To examine long-term healthcare utilization and costs of patients with stable coronary artery disease (SCAD).Linked cohort study of 94 966 patients with SCAD in England, 1 January 2001 to 31 March 2010, identified from primary care, secondary care, disease, and death registries. Resource use and costs, and cost predictors by time and 5-year cardiovascular disease (CVD) risk profile were estimated using generalized linear models. Coronary heart disease hospitalizations were 20.5% in the first year and 66% in the year following a non-fatal  event. Mean healthcare costs were £3133 per patient in the first year and £10 377 in the year following a non-fatal event. First-year predictors of cost included sex , SCAD diagnosis , and co-morbidities (heart failure cost £657 more per patient). Compared with lower risk patients (5-year CVD risk 3.5%), those of higher risk (5-year CVD risk 44.2%) had higher 5-year costs (£23 393 vs. £9335) and lower lifetime costs (£43 020 vs. £116 888).Patients with SCAD incur substantial healthcare utilization and costs, which varies and may be predicted by 5-year CVD risk profile. Higher risk patients have higher initial but lower lifetime costs than lower risk patients as a result of shorter life expectancy. Improved cardiovascular survivorship among an ageing CVD population is likely to require stratified care in anticipation of the burgeoning demand. Patients with SCAD might be considered to have ‘fallen off the radar’ of clinical interest: no longer in cardiac rehabilitation [mainly offered to those immediately after AMI or coronary artery bypass grafting (CABG)], discharged from ongoing specialist care, and with suboptimal drug compliance, adherence, and persistence.7 Such patients, however, vary widely in their risk of subsequent AMI or coronary death ,8 which will clearly have differential resource implications.Improved survival coupled with a decline in the incidence of acute myocardial infarction (AMI)10 there is a paucity of information about the contemporary use and associated costs of healthcare beyond the initial hospital stay. In addition, existing studies in the area have been limited in a number of ways. First, as a result of the nature of their samples, they are not population based and do not reflect contemporary and routine clinical practice.11 Second, they use overly simplistic models, typically restricting their analysis to a subset of SCAD index events and not evaluating how the pattern of healthcare resource use changes following a first post-SCAD myocardial infarction (MI) or stroke.10 Third, no study has evaluated resource utilization and costs according to baseline cardiovascular risk, despite the importance of this information in improving decision-making and ensuring more efficient use of limited healthcare resources. Fourth, longer term and particularly lifetime implications of SCAD have not been quantified.While previous studies of resource use and cost have taken as a starting point AMI,These knowledge gaps have a number of important ramifications. They create uncertainty for those who need to forecast future care needs, restrain the research and development of new technologies and treatments for SCAD, and limit informed clinical decision-making. To address these limitations, our study aimed to (i) determine healthcare utilization and the associated costs in the first year with SCAD and in the year following a first non-fatal event , (ii) study predictors of costs in the first year of SCAD, and (iii) estimate the 5-year and lifetime costs among patients at low and high risk of subsequent events and coronary heart disease (CHD) death.13 and overall mortality14 and have been validated for epidemiological research. A description of the CALIBER approach has been presented elsewhere.15 Classification algorithms combining Read, International Classification of Disease 10 (ICD-10), drug, and procedure codes to define risk factors and endpoints are available at http://www.caliberresearch.org/portal/.The ClinicAl research using Linked Bespoke studies and Electronic Records (CALIBER) e-health database was the data resource for this study. CALIBER links patient records from four different data sources: Clinical Practice Research Database (CPRD), Myocardial Ischaemia National Audit Project (MINAP) registry, Hospital Episodes Statistics (HES), and the Office for National Statistics. The data of CPRD were used to obtain heart rate measurements, demographic variables, and other risk factors. Primary care practices that provide valuable data to CPRD and cover ∼4% of UK population are representative in terms of demographic parameters such as gender, age, and ethnicity15 Patients were only eligible for the study during the period they were actively registered at a CPRD practice that was collecting up-to-standard data , with follow-up ending if a patient transferred out of a CPRD practice. Full details of the cohort are available elsewhere.8Eligible patients were those with a diagnosis of stable angina, patients with a diagnosis of ACS within the study period (unstable angina or AMI), or those with a diagnosis of CHD in which there is no further specification of whether it is angina or MI (other CHD). Study start date was calculated from the date of diagnosis of stable angina or other CHD or from 6 months after an ACS or coronary intervention. The period of 6 months was chosen to differentiate long-term prognosis from the high-risk period that typically follows an ACS or revascularization. Diagnoses were identified in CPRD, HES, or MINAP records according to definitions given in the CALIBER data manual.Healthcare utilization extracted from the data set included primary care consultations, pharmaceutical prescriptions, inpatient stays, and diagnostic tests. Primary care consultations included all contacts between the patient and healthcare professionals captured in the CPRD data set. Prescription data were available from the CPRD data set and distinguished between drugs that were cardiovascular disease (CVD)-related and those that were not. Inpatient stays extracted from HES were based on Health Resource Group (HRG) codes and defined as CHD, CVD (including CHD and broader HRGs), or non-CVD related based on ICD-10 codes. Diagnostic tests as recorded in the primary care data set but not outpatient consultations were also extracted, the latter being due to the absence of outpatient HES data linkage.16–18 For hospitalizations, costs are calculated based on finished consultant episodes in HES. Costs are presented in terms of total healthcare costs , CHD costs , and CVD costs .All costs were calculated from the perspective of the UK National Health Service (NHS) in pound sterling based on 2011/12 prices. Costs were calculated by combining healthcare utilization data from CALIBER with associated unit costs that were taken from published UK sources.Estimates of healthcare utilization and costs were calculated for the first year in the SCAD cohort and the first year following a non-fatal event during the follow-up period  with results reported as means and standard deviations, with medians and interquartile ranges reported in the appendices. Observations that were right censored for any reason other than mortality  were excluded from the analysis.et al.,8 which developed a prognostic model for SCAD patients, on the assumption that predictors of costs were likely linked to prognostic indicators. The covariates included the baseline diagnosis for entry to SCAD, co-morbidities, age, gender, smoking status, and biomarkers. Models were fitted on five multiply-imputed data sets and estimates combined using Rubin's rules.19 The impact of covariates has been transformed back onto the natural scale to allow for ease of interpretation.A generalized linear model with a log link and gamma distribution was used to estimate the impact of baseline covariates on costs in the first year in the SCAD cohort to account for the non-linear impact of covariates and the right skewness in cost data. Covariates were based on those used by Rapsomaniki 20 For the purpose of this article, the results of costs over time are presented for patients based on average covariate patterns for the 5-year risk deciles of a cardiovascular event. Discounted costs are also presented using a discount rate of 3.5% per annum.21Panel data methods with time invariant covariates were used to estimate patient costs over each 90-day period. Also estimated were the impact of events  on the costs in the period of the event and subsequent periods if the event was non-fatal. These costs were then combined with a state transition Markov model to estimate costs over a longer period. The model estimated the probabilities of, and mean times to, the first events of non-fatal AMI, ischaemic or haemorrhagic stroke, CVD- and non-CVD-related death, and subsequent CVD or non-CVD death following a non-fatal first event. These estimates were conditional on baseline covariates and were inferred from patient-level costs, covariates, survival, and events experienced. Full details of this model are available elsewhere with a brief description given in the appendix.Table A1.In total, 94 966 patients were identified who met the inclusion criteria, of which 44% were female. The mean age of men and women included were 67 and 72 years, respectively. For the primary SCAD diagnosis, 47.4% of patients had stable angina, 13.5% unstable angina, 6.7% ST-elevation MI (STEMI), 9.7% non-STEMI (NSTEMI), and 22.6% had CHD not otherwise specified. Full details of the cohort can be found in Appendix Figure 1 summarizes the SCAD cohort and the patient numbers used for each analysis.Table 1 reports healthcare utilization in the first year in the SCAD cohort and in the first year following a non-fatal event during the follow-up period . In the first year in the SCAD cohort, 20.5% of patients (n = 17 532) were hospitalized for CVD, and those who were hospitalized had a mean 1.9 stays (spells) in hospital with a mean length of stay of 4.6 days. In the year following a non-fatal event during follow-up, 66% of patients (n = 4354) were hospitalized for CHD. These patients spent a mean of 2.2 stays in hospital with a mean length of stay of 6.5 days. In the first year in SCAD, patients had a mean of 10.8 primary care appointments, and this increased to 13.7 in the first year following a non-fatal event. In the first year of SCAD, 88.2% of patients were taking cardiovascular medication, which decreased to 83.6% in the year following a non-fatal event. In the first year of SCAD, 5.7% of patients had a revascularization procedure, increasing to 13.5% of patients in the first year following a non-fatal event.Table 2 reports costs for hospitalizations, primary care appointments, diagnostic tests, and drugs in patients in the first year in the SCAD cohort, and in the first year following a non-fatal event during follow-up . The mean total healthcare costs in the first year in the SCAD cohort were £3133 per patient, of which 56.8% (£1780) and 70.2% (£2199) were related to CHD and CVD, respectively. This estimate increased to £10 377 per patient in the year following a non-fatal event during follow-up, of which 66.2% (£6869) and 85.9% (£8916) were related to CHD and CVD, respectively. The majority of healthcare costs were driven by hospitalizations .Figure 2 presents the results of the regression analysis showing the incremental costs associated with different covariates and the associated 95% confidence intervals (CI) for the first year in the SCAD cohort. Non-CVD-related co-morbidities had the largest impact on costs, with a history of renal disease associated with the largest increment of £1998 per patient (95% CI £1715–£2297). A history of heart failure resulted in an additional cost of £802 per patient (95% CI £683–£920). Of the baseline diagnoses for entry to the SCAD cohort, NSTEMI had the largest impact on cost with those patients with NSTEMI costing an additional £656 per patient (95% CI £473–£848) when compared with those with stable angina. Females were significantly less costly than males, being female was associated with a cost decrement of £549 per patient (95% CI −£638 to −£457). Figures A1 and A2 in the appendix present the same results for CVD- and CHD-related costs, respectively.Tables A4, A5, and A6 in the appendix provide estimates of total healthcare costs, CVD-related costs, and CHD-related costs, respectively, for a 90-day period based on a range of baseline characteristics. The tables also show the incremental costs in the period of an event and in subsequent periods for non-fatal events. For example, the background total healthcare costs for a male, mean age 69 years, with no co-morbidities would be £341 in the first 90 days, increasing by £10 for each subsequent 90-day period. If the patient had a non-fatal AMI, he would incur an incremental cost  of £5028 in the 90 days following the AMI with the incremental costs decreasing in subsequent trimesters until 360 days after which there is an incremental cost of £521 per 90 days suggesting significant ongoing lifetime costs of events. Also of note, the incremental costs in the period of death from CVD- and non-CVD-related causes were £2008 and £2240, respectively.Table 3 presents estimates of 5-year and lifetime costs  of the representative patients for each risk group. The covariate profiles used are shown in Appendix Table A7.A patient with SCAD representative of the lowest risk decile (5-year cardiovascular risk of 3.5% and a life expectancy of 26.8 years) would have expected undiscounted costs of £9335 over 5 years, of which 44.7 and 56.8% would be CHD and CVD related, respectively; and undiscounted lifetime costs of £116 888, of which 40.1 and 61.5% would be CHD and CVD related, respectively. A representative patient of the highest risk decile (5-year cardiovascular risk of 44.2% and a life expectancy of 5.51 years) would have expected undiscounted costs of £23 391 over 5 years, of which 53.0 and 72.9% would be CHD and CVD related, respectively; and lifetime undiscounted costs of £43 020, of which 51.9 and 72.5% would be CHD and CVD related, respectively.Figure 3 shows the predicted total, CVD-related, and CHD-related costs and the survival curves over a 25-year period for representative patients of risk deciles 1 (lowest risk), 4, 7, and 10 (highest risk). Higher risk patients with SCAD have higher initial costs, which are overtaken by the lower risk patients as a result of higher mortality in the higher risk groups resulting in less time to accrue costs .This study addresses a fundamental gap in knowledge relevant to clinicians and policymakers: what is the clinical, health service, and cost burden associated with SCAD? Using data from a large, contemporary, and representative population of patients from the England with SCAD, the analysis has shown that substantial healthcare costs are likely to be incurred as a result of improved ACS survivorship and the ageing population. Moreover, 5-year and lifetime costs vary according to CVD risk, which may be readily predicted from the baseline characteristics of patients that are routinely collected. High-risk patients have considerably higher costs over the initial 5 years but lower lifetime costs than lower risk patients as a result of shorter life expectancy.6 Our results clearly highlight the unmet need and the shortfalls of current approaches—with high use of primary care and frequent hospitalizations, there are considerable ongoing costs. The number of primary care consultations, a mean of 10.8 per patient in the first year in the SCAD cohort, is higher than previous estimates for the overall population (5.5 per year).22 In the first year in the SCAD cohort, over a third of patients were hospitalized (and 20.5% for CHD reasons). This is substantially higher than recent estimates for the general population in one area of the UK (14.9%),23 and markedly higher than that in the general population for a similar age .Patients with SCAD might be considered to have ‘fallen off the radar’ of clinical interest. Current guidelines give little information about how frequently and where such patients should be followed up or if and how they should be risk stratified.23 Very high costs in the first year following a non-fatal event during follow-up  (£10 377) are reflective of the healthcare utilization required to treat that event.Healthcare utilization is, however, insufficient as a metric of the impact on the healthcare system. It is also important to consider the cost imposed on the NHS associated with those resources as this indicates the value of resources that cannot be devoted to health-enhancing activities for other patients. Mean costs of £3133 in the first year in the SCAD cohort are much higher than those in patients without chronic conditions (£293), but comparable with other chronic conditions (e.g. diabetes £3036).23 yet clinicians tend to focus only on single diseases.24Patients were stratified by risk to understand the costs accrued in greater detail. This higher resolution analysis allows the identification of where novel treatments and health service interventions have the greatest potential to be cost-effective. Non-CVD co-morbidities were common and had a major influence on costs with e.g. renal disease resulting in a mean extra cost of £1998 per patient in the first year of SCAD. This is an important finding when the presence of multiple co-morbidities has been shown to increase costs11 However, this lower estimate may be reflective of less intensive use of healthcare resources in non-trial settings and should provide a more accurate representation of costs of these events in routine clinical practice. Estimated stroke costs in the first year of the event  were similar to those seen in other studies. For example, the OXVASC study estimated mean total healthcare costs per patient in the first year following stroke at £10524.25The panel data analysis examined the average cost per 90-day period with the disease and also the costs of events. The estimated incremental cost of a non-fatal MI over 1 year  was lower than some previous estimates from trial populations .Furthermore, a study of costs in the first year of SCAD and the first year following an event is of limited use to decision-makers who require more detailed information on the long-term costs and consequences of SCAD. This can be seen from the panel data analysis, which suggested ongoing long-term costs as a result of non-fatal events. By estimating 5-year and lifetime costs by CVD risk group, it was possible to examine the long-term cost implications as a result of the disease and future events. In the shorter term of 5 years, which is shorter than the life expectancy of even the highest risk group , costs increased with cardiovascular risk, and were largely driven by the high number of fatal and non-fatal events among these patients. Over a lifetime, however, patients in the lower risk groups eventually had substantially higher costs than higher risk patients, primarily driven by greater life expectancy and, therefore, costs being incurred over a much longer period. This is a key finding of our research: increased survivorship as well as an increasingly co-morbid and older population will result in significant future healthcare costs. In turn, this has implications for the cost and therefore the cost-effectiveness of established and new SCAD treatments.Many studies have attempted to address the burden of disease in terms of health losses but fewer have examined the impact on financial costs of disease. Our research used SCAD as an exemplar in estimating resource use and costs from ‘real world’ electronic health record data. The methods used here could be readily applied to other chronic diseases to help produce evidence of their resource and cost implications to better inform clinicians and decision-makers. This would reduce uncertainty for those who need to forecast future care needs and allow for better focused research and development of new technologies and treatments for these chronic diseases as well as resulting in better informed clinical decision-making.While our study has a number of strengths including the multi-source electronic health record linkage, there are a number of limitations. In addition to being censored at 2010, after which there have been further improvements in the care and survivorship of SCAD, a key weakness of this study was the exclusion of outpatient appointment costs that cannot currently be linked from HES. As a result, the total healthcare costs of this population are underestimated. Further disaggregation of primary care costs into CHD and CVD related was not possible and therefore in each category all primary care costs have been included and therefore are likely overestimated. The estimation of lifetime costs has also involved extrapolation over a longer time period than is currently observed in the CALIBER data. This extrapolation is subject to considerable uncertainty. The SCAD population is also inherently heterogeneous, and there may be value in further disaggregation of the population in future research.Using a multi-source electronic health record approach, this study provides, for the first time, estimates and predictors of contemporary national healthcare utilization and costs in the first year of SCAD and the first year following an event. It reveals that patients with SCAD incur substantial healthcare utilization and costs, which vary and may be predicted by 5-year CVD risk profiles. While high-risk patients incur substantially higher costs over the short term (5 years), low-risk patients incur higher lifetime costs as a result of greater life expectancy. Improved cardiovascular survivorship and an ageing UK population will require stratified care in anticipation of the burgeoning economic demand. The methods used here could be readily applied to other chronic diseases to better inform clinical decision-making.All authors contributed to the interpretation of the data and several drafts of the manuscript. S.W., M.A., A.M., S.P., and M.S. conducted the statistical analysis and developed the model used to estimate long-term costs. A.D.S., K.A., M.C., C.P.G., A.T., and H.H. provided advice throughout the process. A.T. and H.H. were responsible for the overall grant from the NIHR.


To the Editor: Influenza virus subtype H6 was first isolated from a turkey in 1965 in the United States , a representative isolate of predominant H6 viruses in mainland China, was used for the serologic testing  Table 1.2 test results, seropositivity among workers in live poultry markets was significantly higher than that among large-scale poultry farmers .The HI result was ≥20 for H6N2 virus in 298 of the 15,689 specimens, and the MN result was positive in 63 of the 298 specimens   Table 2.Seropositivity did not differ significantly among male and female persons tested (p = 0.08) . No chil2 test results, seropositivity in southern China was significantly higher than seropositivity in northern China (p = 0.0375)  .Human infection with influenza H6 virus in mainland China has not been reported, but 63 serum specimens tested in our study were positive for the H6 virus. This level of seropositivity is much higher than that for highly pathogenic avian influenza A(H5N1) virus, for which only 2 of the serum specimens we tested were positive (data not shown), but much lower than the seropositivity level for low pathogenicity avian influenza A(H9N2) virus; 3.4% of the samples tested were positive for A/Chicken/Hong Kong/G9/1997(H9N2)–like virus (data not shown). A previous US study has reported H6N2-positive antibodies in veterinarians  virus.


Diarrhea is one of the leading causes of childhood morbidity and mortality. Hospitalization for diarrhea can pose a significant burden to health systems and households. The objective of this study was to estimate the economic burden attributable to hospitalization for diarrhea among children less than five years old in Rwanda. These data can be used by decision-makers to assess the impact of interventions that reduce diarrhea morbidity, including rotavirus vaccine introduction.This was a prospective costing study where medical records and hospital bills for children admitted with diarrhea at three hospitals were collected to estimate resource use and costs. Hospital length of stay was calculated from medical records. Costs incurred during the hospitalization were abstracted from the hospital bills. Interviews with the child’s caregivers provided data to estimate household costs which included transport costs and lost income. The portion of medical costs borne by insurance and household were reported separately. Annual economic burden before and after rotavirus vaccine introduction was estimated by multiplying the reported number of diarrhea hospitalizations in public health centers and district hospitals by the estimated economic burden per hospitalization. All costs are presented in 2014 US$.Costs for 203 children were analyzed. Approximately 93% of the children had health insurance coverage. Average hospital length of stay was 5.3 ± 3.9 days. Average medical costs for each child for the illness resulting in a hospitalization were $44.22 ± $23.74 and the total economic burden was $101, of which 65% was borne by the household. For households in the lowest income quintile, the household costs were 110% of their monthly income. The annual economic burden to Rwanda attributable to diarrhea hospitalizations ranged from $1.3 million to $1.7 million before rotavirus vaccine introduction.Households often bear the largest share of the economic burden attributable to diarrhea hospitalization and the burden can be substantial, especially for households in the lowest income quintile. Diarrhea is the second leading cause of death for children less than five years old, causing approximately 751,000 childhood deaths in 2010 .Health insurance helps reduce the economic burden to households that can arise due to a household member’s illness. Health insurance coverage in Rwanda is high with over 90% of the population subscribing to Mutuelle de Santé, a community-based health insurance scheme (CBHIS). Insurance for the remaining segment of the population is covered by Rwandaise d’Assurance Maladie (RAMA) for civil servants, Military Medical Insurance (MMI) for members of the military and Caisse Sociale du Rwanda (CSR) for private sector employees .Even with health insurance, households still face an economic burden if the child is hospitalized, since health insurance does not cover 100% of the medical costs and households still face other costs . Few studies have estimated the economic burden of each inpatient admission for diarrhea borne by the health system and by households in African countries. In Ghana, the direct medical costs  were estimated to range from $65 to $97 .®, Merck Vaccines, Whitehouse Station, New Jersey) into the routine Expanded Program on Immunization (EPI). The last-dose national coverage rates for rotavirus vaccine were reported to be 99% in 2012 and 100% in 2013 [Rotavirus vaccination holds much promise for substantially decreasing the global health and economic burden of diarrhea ,10. In M in 2013 . As partAs cited above, there have been few studies in Africa assessing the economic burden of diarrhea –8, and lThe majority of Rwandans are enrolled in the CBHIS. Government and donor subsidies help finance the enrollment of poor and vulnerable populations which comprise 25% of the CBHIS subscribers. Annual membership costs for CBHIS range from 3,000 Rwandan Francs (RWF) which is approximately 4 United States dollars,  to 7,000 RWF (approximately $10). For the RAMA insurance for civil servants, the contribution rate is 15% of the basic salary of which 7.5% is paid by the employer and 7.5% by the employee. For MMI insurance for military families the contribution rate is 22.5% of gross salary, of which 17.5% is paid by the government and 5% by each military staff member. These other health insurance schemes donate a percentage of their annual earnings to CBHIS in order to sustain its activities [Under the various health insurance schemes, health services can be obtained from primary health facilities as well as from district and national hospitals, and from accredited private providers. Except in the case of emergency situations, the referral system must be followed for the medical expense to be covered by insurance. The health insurance package of benefits covers outpatient and inpatient care, maternity care, essential drugs, medical imaging, and laboratory tests. Households also contribute copayments and the level of copayment varies by specific service. For example, there is no copayment for assisted birth delivery and surgery. For other services the households pay either 10% or 15% while insurance pays 90% or 85% of the cost, respectively; this varies by the type of health insurance scheme. The Ministry of Health compiles a schedule of tariffs for each medical service, drug, and consumable which are then used by the providers to charge for services provided .The financial bill after a hospitalization is itemized for each type of medical service, drug, or consumable the patient received. Each line item in the bill includes the date, code in the tariff schedule, the type of service/drug/consumable received, the quantity received, the unit price, and the total cost for the line item. The bill also includes total costs to be paid by insurance and the OOP medical costs for the patient. Formal contracts are established between the health insurance schemes and the accredited public and private health care providers. Health care providers are reimbursed after they send a claim to the health insurance company .This was a prospective, cross-sectional costing study. Data were collected through a review of patients’ medical and financial records, which were obtained from each of the hospitals involved in this study and through household interviews with the caregivers of the patients enrolled in the study. Sample questionnaires contained in the World Health Organization’s guidelines for estimating the economic burden of diarrhea disease  were modA purposive sampling approach was used to select the hospitals included in the study. The three hospitals included in the study were: Muhima Hospital, a public district hospital located in Kigali, the capital city of Rwanda, which serves an urban population of approximately 285,000; Rwamagana Hospital, a public district hospital located in the Eastern Province, which serves a rural population of approximately 310,000; and Kigali Teaching Hospital, a referral hospital also located in Kigali, which serves a population of approximately 1 million. These hospitals were selected because they were study sites for the other post vaccine introduction studies that were being undertaken.Children enrolled in our study met three inclusion criteria: the child was less than five years old and presented to one of the study sites with non-bloody diarrhea as the primary reason for consultation; the child was admitted into hospital and treated with oral rehydration or intravenous fluids; and the caregiver of the child consented to having the child included in the study and signed the informed consent form.To inform sample selection, we used data on the number of non-bloody diarrhea hospitalizations which we obtained from the Rwanda Health Management Information System (HMIS). The HMIS is a health reporting portal and all public health centers and district hospitals in Rwanda submit monthly reports on specified health indicators into the HMIS. The HMIS does not include data from the national referral hospitals or private health facilities . Using tThis study was approved by the Rwanda National Ethics Committee and the PATH Research Ethics Committee. Data collection occurred between November 2013 and June 2014.The study included three data collectors, one assigned to each of the study sites. Each study site also had a focal nurse who identified children eligible for the study and alerted the data collector about the admission of a child meeting the study’s enrollment criteria. The data collector’s first interaction with the child’s caregiver (parent or guardian) occurred while the child was still hospitalized. During this encounter the data collector explained the study to the caregiver and asked them if they wanted the child to be enrolled into the study. If the caregiver agreed, they had to provide written consent. The data collector then returned to the hospital after the child was discharged and extracted information from the child’s medical record. Information extracted from the medical record included: the date of admission and date of discharge, outcome on discharge, diagnoses made, diagnostic tests done, and medications administered while hospitalized and prescribed for use after discharge. The data collector also visited the hospital’s accounting department to collect information on financial bill for the hospital stay. The hospital financial bill was itemized, including information on services received, quantity and unit costs of resources used, and total charges. The data collector extracted the charges and grouped them into three categories: medications, diagnostic tests, and all other hospital charges . These categories were clearly defined on the hospital bill.The data collector followed up with the caregiver between 7 and 14 days after the child was discharged to interview them about the costs of care before the child was hospitalized and after the child was discharged. The data collectors visited most of the caregivers in their homes and conducted in person interviews, but a few caregivers were interviewed by phone. The caregiver reported the costs incurred for any medications, diagnostic tests, and consultations before and after the child’s hospitalization. They also collected household OOP costs incurred because of the child’s illness and obtained other socioeconomic information about the household. Household OOP costs included transport costs for the caregiver and child on the day the child was hospitalized and transport and accommodation costs for household members who visited the child while he/she was hospitalized. Information was also collected on the type of activities the caregiver would have been doing had the child not been hospitalized, their profession, daily or monthly income, and number of days of income lost because of taking care of the sick child. Visitor data, including their profession, number of days of income lost, and daily or monthly income was also collected. Data to characterize the household was obtained, including household monthly income, type of toilet facility, and source of drinking water.All data were entered into a patient-level database in Excel . Costs were entered in RWF and an exchange rate of 668 RFW per US$ was used to convert all costs to US$ . Data anExcluded from the study were children who died, children whose caregiver absconded from the hospital without paying the medical bills, and children whose caregivers later chose to withdraw from the study after initial consent.Direct medical costs incurred before, during, and after hospitalization for the diarrhea episode were calculated for each child as:Some of the caregivers did not have bills for the outpatient care the child received before and after hospitalization. These caregivers self-reported the costs of care and we asked them to also report the amount they paid and the amount paid by insurance.The direct non-medical costs borne by the household were estimated as:where i = 1,2,….n, where n is the number of household members with lost income.Household income lost due to the child’s illness were estimated as:Household members reported either a monthly salary or a daily wage. In order to convert the monthly salaries into daily wages, we assumed that there were 22 work days per month and divided the monthly salary by 22 to estimate the daily wage rate.Descriptive statistics to characterize the child and the child’s household were presented as the percentage for each study site. We also estimated the average length of stay (LOS) in hospital and the standard deviation. Mean, median and standard deviation of the costs were reported. We also calculated the 95% confidence intervals for the costs borne by insurance and households. Hypothesis tests (to test if the mean LOS and costs for each study site were different from the mean costs for the pooled data) were conducted using Students t-test, with p <0.05 showing significant difference for the two-tailed test. In addition, we calculated the household costs  as a percentage of the household monthly income and reported these statistics by income quintile for our sample.Using data on the annual number of diarrhea hospitalizations obtained from the HMIS , we estiThe annual economic burden borne by the health insurance payers was estimated as:Where t is the year for which the number of hospitalizations is reportedThe annual economic burden borne by the households was estimated as:The annual economic burden borne by insurance and households were then summed to estimate the annual economic burden for all payers for admissions to health centers and district hospitals. These annual economic burden calculations were also done using the lower and upper bounds of the 95% confidence intervals of the average costs per diarrhea illness resulting in a hospitalization.A total of 214 children were enrolled in the study; the final sample used in the analysis was 203, as some children were discontinued from the study because they died (n = 5), they absconded from the hospital and therefore had incomplete medical or financial data (n = 5), or their caregivers chose to withdraw them from the study (n = 1). Enrollment targets were met for Muhima and Rwamagana District Hospitals but not for Kigali Teaching Hospital. The low enrollment number at Kigali Teaching Hospital was due to very low numbers of children presenting with diarrhea as this is a referral hospital, and the fact that sections of the pediatric unit were closed for renovations during part of the data collection period.The sample description is shown in Mothers were the main caregivers (96%) for the children during hospitalization and the majority of the caregivers (65%) reported that their highest level of education was primary school. The two most common activities by the caregivers were cultivating/farming (24%) and housework (22%).About 70% of the children had been ill for 1 to 3 days before hospitalization and 80% of them had received care from a health center or community health worker before hospital admission (data not shown in tables). The mean LOS in the hospital was 5.3 days for the pooled sample and the median LOS was 4 days. Only the mean LOS at Kigali Teaching Hospital (2.5 days) was significantly different from the mean LOS for the pooled sample (p = 0.04), most likely because of the small sample from this hospital. Only 12 (6%) of the children received outpatient care after being discharged from the hospital.For the 80% of children who sought care from a health center or community health worker before hospitalization, the average medical costs of this outpatient care were $6.12 ± $4.32 with the largest share of the costs being consultation fees. The average medical costs for the 12 children who sought care after being discharged were $3.94 ± $4.56. When averaged across the entire sample, the average medical costs for care received before and after hospitalization were $5.30 ± $4.62.Medication costs were estimated at $14.79 for the pooled sample; there were no significant differences in the costs for the individual sites compared to the pooled data. The most common medications prescribed to each child in addition to rehydration solution were vitamins (64%), and antibiotics (62%). Other costs, including consultations, bed day costs, and consumables, were the largest share of costs .Total direct medical costs for the diarrhea illness were estimated at an average of $44.22 ± $23.74 (median $37.68) for the pooled sample. There were no significant differences in the total direct medical costs for each site compared to average costs for the pooled sample. However, the median costs for Kigali Teaching Hospital were almost $20 higher than median costs for the other two hospitals, despite the shorter length of stay for children admitted to this hospital. Insurance covered almost 80% of the costs, and median OOP direct medical costs ranged from $4.42 to $6.58 per household across the three hospitals.For each diarrhea illness that resulted in a hospitalization, we estimated the economic burden to be $101 per illness, which includes $44.22 in direct medical costs, $14.85 in direct non-medical costs and $41.78 in lost income. Insurance bore $34.83  or approximately 80% of the direct medical cost while households bore the remaining 20% of the direct medical costs. When including all the costs that are borne by the households we found that the households bore $66.08  which was 65% of the economic burden per diarrhea illness that resulted in a hospitalization. In the four years prior to rotavirus vaccine introduction, there were between 13,153 and 17,254 diarrhea admissions in public health centers and district hospitals in Rwanda. The estimated annual total economic burden attributable to these diarrhea admissions ranged from approximately $1.3 million to $1.7 million, when using the average costs . UnpubliAlthough previous studies have also evaluated the economic burden attributable to diarrhea hospitalization –8, 14–19Most Rwandans have insurance 93%), which covered approximately 80% of direct medical costs for a diarrhea illness that resulted in a hospitalization, leaving households responsible for approximately 20% of these costs. The economic burden resulting from a child’s diarrhea illness which results in hospitalization accounts for between 21% and 110% of the household’s monthly income, with the largest burden falling on the households in the lowest income quintile. Therefore, these costs can be substantial especially for low income households. Other studies have characterized the heavy economic burden of diarrheal disease in low-income households 3%, which, 20–21.Before rotavirus vaccine introduction, hospital admissions due to non-bloody diarrhea among children less than five years old had cost Rwanda an annual estimated amount of $1,327,269 or the equivalent of $0.74 per child less than five years old using 2010 population data . The majThis study found that the total cost, including direct medical costs, indirect medical costs, and lost income, associated with a diarrhea illness that results in a hospitalizations is similar (no different than the pooled average) across rural, urban, and referral district hospitals. Therefore, this economic cost data could be extrapolated to all diarrheal hospitalizations at referral hospitals and used in models to project costs averted as a result of rotavirus vaccine introduction as well as used as an input in cost-effectiveness studies.This study also highlights the value of a well-functioning health insurance system. The availability of financial bills for each patient in our sample reduced or eliminated the likelihood of our study underestimating the direct medical costs, a risk that can arise when researchers have to abstract the quantities of resources used in each hospitalization, from paper medical records, to estimate the costs. These paper medical records can be incomplete or illegible, which then becomes challenging for researchers to account for all the resources used in the care of the patient.This study has several limitations. The direct medical costs reported in this study may underestimate the total costs of care for children with diarrhea because health services are subsidized by the Government of Rwanda, and therefore the data reported in the financial bills may not reflect the full cost of care for each diarrhea case. Also, the study did not estimate facility overhead costs or infrastructure costs, which may not be included in the financial bills, although some of these costs may be included in the bed-day costs. In addition, this study did not consider costs associated with hospitalizations at health clinics, which according to a prior study accounted for 60% to 72% of diarrhea-related hospitalizations in Rwanda . If the In conclusion, children with diarrhea were hospitalized for an average of five days. The economic burden per diarrhea illness that resulted in a hospitalization was $101 and 65% of this cost was borne by households. All the costs we estimated did not significantly differ across the three hospitals included in our study. The average annual economic burden to Rwanda from these hospitalizations ranged from $1.3 million to $1.7 million for the period just prior to the introduction of the rotavirus vaccine in the national immunization program, and declined by approximately 20% to 40% in 2014, 2 years after vaccine introduction, even though the entire reduction cannot be attributable to the vaccine introduction.


Anopheles gambiae complex display strong preference for human bloodmeals and are major malaria vectors in Africa. However, their interaction with viruses or role in arbovirus transmission during epidemics has been little examined, with the exception of O’nyong-nyong virus, closely related to Chikungunya virus. Deep-sequencing has revealed different RNA viruses in natural insect viromes, but none have been previously described in the Anopheles gambiae species complex. Here, we describe two novel insect RNA viruses, a Dicistrovirus and a Cypovirus, found in laboratory colonies of An. gambiae taxa using small-RNA deep sequencing. Sequence analysis was done with Metavisitor, an open-source bioinformatic pipeline for virus discovery and de novo genome assembly. Wild-collected Anopheles from Senegal and Cambodia were positive for the Dicistrovirus and Cypovirus, displaying high sequence identity to the laboratory-derived virus. Thus, the Dicistrovirus  and Cypovirus  are components of the natural virome of at least some anopheline species. Their possible influence on mosquito immunity or transmission of other pathogens is unknown. These natural viruses could be developed as models for the study of Anopheles-RNA virus interactions in low security laboratory settings, in an analogous manner to the use of rodent malaria parasites for studies of mosquito anti-parasite immunity.Mosquitoes of the  Flaviviridae, Togaviridae, Bunyaviridae, Rhabdoviridae, Mesoniviridae) and the taxon Negevirus have been described in Anopheles mosquitoes [Insect specific RNA viruses from various families , a pathogenic arbovirus transmitted to humans [Aedes mosquitoes [Anopheles has been limited to ONNV infections [Anopheles RNA viruses that could be used as low-biosecurity model systems would facilitate studies of Anopheles antiviral immunity and mosquito-virus interactions.Among o humans , which isquitoes , 22. Confections , 23–27, Anopheles colony where they were discovered. Oral infection experiments were used to evaluate the species-specificity of these newly discovered viral strains and their ability to be transmitted to the progeny. Finally, we detected both viruses in wild Anopheles mosquitoes captured in Cambodia and in Senegal, confirming that they are components of the natural virome.In the current study, we used small RNA deep-sequence datasets to reconstruct more than 90% of a novel Dicistrovirus, and several segments of a previously unknown Cypovirus. We verified their infection prevalence within the Anopheles colony, widely used to study vector interactions with different pathogens, carries previously undetected natural insect viral infections that could potentially influence the results of such studies. These novel insect viruses also could become valuable tools to study Anopheles antiviral immunity, as well as the underlying mechanisms of vector interactions with RNA viruses, without the requirement for high biosecurity infrastructure or procedures.The results indicate that a laboratory An. coluzzii (Ngousso colony) mosquito midguts were analyzed using the Metavisitor pipeline [Cypovirus, sub-family: Spinareovirinae, family: Reoviridae), and a Dicistrovirus .Four datasets of small RNA sequence generated from pooled pipeline , a suitepipeline . Two novCulex restuans Cypovirus (CrCPV)[Uranotaenia sapphirina Cypovirus (UsCPV)[We detected a transcript (accession number KU169880) of 892 nt aligning to segment 10 of s (CrCPV) and to Us (UsCPV) with 91%s (UsCPV) indicateBombyx mori Cypovirus 1 (ACX54961.1) over 98% of its sequence. Realignment of the original small RNA reads to the contig shows a dicing profile with the read length peaking at 21nt in all four datasets (A second contig of 3889 nt was reconstructed (accession number KU169879) containing an ORF coding for a 1246 aa protein that has 34% identity with the RNA dependent RNA polymerase (RdRp) of datasets . This isCypovirus species (Anopheles Cypovirus (AnCPV). We hypothesize that AnCPV is a segmented (10 to 11 segments) double stranded RNA virus of the genus Cypovirus, sub-family Spinareovirinae, family Reoviridae. Based on the phylogenetic tree on the polyhedrin gene, this virus could be a novel member of the Cypovirus type 17 [Phylogenetic analysis of the predicted polymerase and polyhedrin peptide sequences were performed using the available sequences for the different  species . Neighbo type 17 .An. coluzzii larval midguts displayed gross pathological signs of Cypovirus infection, detectable as localized clusters of tightly packed inclusion bodies in the midgut epithelium that appear visually as iridescent blue-white features [Light microscopy of features  (white aDrosophila C virus (DCV) and Cricket paralysis virus (CrPV) from the Distroviridae family. Using additional long paired-end RNAseq datasets (accession number ERS977505), we were able to reconstruct a 8919 nt viral genomic sequence that completely includes these contigs and contains two ORFs (accession number KU169878). The first ORF (5’ ORF) codes for an 1802 aa (nucleotides 391 to 5799 in the reconstructed genome) polyprotein with 48% blastp identity to DCV nonstructural polyprotein. The second ORF (3’ ORF) codes for a predicted 852 aa protein (nucleotides 6067 to 8625 on the reconstructed genome) and displays 54% blastp identity to DCV structural polyprotein. This dicistronic organization of the reconstructed genome is typical of Dicistroviridae with an ORF1 that encodes for non-structural proteins and an ORF2 that encodes for virion proteins . DicistRealignment of the small RNA reads on the reconstructed genome revealed, as for AnCPV, a dicing profile of virus replication intermediates, yielding virus-derived siRNAs with a read length peaking at 21 nt in all four datasets . These rDicistroviridae shows that the discovered virus is related to known Dicistoviruses and groups with the Cripavirus genus (Anopheles C virus (AnCV).Phylogenetic tree reconstruction of non-structural proteins from us genus . Similarus genus . Becausep-value of 9.4e-33 for the RF0458 CrPV IRES family  of the same class and family as observed in other Dicistroviruses in order to translate the second ORF. Searching the RNA family database (Rfam) , we idenS family . MultiplS family  showed RS family  and covaS family . These rCloned virus fragments were used to synthesize RNA probes for hybridization to a Northern blot of the total RNA used for small RNA sequencing . The proAn. coluzzii colony, we examined individual mosquitoes and individual mosquito organs by RT-PCR using virus-specific primers  and An. gambiae (Mbita) tested by RT-PCR were consistently infected with both viruses, but the significance of the observation is unclear because they were all maintained in the same insectaries as the Ngousso An. coluzzii colony at Institut Pasteur Paris. Samples tested from a colony of An. dirus (Pursat) maintained at Institut Pasteur Cambodia were also positive for both viruses, but the history of other insectaries where the colony was previously maintained is unknown .Other colonies of An. stephensi (SDA500) maintained in the same Institut Pasteur Paris insectary as the An. coluzzii Ngousso colony, nor in two Aedes aegypti isofemale lines. Since oral transmission is the natural route of DCV infection [An. stephensi and the two Ae. aegypti lines and divided first instar larvae of each into two trays. One tray of larvae was supplemented with homogenized An. coluzzii Ngousso fourth instar larvae, carrying AnCV and AnCPV viruses, while the other tray of larvae was fed normally and maintained in the same insectary as a control. An. stephensi and Ae. aegypti fourth instar larvae and adult females were then collected and assayed for AnCV and AnCPV as pools or individually. Larvae were washed in multiple changes of clean water and starved over several hours before processing in order to minimize the possibility of external contamination or midgut content contamination. This per os exposure did not infect the Ae. aegypti mosquitoes, but successfully infected An. stephensi mosquitoes with both AnCV and AnCPV at a prevalence of 59% and 100% respectively, while mosquitoes issued from the control trays remained negative  were allowed to blood feed and lay eggs (F1 generation). Both F1 larvae as well as resulting adult females displayed AnCPV and AnCV infections  and their exclusion from a colony of one other species (An. stephensi). Because of the possibility of horizontal transmission in the insectary, data from colonies are not informative for infection in nature, but it does at least indicate the species range of permissive hosts. Importantly, the viruses were present in wild Anopheles mosquitoes captured in Senegal and Cambodia, thus establishing them as components of the natural virome.By deep sequencing of small RNAs we have identified two novel insect viruses, a Cypovirus and a Dicistrovirus, in a laboratory colony of Anopheles Cypovirus (AnCPV), which contains a genomic segment coding for the polyhedrin protein characteristic of this viral family and a genomic segment coding for an RNA-dependent RNA polymerase that shows conservation with the genomes of known Cypoviruses. This is, to our knowledge, the first reported member of the Cypoviridae family infecting Anopheles mosquitoes, complementing previous studies identifying Cypoviruses in other mosquito genera [Anopheles C virus (AnCV) that displays genetic architecture consistent with other members of the family Dicistroviridae, with two open reading frames, 5’ non-structural polyprotein and 3’ structural polyprotein, as well as conserved RNA secondary structure and sequence of the central IRES. This virus was further confirmed to belong to the Dicistroviridae family based on the amino-acid sequence conservation of the AnCV RNA-dependent RNA polymerase with its orthologs of the Drosophila C virus (DCV) and Cricket paralysis virus (CrPV).We identified o genera , 31. We Anopheles but not Aedes mosquitoes, and appears not to induce high mortality nor detectable paralysis phenotypes in mosquitoes infected per os. These data suggest that infection phenotype and range of this virus resemble that of DCV and not CrPV. We cannot exclude that in our model, the virus and the laboratory colony could have co-evolved to minimize pathogenic symptoms. The virus-negative An. stephensi colony, when infected with AnCV per os, also did not display detectable pathogenic symptoms. This scenario could be tested with isolated viruses by infection experiments using different concentrations of virus to study symptoms and pathogenicity. Isolated and culturable viruses would also allow studies of virion morphology, mechanisms of entry, host range, obtaining complete genomic sequences, and generating infectious clones. The main mosquito organs were found to be positive for both viruses. Development of a quantitative real-time PCR assay would be useful in order to measure genome copies in different organs.The novel Dicistrovirus AnCV can infect some DCV, first isolated in 1972 , and CrPAnopheles mosquitoes as the model insect virus DCV has for Drosophila. For example, DCV leads to a strong immune response and to fly death within several days when intra-thoracically injected [It will be interesting to determine whether AnCV has similar phenotypes for injected , but proinjected . Althouginjected .An. gambiae species complex is an initial step that will facilitate work on mosquito-virus interactions and anti-viral immunity, including the influence of the microbiota as well as potential reciprocal interactions with other pathogens such as malaria parasites.The first infectious clone of a Dicistrovirus, CrPV , was recAn. coluzzii Ngousso colony previously established from Yaoundé, Cameroon [An. gambiae M molecular form. Other colonies used and raised in the Institut Pasteur Paris insectaries were An. gambiae Mbita from Kenya , Fd03 initiated from Mali, Fd05 and Fd09 from Burkina Faso, and An. stephensi colony SDA500. In addition, two Aedes aegypti isofemale lines from Thailand were used [An. dirus Pursat colony reared in the insectaries of Institut Pasteur Cambodia, and never grown at Institut Pasteur Paris, were also used.Deep sequencing of small RNAs used mosquitoes from the Cameroon , and forere used . All mosere used . FinallyAnopheles mosquitoes were used for testing of virus presence in nature. Mosquitoes were captured in Ratanakiri and Kampong Chnang, Cambodia and Kedougou, Senegal were visually identified as Anopheles spp., and immediately transferred into RNAlater at 4°C, and then stored at -80°C. Permission to collect mosquitoes was obtained by Institut Pasteur Cambodia from authorities of Ratanakiri and Kampong Chnang, and by Institut Pasteur Dakar from authorities of Kedougou. RNA was extracted from individual mosquitoes using the Nucleospin miRNA kit (Macherey-Nagel) following the supplied protocol. RT-PCR assays for AnCV and AnCPV amplicons were performed as described below (using 45 PCR cycles), with blank reactions and negative control RNA samples. Amplicons from positive samples were Sanger sequenced and aligned with virus sequences assembled from deep sequencing of An. coluzzii Ngousso mosquito RNA. SNPs were verified using the trace files.Wild An. coluzzii Ngousso colony mosquito midguts were homogenized in TRIzol (Ambion) and total RNA was extracted following the supplied protocol. Small-RNA libraries were prepared from the small RNA fraction using TruSeq Small RNA Library reagents (Illumina). Libraries were sequenced on an Illumina Hiseq 2500 in a multiplexed 51 +7 bases single read using a TruSeq SR cluster kit v3 cBot HS, and a TruSeq SBS kit v3 HS 50 cycles (Illumina). Primary analysis of the sequences was performed with Casava software (v1.8 Illumina). Library preparation and sequencing was performed by ARK Genomics at the Roslin Institute (University of Edinburgh). Data are available in the EBI European Nucleotide Archive (http://www.ebi.ac.uk/ena/) under study accession number ERP012577 and sample accession numbers: ERX1180340, ERX1180341, ERX1180342 and ERX1180343.Pools of 30 http://www.ebi.ac.uk/ena/) under accession numbers: ERX1220645 and ERX1220646.Long paired-end RNA libraries were prepared and sequenced by the Institut Pasteur Transcriptomics and Epigenomics core facility using standard long paired-end rapid run protocol on an Illumina Hiseq 2500. Primary analysis of the sequences was performed with Casava software (v1.8 Illumina). Long paired-end sequencing data are available in the EBI European Nucleotide Archive , and R-chie (http://www.e-rna.org/r-chie) in “single covariance” mode to plot the covariance between the nucleotides in the structure [Using the structure of the IRES RF0458 family as a scaffold and the multiple alignment between the seeds of this IRES family and the potential IRES sequence of AnCV  ..http://wRNA from individual mosquitoes, individual organs, saliva or pools of either was extracted using either TRIzol (Ambion), NucleoSpin RNA II or NucleoSpin 96 RNA kits (Macherey-Nagel) according to the supplied protocol. RT-PCR was performed using Superscript One-Step RT-PCR kit (Invitrogen), primer  annealinin silico reconstructed contigs was observed. RNA probe hybridization to the Northern blot was performed at 68°C.RNA migration and Northern blotting used standard procedures. Northern blot sense and antisense probes were synthesized by SP6 transcription of the PCR fragments incorporated in the PGEM-t easy vector. Multiple vector clones were verified by classical Sanger sequencing and no variation from the http://serialbasics.free.fr).Basic multiple alignment of the polyhedrin segment sequences was performed using Clustal O (v1.2.1) and sequence accession numbers DQ212785.1 and AY876384.1. Basic amino acid comparison for the polyhedrin protein of AnCPV  was performed against ABA61432.1 and AAW80667.1 using Serial Cloner  were used to translate nucleic acid sequences to the corresponding peptide sequences.MUSCLE multiple alignment and evolutionary analyses were conducted in MEGA  with 1000 bootstrap replicates. Because non-conserved regions could possibly influence the phylogenetic tree reconstructions, gaps were removed using the 'Complete Deletion' option in MEGA. Phylogenetic reconstructions using neighbor joining (NJ), maximum parsimony (MP) or maximum likelihood (ML) showed the same topology for each dataset. Trees constructed with partial deletion are also included . EMBL-EBThe multiple alignments for Dendrolimus punctatus cypovirus 22), NP_149147.1 (Lymantria dispar cypovirus 1), ABB51571.1 (Heliothis armigera cypovirus 14), AF291683_3 (Trichoplusia ni cytoplasmic polyhedrosis virus 15), DQ192251.1_1 (Operophtera brumata cypovirus 19), AJC97790.1 (Thaumetopoea pityocampa cypovirus 5), AHJ14791.1 (Inachis io cypovirus 2), ACA53380.1 , ADH10220.1 (Antheraea mylitta cypovirus 4).Accession numbers for the RdRp alignment: YP_009111328.1 (Heliothis armigera cypovirus 8), AJC97797.1 (Thaumetopoea pityocampa cypovirus 5), NP_066381.1 (Trichoplusia ni cypovirus 15), ABB17224.1 (Operophtera brumata cypovirus 19), AAY96324.1 (Heliothis assulta cypovirus 14), ACX54964.1 (Bombyx mori cypovirus 1), ABB17220.1 (Operophtera brumata cypovirus 18), AAW80667.1 (Uranotaenia sapphirina cypovirus 17), YP_009002596.1 (Inachis io cypovirus 2), ABW87644.1 , ABH85367.1 (Simulium ubiquitum cypovirus 20), AAP44510.1 (Antheraea proylei cypovirus 4), AIY60608.1 (Dendrolimus punctatus cypovirus 22).Accession numbers for the polyhedrin alignment: AAA50751.1 (Anopheles C virus), NP_647481.1 , NP_044945.1 (Drosophila C virus), AF486073_1 , YP_008888535.1 (Formica exsecta virus 1), YP_164440.1 (Solenopsis invicta virus 1), NP_851403.1 (Kashmir bee virus), ABY57949.1 , NP_620555.1 , AGF84786.1 , AAT81157.2 (Taura syndrome virus), NP_046155.1 , YP_006666503.1 (Macrobrachium rosenbergii Taihu virus), YP_004063985.1 (Mud crab dicistrovirus), AGW80519.1 (Himetobi P virus), NP_620564.1 (Black queen cell virus).The amino acid sequences used for the multiple alignments for S1 Fig(A) Maximum likelihood tree of RNA-dependent RNA polymerase (RdRp) sequences of Cypoviruses, (B) Maximum likelihood tree of Polyhedrin sequences of Cypoviruses. Multiple protein alignment was done using the partial deletion option in MEGA. ML bootstrap values (above 0.75) are indicated above branches.(PDF)Click here for additional data file.S2 FigDicistroviridae family are indicated on the tree.Neighbor-joining tree of non-structural polyprotein sequences of Dicistrovirus. Multiple protein alignment was done using the partial deletion option in MEGA. NJ bootstrap values above 0.75 are indicated above branches. The two genera of the (PDF)Click here for additional data file.S3 FigF and R suffix represent respectively forward and reverse sequencing orientations. Sanger sequencing raw files are in S3 Zipped Archive.(PDF)Click here for additional data file.S4 FigF and R suffix represent respectively forward and reverse sequencing orientations. Sanger sequencing raw files are in S3 Zipped Archive.(PDF)Click here for additional data file.S1 File(XML)Click here for additional data file.S2 FileThese are the inputs for the R-Chie and TRANSAT online tools.(TXT)Click here for additional data file.S3 File(TXT)Click here for additional data file.S4 File(TXT)Click here for additional data file.S5 File(TXT)Click here for additional data file.S6 File(XLSX)Click here for additional data file.S1 TableOnes and zeros highlight RT-PCR positive and negative results, respectively. Yellow color represents AnCV and AnCPV negative mosquitoes while blue and green colors highlight mosquitoes positive only in the midgut or carcass for either virus.(XLSX)Click here for additional data file.S2 TableFinal suffix indicates forward, F, or reverse, R, sense of primers.(DOCX)Click here for additional data file.S1 Zipped Archive(ZIP)Click here for additional data file.


Both the range of motion (ROM) technique and the tibial tubercle landmark (TTL) technique are frequently used to align the tibial component into proper rotational position during total knee arthroplasty (TKA).The aim of the study was to assess the intra-operative differences in tibial rotation position during computer-navigated primary TKA using either the TTL or ROM techniques. The ROM technique was hypothesized to be a repeatable method and to produce different tibial rotation positions compared to the TTL technique.A prospective, observational study was performed to evaluate the antero-posterior axis of the cut proximal tibia using both the ROM and the TTL technique during primary TKA without postoperative clinical assessment. Computer navigation was used to measure this difference in 20 consecutive knees of 20 patients who underwent a posterior stabilized total knee arthroplasty with a fixed-bearing polyethylene insert and a patella resurfacing.p < 0.001). The trial tibial baseplate was on average 4.56 degrees externally rotated compared to the tubercle landmark. This difference was statistically significant (p = 0.028). The amount of maximum intra-operative flexion and the pre-operative mechanical axis were positively correlated with the magnitude of difference between the two methods.The ROM technique is a repeatable method with an interclass correlation coefficient (ICC2) of 0.84 (It is important for the orthopaedic surgeon to realise that there is a significant difference between the TTL technique and ROM technique when positioning the tibial component in a rotational position. This difference is correlated with high maximum flexion and mechanical axis deviations. Rotational alignment of the components in total knee arthroplasty (TKA) is an important factor for both survival and the performance of the prostheses , 2. The However there is more discussion about the rotational alignment of the tibial component in part because of the difficulty of clinically assessing tibial component rotation. Furthermore, a whole range of anatomical landmarks can be used, including the medial border of the tibial tuberosity, the medial third of the tibial tuberosity, the anterior tibial crest, the posterior tibial condylar line, the second ray and the first web space of the foot. Aligning the tibial component to the tibial tubercle is one of the most popular landmark methods –9. The dSeveral studies have demonstrated variability in the relationships between different landmarks and techniques for establishing rotational alignment of tibial components in total knee arthroplasty , 16–18. A prospective, observational study of 20 consecutive primary posterior stabilized TKAs in 20 patients with fixed-bearing polyethylene inserts  was performed by a single surgeon (MvS).Data collection began with 10 consecutive TKAs to determine whether there was a difference between the alignment techniques and if so to gather data to perform a power analysis. Using the acquired data, we determined that a total of 20 subjects were needed to achieve 90 % power assuming a minimum detectable difference of 5.0 degrees, a standard deviation of 7.7 degrees and a significance level of alpha < 0.05.N = 15) was 7.0° ± SD 4.05, of the valgus knees (N = 5) was −6.4° ± SD −4.15.The mean pre-operative mechanical leg axis was 3.65° ± SD 7.15 of varus. The mean pre-operative mechanical leg axis of the varus knees , therefore the patient was not required to provide consent regarding the use of the material.Furthermore, every patient in the Maastricht University Medical Centre is provided with information regarding these kinds of studies. If they do not wish to contribute to these studies, this information will be included in their file. The patient involved in this study did not make an objection against the use of his/her material for research purposes.The patient demographics are summarized in Table The Stryker Knee Navigation System  was used in this study. The prosthesis  used in these surgeries allows five degrees of rotation between the tibial insert and the femoral component. In all cases, a tourniquet was applied for the entire duration of the surgery. After a standard midline skin incision and a medial parapatellar arthrotomy, the active wireless trackers of the navigation system were fixed to the femur and tibia. The required landmarks were entered into the navigation computer, and the rotation centre of the hip was determined by a special algorithm executed in customized software. The transepicondylar axis of the distal femur and the Whiteside’s line were set in each case exactly perpendicular to each other to improve the accuracy of the positioning the femoral component. The femoral component was aligned parallel to the transepicondylar axis. The AP axis of the proximal tibia was determined by placing the tip of the pointer on the centre of the line between the intercondylar eminences and aligning it to the medial 1/3 of the tibial tubercle . This AP axis was saved in the navigation program as 0 degrees of rotation. The proximal tibial and distal femoral cuts were performed and examined with the navigation system. The tibial posterior slope was set according to the patient’s natural slope. The polyethylene insert  had an additional four degrees of posterior down-slope. The rotation of the femoral component was oriented according to the transepicondylar line and the AP axis (Whiteside’s Line) of the distal femur as currently advised in literature , 17. AftThe tibial component was inserted and checked for smooth movement on the tibial cut surface. The knee was then put through five full flexion-extension cycles while the surgeon held the ankle only. During the ROM cycles, no hands were touching the knee to prevent manual manipulation, and no varus/valgus stress was applied. The movement was followed on the navigation computer to confirm that no varus/valgus stress was applied. After performing the ROM cycles, the rotational position of the trial tibial component was recorded as indicated by the navigation computer. Positive values indicated that the trial component was in internal rotation according to the given TTL axis, and negative values indicated that the trial component was in external rotation. While this measurement was being acquired, the patella was lying in the patellar groove to facilitate optimal patellofemoral tracking and to prevent lateral pull on the patella tendon that could cause the tibia to rotate externally. The rotational position was noted (measurement 1). After removing and reinserting the components, the ROM technique was applied two additional times with five full flexion-extension movements and corresponding subsequent measurements from the navigation system (measurement 2 and 3).After completing the operative procedure, the final tibial tray was cemented up to 1/3 of the medial border of the tibial tubercle.p < 0.05 was considered statistically significant. The mean of the 3 ROM measurements was used to evaluate the difference between the ROM and TTL techniques. We evaluated potential factors associated with the difference between the ROM and TTL technique including, leg axis, intra-operative flexion, intra-operative extension and posterior slope.  were included in multivariable regression analyses. In multivariable regression analyses p-values < 0.05 were considered significant. Regression coefficients with their 95 % confidence intervals are reported.The statistical analyses were performed with SPSS statistical software . The reproducibility of the ROM-technique was evaluated using the intra-class correlation coefficient (ICC). For each target, there was one ‘rater’(MvS) who performed the three consecutive attempts at positioning the tibial component using the ROM technique. Since the exact same rater made ratings on every patient and it was assumed that both patients and observer were drawn randomly from larger populations, the ICC2 was used . The ICCp < 0.001). The ICC2 = 0.84 of tibial component positioning using the ROM technique indicating nearly perfect repeatability. Because the ROM technique was nearly perfectly reliable, the means of the 3 ROM measurements were used to evaluate the difference between the two techniques. With the ROM technique, the tibial component was on average 4.56 (± SD 8.59) degrees externally rotated compared to the tubercle landmark. This difference was statistically significant p = 0.028.The tibial component can be reliable positioned in terms of rotation using the ROM technique, as demonstrated by an ICC2 = 0.84 (95 % CI (0.70–0.93); p = 0.019), intra-operative flexion (0.57 (95%CI 0.13 – 1.00); p = 0.014) and intra-operative extension (1.41 (95%CI 0.50 – 2.32); p = 0.005) were associated with a greater difference between tibial component positioning using the ROM and TTL techniques. ; 95%CI 0.1 – 1.00; p = 0.221).In the varus knees, the tibial component was on average 5.9 (± SD 8.7) degrees externally rotated; in the valgus knees, the mean external rotation was 0.4 (± SD 7.6) degrees relative to the tibial tubercle. The differences between the rotational alignments using the ROM and TTL techniques in both the varus and valgus knees were not statistically significant (p = 0.979) was not significantly related to the difference in the rotational alignment of the tibial component according to the tibial tubercle in either the ROM or TTL techniques.The cut posterior slope (0.04 (95%CI −3.05 – 3.13); This study revealed that the ROM technique is a repeatable method for aligning the tibial component. Using the ROM technique, the tibial component was on average 4.56 (± SD 8.59) degrees externally rotated compared to the tubercle landmark. Our result is in contradiction with Ikeuchi et al.  They fouUsing anatomical landmarks for the rotational alignment of the femoral and tibial components is a widely accepted method. Alignment to the medial 1/3 of the tibial tubercle  is baseRetrieval and biomechanical studies have indicated that femoro-tibial rotational mismatches cause increased contact stress on the tibial insert and patellar component that leads to accelerated polyethylene wear –33. SteiTo measure the rotational difference between the ROM and TTL techniques in degrees may not be easy during TKAs that are performed without navigation. Computer navigation is an ideal method for measuring the difference in trial tibial tray position between the TTL and ROM techniques given its reported accuracy of one degree . Furtherp = 0.019). With increasing pre-operative varus alignment, the ROM technique results in increasing external rotation of the tibial component.Huddleston et al.  found thp = 0.014) and extension (p = 0.005) during surgery were also correlated with the difference between the ROM technique and the tubercle landmark in this study, which indicates that the use of the ROM technique for a patient with great preoperative flexion would result in a more internally rotated tibial component position compared with a patient with less preoperative flexion.The maximum degrees of flexion (The number of patients (observations) in our study is rather low. Various studies have suggested that for each variable studied in multiple regression analysis at least 10 observations are required –40 althoClinical and radiological data were not collected postoperatively. Therefore, no results are available regarding potential differences in clinical outcomes between the two techniques. It is known that a tourniquet affects the intra-operative patello-femoral tracking , 43. TheAlthough tibial rotational alignment is also effected by ligament balancing, we did not measure the gaps intra-operatively.The design of the prosthesis (CR or PS version) and the design of the tibial tray  may also have influence on the outcome .The ROM technique is a repeatable intra-operative method for determining the rotational position of the tibia trial component. Because the best method to determine the intra-operative position of the tibia component is still under debate, TKA surgeons should be aware that there is a difference between the ROM and TTL methods, particularly in patients with high peri-operative ranges of motion and/or high pre-operative varus/valgus alignment.


Objective of the present study was to isolate bovine herpes virus Type 1 (BHV-1) from semen of infected bull and to adapt it onto embryonated eggs and Madin–Darby bovine kidney (MDBK) cell line. Further, the virus was identified by agar gel immunodiffusion (AGID) test.Semen samples were collected from five BHV-1 positive bulls previously confirmed for the presence of antibodies against BHV-1 using avidin-biotin enzyme linked immunosorbent assay test. The virus from semen samples was adapted in chorioallantoic membrane (CAM) of 11-day-old embryonated chickens eggs and in MDBK cell line. The presence of BHV-1 in infected CAM and cell culture fluid was confirmed by AGID test.viz. rounding of cells with shrinkage, followed by aggregation or clumping of cells which progressed rapidly and appeared as “bunch of grapes” at 72 h post inoculation. Few cells become elongated when compared with uninfected controls. A homogenate of CAM with distinct pock lesions and infected cell culture fluid developed precipitation line within 48 h against specific anti-BHV-1 immune serum by AGID test.Virus infected CAM showed edema, congestion and thickening at first passage level. Small foci ranged from 1 to 2 mm in diameter, scattered all over the membrane were observed at first passage. More severe changes were observed in CAM after serial passaging. The large pock lesions, round in shape with opaque raised edge and depressed gray central area of necrosis ranged from 3 to 5 mm in diameter were developed at fourth passage. Blind passages in MDBK cell culture were made. The MDBK cell line at second passage level showed characteristic cytopathic effect BHV-1 was easily adapted in CAM of chicken embryos and in MDBK cell line. Virus infected CAM and cell culture fluid showed precipitin band by AGID test. Varicellovirus, Alphaherpesvirinae subfamily of Herpesviridae family [Bovine herpes virus Type 1 (BHV-1), an important pathogen of cattle, reported worldwide [e family . The vire family . The vire family . Moreovee family ,6. In gee family . The BHVe family .The virus is excreted through nasal and ocular secretion, semen and aborted placenta. Subsequent an acute infection, the virus get latent in the sensory ganglia of the animal . BHV-1 iBHV-1 can be readily isolated in cell culture of primary or secondary bovine kidney, lungs, testis, turbinate bone, trachea and established cell lines such as Madin–Darby bovine kidney (MDBK) cell line . The virAll the procedures have been conducted accordance with the approval from Institutional Animal Ethics Committee.et al. [A total of 464 serum samples were screened for presence of antibody against BHV-1 by using avidin-biotin enzyme linked immunosorbent assay (A-B ELISA) as per the standard protocol outlined in the user's manual supplied with kit was procured from Project Directorate on Animal Disease Monitoring and Surveillance, Hebbal, Bengaluru, India. Out of 422 cattle sera 158 were found positive, and 3 sera out of 42 were found positive in buffaloes. Fresh semen sample was collected from five infected bulls  showing high titer of antibody against BHV-1. Samples were collected by per rectal massage of ampullae and seminal vesicle . All samet al.  as quickFor virus isolation, 11-day-old embryonated chickens eggs were inoculated through CAM route . Five seThe BHV-1 was adapted in MDBK cell line, following the procedure described by the OIE . The preBHV-1 antibodies in serum were assessed by A-B ELISA.In the present investigation, the semen from BHV-1 infected bulls was inoculated in chicken embryos via CAM. The CAM showed edema, congestion and thickening at first passage level. Small foci ranged from 1 to 2 mm in diameter scattered all over the membrane were observed at the second passage .The largviz. rounding of cells with shrinkage, followed by aggregation or clumping of cells which progressed rapidly and appeared as “bunch of grapes” at 72 h post inoculation (PI) (Blind passage in MDBK cell culture was made to attempt virus isolation. The MDBK cell line at second passage level showed characteristic CPE ion (PI) . Few celion (PI) .Homogenate of CAM with distinct pock lesions and infected cell culture fluid was tested to identify the presence of virus by AGID test using specific anti-BHV-1 immune serum developed precipitation line within 48 h.et al., Madbouly and Hussein [via CAM route.Five semen samples from BHV-1 suspected bulls  were collected and assessed using A-B ELISA test and antibody against BHV-1 was found positive in the samples. Seropositivity for IBR antibodies highlighted the circulation of virus among the livestock population of Odisha . At init Hussein ,19 when et al. [nd day PI was reported by Mahmoud et al. [The BHV-1 infected and control monolayers were stained with MGG stain, and the CPE were examined microscopically. CPE was developed on second passage level which includes rounding, aggregation and syncytia formation. The result of present study are in accordance to the finding of Deka et al.  who obsed et al. . MDBK ced et al. -24. The The virus produced pocks on CAM of chicken embryos and characteristic CPE was observed in MDBK cell line. Homogenate of CAM and infected cell culture fluid developed precipitation line within 48 h by agar gel immunodiffusion.DS carried out the experiment and drafted the final manuscript. SS designed the experiment. NR, VRG and FS helped in the sample collection and analysis. All authors read and approved the final manuscript.


Canis aureus) and other canids have not been described before. In this study, we combined the use of biparental  loci) and uniparental (mtDNA control region and a Y-linked Zfy intron) genetic markers to assess the admixed origin of three wild-living canids showing anomalous phenotypic traits. Results indicated that these canids were hybrids between golden jackals and domestic dogs. One of them was a backcross to jackal and another one was a backcross to dog, confirming that golden jackal–domestic dog hybrids are fertile. The uniparental markers showed that the direction of hybridization, namely females of the wild species hybridizing with male domestic dogs, was common to most cases of canid hybridization. A melanistic 3bp-deletion at the K locus (β-defensin CDB103 gene), that was absent in reference golden jackal samples, but was found in a backcross to jackal with anomalous black coat, suggested its introgression from dogs via hybridization. Moreover, we demonstrated that MHC sequences, although rarely used as markers of hybridization, can be also suitable for the identification of hybrids, as long as haplotypes are exclusive for the parental species.Interspecific hybridization is relatively frequent in nature and numerous cases of hybridization between wild canids and domestic dogs have been recorded. However, hybrids between golden jackals ( Moreovsimensis ), or maysimensis –6).Canis aureus) is a medium-sized species, currently distributed in northern and northeastern Africa, southeastern Europe and large parts of southern Asia )1, F2, BC1J and BC1D hybrids using a threshold of 0.98. Only 6% and 10% of second backcrosses with jackals and dogs, respectively, were undetected using the Admixture model and no other a priori information in the Bayesian assignment procedure. Thus, the 15 STRs seem to be reliable and powerful enough to detect hybrids between golden jackals and dogs.In order to evaluate the power of the STR loci in the determination of golden jackal–dog hybrids, we also used simulated genotypes for six different hybrid classes. The analysis of the simulation results revealed that these 15 STRs are variable enough to detect 100% of parentals and FZfy intron on the Y chromosome appears to be diagnostic, since these markers proved to be fixed for different haplotypes in golden jackals and dogs , hybridization took place between female golden jackals and male dogs, which is congruent with the sexual asymmetry present in most hybridizations between domestic dogs and wild Canis species, e.g. grey wolf [K locus (K+/KB) in the backcross to that of a golden jackal 60c with black coat coloration, the absence of the melanistic K locus mutation in all reference jackals and its presence in 14 of the dog samples . Allrey wolf  and Ethirey wolf , though rey wolf . The fin samples  suggest  coyotes ,27. Furt coyotes , accordiThe MHC loci further confirmed that the three anomalous individuals described here were hybrids. Three out of four DLA-DRB1 alleles, one out of two DLA-DQA1 alleles and both DLA-DQB1 alleles found in golden jackals in this research  have notGasterosteus aculeatus) [Hybridization might play an important role in the process of animal evolution, especially in rapidly changing environments. Hybridizing with dogs, golden jackals might increase their variability and thus facilitate their long-term adaptation. In addition, adaptive introgression may be facilitated for genes evolving under multi-allelic balancing selection, such as the vertebrate MHC system, where increased resistance to infectious diseases from adapted MHC variants might be transferred to closely related recipient species, as long as fertile hybrids can be formed ,70. Thisuleatus) . It is tuleatus) .Conversely, hybridization and introgression may also have harmful effects on the fitness of animal populations in the wild, causing loss of genetic diversity due to genetic homogenization and/or outbreeding depression in local populations . In addiIn summary, in this paper we document the first occurrence of three cases of golden jackal–dog hybrids. However, the frequency of hybridization events, the extent of possible genetic introgression of dog genes into European golden jackal populations and the consequences on genetic diversity and population fitness  still remain to be investigated.


Apis mellifera filamentous virus), and cutting edge technologies  applied in the field.Pollination of flowering plants is an important ecosystem service provided by wild insect pollinators and managed honey bees. Hence, losses and declines of pollinating insect species threaten human food security and are of major concern not only for apiculture or agriculture but for human society in general. Honey bee colony losses and bumblebee declines have attracted intensive research interest over the last decade and although the problem is far from being solved we now know that viruses are among the key players of many of these bee losses and bumblebee declines. With this special issue on bee viruses we, therefore, aimed to collect high quality original papers reflecting the current state of bee virus research. To this end, we focused on newly discovered viruses , or a so far neglected virus species ( Although the number of managed honey bee colonies has increased globally by more than 45% over the last 60 years ,2,3, theIn reaction to this insight, about ten years ago virologist Michel Aubert started a European initiative called BRAVE (Bee Research and Virology in Europe) to identify research gaps and research needs in the field of bee virology. The aim was to collect the knowledge on honey bee viruses available at that time in order to create a basis for state-of-the-art research because “honey bees must be protected against virus diseases” . This efApis mellifera filamentous virus (AmFV). Interestingly, amongst the 23 honey bee viruses described to date [Rickettsia when it was first described in the early sixties of the last century [Rickettsia melolontha [Nosema apis infected bees, suggesting that N. apis infection is a prerequisite for an AmFV infection to become established [Apis mellifera Filamentous Virus Genome” by Gauthier and co-workers in this issue [In silico analysis of the AmFV genome also revealed many so far unknown ORFs without data base matches. This result underlines the uniqueness of this virus amongst all other honey bee viruses and emphasizes the importance of molecular data on honey bee viruses in order to further our understanding of their evolution and complex relationship with their host(s).Two original papers in this special issue deal with a rather neglected but widely distributed virus,  to date ,8,9,10,  to date . Origina century ,13 and, lolontha . AmFV walolontha . Accordiablished . Infectiablished ,16. Howeablished . To undeis issue , the autN. apis infections [Apis mellifera Filamentous Virus (AmFV) Infections in Honey Bees and Relationships with Other Parasites” [Varroa destructor suggesting that V. destructor does not act as a vector for AmFV. Contradictory to historical data [Nosema spp., hence, preexisting Nosema spp. infections do not seem to be a prerequisite for AmFV infections to become established. Taken together, the study by Hartmann and co-workers does not substantiate a major role for AmFV infections in honey bee colony collapse, although it cannot be ruled out that a general weakening of individual bees through AmFV infections might make them more susceptible to other pathogens.AmFV is supposed to be of low virulence and establishment of infection was thought to be dependent on pre-existing fections  althoughrasites”  was overcal data , there wApis mellifera and Varroa destructor” by de Miranda and co-workers [Tymoviridae in the order Tymovirales. The Tymoviridae comprise three genera  and BeeMLV is closest to the genus Maculavirus. BeeMLV has a polyadenylated RNA genome with about 6500 nucleotides. This entirely new virus was found replicating in honey bees and presumably also in V. destructor. The high titers of BeeMLV in infected adult and pupae samples indicate that honey bees are definitely a host but V. destructor may be a biological vector for this virus rather than a true host. Further investigations are needed to unravel whether BeeMLV is an endemic or an incidental virus, to identify the host spectrum of this virus, and to analyze its transmission routes as well as pathology in and virulence for bees and bee colonies.The study “Genome Characterization, Prevalence and Distribution of a Macula-Like Virus from -workers  is focusSinaivirus clade [V. destructor, albeit viral replication could not be detected [V. destructor [Most recently, the Lake Sinai virus (LSV) group has been discovered as very prevalent and widely distributed bee infecting viruses . Phylogeus clade . LSV wasdetected . In theidetected  analyzedstructor . They alstructor  further Although chronic bee paralysis virus (CBPV) is long since known and its pathology as well as transmission routes are well described ,26,27,28Bombus terrestris”, Piot and co-workers [Members of the ABPV/KBV/IAPV  group of bee pathogenic viruses are consistently implicated in colony losses in many regions of the world ,37,38,39-workers  performe-workers  suggestiThis special issue on honey bee viruses demonstrates impressively how diverse this field is. There still exist many unknown honey bee pathogenic viruses which await discovery, either occasionally or more target-oriented with sequencing approaches, and subsequently analyzed. Furthermore, the existing knowledge on the well-characterized honey bee viruses has to be considerably extended. Therefore, much work is still to be done to get a comprehensive understanding of the virology of the honey bee.


Bunyaviridae.We describe here the nearly complete open reading frame (ORF) of five Gamboa virus strains isolated in Panama and Argentina. The viruses with complete ORF showed the regular genome organization observed in other orthobunyaviruses with exception to the presence of NSs protein. All predicted proteins showed homology with viruses belonging to members of the family  Bunyaviridae is one of the largest families of RNA viruses and comprises more than 350 named viruses that are currently assigned to five genera, namely, Orthobunyavirus, Phlebovirus, Hantavirus, Nairovirus, and Tosposvirus. Some of the viruses in the family are still ungrouped. Bunyaviruses are spherically shaped enveloped viruses with a mean diameter of 80 to 120 nm. The genome is composed of a tripartite ssRNA . In general, six proteins are encoded by the three RNA segments: three are structural (Nucleocapside N and two glycoproteins Gn and Gc), and three are nonstructural . Viruses in this family infect plants and animals; those in the genus Tospovirus infect plants. The vectors for these viral agents are variable and include mosquitoes, sandflies, ticks, midges, and thrips (The family Gamboa-like viruses (GAMV) have been isolated mainly from mosquitoes Aedeomyia squamipennis in Honduras, Panama, Argentina, Ecuador, Suriname, and Brazil leading to the establishment of a new serogroup , the Gamboa virus strain MARU 10962 ; the Alajuela virus strain MARU 11079 , and the Calchaqui virus AG 83-1347  were isolated in Argentina. The Calchaqui virus had 62 gaps in a 3′ end open reading frame (ORF) that was masked with degenerated nucleotides (NNNN…). Regardless of the size heterogeneity of the genomes, in general, the ORFs for S, M, and L were 717 nt (geneN)/393 nt (NSs); 4,779 nt (M polyprotein); and 6,822 nt (polymerase). Sequencing steps were carried out at the Genomic Core of the Center for Technological Innovation, Evandro Chagas Institute, Brazilian Ministry of Health, Ananindeua, Brazil.Five GAMV isolates were subjected to RNA extraction and nearly complete genomes were obtained using a combination of two next generation platforms (GSFLX 454 and Ion PGM torrent)  Among fode novo hybrid assembly strategy using both Ion torrent and GS FLX 454 reads simultaneously with the software Mira version 4.0. Visual inspection was performed with the software Geneious version 6.1.4. The total genome recovered with a mean coverage of >100 fold. The six main genes N-NSs (S segment), Gn-NSm-Gc (M segment), and polymerase (L segment) were recognized. This is the first report of complete ORF genome sequences for Gamboa viruses.The genome was obtained employing a Gamboa virus strain GML 435718 (accession numbers KM272174 to KM272176), Gamboa virus strain GML 903023 (KM272177 to KM272179), Gamboa virus strain MARU 10962 (KM272180 to KM272182), Calchaqui virus AG 83-1347 (KM272183 to KM272185), and Alajuela virus strain MARU 11079 (KM272186 to KM272188).The complete genome sequence has been deposited in GenBank as follows: