Gallium(III) is an iron analog capable of inhibiting essential iron-dependent pathways, exerting broad antimicrobial activity against multiple microorganisms, including Staphylococcus spp. Herein, we evaluated PMMA and CaSO4 as carriers for delivery of gallium(III) nitrate (Ga(NO3)3) to infected surgical sites by assessing the release kinetics subsequent to incorporation and antimicrobial activity against S. aureus and S. epidermidis. PMMA and to a lesser extent CaSO4 were observed to be compatible as carriers for Ga(NO3)3, eluting concentrations with antimicrobial activity against planktonic bacteria, inhibiting bacterial growth, and preventing bacterial colonization of beads, and effective against established bacterial biofilms of S. aureus and S. epidermidis. Collectively, our in vitro results indicate that PMMA is a more suitable carrier compared to CaSO4 for delivery of Ga(NO3)3; moreover they provide evidence for the potential use of Ga(NO3)3 with PMMA as a strategy for the prevention and/or treatment for orthopaedic infections.Antibiotic-loaded bone cements, including poly(methyl methacrylate) (PMMA) and calcium sulfate (CaSO Staphylococcus aureus (MRSA) and Staphylococcus epidermidis  as well as the cumulative release over time, as expressed as percentage released from the total original loaded amount into PMMA or CaSO4, were plotted.Quantification of gallium (Ga) was accomplished using Inductively Coupled Plasma Mass Spectrometry (ICP-MS) of acid digested samples. Briefly, 150 3)3 was determined using a modified version of the broth microdilution assay in 96-well round bottom plates as previously described [8 CFU/mL), washed, and resuspended in diluted MHB II (0.3 g/L) broth to a final bacterial concentration of 106 CFU/mL. One hundred microliters was then transferred to individual wells of a round bottom plate (~105 CFU/well), containing 100 μL of Ga(NO3)3 at increasing concentrations, 0.25–512 μM, diluted in MHB II (0.3 g/L) broth at a 2x concentration. Bacteria were incubated overnight at 37°C under static conditions, and following overnight incubation, the optical densities (A600 nm) were measured.The inhibitory concentration of Ga3 loaded beads at the various concentrations against planktonic bacteria in broth cultures, beads (3/group) were added to sterile 15 mL conical tubes containing 5 mL of a bacterial culture adjusted to 106 CFU/mL in MHB II (0.3 g/L) broth. Cultures containing the Ga(NO3)3 beads were coincubated at 37°C with agitation for up to 7 days, with cultures removed and exchanged every 24 hours. At days 1, 3, and 7, 100 μL was removed and bacterial viability within cultures was determined by plating serial dilutions onto MHB agar plates. As a control group for the experiment, unloaded (0.0%) PMMA and CaSO4 beads were used. Data was represented as the log reduction relative to a bacterial culture grown in MHB II (0.3 g/L) broth without PMMA or CaSO4 beads.To evaluate the antimicrobial ability of Ga(NO3)3 loading on the bacterial colonization of PMMA and CaSO4 beads simultaneously with elution, Ga(NO3)3 loaded and unloaded beads (6/well) were placed into 6-well plates and cultured up to 7 days as previously described [6 CFU/mL of bacteria was added to the individual wells containing the PMMA or CaSO4 beads containing the increasing concentrations of Ga(NO3)3. Every 24 hours, broth cultures were removed and replaced with fresh bacterial cultures exposing the beads to continuous bacterial challenge. At days 1, 3, and 7, beads were removed from plates, washed with sterile 1x PBS, and placed into individual wells of a 96-well plate, and the plates containing beads were sonicated to remove attached bacteria. The number of viable bacteria removed from beads was determined by plating serial dilutions onto MHB agar plates as previously described [To evaluate the effect of Ga(NOescribed . Brieflyescribed .3)3 using the minimum biofilm eradication concentration (MBEC) P&G plates  as previously described with some minor modifications [μL of bacteria diluted to 106 CFU/mL was added to individual wells of the MBEC plates and incubated for 48 hours at 37°C with shaking at 150 rpm . Following incubation, the plate tops containing the pegs with established biofilms were rinsed in sterile 1x PBS, placed in a challenge plate containing either Ga(NO3)3, 0.25–512 μM, or Ga(NO3)3 loaded beads (2.4–13%) in MHB II (0.3 g/L) broth and incubated for an additional 24 hours. After treatment, pegs were then rinsed and sonicated for 15 minutes at 40 kHz  into a 96-well plate containing PBS. Bacterial viability was determined by plating serial dilutions onto MHB agar plates.Biofilms were developed and evaluated for susceptibility to Ga3 on planktonic growth of S. aureus ATCC 29213 and S. epidermidis ATCC 12228 was performed using a modified version of the broth microdilution assay in iron deplete media to assess antimicrobial activity and ensure bacterial susceptibility of the strains used in the study. Antimicrobial activity of Ga(NO3)3 on planktonic, that is, culture grown, bacteria was observed to be both strain- and concentration-dependent with significant decreases in bacterial growth at concentrations ≥16 μM and ≥4 μM for S. aureus and S. epidermidis, respectively (3)3 ≥64 μM were observed to completely inhibit bacterial growth of both strains tested. In addition to the activity on planktonic bacteria, Ga(NO3)3 was also observed to have antimicrobial activity against biofilms of S. aureus and S. epidermidis, albeit at much higher concentrations, ≥128 μM and ≥256 μM, respectively, compared to their planktonic counterparts 3 we evaluated the effect of loading various concentrations on the curing time of these materials; moreover we characterized the release kinetics of gallium(III) over time. Incorporation of Ga(NO3)3 into PMMA at concentrations ≥9.09% (w/w) extended the time for the curing of PMMA roughly up to 1 hour compared to the approximately ~15 minutes required for curing of vancomycin loaded PMMA (2.4% w/w). Of note, while the PMMA beads were not completely cured, even after 30 min, the material was workable and maintained structural integrity with handling. In contrast to PMMA, incorporation of Ga(NO3)3 into CaSO4 up to the 13% (w/w) was not observed to have any impact on the curing time, relative to beads loaded with vancomycin.To evaluate the potential use of PMMA and CaSO3)3 from both PMMA and CaSO4 was characterized by a rapid initial release followed by slower sustained release. Ga(NO3)3 release from PMMA had a large initial burst, releasing 55%, 34%, 19%, and 22% of the total amount loaded within the first day and reaching mean concentrations of 470 ± 9, 592 ± 11, 636 ± 10, and 1149 ± 11 μM, for the 2.4%, 4.7%, 9.09%, and 13% (w/w), respectively 3 was much lower and sustained for up to 7 days releasing 59%, 37%, 23%, and 25% and reaching mean levels of 6 ± 5, 5 ± 3, 7 ± 4, and 14 ± 5 μM, for 2.4%, 4.7%, 9.09%, and 13% (w/w), respectively. Similarly, Ga(NO3)3 release from CaSO4 also had a large initial burst releasing 21%, 26%, 28%, and 28% within the first day and reaching mean concentrations of 178 ± 13, 458 ± 11, 929 ± 13, and 1488 ± 15 μM, for 2.4%, 4.7%, 9.09%, and 13% (w/w), respectively 3 release from CaSO4 was detected up to the 7 days evaluated, releasing 39%, 39%, 35%, and 36%, and reaching mean levels of 21 ± 4, 16 ± 3, 13 ± 3, and 17 ± 8 μM, for 2.4%, 4.7%, 9.09%, and 13% (w/w), respectively.Release of Ga S. aureus and S. epidermidis, bacterial cultures were exposed to PMMA or CaSO4 beads loaded with either 2.4%, 4.7%, 9.09%, or 13% (w/w) Ga(NO3)3 in diluted MHB II broth 3 loaded PMMA beads had antimicrobial activity against both S. aureus and S. epidermidis over time 3 into PMMA at 2.4%–4.7% (w/w) was associated with a 4- to 6-log reduction during the first three days, whereas no significant antimicrobial activity was observed by day 7. Incorporation of Ga(NO3)3 into PMMA at concentrations of 9.09%–13% (w/w) had the most dramatic effects reducing bacterial cultures between 6- and 10-logs during the first day, with ≥3-log reductions up to 7 days 3 loaded beads at all of the concentrations tested was observed to have a much greater effect, reducing bacterial cultures, between 4- and 6-log reduction, up to the 7 days evaluated (3)3 into CaSO4 was also observed to have antimicrobial activity against S. aureus and S. epidermidis 3 loaded CaSO4 was also dependent on the total loaded concentration for S. aureus, albeit only the higher concentrations of Ga(NO3)3 loading, between 9.09 and 13.0% (w/w), were observed to achieve better bacterial reductions, whereas the lower concentrations, 2.4–4.7% (w/w), had less of an effect (3)3 released at the later time points. In contrast to the effect observed with gallium loaded PMMA, antimicrobial activity against S. epidermidis was much more variable, with significant antimicrobial activity observed for beads loaded with ≥4.7% (w/w)  .3)3 loaded PMMA and CaSO4 beads to hinder bacterial surface colonization. The incorporation of Ga(NO3)3 into PMMA markedly reduced bacteria colonization at days 1 and 3 for S. aureus and S. epidermidis and up to 7 days for S. epidermidis 3 into CaSO4 at even the highest concentrations was only observed to inhibit colonization of S. aureus after 1 day, but not thereafter (3)3 ≥4.7% (w/w) into CaSO4 was observed to significantly reduce colonization of the bead surface by S. epidermidis up to the 7 days evaluated 3 loaded beads to reduce bacterial colonization, we assessed whether Ga(NO3)3 loaded beads also retained activity against established biofilms of S. aureus and S. epidermidis. Following the 24 h exposure of the preformed biofilms to Ga(NO3)3 loaded PMMA beads resulted in a 2- to 4-log reduction of viable bacteria within S. aureus and S. epidermidis biofilms compared to untreated controls, which was highly dependent on the percentage loaded into PMMA (4 loaded beads were observed to have a 2- to 3-log reduction of viable bacteria (Given the ability of Ga(NOnto PMMA . Likewisbacteria .3)3, has been shown to have antimicrobial activity against both Gram positive and Gram negative bacteria in vitro and in vivo [3)3 has been approved by the FDA for the treatment of pathological bone loss disorders [3)3 could be incorporated into and released from PMMA and CaSO4 beads for local delivery into wounds as a potential treatment strategy for orthopaedic related infections.Orthopaedic related infections continue to be a significant complication, contributing to the increased overall healthcare associated costs as well as patient associated morbidity , 3, 6, 7 in vivo , 34, 35.isorders –30, the 3)3 has been shown to have significant antimicrobial activity against a number of clinically relevant bacteria, including Staphylococcus spp., P. aeruginosa, and A. baumannii [4, are commonly used for local delivery to infected sites to achieve high local concentrations. While these two different carriers offer certain advantages over the other, a major limiting factor is the compatibility of the antimicrobial and/or agent being incorporated [3)3 was compatible for incorporation into and release from both PMMA and CaSO4. Similar to the release of other previously characterized antibiotics from PMMA [3)3 was characterized by a large initial burst, with mean detectable concentrations between 300 and 1500 μM, followed by a sustained release over the seven-day period. For PMMA, incorporation of the lower percentages, 2.4–4.7% (w/w), was observed to have a greater cumulative release  compared to those beads loaded with the higher percentages (23–25%). Notably, although Ga(NO3)3 release was also observed from CaSO4, cumulative release was much lower in comparison (21–28%) and not observed to increase with increasing amount of Ga(NO3)3 incorporated. Although the exact reasons for this are not entirely clear, there is a possibility that CaSO4 may have interacted with the gallium and subsequently inhibiting its release. From this assessment of elution kinetics alone, our results indicate that PMMA may be a more suitable carrier for local delivery of Ga(NO3)3. Of note, while no effects of incorporation of Ga(NO3)3 on setting time were observed with CaSO4 directly, incorporation of increasing concentrations of Ga(NO3)3 (≥9.09% w/w) into PMMA did extend the curing time significantly (~1 hour). Because antibiotic impregnated beads are prepared during surgical procedures, just prior to placement within wounds, our observations indicate potential limitations to the use of the Ga(NO3)3 with PMMA, in particular concentrations >9.09%. Importantly, while this is the first study to evaluate the incorporation and release of Ga(NO3)3 from bone cements, along the lines of our findings are previous reports showing the utility of bone cements, in particular PMMA, for delivery of antimicrobial agents other than antibiotics, including antimicrobial peptides [3)3 despite limitations.Given the limitations of this treatment modality, recently there has been a resurgence of efforts to optimize this intervention through use and/or incorporation of unique antimicrobial agents alone or as combinations , 41, 42,aumannii –26. Bonerporated . As demorom PMMA , 46, 47,peptides  and antipeptides , 48, dem4 should be appropriate for the organism(s) suspected of causing the infection while also being eluted locally at concentrations sufficient to achieve antimicrobial activity. Consistent with results from the release kinetics studies, the Ga(NO3)3 loaded PMMA and CaSO4 beads were observed to have antimicrobial activity against the planktonic cultures of S. aureus and S. epidermidis, as demonstrated in 3)3 loaded beads was observed to be most effective during the first 3 days, coinciding with the higher elutions of gallium(III) well above the inhibitory concentration against planktonic bacteria, but rapidly losing activity at 7 days, coinciding with release of levels of gallium(III) at levels below this (≤13 μM). In contrast to the antimicrobial activity observed with the gallium loaded PMMA beads, the antimicrobial activities of the gallium loaded CaSO4 beads was much more variable demonstrating limited activity against the bacteria tested herein as compared to PMMA. As indicated above, the differences in antimicrobial activity of Ga(NO3)3 may in part have been explained by interactions of the gallium with CaSO4, thereby limiting activity, which would be consistent with the lower cumulative release gallium as well as the reduced antimicrobial activity despite detection of released gallium. Our results demonstrating the activity of Ga(NO3)3 against staphylococcal species are in line with previous studies [3)3 as a treatment strategy for orthopaedic infections.To be an effective treatment, agents incorporated into PMMA or CaSO studies , 26 and 3)3 loaded beads could limit bacterial colonization  of beads; moreover we assessed whether they retained activity against established bacterial biofilms. As demonstrated herein, the Ga(NO3)3 loaded PMMA beads, and to a lesser extent the gallium loaded CaSO4 beads, were observed to reduce bacterial colonization. These findings are particularly important for PMMA, as the beads are nonabsorbable and potentially can become a foreign body that is subject to colonization by bacteria following elution of the incorporated agent [3)3 beads to limit bacterial colonization did diminish over time, our findings are similar to those observed for antibiotic loaded beads, including vancomycin and tobramycin [ in vivo settings, the model used to evaluate surface colonization was performed under highly stringent conditions with daily subjections to bacterial inoculums (~106), which would likely be much greater than that encountered within a wound containing a single infective dose. Future studies evaluating colonization under in vivo conditions are warranted given the limitations of our in vitro study.While antibiotic-loaded bone cements have been shown to be highly effective against planktonic bacteria, use of this treatment modality against biofilms are often limited , 18, 49.ed agent . While tbramycin . Importa3)3 released from PMMA and CaSO4 3 released from PMMA as well CaSO4 did not completely eradicate bacteria within the biofilms, our findings demonstrate that Ga(NO3)3 does retain some activity against biofilms in addition to the planktonic bacteria. This is particularly important because biofilms are thought to play a major role in surgical site infections [In addition to the antimicrobial activity against planktonic bacteria, Ga3 for treatment of orthopaedic related infections, our current study does have several limitations. First, this study was entirely conducted in vitro under ideal conditions that do not accurately recapitulate in vivo conditions. As such, to extend the impact of these findings, the results from this study require further evaluation to determine the effectiveness of Ga(NO3)3 impregnated PMMA and CaSO4 beads to reduce microbial burden within an in vivo environment. Secondly, although antimicrobial activity following exposure to Ga(NO3)3 was observed against planktonic, and to a lesser extent against established biofilms of S. aureus and S. epidermidis, it is important to note that this activity was largely dose-dependent and did not result in sterility. This poses a particular clinical problem, as those organisms remaining following treatment could contribute to relapse of infection within the surgical sites. In light of recent studies demonstrating enhanced activity of conventional antimicrobials in the Ga(NO3)3, future studies evaluating the use of Ga(NO3)3 as a combined therapy, rather than a monotherapy, could address this limitation and extend its clinical applications [4. As differences in elution kinetics have been observed between the different types of commercially available bone cements [3)3 from different sources to identify optimal delivery devices for release into surgical sites. Lastly, while Ga(NO3)3 is currently approved for therapy of cancer-related hypercalcemia, the current therapeutic regimens, based on intravenous infusion, allow for serum levels of 10–20 μM [ S. aureus and S. epidermidis. While there have been studies extensively evaluating and determining Ga(NO3)3 to have minimal toxicity in vitro, the much higher levels of Ga(NO3)3 from PMMA and CaSO4 well above those levels previously tested warrant further investigation to evaluate biocompatibility and effect on cell function, specifically on osteoblasts and osteoclasts, to determine the limitations of direct application to infected surgical sites.While our study provided preliminary evidence indicating the compatibility with bone cements and potential use of Ga3 can be loaded into and eluted from PMMA and CaSO4 at concentrations effective against both planktonic and biofilms of Staphylococcus spp., commonly associated with orthopaedic related infections. Collectively, our in vitro findings suggest that local delivery of Ga(NO3)3 may be an effective strategy for the prevention and/or treatment of orthopaedic related infections. Future studies utilizing animal models are needed to fully characterize the clinical role for this treatment modality.The use of antibiotic loaded bone cements is a standard of care used for the prevention and/or treatment of orthopaedic infections. Herein, we show that Ga(NO


In 2010, ischemic heart disease was the leading cause of disability-adjusted life-years (DALYs) worldwide. More specially, the prevalence of acute myocardial infarctions (AMI) is increasing in the aged population as mortality decreases; South Korea is no exception. This study aims to examine the economic burden of AMI in the Korean population between 2007 and 2012. AMI-related costs were assessed from a societal perspective. A prevalence-based cost-of-illness framework was used for this analysis. The subjects included all South Koreans with AMI-related ICD-10 codes . Data on direct  costs and indirect  costs were collected from the Korean National Health Insurance Service’s claims data. The human capital approach was used to calculate indirect costs. The total estimated cost of AMI in 2012 was $1,177,649,323 USD. The majority (52%) of this amount was made up of medical costs, followed by productivity losses due to mortality and morbidity . Although the total cost declined by approximately 18% compared to 2007 , the cost of AMI in the over 60 age group amounted to 47% of the total cost of AMI in 2012. AMI led to a high economic burden in 2012. This study, which identified not only the size, but also the trends of AMI-related costs, will provide information to evaluate effects of governmental health projects and the effective allocation of public research funds. Acute myocardial infarction (AMI) is a disease associated with high mortality worldwide . HoweverThis cost-of-illness (COI) study provides valuable data, which is comparable to the relative socioeconomic burdens of other disease. It can also be used as objective evidence when policy makers establish policy priorities and allocate resources related to the healthcare system. The objectives of this study were to quantify the economic burden of AMI caused by an increased in the use of health resources in combination with lost productivity between 2007 and 2012, and to provide a detailed breakdown of the costs attributed to AMI by various sex- and age-related groups.th revision) codes I21 (acute MI), I22 (subsequent MI), I23 (certain current complication following acute MI), I25.0 , and I25.1 (atherosclerotic heart disease). Though I25.0 and I25.1 are associated with chronic conditions, we have included those codes in our study in an effort to reflect typical Korean features related to entering AMI codes. In Korea, several medical institutions frequently make claims for health insurance premiums using codes I25.0 and I25.1 when they conduct screening tests (a creatine kinase muscle-brain (CK-MB) and troponin) and administer treatments (coronary artery bypass graft (CABG), percutaneous coronary intervention (PCI)) to AMI patients. Additionally, a previous study showed that the positive rate of MI associated with both codes I25.0 and I25.1 was high [This study evaluated the costs related to AMI in a population-based cost analysis. We used a prevalence-based approach and a macro-costing method. AMI is defined here by the World Health Organization International Classification of Diseases  from the set. The intention behind dropping this data was to prevent an overestimation of expenses related to AMI by excluding comorbidity diseases, which often cost a great deal to treat .We estimated direct costs  and indirect costs  based on a societal perspective.AMI medical costs were divided into three categories: health insurance costs, out-of-pocket medical expenses and drugs. We used health insurance claims data to calculate health insurance costs related AMI throughout 2007 and 2012. Because there is only one insurance system in Korea, the National Health Insurance Service (NHIS) data represents medical information of the entire South Korean population. To account for private expenditures, we used data from health insurance patients’ medical expenditure surveys from 2007 and 2012 . Non-medWe used a human-capital approach to estimate indirect (productivity loss) costs . ProductWe use an annual time frame to assess change in economic burden between 2007 and 2012. The results of epidemiologic analysis  were presented as the trends from 2007 to 2012 and the total cost of AMI was presented by comparing with the results in 2007 and 2012. Though this study used the same basic framework as has previously been used to measure the costs of chronic diseases , 15, empDirect costs include medical costs and non-medical costs. Medical utilization has been divided into three categories: outpatient care, hospital inpatient care and drugs. Non-medical costs include transportation costs and caregiver costs.Medical costs consisted of reimbursement from NHIS and out-of-pocket expenses. First, we calculated both outpatient and inpatient reimbursement using NHIS claims data. Next, using the proportion of out-of-pocket costs for patients with heart disease  categorized by treatment type in the NHIS patient medical costs survey, we estimated out-of-pocket expenses. Once reimbursement and out-of-pocket expenses for outpatient and inpatient care were estimated, the total medical cost was added up.In case of drug expenditures, inpatients costs already included drug costs incurred during hospitalization. Therefore, we only needed to consider outpatients’ drug expenses, which were calculated using the proportion of outpatient, inpatient and outpatients’ drug costs provided by an annual NHIS statistical report .InNHI = NHI payment for hospitalization expenditures, OutNHI = NHI payment for outpatient expenditure, OP = Out-of-pocket payment, OutD = Outpatient payment for Drug, i = 0, 1, …, n (age), and j = 1 or 2 (sex)We defined non-medical costs as transportation costs and caregiver costs. To estimate the transportation costs, we multiplied the number of visits to a medical institution for AMI treatment by travel expense per visit. We used a single fare per visit to outpatient and inpatient institutions from KNHNES in 2005, and final transportation expenditures were estimated in consideration of inflation rates each year. Caregiver costs incurred through patients’ length-of-stay in hospitals and the hours of visits for outpatient care-related AMI were also estimated. The caregiver costs during hospitalization were calculated by multiplying patients’ length-of-stay in hospital by the average city daily workers’ wage. When we estimated the caregiver costs related to outpatient visits, we assumed that patients under the age of 20 and over the age of 70 would require a guardian and approximately 8 hours would be needed to accompany the patient to outpatient services.InV = number of hospitalization visits, OutV = number of outpatient visits, InT = round-trip transportation costs per hospitalization visit, OutT = round-trip transportation costs per outpatient visit, InD = length of hospitalization, CG = caregiver costs per day, i = 0, 1, …, n (age), and j = 1 or 2 (sex)Indirect costs included lost productivity due to AMI-related mortality and morbidity. We estimated productivity costs attributable to mortality as earnings lost due to premature death. For that, we assumed that the ages of the economically productive population were between 15 and 69 years. To estimate productivity loss after premature death due to AMI, we multiplied the number of deaths (categorized by sex and age) by annual average income. We also took the employment rate at the time into account to estimate the final value.t+k = average annual income at (t+k), Pt+k = employment rate, t = age at death, k = 1, 2, …, n (n is the difference between life expectancy and age at the time of death), r = discount rate, i = 0, 1, …, n (age), and j = 1 or 2 (sex)N = number of death, YMorbidity costs due to AMI were calculated and divided into two categories, outpatient and inpatient. We multiplied patients’ length-of-stay in hospital by the daily average wage (in terms of sex and age) to estimate productivity loss due to AMI inpatient care. Though productivity loss related to outpatient care was calculated in the same way, for the purposes of this study, we assumed that 8 hours of productivity was lost due to each outpatient care visit.InD = length of hospitalization, IC = average daily income, E = employment rate, OutV = number of outpatient visits, i = 0, 1, …, n (age), and j = 1 or 2 (sex)The prevalence of AMI continued to rise during the study period. In terms of sex, the number of male patients with AMI  is more than 1.73 times the number of females  in 2012. Even the increase in patients with AMI between 2007 and 2012 includes more male (1.34 times) than female (1.13 times) patients. Mortality due to AMI  decreased further each year from 2007 to 2011. However, the number of deaths due to AMI rose in 2012 to a number similar to that of 2008. In terms of the number of deaths due to AMI by age groups, mortality continued to decline 0.80 times in the under 60 age group between 2007 and 2012 .We estimated the total economic cost of AMI in South Korea to be more than $1,177,649,323 USD in 2012 . This acWe estimated the total cost of AMI in South Korea at to be $1.178 billion USD in 2012, of which, $607 million USD was incurred by direct medical utilization. The other 48% of the economic burden of AMI, however, was incurred in non-health-care systems, with almost $461 million USD in lost productivity attributable to early death. The total costs due to AMI accounted for 1.28% of National Health Expenditure (NHE) in 2012 and this cost showed a 0.7% decrease from the figure in 2007 . The proTotal AMI costs had decreased in 2012 compared with the costs in 2007. We can predict that the decline of the economic burden due to AMI happened since the decreasing rate of AMI mortality exceeded the growth rate of AMI prevalence. In addition, the decline in AMI mortality may be caused mostly by improvements in acute cardiac treatment . HoweverAccording to the economic burden by sex, male AMI costs were higher than female’s costs. Especially, direct costs were 1.85 times and indirect costs (productivity losses) were 15.21 times higher than females in 2012. The higher costs in male were caused by both higher prevalence and mortality of AMI than females. The AMI-related risk factors in males were reported to be smoking and stress , 22, 23.Recently, cost-of-illness studies have continued to progress in order to estimate the impact of diseases with similar frameworks in Korea. Stroke burden evaluations in 2010 estimated direct costs to be $3.528 billion USD, which was much higher than the direct cost of AMI in our 2012 evaluation . AdditioWe found that in other developed countries, the distribution of AMI economic burden was similar to that of our study , 28–30. Even though disease-related costs were estimated, we cannot discuss whether a country is spending too much or too little on a disease. Rather, the main aim of a COI study is to help inform decisions concerning allocation of research funding, by providing a measure of the economic burden of particular health problems . In addiWe used NHIS claims data as a main data source to estimate the cost of AMI. Since NHIS claims data include most of information necessary to calculate direct and indirect costs, such as main and sub disease codes, NHI costs, distinguishable variables for inpatient or outpatient care, and other variables, this data source provided key information. In addition, since statistics of mortality from the Korea National Statistical Office provide information about cause of death, we are confident that number of deaths-by-cause reported in this study is accurate. KNHNES data, which was used to calculate transportation expenses, has been established by the Korea Center for Disease Control and Prevention and is comprised of survey data based on a representative, extracted sample. To calculate caregiver costs, we used the public notice amount as the estimated daily worker’s wage based on legal criteria for payment of caregivers. The variables related to indirect costs calculations, such as daily average wage, annual work days and employment rate, were derived from statistics on employment and labor by the Ministry of Employment and Labor. Therefore, the data used in this study was established based on representative data sources.It is important to note that our study had several limitations. First, our estimates are likely to be low, or conservative. We could not include some costs related to the healthcare expenditures, such as public health promotion activities and health education due to data source limitation. However, other studies have shown that these costs represent a small proportion of total costs , 34. FurOur study is significant because it analyzed trends over the last six years of economic burden and the epidemiological index of AMI using recent data. These analyses demonstrate the importance of improving the quality of disease management, not only to promote awareness of the current epidemiologic status of this disease, but also so that decision makers can implement evidence-based policies. In this respect, our study provides reliable COI data and indirectly reflects the performance of national projects related to prevention of cardiovascular disease. We believe that our study will offer meaningful information to the decision makers of the Korean government.


We review findings on the following enteric microbial tandems: norovirus – Enterobacter cloacae, mouse mammary tumor virus (MMTV) – bacterial lipopolysaccharide (LPS), poliovirus and reovirus – intestinal bacteria. The close bacterium–virus interplay may also present options to develop unique therapeutic strategies for those infected, and to prevent further virus spread, and thus minimize the risk for the community.Although the disciplines of bacteriology and virology frequently come together in the setting of a diagnostic medical microbiology laboratory, the two scientific fields are usually miles apart. The microbiologists basically form two non-overlapping groups of scientists, the bacteriologists and virologists, which go to separate meetings and do not easily intermingle. Some recent research findings about elegant virus–bacterium interactions may change this situation. Obviously, interactions between these two microbes can occur only when they colocalize, which most likely occurs in the gut/intestines where 10 Caliciviridae family of small positive stranded RNA viruses are infamous for causing outbreaks of gastroenteritis  in nursing homes and on cruise ships. Noroviruses have been estimated to be responsible for 85% of these outbreaks, and there is no cure or vaccine available  rescued norovirus infectivity.Jones et al. recently reported that human norovirus needs help from  B cells . The keyorovirus . This inorovirus , 8. To d B cells . The effin vitro, and treatment of norovirus-infected mice with antibiotics reduced the viral load. A new twist to this story was added very recently in two studies, suggesting that the gut microbiota may interfere with antiviral innate immunity by quenching interferon-lambda (IFN-λ) signaling by an as-yet undiscovered mechanism  and human T-cell leukemia virus (HTLV), but there is no human counterpart of MMTV. To establish a new infection, this virus has to pass through mucosal surfaces that are rich in microbiota. It was recently reported that depletion of the commensal microbiota in pregnant mice by broad-spectrum antibiotics has a profound modulating effect on MMTV transmission to the first offspring, even though the mothers continued high level MMTV production in the milk , which is vertically transmitted from mothers to their offspring via mother milk. MMTV is a member of the the milk . In a cothe milk . FurtherThe putative role of LPS was studied in more detail. MMTV ultracentrifugation led to comigration of LPS in the gradient. LPS concentration by MMTV likely occurs in specific compartments without affecting the global IL-10 levels, an immunosuppressive cytokine. Furthermore, this interaction potentiated the ability of LPS to induce IL-10 production via engagement of the toll-like receptor 4 (TLR4)-IL-6 cascade. TLR4-dependent IL-10 production was already known to be required for MMTV persistence , but it Picornaviridae. Poliovirus spreads via the fecal–oral route and will thus encounter a multitude of microbes in the human gastrointestinal tract, before it can establish infection. In fact, orally acquired poliovirus performs a single replication cycle in the gastrointestinal tract before dissemination throughout the body. In 95% of cases, only a transient primary viremia occurs and the poliovirus infection remains asymptomatic. In about 5% of cases, the virus spreads and replicates in other sites such as reticuloendothelial tissue and muscle, causing minor symptoms like fever, headache, and sore throat. Paralytic poliomyelitis occurs in <1% of poliovirus infections and occurs when the virus spreads into the central nervous system.Poliovirus, the causative agent of poliomyelitis, is a human enterovirus and member of the family of the Early studies indicated that the intestinal microbiome promotes poliovirus replication and pathogenesis. For instance, antibiotic-treated mice were less susceptible to poliovirus disease and demonstrated minimal virus replication in the intestine . HoweverThe authors subsequently identified a specific amino acid residue substitution in the viral capsid that affects virion stability and the sensitivity to LPS-mediated stabilization, possibly by a direct effect on LPS binding . The ThrReoviridae is a family of segmented, double-stranded RNA viruses like rotavirus that can affect the gastrointestinal and respiratory tracts. “Reo” is derived from respiratory enteric orphan viruses, which relates to the fact that a disease association was not immediately apparent for some of these viruses. Most cases in adults are mild or subclinical, but rotavirus can cause severe diarrhea and intestinal distress in children.The A link with the microbiota first became apparent in the above referenced poliovirus study in antibiotic-treated mice, which were not only protected against poliovirus, but also against the pathogenic effects of reovirus infection . AlthougE. cloacae to facilitate attachment to the target B cells. MMTV virion particles are stabilized by interaction with LPS, thus boosting the transmission rate. Poliovirus studies indicate that bacterial-primed virus shows enhanced receptor binding. The reovirus studies are not yet very conclusive, but the antibiotic effect on disease induction is apparent. For all cases, some important mechanistic details are still lacking that will be addressed in future research.Microbiota are advantageous for the host for several reasons, e.g., for homeostatic functions of the mucosa and for limiting bacterial pathogen colonization. However, recent reports suggest that viral pathogens may utilize the microbiota to boost their replication and/or transmission. Four examples of pathogenic viruses that exploit the surrounding microbial community were discussed. It is likely that commensal bacteria may contribute to the transmission of an array of viruses, not only enteric pathogens, and that this list will grow in the near future. The main players and their functional interplay are summarized in Table Although a limited number of virus–bacterial tandems have been studied thus far, it appears that orally transmitted viruses do efficiently exploit the gut microbiota in one way or the other for transmission. Microbiota is predominantly located in the gut and we reviewed virus–bacterial interactions that occur at this location, but bacteria are also found at numerous other sites on the human body, including the skin, respiratory tract, and urogenital tract. Different viruses do navigate one or more of these sites, arguing that functional virus–microbiota interactions may be much more common than previously anticipated. Next generation sequencing techniques are likely to revolutionize the study of this type of cross-kingdom virome–microbiota interactions, e.g., the interplay of the human papillomavirus with cervicovaginal microbiota . AlthougMicrobiota effects may also be indirect via their interaction with the innate and adaptive immune systems . CertainUnderstanding how the microbiota interacts with viruses at the molecular mechanistic level may inform future vaccine strategies and the development of novel antiviral drugs. In fact, given this close viral–bacterial collaborations, antibiotic treatment may – on top of the antibacterial effect – impose an indirect antiviral effect. The therapeutic use of bacteriophages may, through their impact on components of the microbiota, indirectly affect their viral counterparts that infect mammalian cells.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Glossina pallidipes salivary gland hypertrophy virus  is a dsDNA virus exclusively pathogenic to tsetse flies . The 190 kb GpSGHV genome contains 160 open reading frames and encodes more than 60 confirmed proteins. The asymptomatic GpSGHV infection in flies can convert to symptomatic infection that is characterized by overt salivary gland hypertrophy (SGH). Flies with SGH show reduced general fitness and reproductive dysfunction. Although the occurrence of SGH is an exception rather than the rule, G. pallidipes is thought to be the most susceptible to expression of overt SGH symptoms compared to other Glossina species that are largely asymptomatic. Although Glossina salivary glands (SGs) play an essential role in GpSGHV transmission, the functions of the salivary components during the virus infection are poorly understood. In this study, we used mass spectrometry to study SG proteomes of G. pallidipes and G. m. morsitans, two Glossina model species that exhibit differential GpSGHV pathologies . A total of 540 host proteins were identified, of which 23 and 9 proteins were significantly up- and down-regulated, respectively, in G. pallidipes compared to G. m. morsitans. Whereas 58 GpSGHV proteins were detected in G. pallidipes F1 progenies, only 5 viral proteins were detected in G. m. morsitans. Unlike in G. pallidipes, qPCR assay did not show any significant increase in virus titers in G. m. morsitans F1 progenies, confirming that G. m. morsitans is less susceptible to GpSGHV infection and replication compared to G. pallidipes. Based on our results, we speculate that in the case of G. pallidipes, GpSGHV employs a repertoire of host intracellular signaling pathways for successful infection. In the case of G. m. morsitans, antiviral responses appeared to be dominant. These results are useful for designing additional tools to investigate the Glossina-GpSGHV interactions. Glossina pallidipes salivary gland hypertrophy virus  is a dsDNA virus whose 190 kb genome encodes more than 60 confirmed proteins  hytrosavirus , the vector of a group of neglected tropical diseases called the African trypanosomiases  composed of 61 virally-encoded proteins  whose interactions influence the expression or lack of overt SGH symptoms. We tested the hypothesis by comparing the SG proteomes of GpSGHV-infected vs. mock-infected G. pallidipes and G. m. morsitans flies. The host  proteins identified in this study are potential targets for control of GpSGHV infections in tsetse fly mass production facilities. For instance, antiviral strategies could be developed to block virus replication and egress , Seibersdorf, Austria. For each treatment described below, groups of experimental flies were kept in holding cages (diameter of 20 cm and height of 5 cm) at a density of 75 flies per cage and a mating ratio of 1:4 . The holding cages had netting on top and bottom for fly feeding and pupae collection, respectively. The experimental flies were reared at 23 ± 1°C, 75–80% relative humidity, 12 h scotophase and fed on defibrinated bovine blood meals  based on available data that the incidence of SGH symptoms reaches 100% at the G4  under the same insectaria conditions and handled as the parents. All the treatments described here were replicated at least three times.The G. pallidipes fly and stored in 1 ml of ice-cold sterile saline (pH 7.4). The SGs were then homogenized and clarified by brief centrifugation  to remove tissue debris. The supernatants were sterilized by passing through a 0.45-μm filter unit and the virus titters present in the filtrate were estimated by a quantitative polymerase chain reaction (qPCR) as described by Abd-Alla et al.  female G. m. morsitans and G. pallidipes flies were artificially (intra-hemocoelic) injected with the virus preparations using a protocol described by Boucias et al.  male a et al. . By thiss et al. . Briefly1 male progenies produced by the mock- and virus-infected mothers were screened using a diagnostic PCR protocol described by Abd-Alla et al.  were selected for mass spectrometry measurements. For negative controls, samples from 4-week-old male flies were selected from the mock-infected fly groups (confirmed to be PCR-negative). Ten flies from each of the replicated treatments described above were selected for subsequent SG dissections.To confirm GpSGHV infections, the 4 week-old Fa et al. . For thia et al. , 2011b, a et al. . The acta et al. . For vir1 progenies described above were dissected 2 days after the flies had their last blood meals to allow for full digestions  of normalized LFQs were used. Logarithms of the total intensity corrected for the number of measurable peptides  were used to compare the levels of different proteins from the same sample , a result which was in agreement with our recent report  compared to the virus-infected G. m. morsitans F1 progenies  were host (Glossina)-specific, while 8.5% (n = 73) and 3.8% (n = 32) were from GpSGHV and bacterial endosymbionts , respectively. We then filtered out proteins with single and modified peptides, which resulted in 606 proteins, of which 540, 58, and 9 proteins were from the host, GpSGHV and W. glossinidia, respectively. All the identified proteins are detailed in Supplementary Material  were aligned around the y-axis (circled). On the other hand, a cohort of SG proteins were detectable in the proteome of GpSGHV infected G. pallidipes, but were hardly detectable in the proteome of the mock-infected flies , 65.1% (n = 123) of which were up-regulated , 96.9% of which were up-regulated  pathways, integrin-liked kinase pathway, transketolase pathway, hippo signaling pathway and diverse signaling pathways , the ubiquitin/26S proteasome system, V-ATPase system (virus degradation via acidification of endosomes and or/lysosomes), the phagocytic engulfment system (to clear virus infection) and adaptive mitochondrial-mediated immune responses (interfere with production of progeny virus)  were detectable in Wigglesworthia proteins were in the detected in the SG proteome of G. m. morsitans . Further, the injected virus appears incapable of infecting and inducing overt SGH symptoms in fully differentiated SG cells in adults (as evidenced from our study).In the current study, the effects of GpSGHV infections in G. m. morsitans could represent DNA replication but may not represent production of infectious viral particles. The comparable virus titters between the virus-infected G. m. morsitans and the mock-infected G. pallidipes suggest that the virus may be undergoing only partial replication in adult cells to maintain steady-state titters throughout the adulthood. This is in agreement with previous studies in G. pallidipes whereby, utilizing a diagnostic PCR, Abd-Alla et al. , virus density levels (by qPCR), and protein expression (by LC-MS/MS) imply that either G. m. morsitans, at least in the SGs, is far less permissive to virus replication or that the virus undergoes limited replication in G. m. morsitans (whereby only a subset of genes are expressed) compared to G. pallidipes. Potentially, unlike in G. pallidipes where SGH symptoms can be overt, we are of the opinion that GpSGHV infection is entirely latent in G. m. morsitans as previously proposed by Kariithi et al.  protein is important for the tricarboxylic acid cycle (TCA), gluconeogenesis and nitrogen metabolism  encode viral suppressors of RNA silencing (VSRs) that hijack the UPS components to promote degradation of key components of the host's RNA-interference (RNAi) system, thereby promoting virus replication  has significant roles. In the current study, we detected the main components of the UPS, i.e., E3 ligase (D3TNJ0) and 26S proteasome (D3TRZ3) Table . The UPSVerchot, . DNA virVerchot, , baculovVerchot, , densoviVerchot, , whispovVerchot, , and plaVerchot, . As statBombyx mori nucleopolyhedrovirus (BmNPV)-infected cells significantly inhibited viral proliferation. Potentially, the acidification of endosomes and lysosomes by V-ATPase renders these organelles competent for viral degradation. It is therefore not surprising that in the current study, V-ATPase were up-regulated in the SG proteome of G. m. morsitans as opposed to that of G. pallidipes  detected in the current study , but down-regulated in G. m. morsitans   Table . Howevern) Table . For insn) Table , while an) Table  are invon) Table . Other pes Table . PotentiG. pallidipes. As discussed above, the TKTL pathway may also be involved in the expression of overt SGH symptoms in G. pallidipes. The current data provide potential targets for development of rationally designed antiviral strategies in large tsetse fly rearings for sterile insect technique.Induction of the SGH symptoms is possibly a reflection of active production of viable progeny virus particles. During active virus progeny production, enveloped viruses are known to depend on the endoplasmic reticulum (ER) for maturation of viral envelope glycoproteins, and proteins involved in the formation of replication complexes, assembly, envelopment and genome packaging  validations to understand the relevance of the differences in their expression patterns. The current study is a critical baseline data in a new Coordinated Research Project (CRP) initiated by IAEA, aimed at gaining a deeper knowledge of the Glossina/symbiont/GpSGHV tripartite interactions and how these interactions affect Trypanosoma parasite transmission (Van Den Abbeele et al., Glossina. Candidate proteins experimentally validated as essential for efficient GpSGHV pathogenesis are ideal targets for developing rationally designed antiviral strategies to control the virus infections in tsetse mass rearing facilities. In a larger view, our data are important for future studies on molecular and biochemical routes employed by members of the new entrants into the family of insect viruses, the Hytrosaviridae.The data presented in this study provide hints as to why HK, İİ, JV, MV, and AA participated in the design of the study. AA and IM set up the bioassays. HK and İİ processed and quantified the salivary gland proteins. SB performed the LC-MS/MS measurements. SB and HK analyzed the proteomics data sets. HK and EM annotated/characterized the proteins. HK wrote the manuscript. JV, MV, AA, İİ, EM, EO, and SN contributed in writing the paper, providing critical comments and suggestions. All the authors read and approved the final manuscript.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.


ALL). Patients between one and 26 years of age with newly diagnosed ALL, who were treated at Primary Children's Hospital (PCH) in Salt Lake City, Utah were included. Treatment and hospitalization data were retrieved from system‐wide cancer registry and enterprise data warehouse. PCH is a member of the Children's Oncology Group (COG) and patients were treated on, or according to, active COG protocols. Treatment‐related hospital costs of ALL were examined by computing the average annual growth rates (AAGR). Longitudinal regressions identified patient characteristics associated with costs. A total of 505 patients  were included. The majority of patients had B‐cell lineage ALL, 6.7% had T‐ALL, and the median age at diagnosis was 4 years. Per‐patient, first‐year ALL hospitalization costs at PCH rose from $24,197 in 1998 to $37,924 in 2012. The AAGRs were 6.1, 13.0, and 7.6% for total, pharmacy, and room and care costs, respectively. Average days (AAGR = 5.2%) and admissions (AAGR = 3.8%) also demonstrated an increasing trend. High‐risk patients had 47% higher costs per 6‐month period in the first 5 years from diagnosis than standard‐risk patients (P < 0.001). Similarly, relapsed ALL and stem cell transplantations were associated with significantly higher costs than nonrelapsed and no transplantations, respectively (P < 0.001). Increasing treatment‐related costs of ALL demonstrate an area for further investigation. Value‐based interventions such as identifying low‐risk fever and neutropenia patients and managing them in outpatient settings should be evaluated for reducing the hospital burden of ALL.This retrospective study examined the longitudinal hospital outcomes  associated with the treatment of pediatric, adolescent, and young adult acute lymphoblastic leukemia ( In the United States, over 3000 children under the age of 19 will be diagnosed with acute lymphoblastic leukemia (ALL) in 2015 While there is a lack of adequate information on the costs associated with childhood ALL, pediatric and adolescent cancer‐related hospitalizations, on average, cost five times as much as hospitalizations for other pediatric conditions in the United States This study examined treatment‐related hospital outcomes  among pediatric, adolescent, and young adult ALL patients treated at Intermountain Healthcare's (IH) Primary Children's Hospital (PCH), Salt Lake City, UT. Temporal changes in hospital costs, cost components , admissions, and days were examined to understand the drivers of rising costs from 1998 to 2012. Patient and diagnostic factors  associated with hospitalization outcomes within 5 years of diagnosis were also evaluated.Patients were diagnosed between one and 26 years of age, from January 1, 1998–December 31, 2012. As available electronic data prior to 1998 was less comprehensive, we restricted our analysis to these years. Patients received full or part of their treatment at PCH, which serves as the sole children's hospital for Utah and six other neighboring states We examined hospitalization outcomes including costs, days , and admissions associated with in‐patient stays at PCH for our patient cohort. Hospitalization data were from January 1, 1998 to December 31, 2013 to allow for at least one year of follow‐up for our sample. An activity‐based accounting approach is used to compute costs and volume of healthcare activities delivered at PCH. Costs are calculated by taking the total operating expenses and applying them to volume metrics. Total costs are distributed across components such as (1) room and care (includes daily room and nursing costs), (2) therapy , and radiation), (3) diagnostics (lab and imaging), and (4) pharmacy (in‐patient medication). All costs were adjusted for inflation using the Producer Price Index for Hospitals with 2013 as the base year to examine real changes in costs Patient demographics, diagnosis, treatment, and cancer‐specific outcomes were summarized. We examined annual per‐patient hospitalization outcomes  and cost components for the first year following cancer diagnosis, typically the therapy‐intensive year, for patients diagnosed from 1998 to 2012. Per‐patient outcomes were also plotted separately for high‐ and standard‐risk ALL (refer to Appendix S1 for the definition of type of leukemia).Longitudinal regressions examined associations between patient factors with per‐period (increments of 6 months from the date of diagnosis) costs, days, and admissions within the first 5 years of diagnosis P‐values are two sided and considered significant at α = 0.05. Data are presented as medians (ranges) unless otherwise specified.The data were analyzed with the software packages Stata 13  and R 3.0.2  Most patients (75.3%) were diagnosed at <10 years of age (Table P = 0.50). High‐risk patients also encountered more growth in hospital days  and admissions .The average or per‐patient first‐year hospitalization costs rose from $24,197 in 1998 to $37,924 in 2012, with an annual average growth rate (AAGR) of 6.1% , spent more days in the hospital , and had more hospital admissions  compared to standard‐risk patients (Table P < 0.001) after relapse, along with hospital stays over four times longer , and four times more admissions  than patients who did not relapse. Patients who received SCT were likely to have greater costs  during SCT or post‐SCT periods, and spend more days in hospital  in comparison to patients who did not receive SCT. Compared to patients who survived, those who were nearing death were more than twice as likely to accrue higher hospitalization costs  in the period prior to death, yet there were no significant differences in days  or admissions . Importantly, on average, the first 6 months costs , days , and admissions  were highest than within any 6‐month period in the 5 years of diagnosis.Conditional on nonzero costs, high‐risk patients had higher per‐period costs . Drug‐acquiring costs have increased over time, which have been observed in previous studies Our study is important because we investigate increasing costs, rather than charges, of hospitalizations associated with the treatment of ALL from the perspective of a large hospital. At PCH, much of the patient care has been shifted to outpatient clinics and home care, yet high‐risk ALL patients may be hospitalized more frequently for high‐dose methotrexate administration in an attempt to prevent relapse after remission induction therapy This article has certain limitations. Our analyses were retrospective and restricted to one institution. ALL treatments implemented worldwide may define risk differently, although these risk algorithms include the NCI risk criteria used in this study. Patients in our cohort may have been hospitalized at other facilities, which could lead to an underestimation of true costs. We were unable to define treatment costs in terms of medication type . Likewise, we could not examine the differences in costs according to leukemia subtype . We did not incorporate minimal residual disease in risk classification since it was not routinely used for all patients in this study To conclude, we have shown that the costs of high‐risk ALL patients are 47% more, per period, than for standard‐risk patients. Value‐based interventions and outpatient supportive care should be evaluated to reduce the ALL burden. We recommend cost‐based comparisons of protocols such as Dana‐Farber Cancer Institute ALL Consortium Protocol 05‐001Authors have no conflict of interest to report.Appendix S1. Methods.Table S1. Acute lymphoblastic leukemia treatment protocols followed by the primary children's hospital from 1998 to 2012.aFigure S1. Per‐patient first‐year hospitalization cost for pediatric, adolescent, and young adult acute lymphoblastic leukemia from 1998 to 2012.Click here for additional data file.


Manihot esculenta Crantz), a food security crop in sub-Saharan Africa, is threatened by the spread of cassava brown streak disease (CBSD) which manifests in part as a corky necrosis in the storage root. It is caused by either of two virus species, Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV), resulting in up to 100% yield loss in susceptible varieties.Production of cassava  contains supplementary material, which is available to authorized users. Cassava brown streak disease (CBSD) has been identified among the seven most serious threats to world food security . Leaf syCassava brown streak virus (CBSV), and Uganda cassava brown streak virus (UCBSV), both picorna-like (+) ssRNA viruses from the genus Ipomovirus, family Potyviridae [Bemisia tabaci [Bemisia tabaci [CBSD is caused by at least two distinct virus species; yviridae ,8. Thesea tabaci . For thea tabaci  it occura tabaci ,5. In tha tabaci , westerna tabaci . More rea tabaci , Rwanda a tabaci  and the a tabaci , indicata tabaci ,15.M. glaziovii and M. melanobasis (now regarded as M. esculenta subsp. flabellifolia [Breeding for resistance to cassava mosaic disease (CMD) and CBSD was initiated in 1937 in Amani, Tanzania and due to insufficient levels of resistance in cultivated cassava, a strategy to incorporate resistance from wild species, particularly from llifolia ), througllifolia ,18. Sevellifolia ,19. Todavisa versa, as well as combinations of milder versions of both leaf and root symptoms [Severity of CBSD symptom expression varies considerably with cassava varieties and with the environment ,18. Somesymptoms ,20. Recesymptoms . This susymptoms  and suggGenotypes NASE 14, NASE 1, Kiroba and NASE 19 did not show shoot symptoms during the duration of the experiment (Table One of six Namikonga plants showed the mildest of symptoms Class 2) Tables  and 2. I Tables  Interestingly 15 of NASE 14 plants were asymptomatic for both shoot and root symptoms, five showed mild symptoms and two showed very high severity (4 or 5) and incidence (90–100) on roots. This was coupled with reduction in growth and in some cases dieback.t values of both UCBSV and CBSV observed in the selected genotypes at 3,5,7,9 and 11 MAP are presented in Additional file Both UCBSV and CBSV were detected in all varieties at some stage during the growing season. None of the varieties were immune. Amplification plots are shown in Figure Though both virus species were detected in all the genotypes, the viral load differed among genotypes. At the final sampling time-point 11MAP), genotype NASE 14, had the least relative viral load for both UCBSV and CBSV i.e. 1.16 and 0.00071 folds (ΔΔCt), respectively  was calculated between log10 of the virus titre fold change and mean shoot symptom score at 3,5,7,9 and 11MAP , Albert (0.96) and NDL06/132 (0.92) for UCBSV and AR40-6 (0.97) for CBSV. Weak relationships and low r2 values were obtained for TZ/130 (0.17) and AR40-6 (0.16) for UCBSV and for TZ/130 (0.33) and Kibaha (0.35) for CBSV. In terms of root necrosis and log10 fold change in virus titre, Namikonga and to some extent Kiroba both had relatively low virus loads and root necrosis incidence and severity. NASE 14 and NASE 19 had low virus titres but high root necrosis incidence (31.7% and 67% respectively) and severity (both with maximum scores of 5). NASE 1 on the other hand had a high relative virus load of 133826 for CBSV at 11MAP but no shoot symptoms and a root necrosis incidence of 18% with a mean severity score of 1.05 and maximum of 2 , Uganda  and The International Institute of Tropical Agriculture, Nigeria, (TME 204). The experiment was conducted with virus-free cassava stakes over 12 months. None of the varieties tested were immune to CBSV or UCBSV. Mixed infection of both UCBSV and CBSV was evident in all cassava genotypes. Genotypes varied in symptom expression and relative virus load of UCBSV and CBSV which also varied over time, indicating differential genotype response to virus infection.In accordance with previous work  considerTo date the focus in breeding has been on reducing roots necrosis, and the expression of shoot symptoms has been considered acceptable if root symptoms are absent, infrequent or very mild . HoweverVariation in root necrosis was observed as expected and was consistent with previous observations of CBSD . NamikonM. melanobasis (now regarded as M. esculenta subsp. flabellifolia [Interestingly NASE14 remained asymptomatic for CBSD for both shoot and roots while the few that succumbed to infection showed very high root severity (4 or 5) and incidence (90 -100%). This was coupled with reduction in growth and in some cases dieback. This response will have to be confirmed through fingerprinting of individual plants to ensure uniformity in genotype and diagnostics on individual plants to dismiss the possibility of ‘escapes’. It can be hypothesized that there could be a threshold at which the virus overcomes the plant defense mechanism thereby causing necrosis. This hypothesis should be further investigated in ‘degeneration’ trials. Such studies will be important in determining resistance durability and in designing seed systems for cassava planting materials. Similarly derivatives of llifolia ), were ollifolia . This waThe large differences in virus load of UCBSV (low) and CBSV (high) in TZ/130, AR40-6 and NASE 1 could be due to competition among the viruses with CBSV outcompeting UCBSV, differences in pathogenicity or differential reaction of genotype to each virus. Higher virus loads of epidemic CBSV than endemic UCBSV in cassava varieties and herbaceous hosts have been observed previously  and moreRelative virus loads changed through the growing season with NASE 14 and Namikonga showing a decline in relative UCBSV and CBSV loads at 7MAP and Kiroba at 7MAP and 9MAP respectively. Kiroba had tested positive for UCBSV at 5 and 7MAP but this could not be detected at 9 and 11MAP Table . A similSymptom expression has been shown to correlate with virus load in different organs of two genotypes  althoughVirus resistance terminology is a contentious issue on which there is no general agreement and a number of definitions exist ,34. Acco Namikonga showed resistance to field disease symptoms and kept virus loads low relative to the susceptible genotypes. Namikonga remained symptomless apart from one plant that showed root necrosis with maximum score of 2 (very minor discoloration). Relative virus load declined from 7 to 9 and 11MAP for both UCBSV and CBSV respectively in Namikonga. This indicates an ability to restrict virus accumulation and resist root necrosis development. Based on relative virus load, under glasshouse conditions with graft inoculation ‘Namikonga’ has been classified as ‘resistant’  and our  This category comprises genotypes that appear to keep virus loads low, but express a range of symptoms from slightly more severe, at a slightly higher incidence, than Category 1 (Kiroba) to those that show root necrosis up to Class 5. Kiroba had an average root necrosis of 1.07, maximum score 3 and an incidence of 14.3%, but kept virus loads low. A decline in virus loads was observed from 7MAP for UCBSV, and dramatically from 9MAP for CBSV. Kiroba has previously been classified as ‘tolerant’ due to intermediate virus loads . Here Ki This category comprises genotypes that harbor high virus loads but show relatively mild symptoms with low incidence. NASE 1 showed mild symptoms with no shoot symptoms, a maximum root necrosis of 3 with 73.3% of plants remaining symptom free. Similarly TZ/130 showed mild symptoms with 17% shoot symptoms and a maximum score of 2, and a mean root necrosis score of 1.2, and a maximum of 4. AR40-6 could also be considered in this category with maximum root necrosis of score 3, and a mean of 1.09, although it did show a high level of shoot symptom incidence (52%) and a maximum score of 3. NDL06/132 also had a high incidence of shoot symptoms (67%), but moderate root symptoms . The four varieties did harbor high levels of CBSV and thus seemed to be able to restrict symptom expression to some extent but not CBSV load. NDL06/132 also had a high UCBSV load. This again brings into question the relationship between symptom expression and relative virus load observed by ,21,23. Kibaha, Albert and TME 204 were susceptible both in terms of field symptoms (both shoot and root necrosis) and virus load, having high relative virus loads for both UCBSV and CBSV.CBSD shoot and root necrosis incidence and severity and relative virus load suggest that at least two main mechanisms may be operating, one that seems to restrict symptom expression under high virus load, and the other that seems to inhibit virus accumulation. The ability of some varieties to impair the replication of CBSVs has been observed in cassava ,23, althRelating these four categories to conventional terminology, Category 1 can be equated to ‘resistance’, Category 2 can be considered ‘tolerant (restricted virus load)’, Category 3 ‘tolerant (restricted symptom incidence and severity)’ and Category 4 as ‘susceptible’. It is envisioned that classifying genotypes in this way will not only make biological sense to ‘field breeders’, but, by providing transparency in terms of symptoms and virus load, will help breeders in making choices of parents for crossing. For example, it may be prudent to cross a variety showing resistance to symptom expression with one showing restricted virus accumulation. It is worth noting that only leaf samples were used for analysis of virus accumulation. Therefore it is possible that those genotypes that show reduced root necrosis  allow virus accumulation in the leaves but restrict the translocation of the virus to the roots. This requires further investigation. In addition, samples were pooled across plants, which obscures among plant variation.The above results indicate at least two possible mechanisms of resistance/tolerance to CBSVs. This is consistent with earlier findings. Namikonga and possibly Kiroba are direct derivatives of the Amani breeding program, whereas NASE 14 and NDL06/132 have Amani breeding germplasm in their pedigrees Table . The AmaThe breeding strategy was likely to have resulted in the combination of resistance genes from several sources . Inter-cCBSD resistance has been reported to be quantitative and recessive with both additive and non-additive genetic effects ,32. Howepersonal communication).This was a preliminary study to investigate virus load in genotypes with contrasting symptoms under field conditions. It was based on responses in a single growing season (12 months) and thus broadening our understanding on the concept of virus resistance  and disease resistance (symptom expression). It is important that disease observations and virus load are measured over several years and across a broader range of environments. Studies to identify quantitative trait loci are underway to further extrapolate resistance mechanisms as are differential gene expression studies based on RNASeq  . Other genotypes are able to restrict virus accumulation or have a system of recovery . Some genotypes may possess a combination of these different mechanisms (Namikonga). Historical evidence from the Amani breeding program, based on backcrossing from inter-specific crosses and inter-crossing of inter-specific derivatives supports the hypothesis and evidence for different mechanisms of resistance including those that restrict virus accumulation and those that restrict symptom expression. A substantial amount of research still needs to be undertaken to fully understand the bases of resistance. This information will be useful to plant breeders in informing breeding strategies and restricting virus spread. For durable resistance, various mechanism can be combined or exploited by considering both virus and disease resistance in different genotypes.Eleven cassava genotypes selected from Uganda and Tanzania were screened for field resistance to both UCBSV and CBSV in Uganda. Tanzanian genotypes reported to be resistant/tolerant in Tanzania were AR40-6, NDL06/132, Kiroba and Namikonga , and Ugandan genotypes reported to be tolerant in Uganda were NASE 14 (MM96/4271), 72-TME 14 (NASE 19), NASE 1 and TZ/130 Table . GenotypThe trial was monitored for above ground symptoms during the crop growth period and symptoms in the roots after harvest. Symptoms on shoots (leaves and stems) were recorded on each plant at three, five, seven and nine months after planting (MAP). A severity score of 1–5  was adopSeverity scores for root necrosis were also taken on all roots harvested per plant at 12MAP. At harvest, each root was cut across into slices approximately 5 cm apart, and the maximum severity score taken for each root where 1- no necrosis, 2- mild necrotic lesions (1-10%), 3-pronounced necrotic lesions (11-25%), 4-severe necrotic lesions (26-50%) and 5- very severe necrotic lesions (>50%). A root disease severity mean value was calculated on a per plant basis, and then averaged over plants to give a mean value for each genotype. Per plant mean root necrosis incidence was quantified as a ratio of the number of roots showing root symptoms to the total number of roots harvested per plant. This was averaged to give a value per genotype.In addition, at 12 MAP fresh shoot biomass (stems and leaves) and roots per plant were weighed separately and harvest index calculated on a plot basis as the ratio of storage root weight to total plant biomass and storage root weight . This waAt 3MAP, six plants per genotype that showed leaf symptoms were tagged for leaf sampling, whereas sampling of six plants of symptomless genotypes was done through random selection. At 3,5,7,9 and 11 MAP a mature leaf (second level from the bottom) was sampled from each tagged plant and stored at −84°C. At the beginning of the trial, many of these genotypes did not show foliar symptoms for the first 3 MAP. Leaves were therefore pooled together to avoid or reduce false negative probability for detection and quantification of CBSV/UCBSV in cassava tissues  and also−1 by addition of an appropriate amount of sterile water.The pellet was re-suspended in 200 μl of TE buffer containing 1% SDS after which 100 μl of 5 M NaCl and 300 μl of ice cold iso-propanol was added and the mixture incubated at −20°C for 30 min. After incubation, the mixture was centrifuged for 10 min at 13,000 g to pellet the nucleic acid. The pellet was then washed by adding 500 μl of 70% ethanol and centrifuged for 4 min at 4°C. The ethanol was decanted off and the pellet dried and re-suspended in 50 μl of nuclease –free sterile water. RNA quality and quantity was measured using a Nanodrop ND-1000. Due to differences in RNA quantity, the samples were normalized to a working concentration of 100 ngμlThe RT-PCR assay used was based on TaqMan chemistry using primer and probe sequences reported by  except t-∆∆Ct method as described by [target-CtCox)time x – (Cttarget-CtCox)3 months and where x is time . All genotypes that had Ct value of 40 for UCBSV or CBSV were considered to be free of these viruses. The fold changes were transformed to log10 and plotted against time (MAP) to monitor the relative accumulation of virus in different genotypes with time. In addition log10 fold changes were regressed against mean shoot symptom scores at 3,5,7,9 and 11MAP and the coefficient of determination (r2) calculated.All real-time PCR reactions were performed on an Applied Biosystems’ One Step Plus® sequence detection system (Applied Biosystems). The generated cycle threshold (Ct) values were used to determine the fold change in expression of a target gene relative to that at 3MAP for both CBSV and UCBSV using a comparative 2ribed by  where ∆∆


Sandfly fever Naples species. Although these new viruses belong to genetic groups that include several human pathogens, it is not yet clear if Toros and Zerdali viruses can infect humans and cause disease such as sandfly fever. Consequently, the availability of these genetically characterized infectious viruses will enable seroprevalence studies to establish their medical importance in this region and to assist the health agencies to develop appropriate and effective disease control strategies.Southern Anatolia in Turkey at the border with Syria, where many refugee camps are settled, is endemic for sandfly-borne leishmaniasis. Sandfly-borne phleboviruses are also known to circulate in this region, although their relevance in terms of medical implications is virtually unknown. Therefore, the specific objectives of our study were firstly to identify isolate and characterise potentially pathogenic phleboviruses in sandflies; secondly to determine the complete genomic sequence of any viruses that we were able to isolate; and thirdly, to further our understanding of the potential medical importance and epidemiological significance of these viruses. To achieve these objectives, we organised field campaigns in 2012 and 2013. Two new phleboviruses  were isolated and characterized by complete genome sequencing and phylogenetic analyses. Toros virus was genetically most closely related to Corfou virus within the Sandfly fever Sicilian group. Zerdali virus was most closely related to Tehran virus within the Many studies have presented virus sequences which suggest the existence of a variety of putative new phleboviruses transmitted by sandflies in the Old World. However, in most of these studies, only partial sequences in the polymerase or the nucleoprotein genes were characterised. Therefore to further our understand of the presence and potential medical importance of sandfly-borne phleboviruses that circulate in southern Anatolia, we initiated field campaigns in 2012 and 2013 designed to identify, isolate and characterise phleboviruses in sandflies in this regionSandfly fever Naples species), whereas Toros virus was closest to Corfou virus.An entomological investigation encompassing 8 villages in Adana, Mediterranean Turkey was performed in August and September 2012 and 2013. A total of 11,302 sandflies were collected and grouped into 797 pools which were tested for the presence of phleboviruses using specific primers for RT-PCR analysis and also cell culture methods for virus isolation. Seven pools were PCR positive, and viruses were isolated from three pools of sandflies, resulting in the identification of two new viruses that we named Zerdali virus and Toros virus. Phylogenetic analysis based on full-length genomic sequence showed that Zerdali virus was most closely related with Tehran virus  co-circulate in Adana city, in Mediterranean Turkey. While Adana virus was recently described as a new member of the SALV species, Zerdali and Toros viruses are described here as new phleboviruses genetically closely related to SFNV and SFSV, respectively. In this study, isolated and characterised these two new viruses by determining their complete genome sequence and by phylogenetic analysis. This study demonstrates that 3 distinct viruses can co-circulate in the same geographic area and based on their phylogenetic relationships and association with sandflies are likely to be transmitted by these arthropod vectors. Our molecular and phylogenetic data are important for establishing group-specific molecular detection assays in order to further understand of the possible impact of these viruses in animal and human health in this region of Turkey. Phlebovirus (family Bunyaviridae) currently contains 9 viral species Sandfly fever Naples (SFNV), Salehabad (SALV), Rift Valley fever (RVFV), Uukuniemi (UUKV), Bujaru (BUJV), Candiru (CRUV), Chilibre (CHIV), Frijoles (FRIV) and Punta Toro (PTV) including 33 distinct serotypes, and 32 tentative serotypes as defined in the 9th Report of the International Committee on Taxonomy of Viruses (ICTV) [The genus s (ICTV) . Neverths (ICTV) , others s (ICTV) , 6, whers (ICTV) , 8.Phlebotomus) when they take a blood meal. Some Old World sandfly-borne phleboviruses may cause self-limiting febrile illnesses (sandfly fever) or neuro-invasive infections. They are widely distributed in the Mediterranean Basin, in Africa, in the Indian subcontinent, in the Middle-East, and in far-eastern former USSR republics [In the Old World, sandfly-borne phleboviruses are transmitted between vertebrates mainly by female sandflies  and SFNV were present in the Mediterranean and Aegean regions of Turkey , 12. RecSandflies were captured during August and September in 2012 and in 2013 in Adana city located in the Mediterranean region of Turkey  using CDSandfly pools were processed as previously described  in a finTwo real-time RT-PCR assays (Rt-RT-PCR) were designed for specific detection of the new strains in the N gene: TORV-N-FW (AACTCTGACTCGTGTGGCTG), TORV-N-REV (GCCTTGGGTATGTCTGACCA), and TORV-N-Probe (6FAM-AGGCAATAGAAGTTGTGGAGAAC-TAMRA); ZERV-N-FW (ACTTCCTGTTACTGGAACAACAAT), ZERV-N-REV (CCATGAGCATCTGCAATAACTTC), and ZERV-N-Probe (6FAM-ATGATGCATCCTAGTTTTGCAGGA-TAMRA). Reaction conditions and cycling programs were previously described .2-flask of Vero cells, incubated at room temperature for 1 hr, and supplemented with 3mL of EMEM . The flasks were incubated at 37°C in 5% CO2 atmosphere and examined daily for cytopathic effects (CPE).Fifty μL (derived from sandflies ground in the 600μL of EMEM as aforementioned) were inoculated onto a 12.5 cmFor detailed characterisation, Zerdali virus (ZERV) passage 5, Toros virus (TORV) strain 292, passage 3, and TORV strain 213, passage 7 were subjected to complete genome characterisation using Next Generation Sequencing (NGS). Briefly, 140μL of infectious cell culture supernatant medium was incubated with 30 U of Benzonase (Novagen 70664–3) for 7 hr at 37°C.This material was then purified using the Viral RNA Mini Kit (Qiagen). Tagged random primers for reverse transcription (RT) and tag-specific and random-primers were used for PCR (Applied Biosystems). The resulting PCR products were purified ; 200ng of DNA were used for sequencing using the Ion PGM Sequencer . NGS reads, of 30 nucleotides minimum length, were trimmed using CLC Genomic Workbench 6.5, with a minimum of 99% quality per base and mapped to reference sequences. Parameters were set such that each accepted read had to map to the reference sequence for at least 50% of its length, with a minimum of 80% identity to the reference. From the contigs obtained, viral sequences were identified by best BLAST similarity against reference databases. Sequence gaps were completed by amplification and sequencing overlapping regions using either Sanger sequencing or NGS. The 5' and 3' extremities of each segment were sequenced using a primer including the 8-nt conserved sequence as previously described .Complete genome sequencing was also performed for Corfou virus (CFUV) strain PaAr814 using the frozen cell culture supernatant medium following the methods above for comparison with the newly discovered TORV strains since they were shown to be closely related but only the complete S , the parComplete sequences of each of the 5 genes  were aligned without indels together with homologous sequences of selected phleboviruses retrieved from the Genbank database using CLUSTAL within the MEGA 5 program . NucleotThe Recombination Detection Program v.4.27 (RDP4) was used for recombination analysis using the nucleotide alignments. Recombination events, likely parental isolates of recombinants, and recombination break points were analyzed using RDP, GENECONV, Chimaera, MaxChi, BOOTSCAN, and SISCAN algorithms implemented in the RDP4 program with default settings .To attempt identification of the sandfly species present in the TORV and ZERV positive pools, PCR was performed using 3-μL of nucleic acid extract of the pool to amplify the cytochrome c oxidase I (COI) gene using the following primers; LCO1490: GGTCAACAAATCATAAAGATATTGG and HCO2198: TAAACTTCAGGGTGACCAAAAAATCA . The PCRHowever, we would like to acknowledge that a valid protocol would be to cut off the male genitalia using a cold-stage microscope in the laboratory, so that the specimens can be identified morphologically. This would be faster and cheaper than PCR amplification followed by NGS or Sanger sequencing of the COI gene.A total of 11,302  sandflies were collected in August and September 2012 and 2013 from eight villages  located The pool #37  was positive using primers SFNV-S1/S2 [The TORV specific rt-RT-PCR confirmed that pools #213 and #292 were positive . Pool #10  was also positive for TORV. Four-fold dilutions of the RNA were positive until the dilution 1:256 for the pools #213, #292, and #10.The ZERV specific rt-RT-PCR confirmed pool #37 was positive , and detected ZERV RNA in 3 additional pools . Four-fold dilutions of the pool #37 RNA on the one hand and of pools #128, #342 and #374 on the other were positive until dilutions 1:4,096 and 1:1,1024, respectively. Pools#128, #342 and #374 consisted of sandflies trapped in 2012 in the respective villages of Damyeri, Camili and Koyunevi .The rate of infection for TORV was 0.026%, for ZERV 0.035%, and for both TORV and ZERV 0.062% assuming that only one sandfly was infected in each pool.Vero cells inoculated with pool #292 showed a clear CPE at day 6 post-inoculation (pi). Pool #213-inoculated Vero cells did not produce CPE during 4 serial passages. However, virus replication was demonstrated by RT-PCR  starting from passage 3. CPE appeared at day 4 pi at passages 4 and 5 and virus replication was confirmed by RT-PCR. In a similar manner, pool #37- inoculated Vero cells provided a clear CPE at day 4 pi of passage 3 (RT-PCR was positive at passage 2). Neither virus isolation nor positive RT-PCR was obtained after 5 serial passages for pools #10, #128, #342, and #374.www.european-virus-archive.com/) where they are publicly available for academic research at non-profit costs.Freeze-dried suspensions of ZERV-strain #37 (passage 8), TORV-strain #292 (passage 5) and TORV-strain #213 (passage 8) have been included in the collection of the European Virus Archive (The complete genomes of both strains (#213 and #292) of the TORV consisted of 6,456 nts, 4,326 nts and 1,702 nts for the L, M and S segment, respectively . The polymerase gene encoded a 6,270-nt long ORF , whereas the glycoprotein gene encoded a 4,077-nt long ORF . The small segment encoded a 738-nt and a 780-nt long ORF which when translated corresponded to the nucleocapsid protein (246 AA) and a non-structural protein (260 AA), respectively.The complete genome of the ZERV (strain #37) consisted of 6,403 nts, 4,202 nts and 1,907nts for the L, M and S segment, respectively . The polymerase gene encoded a 6,285-nt long ORF , whereas the glycoprotein gene encoded a 4,002-nt long ORFs . The small segment encoded a 942-nt and a 762-nt long ORF which were translated to a nucleocapsid protein (314AA) and a non-structural protein (254AA), respectively.The complete genome of the CFUV consisted of 6,453nts, 4,329nts, and 1,704nts for the L, M and S segment, respectively . The polymerase gene encoded a 6270-nt long ORF , whereas the glycoprotein gene encoded a 4,077-nt long ORFs . The small segment encoded a 738-nt and a 780-nt long ORF which were translated to a nucleocapsid protein (246AA) and a non-structural protein (260AA), respectively.Pairwise distances of the nt- and AA- sequences are presented in AA distances between TORV and SFSV-like viruses  were ≤25.2% (N), ≤37.8% (NS), ≤43.3% (M), ≤40.7% (Gn), ≤33.7% (Gc) and ≤20.6% (L), whereas AA distances between TORV and other phleboviruses were much higher: ≥ 42.9% (N), ≥71.4% (NS), ≥58.7% (M), ≥53.3% (Gn), ≥ 47.4% (Gc) and ≥43.9% (L).AA pairwise distances between ZERV and viruses of the SFNV species  were ≤17.3% (N), ≤58.0% (NS), ≤42.7% (M), ≤41.8% (Gn), ≤29.5% (Gc) and ≤17.4% (L), whereas AA distances between ZERV and other phleboviruses were much higher: ≥ 40.6% (N), ≥80.9% (NS), ≥66.3% (M), ≥64.8% (Gn), ≥55.0% (Gc) and ≥44.5% (L).Gene by gene comparative analysis showed that distances observed between ZERV and viruses belonging to the SFNV species were consistently lower than the lowest distances observed between ZERV and non SFNV-phleboviruses. The same relationship was observed with distances between TORV and SFSV-like viruses on the one hand, and TORV and non-SFSV-like viruses on the other.These findings are supportive for (i) the inclusion of TORV in the SFSV species complex , (ii) the inclusion of ZERV in the SFNV species complex .Regardless of the gene used for phylogenetic analysis, and the tree-building programme (i.e. NJ or ML) TORV clustered with SFSV, CFUV and the other SFS-like viruses  with bootstrap values ≥ 99% Figs , 5 and 6ZERV consistently grouped together with THEV and Naples virus strain Yu_8–76 (subgroup I), with bootstrap values at ≥ 99 for L, Gn and Gc, and with lower values for N and Ns. Within this species, there were 3 other subgroups: (i) subgroup II: Toscana viruses; (ii) subgroup III: Naples viruses (except for Yu_8–76 included in subgroup I); (iii): subgroup IV: Granada, Massilia and Punique viruses.Partial sequences were obtained in the polymerase gene for pool #128 (505 nt) and #10 (379 nt), and in the nucleoprotein for pools #128 (280 nt), #342 (438 nt), and #374 (245 nt). Partial polymerase sequences of the pools of #128 and #10 were identical  with ZERV and TORV, respectively. The partial nucleoprotein sequence from the pool #128 was also identical with the ZERV . However, the partial nucleoprotein sequences from the pool #342 (6 nt and 3 AA different) and #374 (49 nt and 2 AA different) were not identical with ZERV although originating from neighbouring localities ; this suSergentomyia sp. sequences which had ≥85% similarity with S. dentata from Adana . Therefore we indicated these sequences as Sergentomyia sp.Genotyping was performed for 7 pools. The species composition of the pools and number of reads are shown in Salehabad species [Although there are published serological data  and a re species . These r species  enabled  species ,33, 34.Phlebotomus major sensu lato [P. perfiliewi sensu lato and P. tobbi, both belonging to the Larroussius subgenus as P. major sensu lato. Similarly, SFTV was detected in P. major sensu lato [P. papatasi that belongs to the Phebotomus subgenus which is distinct from the Larroussius subgenus [Larroussius subgenus whereas other SFSV viruses are associated with P. papatasi with the exception of SFTV association with P. major sensu lato. Vector-virus association needs to be studied in a more detailed manner in order (i) to determine unambiguously the sandfly species transmitting these newly described viruses, (ii) and to verify our hypothesis that virus subgroups within viral species might be linked to specific vectors belonging to distinct taxonomic entities.Regardless of the gene used for analysis TORV and CFUV were grouped together in a sublineage that is clearly distinct from that including all other SFS-like viruses. CFUV was isolated from nsu lato  trapped nsu lato . In contsubgenus . TherefoAdditional field studies combined with experimental studies using sandfly colonies need to be initiated to understand the parameters driving vector capacity and competence for different strains of viruses.ZERV was consistently grouped together with THEV and Naples virus strain YU-8-76. Interestingly, THEV and the Serbian isolate Yu 8/76 apparently do not require expression of the NSs ORF, since their replication is not impaired by the presence of either an early stop codon or a large truncation , whereasP. papatasi sandflies in Iran in 1959 [P. perfiliewi sensu lato trapped in Serbia in 1976 [P. papatasi, whereas subgroups II and IV appear to be associated with vectors belonging to the subgenus Larroussius. Subgroup I may be associated with Larroussius except THEV isolated in P. papatasi. Only P. tobbi was found to be present in all of the four ZERV positive pools. The same comment formulated above concerning the need for experimental studies to understand species-related competence and specificity of sandflies applies here.Within the SFNV species, it is possible to discriminate 4 sublineages (I to IV); we propose to assign ZERV to sublineage I, where it was most closely related with THEV Figs  and 6. T in 1959 , whereas in 1976 . SubgrouGenetic and phylogenetic analyses support the fact that both ZERV and TORV should be considered as new strains within pre-existing SFNV species and the yet to be recognised species including SFSV and CFUV, respectively.Bunyaviridae Study Group of the ICTV.To date, SFSV and CFUV are listed as tentative species by the ICTV . This stDuring this two-year study, 48% of the sandflies were trapped from Damyeri village where the ecological conditions were no different from those observed in other sampling stations. However, in Damyeri, the number of domestic animals  was much higher than in other localities and these animals were constantly in the close vicinity of houses producing droppings which are known to be favoured breeding sites for sandflies or we set the traps very close to the possible breeding sites by chance therefore we got higher numbers of sand flies for this village. Thus, human exposure to sandflies might be greater in Damyeri than other sampling stations.Phlebotomus perniciosus larvae [Whether TORV and ZERV can infect humans and cause diseases such as sandfly fever is currently unknown and remains to be investigated. After the outbreak of sandfly fever occurred in Adana in 2008, specific IgM against SFSV and/or SFCV was detected in acute cases by mosaic-immunofluorescence test, although the cause of the epidemic was not formally established through virus isolation or molecular detection with sequence confirmation . The regs larvae  may alsoPhlebovirus genus still relies on neutralisation-based antigenic relationships, virus isolation is also essential. Nevertheless, the criteria for taxonomy appear to be evolving towards full-length genome comparative analysis.Recent studies have detected sequences compatible with the existence of many putative new phleboviruses transmitted by sandflies in the Old World. In most of the cases, they were only partial sequences in the polymerase or the nucleoprotein genes. To date these limited genetic data, preclude classification by ICTV. This situation applies for viruses that may belong to (i) the SFNV species such as FERV , ProvincIn conclusion, the results obtained in this study together with previously published results  demonstrS1 Table(XLSX)Click here for additional data file.S2 Table(MAS)Click here for additional data file.