The co-infection of diverse viruses in a host plant is common; however, little is known about viral populations and their quasispecies in the host.Here, we report the first pepper viromes that were co-infected by different types of viral genomes. The pepper viromes are dominated by geminivirus DNA-A followed by a novel carlavirus referred to as Pepper virus A. The two pepper cultivars share similar viral populations and replications. However, the quasispecies for double-stranded RNA virus and two satellite DNAs were heterogeneous and homogenous in susceptible and resistant cultivars, respectively, indicating the quasispecies of an individual virus depends on the host.Taken together, we provide the first evidence that the host plant resistant to viruses has an unrevealed antiviral system, affecting viral quasispecies, not replication.The online version of this article (doi:10.1186/s12864-017-3838-8) contains supplementary material, which is available to authorized users. Capsicum species, which are members of the Solanaceae family, have been cultivated worldwide and consumed as fresh fruits or powdered spices [Potyvirus, Cucumovirus, Polerovirus, and Crinivirus have been reported [Begomovirus, in the family Geminiviridae, are frequently reported. Begomoviruses transmitted by Bemisia tabaci are very destructive and emerging viruses in the production of pepper [Pepper plants in the d spices . A wide d spices . Out of d spices . Among treported . Of the f pepper .Pepper vein yellows virus and Bell pepper endornavirus (BPEV), and a novel virus, Pepper yellow leaf curl virus, have been identified by Illumina NGS techniques [Next-generation sequencing (NGS) enables us to identify known as well as novel viruses in various plant species . In the chniques –8.Tomato yellow leaf curl virus (TYLCV), also exhibit high mutation rates, suggesting quasispecies of single-stranded (ss) DNA viruses in the host [A wide range of viruses display a significant level of genetic diversity within the host by replicating with high mutation rates . Viral pthe host .Several previous studies have reported that many plants are often co-infected by different plant viruses and viroids –16. PlanRecently, we screened available pepper transcriptomes to identify BPEV-associated sequences . Out of Croton yellow vein mosaic virus alphasatellite (CYVMVA), Aphid lethal paralysis virus (ALPV), Pepper leaf curl Bangladesh virus (PepLCBV), Pea streak virus (PeSV), Chilli leaf curl virus (ChiLCV), Pepper leaf curl virus betasatellite (PepLCVB), Tobacco vein clearing virus (TVCV), and Bell pepper endornavirus (BPEV) – were commonly identified in four datasets. Several viruses were specifically identified in each dataset. We combined all virus-associated contigs from four different datasets . Of five ssRNA viruses, ALPV is an aphid-infecting virus (ALPV) in the family Dicistroviridae, and the other four viruses – Aconitum latent virus (AcLV), PeSV, Potato virus P (PVP), and Gaillardia latent virus  – are members of the genus Carlavirus.The two different transcriptomes were derived from Pusa Jwala (PJ) and Taiwan2 (TW) cultivars, which were susceptible and resistant to various viruses, respectively , 21. Detets Fig. . BPEV-asets Fig. . They caTo assemble viral genomes using transcriptome data, we mapped the identified virus-associated contigs on the respective reference virus genome. Of the identified viruses, BPEV-associated contigs were enriched. The sizes of BPEV-associated contigs ranged from 2153 nt to 203 nt Fig. . In geneWe identified several contigs associated with DNA virus components. A total of 13 and 16 contigs from PJ and TW, respectively, were closely associated with ChiLCV DNA-A Fig. . The lenCarlavirus in the family Betaflexiviridae. Phylogenetic analyses revealed that PepVA is closely related to Cowpea mild mottle virus, followed by Cucumber vein-clearing virus and Hippeastrum latent virus  for each virus using assembled consensus genome sequences in an individual cultivar  primers to amplify the mRNAs of pepper. Thus, the identification of ssRNA with poly(A) tail, such as PepVA, was not surprising. However, the identification of RNA viruses without poly(A) tail, such as BPEV, as well as the identification of DNA viruses, such as TVCV, and several geminiviral DNA-A, alphasatellite, and betasatellite sequences from the plant transcriptomes is the first reported to our knowledge. Recently, several studies have shown that RNA viruses without poly(A) tail can be recovered by RNA-Seq data , 19. PreCarlavirus. Due to the poly(A) tail of PepVA, it was possible to assemble two complete genomes of PepVA from PJ and TW using only transcriptome data.We identified ten contigs with no close relatives to known viruses. However, conserved domain prediction and phylogenetic studies have identified a novel virus tentatively named PepVA belonging to the genus Recent studies have demonstrated that plant transcriptome data can be usefully applied in the assembly of complete or nearly complete genomes for several viruses and viroids , 14, 25.Bemisia tabaci [The same list of diverse viruses has been infected in two different pepper transcriptomes, indicating that the two cultivars were grown in the same field. The presence of ALPV suggests the sampled pepper plants were severely damaged by aphids, which are vectors for many plant viruses . In addia tabaci . The co-a tabaci , 35. In a tabaci . Based oIn general, the co-infection of various viruses in the host might be harmful for the host. For example, a previous study showed that the mixed infection of two geminiviruses in the pepper plants exhibited a synergistic interaction with increased disease symptoms . AlthougPepper leaf curl virus (PepLCV) infection in field conditions [The two different pepper cultivars, PJ and TW, have been demonstrated to be susceptible and resistant to diverse viruses, including nditions , 21. UneIn this study, we demonstrated that two different pepper cultivars were co-infected by four different viral genomes – ssRNA, dsRNA, ssDNA, and dsDNA. Furthermore, we quantified the amount of viral RNAs for an individual virus and examined the copy numbers for the respective virus. Although two transcriptomes were sequenced by different methods, there were no big differences between two cultivars for viral populations. In addition, we found that the amounts of two satellite DNAs, dsDNA, and dsRNA replications were low, indicating that their replication was very low in the pepper host.Taken together, we provide a wide range of new results associated with virus identification, viral genome assembly, viral populations, and virus quasispecies in two different plant cultivars using only pepper transcriptome data. Based on our results, it might be of interest to find a correlationship between the co-infection of different viruses and disease symptoms in the pepper host.Capsicum annuum) cultivars named Pusa Jwala (PJ) and Taiwan-2 (TW) and grown in India were used in this study. PJ and TW were identified as susceptible and resistant, respectively, to diverse viruses, including capsicum chlorosis, cucumber mosaic, and groundnut bud necrosis, which were screened by the ELISA test. Four different tissues composed of the root, stem, leaf, and fruit were pooled and used for total RNA extraction. The two prepared libraries were sequenced by the HiSeq2000 system. The PJ library was single-end sequenced, while the TW library was paired-end sequenced.Two different pepper (http://www.ncbi.nlm.nih.gov/sra) in NCBI with their respective accession numbers. Initially, the raw data were de novo assembled by the Velvet (ver. 1.2.10) and Oases (ver. 0.2.08) programs [Two raw datasets were downloaded from the SRA (Sequence Read Archive) database (programs  and the programs .http://www.ncbi.nlm.nih.gov/genome/viruses/). We used MEGABLAST, which is optimized for highly similar sequences with the e-value 1E-5 as a cutoff. The identified viruses were manually filtered to remove endogenous virus-like sequences and bacteriophages. Finally, virus-associated contigs from each dataset were used for further analyses.Four different transcriptome datasets assembled by two different de novo assemblers were subjected to a BLAST search against the virus reference database  protein database and homologous viral protein sequences were subjected to sequence alignment. The amino acid sequences for an individual viral protein were aligned by ClustalW followed by manual editing. Phylogenetic trees were constructed by 1000 bootstrap replicates using the Neighbor-Joining method with the Poisson correction of distance using the MEGA6 program . For BPETo detect single nucleotide variations (SNVs) for the identified viral genome in each cultivar, we mapped raw sequence data on the individual consensus viral genome sequence using BWA with default parameters . The aliTo analyze the amount of viral RNAs in each transcriptome, we performed MEGABLAST against the identified consensus viral sequences. To calculate the viral copy number for each virus, the obtained number of viral reads was multiplied by 101 and then divided by the total length of the individual genome.Additional file 1: Table S1.Table S2. Summary of MEGABLAST results to identify virus-associated contigs from PJ transcriptome. Table S3. Summary of MEGABLAST results to identify virus-associated contigs from TW transcriptome. Table S4. Summary of identified viruses in PJ and TW transcriptomes with numbers of associated viral contigs. Table S5. Single nucleotide variations of BPEV in the PJ transcriptome. Table S6. Single nucleotide variations of BPEV in the TW transcriptome. Table S7. Single nucleotide variations of PepVA in the PJ transcriptome. Table S8. Single nucleotide variations of PepVA in the TW transcriptome. Table S9. Single nucleotide variations of ChiLCV DNA-A in the PJ transcriptome. Table S10. Single nucleotide variations of ChiLCV DNA-A in the TW transcriptome. Table S11. Single nucleotide variations of PepLCV betasatellite in the PJ transcriptome. Table S12. Single nucleotide variations of PepLCV betasatellite in the TW transcriptome. Table S13. Single nucleotide variations of CYVMV alphasatellite in the PJ transcriptome. Table S14. Single nucleotide variations of CYVMV alphasatellite in the TW transcriptome. Table S15. Number of viral sequence reads and copy number for identified viruses. (XLSX 131 kb)Summary of de novo-assembled transcriptomes for two libraries using Trinity program. Additional file 2: Figure S1.Figure S2 Sequence alignment for PepLCVB. C1 nucleotide and protein sequences were obtained from PJ and TW transcriptomes and used for sequence alignment with C1 reference sequence. Alignment was conducted and visualized by ClustalW. Figure S3 Sequence alignment for CYVMVA. Replication gene and protein sequences for CYVMVA were obtained from PJ and TW transcriptomes and used for sequence alignment with the alphasatellite replication reference sequence. Alignment was conducted and visualized by ClustalW. Figure S4 Phylogenetic relationships of ORF2, ORF3, and ORF5 with respective other homologous viral proteins for PepVA. (PDF 1867 kb)Alignment of contigs associated with TVCV. Green and orange colored lines and bars indicate contigs assembled by Velvet and Trinity, respectively. The genome organizations of TVCV were manually drawn based on the reference viral genome annotation. Putative ORFs were indicated. Abbreviations: movement protein (MP) and transactivator factor (TF).


An outbreak of a disease in camels with skin lesions was reported in Israel during 2016. To identify the etiological agent of this illness, we employed a multidisciplinary diagnostic approach. Transmission electron microscopy (TEM) analysis of lesion material revealed the presence of an orthopox-like virus, based on its characteristic brick shape. The virus from the skin lesions successfully infected chorioallantoic membranes and induced cytopathic effect in Vero cells, which were subsequently positively stained by an orthopox-specific antibody. The definite identification of the virus was accomplished by two independent qPCR, one of which was developed in this study, followed by sequencing of several regions of the viral genome. The qPCR and sequencing results confirmed the presence of camelpox virus (CMLV), and indicated that it is different from the previously annotated CMLV sequence available from GenBank. This is the first reported case of CMLV in Israel, and the first description of the isolated CMLV subtype. Orthopoxvirus (OPV), and Camel contagious ecthyma virus (CCEV), the causative agent of Auzdik disease, which belongs to the genus Parapoxvirus (PPV). The third virus is camelus dromedary papillomavirus, the causative agent of camel papillomatosis  × 40, melt curve: 70.0 °C to 84.4 °C, increment of 0.4 °C for 1 min. A second PCR, termed 137637, was designed during the course of this study, and directed to amplify a different region in the CMLV genome. Two primers were designed to target a 192 bp region located downstream to the gene encoding RNA polymerase subunit RPO132 (acc. No. NC_003391). This region is approximately 61Kb upstream to the C18L region. The primers sequence was as follows: CMLV_137637_FWD 5′-AGTTGATCTCAAGCCGTC-3′, and CMLV_137829_REV 5′-GAGGTGTAACAGCCACTTG-3′. The reaction mix and conditions were as described for the C18L reaction. The sensitivity of the test was assayed using decimal dilutions of a purified control PCR product that contained the reaction target sequence. The primers that were used to generate the control PCR product were as follows: CMLV_137401F 5′-GTTTTAAAAGACTCAGAAGAGG-3′, and CMLV_138164R 5′-AACGTGAATTGGAATCTGAACG-3′. The conditions for the control product amplification were as described for the standard PCR run above.DNA was extracted from blood and skin samples using the iNTRON viral extraction kit , 2–4 μL of the DNA extraction, 0.4 μM of the final concentration of each primer, and ddH2O to a final reaction volume of 20 μL. The following conditions were used in a ProFlex thermocycler : 98 °C 1′,  x 35, 72 °C 5′. PCR products were resolved on agarose 1.2% gel, and visualized using Olerup SSP GelReddye (http://www.olerup.com/). For the identification of CPXV and VACV DNA, the extension time was extended to 1′25″. The PCR products were purified either following gel separation or directly from the reaction tube, and were sequenced.The mix for each reaction was assembled as follows: 10 μL of Takara EmeraldAmp mix  of 10-day old embryonic chicken eggs were infected with skin extracts, as described above. Egg death occurred four days post-injection, at which time white pocks were observed on the membrane A. The po5/mL.The resuspended pock material was used to infect Vero cells. Cytopathic effects (CPE) were observed four days post-infection C. Virus Members of the OPV family share common antigens ; thus, tMoreover, viral factories stained by DAPI and anti-OPV antibodies, which are characteristic of OPV-infected cells, can be seen in the infected cells A-3. ThesSyncytia (cell–cell fusion) formation is characteristic of several pathogenic OPV members, including CMLV, ectromelia virus (ECTV), and monkeypox (MPXV) . In ordeIn order to enable a definitive identification of the etiological agent of the disease, PCR was employed, with both rapid SYBR-based qPCR, and the amplification of several genomically distant regions for sequencing. For preliminary detection and identification, two separate qPCR were used, identifying different regions in the CMLV genome. The C18L test  and the In order to determine the sensitivity of the new qPCR, we performed a calibration test to compare its sensitivity with that of the C18L test . The twot values ranging from 9.87 to 31 . This reaction specifically identified CMLV and distinguished it from other OPV, namely CPXV and VACV. This was accomplished by amplifying a region whose length is 420 bp in the CMLV genome, 1140 bp in the CPXV genome (accession LN864565), and 1048 bp in the VACV (accession AY313848). By keeping the extension time of the reaction below 25 s, only the CMLV presence could be detected A.However, by extending this stage to one minute and 25 s, two longer products were generated, demonstrating that under permissive conditions, this test could differentially detect CMLV and other related viruses B. This rThe sequenced samples shared a high degree of identity, and were different from the annotated CMLV isolates available from GenBank . CollectThis study reports the first documented outbreak of camelpox disease in Israel, and demonstrates the diagnostic procedures used for its diagnosis, which include morphological, cellular, immunological, and molecular methods. The employment of several approaches based on different features of the causative agent significantly improved the reliability of the diagnosis . Since tThe disease occurs mainly in adult females camels, and is manifested by skin lesions on hairless skin regions such as the face and neck, whereas in several cases, lesions appear throughout the body. Symptoms such as fever, weakness, lack of appetite, and abortion were observed with no mortality. Since camels serve as transport and milk-producing animals, such a disease that is infectious and easily transmitted will have profound economic implications on societies that rely on camels.Eight skin and blood samples from sick camels were examined in this study. A disease involving exanthematous skin conditions in camels raises several possibilities regarding the causative agent: camelus dromedary papillomavirus, CCEV, CMLV, or co-infection with CCEV and CMLV. This necessitated the use of a differential diagnosis using several methods, the first of which was diagnostic TEM . It shouFurther examination and characterization was carried by the infection of CAM and Vero cells. While the infection of SPF-ECE resulted in typical pocks formation, the infected Vero cells were positive by immunofluorescence using an OPV-specific antibody. In order to determine the virus identity at the species level, PCR followed by sequence analysis was used. Of the various OPV, the primary candidate was CMLV. To establish this, we employed the C18L test , and dueThe recent CMLV-annotated genomes suggested the presence of a different CMLV. The three endpoint PCR tests aimed to establish the subspecies identity of the isolated virus, and showed that all of the tested samples were of the same subspecies, which was distinct from the other four annotated subtypes currently available from GenBank. The regions selected for the analysis contained variations and insertions that enabled a clear distinction between CMLV and other OPV species, such as monkeypox, cowpox, and rabbitpox viruses .Recent studies published after the completion of this manuscript compared a large number of samples using the HA and B2L genes  and the orthopoxvirus human infections, raising the need for adequate diagnostic approaches, as outlined in this study.This study describes the first diagnosed outbreak of camelpox virus in Israel. Although camelpox virus infects camels, it is genetically the closest relative of the variola virus, the causative agent of smallpox. Although rare, human infections of camelpox have been previously described. The findings in this study adds to the recent emergence of


Public health decision makers not only consider health benefits but also economic implications when articulating and issuing lifestyle recommendations. Whereas various estimates exist for the economic burden of physical inactivity, excess body weight and smoking, estimates of the economic burden associated with our diet are rare. In the present study, we estimated the economic burden attributable to the inadequate consumption of vegetables and fruit in Canada.We accessed the Canadian Community Health Survey to assess the inadequacy in the consumption of vegetables and fruit and published meta-analyses to assemble risk estimates for chronic diseases. Based on these inadequacy and risk estimates, we calculated the population-attributable fraction and avoidable direct and indirect costs to society. Direct costs include those for hospital care, physician services and drugs in 2015.About 80 % of women and 89 % of men consume inadequate amounts of vegetables and fruit. We estimated this to result in an economic burden of $CAN 3·3 billion per year, of which 30·5 % is direct health-care costs and 69·5 % is indirect costs due to productivity losses. A modest 1 percentage point annual reduction in the prevalence of inadequate vegetables and fruit consumption over the next 20 years would avoid approximately $CAN 10·8 billion, and an increase of one serving of vegetables and fruit per day would avoid approximately $CAN 9·2 billion.Further investments in the promotion of vegetables and fruit will prevent chronic disease and substantially reduce direct and indirect health-care costs. In Canada, chronic diseases account for 89 % of all deaths and for more than $CAN 93 billion in direct and indirect health-care costs per year. Dietary intake, sedentary lifestyle, body weight and cigarette smoking have long been established as modifiable risk factors for chronic disease. Despite the issuing of public health recommendations to reduce the burden of chronic diseases, recent studies revealed that 74 % of Canadians consume less than the recommended daily number of servings of vegetables and fruit, 85 % of adults are not meeting recommendations for physical activity, 62 % have excess body weight and 18·1 % use tobacco.Chronic diseases are the leading cause of morbidity and mortality worldwide1. In (–). For Canada, Krueger et al. estimated the economic burden attributable to physical inactivity, excess body weight and tobacco smoking to be $CAN 10·8 billion, $CAN 23·3 billion and $CAN 18·7 billion per year, respectively. Estimates of the economic burden associated with dietary intake, however, are uncommon. For Australia, the economic burden attributable to low vegetables and fruit consumption was estimated to be $AU 269 million in 2008, and for the UK the costs for treatment and management of chronic diseases attributable to a poor diet to be £4·9 billion in 2006/2007. No such estimates exist for the Canadian setting, whereas country-specific estimates are essential to reflect differences in lifestyle, culture, food choices, dietary recommendations and health-care costs. Since the majority of Canadians are not meeting the recommendations for vegetables and fruit, the associated economic burden is expectedly substantial. An estimate of this burden may help prioritizing public health intervention strategies. The objective of the present study was to estimate the economic burden attributable to the inadequate consumption of vegetables and fruit in Canada.Public health decision makers not only consider potential health benefits but also financial implications when articulating and issuing their recommendations. Various studies have estimated the economic burden in terms of costs for treatment and management of chronic diseases and for productively losses resulting from physical inactivity, excess body weight and smoking–16. . In doing so, we considered the consumption of vegetables and fruit by Canadians, the risk that inadequate consumption poses for chronic diseases and the costs for the treatment and management of these diseases, as detailed below.We estimated the economic burden associated with inadequate consumption of vegetables and fruit with a population-attributable fraction (PAF) approach and a societal perspective18. In. The CCHS collects data from participants 12 years of age or older living in the ten provinces and the three territories of Canada and incorporates population weighting to provide estimates that are representative for the Canadian population. Since 2007, data for the CCHS are collected annually instead of every two years with a target sample size of 65 000 respondents each year. We accessed the 2014 responses of 63 964 Canadians to validated questions on the consumption frequency of vegetables and fruit. These responses had been summarized by means of a single variable representing the total number of servings of vegetables and fruit consumed daily. Since risk estimates are available for increases per 80 g (1 serving) per day in vegetables and fruit intake (see next section), we retrieved age- and gender-specific distributions of vegetables and fruit consumption in the intervals of 0–<1, 1–<2, 2–<3, 3–<4, 4–<5, 5–<6, 6–<7, 7–<8 and 8+ servings/d. Since the CCHS covers only participants aged 12 years or older, we applied the distribution of vegetables and fruit consumption observed in the 12–14 years age group to those under 12 years old. In Canada, males are recommended a minimum of 4–6, 8, 8–10 and 7 servings vegetables and fruit/d for the age groups 2–<14, 14–<19, 19–<51 and 51+ years, respectively. For females, this is a minimum of 4–6, 7, 7–8 and 7 servings vegetables and fruit/d for the age groups 2–<14, 14–<19, 19–<51 and 51+ years, respectively. To align these age-specific recommendations with the available age categories for health-care costs (see below), we adapted this for males to a minimum of 5, 8 and 7 servings vegetables and fruit/d for age groups <15, 15–<55 and 55+ years, respectively. For females, we considered a minimum of 5 and 7 servings vegetables and fruit/d for age groups <15 and 15+ years, respectively.The Canadian Community Health Survey (CCHS) is a large cross-sectional survey that collects information related to health status, health care and health determinants for the Canadian population19. Th. Based on these sources, we considered risk estimates for type 2 diabetes, CVD  and cancers . Because we were interested in the effect of the combination of vegetables and fruit, in cases where a meta-analysis reported separate effects of vegetables only and of fruit only, we considered the lower risk estimate.We accessed reports by the WHO and the World Cancer Research Fund/American Institute for Cancer Research, complemented with published meta-analyses, to assemble established estimates of the risk of inadequate vegetables and fruit consumption for chronic diseases,–24The PAF represents the proportional reduction in chronic disease that would occur if all Canadians would consume the recommended number of servings of vegetables and fruit. The PAF applies the risk of inadequate vegetables and fruit consumption for chronic disease to the distribution of vegetables and fruit consumption in the Canadian population in the formula26wPi is the proportion of individuals in interval i;i  refers to the consumption of 0–<1, 1–<2, 2–<3, 3–<4, 4–<5, 5–<6, 6–<7, 7–<8 and 8+ servings/d;RR is the relative risk for each serving increase in vegetables and fruit consumption;i relative to the recommended number of servings;Xi is the mid value of interval i;L is the recommended number of servings; andn is the number of intervals below the recommended number of servings.We calculated the PAF using the distribution of vegetables and fruit consumption (described above under ‘Consumption of vegetables and fruit in Canada’) and the relative risk for disease (described above under ‘Risk of inadequate vegetables and fruit consumption for chronic disease’) for each of type 2 diabetes, IHD, ischaemic stroke, and lung, colorectal, oesophagus, stomach/gastric, bladder, oral, larynx and breast cancer.. For costs for type 2 diabetes, we further applied the proportion of 0·96 to the costs of all diabetes (type 1 and type 2 combined). We multiplied these proportions by the total costs  in 2015 for hospital care, for physician services and for drugs, available through the Canadian National Health Expenditure Trends report, to compute the direct health-care costs. These costs were then multiplied by the PAR to estimate the avoidable direct health-care costs. We estimated indirect costs following the human capital approach applied in the Economic Burden of Illness in Canada (EBIC 1998): we calculated the ratio of indirect costs to direct costs for each disease of interest, assumed these ratios to be stable over time, and used the product of the ratio and the estimated avoidable direct health-care costs for each of the diseases of interest to represent the avoidable indirect health-care costs. The economic burden represents the sum of avoidable direct health-care costs and avoidable indirect costs for all diseases of interest.We obtained the proportions of costs for hospital care, physician services and drugs for each of the diseases of interest from the Economic Burden of Illness in Canada (EBIC 2008) online tool27. FoWe carried out 50 000 Monte Carlo simulations to obtain 95 % confidence intervals for the economic burden estimates. In these simulations, we assumed the reported relative risks to follow lognormal distributions and estimated variance parameters from the reported 95 % confidence intervals.et al. had previously applied a similar scenario for decreases in physical inactivity, body weight and tobacco use, assuming the projected Canadian population from 2015 to 2035.The economic burden is an estimate of the potential cost savings if all 35·8 million Canadians were to eat adequately in terms of vegetables and fruit. As an alternative scenario, we calculated the potential cost savings for a scenario whereby all Canadians with an inadequate consumption of vegetables and fruit would increase this consumption by one extra serving daily. As a third scenario we estimated the potential cost saving of a modest 1 percentage point annual decrease in the proportion of individuals with inadequate consumption of vegetables and fruit. For this scenario, we assumed that all Canadians with inadequate vegetables and fruit consumption had an equal probability to become adequate in their consumption of vegetables and fruit. Krueger et al.17 hadThe above estimates of the economic burden represent the sum of direct and indirect costs that would be avoided if all Canadians would have diets adequate in vegetables and fruit. In a scenario where all Canadians with inadequate diets would increase their vegetables and fruit consumption by 1 serving/d, the avoided cost would total $CAN 362 million in the first year and would reach $CAN 551 million in 2035 for a total of $CAN 9·2 billion in cumulative avoided costs over 20 years . In the , of which an estimated $CAN 19·8 billion was for the treatment and management of diabetes, CVD and cancer. About 5 % of these costs for diabetes, CVD and cancer could have been avoided if Canadians had complied with existing recommendations for vegetables and fruit. With a Canadian population of approximately 35·8 million, the avoidable costs for health care and productivity loss approximate $CAN 92 per capita per year.The present study estimated that the cost to Canada attributable to the inadequate consumption of vegetables and fruit totals $CAN 3·3 billion per year, with 30·5 % in direct health-care costs and 69·5 % in indirect costs due to productivity losses. Canada spent an estimated $CAN 219·1 billion on direct health-care costs in 201529, ofet al. estimated the economic burden attributable to a low consumption of vegetables and fruit to be $AU 269 million in 2008, which approximates to per capita avoidable costs of $CAN 11 per Australian per year. Differences with our per capita estimate of $CAN 92 per year may originate from differences in lifestyle, culture, food choices, dietary recommendations and health-care costs between the two nations, as well as from methodological differences between the studies. With respect to the later, for example, Cadilhac et al. estimated avoided costs associated with a 172 g/d increase in the consumption of vegetables and fruit, whereas we estimated costs associated with inadequate consumption of vegetables and fruit using national dietary recommendations as a reference. Scarborough et al. estimated the costs associated with the treatment and management of diabetes, CVD and cancer attributable to poor diet to be £4·9 billion in 2006/2007, which approximates to per capita avoidable costs of $CAN 133 per UK citizen per year. It is important to note that Scarborough et al. took a broader perspective by considering everyone consuming 600 g vegetables and fruit/d, having total cholesterol levels of 3·8 mmol/l and a BMI of 21 kg/m2 as the counterfactual of poor diet, thus capturing more costs than those attributable to low vegetables and fruit consumption only. Because Scarborough et al. estimated the costs attributable to poor diet, and Cadilhac et al. costs associated with low vegetables and fruit consumption, the present study represents the first study to estimate costs associated with the inadequate consumption of vegetables and fruit using national dietary recommendations as the criterion for adequate consumption.Cadilhac n in 200815, whtrans and saturated fats are listed as the risk factors with convincing evidence. Calculations that consider each of these dietary risk reducers and risk factors will produce higher estimates for the economic burden of poor diets relative to the estimates that consider solely the inadequate consumption of vegetables and fruit, the approach of the present study. Where some nations have distinct recommendations for vegetables and for fruits, Canadian recommendations specify the combined number of servings of vegetables and fruit. This poses challenges for the estimation of the economic burden. For example, in their meta-analysis, Li et al. revealed a statistically significant protective effect of fruit and of green leafy vegetables on the development of type 2 diabetes, but no statistically significant effect of the combined number of servings of vegetables and fruit on type 2 diabetes. A calculation that considers fruit and green leafy vegetables, rather than the grouping of vegetables and fruit, will provide more accurate estimates of the economic burden. However, the reality is that Canadian public health decision makers settled on a recommendation for the combination of vegetables and fruit, and thus it is desirable to provide an economic burden associated with this recommendation. Excluding type 2 diabetes, as well as oesophageal cancer and stomach/gastric cancer for which the evidence is also mixed, revealed a substantially lower estimate, although more precise, for the economic burden ($CAN 2·3 billion per year). Some may, however, consider the latter estimate too low as it does not capture the risk-reducing effects of fruit and green leafy vegetables.The effects of diet on chronic diseases are multifactorial and include both risk reducers and risk factors. In addition to vegetables and fruit, the WHO lists fish and fish oils, potassium and moderate alcohol intake as risk reducers for which research has provided convincing evidence, and dietary fibre, wholegrain cereals, nuts, folate and unsaturated fatty acids as risk reducers with less convincing evidence. Diets high in sodium, high in alcohol and containing  evidence33. Caet al. estimated the economic burden of smoking tobacco, excess body weight and physical inactivity to be $CAN 23·3 billion, $CAN 19 billion and $CAN 11 billion, respectively. In their approach, Krueger et al. were more inclusive in their consideration of health-care costs. Specifically, where we considered costs for hospital, physician services and drugs, Krueger et al. also included costs for health research and for other health-care professionals  and expenditures. In 2015, the costs for items we considered constituted about 86 % of the costs for the items considered by Krueger et al.. If we had been as inclusive as Krueger et al., our estimate for the economic burden of inadequate consumption of vegetables and fruit would have been $CAN 3·8 billion per year. The estimate of the economic burden of inadequate vegetables and fruit consumption should be considered an economic burden in addition to the estimates for smoking tobacco, excess body weight and physical activity by Krueger et al., because the relative risks used in our estimation originated from meta-analyses of mostly clinical trials and observational studies that were adjusted for tobacco use, excess body weight, and physical activity among other confounders. By expanding the estimates by Krueger et al. with an estimate for the economic burden of inadequate intake of vegetables and fruit, we provide policy makers with further evidence-based options for interventions aimed at preventing chronic diseases and reducing associated health-care expenditures.Krueger pectively17. In, this would translate into approximately $CAN 10 billion per year in costs to consumers. Our estimate of $CAN 3·3 billion in avoidable costs to society provides an appealing incentive for further investments in the promotion of vegetables and fruit. These investments should not only target education and behavioural change of consumers, but also strategize vegetables and fruit production. Investments and changes in the consumption of vegetables and fruit are not expected to come quick. Our scenario analyses showed that a gradual target of a modest 1 percentage point annual reduction in the proportion of individuals consuming less than the recommended servings of vegetables and fruit daily would avoid approximately $CAN 10·8 billion in 20 years and an increase of one serving of vegetables and fruit daily would avoid approximately $CAN 9·2 billion in 20 years.We estimated that Canadians collectively consume 171 million servings of vegetables and fruit daily and that an additional 92 million servings are needed for consumption to increase to adequate levels for all Canadians. As many vegetables and fruit in Canada can be purchased for a price of approximately $CAN 0·3035, th. Other limitations of the present study relate to the PAF approach that did not consider a latency period, which may lead to an overestimation of the cost savings of the two intervention scenarios. As a further limitation we note that although we used direct cost estimates provided in the Canadian National Health Expenditure report, the allocation of these costs to specific disease categories was based on the assumption that the distribution of costs by disease category and the ratio of direct to indirect costs do not change over time. However, it is likely that disease-specific costs may change due to changes in treatments over time. Third, we adapted some of the age-specific recommendations for vegetables and fruit to align them with the available age categories for health-care costs. For some age/gender groups we used the lower bound of the recommended range. Had we used a mid-range value or otherwise a higher value for the recommendation, our estimates of the economic burden would have been higher. Lastly, since the CCHS included only participants aged 12 years or older, we assumed that the distribution of vegetables and fruit consumption observed in the 12–14 years age group applied to those under 12 years of age. The overall error this may have introduced is assumed to be very small as the contribution of the ‘under 15 years age group’ is less than 0·2 % of the estimated burden (Our estimates of the economic burden of inadequate vegetables and fruit consumption are based on those chronic diseases for which there are published meta-analysis showing a significant relationship with vegetables and fruit. Our estimates did not consider chronic diseases for which the relationship with vegetables and fruit has not been studied or for which there is insufficient evidence. The evidence on importance of vegetables and fruit in the causation of cancers is evolving, with recent publications suggesting a more modest risk than earlier publications. For the above reasons it is essential that our estimates be updated when new and better evidence on the health benefits of vegetables and fruit becomes available. Established limitations of dietary research apply to the present study: the assessment of dietary intake depends on self-report, which is prone to error, and reporting of food frequency does not translate accurately into absolute consumption. In addition, the CCHS summary variable on consumption of vegetables and fruit includes potatoes, which has been shown not to provide the same benefit in terms of preventing chronic disease as other non-starchy vegetables,37. d burden .In conclusion, we estimated that $CAN 3·3 billion of annual health-care costs in Canada are attributable to inadequate consumption of vegetables and fruit. Further investments in the promotion of vegetables and fruit will prevent chronic diseases and substantially reduce direct and indirect health-care costs.


Aedes mosquito–borne alphavirus, is endemic to Brazil and other South America countries. We investigated dengue- and chikungunya-negative febrile patients visiting rural areas near Goiânia, Goiás, and found a high proportion (55%) of MAYV IgM. Our findings suggest the presence of highly endemic foci of MAYV in central Brazil.Mayaro virus (MAYV), an  Aedes mosquito–borne alphavirus of the New World, transmitted mainly by tree canopy–dwelling Haemagogus spp. mosquitoes, was discovered in Trinidad in 1954. MAYV causes a dengue-like acute febrile illness with arthralgic manifestations (Mayaro virus (MAYV) is an http://www.saude.gov.br) are notified to the Center for Strategic Information in Health Surveillance of Goiânia city for further investigation. At the end of 2014, after chikungunya virus (CHIKV) spread in South America, the Brazil Ministry of Health enhanced the surveillance system for dengue-like illness. Accordingly, febrile patients attending primary care centers are tested for dengue virus (DENV) infection by using viral antigen (NS1), virus isolation, or reverse transcription PCR (up to 5 days after symptom onset), and/or ELISA for IgM (after the sixth day). DENV-negative patients fitting the clinical and epidemiologic criteria for chikungunya set by the Ministry of Health , to be evaluated for CHIKV by an IgM-capture ELISA . A subsample of CHIKV-negative serum collected during December 2014–June 2015 was tested by hemagglutination inhibition (HI) ; of these, 15 (56%) were confirmed as positive by IgM MAC ELISA for MAYV, and 1 sample was borderline. The median interval between symptom onset and serum collection was 37 days (range 12–167 days).Patients were a median of 45 years of age (range 30–70 years). Fourteen were women. Patients’ educational level was high; 11 (73%) patients had >8 years’ education, and 47% had >12 years’ education.All the patients with an antibody profile suggesting recent MAYV infection resided in Goiânia but had traveled to rural areas in the 15 days before symptom onset. Twelve patients had visited farms or small holdings in the forests around towns located 34 km (Hidrolandia) and 46 km (Bela Vista) from Goiânia  Figure. http://www.paho.org/hq/index.php?option=com_docman&task=doc_view&Itemid=270&gid=34247&lang=en), is represented by a wet area hosting small primates, which may play a role as virus amplifier.The high frequency of MAYV IgM detection among febrile patients in Goiânia is surprising. Identification of recent infections most likely acquired in rural areas and forests around the city of Goiânia indicates the existence of active foci where a sylvatic cycle of MAYV is established. The MAYV belt around Goiânia, where epizootics of jungle yellow fever were also reported (Our conclusions are subject to several limitations. First, selection bias could affect findings regarding the proportion of MAYV recent infections because of the lack of systematic testing for dengue-like illness. Second, we did not conduct PCR to identify acute infections because the samples were collected after the viremic phase. Third, plaque-reduction neutralization testing was not performed, and because of the lack of convalescent serum, IgG seroconversion or titer increase were not evaluated; however, MAC ELISA is considered a valid technique for diagnosing recently acquired infection with MAYV , higher In conclusion, infection with MAYV occurs more frequently than expected in central Brazil. Mayaro fever should be considered in the differential diagnosis with DENV, CHIKV, and Zika virus infections in areas characterized by arbovirus cocirculation.Goiânia, Goiás, Brazil, and the area where most cases of Mayaro virus infection were detected, June 1, 2014–June 30, 2015.


A new 11,877-nucleotide cytorhabdovirus sequence with 6 open reading frames has been identified in a maize sample. It shares 50 and 51% genome-wide nucleotide sequence identity with northern cereal mosaic cytorhabdovirus and barley yellow striate mosaic cytorhabdovirus, respectively. Rhabdoviridae includes viruses with plant, insect, fish, and mammalian hosts. Members of the genera Cytorhabdovirus and Nucleorhabdovirus are plant-infecting viruses that also infect their insect vectors .Total RNA was isolated from lyophilized leaf tissue from two maize plants collected from a field in Lima, Peru, testing positive for infection with maize chlorotic mottle virus (MCMV) . Sample Antisense ORFs 1 to 6 were predicted in the virus genome sequence at nt 7 to 1344, 1449 to 2345, 2462 to 2971, 3093 to 3596, 3663 to 5123, and 5405 to 11605. This is consistent with predicted ORFs of other described plant-infecting rhabdoviruses that contain 6 to 8 ORFs from the antisense RNA encoding the N, P, 3, 4 (variable), M, G, and L proteins. Predicted proteins were aligned to corresponding cytorhabdovirus proteins  using ClustalW, with a Gonnet matrix for similarity calculations. The putative L protein is 60% identical and 76% similar to the L protein of BYSMV, 59% identical and 76% similar to NCMV L, and 70% identical and similar to the aligned portion of partial L protein of maize yellow striate virus (GenBank accession no. JQ715419). Translated ORFs 1 to 5 of the maize-associated rhabdovirus are 29.8 to 42.1% identical (50.2 to 60.2% similar) to corresponding proteins of BYSMV and 30.3 to 43.5% identical (48.4 to 62.1% similar) to NCMV proteins.Previously described maize-infecting rhabdoviruses include maize mosaic virus , maize fKY965147.The genome sequence of maize-associated cytorhabdovirus has been deposited at GenBank under the accession number


We report reoccurrence of highly pathogenic avian influenza A(H5N2) virus clade 2.3.4.4 in a wild mallard in Alaska, USA, in August 2016. Identification of this virus in a migratory species confirms low-frequency persistence in North America and the potential for re-dissemination of the virus during the 2016 fall migration. Historically, apparently effective geographic barriers (Bering and Chukchi Seas of the North Pacific Ocean) appeared to limit dissemination of Asian-origin, highly pathogenic avian influenza virus (HPAIV), such as influenza A(H5N1) virus A/goose/Guangdong/1/1996 (Gs/GD), between the Old and New Worlds  fly to Alaska, USA, to breed. Thus, Alaska is a potentially major location for intercontinental virus transmission  virus, hereafter known as 8887/2016(H5N2) virus, to trace its origin and understand its genetic relationship to HPAIV H5N2 isolated in 2014–2015 .http://www.offlu.net/fileadmin/home/en/resource-centre/pdf/Influenza_A_Cleavage_Sites.pdf). Homology BLAST searches showed that all genes had >99.2% nucleotide similarity with genes of H5N2 virus outbreak strains collected during late February–March 2015 (We considered 8887/2016(H5N2) virus an HPAIV on the basis of amino acid sequence at the hemagglutinin proteolytic cleavage site (PLRERRRKR/G), as shown for other Gs/GD HPAIV H5Nx subtypes in subclade 2.3.4  virus formed a cluster with viruses from initial detections in the midwestern United States, including a snow goose in Missouri, a backyard poultry farm in Kansas, and a turkey farm in Minnesota . Our epi–3  substitutions/site/year. In the root-to-tip regression plot of maximum-likelihood phylogeny, we found that 8887/2016(H5N2) virus fell below the regression line, which indicated sequences that are slightly less divergent than average of 2014–2015 H5N2 outbreak viruses . Consistent clustering of 8887/2016(H5N2) virus with other H5N2 outbreak viruses in phylogenies for each gene suggests that the 8887/2016(H5N2) virus probably evolved through genetic drift from common ancestors of outbreak viruses in the absence of further reassortment  Figure 2 viruses  Figure 3The last reported detection during the influenza outbreak in the United States in 2015 was from a Canada goose in Michigan on June 17. There were 2 detections by PCR  from mallards in July (bird banding effort in Utah) and November (hunter harvest in Oregon) during surveillance in 2015–2016. Sequence of the HPAIV H5N2 from a wild mallard during surveillance in 2016–2017, evidence for continued evolution of this virus lineage, widespread detections of HPAIV H5N2 in healthy wild birds  virus clade 2.3.4.4 in wild birds, Alaska, USA, 2016.


The virulence of Newcastle disease virus varies greatly and is determined by multiple genetic factors. In this study, we systematically evaluated the roles of the polymerase-associated  protein genes in genotype VII NDV virulence after confirming the envelope-associated (F and HN) proteins contributed greatly to NDV virulence. The results revealed that the polymerase-associated protein genes individually had certain effect on virulence, while transfer of these three genes in combination significantly affected the chimeric virus virulence, especially when the L gene was involved. These results indicated that the L protein was a major contributor to NDV virulence when combined with the homologous NP and P proteins. We also investigated viral RNA synthesis using NDV minigenome systems to assess the interaction between the NP, P, and L proteins, which showed that the activity of the polymerase-associated proteins were directly related to viral RNA transcription and replication. Avulavirus within the family Paramyxoviridae and its genome is a non-segmented single-stranded RNA molecule of 15,186, 15,192, or 15,198 nucleotides (nt) in length (Newcastle disease (ND) is a severe infectious disease of poultry caused by the Newcastle disease virus (NDV) that affects more than 250 species of birds. NDV belongs to the genus n length . The genn length .Based on their pathogenicity in chickens, NDV strains can be categorized into three different pathotypes: lentogenic (low virulence), mesogenic (moderate virulence) and velogenic (high virulence) . The virNewcastle disease viruses can be classified into different genotypes based on the sequence and phylogenetic analysis of the F protein gene. Since the 1990s, genotype VII NDV strains have frequently been reported in many regions including Asia, Europe, the Middle East and South Africa , indicatIn this study, we evaluated the contributions to genotype VII NDV virulence of the envelope-associated protein genes (F and HN) in combination, as well as the polymerase-associated protein genes  individually or in combination. In addition to the two parent strains , we recovered 16 chimeric viruses using reverse genetics. The pathogenicity of the chimeric viruses in 1-day-old chicks as well as in 4-week-old chickens, the replication kinetics in DF-1 cells and chicken embryos, and the RNA synthesis in NDV minigenome systems were analyzed. Our results reveal that both the envelope-associated and polymerase-associated proteins contribute to the virulence of genotype VII NDV, and the activity of the polymerase-associated proteins is directly related to viral RNA synthesis.Specific-pathogen-free (SPF) chicken eggs and chickens were purchased from Merial Vital Laboratory Animal Technology Co., Ltd . All birds were kept in isolators at the China Agricultural University throughout the experiments and the animal rearing facilities were approved by the Administration Committee of Laboratory Animals under the auspices of the Beijing Association for Science and Technology .The study was carried out in strict accordance with the Guidance for the Care and Use of Laboratory Animals formulated by the Ministry of Science and Technology of the People’s Republic of China. The experimental protocol, including the possibility of animal death without euthanasia, was specifically considered and approved by the Animal Welfare and Ethical Censor Committee at China Agricultural University . All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.2. NDV strain SG10 was isolated from an outbreak of ND in chickens and identified as velogenic genotype VII virus with the intracerebral pathogenicity index (ICPI) of 1.79 and the mean death time (MDT) of 45 h (h) (A chicken embryo fibroblast cell line (DF-1) was grown in Dulbecco’s modified Eagle’s medium (DMEM)  containing 10% (v/v) fetal bovine serum (FBS) (Gibco) and maintained in DMEM containing 5% FBS. Baby hamster kidney (BHK-21) cells stably expressing T7 RNA polymerase (BSR T7 cells) were grown in DMEM containing 10% FBS and 1 mg/ml G418 . Both cell lines were grown at 37°C in an incubator  under 5% CO45 h (h) . NDV strFigure 1) . The leader region was preceded by a T7 RNA polymerase promoter that initiated the encoded antigenomic RNA with three G residues at its 5′ end. The trailer region was flanked by a hepatitis delta virus ribozyme sequence that cleaved after the last NDV-specific residue . Of the seven restriction sites in the NDV sequence, the MluI and the NotI restriction sites were introduced during RT-PCR and the others occurred naturally on the genome. In addition, in order for the restriction sites to be unique, the naturally occurring SalI, XbaI and HindIII sites in the P, M and L open reading frames (ORFs) were eliminated without changing the amino acid coding to act as genetic markers. The constructed plasmid was designated pOK-rLaSota and was confirmed to be identical to the biological virus by complete nucleotide sequencing.The construction of full-length antigenomic cDNA of NDV strain SG10 (rSG10) has been described previously (igure 1) . To consKpnI and HindIII to generate a single DNA fragment without the F and HN genes. Second, the F and HN ORFs of rSG10 were engineered by PCR amplification as two DNA fragments each flanked by 5′ and 3′ UTRs of the respective rLaSota F or HN genes. To maintain the genome length as a multiple of six nucleotides, three extra nucleotides were introduced at the 3′UTR of the HN gene as necessary. The other three DNA fragments with homologous ends, one extending from the KpnI site to the F initiation codon, one from the F termination codon to the HN initiation codon and the other from the HN termination codon to the HindIII site, were generated by PCR amplification with compatible primers. Finally, these six DNA fragments with overlapping homologous ends of various lengths were effectively joined together using the Cloning Kit. The complete F and HN ORFs of NDV strain LaSota were inserted in combination into the SG10 backbone in place of the corresponding SG10 sequence using the same strategy. The full-length rLaSota cDNA plasmid containing the rSG10 F and HN genes was designated rLaSGFHN, whereas the full-length cDNA plasmid of rSG10 containing the rLaSota F and HN genes was designated rSGLaFHN.The F and HN ORFs of NDV strain rSG10 were inserted in combination into a full-length antigenomic cDNA of strain rLaSota in place of the original NDV F and HN ORFs using the presence of unique restriction enzyme sites and the Seamless Assembly Cloning Kit (Invitrogen). First, the full-length antigenome of rLaSota was digested with The NP, P, and L ORFs were reciprocally exchanged between the LaSota and SG10 antigenome cDNAs using the relevant restriction enzyme sites noted above and the Seamless Assembly Cloning Kit (Invitrogen). Seven chimeras based on a full-length antigenomic cDNA of rLaSota was constructed in the same strategy: rLaSGNP, rLaSGP, rLaSGL, rLaSGNPP, rLaSGNPL, rLaSGPL, and rLaSGNPPL, as well as a parallel set of seven constructs based on a full-length antigenomic cDNA of rSG10: rSGLaNP, rSGLaP, rSGLaL, rSGLaNPP, rSGLaNPL, rSGLaPL, and rSGLaNPPL. All the exchanged regions in the full-length cDNAs were sequenced by dideoxynucleotide sequencing to confirm the presence of the desired genes.N-tosylphenylalanine chloromethyl ketone (TPCK)-treated trypsin  as a source of exogenous protease . Virus titers in the collected supernatants were quantified via limiting dilution in DF-1 cells using the endpoint method of 50).The growth kinetics of rLaSota and rSG10 and their chimeric viruses were determined under multiple-cycle growth conditions in DF-1 cells and in SPF chicken embryos. Duplicate wells of six-well plates containing DF-1 cells were infected with virus at a multiplicity of infection (MOI) of 0.01. After 1 h of adsorption, the cells were washed with DMEM and then covered with DMEM containing 5% FBS and placed at 37°C under 5% CO6 TCID50 of each virus per embryo. Three embryos were chilled at regular 12 h intervals until 72 hpi, allantoic fluid samples were harvested, and the titers (TCID50) of virus in the collected samples were determined by the method described above.To analyze the growth kinetics of chimeric viruses in chicken embryos, the allantoic cavities of 9-day-old SPF chicken embryos were inoculated with 106 50% egg infectious dose (EID50) of virus per bird. The birds were monitored daily for clinical signs for 14 dpi. Two birds were euthanized daily from days 1–5 pi for detection of gross lesions, and samples were collected into two parts. One part  was used for virus titration and the other part  was fixed in 10% buffered formalin for histopathology. Necropsies were carried out, and external and internal abnormalities were recorded.Two viruses, rSGLaNPPL and rLaSGNPPL, and their parent strains (rSG10 and rLaSota) were used to determine the pathogenicity in chickens. Four-week-old SPF white Leghorn chickens were assigned randomly to five groups of 20 birds each  and each group was inoculated by the oculonasal route with either one of the four viruses or with 0.9% NaCl as the negative control. The target dose of the inoculum was 1050 per gram using the Reed and Muench method , at 37°C for 1 h. The virus titers were determined as TCIDh method .For histopathology, tissue samples were fixed in 10% neutral buffered formalin for 48 h. All sampled tissues were routinely embedded in paraffin and 5-μm sections were cut for hematoxylin and eosin staining and examined for lesions using light microscopy. The tissue sections were scored according to the severity of the observed lesions. The absence of injury was classified as -, while mild, moderate and severe injuries were classified as +, ++, and +++, respectively.KpnI and XbaI  gene (in an antisense orientation) flanked at the 5′ side by the trailer region and at the 3′ side by the leader region (LaSota and SG10), followed by the hepatitis delta virus ribozyme and the transcription termination signal, were synthesized and cloned into plasmid pGL3-Basic with and XbaI . The conThe BSR T7 cells were seeded in 24-well culture plates. The cells were cotransfected with either pLaSota-FLuc or pSG10-FLuc (340 ng) and helper plasmids expressing NP (340 ng), P (170 ng), and L (170 ng) proteins originating either from the LaSota strain or from the SG10 strain as well as a Renilla luciferase reporter gene . At 20–22 hpi, the cells were washed in PBS once, and 100 μL passive lysis buffer  was added. Cells were vigorously mixed for 15 min, and 20 μL of lysate from each well were used for dual luciferase assay by dual luciferase assay kit (Promega). The relative luciferase activity unit was defined as the ratio of FLuc to RLuc activities . Six sept-test in GraphPad Prism Software Version 6.0 . Statistically significant differences between experimental groups were determined using the analysis of variance (ANOVA) method. The significance was considered as follows: significant, p = 0.01–0.05 (∗); very significant, p = 0.001–0.01 (∗∗); extremely significant, p = 0.0001–0.001 (∗∗∗).All statistical analyses were performed using the unpaired Figure 1). The virulence of rLaSota was evaluated by the ICPI test in 1-day-old SPF chicks. The ICPI value of rLaSota was 0, which was the same as its biological parent LaSota (date not shown). These results showed that the cDNA-derived strain rLaSota and its parent had the same biological characteristics.A cDNA clone encoding the antigenome of strain LaSota (rLaSota) was constructed from six cDNA segments that were synthesized by RT-PCR from virion-derived genomic RNA  to the virulence of genotype VII NDV, the genes encoding these proteins were exchanged between strains rLaSota and rSG10 using reverse genetics. Fourteen chimeric viruses were recovered. Seven chimeric viruses were derivatives of strain rLaSota in which the NP, P and L genes were replaced individually or in combination with those of rSG10. Seven chimeric viruses were derivatives of rSG10, in which the NP, P and L ORFs were replaced individually or in combination with those of rLaSota. All chimeric viruses were amplified by RT-PCR and sequenced, which confirmed the correct structure of each exchange and a lack of adventitious mutations.Table 1). These values were consistent with the designations of rSG10 as velogenic and rLaSota as avirulent. Replacement of the envelope genes (F and HN) of the rSG10 strain in combination with those of the rLaSota strain (rSGLaFHN) resulted in a significant decrease in virulence, the MDT and ICPI values being >120 h and 0.86, respectively. Derivatives of rLaSota bearing the envelope protein genes (F and HN) of rSG10 (rLaSGFHN) in combination showed an obvious increased virulence with MDT and ICPI values of 79 h and 1.49 respectively (Table 1). The results indicated that exchange of these envelope-associated protein genes in combination between the low-virulence strain LaSota and the highly virulent genotype VII strain SG10 had a marked effect on the viral virulence.To determine the contributions to genotype VII NDV virulence of the F and HN protein genes in combination, the virulence of the two chimeric viruses along with their respective parental viruses was evaluated by determining the ICPI and MDT tests. The respective MDT values of parental rSG10 and rLaSota were 57 h and >120 h in embryonated eggs, and they had ICPI values of 1.86 and 0.00, respectively (Table 2). Derivatives of rLaSota bearing the polymerase-associated protein genes of rSG10 had respective MDT/ICPI values as follows: rLaSGNP, >120 h/0.00; rLaSGP, >120 h/0.00; rLaSGL, >120 h/0.00; rLaSGNPP, >120 h/0.34; rLaSGNPL, >120 h/0.14; rLaSGPL, >120 h/0.12; and rLaSGNPPL, >120 h/0.63 (Table 2). The results indicated that the individual replacement of the polymerase-associated genes between the SG10 strain and LaSota strain did not affect the virulence of NDV except for the rSGLaL. The simultaneous replacement of the three polymerase-associated genes (NP + P + L) had an evident effect on viral virulence, as shown by a decrease in the ICPI value from 1.86 for rSG10 to 1.32 for rSGLaNPPL and an increase in the ICPI value from 0.00 for rLaSota to 0.63 for rLaSGNPPL (Table 2).We further examined the roles of the polymerase-associated proteins genes  in NDV virulence. Derivatives of rSG10 bearing the polymerase-associated protein genes of rLaSota had respective MDT/ICPI values as follows: rSGLaNP, 72 h/1.78; rSGLaP, 52 h/1.89; rSGLaL, 83 h/1.68; rSGLaNPP, 58 h/1.90; rSGLaNPL, 86 h/1.48; rSGLaPL, 84 h/1.40; and rSGLaNPPL, 89 h/1.32 (p > 0.05) except for rLaSGNP which showed an obvious increase at 60 hpi (p < 0.05) . However, the chimeric viruses rLaSGNPL and rLaSGNPPL, in which the NP + L or NP + P + L genes of the rLaSota virus in combination were replaced with those of the rSG10 virus, showed much higher virus titre than the parental rLaSota virus at 36 h and 48 hpi (p < 0.05) . These results indicated that, among the polymerase-associated protein, the NP and L proteins had an obvious influence on the level of virus replication and this influence was greatest when induced by the interaction of homologous NP, P, and L proteins.The growth kinetics of 14 chimeric viruses in which the NP, P, and L genes were replaced individually or in combination between the low-virulence strain LaSota and the highly virulent genotype VII NDV strain SG10, along with their respective parental viruses, were compared using multicycle growth curves in DF-1 cells. When the growth of strains rLaSGNP, rLaSGP and rLaSGL was compared with that of the parental rLaSota, no significant growth differences were observed (p < 0.05 or 0.01), while the chimera rSGLaL showed the lowest replication at 48 hpi (p < 0.05) . The chimeric virus rSGLaNPL, in which the SG10 NP and L genes in combination were replaced with those of the LaSota virus, showed decreased replication at 24 hpi (p < 0.05), while another chimeric virus rSGLaNPPL also showed significantly decreased replication from 24 hpi to the end of the observation period (p < 0.05) .Some constructs of rSG10 bearing rLaSota-derived NP, P, and L genes, alone or in combination, showed a decreased replication compared with parental rSG10. The chimera rSGLaNP showed the lowest replication from 24 hpi to the end of the observation period . For chimeric viruses with LaSota backbone, rLaSGNP, rLaSGP and rLaSGL grew at the same rate as their parental rLaSota virus. The other chimeric viruses, of which the rSG10 virus-derived NP + L, NP + P, or NP + P + L genes in combination were replaced with those of the rLaSota virus, showed a slightly increased growth compared to the parental rLaSota . This indicated that the presence of the polymerase-associated genes  of rSG10 virus individually in the rLaSota backbone did not alter the replication of chimeric viruses, while the combinations had a slight effect in 9-day-old chicken embryos. For the chimeric viruses with SG10 backbone, rSGLaL showed decreased replication compared to the parental rSG10 virus at 48 hpi, while the embryos infected with rSG10, rLaSGNP and rLaSGP had all died at this time. The chimeric viruses rSGLaNPL and rSGLaNPPL showed a significantly decreased replication at 24 hpi (p < 0.05) . These results indicated that changing the NP or P gene did not alter the replication of the chimeric viruses compared with that of their parental viruses, except that rSGLaL, rSGLaNPL and rSGLaNPPL showed decreased replication at an early or later stage of infection.The 6 EID50 of virus through the natural route of infection. No obvious clinical signs were observed in any of the birds inoculated with rLaSota, and slight punctate hemorrhages and catarrhal exudates were observed in the throat and trachea at days 4–5 pi (Table 3). Birds inoculated with rLaSGNPPL exhibited slight depression, punctate hemorrhages and catarrhal exudates in the throat and trachea, and hemorrhages and enlargement of the thymus at 4–5 dpi. The pathogenicity of the rSGLaNPPL and its parental virus differed greatly, the birds infected with rSGLaNPPL developing mild illness with moderate lesions and no mortalities, whereas the birds infected with rSG10 developed severe illness with severe lesions by days 3–5 pi and a 100% mortality by days 5.Based on previous results, the pathogenicity of two chimeric viruses (rSGLaNPPL and rLaSGNPPL) and their parental viruses (rSG10 and rLaSota) were evaluated further in 4-week-old SPF chickens by inoculating each bird with 10Figure 4). The results showed that rLaSota could replicate in certain organs, such as trachea and occasionally in brain or duodenum. While rLaSGNPPL showed increased replication ability, and the virus replicated to moderate or high titers in many of the sampled tissues, such as proventriculus, brain, lung, duodenum and cecal tonsil and especially in trachea, the replication ability showed significant differences (p < 0.001) compared to rLaSota. For rSG10 and rSGLaNPPL, all sampled tissues in the rSG10-inoculated birds showed high viral titers from 3 dpi. The virus titers in most of the sampled tissues were significantly lower in rSGLaNPPL-inoculated birds than in birds inoculated with the parental rSG10 (p < 0.001), indicating a decreased replication ability of rSGLaNPPL compared with the parental virus.Two chickens from each group were sacrificed daily on days 1–5 pi for virus titration by indirect immunofluorescence in DF-1 cells . For the chimera rLaSGNPPL and its parental virus rLaSota, no obvious histological changes were observed in sampled tissues except for focal congestion in the lungs of the group inoculated with rLaSGNPPL (empty arrows) . The parental rSG10 caused the following moderate to severe histological changes in all of the sampled tissues: lymphocytic infiltration of the lamina propria (black arrows); submucosal congestion of the tracheal mucosa (empty arrows); hemorrhage, RBC infiltration (black arrows) and congestion (empty arrows) in the lung; severe mucosal epithelial shedding in the proventriculus (empty arrows); congestion of the lamina propria (black arrows); necrosis and shedding of lymphocytic cells in the mucosa of cecal tonsil (empty arrows); increased and gathered microglial cells in the brain (black arrows); and multifocal confluent coagulative necrosis (black arrows) in the spleen . However, the chimera rSGLaNPPL exhibited decreased virulence with mild or moderate lesions in the trachea, lung, proventriculus and cecal tonsil, as well as no obvious changes in the brain and spleen compared with the parental rSG10 virus.Two chickens from each group were sacrificed at days 4 pi for histopathological analysis  were constructed and a dual luciferase assay was carried out.p > 0.05) with that of the LaSota replication proteins only. The FLuc activity was reduced in the presence of L, NP + L, and P + L proteins from SG10 (p < 0.001). However, the two combinations, NP + P and NP + P + L of SG10, caused a highly significant increase in FLuc activity compared with the native LaSota strain (p < 0.01) . For the second pSG10-FLuc minigenome, when we replaced the SG10 helper plasmids, NP, P, L, NP + P, NP + L, P + L, NP + P + L with their LaSota strain counterparts, they showed a similar influence on the expression of the reporter gene, resulting in a significant decrease in replication of the FLuc gene (p < 0.001) . These results revealed that the activity of the SG10 replication complex was higher than that of the LaSota replication complex, and the combined action of all three homologous replication proteins was required to reach the highest level of activity. The two minigenomes demonstrated different levels of reporter gene expression when driven by the LaSota replication proteins and those of the SG10 virus. This indicated that the nucleotide differences in the leader or trailer sequences that code for the genomic and antigenomic promoter (63.8% homologous) of LaSota and SG10 had a great influence on the efficiency of RNA synthesis.For the pLaSota-Fluc minigenome, using the SG10 helper plasmids, NP or P proteins individually, no significant differences were observed in comparison , there being several examples of incongruity between different protein genes and virulence, as well as evidence that the contribution of these genes to virulence might be related to the particular NDV strain involved . When thin vitro or in vivo of each chimeric virus mimicked the change in its virulence, indicating that the change in pathogenicity may be correlated with the changed replication level of the chimeric virus. These findings are in agreement with some previous studies  in combination between rLaSota and rSG10 affected the virulence of the chimeric virus, especially when the L gene was involved. For rLaSGNPPL, in which the NP, P, and L genes of the rLaSota virus were replaced with those of the rSG10 virus, the ICPI value was 0.63, which had obviously increased when compared with that of its parental virus rLaSota (0.00). Again, the rSG10 derivatives rSGLaNPL, rSGLaPL and rSGLaNPPL, bearing the polymerase-associated protein genes in combination from rLaSota, shared a decreased virulence with ICPI values 1.48, 1.40, and 1.32, respectively. In addition, the change in the replication kinetics  studies . Our resWe further determined the pathogenicity of two chimeric viruses (rSGLaNPPL and rLaSGNPPL) and their parental viruses in 4-week-old SPF chickens inoculated via a natural infection route. There was a marked difference in the pathogenicity and tissue tropism between the parental viruses and the two chimeric viruses. The NDV strain rSG10 caused a 100% mortality at 5 dpi, and the tissue titers remained at a high level up to the end of the sampling at 5 dpi, whereas the chimeric virus rSGLaNPPL maintained a 0% mortality until 14 dpi and it replicated at a lower level than its parental virus in all sampled tissues. In contrast, the parental rLaSota failed to cause clinical manifestations and the tissue titers were limited in the trachea or occasionally in the duodenum. Its chimeric virus rLaSGNPPL also failed to cause any mortalities, but showed an increased tissue tropism or replication efficiency in some tissues, including trachea, lung, proventriculus, duodenum and brain. The results suggested that exchange of the polymerase-associated genes together (NP + P + L) had a substantial effect on the tissue tropism of the two viruses, which may be associated with the variations of virulence.It has been reported for NDV that the levels of RNA synthesis are associated with virulence . In thisIn summary, we evaluated the role of the envelope-associated and the polymerase-associated protein genes in NDV virulence individually or in combination by exchanging genes between the prevalent genotype VII strain SG10 and the lentogenic genotype II strain LaSota. Our study further confirmed that the virulence of NDV is determined by multiple viral proteins. The F and HN proteins are the major contributor to virulence, and the L protein as well as NP and P protein also have an obvious influence on virulence. The results have distinct implications in furthering a comprehensive understanding of the virulence factors associated with NDV and other paramyxoviruses. Based on the obtained structure of the mononegavirales L protein , furtherConceived and designed the experiments: X-hY, J-hJ, and G-zZ. Performed the experiments: X-hY, J-lC, J-hJ, and YS. Analyzed the data: X-hY, JX, JZ, G-zZ. Contributed reagents/materials/ analysis tools: G-zZ. Wrote the paper: X-hY, JZ, and G-zZ.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.


Health care systems around the world have started to develop pharmacists prescribing for minor ailments (PPMA) programs. These programs aim to improve the efficiency of care, reduce physician visits, and increase the accessibility to prescription medication (Rx). This study performed an economic impact analysis of the pharmacists prescribing for minor ailments program in Saskatchewan.We measured costs for the program and the alternative scenario (i.e. no PPMA program) from a public payer and societal perspective, using primary data on pharmacists prescribing consultations in Saskatchewan. Furthermore, we calculated public payer and societal savings, and return on investment ratios for the program, as well as projecting the costs and benefits over the next 5 years.Overall, we found that from a societal perspective, the Saskatchewan PPMA program saved the province approximately $546,832 in 2014, while according to the public payer perspective, the program was only marginally cost-saving in 2014. After 5 years of implementation, from a societal perspective, cumulative cost savings were projected to be $3,482,660, and the return on investment ratio was estimated to be 2.53.Our results demonstrate that this type of program may prove cost-saving and lead to improved access to the health care system in Canada, especially if savings to society are considered. This type of PPMA program may prove economically feasible and beneficial in many countries considering expanding pharmacists scope of practice.The online version of this article (doi:10.1186/s12962-017-0066-7) contains supplementary material, which is available to authorized users. Health care systems around the world have started to develop pharmacists prescribing programs with the aim to improve the efficiency of care, reduce physician visits, and increase the accessibility to prescription medication , 2. AlthScotland and Northern Ireland were some of the first countries to implement a pharmacists prescribing program, with pharmacists being able to prescribe independently in some areas since 2006 . CurrentIn Saskatchewan, beginning March 2011, pharmacists were able to prescribe for a variety of minor ailments from a list of medications previously available only from a physician. At present, pharmacists can consult and prescribe for 17 minor ailments including, acne, cold sores, allergic rhinitis, oral aphthous ulcer, oral thrush, diaper dermatitis, atopic dermatitis, dysmenorrhea, gastroesophageal reflux disease (GERD), headaches, hemorrhoids, musculoskeletal pain, skin infections (impetigo and folliculitis) and tinea skin infections . Saskatchewan currently pays $18 per pharmacist consultation for pharmacists to prescribe for these ailments, the first provincial government to cover such costs. Part of the motivation for starting the Saskatchewan PPMA program (and similar programs), was to decrease costs to the health care system while increasing accessibility and patient satisfaction .Research shows that pharmacists prescribing programs may allow general practitioners (GPs) to focus on more complex cases. Currently, between 10 and 20% of GPs workload and 5% of ER consultations in some countries are for minor ailments . ChanginPPMA programs are not the only alternative to GP visits for minor ailments. One common option is patients’ self-care. For instance, the European Union set up a pilot project to examine and support self-care initiatives in the EU . Self-caA recent systematic literature review of 2010–2015 economic evaluations of pharmacy services in hospital and community-based settings in publicly funded health systems worldwide found that these pharmacy services provided clinical benefits which include improved patient outcomes and reduction in adverse use of medications . The finMoreover, much of the empirical and evaluative work has focused on the UK, and very few analyses from other countries are available to offer a comparative viewpoint . No studThe objectives of this paper are to perform an economic impact analysis of the pharmacists prescribing for minor ailments program in Saskatchewan, including (1) the current costs and savings of the program from both the public payer and societal perspective and (2) the costs and savings of the program for a time horizon of five years after implementation.This study performed an economic impact analysis of the Pharmacists Prescribing for Minor Ailments Program (PPMA) in Saskatchewan using primary data on pharmacists prescribing consultations recorded since program inception and the Saskatchewan Minor Conditions Survey, an online questionnaire administered to individuals who had consulted a pharmacist as part of the PPMA in Saskatchewan . FurtherFinally, using the societal perspective, we estimated a projection of costs and benefits over five years. We present a detailed description of the methods in the following sections. For a full list of parameters and the data sources see Table Using a public payer perspective, we considered only direct costs to the health care system associated with the PPMA, including pharmacist remuneration for service delivery based on the current pharmacist remuneration fee of $18 per consultation and the cost of publically funded prescriptions. Using consultation records kept by the health department from 2014, we estimated there were 10,739 pharmacy consultations for minor ailments per year in Saskatchewan.Prescription drugs used for each minor ailment and their corresponding drug acquisition costs were obtained from Drug Plan and Extended Benefits Branch of Saskatchewan Health . “AccordFor recording of transactions by the Saskatchewan health department and subsequent remuneration, the minor ailment consultation must end in a prescription. Therefore we assumed all consultations reported ended in a prescription. Subsequently, to calculate the cost of prescription to the health care system each year, we multiplied the average cost per prescription by the total number of consultations per year  and by 46%, which represented the percentage of prescription costs paid by the government of Saskatchewan in 2013  . We calcThe Saskatchewan Minor Conditions Survey asked patients who had used the PPMA program “If you had not asked for help, what would you have done instead?”. Using these results we determined where patients with minor ailments would have sought care if the PPMA program was not available. The survey concluded that 35% of patients would have seen a GP, 3% would have gone to the ER, 43% would have self-treated with OTC medication and 14% would have done nothing or used something at home , which sTo calculate the costs of running the PPMA program under the societal perspective, we included both the direct health care costs outlined above and the indirect costs to society and the individual patient costs . The total lost time for a pharmacy consultation was assumed to be 2 min waiting time and 15 min duration of consultation . TherefoIn order to calculate societal costs for the alternative scenario, we estimated the direct costs of the alternative, along with the average cost of OTC medications, as well as indirect costs for travel to and wait time for GP visits or the emergency room and productivity loss from inadequate care for minor ailments. First, using expert opinion and a survey of the average cost of OTCs for the five most common minor ailments covered in the PPMA program, we estimated OTC costs at $12 per medication. Cost per OTC medication along with the total number of patients who would have used OTCs in the absence of the PPMA program, projected the total cost of OTCs at $55,413 per year. Moreover, we were able to estimate the productivity loss as a result of the potential lower effectiveness of OTCs as compared to prescription drugs. For this analysis we considered five pharmacist-consulted minor ailments , which accounted for 63% of pharmacist consultations in Saskatchewan and identified as being minor ailments quite likely to cause time off work and may require prescription medication for resolution. Therefore, we only considered there to be productivity losses for the 57% of individuals who would have self-treated or treated with OTCs as part of the alternative, as everyone in the PPMA program received a prescription. First, we extracted the number of days absent per year for each minor ailment from the literature –30. SecoFurthermore, we estimated the total time a patient spends at a family physician or walk-in clinic for a minor ailment would be 105 min (90 min wait time and 15 min per consultation). These estimates were based on data obtained through consultations with walk-in clinics in Saskatoon. Moreover, we estimated that 4.6 h was the average time a patient waits for care at emergency department units in Canada . To calcFinally, we obtained the average length of time a person spends travelling to the GP clinic, ER department and pharmacy for OTC medications, from the Geographic Distribution of Physicians in Canada , which sWe calculated the total costs and savings of running the PPMA program in 2014 from both the public payer  and societal (direct and indirect costs) perspective by taking the difference between total costs of the PPMA program and the total costs of the alternative scenario (i.e. no pharmacist prescribing).We calculated the return on investment ROI ratio of the PPMA using as follows:In this formula, we assumed the initial investment costs were the number of pharmacy consultations multiplied by the consultation fee under the public pay perspective; and these costs in addition to pharmacist training costs under the societal perspective.In order to establish the future benefits and costs of the pharmacy prescribing program, we extended the incremental annual direct and indirect costs and benefits of the PPMA program over 2015–2019. Using data on the number of pharmacist consultations in Saskatchewan for 2013, 2014 and 2015, we found that the number of pharmacist consultations has been increasing over these years but at a decreasing rate. The increase in the total number of consultations from 2013 to 2014 was 45% and from 2014 to 2015 was 20%. Therefore, we assumed that the number of pharmacist consultations would continue to increase but that the rate of increase will continue to decrease over the years. We assumed the increase rate would be 8.9, 4, 1.8 and 0.08% in the next four year (2016–2019). We held all other parameters constant, however we discounted any future costs and benefits based on the CADTH recommendations of 5%. This analysis allowed us to estimate a cumulative present value of cost and benefit over 5 years  based on their likely impact on the results and the level of uncertainty in how we derived the estimate. Moreover, using scenario analysis we calculated the worst and best possible return on investment for costs and savings from the PPMA program, both for the current year and the projection over the next 5 years. See Table Overall, we found that PPMA program saved Saskatchewan approximately $801,347 and $201,552 in 2014 from societal and public payer perspectives, respectively. However, the public payer perspective was only marginally cost-saving in 2014, averting $8250 in public costs  account for 47.7% of the total costs of the program. The societal costs comprised of training costs that accounted for 19.53% of total costs, prescription costs (i.e. private insurance or out-of-pocket payments), opportunity costs (i.e. wait and consultation time), and travel costs to the pharmacy of which accounted for 55.86, 23.94 and 0.67% respectively, of the total societal costs.For the alternative scenario (absence of the PRMA program), we estimated that total costs for minor ailments from a public payer perspective for one year was $350,715. We found that 71.16% of the public-pay costs were associated with GP visits, 12.68% were from ER visits and 16.16% were attributable to government paid prescription costs. Finally, considering societal costs in the alternative scenario, we found that productivity losses from OTCs versus prescription medications accounted for the highest costs at 61.68%, with wait time for GP or ER, cost of OTC meds, travel costs and prescription costs accounting for 23.61, 6.50, 0.040 and 7.81%, respectively, of total patient costs (i.e. costs borne by patients).Currently, there are major questions surrounding how health systems can address minor health conditions in more efficient and effective ways, including through the use of preventative medicine and by administering care in less expensive settings –43. To aStudies show that alternatives to physician care  generally have a similar level of quality to physician offices and ER visits, and are often more convenient and cheaper for patients –46. SimiA study by the Ontario Pharmacists Association (OPA) determined that the implementation of a PPMA program aimed at ‘five key practice areas’, including “counselling and prescribing for smoking cessation, administering flu vaccinations, adapting patients’ drug therapy, renewing prescriptions for stable chronic conditions, and prescribing for minor ailments” could save the provincial health system $143 million over the next 5 years . MoreoveOur estimated savings from the public payer perspective were more conservative than those from Baqir et al. , likely Previous research in Canada shows respondents would generally have a willingness to pay for this service, if it was not covered by the government or insurance, of approximately $18.95 per patient per consultation . This isThere were some benefits of the PPMA we were not able to capture as part of our costing analysis, particularly the potential for improved accessibility to health care advice and a prescription medications. Studies show that many people with minor ailments often do not seek appropriate care –54. One The uptake of a PPMA program in different locations depends on a large number factors and seems to vary significantly. For instance, while Nova Scotia saw their pharmacists prescribing program had particular benefits in rural areas where there was limited access to GPs, a study by Wagner et al.  in ScotlThere are also potential limitations to the PPMA program not captured in this economic analysis. For instance, currently the program only compensates pharmacists when they prescribe a medication, therefore theoretically this program could encourage the unnecessary use of prescriptions. In effect, the pharmacist fees could create a potential conflict of interest for pharmacists who could earn money for both the prescribing and the dispensing of prescriptions , 56. AnoIt is important to note some of the limitations of our analysis. For instance, we did not include the productivity loss due to lack of treatment for all the applicable minor ailments under the program, including acne, insect bites, oral aphthous ulcer, oral thrush, diaper dermatitis, atopic dermatitis, dysmenorrhea, hemorrhoids, skin infections and tinea skin infections. However, as these are not as commonly associated with productivity loss or were relatively rare conditions within this particular prescribing environment, we decided to stay conservative with our savings estimate. Furthermore, we were not able to measure the costs of continuing to run the PPMA program, as these estimates were not available to us. We also did not measure directly the cost of re-consultation rates (i.e. rate that a patient goes to see a doctor after consulting a pharmacist). However, the evidence indicates that re-consultations are very similar whether an individual originally consulted a pharmacist or a GP , 57 and This study investigated costs and savings associated with the PPMA program, which was the only feasible study design given the recent implementation of the program and data availability. However, future research should also focus on conducting full economic evaluations of PPMA programs to evaluate both the costs and benefits of having or not having these programs. Currently, legislation for prescribing by pharmacists exists in only a few countries and its expansion to a larger number of countries may partially depend on the economic evaluations of existing PPMA programs.As countries develop pharmacists prescribing programs with the aim of improving the efficiency of care, reducing pressure on the healthcare system, and increasing accessibility to prescription medications , 2, the


Aphis glycines, is a major pest in soybeans, resulting in substantial economic loss. We analyzed the A. glycines transcriptome to identify sequences derived from viruses of A. glycines. We identified sequences derived from a novel virus named Aphis glycines virus 2 (ApGlV2). The assembled virus genome sequence was confirmed by reverse transcription polymerase chain reaction (RT-PCR) and Sanger sequencing, conserved domains were characterized, and distribution, and transmission examined. This virus has a positive sense, single-stranded RNA genome of ~4850 nt that encodes three proteins. The RNA-dependent RNA polymerase (RdRp) of ApGlV2 is a permuted RdRp similar to those of some tetraviruses, while the capsid protein is structurally similar to the capsid proteins of plant sobemoviruses. ApGlV2 also encodes a larger minor capsid protein, which is translated by a readthrough mechanism. ApGlV2 appears to be widespread in A. glycines populations and to persistently infect aphids with a 100% vertical transmission rate. ApGlV2 is susceptible to the antiviral RNA interference (RNAi) pathway. This virus, with its unique genome structure with both plant- and insect-virus characteristics, is of particular interest from an evolutionary standpoint.The invasive soybean aphid,  Aphis glycines Matsumura was introduced into North America in 2000 and has since become a primary pest of soybeans . To determine whether degradation of ApGlV2 is regulated by the RNAi pathway, we sequenced the sRNA of the Iowa observed .A. glycines was confirmed by sRNA sequencing with virus-derived sRNA identified from both RNA strands, indicative of RNAi-mediated degradation of the viral dsRNA produced during replication. The impact of ApGlV2 infection on the soybean aphid could not be assessed due to the lack of an ApGlV2-free soybean aphid colony.The novel insect virus, Aphis glycines virus 2 (ApGlV2), has a unique genome structure that encodes an RdRp similar to that of insect viruses and a capsid protein resembling those of luteovirids. ApGlV2 infected all individuals in all soybean aphid populations examined and is vertically transmitted. ApGlV2 replication in Viral RdRps are the most conserved viral proteins and share several conserved motifs in a specific order ,39,40. TPermutotetraviridae) and a new genus (Alphapermutotetravirus) [ApGlV2 represents a new type of insect virus that also has a permuted RdRp. A permuted RdRp was first found in the insect tetraviruses (EEV and TAV), with the RdRp closely related to those of birnaviruses . On the ravirus) ,26. A peravirus) , which iravirus) . The perravirus) , differ ravirus) ,45.In addition to negeviruses, and permutotetraviruses, the recent discovery of novel insect viruses suggests that permuted RdRps have evolved in divergent insect viruses. Sequence comparison suggests that the RdRp of DAV, ApGlV2 and other unassigned viruses ,48,49 , the 5′RACE from the CP start codon region would be expected to have gone further than 111 nt. The detection of differing lengths of 5′ UTR is not uncommon for 5′RACE reactions. It is also unlikely that the reverse transcriptase fell off the RNA template during RACE due to secondary structures in the RNA as the entire genomic sequence was confirmed by RT-PCR. Although our results support the hypothesis that the structural proteins of ApGlV2 are encoded by an sgRNA, this remains to be confirmed.The organization of the structural proteins of ApGlV2 resembles that of luteovirids. Luteovirids are exclusively transmitted by aphids in a persistent and circulative manner but do not replicate in aphids. Low sequence identity of ApGlV2 CP to those of sobemoviruses and other plant viruses suggests that these viruses only share similar virion structures. In addition, except for the polyproline sequences at the N-terminus, the ApGlV2 RTD has no sequence homology to the RTD of luteovirids, an indication that the function for ApGlV2 RTD may differ from that of luteovirids.Polymyxa betae . In addition, 5′-RACE of sgRNA showed variable results, suggesting 5′-RACE is not reliable for confirming the 5′-end sequences of ApGlV2. Similar 5′-RACE resulting in shorter 5′-UTR sequences than those of the assembled viral genomes have been reported previously ,62,63, dIn conclusion, ApGlV2 represents a new virus type with a unique genomic structure, which encodes a permuted RdRp similar to several insect viruses, and structural proteins similar to plant viruses. ApGlV2 RNA is subject to degradation by the RNAi pathway. This study highlights the potential for the use of next-generation sequence data for identification of virus-derived sequences  and some