Aedes aegypti, the mosquito vector of dengue viruses, utilizes its innate immune system to ward off a variety of pathogens, some of which can cause disease in humans. To date, the features of insects' innate immune defenses against viruses have mainly been studied in the fruit fly Drosophila melanogaster, which appears to utilize different immune pathways against different types of viruses, in addition to an RNA interference–based defense system. We have used the recently released whole-genome sequence of the Ae. aegypti mosquito, in combination with high-throughput gene expression and RNA interference (RNAi)-based reverse genetic analyses, to characterize its response to dengue virus infection in different body compartments. We have further addressed the impact of the mosquito's endogenous microbial flora on virus infection. Our findings indicate a significant role for the Toll pathway in regulating resistance to dengue virus, as indicated by an infection-responsive regulation and functional assessment of several Toll pathway–associated genes. We have also shown that the mosquito's natural microbiota play a role in modulating the dengue virus infection, possibly through basal-level stimulation of the Toll immune pathway. Aedes aegypti mosquito is largely responsible for the transmission of dengue viruses that cause disease in humans. The virus is taken up with an infected blood meal from which it will first infect the mosquito gut tissue. From the gut it will migrate to other parts of the mosquito, including the salivary glands, from where it can be transmitted to another human upon a second blood meal. In this study we show that the mosquito utilizes its innate immune system to control dengue virus infection. Infection with the virus will result in the activation of mosquito immune responses that are mainly controlled by the Toll pathway. These responses entail antiviral activities that limit infection with the virus. We also show that the mosquito's natural microbial flora play a role in modulating the dengue virus infection, possibly through the stimulation of the mosquito's immune system.The  Flaviviridae, genus Flavivirus. They are transmitted between humans primarily by the mosquito Ae. aegypti and by Ae. albopictus as a secondary vector The dengue viruses, whose geographic distribution resembles that of malaria, has become the most important arboviral pathogen in recent years because of its increasing incidence in the tropics and subtropics as well as its high morbidity and mortality. The public health impact of dengue is enormous, given that 2.5 billion people live in dengue-endemic areas and are at daily risk of infection The extrinsic incubation period of dengue viruses in the mosquito is 7–14 days and is dependent on the mosquito strain, virus genotype, and environmental factors such as humidity and temperature D. melanogaster have shown that arthropod immune responses are largely regulated by two main pathways, the Toll and immune deficiency (Imd) pathways In arthropods, innate immunity plays an important role in limiting pathogen infection, both through the production of effector molecules such as antimicrobial peptides and through phagocytosis and encapsulation, secretion of physical barriers, and melanization D. melanogaster, Anopheles gambiae and Ae. aegypti; in contrast, the upstream pattern recognition receptors and the downstream effectors have differentiated quite significantly among the three species, probably as a result of different microbial exposures Activation of the Toll pathway by microbes through pattern recognition receptors (PRRs) leads to a cascade of events that result in the degradation of the negative regulator Cactus, translocation to the nucleus of transcription factors such as Dif, and a rapid increase in antimicrobial compounds and other effectors The Rel family transcription factors, Dif and Relish in Drosophila or their corresponding Rel1 and Rel2 in mosquitoes, can be studied through RNA interference (RNAi)-mediated silencing of the negative regulators Cactus and Caspar, respectively D. melanogaster, the RNAi-mediated defenses appear to be key players in the defense against a broad range of viruses D. melanogaster has been shown to use its RNAi machinery and the Toll pathway to limit Drosophila×virus infection (a member of the Dicistroviridae) Drosophila C virus infection (a member of the Birnavirus family) D. melanogaster RNAi machinery in the defense against two diverse animal viruses: a flock house virus and a cricket paralysis virus At present, relatively little is known about the anti-viral defense systems in insects. In Ae. aegypti, Sindbis virus  infection has been shown to induce the Toll pathway-related Rel1 transcription factor and three transcripts of the ubiquitin-ligase pathway genes, which are known regulators of NFkB-like proteins Ae. aegyptiAlphavirus; Togaviridae) in An. gambiaeA. gambiae, including a 70-kDa heat shock protein factor that later was shown to influence the virus's ability to propagate in the vector Similarly, limited knowledge on the antiviral response in mosquitoes is available. In Ae. aegypti genome sequence Ae. aegypti to the infection of dengue virus serotype 2 (DENV-2), and show that DENV-2 induces a set of genes corresponding to the Toll and JAK-STAT pathways. Activation of the Toll and Imd pathways in Ae. aegypti through RNAi-mediated silencing of Cactus and Caspar caused a reduction in dengue virus infection level that appeared to be controlled primarily by the Toll pathway. Repression of the Toll pathway through MYD88 gene silencing resulted in higher dengue virus infection levels. We also present compelling evidence for an inhibitory effect of the mosquito's natural microbiota on virus infection and discuss the implications of these findings and the potential role of the mosquito's microbial exposure and innate immune system in modulating dengue virus transmission.The recently available Ae. aegypti mosquito to systemic dengue infection at the gene-specific level in the midgut and remaining carcass by using a genome-wide transcriptional profiling approach. A comparison of the transcript abundance in the two body compartments of mosquitoes that were fed 10 days earlier on dengue-infected blood or naïve blood revealed broad responses to virus infection that entailed a variety of physiological systems , multiple Toll receptors , Spätzle ligands , Gram-negative binding proteins (AAEL007626 and AAEL003889), and the antimicrobial peptides DEFD, CECA, D, E & G . In total, Cactus gene silencing resulted in the up-regulation of 460 and down-regulation of 1423 genes belonging to different functional classes, with a predominant representation by immune genes . The regulation of a variety of other functional gene groups by Rel1 is indicative of the multiple functional roles of the Toll pathway, including its contributions to immunity and development Differential gene regulation in Caspar-depleted mosquitoes was much less pronounced, with only 35 genes being induced and 137 being repressed. Those induced by Caspar silencing included TEP13 and the antimicrobial peptides DEFE and gambicin (AAEL004522 and AAEL003849). Rel1 and Rel2 are most likely regulating additional genes that were not detected because of the limited sensitivity of microarray-based gene expression assays.A comparison of the dengue infection-responsive gene repertoire to that of Cactus gene-silenced mosquitoes showed a significant overlap, with 41% (18 of 44) of the immune genes being up-regulated by both the virus infection and Cactus gene silencing . In contD. melanogasterD. melanogaster in response to Drosophila C virus infection Further dissection of the expression cluster V defined three main groups: Toll pathway-, JAK-STAT pathway-, and signal modulation- related genes. The signal modulation cascade genes included four C-type lectins (CTLs) and six serine protease inhibitors (SRPNs). A plausible hypothesis is that both the Toll and JAK-STAT pathways may be regulated at least in part by the same signal modulation cascade that includes serine proteases and serpins. Consistent to this hypothesis, evidences suggest that the JAK-STAT pathway could be indirectly activated by the Toll cascade in D. melanogaster, in which the Toll pathway, but not the Imd pathway, has been shown to be involved in limiting X-virus infection. Together with the gene expression data discussed above, our results suggest that the infection of mosquitoes with dengue virus induces the Toll pathway, which then exerts an anti-dengue effect.The prominent activation of the Toll pathway (Rel1)-regulated genes in response to dengue infection strongly suggested that this pathway is involved in the mosquito's anti-dengue defense. To investigate this hypothesis, we tested the effect of both Cactus and Caspar gene silencing on virus infection in the midgut and carcass at 7 days after an infectious blood meal. This cactus gene silencing reduced the extent of dengue infection in the midgut by 4.0-fold (P<0.05), while Caspar gene silencing had no effect on viral infection when compared to the GFP dsRNA control . The lowAe. aegypti on the mosquito's immune gene expression and susceptibility to dengue virus infection, we compared transcription and infection levels between normal septic mosquitoes and mosquitoes from which the bacterial flora had been eliminated through antibiotic treatment  has also been observed in response to Sindbis virus infection in a previous study D. melanogaster, infection with either the Drosophila×virus or E. coli has been shown to induce the same antimicrobial peptide genes, suggesting that these two diverse classes of pathogen can activate the same immune response pathway D. melanogaster with the Drosophila C virus also resulted in the up-regulation of several putative Toll pathway-related genes, such as Spätzle, Dorsal, the immune induced molecule 2, CG16836, PGRP-SA, a GNBP-like molecule encoded by CG12780, Drosomycin, and nine other putative antimicrobial peptides D. melanogaster GNBP functions as a co-receptor for Gram-positive bacteria and is involved in Toll immune pathway activation. It will be interesting to elucidate the functional significance of the observed GNBP induction in response to dengue infection in Ae. aegypti. For instance, the GNBPs might function as receptors that interact with the virus or cellular debris released during virus particle release, either activating an immune response or directly neutralizing the virus particles.The activation of the Toll immune signaling pathway by dengue infection is strongly supported by the up-regulation of Rel1 and several of its upstream putative PRRs and its downstream antimicrobial peptides . Up-reguActivation of Toll pathway-controlled Rel1 transcription factors through the RNAi-mediated depletion of Cactus resulted in suppression of the viral infection in the midgut, while activation of the Imd pathway-controlled Rel2, mediated through Caspar depletion, had no effect on infection. Although Rel1 over-expressing transgenic mosquitoes have been developed, we chose to use this RNAi-based approach to activate these factors in order to achieve systemic pathway activation and enrichment of immune factors prior to infection. The Rel1 transgenes are under the control of a blood meal-induced fatbody-specific promoter that only activates transcription at around 12 to 20 h after a blood meal in a very specific tissue compartment, which may not be the main destination for the dengue virus  D. melanogaster is involved in the defense against the Drosophila X virus The lack of an apparent link between the Imd pathway and the anti-dengue defense at 7 to 10 days after an infectious blood meal does not, however, exclude the possibility that this pathway is involved in the anti-viral response at some other stage of infection or in a specific tissue or cell type. However, our results are consistent with the previous report that the Toll pathway, but not the Imd pathway, in Drosophila C virus infection in D. melanogaster, where it is involved in limiting viral infection but, while required, is not sufficient to mount a potent antiviral response Our gene expression data also pointed to an activation of the less characterized JAK-STAT pathway, which has been shown to be activated in response to D. melanogaster: an inducible response that involves the activation of immune signaling pathways and an intrinsic defense system based on RNAi D. melanogaster responses to Drosophila C virus infection Two types of anti-viral defense have been proposed to operate in An. gambiae mosquitoes significantly reduces the microbial midgut flora, resulting in a decreased expression of immune genes and an increased susceptibility to Plasmodium infection Plasmodium development has recently been shown to be significantly influenced by the mosquito's basal level immunity rather than an infection-stimulated induction of immune genes An. gambiae genes implicated in the defense against Plasmodium have also been shown to play a role in its anti-bacterial defense Culex. bitaeniorhynchus, tetracycline treatment resulted in an increased susceptibility to the Japanese encephalitis virus Ae. aegypti were less resistant to dengue virus, and they expressed lower levels of certain immune genes that are also controlled by the Toll pathway.A major role of immune signaling pathways in insects is to protect the organism against the continuous exposure to opportunistic microbes as well as controlling the natural microbiota, such as the gut flora. Several studies have shown the importance of the host microbiota in inducing a basal level of immune activity that enhances the insect's resistance to pathogens. For instance, antibiotic treatment of Plasmodium effector genes in A. gambiae is unlikely to occur between the anti-microbial and anti-dengue defense systems because of the drastically difference in surface molecules and life style between virus particles and bacteria or parasites; nevertheless, the defenses appear to be regulated by the same immune signaling pathways. Finally, it is unlikely that the presence of low levels of antibiotics in the mosquito hemolymph contributed directly to changes in gene expression or increased virus infection. Antibiotic treatment has for instance no effect on virus propagation in the C6/36 cell line, and experiments in sterile A. gambiae mosquito cell lines have not shown any direct effects of antibiotic treatment on immune gene expression .These findings suggest that it is plausible to hypothesize that the mosquito's endogenous microbiota and natural microbial exposure stimulate a certain level of immune gene expression through the activation of the Toll pathway which, in turn, mediates antiviral activity. Alternatively, the microbiota could affect viral infection in a more direct way by altering the biochemical environment of the midgut or by directly interacting with the virus particles. However, the latter is not supported by our preliminary data on virus-bacteria interaction, while a possibility still exist that the bacteria hinder virus interaction with the midgut epithelium . RegardlAe. aegypti. Dengue infection can activate this pathway, which in turn induces a mechanism that suppresses the virus infection. Consistent with this observation, a low basal immunity in aseptic mosquitoes is correlated with a high virus infection level. Our results provide support for future experiments to dissect the biological network involved in the defense against dengue virus infection in Ae. aegypti. For example, it will be interesting to study the potential links between the Toll, JAK-STAT and RNAi pathways and their relevant contribution to the resistance of dengue infection in mosquitoes. In addition, how the virus is recognized to activate the Toll pathway and what are the downstream anti-viral effector molecules are important questions to be answered. In the present study, we have utilized a specific New Guinea genotype of the DENV-2 serotype that is known to be highly virulent. It will be interesting to assess the potential similarities and differences in the response of the mosquito to different dengue virus serotypes and genotypes that are known to differ in their virulence and tropism. Conversely, a comparison of mosquito strains with different susceptibilities to a particular virus genotype may also further our understanding of this complex vector-pathogen system.In summary, the results presented here show that the Toll pathway is involved in controlling dengue virus infection in Ae. aegypti, as indicated by an infection-responsive regulation and functional assessment of several Toll pathway-associated genes. We have also shown that the mosquito's natural microbiota play a role in modulating the dengue virus infection, possibly through basal-level stimulation of the mosquito's antiviral immune system.In summary, we demonstrate a significant role of the Toll pathway in regulating resistance to dengue virus in Ae. aegypti mosquitoes of the Rockefeller/UGAL strain were maintained on sugar solution at 27°C and 95% humidity with a 12-hr light/dark cycle according to standard rearing procedures. The Ae. albopictus cell line C6/36 was grown in minimal essential medium (MEM) with 10% heat inactivated FBS, 1% L-glutamine, and 1% non-essential amino acids at 32°C with 5% CO2.2 flasks of C6/36 cells at 80% confluency with a multiplicity of infection (MOI) of 3.5 virus particles/cell. Infected cells were incubated for 5–7 days. Cells were harvested with a cell scraper and lysed to release the virus particles by repeated freezing and thawing in dry CO2 and a 37°C water bath. The virus suspension was mixed 1∶1 with commercial human blood. A flask with uninfected C6/36 cells were maintained under similar conditions and used to create the noninfectious blood meal that served as our control. The blood meal was maintained at 37°C for 30 min prior to feeding 3- to 4-day-old mosquitoes (http://www.jove.com/index/Details.stp?ID=220). Primary PFU data are presented in The New Guinea C strain of DENV-2 was propagated in C6/36 cells according to standard conditions For microarray assays, mosquitoes at 10 days after blood meal were dissected to collect the midguts and carcass in RNALater, with 10 to 15 individuals in a single replicate. Three or four replicate biological assays were performed. Total RNA was extracted using the RNeasy kit (QIAGEN), and RNA concentrations were measured using Nanodrop; for virus titer measurement, mosquitoes at 7 days after blood meal were briefly washed in 70% ethanol, then rinsed in sterile distilled water. The midgut and carcass were dissected in sterile PBS and transferred separately to microcentrifuge tubes containing 150 µl of MEM, then homogenized with a Kontes pellet pestle motor in a sterile environment.t-tests at a significance level of p<0.05 using TIGR MIDAS and MeV software. Expression data from all replicate assays were averaged with the GEPAS microarray preprocessing software prior to logarithm (base 2) transformation. Self-self hybridizations have been used to determine the cut-off value for the significance of gene regulation on these type of microarrays to 0.8 in log2 scale, which corresponds to 1.74- fold regulation. For genes with P<0.05, the average ratio was used as the final fold change; for genes with P>0.05, the inconsistent replicates (with distance to the median of replicate ratios large than 0.8) were removed, and only the value from a gene with at least two replicates were further averaged. Toll and Imd signaling pathways were built on the basis of a recent bioinformatics prediction Ae. aegypti gene ontology by the GeneMapp development team. Three independent biological replicate assays were performed. Numeric microarray gene expression data are presented in Transcription assays were conducted and analyzed as reported previously with a full genome Agilent-based microarray platform Real-time qPCR assays were conducted as previous described http://www.jove.com/index/Details.stp?ID=230). Three to four days after injection and validation of gene-specific silencing, mosquitoes were fed on a DENV-2-supplemented blood meal. Dissection of mosquito midguts, thoraxes, and heads were done on the seventh day PBM. Each tissue was homogenized separately in the same medium as used for C6/36 cells (MEM) and used for virus titration. Three independent biological replicate assays were performed for each gene. The following primers were used for the synthesis of Cactus, Caspar and MyD88 dsRNA using the T7 megascript kit (Ambion): Cactus_F: TAATACGACTCACTATAGGG CGAGTCAACAGAACCCGAGCAG, Cactus_R: TAATACGACTCACTATAGGG TGGCCCGTCAGCACCGAAAG, Caspar_F: TAATACGACTCACTATAGGG GGAAGCAGATCGAGCCAAGCAG, Caspar_R: TAATACGACTCACTATAGGG GCATTGAGCCGCCTGGTGTC, MyD88_F: TAATACGACTCACTATAGGGGGCGATTGGTGGTTGTTATT, MyD88_R: TAATACGACTCACTATAGGGTTGAGCGCATTGCTAACATC,RNA interference (RNAi)-based gene-silencing assays were conducted according to standard methodology http://www.jove.com/index/Details.stp?ID=220): The virus-containing homogenates were serially diluted and inoculated into C6/36 cells in 24-well plates. After incubation for 5 days at 32°C and 5% CO2, the plates were assayed for plaque formation by peroxidase immunostaining, using mouse hyperimmune ascitic fluid  and a goat anti-mouse HRP conjugate as the primary and secondary antibody, respectively. Numeric PFU data are presented in Virus titers in the tissue homogenates were measured as previously reported , 5569526 (REL1A), 5578608 (Caspar), 5569427 (REL2), 5579094 (DEF), 5579377 (CEC), 5578028 (Attacin), 5565542 (Diptericin), 5579192 (GNBPB1), 5564897 (PGRGLC), 5564993 (Gambicin), 5569574 (MyD88), 5579458 (LYSC), 5576410 (Ikkg), 5565422 (GNBPA2), 5580019 (AAEL009645), 5572476 (AAEL009822), 5576330 (AAEL000393), 5576380 (DOME), 5573010 (SPZ5), 5578273 (TOLL1B), 5577966 (TOLL9A), 5576030 (TEP13), 5565197 (TEP15), 5572428 (TEP20). 5563609 (TEP22), 5568254 (FREP), 5577659 (CLIPB13B).Figure S1In vitro exposure of dengue virus to midgut bacteria does not affect the virus viability. Incubation of the dengue virus with either sterile PBS, bacteria exposed supernatant or a bacteria suspension did not result in any significant difference in virus viability. Ten midguts from seven days old septic female mosquitoes were dissected and homogenized in 100 ul sterile PBS prior to plating on a LB agar plate for bacterial growth. Bacteria colonies were washed off the plate with PBS and collected into a 1.5 ml tube. After a 10 minutes centrifugation at 1,500 g the bacteria-free supernatant and the bacteria pellet were collected. The bacteria pellet was re-suspended into PBS to get the bacteria solution. Then, equal amount of virus were incubated for 3 hrs at room temperature with the bacteria, the bacteria free supernatant and the sterile PBS prior to titer determination with plaques assay. Three replicates were performed for each treatment.A. The bacteria flora in the mosquito lumen does not influence the viability of the dengue virus. Seven days old antibiotic treated aseptic or non-treated septic mosquitoes were fed with the same mixture of DENV-2 and blood. Two hour after the blood meal, midguts were dissected and their content was immediately diluted with 100 ul sterile PBS. Three replicates of five guts each were collected. After a brief homogenization and centrifugation, the supernatants were used to determine the virus titer with the standard plaque assay. B. (0.07 MB JPG)Click here for additional data file.Table S1The functional groups of the total 432 genes that were regulated by DENV-2 infection in the mosquito carcass at ten days after an infected blood meal, compared to that of non-infected blood fed control mosquitoes. Functional group abbreviations: IMM, immunity; RED/STE, redox and oxidoreductive stress; CSR, chemosensory reception; DIG, blood and sugar food digestive; PROT, proteolysis; CYT/STR, cytoskeletal and structural; TRP, transport; R/T/T, replication, transcription, and translation; MET, metabolism; DIV, diverse functions; UNK, unknown functions.(0.38 MB DOC)Click here for additional data file.Table S2The functional groups of the total 63 genes that were regulated by DENV-2 infection in the mosquito midgut at ten days after an infected blood meal, compared to that of non-infected blood fed control mosquitoes. Functional group abbreviations: IMM, immunity; RED/STE, redox and oxidoreductive stress; CSR, chemosensory reception; DIG, blood and sugar food digestive; PROT, proteolysis; CYT/STR, cytoskeletal and structural; TRP, transport; R/T/T, replication, transcription, and translation; MET, metabolism; DIV, diverse functions; UNK, unknown functions.(0.08 MB DOC)Click here for additional data file.Table S3Averaged data from three biological replicate real time qPCR assays of the expression of defensin, cecropin, Cactus, and Rel1in Cactus, and Cactus & Rel1 depleted mosquitoes (A) and in Caspar, and Caspar & Rel2 depleted mosquitoes (B). C. Fold change in the expression of selected immune genes in aseptic mosquitoes compared to septic mosquitoes. S.E., standard error.(0.06 MB DOC)Click here for additional data file.Table S4A. Averaged data from three independent biological replicate plaque assays of the virus titer in the midguts of the Cactus, Caspar, MYD88 and GFP dsRNA treated mosquitoes. B. Results from three independent biological replicate plaque assays of the virus titer in the midgut of antibiotic treated aseptic and non-treated septic mosquitoes. S.E., standard error; S, significant; NS, Non-significant.(0.04 MB DOC)Click here for additional data file.Table S5The prime sequences used for the real-time qPCR assays.(0.04 MB DOC)Click here for additional data file.Table S6The expression data of all the genes that are shown in the hierarchical cluster matrix .(0.30 MB DOC)Click here for additional data file.Text S1This section refers to other dengue infection responsive genes.(0.05 MB DOC)Click here for additional data file.


In addition, the mutant virus demonstrated lower binding efficiency to plasma membrane preparations from all three cell types compared to the parental virus. Antisera raised against the parental virus reacted equally to both homologous and heterlogous viruses, however, antisera raised against the mutant virus showed 4-8 folds lower reactivity to the parental virus.The hemagglutinin (HA) protein of influenza virus mediates essential viral functions including the binding to host receptor and virus entry. It also has the antigenic sites required for virus neutralization by host antibodies. Here, we characterized an H3N2 triple reassortant (TR) influenza virus (A/turkey/Ohio/313053/04) with a mutation at the receptor binding domain (Asp190Ala) that occurred upon virus transmission from turkeys to pigs in an experimental infection study. The mutant virus replicated less efficiently than the parental virus in human, pig and turkey primary tracheal/bronchial epithelial cells, with more than 3-log Influenza A viruses infect a wide range of animal species including mammals and birds . All subThe HA is a major surface glycoprotein on influenza virus envelope and is essential for binding to host receptors and virus entry . In addiACF25543) or Ala (NCBI gene bank accession number: ACD35865) at the corresponding position.In this study, we characterized an H3N2 triple reassortant (TR) influenza virus with a mutation at the RBD (Asp190Ala) that occurred upon virus transmission from turkeys to pigs in an experimental infection study . H3N2 TRIn general, avian viruses express Glu  and human viruses expresses Asp  at position 190 of the RBD ,13. Ala The H3N2 TR virus used in this study, A/turkey/Ohio/313053/04 (TK04), was previously isolated at our laboratory  and has BsmBI restriction enzyme and cloned into pHH21 vector between promoter and terminator sequences of RNA polymerase I. Eight plasmids harboring the eight gene-segments were transfected along with four expression plasmids  into 293T cells with the help of Lipofectamine-2000 reagent . Supernatant from transfected cells was collected at 36 hours post transfection (hpi) and was subsequently inoculated into 10-day-old ECE for virus isolation. Single amino acid change at residue 190 of the RBD (Asp to Ala) was generated using QuikChange® Site-Directed Mutagenesis kit  based on manufacture protocol. In addition, we generated a virus with a mutation at residue 627 of PB2 gene (Glu627Lys) that has been shown to affect replication and transmission of influenza viruses in different species [Utilizing the 12-plasmid reverse genetics system, we rescued the TK04 virus as previously described ,19. BriePrimary human tracheal/bronchial epithelial cells (HAEC) were purchased from Cell Application  and were maintained in tracheal/bronchial epithelial cells growth medium purchased from the same company .2 and non-adherent epithelial cells were collected and seeded into new culture flask for further growth. Cells were passaged up to five times prior to use in experiments.Primary pig and turkey tracheal/bronchial epithelial cells  were generated based on previously published protocols with slight modifications -23. Brie50. Serum free DMEM media served as negative control. Plates were rocked every 15 minutes and inoculum was removed after 45 minutes followed by adding DMEM media supplemented with 1 μg/ml TPCK-treated trypsin on top of the cells. Supernatant from inoculated cells was collected at 24, 48, and 72 hpi and titrated in Madin-Darby canine kidney (MDCK) cells based on previously published protocol [For the kinetic study, 70-80% confluent cells seeded in 6-well plate were infected with either virus at 0.01 TCIDThe cross hemagglutinin inhibition (HI) test was employed to evaluate the cross reactivity between parental (190Asp) and HA-mutant (190Ala) TK04 viruses. Additionally, cross reactivity was evaluated between TK04 parental and mutant viruses, and other H3N2 TR viruses isolated from turkeys in the United States (U.S.). This includes: A/turkey/North Carolina/03, A/turkey/Illinois/04, A/turkey/Minnesota/05, and A/turkey/North Carolina/05.6 TCID50/ml) for three times in 2-weeks interval. HI test was carried out as previously described [Antisera against TK04 viruses were produced by vaccinating two 2-week-old chickens with an inactivated virus vaccine  on x-axis. To inhibit neuraminidase activity, all experiments were performed in the presence of Zanamivir hydrate  at a final concentration of 0.25 μm. Recorded results are the average of three independent experiments.Plasma membranes were prepared from HAEC, PEC, and TEC based on formerly published protocol -29. SoliIn 1998, a new subtype of influenza A viruses, H3N2 TR, emerged in pig population in the U.S. and transmitted to other species including humans, turkeys, minks and waterfowls ,31-33. Iin vitro utilizing reverse genetics created viruses.One change, Asp to Ala, occurred at residue 190 of the RBD  in human, pig and turkey primary tracheal/bronchial epithelial cells. Virus with a mutation at residue 627 of the PB2 gene (Glu627Lys) was used as control, where such mutation has been shown to affect replication and host range specificity of influenza viruses.10 difference in virus titer between 190Asp and 190Ala viruses recorded at 72 hpi . Evident variation in virus titer was manifested since 24 hpi, with more than 3-logWe then assessed the effect of Asp190Ala mutation on binding efficiency of the TK04 virus to PMP from primary tracheal cells of human, pig and turkey origin Figure . Both viNext, we evaluated the effect of Asp190Ala mutation on antigenicity of H3N2 TR virus using the conventional cross-HI test (Table ACD35865), and thus, supporting the effect of such mutation on the antigenicity of H3N2 TR viruses.To further evaluate the above results, we included a wider range of turkey H3N2 TR viruses in the cross reactivity test. Again, anti-190Asp antisera reacted better against most turkey viruses compared to anti-190Ala antisera " between the different viruses using the Archetti and Horsfall formula . The parAlthough antibodies to the HA-antigenic sites have been shown to affect receptor binding specificity and neutralization sensitivity, mutations solely to the RBD have not been shown to alter immunogenicity . In thisGlu  is typically expressed in avian viruses at residue 190 of the HA molecule, while human viruses express Asp  at the corresponding position. Both amino acids are negatively charged, while Ala is a neutral amino acid. We assume that Ala at the corresponding position (Figure in vivo. Additionally, it is of importance to see the effect of the above mutation on the receptor binding specificity of H3 subtype viruses for its potential effect on interspecies transmission of influenza viruses.In conclusion, the Asp190Ala mutation that occurred upon virus transmission from turkeys to pigs could have been a transient or rare occurring mutation that resulted in a less fitted virus, explaining the rareness of Ala at this position in swine and turkey H3N2 influenza isolates. More work is needed to evaluate the replication and antigenicity of 190Ala mutation The authors declare that they have no competing interests.YMS is the leader of the study group. HMY carried out the experiments and wrote the manuscript. MK generated the pig and turkey epithelial cells and helped in the infection studies. HMY, CWL, and YMS designed the experiments and analyzed the data. All authors read and approved the final manuscript.


Rousettus aegyptiacus) based on detection of Marburg virus RNA in 31/611 (5.1%) bats, virus-specific antibody in bat sera, and isolation of genetically diverse virus from bat tissues. The virus isolates were collected nine months apart, demonstrating long-term virus circulation. The bat colony was estimated to be over 100,000 animals using mark and re-capture methods, predicting the presence of over 5,000 virus-infected bats. The genetically diverse virus genome sequences from bats and miners closely matched. These data indicate common Egyptian fruit bats can represent a major natural reservoir and source of Marburg virus with potential for spillover into humans.In July and September 2007, miners working in Kitaka Cave, Uganda, were diagnosed with Marburg hemorrhagic fever. The likely source of infection in the cave was Egyptian fruit bats ( Rousettus aegyptiacus) as a natural host of Marburg virus based on multiple lines of evidence which include, for the first time ever, the isolation of virus directly from wild-caught and apparently healthy bats. The species R. aegyptiacus is common throughout Africa with distribution into the eastern Mediterranean and Middle East. Our finding of active virus infection in approximately 5% of R. aegyptiacus bats and their population exceeding 100,000 in Kitaka cave in Uganda suggests there are likely over 5,000 Marburg virus–infected bats in this cave, which is only one of many such cave populations throughout Africa. Clearly, these bats could serve as a major source of virus with potential to initiate human epidemics, and the implications for public health are striking. Additionally, we found highly divergent (21%) genome sequences among viruses circulating in these bat populations, a level of diversity that would result from a long-term association with a suitable reservoir host of large population size.Marburg virus, similar to its close cousin Ebola virus, can cause large outbreaks of hemorrhagic fever (HF) in rural Africa with case fatalities approaching 90%. For decades, a long-standing enigma has been the identity of the natural reservoir of this deadly virus. In this report, we identify the cave-dwelling Egyptian fruit bat ( Marburgvirus and Ebolavirus genera (family Filoviridae) cause outbreaks of hemorrhagic fever in Africa characterized by person-to-person spread and high case fatality. Humans have on occasion acquired infection from contact with tissues of diseased nonhuman primates and perhaps herbivores, but the susceptibility of these animals to fatal infection renders it unlikely that they could serve as filoviruses reservoir hosts.Viruses of the Rousettus aegyptiacus, and two species of insectivorous bat in the mine, and the outbreak ceased when the mine flooded, but no live virus was isolated from bats R. aegyptiacus bats in the same country in the absence of a corresponding outbreak of disease Although the source of filoviruses in nature has not been definitively identified, the cumulative evidence suggests that bats are involved. The infected monkeys consigned from Uganda to Europe in 1967, which resulted in the first recognized outbreaks of Marburg hemorrhagic fever (MHF), were caught on the shores of Lake Victoria and on islands where fruit bats are prevalent R. aegyptiacus and insectivorous Hipposideros species bats were present in this mine. Ecological investigations were conducted in August 2007 and May 2008, and the findings are presented here.In July 2007, a small outbreak of MHF occurred in workers mining lead and gold in Kitaka Cave near Ibanda village in western Uganda. Large numbers of Kitaka Cave was first mined in the 1930s and eventually became a large producer of lead ore in Uganda, but was closed in 1979. It was reopened in January 2007, and in July a miner working in the cave fell ill and died with disease confirmed at Centers for Disease Control and Prevention, USA (CDC) to be MHF  Hipposideros spp. bat  of R. aegyptiacus and two species of insectivorous bats were found positive for Marburg virus RNA in Goroumbwa Mine, DRC, in 1999 By equating RNA-positivity with virus infection, it is possible to derive preliminary conclusions on the dynamics of Marburg virus activity in bat populations. Although there was a similar frequency of RNA-positivity in bats collected in August 2007 and May 2008, the fact that a total of 31/611 (5.1%) spp. bat , suggestR. aegyptiacus bats (data not shown), all adults, clearly positive for Marburg virus-specific IgG antibody , two of which (#s 273 and 278) were also weakly positive by Q-RT-PCR. The testing found 455/546 (83.3%) bats to be clearly negative, while another 78 R. aegyptiacus bats had indeterminate antibody levels . None of the Hipposideros spp. bats had detectable IgG to Marburg virus. It is unclear why only a low percentage of the R. aegyptiacus bat population is found positive for Marburg virus reactive IgG antibody. Perhaps a greater proportion of the population was previously infected, but antibody levels are below the conservative IgG assay cut-off used here. This would be consistent with low Marburg virus reactive antibody levels reported in previous bat studies Serologic testing found 13/546 (2.4%) R. aegyptiacus was similar in the two collections, and there appeared to be no gender bias in the evidence for Marburg virus infection  of the collection. The prevalence of Marburg virus RNA detected in the juveniles, 8/78 (10.3%), was significantly higher than in adult R. aegyptiacus bats, 14/333 (4.2%)  of the collection, but only 1/60 (1.6%) of the juveniles were RNA-positive , and the same was true for larger numbers of parasitic and cave-associated arthropods tested in the investigations in the DRC in 1999 R. aegyptiacus bats.It can be concluded that there was no evidence of vertical transmission of infection in For ebolavirus, it has been suggested that outbreaks in nonhuman primates follow seasonal patterns which may reflect changes in diet or reproductive status of reservoir hosts, and that infection of the primates could be initiated through consumption of fruit contaminated with blood and placentas during parturition of infected bats R. aegyptiacus bats and one Hipposideros spp. bat which produced positive PCR results, and 49 bats which were uniformly negative in Q-RT-PCR plus NP and VP35 RT-PCR assays, revealed no lesions which could specifically or consistently be ascribed to Marburg virus infection. Viral antigens were detected by IHC in the livers of two bats which yielded Marburg virus isolates in culture , and from these data it was calculated that approximately 112,000 The results of Bayesian analysis of the nucleotide differences among full-length virus genome sequences of the isolates from the two miners (01Uga2007 and 02Uga2007), plus the five isolates from bats  found no correlation between genetic and geographic distances (p>0.3). The geospatial separation of the closely related Marburg virus lineages is most consistent with mobility of their natural host, a dynamic easily accomplished by the enormous meta-population of R. aegyptiacus colonies would provide valuable insights into the dynamics of immune status, as well as the shedding, transmission and persistence of Marburg virus in bat populations, and help to determine if the proportions of infected individuals relative to age are periodic or stochastic. The studies should be supplemented by experimental infections to observe the dynamics of infection within individual bats. Given the detection of infectious ebolavirus in privileged sites, such as testes, up to three months after onset of symptoms in human infections Longitudinal studies of naturally infected Blood samples collected during acute illness and submitted as diagnostic samples to the Centers for Disease Control and Prevention (CDC), Atlanta, USA, were tested for Marburg virus antigen and IgG antibody by enzyme-linked immunoassay as described previously According to an institutionally reviewed IACUC protocol, bats were captured with mist nets or harp traps at the opening of the mine, euthanized with Isoflurane, and samples of liver, spleen and placenta (where applicable) collected by dissection, using safety precautions described previously R. aegyptiacus and the insectivorous Hipposideros spp. bats, were sampled during the first field trip in August 2007. Smaller numbers were sampled during the second field trip which was undertaken in May 2008, during the putative breeding season of R. aegyptiacus bats in Uganda, mainly to seek evidence of continued circulation of virus and possible vertical transmission of infection. Opportunity was taken to collect oral swabs from the bats sampled in May to determine the likelihood of virus transmission through saliva or respiratory aerosols. A mark and recapture study was also conducted in May to estimate the size of the R. aegyptiacus population, and to possibly allow for later determination of foraging and migration distances of the bats. A total of 1,329 R. aegyptiacus bats were tagged with coded aluminum necklaces or leg bands over a period of two weeks, and recaptures which were recorded once the number of marked bats reached 1,000, were used in the Jolly-Seber model for estimating the abundance of an open population To maximize the chances of isolating virus, large numbers of each of the two species of bat found in the mine, the fruit bat Nycteribiidae) found in the pelage of bats during dissection, and 100 adult and nymphal argasid ticks (Carios faini) taken from crevices in the rocks near bat roosting sites. Apart from dermestid beetles, spiders, crickets, moth flies and cockroaches, the only other fauna seen in the cave consisted of a target rat (Stochomys longicaudatus) and forest cobras (Naja melanoleuca).Limited numbers of arthropod parasites of bats were collected and frozen, including 25 wingless flies  were extracted using non-cellular lysis buffer (Applied Biosystems) All human and bat samples were screened by Q-RT-PCR, designed to detect RNA of all known lineages of Marburg virus, and bat samples found positive (Ct<40) were re-analyzed by extracting RNA from frozen tissue using RNAeasy mini-kits (Qiagen) after overnight incubation at 4°C in lysis buffer. The extracts were subjected to the Q-RT-PCR and conventional RT-PCR based on the NP and VP35 genes. Tissues from 39 bats found negative in the initial Q-RT-PCR were also re-extracted and subjected to Q-RT-PCR and NP and VP35 gene RT-PCR. Nycteribid flies and argasid ticks were individually ground in cellular lysis buffer and extracted RNA tested by Q-RT-PCR.2 flasks and incubated for 14 days at 37°C/5% CO2 in MEM/2% fetal calf serum with a media change after day 7. Cultures were monitored daily for CPE with cell scrapes at days 7 and 14 tested by IFA. For bat samples, 10% suspensions of freshly thawed ∼250 mg frozen tissue sections were homogenized on ice in viral transport medium  with a plastic pestle and ∼250 mg sterile alundum (Fisher cat# A634-3) in 15 ml conical tubes. The homogenate was clarified by low speed centrifugation and 100 µl of supernatant fluid was inoculated onto Vero E6 cell cultures in 25 cm2 flasks at 37°C/5% CO2 for 1 hr with gentle rocking followed by media replacement with MEM/2% fetal calf serum. Inoculated flasks were monitored daily for 14 days (with media change after day 7) for the appearance of CPE and by IFA of cell scrapes on days 7 and 14. Cultures positive by IFA for Marburg virus were additionally analyzed by RT-PCR (see below).For human samples, 100 µl of blood was inoculated onto Vero E6 monolayers in 25 cmSequencing of Marburg virus whole genomes and partial gene sequences (NP and VP35) were performed as previously described Blood samples from bats were tested by enzyme-linked immunoassay for the presence of IgG antibody reactive with Marburg virus as described previously Genbank accession numbers are described in Modeltest 3.730 In addition, Bayesian phylogenetic analyses were conducted for each of the datasets in MrBayes 3.2 To determine if marburg virus infection caused lesions in infected bats, sections were cut from paraffin-embedded blocks prepared from formalin-fixed liver and spleen samples from 32 bats found positive by Q-RT-PCR, and examined in parallel with the tissues of 39 bats found negative in both the Q-RT-PCR and conventional RT-PCR. Hematoxylin and eosin (H&E) stained sections of the tissues were examined for lesions, and sections stained by an immunoalkaline phosphatase technique Table S1Genbank Accession numbers used for phylogenetic analysis(0.08 MB DOC)Click here for additional data file.


CHRNA5-CHRNA3-CHRNB4 cholinergic nicotinic receptor subunit genes. In particular, association with the nonsynonymous CHRNA5 SNP rs16969968 and correlates has been replicated in several independent studies. Extensive genotyping of this region has suggested additional statistically distinct signals for nicotine dependence, tagged by rs578776 and rs588765. One goal of the Consortium for the Genetic Analysis of Smoking Phenotypes (CGASP) is to elucidate the associations among these markers and dichotomous smoking quantity (heavy versus light smoking), lung cancer, and chronic obstructive pulmonary disease (COPD). We performed a meta-analysis across 34 datasets of European-ancestry subjects, including 38,617 smokers who were assessed for cigarettes-per-day, 7,700 lung cancer cases and 5,914 lung-cancer-free controls , and 2,614 COPD cases and 3,568 COPD-free controls . We demonstrate statistically independent associations of rs16969968 and rs588765 with smoking . Because the risk alleles at these loci are negatively correlated, their association with smoking is stronger in the joint model than when each SNP is analyzed alone. Rs578776 also demonstrates association with smoking after adjustment for rs16969968 (p<10−6). In models adjusting for cigarettes-per-day, we confirm the association between rs16969968 and lung cancer (p<10−20) and observe a nominally significant association with COPD (p = 0.01); the other loci are not significantly associated with either lung cancer or COPD after adjusting for rs16969968. This study provides strong evidence that multiple statistically distinct loci in this region affect smoking behavior. This study is also the first report of association between rs588765 (and correlates) and smoking that achieves genome-wide significance; these SNPs have previously been associated with mRNA levels of CHRNA5 in brain and lung tissue.Recently, genetic association findings for nicotine dependence, smoking behavior, and smoking-related diseases converged to implicate the chromosome 15q25.1 region, which includes the  Nicotine binds to cholinergic nicotinic receptors, which are composed of a variety of subunits. Genetic studies for smoking behavior and smoking-related diseases have implicated a genomic region that encodes the alpha5, alpha3, and beta4 subunits. We examined genetic data across this region for over 38,000 smokers, a subset of which had been assessed for lung cancer or chronic obstructive pulmonary disease. We demonstrate strong evidence that there are at least two statistically independent loci in this region that affect risk for heavy smoking. One of these loci represents a change in the protein structure of the alpha5 subunit. This work is also the first to report strong evidence of association between smoking and a group of genetic variants that are of biological interest because of their links to expression of the alpha5 cholinergic nicotinic receptor subunit gene. These advances in understanding the genetic influences on smoking behavior are important because of the profound public health burdens caused by smoking and nicotine addiction. Smoking is associated with many different diseases. Lung cancer is the illness most identified with smoking, and its prevalence over time mirrors per capita tobacco consumption CHRNA5-CHRNA3-CHRNB4 cluster of cholinergic nicotinic receptor subunit genes. The nicotine dependence locus tagged by the single nucleotide polymorphism (SNP) rs16969968 and correlates has been replicated for smoking related traits including cigarettes-per-day and heavy smoking 2>0.8 with rs16969968) implies that these statistical signals tag the same locus in European-ancestry populations. The SNP rs16969968 results in an amino acid change (D398N) in the alpha5 receptor subunit protein and has been shown to affect receptor function Recently, genetic findings for nicotine dependence and smoking related diseases converged to implicate the chromosome 15q25.1 region, which includes the CHRNA5-CHRNA3-CHRNB4 region has provided potential evidence for at least two additional distinct signals for nicotine dependence 2 = 0.24 in the HapMap CEU panel), though the linkage disequilibrium (LD) coefficient |D'| is 1. A third important locus in this region is a group of highly correlated SNPs, tagged by rs588765, which are associated with mRNA levels of CHRNA5 in brain tissue Extensive genotyping of the CHRNA5-CHRNA3-CHRNB4. Specifically, we focused on the four distinct loci discussed above, which have low correlation with each other and have demonstrated evidence for involvement in nicotine dependence. Analyses were undertaken to investigate two questions: first, are there multiple statistically distinct genetic loci in this region that exert independent effects on smoking, and second, are similar patterns of genetic risk shared across smoking, lung cancer, and COPD.With the support of the National Institute on Drug Abuse (NIDA), we formed the Consortium for the Genetic Analysis of Smoking Phenotypes (CGASP), which includes smoking, lung cancer, and COPD researchers, to enable the pursuit of several research goals. For this first analysis project we focused on the chromosome 15q25.1 region containing This study was conducted according to the principles expressed in the Declaration of Helsinki and obtained informed consent from participants and approval from the appropriate institutional review boards.All subjects included in these meta-analyses were current or former smokers of European ancestry. Results from 34 datasets, which include a total of 38,617 unrelated subjects who were assessed for cigarettes-per-day, contributed to the meta-analyses. Eight of the datasets were drawn from family-based studies and contributed only a subset of unrelated individuals to these analyses. The traits examined were smoking quantity, lung cancer, and COPD. Two smoking traits were derived from measurements of cigarettes smoked per day (CPD): a 4-level categorical trait  and a dichotomous trait contrasting subjects from the lowest smoking category (CPD≤10: light-smoking “controls”) to those in the two highest categories combined (CPD>20: heavy smoking cases). The dichotomous trait of heavy versus light smoking was our primary trait for analysis. For one study (NAG-Finland), which used different boundaries to record CPD as detailed in the supplemental material, the distribution of CPD was examined to harmonize the phenotypes and select alternative boundaries. The numbers of subjects in each smoking category, total and by study, are given in 2 with each other). For each locus, we chose one target SNP for analysis: rs16969968 (locus 1), rs578776 (locus 2), rs588765 (locus 3), and rs12914008 (locus 4); the pairwise correlations between any two of these loci are r2<0.5  and R In each dataset, associations between the loci and the traits were evaluated using logistic regression. Our primary analysis model coded genotypes additively as the number of copies of the minor allele according to the HapMap CEU reference population. This allele is referred to as the “coded allele” (C) and the major allele is referred to as the “reference allele” (R). To confirm the appropriateness of the additive model, for each locus a 2 degree of freedom model including the additive term and a heterozygote deviation term was evaluated. The analyses of the 4-level CPD trait used generalized logistic regression to obtain separate effect estimates (beta coefficients) for each category with respect to the lowest smoking category as the referent. All these association analyses included sex and age as covariates. In addition, lung cancer and COPD analyses included categorical cigarettes-per-day as an unordered covariate.Association results from each dataset, including the beta coefficient and standard error, were provided to the coordinating team. Meta-analysis was carried out using PLINK 2 if the alleles that tend to co-occur on the same haplotype have very different allele frequencies. Previous results in the COGEND data suggest that there may be independent or synergistic effects on nicotine dependence between locus 1 and locus 3 As noted earlier, locus 1 (representing rs16969968) is a highly replicated association finding and furthermore rs16969968 has been shown to have functional effects on the resulting alpha5-containing receptor To test whether additional loci contribute to dichotomous smoking quantity over and above the effect of rs16969968, we included both locus 1 and each of the other loci in the logistic regression models adjusting for sex and age, with and without a SNP×SNP interaction term. For lung cancer and COPD the models also included categorical cigarettes-per-day as an unordered covariate. These results were then meta-analyzed as described above. The SNP×SNP interaction term was never significant in the meta-analysis (p>0.3), so we report results from the joint models without interactions. To allow comparison between single-SNP and joint results on comparable data, for each locus pair we also repeated the univariate single-SNP meta-analyses on the subset of datasets that had genotypes available at both loci. For dichotomous smoking quantity we also tabulated pair-wise joint genotype by case status counts for locus 1 (rs16969968) versus each of the other three loci across the contributing datasets that had both loci.−4 corresponding to an experiment-wide alpha of 0.05. The results tables report uncorrected p-values which we compared to this threshold to determine statistical significance.Across the four target loci, multiple traits (4), the multiple models (additive and additive+heterozygote deviation), and the 2-SNP joint analyses (3 loci), our study was designed to perform fewer than 80 tests. A conservative Bonferroni correction would result in an uncorrected p-value threshold of 6.25×10We calculated allele frequencies within each sample to confirm that the coded allele  was indeed the minor allele as expected in these European-ancestry subjects. All reported results are based on additive models. The additive model is appropriate because none of the tests for deviation from the additive assumption were significant. For each analysis, the tables and figures report the number of individuals successfully genotyped for the relevant SNP or SNPs.−31, OR = 1.33, 95% confidence interval (1.26–1.39)), and also the ORs in each contributing dataset.−25 and an OR of 0.78 (0.74–0.81), indicating a protective association for the minor allele as has previously been reported  yields a meta-analysis p-value of 1.38×10reported . Locus 3reported . Locus 4The categorical CPD analysis, which includes all 4 CPD levels in a generalized logit model, allows us to evaluate genetic effects for each CPD category with respect to the lowest smoking class (CPD≤10). −8 to 2.12×10−12 to 5.47×10−40. A similar ordinal effect is seen for locus 2 (rs578776), with the odds ratio decreasing from 0.88 to 0.79 to 0.77. For locus 3 (rs588765) we see an effect only with the highest smoking category (CPD>30). For locus 4 no effect is seen across smoking categories, consistent with the dichotomous CPD results.For locus 1 (rs16969968), we see an ordinal effect with increasing CPD; that is, the odds ratio increases from 1.15 to 1.29 to 1.40 for categories 2, 3 and 4, with a corresponding decrease in p-value from 3.17×10To dissect the potential distinct effects of these loci on heavy versus light smoking, we carried out meta-analyses of joint SNP models that included sex, age, locus 1 and each of the other loci, coded additively.−22, OR = 1.27 (1.21–1.33) and locus 2 gives p = 4.50×10−7, OR = 0.87 (0.83–0.92). When each SNP is placed individually in the model and meta-analyzed across the 32 datasets that provided data for both loci, locus 1 gives p = 1.41×10−32, OR = 1.34 while locus 2 gives p = 1.38×10−25, OR = 0.76. The risk-increasing alleles at locus 1 (C) and locus 2 (R) are positively correlated, even though the minor alleles are negatively correlated.In the joint analysis of locus 1 and locus 2, there is suggestive evidence of distinct effects, but the association at locus 2 is no longer genome-wide significant in the presence of locus 1. Both SNPs become less significant compared to their single locus models: in the joint model, locus 1 gives p = 2.15×10−36, OR = 1.47 (1.38–1.56); locus 3 (rs588765) gives p = 6.03×10−9, OR = 1.17 (1.11–1.23). Thus locus 3 attains genome-wide significance (p<5×10−8) after adjusting for the effect of locus 1. Note that adjusting for locus 1 changes the direction of effect for locus 3 (OR>1) compared to the single-SNP results. In the 33 datasets that have both loci genotyped, we obtain p = 5.39×10−29, OR = 1.32 for locus 1 alone, and p = 0.00027, OR = 0.93 (0.89–0.97) for locus 3 alone. The evidence for association in the joint model is stronger than when each SNP is analyzed alone. In fact, when locus 1 is not taken into account, the effect of locus 3 is potentially masked, and the effect of the minor allele is in an opposite direction (protective versus risk).In joint analysis of locus 1 and locus 3, locus 1 (rs16969968) yields a p-value of 3.52×10To further examine these interesting results for locus 1 and locus 3, we show the number of heavy and light smokers in each joint genotype class, and corresponding odds ratios using the genotype that is homozygous for both reference (major) alleles as the reference group . The refThe first important observation is that there are very few subjects in certain cells, namely the cells corresponding to RC/CC at locus 1/locus 3, CC/RC, and CC at both loci. This table therefore reveals that the risk alleles at locus 1 (C) and locus 3 (C) are negatively correlated, and explains why the effect of rs588765 is seen only after adjusting for rs16969968. This pattern also reflects the high |D'| between the loci.The second observation is that for the remaining, well populated cells, the coded allele at locus 3 increases risk on the background of a fixed genotype at locus 1 . Similarly, for a fixed genotype at locus 3, the coded allele at locus 1 increases risk . Thus for each locus, the effect seen in the joint, 2-SNP logistic regression is confirmed in the most informative stratum at the other locus.−38, OR = 1.35 (1.29–1.41) and locus 4 gives p = 5.55×10−3, OR = 1.17 (1.05–1.31). While the effect for locus 4 is stronger than was seen in single-SNP analysis, it does not meet our multiple test threshold for significance. In single-SNP analysis of the 25 datasets that have genotypes at both loci, locus 1 alone gives p = 7.56×10−35, OR = 1.33; locus 4 is non-significant .For locus 1 and locus 4 in the joint model, locus 1 gives p = 1.01×10−21). The summary odds ratio of 1.31 (1.24–1.38) closely matches the dichotomous CPD odds ratio of 1.33 (1.26–1.39). In −10; OR = 0.82 (0.77–0.87)) (Locus 2 (rs578776) also shows evidence of association with lung cancer in single-SNP analysis (p = 9.74×107–0.87)) . Locus 37–0.87)) ; as withSimilar to our analyses of categorical CPD, we carried out joint analyses of locus 1 with each of the other 3 loci, with covariates for sex, age and dummy-coded CPD. After adjusting for the effect of locus 1, none of the other loci reached our multiple-test-corrected significance threshold.−13, OR = 1.26 (1.19–1.34) and locus 2 gave p = 0.012, OR = 0.91 (0.85–0.98). In joint analysis of locus 1 and locus 3, locus 1 yields p = 2.24×10−19, OR = 1.39 (1.30–1.50) and locus 3 gives p = 0.0050, OR = 1.11 (1.03–1.19), showing the same change from protective to risk for the minor allele as was observed in the dichotomous CPD analysis. Finally, in the last pairing, locus 1 gives p = 2.66×10−22 OR = 1.33 (1.26–1.41) and locus 4 gives p = 0.028, OR = 1.26 (1.02–1.55).For locus 1 and locus 2 jointly in the model, locus 1 gave p = 2.68×10CHRNA5-CHRNA3-CHRNB4 gene cluster demonstrate independent effects on smoking behavior (heavy (CPD>20) versus light (CPD≤10) smoking). We selected loci for study based on prior statistical and/or functional evidence for involvement. The second goal was to test whether similar patterns of association are seen across these loci in the smoking-related diseases of lung cancer and COPD. This meta-analysis marks the first large-scale effort to line up association results for these related traits – smoking, lung cancer, and COPD – using a uniform analysis protocol. Our results contribute important new insights about genetic risk for these traits. In particular, we demonstrate strong evidence that smoking behavior is influenced by multiple distinct loci in this region, including two loci that are associated with relevant biological effects in functional studies.The first goal of this meta-analysis project was to test whether distinct loci in the CHRNA5 amino acid change rs16969968 and correlates, demonstrates highly significant association with smoking behavior . Our strong evidence for the involvement of locus 1 with smoking across these samples marks the robustness of its genetic effect. The contributing datasets for the smoking analyses range from samples ascertained for nicotine dependence, lung cancer, or COPD, to adolescent samples, to populations ascertained for a variety of diseases including schizophrenia, alcohol or other substance dependence, breast cancer, type 2 diabetes, and heart disease. This meta-analysis represents a very diverse group, and yet the association between rs16969968 and smoking behavior is consistent.First, our results show that locus 1, representing the −9 (OR = 1.17) when we adjust for locus 1 in a logistic regression model. It is notable that the association between locus 3 and CPD is not as apparent in the single-SNP analysis that does not control for locus 1 . The negative correlation between the risk alleles at locus 1 and locus 3 (r = −0.64) masks the effect at the latter locus in single-SNP analysis, a phenomenon known as suppression CHRNA5 in brain and lung tissue from European-ancestry subjects CHRNA5 mRNA levels is involved in risk, and highlight locus 3 as an important group of SNPs for further investigation.The second, and novel, finding from this meta-analysis is the evidence for an additional, distinct, locus in this region that is associated with heavy/light smoking and is genome-wide significant. We demonstrated that locus 3, representing rs588765 and correlates, attains a p-value of p = 6.03×10−25, OR = 0.78). However the association is much weaker  in the joint logistic regression model that includes locus 1 and locus 2. One interpretation is that part of the single-SNP association at locus 2 is driven by the effect of locus 1 (perhaps related to the high |D'|). Nevertheless, there is evidence for residual signal at locus 2.A third observation from this study is that locus 2 (rs578776 and correlates) shows evidence for involvement in heavy/light smoking. Locus 2 is genome-wide significant in the single-SNP analysis of dichotomous CPD without adjustment for locus 1, with the minor allele elevated in controls  non-synonymous SNP in 2 = 0.07 between rs578776 and rs588765 in HapMap CEU release 23); however their correlation sharply increases when locus 1 is taken into account .This meta-analysis therefore highlights locus 1, locus 2, and locus 3, and indicates dependencies in their effects on risk for heavy smoking. Haplotypes based on these three loci have been described Our association results together with the correlation patterns of these three loci suggest that future haplotype or diplotype analyses across large datasets could clarify the relative contributions of these loci. Our evidence that multiple distinct genetic loci affect smoking quantity is consistent with previous reports of risk and protective haplotypes for nicotine dependence in the Utah and LHS samples in vitroCHRNA5 in brain and lung tissue Taken together, our meta-analysis results argue strongly for the existence of at least two statistically distinct loci in this region that affect risk for heavy smoking. In particular, both locus 1 and locus 3, which have known functional effects, are genome-wide significant in joint, mutually-adjusted analysis. The minor allele at locus 3 shifts from a marginally significant protective factor when considered alone to a robust risk factor when considered in combination with locus 1. The statistical evidence and negatively correlated alleles at locus 1 and locus 3 are consistent with at least two mechanistic models: distinct effects of two loci where the minor allele at each locus increases risk across a constant background at the other locus, or a haplotype dose effect where alleles at the two loci act in concert on the same haplotype strand. In the latter model, the minor-major and major-minor haplotypes each increase risk relative to the major-major haplotype, as can be seen in −21, OR = 1.31). This result suggests possible direct genetic effects of locus 1 on this cancer, at least in the presence of smoking. However, CPD is not a sufficient proxy for carcinogen exposure An important open question is the degree to which the associations between chr15q25 variants and lung cancer are due to their effects on smoking. When comparing smoking and lung cancer single-SNP results, the patterns of association (odds ratios and directions of effect) were similar across the loci studied. Locus 1 is associated with lung cancer even when controlling for amount smoked per day  of the effects did not increase. There are several possible reasons for this, including: chance, the smaller sample size for lung cancer, or qualitative differences in the relationship between these loci and smoking behavior versus the relationship between these loci and lung cancer (after adjusting for smoking quantity). This highlights the challenges posed when attempting to dissect the contributions of multiple loci of modest effect on complex, correlated traits. Further studies, and larger sample sizes, are needed.For COPD, when controlling for cigarettes-per-day we did not find evidence for association with any of the loci after correction for multiple tests. For locus 1, the odds ratio of 1.12 (1.01–1.23) is lower than for smoking and lung cancer. The COPD analyses were based on smaller samples than those available for CPD or for lung cancer.Very recently, three other large smoking genetics consortia published their meta-analysis findings that confirm locus 1  as the locus most associated with smoking quantity, genome-wide 2>0.96 with rs16969968). After conditioning on rs55853698, the strongest residual signal was detected at a locus 2 SNP, rs6495308 ; they do not report the association result for rs588765 in the conditioned analysis, although it must have been less significant than 3.96×10−5. In their single-SNP analysis, rs6495308 (locus 2) gave a p-value of 2.2×10−10. Their results for locus 2 are therefore consistent with our observation that in joint analysis of locus 1 and locus 2, the significance at locus 2 is reduced compared to the single-SNP analysis. They do not report on whether the evidence for locus 1 and locus 3 strengthens in the joint analysis compared to single-SNP analysis, as we observed in the CGASP datasets. They do note that there is no obvious residual association with a third SNP after conditioning on either the pairing of locus 1 (rs16969968) and locus 3 (rs588765), or the pairing of locus 1 (rs55853698) and locus 2 (rs6495308). That result is consistent with the correlation and haplotype structure of these three loci discussed previously.In contrast to our novel finding in CGASP of genome-wide significance for locus 3 when analyzed jointly with locus 1, none of the other consortia report strong evidence for locus 3 when paired with locus 1. In the Oxford-GSK study −5) and rs2036534 (p = 9.1×10−5), neither of which is genome-wide significant. Rs2036534 tags locus 2 (r2 = 0.74 with rs578776 in HapMap CEU) while rs2869046 is only weakly correlated with locus 3 (r2 = 0.46).In the ENGAGE study −9), rs9788682 (p = 1.06×10−8), and rs7163730 (p = 1.22×10−8), which attain genome-wide significance. These SNPs are each correlated with locus 2, and much less correlated with locus 3 . It is possible that differences in samples, phenotype definitions, or analysis methods may be contributing to the differences between our strong findings for locus 3 and the three other consortium reports. To further understand the genetic contributions in this region, more work is needed, and not only statistical evidence but also biological evidence will be important.In TAG CHRNA5 SNP rs16969968 as well as rs1051730 and rs55853698, with smoking heaviness across very diverse datasets. Our study also demonstrates strong evidence that at least one additional distinct locus in this region affects risk for heavy smoking. In particular, we have identified for the first time that locus 3 – representing the CHRNA5 expression-associated SNPs rs588765 and correlates – surpasses GWAS-level significance for association with heavy smoking in European-ancestry subjects; this effect is detectable after adjusting for the effect of rs16969968. This new result for locus 3 raises the corresponding SNPs (rs588765 and correlates) to the level of interest already accorded to the two loci which have previously been detected at GWAS-level significance in single-SNP analyses: locus 1 (rs16969968 and correlates) and locus 2 (rs578776 and correlates). Our result also has implications for all genetic association studies, as it illustrates that joint analysis of SNPs is an important tool for identifying genome-wide significant effects that, soberingly, may be obscured in single SNP analyses.In summary, our meta-analysis demonstrates significant, robust association of locus 1, representing the non-synonymous Our study used multiple highly correlated SNPs to represent each of the 4 tested loci, depending on availability in each dataset, and all subjects were of European ancestry. Hence this study is not designed to determine which SNP(s), among the highly correlated SNPs for each locus, are most likely to be biologically involved. Future work, involving large-scale meta-analysis of other populations (e.g. Asian or African ancestry) to capitalize on LD differences between populations, comprehensive functional annotation of genetic variants, DNA re-sequencing and variant discovery, and functional and animal studies may help narrow down these large sets of correlated SNPs to the most promising causal alleles.Figure S1CHRNA5-CHRNA3-CHRNB4 region containing the target SNPs rs16969968 (locus 1), rs578776 (locus 2) rs588765 (locus 3), and rs12914008 (locus 4). The SNPs used in this study to represent each locus are drawn with dotted lines connecting them to each other.The (1.09 MB TIF)Click here for additional data file.Figure S2Forest plot for dichotomous CPD and locus 4.(0.38 MB TIF)Click here for additional data file.Figure S3Forest plot for lung cancer and locus 4.(0.30 MB TIF)Click here for additional data file.Table S1Correlation (r-squared) between the four target SNPs representing loci 1, 2, 3, and 4 (HapMap CEU Release 23).(0.05 MB DOC)Click here for additional data file.Table S2Correlation (r-squared) between the target SNPs and their proxies (HapMap CEU Release 23).(0.06 MB DOC)Click here for additional data file.Table S3Genotyped SNPs and overall allele frequencies, by sample dataset.(0.10 MB DOC)Click here for additional data file.Text S1Descriptions of contributing datasets. Numbered according to appearance in (0.17 MB DOC)Click here for additional data file.


Saffold virus may be the first human cardiovirus species. Cardioviruses cause serious disease, mainly in rodents, including diabetes, myocarditis, encephalomyelitis, and multiple sclerosis–like disseminated encephalomyelitis. Recently, a human virus isolate obtained 25 years ago, termed Saffold virus, was sequenced and classified as a cardiovirus. We conducted systematic molecular screening for Saffold-like viruses in 844 fecal samples from patients with gastroenteritis from Germany and Brazil, across all age groups. Six cardioviruses were identified in patients <6 years of age. Viral loads were 283,305–5,044,412,175 copies/g of stool. Co-infections occurred in 4 of 6 children. No evidence for outbreak-like epidemic patterns was found. Phylogenetic analysis identified 3 distinct genetic lineages. Viral protein 1 amino acids were 67.9%–77.7% identical and had a distance of at least 39.4% from known cardioviruses. Because closely related strains were found on 2 continents, global distribution in humans is suspected. Saffold-like viruses may be the first human cardiovirus species to be identified. Picornaviridae comprises 9 genera with >142 species and 200 serotypes, many of which are highly pathogenic for humans and animals. The genus Cardiovirus contains 2 animal-pathogenic species— Encephalomyocarditis virus and Theilovirus—that occur mainly in rodents and swine. The type species is Encephalomyocarditis virus, which includes strains of murine encephalomyocarditis virus (EMCV), Mengo virus, and Maus Eberfeld virus. The species Theilovirus is represented by Theiler’s murine encephalomyelitis virus  and rat encephalomyelitis virus. Both species show clinical association with encephalomyelitis in mice, and EMCV shows an additional association with myocarditis  targeted at domains conserved between the Saffold prototype virus, various TMEV strains, and EMCV. We screened 844 patients from all age groups with acute enteritis, including 39 controls, in 3 independent cohorts from 2 countries (Germany and Brazil) on 2 continents. Viral loads were determined by specific real-time RT-PCR. Phylogenetic analysis showed 3 independent lineages of circulating Saffold-like viruses (SafVs). Analysis of amino acid identities and considerations regarding transmission patterns suggest that SafV most likely constitutes a new cardiovirus that is associated with humans worldwide.<6 years old. Diarrhea in these patients was defined as excretion of at least 2 loose and malodorous stools during 24 hours for breastfed infants and of at least 2 loose stools in a 24-hour period for all other patients. Patients were excluded if they had inflammatory bowel disease, celiac disease, cystic fibrosis, food intolerance, or a known malignant disease. The cohort included stool samples from 39 control patients of compatible ages with conditions other than enteritis. Written informed consent was obtained from all patients or their parents. The study protocol and data handling were approved by the local ethics committee. In both groups, patients with norovirus, adenovirus, enterovirus, astrovirus, or rotavirus infection were excluded.From January through December 2004, 538 stool samples were collected from patients in urban areas in northern Germany in a prospective study on acute, community-acquired diarrhea. All patients were outpatients who had been examined by general practitioners; 96 (17.8%) were Cohort 2Archived stool samples from 118 patients with acute enteritis were obtained from the routine diagnostic laboratory of a municipal health service in Hamburg, Germany. This cohort contained 3 subcohorts classified with regard to patient age and context of sampling: 1) children sampled during childcare center outbreaks (n = 51); 2) adults sampled in the context of outbreaks of gastroenteritis mainly in association with catering and canteen food (n = 35); and 3) senior citizens sampled because of outbreaks of enteritis in retirement homes (n = 32).Cohort 3This cohort contained 188 samples from infants and children in Brazil with acute diarrhea, defined as >3 watery stools in the previous 24 hours and within 13 days before admission. Patients were seen as outpatients or were hospitalized because of severe dehydration from February through December 2006 at the University Hospital Professor Edgar Santos in Salvador de Bahia, Brazil. Informed consent was obtained from the mothers of all patients enrolled in the study. The study was approved by the institutional ethics committee. All analyses were performed at the Infectious Disease Research Laboratory, University Hospital Professor Edgar Santos.Co-infection in the Brazil cohort was assessed by using recently published methods of real-time RT-PCR for norovirus, rotavirus, enterovirus, parechovirus, adenovirus, and astrovirus . Both protocols used an input of ≈200 mg of stool prediluted 1:10 in phosphate-buffered saline. Suspensions were vortexed and centrifuged, and 200 or 140 μL of supernatant, respectively, was extracted according to the manufacturer’s instructions.Primers  were desVarious combinations of primers and probes were designed manually upon inspection of the SafV prototype sequence EF165067 (Based on the published genome of Saffold virus EF165067 and the 5′ untranslated region sequences obtained from our positive samples (nested PCR), primers spanning the complete viral protein 1 (VP1) gene were designed. cDNA was produced by using the Superscript III Kit  and an ≈4-kb fragment was amplified by using the Expand High Fidelity Plus Kit . This PCR product was sequenced directly from both sides by using primer walking. All primer sequences are available upon request.The 800-bp 5′-noncoding region fragment from sample BR/118/2006 was ligated into pCR 2.1 (Invitrogen) and TOPO-cloned. Plasmids were purified, sequenced, and reamplified with plasmid-specific primers. Reamplification products were transcribed into RNA with a MegaScript T7 kit . After DNase I digestion, RNA transcripts were purified with QIAGEN RNeasy columns and quantified photometrically. Sensitivity of real-time RT-PCR was determined to be in the single-copy range when purified and quantified in vitro transcripts were amplified.The following sequences were used for analysis and primer design: Saffold virus (EF165067), TMEV strain DA (M20301), TMEV strain GDVII (M20562), TMEV strain BeAn (M16020), Vilyuisk virus (M94868), Theiler-like virus of rats NGS910 (AB090161), Mengo virus (L22089), and EMCV (X87335). Several other subgenomic sequences of TMEV and EMCV were added in alignments for PCR primer design. At the time of preparation of this article, the polyprotein sequence of a Canadian virus isolate related to Saffold virus was described, AM922293 .A broad range of viral loads was observed . Co-infections with enteric viruses occurred in both patients from Brazil and in 2 of the 4 patients from Germany. Average viral loads in patients with and without co-infections were 1.7 × 10Only 1 child had concomitant respiratory symptoms. A nasopharyngeal aspirate from this child taken at the same time as the positive stool specimen was tested by real-time RT-PCR for SafV; results were negative.To appreciate the genetic range of cardioviruses in our patients, we first sequenced an 800-bp fragment containing 80% of the viral 5′-noncoding region from all 6 samples. The complete P1 gene could then be sequenced from 4 samples. Samples D/PN11/2004 and BR/176/2006, which showed the lowest virus concentrations, did not yield P1 gene PCR products on several trials.>2 genetic lineages were identified (In addition to the lineage containing the prototype Saffold virus (hereafter referred to as the Saf-1 lineage), entified . A seconPicornaviridae, the degree of nucleotide and amino acid identity in the P1 protein gene or in VP1 alone is used as a criterion of taxonomic classification. In several genera of In parallel with a recent report on the detection of SafV cardioviruses in 3 children . Support of a genuine human association is also provided by the occurrence of closely related members of the same lineage (Saf-2) in Brazil and in Germany. Such wide distribution requires efficient transmission of virus from human to human. In combination with findings of the virus in the United States , makes it appear not unlikely that SafV may be classified as a new cardiovirus species in the future. However, more genetic, ecologic, and functional analysis must be done before such a conclusion can be reached.The amino acid distance between isolates from the 2 established cardiovirus species, Theilovirus and Encephalomyocarditis virus. Research into the human disease association of SafV should therefore receive high priority in the clinical virology community.In recent years, several novel viruses have been discovered in humans, mostly by advanced molecular screening (


One-sentence summary for table of contents: A new nairovirus isolated from ticks collected from cattle hides was characterized. Bunyaviridae, genus Nairovirus. Six isolates of the virus were obtained from pools of Amblyomma gemma and Rhipicephalus pulchellus ticks collected from hides of cattle in Nairobi, Kenya, in October 1999. We report results of further characterization of this virus, including growth kinetics in cell culture and full-length genome sequencing and genetic characterization, which show it to be distinct from DUGV. We suggest that this is a new virus in the family Bunyaviridae, genus Nairovirus, and we propose that it be designated Kupe virus.We have previously described isolation and preliminary identification of a virus related to Dugbe virus (DUGV), family  Nairovirus in the family Bunyaviridae comprises 7 species groups containing primarily tick-borne viruses, some of which have been identified as human or animal pathogens. The genome of the nairoviruses consists of 3 segments of negative-sense, single-stranded RNA, small (S), medium (M), and large (L), which encode the nucleocapsid protein, glycoproteins (Gn and Gc), and viral polymerase, respectively. Additionally, an M segment–encoded nonstructural protein, NSM, was recently identified in the nairovirus Crimean-Congo hemorrhagic fever virus (CCHFV) .In a recent survey of ticks infesting market livestock in Nairobi, Kenya, we identified 26 isolates of DUGV and additionally obtained several isolates of a virus that was identified as a nairovirus related most closely to DUGV , BHK (baby hamster kidney), SW-13 , HeLa , HUH-7 (human hepatocarcinoma), and C6/36  cells in culture. Growth kinetics of the 2 viruses were compared in a 13-day growth curve in which cells were infected at a multiplicity of infection of 0.01 and aliquots removed daily. Virus titers were assayed on Vero cell monolayers in 6-well plates by using a published double-overlay method (Growth of Kupe virus and DUGV was tested in Vero (African green monkey kidney), LLC-MKViruses to be sequenced were amplified in Vero cells, and viral RNA was extracted from cell culture supernatant by using the QIAamp Viral RNA Mini Kit . Reverse transcription–PCR was conducted by using the Titan One Tube Reverse Transcription–PCR system . Amplified products were purified by agarose gel electrophoresis, and DNA fragments were extracted by using the MinElute Gel Extraction Kit (QIAGEN). Purified DNA fragments were sequenced by using the BigDye 3.1 kit  and analyzed by using a model 3130 automated sequencer (PE Applied Biosystems). Both strands of the DNA were sequenced.The full-length genome of Kupe virus isolate K611 was sequenced, beginning with fragments amplified by Nairobi sheep disease virus (NSDV)–specific primers or DUGV-specific primers from each segment. Full-length sequence was obtained by using a previously described method of primer walking and the 5′/3′ Rapid Amplification of cDNA Ends (RACE) Kit (Roche), which was used to determine the sequence of the segment ends (http://ca.expasy.org/prosite), psi-BLAST and CDS-BLAST (www.ncbi.nlm.nih.gov/BLAST), NetOGlyc 3.1, and MOTIFS in the Wisconsin Package version 11.1.2 (www.megasoftware.net).The nucleotide sequence of each segment of the Kupe virus genome was analyzed for open reading frames (ORFs) by using the EditSeq module of Lasergene  and translated into deduced amino acid sequence. Identification of protein motifs and potential sites for glycosylation was accomplished by using Prosite .Growth kinetics of Kupe virus and DUGV were compared in 7 cell types . NeitherThe 3 genomic RNA segments of Kupe virus, isolate K611, were completely sequenced, ORFs were identified, and deduced amino acid sequences were determined. Similar to other viruses in this family, the ends of each RNA segment contain a conserved sequence, the terminal 9 nt of which are identical to those found in all segments of DUGV, CCHFV, and HAZV and in the S segment of NSDV (sequence of other NSDV segments not available). The S segment of Kupe virus has 1,694 nt, an ORF of 483 aa, and 5′ and 3′ noncoding regions (NCRs) of 49 nt and 193 nt, respectively. The DUGV S segment has 1,716 nt, 5′ and 3′ NCRs of 51 nt and 213 nt, and an ORF of 483 aa  and DUGV  glycoproteins; similar peptides are found in the Kupe virus ORF (RRIL375 and RRLL898) (247) has been identified in CCHFV upstream of the amino terminus of Gn that has been shown to produce an additional glycoprotein; however, DUGV and Kupe virus do not share this motif .Previous studies of CCHFV and DUGV suggest that precursors of Gn and Gc glycoproteins are produced and then post-translationally cleaved to form mature glycoproteins. Potential tetrapeptide cleavage sites for SKI-1/S1P protease or a related protease have been identified immediately upstream of the N-termini of the CCHFV  and ORF  of Kupe virus is almost twice that of other bunyaviruses . CFull-length M segment sequences are available for only 3 of the known nairoviruses: DUGV (strain ArD 44313), HAZV, and CCHFV. Comparison of these viruses with Kupe virus M segment sequence showed 61.9%, 54.7%, and 52.1% nt identity and 57.0%, 47.7%, and 43.0% aa identity, respectively . AdditioFull-length L segment sequences are available only for DUGV (strain ArD 44313), HAZV, and CCHFV. Comparison of these sequences with Kupe virus sequence showed 77.4%, 62.8%, and 61.5% nt identity and 89.0%, 66.3%, and 63.7% aa identity, respectively . As expeNairovirus genus published by Honig et al.  were also made between Kupe virus and sequences of 14 other viruses representing 7 groups of the g et al. . An addiNairovirus, current classification of the diverse group of viruses in the genus is in relative agreement with available genetic analyses , suggest otherwise.A. gemma and R. pulchellus, the species from which Kupe virus was isolated (A. variegatum, A. gemma, A. lepidum, and R. pulchellus (A. variegatum and A. lepidum were collected and tested in that study, no isolates of Kupe virus were found in those species, which suggested that vector hosts for DUGV and Kupe virus may differ (Little is known about the ecology of Kupe virus other than its isolation from ticks infesting cattle. DUGV has been reportedly isolated from several tick species, including Kupe virus and DUGV were observed to replicate and cause CPE in a variety of cultured mammalian cell types. Kupe virus was observed to have a more rapid increase and subsequent decrease in viral titer, an earlier onset of visible CPE, and greater destruction of the cell monolayer in most of the mammalian cells tested. These findings show that this virus is more virulent than DUGV in the mammalian cells tested.Taxonomic classification of viruses is an evolving discipline that in early years was based primarily on morphologic characters. More recently, better classification has been obtained by using antigenic relationships and information gained from genetic characteristics. The International Committee on Taxonomy of Viruses has defined a virus species as “a polythetic class of viruses that constitute a replicating lineage and occupy a particular ecological niche” (Nairovirus. We also noted differences in other genetic characteristics between Kupe virus and DUGV, including M segment N-gly sites, L segment variable region, and NCR length variations. This evidence, combined with increased virulence of Kupe virus in cultured mammalian cells and potential differences in vector hosts, shows that Kupe virus is substantially different from, although closely related to, DUGV and is a new virus in the genus Nairovirus. However, further studies are necessary to determine the hosts, vectors, and geographic range of Kupe virus along with its virulence as a human or animal pathogen. Such information will aid in appropriate classification of this new virus.For Kupe virus, nucleotide and amino acid sequence variation between the S and M segments of Kupe virus and DUGV, or any other genetically characterized nairovirus, was greater than expected between strains of a single virus in the genus