This paper aims to estimate the service and social costs of headache presenting in primary care and to identify predictors of headache costs. Patients were recruited from GP practices in England and service use and lost employment recorded. Predictors of cost were identified using regression models. Service and social costs were available on 288 and 282 patients, respectively. Average service costs over 3 months were £117 whilst total costs (including lost production) were £582. Patients referred to neurologists had service costs that were £82 higher than those not referred (90% CI £36–£128). Costs including lost employment were higher by £150, but this was not significant (90% CI -£139–£439). The annual mean service and social costs, weighted to represent population rates of referral, were £468 and £2328, respectively. Higher costs were significantly related to pain. Age was linked to higher service costs and lower social costs. The figures extrapolated to the whole of the UK suggest £956 million due to service use and £4.8 billion including lost employment. These are likely to be underestimates because many people experiencing headaches do not consult their GP. Headache, including migraine, is a common problem and is in the top ten causes of disability . It is uClearly headache can cause distress for individuals and limit their activities. This, combined with the demand for treatment, suggests that there is an economic burden associated with headache . Direct Most studies have reported costs for migraine, but this is only a subset of all headaches treated in primary care settings. We have previously compared the demographic and clinical characteristics of patients referred by GPs to neurologists with those remaining in primary care. It was found that referral to neurologists was not related to headache severity, but was associated with an increased number of consultations with GPs and increased fear and anxiety expressed by patients regarding their headaches, as well as with GPs lack of clinical confidence and patient pressure , 10. TheThis was a primary care-based study set in England and the study methodology has been described in detail elsewhere . PatientEligible patients were invited to be interviewed by research workers in their homes or at the general practice. The interviews consisted of a number of measures including the migraine disability assessment score (MIDAS) , the HeaIt is important to take a comprehensive approach when estimating the economic consequences of a particular condition . HeadachHeadache can have an impact through people taking time off work, or having reduced work effectiveness. The MIDAS questionnaire includes two questions covering the effect of lost work time due to headache. The first of these asks for the number of whole days lost from work in the past 3 months. The second asks for the number of days where productivity is reduced by more than 50%. In the absence of further information, we have conservatively assumed this to be equal to half-a-day’s lost work. The economic cost of lost work time was calculated by multiplying the lost days by the earnings that patients in the sample received . Not all patients stated their earnings and in these cases we obtained average figures for their job type and gender from official data . Where tService use and costs were reported for each group. The overall costs are not representative of the population of people consulting GPs due to the overrepresentation of those referred to specialists. Therefore, weighted costs were also computed, based on the finding by Latinovic et al.  that oveFurther regression models were then constructed to identify the demographic and clinical characteristics that were associated with differences in costs in addition to any difference due to whether the patient was referred or not. Independent variables in the model related to headache severity , psychological distress (anxiety and depression scores from HADS) , illnessDuring the sampling periods there were 533 patients who consulted a GP for headache and a further 93 who were referred to secondary care services. Of these 626 patients, 569 met inclusion criteria and 303 consented to take part . These patients were predominantly female  and of similar mean age (39 vs. 41). Ridsdale et al. [Most patients had consulted their GP during the previous 3 months, but for the majority this was not due to headache Table . The aveThe mean service and total costs for the whole sample are £128 and £601, respectively. For the non-referred patients the figures are £115 and £579, respectively, whilst for those referred they are £197 and £729, respectively. If we weight the costs for non-referred and referred patients by 0.98 and 0.02, respectively, the mean service cost becomes £117 whilst the mean total cost is £582. If representative, this would suggest annual service costs of £468 and total costs of £2,328 per person.Mean service costs (i.e. excluding lost productivity) for the 3-month period were £82 higher in the referred group, a difference that was statistically significant (90% CI £36–£128). Those who were referred also had more time off work due to headache and this increased the cost difference to £150. However, there was substantial variation in lost work time and this cost difference was not statistically significant (90% CI -£139–£439). Productivity costs accounted for 81% of the costs in the non-referred group and 72% of the costs in the referred group.Figures Men had lower total costs than women, although the difference was not significant. This result was surprising as the data revealed that men had slightly more days when they could not work (according to the MIDAS questions) than women (4.5 vs. 4.0 days). Also daily wages were slightly higher for men (£127 vs. £103). Further analyses did though explain the lower total costs for men. For women in employment, there was a positive correlation (0.15) between their daily wages and days off work. However, for men the correlation was negative and larger in magnitude (−0.32). This implies that men who had days off work were likely to be paid less and, therefore, this reduces the overall cost of lost employment, and consequently total costs, compared to women.Headache is a common health problem affecting over 90% of the population during their lives. Most people self-manage symptoms with over-the-counter medication, with 4% of adults consulting their GP for headache each year . If we eThe results presented in this paper are of importance to a specialist audience because, whilst most patients with headache receive care from GPs, patients with headache account for around one-third of referrals to neurologists . In addiClearly, the main economic effect of headache is the impact that it has on productivity. We found that 81% of the total costs of headache were due to lost work time. This may be an underestimate of the broader social costs––there would clearly be an impact on work in the home also, as well as on leisure activities. Other studies have found higher proportions accounted for by indirect costs , 6, and Service costs were found to be higher for patients who had been referred to secondary care services. These extra costs consisted of more GP contacts, contacts with neurologists and scans. The study found that one-quarter of the referred sample had received input from neurologists by the time the first questionnaire was administered. Therefore, the cost difference would be even greater in the longer term once all referred patients had received specialist advice or care.The regression model found that higher service costs were significantly associated with age, which likely reflects increasing morbidity with age. Greater pain severity was also associated with higher costs. The only IPQ-R subscale that was significantly related to service cost was beliefs about the consequences of the headache. The more substantial the consequences were perceived to be, the higher the costs. Referrals to neurologists for headache has been associated with dissatisfaction in up to one-third of patients , and thiEach of the above relationships could be expected and indicate that patients with higher levels of need are in receipt of most resources. Total costs were, however, inversely related to age, which is due to the fact that older patients are less likely to be in employment and to experience lost work days. As before, pain severity was associated with higher costs.The weighted mean cost for neurologist contacts was only £9, but across the population this would equate to around £18 million. In a recent study, we have shown that if GPs receive specific training in headache management, then patients can be seen for a lower cost, and they are more satisfied with the service . HoweverThe study has a number of limitations. First, a limited number of services were included in the costings and as such the figures may be an underestimate. Second, we have included GP and neurologist contacts and interventions used by people with headache, but not necessarily due to headache. It is clear that comorbidity is common and does have a cost impact , but it This paper has found that the mean costs associated with headache presenting in primary care are £601 over a 3-month period. Most of this is due to lost employment. Service costs are significantly higher for patients referred to neurologists and those with greater symptom severity.


Crimean-Congo hemorrhagic fever (CCHF) is a severe tick-borne disease well recognized through Europe and Asia where diagnostic tests and medical surveillance are available. However, it is largely neglected in Africa, especially in the tropical rainforest of Central Africa where only sporadic human cases have been reported and date back to more than 30 years. We describe here an isolated human case that occurred in the Democratic Republic of the Congo in 2008 and performed phylogenetic analysis to investigate whether it resulted from a regional re-emergence or from the introduction of a novel virus in the area.Near complete segment S and partial segment M sequences were characterized. Bayesian phylogenetic analysis and datation were performed to investigate the relationship between this new strain and viral strains from Africa, Europe and Asia. The new strain is phylogenetically close to the previously described regional genotype (II) that appears to be specific to Central Africa. Phylogenetic discrepancy between segment S and M suggested genetic exchange among local sublineages, which was dated back to 130–590 years before present.The phylogenetic analyses presented here suggest ongoing CCHF virus circulation in Central Africa for a long time despite the absence of reported human cases. Many infections have most probably been overlooked, due to the weakness of healthcare structures and the absence of available diagnostic procedure. However, despite the lack of accurate ecological data, the sporadic reporting of human cases could also be partly associated with a specific sylvatic cycle in Central Africa where deforestation may raise the risks of re-emergence. For these reasons, together with the high risk of nosocomial transmission, public health authorities' attention should be drawn to this etiological agent. Crimean-Congo hemorrhagic fever virus (CCHFV) is transmitted to humans through tick-bite or contact with infected blood or tissues from livestock, the main vertebrate hosts in a peri-domestic natural cycle. With numerous outbreaks, a high case fatality rate (3%–30%) and a high risk for nosocomial transmission, CCHFV became a public health concern in Europe and Asia. However virus surveillance in Africa is difficult due to the limited sanitary facilities. Especially, CCHFV occurrence in Central Africa is very poorly described and seems highly in contrast with the temperate to dry environments to which the virus is usually associated with. We described a single human infection that occurred in Democratic Republic of the Congo after nearly 50 years of absence. The phylogenetic analysis suggests that CCHFV enzootic circulation in the area is still ongoing despite the absence of notification, and thus reinforces the need for the medical workers and authorities to be aware of the outbreak risk. The source of infection seemed associated with a forest environment while no link with the usual agro-pastoral risk factors could be identified. More accurate ecological data about CCHFV enzootic cycle are required to assess the risk of emergence in developing countries subjected to deforestation. Bunyaviridae, genus Nairovirus) is a tick-borne virus. It causes severe illness throughout Africa, Asia, Southeast Europe and the Middle East, with case fatality rates ranging from 3% to 30%. Its worldwide distribution closely matches that of its main arthropod vector, ixodid ticks belonging to the genus Hyalomma. Human infection occurs through tick bites, contact with infected livestock, or nosocomial transmission. The CCHFV negative-stranded RNA genome is divided into a small (S), medium (M) and large (L) segment.Crimean-Congo hemorrhagic fever virus ) in group II, South Africa and West Africa in group III, Middle East and Asia  and has re-emerged in countries located southwest of the Russian Federation after an absence of nearly 30 years In 2008, CIRMF  identified CCHFV in a serum sample received for etiological diagnosis of a case of hemorrhagic fever in DRC. This is the only identification of CCHV in DRC for more than 50 years. To determine whether it was due to introduction of a novel virus or to re-emergence of a local genotype, we determined the phylogenetic relationships between this virus (hereafter referred to as Beruwe-2008) and previously described isolates. Phylogenetic analysis showed that the Beruwe-2008 strain belonged to the genotype previously identified in this area and thus suggested that it had re-emerged. Local CCHFV persistence may have been supported by a sylvatic natural cycle specific to Central Africa, indicating that countries subject to major deforestation may note an increasing number of human infections.Laboratory investigations were performed subsequently to the WHO request for surveillance and early alert of hemorrhagic fever outbreak in Central Africa. Because of the emergency settings associated with the suspicion of such acute illnesses, no ethics committee approval or written consent was deemed necessary. The blood sample was taken by national healthcare workers of the Lubutu hospital where the patient came for medical care. He was informed that his blood sample will be further used for diagnostic investigation and gave his verbal consent. The patient described here is anonymous. The blood sample was next sent to the Institut National de Recherche Biomédicale  and then to CIRMF upon WHO authorities. The study was approved by the scientific committee of CIRMF.The patient was a 26-year-old man living in Beruwe (Nord Kivu province) in DRC, 325 km from Kisangani . He becaThe patient's serum was manipulated in biosafety level 4 (BSL-4) conditions. The serum was first tested for Ebola and Marburg viruses. As results were negative, investigations were next performed for CCHFV. RNA was extracted with the QIAamp viral RNA mini kit  according to manufacturer's instructions. Reverse transcription (RT) and real-time PCR amplification were performed with the High Capacity cDNA RT kit and Taqman universal PCR master mix , and previously reported primers and probes As viral isolation on Vero cells was unsuccessful, viral RNA was extracted from the patient's serum as described above, and was used for RT-PCR amplification with Platinum Taq DNA polymerase (Invitrogen). Primers were derived from nucleotide alignments . Three oA total of 44 complete sequences for segment S and 38 complete sequences for segment M were retrieved from GenBank .Pairwise nucleotide comparison of the Beruwe-2008 segment S sequence with those of the most closely related strains Congo3010-1956 (DRC) and Semunya-1958 (Uganda) – showed 92.4% and 92.0% similarity, respectively. In segment M the pairwise identities were 96.1% and 93.8% respectively. Identity between the Beruwe-2008 strain and strains belonging to other genetic groups ranged from 82.2% to 87.6% in segment S and from 72.5% to 81.3% in segment M .Bayesian phylogenetic analysis with a molecular clock assumption was applied to segment S  and M F datasetsOur dating analysis of the S segment resulted in time estimations slightly more recent than previously reported, but nonetheless within the same range and in keeping with an ancient origin of CCHF viruses CCHV genotype II has been identified only in DRC and Uganda, while different CCHV lineages have been identified in neighboring countries to the north. Multiple genotypes have been identified in CAR, belonging to groups IV and III Of note, the MRCA estimates presented here are in agreement with ancient divergence of this lineage (around 1000 years ago), but whether or not this split-off was linked to virus adaptation to Central Africa cannot be assessed. However, as the MRCA of the three strains was dated back to 683 to 243 years BP emergence and of the high risk of nosocomial transmission.Table S1GenBank accession numbers for the sequences used in this study. Countries, strains, date of sampling and hosts are reported along with the associated GenBank accession numbers for segment S and segment M.(DOC)Click here for additional data file.


Farm-level biosecurity provides the foundation for biosecurity along the entire production chain. Many risk management practices are constantly in place, regardless of whether there is a disease outbreak or not. Nonetheless, the farm-level costs of preventive biosecurity have rarely been assessed. We examined the costs incurred by preventive biosecurity for Finnish poultry farms.We used a semi-structured phone interview and obtained results from 17 broiler producers and from 5 hatching egg producers, corresponding to about 10% of all producers in Finland.Our results indicate that the average cost of biosecurity is some 3.55 eurocent per bird for broiler producers (0.10 eurocent per bird per rearing day) and 75.7 eurocent per bird for hatching egg producers (0.27 eurocent per bird per rearing day). For a batch of 75,000 broilers, the total cost would be €2,700. The total costs per bird are dependent on the annual number of birds: the higher the number of birds, the lower the cost per bird. This impact is primarily due to decreasing labour costs rather than direct monetary costs. Larger farms seem to utilise less labour per bird for biosecurity actions. There are also differences relating to the processor with which the producer is associated, as well as to the gender of the producer, with female producers investing more in biosecurity. Bird density was found to be positively related to the labour costs of biosecurity. This suggests that when the bird density is higher, greater labour resources need to be invested in their health and welfare and hence disease prevention. The use of coccidiostats as a preventive measure to control coccidiosis was found to have the largest cost variance between the producers, contributing to the direct costs.The redesign of cost-sharing in animal diseases is currently ongoing in the European Union. Before we can assert how the risk should be shared or resort to the 'polluter pays' principle, we need to understand how the costs are currently distributed. The ongoing study contributes towards understanding these issues. The next challenge is to link the costs of preventive biosecurity to the benefits thus acquired. Biosecurity can be defined as the exclusion, eradication and effective management of risks posed by pests and diseases to the economy, environment and human health . Risk maAlthough a few studies have assessed preventive actions against alternative strategies (reviewed in ), the faa [The farm-level costs are important for at least four reasons. First, many management practices are in place even in the absence of an outbreak, but an outbreak may increase these costs. Costly preventive actions may reduce the resources required and the costs of eradication in the event of a disease outbreak, although this impact has rarely been studied. Second, farm-level biosecurity provides the foundation for biosecurity in the entire production chain. This is particularly important in a system such as the EU, where the production strategy is based on biosecurity and safety in the entire production chain. Third, the farm-level costs in part determine the incentives which producers have in providing biosecurity, which is to a large extent a weakest (or weaker) link public gooda . Identifa . One faca .We contribute towards filling the information gap regarding the current costs of biosecurity at farm level by examining the costs incurred by preventive biosecurity for Finnish poultry farms. Our aims were: 1) to estimate the total level of monetary and labour costs of preventive biosecurity in poultry farms, 2) to identify the largest and smallest cost components within the farms and 3) to obtain information regarding the variance of producer investments in specific components of biosecurity and to examine what explains this variation. The information obtained could, for instance, be used in designing cost-efficient risk management measures.Salmonella in Finnish poultry is normally below 1%, and as for other poultry diseases, in 2009 there were five cases of Marek's disease, and no cases of Gumboro, avian influenza or Newcastle disease [The situation regarding poultry diseases in general is relatively good in Finland compared to many other countries in Europe and worldwide ,21. All This study ran from February to December 2007 and involved interviews with 22 poultry farms (17 broiler producers and five hatching egg producers). All the producers participated in the study voluntarily. They were selected with the assistance of Finnish poultry processors and their veterinarians who provided the contact details as well as gave assistance in persuading the producers to participate. The production of poultry in Finland is vertically highly integrated, meaning that each farm is associated with a particular poultry processor. They have a comprehensive agreement regarding the details of production, including some aspects of biosecurity, but these contracts are not publicly available. As a result, the processor has a relatively large influence on the actions of the producer. In the study, the proportion of the farms for each of the three processors is consistent with their market share.The data were acquired through a telephone survey of the farms and concerned the 2006 production year. The questionnaire was designed with the extensive help and consultation of experts in poultry production, and it dealt with different types of actions related to biosecurity at the farm level. Only actions primarily taken for disease management purposes were included in the questionnaire. The questions were divided into 14 categories, presented in Table The questionnaire (available upon request from the authors) was sent to the producers roughly one week in advance of the actual interview call. Personal interviews were required in order to present the complex issues coherently to the producers, as well as to avoid double counting of different types of costs . The intThe answers given by the producers were in either euro (for direct costs or purchased services) or in hours of labour. A summary of the data is presented in Table The biosecurity costs were calculated for each of the categories using Microsoft Excel . The costs were divided into direct monetary costs and costs in terms of labour. These were also transformed into costs per bird per year, where the annual number of birds produced was used.In addition, we undertook ordinary least squares regression analysis with SPSS statistical software  to examine how the costs are related to the unit size and which factors are primarily responsible for the cost variation between the farms. On the basis of background data on the correspondents, we postulated the following relationship to explain the variation in costs:i measured in terms of the number of birds produced annually (ibirds) as well as on the processor with whom the producer has a contract. There were three processors in our sample, and we included dummy variables for two of them, treating the third processor as the reference level. We also postulated that the gender of the producer may matter and denoted female producers with a dummy variable. The last term i εis the error term.The variation in the total cost per bird was assumed to depend on the size of the production unit Three alternatives for the dependent variable were considered: 1) the total cost per bird, 2) the direct monetary cost per bird and 3) the labour cost per bird. The total cost is the sum of the direct monetary cost and the labour cost. Work costs were measured in hours of labour and converted into euro using an hourly wage rate of €12 per hour, the figure used in the Farm Accountancy Data Network (FADN). Additionally, in one analysis, the density of birds was used as an explanatory variable instead of the number of birds. Regression analysis was only performed for the broiler producers, as the small sample size for the hatching egg producers did not allow statistical testing.b, the figures were 0.10 eurocent per bird per rearing day for broilers and 0.27 eurocent per bird per rearing day for hatching egg producers. The general results are presented in Table The average total cost of preventive biosecurity for the broiler producers in our sample population was 3.55 eurocent per bird . For hatching egg producers, the expenses were higher, the mean expense being 75.7 eurocent per bird (39.3-115.5 eurocent per bird). The small number of hatching egg producer holdings did not allow for reliable statistical testing. When transformed into costs per rearing day, by dividing the cost by the average number of rearing daysFor broiler producers, the direct monetary cost was on average 2.51 eurocent per bird (71% of costs) and the labour cost 1.04 eurocent per bird (29%). For hatching egg producers, the corresponding figures were 49.5 eurocent (65%) and 26.2 eurocent (35%). On a per bird per rearing day basis, the corresponding figures were 0.07 and 0.03 eurocent for broiler producers and 0.18 and 0.09 eurocent for hatching egg producers.th and 75th percentiles of the costs. The whiskers show the 5th and 95th percentiles.The costs were further divided into categories according to the categorisation presented in Table ®) for the prevention of intestinal disturbances in newly hatched chicks. The two other larger components were pest control (Category 3) and operational hygiene (Category 10).The figure illustrates that only a few categories produce the majority of the expenses. The main constituent of the costs was preventive medication (Category 2), which includes the use of coccidiostats in broiler feed as a preventive measure to control coccidiosis and the use of a competitive exclusion product  constituted the largest cost component, followed by preventive medication including vaccination (Category 2) and insurance against animal diseases (Category 13). The smallest cost components for both the broiler and the hatching egg producers included the biosecurity plan (Category 1), contracts for purchases and sales (Category 7), construction plans and investments (Category 8) and health monitoring programme (Category 9), the last of which can hardly be affected by the producer. Pest control (Category 3), education (Category 5) and operational hygiene (Category 10) had a similar share of total expenses for both the broiler and the hatching egg producers, each constituting about 6-10% of total biosecurity expenses.Salmonella (0.15 eurocent). Salmonella insurance and an extra level of production hygiene also stood out (both about 0.25 eurocent) and, in these cases, there were large numbers of farms with zero expense in these categories.The costs naturally varied among the producers. The largest cost variances in terms of monetary costs were found in the use of preventive medication , disposable requisites for pest control such as pesticides (0.25 eurocent), and insurance other than Similarly, for labour expenses, by far the largest cost variance (90% of variation) was related to the time taken to shower for disease prevention purposes, which varied between the farms by about 1.5 seconds, or by 0.50 eurocent, per bird. The other primary reasons for variance in labour costs were pest control, as well as the time which the producer was out of the production facilities due to quarantine , education, production monitoring and health monitoring programmes, and control and inspections .Regarding the impact of the unit size and other factors (equation 1), the results of the base regression using the three dependent variables are presented in Table When bird density was substituted for bird number in the regression, both processor dummies became positive and significant at the 95% level. The bird density variable itself was positive and significant at the 95% level. Bird density had no statistical impact on the direct monetary costs of biosecurity (p = 0.797), but it had a statistically significant impact on biosecurity labour costs (p = 0.039) as well as on the total cost (p = 0.027). As the sign of the coefficient is positive, it suggests that one more bird per square metre increases the labour cost of biosecurity per bird by about 0.20 eurocent.This study represents one of the first attempts to determine the total farm-level costs of biosecurity during a disease-free period. Our results indicate that the average cost of biosecurity is some 3.55 eurocent per bird for broiler producers (0.10 eurocent per bird per rearing day) and 75.7 eurocent per bird for hatching egg producers (0.27 eurocent per bird per rearing day). For a batch of 75,000 broilers, the total cost would be €2,700. This represents some two per cent of the total production costs and is similar in magnitude to the cost of logistics (loading and transportation) (unpublished information). The results also indicate that the work time devoted to biosecurity represents some 8% of the total work time on broiler farms and about 5% on broiler breeder farms. The costs are in the same range as the cost of vaccines and other veterinary services in England, where they were found to amount to 1.2% of total expenses and to 1.4 pence (about 1.9 eurocent) per bird . The resThe higher cost of biosecurity per bird for the hatching egg producers is logical, since the birds spend a much longer period of time in the production facilities. However, also the cost per bird per rearing day was about three times higher for the hatching egg producers. This may suggest that the birds are more valuable to the producer and hence worth investing more. We have not compared the costs of biosecurity to the income generated by the birds in this paper. Also the higher amount of traffic in hatching egg production may explain the higher cost.Some basic conclusions can be drawn from the regression results. First of all, regression 1 Table  suggestsFurthermore, it appears that the impact of processor B cannot be distinguished from that of processor C, but the farms associated with processor A (the reference level) have an approximately one eurocent lower cost of preventive biosecurity than the farms associated with the other two processors. This effect was maintained even when new variables (such as bird density) were included in the model. When examined categorically, the impact was particularly strong in Categories 2 (preventive medication), 3 (pest control) and 6 . Whether this is due to more cost-effective actions, less stringent requirements of the classified production contracts or different disease pressure cannot be ascertained with this study, as we have no information on the disease history of the farms. However, since poultry production in Finland is to a large extent controlled by the processors, it is clear that the attitudes, guidelines and instructions given by the processor have an important impact at the farm level.Female producers (24% in the sample) were also found to invest more in biosecurity. It has been observed in many studies that women are more sensitive towards risks in general ,24 and, 2. When the impact of bird density on the costs was examined, it was found that bird density was positively related to the labour costs of biosecurity. This suggests that when the bird density is higher, greater labour resources need to be invested in their health and welfare and hence disease prevention. This may be due, for instance, to the fact that the potential disease pressure is higher when the bird density is high.In the dataset, bird density varied from 19 to 24 birds per mIn the analysis, the use of coccidiostats was found to have the largest cost variance between the producers, contributing to direct costs. The reason for this remains unclear, as the separate regressions undertaken for this cost component could not reliably relate it to any of the explanatory factors. The answer may have to do with the disease history of the farm, but that information was not available for this study.The data for this study were collected by semi-structured interviews, which were all carried out by the same person in order to ensure inter-farm comparability. We believe that personal interviews, despite being time-consuming, are the best way to gather relatively complex data on disease prevention in a comparable way. A written questionnaire could result in more responses and thus more data to test, but the quality of the data would suffer, hence resulting in problems with statistical testing. As a result of this type of data acquisition, the amount of data collected cannot be extremely large. However, our dataset covers about 10% of Finnish producers for both production types (broiler and hatching egg). We also believe that the quality of the data more than compensates for the modest quantity.Although the main objective in the current study was to examine the magnitude and constituents of preventive biosecurity at the level of poultry production farms, we also had some methodological issues in mind. In the future, the current methodology and the questionnaire could be applied: 1) to investigate the costs at other points in the poultry production chain as well as to determine how much the chain as a whole is investing in biosecurity and 2) to explore the costs for different types of animal production chain, including the production of pork and beef. However, for application in other countries, some modifications need to be made. Poultry production legislation and practices vary somewhat within the EU, and national regulations may differ. Hence, the questionnaire used in this study may require some revision to be applicable elsewhere.The average size of the farms in the sample is somewhat larger than the average size of all broiler farms in Finland. However, as the trend is towards increasing farm sizes, we believe that the results are reasonably close to the current situation and are representative of the situation which we will shortly be facing. The results also suggest that if increasing farm sizes also lead to increased bird density, then greater resources will need to be invested in preventive biosecurity.The obvious next question is whether the incurred costs effectively prevent the introduction of diseases. Furthermore, we need to know how the unit size affects the optimal level of investment on a per bird basis on preventive biosecurity. These questions cannot be answered by the results presented here. However, identifying the costs associated with preventive biosecurity is a necessary first step in understanding that biosecurity is not a free lunch.The redesign of cost-sharing in animal diseases is currently ongoing in the EU. Before we can assert how the risk should be shared or resort to the 'polluter pays' principle, we need to have an idea of how the costs are currently distributed. We investigated the costs paid by the producers for preventive biosecurity, determined the largest cost components and explained the variation in these costs. Our results indicate that the average cost of biosecurity is some 3.55 eurocent per bird for broiler producers and 75.7 eurocent per bird for hatching egg producers. For broiler producers the main constituents of the costs were preventive medication, pest control and operational hygiene. The total costs per bird are dependent on the annual number of birds: the higher the number of birds, the lower the cost per bird. This impact is primarily due to decreasing labour costs rather than direct monetary costs.The authors declare that they have no competing interests.KMS designed the questionnaire, undertook the interviews, processed and analysed the data, and drafted the manuscript. JH helped in designing the questionnaire, analysed the data and drafted the manuscript. NT helped in designing the questionnaire and in drafting the manuscript. HR initiated the study, helped in designing the questionnaire and in analysing the data, and drafted the manuscript. All authors read and approved the final manuscript.a) Biosecurity is a public good because it is non-excludable in production and non-rival in consumption. Non-excludability means that once protection against the disease is provided, no agent (e.g. no farm) can be excluded from enjoying the benefits. For instance, if disease entry is prevented, nobody can be prevented from enjoying that. Non-rivalry means that one agent's consumption of protection does not reduce the amount of protection enjoyed by others. These are the two elements of a pure public good. Moreover, biosecurity is often of the 'weakest link' type, because its effectiveness depends on the weakest link in the protection chain. It does not matter how well other parts of the chain provide protection if the disease gets into the country through the weakest control point. A milder version of this argument (a weaker link) can be found in . In thisb) The rearing times in Finland at the time of this study were approximately 33-39 days for broilers and about 40 weeks, i.e. 280 days, for the parents .


African swine fever (ASF), a highly contagious fatal acute haemorrhagic viral disease of pigs currently has no treatment or vaccination protocol and it threatens the pig industry worldwide. Recent outbreaks were managed by farmers with ethnoveterinary preparations with various claims of effectiveness.We identified 35 compounds using GC-MS protocol and ASF virus (NIG 99) was significantly reduced by some extracts and fractions of the plant. However, the plant was poorly extracted by water and cytotoxicity was found to be a major problem with the use of the plant since its extracts also reduced the primary cells used in the assay.in-vitro and in-vivo will be necessary.It is confirmed that the plant has antiviral potentials against ASF virus and farmers’ claims seem to have certain degree of veracity, but finding the best means of exploring the potential of the plant while reducing its cytotoxic effect  African swine fever (ASF), a highly contagious fatal acute haemorrhagic viral disease of pigs results in major economic losses and has substantial food security implications. The disease continues to devastate animal resources (pigs) in parts of Sub-Saharan Africa and other infected regions of the world -3. ThougCurrently, in the event of an outbreak and its possible spread within pig herds, the standard practice remains the zoning, culling of the herd (stamping out) and payment of compensation to prevent epizootics , HoweverAncistrocladus uncinatus; and there have even been claims of complete freedom from illness following oral administration of Ancistrocladus uncinatus preparations. This Liana plant species was previously described by Hutch and Dalziel in South-Eastern Nigeria as reported by Cheek  , Benzene-Ethanol-Ammonia [90:10:1] , and Chloroform-Ethyl acetate-Formic acid [5:4:1]  using the method of Kotze and Eloff  . The sysRe-infectivity assay was performed to determine whether the observed effect of the plant on the virus was virucidal or virustatic and to correlate the PCR results with the cell culture; briefly, 100 μl of the recently harvested virus-extracts/fractions as well as the positive (ASF NIG/99 virus) and the negative (wash buffer) controls were filtered using the 0.22 μ filter and inoculated again into freshly prepared PBMC. The culture plates were incubated as described above and microscopically inspected 72, 96 and 120 hours post-infection to determine the residual virus infectivity following exposure to the extracts/fractions.Development of rapid and effective Diagnostic and Control tools for African Swine Fever). No human or animal subject was used directly in the project.Ethical clearance and approval were obtained as part of the overall approval for the project at the National Veterinary Research Institute, Vom, Nigeria under the Project Code 025060410100000 (We do not have any competing interest that should prevent the publication of this article.FOF, LH, ADSB conceived the project and draw up the protocols. MMF identified and prepared the plant for laboratory analysis with OOO+. FOF and OOO carried out the TLC and extraction procedures; OOO+, MMF and FOF carried out the phytochemical screening and GC-MS; FOF did the cell culture and molecular biology works. LH, ADSB and FOF carried out the analyses. All authors contributed to the drafting of the final manuscript and approved it for submission.Gas chromatography-mass spectrometry result of the leaf, stem bark and root of Ancistrocladus uncinatus.Click here for fileGas chromatography-mass spectrometry result of the leaf of Ancistrocladus uncinatus.Click here for fileGas chromatography-mass spectrometry result of the stem bark of Ancistrocladus uncinatus.Click here for filePreparation of Primary Bone Marrow Cultures.Click here for file


To the Editor: Hepatitis E virus (HEV) is the only virus in the genus Hepevirus, which is the only member of the family Hepeviridae. HEV is the causative agent of acute hepatitis E, a moderately severe enteric disease with death rates <4% in the general population and ≈30% in pregnant woman ; results were positive. To confirm the presence of HEV, RNA was extracted from 140 μL serum and fecal suspension by using QIAamp Viral RNA Kit , then retrotranscribed with Superscript II . HEV infection was confirmed by using nested PCR amplification of a conserved sequence within the HEV open reading frame (ORF) 2–3 overlapping region (Phylogenetic reconstruction of the ORF2–3 overlapping region showed that the HEV isolated in Uruguay clustered in genotype 1 with high bootstrap values , panel A


Acute respiratory infections are a major cause of mortality and morbidity worldwide. Information on viral etiology in ALRI from Lao PDR is limited. The aim of the present study was to use Multiplex PCR/RT-PCR methods for the detection of the major respiratory viruses.Nasal/throat swab specimens were collected from patients enrolled in the ALRI surveillance programme within 2 hospitals, one in Vientiane Capital  and the second one in Luang Prabang. From each sample, viral RNA was extracted and amplified by using 5 multiplex PCR/RT-PCR targeting 18 respiratory viruses.Between 2009 and 2010, Multiplex PCR / RT-PCR detected respiratory viruses in 111 (54.7%) of 203 samples. Single virus was detected in 44.8% (91/203) and virus co-infections were observed in 9.9% (20/203). Rhinovirus (40.5%), human Respiratory Syncytial virus , and Influenza A virus (9.0%) were the most frequently detected viruses. Adenovirus and human Metapneumovirus were detected in 8.1% and 6.3% of ALRI specimen, respectively. Influenza C virus and SARS-coronavirus were not detected during the study period. Children < 5 years represented 50% of patients identified.Our results provide new documentation about etiology of respiratory virus diseases in Lao People's Democratic Republic. In this context, multiplex PCR/RT-PCR offers a sensitive and reasonably priced diagnostic method for common respiratory viruses.


IFNG locus during developmental processes leading to its transcriptional activation and silencing.Transcriptional activation and repression of genes that are developmentally regulated or exhibit cell-type specific expression patterns is largely achieved by modifying the chromatin template at a gene locus. Complex formation of stable epigenetic histone marks, loss or gain of DNA methylation, alterations in chromosome conformation, and specific utilization of both proximal and distal transcriptional enhancers and repressors all contribute to this process. In addition, long non-coding RNAs are a new species of regulatory RNAs that either positively or negatively regulate transcription of target gene loci. IFN-γ is a pro-inflammatory cytokine with critical functions in both innate and adaptive arms of the immune system. This review focuses on our current understanding of how the chromatin template is modified at the  The cytokine, IFN-γ, plays key roles in controlling infection by intracellular pathogens, including bacteria and viruses, as well as malignant transformation and growth , or IL17 (Th17) .At the chromatin level, this differentiation process arises from marked epigenetic changes spanning a region greater than 100 kb surrounding the gene that encodes IFN-γ , phosphorylation, ubiquitination, and sumoylation and these modifications are associated with activation and silencing of specific genes  elements across the IFNG. Peripheral NK cells also possess a pre-existing H4-Ac pattern of epigenetic modifications across the IFNG locus and presumably H3K4-Me modifications  responsible for DNA Me. It is generally thought that DNMT1 is necessary to maintain DNA Me at the IFNG locus and has shown how this “roadmap” changes in cells as they differentiate to actively transcribe or actively silence IFNG. However, it cannot be inferred from these analyses whether CNS’s undergoing epigenetic modifications in response to these differentiation signals contribute to transcriptional activation or repression and how this is orchestrated. Various strategies have been employed to dissect functional properties of CNS’s across the IFNG locus undergoing epigenetic modifications in response to differentiation signals. These include analysis of enhancer activity by using reporter constructs in cell lines, use of these reporter constructs as transgenes in mice, and use of bacterial artificial chromosomes (BAC) as transgenes in mice  plays a key role in early epigenetic remodeling of the IFNG, CNS + 18–20, has been examined in both reporter assays and in BAC transgenic models. In reporter assays, this CNS lacks enhancer activity but cooperates with CNS-2 to increase CNS-2 enhancer activity  or by the cytokine combination, IL-12 and IL-18. NKT cells do not require CNS-2 for active IFNG transcription. In contrast, NK cells do not exhibit an absolute requirement for any individual CNS but rather exhibit partial requirements for CNS-16, CNS-2, and CNS + 18–20 to produce IFN-γ. Thus, CNS-16 is a bi-functional enhancer repressing IFNG transcription in Th2 cells yet activating IFNG transcription in NK cells. Another feature of this modular design is that defects in transcription revealed by CNS deletion are independent of stimulus, TCR stimulation versus cytokine stimulation (IL-12 and IL-18) but are cell-type specific. A schematic of different CNS functions in T cells and NK cells is shown in Figure Analysis of CNS requirements for IL26 is deleted from rodent genomes, linkage of IL26 and IFNG is preserved throughout evolutionary time from zebrafish to humans   are located within the CDKN1A locus and are regulated by p53, a potent tumor suppressor. LincRNA-p21 negatively regulates expression of pro-apoptotic genes conferring cell cycle regulation, whereas PANDA responds to DNA damage by sequestration of the NF-YA transcription factor resulting in cell survival  also named NEttoie Theiler’s Pas Salmonella (NeST) is the first identified enhancer lncRNA of the immune system to regulate expression of a master cytokine such as IFN-γ . Initially described in the context of an intracranial Theiler’s virus infection, the inability to control chronic viral infection in mice maps to a genetic deficiency within the TMEVPG1 gene. Analyses of epigenetic regulatory marks across the TMEVPG1 promoter indicate that it is an area of active transcription by enrichment of H3K9 and H4-Ac as well as H3K4 mono- and tri-Me. The TMEVPG1 gene is conserved among placental mammals with increasing rates of sequence conservation between human and mouse orthologs within the first exon and intron. Further, several Th1-specific DNase I HS sites are found upstream to and at the TMEVPG1 promoter supporting the permissivity of the locus to Th1-specific expression.Theiler’s Murine Encephalitis Virus Possible Gene 1  joins HOTTIP as a lncRNA that associates with WDR5 to promote H3K4-Me of histones, a mark associated with transcriptional activation, presumably at the neighboring IFNG locus.Functionally, IFNG locus is a prime example of this phenomenon. Alterations in conformation of the IFNG locus proceed through discrete stages. In unstimulated naïve T cells, the IFNG genomic locus associates with the Th2 cytokine genomic locus and exists in an open conformation (Spilianakis et al., IFNG genomic locus from the Th2 cytokine genomic locus and to initiate alterations in the conformation of the IFNG locus (Eivazova and Aune, IFNG locus to adopt a new conformation. The general view is that this new conformation recruits distal CNS with their associated transcription factors and epigenetic modifying machinery to IFNG to establish long-range epigenetic modifications that ultimately facilitate recruitment of RNA Pol2 to the promoter and active transcription.It is now recognized that alterations in chromosome conformation is a common mechanism to bring chromatin domains within close proximity to one another or to exclude chromatin domains from each other (Sajan and Hawkins, IFNG locus and these are conserved between humans and mice (Hadjur et al., IL26, one is located proximal to TMEVPG1 and one is located within an IFNG intron. CTCF is typically known as a transcriptional repressor and is also involved in insulator activity and regulation of chromatin architecture (Phillips and Corces, IFNG. It has been proposed that this defines the boundaries of the IFNG locus and serves to insulate IFNG from the negative effects of adjacent heterochromatin. However, adjacent TMEVPG1 is co-expressed with IFNG in effector Th1 cells. Adjacent IL26 is co-expressed with IFNG under some conditions. Thus, an alternate hypothesis is that the CTCF sites recruit adjacent TMEVPG1 and IL26 into the IFNG locus, which would serve two purposes. First, localizing the TMEVPG1 gene close to the IFNG gene would permit TMEVPG1 RNA to more easily associate with the IFNG locus. Second, localizing the IL26 gene close to the IFNG gene would permit sharing of the transcriptional and enhancer machinery necessary to express both genes. Thus, these CTCF sites may not serve to insulate IFNG from IL26 and TMEVPG1 but rather to bring them into close proximity to each other to facilitate their function and co-expression. Figure IL26-IFNG-TMEVPG1 locus may look in two-dimensional and three-dimensional space.CCCTC-binding factor or CTCF is a zinc finger transcription factor that binds the core CCCTC sequence (Filippova et al., Over the past decade, many studies have contributed to our increased understanding of the complexities of the regulation of gene transcription. Given the fundamental roles that cytokines play in both the innate and adaptive immune systems and the fact that their activities are mostly regulated at the transcriptional level, it is easy to see how the genes that encode these proteins must be under very tight regulation. Too little production of a cytokine such as IFN-γ could fail to control pathogen infection leading to bacteremia, viremia, or even death. Conversely, excess production of cytokines such as IFN-γ could produce excess inflammation, autoimmunity, or death.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To determine geographic range for Ebola virus, we tested 276 bats in Bangladesh. Five (3.5%) bats were positive for antibodies against Ebola Zaire and Reston viruses; no virus was detected by PCR. These bats might be a reservoir for Ebola or Ebola-like viruses, and extend the range of filoviruses to mainland Asia. Filoviridae contains 2 genera: Marburgvirus, which contains Marburg virus (MARV), and Ebolavirus, which contains 4 viruses: Zaire Ebola virus (ZEBOV), Sudan Ebola virus, Reston Ebola virus (REBOV), and Côte d’Ivoire Ebola virus, and 2 tentative species (Bundibugyo Ebola virus and Lloviu Ebola virus)  . PathogeRhinolophus and Miniopterus) and frugivorous bats (family Pteropodidae). Fruit bats of the genus Rousettus have been implicated as a reservoir of filoviruses in Africa  are the primary natural hosts, including Old World insectivorous bats  during April 2010–March 2011 from the Faridpur, Rajbari, Lalmonirhat, and Comilla Districts in Bangladesh. All bats were identified to species in the field, except Cynopterus spp. bats, because of cryptic diversity in this group; we are awaiting genetic species confirmation. Bats were captured in mist nets near roosts or at feeding sites and were handled in accordance with the Tufts University  Institutional Animal Care and Use Committee protocol (no. G2011-106).We captured and sampled 276 bats . To inactivate potentially infectious agents, serum samples were heated at 56°C for 20 min before shipment. All samples were screened by using a 1:1 mixture of purified recombinant nucleoproteins (0.2 mg/mL) of REBOV and ZEBOV (R + Z ELISA), which were expressed in an dine tag ,11.Potentially positive serum cutoff values were determined to be >0.454 for the R + Z ELISA by using maximum-likelihood estimation, gamma distribution, and 95% risk for error . PotentiTotal nucleic acids were extracted from samples  by using the easyMAG NucliSENS platform (bioMérieux) at Columbia University . Samples were tested for filovirus RNA (RNA polymerase gene) by using a consensus PCR protocol validated to amplify 19 diverse filovirus strains. This PCR has a sensitivity of 50–500 RNA copies with synthetic transcripts and has been further validated with blood samples .R. lescehnaulti, 6 (8%) of 75 Cynopterus spp., and 4 (7%) of 56 M. lyra bats were potentially positive after initial screening. Five (3.5%) of 141 (95% CI 1.5%–8.0%) R. leschenaultii bats were confirmed as seropositive after testing by ELISAs and Western blotting (R. leschenaultii bats were pregnant and 8 (23%) of 34 carried pups. We sampled 3× as many males as females; all 5 confirmed virus-positive animals were healthy adult males (Fifteen (11%) of 141 blotting . Bats welt males . All 698lt males . All conlt males . SimilarOur study provides evidence of Ebola virus infection in wildlife from mainland Asia and corroborates the observation that filoviruses are harbored across a much larger geographic range then assumed . PrefereRousettus spp. bats have reported higher values    ,7, and 5o India) ; and morWe demonstrated that serologic and virus surveys of bats can be informative for identifying potential virus hosts. Previous studies amplified ZEBOV nucleic acid from bat feces . We alsoPteropus giganteus bats (R. leschenaultii bats and other small fruit bat species visit date palm trees 10× more frequently than Pteropus spp. bats (R. leschenaultii bats carry. It also highlights the need for more research to understand this ecologic system and for better implementation of low-cost barriers to reduce bat–human contact during periods of date palm harvesting (In Bangladesh, human outbreaks of Nipah virus have been linked to drinking date palm sap contaminated with bat excreta, presumably from eus bats . R. lescpp. bats . This firvesting .


Togaviridae in the genus Alphavirus, is a mosquito-borne enveloped RNA virus that was identified using the Virus-Discovery-cDNA RAPD (VIDISCR) method.The identification of new virus strains is important for the study of infectious disease, but current (or existing) molecular biology methods are limited since the target sequence must be known to design genome-specific PCR primers. Thus, we developed a new method for the discovery of unknown viruses based on the cDNA - random amplified polymorphic DNA (cDNA-RAPD) technique. Getah virus, belonging to the family Aedes albopictus) collected in Yunnan Province, China. The non-structural protein gene, nsP3, the structural protein gene, the capsid protein gene, and the 3'-untranslated region (UTR) of the novel Getah virus isolate were cloned and sequenced. Nucleotide sequence identities of each gene were determined to be 97.1–99.3%, 94.9–99.4%, and 93.6–99.9%, respectively, when compared with the genomes of 10 other representative strains of Getah virus.A novel Getah virus was identified by VIDISCR from suckling mice exposed to mosquitoes (The VIDISCR method was able to identify known virus isolates and a novel isolate of Getah virus from infected mice. Phylogenetic analysis indicated that the YN08 isolate was more closely related to the Hebei HB0234 strain than the YN0540 strain, and more genetically distinct from the MM2021 Malaysia primitive strain. Alphavirus belong to the group IV Togaviridae family and include nearly 30 virus species [Alphavirus[Viruses in the genus  species . Alphavi species . Getah vlphavirus. To datelphavirus-6.in vitro but some may cause a cytopathic effect. Furthermore, specific techniques that require sequence identification are not applicable. To overcome these limitations, we developed a new method for virus discovery: Virus-Discovery-cDNA RAPD (VIDISCR), based on the cDNA-random amplified polymorphic DNA technique (cDNA-RAPD) [The identification of novel virus species is important for the identification and characterization of disease. However, present research methods are mostly applicable for known viruses but few methods exist to characterize unknown viruses. Current molecular biological techniques for the identification of new virus species are troublesome since some viruses do not replicate NA-RAPD) -11. VIDIAcute encephalitis syndrome (AES) was observed in suckling mouse with growth retardation, panting, abdominal breathing, and arthritis (data not shown). Negative-staining electron microscopy (EM) of the supernatant from infected suckling mouse brain (named YN08) revealed virus-like particles Figure . These pThe VIDISCR method was developed based on the cDNA-RAPD technique ,9,11. VITogaviridae family with 98% identity to GETV using the basic local alignment search tool (BLAST).The supernatant of the suckling mouse brain tissue infected with YN08 was analyzed by VIDISCR. The supernatant of uninfected suckling mouse brain tissue was used as a negative control. Unique amplified DNA fragments were present in the test sample but not in the control where the 11 reactions gave prominent DNA fragments in 20 VIDISCR selective PCR reactions , MAG (Russia), ALPV_M1, (China) GETV_M1 (China), MPR (Mongolia), S_KOREA (South Korea), HB0234 , YN0540 , and SAGV (Sagiyama virus from Japan). The YN08 isolate non-structural protein gene nsP3, the structural protein gene (capsid protein gene), and 3’-UTR sequence identity were 97.1–99.3%, 94.9–99.4%, and 93.6–99.9%, respectively, by alignment with 10 strains of Getah virus found worldwide. Analysis of all sequences  included in this study showed the highest nucleotide sequence identity between YN08 and GETV HB0234 strains.The YN08 isolate nsP3 nucleotide sequences identity ranged from 98.00 to 99.31%, while amino acid sequence identity ranged from 98.89 to 99.44% Table  between Alphaviruses possess a highly conserved 3’ sequence element  that immediately precedes the poly(A) tail . Both thAlignment with the other nine strains of Getah virus indicated that the 3’-UTR sequence homology between YN08 isolate and other Chinese isolates  ranged from 99.65 to 99.77% , the previously published genetic sequences of GETV and other alphavirus capsid protein genes and 3’-UTR sequences obtained from GenBank were used to construct phylogenetic trees. The phylogenetic analyses clearly showed that YN08 is more closely related to the Hebei HB0234 strain than the YN0540 strain, and more distantly related to the MM2021 Malaysia primitive strain  supplemented with 2 mM glutamine, 0.12% NaHCOVirus controls, including SV40 and SV5, were cultured on Vero E6 cells. Culture supernatants of SV40 and SV5 viruses were analyzed by VIDISCR to assess the general applicability of the technique. The unknown YN08) virus was cultured in the capsule of brain of 2–3 day old Kunming suckling mice. Pooled brain tissues contai-ning virus were added to 2 mL MEM, followed by homogenization in a pre-cooled sterile plastic grinding tube. To remove residual cells and mitochondria, 110 μL brain homogenate supernatant was centrifuges for 10 min at maximum speed 17 000 × g) in a microcentrifuge at 4°C. To remove chromosomal DNA and mitochondrial DNA from the lysed cells, 100 μL of supernatant was transferred to a fresh tube and treated with DNase I for 45 min at 37°C (Takara) [ 000 × g  virus waA reverse transcription reaction was performed with random hexamer primers (Takara) and Moloney murine leukemia virus reverse transcriptase . Second-strand DNA synthesis was carried out using Sequenase II (Takara) without further addition of primers. A phenol-chloroform extraction was followed by ethanol precipitation. The cDNA-RAPD assay was performed as previously described -11, withGETV was observed by EM. Preparation of the sample from a 1/10 volume of the brain extract from suckling mice included extraction with chloroform and incubation of the mixture for 30 min at 4°C. The extract was then centrifuged at 13 800 × g for 30 min. The precipitate was resuspended in 5 mM phosphate buffered saline  and negatively stained with 2% phosphotungstic acid. Specimens were examined using a transmission electron microscope  at 80 kV.The Getah virus nsP3 non-structural protein gene, the structural protein gene capsid gene and 3’-UTR primer sequences used were as follows : nsP3 gehttp://www.megasoftware.net/). Previously published GETV sequences used in this study include sequences YN08 isolates MM2021 , MAG , ALPV_M1 x, GETV_M1 , MPR , S_KOREA , HB0234, YN0540 , and SAGV .Sequence data were analyzed using computer programs such as DNAMAN and DNASTAR. Phylogenetic analyses were performed by the neighbor-joining method using MEGA (version 5.05; The authors declare that they have no competing interests.Tingsong Hu, Ying Zheng and Yan Zhang participated in the design and conducted the majority of the experiments in the study and drafted the manuscript. Gangshan Li, Wei Qiu and Jing Yu carried out the molecular genetic studies, participated in the sequence alignment. Qinghua Cui,Yiyin Wang, Caoxiong Zhang and Xiaofang Zhou contributed to the interpretation of the findings and revised the manuscript. Ziliang Feng and Weiguo Zhou performed the analyses of transmission electron microscope. Quanshui Fan and Fuqiang Zhang participated in the design of the study and performed the statistical analysis. All authors read and approved the final manuscript.


Clostridium difficileinfection is a significant issue in healthcare facilities, and proper hand hygiene is recommended to help prevent C. difficile transmission. It is known that alcohol based-handrubs are ineffective at killing C. difficile spores and recent studies demonstrate that the efficacy of hand washing is limited.C. difficile spores.The objective of this study was to evaluate several aggressive chemistries including chlorinated lime (the Semmelweis hand disinfection procedure) for reduction of C. difficile spore removal and inactivation. Approximately 1x106 spores of non-toxigenic C. difficile ATCC #700057 were distributed onto the palms of subject’s hands. A series of hand hygiene procedures were evaluated including a 30-second non-antimicrobial handwash and a 5 minute hand disinfection procedure with a scrub brush using 4% chlorinated lime, 2000 ppm peracetic acid, or 1000 ppm acidified bleach. Log10 reductions from baseline for each product were compared using ANOVA and post-hoc analysis (P<0.05) to identify statistically significant differences.A modification of the ASTM method E1174 was used to evaluate 10 reductions of 0.66, 0.79, 1.64, and 2.45, respectively. Although log10 reductions were low, those for chlorinated lime and peracetic acid were statistically superior to acidified bleach and the non-antimicrobial handwash.The handwash, acidified bleach, peracetic acid, and chlorinated lime achieved logC. difficile spores from hands is very difficult. The two best chemistries, peracetic acid and chlorinated lime, still only achieved log reductions of <2.5 log10, despite aggressive and lengthy application procedures not feasible for healthcare workers. These data reinforce the need for contact precautions including gloving when caring for a C. difficile infected patient; and the importance of cleaning and disinfection to reduce environmental spore contamination. Further research is needed to identify hand hygiene approaches to effectively eliminate C. difficile from hands and to reduce patient safety risk.These data further reinforce that elimination of None declared


The design of new technologies for treatment of human disorders such as protein deficiencies is a complex and difficult task. Particularly, the construction of artificial organs, based on the immunoisolation of protein-secreting cells, requires the use of suitable materials which have to be biocompatible with the immunoisolated cells and avoid any inappropriate host response.in vivo behavior of mechanically resistant hybrid beads which can be considered as a model for artificial organ for cell therapy. This hybrid system was designed and fabricated via the encapsulation of living cells (HepG2) within alginate-silica composites. Two types of beads  or alginate/silica hybrid subsequently covered by an external layer of pure alginate (ASA)), with or without HepG2 cells, were implanted into several female Wistar rats. After four weeks, the potential inflammatory local response that might be due to the presence of materials was studied by histochemistry. The results showed that the performance of ASA beads was quite promising compared to AS beads, where less abnormal rat behaviour and less inflammatory cells in histological sections were observed in the case of ASA beads.This work investigates the The current study highlights that alginate-silica composite materials coated with an extra-alginate shell offer much promise in the development of robust implantation devices and artificial organs. The success of this technique is due to its flexibility in term of constructing materials with good mechanical and thermal stability, tuned pore size, as well as an adapted morphology. Nevertheless, the encapsulation of animal cells is a challenging task. In particular, immuno-isolation is a key factor to successfully develop cell therapy technologies where cells are protected against rejection by the immune system whilst allowing nutrients and metabolites to be evacuated. This protection can only be conferred by a biocompatible and semi-permeable membrane. Although previous works generally report a molecular weight cut-off (MWCO) around 150 kDa Silica hydrogels have emerged as the prime materials to entrap living species since they can be synthesised through mild conditions  that are synthesised via a conventional synthesis method reported elsewhere Very recently, we reported the encapsulation of HepG2 cells within resistant mineralised beads composed of two parts: an alginate-silica core and a Ca-alginate layer (ASA) The composition, porosity and robustness of materials play a major role to obtain implanted artificial organs with long-term efficiency. The use of polysaccharide hydrogels is commonplace, with alginate having been employed the most because of its recognised biocompatibility. Nevertheless, silica materials derived by the sol-gel process offer several advantages over biopolymers, such as negligible swelling in aqueous media, controllable porosity, chemical inertness, and hardness. In this work, two kinds of hybrid materials, which combined the advantages of alginate (e.g. biocompatibility) with those of silica (e.g. robustness), were synthesised under mild conditions (room temperature and atmospheric pressure).in vitro biocompatibility tests have shown an absence of a proinflammatory response from cells in direct contact with alginate or silica nanopowder in vivo behavior of materials, the hybrid beads were characterised by different techniques. The mechanical strength of the materials was firstly evaluated by stirring 100 beads at 60 rpm in 100 ml of a modified PBS buffer supplemented with EDTA (50 mM) and counting the fractured beads at selected intervals of time. This analysis medium was selected to mimic the major in vivo mechanism of degradation of alginate-based biomaterials due to the gradual exchange of calcium ions with sodium ions. As shown in Previous in vitro results suggested that these materials were biocompatible in vivo experiments were performed to evaluate the inflammatory reaction within the surrounding tissues of these implanted beads.As preliminary vivo experiments were conducted to compare the performance of AS beads and ASA beads by analysing the potential lesions of surrounding tissues of the beads four weeks after subcutaneous implantation within the back midline of female rats. Throughout the experiment, the body weight gain ratio was similar in both control and treated groups  showed signs of scratching. This ratio was similar to the control group, thereby suggesting that inflammation was related to surgery rather than implanted ASA beads. These observations led to similar conclusions as those drawn by Arcangeli Histological sections of the subcutaneous and muscle tissues surrounding the implanted beads were performed to look for the presence of mast cells and polymorphonuclear leukocytes (PML). These cells are found near blood vessels in normal tissues, as sentinel cells. These sentinels are identifiable owing to their purple nucleus surrounded by a strong-red staining, especially for PML which can display various shapes, usually bi- or tri-lobed. Pictures from the control group in in vivo experiments suggest that the ASA beads appeared to have a more pronounced biocompatibility than AS beads. Furthermore, ASA beads were still intact one month post-implantation, as shown by the SEM micrographs from subgroup 4  and alginate/silica hybrid subsequently covered by an external layer of pure alginate (ASA). Consequently, AS beads are essentially distinguishable from ASA beads by their surface chemistry. The results show that the mineralised ASA beads covered by an external alginate crust are promising hybrid materials since fewer animals displayed localised inflammatory, compared to conventional alginate-silica composites (AS beads). As integrity of ASA beads was preserved during this in vivo study, we are very optimistic about their efficiency for further clinical applications.2) by Merck. The HepG2 cell line was purchased from the American Type Culture Collection (ATCC No HB-8065). The Dubelcco’s Modified Eagle Medium (DMEM) and the foetal bovine serum were obtained from Invitrogen . Before used, commercial sodium alginate powder was purified according to the method published by de Vos Calcium chloride , sodium alginate powder , ethylene glycol tetraacetic acid , hydrochloric acid , chloroform (99%), butanol (99%) were provided by Sigma-Aldrich; and sodium silicate  with 18 ml DMEM medium and 10% foetal bovine serum, and incubated at 37°C in a humid atmosphere of 5% CO2.Encapsulation was carried out using human hepatoma HepG2 cells (Hepatocellular Liver Carcinomia Cell line) cultivated in 75 cmPrior to encapsulation, cells were first washed with a sterile phosphate buffer solution (PBS), trypsinised using trypsin/EDTA and centrifuged at 1000 rpm for 4 min. The supernatant was discarded and fresh DMEM medium+serum were added to resuspend the HepG2 cells.6 cells ml−1 and the diameter of AS beads was 2.85(±0.01) mm.The first route of encapsulation used the method described by J. Livage and coworkers with some modifications 6 cells ml−1) were first encapsulated within Ca-alginate beads (1.6% wt), by dropping a cell culture/Na-alginate mix into a CaCl2 solution (110 mM) using a syringe with a 24G-needle. The alginate capsules were then placed in a sodium silicate solution (1.5 M) for a short period of time (30 s). The mineralised beads were then collected and an external Ca-alginate crust was formed by re-suspending the beads in 1.5% sodium alginate solution for 3 min. Finally, the hybrid materials were dipped into a CaCl2 solution (110 mM) in order to crosslink the outer sodium alginate layer. The diameter of ASA beads was 2.55(±0.09) mm.Secondly, the alginate-silica/alginate beads were synthesised according to the 3-step procedure previously reported Using an optical microscope, the efficiency of entrapment was checked by measuring the numbers of HepG2 cells that could grow out of the beads. In both cases, no cell was detected.The present study is conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the protocol was approved by the Committee on the Ethics of Animal Experiments of the University of Namur (Permit Number: FUNDP AR 10/136).The experiments were performed on 8 weeks old female Wistar rats bred in our animal facility under controlled environmental conditions. The animals had free access to water and were fed with a standard rat chow diet.Prior implantation step, cell viability was assessed and 90% of cells, homogenously dispersed within the AS or ASA beads, were still alive. The rats were anesthetised with a mix of ketamine/xylazine  and placed on a heated table to maintain body temperature between 37 and 38°C. A skin incision was performed along the back midline between the shoulder blades. Two beads were then implanted subcutaneously on each side of the surgical site. The incision was closed using Ethicon Vicryl suture 4–0 thread and the rats were allowed to recover. Throughout the four weeks experiment, daily observations were made related to the welfare and behaviour of the animals. This assessment consisted to manipulate the animals, observe their rodent habits, detect fear, anxiety, stress, discomfort and identify wound or trace of itching around the surgical site. Treatment with astrexine  applied on the skin surface was only performed when signs of inflammation were detected by daily examination. Four weeks later, the rats were submitted to euthanasia  to allow collection of the beads as well as sampling of the surrounding tissue, in order to observe potential local inflammation.Three groups were considered:Control group (n = 3): the rats were only submitted to anaesthesia, surgery and recovery;AS bead implants (n = 10) and ASA bead implants (n = 10) groups. These two groups were subdivided in two subgroups, rats were randomly implanted with either blank beads containing no cells  or with beads containing HepG2 cells to evaluate the immuno-isolation .Muscle and subcutaneous tissues collected around the beads were fixed in Duboscq-Brazil fluid, embedded in paraffin and cut into 6 µm thick sections, for histological staining purposes. To allow histological examination, a trichromic stain was employed using the HES protocol (Haematoxylin – Eosin– Safranin) : the slides were submitted to successive baths of toluene, methanol, water, haematoxylin, water, hydrochloric acid+ethanol, water, eosin, water, ethanol, isopropanol, safranin, isopropanol and toluene using a continuous linear stainer COT 20 (Medite Inc.). Each bath lasted 150 s with a dripping step of 10 s between baths. Coverslips were attached with DPX , and Xylene) and the slides were observed using a Multizoom AZ100 microscope (Nikon) with a DS-Ri1 camera in bright-field mode.2.Using immunohistochemistry, macrophages were identified using ED-1 antibodies. Briefly, deparaffinized sections were pretreated with a citrate buffer and endogenous peroxidase activity was eliminated by a treatment with hydrogen peroxide for 5 minutes. Sections were then successively incubated with avidin, biotin and casein proteins to block nonspecific antibody binding sites. Primary antibody  was incubated during 30 minutes. Thereafter, sections were incubated with biotinylated anti-mouse immunoglobulins (1∶50). Finally, the avidin-biotin method was employed to stain tissue sections


We isolated a novel influenza virus A(H1N2) strain from a pig on January 13, 2012, in Gunma Prefecture, Japan. Phylogenetic analysis showed that the strain was a novel type of double-reassortant virus derived from the swine influenza virus strains H1N1pdm09 and H1N2, which were prevalent in Gunma at that time. Influenza A viruses can be transmitted between humans, swine, and birds; virus subtypes have the potential to reassort and generate new viruses by cross-breeding in the various hosts  and Tree Explorer version 2.12 (http://en.bio-soft.net/tree/TreeExplorer.html)  were collected from different pig farms ≈60 km apart. The epidemiologic association may be low among the samples, because the quarantine inspection system is well established in Japan. All samples were collected from pigs 6 months of age; therefore, the potential for infection with the virus could have been low. Additional and larger studies investigating the emergence of the parent virus of the strain may be needed.Vijaykrisna et al. found a new reassortant virus among avian-type, swine-type, and pH1N1 viruses  Virus


In South Africa, the Xpert MTB/Rif assay and the GenoType MTBDRplus) for tuberculosis testing and drug-resistance testing in South Africa, and compared with the costs of conventional approaches involving sputum microscopy, liquid mycobacterial culture, and phenotypic drug sensitivity testing.We conducted a detailed reference laboratory cost analysis of new rapid molecular assays .From a laboratory perspective, Xpert MTB/RIF cost $14.93/sample and the MTBDRplus are comparable in cost to mycobacterial culture and conventional drug sensitivity testing. Overall, reference laboratories must balance costs with performance characteristics and the need for rapid results.At current public sector pricing, Xpert MTB/RIF and MTBDR Two cplus is a line probe assay that has shown good diagnostic accuracy for the detection of M. tuberculosis in smear-positive cases and isoniazid and/or rifampin resistance in several validation studies, and test results can be available in as few as 1–2 days [M. tuberculosis and rifampin resistance and offers results within hours. Xpert has the advantage of high sensitivity when performed on smear microscopy-positive sputum samples and requires little laboratory processing, overhead, or labor [MTBDR1–2 days -6. Xpertor labor .plus offer rapid results and similar performance characteristics, each has limitations and neither may be a complete replacement for conventional culture and DST [plus is a technically complicated assay requiring substantial laboratory resources, and both tests require expensive equipment and reagents. In August 2012, however, public sector prices for Xpert consumables were significantly reduced [plus and Xpert. We further explored the costs of incorporating these assays as stand-alone tests for TB diagnosis, or alternatively in conjunction with existing diagnostics.South Africa is a middle-income country that has sought to scale-up laboratory services to implement these new tests. However, while Xpert and MTBDR and DST ,3,6,7. B reduced . To dateplus, Ziehl-Neelsen smear microscopy using a light microscope, and florescence smear microscopy using auramine-O staining and a light emitting diode microscope [M. tuberculosis from non-tuberculous mycobacteria, and cultures positive for M. tuberculosis were subjected to phenotypic MGIT SIRE DST. Xpert testing was conducted per manufacturer instructions and performed using a 4-module GeneXpert (Cepheid) instrument with automated readout. MTBDRplus was performed according to manufacturer instructions and consisted of DNA extraction, amplification, and hybridization steps, with hybridization performed using a GT Blot instrument (Hain Lifescience).Costs associated with mycobacterial testing were analyzed from a laboratory perspective at the National Health Laboratory Services (NHLS) National TB Reference Laboratory in Johannesburg, South Africa. Costs were collected for testing conducted using Xpert, MTBDRcroscope . Costs wcroscope , liquid M. tuberculosis based on laboratory records. Unit costs of key equipment and consumables are shown in Table Costs were analyzed using an “ingredients” approach that involved multiplying the quantity of inputs used by their unit prices; costs and wages were gathered using detailed laboratory records. The amount of staff time, consumable supplies and equipment quantities utilized for each test were determined through direct observation of testing procedures, and included costs associated with quality assurance and quality control. Overhead laboratory costs included indirect labor costs, office and lab supplies and furniture, general operations costs, and physical infrastructure costs . Overhead costs were allocated based on the volume of testing and amount of physical infrastructure utilized by each diagnostic system. Equipment costs were annualized over their useful lifespans. South Africa is eligible to pay prices negotiated by the Foundation for Innovative Diagnostics (FIND), and costs for consumables and equipment reflect this pricing structure ,11,12. Lplus, $3.40 for fluorescence smear microscopy, $2.25 for Ziehl-Neelson light microscopy, $12.16 for MGIT culture, and $26.19 for DST using MGIT SIRE. We calculated a total cost of $16.88 per specimen tested for the combination of fluorescence smear microscopy plus the liquid culture testing scenario . This combination of fluorescence microscopy plus the liquid culture testing scenario was over $6 cheaper per sample than using MTBDRplus on all sputa, but was more expensive than Xpert (Table Base-case laboratory costs for TB diagnostics are shown in Table plus($3.46 per test). Overall, if the volume of testing in the laboratory were reduced by 50%, the cost of Xpert would rise to $16.50 per test.The cost of Xpert was largely attributable to consumables , and was driven by the cost of Xpert cartridges , but also had high labor costs ($3.46 per test) and overhead costs ($4.28 per test) due to the time involved and extensive laboratory facilities needed for test performance.By comparison, MTBDRplus in addition to the liquid culture testing scenario on all sputum samples would nearly double laboratory costs per sample  compared to a strategy of using only the liquid culture testing scenario . This strategy would lead to an incremental cost of less than $3 per sample, compared to using smear microscopy plus the liquid culture testing scenario .An additional strategy considered by some labs may be to utilize molecular assays primarily as a replacement for conventional DST. Such a strategy would cost $16.51 if Xpert were used in place of DST  and $17.75 if MTBDRplus were used  but offers the benefit of rapid isoniazid resistance testing in addition to rifampin resistance testing. Costs of molecular testing were most influenced by consumable costs which accounted for 60-80% of total costs associated with Xpert and MRTBDplus. Xpert additionally offers the benefit of reduced staff time needed for testing, with potential to increase volume of testing or increased opportunities and time to perform other diagnostic tests or laboratory activities.Scale-up of laboratory capacity for detection of TB and drug resistance is urgently needed, but may be costly. Current reference standard approaches involving mycobacterial culture and DST are slow, and are resource intensive for laboratories to perform. The Xpert MTB/Rif and MTBDRe assays . PreviouM. tuberculosis and drug-resistance. However, reliance on either Xpert or MTBDRplus alone for diagnosis of TB and drug-resistant TB in place of liquid culture and DST has limitations. Each has suboptimal sensitivity in individuals with smear-negative TB, and neither allows testing of second line drugs; Xpert also does not allow assessment of isoniazid mono-resistance [M. tuberculosis and drug-resistance, laboratories may choose to perform conventional culture and DST in addition to newer molecular assays. We found that intensive implementation of molecular testing in conjunction with conventional diagnostics for all sputa in order to optimize both sensitivity and speed of diagnosis would lead to significant laboratory cost increases (70-120% increase), making this option potentially unaffordable for laboratories in some settings.Overall, laboratories and TB control programs must balance costs with performance characteristics and the need for rapid results . Both mosistance ,7. To maplus for smear-positive samples  leads to only modest increases in laboratory costs . Such a strategy would allow rapid species-level assessment of M. tuberculosis and rapid identification of drug-resistant TB in those likely to be most infectious, while also allowing performance of reference standard testing on all patients. Similarly, we found only relatively small increases to laboratory costs if molecular testing were used as a rapid alternative to conventional indirect phenotypic DST.To assist laboratories in allocation of resources, we examined the costs of multiple diagnostic algorithms. Alternative diagnostic algorithms with selective application of molecular assays may be considered in some laboratories. We found that selectively performing Xpert or MTBDRplus as part of future diagnostic strategies.Our study has several limitations. Not all countries qualify for negotiated reduced pricing, and costs associated with labor (i.e. wages) and overhead can vary geographically. Nonetheless we provide a detailed cost-breakdown to aid generalizability and allow laboratories in other settings to estimate costs associated with implementing these emerging rapid TB diagnostic tests and provide cost estimates for Xpert testing without negotiated prices. This study was a cost-analysis from a reference laboratory perspective. Cost of transportation to a reference laboratory were not included in this analysis and may represent a significant expense and vary geographically; alternatively, implementation of Xpert at a lower level of the health system may avert specimen transport costs associated with mycobacterial culture and conventional DST that must be performed in more established laboratories. TB control programs making decisions on diagnostic algorithms must additionally consider costs associated with clinical evaluation and TB treatment in addition to laboratory costs, as well as consider the local prevalence of TB and drug-resistance and the need for rapid diagnosis. Nevertheless, our study provides important information regarding the likely diagnostic costs associated with incorporating Xpert and MTBDRplus laboratory costs are comparable to those of conventional diagnostics and should be considered as part of TB diagnostic algorithms; we additionally offer insight into the costs of alternative algorithms that some laboratories may be considering. Our results provide important information to aid future studies evaluating cost-effectiveness and implementation of emerging TB diagnostic algorithms and TB case-finding strategies.On the other hand, our study has several strengths. We incorporated recent negotiated price reductions to aid generalizability to other low and middle-income settings, and report the component of costs attributable to consumables, equipment, and labor for each TB diagnostic system. We found that the Xpert and MTBDRplus. We present detailed cost information related to implementation of these rapid molecular assays to guide laboratories seeking to scale up TB diagnostics. Overall, laboratories and TB programs must balance costs with performance characteristics and the need for rapid results. Intensive implementation of molecular assays as an addition to conventional automated liquid culture and DST may lead to significant laboratory cost increases; selective implementation of molecular assays could be considered for some settings.The cost of newer molecular diagnostic tests are comparable to conventional diagnostic methods, when paying reduced negotiated pricing for Xpert and MTBDRThe authors declare that they have no competing interests.MS conducted data collection, data analysis, and led manuscript writing and study design. VC, GC, GC assisted with data collection and manuscript writing. SD assisted with manuscript writing, study design, and conceived the study. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/13/352/prepub


Here, we present the complete genome sequence of a cytomegalovirus Towne-BAC  isolate that we first genetically engineered to mutate the UL96 gene and then serially passaged in human fibroblasts to allow for the accumulation of compensatory mutations. A total of 17 single-base substitutions were discovered in the passaged genome compared to the reference sequence (KF493877).  This mutant virus (DUL96C) manifested a smaller plaque size and significantly reduced growth rate compared to the wild-type virus (Towne-BAC) (data not shown). The mutant virus was serially passaged in fibroblasts until a bigger plaque size and an approximately 10,000-fold increased virus growth rate was achieved (data not shown). To define the mutations that accumulated during passaging of DUL96C virus, which could account for a compensatory effect on virus growth, we sequenced the genome of the passaged virus (UL96P10) and compared it to the Towne-BAC genome sequence (KF493877) , using a maximum number of ambiguities of 2, and discarding reads of <15 bp. The de novo assembly used a word size of 45, a bubble size of 90, and a minimum contig length of 1,000 bp. Paired distances were autodetected and reads were mapped back onto contigs. All other parameters were default. A total of 21.2 million paired-end reads were collected. The number of scaffolds, scaffold N50, and total sequence length were 4, 49, and 232 kb, respectively. Each scaffold from each virus had an average coverage of >7,000. Gaps in the genome sequence were filled by PCR and sequencing of resultant amplicons. Open reading frames were identified using an NCBI-BLAST search and subsequently annotated in Sequin 12.30.For the purpose of genome sequencing, individual virus plaques of UL96P10 virus were purified and grown into virus stocks. Viral DNA was purified from pelleted virions from the cleared cell culture media using a DNeasy tissue kit . The quantity and quality of purified dsDNA were determined using a Qubit fluorometric assay (Invitrogen). A Nextera XT DNA sample preparation kit (Illumina) was used to prepare the multiplexed paired-end libraries (2 × 150 bp) before sequencing using an Illumina MiSeq. The fastq files were imported into CLC Genomics workbench 6.0. We trimmed the reads for quality by using a limit of 0.05 , excluding the myc epitope tag engineered at the N terminus of UL96. The differences were located in the following regions of the genome: UL96, RNA4.9, upstream of TRS1, upstream of RNA4.9, downstream of UL97, and downstream of UL145.KF493876.This whole-genome sequence of the Towne-BAC passaged virus has been deposited at GenBank under the accession no.


Culex pipiens mosquitoes. Phylogenetic analysis demonstrated that this sequence cannot be assigned to previously described lineages of WNV.To ascertain the presence of West Nile virus (WNV), we sampled mosquitoes in 2006 in locations in southern Spain where humans had been infected. WNV genomic RNA was detected in 1 pool from unfed female  West Nile virus (WNV) has been described in Africa, Europe, the Middle East, Asia, Australia, and, most recently, the Americas. Over the last few years, many reports about WNV have been published after the outbreaks in Romania, Morocco, Italy, Russia, and Israel, but especially with the introduction and spread of the virus in the Americas. Currently, the virus has a wide geographic distribution, and WNV infection is considered an emerging zoonosis  in Czech Republic , of as yet unknown human pathogenicity, was isolated from Previous serologic surveys conducted with small rodents and humans in different areas of Spain have shown evidence of WNV circulation  and 10% of fetal bovine serum and then were stored at –80°C until they were tested for flavivirus. The homogenate was centrifuged at 13,000 rpm for 5 min at 4°C, and the screening was performed with a generic nested reverse transcription PCR  and Doñana (freshwater marshes), both located in southwestern Spain. Mosquitoes were captured in 2006 with U.S. Centers for Disease Control and Prevention light traps supplied with COCx. pipiens complex females, captured in June 2006 in Palos de la Frontera .This study comprised 35,424 mosquito specimens grouped in 1,641 pools and representing 14 species . ApproxiA fragment of 1,813 nucleotides (nt) from the nonstructural protein 5 (NS5) gene from this WNV genome (GenBank accession no. GU047875) was amplified by using 3 WNV-specific nested-PCRs designed in this study. The phylogenetic analysis resulted in a tree in which, as expected, this sequence fell under the branch of WNV, with a value of certainty of 100% , panel AAedes albopictus cells), RK-13 (rabbit kidney cells), and Vero (African green monkey cells) monolayer cells grown at a constant temperature for each cell line . Cell cultures were incubated under the same conditions for 7 days, and 3 blind passages were carried out. Signs of cytopathic effect were checked daily, and the culture supernatants were tested by reverse transcription–PCR. Neither cytopathic effect nor amplification was obtained. No virus was isolated from any of the 3 cell cultures.To isolate the virus, the positive pool was diluted 1:20 in the minimal essential medium, and 200 µL were injected onto C6/36 . The tree topology shows a common evolutionary branch between the Spanish WNV genome (HU2925/06) and lineage 4, which clusters close to lineage 3. The lineages 3 and 4 were detected recently in Europe , and they have not been previously associated with natural disease in vertebrates. In addition, the phylogenetic analysis performed on 800 nt fragments of the NS5 gene indicated that the Spanish strain was not the same that KUN MP502–66 and KOUV, and that KUN MP502–66 seems to be a different lineage.This report and the recent description of WNV lineage 1 in wild birds (