Bombyx mori published in 2008 due to repetitive sequences, resulting in gaps and an incomplete view of the locus. To obtain the complete sequence of the chorion locus, expressed sequence tags (ESTs) derived from follicular epithelium cells were used as probes to screen a bacterial artificial chromosome (BAC) library. Seven BACs were selected to construct a contig which covered the whole chorion locus. By Sanger sequencing, we successfully obtained complete sequences of the chorion locus spanning 871,711 base pairs on chromosome 2, where we annotated 127 chorion genes. The dataset reported here will recruit more researchers to revisit one of the oldest model systems which has been used to study developmentally regulated gene expression. It also provides insights into egg development and fertilization mechanisms and is relevant to applications related to improvements in breeding procedures and transgenesis.The silkmoth chorion was studied extensively by F.C. Kafatos’ group for almost 40 years. However, the complete structure of the chorion locus was not obtained in the genome sequence of  The structural genes for chorion proteins comprise a multigene family whose members are grouped under α and β branches based on their evolutionarily conserved central domains2. Chorion proteins are further classified into six subgroups, early A, early B, middle A, middle B, late high-cysteine A (HcA) and late high-cysteine B (HcB), according to their timing of developmental expression and amino acid composition3. Based on genetic linkage mapping, the chorion genes are located between the larval marker p at the proximal end of chromosome 2 and the cocoon color marker Y4–6. The recent silkworm genome assembly7 indicates that the chorion locus is localized at  on chromosome 2, although it is largely interrupted by gaps due to highly repetitive sequences.Silkmoth chorion proteins, the main components of the eggshell, are sequentially synthesized and secreted by follicular epithelium cells with a high degree of developmental programmingEcoRI8, designated RPCI-96 (RP96), and is available from BACPAC Resources of the Children’s Hospital Oakland Research Institute (BACPAC Resources Center [bacpac.chori.org/]). Here we undertook the following strategy to obtain complete sequences of the chorion locus . We identified ESTs of chorion genes by BLASTx search in public protein databases including nr of NCBI.10. BAC clones derived from the chorion locus were screened by hybridization of BAC high density replica (HDR) filters arrayed in duplicate with RPCI-96 BAC clones (BACPAC Resources Center [bacpac.chori.org/]) using the ESTs of 10 chorion genes selected as representatives of the three chorion families which provided strong signals in hybridization with multiple BACs, among which some were cross-hybridized with different chorion families. A list of ESTs used for BAC screening is presented in 8.The silkworm BAC library (RPCI-96) used in this paper was obtained from BACPAC Resources Center, Children’s Hospital, Oakland Research Institute and previously describedhttp://sgp.dna.affrc.go.jp/KAIKObase/), and BAC 544H24, because we already knew that its full sequence was aligned with the 3′ part of the chorion locus and the neighboring region7. We performed contig construction for these BAC clones with the fingerprinting method described previously10. This resulted in two contigs; one was composed of four BACs covering the 5′ half of the chorion locus, while the other was composed of three BACs aligning with the 3′ half of the chorion locus , in .Six BAC clones from 384 well plateseach BAC . Then BA13. After trimming vector sequences using cross_match, all paired-end reads were assembled with the programs Phrap 1.0808122214 and Consed 16.015. The position of mis-assembled clone sequences could be adjusted according to the size of the clones (insertion segment) by both assembly programs. The small gap in assembly sequences was filled by primer walking. The software program fgenesh16 was used to predict the chorion genes.The low-quality bases (QV<20) were removed by PhredDDBJ AB999997 (Data Citation 1).The complete sequence of the chorion locus appears under 3. Thus, we selected representatives for the three types of chorion gene ESTs to screen the BAC library  of a highly repetitive genome. The complete sequence of the chorion locus and detailed gene annotation data are provided for users to study developmental regulation of gene expression using the silkmoth chorion gene model.The complete sequences of chorion locus data described here can be downloaded from How to cite this article: Chen, Z. et al. Construction, complete sequence, and annotation of a BAC contig covering the silkworm chorion locus. Sci. Data 2:150062 doi: 10.1038/sdata.2015.62 (2015).


In recent years novel human respiratory disease agents have been described for Southeast Asia and Australia. The causative pathogens were classified as pteropine orthoreoviruses with a strong phylogenetic relationship to orthoreoviruses of bat origin.In this report, we isolated a novel Melaka-like reovirus (named “Cangyuan virus”) from intestinal content samples of one fruit bat residing in China’s Yunnan province. Phylogenetic analysis of the whole Cangyuan virus genome sequences of segments L, M and S demonstrated the genetic diversity of the Cangyuan virus. In contrast to the L and M segments, the phylogenetic trees for the S segments of Cangyuan virus demonstrated a greater degree of heterogeneity.Phylogenetic analysis indicated that the Cangyuan virus was a novel orthoreovirus and substantially different from currently known members of Pteropine orthoreovirus (PRV) species group.The online version of this article (doi:10.1186/s12866-014-0293-4) contains supplementary material, which is available to authorized users. Rhabdoviridae, Paramyxoviridae, Coronaviridae, Togaviridae, Flaviviridae, Bunyaviridae, Reoviridae, Arenaviridae, Herpesviridae, Picornaviridae, Filoviridae, Hepadnaviridae and Orthomyxoviridae [Many emerging infectious diseases are caused by zoonotic transmission, and the consequence is often unpredictable. Zoonoses have been well represented with the 2003 outbreak of severe acute respiratory syndrome (SARS) due to a novel coronavirus ,2. Bats oviridae .Reoviridae (respiratory enteric orphan viruses) comprise a large and diverse group of nonenveloped viruses containing a genome of segmented double-stranded RNA, and are taxonomically classified into 10 genera [Orthoreovirus contain a genome with 10 segments of dsRNA; 3 large (L1-L3), 3 medium (M1-M3), and 4 small (S1 to S4) [The 0 genera -13. Orth0 genera . Members1 to S4) .The discovery of Melaka and Kampar viruses, two novel fusogenic reoviruses of bat origin, marked the emergence of orthoreoviruses capable of causing acute respiratory disease in humans ,16. SubsRousettus leschenaultia) in Yunnan province, China. In the absence of targeted sequencing protocols for a novel virus, we applied the VIDISCR (Virus-Discovery-cDNA RAPD) virus discovery strategy to confirm and identify a novel Melaka-like reovirus, the “Cangyuan virus”. To track virus evolution and to provide evidence of genetic reassortment PCR sequencing was conducted on each of the 10 genome segments, and phylogenetic analysis performed to determine genetic relatedness with other bat-borne fusogenic orthoreoviruses.In the present study we isolated a novel reovirus from intestinal contents taken from one fruit bat ( Rousettus leschenaultia) were collected . After the first passage in Vero E6 cells, Cangyuan virus began to cause syncytial CPE 24 hours post-inoculation; notably earlier than for Melaka virus and other orthoreovirus  after 24 hours of the first inoculation at 37°C Figure . The virs Figure B [9,16,1Reoviridae, genus Orthoreovirus  collected from Cangyuan city were screened for anti-Cangyuan virus neutralizing antibody. According to the Neutralizing Antibody Titers determined in this study, the serum of two bats had a neutralizing antibody titer of 1,280 against Cangyuan virus and the serum of five bats  a titer of 640 against Cangyuan virus. Our studies indicated a 26% (13/50) prevalence for antibody titers >1:160 for Cangyuan virus-specific antibodies in fruit bats (Rousettus leschenaultia). The control serum had a neutralizing antibody titer <10. At present, it is not clear whether Cangyuan virus is carried by a specific fruit bat species or by multiple bat species circulating in the region. A preliminary survey of bat sera collected in Cangyuan city of Yunnan province from our previous studies indicated a low prevalence of Cangyuan virus-specific antibodies in at least two different insectivorous bat species: Rhinolophus luctus  and Small leaf-nosed bat (Hipposideros pomona) .Serum samples from 50 fruit bats  and rectal swab sample, and the Ct (Threshold Cycle) of QPCR were 19.86 ± 0.056, 19.52 ± 0.041, 19.64 ± 0.061 respectively. The Ct of another bat’s PCR were 23.07 ± 0.253, 22.53 ± 0.171 in the intestine sample and rectal swab sample, respectively.81.6% –94.2%, 83.8%–97.9%, 85.9%–97.6%  is especially interesting and may indicate two important findings ,15-17. FIn conclusion, phylogenetic analysis demonstrated that the Cangyuan virus was closely related to the Malaka and Kampar viruses and that a prevalence of Cangyuan virus-specific antibodies was present in fruit bat families circulating in Yunnan province, China. This further highlights the urgent need to systematically survey bat-borne viruses in south China so as to enable us to conduct more effective risk assessment, to provide forecast and devise better prevention and control strategies for potential future outbreaks.Rousettus leschenaultia) were collected from across eight different locations in Cangyuan city of China’s Yunnan province. All of the locations including three lychee, two mango orchards and three caverns were in close proximity to human residences. All bats were euthanized by intracardiac injection of sodium pentobarbital. Following euthanasia, serum samples were collected by cardiac puncture. The serum samples were used for neutralizing-antibody tests. Necropsies were performed directly following euthanasia. The necropsy findings demonstrated that these bats were apparently healthy and no special pathological lesions was observed (data not show). The intestine samples were used for virus isolation and the kidney, heart, lung, liver, spleen, rectal swab and brain samples were processed for PCRs testing. The tissue samples stored at -80°C.Fifty fruit bats  for 10 min at 4°C, treated with 100,000 U/ml penicillin and 100 μg/ml streptomycin, and then inoculated into Vero E6 cells for 1 hour under standard cell-culture conditions and washed the cells three times with PBS  after inoculation. The infected cells were sub-cultured three times and observed daily for the occurrence of cytopathic effects (CPE). Upon CPE in the third subcultivation, the supernatant was passaged in 80–90% confluent fresh Vero cells in a 175 cm2 flask for one week. Aliquots were stored at -80°C. One aliquot was titrated on Vero E6 cells to estimate the viral titer. After the second passage, Virus titrations were performed by end-point titration in VeroE6 cells and the TCID50/0.1 ml was calculated from 5 replicates by the method of Spearman-Karber.Virus isolation was carried out in Vero E6 cells. To refer , kohl etCangyuan virus was observed by EM. For negative staining, paraformaldehyde-fixed (2%) purified Cangyuan virus was adsorbed onto carbon-coated parlodion-filmed nickel grids and negatively stained with 2% phosphotungstic acid. Specimens were examined using a transmission electron microscope  at 80 kV.www.megasoftware.net). The Phylogenetic data have been deposited in TreeBase (Study Accession URL: http://purl.org/phylo/treebase/phylows/study/TB2:S16635). RT-QPCRs testing were repeated on the 50 fruit bats original samples including the Kidney, heart, lung, liver, spleen, intestine, rectal swab sample, and brain samples with the L2 segment primers . The reaction was performed using the One Step SYBR® PrimeScript™ PLUS RT-PCR Kit  CO., LTD) by lightcycler2.0 (Roche). To test the viral growth curve of the Cangyuan virus, the virus samples were harvested at 2, 4, 8, 12, 16, 20, 24 and 28 hours after infection and quantified with the already established RT-QPCR. To test whether the Cangyuan virus is the virus (or one of the viruses) replicating in the cells and responsible for the observed CPE, the culture supernatants 0.1 mL  was serially diluted until 10−3 and infected Vero E6 cells. After the 24 hours, the culture supernatants were analyzed by RT-QPCRs with the L2 segment primers.Culture supernatants of Cangyuan virus was analyzed by VIDISCR. Virus particles were harvested from cells by three freeze-thaw cycles and the resulting suspension purified from cell debris by low-speed centrifugation. Nucleic acids were extracted using the AxyPrep Body Fluid Viral DNA/RNA Miniprep Kit . Reverse transcription of the viral RNA was performed by using the RevertAid™ First Strand cDNASynthesis Kit . The VIDISCR assay was performed as previously described . To furtTo refer the Chua’s report , Serum s


Drosophila melanogaster. Nora virus was purified from infected flies and used to challenge mice for the production of antisera. ORF3, ORF4a, ORF4b, and ORF4c were individually cloned and expressed in E. coli; resultant recombinant proteins purified and were used to make monospecific antisera. Antisera were evaluated via Western blot against whole virus particles and Nora virus infected fly lysates. Viral purification yielded two particle types with densities of ~1.31 g/mL (empty particles) and ~1.33 g/mL (complete virions). Comparison of purified virus polypeptide composition to Nora virus infected D. melanogaster lysate showed the number of proteins in infected cell lysates is less than purified virus. Our results suggest the virion is composed of 6 polypeptides, VP3, VP4a, two forms of VP4b, and two forms of VP4c. This polypeptide composition is similar to other small RNA insect viruses.Nora virus is a single stranded RNA picorna-like virus with four open reading frames (ORFs). The coding potentials of the ORFs are not fully characterized, but ORF3 and ORF4 are believed to encode the capsid proteins  comprising the virion. To determine the polypeptide composition of Nora virus virions, polypeptides from purified virus were compared to polypeptides detected in Nora virus infected Drosophila melanogaster flies as a cell line suitable for its replication is not yet identified. D. melanogaster can be infected by a number of viruses. Some are species specific, such as Drosophila A, C, and X viruses, Nora virus, and Sigma virus. Other viruses, such as Cricket Paralysis Virus (CrPV), Flock House virus, and Invertebrate Iridescent virus, infect other species of insects, as well as D. melanogaster [ Drosophila a suitable model for virus replication studies.Nora virus is a picorna-like virus containing a single stranded positive-sense RNA genome ~12,000 bases long. It is related to a few other viruses including an isolate found in the mountain pine beetle, Lotus plant, a parasitoid wasp, and a freshwater mussel [nogaster . This ma Drosophila flies. The objective of this study was to identify the number of polypeptide species that comprise the Nora virus virion by examining the proteins made in infected D. melanogaster flies as well as from purified virus utilizing antisera made against whole virus as well as against the individual structural proteins.The Nora virus genome consists of four open reading frames (ORFs). The first three of these overlap each other in alternative reading frames. The fourth open reading frame, based on its position in the genome, appears to be independently read from the other three. Characterization of the virion suggests it is composed of 4 primary proteins, named VP3, and VP4a, VP4b, and VP4c, specified by ORF3 and ORF4 of the viral genome, respectively. The proteins produced from ORF4 are initially translated into a polyprotein and subsequently released by proteolytic processing. In addition, there are several minor protein components of the virion that appear to be derived from these primary proteins . What isSome of the most important aspects of characterizing a new virus are to determine the size, structure, and polypeptide composition of the virion. This involves a number of steps. First, a purification procedure that yields pure virus particles must be established. Second, SDS-PAGE analysis of the purified virus proteins is performed. If the virus is produced in sufficient quantity, Coomassie blue staining is adequate; if not then radioactive labeling of the viral proteins may be needed for detection. Alternatively, antisera can be produced against whole virus particles and the proteins can be detected by Western blot analysis. Antibody reagents have the additional advantage of being useful in virus detection in the infected cell. If the viral genome is sequenced, the predicted viral proteins can individually be cloned, expressed as recombinant proteins, and the recombinant proteins can be used to produce monospecific antisera. Mass spectrometry can also be performed on viral proteins purified from SDS-PAGE gels and compared back to the known nucleotide sequence of the genome to identify viral protein components , 4–7. D. melanogaster. Western blot analyses of proteins from purified virus and from infected cells indicate that the Nora virus virion is composed of 6 proteins, VP3, VP4a, two forms of VP4b, and two forms of VP4c. Additional components of the virion [Characterization of the Nora virus virion shows that two types of particles are readily made in infectede virion  are mostD. melanogaster strain witi RelE23 infected with Nora virus were a kind gift from Dan Hultmark and Jens-Ola Ekström . Infected flies were reared under standard conditions at 25°C in a 12-hour light, 12-hour diurnal cycle. The identical uninfected strain was reared under identical conditions in a separate incubator and was confirmed as uninfected by reverse transcription polymerase chain reaction [reaction . InfecteApproximately 3,000 Nora virus infected flies were placed in 10 mM Tris, pH 7.4, and 100 mM NaCl (NT buffer) and homogenized on ice with a motor-driven pestle. The resulting lysate was sonicated on ice for four 15-second pulses with 45 seconds between pulses. The debris was removed by two successive centrifugation steps at 12,000 ×g at 4°C for 20 minutes. The supernatant fractions were pooled and layered onto the top of a cesium chloride step gradient consisting of a 10 mL bottom layer of 1.5 g/mL CsCl and a 10 mL top layer of 1.2 g/mL CsCl. The gradient was spun in a SW28 rotor at 24,000 rpm for 24 h at 20°C. Following the centrifugation of the step gradient, a single virus band appeared at the interface between the two CsCl layers. The virus was recovered from the step gradient using a syringe to puncture through the side of the tube. The crude virus was centrifuged a second time by placing the CsCl solution containing the virus in an SW55 rotor at 28,000 rpm for 24 h at 20°C. Under these conditions, the CsCl forms its own gradient and the virus was separated into two distinct bands, with the virus banding at ~1.33 g/mL (lower band) and 1.31 g/mL (upper band), respectively. The virus bands were harvested independently and dialyzed against 10 mM Tris, pH 8.0, and 250 mM NaCl to remove the CsCl. The virus was stored either at 4°C or at −80°C, depending on the downstream application.μL drop of each sample collected from the gradient was placed on the grid for one minute. The excess liquid was wicked off with a piece of Whatman 50 filter paper, and the grid was allowed to air-dry for two minutes. A droplet of Nano-Van solution  was placed on the grid for one minute. The excess liquid was wicked off as above, and the grid was allowed to dry for two minutes. The grids were examined in a Tecnai G2 Transmission Electron Microscope operated at 80 Kv.Samples stored at 4°C were sent to the Electron Microscopy Core Facility at the University of Nebraska Medical Center. Grids used for electron microscopy were 200 mesh copper grids coated with formvar and silicon monoxide . A 10 Viral RNA was quantitated from upper band and lower band virus samples from the isopycnic CsCl gradients  as follo E. coli. An In-Fusion® HD Cloning Kit (Clontech) was used for cloning optimized genes ORF1, ORF3, ORF4a, ORF4b, and ORF4c, into the linearized pET28a vector and they were transformed into competent Stellar cells (Clontech) per the manufacturer's instructions. Colonies were screened by PCR and colonies testing positive for the various Nora virus genes were grown for plasmid DNA preparation using a Qiagen Plasmid preparation kit. Plasmid DNA was used to transform One Shot® BL21 (DE3) pLysS Chemically Competent E. coli cells (Invitrogen). These cells were subsequently used for expression of the Nora virus genes.Nora virus gene sequences were obtained commercially (GenScript) and were codon optimized for their expression in E. coli BL21 (DE3) cells were placed in 5 mL of LB-Kanamycin (50 μg/mL) and incubated overnight at 37°C with shaking. Flasks containing 50 mL of LB-Kanamycin broth were inoculated with 500 μL of the overnight cultures and incubated at 37°C until midexponential phase (OD600 nm = 0.5) was reached. Isopropyl β-D-thiogalactoside (IPTG) was added to a final concentration of 1.0 mM to each culture. One-milliliter samples were removed from each culture at 0 hours, 1 hour, 4 hours, and overnight following the IPTG addition. Pellets were collected by centrifugation at 13,000 rpm for three minutes at room temperature and resuspended in 200 μL distilled water, and an equal volume of 2x Laemmli sample buffer (BioRad) was added and boiled for approximately two minutes. Twenty microliter samples were run on Mini-PROTEAN® TGX™ Gels (BioRad) and stained with Coomassie brilliant blue (BioRad). For large scale protein preparations, two milliliters of overnight cultures was placed in 200 mL fresh LB-Kanamycin. Cells were incubated at 37°C until midexponential phase (OD600 nm = 0.5) and IPTG was added to a final concentration of 1.0 mM to each culture and allowed to grow for an additional 24 h at 37°C incubation.TransformedE. coli cells were collected from 200 mL cultures (see above) by centrifugation at 8,000 ×g for 10 minutes at 4°C. Supernatant fractions were removed and 10 volumes (g/mL) of lysis/wash buffer  were added to resuspend the pellets. Lysates were sonicated on ice for 15-second pulses with 35 seconds between pulses until a total amount of sonication time of 4 minutes was obtained. Lysates were centrifuged at 12,000 ×g for 10 minutes at 4°C. Supernatant fractions were removed and filtered through a sterile 0.22 μm syringe filter before placing them on the IMAC column.™ IMAC (BioRad) resin was equilibrated with 5 column volumes of equilibration/wash buffer 1 . The cartridge was washed with 6 column volumes of both wash buffer 1 and wash buffer 2 . Proteins were eluted with 10 column volumes of elution buffer  and analyzed by SDS-PAGE with Coomassie blue staining. Purified protein samples were pooled for determination of protein concentration using Pierce™ BCA Protein Assay Kit (Thermo Scientific) following kit instructions.Purification of his-tagged proteins  was performed for each protein individually through separate 1.0 mL IMAC cartridges under denaturing conditions or for all the proteins together through a 5.0 mL IMAC cartridge under denaturing conditions. A column packed with Profinity ad libitum. All mice were prebled via the retroorbital route [μL. An equal volume of Freund's complete adjuvant  was added and an emulsion was formed by passing the mixture back and forth through a double-hub needle. Two hundred microliters of the emulsion containing the virus or protein was administered to each mouse via a subcutaneous route on day one. Four weeks later, a test bleed from the mice was taken and a booster injection identical to the primary injection was given, with the exception that the inoculum was prepared using Freund's incomplete adjuvant . Every two weeks following the booster injection, additional test bleeds were taken until the titer waned. In some instances, a tertiary injection was given and additional test bleeds were taken as described.All experimental procedures were reviewed and approved by the UNK Institutional Animal Care and Use Committee (IACUC # 091311). Mouse antisera against purified virus or the individual recombinant proteins were prepared as follows. CD-1 outbred retired breeder female mice (Charles Rivers) were used for production of antisera. Mice were housed, 5 per cage, in a holding facility maintained at constant temperature (25°C) under a 12 : 12 light/dark cycle. Food and water were providedal route . Twenty ™ Gels (BioRad) as described above and subsequently transferred to nitrocellulose membranes using a semidry blot format . Following protein transfer, the membranes were blocked with blocking buffer ). The various antisera, either against whole virus or the individual proteins, were diluted in blocking buffer at dilutions varying from 1 : 500 to 1 : 2000 and incubated with the membranes for at least 1 h at room temperature or overnight at 4°C. Following incubation with the primary antibody, the membranes were washed three times for 10 minutes each with TBST. After washing, the secondary antibody  was diluted in blocking buffer at 1 : 10,000 and incubated with the membrane for at least 1 h at room temperature. The membranes were washed twice with TBS and developed using NBT/BCIP . Blot development was stopped after several minutes with 2 rinses of distilled water.Viral proteins from whole virus (upper and lower bands), recombinant proteins, or infected cell lysates were run on Mini-PROTEAN® TGXρ = 1.33 g/mL) was previously reported [ρ = 1.31 g/mL) typically contained less virus particles than the lower band as determined by protein assay. When the particles from each band were observed independently by electron microscopy, the average particle sizes were different. The lower band virus averaged a diameter of 29.7 nm (n = 47) and the upper band averaged a diameter of 31.8 nm (n = 39).As a first step to fully characterizing the protein composition of Nora virus, we modified the Nora virus purification procedure  to inclureported . The uppOne potential reason the two particle sizes were observed was the upper band virus particles represented empty capsids. This could result in a larger particle size because the RNA may assist the particles to condense upon assembly as reported for other RNA viruses . It woul ORF2, was amplified by PCR from codon optimized genes (see  pET28a linearized vector and ligated together using an In-Fusion® HD Cloning Kit (Clontech). The subsequent plasmids were purified and transformed into BL21 DE3 E. coli cells for expression. The proteins were purified from the E. coli cultures using the histidine tag added to them by sequences from the pET28a vector. The results of the purification are shown in  E. coli protein, as it was not detected by antiserum prepared against whole virus particles  that were not observed from the individual viral protein purifications. We suggest these may be complexes of individual or multiple viral proteins, as they are detected by whole virus antiserum in Western blots see . In addiThe protein composition of the upper and lower band virus particles was further characterized by preparing antisera against a mixture of these particles and subsequently performing a Western blot analysis . In the The antisera made against whole virus particles were shown to react strongly with each of the recombinant-produced proteins, with the exception of VP4b (weak reaction) and ORF1 (no detectable reaction). It is not clear why VP4b gave a weak reaction, as the monospecific sera made against VP4b react very strongly with purified virus . This ma D. melanogaster infected (Dm-I) or uninfected (Dm-U) cell lysates. In each case, the recombinant proteins migrate at a larger size than those from purified virus or infected cells do. This is due to the presence of a HIS-tag on the recombinant proteins. The exception to this is VP4b. The protein from infected cells and purified virus migrated at a larger size than the rVP4b. We suggest VP4b may be posttranslationally modified in some as yet uncharacterized manner, increasing the predicted molecular weight of the protein.As a final step to characterize the protein composition of the two types of Nora virus particles, antisera were prepared against each of the recombinant proteins . Western blots were developed with each of the antisera . The samORF1 monospecific antiserum did not detect proteins in purified virus particles but did detect a single protein species in infected cells (data not shown). This result was consistent with previous reports of Nora virus virion protein composition [ D. melanogaster Nora virus. Most insect RNA virus virions are composed of relatively few proteins. For example, CrPV is composed of 4 capsid proteins of molecular weights 33, 31, 30, and 8 kDa [ Drosophila C virions consist of 5 viral proteins, again in the size range of CrPV [ ORF3 and the other 3 are derived from ORF4 via proteolytic cleavage of a polyprotein precursor [An initial step necessary to understand the function of the protein components of a virus is to determine precisely what proteins are virion components and what proteins specified by the genome are nonstructural proteins. The results of this study have clarified this for thend 8 kDa . Drosoph of CrPV . Many ot of CrPV ). The pr of CrPV  suggestsThe purification procedure for Nora virus was modified from that previously reported  to incluThe production of antisera against whole virus and against the individual viral proteins was used to determine the protein composition of the Nora virus particles. Antibodies against purified virus did not detect ORF1 in virus particles . We the C virus . However


Caenorhabditis elegans, more than 90% of the shape variance can be captured using just four principal components. However, it remains unclear if other methods can achieve a more compact representation or contribute further biological insight to worm locomotion. Here we take a data-driven approach to worm shape analysis using independent component analysis (ICA), non-negative matrix factorization (NMF), a cosine series, and jPCA  and confirm that the dimensionality of worm shape space is close to four. Projecting worm shapes onto the bases derived using each method gives interpretable features ranging from head movements to tail oscillation. We use these as a comparison method to find differences between the wild type N2 worms and various mutants. For example, we find that the neuropeptide mutant nlp-1(ok1469) has an exaggerated head movement suggesting a mode of action for the previously described increased turning rate. The different bases provide complementary views of worm behavior and we expect that closer examination of the time series of projected amplitudes will lead to new results in the future.High-throughput analysis of animal behavior is increasingly common following the advances of recording technology, leading to large high-dimensional data sets. This dimensionality can sometimes be reduced while still retaining relevant information. In the case of the nematode worm  Caenorhabditis elegans  has shown that the effective dimensionality of worm locomotion is low, as there are correlations between bends along different parts of the body  and NMF. To obtain the variance of the basis shapes, we resampled the same collection 100 times obtaining 3000 N2 shapes each time. For analysis, a testing set of 3000 N2 shapes was projected onto each basis shape to retrieve the corresponding amplitudes. To ensure that all of the mutants were represented in the test between PCA and the sinusoidal basis shapes, we sampled one shape from each of the 9964 recordings in the dataset. Each worm shape was reconstructed using either four principal components or the sinusoidal basis and the squared difference between the reconstructed and original shapes were determined in each case. PCA and NMF were conducted using built-in functions of MATLAB, while freely available methods were used for ICA  to obtain the projected amplitudes for each worm at each time-point. The projected amplitudes have arbitrary units because the basis shapes are normalized. We then took the mean absolute value of each projected amplitude as a simple feature characterizing each worm’s average shape. For jPCA, we measured the mean amplitude of the anterior oscillation in each individual, i.e.,1 and A2 are the projections onto the first two eigenshapes, and t is the frame number. These were compared between each genotype and the wild type (N2) using a Mann-Whitney U-test. Bonferroni correction was used to control for multiple comparisons.where A(snf-6)eg28, VC1295 egg-5(ok1781) and RB1340 nlp-1(ok1469). Induced reversal experiments were carried out as described in Alkema et al. .As previously described  across all 335 genotypes in the database using the basis shapes derived from the training set of wild type N2 shapes. This set of basis shapes captures 97.6% of the variance in N2 and 97.1% in mutants. At least one feature was significantly different compared to the wild type N2 strain in 172 genotypes , the difference is small (the top four PCA components capture 97.1% of the variance) and may be negligible for some applications. Using a set of analytically defined modes may prove useful in theoretical applications.Data-driven dimensionality reduction methods are inherently dependent on the dataset used to train them, meaning that the basis shapes produced will be different if a different training set is used. If the training set is large enough, variation will be small, but if only a small number of trajectories are available for a given condition then the derived shapes could vary significantly from sample to sample. Using a set of pre-determined basis shapes would avoid this issue, but to be useful they must explain most of the shape space variance across different individuals. Given the sinuous set of basis shapes derived using both PCA and ICA, we defined a Fourier cosine series as a set of basis shapes and tested if it could capture worm shapes compactly Figures . The firThe methods considered above are time independent: they only take into account the distribution of shapes. In contrast, jPCA uses time series trajectories of worm motion, maps the shape space with PCA and then reorients these components to identify components that show strong oscillations  mutants, which have been reported to maintain their head tip oscillation during touch-evoked reversals  touch-evoked reversals, but we do find that the magnitude of the oscillation is lower in tdc-1 during spontaneous forward locomotion. This suggests that the jPCA anterior oscillation is not the same as the small oscillation that worms exhibit at the very tip of their heads. Despite this, the jPCA anterior oscillation does show a difference between spontaneous and touch-evoked reversals: both wild type and tdc-1 worms show a stronger anterior body oscillation during touch-evoked reversals  significantly different compared to wild type N2 in the size of the anterior oscillation. Two examples are shown in Figure s Figure . FinallyWe used four different dimensionality reduction methods to obtain a number of new features that can be used to describe different groups of worms. The new features are straightforward to use and show interpretable differences between mutants.C. elegans shape space compared to PCA, confirming the previous lower-bound dimensionality of four for the worm shape space  that are influenced by food availability and nutritional status (You et al., Behavior is a dynamic process often involving shifts between different states, single events and cyclic episodes. The amplitudes of the shapes projected onto the different bases also change over time, but this was not taken into account when we interrogated the database. Instead, we used the magnitude averaged over the entire recording that reflects the general shape of the worm, which was sufficient to detect many significant differences. However, thorough time series analysis would likely reveal more details about the locomotion trajectories. Oscillations are ubiquitous in all four bases, but each feature also has a rich dynamical profile with different properties and comparison between these has the potential to provide different and complementary information. One example could be the characterization of the spontaneous switch between the feeding states of the worm. Worm behavior has often been described using states defined by the experimenter. Using recording equipment and automated feature extraction was initially conceived to help with the process of group assignment and definition (de Bono and Bargmann, BG performed data analysis, BG and AEXB conceived the research and wrote the manuscript.Funding for this work was provided by the Medical Research Council through grant MC-A658-5TY30 to AEXB.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


It is commonly accepted that there are many unknown viruses on the planet. For the known viruses, do we know their prevalence, even in our experimental systems? Here we report a virus survey using recently published small (s)RNA sequencing datasets. The sRNA reads were assembled and contigs were screened for virus homologues against the NCBI nucleotide (nt) database using the BLASTn program. To our surprise, approximately 30% (28 out of 94) of publications had highly scored viral sequences in their datasets. Among them, only two publications reported virus infections. Though viral vectors were used in some of the publications, virus sequences without any identifiable source appeared in more than 20 publications. By determining the distributions of viral reads and the antiviral RNA interference (RNAi) pathways using the sRNA profiles, we showed evidence that many of the viruses identified were indeed infecting and generated host RNAi responses. As virus infections affect many aspects of host molecular biology and metabolism, the presence and impact of viruses needs to be actively investigated in experimental systems. Viruses infect all prokaryotic and eukaryotic species and are one of the major sources of disease-causing agents. However, our knowledge of the world’s virosphere and viromes in ecosystems is still very limited New technologies, particularly those associated with Next Generation Sequencing (NGS), now offer powerful tools to detect the presence of viruses in any biological samples. For example, metagenomics protocols have been successful on making virus survey and discovery in many case studies To assess the presence of viruses and infections in experimental systems, we used sRNA datasets of 94 recently published papers of 10 model species to search for known viruses by homology based BLAST  program http://www.ncbi.nlm.nih.gov/gds/advanced) of the NCBI http://www.nchi.nlm.nib.gov/geo) Database  OR short RNA) OR sRNA) AND “high throughput sequencing” [Platform Technology Type]) AND model organism [Organism]. Only files with size <2 GB were downloaded and analysed. All datasets used were associated with papers published before 17 April, 2013. The NCBI accession numbers of all data libraries used are provided in supplementary files (http://www.ncrna.org/frnadb/), a database for comprehensive non-coding RNA sequences To identify and download sRNA sequence libraries, we used the advanced search function (Database . The smary files . The seqhttp://soap.genomics.org.cn/SOAPdenovo-Trans.html) and Velvet http://www.ebi.ac.uk/~zerbino/velvet/) programs with different series of kmer parameters . The outputs from SOAPdenovo-trans and Velvet assembly were re-assembled once again using the Velvet de novo assembly program, using combined kmer series . The assembled contigs (≥50 nt in length) were extracted using an in-house Perl script. Finally, contigs were filtered for redundancy using the CD-HIT program https://code.google.com/p/cdhit/downloads/list).Small RNA sequences from each individual library were assembled using the SOAPdenovo-trans  was used to map all reads back to each contig with parameters as “-N 0 -L 16 -i S,1,0.75–local -a -I 16”. The output file was analyzed by the Samtools software package http://samtools.sourceforge.net), the BEDtools http://code.google.com/p/bedtools) program and in-house Perl scripts to calculate the read coverage of each contigs. Only contigs with >95% read coverage were used for further analyses.To validate the assembled contigs, the Bowtie 2 program ftp://ftp.ncbi.nlm.nih.gov/blast/db/FASTA/) using the BLASTn program The assembled contigs were used as query sequences to screen against a local copy of the NCBI nucleotide (nt) database  to the detected viral contigs using Bowtie2, Samtools and BEDtools programs. Read counts were converted to counts per million (CPM). Size distribution of virus-specific reads of each overlooked virus from each sample library was calculated using Perl scripts and represented into heatmap using R 2.15.1 program. This analysis was used to identify the RNAi pathways responsible for the production of the identified vsiRNAs. To determine pathway variation, χTo support the results of vsRNA profile, the Bowtie 2 program was used to map all reads of each sample to the virus genomes without any mismatch. The output file was processed by in-house Perl scripts and the R 2.15.1 program to display the vsRNA positions on virus genomes.http://www.nchi.nlm.nib.gov/geo) database. The short reads were assembled for each library and in total, 4,195,253 contigs ≥50 nt were obtained. To avoid possible assembly artefacts, only contigs with greater than 95% coverage by the original sequence reads were used in this work. The contigs were screened against the NCBI NT database using the Standard Nucleotide BLAST (BLASTn) program. If a contig had the highest BLAST score against a viral sequence with a minimum 80% identify and 95% of the contig length was matched to the subject viral sequence, the contig was deemed as an identifiable virus hit. In total, 461 contigs  was positive for a plant virus  associated with Oryza sativa, two vsRNA phenotypes were observed, i.e., 21 & 22-nt domination vs 24-nt domination  were calculated and are represented in D. melanogaster  samples  was detected in sRNA libraries associated with Arabidopsis. In particular, a few thousand reads were matched to the viral contigs in libraries GSM889279 and GSM889268  sequences were detected in both Arabidopsis (T. aestivum)  . The vsRMetagenomics strategies based on the NGS technology provide powerful tools for making virus discoveries e.g., . Among tThis study also demonstrated the feasibility of screening virus infections using the sequencing datasets produced from experiments not designed for virology studies. By assembly of sequence reads followed by standard BLAST screening, virus hits can be readily detected. Although assessing whether or not an unexpected virus infection may affect the quality of an experiment and the interpretation of results must be decided on a case-by-case basis, knowing that there may be a viral factor involved should generally be considered an improvement to the overall experiment. From a virology point of view, screening for viruses will help to extend our knowledge of the virus-host range, and to understand the host antiviral RNAi and PTGS immunity if sRNA libraries are used. Information about the presence of viruses would also be useful in a broader context because changes to the sRNA population may affect host metabolism and development Figure S1Flow chart of bioinformatics procedure.(TIFF)Click here for additional data file.Figure S2Distribution of viral contigs mapped to the virus genomes. Each viral genome fragment was shown as a black bar and each viral contig was represented as a green bar.(TIFF)Click here for additional data file.Figure S3Positional distribution of viral reads showing vsRNA coverage on the virus genome. Each viral genome fragment was shown as a black bar and the viral reads were represented as red dots.(TIF)Click here for additional data file.Table S1Small RNA libraries used and viruses detected.(XLS)Click here for additional data file.Table S2BLASTn results of viral contigs mapped to the virus reference genomes.(XLS)Click here for additional data file.Text S1Viral contig sequences in the Fasta format.(XLSX)Click here for additional data file.


CASP1-mutations with incomplete penetrance. These CASP1-variants cause reduced enzymatic activity of procaspase-1 and less IL-1ß secretion.Caspase-1  plays an important role in mediating proinflammatory innate immune responses, especially by activation of pro-IL-1ß within inflammasomes. Some patients with recurrent febrile episodes and systemic inflammation of yet unknown origin harbor CASP1-variants have different protein interaction clusters and thus enhance alternative signaling pathways.The paradox of reduced IL-1β secretion but increased inflammation led to the hypothesis, that We established an in vitro model of transduced immortalized murine macrophages, expressing either wild type (WT) or enzymatically inactive (C284A) procaspase-1 fusion-reporter proteins and characterized them after NLRP3-inflammasome stimulation.As expected, variant procaspase-1 (C284A) macrophages did not secret IL-1ß and pyroptosis was reduced. In addition, the usage of fluorophore-tagged fusion proteins revealed a longer and more intense interaction of the enzymatically inactive procaspase-1 (C284A) with ASC (apoptosis-associated speck-like protein containing a CARD) compared to WT. Variant procaspase-1 (C284A) and ASC formed macromolecular complexes in the cytosol , that were significantly larger than those formed in cells expressing fluorophore-tagged WT procaspase-1. We could confirm our results by adding the caspase-1 inhibitor YVAD-CMK to Casp1-WT macrophages: the pyroptosomes became larger, more intense and more stable over time. Furthermore, life-cell-imaging detected for the first time, that pyroptosomes of enzymatically inactive procaspase-1 were spread by cell division.Variant procaspase-1 stabilizes inflammasome/pyroptosome formation. This may enhance inflammation via two IL-1ß-independent mechanisms: The pyroptosome causes a proinflammatory stimulus through increased recruitment and interaction of further proinflammatory proteins . Moreover, this stimulus might be amplified via pyroptosome- and cell division.


Molecular identification and genetic diversity of open reading frame 7 (ORF7) of field isolated porcine reproductive and respiratory syndrome virus (PRRSV) in North Sumatera, Indonesia, in the period of 2008-2014.A total of 47 PRRSV samples were collected from the death case of pigs. The samples were collected from different districts in the period of 2008-2014 from North Sumatera province. Two pairs of primer were designed to amplify ORF7 of Type 1 and 2 PRRSV based on the sequence of reference viruses VR2332 and Lelystad. Viral RNAs were extracted from samples using PureLink™ micro-to-Midi total RNA purification system (Invitrogen). To amplify the ORF7 of PRRSV, the synthesis cDNA and DNA amplification were performed by reverse transcription polymerase chain reaction (RT-PCR) and nested PCR method. Then the DNA sequencing of PCR products and phylogenetic analysis were accomplished by molecular evolutionary genetics analysis version 6.0 software program.RT-: PCR and nested PCR used in this study had successfully detected of 18 samples positive PRRS virus with the amplification products at 703bp and 508bp, respectively. Sequencing of the ORF7 shows that 18 PRRS viruses isolated from North Sumatera belonged to North American (NA). JXA1 Like and classic NA type viruses. Several mutations were detected, particularly in the area of nuclear localization signal (NLS1) and in NLS2. In the local viruses, which were related closed to JXA1 virus; there are two differences in amino acids in position 12 and 43 of ORF7. Our tested viruses showed that the amino acid positions 12 and 43 are Asparagine and Arginine, while the reference virus  occupied both by Lysine. Based on differences in two amino acids at position 12 and 43 showed that viruses from North Sumatera has its own uniqueness and related closed to highly pathogenic PRRS (HP-PRRS) virus (JXA1).The results demonstrated that North Sumatera type PRRS virus has caused PRRS outbreaks in pig in North Sumatera between 2008 and 2014. The JAX1 like viruses had unique amino acid residue in position 12 and 43 of asparagine and lysine, and these were genetic determinants of North Sumatera viruses compared to other PRRS viruses. Porcine reproductive and respiratory syndrome virus (PRRSV) is an infectious disease in pigs which is caused by a virus and brings about an economic loss in pig breeding industry . The firIn 2006, PRRS virus became the important disease in China because of its high morbidity and mortality, which caused the death of millions of pigs. The result of the analysis on clinical symptoms, pathogenicity, and molecular virus showed that this virus was very different from classic PRRS virus so that this virus was then known as highly pathogenic PRRS (HP-PRRS) ,5. HP-PROpen reading frame 7 (ORF7) had 123-128 amino acid which consists of N-terminal RNA binding domain and C-terminal dimerization domain . The ORFIn Indonesia, PRRS disease has not been known by many people. PRRS virus infection occurred in North Sumatera in 2008, but official information about this disease was difficult to obtain. The limitation of the data of the dynamic of PRRS disease in Indonesia had encouraged the researcher to conduct a study on it. In the present study, we determined the complete ORF7 sequences of 18 PRRS viruses from North Sumatera Province. The aims of this study were (1) To find out the genetic database for the ORF7 gene in North Sumatera PRRSV viruses; (2) to compare North Sumatera PRRSV ORF7 sequences with the ORF7 sequences of the reference viruses, (3) to determine the phylogenetic relationships between North Sumatera PRRSVs and reference viruses.This research was conducted after approval by institutional research committe of Gadjah Mada University.A total of 47 samples were collected from the death case of pigs. The samples were collected from different districts in the period of 2008-2014 in North Sumatera.http://primer3.ut.ee/) was used to help in designing the primers for this study. The specific primers ORFF-5’-GCCCTAATTGACTAGGTGACT-‘3 and ORFR-5’-GCCCTAATGACTAGGTGACT-‘3, were designed on the ORF6 and 3’UTR sequences to generate a 703 bp fragment including the whole ORF7 sequence. The second PCR was performed using the primers described by Guarino [In this study, two pairs of primer which was able to amplify ORF7 in Type 1 and 2 PRRS viruses. The two pairs were designed by using isolate reference VR2332, Lelystad, and also used multi alignment from the sequences of the two virus types, which were available in GenBank data. Primer3 software  according to the instructions of the manufacturer. In RT, we used of SuperScript III First-Strand Synthesis (Invitrogen). RT contained the following ingredients: 5 µl of RNA, 1-µl (50 µM) primer oligo (dT) 20, 1-µl (10 mM) dNTP, and 4 µl diethylpyrocarbonate. The RNA was denatured at 65°C for 5 min and then cooled on ice. Master mix consisting of 10 µl cDNA synthesis mix was added at 10 µl sample and then incubated in a thermal cycler at 50°C for 50 min, 85°C for 5 min, and then stored at −20°C.® PCR SuperMix. The PCRs were run as follows, pre-denaturation at 95°C for 2 min, and 40 cycles of denaturation at 95°C for 45 s, annealing at 50°C for 1-min, extension at 72°C for 45 s, and final extension at 72°C for 5 min.The PCRs contained the following ingredients, 2 µl sample, 2 µl (20 µM) of forward primer, 2 µl (20 µM) of the reverse primer, and 45 µl PlatinumFor nested PCR, we used KAPA2G™ Fast Ready Mix 2X (KapaBiosystems). The PCRs contained the following ingredients, 12.5 µl of 2x KAPA2G Fast Ready Mix, 1.25 µl (10 µM) of forward primer, 1.25 µl (10 µM) of reverse primer, 5 µl of PCR product, and the addition of RNA-se free water until 25 µl. The PCRs were run as follows, pre-denaturation at 95°C for 2 min, and 40 cycles of denaturation at 95°C for 20 s, annealing at 49°C for 20 s, extension at 72°C for 30 s, and final extension at 72°C for 3 min.® 3700 DNA Analyzer. All products were sequenced in both directions. Identification of the resulting nucleotide sequence of ORF7 in local viruses was tested with BLAST in order to assure the specificity of the PCR. Multiple nucleotide ORF7 virus sequence was aligned by using Clustal W and compared with some isolate references .PCR products were sequenced using the Big Dyes TN kit  following the manufactories instructions, run, and analyses on an ABI PRISMTo analyze the ORF7 genes of isolates from North Sumatera, Indonesia, Phylogenetic reconstructions were generated using the neighbor-joining method by the computer program Molecular Evolutionary Genetics Analysis Version 6 based on the formulas of Kimura 2-parameter. The robustness of the phylogenetic analysis was determined by bootstrap analysis with 1000 replications.About 10 of 47 samples were detected PRRSV-positive using external primers with the amplicon size of 703bp. Of the 37 samples, which found negative by the external primer, 8 were PRRSV-positive by nested PCR using P71F and P71R primers with the amplicon size of 508bp. The result of amplicons ORF7 from Two-Step PCR and nested PCR through gel electrophoresis could be seen in In this study, there were 18 complete sequences of ORF7, which came from 9 districts in North Sumatera. The specific primers ORFF and ORFR were designed to amplify gene target, which flanks from the partial of ORF6 to 3’UTR as long as 703 bp of DNA fragment, this flanking region including the whole of ORF7 sequence. The whole ORF7 of the North Sumatera PRRSV sequences have the same nucleotides length in size of 369nt, which encoded 123 amino acids. There was no deletion and insertion in all local virus sequences. The nucleotide analysis indicated several points of silent mutations or missense mutations, compared with classic PRRS viruses  and HP-PRRS virus (JXA1).N protein of PRRS virus has been recognized to have some areas . The areN protein in PRRS virus had multifunction ; one of N protein in Type 2 virus had amino acid as binding domain place with fibrillarin in the position of amino acid 30-37 (IAQQNQSR). The result of the sequence of all local viruses, which indicated domain binding position with fibrillarin had conserved site . The intPhylogenetic analysis demonstrated that the 18 North Sumatera PRRS viruses were divided into two groups . The firThe second group was considered of viruses that clustered in the same lineage with the VR2332 virus. The virus shared 93.0-100% nucleotide similarities in the ORF7 and 93.4-100% in the deduced amino acid sequences compared with VR 2332 virus. This indicates that Type 2 PRRS virus had spread widely in North Sumatera Province. The sequences of ORF7 compared with modified live vaccine (MLV) virus, showed 93.0-100% similarity in both nucleotide and amino acid sequences. High phylogenetic relatedness was found among the one virus (11.363.3 Medan) belonging to the second group in this study. The similarity with comparison with MLV virus was about 100% in both nucleotide and amino acid. This virus was a possibility that the vaccine virus has been circulating in North Sumatera.The existence of North Sumatera viruses was related close to classic virus VR2332 and MLV, which indicated that there was the possibility for the virus which came from vaccine had caused infection in breeding pigs in North Sumatera. The implementation of vaccination, which was not in line with the rule or vaccinating sick animals, would bring about sickness in breeding pigs, which would eventually the incidence of PRRS case occurrence in pigpens and spread to other pigpens. Geographically, Nias District was bordered with the Indian Ocean and the mainland of Sumatera, but virus, which came from Nias and several other local viruses were close to the vaccine virus. Another assumption about the existence of the classic virus was that PRRS virus had long settled in North Sumatera, along with the case of HP-PRRS China virus which spread to Southeast Asia, including North Sumatera. At the same time, the classic virus was also able to infect pigs, which brought about sickness. In order to know more about this case, it was necessary to study epidemiology deeply so that this case could be solved properly.After analyzing local viruses which were found in North Sumatera, there were some important things, which could be concluded. First, PRRS virus, which spread in North Sumatera was included in Type 2 PRRS virus (NA); second, in its development, this virus was divided into two groups: Virus group which was close to HP-PRRS virus and virus group which was close to PRRS classic virus; and third, in the HP-PRRS virus group there were amino acids 12N and 43R which could be used as the marker of PRRS virus which came from North Sumatera. The two amino acids were not found in other virus references and HP-PRRS China viruses. Further profound studies in various aspects could be conducted on these local viruses, particularly the function of amino acids 12N and 43R as one of virus virulence factors.FF was responsible for the collection of samples ADIC Medan at 2008-2014. AH did the PCR identification and RW phylogenic analysis. CRT drafted and revised the manuscript. All authors read and approved the final manuscript.


In recent years, a number of serious disease outbreaks caused by viruses and viroids on greenhouse tomatoes in North America have resulted in significant economic losses to growers. The objectives of this study were to evaluate the effectiveness of commercial disinfectants against mechanical transmission of these pathogens, and to select disinfectants with broad spectrum reactivity to control general virus and viroid diseases in greenhouse tomato production.Pepino mosaic virus (PepMV), Potato spindle tuber viroid (PSTVd), Tomato mosaic virus (ToMV), and Tobacco mosaic virus (TMV). The efficacy of each disinfectant to deactivate the pathogen’s infectivity was evaluated in replicate experiments from at least three independent experiments. Any infectivity that remained in the treated solutions was assessed through bioassays on susceptible tomato plants through mechanical inoculation using inocula that had been exposed with the individual disinfectant for three short time periods . A positive infection on the inoculated plant was determined through symptom observation and confirmed with enzyme-linked immunosorbent assay  and real-time reverse transcription-PCR (PSTVd). Experimental data were analyzed using Logistic regression and the Bayesian methodology.A total of 16 disinfectants were evaluated against Statistical analyses using logistic regression and the Bayesian methodology indicated that two disinfectants (2% Virkon S and 10% Clorox regular bleach) were the most effective to prevent transmission of PepMV, PSTVd, ToMV, and TMV from mechanical inoculation. Lysol all-purpose cleaner (50%) and nonfat dry milk (20%) were also effective against ToMV and TMV, but with only partial effects for PepMV and PSTVd.With the broad spectrum efficacy against three common viruses and a viroid, several disinfectants, including 2% Virkon S, 10% Clorox regular bleach and 20% nonfat dry milk, are recommend to greenhouse facilities for consideration to prevent general virus and viroid infection on tomato plants. Solanum lycopersicum L.) is one of the world’s most economically important vegetables. According to the FAO statistics [Tomato , in the genus Potexvirus and family Alphaflexiviridae [Solanum muricatum) from Peru in 1980 [In the last decade, one of the most widespread viruses in greenhouse tomato productions in Europe and North America has been iviridae ,6. PepMV in 1980 . It was  in 1980 , America in 1980 –21, and  in 1980 –24.Potato spindle tuber viroid (PSTVd), Tomato chlorotic dwarf viroid (TCDVd), Mexican papita viroid (MPVd), Citrus exocortis viroid (CEVd), Columnea latent viroid (CLVd), and Tomato apical stunt viroid (TASVd). Symptoms incited by these pospiviroids on tomatoes include plant stunting, leaf chlorosis and necrosis, with smaller size fruits to no fruit at all.In recent years, viroid disease outbreaks on tomatoes have been reported frequently both in Europe ,26 and NVirgaviridae, Tomato mosaic virus (ToMV) and Tobacco mosaic virus (TMV), are frequently observed in greenhouse tomatoes [In addition to the endemic PepMV and the emerging pospiviroids, two tobamoviruses in the family tomatoes .Grafting is a common practice in greenhouse tomato seedling production. Contaminated tomato seeds used as scion and/or rootstock for grafting could also be an important source of initial virus inoculum ,32. OnceIn recent years, several studies conducted to identify disinfectants that reduce the infectivity of plant viruses and viroids have been reported for ornamental plants –35, cucuIn experiments to assess the virus infectivity remained in the treated solutions, two different inoculation methods were applied, cut- and rub-inoculation. In the first two cut-inoculation experiments for PepMV, since expression of typical disease symptoms (mosaic) from PepMV-infected tomato plants was not obviously visible, ELISA was used to determine the PepMV infection. Preliminary data indicated that there was a significant delay in the onset of PepMV infection from the cut-inoculated plants because extra time was needed to regenerate side shoots on the test plants after the primary shoot was removed by cut-inoculation. It took approximately 5 weeks post inoculation for PepMV to be detectable by ELISA achieving a 100% infection rate in plants from the positive control. In comparison, it typically took approximately only 1–3 weeks for PepMV to be detectable by ELISA in rub-inoculated plants. Such delay in the onset of PepMV infection to test plants through cut-inoculation hindered evaluation efficiency. In addition, the first two cut-inoculation experiments generated a higher infection rate (>50%) on test plants from all disinfectant solutions  completely deactivated the PepMV infection. Only 1 or 2 in a total of 9 test plants tested positive from disinfectant solutions in nonfat dry milk, Trisodium phosphate, Lysol all-purpose cleaner, or Clorox regular bleach. The data from experiments 3 and 4 were similar , the impact of application rate to TMV infection were observed  were examined against TMV infection. Each treatment was performed through mixing an equal volume of TMV inoculum with a 2X stock solution of a respective disinfectant. In the case of Clorox regular bleach, the 2X stock solution (20% Clorox regular bleach) was stored at room temperature 20-30°C) for 30 days. Virkon S, 4% stock solution (2X) was prepared and stored for two time periods, 10 days and 30 days. At each storage time point, treatments were carried out by mixing an equal volume of a prepared virus inoculum with a 2X stock solution for 30 seconds just before used for a bioassay. Results showed that in comparison to that of a freshly prepared solution, the ability to deactivate TMV infectivity of Clorox regular bleach in storage for over 30 days was equally effective  and one viroid (PSTVd) through mechanical inoculation on tomato seedlings. This study demonstrated consistently the broad spectrum effects from two common disinfectants (2% Virkon S and 10% Clorox regular bleach) in deactivating the infectivity of PepMV, PSTVd, ToMV, and TMV  in treatment of inocula by disinfectants were selected to mimic the short time intervals in cutting tissues between plants during tomato crop work in a greenhouse, such as grafting, deleafing, or fruit harvesting. However, there were no significant differential responses observed among the three time intervals. These results suggested that the effects of those effective disinfectants against virus and viroid infectivity were immediate upon mixing and exposure. With such swift action from the effective disinfectants, there is no need to wait after dipping for a disinfectant to achieve its full function.Growers are concerned about how long a diluted disinfectant could be stored in a greenhouse without losing its efficacy. Our tests demonstrated that there was no major reduction in the effect of a disinfectant in deactivating TMV infectivity when those ready-to-use solutions of Virkon S and Clorox regular bleach were stored for more than 30 days. It is not surprising given the long shelf life of these highly stable products. However, caution should be considered when using disinfectants with high amount of organic matter, such as a large quantity of leaf sap, which could compromise the disinfectant activity .Cymbidium mosaic virus (CyMV) on a local lesion host and commercial bleach inactivated both CyMV and Odontoglossum ringspot virus (ORSV) at 10% or 20% concentration. Kamenova and Adkins [Hibiscus latent Fort Pierce virus infection in hibiscus. In searching for effective disinfectants in ornamental production to control TMV transmission during propagation, Lewandowski et al. [Tomato chlorotic dwarf viroid (TCDVd)-contaminated scalpels. Wintermantel [Results obtained in screening of 16 disinfectants against three viruses in tomato were consistent with some conclusions achieved in previous studies from other plant-virus systems. Hu et al. (33) found that undiluted skim milk inactivated d Adkins  reportedi et al.  discoveri et al.  examinedi et al.  found thermantel  demonstrermantel .With their broad spectrum effects against infection on treated tomato plants for three viruses and a viroid, 2% Virkon S, 10% Clorox regular bleach and 20% nonfat dry milk could be recommended to greenhouse vegetable growers to protect plants from virus infection in various crop work activities, including grafting, deleafing, and fruit harvesting. A suitable disinfectant for greenhouse tomato production should satisfy several criteria, including: short contact time, broad efficacy against viruses and viroids or even other plant pathogens, safe for workers, not corrosive to infrastructure, not phytotoxic to plants, and economic. Although Virkon S is relatively expensive and also corrosive, it is the most promising disinfectant and has already been proven effective against human and animal viral pathogens ,41. ClorThe PepMV isolate (TX10-01) was isolated from a diseased tomato plant collected in Texas in 2010 and determined by sequence analysis to be genotype CH2 . ToMV  were provided by Monsanto. Experiments were conducted in a greenhouse . For each replicated experiment (one virus or a viroid treated with 16 disinfectants and controls), over 220 seeds were individually sowed in 5-inch pots filled with soil-less potting mix  in a greenhouse. The test plants were maintained in each individual pot on trays to keep them separated in space to prevent potential cross contamination. Normal plant maintenance activities, including daily watering, weekly fertilizing and occasional spraying to control insects, were performed.Certified healthy tomato seeds  using a Homex-6 tissue homogenizer . PSTVd inoculum was prepared using the above listed saline phosphate buffer or 1× TE buffer . The freshly prepared virus or viroid inoculum was maintained on ice prior to use.The virus and viroid sources were maintained on tomato plants in individual insect-proof domes in a greenhouse. Virus inoculum was prepared by grinding symptomatic tomato leaves (1:5 w/v) in a plastic bag containing saline phosphate buffer, pH 7.0  prior to use. Equal volumes (0.3 ml) of the prepared disinfectant stock solution and virus/viroid inoculum were mixed in a 1.5 mL Eppendorf tube. Each disinfectant and inoculation combination was left at room temperature for 0–10 sec, 30 sec or 60 sec prior to use for inoculation.For PepMV only, the first 2 experiments were done using cut-inoculation, which simulated to the deleafing process. The cut-inoculation was done by dipping a clean surgical blade to the treated solutions as stated above and cut off a leaf or a shoot on 3 tomato seedlings at each exposure time intervals .Rub-inoculation was conducted by rubbing gently with a cotton-swab (Q-tip) soaked in the treated solution onto 3 tomato seedlings lightly dusted with Carborundum  at 2–3 leaf stage. Each treated inoculum was inoculated on 3 tomato seedlings for each exposure time period . In each experiment, 3 plants at each time point were inoculated with the same virus or viroid inoculum that were mixed with an equal volume of water as positive controls. Same number of mock-inoculated plants with the inoculation buffer were served as negative controls. The entire process from inoculum preparation to the completion of an inoculation experiment took 2–3 hours. As a control, the infectivity of a virus inoculum that remained in the solution at the end of each experiment was tested by inoculating 3 additional tomato seedlings to confirm symptom expression. The virus/disinfectant solution was washed off gently with tap water from the inoculated plants after application.Tests on PSTVd, ToMV and TMV, consisted of 3 repeat experiments and were done using the rub-inoculation method. For PepMV, 2 experiments were done with the cut-inoculation method and 2 experiments were done with the rub-inoculation method. The data from two inoculation methods were compared for PepMV. The inoculated plants were randomized on a bench and maintained in a greenhouse for 4–6 weeks post inoculation for symptom observation. ELISA for virus detection and qRT-PCR for PSTVd were performed as described in the following to confirm each virus or viroid infection. The experimental plant materials were bagged, autoclaved, and disposed of after completion of each experiment. For each pathogen, the experiment to each disinfectant was repeated for 3 times. Due to the limitation in greenhouse space and to prevent potential cross contamination, only plants inoculated by the same type of pathogen at each time were placed in the same greenhouse. The entire experiments were carried out from April 2012 to June 2013 in an environmental controlled greenhouse.To assess the efficacy of selected disinfectants in storage, 2X stock solutions of Clorox regular bleach (20%) were stored at room temperature (25°C) for 30 days and then used to treat the TMV inoculum with an exposure time for 30 sec. In comparison, a freshly prepared 20% Clorox regular bleach was used as a control and a non-treated TMV inoculum was used to assess the virus infectivity in the inoculum. For Virkon S, the 2X stock solution (4%) was assessed after storage for 10 to 30 days, in comparison with a freshly prepared stock solution to treat the TMV inoculum with a 30 sec exposure. A non-treated TMV inoculum was included as a positive control. Preparation and maintenance of plants for inoculation and the method on rub-inoculation were described as above.To assess virus infection on the test plants for PepMV, TMV, and ToMV, a standard ELISA method was used following the manufacturer’s instructions  with minor modifications. For sample processing, a small leaf tissue (ca. 150 mg) was collected from individual inoculated plant in a plastic bag. After addition of 2.0 ml 1X tissue extraction buffer   into each bag, the tissue was grounded thoroughly with a Homex-6 homogenizer . The ELISA plate was pre-coated with 100 μl of primary antibody for PepMV , ToMV  or TMV  in suitable dilution in the coating buffer overnight at 4°C. After washing with 1x PBS-Tween buffer for 3–4 times, the above prepared leaf crude extract (100 μl) was added to the wells of the antibody-coated plate and incubated for 2 h at 37°C or overnight at 4°C. A positive control (virus-infected), negative control (not virus-infected), and non-template control (extraction buffer only) were included in each plate. After rigorous washing, the plate was filled with 100 μl of secondary antibody-Alkaline phosphatase enzyme conjugate (1:200 dilution in the conjugate buffer) and incubated for 2 h at 37°C. Color development was generated by incubation with 100 μl of p-nitrophenyl phosphate (PNP) substrate and absorbent readings were measured at 405 nm with an ELISA plate reader . Absorbance values at least twice over that of the negative control were considered positive.Real-time RT-PCR reaction was used to determine the infection of PSTVd and carried out using a Takara One Step Ex Taq qRT-PCR kit  on an Mx3000P qPCR machine . RNA template was prepared with a simple dilution of crude tissue extract method . ApproxiTo determine whether there was any significant effect between disinfectants on each target pathogen, the number of plants infected for each disinfectant solution and exposure time were pooled and analyzed using Logistic regression and the Bayesian methodology . To do s


Determining the complex relationships between diseases, polymorphisms in human genes and environmental factors is challenging. Multifactor dimensionality reduction (MDR) has been proven to be capable of effectively detecting the statistical patterns of epistasis, although classification accuracy is required for this approach. The imbalanced dataset can cause seriously negative effects on classification accuracy. Moreover, MDR methods cannot quantitatively assess the disease risk of genotype combinations. Hence, we introduce a novel weighted risk score-based multifactor dimensionality reduction (WRSMDR) method that uses the Bayesian posterior probability of polymorphism combinations as a new quantitative measure of disease risk. First, we compared the WRSMDR to the MDR method in simulated datasets. Our results showed that the WRSMDR method had reasonable power to identify high-order gene-gene interactions, and it was more effective than MDR at detecting four-locus models. Moreover, WRSMDR reveals more information regarding the effect of genotype combination on the disease risk, and the result was easier to determine and apply than with MDR. Finally, we applied WRSMDR to a nasopharyngeal carcinoma (NPC) case-control study and identified a statistically significant high-order interaction among three polymorphisms: rs2860580, rs11865086 and rs2305806. Complex interactions among genes and environmental factors are known to play a role in common human disease etiology. However, the identification and characterization of gene-gene interactions for common complex human diseases remain a challenge for human geneticists. Traditional statistical methods are not well suited for detecting such interactions, especially when the data are highly dimensional (having many attributes or independent variables) or when interactions occur between more than two polymorphisms . To addrNasopharyngeal carcinoma (NPC) is a squamous cell carcinoma that arises in the epithelial lining of the nasopharynx . This neIn this paper, we introduce a novel weighted risk score-based multifactor dimensionality reduction (WRSMDR) method for detecting and characterizing high-order gene-gene interactions in case-control studies. This WRSMDR method uses the Bayesian posterior probability of each genotype combination as a quantitative measure of disease risk and computes the proportion of each genotype combinations in all samples as the weight. WRSMDR exhaustively searches all possible combinations of polymorphisms to identify the one that can divide the samples into the best risk sub-groups. We first evaluated WRSMDR using simulated multi-locus data with epistatic effects and then compared it to the original MDR method. Next, we applied the WRSMDR method to identify multiple single-nucleotide polymorphisms (SNP) associated with nasopharyngeal carcinoma.Specific detection rate = the proportion of simulated datasets in which the true model was detected as the overall best model .Detection rate = the proportion of simulated datasets in which a multi-locus model, including the true model, was detected as the overall best model .Error rate = the proportion of simulated datasets in which the overall best model did not include the true model.No detection rate = the proportion of simulated datasets in which the method did not detect any statistically significant model.p > 0.05). The detection rates of the WRSMDR and MDR methods were 100% for the two- and three-locus simulated datasets. For the balanced four-locus datasets, the specific detection rates of the WRSMDR and MDR were 92% and 46%, respectively, and this difference was statistically significant (p < 0.05). The detection rates of the WRSMDR and MDR were 97% and 56%, respectively, and this difference was also statistically significant (p < 0.05). For the 100 balanced simulated four-locus datasets, the WRSMDR method did not produce statistically significant results in two datasets and produced error results in one dataset; the MDR method produced error results in 44 datasets. For the imbalanced datasets of three-locus models, there was no significant difference between the detection rate and the specific detection rate of the two methods (p > 0.05). For the imbalanced two- and four-locus models, the specific detection rate and the detection rate of the WRSMDR method were higher than the rates of the MDR method, and their respective differences were statistically significant (p < 0.05). In the simulated datasets, the WRSMDR method demonstrated reasonable power to identify high-order gene-gene interactions, and it was more effective than the MDR method at detecting four-locus models.We applied the WRSMDR and MDR methods to balanced and imbalanced simulated datasets, and the results are shown in p-value, consistency and weighted risk score from the WRSMDR analysis of the NPC dataset for each two- to five-locus combinations. One three-locus model, which had the maximum consistency and the maximum weighted risk score, p < 0.001, was selected by using the WRSMDR method. This three-locus model included rs2860580, rs11865086 and rs2305806. We also applied the MDR method to explore the NPC data in a further step. The result is shown in The three SNPs are located in the HLA-A, MAPK3 and VAV1 genes, which play important roles in the NK cell pathway. NK cells are lymphocytes distinct from B- and T-cells that induce the perforin-mediated lysis of tumor cells and virus-infected cells, and the NK cell pathway regulates the role of NK cells in the immune response. The highest NPC risk among the three-locus genotype combinations was three times greater than the cumulative risk of the disease, which indicates that this pathway may be associated with NPC. To the best of our knowledge, this is the first report describing a three-locus interaction associated with NPC, and the results of this study therefore provide new insights into the pathogenesis of NPC.p < 0.01 may be selected. These measures provide WRSMDR with reasonable power to identify interactions among two or more loci in relatively small samples.The WRSMDR method provides several advantages. First, similar to the original MDR method, the WRSMDR method is a non-parametric approach and assumes no particular genetic model; Second, the WRSMDR method provides a more robust quantitative measure of disease risk and reveals more information regarding the effect of certain genotype combinations on the disease risk, and this also represents an important difference from the MDR method, which only discretized the risk into high and low. Our results showed that the WRSMDR method had more power than the MDR method in detecting four-locus gene-gene interactions in the simulated datasets. For the balanced four-locus datasets, the specific detection rates of the WRSMDR and MDR method were 92% and 46%, respectively. For the imbalanced four-locus datasets, the specific detection rates of the WRSMDR and MDR method were 94% and 68%, respectively. The reason for this difference may lie in the fact that the MDR method is vulnerable to false positive and false negative errors when the sample size is small or when the number of simultaneously detected loci is large. In the case of this scenario, the number of cases and controls with a certain genotype combination is very small, and a small change in the frequency can change the classification to the opposite result. With the WRSMDR method, the quantitative measure of the disease risk was effected less than binary classification values in this scenario; Third, the WRSMDR method uses a weighted risk score rather than classification accuracy as the evaluation measure of the multi-locus interaction. The goal of MDR is to search a locus combination with maximum classification accuracy. For imbalanced datasets, classifiers seeking an accurate performance are not suitable. The imbalanced dataset can cause a seriously negative effect on classification accuracy . For extHowever, similar to MDR, WRSMDR has the limitation of being computationally intensive. A genome scan with hundreds or thousands of polymorphisms requires robust machine learning algorithms, as all of the possible multi-locus combinations cannot be exhaustively searched. This requirement, however, is a limitation of any multi-locus method that does not first condition on a particular locus showing an independent main effect  . ConsequP(D/G), with D and G representing the disease status and the genotype combination, respectively. P(D/G) is a measure of the disease risk for a genotype combination, with larger values corresponding to a higher risk of the genotype leading to disease. With a known cumulative risk of the disease and the genotype frequencies in cases and normal controls, this value can be calculated from the Bayesian posterior probability formula as follows:With the WRSMDR method, the disease probability of an individual carrying a multi-locus genotype combination was used to assess the susceptibility of the genotype combination, which was denoted as P(D) represents the cumulative risk of the disease, P(N) = 1 − P(D). P(G/D) and P(G/N) represent the genotype combination frequencies in the case and control populations, respectively, and these values can be estimated from the case-control genotype dataset. The formula is as follows:In this equation, dd and dn represent the number of individuals who carry the genotype combination in the case and control groups, respectively, and d and n represent the size of the case and control groups, respectively.In this equation, k-way gene-gene interactions. There are 3k different genotypic combinations at most. We denote jG  as the genotype combinations of k loci, and we denote the relative risk of individuals with genotype jG as follows:Suppose we want to investigate The extent of increased or decreased risk can be defined as follows:jG can be defined as follows:The weight of genotype Based on the “common disease-common variant” hypothesis, we omit the genotype combinations with weights less than 0.01, and the weighted risk score is defined as follows:k-locus combinations  were computed based on the randomly selected samples, and the combination with the maximum score within each of the k-locus combinations was selected. After ten repeats for each k-locus combination, the multi-locus combination that demonstrated the maximum consistency, that is, the one appearing most frequently, was chosen as the candidate susceptibility k-locus combination. The average score of each of the selected k-locus combinations was then computed; In Step 2, the significance level of each candidate k-locus combination was assessed with the permutation test. Specifically, for every 1000 permutation datasets simulating the null hypothesis of no association, the weighted risk score of the multi-locus combination was computed using the above method. The p-value was then determined according to the proportion of the permutation datasets with a greater weighted risk score than the average score. The null hypothesis was rejected when the Monte Carlo p-value derived from the permutation test was less than 0.01; In Step 3, among the candidate locus combinations, the one with p < 0.01, the maximum consistency, and the maximum average weighted risk score was selected as the final susceptibility multi-locus combination. Instead of performing a direct search of the multi-locus combinations with the maximum score among the SNP set, which is liable to result in some false-positive loci in the detected multi-locus combinations, we used a random sampling method to repress noise in the identification of the susceptibility of multi-locus combinations. This procedure included three steps. In Step 1, 90% of the samples were randomly selected. The weighted risk scores of i.e., case or control). The six datasets consisted of 10 SNPs, including 2, 3 and 4 predictive SNPs and up to 8 randomly generated non-predictive SNPs.To evaluate the WRSMDR method, we simulated six sets of 100 replicates using three different multi-locus genetic models. Three sets were balanced, and the simulated dataset was composed of 400 cases and 400 controls. The other three sets are imbalanced, and the simulated dataset was composed of 1200 cases and 400 controls. All the genetic models and datasets were generated using the Genetic Architecture Model Emulator for Testing and Evaluating Software (GAMETES) . In partThis NPC data were based on a large GWAS of NPC that was performed on Southern Chinese individuals by genotyping 620,901 SNPs in 1615 cases and 1025 controls of persons of Han Chinese descent from Guangdong and an additional 1008 Singapore Chinese controls, who share the same ancestral origin with Han Chinese individuals in Southern China . After sPrior to applying WRSMDR to the NPC dataset, the method was evaluated using the simulated multi-locus datasets. For every 100 replicates generated by each of the three multi-locus epistasis models, we applied the WRSMDR algorithm as described in the subsection “WRSMDR”. An exhaustive search of all possible two- to five-locus models was performed. Then, the WRSMDR method was applied to the NPC dataset with the cumulative risk of the disease equal to 0.00025 . FinallyIn this study, we introduced WRSMDR as a method for detecting gene-gene interactions in case-control studies. We compared the WRSMDR and MDR methods in simulated datasets. Our result showed that the WRSMDR method had reasonable power to identify high-order interactions in simulated datasets. In particular, for the four-locus datasets, the detection rate and specific detection rate of the WRSMDR method were higher than the MDR method, whereas the error rate of the WRSMDR method was lower than the MDR method; these differences were statistically significant. The WRSMDR method was more effective than the MDR method at detecting four-locus models in the simulated datasets. Moreover, the WRSMDR method reveals more information regarding the effect of genotype combinations on the disease risk. We then applied WRSMDR to identify gene-gene interaction effects in the NK cell pathway related to the risk of NPC, and we found a statistically significant, high-order interaction among three polymorphisms. For ease of use, the source code and binaries are freely available for download at . However