Caenorahbditis nematode isolates and can now be used to explore host antiviral pathways, nematode ecology, and host-pathogen co-evolution.Novel viruses have been discovered in wild  C. elegans as a model to define host-viral interactions has been limited by the lack of viruses known to infect nematodes. From wild isolates of C. elegans and C. briggsae with unusual morphological phenotypes in intestinal cells, we identified two novel RNA viruses distantly related to known nodaviruses, one infecting specifically C. elegans (Orsay virus), the other C. briggsae (Santeuil virus). Bleaching of embryos cured infected cultures demonstrating that the viruses are neither stably integrated in the host genome nor transmitted vertically. 0.2 µm filtrates of the infected cultures could infect cured animals. Infected animals continuously maintained viral infection for 6 mo (∼50 generations), demonstrating that natural cycles of horizontal virus transmission were faithfully recapitulated in laboratory culture. In addition to infecting the natural C. elegans isolate, Orsay virus readily infected laboratory C. elegans mutants defective in RNAi and yielded higher levels of viral RNA and infection symptoms as compared to infection of the corresponding wild-type N2 strain. These results demonstrated a clear role for RNAi in the defense against this virus. Furthermore, different wild C. elegans isolates displayed differential susceptibility to infection by Orsay virus, thereby affording genetic approaches to defining antiviral loci. This discovery establishes a bona fide viral infection system to explore the natural ecology of nematodes, host-pathogen co-evolution, the evolution of small RNA responses, and innate antiviral mechanisms.An ideal model system to study antiviral immunity and host-pathogen co-evolution would combine a genetically tractable small animal with a virus capable of naturally infecting the host organism. The use of  C. elegans is a robust model organism that is broadly used in biology. It also has great potential for the study of host-microbe interactions, as it is possible to systematically knockout almost every gene in high-throughput fashion to examine the potential role of each gene in infection. While C. elegans has been successfully applied to the study of bacterial infections, only limited studies of antiviral responses have been possible since no virus capable of infecting any Caenorhabditis nematode in laboratory culture has previously been described. Here we report the discovery of natural viruses infecting wild isolates of C. elegans and its relative C. briggsae. These novel viruses are most closely related to the ssRNA nodaviruses, but have larger genomes than other described nodaviruses and clearly represent a new taxon of virus. We were able to use these viruses to infect a variety of laboratory nematode strains. We show that mutant worms defective in the RNA interference pathway, an antiviral system known to operate in a number of organisms, accumulate more viral RNA than wild type strains. The discovery of these viruses will enable further studies of host-virus interactions in C. elegans and the identification of other host mechanisms that counter viral infection.The nematode  D. melanogasterC. elegansC. elegans of bacterial pathogens such as Pseudomonas, Salmonella, or Serratia has been highly fruitful, in some instances revealing the existence of innate immune pathways in C. elegans that are also conserved in vertebrates C. elegans intestinal cells by microsporidia makes it a promising model for microsporidia biology C. elegans to understand anti-viral innate immunity, however, have been hampered by the lack of a natural virus competent to infect and replicate in C. elegans.Model organisms such as C. elegans have been pursued using artificial methods of introducing viruses or partial virus genomes into animals In the absence of a natural virus infection system, some efforts to define virus-host responses in C. elegans have proven hard to find until recent years. The identification of C. elegans habitats and the development of simple isolation methods  C. elegans. Here we report the discovery of natural populations of C. elegans and of its close relative C. briggsae that display abnormal morphologies of intestinal cells. These abnormal phenotypes can be maintained in permanent culture for several months, without detectable microsporidial or bacterial infection. We show that these populations are infected by two distinct viruses, one specific for C. elegans (Orsay virus), one for C. briggsae (Santeuil virus). These viruses resemble viruses in the Nodaviridae family, with a small, bipartite, RNA(+sense) genome. Infection by each virus is transmitted horizontally. In both nematode species, we find intraspecific variation in sensitivity to the species-specific virus. We further show that infected worms mount a small RNA response and that RNAi mechanisms act as antiviral immunity in nematodes. Finally, we demonstrate that the C. elegans isolate from which Orsay virus was isolated is incapable of mounting an effective RNAi response in somatic cells. We thus find natural variation in host antiviral defenses. Critically, these results establish the first experimental viral infection system in C. elegans suitable for probing all facets of the host antiviral response.Natural populations of Caenorhabditis strains were isolated that displayed a similar unusual morphology of the intestinal cells and no visible pathogen by optical microscopy. Intestinal cell structures such as storage granules disappeared  and C. briggsae JU1264 , were selected for detailed analysis. Bleaching of adult animals resulted in phenotype-free progeny from both strains, demonstrating that the phenotype was not vertically transmitted (embryos are resistant to the bleaching treatment)  . Additioeatment) . We founeatment)  with 10 Caenorhabditis were infected by a virus, small virus-like particles of approximately 20 nm diameter were visible by electron microscopy of the intestinal cells .In support of the hypothesis that these wild al cells , S1. SucC. briggsae JU1264 isolate compared to the uninfected control , nor a change in brood size ,B. Howev control .Nodaviridae. Nodaviruses are bipartite positive strand RNA viruses. The RNA1 segment of all previously described nodaviruses is ∼3.1 kb and encodes ORF A, the viral RNA-dependent RNA polymerase. Some nodaviruses also encode ORFs B1/B2 at the 3′ end of the RNA1 segment. The B1 protein is of unknown function while the B2 protein is able to inhibit RNAi An unbiased high-throughput pyrosequencing approach was used to determine whether any known or novel viruses were present in the animals. From JU1264, 28 unique sequence reads were identified initially that shared 30%–48% amino acid sequence identity to known viruses in the family Pyrosequencing of JU1580 demonstrated the presence of a second distinct virus that shared the same general genomic organization as the virus detected in JU1264. Partial genome sequences of 2,680 nucleotides of the RNA1 segment and 2,362 nucleotides of the RNA2 segment were obtained and confirmed by RT-PCR. The putative RNA-dependent RNA polymerases of the two viruses shared 44% amino acid identity by BLAST analysis. Like the virus in JU1264, the virus in JU1580 was predicted to encode a capsid protein at the 5′ end of the RNA2 segment as well as a second ORF in the 3′ half of the RNA2 segment. The ORF δ encoded proteins from the two viruses shared 37% amino acid identity when compared using BLAST. Thus, the genomic organization of these two viruses, while sharing substantial commonality with known nodaviruses, also displayed novel genomic features. Phylogenetic analysis of the predicted RNA polymerase and capsid proteins demonstrated that the virus sequences in JU1580 and JU1264 were highly divergent from all previously described nodaviruses and most closely related to each other . We prop3) to the next plate twice a week continued to yield positive RT-PCR results (unpublished data).RT-PCR assays were used to analyze RNA extracted from JU1580, JU1264, their corresponding bleached control strains JU1580bl and JU1264bl, and the same strains following reinfection with viral filtrates. Orsay virus RNA could be detected by RT-PCR in the original JU1580 culture, disappeared in the bleached strains, and stably reappeared following re-infection with the corresponding viral filtrate . The samNorthern blotting confirmed the presence of Orsay and Santeuil virus RNA sequences in the infected animals. Hybridization with a DNA probe targeting the RNA1 segment of Santeuil virus yielded multiple bands in JU1264 animals but not in the corresponding bleached control strain. The strongest band detected migrated between 3.5 and 4 kb consistent with the 3,628 nt sequence we generated for the putative complete RNA1 segment . MultiplTo demonstrate virus replication in the infected animals, we performed Northern blotting using strand-specific riboprobes. For positive sense RNA viruses like nodaviruses, the negative sense RNA is only synthesized during active viral replication. It is not packaged in virions and typically exists in much lower quantities than the positive strand. Robust levels of the positive strand of the Santeuil virus RNA1 segment were detected . NortherIn order to determine the localization of Orsay viral RNA in infected animals, we performed RNA fluorescent in situ hybridization (FISH) using a probe complementary to the positive sense RNA1 segment of Orsay virus. Viral RNA was robustly detected in intestinal cells of JU1580bl animals infected 4 d previously with Orsay viral filtrate .One key defense mechanism of plants and animals against RNA viruses is the small RNA response rde-1 gene—which encodes an Argonaute protein required for the initiation of exogenous RNAi rde-1 strain produced infectious viral particles, as reinfection of the cured JU1580 strain by filtrates of infected rde-1 animals yielded positive RT-PCR results (unpublished data). In addition, mutation of other exogenous RNAi pathway genes including rde-2, rde-4, and mut-7  but competent for RNA inactivation of a germline-expressed gene (pos-1) , which was inactivated by GFP RNAi in the C. elegans N2 reference background, but only modestly repressed in the JU1580bl isolate  relative to the reference N2 strain. Using external application of dsRNAs by feeding, JU1580bl was found to be highly resistant to RNAi of a somatically expressed gene ( (pos-1) . C. eleg isolate . We confgermline . Thereforde-1 were exposed to Orsay virus filtrate. A subset of adult animals from each plate was bleached and their adult offspring collected 4 d later. No evidence for vertical transmission was observed by qRT-PCR for Orsay virus RNA in any strain . The same range of intestinal symptoms was observed in animals that were infected by the Orsay and Santeuil viruses, further suggesting that these viral infections were causing the cellular symptoms. We observed putative viral particles of the size expected for nodaviruses, and a strong RNA FISH signal in intestinal cells and the somatic gonad of infected animals demonstrating that the virus is present intracellularly. It is likely that further sampling of natural populations of Caenorhabditis will yield other viruses of this and other groups. In fact, these symptoms were seen repeatedly in C. briggsae animals sampled from different locations in France, and in one instance, a Santeuil virus variant has been identified (unpublished data).Here we report the first molecular description, to our knowledge, of viruses that naturally infect nematodes in the wild. The two novel viruses we identified, while clearly related to known nodaviruses, possess unique genomic features absent from all other previously described nodaviruses. These viruses may thus define a novel genus within the family A characteristic feature of these two viruses is the presence of the novel ORF δ. Conservation of sequence length and identity of the ORF δ in these two viruses, and the absence of this ORF in all other described nodaviruses, suggests that this predicted protein is likely to be important for the ability of the virus to infect or replicate in nematodes. Its function is currently unknown, but it is tempting to speculate that this protein may play a role in antagonizing an innate antiviral pathway.C. elegans by the Orsay nodavirus provides an exciting prospect for studies in virology, host cell biology, and antiviral innate immunity. Genetic screens to identify anti-viral factors in model organisms have been limited in large part by the lack of natural infection systems. Although Drosophila has been used with great success to examine host-virus interactions for various insect viruses C. elegans and a virus that persists in culture through horizontal transmission, causing high damage in intestinal cells yet remarkably little effect on the animal, which continues moving, eating, and producing progeny, although at a lower rate.The infection of rde-1 mutants in N2 background was highly reproducible. The fact that the reference wild type N2 strain may only sustain a very low yet detectable viral titer makes it a particularly favorable genetic background in which to screen for genes involved in interaction with the virus.De novo infection of naïve animals can be affected by the simple addition of either dead infected animals or homogenized lysates made from infected animals to culture dishes. This is sufficient to seed sustained complete cycles of viral replication, shedding, and infection. With this system, it is now possible to embark on whole genome genetic screens to identify host factors that block any facet of the viral life cycle. Using the current experimental conditions, infection of JU1580bl and C. elegans as in other animals. In C. elegans, the intestinal cells are large and easily amenable to observations by optical microscopy. The viral parasites affect the organization of the polarized epithelial intestinal cells and will likely provide interesting mechanisms and tools to study their cell biology. Clear reorganization occurs in the intermediate filaments that line the apical brush border, as well as in the lipid storage granules, the nuclear membrane, and other intracellular compartments.The intestine is a tissue that is particularly exposed to microbes through ingestion, and is a main entry point for pathogens in The abnormal state of the intestinal cells may slow down progeny production by decreasing the food intake. Alternatively, the presence of viral RNA in the somatic gonad may explain the delay in progeny production, although no gonadal cellular phenotypes have been observed. The presence of viral RNA in the somatic gonad is particularly interesting given the lack of vertical transmission.C. elegans RNAi in counteracting viral infection, these studies utilized either a transgenic system of viral RNA expression rde-1 mutants suggest that perturbing germline RNAi is not sufficient to enable vertical transmission.Although prior studies have clearly demonstrated a role for C. elegans genetic backgrounds will allow the identification of a set of host genes that modulate viral sensitivity during evolution of natural host populations. Based on the defect in exogenous RNAi of the JU1580 strain, we speculate that this set will include, but is unlikely to be limited to, genes involved in exogenous RNAi pathways. Support for the role of other genes outside the RNAi pathway comes from our data on natural isolates. Despite the fact that the magnitude of the somatic RNAi defect of the natural isolate PS2025 was comparable to that of JU1580, no evidence of viral RNA accumulation or morphological symptoms was observed following addition of Orsay virus filtrate. Whether PS2025 lacks one or more crucial receptors for viral infection or has alternative antiviral pathways that suppress viral replication is currently unknown.The quantitative difference in Orsay nodavirus sensitivity between the N2 and JU1580 wild C. elegans and C. briggsae, respectively. Moreover, the C. elegans rde-1 mutation in the N2 background confers susceptibility to the Orsay virus, but not to the Santeuil virus  or the apparent disappearance of the nucleus; 3, the loss of cytoplasmic viscosity visible as a very fluid flow of cytosol within the cell; and 4, the fusion of intestinal cells. Some of these traits may sometimes appear in uninfected animals. We systematically tested for a significant increase after infection of the proportion of animals with symptoms (Fisher's exact test). Note that some of these symptoms can also be caused by microsporidial and bacterial infections. Thus, the diagnostic of a viral infection based on the cellular symptoms requires an otherwise clean culture.When observed with a transillumination dissecting microscope, infected animals displayed a paler intestine than healthy worms. This lack of intestinal coloration occurred all along the entire intestinal tract in 20, pelleted and incubated in 10 ml of 10 µg/ml Hoechst 33342 in ddH20 for 45 min with soft agitation, protecting the tube from light with an aluminum foil. The animals were then pelleted and transferred to a new culture plate seeded with E. coli OP50. After 2 h, they were mounted and observed with a fluorescence microscope.Animals were washed off a culture plate in 10 ml of ddH4 at 4°C, washed, embedded in 2% low melting point agar, dehydrated in solutions of increasing ethanol concentrations, and embedded in resin . High-pressure freezing was performed using a Leica PACT2 high-pressure freezer A few adults were washed in 0.2 ml of M9 solution, suspended in 2% paraformaldehyde +0.1% glutaraldehyde, and cut in two on ice under a dissecting microscope for better reagent penetration C. elegans JU1580 and single L3 larvae for C. briggsae JU1264. The parent animal then transferred every day to a new plate until the end of progeny production. The plates were incubated at 20°C for 2 d and kept at 4°C until scoring. The few cases where the parent died before the end of its laying period were not included. Some progeny died as embryos in both infected and non-infected cultures . The timing of progeny production was analyzed in R using a Generalized Linear Model using infection status, day, individual (nested in infection status), and Infection Status×Day as explanatory variables, assuming a Poisson response variable and a log link function. Individual, day and Infection Status×Day were the significant explanatory variables for both JU1264 and JU1580 (p<0.001).The time course was started by isolating single L4 larvae for rde-1(ne219), or JU1580bl. At the same time  was used so that both species developed at similar speeds. Maintenance over more than 4 d after re-infection was performed by transferring a piece of agar (approx. 0.1 cm3) every 2–3 d to a new plate with food.Nematodes were grown on 10 plates (90 mm diameter) until just starved, resuspended in 15 ml of 20 mM Tris-Cl pH 7.8, and pelleted by low-speed centrifugation . The supernatant was centrifuged twice at 21,000 g for 5 min (4°C) and pellets discarded. The supernatant was passed on a 0.2 µm filter. 55 mm culture plates were prepared with 2–5 young adults of N2, ame time , and S3,ame time , 7A, 30 Phenol-chloroform purified DNA and RNA from infected JU1580 and JU1264 animals were subject to random PCR amplification as described 5′ RACE was performed according to standard protocols (Invitrogen 5′ RACE kit). 3′ RACE was performed by first adding a polyA tail using PolyA polymerase (Ambion) and then using Qiagen 1-step RT-PCR kit with gene specific primers and an oligo-dT-adapter primer. Products were cloned into pCR4 and sequenced using standard Sanger chemistry.C. elegans genome (WS190) downloaded from the UCSC Genome Browser website (http://genome.ucsc.edu/) 4–6 90 mm plates with 15–20 adults (JU1580 or bleached JU1580) were grown for 4 d at 20°C. Mixed stage animals from all plates were collected, pooled, and frozen at −80°C. Total RNA was extracted using the mirVana miRNA isolation kit (Ambion). Small RNAs were size selected to 18–30 bases by denaturing polyacrylamide gel fractionation. A cDNA library that did not depend on 5′-monophosphates was constructed by tobacco acid pyrophosphatase treatment using adapters recommended for Solexa sequencing as described previously Epinephelus tauvina nervous necrosis virus NC_004136, Flock house virus NC_004144, Macrobrachium rosenbergii nodavirus RNA-2 NC_005095, Nodamura virus RNA2 NC_002691, Pariacoto virus RNA2 NC_003692, Redspotted grouper nervous necrosis virus NC_008041, Striped Jack nervous necrosis virus RNA2 NC_003449, Tiger puffer nervous necrosis virus NC_013461, Wuhan nodavirus ABB71128.1, and American nodavirus ACU32796.1. 1,000 bootstrap replicates were performed.The predicted amino acid sequences from Orsay and Santeuil nodaviruses were aligned using ClustalW to the protein sequences of the following nodaviruses. Capsid Protein: Barfin1 flounder nervous necrosis virus NC_013459, Barfin2 flounder virus BF93Hok RNA2 NC_011064, Black beetle virus NC_002037, Boolarra virus NC_004145, Epinephelus tauvina nervous necrosis virus NP_689433.1, Flock house virus NP_689444.1, Nodamura virus NP_077730, Pariacoto virus NP_620109.1, Redspotted grouper nervous necrosis virus YP_611155.1, Striped Jack nervous necrosis virus NP_599247.1, Tiger puffer nervous necrosis virus YP_003288759.1, Macrobrachium rosenbergii nodavirus NP_919036.1, Wuhan_Nodavirus AAY27743, and American nodavirus SW-2009a ACU32794.1. 1,000 bootstrap replicates were performed.RNA Polymerase: Barfin flounder nervous necrosis virus YP_003288756.1, Barfin flounder virus BF93Hok YP_002019751.1, Black beetle virus YP_053043, Boolarra virus NP_689439, 2O. 5 µg of RNA were reverse transcribed using SuperscriptIII (Invitrogen) in a 20 µl volume. 5 µl were used for PCR in a 20 µl volume . For the Orsay nodavirus, the reverse transcription used the GW195 primer (5′ GACGCTTCCAAGATTGGTATTGGT) and the PCR oTB3 (5′ CGGATTCTCGACATAGTCG) and oTB4 (5′GTAGGCGAGGAAGGAGATG). For the Santeuil nodavirus, reverse transcription used oTB6RT (5′ GGTTCTGGTGGTGATGGTG) and PCR oTB5 (5′ GCGGATGTTCTTCACGGAC) and oTB6 (5′ GTCAGTAGCGGACCAGATG).Nematodes from two culture plates were resuspended in M9 and then washed three times in 10 ml M9. RNA was extracted using Trizol (Invitrogen) (5–10 vol∶vol of pelleted worms) and resuspended in 20 µl in RNAse-free ddH2O. 0.1 µl was used for RT-PCR using the OneStep RT-PCR Kit (Qiagen). Primers annealed to viral RNA1 (GW194 and GW195).Animals from one 55 mm culture plate plus viral filtrate (see infection procedure) were washed twice in M9. RNA was extracted using 1 ml Trizol (Invitrogen) and resuspended in 10 µl DEPC-treated Hama-1, and then viral RNA1 (primers GW194: 5′ ACC TCA CAA CTG CCA TCT ACA and GW195: 5′ GAC GCT TCC AAG ATT GGT ATT GGT) levels were compared to those present in re-infected bleached JU1580 animals.cDNA was generated from 1 µg total RNA with random primers using Superscript III (Invitrogen). cDNA was diluted to 1∶100 for qRT-PCR analysis. qRT-PCR was performed using either QuantiTect SYBR Green PCR (Qiagen) or ABsolute Blue SYBR Green ROX (Thermo Scientific). The amplification was performed on a 7300 Real Time PCR System (Applied Biosystems). Each sample was normalized to 32P dATP using the Decaprime kit (Ambion). Blots were hybridized for 4 h at 65°C in Rapid hyb buffer  and washed in 2XSSC/0.1%SDS 5 min×2 at 25°C, 1XSSC/0.1%SDS 10 min×2 at 25°C, 0.1XSSC/0.1%SDS 5 min×4 at 25°C, and 0.1XSSC/0.1%SDS 15 min×2 at 42°C and 0.1XSSC/0.1%SDS 15 min×1 at 68°C. For strand specific riboprobes, 32P labeled RNA was generated by in vitro transcription with either T7 or T3 RNA polymerase (Ambion) in the presence of α-32P UTP. The target plasmid contained a cloned region of the Santeuil nodavirus RNA1 segment (nt 523–1022) and was linearized with either PmeI or NotI, respectively. For the riboprobes, blots were hybridized at 70°C and then sequentially washed as follows: 2XSSC/0.1%SDS 5 min×2 at 68°C, 1XSSC/0.1%SDS 10 min×2 at 68°C, 0.1XSSC/0.1%SDS 10 min×2 at 68°C, and 0.1XSSC/0.1%SDS 20 min×1 at 73°C. The Santeuil RNA1 segment migrates at approximately the same position as the 28S ribosomal RNA. Under the extended exposure time (72 h) needed to visualize the negative sense genome, low levels of non-specific binding to the 28S RNA become apparent  and the RNA2 segment of Santeuil nodavirus (nt 1833–2308) were generated by random priming in the presence of α-apparent .pos-1 and unc-22 RNAi using bacteria as the dsRNA source, bacterial clones from the Ahringer library expressing dsRNAs C. elegans on agar plates. For the pos-1 experiment, bacteria were concentrated 10-fold by centrifugation prior to seeding the plates. A C. briggsae Cbr-lin-12 fragment C. elegans. Three or four L4s were deposited on an RNAi plate, singly transferred the next day to a second RNAi plate, and their progeny scored after 2 d (pos-1) or 3 d (unc-22) at 23°C.For unc-22 dsRNA synthesis and injection, the unc-22 fragment in the Ahringer library clone was amplified by PCR using the T7 primer and in vitro transcribed with the T7 polymerase using the Ambion MEGAscript kit, according to the manufacturer's protocol Cel-unc-22 dsRNAs were injected at 50 ng/µl into both gonadal arms of young hermaphrodite adults of the relevant strain. The animals were incubated at 20°C. The adults were transferred to a new plate individually on the next day, and the proportion of twitching progeny scored 3 d later, touching each animal with a platinum-wire pick to induce movement.For let-858::GFP and the pharyngeal marker myo-2::DsRed as an extrachromosomal array. Bacteria expressing dsRNA against GFP cDNA were used to feed animals on agar plates. An empty vector was used as a negative control. Two or three L4s were deposited on a 55 mm RNAi plate, grown at 20°C for 3 d, and the GFP/DsRed expression levels in their offspring measured using flow cytometry (Union Biometrica) as described previously p<0.001.For GFP RNAi, transgenic N2 and JU1580 strains were generated expressing the ubiquitously expressed A segment of Orsay virus RNA1 was generated with primers GW194 and GW195 and cloned into pGEM-T Easy (Promega). Fluorescein labeled probe was generated from linearized plasmid using the Fluorescein RNA Labeling Mix (Roche) and MEGAscript SP6 transcription (Ambion). JU1580bl animals were infected with Orsay virus filtrate and grown for 4 d at 20°C on 90 mm plates. Control animals were grown under the same conditions in the absence of virus. In situ hybridization was performed essentially as previously described Genbank Sequences: Accession numbers for Orsay and Santeuil virus contigs: HM030970–HM030973. Small RNA sequencing data at GEO: GSE21736.Figure S1Putative viral particles in transmission electron microscopy of intestinal cells of infected C. elegans JU1580 adult hermaphrodites. On the right are shown higher magnifications of the parts delimited by a black rectangle in the left micrograph, showing putative viral particles (arrows). (A) Putative viral particles are found in an intracellular multi-membrane compartment. The particles in the upper left of the inset of (A) are ribosomes; the putative viral particles in the lower two-thirds are characterized by a slightly larger and more regular ring appearance (ca. 20 nm diameter). (B) Putative viral particles similar to those in (3.41 MB PPT)Click here for additional data file.Figure S2Infection does not alter brood size, but results in delayed progeny production in C. briggsae JU1264.  Boxplots of the distribution of brood size in naturally infected and bleached (“bl”) cultures of C. elegans JU1580 (A) and C. briggsae JU1264 (B) at 20°C. The line indicates the median, the box the lower and upper quartiles, and the whiskers the 10th and 90th percentiles. Brood size is not significantly different in infected versus non-infected cultures .  Progeny number over time in JU1580 (C) and JU1264 (D). Infection results in a significant change in the timing of progeny production . Note that time 0 corresponds to the L4 stage in the experiment in  and the L3 stage in .(0.33 MB PDF)Click here for additional data file.Figure S3Scoring of morphological symptoms after exposure of various wild isolates and rde-1 mutants. (A) Specificity of infection by the Orsay nodavirus. Each Caenorhabditis strain was mock-infected (−) or infected with a virus filtrate (+). The proportion of worms with morphological infection symptoms after 7 d at 23°C is shown for the same experiment as in rde-1 mutants in the C. elegans N2 background. Morphological symptoms were scored 5 d after infection at 23°C by the Santeuil virus filtrate. * p<0.05; *** p<0.001.(0.29 MB PDF)Click here for additional data file.Figure S4Quantification of GFP transgene expression and silencing. (Top) Reporter genes are expressed in a larger proportion of animals from N2 compared to JU1580. Shown are the number of animals according to binned log2 DsRed intensities. Each line corresponds to one flow cytometry run with colors indicating strain and treatment as explained in the color legend. The extrachromosomal array was inherited more efficiently in N2 than in JU1580, making it necessary to analyze only those animals carrying the array. (Bottom) Each data point corresponds to the difference between treatment with GFP RNAi and empty vector in N2 (blue) and JU1580 (red) observed for animals with log2 DsRed intensity in a given bin. Dots indicate the difference between the means of median log2(GFP/DsRed) ratios for treatment with GFP RNAi and empty vector. Vertical bars indicate standard errors. The cutoff for DsRed intensities is indicated in both panels by a vertical dotted line.(0.29 MB AI)Click here for additional data file.Figure S5Quantitative analysis of vertical transmission of Orsay virus. N2, JU1580bl, and rde-1 (5 replicates each) were infected with Orsay virus. After 4 d 25 adults from each plate were bleached onto a new, uninfected plate. The remaining adults were collected for RNA extraction. The offspring from the bleached adults were collected for RNA extraction after 4 d. As a control, virus was added to plates and incubated in the absence of animals for 4 d. Viral RNA levels were determined by qRT-PCR and normalized to gapdh. Viral RNA level is shown on a log scale, using a reference value of 1 for the infection of JU1580.(0.27 MB AI)Click here for additional data file.


To the Editor: Saffold virus (SAFV) is a new human virus belonging to the genus Cardiovirus of the family Picornaviridae  cannot be excluded. Accepting that SAFV genotypes correspond with existence of different serotypes, the infection rate in the first years of life must be quite high, similar to that for human parechoviruses and enteroviruses, which are found in 15%–18% of stool samples from children <5 years of age (,The outcome can be explained by a short duration of virus excretion in stool, which, however, is unlikely for an infection spreading by the fecal–oral route. Alternatively, it may be that the virus is unstable in stool and rapidly degrades, such that fecal samples are inadequate for diagnosis of the infection. Other specimens, however, such as respiratory samples, yielded also low numbers of positive findings (


Yet this strategy has limited effect on subsequent breast milk transmission, making the case for new treatment approaches to extend maternal/infant antiretroviral prophylaxis through the period of lactation. Maternal and transmitted infant HIV-1 variants frequently develop NVP resistance mutations following sdNVP, complicating subsequent treatment/prophylaxis regimens. However, it is not clear whether NVP-resistant viruses are transmitted via breastfeeding or arise We performed a detailed HIV genetic analysis using single genome sequencing to identify the origin of drug-resistant variants in an sdNVP-treated postnatally-transmitting mother-infant pair. Phylogenetic analysis of HIV sequences from the child revealed low-diversity variants indicating infection by a subtype C single transmitted/founder virus that shared full-length sequence identity with a clonally-amplified maternal breast milk virus variant harboring the K103N NVP resistance mutation.In this mother/child pair, clonal amplification of maternal NVP-resistant HIV variants present in systemic and mammary gland compartments following intrapartum sdNVP represents one source of transmitted NVP-resistant variants that is responsible for the acquisition of drug resistant virus by the breastfeeding infant. This finding emphasizes the need for combination antiretroviral prophylaxis to prevent mother-to-child HIV transmission. In 2011, 330,000 infants acquired HIV infection from their mothers -6. WHO cde novo in the infant [Although a large fraction of infants harbor NVP-R variants following perinatal NVP administration, it is not clear whether NVP-R variants are transmitted to the infant postnatally or if they arise e infant -11,16. Pe infant ,14,16. Ue infant . Here weenv) sequences derived from child’s plasma at the time of HIV diagnosis  since a most recent common ancestor (MRCA) was 43 days , a time consistent with postpartum transmission of a single virus. In addition, when sets of 5’-half and 3’-half infant HIV genomes were analyzed, each set conformed to model predictions, as described above for env-only sequences  who received intrapartum sdNVP prophylaxis 200 mg) to prevent MTCT the day before delivery, gave birth to an uninfected child and was followed longitudinally for 12 weeks  sequences from 4 weeks postpartum breast milk (right and left breast for env sequences) coalescing to a consensus identical to the infant consensus virus, thus suggesting common ancestry. Most milk sequences differed from consensus by 0, 1, 2, and 3 nucleotides, while sequences with >4 nucleotide changes represented viral recombinants with more divergent virus sequences . Clonally-amplified pol and rev/env milk sequences were linked by an amplified 9 Kb milk variant  and mammary (6 out of 6) compartments had the K103N mutation  and left breast (22/44 sequences), respectively  as previously shown [rd trimester of gestation, one out of eight pol sequences derived from maternal plasma collected after delivery (approximately one day after sdNVP) had the K103N mutation (clone pE7). The one-log reduction in plasma viral load that occurred between the 3rd trimester and parturition samples  was an indication of the antiretroviral response between NPV administration at the onset of labor and sample collection one day later. Although the depth of sequencing was not sufficient to determine whether clone pE7 which was present at parturition preexisted as a low-frequency variant before NVP prophylaxis, mathematical models [env sequences (shown inside circles in Figure Because sdNVP prophylaxis is known to select for NVP-R variants -11, we is Figure . Withoutth blood  out of 2ly shown . Althougl models  and a poFew studies have addressed the origin of NVP-R variants observed in postnatally-infected infants. Studies proposing transmission of NVP-R variants in infants based their conclusion on the results of a first time HIV positive test done at late times during the breastfeeding period under the assumption that NVP had been cleared from tissues ,14. Yet,in vitro studies indicated that K103N mutation has no effect on replication fitness, while the Y181C mutation results in a much less fit virus when introduced in the backbone of the subtype C molecular clone MJ4 [Breast milk HIV quasispecies include multiple independent lineages with evidence of intermixing with plasma virus ,29-31, wlone MJ4 . Our datIn summary, although we are reporting a single MTCT pair analysis, our data are in line with other studies that emphasize the need to implement MTCT prophylaxis strategies that reduce the risk of development and clonal amplification of drug resistant variants in maternal blood and breast milk to prevent MTCT of drug-resistant HIV variants. Our work suggests that administration of sdNVP prophylaxis can lead to postnatal transmission of selected NVP-R variants, and support the global implementation of effective multidrug maternal/infant prophylaxis strategies to prevent infant acquisition of drug-resistant HIV variants that will complicate pediatric ARV treatment strategies.The authors declare that they have no competing interest.SP designed the investigations, led the overarching clinical study, and contributed to manuscript writing; MS performed maternal virus amplification and sequencing and sequence analysis; FG led infant virus sequencing and analysis; FC performed infant virus amplification and sequencing; GL performed the phylogenetic analysis; LK led patient enrollment and clinical site oversight; BH and GM contributed to sequence analysis and interpretation; JS-G led the virus sequencing, analysis, and interpretation and wrote the manuscript. All authors read and approved the final manuscript.Material and methods.Click here for file


Flavivirus includes several pathogenic agents that cause severe illness in humans. Re-emergence of West Nile virus in Europe and continuous spread of certain flaviviruses such as dengue, yellow fever and Japanese encephalitis viruses represent a global danger to public health. Therefore, a rapid and accurate molecular method is required for diagnostics and epidemiological surveillance of flaviviruses.The genus A Pan-Flavi quantitative RT-PCR assay using a Locked-Nucleic Acid probe targeting the flavivirus NS5 gene was developed and optimized to detect a wide range of flaviviruses simultaneously. The specificity and sensitivity of the Pan-Flavi assay were tested using RNA of different flaviviruses and non-flaviviruses. Furthermore, the assay was compared directly to flavivirus species-specific assays for the ability to detect flaviviruses sensitively.Two degenerate primers and one Locked-Nucleic Acids probe were designed to amplify most of the flaviviruses. To increase the specificity and fluorescence signal of the Pan-Flavi assay for detection of yellow fever virus and dengue virus 4, additional primers and probes were included. Viral RNA of thirty different flaviviruses was detected, verifying the broad range specificity. The testing of this assay was successful, using standard plasmid and RNA dilutions of yellow fever virus vaccine strain, dengue virus 1 and tick-borne encephalitis virus, with a sensitivity limit of 10–100 genome copies/reaction. Also comparatively good results were achieved for detecting different flaviviruses by the Pan-Flavi assay when compared to the flavivirus species-specific assays.The assay is rapid, broad-range flavivirus-specific and highly sensitive making it a valuable tool for rapid detection of flaviviruses in livestock samples, epidemiological studies or as useful complement to single flavivirus-specific assays for clinical diagnosis. Flavivirus of the family Flaviviridae consists of more than 70 virus species including many arthropod-borne viruses. It contains highly pathogenic agents such as the name-giving member yellow fever virus (YFV), West Nile virus (WNV), Japanese encephalitis virus (JEV), tick-borne encephalitis virus (TBEV) and dengue virus (DENV), which can cause encephalitis or hemorrhagic fever and pose some of the most challenging emerging diseases in humans [The genus n humans .Flaviviruses comprise three epidemiologically distinct groups; the mosquito-borne group, the tick-borne group and the unknown vector viruses . They arFlaviviruses pose a threat to public health worldwide, especially in developing countries. In recent years, there has been a dramatic increase in cases of DENV infections globally. The disease is endemic in more than 100 countries in Africa, the Americas, the Eastern Mediterranean, South-east Asia, and the Western Pacific. About 50–100 million cases of DENV infection are reported worldwide every year, resulting in 500,000 cases of hemorrhagic fever requiring hospitalization and 25,000 fatalities . Yellow Culex and Aedes flaviviruses were discovered throughout the world [A number of flaviviruses have repeatedly demonstrated their ability to expand their geographical range. Some current examples are the introduction of Usutu virus and Bagaza virus into central Europe ,13, or Zhe world -18. Aparhe world , T’Ho vihe world , Lammi vhe world , and Duche world . In contThe clinical diagnosis of flavivirus infections is not unambiguous due to unspecific symptoms varying from mild, febrile illness to viral hemorrhagic fever. Many of these viruses have a common transmission vector and can co-circulate in the same area. All these factors make a precise identification of the pathogen difficult.Today most diagnostic laboratories use serological assays for flavivirus testing. These tests are commonly based on the enzyme-linked immunosorbent assay (ELISA) and detect virus-specific IgM and IgG antibodies. Antibodies are undetectable prior to 5–7 days after onset of infection which hampers the usefulness of the serological methods. Molecular techniques, in contrast, can be used already in the acute phase and are known to be rapid, highly specific and sensitive.Since the early 1990s, several group-specific and generic molecular assays for detection of flaviviruses have been developed as reviewed previously -25. A nuThe aim of this study was to develop a rapid, sensitive and reliable TaqMan probe-based quantitative RT-PCR (qRT-PCR) assay for simultaneous detection of several flaviviruses, using the conserved NS5 gene region. The introduction of Locked-Nucleic Acid (LNA) bases in the probe increases robustness, specificity and sensitivity of the assay and has been shown to allow quantification of the viral load.Primers from publication -28 were In total, six combinations assembled from two degenerate primer sets  and three probes  were tested using RNA from flaviviruses and other viruses to check the performance of these Pan-Flavi assays. Combinations with Flavi all probe 3 showed earlier Ct values  from DENV, WNV, YFV, TBEV and JEV were detected quantitatively by using virus species-specific qRT-PCR assays, and the results were compared with those obtained by the Pan-Flavi assay for these viruses Table . All fouIn summary, the performance of the Pan-Flavi assay was comparable to virus species-specific assays except for minor differences for some strains of flaviviruses.The ongoing emergence of flaviviruses poses major public health concerns worldwide. Techniques used for molecular testing and surveillance of flaviviruses should be able to detect and identify a wide range of flavivirus species rapidly, with a high level of specificity and sensitivity. For this purpose, an LNA probe-based qRT-PCR (Pan-Flavi assay) for detection of RNA of various flaviviruses was developed. We tested several important human-pathogenic flaviviruses to demonstrate the ability to detect relevant members of the flavivirus genus. The Pan-Flavi assay was also tested with other viruses, in particular phleboviruses, alphaviruses and influenza viruses, to determine absence of cross-reactivity.In recent years, generic approaches targeting highly conserved NS3 and NS5 genes have been described for the detection of flaviviruses as summarized previously ,26. ThesIn the past 10 years, the primer sets targeting a conserved region of NS5 have been developed and optimized for a broad detection of flaviviruses. This approach was not only useful to detect known flaviviruses, but has also led to discovery of novel flaviviruses ,35,36.in-silico analysis provides only a limited prediction for the primer specificity, which still requires to be confirmed by laboratory testing. Designing of a single probe for detection of all flaviviruses is challenging because of the genetic diversity and the restricted length of conserved regions. One approach is the use of oligonucleotide mixtures covering many target sequence variants, which was applied for detection of DENV and Crimean-Congo Hemorrhagic fever virus (CCHF) [For the novel Pan-Flavi assay, the degenerate primer sequences share the conserved NS5 region Figure  targeteds (CCHF) ,38. Othes (CCHF) ,40. Firss (CCHF) ,41-45. OAt present, RT-PCR in nested or hemi-nested format is used most frequently. It requires sequencing for the identification of viruses and needs approximately one day for experimentation. In addition, this carries a high risk of contamination caused by open handling of PCR products, increasing the potential for false positives. In contrast, the Pan-Flavi assay presented here requires only some 50 minutes for specific and sensitive detection of several flaviviruses in one reaction. The detection sensitivity of YFV, DENV, TBEV, JEV and WNV, using the described Pan-Flavi assay, was close to or as good as the species-specific qRT-PCR assays. These differences may have resulted from deviant reactions conditions, enzyme kits and instruments, which were used as published or in-house established, and it is to mention, that WNV and JEV assays were performed in a two-step PCR which is known to be more sensitive than the one-step procedure .The sensitivity of the Pan-Flavi assay to detect RNA was demonstrated by using 10-fold serial dilutions of viral RNA from DENV-1, YFV and TBEV. The assay was able to detect 10–100 viral RNA copies per reaction, depending on the respective flavivirus. The assay allows the quantification of flavivirus RNA by use of standard plasmids or standardised in-vitro RNA, providing information regarding viral load and can be easily established in other laboratories. Therefore, the Pan-Flavi assay could be used to confirm the presence of flaviviruses in a sample without post amplification steps or complement species-specific flavivirus assays. The amplicon length is about 260 bp, which is sufficient to distinguish and identify different the virus by (pyro) sequencing or using for example an array.The Pan-Flavi assay allows rapid detection of a broad range of flaviviruses including local-endemic and exotic species with a high level of sensitivity and is cost-effective, making it suitable for screening of livestock samples, monitoring viral activity, outbreak investigation as well as surveillance studies of flaviviruses, and might help finding and identifying novel flaviviruses. We expect that the assay can be also helpful for testing clinical samples from patients with symptoms especially in an early phase of infection and without knowledge about locally prevalent flaviviruses or travel history in order to avoid parallel testing on several flaviviruses, although a negative test will of course not prove absence of a flavivirus infection.Re-emergence of West Nile virus in Europe and continuous spread of certain flaviviruses such as dengue, yellow fever and Japanese encephalitis viruses represent a global danger to public health. The Pan-Flavi assay presented here has been developed and evaluated in comparison with flavivirus species-specific qPCR assays to detect a broad range of flaviviruses simultaneously in a single-tube. This assay represents a valuable tool for the detection of flaviviruses in livestock samples, and will support molecular diagnosis as well as surveillance studies of various vectors and non-human hosts.All flavivirus and non-flavivirus strains listed below were derived from cell culture and provided by the Robert Koch Institute, Berlin, Germany. The following inactivated and stable virus preparations were used in this study: DENV-1 VR344 (Thai 1958); DENV-2 VR345 (TH-36 strain); DENV-3 VR216 (H87 strain); DENV-4 VR217 (H241 strain); WNV Uganda strain (AY532665); WNV Israel , kunjin virus , usutu virus (AY453411); JEV (ATCC SA14-14-2); Saint Louis encephalitis virus (SLEV) (ATCC VR-1265); TBEV strain K23 (AF091010); YFV strain 17D (X03700); YFV strain ASIBI (AY640589); YFV strain Brazil; YFV strain Ivory Coast; Russian Spring Summer Encephalitis virus (RSSEV); chikungunya virus (LR 2006); chikungunya virus African isolate; sindbis virus; Rift Valley Fever virus and influenza A virus subtype H5N1 (A/dk/Germany R603/06 H5N1).Two different standard preparations of H1N1 influenza viruses  used in this study were provided by the European Network for Diagnostics of Imported Viral Diseases (ENIVD). RNA of Nounane virus was kindly provided by Dr. Fabian Leendertz from NG2, Robert Koch Institute, Berlin. RNA of eleven African flaviviruses Table  were kinViral RNA from above described viruses was isolated from 140 μl aliquots of cell culture supernatants, using the QIAamp Viral Mini Kit  according to the manufacturer’s instructions. RNA was eluted in 100 μl of elution buffer and stored at -80°C until further use.′–5′ bridge, increasing the duplex stability, which in turn allows the use of shorter sequence probes matching the conserved sequence motif.The flaviviral NS5 gene contains conserved regions often used for PCR testing. Our laboratory results but also the literature  revealedThe Pan-Flavi qRT-PCR assay (Pan-Flavi assay) was run with the Transcriptor One-Step RT-PCR Kit  according to the manufacturer’s instructions. In brief, 5 μl of RNA, 10 pmol of primers Flavi all sense, Flavi all antisense 2 and DEN4 F, and a mixture of three probes (Table 6 copies/μl) were run in duplicate for quantification. Standard plasmid was prepared by cloning the PCR product obtained from YFV using a TOPO TA Cloning Kit  according to the manufacturer’s instructions and quantified by spectrophotometric method.Serial dilutions of a standard  of WNV as described previously . qRT-PCRYFV-specific primer combination YFV FP/RP and probe YFV LNA2 were used to detect and quantify genomic RNA of YFV as described previously . The assDENV-genomic RNA was tested and quantified by an in-house qRT-PCR as described previously . The ass2, 300 nM of each primer (JEV9250 F: CgTCCAAAAgCTgggATACAT and JEV9334 R: gTCCCAYCCggCggTRTC), 100 nM of probe (FAM-TCCgTgAYATAgCAggRAAgCAAgg –BBQ) and 2.5 μM of dNTPs, using a Stratagene Mx3000 instrument and the following conditions: 15 min at 95°C; 45 cycles of 15 s at 95°C and 30 s at 60°C. Ten-fold serial dilutions of the standard (plasmid 10–106 copies/reaction) were included in order to determine the limit of detection.A JEV-specific in-house qPCR assay was used for detection and quantification of JEV GE. qPCR was performed in two-step format on a Stratagene MX 3000 cycler. cDNA was generated using SuperScript II (Invitrogen) and was stored at -20°C until use. The test was performed using Platinum Taq polymerase (Invitrogen) in a total volume of 25 μl containing 5 μl of cDNA, 2 μl of 10x Reaction Mix, 4 mM MgClThe pan-flavi assay was evaluated using forward primers Flavi all S and DEN4 F, reverse primer Flavi all AS2 and Flavi all probe 3 mix. Based on an alignment of latterly available NS5 gene flavivirus sequences, we substituted primer DEN4F by Flavi all S2 and Flavi all AS2 by Flavi all AS4 to include some novel flaviviruses (Table RT: Reverse transcription; Tm: Melting temperature; Ct: Threshold cycle; GC: Genome copies; DENV: Dengue virus; YFV: Yellow fever virus; TBEV: Tick-borne encephalitis virus; WNV: West nile virus; JEV: Japanese encephalitis virus; LNA: Locked-nucleic acids.The authors declare that they have no competing interests.PP, MK conceived and designed the experiments; OL, MK, PP participated in the sequence alignment/assay design; PP, TK, UL, OF performed the Experiments; PP, OL, MK analysed the data; AS, OL, MN contributed reagents/materials/analysis tool, PP wrote paper. All authors have read and approved this manuscript.Pranav Patel is working as a scientist at Department of Centre for Biological Security 1, Robert Koch Institute, Berlin, Germany and has vast experience on developing rapid diagnostic assays and platforms for detection of emerging pathogens.


Here we present a novel approach entitled Magnetic Forced Hybridization (MFH) that provides the means for efficient and direct hybridization of target nucleic acids to complementary probes immobilized on a glass surface in less than 15 seconds at ambient temperature. In addition, detection is carried out instantly since the beads become visible on the surface. The concept of MFH was tested for quality control of array manufacturing, and was combined with a multiplex competitive hybridization (MUCH) approach for typing of Human Papilloma Virus (HPV). Magnetic Forced Hybridization of bead-DNA constructs to a surface achieves a significant reduction in diagnostic testing time. In addition, readout of results by visual inspection of the unassisted eye eliminates the need for additional expensive instrumentation. The method uses the same set of beads throughout the whole process of manipulating and washing DNA constructs prior to detection, as in the actual detection step itself. Over the past decade magnetism and magnets, both conventional and electromagnets, have found a growing field of application in the areas of biotechnology and medical technology. Combining the forces of magnetism with micro- and nanotechnology has further miniaturized the modes of application Today the movement of magnetic particles in magnetic fields, most commonly beads of nanometer or micrometer sizes, has been well characterized. The force on the magnetic particle is dependent on the diameter of the particle and the strength of the magnet as well as the magnetic susceptibility of the material. In general terms, provided a certain diameter of the magnetic particle and strength of the magnet the magnetic force on the particle markedly overcomes those forces on the particle created by common diffusion In the biological sciences magnetic particles are utilized for several different purposes such as transporting, carrying or collecting biomolecules in solution. For instance magnetism has proven useful in protocols for washing Here we present a novel approach entitled Magnetic Forced Hybridization (MFH) that provides the means for efficient and direct hybridization of target nucleic acids to complementary probes immobilized on a glass surface in less than 15 seconds, without need for heating. The approach is demonstrated for the purposes of quality control of microarrays, and is combined with a method for multiplex competitive hybridization (MUCH) This method development project used only synthetic and plasmid derived DNA for the HPV assay. For the quality control assay canine DNA extracted for a previous study was used.For the array quality control application canine DNA extracted for previous study was used. The sample was amplified targeting a 100 bp mitochondrial region as previously described TTTGTTACTGTGGTAGATACTAC and GP6+ GAAAAATAAACTGTAAATCATATTC) where the GP6+ was biotinylated. The amplification reactions were performed as previously described The HPV plasmids (HPV 18 and 45) were normalized to 100 ng/ml by using a ND-100 spectrophotometer . The L1 region of the HPVs were PCR amplified using the general primer set GP5+/GP6+  of 1 µm streptavidin-coated beads  and 90 µl 2× Bind and Wash solution  was prepared as previously optimized For the quality control assay, following removal of the non-biotinylated strand the beads were washed twice in 40 ul 1xPBS 0.2% Tween(20), and were dissolved in 30 ul 1xPBS 0.2% Tween(20).2, pH 7.6), after which the beads were resolved in 30 µl 1× AB. The immobilized single-stranded DNA was then divided into two reaction tubes (tube 1 and tube 2). Each reaction tube was subjected to a multiplex competitive hybridization (MUCH) using one out of two sets of probes. The concentration of each MUCH probe was 0.1 µM. Each MUCH probe contains a 5′-signature tag sequence that is complementary to a specific tag on the array 2HPO4, 2 mM KH2PO4, pH 7.4, with 0.2% Tween(20) added), after which the beads were resolved in 30 µl 1× PBS 0.2% Tween(20). The content of the two reaction tubes were then mixed.For the HPV diagnostic assay, after removal of the non-biotinylated strand the beads were washed twice in 40 µl 1× AB .The results were detected by visual inspection of the surface for a pre-determined pattern. The pattern was based on how the tags were placed when generating the surface. The surface were also photographed and/or characterized by an Agilent scanner (Agilent Technologies) to determine to which locations on the surface the DNA-beads had attached through hybridization.The array chips were scanned in an Agilent microarray scanner. The signals in the respective probe areas for HPV 18, HPV 45 and HPV 33  were obtained using Genepix Pro software  and integrating the average signal inside and outside the respective probe areas. From integrating the positive signal and the non-specific signal (HPV 33 probe), the signal to noise ratios could be calculated.This eliminates the need for conventional hybridization, based on traditional diffusion carried out at moderate to high temperatures (above 50 degrees Celsius) and under long time-spans (1 hr –72 hrs). In addition, the method facilitates upstream template preparation since the same beads are used through the entire protocol.The most straightforward application of MFH is to hybridize an amplicon that has been made single-stranded directly to an array surface. If all probes on the array carry a common sequence, the method can be applied to test the quality of array manufacturing. The theoretical printed pattern and the results of a quality control can be seen in In the context of highly complex samples MFH can be combined with additional methods for genetic discrimination, such as multiplex competitive hybridization (MUCH). In this study two sets of type-specific MUCH probes that align to the L1 region of 8 HPV types  were used Since there are small differences between the HPV types, the competitive MUCH approach, as an upstream protocol, is necessary to discriminate the different HPV sequences and obtain reliable results. The address tags on the MUCH-probes, however, have minimum sequence similarity and are used in the MFH that is performed at ambient temperature.In Hybridization of a sample molecule to a surface is a process that has traditionally required a period of anywhere from 1 hour to 72 hours. With the presented method for Magnetic Forced Hybridization (MFH) this dogma no longer holds true.Possible applications of MFH include surface array quality control for the assessment of spot quality. Here, the most straight-forward way of using MFH is to introduce a common linker sequence into the printed array-probes. Complementary oligos attached to beads can then hybridize directly to this sequence. This provides a powerful means to quickly oversee the general printing quality and saves time compared to quality control protocols that are based on traditional hybridization. As further presented, the MFH method has applications in diagnostics and treatment where DNA testing of cell samples for presence different Human Papilloma Virus (HPV) strains is needed. Other possible applications include detection and identification of different species, or DNA testing of susceptibility mutations or disease mutations in human genomes.The majority of the current techniques for DNA testing rely on PCR amplification prior to genotyping. This step offers improved sensitivity, allowing typing of complex and non-complex samples in combination with a range of different detection technologies. These include Sanger DNA sequencing These problems are addressed in the present work by introducing a liquid phase multiplex competitive hybridization (MUCH) of target specific probes, followed by hybridization to generic tag arrays In conclusion, in clinical settings, the reduction in time and cost provided by the MFH method has a significant impact. In all diagnostic tests, time is a factor of great importance. With retained precision faster tests are preferred because it means decreased costs and in some cases even saving lives. Thus, a diagnostic test benefits from simple preparation procedures, inexpensive and simple equipment, cheap reagents and ease of use. Point-of-care testing to date is relying on instrumentation that is not available in all laboratories. PCR is central piece of instrumentation which can be removed entirely from the current Magnetic Forced Hybridization (MFH) protocol by the use of commercially available kits for isothermal amplification Table S1Surface bound tags for quality control array and HPV array.(DOC)Click here for additional data file.Table S2Signature tagged much probes.(DOC)Click here for additional data file.


Giardia duodenalis is a common protozoan parasite of humans and animals. Genetic characterization of single loci indicates the existence of eight groups called assemblages, which differ in their host distribution. Molecular analyses challenged the idea that G. duodenalis is a strictly clonal diplomonad by providing evidence of recombination within and between assemblages. Particularly, inter-assemblage recombination events would complicate the interpretation of multi-locus genotyping data from field isolates: where is a host infected with multiple Giardia genotypes or with a single, recombined Giardia genotype.G. duodenalis isolates from humans and animals were performed.Population genetic analyses on the single and multiple-locus level on an extensive dataset of Giardia duodenalis. At the multi-locus level, our statistical analyses are more congruent with clonal reproduction and can equally well be explained with the presence of multiple G. duodenalis genotypes within one field isolate.Our analyses indicate that recombination between isolates from different assemblages are apparently very rare or absent in the natural population of G. duodenalis assemblages is either very rare or absent. Recombination between genotypes from the same assemblage and genetic exchange between the nuclei of a single cyst needs further investigation.We conclude that recombination between  Giardia duodenalis  is the etiological agent of giardiasis, a gastrointestinal infection of humans, companion animals, livestock and wildlife. Symptoms of a G. duodenalis infection range from asymptomatic to severe diarrhea as well as chronic disease [G. duodenalis has a simple life cycle comprising rapidly multiplying, non-invasive trophozoites on the mucosal surface of the intestine, and the production of environmentally resistant cysts that are shed with the host faces. Infectious cysts are transmitted by the faecal-oral route either by direct contact or by ingestion of contaminated food or water [duodenalis is considered as a species complex, whose members show little variation in their morphology, yet can be assigned to eight distinct assemblages (A to H) based on enzyme electrophoretic and genetic studies [vice versa may occur [Giardia cysts are shed into the environment, making it very difficult to determine the primary source of the infection. Genetic characterization has been extensively used to assess the role of animals in the epidemiology of human infection and to develop tools for tracing sources of infection. However, the zoonotic potential of G. duodenalis remains a major and unresolved issue [ disease . G. duodor water . G. duod studies ,4. Assem studies ,6. In enay occur -9. Direced issue ,10,11.G. duodenalis assemblage A and B appears to be high: Irrespective of the genetic marker used, sequences from human and animal field isolates frequently appeared similar, if not identical [G. duodenalis isolates, multi-locus sequence typing strategies were introduced [Giardia field isolates repeatedly constituted a combination of loci derived from different assemblages [Many molecular epidemiological studies have been based on the analysis of a single marker, often from a limited number of isolates. Using single locus approaches, the zoonotic potential of dentical . In ordetroduced -18. Whentroduced . Surprisemblages . The latGiardia field isolate is not a singular clone, but consists of a mixture of different Giardia genotypes. A Giardia field isolate is often not more than a DNA extract, obtained either directly from a stool sample or indirectly after (immune-) isolation of faecal cysts. For this situation, the uptake of genetically different Giardia cysts from the environment by a host, or subsequent infection of an already infected host, likely without overt symptoms, with a different Giardia genotype, must occur. As starting in vitro cultures from field samples prove to be very difficult due to variations in excitation and adaptation, and the ever-present bacterial and fungal contamination, it is hard to affirm the clonality of Giardia field isolates [Giardia isolates are clonal, but the mixing of loci from different assemblages have arisen by (para) sexual recombination, i.e. genetic exchange, between G. duodenalis assemblages. Although G. duodenalis shows no cytological evidence of meiotic and sexual recombination, several studies challenged the idea that G. duodenalis is a strictly clonal organism [G. duodenalis genome of true homologs of genes involved in meiosis in other eukaryotes [G. duodenalis: (i) between the two nuclei at an individual level, (ii) between individuals of the same assemblage and (iii) between individuals of different assemblages.One explanation is that a isolates ,20. Alteorganism ,22. Theskaryotes ,24; (ii)karyotes ,26;  are available for those isolates. All molecular epidemiological data were stored and analyzed in Bionumerics . A selection of these sequences was made using the same strategy as previously described [A European network of public and veterinary health Institutions that focused on zoonotic protozoan parasites  was established . Within The loci beta-giardin (BG), glutamate dehydrogenase (GDH), and triose phosphate isomerase (TPI) were sorted into their different genes, assemblages, and sub-assemblages  as well as alignments along the gene using previously defined references . Tests fSequences were aligned by using MAFFT , distancOccurrence of a recombinant of B assemblage and E assemblage at TPI locus of a human isolate is an evidence of genetic exchange between the two assemblages. Under the hypothesis of selective neutrality, we calculated the probability of our observation; each recombinant type was found in only one sample at the particular locus, to statistically test the hypothesis, we made an assumption that a recombinant and a non-recombinant sequence type are selectively neutral, and calculated the probability that each recombinant type was observed exactly once in the samples of DNA sequences under the assumption of selective neutrality. The probability was calculated using the formula ,n is the number of samples at a given locus, the symbol k is the number of distinct recombinant types and a non-recombinant type, the function s is the stirling numbers of the first kind, and the bracket |.| indicates an absolute value.where the symbol P of observing as many or more individuals of the particular genotype as actually observed in the sample,The extent to which the predominant genotype was overrepresented can be quantitatively evaluated by calculating the probability x = probability of the multi-locus genotype under the null hypothesis of full recombination, estimated by multiplying the observed frequency of the single-locus genotypes; n = number of individuals sampled; and m = number of individuals in the sample with the particular genotype [where genotype .This criterion is the probability of observing as few different multi-locus types as actually observed, given the size of the samples and expected multi-locus type frequencies under the hypothesis of full recombination . The proG. duodenalis, two genotypes were recorded at each locus. Hence we refer to one genotype by a letter 0 and to the other genotype by a letter 1. Linkage disequilibrium D12 between locus 1 and locus 2 was calculated using the equation [This is a standard test for non random association between loci, under the assumptions of random mating, and non-overlapping generations . In our equation ,p00 is the frequency of congruent assemblages 00, p0* is the frequency of assemblage whose locus 1 is the type 0 and locus 2 is either 0 or 1, and p*0 is the frequency of assemblage whose locus 2 is the type 0 and locus 1 is either 0 or 1.where In the case of three-locus typing, linkage disequilibrium was calculated using the equation 1 in ,D23D13 and D12 are pair-wise linkage disequilibrium between the three loci 1, 2, and 3, p000 is the frequency of multi-locus types 000. The symbol * is a wildcard for either genotype 0 or 1. Thus, p00* is the frequency of the assemblages having genotype 0 at the first and at the second loci and genotype at the third locus is either 0 or 1. P-values for linkage disequilibrium were obtained by a Monte Carlo approach as described in the test e but using D as the test statistic.where G. duodenalis was calculated using the observed frequencies of two genotypes at each of the three loci  [In a recent study, recombination between homologous loci from different assemblages was identified in at least three isolates . In thisisolates  to analyK225348) . No inteK225348) , but turK225348) .Lack of inter-assemblage recombination will eventually result in the independent and divergent evolution of alleles from different assemblages . PhylogeG. duodenalis isolates from humans, between assemblages C and D in dog isolates, between A and F in cat isolates and between A and E in isolates from livestock. The presence of a particular multi-locus type in great excess is often the most robust and significant evidence of clonal reproduction, i.e. lack of recombination [G. duodenalis isolate was based on the concatenated assemblage-typing of the three different loci BG, GDH and TPI. Only two multi-locus types are found in great excess in humans, namely AAA (38%) and BBB (59%). Of the six possible combinations of A and B in three-locus genotypes, only 3% are found , was originally developed to index the extent of clonality within bacterial populations [Three formal tests for clonality were performed and all three yielded highly significant results in the four og Table . These rulations . Small eGiardia duodenalis assemblages. In fact, in a considerable number of isolates present in the database, this recombinant was found only once. The test under the assumption of selective neutrality between a recombinant and a non-recombinant type supported that a Giardia recombinant might contribute genetically little to the population structure of Giardia duodenalis. It could be argued that a zoonotic transmission event between a human individual and domestic livestock is unlikely to produce a novel viable G. duodenalis assemblage by a recombination of existing assemblages at TPI locus. Molecular typing of the Giardia isolates at GDH locus in addition indicated that a recombinant at the GDH locus contributes genetically very little to the population structure of Giardia duodenalis. The observation of a recombination event between a GDH DNA-sequence (GQ337967) and a DNA sequence probably derived from its host (sheep) is highly anomalous. A PCR-artifact or sequencing error seems more likely.Occurrence of one recombination event between assemblage B and assemblage E at the TPI locus of a human isolate is an indication that genetic exchange between the two assemblages has occurred. However, detection of a single recombinant is important but not sufficient information to assert the nature of recombination between G. duodenalis is also limited. These population genetic tests have been applied to a number of microorganisms, to support a clonal population structure of Leishmania as well as Trypanosoma, and to reject a clonal population structure of Candida albicans[G. duodenalis, both a single and multi-locus based typing method, we opt for the hypothesis that the population structure of G. duodenalis assemblages is clonal. This conclusion is by no means in conflict with the existence of recombination within the two nuclei of a cyst at the genomic and the cellular level [The lack of observation of selectively neutral or fitter recombinant types was not due to a limited capacity of detecting a recombination event that could be associated with a single locus typing method. The analysis of the three-locus sequence typing data by applying a battery of population genetic tests supported that the contribution of recombination events between the three loci to the population structure of  albicans. Judgingar level ,22, nor Giardia genotypes from different assemblages. This is probably an underestimation, as mixed infections are not always detected. Unequal loads or stability of the microorganisms of interest in the original sample, preferential immuno-purification or preferential binding of primer pairs to one of microorganisms will result in the detection of only one of them. PCR assays based on the use of assemblage-specific primers have been developed to show the frequency of mixed infections [Giardia genotypes from the same assemblage. Besides allelic sequence heterogeneity, the latter may be an additional explanation for the presence of ambiguous nucleotides (“double peaks”) present in the sequences of many isolates. Unfortunately, it is still not possible to distinguish between a clonal isolate and an isolate consisting of a mixture of the same assemblage. A PCR-based approach using a generic set of primers appears to be unreliable for the detection of mixed infections.Our molecular epidemiological database consists of 1312 human and animal isolates with two or more loci. Fifteen percent (n = 197) of these isolates constitute a combination of loci derived from different assemblages. Based on the analyses of this study, we conclude that these isolates are a mixture of different fections ,44. It aG. duodenalis in and between human and animal populations. The relatively poor genetic resolution of the available single loci used for these studies could not be solved by combining several of these loci to a multi-locus genotyping scheme. Multilocus-sequence typing directly on field isolates is hampered by the occurrence of mixed infections in both animals and humans. Our data imply that genetic recombination between genotypes of different assemblages is either very rare or absent in the G. duodenalis population. Thus, multilocus sequence types from field isolates, which consist of more than one assemblage, should be interpreted as a mixed infection. Improved in vitro culturing methods and novel molecular typing methods, such as single cyst PCR, are required to investigate the directionality and frequency of animal to human transmission and to determine the frequency of intra-assemblage recombination events [Molecular epidemiology is probably the best method available to study transmission dynamics of n events .The authors declare that they have no competing interests.TM, ML, SC, and HS performed data collection and sequence analysis. KT and AS performed population genetic and mathematical analysis. HS performed phylogenetic analysis. KT, SC, EL and HS were involved in the study design and interpretation of the results. KT and HS drafted the manuscript and wrote the final version. All authors read and approved the final manuscript.Table S1. Inter-assemblage recombination events within three loci. Click here for file


Genetic variations in DNA double-strand break repair genes can influence the ability of a cell to repair damaged DNA and alter an individual’s susceptibility to cancer. We studied whether polymorphisms in DNA double-strand break repair genes are associated with an increased risk of glioma development.XRCC3, BRCA2, RAG1, XRCC5, LIG4, XRCC4 and ATM) in a case–control study including 384 glioma patients and 384 cancer-free controls in a Chinese Han population. Genotypes were determined using the OpenArray platform.We genotyped 10 potentially functional single nucleotide polymorphisms (SNPs) in 7 DNA double-strand break repair pathway genes  TT genotype , as well as the TC genotype . We also found that the homozygous variant genotype (GG) of XRCC4 rs1805377  was associated with a significantly increased risk of gliomas . Interestingly, we detected a significant additive and multiplicative interaction effect between the LIG4 rs1805388 and XRCC4 rs1805377 polymorphisms with an increasing risk of gliomas. When we stratified our analysis by smoking status, LIG4 rs1805388 was associated with an increased glioma risk among smokers.In the single-locus analysis there was a significant association between gliomas and the LIG4 rs1805388 and XRCC4 rs1805377, alone or in combination, are associated with a risk of gliomas.These results indicate for the first time that  Gliomas are the most common type of primary intracerebral neoplasm in China as well as in the West, and comprise more than 40% of primary brain tumors in humans-3. AlthoDNA double-strand breaks (DSBs) can be generated during V(D)J recombination, class-switch recombination at the immunoglobulin heavy chain (IgH) locus or meiosis and result from a variety of factors including ionizing radiation and reactive oxygen species. InadequXRCC3, BRCA2, RAG1, XRCC5, LIG4, XRCC4 and ATM in the development of gliomas, and further evaluate their gene-gene and gene-environment interactions in the development of glioma.Recent evidence suggests that several single nucleotide polymorphisms (SNPs) in the DSB repair pathway genes may be prognostic biomarkers for GBM survival and modulate gamma-radiation-induced mutagen sensitivity in glioma patients,13. GeneThe study was approved by the Ethics Review Board of Nanjing Medical University. All studies involving human subjects were conducted under full compliance with government policies and the Helsinki Declaration. From 2005 to 2010, a total of 447 patients with histopathologically confirmed gliomas were recruited from the Department of Neurosurgery of Jiangsu Province Hospital  and Beijing Tiantan Hospital Neurosurgery Center (BTHNC). The tumors were graded according to the World Health Organization (WHO) classification. A total′ or 3′-UTRs with a minor allele frequency (MAF) ≥ 0.05 in the general Han Chinese population were selected.Potential functional SNPs within each gene were identified through an extensive mining of the databases of the International HapMap Project and dbSNP. As a result, 10 SNPs in the coding sequence, promoter, splice sites, and 5Genomic DNA was isolated from leukocyte pellets of venous blood by proteinase K digestion and phenol-chloroform extraction. Genotyping was performed using the OpenArray platform . This platform employs a chip-based TaqMan genotyping technology. Sequences of primers and probes are available on request. Genotyping was conducted according to the manufacturer’s standard protocols, and genotype calls were made by OpenArray SNP Genotyping Analysis Software V.1.0.3. To ensure quality control, genotyping was performed without the knowledge of individual case–control status. We randomly selected 5% of the samples to be genotyped again by different investigators and the reproducibility rate was 100%. To validate the genotyping results, selected PCR-amplified DNA samples  were confirmed by DNA sequencing, and these results were found to be 100% concordant.t test (age and pack-years) for continuous variables and the χ2 test for categorical variables . The Hardy-Weinberg equilibrium (HWE) was evaluated in control subjects using a goodness-of-fit χ2 test with 1 degree of freedom. The effect of each SNP on glioma risk was estimated as an odds ratio and 95% confidence intervals (95% CI) by unconditional logistic regression adjusted by age, gender, smoking and drinking status. A score test of linear trend was conducted for each SNP using a three-level ordinal variable. To minimize false positive results generated from the multiple statistical tests used in our analysis, we applied a false discovery rate (FDR) method to the P values for trend[LIG4 rs1805388 and XRCC4 rs1805377 polymorphisms. A more-than-multiplicative gene-gene or gene-environment interaction was evaluated using logistic regression analysis. When the test for multiplicative interaction was not rejected, further test for an additive interaction was done by a bootstrapping test of goodness-of-fit of the null hypothesis, for no departure from the additive model compared to an alternative hypothesis, and for a departure from an additive model by using Stata software . All the other statistical analyses were performed with SAS 9.1.3 software (SAS Institute).Demographic variables between cases and controls were compared using Student’s or trend. To evalP = 0.277), gender (P = 0.715), smoking status (P = 0.465) and drinking status (P = 0.222), suggesting that our frequency matching of the demographic characteristics was satisfactory.The distribution of demographic characteristics of the 384 cancer cases and 384 cancer-free controls available for this analysis are summarized in Table p > 0.05).Primary information of the 10 functional SNPs found in the Chinese population in the dbSNP database is presented in Table LIG4 rs1805388 was associated with a significantly increased risk of gliomas compared to the wild-type CC genotype . Similarly, compared with the common homozygous genotype, carriers with XRCC4 rs1805377 homozygous variant genotype showed a significantly increased risk of gliomas . Furthermore, the association for allele variants were dose dependent for each locus .In the multivariate logistic regression models Table , each ofLIG4 rs1805388 and XRCC4 rs1805377 polymorphisms and tobacco smoking with respect to the risk of gliomas. We detected a significant additive (Pinteraction = 0.013) and multiplicative interaction (Pinteraction = 0.046) effect between LIG4 rs1805388 and tobacco smoking for the development of gliomas. Compared with never-smokers carrying the wild-type genotype of LIG4 rs1805388, those ever-smokers with variant-containing genotype of LIG4 rs1805388 polymorphism had a significantly increased risk to develop gliomas  . In comparison with the reference combination of LIG4 rs1805388 CC and XRCC4 rs1805377 AA, the combination of the LIG4 rs1805388 CT+TT genotype together with XRCC4 rs1805377 AG+GG genotype was found to be significantly associated with glioma . Furthermore, significant more-than-multiplicative (0.005) and more-than additive (0.009) gene-gene interactions of these two loci (LIG4 rs1805388 CT+TT and XRCC4 rs1805377 AG+GG) were found in relation to the risk of gliomas.The LIG4–XRCC4 complex plays a fundamental role in DNA non-homologous end-joining and is present in all eukaryotes. It has been demonstrated that XRCC4 can stimulate LIG4 activity and is required to stabilize LIG4. Thus, we estimated the combined effect of XRCC4  and one non-synonymous SNP in LIG4  are associated with the increased susceptibility to gliomas in a Chinese population.Accumulating evidence demonstrates that the DSB repair pathway plays a critical role in repairing double-strand breaks caused by a variety of exposures. Although genetic variants in DSB repair pathway genes are considered as potential risk factors for various cancers, less evidence exists as to the potential role of the DSB repair pathway genes polymorphisms on glioma susceptibility. To our knowledge, this study is the first to provide a comprehensive evaluation of the relationship between polymorphisms in both NHEJ and HR pathway genes and susceptibility to gliomas. On the basis of our analysis of 384 controls and 384 glioma patients, we observed that one splice-site SNP in XRCC4 rs1805377 and LIG4 rs1805388 polymorphisms have been informative in understanding the potential roles of these two polymorphisms in the development of gliomas. The LIG4 rs1805388 polymorphism results in a nonsynonymous amino acid change from threonine to isoleucine at the N-terminal of the LIG4 protein that is essential for its activity[XRCC4 rs1805377 polymorphism in intron 7 may have functional significance since the nucleotide change potentially abolishes an acceptor splice site at exon 8[Previous researches on the function of the activity. Two linactivity and incractivity. The XRCt exon 8.LIG4 rs1805388 which causes a nonsynonymous amino acid change at the N-terminal of the LIG4 protein and XRCC4 rs1805377 interacted to modulate the risk of gliomas as a joint effect. Tseng et al.[XRCC4 (rs1805377) and LIG4 (rs1805388) genes interacted to modulate the risk of lung cancer  and demonstrated that LIG4 rs1805388 and XRCC4 rs1805377 polymorphisms are linked significantly with high fractional allelic loss (FAL), an indicator of genomic instability. Taken together, considering the functional relevance of these two proteins, an individual SNP or combinations of these two SNPs may change the activity of the LIG4-XRCC4 complex and pose a substantial influence on the development of gliomas.Gene-gene interaction was also studied since XRCC4 and LIG4 proteins form a tight and specific complex that catalyzes ligation of processed DNA ends. Although LIG4 interacts with the coiled-coil region of human XRCC4 via the region that lies between the two C-terminal BRCT domains,20,21, tg et al. performeXRCC4- or LIG4-deficient mouse fibroblasts exhibit marked sensitivity to ionizing radiation, growth defects and premature senescence[XRCC4 or LIG4 null mice die in late embryogenesis accompanied by defective lymphogenesis and massive apoptotic cell death of newly generated postmitotic neurons[LIG4 or XRCC4 in animals can lead to increased rates of neoplastic transformation. Although loss of p53 expression rescues neuronal death and embryonic lethality, XRCC4 or LIG4/p53 double-null mice routinely succumbed to RAG-dependent pro-B lymphomas with translocations/amplifications of c-myc and IgH loci[Y288C mice (a mouse model for human LIG4 syndrome) exhibit multiple defects in lymphocyte development and a hypomorphic LIG4 mutation can confer strong predisposition to lymphoid malignancies[LIG4 haploinsufficiency with decreased NHEJ activity contributes to development of soft tissue sarcomas that possess clonal amplifications, deletions and translocations[LIG4/p53 double-null mice can develop medulloblastoma[XRCC4/p53 doubly deficient in nestin-expressing neuronal progenitor cells can lead to early onset of neuronally differentiated medulloblastomas[XRCC4 was found in grade II, III, IV of astrocytoma compared to normal brain tissues and decreased expression of XRCC4 was significantly associated with a poor prognosis (P < 0.05)[LIG4 or XRCC4 activity plays a role in human carcinogenesis.Overwhelming evidence indicates that our findings are biologically plausible. NHEJ is a multistep process initiated by the XRCC5/XRCC6 dimer  which immediately binds to both broken ends of DNA and recruits the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) forming the trimeric DNA-PK holoenzyme,24. Finanescence,26. The nescence. Further neurons,29. ManyIgH loci,29. Nijngnancies. In addiocations. A defecblastoma. Consistlastomas. Signifi < 0.05). These sLIG4 rs1805388 were associated with an increased risk of gliomas among smokers under a dominant model. Our data indicated the presence of an interaction between the NHEJ pathway genes and smoking status. In addition, smokers with less efficient DSB repair capacity may be more likely to develop gliomas.Since tobacco is a well-confirmed inducer of DNA damage, in particular DSBs, we perfXRCC4 rs1805377 and LIG4 rs1805388 polymorphisms and glioma risk[P value. Second, the frequencies of XRCC4 rs1805377 and LIG4 rs1805388 polymorphisms and patterns of linkage disequilibrium (LD) are very different in two HapMap populations (CEU and CHB). Thirdly, it could be that the association of this variant may be population-specific and the interaction between genes and environmental factors vary in different human populations. Our results require confirmation in further GWA studies of gliomas in Chinese population.Currently, the number of genome-wide association studies (GWAS) has been growing rapidly, leading to the discovery of many new variants associated with complex diseases. Two recent genome-wide association studies (GWAS) of risk of glioma in European populations did not identify an association between the oma risk,37. TherLIG4 and XRCC4 genes, which may provide enhanced risk assessment. Finally, we used a relatively comprehensive analysis of 10 polymorphisms in 7 candidate genes involved in DNA double-strand break repair pathways. However, our current study has some limitations. First, we were not able to explore the exact biological mechanism of how XRCC4 rs1805377 and LIG4 rs1805388 polymorphisms affect the development of gliomas. Second, this study was a hospital-based case–control study; thus, selection bias in the present study may have led to spurious findings. However, the controls and cases were matched on age and sex, which may have minimized selection bias. Third, our findings need to be replicated in other independent studies. Fourth, we did not perform the stratified analysis by glioma grade due to the limited sample size. Therefore, large-scale studies and functional evaluation are warranted to replicate these findings in an independent population that is well-powered to performed stratified analysis by glioma grade.Our study has several strengths. First, all tested SNPs were in Hardy-Weinberg equilibrium in controls. Second, in this study, a standardized genotyping approach was performed and quality control samples indicated a high degree of reproducibility of the genotyping results. Third, we were able to examine the association between the 2 SNPs and the risk of gliomas in a well-described and racially homogeneous population of the same ethnicity. Moreover, we use a pathway-based approach to estimate the combined effect of XRCC4 rs1805377  and LIG4 rs1805388  polymorphisms contribute to the risk of developing gliomas, alone or in combination. This is also the first report to investigate the role of gene-gene and gene-environment interactions of these 10 potential functional SNPs of 7 major NHEJ and HR pathway genes in the development of gliomas. These findings may be helpful in improving our understanding of the etiology of gliomas. XRCC4 rs1805377 and LIG4 rs1805388 polymorphisms may be useful susceptibility biomarkers for gliomas and aid in the development of diagnostic strategy to reduce the burden of gliomas. Future larger scale studies with ethnically diverse populations and functional evaluation are needed to confirm our findings.In conclusion, of the 10 potential functional polymorphisms investigated here, we provide the first evidence that the The authors declare that they have no competing interests.PZhao and PZou participated in collection of data and manuscript preparation. PZou and LZ performed the statistical analysis. WY, CK, YY and TJ provided the samples. PZhao participated in study design and critically revised the manuscript. PZhao and TJ participated in study design and manuscript preparation. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/13/234/prepub