Aedes aegypti, a highly domesticated mosquito species that is notoriously difficult to control. When transinfected into Ae. aegypti, the intracellular bacterium Wolbachia has recently been shown to inhibit replication of DENVs, CHIKV, malaria parasites and filarial nematodes, providing a potentially powerful biocontrol strategy for human pathogens. Because the extent of pathogen reduction can be influenced by the strain of bacterium, we examined whether the wMel strain of Wolbachia influenced CHIKV and YFV infection in Ae. aegypti. Following exposure to viremic blood meals, CHIKV infection and dissemination rates were significantly reduced in mosquitoes with the wMel strain of Wolbachia compared to Wolbachia-uninfected controls. However, similar rates of infection and dissemination were observed in wMel infected and non-infected Ae. aegypti when intrathoracic inoculation was used to deliver virus. YFV infection, dissemination and replication were similar in wMel-infected and control mosquitoes following intrathoracic inoculations. In contrast, mosquitoes with the wMelPop strain of Wolbachia showed at least a 104 times reduction in YFV RNA copies compared to controls. The extent of reduction in virus infection depended on Wolbachia strain, titer and strain of the virus, and mode of exposure. Although originally proposed for dengue biocontrol, our results indicate a Wolbachia-based strategy also holds considerable promise for YFV and CHIKV suppression.Incidence of disease due to dengue (DENV), chikungunya (CHIKV) and yellow fever (YFV) viruses is increasing in many parts of the world. The viruses are primarily transmitted by  Aedes aegypti, a mosquito that due to its close association with humans has historically been difficult to control. An innovative control strategy involving the release of mosquitoes infected with the intracellular bacterium Wolbachia is currently being developed. This approach is based on the recent discovery that Wolbachia reduces infection of mosquitoes with dengue virus, malaria parasites and filarial nematodes. In the current study, we demonstrated that Wolbachia also blocks infection of chikungunya and yellow fever viruses in Ae. aegypti. The degree of virus inhibition depended on the strain of Wolbachia, the route of virus exposure, the virus strain and the titer of virus that the mosquitoes were exposed to. The implementation of Wolbachia-based control strategies has the capacity to transform the way that mosquitotransmitted diseases are controlled in the future.Mosquito-transmitted viruses such as dengue, yellow fever and chikungunya, are responsible for significant morbidity and mortality throughout tropical and sub-tropical regions of the world. These viruses are primarily transmitted by  Mosquito-transmitted viruses cause significant human morbidity and mortality throughout the world and impose particularly heavy health and economic burdens on developing countries. Dengue, caused by infection with any of the four dengue virus (DENV) serotypes, is currently the leading arboviral disease, with millions of cases of classic dengue fever and tens of thousands of deaths annually due to hemorrhagic disease Aedes aegypti, a highly domesticated species that feeds almost exclusively on humans. Its geographic range has expanded with increased urbanization, resulting in increased arbovirus transmission s-methoprene), or adulticiding with indoor residual spraying or ultra-low-volume application. While these approaches can be successful, they are often labor-intensive, can be prohibitively expensive to implement and require a sustained commitment from all levels of government Current disease control measures focus on the suppression of mosquito vector populations to reduce virus transmission. The primary vector of DENVs, CHIKV and YFV is the mosquito Ae. aegypti mosquitoes with Wolbachia pipientis from other insect hosts Wolbachia is a maternally transmitted, endosymbiotic bacterium that manipulates host reproduction to enhance its own transmission Wolbachia can provide its host with nutritional benefits Ae. aegypti transinfected with the wMelPop-CLA strain of Wolbachia from Drosophila melanogasterWolbachia-free control mosquitoes wMelPop-CLA may also impose fitness costs on Ae. aegypti, such as reduced fecundity due to poor blood feeding success An emerging biocontrol approach to reducing transmission of arboviruses is provided by the transinfection of Wolbachia, wMel that is closely related to wMelPop-CLA and also occurs naturally in D. melanogaster was recently introduced into Ae. aegypti as an additional strain for the biocontrol of dengue Wolbachia in natural populations of mosquitoes. Unlike wMelPop-CLA infected mosquitoes, Ae. aegypti infected with wMel did not suffer any significant deleterious fitness costs when compared to uninfected controls wMelPop-infected Ae. aegypti, wMel-infected mosquitoes displayed dramatically reduced replication of DENV-2 Ae. aegypti infected with wMel were deployed in the field in north Queensland, Australia over the 2010–2011 summer Wolbachia was able to reach almost 100% fixation in wild mosquito populations only a few months following release, indicating that Wolbachia-infected mosquitoes present a very promising strategy for the control of dengue that is cost-effective and poses minimal environmental and social harm A second strain of Ae. aegypti infected with wMelPop-CLA showed reduced infection with CHIKV wMelPop-CLA infected mosquitoes could potentially be used for biocontrol in areas where human pathogens other than DENVs occur. However, not all strains of Wolbachia protect equally well, with those phylogenetically most closely related to wMel and wMelPop conferring the greatest degree of protection Drosophila, wMel confers protection against a range of viruses, for example Drosophila C virus, Flock House virus and Cricket paralysis virus wMel infection is able to limit the replication of human pathogens other than DENVs in mosquitoes, information that is critical to evaluating different Wolbachia strains for biocontrol.Although originally developed as a biocontrol tool for DENVs, wMel strain of Wolbachia to limit infection in Ae. aegypti with CHIKV and YFV. We also tested the ability of YFV to replicate in mosquitoes infected with wMelPop-CLA, in order to compare this strain to wMel. We found reduced levels of CHIKV but not YFV infection in wMel-infected Ae. aegypti, although the degree of virus inhibition depended on the mode of infection. By contrast, mosquitoes harboring the wMelPop-CLA strain of Wolbachia showed reduced infection rates with YFV with the extent of reduction virus strain and titer dependent.Here, we tested the ability of the Ae. aegypti were used in the experiments. The transinfection of Ae. aegypti with the wMelPop-CLA and wMel strains of Wolbachia and maintenance of infected lines has been previously described wMel-infected line, MGYP2, and its tetracycline-treated counterpart, MGYP2.tet, were exposed to both YFV and CHIKV. Both lines were in the F16 generation for the YFV experiments and the F24 generation for the CHIKV experiments. The original wMelPop-CLA infected line, PGYP1 52 and F53 generations, while the PGYP1.tet was used in the combined F50 and F52 generations. A Wolbachia-uninfected wild-type line of Ae. aegypti, designated Cairns3, which originated from Cairns, Australia, was used as a positive control for all experiments. The YFV-susceptible Rex-D white-eye Higgs strain of Ae. aegypti that originated from Rexville, Puerto Rico, was used as an additional positive control for the YFV experiments Six different lines of Ae. albopictusAe. albopictus) cells.The CHIKV strain was isolated from a patient visiting Melbourne, Australia in 2006 and contained the alanine to valine mutation in the membrane fusion glycoprotein E1 gene (E1-A226V) that has been linked to increased infectivity in mosquitoes, especially Ae. aegypti lines were exposed to YFV via intrathoracic inoculation and in an infectious blood meal. However, low feeding rates in all lines coupled with unexpectedly low infection rates in control lines that did actually feed, compromised our ability to draw any meaningful conclusions from the YFV oral feeding data. Thus it was excluded from the current paper. For intrathoracic inoculation, immobilized mosquitoes were inoculated with 0.5 µl and 0.22 µl of YFV or CHIKV suspension, respectively. The suspension consisted of stock virus diluted in growth media  which contained antibiotics and antimycotics and was supplemented with 10% fetal bovine serum (FBS) for YFV or 3% FBS for CHIKV. For oral feeding, mosquitoes were allowed to feed on a blood meal consisting of CHIKV diluted in commercially available defibrinated sheep blood  and 1% sucrose. The blood meal was housed within a membrane feeding apparatus that was fitted with pig intestine as the membrane 2 and engorged mosquitoes placed in 900 ml gauze-covered containers. All mosquitoes were maintained on 10% sucrose at 28°C, high relative humidity and 12L∶12D light cycle within an environmental growth cabinet.The CHIKV experiments were undertaken in the Biosafety Level 3 (BSL-3) insectary at Queensland Health Forensic and Scientific Services, Brisbane, Australia, and the experiments with YFV were undertaken in the BSL-3 insectary located at the University of Texas Medical Branch, Galveston, Texas, USA. The MGYP2, MGYP2.tet and Cairns3 lines were exposed to CHIKV using both intrathoracic inoculation and oral feeding. Intrathoracic inoculation was employed because it circumvents the midgut infection and escape barriers, and allows a standard amount of virus to be delivered. The six For the YFV experiments, mosquitoes were processed at either day 10 or 14 post-exposure. Mosquitoes were chilled and the heads removed from the bodies and placed separately in 0.5 ml of GM supplemented with 10% FBS, and antibiotics and antimycotics. Recovery of virus from the head demonstrated that the virus had infected head tissue and was potentially able to be transmitted.in vitro capillary tube system 2 and the legs and wings removed. The saliva was collected by inserting the proboscis of the mosquito into a capillary tube containing GM with 20% FBS. After 30 min, the contents of the capillary tube were expelled into 600 µl of GM with 3% FBS. The salivary glands were then dissected from the body in a drop of GM with 3% FBS. The dissected salivary glands were washed in a drop of 1% bleach For the CHIKV experiments, infection, dissemination and transmission were assessed using a modified Salivary glands were also excised for visualization of CHIKV infection via an immunofluorescence assay (IFA). The glands were dissected in a drop of PBS and washed in another two drops of PBS, before each gland was placed in separate wells of an 18 well microscope slide. Slides were air-dried, fixed in cold acetone for 30 min, before being stored at −80°C.Blood/virus mixtures were titrated as serial ten-fold dilutions in 96 well microtiter plates seeded with confluent monolayers of C6/36 cells. Plates were incubated at 28°C for 5 days or 7 days for YFV- or CHIKV- infected blood/virus mixtures, respectively, before being fixed in PBS/acetone and stored at −20°C. Infection with YFV was detected using an indirect immunofluorescence assay with the monoclonal antibody (Mab) 863 Heads, salivary glands, bodies and body remnants were homogenized and clarified by centrifugation. The CC-EIA was used to detect CHIKV in all mosquito tissues and saliva expectorates. To further reduce bacterial and fungal contamination, the mosquito tissues and saliva expectorates were filtered through a 0.2 µm filter prior to being inoculated onto the C6/36 cell monolayers.Ct) value was <40. The number of YFV RNA copies in heads and bodies was quantified using the TaqMan RT-PCR, with RNA standards produced for the relative quantification of YFV RNA copy numbers normalized to RNA levels of the Ae. aegypti house-keeping gene RpS17 Salivary glands and saliva expectorates were also analyzed for CHIKV RNA using real time TaqMan RT-PCR. YFV viral RNA was detected in the heads and bodies using a YFV-specific TaqMan RT-PCR. Viral RNA was extracted from 140 µl of each of the mosquito components and the saliva expectorates using the QIAmp Viral RNA kit following the manufacturer's protocol , with modifications described in Moreira and others used for the YFV extractions 3; Dako, Glostrup, Denmark). A slide containing wells inoculated with Ross River virus, Barmah Forest virus, Sindbis virus and CHIKV infected cells was included as a positive control. Slides were examined under an Olympus CX41 fluorescent microscope .Fifteen microliters of B10 Mab was added to each well. After incubation at 37°C for 30 min in a humidified container, slides were rinsed in PBS pH 7.4 before being washed for 10 min in PBS pH 7.4. Slides were air-dried before the addition to each well of 15 µl Dako Anti-Mouse FITC Conjugate  in 0.04% Evan's Blue in PBS pH 7.4. Slides were incubated and washed as described above, followed by application of two drops of Dako fluroescent mounting medium  and uninfected mosquitoes (the tetracycline-treated MGYP2.tet and wild-type Cairns3 lines) with CHIKV in two replicate oral feeding experiments. In both experiments, fewer MGYP2 mosquitoes displayed CHIKV infection compared to MGYP2.tet and Cairns3 lines (P<0.01). Similarly, in experiment 2, CHIKV was detected in significantly fewer MGYP2 (30%) bodies compared to MGYP2.tet (70%) and Cairns3 (67%) lines (P<0.01).We challenged s3 lines Table 1.P<0.01) in experiment 1, but only when using the TaqMan RT-PCR assay (P<0.01), but not Cairns3 mosquitoes.Next, we used both CC-EIA and a CHIKV-specific TaqMan RT-PCR assay to track dissemination of the virus to the salivary glands and saliva following oral feeding experiments. CHIKV was detected in significantly fewer MGYP2 (20%) salivary glands compared to those from MGYP2.tet (80%) and Cairns3 (66%) lines . Twelve days following oral exposure, 27% (3/11) and 67% (6/9) of the MGYP2 and Cairns3 lines, respectively, were positive in the IFA. There did not appear to be any consistency in the localization of virus within infected salivary glands from either Ae. aegypti line  Wolbachia strains on body infection rates, using intrathoracic inoculation with two strains of YFV isolated from Nigeria (BA-55) and Bolivia (Cinetrop 28). Four Wolbachia-uninfected mosquito lines were used as controls: Cairns3, Rex-D, MGYP2.tet and PGYP1.tet. Due to high mortality rates in some lines, mosquitoes exposed to 103.5 TCID50/ml of YFV BA-55 were tested at 10 days post virus exposure. Mosquitoes exposed to104.5 TCID50/ml of YFV BA-55 and 104.0 TCID50/ml of YFV Cinetrop 28 were tested at 14 days post exposure.We tested the effect of 3.5 TCID50/ml) and high (104.5 TCID50/ml) titers of YFV strain BA-55, MGYP2 mosquitoes had the same high (100%) rates of body infection as MGYP2.tet, Cairns3 and Rex-D mosquitoes (P<0.001). PGYP1 mosquitoes also displayed significantly lower rates of body infection compared to MGYP2 mosquitoes (P<0.001). At the higher virus titer a similar number (100%) of PGYP1 mosquitoes was infected with YFV as for the PGYP1.tet, Cairns3 and Rex-D lines. A similar pattern was observed for the Cinetrop 28 strain of YFV, with mosquito body infection rates ranging from 96–100% and no significant differences among the mosquito lines, although this strain was tested at only one titer (104.0 TCID50/ml).At both low , at both low and high virus titers (P<0.001). At high virus titer, a higher (78%) number of PGYP1 mosquitoes had virus in the heads and no significant differences in infection rates were found across the mosquito lines. High head dissemination rates (84–100%) were found across all mosquito lines challenged with the Cinetrop 28 strain of YFV, with no significant differences among them.Next, we assessed the impact of the two strains of s titers Table 2.Wolbachia on the number of YFV RNA copies in Ae. aegypti bodies following intrathoracic inoculation with the strains BA-55 and Cinetrop 28, at three different titers. Overall, infection with both Wolbachia strains resulted in a lower median number of virus copies per body in Ae. aegypti compared to control lines. However, PGYP1 mosquito bodies consistently displayed several log fewer copies than MGYP2 lines  in challenges with YFV strain BA-55 at a titer of 103.5 TCID50/ml. However, virus levels were high in both MGYP2 and the Wolbachia-uninfected lines  and significantly less than PGYP1.tet, Cairns3 and Rex-D mosquitoes   but not the MGYP2.tet or Cairns3 lines    , although the number of virus copies was still high  , although virus was present in the majority of PGYP1 heads  when mosquitoes were challenged with the Cinetrop 28 strain  . Median Wolbachia-infected mosquitoes into natural populations has been proposed as a way of reducing DENV transmission while minimizing social and environmental harm Wolbachia-infected mosquitoes may prevent the transmission of other significant arboviruses. Here, we evaluated whether mosquitoes infected with the wMel strain of Wolbachia show limited infection with CHIKV and YFV. To compare the two Wolbachia strains, we tested whether YFV was able to infect and replicate in wMelPop-CLA infected mosquitoes.Mosquito-transmitted viruses such as DENVs, CHIKV and YFV pose a significant health risk for almost half of the world's population. The release of wMel showed significantly reduced rates of CHIKV infection and dissemination to the salivary glands compared to controls, but only in the oral exposure experiments. CHIKV also showed limited dissemination in wMelPop-CLA-infected mosquitoes following oral exposure Wolbachia may be useful candidates for release in CHIKV control programs. By contrast, YFV was much less likely to infect and disseminate in Ae. aegypti infected with wMelPop-CLA compared to wMel strains. The virus was also less likely to replicate in wMelPop-CLA infected mosquitoes, with very high virus loads detected in wMel-infected Ae. aegypti. Our experiments suggest that wMelPop-CLA infected mosquitoes may be the best candidates for YFV biocontrol programs, but were unable to determine the extent of virus replication following oral exposure rather than intrathoracic inoculation. Because virus inhibition with some Wolbachia-virus combinations does not appear to be complete, it is essential that epidemiological models be utilized to establish the threshold of virus inhibition necessary to minimize and prevent transmission in the field.Mosquitoes infected with wMel strain occurs at high levels in Ae. aegypti ovaries and salivary glands but it is not present in as many body tissues as the wMelPop-CLA strain wMel does not accumulate to high levels in the fat bodies, brain and thoracic ganglia, which are involved in secondary replication of arboviruses prior to infection of the salivary glands wMel-infected mosquitoes to allow dissemination to the salivary glands and potentially be transmitted to humans. The presence of wMel in the mosquito did not completely prevent the replication of CHIKV in Ae. aegypti salivary glands, as determined by cell-culture, real-time RT-PCR and IFA analysis.The Wolbachia-infected Ae. aegypti show significant immune upregulation Wolbachia infection activates the Toll immune pathway in Ae. aegypti and the production of antimicrobial peptides which inhibit DENV-2 Wolbachia infection in Drosophila, the endosymbiont's native host, also blocks DENV-2 replication despite the absence of immune upregulation Wolbachia-mediated interference with virus replication, it may not be the whole explanation Wolbachia may compete with arboviruses for key cellular components in situ hybridization has revealed that wMelPop-CLA infection is widespread throughout the body of infected Ae. aegypti, and is prevalent in tissues and organs associated with viral replication, including the fat bodies, brain and ommatidia wMelPop-CLA. As DENV-2 and YFV are closely related flaviviruses, it is possible that a similar pattern of virus exclusion is occurring with YFV in wMelPop-CLA infected cells and tissues. In vitro studies have shown that reduced virus replication correlates with the density of Wolbachia in the cell Wolbachia-mediated interference with virus replication.Host immune pre-activation has been proposed as an explanation for interference with virus replication because Wolbachia limits arbovirus infection and replication in Ae. aegypti has the potential to fundamentally alter vector control strategies wMel-infected Ae. aegypti in two communities near Cairns, northern Australia, resulted in >90% frequency within the population six weeks after the first release Wolbachia-based control strategy was initially proposed to control DENVs, previous work and the current study have demonstrated that releases of Wolbachia-infected Ae. aegypti to control DENVs may have the added benefit of reducing CHIKV and YFV transmission. Alternatively, Wolbachia-infected Ae. aegypti could be released to specifically reduce transmission of these viruses, irrespective of whether DENVs were circulating or not in an area.The discovery that Wolbachia is dependent on the strain of Wolbachia, the strain of virus and the mode of exposure to the virus. Higher YFV intrathoracic inoculation titers allowed more breakthrough of virus in wMelPop-CLA infected mosquitoes. Infection rates, accumulation and dissemination to the head also differed between BA-55 and Cinetrop 28 in the presence of wMelPop-CLA, suggesting that different virus strains may have different replication dynamics in Wolbachia-infected mosquitoes. The CHIKV experiments showed that the TaqMan RT-PCR was generally more sensitive than the CC-EIA in detecting the presence of virus. This was not unexpected, given that the TaqMan RT-PCR does not differentiate between RNA derived from live, packaged infectious virus particles, from that of dead or defective, non-infectious virus. Thus, our experiments illustrate the importance of carefully selecting virus titer, virus strain, mode of delivery and method of virus detection when assessing the potential utility of different Wolbachia strains for biocontrol.We have demonstrated that the level of virus blockage induced by Wolbachia-based control strategies, it will be essential to assess the virus blocking ability of different Wolbachia strains against different virus strains, and if necessary, identify additional Wolbachia strains that could be deployed in the field. Commensurate with this will be the need for ongoing monitoring of Wolbachia-infected Ae. aegypti populations to confirm that the virus-limiting phenotype is maintained in successive generations post release.As part of the implementation of


Nicotiana benthamiana. Southern blot hybridization showed that RecSat could be trans-replicated stably in N. benthamiana plants by TbLCYNV, and impaired the accumulation of helper virus and co-inoculated alphasatellite. Our results provide the first evidence for recombination between two distinct types of satellites among geminivirus complex and highlight recombination as a driving force for geminivirus evolution.Monopartite begomoviruses and their associated satellites form unique disease complexes that have emerged as a serious threat to agriculture worldwide. It is well known that frequent recombination contributes to the diversification and evolution of geminiviruses. In this study, we identified a novel defective satellite molecule (RecSat) in association with Tobacco leaf curl Yunnan virus (TbLCYNV) in a naturally infected tobacco plant. Sequence analysis showed that Recsat comprises 754 nucleotides in size and is a chimera involving alphasatellite and betasatellite sequences, containing both betasatellite-conserved region and alphasatellite stem-loop structure. Recombination analysis revealed that RecSat has arisen from three independent recombination events likely involving Tomato yellow leaf curl China betasatellite, Ageratum yellow vein China betasatellite and Tobacco curly shoot alphasatellite. Co-inoculation of RecSat with TbLCYNV induced symptoms indistinguishable from those induced by TbLCYNV alone in  Begomovirus, the largest and the most economically important genus of the Geminiviridae family, encompasses viruses that are exclusively transmitted by the whitefly Bemisia tabaci and infect only dicotyledonous plants  dCTP-labeled probe. For analysis of TbLCYNV, a probe containing AC2 and parts of AC1 gene (approximately 710 bp) was amplified from pBINPLUS-Y143 with Y143F (5'-AAGTCTGAGGTTGTAAGCGAGTC-3') and Y143R (5'-CCATCAGTATTGTCATAGAGGGAG-3') primers as described previously [Nucleic acids were isolated from upper leaves at 30 days post inoculation (dpi) and viral DNA accumulation was assayed by Southern blot hybridization as described . In brieeviously . For speIn summary, we report for the first time the occurrence of recombination between different types of satellites of geminivirus complex in the field. The recombinant satellite interferes with helper virus accumulation, behaving like a defective interfering DNA. Our study highlights the potential of recombination as a driving force for geminivirus diversification.


We identified a novel rhabdovirus, American bat vesiculovirus, from postmortem tissue samples from 120 rabies-negative big brown bats with a history of human contact. Five percent of the tested bats were infected with this virus. The extent of zoonotic exposure and possible health effects in humans from this virus are unknown. Bats are reservoirs for many emerging viral pathogens, including Ebola viruses, Marburg viruses, henipaviruses, and severe acute respiratory syndrome coronaviruses; >80 bat virus species have been characterized  is commonly detected in bats from the United States. Analyses of several cases of human rabies infections have reported insectivorous bats as the source , such as vesicular stomatitis virus, cause fever and vesicular diseases in animals such as cattle, horses, and pigs. Some vesiculoviruses, including Chandipura virus and vesicular stomatitis virus, are also zoonotic and cause acute diseases in humans.Rabies virus (family us genus . NonrabiEptesicus fuscus) collected in Maryland, USA.The bat virome has not been fully characterized. Most bat virome studies have been conducted by analyzing fecal, anal swab, or pharyngeal swab specimens from healthy bats (http://blast.ncbi.nlm.nih.gov/Blast.cgi), indicating a possible novel virus. PCR and rapid amplification of cDNA ends were performed to obtain the complete genome of this virus  consists of 10,692 nt of negative-sense, single-stranded RNA beginning with a 103-nt 5′ untranslated region, followed by open reading frames encoding for the nucleoprotein, phosphoprotein, matrix protein, glycoprotein, and polymerase protein genes , panel ALung and liver tissues from 60 of the bats used for the pooled metagenomic analyses were screened individually for ABVV by using reverse transcription PCR targeting the polymerase gene . Three  on the basis of a sequence alignment of the polymerase gene of ABVV and the formally classified mammalian vesiculoviruses. When the ABVV-positive bat liver cDNA was used as a control, the nested PCR yielded an amplicon of 704 bases, and its sequence was confirmed by cloning and Sanger sequencing. Future studies may use these pan-vesiculovirus PCR primers to investigate vesiculovirus diversity in other bat species and in other regions.Big brown bats are prevalent in North America, where their geographic range overlaps extensively with that of humans, and considerable interactions occur between big brown bats and humans and their pets. Big brown bats from this region are a known reservoir of rabies virus; our analysis shows that these bats also constitute a sylvatic mammalian reservoir of vesiculoviruses.The characterization of ABVV sheds light on vesiculovirus diversity in bats. The other bat rhabdoviruses—Mount Elgon bat virus, Oita rhabdovirus, and Kern Canyon virus—do not belong to the vesiculovirus clade but cluster together in a separate clade , panel BRhabdoviridae.Detailed methods for postmortem diagnosis of rabies by direct fluorescent antibody assay in >500 bats associated with possible human exposure submitted to the Maryland Department of Health and Mental Hygiene State Laboratory; location of the primers used in this study relative to the American bat vesiculovirus; sequences of the primers used in this study; and Bayesian inference tree based on the polymerase gene depicting relationships among the members of the family


Philomachus pugnax) possess a genetic polymorphism for male mating behaviour resulting in three permanent alternative male reproductive morphs: (i) territorial ‘Independents’, (ii) non-territorial ‘Satellites’, and (iii) female-mimicking ‘Faeders’. Development into independent or satellite morphs has previously been shown to be due to a single-locus, two-allele autosomal Mendelian mode of inheritance at the Satellite locus. Here, we use linkage analysis to map the chromosomal location of the Faeder locus, which controls development into the Faeder morph, and draw further conclusions about candidate genes, assuming shared synteny with other birds.Ruffs (Aves: Faeder locus were obtained from captive-bred pedigrees comprising 64 multi-generation families (N = 381). There was no evidence that the Faeder locus was linked to the Satellite locus, but it was linked with microsatellite marker Ppu020. Comparative mapping of ruff microsatellite markers against the chicken  and zebra finch (Taeniopygia guttata) genomes places the Ppu020 and Faeder loci on a region of chromosome 11 that includes the Melanocortin-1 receptor (MC1R) gene, which regulates colour polymorphisms in numerous birds and other vertebrates. Melanin-based colouration varies with life-history strategies in ruffs and other species, thus the MC1R gene is a strong candidate to play a role in alternative male morph determination.Segregation data on the Faeder locus is linked to Ppu020, which, assuming synteny, is located on avian chromosome 11. MC1R is a candidate gene involved in alternative male morph determination in ruffs.Two unlinked loci appear to control behavioural development in ruffs. The  Philomachus pugnax) possess three distinct permanent alternative male reproductive morphs that differ in territorial lekking behaviour, plumage colour, and size: dark-plumed territorial ‘Independents’, white-plumed non-territorial ‘Satellites’ and small female mimic ‘Faeders’ that lack display plumage and behaviour)ehaviour-7. Develtellites,9. Genetozygotes. Remarkaozygotes   and zebra finch (Taeniopygia guttata) chromosomes[Recently, a microsatellite linkage map for the ruff was constructed, identifying seven linkage groups and a further five single-marker loci homologous to locations on known chicken , 38 satellites  and 381 individuals for the Faeder locus: 43 faeders  and 338 non-faeders .Pedigree, phenotype, and microsatellite information were available from 381 individuals from a captive population of ruffs spanning fourteen breeding years and comprising 64 families . In This research was conducted at Simon Fraser University under approval of the Animal Care Committee.versus Independent; Faeder versus Not Faeder) were tested in CRIMAP v.2.4[Satellite and Faeder loci. For the Satellite locus: independent males (N = 120) were coded as homozygous recessive (ss) and satellite males (N = 35) coded with the dominant S allele (S_), with faeders not coded at this locus. A small number of females (N = 12) were assigned a satellite or independent behavioural morph and putative genotype based on pedigree analysis of their male offspring morph ratios when mated with an independent male . Females mated with an independent male that produced mixed offspring were designated as heterozygotes , and females with a high number of offspring (N = 11–22) who failed to produce any satellites when mated with independents were designated as homozygous recessive at the Satellite locus . In the majority of cases, these morph assignments were confirmed with testosterone-induced behavioural data[Faeder locus: both independent and satellite males were coded as homozygous recessive  and faeder males as (F_), indicating that they carry at least one copy of the F allele (N = 24)[ca 1%[N = 19) were made through principal component analysis of size distributions based on tarsus, culmen, and minimum mass[N = 183) were coded as homozygous recessive (ff), and faeder females coded as (F_) for similar reasons as were the males.Separate autosomal genetic models for the two male behavioural polymorphisms . Since tles N = 3 coded wi)[ca 1%-20, the mum mass. All nonSatellite locus and Faeder locus, and all microsatellite markers (N = 58) used in the ruff microsatellite linkage map[Satellite and Faeder loci were first run separately, then together in CRIMAP. We used comparative mapping[Faeder locus.A test for linkage between the kage map, was per mapping,22 of mi mapping against Satellite and Faeder loci, and the Satellite locus was unlinked to any other marker in twopoint analysis. The latter result may be due in part to the low number of satellites with heterozygous genotypes and high number of independents contained within the pedigree, resulting in a small number of informative meioses at the target Satellite locus. Out of the total 167 individuals with inferred genotypes at the Satellite locus, 129 of these were independents and 38 were satellites. The non-linkage of the two behavioural loci, Satellite and Faeder, to the same marker or, more importantly, to each other, indicates that two independent loci determine alternative morph development in ruffs. Additional genotyping of satellite individuals and/or more detailed pedigree data will further test this two-locus model.No linkage was detected between the Paracerceis sculpta[Several species with three heritable alternative mating phenotypes have been described , but exs sculpta, for whis sculpta. In the Faeder locus was strongly linked to microsatellite marker Ppu020 with a LOD score 8.24 and recombination fraction of 0.03. This locus was not placed on the ruff linkage map but comparative mapping has shown it to be on chromosome 11[In the twopoint analyses, the osome 11. Furtherosome 11.Melanocortin-1 receptor (MC1R) gene, an important pigment-regulating gene in birds and numerous other vertebrates, is located on chromosome 11 in both species. In chicken, the distance between Ppu020 and MC1R is 1.2 Mb and, in zebra finch, the distance is 20.7 Mb  has yet been the subject of a full genome sequencing project.Although ruff microsatellite  mapping. CompariMC1R in ruffs, we conclude that this gene and those in proximity to it are candidates for the Faeder locus. Melanin-based colouration has previously been shown to be associated with morphology, physiology, life-history strategies and behaviour in several bird species , inc, incMC1Rs (e.g.,,30.The authors declare no competing interests.LLF designed the study, performed the laboratory work, completed the data analysis, and drafted and revised the manuscript. DBL, TB and JS helped with the design of the study, the interpretation of the data, and with drafting and revision of the manuscript. JS and TB conceived coding the faeder behaviour locus into the linkage map. SBM contributed to the development of the ruff pedigree and quantifying faeder phenotypes with DBL. All authors read and approved the final manuscript.


Avian influenza (AI) viruses infect numerous avian species, and low pathogenicity (LP) AI viruses of the H7 subtype are typically reported to produce mild or subclinical infections in both wild aquatic birds and domestic poultry. However relatively little work has been done to compare LPAI viruses from different avian species for their ability to cause disease in domestic poultry under the same conditions. In this study twelve H7 LPAI virus isolates from North America were each evaluated for their comparative pathogenesis in chickens, ducks, and turkeys.6 50% egg infectious doses based on seroconversion, although not all animals seroconverted with each isolate-species combination. The severity of disease varied among isolate and species combinations, but there was a consistent trend for clinical disease to be most severe in turkeys where all 12 isolates induced disease, and mortality was observed in turkeys exposed to 9 of the 12 viruses. Turkeys also shed virus by the oral and cloacal routes at significantly higher titers than either ducks or chickens at numerous time points. Only 3 isolates induced observable clinical disease in ducks and only 6 isolates induced disease in chickens, which was generally very mild and did not result in mortality. Full genome sequence was completed for all 12 isolates and some isolates did have features consistent with adaptation to poultry , however none of these features correlated with disease severity.All 12 isolates were able to infect all three species at a dose of 10The data suggests that turkeys may be more susceptible to clinical disease from the H7 LPAI viruses included in this study than either chickens or ducks. However the severity of disease and degree of virus shed was not clearly correlated with any isolate or group of isolates, but relied on specific species and isolate combinations. Avian influenza (AI) virus causes one of the most economically important diseases of poultry worldwide. AI is classified by the world organization for animal health (OIE) into two forms, low pathogenicity (LP) and high pathogenicity (HP), based on virulence in chickens . H7 is oIn the U.S., H7 AI viruses are sporadically recovered from wild birds (WB) and commercial poultry. Many of the outbreaks in commercial poultry  can be tClinical disease signs varied in severity among the virus-host combinations. Mean maximum clinical disease scores  ranged from 0 to 0.7 in chickens Figure . DiseaseMicroscopic lesions were consistent with what has been previously reported for AI virus in chickens, ducks and turkeys. Briefly, lesions observed in tissues from turkeys included viral tracheitis, cilial loss, heterophilic cecitis, and heterophilic rhinitis in the nasal cavity. Patchy cilial loss in the trachea, heterophilic tracheitis, and serositis of the kidney was observed in tissues from ducks. Rare to minimal rhinitis and the presence of heterophils, excessive mucus and slight lymphocytic accumulation were observed in the nasal cavity as well as rare focal heterophilic bronchitis was seen in tissues from chickens.6.3 50% egg infectious doses (EID50) with CK/NY/30749 at 2 days PI and GH/MA/148081 at 4 days PI  and cloacal (CL) swabs from all species were positive for AI viruses by quantitative real-time RT-PCR (qrRT-PCR) with the exception of CL swabs from ducks and turkeys inoculated with ML/OH/421  more virus orally than either chickens or ducks at 2, 7 and 14 days PI. At 4 days PI the amount of virus shed orally by both chickens and turkeys was significantly higher than ducks. Ten days PI OP viral shed by turkeys was significantly higher than chickens. The only statistically significant differences among species in CL shed for all viruses collectively was at 14 days PI when turkeys shed higher titers than either chickens or ducks. All sham inoculated birds were negative for AI virus OP and CL shedding throughout the experiment.4EID50 at 2 days PI  staining for AI virus antigen with an antibody to the influenza A NP protein was conducted with tissues collected 3 days PI selected from birds shedding more than 10At the termination of the experiments, 18-21 days PI, blood was collected from surviving birds and AI virus antibody was evaluated by commercial ELISA for all 3 species. All surviving turkeys except one exposed to CK/MD/MinhMa had detectable AI virus antibody at termination  for A/Turkey/VA/158512/2002 H7N2, which was isolated from the same outbreak as TK/VA/67, to be 102.8EID50 in chickens, 100.8EID50 in turkeys and 103.5EID50 in Pekin ducks [50 for ML/OH/421 has been determined to be 106.6EID50 in chickens, and101.0EID50 in mallard ducks (data not published). Interestingly, ML/OH/421 had some of the lowest shed titers of all the viruses in all three species and was shed at the highest titers by turkeys. Turkeys also had the highest clinical scores of all three species with ML/OH/421. Importantly, it is unknown how the chicken egg passage that was used to propagate these viruses may affect adaptation to chickens and turkeys.Also, consistent with the differences in disease severity among the species, susceptibility studies have reported the 50% bird infectious dose .Since both LPAI viruses and HPAI viruses are reported to primarily cause sub-clinical disease in wild mallard ducks, the induction of mild disease by LPAI virus in Pekin ducks which are most closely related to mallard ducks but which are bred for rapid growth, is important for the commercial duck industry. It has been reported that some Asian lineage H5N1 HPAI virus isolates can cause disease in ducks, but the severity of disease depends on duck age and species ,21. Two-To complement the clinical data and to provide a more complete characterization of the isolates, full genome sequence was produced for all 12 isolates and a basic analysis was conducted. In depth analysis of the HA and NA genes of the H7N2 isolates has already been reported ,11. Ther6EID50 by the simulated respiratory route based on seroconversion. Turkeys consistently presented with the most severe disease and highest OP and CL shed titers, which indicate that, broad breasted white turkeys may be more susceptible to disease from some LPAI virus than white leghorn chickens or Pekin ducks.Twelve LPAI viruses of the H7 subtype were evaluated in chickens, turkeys, and ducks for clinical disease and virus shed. All 12 viruses could infect all three species at the dose of 10Twelve North American origin LPAI viruses were selected to represent different H7 HA genetic groups, different species of origin and different dates of origin within the available H7 subtype domestic LPAI viruses Table . VirusesGallus gallus domesticus), broad breasted white turkeys  and Pekin ducks (Anas platyrhynchos domesticus). Chickens were obtained at 2 weeks of age from a commercial supplier of SPF animals  and were housed in isolators until they were exposed to the virus at 4 weeks of age. There are no sources of turkeys and ducks which are maintained as SPF or free from viral or bacterial respiratory diseases therefore turkeys and ducks were obtained from commercial hatcheries at hatch and were housed in isolators until they were exposed to virus at 2 weeks of age. All birds were obtained from flocks with no antibody or prior exposure to AI virus. The birds were housed in glove-port isolators  with ad libitum access to feed and water before and after exposure to the viruses. Ducks and turkeys were exposed to the virus at 2 weeks instead of 4 weeks of age to accommodate the larger size and faster growth rates of these species in the animal facilities. Since the immune systems of all three species at these ages are considered to be relatively immature, this difference is not expected to impact species associated differences in susceptibility to LPAIV infection and disease. Animals were cared for in accordance with established humane procedures and biosecurity guidelines.Pathogenesis studies were conducted with specific pathogen free (SPF) white leghorn chickens , 2 = moderate to severe , 3 = Dead. Oro-pharyngeal and CL swabs were each collected at days 2, 4, 7, 10 and 14 post inoculation (PI) to evaluate virus shed by quantitative real-time RT-PCR (qr-RT-PCR). Three days PI, 3-5 birds from each group were euthanized and necropsied to evaluate gross lesions. Tissues  were collected for microscopic evaluation. Serum was collected from ducks at 18 days PI and from chickens and turkeys at 21 days PI to confirm infection status.Thirteen to 15 of each species were inoculated with 10RNA was extracted with the MagMAX-96 Viral Isolation Kit  with the KingFisher  magnetic particle processor in accordance with the manufacturer's instructions. Quantitative real-time RT-PCR was conducted with a primer-probe set that targeted the matrix gene as described previously  using th4EID50 2 days PI. Tissue sections were cut (4 μm thick) from paraffin-embedded tissue samples and mounted on charged glass slides . Deparaffinization, antigen retrieval and blocking procedures have been previously described [Because of the sensitivity limitations of immunohisotochemical (IHC) staining for AI virus antigen, tissues were only processed for IHC from birds with OP or CL shed titers greater than 10escribed . A 1:2,0Antibody collected at 18 (ducks) or 21 (chickens and turkeys) days PI from surviving birds was used to confirm infection status. Sera were tested by commercial ELISA . Sera were tested at the manufacturer's recommended dilution of 1:500 and also at 1:100 and 1:50. The dilution of 1:100 was selected for final analysis because at this dilution there were no false-positives among the sera from negative control birds and there appeared to be better sensitivity.6.Full genome sequencing of all isolates was performed as previously described . GenbankMean maximum clinical scores were compared by poultry species for each isolate and virus shed was evaluated by species for each day PI and swab type (OP or CL). All comparisons were conducted with the Student's T-test and the Mann-Whitney rank-sum test if the normality test failed . All statistics were evaluated with a significance threshold of p ≤ 0.0550: 50% bird infectious dose; PI: post inoculation; EID50: 50% egg infectious dose; HA: hemagglutinin; HP: high pathogenicity; IHC: immunohistochemistry; LBM: live bird market; LP: low pathogenicity; NA: Neuraminidase; qrRT-PCR: quantitative real-time reverse-transcription polymerase chain reaction; SPF: specific pathogen free; WB: wild bird.AI: avian influenza; BIDThe authors declare that they have no competing interests.ES was involved in virus selection and experimental design and conducted sequence and phylogenetic analysis. JG was involved in virus selection and experimental design. BL and LP conducted animal experiments, collected specimens, ran qrRT-PCR, and serological assays. CP evaluated tissue sections for microscopic lesions. MJP conducted the immuno-histochemical staining and evaluation. ETM contributed to data analysis and conducted minimum infectious dose studies. All authors contributed to data analysis and manuscript preparation.Phylogenetic trees of the A) N2, B) N3, C) N8, D) N9 genes of viruses included in this study. Trees were constructed with BEAST v. 1.4.8 [7.v. 1.4.8  using HKClick here for filePhylogenetic trees of the A) NS, B) M C) NP D) PA, E) PB1 and F) PB2 genes of viruses included in this study. Trees were constructed with BEAST v. 1.4.8 as described for additional file Click here for fileGenBank accession numbers for all genes for isolates evaluated in this study. Table of GenBank accession numbers by gene and isolate.   Accession number for genes sequenced for this study are shown in boldface type.Click here for file


In Response: We welcome the critiques of Nasci, et al. (The underlying data were obtained with an ELISA specific to severe fever with thrombocytopenia syndrome virus (SFTSV). The conclusion was based on our knowledge, at the time our manuscript was submitted, that in North America no other known phleboviruses of this expanded Uukuniemi group that contains SFTSV and HLV were reported to be cross-reactive with SFTSV. When tested by using our reagent, SFTSV was not cross-reactive with Rift Valley fever virus. Related phleboviruses of this group  have not been reported in North America  are distantly related and have ≈11% amino acid sequence homology to SFTSV. The recently characterized Lone Star virus appears to be the closest relative to SFTSV and HLV and may cross-react with SFTSV and HLV, as also suggested by Nasci et al., but this virus is apparently known only from 1 isolate obtained in 1967 (


This study searches the National Health Insurance Research Database (NHIRD) used in a previous project, aiming for reconstructing possible cerebrovascular disease-related groups (DRG),and estimating the costs between cerebrovascular disease and related diseases.We conducted a nationwide retrospective cohort study in stroke inpatients, we examined the overall costs in 3 municipalities in Taiwan, by evaluating the possible costs of the expecting diagnosis related group (DRG) by using the international classification of diseases version-9 (ICD-9) system, and the overall analysis of the re-admission population that received traditional Chinese medicine (TCM) treatment and those who did not.The trend demonstrated that the non-participant costs were consistent with the ICD-9 categories (430 to 437) because similarities existed between years 2006 to 2007. Among the TCM patients, a wide variation and additional costs were found compared to non-TCM patients during these 2 years. The average re-admission duration was significantly shorter for TCM patients, especially those initially diagnosed with ICD 434 during the first admission. In addition, TCM patients demonstrated more severe general symptoms, which incurred high conventional treatment costs, and could result in re-admission for numerous reasons. However, in Disease 7 of ICD-9 category, representing the circulatory system was most prevalent in non-TCM inpatients, which was the leading cause of re-admission.We concluded that favorable circulatory system outcomes were in adjuvant TCM treatment inpatients, there were less re-admission for circulatory system events and a two-third reduction of re-admission within ICD-9 code 430 to 437, compared to non-TCM ones. However, there were shorter re-admission duration other than circulatory system events by means of unfavorable baseline condition. Cerebrovascular diseases pose a critical threat to human health. The prevalence of cerebrovascular diseases in the study population older than 45 years old was 17.5/1000  Traditional Chinese medicine (TCM) has been used to treat cerebrovascular disease for millenary. Treatments include passive massage and Tai-Chi rehabilitation. Acupuncture and moxibustion have been used for at least two thousand years Since 2006, the Pilot Scheme of National Health Policy in Stroke Adjuvant Acupuncture and Traditional Chinese Medicine (TCM) Therapy has been applied. Our previous reports demonstrated that verum acupuncture, in moderate to severe ischemic stroke patients, might play a protective role against comorbidity and mortality during admission, as well as for 6 months after discharge. However, the studies did not find improvements in neurological deficits We analyzed inpatient costs using the 2006 to 2007 research database. Ambulatory care costs were analyzed using the profiles international classification of diseases version-9 (ICD-9) code 430 to 437 from the research database of the National Health Insurance (NHIRD) to determine attributes such as in-patient duration-time, age, and sex. The investigation project was approved by the ethical committee of Chang Gung Memorial Hospital (Institution Review Board No.99-1800B). The apriori association rule was used to perform data mining using the primary ICD-9 code to identify cerebrovascular disease-related groups. To analyze expenses, we first combined the identity number (ID) and the first date of admission (IN_DATE) column as an index key to distinguish duplicative data based on a screen of the ICD code (430–437). Second, we classified medical institution types based on column of contract hospital type (HOSP_CONT_TYPE). Third, if the department type for selecting if Chinese Medical Treatment records (FUNC_TYPE) column number 60–69 existed, we considered it as a TCM treatment. In Taiwan, acupuncture and Chinese Traumatology manipulation are classified as one TCM therapeutic method. Finally, to determine whether combined TCM and western medical treatment data existed, we used the ID column to compare the medical records.Because the detail document (DD document) of the hospital medical costs database is screened under the main diagnosis code bar unit and filtered under a cerebrovascular code from the research database of the Bureau of National Health Insurance, the first time follow-up patient data were analyzed by (1) merging the ID digital number field and the hospital date as the index key, which was differentiated using repeat gender data, (2) distinguishing between medical institution and special category field outpatient data, (3) regarding the column of interview specialty on medical character, if a TCM code (60 to 69) appeared, we defined it as a rational to distinguish between TCM and western medicine patient treatments, (4) finally, comparing ID digital numbers to identify if any duplications existed in the combination of TCM and western medicine treatments. The analysis of outpatient costs included outpatient visits, and character counting, and was scaled to 100,000 inpatients and the details of the drug and diagnosis and treatments, and pharmacy services costs, as well as information on the amount and average amount of costs related to partial cash payments for treatments. A decomposition analysis of medical cost was conducted to investigate cost trends compared to average medical costs per annum per person. All medical causes of the disease were influenced by age. Therefore, high costs are an accepted factor for aging populations, and result in a rise in total costs. In addition, the average age of the population is growing, which influences average costs. Therefore, the first step of the medical treatment cost analysis was to consider the average age of the population and include an analysis of the population structure. We filtered inpatients out using the ICD-9 code (430 to 437), and included all inpatients that received TCM treatments (case group), and also evaluated the re-admission inpatients database for the 2 years covered by this study. The random-control population was selected from inpatients who did not receive TCM treatments. The multIn 2006, the inpatients’ first ICD-9 code between 430–437 as the primary analysis population, random-control group matched for sex, age by propensity score matching procedures.  The adjuAnalysis showed that the adjuvant TCM treatment group had less odds (68.4%/31.6% = 2.16) than non-TCM treatment group (87.4%/12.6% = 6.93), favorable odds on non-TCM treatment group by re-admission ICD-9 code 430 ∼ 437 , 95% confidence interval not containing 1, P<0.01. A two-third reduction of re-admission within ICD-9 code 430 ∼ 437 favored the adjuvant TCM treatment group, the difference was statistically significant.We estimated that overall, only 4% of the annual budget was spent on TCM treatments, and the rest was spent on conventional treatments. We investigated three municipals— Kaohsiung, Taichung and Taipei, they are on behalf of the south, the middle and the north part of Taiwan. The medical expenditure trend indicated that the costs for non-TCM treatment inpatients were consistent in the ICD-9 category (430 to 437), and were similar for both years. However, wide variation and more costs were incurred by TCM participants than non-TCM ones during these 2 years.The overall secondary costs to western medicine treatment for all inpatients were more in 2006, as indicated by ICD 430, 432, 434, and 436, than in 2007. In 2007, ICD 433 and 437 reported higher costs than in 2006. In 2007, the trend demonstrated that adjuvant TCM treatment inpatients paid less for conventional treatments, with the exception of ICD 437, which appeared to be higher in the first instance.This study demonstrated that for inpatients receiving TCM treatments for the DRG1501-02 Project, the annual costs during 2007 were significantly lower than in 2006. By contrast, for DRG1601-1702, if the TCM associated medical costs were excluded, the medical treatment costs for the 2 years were similar.During the first hospitalization, the percentage of adjuvant TCM treatments was 0.82% in 2006 and 1.70% in 2007. In addition, the re-admission rate of TCM inpatients was 17.84% in 2006 and 19.04% in 2007. During the first hospitalization, 99.18% (2006) and 98.3% (2007) of the whole population were non-TCM inpatients, and the re-admission rate was 10.56% in 2006 and 9.43% in 2007. The average re-admission duration was significantly shorter for TCM participants, especially for the initial ICD 434 cases. However,Among inpatients who were diagnosed with ICD-9 code (430 to 437) upon first admission, the average re-admission duration was significantly shorter for TCM inpatients. In addition, the inpatients being introduced to TCM demonstrated more severe general symptoms, which were costly to deal with conventional treatments, and the severity of underline diseases might be causative re-admission. However, adjuvant TCM treatment inpatients appeared to have significantly lower circulatory system events while re-admission. However, there were shorter re-admission duration other than circulatory system events by means of unfavorable baseline condition.The disease diagnosis related group (DRG) measure enforces equal medical fee payments for diagnosis-related groups and helps to reduce unnecessary medical costs that burden the system. Recently, Taiwan has been planning to implement the DRG. However, after numerous meetings, consultations, commissioning of scholars and experts to lead relevant research projects, some public interest-related support measures have been developed for the short-term, but no guarantee exists that public health interests are not negatively influenced by the waste of additional medical resources. Since 2009, DRG measures have focused on the gradual implementation of health payments with a pilot project. This study’s design was based on a previous study focusing on data from 2006 and 2007 Taiwanese National Health Insurance Research Database, and included study participants that were diagnosed under the international standard code for diagnosis of disease (ICD-9) in the cerebrovascular disease Group (430 to 437) for analysis. In April 2006, the National Health Council issued a Pilot Scheme of Health Policy in Stroke Adjuvant Acupuncture and TCM Therapy Among inpatients diagnosed with ICD-9 code 430 to 437 upon first admission, the adjuvant TCM treatment patients appeared to be significantly less re-admission for circulatory system events. A two-third reduction of re-admission within ICD-9 code 430 ∼ 437 favored the adjuvant TCM treatment patients.File S1Appendix S1.(DOCX)Click here for additional data file.


Giardia intestinalis and Trichomonas vaginalis.Eukaryotic cells possess a complex network of RNA machineries which function in RNA-processing and cellular regulation which includes transcription, translation, silencing, editing and epigenetic control. Studies of model organisms have shown that many ncRNAs of the RNA-infrastructure are highly conserved, but little is known from non-model protists. In this study we have conducted a genome-scale survey of medium-length ncRNAs from the protozoan parasites Giardia RNase MRP RNA, which is a key ribozyme involved in pre-rRNA processing. We have also uncovered 18 new H/ACA box snoRNAs, expanding our knowledge of the H/ACA family of snoRNAs.We have identified the previously 'missing' Giardia intestinalis and Trichomonas vaginalis, like their distant multicellular relatives, contain a rich infrastructure of RNA-based processing. From here we can investigate the evolution of RNA processing networks in eukaryotes.Results indicate that  G. intestinalis and T. vaginalis. We can compare the ncRNAs from these two protists with other model eukaryotes , to understand more about the general nature of the RNA infrastructure within eukaryotes.The current view of cellular RNA organization indicates an RNA infrastructure ,2, whichGiardia have identified key eukaryotic ncRNAs such as the RNase P [Trichomonas ncRNAs show that the currently known ncRNAs also exhibit typical features of eukaryotes [Giardia, and the different types of snoRNAs in Trichomonas  and Trichomonas vaginalis , to clarify uncertainties about conserved features of the RNA infrastructure.Previous studies in  RNase P , snoRNAs RNase P ,10, spli RNase P , miRNAs  RNase P -14 and a RNase P . Studieskaryotes ,16-18 inkaryotes  and somekaryotes ,14. HoweG. intestinalis and T. vaginalis, and identified from this data small ncRNAs such as microRNAs in the two protists [G. intestinalis, and clarify annotations of RNase P and MRP from both G. intestinalis and T. vaginalis. We also identify new H/ACA box snoRNAs from these species. Our study clearly demonstrates that high-throughput sequencing cannot only screen for small regulatory RNAs, but can also be used for the characterization of longer ncRNAs from diverse organisms. Results from our work support that G. intestinalis and T. vaginalis possess a rich network of RNA processing components expected in the consensus eukaryotic RNA infrastructure.We have previously sequenced ncRNAs sized from 10 to 200 nucleotides (nt) from protists . Using tG. intestinalis and T. vaginalis were purified, size fractionated and sequenced according to Methods. Constructing contigs of short to medium RNAs requires a different approach from standard assembly protocols. Most ncRNAs do not have long poly-A tails; therefore small-RNA sequencing is a way of recovering medium-length ncRNAs when used in combination with consensus mapping. We successfully generated RNA contigs from G. intestinalis and T. vaginalis and compared the updated mapping software Bowtie [The total RNAs from e Bowtie  to the oe Bowtie . OverallDe novo assembly tools such as Velvet [de novo assembly of small contigs. Our study was carried out on G. intestinalis WB strain (Genome Assemblage A), and the genome assemblages of the other two strains  were used for comparison in some of the subsequent analysis.With the length cutoff of the new contig datasets set above 50nt we discarded mature miRNAs and siRNAs. s Velvet  and Abyss Velvet  producedG. intestinalis and T. vaginalis genome annotation to check that rRNAs and tRNAs were represented as expected. From the G. intestinalis sequences all 80 annotated rRNAs and tRNAs from the WB isolate were covered by contigs generated with Bowtie mapping. However, many of these contigs contain Illumina short reads mapped to multiple sites in the genome and therefore were assigned equally to each of the possibilities. Contigs were trimmed in length from the 5' end, to a minimum length of 20 nt, 36 nt and 50 nt to use as sequence tags and mapped against the annotated ncRNAs. Our original trimmed sequence datasets were also mapped against the annotated ncRNAs. All G. intestinalis and T. vaginalis rRNAs were found by sequence and contigs. In G. intestinalis all tRNAs and rRNAs were found from Assemblage A with 3 tRNAs not found by contig but found by sequence in Assemblage B, (P15)  and four not found by sequence or contig . A comparison against Assemblage E (Isolate P15) had 4 tRNAs  not found by sequence or contig. Given that our sequences came from the same strain as Assemblage A, not finding some sequences in the other strains is not surprising. T. vaginalis had many annotations for its rRNAs and tRNAs and the majority were found by both contig and sequence  ~10 proteins. It catalyzes the nucleotide cleavage reaction at the A3 site  on the pre-rRNA transcript. MRP RNA is evolutionarily related to the eukaryotic RNase P RNA which processes the 3'- end of pre-tRNA transcripts . The RNastinalis  imposed  MRP RNA .G. intestinalis MRP and a representative of other eukaryotic MRPs showed a high degree of sequence similarity  and as expected, our sequence had 100% identity to isolate A RNAs are a group of ncRNAs of variable length (from 60 up to 1000 nt in yeast), which are involved in processing of several types of transcripts . Most ofaginalis ,14. C/D G. intestinalis and T. vaginalis were aligned with human rRNAs to locate conserved pseudouridylation sites, which were then incorporated into the search for the complementary target-binding regions in candidate H/ACA box snoRNAs. After generating the negative controls, the RNA contigs were searched using the established parameters from control runs (see Methods). Table G. intestinalis and 10 T. vaginalis new snoRNAs were identified using this method. The RNA sequences and corresponding genomic positions are in Additional file G. intestinalis snoRNAs (Gi/ACA.1 and Gi/ACA.2), and two sets of the new T. vaginalis snoRNAs  are overlapping in the same genomic region on the plus and minus strand respectively. The structures of the target binding regions of new snoRNAs are shown in Figure As a first step in identification of new H/ACA box snoRNAs, the rRNAs of G. intestinalis snoRNAs all have different target pseudouridylation sites on rRNAs, whereas two of the T. vaginalis new snoRNAs (Tv/ACA.3 and Tv/ACA.10) share the same target (LSU rRNA U2214). In addition, Gi/ACA.5, Tv/ACA.3 and Tv/ACA.10 target a conserved site on the large subunit rRNA, the same as Gi/ACA.6 and Tv/ACA.2. The target regions are highly conserved between G. intestinalis and T. vaginalis, but the sequences of the snoRNAs targeting these sites do not show substantial sequence similarity. A BLAST [G. intestinalis H/ACA box snoRNAs was performed against the genomes of the other two isolates of Giardia strains (Assemblage B and E) to look for homologous sequences. Three G. intestinalis new snoRNAs have homologous sequences in either Assemblage B or E, and 2 have homologous sequences in both Assemblage B and E. Overall the sequences are highly conserved and the nucleotides complementary to the rRNA target sequences show minimal changes across the three Giardia strains.The new  A BLAST  search oRecent studies on ncRNAs throughout eukaryotes have expanded our understanding of the RNA-processing infrastructure  with theG. intestinalis RNase MRP RNA is an important achievement in searching for conserved key ncRNAs of the central RNA-processing pathway. Sequence and structural analysis of the G. intestinalis MRP RNA has shown all the conserved characteristics of eukaryotic MRP, as referred to in Figure G. intestinalis P and MRP RNAs indicates that the protein-RNA relation in G. intestinalis P and MRP does not differ significantly from other eukaryotes. Identification of the MRP RNA from G. intestinalis has filled the gap left from previous studies of MRPs. In looking at the structure of the G. intestinalis MRP we can see how consensus models based on the eukaryotic P3 region could not have detected either the sequence or the structure. The typical P3 region of G. intestinalis resembles more the bacterial model of RNase P RNA than the eukaryotic P3 model for both RNase P and RNase MRP RNAs. However, the rest of the structure fits the eukaryotic RNase MRP RNA model. This demonstrates that with protists in particular, the standard eukaryotic models for ncRNAs may not necessarily apply.Characterization of the G. intestinalis and T. vaginalis all have only one predicted target-binding site regardless the number of stem-loops. Having one target-binding site is also seen in the H/ACA box snoRNAs found in Trypanosomes [G. intestinalis and T. vaginalis. Understanding more about general structural and sequence motifs, will aid us in further searches for H/ACA box snoRNAs.The novel H/ACA box snoRNAs identified from anosomes . Identifanosomes , but to anosomes . TherefoG. intestinalis contigs . However, despite strong G-C rich complementary binding, the potential editing site does not have an un-bound nucleotide immediately 3' to the uridine, as typically observed in all H/ACA box snoRNAs found so far. Furthermore, there are only 3 base pairs on the 5' side of the base-pairing region instead of 4 to 10 base pairs typically observed in known snoRNAs. In addition, there has been no evidence of pseudouridine modification in RNase P. Therefore, any conclusion that this ncRNA is acting as an orphan snoRNA on RNaseP cannot be made at this stage. Apart from the above, the rest of these 29 H/ACA-like RNAs do not appear to have rRNA targets.Many snoRNAs do not have identified targets and are therefore termed orphan snoRNAs . Althougs Figure . ResultsWe demonstrate in our study that high-throughput sequencing of ncRNAs larger than ~21-25nt  is possible, and that the assembly of short read sequences can lead to the characterization of medium-length ncRNAs. We have found this technology to be a boost to the study of ncRNAs from non-model eukaryotes and especially those distantly related to well characterized species . A limitation of this study is that the sequencing length of 36nt (standard in ncRNA sequencing) is short and that longer sequences would enable the identification of ncRNAs ~70-150nt in single reads, thus not requiring assembly.G. intestinalis, which answers positively the previously open question as whether MRP exists in all extant eukaryotes with the A3 site. In addition, a number of new H/ACA box snoRNAs have been identified in G. intestinalis and T. vaginalis, with a reduced structure compared to model species, but still possessing the characteristic target-binding pattern and sequence motifs. It is becoming evident that not only are the components of RNA-processing network highly conserved within eukaryotes, but also the pattern of transcription across the genome appears to be shared among distant lineages. Overall, our results imply that it is increasingly likely that the main classes of RNA processing and regulation were present in the last common ancestor of eukaryotes [In conclusion, we constructed new RNA contigs with updated genomes and identified the RNase MRP of karyotes . We demoOur general strategy has been to search for the major classes of RNA in all major groups of eukaryotes and investigate the evolution of their mechanisms ,43,44. INote that the sample preparation and sequencing was prepared as for previous publications from this data . We haveG. intestinalis trophozoites (WB strain) were grown in TY1-S-33 media, and T. vaginalis was maintained in T. vaginalis broth (Fort Richard) at 37°C. Cells were harvested by centrifugation . Growth media was removed and cells were washed once in PBS buffer. Total RNA was extracted using Trizol (Invitrogen) according to the protocol provided by the manufacturer, and further purified by phenol: chloroform extraction. The purified RNA was dissolved in double distilled water. For sequencing, 10 μg of DNase treated, 5'- de-capped total RNAs were separated on a 15% denaturing acrylamide 8 M urea gel and RNAs ranging from 10 to 200 nt were cut out from the gel and prepared according to Illumina's small RNA preparation protocol. This effectively includes an RT-PCR step that converts RNA to DNA for further sequencing. 8 and 12 pmol (in each lane) of G. intestinalis and T. vaginalis cDNA were sent for sequencing on an Illumina Genome Analyzer for 36 cycles. Pipeline analysis was performed with the Illumina Pipeline version 1.4.G. intestinalis (version 1.2) and T. vaginalis (version 1.1) and short consensus 'RNA contigs' were generated using Maq version 0.7.1 [G. intestinalis version 2.3 and T. vaginalis version 1.2). For these new contigs we used Bowtie [G. intestinalis RNA contigs using Infernal 1.0 [Giardia strains using BLAST[During our previous study  full lenon 0.7.1 . We initon 0.7.1  (G. inted Bowtie  for mappd Bowtie  for convd Bowtie , and thernal 1.0 . Secondarnal 1.0  and the ing BLAST.G. intestinalis and T. vaginalis. rRNAs of G. intestinalis and T. vaginalis were first aligned with human rRNAs, then conserved sites in rRNAs with known pseudouridylation in human were selected as possible target sites. These sites were then used for snoRNA prediction using SnoGPS [G. intestinalis and T. vaginalis contigs were shuffled with the same nucleotide frequency 100 times and the resulting sequences were used as control input for SnoGPS program. First, the standard two stem-loop model was used, and this search permitted G-U base pairs in snoRNA-target binding. However, previous studies have shown that snoRNAs in G. intestinalis could also adopt an archaeal one-stem structure [The search for new H/ACA box snoRNAs used SnoGPS 2.0  with defg SnoGPS . Initialtructure , therefoAll new sequences are in preparation to be included into Rfam .XSC carried out the culturing and RNA isolation, ran the snoRNA analysis and drafted the manuscript. LJC carried out the MRP analysis, coordinated the project and helped to draft the manuscript. DP conceived of the study, and participated in its design and helped to draft the manuscript. All authors read and approved the final manuscript.Supplementary Tables.Click here for fileG. intestinalis H/ACA snoRNAs.Click here for fileT. vaginalis vaginalis H/ACA snoRNAs.Click here for file


Meleagris gallopavo) with gastrointestinal disease from a farm in Indiana. Its genome organization is as follows: 5′ untranslated region (UTR)IRES-II  3′ UTR-poly(A). TuASV shares only 34% (P1), 36% (P2), and 35% (P3) amino acid identities with avihepatoviruses, indicating that it potentially represents a novel picornavirus genus.A novel picornavirus, turkey avisivirus (TuASV), was identified from the feces of turkeys ( Picornaviridae consists of 12 formal genera, but potential new genera infecting humans and domestic and wild animals have been recently described (Picornaviridae website [http://www.picornaviridae.com]). Turkey production is an important food industry worldwide, valued at $5 billion in the United States in 2011  based on its relatedness to the duck hepatitis A virus of the genus Avihepatovirus, and for consistency with a publication in press describing a closely related virus named TuASV-1 (strain turkey/M176-TuASV/2011/HUN); TuASV-1 was found in Hungarian turkeys with stunting syndrome and/or poultry enteritis . The 5′ UTR was 609 bases long, containing a type II internal ribosome entry site (IRES-II), followed by a 6,495-base ORF  encoding the polyprotein P1, P2, and P3 regions. The 3′ UTR was 266 bases long, followed by poly(A). Both 2A proteins (2A2 and 2A3) and the 2C protein contained the helicase nucleotide-binding motif GXXGXGK(T/S). Surprisingly, the closely related Hungarian viruses contained a third 2A protein (2A1) that is missing in the U.S. strain.The virus was provisionally named turkey avisivirus , 36% (P2), and 35% (P3) amino acid identities with duck hepatitis virus (DHV) and lower identities with other picornaviruses. Based on the International Committee on Taxonomy of Viruses (ICTV) criteria, members of the same picornavirus genus share, for P1, P2, and P3 regions, >40%, 40%, and 50% amino acid identities, respectively. Therefore, TuASV-USA-IN1 likely reflects the presence of a new genus in the family KC614703.The complete genome sequence of TuASV-USA-IN1 has been deposited in GenBank under accession no.


Erve virus (ERVEV) is a European Nairovirus that is suspected to cause severe headache (thunderclap headache) and intracerebral hemorrhage. The mode of transmission to humans (ticks or mosquitoes) is still unknown. Currently, no standardized testing method for ERVEV exists and only a small partial sequence of the polymerase gene is available. Here, we present the first complete genome sequence of ERVEV S, M, and L segments. Phylogenetic comparison of the amino acid sequence of the L-protein (RNA-dependent RNA polymerase) revealed only 48 % homology to available L-protein sequences of other Nairoviruses like Crimean-Congo hemorrhagic fever virus, Nairobi sheep disease virus, Hazara virus, Kupe virus, and Dugbe virus. Among themselves, these Nairoviruses show 62–89 % homology in the L-protein sequences. Therefore, ERVEV seems to be only distantly related to other Nairoviruses. The new sequence data can be used for the development of diagnostic methods and the identification of the natural vector. Crocidura russula) collected in the Erve River Valley in northwestern France after intracerebral inoculation of young mice, which showed signs of acute encephalitis, edema, capillary congestion, cuffing, and disseminated necrosis of neurons [Nairovirus genus of the Bunyaviridae family [Crocidura sp. in Senegal. Both viruses reacted in IFA with a polyvalent serum that contained antibodies to different Nairoviruses like Crimean-Congo hemorrhagic fever virus (CCHFV), Hazara virus (HAZV), Ganjam virus (GANV) a strain of Nairobi sheep disease virus (NSDV) found in India, Dugbe virus (DUGV), and the so far unassigned Bhanja virus (BHAV), but they did not react with antibodies to either of these viruses used to prepare the polyvalent serum [Borrelia burgdorferi infections have not been detected, indicating that it is rather unlikely that ERVEV is transmitted by Ixodes or Dermacentor ticks [Erve virus (ERVEV) was isolated in 1982 from tissues of a white-toothed shrew , centrifuged at 700×g  for 10 min then at 2,800×g  for 5 min and filtered through a 0.2-μm sterile filter. To enrich for viral particles, 20 ml supernatant was mixed with 1.48 ml 5 M NaCl and 10.8 ml 30 % PEG8000 in NTE , incubated on a shaker for 30 min at 4 °C, and subsequently centrifuged at 6,000 rpm for 60 min and 4 °C. The virus pellet was resuspended in 500 μl PBS. RNA extraction was performed as described [ERVEV was passaged 5 times in Vero E6 cells maintained in DMEM medium supplemented with 2 % fetal bovine serum, 2 mM Glutamine, 10 mM Penicillin, and 10 mM Streptomycin in a 175-cmTo be able to cover the 3′ terminal parts of the segments, 500 ng self-complementary FLAC adapters were ligated to 500 ng purified viral ssRNA as described for dsRNA Orbiviruses , 13, 20.300 ng of the whole genome amplified dsDNA was used for Titanium Shotgun Rapid Library Preparation and pyrosequencing as described in the FLX Titanium Protocol (Roche) and in , but omiAssembly of the sequenced ERVEV genome segments was done by means of the Genome Sequencer FLX System Software Package version 2.3  in combination with the commercially available SeqMan Pro Software version 9.1.0 .Additional Nairovirus genomes were downloaded from GenBank  and SwisDENDROSCOPE [For phylogenetic analysis of the ERVEV proteins, ERVEV-specific sequences were aligned to a selection of available corresponding Nairovirus sequences from GenBank by means of the ClustalW algorithm (neighbor-joining method) of commercially available MegAlign software version 9.1.0 , MEGA version 5.05  and DENDDROSCOPE  with a 1Using combined FLAC anchor primers for the 3′ end and 5′ RACE adaptors pyrosequncing determined the full-length sequences of the 3 genome segments of ERVEV in 1 lane of a 4-lane picotiter plate in a pool of 6 different RL-MID-tagged virus libraries in one sequencing run. The ERVEV library yielded 42,025 reads  with 19,455 specific reads (= 46 %) or 5,174,304 bases  = 289). All 3 genome segments were covered by 100 %.Table Sequence distance analysis for all available Nairovirus protein sequences in comparison to putative CCHFV protein sequences showed a low degree (<49 %) of similarity of the 3 putative ERVEV protein sequences with those of CCHFV Fig. a–c. NeigSince there are more partial sequences available for other Nairoviruses than complete genomes, we assembled all available partial sequences of the RdRP into a neighbor-joining phylogenetic tree Fig. a, which Innate immune cytokines such as type I interferon (IFN-α/β) and tumor necrosis factor alpha (TNF-α) play fundamental roles in the early response to viral infection. The gene expression of both cytokines depends on the host proteins Ubiquitin (Ub) and interferon-stimulated gene product 15 (ISG15) which reversibly conjugate to proteins via a conserved LRLRGG C-terminal motif. This common structure motif can be targeted by viruses to simultaneously avoid both IFN-α/β and TNF-α effects. Ub and ISG15 are synthesized as inactive precursors that undergo cleavage to expose the LRLRGG sequence required for conjugation to target molecules. The coordinated activities of an enzymatic cascade comprising an activating enzyme (E1), a conjugating enzyme (E2), and a ligase E3) result in the conjugation of Ub or ISG15 to the ε-amino group of a lysine residue present in the target protein. This conjugation can be reversed by the activity of deconjugation enzymes. In case of Ub, five classes of de-ubiquitinating proteolytic enzymes have been described . One of  result iBorrelia burgdorferi infections was observed [ERVEV is a Nairovirus described in France, Germany, the Netherlands, and Czech Republic . It is sobserved . Taken tThe new sequence data can now be used for the development of diagnostic methods especially nucleic acid detection methods or expression cloning and protein purification for serological assays. The developed diagnostic systems could help to identify the natural vector and to clarify the importance of this human pathogen.


We report the complete genome sequence of a reassortant H14N2 avian influenza virus isolated in 2011 from a northern shoveler in California. This introduced Eurasian subtype acquired seven segments from North American viruses and circulated in the Pacific Flyway 1 year after its detection in the Mississippi Flyway. Anas platyrhynchos) and a herring gull (Larus argentatus) sampled at the Caspian Sea in 1982  in the U.S. Mississippi Flyway, demonstrating the first known movement of H14 into the Western hemisphere  belonging to the H14 subtype were sequences from mallards (misphere .Anas clypeata) sampled on 29 October 2011 in the Suisun Marsh near San Francisco Bay, CA. This virus, A/northern shoveler/California/2696/2011(H14N2), was compared to all H14 sequences available on GenBank (n = 13).We isolated an H14N2 virus from the cloacal swab of a Northern shoveler  shares 97% nucleotide identity with six H14 viruses isolated in North American in 2010 and 89% identity with the seven H14 viruses isolated in Central Asia in 1982. Although the H14 subtype can support a highly pathogenic phenotype  revealed extensive reassortment of all segments other than HA , sharing 99% identity with contemporary North American isolates circulating in both the Pacific and Mississippi Flyways.The isolation of A/northern shoveler/California/2696/2011(H14N2) supports the hypotheses that H14 viruses circulated in North America from 2010 to 2011 and that mixed infections readily produced reassortant viruses in different flyways separated by 2000 km within a single year , 6.CY146897 through CY146904.The GenBank accession numbers for the H14N2 complete genome sequence are


Dengue virus infection is a serious health problem infecting 2.5 billion people worldwide. Dengue is now endemic in more than 100 countries, including Pakistan. Each year hundreds of people get infected with dengue in Pakistan. Currently, there is no vaccine available for the prevention of Dengue virus infection due to four viral serotypes. Dengue infection can cause death of patients in its most severity, meanwhile many antiviral compounds are being tested against dengue virus infection to eradicate this disease but still there is a need to develop an efficient, low-cost and safe vaccine that can target all the four serotypes of dengue virus. This review summarizes dengue molecular virology, important drug targets, prevalence in Pakistan, diagnosis, treatment and medicinal plant inhibitors against dengue. Aedes aegypti or Aedes albopictus mosquito [Dengue virus (DENV) infection is an important arthropod-born viral infection infecting about 2.5 billion people worldwide, of which approximately 975 million belong to large and small cities of tropical and sub-tropical countries in Southeast Asia, the Pacific and the America ,2. The pmosquito ,7. Theremosquito ,9. Dengumosquito . Predomimosquito . Pakistamosquito . To dateTo date, many research studies have been conducted to understand the life cycle of dengue virus, especially the viral protein processing and the genome replication. Dengue virus binds to its receptor, and this process is mediated by envelop protein (E). In mammalian cell, DEN 1–4 serotypes bind with Heparan sulfate, nLc4Cer, DC-SIGN/L-SIGN and Mannose receptors. DEN-2 serotype also binds with HSP70/HSP90, GRP78, CD14-associated protein and two unknown proteins having trypsin resistance and trypsin sensitive properties. DEN 1–3 serotypes as well bind with Laminin receptor. DEN 2–4 serotypes also bind with an unknown protein having the property of serotype specific binding , a membrane associated protein (M) and Envelop protein (E). These proteins are involved in defining the structure of the virus.DENV core protein is a highly basic protein responsible for assembly of nucleocapsid through interaction with RNA but only a little is known about C protein. Structure of the protein revealed that the C protein dimer had high net charge and there is asymmetric distribution of basic residues over the surface of protein . Anti DEMembrane associated protein is a membrane glycoprotein works as a part of nucleocapsid and assists to envelop protein to form mature virions. Antibodies against prM protein were developed for Japanese encephalitis virus (JEV), West Nile virus (WNV) and dengue virus. Antibodies against the prM protein of JEV do not react with the prM of dengue viruses or West Nile virus. prM protein can be used to investigate differentiating antibody responses to difference flaviviruses . C-prM wEnvelop protein is present on the surface of the virus and is extremely involved in virus attachment with host cell through cell receptors like heparin sulphate DC-SIGN. It is most important protein for the entry of virus in cell. This protein has three domains, domain I , domain II (dimmers) and domain III (binding domain). Dimmer domain links structural and binding domain ,27. The DENV genome encodes seven non-structural proteins, including NS1, NS2a, NS2b, NS3, NS4a, NS4b and NS5. These proteins are responsible for viral replication and other cellular functions.st day to day 9 of infection. This was even detected when viral RNA was negative in RT-PCR. Its level differed in individuals ranging from a couple of nanograms per milliliter to several micrograms per milliliter. Thus, detection of NS1 protein may allow diagnosis of dengue infection in early stages [NS1 is expressed on the surface of the infected cell and can serve as an antibody target for dengue infection . NS1 proy stages . In a sty stages . NS1 medy stages .NS2A protein is majorly involved in RNA replication and viral assembly. A mutation in NS2A protein blocked virus replication. NS2A also inhibits the IFN-β promoter-driven transcription, thus serves as interferon (IFN) antagonist ,35. A siNS2B works as a cofactor in NS2B-NS3 serine protease. NS2B is required for the cleavage of NS2A/NS2B and NS2B/NS3.NS2B is also required for NS3/NS4A cleavage and for other internal cleavages .Wang andNS3 of DEN-2 is second largest non-structural protein having serine protease at N-terminus and NTPase helicase RNA triphosphate (RTPase) at C-terminus end. . NS3 is NS4A is the least characterized protein. At its C-terminus end, it is highly hydrophilic and serves as a signal to translocate NS4B to the endoplasmic reticulum lumen. In membrane bound viral replication complex (RC), NS4A forms components that help in maintaining this complex . NS4A haNS4B interacts with NS3 to regulate RNA replication. When treated with IFN-β or IFN-γ, NS4B expressing cells did not exhibit signal transducer and activator of transcription (STAT 1), thus indicated that NS4B may be involved in blocking IFN signaling and can be implicated as IFN-signaling inhibitor . FunctioNS5 has a methyltransferase (MTase) domain at N-terminus end and a polymerase domain at C-terminus end. NS5 has a role in RNA replication due to presence of RNA dependent RNA polymerase activity. It is also involved in de novo initiated RNA synthesis .Charge-tDengue has a worldwide history of about 200–400 years, mostly causing infection in tropical and sub-tropical areas. Dengue virus is believed to come in Pakistan with tyres at Karachi sea port carrying eggs of infected mosquitoes. To date, dengue virus infection has caused several outbreaks in Pakistan ,58. Untihttp://www.paperpk.com/news/index.php/new-dengue-cases-punjab-2012/). The year 2007 and 2011 has been worst years in regard of dengue virus infection in Pakistan. Dengue virus circulates in Pakistan throughout the year with a peak incidence in the post monsoon period. Recent flood in Pakistan made the situation worse [During 2011, the disease rapidly assumed the proportions of epidemic, specifically in Punjab and particularly Lahore where, in September, more than 250 people were reported dead and, according to the Punjab Health Department, over 12,000 people were infected during January to September 2011. Sindh, too, was hit by the dengue virus. According to the Sindh Provincial Dengue Surveillance Cell, nearly 400 people were infected with the virus in few months with over 300 people in Karachi alone, and the death toll stood at five at the end of September 2011. In February 2012, 73 confirmed cases of dengue in Lahore and 13 other regions of the state of Punjab were reported (Dengue virus can cause mild fever to Dengue fever (DF) or more severely DHF/DSS. DF causes fever in 3–5 days of infection, in which patient can feel headache, joint pain and rashes on the body with thrombocytopenia and relative leucopenia. Both DHF and DSS have same symptoms as of DF but they also have increased vascular permeability and hemorrhage, can lead to death. Dengue infection is mostly clinically diagnosed through symptoms. However, diagnosing dengue infection through symptoms is not reliable, and it needs laboratory studies to confirm the presence of dengue virus ,72. DiffLaboratory diagnosis of dengue virus infection can be made by the detection of particular virus, viral antigen, genomic sequence, and/or antibodies. IgM captured ELISA, virus isolation in mosquito cell lines and live mosquitoes, dengue specific monoclonal antibodies and detection of viral RNA by nucleic acid amplification tests (NAAT) have all represented major advances in dengue diagnosis. There are two stages of diagnosing dengue infection. In stage 1, fever is accompanied by NS1 antigens in blood and in the stage 2, post-febrile period in which IgM and IgG antibodies are in excess. In acute phase of dengue, patients generally suffer from flu-like fever and seek medical attention in first two days. During this phase, diagnosis is possible by detecting viral RNA/proteins in patient blood. If after two days fever is not recovered, then patient should be advised for a complete blood checkup (CBC). After the checkup, if platelet count is below normal range , and WBC count is below normal range  of blood) then patient should go for dengue antigen test. Patients with DHF/DSS should go for serological test. Using serotype specific NS3 primers, infection was identified in 80% patients . Dengue http://en.wikipedia.org/wiki/2011_dengue_outbreak_in_Pakistan). The Second is vaccination, which is currently not available and the Third is drug therapy but no antiviral drugs are available to target dengue virus [http://www.denguevirusnet.com/treatment.html).To date, there is no specific treatment available for dengue virus infection. There are three ways to solve the problem of dengue infection. The First is to use a preventive measure by avoiding contact with infected mosquitoes. Aedes mosquitoes bite during daytime and its contact can be avoided by: properly managing waste and improving storage of water, removing all sources of stagnant water, using household pesticides to kill mosquitoes, using mosquito coils and nets, wearing long-sleeved shirts, socks and trousers and using insect repellent to avoid mosquitoes. Insecticide treated nets (ITNs) are available to protect young children, pregnant women, old people, in addition to others who may rest during the day . Governmue virus . HoweverPlants have been traditionally used to cure a number of human diseases. To date, few plants derivatives due to their medicinal properties have successfully been tested against viral diseases. The first step of DENV cycle is to attach with host via host receptors. Envelop protein (E) is involved in viral and host attachment. Thus, dengue infection can be inhibited by targeting envelop protein/inhibiting host-viral interactions. NS2-NS3 protease and NS5 also serve as important antiviral drug targets due to their role in viral replication and other cellular processes. To date, many medicinal plants have been tested against DENV and some of them showed significant inhibition effects in the DENV replication cycle.3/μL to 168x103/μL, White blood cells from 3.7x103/μL to 7.7x103/μL and neutrophils from 46% to 78%. Thus carica papya can be used to target dengue fever [6/36  cells. Aqueous neem extracts at its maximum nontoxic concentration of 1.897 mg/ml, completely inhibited 100–10,000 TCID50 of virus as indicated by the absence of cytopathic effects [50) value of 55 μg/mL and selectivity index (SI) of 4.49. In Vero cells, rutin and naringenin did not inhibit DENV-2 replication [Antiviral effects of methanolic extracts of Andrographis paniculata, Citrus limon, Cymbopogan citrates, Momordica charantia, Ocimum sanctum and Pelargonium citrosum on dengue virus serotype 1 (DENV-1) were investigated by Tang and colleagues. A. paniculata showed the most antiviral inhibitory effect followed by M. chrantia in in-vitro assays. Both these plants can be advantageous in developing novel antiviral compounds . Carica ue fever . Extract effects . Talaric effects . Rehman  effects . DEN-2 i effects . Kiat an effects . A serie effects . Zandi a effects . Three flication . Recentllication .Dengue infection has emerged as a major health concern in Southeast Asia, the pacific and America. Dengue virus has become a serious issue in Pakistan as it has caused many endemics starting from 1994 to 2011. Developing tetravalent vaccine against all four dengue serotypes is quite challenging. To date, there is no licensed vaccine available for dengue virus. Therefore, there is an urgent need to develop an alternative solution to combat this endemic infection. Currently, there are no antiviral compounds available against dengue virus and there is a need to develop antiviral compounds that can target all four serotypes of dengue with same efficiency. Dengue virus enveloped protein is involved in virus cell entry; NS3 and NS5 are involved in viral replication and other cellular processes; therefore, these can serve as an important drug target to combat this life-threatening disease. Several medicinal plants have been tested against dengue virus entry and replication; many of them showed significant inhibitory effects. Nevertheless, it will be very exciting to see these medicinal plants as potential DENV inhibitors to progress through clinical developments and, hopefully, provide dengue patients with much needed, more effective therapies.All authors have no kind of institutional and financial competing interests.UAA designed the study and SI wrote the manuscript. Both the authors read and approved the final manuscript.Sobia Idrees (MPhil student), Usman A Ashfaq (PhD molecular Biology and Group leader, Human Molecular Biology Group, Department of Bioinformatics and Biotechnology, GCU, Faisalabad.