Anas platyrhynchos) — a partially migratory species — we identified resident and migratory host populations using stable hydrogen isotope analysis of flight feathers. We investigated the role of migratory and resident hosts separately in the introduction and maintenance of H3 LPAIV during the epizootic. To test this we analysed (i) H3 virus kinship, (ii) temporal patterns in H3 virus prevalence and shedding and (iii) H3-specific antibody prevalence in relation to host migratory strategy. We demonstrate that the H3 LPAIV strain causing the epizootic most likely originated from a single introduction, followed by local clonal expansion. The H3 LPAIV strain was genetically unrelated to H3 LPAIV detected both before and after the epizootic at the study site. During the LPAIV epizootic, migratory mallards were more often infected with H3 LPAIV than residents. Low titres of H3-specific antibodies were detected in only a few residents and migrants. Our results suggest that in this LPAIV epizootic, a single H3 virus was present in resident mallards prior to arrival of migratory mallards followed by a period of virus amplification, importantly associated with the influx of migratory mallards. Thus migrants are suggested to act as local amplifiers rather than the often suggested role as vectors importing novel strains from afar. Our study exemplifies that a multifaceted interdisciplinary approach offers promising opportunities to elucidate the role of migratory and resident hosts in infectious disease dynamics in wildlife.Migratory and resident hosts have been hypothesized to fulfil distinct roles in infectious disease dynamics. However, the contribution of resident and migratory hosts to wildlife infectious disease epidemiology, including that of low pathogenic avian influenza virus (LPAIV) in wild birds, has largely remained unstudied. During an autumn H3 LPAIV epizootic in free-living mallards ( Migratory and resident (i.e. sedentary) hosts are thought to fulfil different, non-mutually exclusive, roles in infectious disease dynamics in wild animal populations, although empirical evidence is largely lacking. For one, migratory hosts may transport pathogens to new areas, resulting in the exposure and potential infection of new host species, thereby contributing to the global spread of infectious diseases Additionally, migratory hosts may amplify pathogens upon arrival at a staging site, either because they are immunologically naïve to locally circulating pathogens Anseriformes and Charadriiformes) are infected with LPAIV Wild bird populations are considered the reservoir hosts of low pathogenic avian influenza A viruses (LPAIV). Predominantly birds from wetlands and aquatic environments , and connected host migratory strategy with (i) H3 virus kinship, (ii) H3 virus prevalence and shedding, and (iii) H3-specific antibody prevalence. H3 LPAIV is a dominant subtype in wild ducks in the northern hemisphere We build on the study of van Dijk et al. Capturing free-living mallards was approved by the Dutch Ministry of Economic Affairs based on the Flora and Fauna Act (permit number FF/75A/2009/067 and FF/75A/2010/011). Handling and sampling of free-living mallards was approved by the Animal Experiment Committee of the Erasmus MC (permit number 122-09-20 and 122-10-20) and the Royal Netherlands Academy of Arts and Sciences (KNAW) (permit number CL10.02). Free-living mallards were released into the wild after sampling. All efforts were made to minimize animal suffering throughout the studies.Anas genus, harbouring almost all LPAIV subtype combinations found in birds to date Mallards are considered a key LPAIV host species, together with other dabbling duck species of the During the 2010 LPAIV epizootic described here, free-living mallards were caught in swim-in traps of a duck decoy During the LPAIV epizootic (i.e. from August until December 2010) studied here, the duck decoy was visited, on average, seven times per month capturing approximately 11 birds per visit. Each captured mallard was marked using a metal ring with an unique code, aged  and sexed based on plumage characteristics 2H) ratio in feathers to be correlated with δ2H of local precipitation 2H in feathers is found in mallards 2H and additional criteria, van Dijk et al. 2H values of local (−103.5 to −72.6‰) and distant migrants (−164.5 to −103.7‰) and using a European feather δ2H isoscape of mallards In the study of van Dijk et al. 2H values are reported in units per mil (‰) relative to the Vienna Standard Mean Ocean Water-Standard Light Antarctic Precipitation (VSMOW-SLAP) standard scale.For full details on the stable hydrogen isotope analysis, see van Dijk et al. As part of the national wild bird AIV surveillance program — including the 2010 LPAIV epizootic — LPAIV infection of free-living and hunted mallards was assessed using cloacal and oropharyngeal swab samples. RNA from these samples was isolated using the MagnaPure LC system with a MagnaPure LC total nucleic acid isolation kit  and analysed using a real-time reverse transcriptase-PCR (RT-PCR) assay targeting the matrix gene. Matrix RT-PCR positive samples were used for the detection of H5 and H7 influenza A viruses using HA specific RT-PCR tests 5′-GAACAACCGGTTCCAGATCAA -3′) and 40 pmol RF3227 (5′- TGGCAGGCCCACATAATGA-3′) and 10 pmol of the double-dye labelled probe RF3228 (5′-FAM-TCCTRTGGATTTCCTTTGCCATATCATGC-BHQ-3′). Primers and probe were designed with the software package Primer Express version 3.01 , based on avian H3 nucleotide sequences obtained from Genbank (www.ncbi.nlm.nih.gov).Matrix RT-PCR positive samples collected during the 2010 LPAIV epizootic for which virus culture was not successful, were screened for the presence of H3 influenza A viruses using a H3 specific RT-PCR test (n = 126). Additionally, matrix RT-PCR positive samples collected half year prior to the LPAIV epizootic (November 2009-July 2010) were screened for the presence of H3 influenza A viruses to determine whether H3 LPAIV was detected in mallards prior to the epizootic (n = 20). Amplification and detection were performed on an ABI 7500 machine with the taqman Fast Virus 1 Step Master mix reagents  and 5 µl of eluate in an end volume of 30 µl using 10 pmol Oligonucleotides RF3226 (T) value, i.e. first real-time matrix RT-PCR amplification cycle in which matrix gene amplification was detected. The CT-value is inversely proportional to the amount of viral RNA in a sample.The degree of virus shedding from the cloaca and the oropharynx during the LPAIV epizootic was based on the cycle threshold (C5′- GGATCTGCTGCTTGTCCTGT-3′ and 5′- GRATAAGCATCTATTGGAC-3′), as described previously To investigate H3 LPAIV diversity in time and space among resident and migratory mallards during the LPAIV epizootic, we performed a genetic analysis focussed on the HA segment, one of the two most variable gene segments of LPAIV. Nucleotide sequences of the HA gene segment were obtained from virus isolates that were previously characterized by hemagglutination inhibition (HI) assay as H3 LPAIV. The RT-PCR and sequencing of the HA segment was performed using HA specific primers , from which only European virus sequences with a known isolation date were retrieved (n = 28). Duplicate and incomplete sequences were removed. Nucleotide sequences were aligned using the software MAFFT version 7 .A total of 86 HA gene segments of 1576 nt in length were included in the genetic analysis. The genetic analysis comprised H3 nucleotide sequences obtained from (i) residents and migratory mallards during the 2010 LPAIV epizootic (n = 23), (ii) additional H3 LPAIV isolates from the national wild bird surveillance program of Erasmus MC (n = 35), and (iii) a BLAST analysis using public databases available as of 29 November 2013 . During the 2010 LPAIV epizootic, H3 nucleotide sequences were obtained from 23 viruses, isolated from residents (n = 3), from local migrants (n = 13), from distant migrants (n = 2), and from birds of which the migratory strategy could not be assessed (n = 5). This was supplemented with 12 H3 nucleotide sequences obtained from viruses isolated from mallards sampled in the duck decoy in different years, notably in 2008 (n = 11) and 2011 (n = 1). There were 31 H3 nucleotide sequences from virus samples collected at other sampling locations in the Netherlands and elsewhere in Europe between 1999 and 2011. Of these virus samples, 18 originated from locations within the province of Zuid-Holland (5–30 km from the duck decoy), i.e. from Berkenwoude (n = 13) , Lekkerkerk (n = 2) , Oudeland van Strijen (n = 2)  and Vlist (n = 1) . Eleven viruses were isolated from birds in coastal regions in the Netherlands (i.e. 115–200 km from the duck decoy), i.e. Schiermonnikoog (n = 1) , Vlieland (n = 1) , Westerland (n = 8)  and Wieringen (n = 1) . Outside the Netherlands, two H3 sequences were from viruses isolated in Hungary in 2009. The remaining 20 H3 nucleotide sequences originated from multiple locations throughout Europe  and Russia.http://tree.bio.ed.ac.uk/software/figtree). Overall rates of evolutionary change (i.e. number of nucleotide substitutions per site per year) and time of circulation to the most recent common ancestor (TMRCA) in years was estimated using the BEAST program version 1.8.0 (http://beast.bio.ed.ac.uk/). To accommodate variation in the molecular evolutionary rate among lineages, the uncorrelated log-normal relaxed molecular clock was used. Isolation dates were used to calibrate the molecular clock. Three independent Bayesian Markov Chain Monte Carlo (MCMC) analyses were performed for 50 million states, with sampling every 2,000 states. Convergence and effective sample sizes of the estimate were checked with Tracer v1.6 (http://tree.bio.ed.ac.uk/software/tracer/). Uncertainty in parameter estimates was reported as the 95% highest posterior density (HPD) A Maximum Likelihood (ML) phylogenetic tree was generated using the PhyML package version 3.1 using the GTR+I+G model of nucleotide substitution, performing a full heuristic search and subtree pruning and regrafting (SPR) searches. The best-fit model of nucleotide substitution was determined with jModelTest To assess whether mallards had H3-specific antibodies during the 2010 LPAIV epizootic, all sera were first tested for the presence of AIV antibodies specific for the nucleoprotein (NP) using a multispecies blocking enzyme-linked immunosorbent assay , following manufacturer's instructions. Each plate contained two positive and two negative controls. Samples were tested in duplicate. An infinite M200 plate reader  was used to measure the absorbance  at 620 nm. Samples were considered positive for the presence of NP antibodies when signal-to-noise ratios  were <0.5. NP antibody positive serum samples were subsequently tested for the presence of H3-specific antibodies using the HI assay according to standard procedures Birds were considered LPAIV positive when either cloacal or oropharyngeal swabs were positive. To exclude samples of birds that had been sampled twice within the same infectious period during the 2010 LPAIV epizootic, we used an interval of at least 30 days between the day that a bird tested LPAIV positive and the next sampling day. Mallards may shed virus up to 18 days During the LPAIV epizootic, 709 cloacal and oropharyngeal swabs were collected from 472 mallards of which 129 individuals were recaptured. Of these swabs, 84 tested positive for H3 LPAIV, 35 tested LPAIV positive but H3 negative (i.e. matrix-positive H3-negative), and 583 swabs tested LPAIV negative. Of 7 matrix-positive swabs we were unable to determine H3-positivity. To test H3 virus prevalence and shedding, we included H3-positive and H3-negative swabs (i.e. matrix-negative and matrix-positive). Swabs from birds of which the migratory strategy could not be assessed (n = 269) or with undefined age and sex (n = 13) were excluded. The exclusion of birds of which the migratory strategy could not be assessed did not affect the temporal pattern of H3 LPAIV prevalence. In total we included 420 cloacal and oropharyngeal swabs from 305 individual birds, of which 55 birds were sampled more than once .During the LPAIV epizootic, 428 serum samples were collected from 364 mallards of which 52 individuals were recaptured. Of these serum samples, 9 tested positive for H3-specific antibodies, 98 tested positive for AIV antibodies but negative for H3-specific antibodies (i.e. NP-positive H3-negative), and 321 sera tested negative for AIV antibodies. To investigate H3-specific antibody prevalence, we included H3-specific antibody positive and H3-specific antibody negative sera (i.e. NP-negative and NP-positive). Sera from birds of which the migratory strategy could not be assessed (n = 96) or with undefined age and sex (n = 5) were excluded. Thus in total we included 320 sera samples from 281 individual birds, of which 30 birds were sampled more than once .T-value) with migratory strategy and month as fixed factors. A Tukey's post hoc test was performed to detect differences in H3 LPAIV prevalence between the three categories of migratory strategy and months. All analyses were conducted using R 2.14.1 A generalized linear mixed model (GLMM) was used in the analysis of H3 virus prevalence, with migratory strategy , age, sex and month as fixed factors, all two-way interactions with migratory strategy, and individual bird as random factor. The interactions between migratory strategy and age, migratory strategy and sex, and migratory strategy and month were tested to assess whether H3 virus prevalence differed per age class, sex and month for the three categories of migratory strategy. The fixed factors age and sex were merely included in the models to conduct the interactions. A general linear model (GLM) was used to test for differences in prevalence of H3-specific antibodies, with migratory strategy and month as fixed factors. Linear models (LMs) were used to determine differences in the degree of virus shedding of H3 LPAIV-particles based on viral RNA from the cloaca and the oropharynx  and the beginning of winter (December), with some exceptions in March 2009 and June 2011 . Detectith of August 2010  and with other LPAIV subtypes, namely H4, H6 and H10 , three residents and one local migrant were infected with H3 LPAIV, with their populations constituting respectively 88% and 12% of the sampled mallard population. Two weeks later (26th of August), the first distant migrant infected with H3 LPAIV was detected . In September and October, most mallards infected with H3 LPAIV were local migrants , while local migrants comprised respectively 24% and 40% of the sampled mallard population. In October, 11 residents and nine distant migrants were infected with H3 LPAIV, the latter constituting only 17% of the sampled mallard population. In November, only nine local and five distant migrants were infected with H3 LPAIV . The last month of the H3 LPAIV epizootic, only one distant migrant and two residents were infected with H3 LPAIV, although distant migrants and residents constituted respectively 43% and 32% of the sampled mallard population.Local and distant migrants were more often infected with H3 LPAIV  than residents . Nor were there any differences in the monthly amount of H3 virus shed from the cloaca and oropharynx during the H3 LPAIV epizootic .H3 virus shedding from the cloaca and oropharynx did not differ between the three mallard populations . During the H3 LPAIV epizootic, H3-specific antibodies were detected on four sampling dates. On the 5th of August, before the start of the H3 LPAIV epizootic, one distant migrant had H3-specific antibodies . During the H3 LPAIV epizootic, the first resident with H3-specific antibodies was sampled on the 21st of September, with 9% of the sampled mallard population comprised of residents. After the peak of the H3 LPAIV epizootic (1st of November), two local migrants, one distant migrant and one resident had antibodies specific for H3 LPAIV. That day, local migrants constituted the largest proportion of the sampled mallard population (71%). At the end of the epizootic (21st of December), only migrants  had specific antibodies against H3 LPAIV .During the 2010 LPAIV epizootic, NP-specific LPAIV antibody prevalence increased from September onwards to 60% in December . During th of February 2010) differed from the H3 LPAIV strains of the epizootic , and was therefore unlikely to have seeded the outbreak. Furthermore, the H3 LPAIV strains isolated during the H3 LPAIV epizootic were not closely related to isolates obtained from mallards at our study site in autumn 2008 (sequence identity 0.958–0.967), or November 2011 . The H3 LPAIV strain detected in a single mallard at our study site prior to the H3 LPAIV epizootic . What is remarkable though is that one week after detection of the first H3 LPAIV infections, no migrants were infected while a large proportion of the sampled mallard population consisted of migrants (∼40%). Either migratory birds were not, or to a lesser extent, susceptible to H3 LPAIV infection, or contact rates and the amount of H3 virus particles in the surface water were still too low to infect arriving migrants. Interestingly, the peak of virus infection in October in the resident population was mainly induced by recaptured resident birds (i.e. captured multiple times) . H3 viruth of August). We cannot exclude that this individual was infected with H3 LPAIV either during migration, at a stop-over site or at the breeding grounds. Hypothetically, this individual could have been infected with H3 LPAIV when transiting through southern Sweden . Resident mallards with H3-specific antibodies most likely have been infected by the H3 LPAIV of the epizootic. Only 20% (1 of 5) of residents that had been infected with H3 LPAIV during the epizootic had H3-specific antibodies when recaptured -specific antibodies in mallards (Anas platyrhynchos) during the H3 low pathogenic avian influenza virus epizootic in 2010.(PDF)Click here for additional data file.Figure S2Prevalence of H3 influenza virus in resident and migratory mallards during H3 epizootic. This figure shows H3 low pathogenic avian influenza virus (LPAIV) prevalence in resident mallards (i.e. primary captured and recaptured), local and distant migratory mallards, during the H3 LPAIV epizootic in 2010.(PDF)Click here for additional data file.Table S1The H3 influenza virus strain names and accession numbers used in this study. This table includes all H3 influenza virus strain names and accession numbers used in this study.(PDF)Click here for additional data file.Table S2The sequence information of H3 influenza viruses from GISAID's EpiFlu Database. This table includes details of H3 influenza viruses downloaded from GISAID's EpiFlu Database.(PDF)Click here for additional data file.Table S3Sample collection for influenza virus and antibody detection from free-living mallards. This table includes number of samples collected for influenza virus and antibody detection from free-living mallards (Anas platyrhynchos) during the H3 low pathogenic avian influenza virus epizootic.(PDF)Click here for additional data file.


Cilevirus, Higrevirus and Blunervirus, as well as to those within the family Virgaviridae. Genetic distances and phylogenetic analyses suggest that Nelorpivirus and Sandewavirus might form taxonomic groups on genus level that may define alone or together with Cilevirus, Higrevirus and Blunervirus a viral family.Pools of mosquitoes collected in Côte d’Ivoire and Mexico were tested for cytopathic effects on the mosquito cell line C6/36. Seven pools induced strong cytopathic effects after one to five days post infection and were further investigated by deep sequencing. The genomes of six virus isolates from Côte d’Ivoire showed pairwise nucleotide identities of ~99% among each other and of 56%–60% to Dezidougou virus and Wallerfield virus, two insect-specific viruses belonging to the proposed new taxon Negevirus. The novel virus was tentatively named Goutanap virus. The isolate derived from the Mexican mosquitoes showed 95% pairwise identity to Piura virus and was suggested to be a strain of Piura virus, named C6.7-MX-2008. Phylogenetic inferences based on a concatenated alignment of the methyltransferase, helicase, and RNA-dependent RNA polymerase domains showed that the new taxon Negevirus formed two monophyletic clades, named Nelorpivirus and Sandewavirus after the viruses grouping in these clades. Branch lengths separating these clades were equivalent to those of the related genera  Bunyaviridae, Flaviviridae, Togaviridae, Reoviridae, Rhabdoviridae and Asfarviridae. Within recent years, novel viruses that seem to infect insects only and not to be able to replicate in vertebrates have been discovered in mosquitoes. These insect-specific or insect-restricted viruses belong not only to viral families that contain arboviruses [Mosquitoes transmit a great diversity of viruses that can cause severe disease in humans, e.g., Yellow fever virus, Dengue virus, and Chikungunya virus ,2,3. Patoviruses ,10,11,12oviruses ,14,15 suCilevirus (unassigned family) [Higrevirus (unassigned family), and Blueberry necrotic ring blotch virus (BNRBV), proposed to be the type species of the genus Blunervirus (unassigned family) [Virgaviridae [One of these new taxa of insect-specific viruses is the recently proposed new taxon Negevirus . Negevir family) . Other r family) . Phylogeaviridae ,21,22,23Gouléako and Taï National Park in Côte d’Ivoire from which the mosquitoes originated, as well as of 33 strains of Piura virus (PIUV) from mosquitoes collected in Mexico.Here, we report the detection of a novel virus, named Goutanap virus (GANV), after the village Aedes albopictus (C6/36) and African green monkey (Vero E6/7) cells were infected with mosquito homogenates and observed for cytopathic effects (CPE). CPE positive cultures were used to infect confluent C6/36 cells in 175 cm2 cell culture flasks. Virus particles were pelleted by ultracentrifugation through a 36% sucrose cushion, re‑suspended in 200 µL PBS and stored at −80 °C until further analysis.Mosquitoes were collected in the area of the Taï National Park in Côte d’Ivoire  and of tViral nucleic acids from Ivorian samples were extracted using TRIzol  and double-strand cDNA was synthesized using the cDNA Synthesis System Kit  following the manufacturer’s instructions. One hundred nanograms (ng) of cDNA per sample were fragmented as described in the Ion Xpress Plus gDNA Fragment Library Preparation Manual (Life Technologies). Fragment ends were repaired, barcode containing adapter oligonucleotides were ligated and emulsion PCR (emPCR) was performed according to the Ion Torrent protocol (Life Technologies). Next Generation Sequencing (NGS) was performed on a 316 chip using the Ion Torrent platform (Life Technologies). Viral nucleic acids from Mexican samples were extracted using the RNeasy Mini Kit  and cDNA synthesis was performed with the Superscript OneCycle cDNA Kit (Life Technologies) according to the manufacturer’s instructions. Fragmentation of 100 ng cDNA per sample was done by nebulization and a library was constructed according to the GS Junior Titanium Series Rapid Library Preparation Method Manual (Roche). Fragment end repair, adaptor ligation and emPCR (Kit Lib-L) were done following the standard Roche protocols. NGS was performed on a Roche 454 GS Junior system.Virgaviridae . cDNA was synthesized using random hexamer primers and Superscript III Reverse transcriptase (Life Technologies). PCR was performed using Platinum Taq polymerase , the primers MX_C6.7_F1 (5'-CTTGAGATTAAGTTCTTCGAGTTTGAGC) and MX_C6.7_R1 (5'-AAAGGCGGTGTGATCGGTG) and 1 µL template cDNA. PCR products were visualized by agarose gel electrophoresis and Sanger sequenced .The genome sequences of Goutanap virus were assigned to GenBank accession numbers KM249339 and KF588035 to KF588039. The genome sequence of Piura virus C6.7-MX-2008 was assigned to GenBank accession number KM249340. Sequence fragments of the RdRp domain of other Piura virus isolates were assigned to GenBank accession numbers KM258581 to KM258599 and KM924386 to KM924398.Seven mosquito pools induced strong CPE one to five days post infection in C6/36 cells and were further investigated by NGS . GenomesCulex mosquitoes but also species of the genera Aedes, Mansonia, Psorophora, and Wyeomyia showing that PIUV infects a large diversity of mosquito species . PIUV Palenque C6.7-MX-2008 clustered together with PIUV P60, Negev virus (NEGV), Ngewotan virus (NWTV), and Loreto virus (LORV)  [et al. (2014) [et al. (2013) [Phylogenetic analyses based on conserved RdRp domains grouped GANV in a basal phylogenetic position to the two sister species WALV and DEZV A. The ths (LORV) A. This c. (2014) , Nabeshi. (2014)  and Vasi. (2013) . Intra- Virgaviridae, a closely related family . However, the clustering of Blunervirus with other plant-infecting viruses might as well result from a long-branch attraction effect that could resolve upon inclusion of further (as yet unknown) taxa on this solitary branch.However, based on genome organization and host association, the topology in Virgaviridae the tree topology suggests that Nelorpivirus and Sandewavirus might form taxonomic groups on genus level. Differences in genome architecture may not suffice to define whether Nelorpivirus and Sandewavirus alone or in combination with Cilevirus, Higrevirus and Blunervirus define a viral family, as differences in the number of genomic RNAs of different genera are known in other viral families, e.g., Viragviridae [Reoviridae [According to phylogenetic distances separating established genera within the family gviridae ,23 and Roviridae .et al. (2013) [Nelorpivirus and Sandewavirus). All negeviruses share one type of genome organization  as opposed to outgroup taxa, which in turn have heterogeneous genome organizations, predicting the taxon Negevirus to constitute a candidate virus family or subfamily. The high infection rate of Nelorpivirus and Sandewavirus in mosquito populations and their wide geographic distribution invites further studies on the influence of negeviruses on mosquito populations as well as the transmission of mosquito-borne disease.In summary, phylogenetic analyses suggest that the taxon Negevirus as presented in Vasilakis . (2013)  is a mon


We conducted phylogeographic modeling to determine the introduction and spread of Guaroa virus in South America. The results suggest a recent introduction of this virus into regions of Peru and Bolivia over the past 60–70 years and emphasize the need for increased surveillance in surrounding areas. Bunyaviridae, genus Orthobunyavirus) infection in humans frequently results in febrile illness, and limited serologic surveillance indicates that the virus infects a substantial portion of the rural population in tropical regions of Central and South America  or from the World Reference Center for Emerging Viruses and Arboviruses . These viruses had been isolated during 1956–2011 and originate from 4 of the 5 countries in which GROV has been isolated: Columbia, Brazil, Peru, and Bolivia  Table. CViral RNA was extracted from the isolates by using the QIAamp Viral RNA Mini Kit , and reverse transcription PCR reactions were performed by using the Superscript III Reverse Transcriptase  and the iProof High-Fidelity PCR Kit . Coding region sequencing was based on partial sequences of strain BeH22063  and completed where necessary by using primer walking. Terminal noncoding sequences were obtained by using ligation-anchored PCR, as previously described  Table; phttp://beast.bio.ed.ac.uk/) with multiple sequence alignments containing nucleoprotein open-reading frame sequences from the GROV and WYOV groups. The resulting trees, along with tables containing the geographic coordinates of the samples, were then input into SPREAD v1.0.6 (https://www.google.com/earth), and exported as video files (We then used the large GROV dataset and one for the closely related Wyeomyia virus (WYOV) group to conduct a phylogeographic analysis . We calcD v1.0.6  Figure aeo files . BecauseThe results of our analysis indicate that the GROV/WYOV common ancestor was introduced into South America ≈250 years ago  at a site along the Amazon River; the identified site is within ≈250 km of Manaus, Brazil, which has been a major population center in this area since the early days of European colonization, possibly even longer . This esFrom this central introduction point of the GROV/WYOV common ancestor, the GROV group and the WYOV group, which is further made up of 2 distinct lineages (the WYOV and Anhembi virus [ABMV] lineages), exhibit differences in their patterns of spread. The ABMV lineage spread in a predominantly southward direction within Brazil , panel AThe observed spatial patterns are also notable in that they suggest possible mechanisms of virus spread within the endemic region. The northwest/southeast axis of spread seen with the WYOV and ABMV lineages closely reflects what was reported for YFV (Overall, our study has led to the generation of a substantial set of full-length GROV sequences, which we anticipate will aid in future efforts to develop improved diagnostic approaches for this and related viruses, and has enabled us to model the spread of GROV within the virus-endemic area. In doing so, we have identified Peru and Bolivia as regions of recent and active GROV spread that should be considered areas for future virus surveillance efforts. In addition, our data implicate both human-associated factors and possibly bird populations in the spread of this virus within South America.Source and sequence information for virus strains used in this study and phylogeographic analysis of the predicted spread of Guaroa virus and Wyeomyia and Anhembi lineage viruses over time in South America.


M1TM, 70–150 μm) capable of an increased bead delivery in the distal vasculature, corresponding to greater tumour coverage and drug dose delivered. Both unloaded and drug loaded DC BeadM1 were shown to have a greater density of distal volume of penetration although the ultimate distal level of penetration was the same as that of the 100–300 µm beads in an in vitro penetration model. Elution of doxorubicin was slower than irinotecan elution, but it was similar when comparing the same drug elution from 70 to 150 µm compared to 100–300 µm beads. Radiopaque versions of 70–150 and 100–300 µm beads were prepared in order to evaluate distribution ex vivo using µ-CT and doxorubicin distribution using epifluorescent microscopy. Liver distribution of the radiopaque versions of the beads was shown to be more distal and efficient at filling smaller vessels with the DC BeadM1 and correspondingly more beads were found per vessel histologically with a larger area of drug coverage with the smaller size range. This study indicates that the smaller (70–150 μm) beads should permit an increased dose of drug to be administered to both hypervascular and hypovascular tumours as compared to 100–300 µm beads.Clinical use of DC Bead™ loaded with doxorubicin (DEBDOX™) or irinotecan (DEBIRI™), for the treatment of primary and secondary tumours of the liver respectively, is showing great promise. Recently there has been a tendency to select smaller bead size ranges to treat tumours in an effort to allow more drug dose to be administered, improve tumoural penetration and resultant drug delivery and tumour coverage. Herein we describe the development and performance characterisation of a new DC Bead size range (DC Bead More recently, drug-eluting bead TACE (DEB-TACE) has emerged as an alternative to cTACE . At 9 and 12 months the CR rates were no longer significant due to disease progression (PD) in untreated areas . Heithaus et al. recently reported on midterm outcomes from 94 DEB-TACE sessions using 70–150 µm beads in which disease control (DC = CR + PR + SD) was achieved in 95 % of focal and 96 % of small tumors versus 61 % of multifocal (P = 0.04) and 55 % of large tumors (P = 0.0009). They concluded the treatment was safe with no grade 4/4 complications, 88 % of patients discharged within 24 h and demonstrated antitumoural response and survival benefits [P > 0.05) although hair loss was significant  [Spreafico et al. treated 45 consecutive patients with HCC using 70–150 µm beads and obtained an OR rate [CR + partial response (PR)] of 77.7 % with a median time to best response of 3 months . In 13 patients, the treatment served as a bridge/downstaging to LT/surgery and pathological analysis of the removed tumors showed that more than 90 % necrosis was achieved in 10 of 28 nodules. The small beads were well tolerated, and the grade 3/4 adverse event rate was low (1 of 65 procedures) [benefits . Rao et  = 0.01) . Thus itAs clinical experience is mounting for the use of DEB for the treatment of hepatic malignancies, techniques for bead administration  versus stasis  ) have a


Geminiviridae) by targeting different viral genome sequences in infected Nicotiana benthamiana or Arabidopsis plants. The DNA viruses targeted include tomato yellow leaf curl virus and merremia mosaic virus (begomovirus); beet curly top virus and beet severe curly top virus (curtovirus); and bean yellow dwarf virus (mastrevirus). The technique has also been used against the RNA viruses zucchini yellow mosaic virus, papaya ringspot virus and turnip mosaic virus (potyvirus) and cucumber vein yellowing virus  by targeting the translation initiation genes eIF4E in cucumber or Arabidopsis plants. From these recent advances of major importance, it is expected that NGS and CRISPR-Cas technologies will play a significant role in the very near future in advancing the field of plant virology and connecting it with other related fields of biology.Next-generation sequencing (NGS) has been applied to plant virology since 2009. NGS provides highly efficient, rapid, low cost DNA, or RNA high-throughput sequencing of the genomes of plant viruses and viroids and of the specific small RNAs generated during the infection process. These small RNAs, which cover frequently the whole genome of the infectious agent, are 21–24 nt long and are known as vsRNAs for viruses and vd-sRNAs for viroids. NGS has been used in a number of studies in plant virology including, but not limited to, discovery of novel viruses and viroids as well as detection and identification of those pathogens already known, analysis of genome diversity and evolution, and study of pathogen epidemiology. The genome engineering editing method, clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system has been successfully used recently to engineer resistance to DNA geminiviruses (family,  The field of virology was born in the late 1890s when it was found that the tobacco mosaic disease is caused by a novel form of infectious agent named “ultravirus” and referred to as ‘contagium vivum fluidum’  technology. Similarly, despite progress in understanding virus or viroid–plant interactions, obtaining plants resistant to these pathogens by conventional breeding or transgenic strategies are often complicated and faces the problem of resistance durability. Virus or viroid genome and/or plant gene editing is expected to play a significant role in developing transgene-free plants resistant to viruses or viroids. Advances in NGS capabilities and the rapid development and widespread adoption of a simple, inexpensive, easy to use and very effective genome engineering editing method known as “clustered regularly interspaced short palindromic repeats and their associated Cas proteins (CRISPR-Cas)” are revolutionizing the fields of genetics, genomics, molecular biology and others. In this article we discuss the progress made in plant virology during the last 7 years by NGS and very recently by CRISPR-Cas systems.de novo discovery and assembly of plant virus or viroid genome reads, has been used since 2009, first in discovering novel DNA and RNA viruses .Next-generation sequencing of vsRNAs or vd-sRNAs has also been used in various studies on viruses or viroids, which include, but are not limited to, their characterization, profiling, distribution, accumulation, biogenesis, as well as their use in extending the known pathogen host range, and in virus strain differentiation, systemic movement, virus or viroid-host interaction, viroid evolution, and pathogenesis, mutation, mRNA targeting, and others  of interest but can also be compared to the host genome in an effort to identify genes that may be down-regulated upon infection as a consequence of their local homology with the infecting virus or viroid. In parallel, the identification in otherwise healthy plants of sRNAs with homology to viral satellite RNAs has provided recently a tentative scenario for their evolution from the host plant genome, offering a possible solution to the long lasting conundrum of the origin of these infectious agents .Macluravirus, family Potyviridae, and that ranunculus latent virus should be considered as a strain of ArLV but not a distinct species (Solanum tuberosum) and turnip mosaic virus in rengarenga (Arthropodium cirratum), which is a new host . Thus, NGS has the potential to be used as a primary diagnostic tool for plant viruses and viroids as the cost of sequencing platforms has become more competitive and affordable. Currently, the cost of NGS-based diagnostics is still high as compared to that of a PCR or serological assay, so that the technique is limited to situations where exhaustiveness is critical, such as quarantine or when trying to identify a causal agent, or to situations involving high value added samples, such as nuclear stock mother plants used for production of certified planting materials. It should be stressed that nucleic acids purifications and sequencing bank preparation protocols may have to be optimized and fine-tuned/adapted to particular plant species that may contain inhibitory substances that may otherwise interfere with the sensitivity of the detection procedure.NGS has provided a very powerful alternative for detection and identification of plant viruses and viroids without The volume and diversity of international movements of plant materials, including exchanges of germplasm or newly bred cultivars, have increased considerably during the last few decades, which have also created additional pathways for the introduction of plant viruses and viroids in new areas or for the emergence of novel virus or virus-like agents. Preventive measures for blocking the introduction and spread of these pathogens include the implementations of plant quarantine (phytosanitary regulation) and certification programs. These programs are tools that can be used to protect or improve the health status of cultivated plants . NGS hasRecently, the US Department of Agriculture, Animal and Plant Inspection Service (APHIS), Plant Protection and Quarantine (PPQ) formed an internal working group to discuss the application of NGS to PPQ policy and operations. There are a number of challenges, however, that have to be addressed on the use of NGS in quarantine before a regulatory policy can be implemented. These include standardization of testing methods, interpretation of test results, biology of the discovered new pathogen, constructing a reliable database of whole genome sequence of pathogens of quarantine importance and others . It is expected that these challenges will be soon resolved due to advances in NGS capabilities and the rapid adaptation of this technology in plant pathogen diagnostics. NGS can become instrumental in releasing plants in quarantine and certification programs at a faster rate than current strategies while improving our ability to prevent the introduction of foreign plant viruses and/or viroids into new countries. Thus, NGS has the potential to be utilized in plant quarantine and certification programs standard assays in North America and Europe in the near future as, when compared with routine conventional assays, it could reduce significantly the number of non-detected viruses and viroids.Phaeodactylum tricornutum have been associated with vaginitis  were detected and identified in human oropharyngeal samples of healthy normal adults without any physical or mental disorder/illness . Mice inaginitis . Women walbicans .Similarly, the observation that the RNA viral community of the human feces is dominated by plant viruses  came as More insights into the role of PMMV, TSWV, algal viruses and other higher plant and algal viruses in human diseases may be revealed by metagenomic studies using NGS of human gut viromes and other organs of patients . Thus, tPhytophthora infestans, which caused the Irish potato famine of 1845–1847  systems act as archaea and bacteria immune systems against invading foreign DNAs . The CasRNA-guided cleavage paired with donor-guided repair allows easy introduction of any desired modification in a living cell. The possibility of re-directing the dsDNA targeting capability of CRISPR-Cas9 for RNA-guided ssRNA binding and/or cleavage  has been reported , and eveThe double-strand breaks created by Cas9 induce insertion or deletion (indel) mutations in the targeted gene or genome sequence of an organism , which aDuring the last few years, the CRISPR-Cas based systems have become the method of choice for genome editing by introducing or correcting genetic mutations in a wide variety of biological contexts: cell lines, animals (including humans) and plants , as wellGeminiviridae) are circular ssDNA viruses with genomes of 2.3 to slightly above 5 kb, distributed worldwide and transmitted by insects, which cause serious damage to many economically important dicotyledonous and monocotyledonous crops (Geminiviruses (family us crops . They reus crops . Currentus crops . All theBegomovirus) and bean yellow dwarf virus  in Nicotiana benthamiana (Curtovirus) in N. benthamiana and in Arabidopsis  and merremia mosaic virus (genus Begomovirus) when sgRNAs specific for the IR sequence of each virus were used , and resistant to papaya ringspot virus-W and zucchini yellow mosaic virus . As expected, the plants remained susceptible to viruses that do not appear to highjack the host eIF4E to complete their cycle, such as cucumber mosaic virus  or cucumber green mottle mosaic virus . Arabidopsis to turnip mosaic virus, a potyvirus, using the CRISPR/Cas9 technology targeting the plant elF (iso) 4E gene.It had been shown that engineering mutations in the host plant eIF4E through the use of the Targeting-Induced Local Lesions in Genome (TILLING) strategy could provide resistance against potyviruses in a range of plants . The CRIFor the last seven years, NGS and bioinformatics have provided rapid and low cost DNA and/or RNA sequencing for plant viruses and viroids. Full genomes or virus- or viroid-specific small RNAs, which cover essentially the whole genome, have been sequenced for discovery of novel pathogens, as well as for pathogen detection and identification, replication, ecology, epidemiology, and pathogen-host interactions. Many novel plant RNA and DNA viruses were successfully discovered but only a couple of novel viroids were revealed. Known viruses and viroids were successfully deteted and identified by NGS. Prior knowledge of virus or viroid sequence was not needed, which has made NGS a universal rapid and accurate method for pathogen discovery and diagnostics. Using NGS in a quarantine program, a novel virus in imported sugarcane was discovered that previously escaped detection by standard detection methods. Similarly, using NGS in a certification program, the causal agent of severe hop stunt disease has been very recently discovered and now the disease is under control. In the very near future NGS may be utilized in plant quarantine and certification programs to significantly increase their capacities and reliabilities. NGS is playing a significant role in connecting plant virology with other related fields of biology such as genome editing. Very recently, CRISPR-Cas9 system has been used in developing plants resistant to five DNA geminiviruses and four RNA potyviruses. Since CRISPR/Cas9 system has the capabilities to work on DNA or RNA sequences, expanding the use of these capabilities in research on DNA/RNA viruses or viroids, in association with NGS, may open widely the door for studying control of diseases caused by these pathogens at the genomic level. These advances are propelled in part by synergies between two powerful technologies: NGS and genome engineering.AH conceived and wrote the manuscript, and RF, TC, and MB revised it and made useful suggestions. AH prepared the final version of the manuscript and all authors approved it.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer VP declared a shared affiliation, though no other collaboration, with one of the authors RF to the handling Editor, who ensured that the process nevertheless met the standards of a fair and objective review.


In 2010, the Centers for Disease Control and Prevention funded 50 communities to participate in the Communities Putting Prevention to Work (CPPW) program. CPPW supported community-based approaches to prevent or delay chronic disease and promote wellness by reducing tobacco use and obesity. We collected the direct costs of CPPW for the 44 communities funded through the American Recovery and Reinvestment Act (ARRA) and analyzed costs per person reached for all CPPW interventions and by intervention category.From 2011 through 2013, we collected quarterly data on costs from the 44 CPPW ARRA-funded communities. We estimated CPPW program costs as spending on labor; consultants; materials, travel, and services; overhead activities; and partners plus the value of in-kind donations. We estimated communities’ costs per person reached for each intervention implemented and compared cost allocations across communities that focused on reducing tobacco use, or obesity, or both. Analyses were conducted in 2014; costs are reported in 2012 dollars.The largest share of CPPW total costs of $363 million supported interventions in communities that focused on obesity ($228 million). Average costs per person reached were less than $5 for 84% of tobacco-related interventions, 88% of nutrition interventions, and 89% of physical activity interventions. Costs per person reached were highest for social support and services interventions, almost $3 for tobacco‑use interventions and $1 for obesity prevention interventions.CPPW cost estimates are useful for comparing intervention cost per person reached with health outcomes and for addressing how community health intervention costs vary by type of intervention and by community size. Preventing chronic disease and its associated illness, death, and cost requires a comprehensive approach that includes population-wide approaches to make healthful living easier . In 2010The ARRA-funded CPPW awards were intended to reduce tobacco use and exposure to secondhand smoke in 14 communities, prevent obesity in 23 communities, and prevent both tobacco use and obesity in 7 communities. These awardees consisted of 14 large cities, 11 urban areas, 16 small cities or rural counties, and 3 tribal nations.Our study’s purpose was to analyze CPPW program costs as part of a multicomponent evaluation –5. We usWe collected CPPW cost data quarterly from the 44 ARRA-funded communities via a web-based Cost Study Instrument (CSI) . We provEach community developed a CPPW community action plan that included multiple community-defined objectives to improve population health. We asked communities to report quarterly costs for each resource category  and enter the percentage of costs allocated to each CPPW objective. Because objectives were unique to each community, we also asked communities to indicate which of a common set of CPPW strategies supported each objective. CPPW strategies were collectively known as media, access, point of promotion/decision, price, and social support and services (MAPPS). Each MAPPS strategy could have supported multiple community objectives, and each objective had one or more MAPPS strategies linked to it. A media strategy was hard-hitting counteradvertising for tobacco; an access strategy was limiting availability of unhealthy food and drink.We estimated community costs at the most disaggregated level by generating objective- and MAPPS strategy-level costs. For example, if a community had 10 objectives, and each objective had 2 MAPPS strategies linked to it, we estimated costs for 20 objective and strategy combinations. Communities also allocated costs to administration or evaluation. We assigned administrative costs to program cost estimates in proportion to spending on each objective or strategy. We also estimated evaluation costs but excluded them from program cost estimates.CPPW interventions were defined after the programs were under way and were usually more specific than MAPPS strategies. For example, “healthy vending” was an intervention linked to the broader MAPPS strategy “healthy food/drink availability.” We estimated CPPW intervention costs by assigning objective/strategy costs to each intervention that was linked to a community objective. First, we calculated each awardee’s total CPPW spending and compared it with the total value of its CPPW award to validate awardees’ reported spending. “Spending” is the actual dollar outlays for CPPW, which included evaluation expenditures. We next estimated total CPPW program costs as the sum of spending on labor; materials, travel, and supplies; overhead; and partners, but excluding evaluation costs because such costs are for research and do not reflect costs to deliver CPPW interventions. Grantees were required to evaluate their programs, but without specific guidance on how much to spend. We then added the value of in-kind donations to ensure that our costs reflect the full value of resources used for CPPW. We used CPPW program cost estimates  in all cost analyses.We analyzed costs from the provider’s perspective, excluding costs incurred by program participants. Analyses were conducted in 2014 using costs collected from 2011 to 2013, which covered the full period of CPPW (2010–2013). We adjusted costs to 2012 dollars using gross domestic product price indices . Any cosEach intervention could have supported multiple objectives. We estimated community intervention costs as the sum of all objective costs assigned to an intervention. For example, if the intervention “Create safe places for physical activity” was used to support 3 community objectives and no other interventions supported those objectives, then we summed the costs for those 3 objectives to estimate the intervention cost. If multiple interventions supported an objective, we calculated intervention costs by weighting the relevant objective costs .We summed community intervention costs across all communities to estimate aggregate costs for each CPPW intervention. To estimate the cost per person reached, we needed estimates of the number of people reached by each intervention. Because data on reach was not collected at the intervention level, we estimated intervention reach using objective reach data that communities submitted to the Centers for Disease Control and Prevention (CDC). These data were supplemented with narratives on program accomplishments written by CDC program staff, then reviewed and validated by CPPW program officers, subject matter experts, and evaluation contractors. Reviewers used US Census Bureau, school, and other local data sources to validate reach estimates. If an intervention was linked to only one objective, we assumed intervention reach was equal to objective reach. If an intervention was linked to multiple objectives, we used the maximum objective reach to approximate intervention reach. Objectives were marked as complete or incomplete by the end of the grant period. We used an estimate of “interim reach” for incomplete objectives, but we dropped interventions for which data on interim reach were not available. Of the 768 community interventions for which we collected cost data, 120 were eliminated because of missing reach data; costs assigned to the dropped interventions totaled $33 million.We calculated intervention costs per person reached as aggregate intervention costs across implementing communities divided by aggregate intervention reach. We also averaged community intervention costs per person reached, thus giving equal weight to costs regardless of community size. In all analyses, a community was considered to have worked on an intervention only if it had non-zero costs and if intervention reach could be estimated from objective reach data. We also averaged intervention costs by MAPPS category.We plotted CPPW intervention costs per person reached against intervention reach to examine whether per person costs declined as the number of people reached increased and to explore differences by community type. Community types were tribal, state-coordinated, urban, or large city, where state-coordinated communities supported work in 2 separate cities or rural areas of up to 500,000 people, urban areas had populations of 500,000 to 1 million, and large cities had populations of more than 1 million. The award amount for the 44 CPPW ARRA communities was $379 million. Total spending for these communities was $376 million; of that, $33 million was for evaluation. Total CPPW program costs, which are equal to total payments minus the amount for evaluation ($33 million) plus the value of in-kind costs ($21 million), were $363 million — $136 million for tobacco and $228 million for obesity prevention. CPPW communities spent an average of 9% of their total awards (ranging from 3% to 18%) on evaluations.CPPW partner costs were the largest share of total costs (52%) (data not shown). The next largest share was for labor (20%), followed by materials, travel, and services (12%); administration (7%); and consultant expenditures (3%). Communities that focused on obesity had a higher percentage of in-kind contributions (7%) than communities that focused on tobacco (4%). Communities that focused on tobacco had a higher share of costs for materials, travel, and services (15%) than communities that focused on obesity (9%), probably reflecting purchase of tobacco use cessation services and nicotine replacement therapy.By analyzing tobacco intervention costs per person reached, we estimated costs of $1.02 per person reached for “usage bans,” but costs varied from $0.16 to $115.37 across the 20 communities that implemented this intervention . The tobCosts per person reached tended to be lower for nutrition and physical activity interventions than for tobacco interventions . For exaThe physical activity media intervention “Media to support improved physical activity to prevent obesity” had a low average cost per person reached of $0.76 across the 28 communities that implemented the intervention, but costs varied from $0.17 to $2,784.74 . AlthougWe also examined costs per person reached for each MAPPS category . Across We report costs per person by intervention reach for selected tobacco, nutrition, and physical activity interventions. For all 3 interventions examined, we found that per person costs declined as intervention reach increased. The CPPW cost study was one of the first to use a systematic, prospective approach to estimate costs of evidence-based community interventions across multiple diverse communities. On average, half of CPPW costs (52%) went to partner organizations that worked with awardees, although partner contributions varied across communities. Because a condition of receiving a CPPW award was having a high level of readiness to implement interventions in a short time, we expected to see a high degree of partner support to supplement the more limited capacity of awardees. In-kind resources also were an important contribution to CPPW programs, as awardees were encouraged to use other resources to promote sustainability, including foundation funding, other US government funding sources, and state appropriations. On average, 6% of total CPPW costs were donations of labor or nonlabor contributions.Intervention costs per person reached generally declined as the number of people reached increased. Although this finding is consistent with economies of scale, other factors cannot be ruled out for 2 reasons. First, the concept of economies of scale refers to how output changes with inputs , and measures of CPPW output besides intervention reach are not yet available. Second, the CPPW Funding Opportunity Announcement suggested upper limits on awards by community size, and actual awards appear to be driven by de facto funding caps. For example, many of the awards for large cities clustered around $15 million, although the large city populations ranged from 1 million to nearly 10 million. Large cities could have responded to the budget caps by focusing on a limited number of interventions, but such did not appear to be the case.Estimated costs were less than $5 per person reached for 84% of tobacco interventions, 88% of nutrition interventions, and 89% of physical activity interventions. Additionally, costs per person reached were less than $1 for about one-half of interventions. Interventions with the highest costs per person reached were tobacco cessation services and subsidized memberships to recreational facilities. The costs required to implement these types of social support and services interventions are largely variable costs, which means that the cost to reach each person served is roughly constant, thus resulting in higher cost per person reached. In contrast, costs for access, point of decision or promotion, and price interventions are mostly fixed, which means they typically do not depend on the number of people reached, leading to lower per-person costs as more people are reached.Other research on the costs of community health promotion interventions is limited. Although several studies analyzed population-based tobacco interventions, those studies reported results as costs per unit of outcome achieved  –15; thusA limitation of our analysis is that costs and reach were obtained at the level of the objective and not the intervention. To compare intervention costs, we assigned objective costs and reach to interventions. If an intervention was used to support multiple objectives in a community, we assigned the largest objective reach to avoid overstating reach. However, if different people were reached by each objective linked to an intervention, our approach understates intervention reach and overstates intervention costs. Nonetheless, this approach is conservative in that our intervention cost estimates probably provide an upper bound for intervention costs.Another limitation is that CPPW reach estimates typically reflected the total number of people reached, even if those people were not smokers or obese. One exception is smoking cessation services, which were provided only to smokers. However, because CPPW interventions were largely focused on prevention for populations at high risk for obesity or tobacco use or both –20, our Our final limitation is that cost data were self-reported by community representatives. We verified quarterly total costs entered in the CSI against quarterly expenditures reported in ARRA financial reports; however, we could not verify the accuracy of allocations to objectives and strategies or in-kind contributions.The initial study goals were 1) to determine how cost data for prevention activities could be collected, 2) to understand the challenges of collecting cost information, and 3) to build an accurate tool for cost data collection. An important challenge was collecting uniform cost data that could be aggregated and standardized across communities, given changes during the study in community activities. As more community prevention programs collect and report intervention costs, it will be important to compile and disseminate cost estimates to inform program design and provide input for cost-benefit evaluations.Although the CPPW program was implemented as one of many ARRA-funded strategies to create jobs, it also supported the adoption of community-based tobacco use and obesity prevention interventions in 44 US communities. Estimated costs to implement CPPW interventions are useful for planning for intervention implementation and evaluating costs relative to CPPW-related short- and long-term health outcomes.


Vascular endothelial cells (ECs) line the inner surface of blood vessels, providing a critical barrier between the vasculature and organ systems. ECs represent an important target for infection of most human viruses, including beta- and gamma-herpesviruses. Infection of the endothelium has profound implications for both the virus and the host. For the virus, infection of ECs can provide a gateway for dissemination to organs and a reservoir for long-term persistence. For the host, virus replication and the ensuing immune response at the endothelium increases tissue permeability and inflammation, contributing to vascular and pulmonary diseases and to the severity of viral disease. Despite the clear importance of the vascular endothelium in viral infection, we know little about infection in ECs.Human cytomegaloviruses (CMV), the ubiquitous and prototypical β-herpesvirus, is remarkable in its ability to infect a wide variety of cell types resulting in a complex persistence in the host. CMV productively replicates in some cell types, such as fibroblasts, and is latent in hematopoietic progenitor and myeloid-lineage cells. The latent infection is defined by the maintenance of viral genomes, but replication is reversibly abated. Still other cell types are sites of long-term virus shedding as they support a chronic infection  where low levels of virus are produced over very long time periods. Cell intrinsic factors influence the ultimate outcome of infection: replicative, chronic, and latent. Specialized cell types provide unique environments for and barriers to infection that CMV must negotiate to ensure persistence.CMV persistence is marked by sporadic and subclinical reactivation events in healthy individuals. In the immunocompromised, reactivation of CMV from latency causes life-threatening disease characterized by pneumonitis, retinitis, gastroenteritis, and hepatitis. CMV, the “troll of transplantation”, is particularly problematic in solid organ and stem cell transplantation. In addition to the risk of CMV disease, CMV is an important risk factor in graft failure and the long-term outcome for transplant recipients. While once considered benign, important long-term costs of subclinical viral persistence are emerging. CMV sero-positivity in healthy individuals carries an increased risk of vascular disease, including atherosclerosis, frailty, and cardiovascular mortality. CMV infection of ECs promotes proinflammatory signaling contributing to angiogenesis and vascular disease –4. The cb’ region of the HCMV genome. We focused on the ULb’ region because it is retained in clinical strains but uniformly lost during serial virus passage in cultured fibroblasts, suggesting that these genes are required for replication in other cell types or for persistence in the host. We identified the UL133-UL138 (UL133/8) polycistronic locus as important for replication in ECs [Clinical isolates of CMV replicate with differing efficiencies in ECs—suggesting the existence of viral determinants important for tropism in ECs beyond virus entry. To identify these viral genes, we generated and screened a library of recombinant viruses containing disruptions in the ULn in ECs , 6. Infen in ECs . MVBs arUL133/8 locus contains four genes: UL133, UL135, UL136, and UL138. While UL133 and UL138 suppress viral replication for latency in CD34+ HPCs, UL135 and UL136 are required for replication in both CD34+ HPCs and ECs. The disruptions to intracellular membrane organization associated with the UL133/8-mutant virus infection in ECs largely segregate between UL135- and UL136-mutant viruses . Our work indicates that UL135 and UL136 function in co-opting cellular trafficking pathways for viral replication, but the mechanisms of their action and cooperation are not yet known. While the failure to reorganize or maintain organization of secretory membranes may ultimately be realized in defects in virion maturation and egress, the primary purpose of viral-mediated membrane organization and the alteration of MVBs may be to regulate trafficking, secretion or signaling.The While CMV encodes determinants specifically to ensure entry and delivery of the genome to the nucleus in ECs , UL135 a


The expansion rate of the separation system showed a significant negative partial correlation with the daily costs of total, internal, and injection medicines as well as medical devices. Moreover, the rate of replacing brand name medicines with generic medicines showed a significant negative partial correlation with the daily costs of total and internal medicines. However, external and injection medicines and medical devices did not because only a few or no generic products of these types were sold in the Japanese market. Otherwise, expansion of the separation system was effective in reducing medicine costs, except in the case of external medicines. This suggests that the cost efficiency effect of the separation system does not function all the time.This study used publicly available data to examine the effect of the separation of dispensing and prescribing medicines between pharmacists in pharmacies and doctors in medical institutions (the separation system) and the generic medicine replacement ratio on the cost of various medicines in Japanese prefectures. For Japanese medical institutions, participation in the separation system is optional. Consequently, the expansion rate of the separation system for each administrative district is highly variable. In our multiple regression analysis, the dependent variables were the costs of  There are several studies that examine the effectiveness of interventions in clinical care by pharmacists. For example, th century, while in China, the government is still hesitating to introduce the system.With regard to pharmacists’ cost-effective activities, several studies have examined the influence of the separation of dispensing and prescribing medicines on medicine costs between pharmacists in pharmacies and doctors in medical institutions. This system  has been introduced and become familiar over several hundred years in European, North American, and Middle Eastern countries. On the other hand, Asian countries, for example the Republic of Korea and Japan, only began the widespread introduction of the separation system in the latter half of 20th century because the government expected it would reduce medicine costs, especially medicine costs, by decreasing the number of duplicate prescriptions and reducing the economic motive for prescriptions. However, little evidence has been reported to support these expectations. It has been pointed out that most economic evaluations of clinical pharmacy intervention suffer from several methodological limitations relating to the absence of a control group without clinical pharmacy interventions , we calculated daily costs of internal, external, and injection medicines as well as medical devices, like injection needles for patients’ own usage as well as those for total costs. The results were expected to provide a clear understanding of the cost efficiency of the separation system for various medicines and medical devices. We performed multiple linear regression analysis as follows: the dependent variables were daily medicine costs for internal medicines, external medicines, injection medicines, and medical devices per prescription. The independent variables were the expansion rate (%) of the separation system and the base ratio (%) of brand-name medicines replaced with generic medicines.We obtained 2013 fiscal data on average medicine cost and dosage days per prescription for each prefecture . We thenNationwide data on prescriptions are available from insurance applications for medicine expenses received from pharmacies, 99.9% of which are covered currently by medical insurance in Japan. In The independent variables were the expansion rate (%) of the separation system and the base ratio (%) of brand-name medicines replaced with generic medicines. We expected these variables to have a considerable influence on daily medicine costs . In addiWe obtained 2013 fiscal data of the expansion rate (%) of the separation system among administrative divisions in Japan . The expThese data were taken from the MHLW website—specifically, they were based on trends in the composition of medical expenses for computation processing in 2013 . From these data, we calculated the rate of brand name medicines replaced with generic medicines  for each prefecture. These data are precise because they are based on data for universal health care and medical insurance, which covers 99.9% of Japanese medical care.Multiple regression analysis was conducted using the various daily medicine costs per prescription as the dependent variable and the spreading rate and generic medicine replacement ratio as independent variables. All statistical analyses were performed using Excel Statistics 2012 .The results show Tables total, internal, external, and injection medicine costs as well as medical device costs.As for partial correlations, Tables total and internal medicines. In addition, for the cost of injection medicines and medical devices, a significant partial correlation was observed only in the expansion rate and not in the generic medicine replacement ratio. For the cost of external medicines, we observed no significant partial correlation for either the expansion rate or the generic medicine replacement ratio.The results reveal a significant partial correlation for the expansion rate and the generic medicine replacement ratio for the items included in the analysis of the costs of the eternal medicines and medical devices. Therefore, the costs of total, internal, and injection medicines are considered to be influenced by the expansion rate of the separation system and the generic medicine replacement ratio.We found significant multiple correlation, except for the costs of total and internal medicines had significant partial correlations with two factors—the expansion rate and the generic medicine replacement ratio. The marketing share of the costs of internal medicines is 84% of total medicine costs in Japan. Therefore, total costs were influenced easily by the costs of internal medicines. In addition, the costs of injection medicines and medical devices had significant partial correlations with the separation system. First, this study revealed that the separation system had a strong influence on reducing costs for total, internal, and injection medicines as well as medical devices  that the Japanese government has begun to consider whether to remove injection medicines had a significant partial correlation with the separation system, which is the same result as that for internal and total medicines. The economic efficiency of the separation system is considered to work in the field of injection medicines. Injection medicines are considered to be strong medicines and require doctors’ decisions, pharmacists’ review, and explanation for use by outpatients. Therefore, the economic efficiency effect for the separation of dispensing and prescribing could function normally.The costs of injection medicine costs. The reason is considered to be the low sales in Japan of injection medicines for outpatients’ use of injections at home, such as those for insulin, growth hormones, and pain-killers for cancer. Few pharmaceutical companies sell and manufacture these medicines. However, if many pharmaceutical companies were to sell injection medicines for outpatients in the future, the generic medicine replacement ratio would have a significant partial correlation with injection medicines.The generic medicine replacement ratio has no significant partial correlation with Medical device costs had a significant partial correlation with the separation system. This is the same as the result for injection medicine costs. Most medical devices adopted in Japanese public insurance are used with injection medicines, like injection needles and catheters, or are considered to require doctors’ decisions, pharmacists’ review, and explanation to outpatients, because these medical devices need expert knowledge for usage. Therefore, the economic efficiency of the separation system is considered to function in this field as it does for injection medicines.medical device costs, it does not influence daily medical device costs.In Japanese regulation, generic medicine can be substituted for brand-name medicine by community pharmacists when doctors have not forbidden substitution and patients have agreed to substitution. Although there is no regulation of generic medical devices in Japan, when considering the need for patients’ agreement, patients in prefectures where the generic medicine replacement ratio is high are considered to have significant interest in the price of medical devices. As the generic medicine replacement ratio is considered to have no significant correlation with total, internal, and injection medicines had significant multiple correlations with the expansion rate and the generic medicine replacement ratio, the particular relationships denoting causes and effects remain obscure. While there are some possible reasons, they are only hypotheses and this study was unable to establish them. For a clear explanation, we have to analyze the actions of patients, doctors, and pharmacists. This remains for further research.Although we found that the daily costs of total, internal, and injection medicines as well as medical devices. However, external medicines had no observable effect in reducing costs. It was notable that in most cases, the economic efficiency of the separation system functioned normally, although this was not the case for external medicines. This suggests that the cost-efficiency effect of the separation system does not function all the time.The results suggest that the separation system is effective in reducing medicine costs for


Pteropus (commonly known as flying-foxes) are the natural host of the virus, and the putative source of infection in horses; infected horses are the source of human infection. Effective treatment is lacking in both horses and humans, and notwithstanding the recent availability of a vaccine for horses, exposure risk mitigation remains an important infection control strategy. This study sought to inform risk mitigation by identifying spatial and environmental risk factors for equine infection using multiple analytical approaches to investigate the relationship between plausible variables and reported Hendra virus infection in horses. Spatial autocorrelation  showed significant clustering of equine cases at a distance of 40 km, a distance consistent with the foraging ‘footprint’ of a flying-fox roost, suggesting the latter as a biologically plausible basis for the clustering. Getis-Ord Gi* analysis identified multiple equine infection hot spots along the eastern Australia coast from far north Queensland to central New South Wales, with the largest extending for nearly 300 km from southern Queensland to northern New South Wales. Geographically weighted regression (GWR) showed the density of P. alecto and P. conspicillatus to have the strongest positive correlation with equine case locations, suggesting these species are more likely a source of infection of Hendra virus for horses than P. poliocephalus or P. scapulatus. The density of horses, climate variables and vegetation variables were not found to be a significant risk factors, but the residuals from the GWR suggest that additional unidentified risk factors exist at the property level. Further investigations and comparisons between case and control properties are needed to identify these local risk factors.Hendra virus causes sporadic but typically fatal infection in horses and humans in eastern Australia. Fruit-bats of the genus  Henipavirus, family Paramyxoviridae) was first described in September 1994 in Australia, when it caused an outbreak of typically fatal disease in horses and two close-contact humans Pteropus (commonly known as flying-foxes) are the natural reservoir of the virus Hendra virus (genus Effective treatment for Hendra virus (HeV) infection in horses and humans is lacking, and while a vaccine for horses has been recently released et al (2008) showed that Hendra virus survival in-vitro decreased with increasing temperature and desiccation A limited number of studies have sought to elaborate the infection and transmission dynamics of Hendra virus in flying-foxes, and identify temporal and spatial risk factors for spill-over of infection from flying-foxes to horses. McFarlane et al (2011) identified a positive association with postcodes containing flying-fox roosts, and with geographic seasonal low rainfall, but found no evident association with horse density and vegetation primary productivity Reported equine Hendra virus cases to date have been restricted to the adjoining eastern Australia states of Queensland and New South Wales. The limited case numbers prior to 2011 has precluded robust spatial analysis to date, however the unprecedented cluster of 18 separate incidents involving 23 cases in 2011 somewhat relieves this constraint, and with our coincident improved knowledge of horse and flying-fox geographic distribution, invites further spatial analysis. In this paper, we model the spatial occurrence of reported Hendra virus infections in horses, and seek to identify key spatial and environmental risk factors.There were about 80 recorded cases of Hendra infection in horses from 40 discrete case properties in Queensland (n = 31) and New South Wales (n = 9) between September 1994 and 30 December 2012. These cases were reported to and investigated by the Queensland Department of Agriculture, Fisheries and Forestry (DAFF) and the New South Wales Department of Primary Industries (DPI) respectively. The 40 epidemiologically unrelated properties, each with one or more confirmed cases We randomly selected 1189 control properties from 118,900 registered horse properties in Queensland, New South Wales and Victoria that had not had a confirmed or suspect case of Hendra virus between September 1994 and 31 December 2012. This provided a case-control ratio of nearly 1∶30, and ensured that case properties were matched regardless of variability in property characteristics. Control property spatial data in the form of property centroid (latitude and longitude) was sourced in de-identified format from DAFF, DPI, and the Victorian Department of Primary Industries.P. alecto and P. poliocephalus was derived from Roberts et al (2011) P. conspicillatus and P. scapulatus occurrence was sourced from WildNet, an electronic database managed by the Queensland Department of Environment and Heritage Protection Flying-fox data are recorded observations of the geographic occurrence of flying-foxes drawn from two sources. Spatial data for the distribution of Spatial climate data  for eastern Australia based on the most recent standard 30-year period 1961–1990 as defined by the World Meteorological Organisation Analysis focused on eastern Australia, comprising the states of Queensland and New South Wales  and the adjoining state of Victoria. Analysis on state-based boundaries  facilitated data collection. To facilitate computation, we used a 1% subset of the 118,900 potential control properties, as indicated above. We also merged the datasets of P. alecto and P. conspicillatus because of their demonstrated paraphyly Spatial autocorrelation  was used to identify spatial clustering and distances at which spatial processing was most pronounced The 40 equine case properties reported up to December 2012 and the 1,189 randomly selected control horse properties are identified in The Global Moran’s I test identified that spatial clustering of spill-over events was evident, and was statistically significant at a distance of 40 km (P<0.001) . This diHot spot (Getis-Ord Gi*) analysis identified multiple areas of significant clustering along the east coast of Australia (Z Score >1.96 SD) . Also inP. alecto and P. conspicillatus had the strongest positive correlation (>0.5) with reported Hendra virus equine cases. The annual minimum temperature had a weak positive correlation (<0.2) and the density of P. scapulatus had a weak negative correlation (>−0.2). Negligible correlations (−0.1<0.1) were identified for P. poliocephalus, relative humidity (as measured at 0900) and horse population. The predicted (P) and residual (SD) values for a Hendra virus spill-over on horse properties are mapped in Of the 15 spatial and environmental variables included in the study , six werHendra virus was initially perceived as a Queensland problem, with the first six reported equine incidents (1994–2004) occurring in this state. That perception changed in 2006 when the first equine case was reported in the neighbouring state of New South Wales. While it is probable that under-reporting of cases occurred historically, it is less likely post-2008, with increased industry and public awareness following the fatal infection of a veterinarian. However, any reporting bias is likely to be constant geographically, and thus unlikely to significantly impact our analysis. A lack of serological surveillance data precludes inclusion of sub-clinical infections. To date, all reported cases have occurred in eastern Queensland and New South Wales, within the variably overlapping geographic ranges of all four mainland Pteropus species Hot spot analysis (Getis-Ord GI*) is a useful tool to identify spatial clusters of both high and low values, and has previously been used to model disease occurrence In addition to identifying areas of evident increased spillover risk, the hot spot analysis also suggested areas of decreased risk, or ‘cold spots’. While the findings are not statistically significant, the putative ‘cold spots’ do correspond geographically to spatial clusters of equine control properties, and support the concept of a spatial risk gradient.P. alecto and P. conspicillatus to be the strongest positive correlation with reported equine case locations in both the OLS and GWR models  suggest that some species of flying-foxes may play a greater role in the transmission of infection to horses P. alecto and P. conspicillatus density with equine case locations supports this thinking, and suggests that these species play a primary role in the infection of horses. The frequent detection of Hendra virus genome in the urine of these two species further supports this contention et al, unpublished).The geographically weighted regression analysis found the combined density of R models . While tet al (2011) P. alecto. Their reported rate of southern range expansion (100 km per decade) plus our finding of the strong correlation between P. alecto/P.conspicillatus density and equine case location, suggests the likelihood of a future southward expansion of Hendra virus equine cases, unless currently unidentified moderating factors exist. The absence of reported equine cases in NSW in the 1990s, the single case in 2006, and the cluster of cases in 2011 is consistent with this scenario.Roberts P. scapulatus and P. poliocephalus have weak negative or negligible correlations with equine cases, reflecting the absence of equine cases in that part of their range not overlapping P. alecto or P. conspicillatus. This lack of positive correlation suggests P. scapulatus and P. poliocephalus play a limited role in the infection of horses, and is consistent with the infrequent detection of Hendra virus genome in the urine of these species et al, unpublished), despite the presence of neutralising antibodies to Hendra virus. While it might be suggested that the spatial correlation between equine case locations and the combined density of P. alecto and P. conspicillatus reflects an underlying latitudinal trend rather than a biological correlation with these two species, two observations argue against this: firstly, the absence of a positive gradient between far north Queensland and southeast Queensland (indeed the reverse is true); and secondly, the evident negative correlation between Hendra virus excretion in P. scapulatus and P. alecto at corresponding latitudes .The densities of P. vampyrus, the ‘Bangladesh’ strain found in P. giganteus, and both strains detected in the geographically intermediate P. lylei in Thailand Thus, an increasingly plausible scenario is the occurrence of species-specific Hendra virus ‘strains’ in Australian flying-foxes with varying infectivity and/or pathogenicity to horses. The related Nipah virus provides a relevant precedent, with the ‘Malaysian’ strain found in et al (2011) found the same a priori potential for spill-over. But given the presence of horses, our findings show that the primary spatial risk factor for spill-over and consequent equine cases is the occurrence of P. alecto or P. conspicillatus. However, the lack of clustering of the residuals in both the OLS and GWR models  who reported that Hendra virus survivability decreases dramatically as temperature increases et al (2014) modelled the effect of temperature on Hendra virus survival in the environment, finding a good temporal fit with the cumulative annual clustering of spill-overs in the Australian winter P. alecto and P. conspicillatus. More broadly, the absence of any identifiable climatic risk factor does not preclude its existence. It might be argued that the period of the current standard 30-yr climate dataset constrained our ability to identify any emerging climatic factors, but more fundamentally there are inherent challenges in terms of which variables to include in an analysis, and on what spatial scale. With a nomadic natural host species able to move hundreds of kilometres in a week, it is entirely plausible that a climatic risk factor might manifest remote to a subsequent spill-over location. Additionally, a climatic risk factor that acts indirectly and/or after a time-lag  poses confronting analytical complexity.Our analysis of climate data sought to identify correlations consistent with the experimental findings of Fogarty P. alecto and P. conspicillatus, has a strong positive correlation with equine case locations. This finding suggests that these species play a primary role in Hendra virus infection of horses. We continue to seek other unidentified risk-contributing variables and to refine our model accordingly.The threat of Hendra virus causes particular anxiety and concern for horse owners in eastern Australia because of the very high case fatality rates in infected horses and close contact humans. Despite the recent availability of a vaccine for horses, it remains important that horse owners continue to implement risk management strategies that minimise horse exposure to Hendra virus. Our findings validate anecdotal assessments of a spatial risk component to Hendra virus infection in horses. They indicate that equine incidents are indeed spatially clustered, indicating underlying spatially occurring risk factors, and that multiple equine infection hot spots exist along the eastern Australia coast. Furthermore, we have shown that the density of two related species of flying-fox,


Leptomonas moramango. This virus belongs to a newly discovered group of bunyavirus-like elements termed Leishbunyaviruses (LBV), the first discovered from protists related to arboviruses infecting humans.We report here the sequences for all three segments of a novel RNA virus (LepmorLBV1) from the insect trypanosomatid parasite  Bunyaviridae comprise >350 species, often arthropod-borne and including serious human, animal, and plant pathogens . Within each pair, one was more abundant, as judged by read depth coverage ; we arbitrarily grouped the more abundant segments as “a” and refer to the viruses as LepmorLBV1a and 1b. The G+C compositions of LepmorLBV1s were similar to each other and to that of the meta-transcriptomic assembly (38 to 41%). We were able to detect 9-nt “panhandle” repeats (Comparisons between the LepmorLBV1a/b segment open reading frames (ORFs) showed significant nucleotide and amino acid identity . L segments displayed a single ORF encoding 1,979 or 1,982 amino acids (aa), bearing motifs found typically in bunyavirus RNA-dependent RNA polymerases (RdRp) , 7. M sePhlebovirus or other genera. Additional affinities to Bunyaviridae include a negative single-stranded RNA genome, terminal repeated panhandle sequences important for replication and transcription, and three segments typically encoding the viral RdRp, envelope glycoproteins, and the nucleocapsid .Phylogenetic analysis of the RdRp domain showed that LepmorLBV1s grouped together with bunyaviruses, relative to other viral outgroups, but as a clearly distinct clade well separated from eocapsid , 5, 7. TL. moramango may bear cytoplasmic virus-like particles (articles  that couarticles .KX280012 to KX280017.The sequences of all segments of LepmorLBV1a and 1b were deposited in GenBank under the accession numbers


Cost is frequently reported as a barrier to cataract surgery, but few studies have reported costs of accessing surgery in Africa. The purpose of this prospective, facility based study was to compare direct non-medical cost with total direct cost of cataract surgery to patients, and to assess how money was found to cover costs.Participants were those aged 17 years and above attending their first post-operative visit after first eye, subsidised, day case cataract surgery. Systematic random sampling was used to select participants who were interviewed to obtain data on socio-demographic details, and on expenditure during the assessment visit, the surgical visit, and the first follow-up visit. Costs were a) direct medical costs , and b) direct non-medical costs . The source of funds to pay for the services received was also assessed.Almost two thirds (63%) of the 104 participants were men. The mean age of men was 64 (±12.5) years, being 63 (±12.9) years for women. All men were married and 35% of women were widows. 84% of men were household heads compared with 6% of women. The median total direct cost for all visits by all participants was N8,245 (US$51), being higher for men than women  (p < 0.09) respectively. Direct non-medical cost constituted 49% of total direct cost. 92% of participants had adequate money to pay, but 8% had to sell possessions to raise the money. 20% of unmarried women sold possessions or took out a loan.Despite the subsidy, cost is still likely to be a barrier to accessing cataract surgery, as the total direct costs represented at least 50 days income for 70% of the local population. Provision of transport would reduce direct non-medical costs. Cataract is responsible for blindness in more than 20 million people  and may Patient costs include direct and indirect costs. Direct costs comprise direct medical costs (fees charged to patients) and direct non-medical costs  . IndirecHealth expenditure is “catastrophic” when it exceeds some proportion of household income or household expenditure, although there is no consensus concerning what proportion . HousehoIn Zamfara State 40% of the 3.6 million population live in the state capital, Gusau. The rural population are mainly subsistence farmers and 70% live on less than a dollar a day . The roaEvery third person attending their first post-operative visit was systematically selected. All had paid the standard fee for cataract surgery on their first operated eye.Assuming that approximately 20 postoperative patients would be eligible each clinic day, this would give 200 eligible participants over the 5 weeks of the study. A sample of this size was considered adequate, giving a reasonably representative sample and an indication of the range of costs incurred.Eligible participants had to be aged 17 years and above and 1) have undergone recent conventional cataract surgery on their first eye 2) be blind in the other un-operated eye  from any cause and 3) attending within 2-4 weeks of cataract surgery.Participants were interviewed, after obtaining written informed consent, with their escort, if present. Two ophthalmic nurses bilingual in English and Hausa conducted the interviews after two days of training. Data were collected using a structured questionnaire on age, sex, marital and socio-economic status e.g. educational level, relationship to head of household, household size, type of sanitation and wealth assessment i.e. ownership of farm/land, type of housing, and asset ownership such as radio or television. The data collection instrument was pre-tested on postoperative patients who were not included in the study.For each visit the following information was obtained: number of escorts, number of meals away from home, mode of transport and accommodation and their costs. Costs of hospital registration, surgery, investigations, medication and other charges were also assessed for each visit. Participants were asked how they found the money e.g. already available, or if they had to borrow money or mortgage or sell assets.Data were entered into a Microsoft Access database and analysed in STATA 13.0. Parametric analysis was performed for normally distributed data, and non-parametric tests for skewed data (e.g. costs). Significant levels are reported at p < 0.05. Total direct patient costs were calculated and the proportion due to direct non-medical costs determined. Analysis was conducted using Nigerian Naira (N) at the exchange rate of US$1 to N161.Ethical approval was obtained from the institutional review board/ethics committee of the London School of Hygiene & Tropical Medicine and the Ministry of Health, Zamfara state. The study adhered to the tenets of the Helsinki Declaration.104 participants were interviewed, 66 (63%) of whom were female  for all visits, including costs for escorts  for escorts and N6,320 (US$39) for participants (p < 0.01) Tables  and 5. CA limitation of the study was that the desired sample size was not reached. This was because the study was undertaken during the rainy season when transport is difficult meaning that fewer cataract surgeries were performed. A larger sample may have been adequately powered to detect statistically significant differences in costs between men and women. Another limitation was that productivity costs (e.g. loss of wages) were not assessed. In subsistence farming communities, as in Zamfara State, time spent away from agricultural activities may also have economic consequences which would add to the financial barriers.In this study more women than men had recently accessed cataract surgery despite several studies indicating that men usually outnumber women. This is reflected in the higher cataract surgical coverage in males reported in studies in other States in Nigeria ,7 and inThe gender difference in costs were as anticipated, reflecting gender differences in social roles, as most women in northern Nigeria have little income to spare for healthcare and have little or no influence on household spending , as deciThe only other study of patient costs for cataract surgery was undertaken in a tertiary level, faith based eye hospital in urban Zambia , but thiDirect non-medical costs accounted for 49% of the total direct costs, most being for transport, reflecting the long distances patients and their escorts travel, usually in private taxis or buses. There were no accommodation cost as patients and their escort wait on hospital premises overnight to reduce costs. In many instances participants did not need an escort after surgery on account of good visual outcomes.The median total direct patient cost of cataract surgery (US$51) represents at least 50 days income for 70% of the population in Zamfara State. A study in Nigeria also found that most glaucoma patients cannot afford treatment as more than half of their monthly income would be spent on care and medication . Other sOne of the outcomes of a good health system is that it protects communities from financial risk . ProtectThis study is the first of its kind to analyse costs of cataract surgery to patients and their families in rural Africa and detailed information was collected for a number of hospital visits over a short period of recall. The findings that direct medical costs were consistent shows accuracy of recall, which is likely to extend to recall of direct non-medical costs.These findings clearly indicate the need for innovative approaches by policy makers and programme planners to make eye care services more accessible. In Zamfara, one way to reduce non-medical costs would be to provide transport. Many eye care programmes in low and middle income settings do not meet the World Health Organisation recommended CSR target of 2000, being only 500 in Zamfara State despite the subsidy and high quality services. There is an urgent need for research into interventions which reduce the cost of cataract surgery to patients and their families in rural Africa, particularly for the most vulnerable such as unmarried women. Interventions could include higher subsidies or a voucher system for the most vulnerable, coupled with free transport.