was only propagated during contact transmission events that allowed the transfer of minority variants, but this occurred even when resistance carried a fitness cost.Transmission experiments were performed in ferrets using engineered influenza viruses based on the pandemic H1N1 2009 virus A/England/195/2009 which transmits efficiently between ferrets by both DC and RD routesWe generated a pair of isogenic viruses, that differed by a single nucleotide polymorphism in the PB2 gene at nucleotide position 318 (numbering from 5′ end of positive strand), A vs U . A seconin vitro. In a multicycle replication assay in MDCK cells, there was no significant difference in viral titres reached at any time point following infection with either wild type A/Eng/195/2009 virus or the double-tagged virus  at a 90%:10% infectivity ratio. The virus mix had a total infectivity of 104 plaque forming units (PFU). The following day each infected animal was paired with an RD sentinel ferret introduced into an adjacent cage. Sentinels were exposed to air from their paired infected donors for 10 days as previously described226To study the virus population All four directly inoculated animals shed robust titres of infectious virus from the nose beginning on day 1 and cleared virus by day 7. RD transmission occurred in all four pairs, although the day at which virus was first detected in sentinel animals varied from day 4 to day 8 post exposure .in vivo fitness cost or gain conferred by the PB2 A318U synonymous mutation and both A1 and A2 virus genotypes replicated robustly.The proportion of each viral genotype in nasal washes from directly inoculated animals was quantified by pyrosequencing virus samples collected on days 1 and 2, when animals are most contagiousIn contrast, the proportions of virus genotypes shed from the four recipient ferrets who acquired their infection through RD exposure varied considerably . All 40 plaques from RD sentinels in chain 1 and 2 were of the majority (A1) genotype and all the plaques from RD sentinel in chain were of the minority genotype (A2). Of the 40 plaques from RD sentinel in chain 3, 4 (10%) contained minority (A2) genotype and the remaining 36 (90%) were majority (A1) genotype . These dIn a second experiment we introduced an additional synonymous nucleotide change to an adjacent region of PB2 .4 PFU of the 70%:30% mixture of A/England/195/09 virus genotypes containing silent tags at nucleotides 318 and 477 in the PB2 gene. The next day a contact sentinel animal (DC1) was introduced into the same cage and an RD sentinel animal (RD) was placed in the adjacent cage. Exposure of both ferrets DC1 and RD to the donor animal was limited to 24 hours (day 1–2 after donor infection) since we have previously shown that transmission occurs efficiently at this timeTwo separate transmission chains involving both contact and RD exposure were set up as illustrated in Pyrosequencing results for nucleotide position 477 are shown in in vivo  plus two mutations in the PA gene segment (one nonsynonymous A404T and one synonymous C1449T) were detected in the first DC animal that were not detected in the donor. Upon further transmission to ferret DC2, the engineered tags at position 318 and 477 in PB2 were not transmitted as a mixture, (as had been shown by pyrosequencing), but a mixed genotype at E118G in PB2 and A404T PA was maintained.Taken together these data suggest that RD transmission between ferrets exerts a tight bottleneck in which mixed genotypes tend to be purified, whereas DC transmission events can propagate genetic mixtures.−μ)), was estimated to be 2.83 infectious genomes  based on both experiments 1 and 2 and to be 3.45 infectious genomes  based only on experiment 1 . The average infectious dose size for RD transmission,  that drop out of the air under Stokes Law before reaching the adjacent cage. Only a proportion of virus is shed from the infectious animal in droplets small enough to remain airborne beyond the distance separating the ferret cages343238A narrow transmission bottleneck has also been reported during natural transmission of HIV, Hepatitis C virus, and Venezuelan equine encephalitis virus, although these viruses are transmitted by different routes than influenza virus41444647in vitro in primary human airway cells. Butler et al. had previously shown that late wave Australian strains of pH1N1 2009 drug-resistant virus could be transmitted to DC-exposed ferrets even in the presence of drug sensitive virusThe contribution of different transmission routes in human influenza outbreaks is difficult to quantify and is poorly understood. It is likely that several different routes including respiratory droplets and aerosols as well as close contact and fomites all play a roleMDCK cells were maintained in Dulbecco’s modified Eagle’s medium  supplemented with 10% FBS and 1% penicillin/streptomycin (Sigma-Aldrich).A/England/195/2009 was a prototypic first wave isolate of the 2009 pH1N1 pandemic from the UK. A/England/687/2010 was a typical third wave isolate of pH1N1 virus from the UK. Reverse genetics systems by which recombinant viruses can be rescued and genetically manipulated were previously described−1 and incubated at 37 °C for 3 days.Plaque assays were performed using MDCK cells as previously describedPCR and sequencing primers for pyrosequencing assays were designed using the Qiagen PyroMark assay design software, against the A/England/195/2009 PB2 sequence (Accession GQ166656). Primer sequences are; PCR forward: 5′ Biotin labelled-GCCGTAACATGGTGGAATAGGAAT; PCR Reverse: TCTTGCCCCCACTTCATTTG; A318U sequencing: TTAGGGTAATGAACTGTACT; A477G sequencing: TTCCATAATCACATCCTG. The assays were designed such that both target single nucleotide polymorphisms were contained in the same PCR amplicon, to eliminate errors due to differential amplification  72 °C: 10 min). Pyrosequencing reactions were performed according to manufacturers’ instructions .To determine the limit of detection of the target nucleotide polymorphism, i.e. the frequency with which it miscalled U for A at nucleotide 318, four independent assays were performed from a pure (100%) stock of the genotype A1 virus (PB2 318A). This gave a mean value of 95.1% A1 virus with a standard deviation of 1.2%. We defined the limit of detection of pure genotype A1 as 92.7% (the mean minus 1.96 standard deviations). Thus any sample with a % genotype A2 less than 7.3%  and xylazine (0.9 mg kg−1) and then inoculated intranasally with virus diluted in phosphate buffered saline (PBS) (0.1 ml per nostril). All animals were nasal washed daily, while conscious, by instilling 2 ml PBS into the nostrils, and the expectorate was collected in modified 250 ml centrifuge tubes. The nasal wash expectorate was used for virus titration by plaque assay and RNA was extracted for pyrosequencing. The limit of virus detection in the plaque assays was 10 PFUml−1.Female ferrets (20–24 weeks old) weighing 750–1000 g were used. After acclimatization, sera were obtained and tested by virus neutralization assay for antibodies against A/England/195/2009, pH1N1. All ferrets were seronegative for influenza antibodies at the start of the experiments. Body weight was measured daily, and strict procedures were followed to prevent aberrant cross-contamination between animals. Sentinel animals were handled before inoculated animals, and work surfaces and handlers’ gloves were decontaminated between animals. Inoculated ferrets were lightly anaesthetized with ketamine  safety committee of Imperial College London, St. Mary’s Campus (centre number GM77), and the Health and Safety Executive of the United Kingdom and carried out in accordance with the approved guidelines. All animal research described in this study was approved and carried out under a United Kingdom Home Office License, PPL 70/7501 in accordance with the approved guidelines.−μ), the probability of infectious dose size X infectious genomes. Given the infectious dose size X, the probability was calculated of M of the X infectious genomes in the infectious dose being the majority strain (i.e. M majority strain genomes and (X-M) minority strain genomes, so M could vary from 0 to a maximum of X, the infectious dose size), given the donor animal was infected with 90% majority strain and 10% minority strain. For experiment 1, given that M of the X infectious genomes were the majority strain, the probability was calculated of Y of the 40 sampled plaques being the majority strain (i.e. Y majority strain plaques and (40-Y) minority strain plaques, so Y could vary from 0 to a maximum 40). The data likelihood for infected ferret i in experiment 1 (L1i) with Y majority strain plaques among the 40 sampled plaques was obtained from the products of these three probabilities such that:The data were used to estimate the average infectious dose size, assuming individual infectious dose sizes (denoted by X) were Poisson distributed, conditional on 1 being the minimum dose size. This was achieved by calculating, for each potential conditional average infectious dose size (μ/(1 − e2j) is simplified in form because ferrets will only be infected with purely the majority strain if all X infectious genomes in the infectious dose were the majority strain:The data likelihood for infected ferret j in experiment 2 (Lj was the average proportion of the majority strain measured in the donor ferret which infected ferret j (for donor ferret in chain 1 this was 77.3% and for donor ferret in chain 2 this was 80.9%). The full data log likelihood as a function of μ, the average infectious dose size, is obtained from ln(∏iL1i)+ln(∏j L2j). Likelihood-ratio-based confidence intervals were obtained from the log likelihood profile for each estimate of μ.where pHow to cite this article: Frise, R. et al. Contact transmission of influenza virus between ferrets imposes a looser bottleneck than respiratory droplet transmission allowing propagation of antiviral resistance. Sci. Rep.6, 29793; doi: 10.1038/srep29793 (2016).


Orthobunyaviruses belonging to the Simbu sero-group occur worldwide, including the newly recognized Schmallenberg virus (SBV) in Europe. These viruses cause congenital malformations and reproductive losses in ruminants. Information on the presence of these viruses in Africa is scarce and the origin of SBV is unknown. The aim of this study was to investigate the presence of antibodies against SBV and closely related viruses in cattle in Tanzania, and their possible association with reproductive disorders.In a cross-sectional study, serum from 659 cattle from 202 herds collected in 2012/2013 were analyzed using a commercial kit for SBV ELISA, and 61 % were positive. Univariable logistic regression revealed significant association between ELISA seropositivity and reproductive disorders (OR = 1.9). Sera from the same area collected in 2008/2009, before the SBV epidemic in Europe, were also tested and 71 (54.6 %) of 130 were positive. To interpret the ELISA results, SBV virus neutralization test (VNT) was performed on 110 sera collected in 2012/2013, of which 51 % were positive. Of 71 sera from 2008/2009, 21 % were positive. To investigate potential cross reactivity with related viruses, 45 sera from 2012/2013 that were positive in SBV ELISA were analyzed in VNTs for Aino, Akabane, Douglas, Peaton, Sabo, SBV, Sathuperi, Shamonda, Simbu and Tinaroo viruses. All 45 sera were positive for one or more of these viruses. Twenty-nine sera (64.4 %) were positive for SBV, and one had the highest titer for this virus.This is the first indication that Aino, Akabane, Douglas, Peaton, Sabo, SBV, Sathuperi, Shamonda and Tinaroo viruses circulate and cause negative effect on reproductive performance in cattle in Tanzania. SBV or a closely related virus was present before the European epidemic. However, potential cross reactivity complicates the interpretation of serological studies in areas where several related viruses may circulate. Virus isolation and molecular characterization in cattle and/or vectors is recommended to further identify the viruses circulating in this region. However, isolation in cattle is difficult due to short viremic period of 2 to 6 days, and isolation in vectors does not necessarily reflect the situation in cattle. Orthobunyavirus, in the family Bunyaviridae and contain three RNA segments. These viruses are naturally capable of genetic reassortment, which can lead to development of new viral strains with altered biological properties. They are transmitted by arthropods, mainly biting midges from the genus Culicoides and mosquitoes [Simbu sero-group viruses belong to the genus squitoes . Most casquitoes .Sathuperi virus and is a possible ancestor of the reassortant Shamonda virus [The Simbu sero-group includes Schmallenberg virus (SBV), a newly emerged livestock virus first identified in Germany in 2011 , 4. Its da virus . The orida virus , 7.A number of Simbu sero-group viruses have been found to be present in different parts of the world, including Africa, Asia, Australia and Israel –10. TheyCulicoides midges and domestic animals. These include Sabo, Sango, Sathuperi, Shamonda, Shuni, Simbu and Yaba viruses [Culicoides midges in 2004 [In Africa, members of the Simbu sero-group have been isolated from  viruses , 14. Neu viruses –17. In A in 2004 , 19.Diagnosis of infections caused by Simbu sero-group viruses has traditionally been accomplished by detection of specific antibodies using virus neutralization assays but more recently enzyme-linked immunosorbent assays (ELISA) have been used and some are available as commercially prepared kits. However, as these viruses were originally clustered on the basis of serological assays, extensive cross reactivity is often observed , 21. Viro Investigate the SBV antibody prevalence before and after the epidemic in Europe.o Investigate the association between seropositivity and reproductive disorders.To investigate the prevalence of SBV-antibodies in cattle in the southern highlands of Tanzania, using a commercially available indirect ELISA kits.To further analyze ELISA results using a series of VNTs for SBV and other Simbu sero-group viruses closely related to SBV.In Tanzania, little is known about the presence of Simbu sero-group viruses and their impact on reproduction in cattle. In addition, the origin and global geographic distribution of SBV is not known. Mixed farming and extensive management systems and the presence of wildlife protected areas may facilitate transmission of these viruses between wild and domestic animals. This environment is conducive for vector activity. The present study was therefore undertaken with the following aims:The present study was part of a larger project on infectious agents causing reproductive disorders in dairy cattle in Tanzania. It was conducted in four districts that included Mbarali district in Mbeya Region and Wanging`ombe, Njombe rural and Njombe urban districts in Njombe Region in the southern highlands of Tanzania between July 2012 and March 2013. The selected farms from Njombe and Mbeya regions practice two different grazing systems. Most of the cattle in Mbeya graze on pastures during the day and are kept indoors at night, while the majority of herds in Njombe are confined in cattle houses. However, cattle houses or barns in both locations are not sealed to vectors. Both dairy cattle, which are mixed breed (Holstein Friesian and Ayrshire with Tanzania short horn zebu), and pastoral local breed of cattle (zebu), were included.A cross-sectional study design was chosen. A total of 202 herds were included in the present study. Of these, 181 dairy herds were small scale with an average size of three cows per herd, two were medium scale dairy herds with about 20 cows each, one was a large scale dairy herd with about 350 cows and 18 were pastoral herds with an average of 15 cows per herd. Inclusion criteria for herds were to have one female above 6 months of age and that the farmer was willing to participate. All animals below 6 months of age were excluded. From herds with less than five cattle above 6 months, all were included in the study. From herds with more than five cattle, five were randomly selected. This made a total of 659 animals. In addition, serum samples from 130 cattle from the same area, collected in connection with another study during 2008–2009, were also included in the present study. Blood samples were collected once from each animal.All herds were visited by qualified personnel and farmers were interviewed using a structured questionnaire. The questionnaire aimed to gather information about reproductive disorders in the herd and individual animals over the last 3 years. The questionnaire was designed to determine the presence or absence of abortions, delivery of weak/malformed calves, stillbirth, dystocia and retained fetal membranes. Abortion was defined as a cow giving birth to a calf at any stage of gestation before term. Stillbirth was defined as giving birth to a dead calf, dystocia was defined as any assisted birth and retained placenta when fetal membranes fail to expel 24 h after delivery. Farmers were informed on the study before commencement of data and sample collection and willingly agreed to participate.Blood samples (about 5 ml) were collected aseptically from the jugular vein in plain evacuated tubes. The samples were left at room temperature for about 12 h to allow serum separation, and then serum was decanted into sterile tubes and transported on ice to the local laboratory and immediately frozen at approximately −20 °C. Between laboratories samples were shipped to the destination laboratories on ice and then kept frozen at −20 °C until analysis.® Schmallenberg Virus Indirect multi-species screening test ID.Vet Innovative Diagnostics) according to the manufacturer’s instructions. For each sample the S/P percentage was calculated: S/P = (OD sample-OD negative control)/OD positive control-OD negative control ×100. Samples presenting S/P values less than or equal to 50 % were considered negative, S/P values between 50 and 60 % were considered doubtful and S/P values greater than 60 % were considered positive.All serum samples were analyzed at the Norwegian Veterinary Institute using a commercial ELISA kit  and negative (n = 10) serum samples from 2012/2013  software. Positively infected animals were reported in proportions. The association between presence or absence of reproductive disorders and herd or individual animal serostatus  were analyzed using univariable logistic regression and reported as OR at 95 % CI, and level of significance at The protocol for field studies and collection of animal material was in accordance with ethical approval by the University Ethics Committee using guidelines from the Code of Conduct for Research Ethics of Sokoine University of Agriculture SUA/VET/012/04. Farmer’s verbal consent was sought before embarking on data and biological material collection.n = 42) were positive while out of 335 individual animals, 194 (58 %) were positive. Seventy one (55 %) out of 130 sera collected in 2008/2009 were positive in the SBV ELISA. Antibodies were observed in sera collected from small, medium and large scale herds, Njombe and Mbarali, dairy and pastoral herds. The results of univariable regression analysis indicated no significant differences between serostatus and location or herd size or animal breed.Out of the 659 serum samples collected in 2012/2013, a total of 405 (61 %) were positive in the SBV antibody ELISA and out of 202 herds, 175 (87 %) had one or more seropositive animals. In Njombe out of 160 herds, 133 (83 %) were positive and out of 324 individual animals, 211 (65 %) were positive. In Mbarali however, all herds (Fifty six 51 %) out of 110 serum samples collected in 2012/2013 were positive with titers ranging from 1:16 to 1:512  were detected in one or more of the 45 serum samples from 2012/2013. Their titers ranged from 80 to 1280 (Table Twenty nine (64.4 %) sera were positive for SBV, out of which seven had high titers ≥160) to this virus. One serum sample had highest titers for SBV, while two had the same titer to SBV and Douglas or Sathuperi virus. A high proportion (91.1 %) of the sera had antibodies against Aino virus and antibodies against Tinaroo, Douglas, Peaton, Shamonda, Sabo and Sathuperi were detected in 75.6, 73.3, 71.1, 55.6, 46.7 and 31.1 % of the animals respectively. There were 24 samples that had high titers to either Aino or Peaton viruses and five sera that had high titers only to Tinaroo virus. Eleven serum samples showed low antibody titers to Akabane virus while a single sample had a titer of 160. However all of these sera had very high titers to at least one of the other viruses  on an individual animal level. There was no association demonstrated between individual animal seropositivity and any reproductive disorder alone. There was also no association observed between herd seropositivity and any specific reproductive disorder.P = 0.059). Antibodies to none of the other viruses were associated with any of the reproductive disorders.With the SBV VNT, there were no significant associations between any reproductive disorders and SBV seropositivity. Of the 45 sera subjected to Simbu sero-group VNT, eight originated from animals with a history of reproductive disorders. With Simbu sero-group VNT, there was only association between Akabane virus antibodies and abortion, and this association was not statistically significant  or to multiple viruses (13 samples). The fact that several samples were negative to SBV and at the same time strongly positive for closely related viruses indicates that cross reactivity may not be the main reason for the seropositivity to SBV in the VNTs.In general, commercially available ELISA assays are sensitive, specific and robust, but cross reactivity with other members of Simbu sero-group has been reported for the assay used in this study previously . The iniThe present study showed an association between SBV ELISA positivity and reproductive disorders. As the ELISA is not SBV-specific, this reactivity may also be due to other Simbu sero-group viruses which are known to cause late abortion, premature birth, still birth and congenital malformations , 36, 37.Antibodies against Akabane virus were associated with abortion, however the association was not statistically significant and the serological evidence of the presence of Akabane virus is inconclusive. It is well known that Akabane virus causes abortion in bovine animals , 28. TheThis is the first serological indication of Simbu sero-group viruses including SBV and their possible association with reproductive disorders in cattle in Tanzania. It is possible that viruses from the Simbu sero-group, other than the ones included in the test, are also present. The origin of the virus that caused the recent SBV epidemic in Europe is still a mystery but this study demonstrates the possibility that the virus may have been present in Tanzania already and other parts of Africa also where the same vectors are abundant. Isolation and further genetic characterization of the viruses, including isolates from different geographical origins, will be essential for understanding the molecular epidemiology and evolution of SBV related viruses.