Closteroviridae. Whereas some of the viruses associated with this disease are transmitted by insect vectors, others are only graft-transmissible. In three regions of California, we surveyed vineyards containing diseased vines and screened symptomatic plants for all known viral species associated with grapevine leafroll disease. Relative incidence of each virus species differed among the three regions regions, particularly in relation to species with known vectors compared with those only known to be graft-transmitted. In one region, the pathogen population was dominated by species not known to have an insect vector. In contrast, populations in the other surveyed regions were dominated by virus species that are vector-transmissible. Our survey did not detect viruses associated with grapevine leafroll disease at some sites with characteristic disease symptoms. This could be explained either by undescribed genetic diversity among these viruses that prevented detection with available molecular tools at the time the survey was performed, or a misidentification of visual symptoms that may have had other underlying causes. Based on the differences in relative prevalence of each virus species among regions and among vineyards within regions, we expect that region and site-specific management strategies are needed for effective disease control.Some diseases manifest as one characteristic set of symptoms to the host, but can be caused by multiple pathogens. Control treatments based on plant symptoms can make it difficult to effectively manage such diseases, as the biology of the underlying pathogens can vary. Grapevine leafroll disease affects grapes worldwide, and is associated with several viral species in the family  The control and management of diseases is predicated on the accurate identification of their etiology. In plant pathology, many diseases are caused by individual pathogens, allowing scientists and stakeholders to devise and implement targeted strategies aimed at reducing disease incidence, as well as limiting economic, environmental, and social impacts that may occur as a consequence of the diseased plants. However, there are also examples of diseases caused by pathogen complexes; therefore, in some cases a set of multiple factors is required for disease symptom expression, while in other examples infection by one member of a group of related pathogens may lead to similar disease symptoms ,2. In thClosteroviridae; this family has four genera, three of which contain viruses associated with GLD [Grapevine leafroll-associated virus 2 (GLRaV-2) is in the genus Closterovirus, which includes aphid-transmitted viruses and other species that lack a known vector, such as the graft-transmissible GLRaV-2 [Ampelovirus, including Grapevine leafroll-associated virus 1 (GLRaV-1), Grapevine leafroll-associated virus 3 (GLRaV-3), and the Grapevine leafroll-associated virus 4 group (herein GLRaV-4LV) are transmitted primarily by mealybug vectors [Grapevine leafroll-associated virus 7 (GLRaV-7) is a member of the newly proposed genus Velarivirus. There are no vectors identified for GLRaV-7, and its role in GLD has been recently questioned [Grapevine leafroll disease (GLD) is caused by a complex of several ssRNA virus species in the family with GLD . Grapevi GLRaV-2 . Differe GLRaV-2 . On the  vectors –10. Ther vectors . It shouestioned ,13. ThusGLD has reemerged as a disease of economic importance during the past few decades, primarily because its incidence was shown to increase in most grape-growing regions of the world in the presence of mealybug vectors . HoweverVitis vinifera, as well as the native grape species, V. californica [California has a few major wine growing regions, along with a handful of regions with smaller production. Major growing regions include the North Coast, Central Coast, and Central Valley, the Sierra Foothills represents a smaller region . GLD hasifornica ,17. Furtifornica . ConsideIn this study, we surveyed three grape-growing regions of California for the presence of GLD-associated viruses. Our results show that relative prevalence of the various GLRaV species differed among wine growing regions within California, and that substantial variability in virus presence occurs among vineyards within regions. Ultimately, our results indicate that management of GLD requires knowledge about the range of viruses associated with afflicted vineyards, rather than relying solely on the visual identification of disease symptoms.Vitis vinifera cvs. Barbera, Cabernet franc, Cabernet Sauvignon, Gamay, Merlot, Petite Syrah, Pinot noir, Syrah, and Zinfandel . Samples were macerated using a Precellys 24 Tissue homogenizer  run at 6,500 Hz for two 10 sec cycles with a 30 sec intermission between cycles. Following maceration, samples were centrifuged for 10 min at 16,000 rpm and 1.5 ml of supernatant was stored in a new microcentrifuge tube at -20°C. For positive controls for all GLRaV species tested in this study, known infected vine cuttings were provided by the Foundation Plant Services at the University of California, Davis  and propagated in the Oxford Tract greenhouse facility at the University of California, Berkeley.All petiole samples were stored at -80°C until RNA extractions were performed from 100 mg of pooled petiole samples using a modified version of the method from a previous survey of Napa Valley in the North Coast Region , based oAll samples were tested for the presence of GLRaV 1–5, 7 and 9 using a multiplex RT-PCR approach modified from Sharma et al. . The majFollowing species level detection; 10% of samples positive for each GLRaV species were randomly selected for sequencing. The same method as above was used to prepare the extractions for PCR. The same primers were used for PCR and sequencing for all GLRaV species, except for GLRaV-3. In order to sequence the same 428 bp region of the GLRaV-3 CP gene from previous studies, the primer pairs from our initial Napa study were used for PCR and a second nested primer pair was used for sequencing . For allTo compare GLRaV prevalence among the three regions, a proportion of GLRaV-positive vines were compared among the three regions surveyed, using a generalized linear mixed model with a binomial distribution. Site was specified as a random variable nested within each region. A single-step post hoc test was used to discern statistically significant differences among regions. The same procedure was followed to compare the prevalence of mixed infections among the three regions. In a previous study a similar survey was performed for the North Coast area of Napa Valley ; due to P < 0.05) of our results would remain consistent after a Bonferroni-Holm correction for multiple tests [To compare all GLRaV species among the three regions, the relative proportion of each GLRaV species at each site was calculated by dividing the number of positive samples of each GLRaV species by the total number of positive samples, and each site was used as one replicate in the analysis. Relative abundance of each virus was compared individually among regions using a generalized linear model with a Gaussian distribution. Relative abundance data were arcsine-transformed prior to analysis to better meet the assumptions of the model. We report statistics that are not corrected for multiple tests, but we note that the statistical significance  , followed by the North Coast, and the lowest prevalence was again in the Central Coast.When considering overall detection of GLRaV -1, -2, -3, -4LV, and -7, there were significantly fewer samples that tested positive in the Central Coast region than in the other two regions, which did not differ from each other (f = 566) . When GLz = 3.113, P = 0.00185, df = 566). When mixed infections were compared among four growing regions with GLRaV-7 excluded, significant differences among regions were found, with mixed infections again being more common in the Sierra Foothills than in the other three regions .Prevalence of mixed infections with all GLRaVs included in the analysis was highest in the Sierra Foothills, while the Central Coast and North Central Valley did not differ significantly from each other . GLRaV-2 also had the highest relative abundance in the Sierra Foothills, and did not significantly differ between the Central Valley and Central Coast . GLRaV-3 had the highest relative abundance in the Central Coast, while its relative abundance did not significantly differ between the Central Valley and Sierra Foothills . Neither GLRaV-4LV nor GLRaV-7 were found in the Central Coast, and relative abundance of neither GLRaV-4LV  nor GLRaV-7  differed significantly between the Central Valley and Sierra Foothills.Relative abundance of individual GLRaVs varied among regions . GLRaV-1F = 36.16, P < 0.0001, df = 38). The relative abundance of GLRaV-2 was highest in the Sierra Foothills, intermediate in the Central Valley and North Coast, and lowest in the Central Coast . GLRaV-3 had the highest relative abundance in the Central Coast and North Coast, followed by the Central Valley, with lower relative abundance in the Sierra Foothills . GLRaV-4LV was not found in the Central Coast, and relative abundance of GLRaV-4LV did not vary significantly among the remaining three growing regions .When considering four instead of three regions but excluding GLRaV-7, our measures of the relative abundance of GLRaVs 1–4 were affected slightly in the Central Valley and Sierra Foothills. GLRaV-1 had the highest relative abundance in the Sierra Foothills, intermediate in the Central Coast, and lowest relative abundance in the North Coast and Central Valley . Within the GLRaV-3 group, there was 100% concordance between the CP and HSP70h primer pairs at one site, indicating that all infections may have consisted of one genetically distinct variant ,21. Of aPlanococcus ficus), a known efficient vector of GLRaV-3 was present at the site [A total 188 samples from nine sites were tested in the Central Valley region (San Joaquin County), with planting dates ranging from 1978 to 2007. Of the sites tested, six were positive for one of the viruses in our panel. Of the samples tested, 40% were positive for at least one virus in our panel, of which roughly 53% of all tested samples were positive for GLRaV-3, the most common virus isolated in the region. GLRaV-1, 2, 4LV, and 7 were also detected in the Central Valley . In one surveyed Cabernet Sauvignon block, planted in 1990, every vine tested was positive for GLRaV-3 when tested with the HSP70h primers but only six of the samples were positive when tested with the CP primer set (n = 21), indicating infections with multiple genetically distinct variants of GLRaV-3 within that block. Additionally, vine mealybug . Every sample in AMEL-4, a site planted with non-certified material, was positive for GLRaV-7, the only such block in our study. Of all samples tested, 30% of all samples were mixed infections, notably higher than the other regions.Sequencing data helped elucidate the reason that the CP primer pair was unable to pick up certain variants while the HSP70h primer was positive. The samples that were positive for the HSP70h primer set but negative for the CP primer grouped closely with the “GLRaV-3g” clade from Sharma et al. . At thatThe results of this study highlight the complexity of GLD and the difficulty in implementing disease management practices that are applicable to a broad geographic range of affected grape-growing regions. The within- and between- region variability in virus prevalence, both in regards to the proportion of plants infected, the number of mixed infections, and the viral species present, indicate that control measures should be implemented after diagnostic assays have identified the virus species present at each site. However, broader regional trends must be confirmed with a larger study, so that heterogeneity in cultural practices, grapevine genotype, presence of vectors, and other factors can be incorporated into such an epidemiological survey.Grapevine red blotch-associated virus [The lack of virus detection in nine of the twenty-five surveyed vineyards is intriguing, as vineyards were selected based on the presence of GLD symptoms. We propose the following possible explanations for this discrepancy. First, we hypothesize that the primer sets used for diagnostics were not capable of amplifying nucleic acids from all virus variants infecting tested plants. PCR primer sets are designed based on available sequence data, and consequently novel or unknown genetically distinct variants of each GLRaV species cannot always be reliably detected. This is highlighted in this study and our previous study , as eached virus ,27. Furted virus ,17,28. Ied virus . For sceWith respect to GLRaV incidence, vineyards had two trends within each region. First, some vineyards had no positive plants while others reached 100% GLRaV incidence. Although the discrepancy may be partly due to the limitations of the detection protocols used, as described above. Second, the identity of GLRaV infecting plants varied within and among regions. We speculate that the prevalence of GLD-associated viruses is not only dependent on localized virus spread mediated by vectors, but that a combination of vector spread and establishment of vineyards with virus-infected plant material occurs to varying degrees in all regions. It is also notable that the number of mixed-species infections was higher in the Sierra Foothills region. This trend was largely driven by the high incidence of GLRaV-2, which has no known vector and has not been associated with disease spread in established vineyards [Although not quantified here, there are substantial differences in the wine grape industry and practices among the surveyed regions. The Sierra Foothills region’s size, climate and relative isolation from other grape-growing regions appear to contribute to lower pest and disease pressure than in the other regions studied. We speculate that this lower pest pressure could result in less frequent removal and replanting of vineyards in that region. The use of certified plant material in these newly planted vineyards may assist in the reduction of GLRaV-2 prevalence. GLRaV-7 also has no known vector and may be similarly reduced by this increased replanting frequency with certified propagation material ,22,29–37Our findings highlight the importance of continued diagnostic testing, including continuing research on grapevine viral diversity, grower education to increase awareness of GLD, and the need to frequently update all stakeholders about advances in current knowledge about GLD and other diseases. Additionally, the regional trends in relative viral prevalence should be taken into account when considering the historical practices in each region and devising future disease management strategies. We acknowledge that grape-growing in California, as well as other regions, is not a uniform agricultural activity and that trends detected in this study should be confirmed with more extensive surveys. Ultimately, a multidisciplinary and collaborative approach that engages researchers, grape growers, and other stakeholders can contribute to effective control of GLD and other economically important diseases in California and worldwide.S1 FileWorksheet “ThreeRegions” includes data from the regions surveyed for this study for GLRaV-1, -2, -3, -4LV, and -7, as well as varietal and planting date information when available. Worksheet “FourRegions” includes data from the three regions for this study and data from the North Coast region that were collected for Sharma et al. 2011, for GLRaV-1, -2, -3, and -4LV. Worksheet “AbbreviationsExplained” lists the abbreviations that were used in the other two worksheets.(XLSX)Click here for additional data file.S2 File18S rRNA gene alone, which was used as a control. 1Primers were designed by Osman et al. . 2Primers for GLRaV-7 were designed for this study, see methods. 3Primers first used in Sharma et. al. 2011 and designed from a consensus of sequences described in Wang et. al. 2010.Primers were designed by Osman et al. . All reactions for virus detection were run in a 3-plex with the first reaction containing the GLRaV-1, GLRaV-2 and GLRaV-3, the second reaction with GLRaV-4, GLRaV-5 and GLRaV-9, a third reaction containing GLRaV-7, CP primer, and a general GLRaV primer, and the fourth reaction with the host plant (DOCX)Click here for additional data file.


The crystal structures of monomeric RNA-dependent RNA polymerases and reverse transcriptases of more than 20 different viruses are available in the Protein Data Bank. They all share the characteristic right-hand shape of DNA- and RNA polymerases formed by the fingers, palm and thumb subdomains, and, in many cases, “fingertips” that extend from the fingers towards the thumb subdomain, giving the viral enzyme a closed right-hand appearance. Six conserved structural motifs that contain key residues for the proper functioning of the enzyme have been identified in all these RNA-dependent polymerases. These enzymes share a two divalent metal-ion mechanism of polymerization in which two conserved aspartate residues coordinate the interactions with the metal ions to catalyze the nucleotidyl transfer reaction. The recent availability of crystal structures of polymerases of the Orthomyxoviridae and Bunyaviridae families allowed us to make pairwise comparisons of the tertiary structures of polymerases belonging to the four main RNA viral groups, which has led to a phylogenetic tree in which single-stranded negative RNA viral polymerases have been included for the first time. This has also allowed us to use a homology-based structural prediction approach to develop a general three-dimensional model of the Ebola virus RNA-dependent RNA polymerase. Our model includes several of the conserved structural motifs and residues described in other viral RNA-dependent RNA polymerases that define the catalytic and highly conserved palm subdomain, as well as portions of the fingers and thumb subdomains. The results presented here help to understand the current use and apparent success of antivirals, i.e. Brincidofovir, Lamivudine and Favipiravir, originally aimed at other types of polymerases, to counteract the Ebola virus infection. Due to their role in replication, transcription, and reverse transcription in the case of reverse-transcribing viruses, RNA-dependent RNA polymerases (RdRp) and reverse transcriptases (RT) are key enzymes in the viral biological cycle. Following the crystallization of the poliovirus RdRp by Hansen et al. , over 20Six conserved structural motifs (motifs A-F) have been identified in the tertiary structures of RdRps and RTs –5,10,11.Motif A is located within the palm subdomain and is formed by a β-strand followed by a helical structure or a loop that continues to the fingers subdomain. At the C-terminus of the β-strand, this motif contains one of the invariant catalytic aspartic acid residues present in DNA- and RNA polymerases .4D- conserved sequence, in which the first aspartate corresponds to the catalytic residue. Structural and thermodynamic evidence shows that the second aspartate in motif A plays a key role in the discrimination of NTP over dNTP by forming a hydrogen bond with the ribose 2’ OH moiety [Single-stranded positive RNA viruses have a characteristic—DXH moiety . The polH moiety .Instead of the second aspartate of motif A, reverse transcriptases have an amino acid with a bulky side chain such as phenylalanine, which is known as “the steric gate” that helps to discriminate between deoxyribonucleotides and ribonucleotides, avoiding the incorporation of the latter by the steric interference of the side chain of this residue with the 2’ OH group of the ribonucleotide ,19. AparMotif B is located in the transition between the fingers and the palm subdomains. Its structure consists of a loop that connects a β-strand of the fingers and the N-terminal helix of the palm subdomain. The residues of this motif have been shown to participate in binding the template and the incoming nucleotide . The N-tSingle-stranded positive- and dsRNA viruses have a conserved threonine within the α-helix that is located in its N-terminal part and faces the active site of the polymerase. In the next helix turn, also facing the active site, ss(+)RNA viruses have a conserved asparagine, which has been shown to aid in the rNTP selection by correctly positioning the catalytic aspartate in motif A . The strMotif C follows motif B in the palm subdomain, and is formed by a β-strand-loop-β-strand structure. The second catalytic aspartic acid residue conserved in both DNA- and RNA polymerases that coordinates the interactions with the metal ions is located within the loop. Motif C is one of the most conserved regions in viral RNA polymerases; all the loops of ss(+), segmented ss(-), ds- and reverse transcribing RNA viruses have two aspartates preceded by a glycine in ss(+) and dsRNA viruses, a serine in segmented ss(-)RNA viruses, and by a methionine in RT viruses. Both aspartates coordinate the interactions with the metal ions . PreviouMotif D follows motif C within the palm subdomain. It is formed by an α-helix and a flexible loop adjacent to the palm’s β-sheet. It is a highly dynamic structure that changes its conformation when the correct nucleotide is bound. It also serves as a structural scaffold for the palm domain, and has been involved in the protonation of the pyrophosphate leaving group after the nucleotidyl transfer reaction ,27. LocaThe second widely conserved residue in motif D is a lysine. It has been proposed that this amino acid, by acting as the general acid that deprotonates the pyrophosphate leaving group, contributes to the rate of nucleotide addition . It is aMotif E is a unique structural feature of RdRp and RTs, which has been called “the primer grip”. It is a β-hairpin that is located facing the palm subdomain’s β-sheet at the junction with the thumb subdomain. As its name implies, this motif has been shown to act in the correct positioning of the 3’ OH end of the primer .The level of primary sequence conservation around motif E seems to be lower in viral RdRps, despite the fact that all the crystal structures exhibit the characteristic β-hairpin located in the same position ,21. One de novo initiation such as the Flaviviridae [Viruses with a protein-priming mechanism such as the Picornaviridae have larger template-binding channels, in which two basic residues with long side chains protruding towards the active site in motif E easily fit , while viviridae ,33, the iviridae , and ssRNA viruses have one conserved glycine. Hass et al.  have shoBesides the conserved structural motifs A to E, which practically define the palm subdomain, one additional conserved motif named motif F has been identified in the fingers subdomain of all the crystallized RNA viral polymerases.This motif extends from the fingers subdomain towards the thumb subdomain as part of the fingertips, directly on top of the palm subdomain active site. This long structure has numerous basic residues that interact with the negatively charged phosphate backbone of the incoming nucleotide, and has been shown to be part of the NTP entrance tunnel in ss(-)RNA viruses ,32,38. TGorbalenya et al.  and Pan Cerny et al  recentlyApart from the conserved structural motifs A-F, Gerlach et al.  identifiThe recently proposed functional regions seem to be conserved in RdRps of ss(+) RNA viruses . Two of Given the availability of crystal structures of polymerases from the four major groups of RNA viruses, i.e., ss(+), ss(-), ds, and reverse-transcribing, we present in this paper a phylogenetic tree built based on comparisons of RdRps and RTs’ tertiary structures, which might help us understand the evolutionary relationships among these enzymes. Prompted by the lack of a crystal of the Ebola virus (EBOV)  polymeraTribolium castaneum telomerase (PDB:3KYL); bat influenza A virus (PDB:4WSB); human influenza B virus (PDB:4WRT); LaCrosse virus (PDB:5AMQ).The RdRp crystal structures employed in this study include: human rhinovirus polymerase (PDB:1XR7); poliovirus (PDB:3OL6); human enterovirus 71 (PDB:3N6L); foot-and-mouth disease virus (PDB:1U09); encephalomyocarditis virus (PDB:4NZ0); Norwalk virus (PDB:3BSO); rabbit hemorrhagic disease virus (PDB:1KHV); Sapporo virus (PDB:2CKW); Japanese encephalitis virus (PDB:4HDH); dengue virus (PDB:2J7U); bovine viral diarrhea virus (PDB:1S48); hepatitis C virus (PDB:1C2P); Qβ-phage (PDB:3MMP); infectious pancreatic necrosis virus (PDB:2YI9); infectious bursal disease virus (PDB:2PUS); φ6 phage (PDB:1HHS); reovirus (PDB:1MUK); simian rotavirus (PDB:2R7Q); human immunodeficiency virus type 1 (PDB:3DLK); moloney murine leukemia virus (PDB:4MH8); human immunodeficiency virus type 2 (PDB:1MU2); Pairwise structural comparisons between the different RdRps and RTs mentioned above were performed with the Secondary Structure Matching (SSM) program  includedA geometric distance measure was then estimated for each of the comparisons using the Structural Alignment Score , which ihttp://tree.bio.ed.ac.uk/software/figtree/) was used to visualize the resulting tree.The program FITCH, included within the PHYLIP package, was used to transform the geometric distance into an evolutionary distance and FigTree (http://www.ncbi.nlm.nih.gov/genome/viruses/). The sequence was edited according to the information provided by the Conserved Domain Database [The search for homologs of the EBOV polymerase and the three-dimensional structure modeling of the EBOV RdRp domain were performed using the PHYRE web server version 2.0 . One ZaiDatabase , leavingThe three-dimensional model and its images were edited with Chimera 1.8 .Secondary structure-based multiple sequence alignments were built using the PROMALS3d web server . For eacFor the Mononegavirales multiple sequence alignment, the different L-protein “Mononegavirales RNA-dependent RNA polymerase” (CDD 250248) domains from the various genera belonging to the Mononegavirales families were chosen. A total of 52 sequences were included for the alignment. These are listed below, and include the Borna disease virus: NP_042024; Lloviu cuevavirus: YP_004928143; Marburg marburgvirus: YP_001531159.1; avian metapneumovirus: YP_443845; avian paramyxovirus 6: NP_150063; Beilong virus: YP_512254; bovine parainfluenza virus 3: NP_037646; bovine respiratory syncytial virus: NP_048058; canine distemper virus: NP_047207; dolphin morbillivirus:NP_945030; fer-de-lance virus:NP_899661; human metapneumovirus: YP_012613; human parainfluenza virus 1:NP_604442; human parainfluenza virus 2:NP_598406; human parainfluenza virus 3:NP_067153; human respiratory syncytial virus:NP_056866; Mapuera virus:YP_001249278; measles virus:NP_056924; mumps virus:NP_054714; Newcastle disease virus B1:NP_071471; parainfluenza virus 5:YP_138518; peste-des-petits-ruminants virus:YP_133828; pneumonia virus of mice J3666:YP_173335; porcine rubulavirus:YP_001331035; rinderpest virus: YP_087126; Sendai virus: NP_056879; simian virus 41:YP_138510; Australian bat lyssavirus:NP_478343; bovine ephemeral fever virus:NP_065409; European bat lyssavirus 1:YP_001285392; Hirame rhabdovirus: NP_919035; infectious hematopoietic necrosis virus:NP_042681; lettuce yellow mottle virus: YP_002308376; maize Iranian mosaic virus:YP_002308459; maize fine streak virus: YP_052849; Mokola virus:YP_142354; northern cereal mosaic virus:NP_597914; potato yellow dwarf virus:YP_004927971; rabies virus:NP_056797; rice yellow stunt virus:NP_620502; snakehead virus:NP_050585; sonchus yellow net virus:NP_042286; spring viraemia of carp virus:NP_116748; taro vein chlorosis virus:YP_224083; vesicular stomatitis Indiana virus:NP_041716; viral hemorrhagic septicemia virus Fil3:NP_049550.RNA-dependent RNA polymerases have been used as evolutionary markers because of their presence in all RNA viruses and their several conserved regions and number of amino acids ,38,47–49A relatively recent important alternative to the construction of primary structure-based phylogenies are evolutionary trees based on the comparison of tertiary structures. This method has proven to be particularly useful when trying to assess the evolutionary relationship between homologous proteins with high levels of sequence divergence . The unrPhylogenetic trees of ss(-)RNA viruses using the most conserved regions of the RdRp ,49 show de novo initiation mechanism [The monophyly of ss(-)RNA viral polymerases is supported by additional biochemical and structural data. Transcription and replication of all ss(-)RNA viruses is similar. First the mRNA, which is modified in a later stage, is synthesized using the genomic RNA as template. A complementary positive strand is also formed, which is used as the template for the synthesis of the genomic negative sense RNA, which is then packed inside the virions. Other shared features of negative-strand RNA viruses are the fact that the polymerase template consists of a ribonucleoprotein complex in which the viral nucleoprotein is bound to the genomic RNA  and, thaechanism ,23,35,36The mRNA of ss(-)RNA viruses must be capped to be recognized by the cellular protein synthesis machinery. Non-segmented RNA viruses have within the L protein a capping domain located bordering one of the faces of the RdRp domain forming the template channel. No structural homologs are known for this domain, and the capping mechanism is different from that of eukaryotic cells and segmented ss(-)RNA viruses. It works by the attack of a guanosine nucleotide to a histidine residue covalently bound to the 5’ end of the RNA, i.e., it is said to have GDP polyribonucleotidyl transferase activity . On the The ss(-)RNA viral polymerases are big complexes endowed with many different functional domains. In the case of the Orthomyxoviridae family, the polymerase is a heterotrimeric complex formed by the proteins PA, PB1 and PB2. The PB1 protein contains the RdRp domain ,23. In tElectron microscopy (EM) studies of the VSV polymerase have revealed certain differences in the overall structure and organization of non-segmented ss(-)RNA viruses  in compaThe different location of the capping enzymes RNA viruses vs C-terminal in the Mononegavirales), the differences in the quaternary structure of the polymerases, and the distinct capping mechanisms indicate a ss(-) RdRp ancestor and later accretion events during which the complementary functional domains were independently acquired.Different attempts to construct evolutionary trees of viral RdRps and RTs based on structural comparisons including this work ,53 yieldWhile the results of Cerny et al. , MönttinDue to the biomedical relevance of ss(-)RNA viruses and the lack of structural information of their RdRp, several attempts had been made to model the polymerase domain based on the homology with other RNA viruses whose polymerases had already been crystallized. A predicted model of an Arenavirus RdRp domain  built usThe current epidemic of the EBOV hemorrhagic fever is by far the biggest outbreak of this disease since its discovery in 1976. The high mortality rates ranging from 40 to almost 90% –57, combEBOV is part of the family Filoviridae which, together with the families Rhabdoviridae, Paramyxoviridae and Bornaviridae, forms the Mononegavirales order. The members of this highly diverse group of viruses all have a linear, monopartite, negative strand RNA genome, share transcription and replication strategies, and exhibit a conserved arrangement of at least five genes that encode for nucleoprotein, phosphoprotein (VP35 in EBOV), matrix protein, glycoprotein and L protein . The filThe L protein of Ebola virus is a multifunctional protein about 2210 amino acids long with a molecular weight of approximately 250 kDa engaged in viral transcription, genome replication, mRNA capping, mRNA methylation and polyadenylation ,64. PochThe description of the VSV L protein structure to a 3.8 A resolution  has provth with a confidence level of 33.9%.The best match yielded by the PHYRE 2.0 server to our EBOV polymerase sequence corresponds to the bat influenza A virus  . Only 25Our predicted three-dimensional model of the EBOV polymerase allowed the identification of the fingers-, palm-, and thumb subdomains structures and in the same sequential order of DNA- and RNA polymerases, i.e., fingers-palm-fingers-palm-thumb. This is consistent with the recently reported structure of the VSV L protein . The finThe EBOV polymerase model presented here shows that motif A is formed by a β-strand followed by a ten amino acids-long loop . The aspThe EBOV polymerase model’s motif B is formed by a loop followed by a long α-helix. The sequence of the loop and the N-terminus of the helix are GGIEGLQQKLWT. According to its position in the model, the third glycine corresponds to the conserved glycine of RdRps and RTs (vide supra). The position in our model of glutamine 564, lysine 565, tryptophan 567 and threonine 568 suggest that they might be involved in the interactions with the incoming nucleotide. These five residues are highly conserved in the Mononegavirales order with a few exceptions . Their hThe predicted motif C has the characteristic structure β-strand-loop-β-strand, and its loop has one aspartate residue within the sequence MGDNQ that matches the second strictly conserved amino acid (Asp593) . The modThe EBOV polymerase model presented here predicts that motif D is formed by an α-helix followed by a long loop . The helGlycine (Gly635), located at the N-terminal end of motif D’s loop , correspThe EBOV polymerase motif E has the characteristic β-hairpin structure and the sequence FIYFGKKQYL . In the KEKELNVGRTFGK. Three of these four basic residues, as well as phenylalanine, are conserved in the Mononegavirales polymerase , which are conserved in all the Mononegavirales , and thade novo initiation mechanism, because the modelling technique we have used only exhibits the enzyme fragment with good confidence levels, and does not include the additional L protein functional domains. This is a strong limitation of our model, since in the ss(-) mononegaviral VSV polymerase structure recently published [The EBOV polymerase model presented here lacks the priming loop present in RNA viral polymerases that use a ublished  the primWe have included our EBOV polymerase model in the RdRps and RTs structure-based phylogenetic tree. As shown in in vitro or in vivo [An overwhelming majority of the recent emergent human epidemics are caused by RNA viruses , and in  in vivo –75 by in in vivo –78, and  in vivo ,80.Lamivudine is a nucleoside-analog reverse-transcriptase inhibitor (NRTI) that also acts as a chain terminator due to its lack of a 3’hydroxyl end . It has in vitro and/or in vivo antiviral activity against a wide array of human-infecting RNA viruses, including ss(-) viruses such as influenza virus [in vivo antiviral activity against Zaire EBOV in a mouse model [Favipiravir is a selective inhibitor of RdRps. Once inside the cell, this nucleotide analog is phosphoribosylated by cellular enzymes and forms favipiravir RTP, which prevents further nucleotide incorporation . This Rdza virus , arenaviza virus , and bunza virus ; ss(+)RNza virus ,90, alphza virus , picornaza virus  and noroza virus . It was se model .Nucleotide/nucleoside analogues are drugs aimed at the active site of RNA- and DNA polymerases that compete with the natural substrates for incorporation into the nascent nucleic acid strands, and may act either as chain terminators or as mutagenic agents ,94. ProtNo polymerase is endowed with absolute template- or substrate specificity ,98–100, in vitro activity against the Mononegavirales RdRp.A structural superposition of the EBOV RdRp with a foot-and-mouth disease virus polymerase bound to a template-primer RNA and ribavirin triphosphate  was drawCrystal structures of ss(-)RNA viral polymerases with NTPs at the active site are required in order to elucidate the exact mechanism of nucleotide binding in these enzymes. However, the high level of conservation of the palm subdomain, together with the similarity of the interactions within the active site in DNA- and RNA polymerases are essential to understand why some nucleotide analogues have broad antiviral spectra such as favipiravir, which has proven to have antiviral activity even against different types of RNA viruses, including ss(-) and ss(+)RNA viruses, or brincidofovir, which has antiviral activity against both DNA- and RNA viruses. Our predicted EBOV polymerase model indicates that this RdRp shares the same basic architecture and mechanism of action, including the structural motifs and some of the residues that participate in nucleotide binding. Therefore, drugs aimed at the active site of different types of polymerases , such asRNA-dependent RNA polymerases are encoded by RNA viruses from different families with a variety of genome organization and replication strategies. All known viral RNA polymerases are homologous monomeric enzymes. Whether this is true or not for all RNA viruses remains to be proven. The availability of more than twenty distinct viral RNA polymerase crystals of different RNA viruses and retroviruses reveals the characteristic right hand architecture typical of the superfamily of DNA- and RNA polymerases, with fingers, palm and thumb functional subdomains . The palAlthough attempts to group RNA viruses based on polymerases sequence data have been criticized , the pioin silico model of the spatial distribution of a 253-amino acid residue data set of the Ebola virus RNA polymerase with a 92% certainty.We have also proposed here a three dimensional model of the EBOV RdRp  using hoA multiple alignment based on secondary structure prediction of the negative single stranded RNA viruses of the Mononegavirales order  allowed The crystal structures of complexes of DNA- and RNA polymerases with nucleotides or nucleotide analogues show that very similar binding mechanisms are involved ,101–105.S1 FigOn the left, colored in red, the fragment of the bat influenza A virus polymerase (edited from PDB 4WSB) in which the PHYRE 2.0 web server yielded confidence levels of 91.7% for the EBOV polymerase. On the right, the PHYRE 2.0 sequence alignment for the corresponding fragments of the EBOV and bat influenza A virus polymerases used for the construction of the three-dimensional EBOV polymerase model.(TIF)Click here for additional data file.S2 FigThe residues predicted to form helical structures are in red; the residues predicted to form β structures are in blue. Only the residues that could be matched to the bat influenza A virus in the Ebola virus polymerase are shown (vide supra). The lines below the multiple alignment match the polymerases subdomains: fingers subdomain, yellow; palm subdomain, green; thumb subdomain, red. The colored frames correspond to the RNA-dependent RNA polymerases conserved structural motifs: motif A, red; motif B, blue; motif C, green; motif D, magenta; motif E, cyan; motif F, orange. The catalytic aspartic acid residues are highlighted in red. The residues with a high degree of conservation in the Mononegavirales are highlighted in yellow.(TIF)Click here for additional data file.S3 FigThe figure shows the superposition of the RdRp domains of bat influenza A virus (colored in brown), LaCrosse virus (colored in blue), vesicular stomatitis virus (colored in purple) and our proposal of the EBOV polymerase model (colored in green). The image exhibits the high level of structural conservation in the palm subdomain and the structural motifs of the fingers and thumb subdomains of the polymerases from single-stranded negative RNA viruses. The superposition was created with the MatchMaker program included in Chimera 1.8 .(TIF)Click here for additional data file.S4 FigThe structural superposition shown here is the same as in (TIF)Click here for additional data file.


Bovine parainfluenza virus type 3 (BPIV3) isolates are classified into three genotypes (BPIV3a to -c). Here, we report the complete genome sequence of the BPIV3c isolate for the first time in Japan. Our results indicate that new primer sets will be required to detect all genotypes of BPIV3 strains. Respirovirus of the Paramyxoviridae family. BPIV3 causes severe respiratory illness in cattle, associated with bovine respiratory disease complex worldwide. Recent phylogenetic analyses revealed that BPIV3 viruses can be classified as genotypes A, B, and C (–Bovine parainfluenza virus type 3 (BPIV3) is an enveloped, nonsegmented negative-sense RNA virus within the genus B, and C –4. Accor, and C –, 2, 5. T, and C –. However, and C –, was obt, and C –, 9. SimiLC000638).The genome sequence of HS9 has been submitted to GenBank (accession number


Sox4 is an important regulator of the pan-neuronal gene expression during post-mitotic cell differentiation within the mammalian brain. Sox4 gene locus has been previously characterized with multiple sense and overlapping natural antisense transcripts Sox4 gene locus, additional experimental data derived from RNA-Fluorescence in situ Hybridization (RNA-FISH), Western blotting, strand-specific reverse-transcription quantitative polymerase chain reaction (RT-qPCR), gain-of-function and in situ hybridization (ISH) experiments. All the additional data provided here support the existence of an endogenous small interfering- or PIWI interacting-like small RNA known as Sox4_sir3, which origin was found within the overlapping region consisting of a sense and a natural antisense transcript known as Sox4ot1.SRY (Sex Determining Region Y)-Box 4 or  This is a rare event within the mammalian genome but is common in the plant.•The data provides a modified method for brain cell fixation and immobilisation on glass slides for effective RNA-FISH analysis.•Sox4 natural antisense transcripts, known as Sox4ot1 and Sox4ot2 in the production of Sox4_sir3 in vitro.Comparison of two different •Sox4 gene locus allows clear, concise and easy visualisation of various features defined in the region by using Artemis software.Compilation of all the information within the 11.1Sox4 gene locus. The Sox4 gene locus is featured by multiple overlapping sense and natural antisense transcripts (NATs) In silico data mining and mapping were also carried out to enrich the features within the locus and the detailed information is summarized in a GenBank file format as Supplementary GenBank File. A snapshot of the annotated Sox4 gene locus visualized using Artemis Genome Browser and Annotation Tool www.ensembl.org), Sox4ot1, Sox4ot2, Sox4_sir3, untranslated regions, coding region and exons/introns.The data reported here consists of information related to the The most important information within the Supplementary GenBank File is the mapped FANTOM Paired-End Ditags (PET) sequences. Twelve pairs of PET sequences were mapped to the locus indicating the presence of 6 different NATs. These NATs were named PET1-6 with 4 of them were successfully cloned and further analysed in Ling et al. 1.2Sox4 sense was generally diffused all over the cytoplasm whereas Sox4 NATs were depicted as aggregates within the cytoplasm. Whenever Sox4 NATs aggregates were observed, Sox4 sense aggregates were found at the same loci within the cytoplasm.The data article also describes the results for RNA-FISH experiments performed on cells isolated from different regions of the mouse brain . All celSox4, RNA FISH was performed on cells obtained from P1.5 olfactory bulbs using probes against the Hmbs housekeeping gene  to quantitatively estimate the intensity of bands in Western blotting experiments. Pixels from each band from two independent experiments were calculated by using a fixed rectangular selection approach (T-test (2-tailed) was used to compare PET/pcDNA3 and control groups for any significant differences but none of the p-values were lesser than 0.05.We used ImageJ software  Sox4 gene locus  and PET6 (NAT that overlaps Sox4_sir3 origin site). The overexpression of PET3 and PET6 both did not alter the level of sense transcript expression. As expected, the expression of the Sox4 NAT at region overlapped by PET3 and PET6 were significantly upregulated . Amplicons were analysed using agarose gel electrophoresis to estimate the size of PET6. The analysis showed that there were 2 forms of PET6 NATs, one is unspliced and the other one is spliced . Sanger mall RNA .1.7in situ hybridization experiments are appropriately controlled to avoid misinterpretation of noisy signals. Locked Nucleic Acid (LNA)-in situ hybridization (ISH) for small RNA is usually controlled with a scramble probe, a mutated antisense probe or a sense probe. As the control for Sox4_sir3 LNA-ISH reported in Ling et al. Sox4_sir3 probe  to determine the relative levels for various Sox4 sense and NATs expression. All RT-qPCR data presented here were conforming to the criteria described elsewhere


Prospective payment arrangements are now the main form of hospital funding in most developed countries. An essential component of such arrangements is the classification system used to differentiate patients according to their expected resource requirements. In this article we describe the evolution and structure of Healthcare Resource Groups (HRGs) in England and the way in which costs are calculated for patients allocated to each HRG. We then describe how payments are made, how policy has evolved to incentivise improvements in quality, and how prospective payment is being applied outside hospital settings. The diversity and complexity of hospital care makes it challenging to devise reimbursement arrangements that ensure that the amount and quality of hospital care meets the needs of the population yet remains affordable. Most countries have adopted some form of prospective payment to encourage efficient provision of care, differentiating payments using local variants of Diagnosis Related Groups (DRGs) such as the Healthcare Resource Groups (HRGs) used in England. In this article we describe the evolution and structure of HRGs in England and the way in which costs are calculated for patients allocated to each HRG. We then explain how payments are made, how policy has evolved to incentivise improvements in quality and how prospective payment is being applied outside hospital settings.a in addition to the International Classification of Diseases (ICD) codes for diagnoses.The origins of HRGs can be traced back to 1981, when the Department of Health commissioned research to assess the ability of North American DRGs to explain variation in the length of stay of English patients ddIndirect costs are indirectly related to the delivery of patient care, but cannot always be specifically identified to individual patients. Overhead costs are the costs of support services that contribute to the effective running of an NHS provider. These costs cannot be traced or easily attributed to patients, and need to be allocated via an appropriate cost driver [t driver .eFixed costs are those that do not change as activity changes . Semi-fixed costs are those that do not change with small changes in activity but that ‘step up’ when a certain threshold is reached (e.g. nursing staff). Variables costs are those that are directly affected by the number of patients treated or seen (e.g. drug costs) [g costs) .f‘Other’ here refers to all other hospital costs that are not part of day-case, inpatient or outpatient activity. It includes community services, critical care services, A&E medicine, radiotherapy and chemotherapy, renal dialysis, and kidney and bone marrow transplantation, for example.gPrior to the Lawlor review there was a two-year lag [year lag .hFor a description of the methods for calculation of Market Force Factors, see reference [eference .iFrom 2015/16: 70 % under the new enhanced tariff option (ETO) https://www.england.nhs.uk/2015/03/06/eto-2015-16/. This money, which would otherwise have been spent by CCGs on admission avoidance measures, is now available to providers to be invested in acute services, including but not limited to winter resilience schemes [2015/16 tariff arrangements FAQ)


Virus-containing material remains stored in an unacceptably high number of facilities worldwide. In 2011, the world was declared free from rinderpest, one of the most feared and devastating infectious diseases of animals. Rinderpest is the second infectious disease, after smallpox, to have been eradicated. However, potentially infectious rinderpest virus material remains widely disseminated among research and diagnostic facilities across the world and poses a risk for disease recurrence should it be released. Member Countries of the World Organisation for Animal Health and the Food and Agricultural Organization of the United Nations are committed to destroying remaining stocks of infectious material or ensuring that it is stored under international supervision in a limited number of approved facilities. To facilitate this commitment and maintain global freedom from rinderpest, World Organisation for Animal Health Member Countries must report annually on rinderpest material held in their countries. The first official surveys, conducted during 2013–2015, revealed that rinderpest material was stored in an unacceptably high number of facilities and countries. Rinderpest, also known as cattle plague, is a highly contagious viral disease of cattle. Until global freedom from rinderpest was declared in 2011, it was one of the most devastating and feared infectious diseases of animals  and the Food and Agricultural Organization of the United Nations (FAO) Advisory Committee  described potentially infective material  as “rinderpest virus–containing material.” The material was defined as follows: rinderpest virus–containing material means field and laboratory strains of rinderpest virus; vaccine strains of rinderpest virus including valid and expired vaccine stocks; tissues, sera, and other clinical material from infected or suspect animals; and diagnostic material containing or encoding live virus. Recombinant morbilliviruses (segmented or nonsegmented) containing unique rinderpest virus nucleic acid or amino acid sequences are considered to be rinderpest virus. Full-length genomic material, including virus RNA and cDNA copies of virus RNA, is considered to be rinderpest virus–containing material. Subgenomic fragments of morbillivirus nucleic acid that are not capable of being incorporated in a replicating morbillivirus or morbillivirus-like virus are not considered as rinderpest virus–containing material . The OIE and the FAO launched a work program to help Member Countries fulfill this commitment.Hereafter, we refer to the above-described material as “rinderpest material.” By adopting 3 resolutions , all OIE Member Countries committed to destroying remaining stocks of rinderpest material or ensuring that the material would be stored securely in a minimum number of approved facilities   was selected, then no further responses were required, and the questionnaire was considered complete.Countries that reported having rinderpest material were required to provide details about the nature and quantity of material held for the following categories:live virus, including field isolates and genetically modified viruses but excluding stocks of approved/registered vaccines;vaccine stocks, including seed stocks;other potentially infectious materials;other noninfectious rinderpest virus–containing materials.Responders were asked to provide information about rinderpest material currently held, material destroyed during the previous 12 months, and material that had been transferred to or from another institute. Questions also asked whether the institute had conducted any manipulation of rinderpest material in the previous 12 months and whether they intended to destroy material or transfer it to another institute for safer keepingAfter responders had submitted the completed questionnaire to the OIE, they still had access to their respective completed questionnaire in a noneditable PDF format. If institutes held a large number of different strains of virus, or types of tissue, then the country could return information in an Excel  spreadsheet format.The OIE Terrestrial Animal Health Code specifies that the deadline for submitting the annual OIE rinderpest report each year is the end of November (For the first survey (completed in 2014), 171 (95%) of 180 OIE Member Countries responded to the survey, and for the second survey (completed in 2015), all 180 OIE Member Countries responded. Additionally, 2 countries that are still (as of September 2015) not OIE Members reported in 2014 but they did not report again in 2015. In 2014, of the 173 countries that responded to the survey, 23 (13.3%) reported holding stocks of rinderpest material; and in 2015, of 180 countries, 24 (13.3%) reported holding stocks of rinderpest material. All countries that reported holding stocks of rinderpest material in 2014 reported still holding rinderpest material in 2015. One country that had reported not holding stocks of rinderpest material in the 2014 survey subsequently discovered that it did hold stocks and reported holding rinderpest material in 2015. For 1 country that reported in both surveys that it held rinderpest material, whether the material (a subgenomic fragment of DNA) constituted the intended meaning of rinderpest material was in doubt. All countries that reported holding stocks of rinderpest material were OIE Member Countries.Of the 24 countries that reported holding rinderpest material, 1 country reported holding it in 5 institutes in 2014; this country subsequently destroyed all the stocks that were held in 1 institute. Another country reported holding rinderpest material in 2 institutes in 2014 and consolidated the material to 1 institute in 2015. All other countries reported holding the material in only 1 institute. By 2015, a total of 27 institutes in 24 countries reportedly held rinderpest material. In 9 facilities, rinderpest material was stored at Biosafety Level 2 (BSL-2), and in 18 facilities the material was stored at BSL-3 or BSL-4. http://www.oie.int/en/about-us/wo/regional-commissions/.The regions with the greatest number of institutes holding rinderpest material were Asia, Pacific, and Oceania . Because of the questionnaire design, it was not possible to differentiate between vaccine seed strains and packaged and manufactured vaccine, unless this differentiation was specified by the country. Also not provided by some countries was complete information on passage history of virus isolates. By 2015, a total of 22 facilities indicated that they stored vaccine seed virus (which would be classified as live virus). According to data provided in 2015, a total of 7 facilities indicated that they stored manufactured and packaged vaccine.The annual rinderpest survey serves several purposes that support and facilitate the destruction and safeguarding of remaining stocks of potentially infective rinderpest material.It identifies the whereabouts of remaining rinderpest material so that action can be taken to ensure that these stocks are destroyed or stored safely.It monitors and evaluates progress of the rinderpest destruction and sequestration program.It locates stocks of rinderpest vaccine that could be mobilized in the event of a recurrence of disease.Because the whole dataset is shared with all OIE Member Countries, it is hoped that transparency will encourage OIE Member Countries to comply with their commitment to destroy stocks or to store them safely.As of June 2015, the survey response rate was 100%, indicating that all OIE Member Countries had fulfilled their obligation to report on remaining stocks of rinderpest virus. Four years after the declaration of global freedom, rinderpest material remains stored in at least 27 facilities in 24 countries. Responses indicate that one third of these stocks are stored in facilities equivalent to BSL-2. Considering the potential consequences of a recurrence of rinderpest, this situation represents an unnecessarily high risk.The data obtained from the surveys may underestimate the real number of facilities holding rinderpest material because there are several potential sources of underreporting. Rinderpest material might be stored in some countries without the knowledge of the reporting authorities, which was confirmed when 1 country submitted a negative report in 2014 and a positive report in 2015. Countries with strong and well-governed official National Veterinary Services should have a system to regulate the shipment, handling, and storage of dangerous pathogens . In theory, these systems should identify where stocks of rinderpest virus are being held; however, on a global level, National Veterinary Services are not universally strong. Many countries have a weak regulatory framework, and even in countries with a strong regulatory framework, mistakes occur. Therefore, a country’s Veterinary Services may be unaware of material that is held in facilities outside of their direct jurisdiction, such as in universities or private laboratories. Also, samples containing rinderpest material may have been poorly identified or not included in a laboratory inventory. Pathogen inventories and quality management systems are unlikely to have been in place in all facilities receiving and storing rinderpest virus several decades ago. Archived pathology and surveillance samples collected from animals for reasons other than rinderpest diagnosis may also contain rinderpest virus if these samples were collected in areas where rinderpest was prevalent and stored under conditions suitable for virus survival.All countries should be encouraged to continue to search for rinderpest material in any places where it may have been held with or without the institute’s knowledge; such places might include laboratories outside the direct control of the official National Veterinary Services, including private laboratories and universities. Institutes that have less contact with National Veterinary Services might even be unaware that rinderpest has been eradicated and unaware of the international commitment to destroy or safeguard remaining stocks. The surprise discovery of smallpox virus at the US National Institutes of Health in 2014 highlights the possibility that unidentified dangerous material may lie in storage unnoticed for years and underscores the need to maintain current and accurate laboratory inventories  purposes. Other countries will have probably kept rinderpest material for the same reasons. However, in a postrinderpest era, the value and justification for maintaining rinderpest material for research are minimal.,The risk for pathogen release from containment laboratories into susceptible animal populations, albeit low, is real. This risk was highlighted in 2007 when a biosecurity breach at a site in the United Kingdom resulted in an outbreak of foot-and-mouth disease among cattle  worldwide are totally destroyed. To address concerns about the loss of historical data, entire genes of rinderpest virus isolates could be sequenced before destruction—a process commonly referred to as sequence and destroy—and archived with data about the epidemiology and pathology of those viruses. The sequence-and-destroy procedure for rinderpest virus will be used as an additional incentive to encourage scientists to destroy high-risk biological material while retaining academic and historical data that may have research value.OIE Member Countries should fulfill their international obligation to continue to report to the OIE on an annual basis so that the OIE can monitor and transparently report progress on sequestration and destruction over time. The OIE and FAO have been working with the World Health Organization to apply lessons learned from the smallpox posteradication era to rinderpest. It is hoped that the experience gained during the rinderpest posteradication era will support future programs for eradication of other diseases.


KUN) were previously shown to enhance virus secretion and virulence, but a mechanism by which this occurred was not determined.Amino acid substitutions I22V and L72S in the prM protein of West Nile virus Kunjin strain (WNVUsing pulse-chase experiments followed by co-immunoprecipitation with anti-E antibody, we demonstrated that the I22V and L72S substitutions enhanced prM/E heterodimerization for both the E-glycosylated and E-unglycosylated virus. Furthermore, analysis of secreted particles revealed that I22V and L72S substitutions also enhanced nucleocapsid incorporation.We have demonstrated mechanistically that improved secretion of virus particles in the presence of I22V and L72S substitutions was contributed by more efficient prM/E heterodimerization. The pre-membrane (prM) and envelope (E) proteins together form the outer structure of West Nile virus (WNV) particles ,2. The oKUN) prM, which when substituted with the corresponding residues of the virulent American New York 1999 strain (WNVNY99) (I22V and L72S) resulted in a more stable antigenic structure of the prM protein , and promoted efficient secretion of prME and virus particles, as well as increased virulence in weanling mice [KUN strain MRM61C, encoding unglycosylated E) with and without the prM22/72 mutations were utilized [35S-methionine/cysteine for 5 mins (pulse). Cell lysates were then collected at chase times of 0, 3, 6 and 20 mins, and co-precipitation of prM was performed using anti-E monoclonal antibody (3.91D) coated onto Protein G Dynabeads (Invitrogen). The 3.91D antibody was chosen because it displays strong binding to E protein under reducing conditions on Western blot, indicating that folding of E is not a requirement for binding [http://imagej.nih.gov/ij/) from two independently conducted experiments, and expressed as prM to E ratio. The prM to E ratio normalizes the amount of prM in relation to E, allowing the accurate quantification of prM/E heterodimerisation efficiency at each time point regardless of E content. prM co-precipitation was detected between 0–3 mins chase for E-CHO (−) prM22/72, and only after 6 mins for E-CHO (−)  and resolved by SDS-PAGE using a NuPAGE 4–12% Bis-Tris Protein Gel (Novex), followed by exposing radiolabelled gel to phosphorscreen  and larger prME particles (corresponding to virions) in the absence of capsid  suggestsKUN prM significantly enhanced the heterodimerisation of prM and E proteins. Additionally, we showed that these mutations in prM enhance nucleocapsid incorporation into virions by an as yet unknown mechanism.In conclusion, our findings revealed that the I22V and L72S mutations in WNV


To the Editor: Chikungunya virus (CHIKV), a mosquito–transmitted virus , was first isolated >60 years ago in Africa and is responsible for epidemics of acute polyarthralgia. During CHIKV epidemics, the transmission cycle is from humans to mosquitoes, with no intervening amplifying host, and the virus can rapidly disseminate, infecting large numbers of persons. Epidemics have been described in Africa, the Middle East, Europe, India, and Southeast Asia. On the basis of detailed clinical descriptions of the disease, chikungunya fever, it appears that CHIKV caused epidemics in the Caribbean  and the southeastern coastal United States during the early 19th century , and Asian  detected CHIKV in human serum specimens from Yap State, Federated States of Micronesia; the specimens were collected during an epidemic of disease clinically compatible with chikungunya fever. In December 2013, the French National Reference Centre for arboviruses confirmed that CHIKV was responsible for an epidemic occurring on St. Martin Island, French West Indies, in the Caribbean  was used to align the complete genome sequences with a variety of CHIKV sequences, representing the 3 genotypes, from GenBank. Nearly identical phylogenetic trees were generated by several methods ; a representative neighbor-joining tree generated and analyzed with 1,000 replicates for bootstrap testing is shown in the By using next-generation sequencing, we determined the complete nucleotide sequence for 1 of the CHIKV specimens detected in BVI and for 2 of the CHIKV specimens detected in Yap. DNA libraries for next-generation sequencing were prepared directly from RNA extracted from serum, and the amplified libraries were sequenced by using the Ion Torrent Personal Genome Machine . The CLC Genomics Workbench  and Lasergene NextGen  were used to analyze and assemble raw sequence reads. ClustalW (In agreement with findings in a recent report characterizing the 2013 CHIKV detected on St. Martin Island (www.ncbi.nlm.nih.gov/nuccore/KC352904.1) as an “imported Chikungunya fever case in Ningbo port”; the virus was isolated and identified in Ningbo, China, but was detected in samples from a traveler from the Philippines (GenBank accession nos. KC352904.1 and KC488650.1). The 2012 Zhejiang CHIKV was detected and characterized in Zhejiang Province, but the virus was from a sailor who traveled around Southeast Asia; therefore, the exact origin of this virus is also unknown  within the Asian genotype . Within The striking similarity between the 2012 CHIKVs from the Philippines and Zhejiang Province suggests a common origin, perhaps the Philippines, where CHIKV transmission was documented in 2012 and 2013. Regardless of the exact origins of these 2 strains, it is clear that the CHIKV strain currently moving throughout the Caribbean originated from a CHIKV strain that was recently circulating between China, the Philippines, and Yap in Southeast Asia.


Macaca mulatta), the removal of a non-alpha matriarch, who had a large set of kin, led to changes in behavior and physiological stress within her matriline. Following her removal, her matriline increased in aggression, vigilance, and social grooming. Additionally, hierarchical stability, measured by levels of rank changes, decreased within her matriline, and levels of intense aggression by high-ranking animals were more frequent, as well as matrilineal wounding. Although ordinal rank was positively associated with higher chronic hair cortisol concentrations (HCCs) in the months before the matriarch’s removal, following her removal, only those who experienced large increases in rank within her matriline displayed higher HCCs. Changes in matrilineal stability, aggression, behavior, and HCCs within the other two matrilines in the troop were not evident, although caution is needed due to the small sample sizes. We conclude that the removal of the non-alpha matriarch led to matrilineal instability, characterized by higher levels of aggression and subsequent vigilance, rank changes, physiological stress, and grooming. We suggest that non-alpha matriarchs with a large number of kin and social support can be integral to the stability of matrilines.In many species, the loss of alpha matriarchs is associated with a number of negative outcomes such as troop fission, eviction, wounding, and reduced vitality. However, whether the dramatic consequences of their loss are due to their role as an old experienced figure or to their alpha status remains unclear. In a retrospective study, we tested that in a semi-free ranging colony of rhesus macaques ( Loxodonta africana), family units are commonly led by the oldest female, who exhibits the necessary social and ecological knowledge to coordinate her family and better recognize dangers [Orcinus orca), menopausal old females use their ecological knowledge to guide their families towards feeding sources, especially during periods of food scarcity [Pan troglodytes), females have been observed acting as mediators to reconcile individuals  during aggressive interactions [Homo sapiens) the presence of grandmothers enhances grandchildren’s survival by increasing their foraging efforts [Among long-living animals, old females can play a crucial role for the survival of their family or social group. This support can come indirectly if these females use the experience they have accumulated over the years to lead the group and protect it from external threats –3 or dir dangers , 6–8. Ac dangers  than youscarcity , a traitscarcity . In chimractions –12, whil efforts –16.Lemur catta), the loss of the troop’s matriarch led to the eviction of distantly related kin [Macaca mulatta), matrilines with matriarchs received fewer wounds than matrilines without matriarchs [Collectively, these studies have highlighted the importance of older females for their social group, and there is evidence that their loss can have dramatic effects on the rest of the group. In elephants, the death of matriarchs results in family unit splitting , 18 and ated kin , while itriarchs . Howevertriarchs , 22, 23.triarchs , 24, 25,triarchs , 26–28 atriarchs . HoweverRhesus macaques are good models for studying the consequences of the removal of non-alpha matriarchs. They form large multi-male multi-female groups with an average group size between 10 and 80 individuals –32. Malend and 3rd) in the troop, and her two younger sisters were the troop’s alpha and beta females . As females in our troop have their first offspring around 3–5 years of age, a family’s matriarch has at least a grandmother status. Because rank in rhesus macaques is determined by birth rank (i.e. mother’s rank) and not age , a matriprediction 1), 2) higher aggression rates (prediction 2), and 3) higher levels of wounding (prediction 3). An increase of rank instability and aggression rates following the matriarch’s removal should, in turn, increase individuals’ vigilant behaviors (prediction 4) and should increase HCCs as an index of chronic stress, especially among high-ranking animals (prediction 5) as their dominance positions are contested [prediction 6). Finally, if social grooming can help alleviate chronic stress, we also predicted that monkeys with the highest frequency of grooming would have relatively lower hair cortisol concentrations (prediction 7). Because the matriarch is likely the most influential within her matriline, we did not predict any significant changes within the other two matrilines, which were presumably unaffected.We tested this hypothesis by retrospectively examining dominance stability, vigilance, grooming, aggressive interactions, and hair cortisol concentrations (HCCs) within H1’s matriline, while also examining the other two matrilines within the troop and overall troop wounding, before and after her removal. We assessed HCC as a measure of chronic hypothalamic-pituitary- adrenocortical (HPA) activity. HCC measurement has several advantages over the collection of other biological samples , including the ability to measure chronic HPA activity without the need of multiple sampling and without the confounds of circadian and environmental variability –38. We hontested . Finallyontested , 40, andad libitum access to water, as well as commercial lab diet  and were given fresh fruits and vegetables twice daily. All procedures described below adhered to the NIH Guide for the Care and Use of Laboratory Animals and were approved by the NICHD Animal Care and Use Committee (ACUC). Furthermore, all applicable international, national, and/or institutional guidelines for the care and use of animals were followed.Subjects were 49 rhesus macaques (age range 3–17 years) born and reared at the Laboratory of Comparative Ethology (LCE) field station at the NIH Animal Center in Poolesville, Maryland, USA. Because there is an age-related decline in HCC in infants and juveniles that later stabilizes , we inclThe troop consisted of three major matrilines, which are related individuals descending from a female line. Matriline 3 has occupied the top rank since 2009, when its major families overthrew the previously dominant matriline 1  leading In late September 2014, the troop’s eldest  matriarch, H1 (18yrs), was removed from the field station due to chronic gastrointestinal issues that resulted in chronic diarrhea and weight loss and necessitated treatment that could not be provided in the field.H1’s offspring remained in the troop after her removal, including six adults  and one juvenile female. However, the two youngest females were permanently removed from the population at the bi-annual health exam in February 2015 as part of routine population management.Behavioral data were collected using modified frequency data sheets  from eigThe 5-min samples were divided into 20, 15-second intervals. For each interval, every behavior that occurred within the 15 seconds was recorded. The frequency of intervals in which the behaviors occurred was then calculated. The maximum number of intervals an animal could perform a behavior therefore in one session was 20 intervals. Focal subjects received one weekly AM session (9:00–12:00) and one weekly PM session (12:00–17:00) for ten consecutive weeks from August to December 2014 by three observers (inter-rater reliability >85%). Data used were part of a long-term data collection procedure for unrelated projects, and therefore, coders were blind to the hypotheses of the study.nd in the male hierarchy, K3), 5) one high-ranking male , 6) and three intermediate ranking females within the matriline . From the focal animals, we recorded data on both social  and non-social  behaviors  the top three ranking females  were routinely recorded as part of the long-term data collection procedures for this colony of animals. For this study, instances of severe aggression from August to December 2014 were examined. Because these types of social interactions are rare, ad libitum sampling is the optimal recording method. Severe aggression was analyzed to determine whether H1’s removal resulted in an increase in this behavior for her matriline compared to matrilines 4 and 1.Hair samples were collected by shaving the back of the animals’ necks  during rDue to the small sample size of matriline 1 (N = 5), data for matrilines 4 and 1 were analyzed together for all analyses.To assess whether focal animals changed frequencies in their behaviors following H1’s removal, we compared rates of each behavior collected approximately three months before and after her removal using paired-sample t-tests for both matriline 3 and the combined matrilines 4 and 1.ad libitum data using paired-sample t tests to examine whether high ranking monkeys increased in intense aggression.Subjects were classified as high or low ranking (within their respective matriline) based on a median split of the Elo-ratings. Levels of intense aggression (i.e. chases and attacks) were compared using both focal and Severe aggression was analyzed as the proportion of events that occurred across each matriline,  and Fisher’s exact tests were used to compare changes in proportion of events for each matriline before and after H1’s removal.HCC values were log transformed to ensure normality prior to analysis. Seven subjects were missing a HCC measurement in August , as they could not be captured during routine exams. Two subjects were also missing a HCC measurement in February . One subject from matriline 3 was also excluded from the August HCC analysis , as the HCC measurement in August was well above six standard deviations from the mean. We tested the association between individuals’ HCCs and their Elo-rating using Pearson’s correlation test for both matriline 3 and matrilines 4/1. Additionally, we ran Spearman’s correlation test  between HCC changes  and Elo-rating changes for high and low ranking monkeys. We predicted that an increase in rank would be associated with an increase in physiological stress among high-ranking monkeys in matriline 3 as their dominance positions were contested.To examine whether frequencies of social grooming were associated with HCCs after the matriarch’s removal, we calculated a grooming frequency across the entire 6 months  for the 15 subjects of whom behavioral data were collected . We then tested the association between grooming frequency and February 2015 HCC (which reflected chronic activity since August) using Spearman’s correlation test.All tests were two tailed with the significance level set at p<0.05. SPSS 22 was used for analyses.prediction 1), we found lower social stability after H1’s removal compared to the three months before: the stability index for matriline 3 increased from 0.039 to 0.128. Matrilines 4&1 had little change in stability (from .017 to .018). Furthermore, our focal observations revealed that in the months immediately following H1’s removal, there was a significant increase in the initiation of aggression (paired-sample t-test: t(9) = -3.20, P = 0.01, (9) = -3.15, P = 0.01, (t(9)) = -2.74, P = 0.02, predictions 2, 4 and 6. No significant changes were found for the initiation of aggression (t(4)= 0.10, P = 0.93), vigilance (t(4) = -1.68, P = 0.17), or grooming (t(4) = -1.33, P = 0.25) for matrilines 4 and 1.As expected  = -3.86, P = 0.002) whereas low ranking animals did not show a significant change (t(13) = -1.629, P = 0.13). There was no significant change in intense aggression for high ranking monkeys (t(9) = -1.65, P = 0.13) or low ranking monkeys (t(9) = -2.25, P = 0.05) within matrilines 4 and 1.The prediction 3), there was a reduction in overall troop severe aggression (from 9 to 6) compared to the period prior to H1's removal. However, when compared to the number of aggressive events experienced by the other matrilines, matriline 3 suffered proportionally more wounds after H1's removal. In the three months after her removal, no severe aggression was recorded for matrilines 4 and 1, while all recorded events occurred within matriline 3 . In the three months prior to her removal, the matrilines had approximately equal numbers of severe aggression (four within matriline 3 and five within matriline 4 monkeys).Contrary to our expectation (t(24) = -1.05, P = 0.31) or matrilines 4 and 1 (t(13) = 0.15, P = 0.89). For matriline 3, there was a significant positive correlation between Elo-rating and HCCs prior to H1’s removal in August  or February .There was no significant change in HCCs from August 2014 to February 2015 for either matriline 3  or low-ranking monkeys , a significant negative association was evident when analyzing all subjects from which grooming data were collected . The lack of any significant relation between rank changes and hair cortisol for the other matrilines suggests that only H1’s matriline was affected by her removal. We found that her matriline experienced both behavioral and physiological changes that resemble the consequences of the loss or takeover of alpha individuals described in both this  and othe ursinus , 50 nakes glaber ; chimpanglodytes ).Papio ursinus [Lycaon pictus [Macaca fascicularis [Haplochromis burtoni [subordinates, who receive continuous harassment from dominants  showing baboons ; but see baboons  for a di baboons . It is pventions . ConsequProcavis capensis [Notably, we did not find any association between dominance rank and HCC in the months following H1’s removal, probably because only a restricted number of individuals experienced heightened levels of stress during social instability, namely those high-ranking monkeys who increased in Elo-rating and intense aggression. Furthermore, since grooming has been shown to decrease animal stress levels –62 it iscapensis ). Our stad libitum sampling and occurred too infrequently during focal observations, and therefore direct comparisons were not available during this study. Therefore, an interesting opportunity exists in the future to study the role and changes in submissive signaling and resultant levels of aggression during a time of social instability such as after the loss of a matriarch.Following H1’s removal, instances of severe aggression were only recorded for her matriline, indicating that high intensity aggression was more frequent within her matriline. These findings are underscored by our observation of increased chases and attacks by the high-ranking moneys of matriline 3 after H1’s removal. The increase in dominance instability, and severe physical aggression, were the likely consequences of monkeys trying to reaffirm their rank. In support of this, one month after the matriarch’s removal, CX had undergone a major rank increase and ranked above all seven of H1’s offspring. During this time, she was also observed with a large tear wound. This wound was likely a byproduct of CX attempting to increase in rank (successfully), as well as the unwillingness of the others to sacrifice their positions (those attempting to reaffirm their position). Unfortunately, wounds are rarely ever seen occurring, so it is impossible to know exactly who inflicted this wound. The fact that no severe aggression was observed within the other matrilines suggests that they were likely not struggling to assert dominance over one another, a notion supported by their relatively stable hierarchies. Furthermore, the increased levels of intense aggression including those resulting in tissue damage may have resulted from changes in submissive signaling. Submissive signals (such as the silent bared teeth display) have been related to lower levels of severe aggression  and are Gorilla gorilla beringei) spend more time monitoring individuals whom they had aggressive interactions [Cebus apella; [As a likely result of the increased levels of aggression and observed tissue damage, there was also an increase in the frequency of vigilance. In a time of rank changes and monkeys attempting to reaffirm their rank, vigilance may have been a buffering mechanism from both directed and redirected aggression. Additionally, monkeys may have increased in vigilance to monitor the social interactions of others. Indeed, female mountain gorillas .Importantly, it is worth noting that the matriarch’s removal happened in the midst of the breeding season and thus the variation in aggression rates and possibly wounding rates we found in our study might be related to increased competition during the breeding season , 72. HowMore generally, our results emphasize the importance of careful consideration in the management of a captive colony when deciding whether some individuals should be permanently removed. Although each individual primate colony is unique and management decisions will differ across facilities, it is important for researchers, colony managers, and veterinarians to consider that group members who play a key role for the stability of the social group should be identified, even if they are not among the alpha individuals. Our study shows how the removal of even non-alpha matriarchs significantly impacts matrilineal dynamics: their absence can trigger a period of social instability and heightened intense aggression. In the case in which socially significant individuals must be removed, extra monitoring is recommended to detecting potential changes in stability. Managers of captive colonies may therefore be able to use data generated via Elo-rating and rank changes  to examine how certain characteristics  of an individual may be important in maintaining stability. This will then in turn allow managers to predict the effects of the removal of certain individuals in the future and guide future management decisions.In conclusion, this study provides evidence that old-aged matriarchs may play a substantial role in family stability in group-living primates. When considering the stability of large family groups, it may be important to recognize the critical role that non-alpha matriarchs provide. Collectively, our findings demonstrate the power of specific individuals, including non-alpha group members, to exert strong influences within their group and the consequences following their loss.S1 Dataset(XLSX)Click here for additional data file.


Southern tomato virus (STV) isolate on tomato plants in a seed production field in Bangladesh was obtained for the first time using next-generation sequencing. The identified isolate, STV_BD-13, shares a high degree of sequence identity (99%) with several known STV isolates worldwide.The complete genome sequence of a  Southern tomato virus (STV) is a double-stranded RNA (dsRNA) virus with a small genome of approximately 3.5 kb containing two overlapping open reading frames (ORFs). Based on its dsRNA genome and the pattern of genome structure, STV was classified in the genus Amalgavirus of the family Amalgaviridae (Tobacco mosaic virus (TMV) and Potato virus Y (PVY) (Pepino mosaic virus (PepMV), Tomato spotted wilt virus (TSWV), and Tomato yellow leaf curl virus (TYLCV) (Cucumber mosaic virus (CMV), and Tomato chlorosis virus  . In Spaiis virus . In summis virus  and sequis virus . From this virus . BLASTn is virus . Such brSouthern tomato virus isolate STV_BD-13 has been deposited in GenBank under the accession number KT634055.The genome sequence of