In contrast, when a Tie2-cre+ transplant was infected by primary viremia in an infected recipient, the recombined EC-derived virus poorly spread to recipient tissues. Similarly, in reverse direction, EC-derived virus from infected Tie2-cre+ recipient tissues poorly spread to the transplant. These data contradict any privileged role of EC in CMV dissemination and challenge an indiscriminate applicability of the primary and secondary viremia concept of virus dissemination.Cytomegalovirus (CMV) is frequently transmitted by solid organ transplantation and is associated with graft failure. By forming the boundary between circulation and organ parenchyma, endothelial cells (EC) are suited for bidirectional virus spread from and to the transplant. We applied Cre/loxP-mediated green-fluorescence-tagging of EC-derived murine CMV (MCMV) to quantify the role of infected EC in transplantation-associated CMV dissemination in the mouse model. Both EC- and non-EC-derived virus originating from infected Tie2- More than sixty years ago Frank Fenner proposed that virus dissemination during acute infection originates from organs replicating virus to high titer (often liver or spleen) early in infection. Although never formally proven, this model has become commonly accepted and was applied to acute virus infections in general. Recently, we challenged this model by showing that - during acute murine cytomegalovirus infection – hepatocyte-derived virus hardly disseminates to other organs. We now applied our well established model of Cre/loxP-mediated green-fluorescence-tagging of MCMV to determine and quantify the role of infected endothelial cells (EC) in transplantation-associated CMV dissemination. We observed an only very poor dissemination of MCMV from the transplant to recipient tissues and vice versa. Interestingly, we observed no evidence for preferential dissemination of EC-derived virus. Significant differences in virus organ titers were found when comparing intravenous infection with transplant-mediated infection. This suggests a preferential dissemination of cell-associated virus in the transplant setting. In summary, our findings argue for a preferential dissemination of cell-associated MCMV but demonstrate that the Fenner model does not apply to MCMV. Human Cytomegalovirus (HCMV), a member of the betaherpesvirus subfamily, represents an important opportunistic viral pathogen in the immune compromised host. Fetuses, AIDS patients, and recipients of both bone marrow and solid organ transplants are at high risk for the development of debilitating and potentially life-threatening CMV disease. Depending on the risk constellation and immunosuppressive regimen, CMV disease can occur in up to 60% of heart or kidney transplant recipients. Therefore, HCMV is the most important viral pathogen especially during the first six months after transplantation ex vivo: polymorphonuclear leukocytes (PMNL), monocytes/macrophages, and detached infected vascular endothelial cells (EC). Although PMNL are thought to be only abortively infected, they might still function as vehicles for infectious virus in vitro infected granulocytes or monocytes in vitroLeukocyte depletion of blood products derived from seropositive donors prior to transfusion efficiently prevents transfer of CMV to seronegative recipients Despite the undisputed and unique potential of EC in CMV infection and pathogenesis, it is still unknown whether infected EC are responsible for systemic virus dissemination during primary infection, contribute to this process, or merely represent an epiphenomenon with no causal involvement in the pathogenesis of organ disease in vivo and then trace the viral progeny of that cell type egfp expression cassette (MCMV-flox). Mice expressing Cre recombinase under control of either the Tie2 or the Tek promoter, which is selectively expressed in vascular EC (Tie2-cre and Tek-cre mice), were infected with MCMV-flox. In this in vivo infection model MCMV-flox is efficiently recombined resulting in MCMV-rec only during virus replication in EC. It is important to note that Cre-mediated recombination of MCMV-flox is equally efficient in Tie2- and Tek-cre mice and only mediated by EC - as shown using bone marrow chimeras - thus providing highly concordant results by both mouse strains To quantify and to address the fate of virus produced by specific cells, we developed a Cre/loxP mediated approach to label virus in defined cell types cre-negative donor mouse into an EC cre-positive host or vice versa. Here, we investigated export of EC-derived virus from heart and kidney transplants to recipients as well as import of EC-derived virus from recipients into heart transplants. This was achieved by counting and comparing the contribution of EGFP-positive EC-derived progeny to the total virus load of organs and tissues.Vascular EC are present in all organs. A way to study the role of EC in virus dissemination from one organ to another is to either introduce organs from an EC EC-derived virus from infected heart or kidney transplants readily disseminated to organs of MCMV-naïve recipients. The bulk of virus produced in and disseminated from heart is EC-derived, whereas in kidneys infected EC only provide a minor contribution. Yet, we found no evidence for any preferential dissemination of EC-derived virus from both types of transplants to other organs. The heart transplant was also tested as a target organ of EC-derived virus produced in recipient tissues. To our surprise, in contrast to the strong dissemination of virus originating from an infected transplant there was only minimal seeding of host EC-derived virus progeny to the transplant. Interestingly, this was independent of whether transplantation was performed prior to or after systemic host infection. In summary, our data argue against a privileged role of EC in virus dissemination.cre mice were transplanted heterotopically into non-infected syngeneic C57BL/6 mice (5 PFU/g organ) of predominantly recombined virus (∼85%) were observed, confirming that the transplantation procedure itself did not affect MCMV replication in general and demonstrating a very high recombination efficiency (cre mice 6 PFU Transplantation of organs from HCMV seropositive donors to seronegative recipients (D+/R-) is a known situation in transplantation medicine and represents the “high risk constellation” because up to 60% of the recipients can develop CMV disease L/6 mice . Four daficiency . This iscre mice into non-infected C57BL/6 mice, recipient organs were analyzed for the presence of disseminated virus . Collectively, the findings after transplantation of two different organs did not support the hypothesis of a predominant role of EC in virus dissemination during the first four days of infection. Only in blood a significantly higher proportion of MCMV-rec was found on day four post kidney transplantation in 3 out of 4 mice. However, as the absolute virus titers were close to the detection limit, any interpretation has to be seen with caution.Next, we studied dissemination of MCMV following kidney transplantation. Kidneys represent the majority of transplanted organs in medicine. Similar to heart transplantation, the transplantation of kidneys from seropositive donors to seronegative recipients is associated with a high risk to develop CMV-related complications ed virus . In conted virus . This locre+ heart to virus dissemination during the situation of systemic infection of C57BL/6 recipients. Under these conditions, all organs, including the transplanted heart, become infected simultaneously. Thus, MCMV-rec, wherever found, must have originated from ECs of the transplant. Note that under such conditions the infection of the transplant does not have a head start. Four days after transplantation mice were systemically (i.v.) infected with MCMV-flox and four days later they were sacrificed and virus titers determined  infection was the extent of virus production in different organs. In contrast to i.v. infection, which resulted in peak titers in the lung and high titers in heart, kidney, liver, spleen and adipose tissue , virus dcre-positive heart to a cre-negative mouse no significant contribution of virus dissemination from the heart transplant to other organs was observed. To study not only cardiac EC but EC in general as a source of virus dissemination, Tie2-cre or Tek-cre recipient mice received a non-transgenic heart. Recipients were then infected i.v. with MCMV-flox  infection, local virus production might simply outnumber secondary import of EC-derived virus. To address this issue and to initiate the activation of immune functions, systemic infection was performed prior to transplantation. Specifically, Tie2-nfection . Strikinnfection  to ∼60% nfection . This avnfection . It is infection  were on nfection . SimilarTotal titers in the transplant decreased when transplantation was delayed from 20 h to 3 days after infection, reflecting the situation at day 5 and 7 p.i., respectively. In two animals transplanted three days after infection, virus titers in the heart transplant even fell below the detection limit of 10 PFU/g organ, probably reflecting enhanced control by the host immune system at day 7. This is supported by the relatively low virus titers in spleen, kidneys and adipose tissues as well as by the lack of detectable virus in blood .In conclusion, we were surprised to see that ongoing virus replication and the accompanying immune response in the transplanted heart did obviously not alter the absolute amount of EC-derived virus originating from recipients' tissues by secondary viremia. These data demonstrate that virus dissemination between organs – originating from both endothelial and non-endothelial cells – has only minor effects on organ viral load following systemic infection.et al. were the first to propose a two-step dissemination model for systemic virus infections. Primary viremia transports the virus from the site of entry to liver and spleen where the virus replicates. Secondary viremia then causes dissemination from liver and spleen throughout the body One hallmark of CMV infection is the ability of the virus to infect many cell types and tissues from which again the virus may spread. Apparently, immune control defines to which extent this potential is realized in a given scenario. Therefore, the various clinical conditions need to be considered to explain CMV pathogenesis. Blood specimens play an important role in CMV diagnostics. Proper usage of the information gained by this analysis should monitor or even predict events that happen in organs. However, it is currently unclear under which conditions CMV is spread via blood. Fenner In the present study the vascular EC were analyzed for their claimed role in contributing to the CMV load in organs, and in disseminating the virus via primary or secondary viremia. Our salient findings are as follows: EC-derived virus significantly, ∼50% of the body virus pool, contributes to total virus load during acute infection. This contribution was quantified for the first time for the major organs. Yet, there was obviously no preference for dissemination of EC-derived virus over virus produced by other cell types. In addition, and similar to hepatocyte-derived virus, EC-derived virus was poorly disseminated via secondary viremia. These data raise doubts on the indiscriminate applicability of the primary and secondary viremia concept to virus infections in general.Properties of EC have enticed scientists to consider them as key production sites for virus dissemination, as they may release free virus particles directly into the blood stream or may detach from the vessel wall and transfer virus to other organs via the blood stream Here, we provide the first quantitative analysis of organ- and cell type-specific virus dissemination. From an infected organ EC-derived virus readily disseminated to the other, uninfected organs. In the specific cases shown here, the infected transplanted organ (heart or kidney) created the condition of a primary viremia initiating from a defined source. EC-derived virus remained a stable fraction in both heart (∼80%) and kidney (∼20%) throughout the first week of infection Infected EC might detach from the vessel wall and circulate. In fact, HCMV-infected EC were considered as a parameter for the diagnosis of HCMV organ involvement and for the study of the pathogenesis of disseminated infection Do our findings formally exclude any prominent role of EC-derived virus? The answer is both yes and no. Yes, we can exclude this role in the mouse model and for the temporal conditions of our experiments. Unfortunately, the more time passes after initial infection of the animal the definition of virus as being EC-derived virus becomes more and more indirect. EC-derived virus progeny keeps the marker independent of the cell type in which the virus replicates in further replication rounds. Thus, with our experimental setup we cannot study later phases of infection when other conditions of virus productivity and dissemination may prevail. However, according to our previous experience, second and third replication rounds contribute less and less to the viral load in the immune competent host due to the onset of immune control Nevertheless, by comparing the virus titers in different organs following transplant-derived and i.v. infection, we observed striking differences. Systemic (i.v.) infection with tissue culture produced virus preparations resulted in a uniform distribution of virus to many organs, whereas transplant-derived virus appeared to preferentially colonize spleen, lung and liver but not heart, adipose tissues and kidneys. This cannot be explained by known differences in organ specific virus kinetics. Therefore, cell-free virus, which is usually used for experimental infection, is apparently able to efficiently colonize all organs, whereas virus leaving an infected organ via a natural route reveals a different kind of spread. What could be the cause of the difference between i.v. infection with a solution enriched in isolated virions and the spread of infection from an infected organ? The most plausible explanation is that the virus leaving an infected organ during systemic infection is predominantly transported in a cell-associated manner. Yet, this difference in organ and tissue distribution shows no preference for EC-derived virus and is altogether marginal with respect to total virus load in an organ.This study was carried out in strict accordance with the recommendations and guidelines for the care and use of laboratory animals according to Tierschutzgesetz . All animal experiments were approved by the responsible state office (Regierung von Oberbayern) under permit number 55.2-1-54-2531-19-07.crecreCre-transgenic mouse strains were maintained on the C57BL/6J background. Experiments were performed with gender matched pairs of mice at 3 to 12 months of age. C57BL/6J mice were obtained from Janvier. Tek-cre mice were obtained from Jackson Laboratories (nr. 4128).M2-10B4  and BALB/c-derived mouse embryo fibroblasts (MEF) were grown as described previously 5 PFU in a volume of 300 µl.All viruses were derived from the molecular clone pSM3-fr cre or Tek-cre mice that were maintained on the genetic background of C57BL/6 mice.Syngeneic transplantations of hearts or kidneys were performed between C57BL/6 mice and Tie2-Heart transplant model: Abdominal-heterotopic cardiac transplants were performed, as previously described Kidney transplant model: The murine kidney transplantation was performed as described previously Virus load in organs was determined by plaque assay as described previously Mice were anaesthetized and the peritoneal cavity was opened. After injection of 50 µl of heparin into the inferior vena cava, abdominal aorta and vena cava were cut cranially of the transplant. After all organs were perfused with 5 ml PBS via the vena cava the heart transplant was removed and perfused separately with 3 ml PBS.The percentage of MCMV-rec compared to total virus organ load per group, mean values, and standard deviations were determined.


Outbreaks of avian influenza in poultry can be devastating, yet many of the basic epidemiological parameters have not been accurately characterised. In 1999–2000 in Northern Italy, outbreaks of H7N1 low pathogenicity avian influenza virus (LPAI) were followed by the emergence of H7N1 highly pathogenic avian influenza virus (HPAI). This study investigates the transmission dynamics in turkeys of representative HPAI and LPAI H7N1 virus strains from this outbreak in an experimental setting, allowing direct comparison of the two strains. The fitted transmission rates for the two strains are similar: 2.04 (1.5–2.7) per day for HPAI, 2.01 (1.6–2.5) per day for LPAI. However, the mean infectious period is far shorter for HPAI (1.47 (1.3–1.7) days) than for LPAI (7.65 (7.0–8.3) days), due to the rapid death of infected turkeys. Hence the basic reproductive ratio,  In recent years, outbreaks of avian influenza in poultry have both caused immense economic loss, and continued to pose threats to human health. In 1999–2000 in Northern Italy, an epidemic of a highly pathogenic avian influenza (HPAI) H7N1 virus caused outbreaks in hundreds of farms, predominantly turkey farms, resulting in the death or culling of over 13 million birds An accurate quantitative understanding of the dynamics of spread of avian influenza within the farm level is vital for developing and assessing possible control measures. Arguably, the key epidemiological parameter is Experimental transmission studies offer an alternative approach to characterise the basic infection dynamics, in which a small number of birds are initially infected, and are then co-housed with uninfected contact birds, with swabs taken on a regular basis to assess infection status of each bird. These are challenging experiments to carry out, but they allow independent exploration of virus strain, host species, and the full shedding history of individual birds may be tracked, not just the mortality data. Previous studies have completed transmission studies with H7N7 HPAI in chickens In this study, we use results from experimental transmission studies of H7N1 in turkeys combined with epidemiological models to estimate transmission rates, the infectious period, and All animal experiments were carried out under UK Home Office project licence number 70/5015  and approved by the local animal ethical committee at AHVLA. All experiments were carried out under SAPO-4 level containment at AHVLA and approved by DEFRA and the Health and Safety Executive.Individual turkeys (British United Turkeys) at three weeks of age were inoculated intranasally with approximately periment , and theperiment  and viraThe well-established SIR mathematical model of infection dynamics has hosts transitioning from (S)usceptible, to (I)nfected, to (R)ecovered or (R)emoved through death or other means. For the infection process, the standard assumption is that force of infection  is proportional to either the absolute number or the proportion of other hosts currently infected. These are termed density-dependent or frequency-dependent mass action, and represented by A continuous time stochastic infection model was used for the transmission process between each consecutive pair of observation time points. Generally these time intervals were one day, but for the 1+41 LP experiment some observations were 12 hours apart, and in the 1+40 LP experiment a missing day was handled as a single two-day transition. Recoveries and deaths were not included in the individual transition probability calculations, so the resulting model within each transition is a pure SI model, and we compute the likelihoods of the observed number of new infections given the initial number of susceptibles and infecteds , and the estimates for the transmission rate were rather broad under either model. For the two larger HP experiments, the number of birds was decreasing through death while new transmission events were still happening, but still the difference between the performance of the models  was small for each experiment on its own. However, the fitted transmission rates for the density-dependent model varied considerably over the three different-sized experiments, and hence forcing a common transmission parameter across all experiments produces a poor fit. In contrast, the frequency-dependent fitted transmission rates were relatively stable across the three experiments, and the confidence intervals are securely overlapping  panel B.Focussing now only on the preferred frequency-dependent infection model, the fitted transmission rates for HP and LP are shown together in The observed infectious periods were not exponentially distributed, so as described above the continuous gamma distribution was fitted . These dCombining the distributions for the transmission rates and the mean infectious periods, we arrive at a distribution for the basic reproductive ratio,   panel C.In this study we have provided estimates of the transmission rate, mean infectious period and the basic reproductive ratio for both highly pathogenic and low pathogenicity strains of infection with H7N1 in turkeys. In contrast to previous transmission studies, we have been able to identify narrower confidence intervals for the key parameters. This resolution has been possible through having experiments with only a single initial infected bird and a larger number of contact birds than used before, thus providing a broad window to analyse transmission events.While daily swabs give a detailed picture of infection status for each bird, model fits are still constrained to the resolution of the experiments: the dynamics are determined only down to the timescale of sampling. The exact time of infection or recovery is unknown, only the time window in which it could have occurred, and so the precise timing must be fitted or assumed. In particular, constraining recovery/death to the observed timepoints means that we may have overestimated the number of infected birds at any given time and thus correspondingly fitted low values of the transmission rates. However, we expect this to only have a small effect on our parameter estimates.The mean latent period in these experiments is shorter than our observational timescale of a day. For four of the five transmission experiments, the birds initially inoculated were shedding virus by 24 hours, and in the fifth, the swab was negative at 24 hours but by 48 hours the inoculated bird was dead. In a separate experiment with ten turkeys initially inoculated with the same HP strain under the same conditions as this study and swabbed every 8 hours, three birds were shedding by 8 hours and all birds were shedding by 16 hours (data not shown). A previous study had assumed a fixed latent period of 48 hours, though in those data all ten of the unvaccinated inoculated birds were shedding virus by 24 hours, which was the first time point after inoculation Though the number of birds in our experiments appears to be the largest in the literature for experimental analysis of transmission of HPAI, the experimental scale is still much smaller than the scale of interest: a commercial turkey farm. Here, we have explicitly tested the two main competing forms of model for population size dependence on transmission We found transmission rates for LP and HP to be indistinguishable, but the infectious period was far shorter for HP, indicating a lower corresponding  . This raAlternative mechanisms for selection of the HPAI virus, beyond a simple within-flock advantage, include of course the possibility that the between-farm transmission risk was higher for HPAI. In addition, our study considers only turkeys. Multiple species were involved in the 1999–2000 epidemic, though turkeys accounted for the majority of infected flocks. While HPAI virus appears to be devastating across a range of species, the LPAI caused more severe pathology in turkeys than chickens Speculatively, prior exposure by the LPAI virus could have conferred some partial protection to HPAI virus, so that birds could be infected but not so quickly killed by the virus. Thus, the H7N1 LPAI outbreaks in 1999 could have laid the groundwork for the successful invasion of the HPAI virus when it emerged. During the incident of H7N7 in Oxfordshire, UK in 2008, in which HPAI virus emerged from LPAI virus on a single farm, HPAI virus spread to poultry sheds housing birds with prior immunity as a result of exposure to the LPAI infection. These birds remained clinically healthy but did shed virus. It is therefore possible that the dynamic in partially immune or immune birds to a LPAI virus to a corresponding HPAI phenotype is such that there will still be infection and shedding, so cryptic infection is possible (Brown, I.H. pers. obs. and Our study was able to specify fitted parameters to a greater accuracy than previously possible for either studies based on observed mortality data from outbreaks, or for experimental transmission studies. We made full use of the time series of infection status to fit transmission rates. This approach will be more accurate than using only the information of final size, particularly when the final size in small experimental groups may be all birds infected for even moderate


Imagine the following scenario: You are head of the medical education unit at a large teaching hospital. You have had a contract with an external organisation that provides assessment services, but that contract has just run out and you now need to decide whether to renew or go with an alternative provider. You consider the costs of switching provider but also have less tangible concerns about going with a lower cost service — even though some of the lower cost alternatives seem excellent. You have had personal experience of an assessment system breaking down in the past. You spend a lot of time worrying about the possible consequences of various decisions even though you know that some potential consequences are extremely unlikely. What should you do? And what should you stop doing?Medical education - like all forms of healthcare professionals’ education - is associated with substantial costs . As a reFor example a medical educator might decide to purchase a license for online learning resources and might decide to purchase the lowest cost option. But the educator might subsequently feel awkward at having purchased the lowest cost option and might spend time unnecessarily pondering over whether they made the right decision for the right reasons. This is the psychic cost, and if the psychic cost is too high it might outweigh any savings resulting from the lower cost purchase. Psychic costs are purely psychological and should not be confused with other costs which are more tangible such as search costs and switching costs. Search costs relate to the amount of time that a purchaser might spend in searching for perhaps a lower cost option and switching costs relate to the amount of time moving from one provider of education interventions — perhaps an online portfolio — to another. Search costs and switching costs can be applied to a range of different purchases in different circumstances .As well as psychic costs, there may occasionally be psychic benefits that result from purchasing decisions. For example an educator might purchase state-of-the-art video conferencing facilities. The facilities might be expensive but the educator may be thinking of the reduced travel that will result and consequently the reduced carbon footprint. The educator might not allocate a financial sum to the reduced carbon footprint but might nonetheless feel a psychic benefit from the decision to purchase.Recognising and reflecting upon psychic costs and articulating such reflections is worthwhile as it will enable educators to make more logical and rational decisions and not be distracted by non-financial or psychic considerations. Many sellers are adept at recognising psychic costs and developing strategies to nullify them in the purchaser. So guarantees might allay the psychic cost of worrying that a piece of just purchased education equipment might break down, and exchange policies might allay the psychic cost of worrying that purchasers will be stuck with unsatisfactory equipment. These psychic costs are exactly that - psychic — but the monetary investments required to allay them are all too real. Purchasers should try to make rational decisions and should consider whether investments designed to mitigate psychic costs are really necessary.However not everyone agrees that psychic costs really exist or that we should take account of them when making decisions. One problem with psychic costs or indeed benefits is that anyone can feel that they have experienced them, and can use them - consciously or unconsciously — to justify decisions. For example an educator could justify paying a high price for an educational intervention as they wished to avoid worry or guilt that they had bought an inferior quality intervention to save costs.So do psychic costs exist and if so what should we do about them? Undoubtedly psychic costs do exist — just as feelings about any other concept or idea exist. However taking account of them is another matter. Ideally we should reflect on our concerns about a decision that involves cost and articulate those reflections and share them with others. As with a lot of worries, getting them out in the open can go a considerable way to putting them into perspective. However we shouldn't assign a monetary value to them or allow them to help us justify decisions that involve costs. Psychic costs exist — but not on the balance sheet.


The sensitivities obtained were in accordance with the levels set by the EU as the measured limits of detection for narasin/salinomycin, lasalocid, diclazuril, nicarbazin  and monensin in eggs were 0.01, 0.1, 0.5, 53 and 0.1 μg/kg and in feed 0.1, 0.2, 0.3, 9 and 1.5 μg/kg, respectively.Coccidiostats are the only veterinary drugs still permitted to be used as feed additives to treat poultry for coccidiosis. To protect consumers, maximum levels for their presence in food and feed have been set by the European Union (EU). To monitor these coccidiostats, a rapid and inexpensive screening method would be a useful tool. The development of such a screening method, using a flow cytometry-based immunoassay, is described. The assay uses five sets of colour-coded paramagnetic microspheres for the detection of six selected priority coccidiostats. Different coccidiostats, with and without carrier proteins, were covalently coupled onto different bead sets and tested in combination with polyclonal antisera and with a fluorescent-labelled secondary antibody. The five optimal combinations were selected for this multiplex and a simple-to-use sample extraction method was applied for screening blank and spiked eggs and feed samples. A very good correlation ( Eimeria, which especially affects poultry. These infections result in economic losses because of decreased growth rate, reduced egg production and increased mortality [Coccidiosis is a protozoan infection of the intestinal tract, caused by protozoa belonging to the genus ortality . To contortality . These cortality .These coccidiostats are not allowed to be used in egg-laying chickens and in broilers 5 days before slaughter. Maximum (residue) levels for the presence of coccidiostats in food (maximum residue limits (MRLs); Commission Regulation (EC) 124/2009  have beeTo monitor these coccidiostats, two different types of methodologies have been developed to detect one or more coccidiostats simultaneously. Firstly chromatography-based methods such as high-performance liquid chromatography , 9 and lThe flow cytometry-based immunoassay (FCIA) applied in the present study is an interesting suspension array format, combining the favourable properties of an immunoassay with the opportunity of combining assays (multiplexing). This FCIA uses paramagnetic microspheres, which are internally dyed with a red and an infrared fluorophore. By varying the ratio of the two fluorophores, up to 100 different colour-coded bead sets can be distinguished and different compounds or conjugates can be coupled on the surfaces of different bead sets.The carboxylated bead surface allows simple chemical coupling of capture reagents such as antibodies, oligonucleotides, proteins, peptides, polysaccharides or receptors. For the coating of the beads with proteins, standard procedures are available . The coaAntibodies can bind to these immobilised compounds or conjugates on the beads and these binding events will be inhibited by the presence of free compounds in the sample in the applied inhibition immunoassay format. The bound antibodies are quantified by a secondary antibody (IgG) labelled with the fluorescent protein R-phycoerythrin (PE). Inside the dual-laser flow cytometer, one light source (red laser) excites the internal dyes that identifies each microsphere, and one (green laser) quantifies the PE reporter dye on the surface of the bead reflected as median fluorescence intensity (MFI). This makes it possible to simultaneously measure up to 100 different compounds in a single well.In the present study, a very attractive high throughput multiplex FCIA was developed for the simultaneous detection of six priority coccidiostats Fig.  in eggs During the development of the five-plex immunoassay, the coccidiostats with and without carrier proteins, were covalently coupled onto different types of carboxylated beads (paramagnetic and non-paramagnetic beads) and compared for their performances in two different flow cytometers and in combination with new and previously developed polyclonal antisera. The five optimal combinations were selected for the multiplex development. Cross-interactions between the assays and cross-reactivities within the assays were examined. For the assay which detects both narasin and salinomycin, special attention was paid to select a combination with an equal sensitivity for both compounds. Different assay parameters such as, concentrations of the reagents, incubation temperatures and time and buffer composition were trialled. Following this optimisation phase, blank and samples spiked egg and feed samples at relevant concentrations, incurred egg and contaminated feed samples were used to optimise the simplified extraction method and to confirm that levels set by the EU could be detected.N-hydroxysuccinimide (NHS) and 0.4 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)) were supplied by GE Healthcare . Ethylenediamine (EDA), dimethyl sulfoxide (DMSO), dimethylformamide (DMF), acetonitrile, methanol, l-glutamic acid gamma-(p-nitroanilide) (GAN), sodium carbonate (Na2CO3), sodium hydrogen carbonate (NaHCO3), sodium dihydrogen phosphate monohydrate, Tween-20 and sodium azide were obtained from VWR International . Goat-anti-rabbit-R-phycoerythrin conjugate (GAR-PE) was from Molecular Probes  and bovine serum albumin (BSA), bovine thyroglobulin (BTG) and ovalbumin (OVA) from Sigma-Aldrich . EDC, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid and N-hydroxysulfosuccinimide sodium salt (sulfo-NHS) were from Pierce . The carboxylated microspheres No. 012, 021, 029, 043, 059 and 070 (SeroMAP™ (non-paramagnetic) and MagPlex® (paramagnetic) beads), in stock bead suspensions of 1.25 × 107 beads mL−1 and stored at 4–6 °C, and sheath fluid were obtained from Luminex . N-succinyl-l-alanyl-l-alanyl-l-alanine 4-nitroanilide (SAN) was from Fluka . Protein A Sepharose 4 Fast Flow from GE Healthcare (Sweden) was used for the purification of the sera.The HBS-EP buffer and an amine coupling kit , FERA  and the Nutreco company MasterLab B.V. .The measurements were performed using two flow cytometry platforms. The Luminex® 100 IS 2.2 system, supplied by Applied Cytometry Systems , consisted of a Luminex 100 analyser and a Luminex XY Platform and operated with StarStation System control software. The FLEXMAP 3D® system, controlled with xPONENT 4.0 Software Solutions, was obtained from Luminex .All washing steps were carried out on a Bio-Plex™ Pro II Wash Station  using a magnetic plate carrier or the vacuum carrier. Plates were incubated on a Dynatech microtiter vari-shaker . During the coupling procedures, the paramagnetic microspheres were captured using a DynaMag-2™  magnetic separator. Mixing for sample extraction was performed in a REAX2  overhead shaker. All centrifuge steps were carried out in an Eppendorf 5810 R centrifuge using the A-4-62 rotor .Polyclonal antisera were obtained from rabbits after immunisations with the different coccidiostats conjugated to the carrier proteins BTG, BSA or transferrin in which three different conjugation chemistries were applied. These chemistries were performed at the Institute of Agri-Food and Land Use of the Queen’s University Belfast  and were also applied for the couplings to OVA or horse-radish peroxidase (HRP).The N,N′-disuccinimidyl carbonate (DSC) reaction was applied for the conjugation of diclazuril, lasalocid and salinomycin to proteins. DSC consists of a carbonyl group containing two NHS esters. The compound is highly reactive towards nucleophiles and, in non-aqueous conditions, it can be used to activate a hydroxyl group to a succinimidyl carbonate derivative. DSC-activated hydroxyl compounds can then react with amine-containing molecules to form stable cross-linked products. For the activation, the drug was mixed with a six times molar excesses of DSC and dimethylaminopyridine, all dissolved in dry acetone. After mixing for 3–4 h at room temperature (RT), the acetone was evaporated to dryness under a stream of nitrogen and the activated drug was re-suspended in PBS (pH 7.5). DMSO or pyridine was used if the product was insoluble in water. The activated drug was added in a molar ratio equivalent to the number of amines groups to the protein (50 mg) dissolved in PBS and incubated for at least 4 h at RT or overnight (ON) at 4 °C.The N,N′-carbonyldiimidazole (CBDI) reaction was applied for the conjugation of lasalocid, monensin, salinomycin, narasin and GAN (a mimic of the active component DNC in nicarbazin) to proteins. The activation of the carboxylate group with CBDI provides an intermediate imide with imidazole as the active leaving group. In the presence of a primary amine-containing compound, the nucleophile attacks the electron-deficient carbonyl, displacing the imidazole and forming a stable amide bond. The drug dissolved in DMF was added to a six times molar excess of CBDI dissolved in acetone and stirred at RT for 4 h and the acetone was then evaporated. The protein (10–20 mg/mL) was dissolved in carbonate/bicarbonate buffer (pH 9.6), and the activated drug was slowly added whilst stirring. The mixture was incubated ON at 4 °C.The carbodiimide reaction was applied for the conjugation of diclazuril and SAN (another mimic of the active component DNC in nicarbazin). In this, EDC reacts with carboxylic acids (hapten or protein) to form highly active O-acetylisourea intermediates which can then react with a primary amine to form an amide bond and release EDC as a soluble isourea derivative. NHS can be used with EDC to increase the stability of the active intermediate. The drug (10 mg) was dissolved in 0.05 M 2-(N-morpholino)ethanesulfonic acid (MES) and 0.5 M sodium chloride (pH 4.7) or pyridine. EDC (10 mg) and NHS (5 mg) dissolved in 0.005 M MES; 0.5 M sodium chloride (pH 4.7) were added to the drug and mixed for 15 min. The protein (20 mg) was dissolved in PBS and added to the activated drug dropwise and allowed to incubate ON at RT.All the protein-drug conjugates were purified by overnight dialysis against 0.15 M saline over 24 h (3 × 4 L).Immunisations were performed at CER  and at least five rabbits were injected with the same immunogen which was received in lyophilised form and was reconstituted to 1 mg/mL with water for injection. For each injection, 0.2 mg of the immunogen was emulsified with NaCl (0.9 %) and Freund’s adjuvant by mixing vigorously. A complete adjuvant was used only for the first immunisation and incomplete adjuvant for all subsequent booster injections. The emulsified antigens were injected subcutaneously at four sites on the New Zealand white-specific pathogen-free rabbits at days 0, 14 and 28 and then every 28 days, with test bleeds taken from the marginal ear vein 10 days after immunisations. These bleeds were collected from the third immunisation onwards. The blood was centrifuged and collected serum was stored at −20 °C until further use.Two methods were used for the evaluation of the presence of antibodies in the sera of the immunised rabbits.2O2. The reaction was stopped by the addition of H2SO4, and the colour intensity was measured at 450 nm.In the first assay format, the microtitre plate was coated with purified sheep anti-rabbit IgG and diluted sera as well as HRP-drug conjugate were added and incubated ON at 4 °C. After washing, colour developed was achieved by given a 30-min incubation of TMB/HIn the second assay format, the diluted sera were coated to the microtitre plate and buffer or standard solution as well as the HRP conjugate were added and incubated for 2 h at 37 °C. After washing, colour development was performed as described for assay format 1.7 beads/mL) was vortexed vigorously for 5 min and then placed in an ultrasonic bath for 1 min. For each bead coupling, 200 μL of the concentrated beads was transferred to an Eppendorf tube and placed in the magnetic separator. Beads were allowed to settle for 3 min and the supernatant was aspirated. The beads were washed by 500 μL MQ water added, mixed for 1 min by vortex, placed in an ultrasonic bath for 1 min and in the magnetic separator for 3 min and aspirating the supernatant. This was the standard washing procedure for the paramagnetic beads. The beads were subsequently washed with 500 μL of 0.1 M NaH2PO4 (pH 6.3), activated for conjugate coupling in 100 μL of 0.1 M NaH2PO4 (pH 6.3) containing 500 μg of EDC and 500 μg of NHS and incubated for 20 min at room temperature. After activation, the beads were captured and washed once with 500 μL PBS . Final conjugate concentrations were adjusted to 125 μg/mL in PBS. The activated beads were then resuspended in 0.5 mL of the conjugate solution. Subsequently, they were incubated for 2 h at RT in the dark with gentle agitation. After the coupling, the conjugate solutions were removed and the beads were washed four times with blocking buffer . The beads were then resuspended in 200 μL blocking buffer and stored ON at 4 °C before use.HRP, BSA or OVA conjugates of lasalocid, monensin, salinomycin, narasin, diclazuril and GAN were coupled to MagPlex® paramagnetic carboxylated beads, and each to a unique bead set, according to a standard Luminex protocol for protein coupling . The con2CO3 and 35 mM NaHCO3 at pH 9.6). To 150 μL of this solution, 150 μL EDC and 150 μL NHS (amine coupling kit) were added and this mixture was incubated for 1 h in the dark before it was added to the activated beads. The concentrated bead suspensions were pretreated as described previously and 200 μL was transferred to an Eppendorf tube and placed in a magnetic separator (paramagnetic beads) or centrifuged for 3 min at 10,000×g for the non-paramagnetic beads. The paramagnetic beads were washed as described above. For the non-paramagnetic beads 500 μL MQ water were added, the mixture was vortexed for 1 min, placed in an ultrasonic bath for 1 min and finally centrifuged for 3 min at 10,000×g. This was the standard washing procedure for the non-paramagnetic beads. The beads were subsequently washed and activated as described previously. The activated beads were captured magnetically or centrifuged and the supernatant removed. The pellet was resuspended in 250 μL of 0.1 M EDA (pH 8.5). After 15 min shaking at RT in the dark, the beads were captured or centrifuged and the supernatant was discarded. The pellet was washed twice with 500 μL PBS. Finally, the beads were re-suspended in the 450 μL of activated coccidiostat solution. The mixture was incubated for 2 h in the dark in a test tube rotator, centrifuged and the supernatant was removed. After the coupling, the drug solutions were removed and the beads were washed and resuspended in blocking buffer as described above.Lasalocid, monensin, salinomycin and GAN were directly coupled to paramagnetic (MagPlex®) or non-paramagnetic beads (SeroMAP™), and each to a unique bead set, using a previously described procedure  in whichg for 10 min. Of the supernatant, 4 mL were transferred to another tube and evaporated to dryness under a flow of nitrogen at 50 °C. After the addition of 0.2 mL of methanol, the sample was vortexed for 10 s. Subsequently, 0.8 mL of HBS-EP buffer was added, the sample was vortexed for 10 s and vigorously shaken for 10 min, followed by a centrifugation at 2,400×g for 10 min.Homogenised whole eggs or grinded feed (2 g) were mixed with 8 mL of acetonitrile by immediately vortexing for 30 s. The sample was vigorously shaken on an overhead shaker for 10 min and centrifuged at 2,400×−1 bead set−1, and antibody dilutions  were made in 0.1 % BSA blocking buffer [Calibration curves of the individual coccidiostats  and monensin) or of a mixture of coccidiostats were prepared in HBS-EP buffer containing 10 % methanol. Bead suspensions, with concentrations of around 100 beads μLg buffer . SubsequA very similar protocol to that applied for the paramagnetic beads was used, except that 96-well filter-bottom plates were utilised which were pre-wetted with 100 μL of 0.1 % BSA in HBS-EP and aspirated before use. A second difference was the washing step where the plate was only washed twice, using the vacuum carrier, and finally, 100 μL of 1/600 diluted GAR-PE was added.For each of the five different coccidiostat assays, antisera from between 5 to 15 rabbits were available for testing and for most assays with different coated beads.n = 5) and narasin-BTG (n = 5), none showed colour development in the ELISA. FCIAs could be made with four of these antisera when beads were coated with their respective conjugates. However, low cross-reactivities were observed with the two structures. The best combination was anti-salinomycin (CF6) combined with narasin-BSA-coated beads, which resulted in a reasonable response  and antibody dilution (1:400), showing a 150 % cross-reactivity for narasin compared with salinomycin with a high sensitivity .Salinomycin and narasin have almost identical chemical structures Fig.  as well n = 6) or low (n = 9) responses were seen with the FCIA using the direct monensin-coated beads. When monensin-OVA-coated beads were used, high responses were observed but with a very low sensitivity . A new monensin-BSA conjugate was therefore produced, coupled to the beads and performed best with the antisera CF141-CF145 (monensin-CBDI-BTG). For further optimisation one of the antisera (CF141) was affinity purified using a protein A column. These modifications had a positive effect on the sensitivity of the assay (IC50 of 0.3 ng/mL) and the maximum response obtained .For the production of polyclonal sera against monensin, two attempts with each five rabbits immunised with monensin-BTG (CF1-CF5) or monensin-CBDI-BTG (CF141-CF145), and one attempt with five rabbits immunised with monensin-CBDI-BSA (CF146-CF150) were done. They did not present any satisfying results in the applied ELISAs. Likewise no (n = 5) immunised with diclazuril-BTG, binding was observed in the ELISA and FCIA however, the IC50 values were high . A previously developed antiserum against carboxy-diclazuril-BTG (CT28) gave a better sensitivity in the ELISA (IC50 of 5.2 ng/mL for free diclazuril). Similar results were seen in the FCIA and the best combination was CT28 with diclazuril-HRP coated beads (IC50 of 1.9 ng/mL).With three of the antisera of the rabbits (n = 15), only a few showed low binding in the ELISA and the FCIA of which one (CF131) showed a good sensitivity (IC50 of 0.5 ng/mL) in combination with lasalocid-OVA-coated beads.Of the rabbits immunised with lasalocid-protein conjugates  and two of them (CF21 and CF22) performed well in the Luminex with the structural mimic GAN-OVA-coated beads (IC50 of 4–9 ng/mL).The rabbits (CF21–CF25) immunised with SAN-BTG showed binding in the ELISA with reasonable sensitivities  calculated from the maximum response (B0) obtained without any analyte.As shown in Fig. To test for cross-reactivities towards the 11 authorised coccidiostats  of the assay were below the MRLs for narasin, diclazuril, nicarbazin and monensin, which was the desired situation. Even after the reduced sensitivity of the nicarbazin (DNC) assay in egg extract, it was still possible to use this assay because of the relatively high MRL. However, for the lasalocid assay the sensitivity was too high which may lead in practice to a higher number of false positive samples Table .Table 4C50 close to the ML. The other four assays had too high sensitivities compared with the levels set within the EU L. Three incurred egg samples which were previously analysed with LC-MS , 39 wereSubsequently six feed samples contaminated with coccidiostat additives were analysed. Each feed sample contained one single coccidiostat at its ML. The results were in accordance with the spiked feed samples except for the cross-contaminated nicarbazin feed. This sample caused an inhibition in the nicarbazin assay but also unexpected inhibition in the narasin assay. It later transpired that for the preparation of the nicarbazin feed the premix Maxiban was used. This premix contains not only nicarbazin but also narasin and therefore the assay result was correct. No false positive nor false negative results were obtained with these real egg and feed samples.g for 10 min before they were analysed. It could be concluded that this method gives the opportunity to do the whole procedure over a single day or to do the extraction on 1 day and analysis of the samples the following day, which could constitute an advantage for some routine control laboratories.The ability to store the extracts was tested. It was found that egg extracts which have been stored at 4 °C for 2 weeks could be measured without any problems. Feed extracts were stored in the same way but the standard deviation between the two measurements increased. The measurements were found to improve if the feed extracts were shaken for 10 min, followed by a centrifugation step at 2,400×r ranging from 0.995 to 0.999 for the different assays). A complete validation for the application in eggs and feed will be performed taking into account internationally recognised standards such as for example the Commission Decision 2002/657/EC [During the multiplex assay development the new Luminex FM3D was introduced and to ensure that the assay could be transferred between the different platforms, compatibility with both instruments was investigated. With the five-plex coccidiostat assay, calibration curves were measured with both instruments Fig.  and a ve2/657/EC , the gui2/657/EC  as well In the present study, a bead-based FCIA screening was developed for the simultaneous detection of six priority coccidiostats: narasin, salinomycin, lasalocid, diclazuril, nicarbazin (DNC) and monensin in egg and feed. The presented bead-based assay format is very flexible and gives the opportunity to add other assays at any time, for example to extend the number of detectable coccidiostats.Different conjugates were prepared and immobilised on the beads. These conjugates had a large influences on the sensitivities and the maximum signals of the assays and explained the difference in performance between the ELISA and the FCIA. One assay had the ability to detect two structurally related coccidiostats . When the singleplex assays were combined, the non-specific binding of antisera with non-associated beads was too high but could be reduced to acceptable levels by purifying two antisera. The direct coupling of the coccidiostats on the beads did not have the desired sensitivity and maximum binding.We succeeded to develop a common and simple extraction procedure for egg and feed samples before analysis by FCIA. The final LODs for narasin/salinomycin, lasalocid, diclazuril, nicarbazin (DNC) and monensin in eggs were 0.01, 0.1, 0.5, 53 and 0.1 μg/kg and in feed 0.1, 0.2, 0.3, 9 and 1.5 μg/kg, respectively. This demonstrated that a five-plex FCIA could detect 6 coccidiostats simultaneously in eggs and feed at their MRLs and MLs, respectively. Spiked and incurred egg and feed samples were analysed and no false positive or false-negative positive results were obtained. A complete validation will be performed. The assays were tested for their compatibility with the Luminex LX100 and FM3D and a very good correlation was observed between both flow cytometers demonstrating adequate transferability between instrumental platforms.


Crimean-Congo hemorrhagic fever (CCHF) activity has recently been detected in the Kordufan region of Sudan. Since 2008, several sporadic cases and nosocomial outbreaks associated with high case-fatality have been reported in villages and rural hospitals in the region.In the present study, we describe a cluster of cases occurring in June 2009 in Dunkop village, Abyei District, South Kordufan, Sudan. Seven CCHF cases were involved in the outbreak; however, clinical specimens could be collected from only two patients, both of whom were confirmed as acute CCHF cases using CCHF-specific reverse transcriptase polymerase chain reaction (RT-PCR). Phylogenetic analysis of the complete S, M, and L segment sequences places the Abyei strain of CCHF virus in Group III, a virus group containing strains from various countries across Africa, including Sudan, South Africa, Mauritania, and Nigeria. The Abyei strain detected in 2009 is genetically distinct from the recently described 2008 Sudanese CCHF virus strains (Al-fulah 3 and 4), and the Abyei strain S and L segments closely match those of CCHF virus strain ArD39554 from Mauritania.The present investigation illustrates that multiple CCHF virus lineages are circulating in the Kordufan region of Sudan and are associated with recent outbreaks of the disease occurring during 2008–2009. The tick-borne virus which causes the disease Crimean-Congo hemorrhagic fever (CCHF) is known to be widely distributed throughout much of Africa, Southern Europe, the Middle East, Central Asia, and Southern Russia. Humans contract the virus from contact with infected people, infected animals (which do not show symptoms), and from the bite of infected ticks. CCHF was recently recognized in the Sudan when several hospital staff and patients died from the disease in a rural hospital. The genetic analysis of viruses associated with the 2008 and 2009 outbreaks shows that several CCHF viral strains currently circulate and cause human outbreaks in the Sudan, highlighting CCHF virus as an emerging pathogen. The Sudanese strains are similar to others circulating in Africa, indicating movement of virus over large distances with introduction and disease outbreaks in rural areas possible. Understanding the epidemiology of zoonotic diseases such as CCHF is especially important in the Sudan given the large numbers of livestock in the country, and their importance to the economy and rural communities. It is imperative that hospital staff consider CCHF as a possible disease agent, since they are at a high risk of contracting the disease, especially in hospitals with limited medical supplies. Crimean-Congo hemorrhagic fever (CCHF), caused by Crimean-Congo hemorrhagic fever virus (CCHFV), is a tick-borne virus with a tripartite RNA genome . CCHFV can be transmitted to humans by the bite of an Ixodid tick and by contact with blood or tissue from infected livestock and humans CCHFV has been detected in a broad geographic zone including much of Africa, southern Europe and Asia, extending from western China to the Middle East and Southern Russia, where focal endemic areas have been identified We conducted the present study to analyze a June 2009 disease cluster involving approximately seven suspect cases of CCHF in Dunkop village, Abyei District, South Kordufan, Sudan. This represents the second CCHF outbreak reported in Sudan's Kordufan region last year. Although clinical samples could only be collected for two of the cases involved in the June outbreak, both cases were laboratory confirmed as acute CCHF by reverse transcription polymerase chain reaction (RT-PCR) targeting the viral nucleoprotein (NP) of the S segment, which is one of the least variable areas of the genome The case definition used for identification of suspect hemorrhagic fever patients included onset of fever and one or more of the following symptoms: hemorrhagic manifestations , bloody diarrhea, severe headache, joint pain, chills, headache, and nausea.Blood samples were collected from two acute hemorrhagic fever (HF) patients in clean, sterile vacutainers. Samples were collected as part of routine diagnostic testing during outbreak response. Ethical clearance was obtained from the Ministry of Health of Sudan and informed consent from all patients was provided through an ethical clearance form which permitted use of the samples for diagnostic and research purposes. In addition to being used for routine malaria screening, blood was allowed to clot, and sera were separated and sent to the Division of Virology, National Medical Health Laboratory, Khartoum, Sudan, for diagnostic screening. Aliquots of serum samples were shipped frozen in Trizol buffer at a ratio of 1∶5 to the Viral Special Pathogens Branch, U.S. Centers for Disease Control and Prevention, Atlanta, GA for viral RNA extraction and subsequent sequencing of the whole virus genome.Due to the hazards associated with cultivation of CCHFV (the presumptive cause of disease), virus isolation attempts were not conducted in Sudan, and regulations prevented shipment of the infectious clinical samples to CDC's high-containment facility in Atlanta. Therefore, identification of the virus was solely dependent on serology and RT-PCR amplification, using primers targeting the S segment of the Al-fulah strain of CCHFV (Genbank accession number GQ862371), followed by direct sequencing.Serologic tests were conducted in Sudan to screen the sera for antibodies to yellow fever and dengue fever using kits provided by NAMRU3, Cairo, Egypt. Rift Valley fever and CCHF virus antibody testing in Sudan was done using ELISA kits from Biological Diagnostic Supplies Limited (South Africa). Screening was performed using either IgM enzyme-linked immunosorbent assays or indirect immunofluorescence assays (IIFT).5′-ATA CGA GTG TGC ATG GGT CA-3′) included bases 286–305 of the positive sense strand of the virus S segment, whereas the reverse primer CCHF2 (5′-TTT GCA ATG TGC TTG AGG AG -3′) included bases 892-912 of the negative sense strand. Use of primers CCHF1 and CCHF2 in an RT-PCR produced a 627-bp primary PCR product. All primers were synthesized on a DNA synthesizer  and purified using oligo-pak oligonucleotide purification columns  as per manufacturer's instructions.Viral RNA was extracted from the patient's serum samples either using QIAamp viral RNA kit  or Qiagen's RNeasy kit as per manufacturer's instructions. The nucleoprotein gene was targeted, using a pair of primers designed based on alignments of nucleotide sequences of Al-fulah strain (Genbank accession number GQ862371) with several CCHFV strains using BioEidit software . The forward primer CCHF1 During June 2009, seven patients meeting the hemorrhagic fever (HF) case definition were identified from Dunkop village. Dunkop village is located 15 kilometers to the east of Abyei in the former state of Western Kordufan, Sudan, an area that is shared in part by the government of South Sudan and the government of National Unity, Sudan. The population in Abyei District includes the nomadic Arab tribes of Misariya from the north and the Nilotic tribes of Dinka from the south. Transport from Dunkop village to the hospital in Abyei town is extremely difficult, if not impossible, during the rainy season, which includes the month of June. When passable, the distance between the primary-care centers and the surrounding villages is usually crossed by walking; therefore, evacuation of suspect cases from Dunkop village to Abyei hospital was not possible for all patients due to poor road conditions and prevailing insecurity at the time. Of the five patients able to travel to Abyei hospital, two died and one was discharged before samples could be collected for laboratory testing. Given the proximity of approximately 250 kilometers from Al-fulah , the locth through 18th 2009, allowing for the possibility of later cases having acquired the infection from the earliest cases identified.Limited epidemiologic and clinical details were obtained where possible from interviews with patients or close relatives, or from hospital records . The ageSymptoms and clinical signs of the disease among the seven suspected patients, included rapid onset of fever , and some or all of the following symptoms: headache; nausea; vomiting (with or without blood); severe muscle, joint, and/or chest pain; agitation; diarrhea; bleeding from the nostrils, mouth, gum, and upper and lower gastrointestinal tracts. Four of the seven cases progressed to coma and fatal outcome.Serologic tests on sera from two suspected HF patients were negative for yellow fever, dengue fever, West Nile fever, and Rift Valley fever, as determined by either IgM enzyme-linked immunosorbent assay (ELISA) or indirect immunofluorescence assay (IIFT). Sera from these patients also tested negative for the presence of CCHF antibodies, as is common in fatal CCHF cases when sera is drawn 1–3 days post onset of illness. A second blood sample was not available from the surviving patient for further serological testing despite having been hospitalized for 2 days before discharge.RT-PCR assays of RNAs extracted from sera of suspected HF patients were negative for Rift Valley fever, yellow fever, and dengue viruses; however, conventional RT-PCR resulted in amplification of a 627-bp PCR product specific for the CCHFV S segment (data not shown). The two RNA specimens were submitted to the Molecular Biology Laboratories at the University of Khartoum and the National Ribat University, and were confirmed as CCHF cases by RT-PCR using the same primers described in this study and CCHF primers described by Schwartz et al Using the method reported earlier Comparison of the virus sequences detected in the 2008 Al-fulah outbreak revealed that few mutations occurred in any of the three segments. Only one nucleotide difference was found among the virus S segments; both the partial Al-fulah 4-2008 and complete Al-fulah 9-2008 M segment sequences had one base change when compared to the reference Al-fulah 3–2008 sequence. The complete L segment sequences for viruses Al-fulah 4–2008 and 9–2008 contained two and one nucleotide differences, respectively, when compared to Al-fulah 3–2008. These results indicated circulation of identical or near-identical virus variants within the outbreak, consistent with the report of most of these cases being linked by nosocomial transmission Phylogenetic analysis of the virus sequences derived from the Abyei outbreak samples demonstrated that this outbreak was also caused by a virus belonging to Group III While viruses from the Al-fulah 2008 and Abyei 2009 outbreaks are both located in Group III, the Abyei strain was genetically distinct from the Al-fulah strain , and 4. In Sudan, sero-epidemiological surveys conducted over the past few decades have suggested the presence of various arboviruses in human populations Recently, CCHFV activities have been reported in the Kordufan region of Sudan. Several suspected sporadic cases and small outbreaks of CCHF associated with high case-fatalities were reported in rural hospitals from October 2008 through June 2009. CCHF was first confirmed to be present in Sudan during the 2008 nosocomial outbreak in a rural hospital in Al-fulah, Western Kordufan Transmission of CCHFV in the recent Sudanese outbreaks likely involved either tick-bites or person-to-person spread. Tick bite transmission or exposure to blood or tissues of infected livestock is more likely to occur during rainy months, when roads become very muddy and transport between villages or cities, becomes extremely difficult. In these conditions, the local residents typically live in close contact with their cattle within their home villages, increasing the risk of acquiring infection from infected livestock or by tick bite. Once CCHF cases are admitted to medical facilities, nosocomial chains of transmission can occur within hospitals and clinics lacking resources or training to fully implement universal barrier nursing conditions and effective infection control practices The genetic diversity of CCHFV, its virulence, and its potential as a bioterrorism agent underscore the importance of genetic characterization of virus strains from all geographically distinct endemic areas. This type of analysis improves the design and development of rapid diagnostics and potential candidate vaccines for this devastating disease.This investigation expands on existing data indicating that Group III CCHFV lineage, known to be endemic in Sudan, South Africa, Mauritania, and Nigeria, is broadly distributed within Africa. Furthermore, our study illustrates that multiple strains belonging to Group III virus lineage from the African continent are currently present in the Sudan and are responsible for the recent outbreaks of the disease in the Kordufan region. The genetic linkage of the recent Sudan outbreak viruses would be compatible with viruses having been introduced to Sudan from South Africa, Senegal, Mauritania or Nigeria as a result of the international trade of livestock or by bird migration. However, as the Sudan is the largest country in Africa and the richest in animal resources, it seems unlikely to import livestock and associated products from these distant regions of Africa. Phylogenetic study of the sequence diversity of all the characterized CCHFV strains clearly indicates that virus genetic lineages are not precisely restricted to defined geographic locations, but considerable movement of viruses is occurring over long distances. These data would suggest that the role of bird migration in disease transmission should strongly be considered in the dissemination of CCHFV.The occurrence of multiple CCHF outbreaks in Sudan in the past few years, and the risks these cases pose for medical staff in resource poor health care facilities indicate the importance of improved surveillance in Sudan for this important disease. The attending physicians, working in those areas of endemicity, should consider this virus in their efforts to diagnose the disease in patients presented to hospitals or clinical centers with symptoms indicative of HF.


Very little is known regarding the persistence of Highly Pathogenic Avian Influenza (HPAI) H5N1 viruses in aquatic environments in tropical countries, although environmental materials have been suggested to play a role as reservoirs and sources of transmission for H5N1 viruses.The survival of HPAI H5N1 viruses in experimental aquatic biotopes  relevant to field conditions in Cambodia was investigated. Artificial aquatic biotopes, including simple ones containing only mud and water, and complex biotopes involving the presence of aquatic flora and fauna, were set up. They were experimentally contaminated with H5N1 virus. The persistence of HPAI H5N1 virus  was determined by virus isolation in embryonated chicken eggs and by real-time reverse-polymerase chain reaction. Persistence of infectious virus did not exceed 4 days, and was only identified in rain water. No infectious virus particles were detected in pond and lake water or mud even when high inoculum doses were used. However, viral RNA persisted up to 20 days in rain water and 7 days in pond or lake water. Viral RNA was also detected in mud samples, up to 14 days post-contamination in several cases. Infectious virus and viral RNA was detected in few cases in the aquatic fauna and flora, especially in bivalves and labyrinth fish, although these organisms seemed to be mostly passive carriers of the virus rather than host allowing virus replication.Although several factors for the survival and persistence of HPAI viruses in the environment are still to be elucidated, and are particularly hard to control in laboratory conditions, our results, along with previous data, support the idea that environmental surveillance is of major relevance for avian influenza control programs. Corbicula fluminea) were capable of removing and reducing the infectivity of avian influenza viruses (AIVs) in water Dreissena polymorpha) were able to accumulate LPAI virus from the surrounding water and to retain the virus in their bodies over an extended period of time before releasing the virus back into freshwater The highly pathogenic avian influenza (HPAI) H5N1 virus is a major public health concern in Southeast Asia, where it has widely spread since its first detection in 1997 Our objectives in this study were: (1) to describe the survival of H5N1 virus in water and mud in experimental setting reproducing as faithfully as possible natural conditions observed in tropical countries; (2) to determine whether aquatic animals such as fish, tadpoles, clams, snails, mussels and aquatic flora may be contaminated and play a role in the persistence of H5N1 virus in water; (3) to determine whether autochthonous aquatic organisms such as bivalves (fresh water mussels) and labyrinth fish (fighting fish) could transmit the virus to each other.All experiments using HPAI H5N1 virus and all animal experiments were performed within the Biosafety Level 3 (BSL-3) laboratory of the Institut Pasteur in Cambodia (IPC), complying with the Animal Committee regulations of Institut Pasteur in Paris, France, in accordance with the EC 86/609/CEE directive, and approved by the Animal Ethics Committee of Institut Pasteur in Cambodia (permit number: AEC/IPC/002/2008).The clade 1, genotype Z, HPAI H5N1 viruses A/Cambodia/408008/2005 (GenBank accession numbers: HQ664938 to HQ664945) and A/chicken/Cambodia/LC1AL/2007 (GenBank accession numbers: HQ200574 to HQ200581) were used to conduct these experiments. The virus stock was obtained after propagation in Specific Pathogen Free (SPF) 9-to-11-day-old embryonated hen eggs, kindly provided by the National Veterinary Research Institute of Cambodia (NaVRI), Ministry of Agriculture, Forestries and Fisheries (MAFF). The amnio-allantoic fluid (AAF) from the second passage on SPF eggs was harvested 48 hours after inoculation and stored at −80°C until further use. Virus titre was determined by calculating the 50% egg infectious dose (EID50) per mL of virus stock. Titration endpoints were calculated using the method of Reed and Muench Mud and water used in the artificial aquatic settings described below were collected from 2 different ponds  and from a lake in areas of Kampong Cham province where an H5N1 virus outbreak had previously occurred. Some of the experiments also involved the use of rain water which was collected and stored in big jars within the IPC external facilities until use. Temperature, pH and conductivity results of all water samples were recorded on site at the time of collection. Additional physico-chemical analyses and all microbiological tests were carried out upon arrival at the laboratory . All watPoecilia reticulata), Siamese fighting fish (Betta splendens), tadpoles , snails (Sinotaia quadrata), clams (Corbicula fluminea), mussels (Pilsbryoconcha exilis), and aquatic plants (Cabomba caroliniana). Fish were bought from a private stockbreeder and the other organisms were collected from local rivers or ponds where H5N1 virus circulation was never reported (no permits required).Freshwater flora and fauna used for the artificial aquatic settings included guppies  as well as tadpoles (D) were investigated , and in complex biotopes that included aquatic flora and fauna (B)  4 EID50/mL of water) was also tested in order to study the virus persistence in case of higher level of contamination.Different virus concentrations were tested in this study. Concentrations of 2×102, 5×103 or 5×104 EID50/mL water), and different temperatures reflecting the parameters measured in the field during the transmission season in Cambodia . Aquariums with a total capacity of 28 litres (38×38×20 cm) were filled with 20 litres water and 5 kilograms mud each, and then were allowed to settle for 24 hours prior to virus inoculation. Water and mud from various origins were also tested: rain, lake, pond 1, pond 2 (as defined above). When flora (about 100 g) and fauna  were included in the experiments (B), collection of samples from each of the species used  was carried out during the first 3 days, and every 3 days from then on. Fifty millilitres of water and 5 grams of mud samples were collected on a daily basis , different inoculum doses  and 5×104 EID50/mL water (C). Water samples of 50 and 10 mL were collected daily during experiments C and D, respectively . After collection, water and mud samples were stored in sterile tubes at −80°C until testing. All aquatic animals were humanely sacrificed, following the Animal Use Protocol defined by the Animal Ethics Committee of Institut Pasteur in Cambodia (permit number: AEC/IPC/004/2008), and subsequently dissected in order to collect the main organs of interest: gills, intestines, fins, scales, brain, and remaining carcass in fish; gills, digestive gland, intestines, and remaining carcass in clams and mussels; gills, intestines, and remaining carcass in tadpoles; all organs in snails. Before dissection, the animals were washed 2 times with sterile distilled water in order to avoid contamination of the organs by the water contained in the aquarium.Prior to testing, organs were weighed and placed into vials containing 1 mL of viral transport medium (VTM)  and stored at −80°C and in 10% formalin solution  stored at room temperature. Plant samples were also weighed and stored in VTM at −80°C until use.Each experiment conducted was coupled with a control experiment, using the exactly same conditions, but without virus.−2 EID50/ mL, as previously determined All water samples were concentrated using the method described by Khalenkov et al. 4 RNA copies/g of mud (approximately 50 TCID50/ g of mud) All mud specimens collected went through an elution step with a 10% beef extract solution at pH 7 followed by a polyethylene glycol-precipitation (PEG) step for virus and RNA concentration, as described previously Precisely, for each mud sample, 5 grams were eluted in 25 mL of elution buffer (10% beef extract solution). One millilitre of the eluted sample was then kept for a first virus detection, while the remaining volume went through the additional concentration step with PEG so as to obtain a final volume of concentrated sample of 1 mL. Mud eluates and concentrates were then used for RNA extraction and qRT-PCR or virus isolation.All solid samples  were weighed and kept in 1 mL VTM before undergoing a homogenization step using the MagNa Lyser Instrument  for 3 runs of 50 seconds at 5000× g. The homogenized samples were then used for further RNA extraction and eventually virus isolation.All samples processed as described above were mixed with a solution containing a mixture of antibiotics and antifungal drugs prior to RNA extraction or virus isolation, in order to reduce the number of contaminating microorganisms in the samples All samples that tested positive by qRT-PCR were inoculated into 9-to-11-days old SPF embryonated hen eggs. Each specimen was inoculated into 3 eggs. One hundred microlitres were injected into the amniotic cavity and 100 µL into the allantoic cavity. The eggs were then incubated for 48 hours at 37°C and chilled overnight at 4°C. The AAF was then harvested and standard hemagglutination (HA) tests were performed to detect the presence of virus before confirmation by qRT-PCR. HA tests were performed in 96-wells microtiter plates with 0.75% guinea pig red blood cells and serial 2-fold dilutions of AAF. When the HA test was negative, the AAF from each of the three eggs was pooled and inoculated into a second and then a third series of 3 eggs. A maximum of three passages were performed for each sample.Immunohistochemical staining of the tissues obtained from the mussels and fish of experiment C and D was carried out for the influenza nucleoprotein using HB65  as described in previously published reports Contaminated rain water was the only type of water from which infectious particles could be recovered , althougNo infectious particle could be isolated from contaminated lake water, although viral RNA was detectable between 1 to 11 days post-inoculation depending on the conditions tested .No infectious particles could be recovered from pond water. In one instance, viral RNA persisted in water for as long as 14 days (end of the experiment)  was associated with the absence of infectious particle isolation in tadpoles and with a shorter persistence of virus RNA at a lower viral load . In cont5 copies on day 1 and 2.30×104 copies per gram of organ on day 8 , RNA was detected in water until day 4 but infectious particles were not found. Infectious virus was not isolated in the infected mussels transferred into clean water on day 6 but the qRT-PCR tested positive until the end of the experiment , no virus could be isolated from the molluscs on day 5, and the water was not contaminated by infectious particles nor by RNA. However, RNA was still detected in infected mussels until day 8 (end of the experiments) and until day 7 in mussels exposed to infected ones  but in fish only until D7, in both infected and exposed animals with 1 log difference in viral loads measured in their organs  and until day 20 (end of the experiment) in both water and fish. Viral loads varied from 2.8×105 copies/g) followed by fins (4.8×105 copies/g), while the values obtained in the other organs varied from 4.5×104 to 2.8×105 copies/g. In contaminated fighting fish, the highest mean viral loads were observed in gills (1.05×106 copies/g) and brain (4.11×105 copies/g), while the viral loads measured in the other organs collected varied from 1.90×103 to 2.93×106 copies/g (7 copies/g for tadpole immersed during 1 day in infected water and between 9.83×104 and 5.82×105 copies/g for tadpole kept 13–14 days in contaminated water)  . In clamImmunohistochemical staining of the tissues did not confirm the presence of the H5N1 virus antigen in any organ of the mussels and fish tested following experiments C and D.This study aimed to recreate simple as well as complex aquatic environments with parameters  as close as possible to those observed in Cambodia, where H5N1 outbreaks are regularly reported, and to observe the survival of the HPAI H5N1 virus in all the different compartments of these artificial aquatic biotopes which have been suggested to be at the origin of asymptomatic or sub-clinical human infections In our experiments, infectious HPAI H5N1 virus could be recovered from water during a maximum of 4 days post-contamination at 25°C but only in rain water. This temperature is commonly observed all year long in Cambodian ponds and lakes, around 20−40 cm beneath the surface, as opposed to the surface where the temperature can easily exceed 30°C. The survival of AIVs in water is known to be shorter when temperature increases Interestingly, although experiments were conducted under identical conditions, the H5N1 virus obtained from a human case did not survive in rain water in the absence of fauna. Other authors reported that in experiments where only pH, salinity and temperatures varied, H5N1 viruses appeared to persist for shorter periods than other avian influenza viruses tested The detection of virus RNA by qRT-PCR did not correlate with the recovery of infectious particles. Indeed, in some experiments, infectious virus could not be isolated while RNA could be detected for several days. In the absence of mud, plants or animals, RNA was detected for periods as long as 11 days at 25°C. In complex biotopes, an increase of the temperature from 25°C to 32°C or 34°C reduced the persistence of the RNA  in removing - by filtration - the virus from the water, and in reducing the infectivity of LPAI virus Dreissena polymorpha) were able to accumulate LPAI virus from the surrounding water, and to retain the virus in their bodies over an extended period of time before releasing the virus back into freshwater When molluscs, fish or tadpoles were introduced in aquariums containing rain water, infectious particles of the strain of human origin could subsequently be recovered and the RNA persistence of both human and avian strains increased significantly (up to 20 days in one experiment). This suggests a probable impact of these aquatic animals on the biological cycle of H5N1 virus. Faust et al. highlighted in 2009 the role of clams (Betta splendens) and tadpoles carried detectable HPAI H5N1 virus particles for 1 day only, while infectious particles were isolated from the seeded water until day 2. Interestingly, fish and tadpoles as well as water specimens tested positive by qRT-PCR for 20 days while RNA persisted for a maximum length of 9 days in aquariums containing only rain water (experiment A.1). After 1 day of transit of the infected fish in clean water, the RNA was transmitted to exposed fish for only a short period of time. But surprisingly, the initially non-contaminated water tested positive by qRT-PCR for an additional 5 days. The histopathology analyses did not show the presence of virus antigen in the animal tissues tested, suggesting that the virus was not replicating in these tissues. Nevertheless, because of the limited sensitivity of this method, this result should be interpreted with caution. Fish and tadpoles seemed to be able to concentrate the RNA but not the infectious virus in their organs, and to efficiently protect this RNA from decay. These animals also released nucleic acids in water, allowing the detection of H5N1 virus by qRT-PCR for longer periods. Their gills probably acted as filtration systems while the RNA detected in their intestines was probably only the result of the passage of the RNA through the digestive tract, presumably together with food. The detection of nucleic acids but not of infectious virus in fish's organ tissues, including in brain, could be the result of a contamination with nucleic acids from contaminated water during the delicate dissection of the tiny animals, although the animals were all washed in sterile distilled water before the dissection.Histopathology suggested that the virus was not replicating in mussels and thus that the detection of infectious particles in these molluscs was probably only the result of their natural capacity to filter water. However, this observation may also only be the result of the lower sensitivity of the immunohistochemical method compared to qRT-PCR. Fish Click here for additional data file.Table S1Physico-chemical and microbiological parameters measured in water samples prior to experimental contamination.(RTF)Click here for additional data file.Table S2Survival of infectious particles and persistence of virus RNA in water specimens of various origins.(DOC)Click here for additional data file.Table S3Survival of infectious particles and persistence of virus RNA in mud specimens of various origins.(DOC)Click here for additional data file.Table S4Survival of infectious particles and persistence of virus RNA in presence of aquatic flora and fauna.(DOC)Click here for additional data file.Table S5Viral load (number of H5 RNA copies/g) measured in different mussel organs obtained in experiment C.2 (virus A/Cambodia/408008/2005).(DOC)Click here for additional data file.Table S6Viral load (number of H5 RNA copies/g) in different fish and tadpole organs obtained from experiments D (virus A/chicken/Cambodia/LC1AL/2007).(DOC)Click here for additional data file.Table S7Survival of infectious particles and persistence of virus RNA in aquatic environments: compiled data.(RTF)Click here for additional data file.


Two travelers returning to Italy from southern Egypt were hospitalized with a fever of unknown origin. Test results showed infection with Alkhurma virus. The geographic distribution of this virus could be broader than previously thought. Flavivirus. It was initially isolated in the late 1990s  is a recently described member of the tick-borne hemorrhagic fever group of the genus Evidence suggests that ALKV infects humans either transcutaneously (by contamination of a skin wound with the blood of an infected vertebrate or through the bite of an infected tick) or orally through consumption of unpasteurized contaminated milk. Transmission to humans has been associated with butchering of sheep and camels. No human-to-human transmission has been reported. ALKV is classified in different countries as a BioSafety Level 3 or 4 agent.ALKV has been detected only in Saudi Arabia, but the closely related Kyasanur Forest disease virus has spread as far as India and the People’s Republic of China  in a touristic village in southern Egypt, near the Sudan border. While visiting a camel and dromedary market in Shalatin on April 29, he was bitten on the foot by an unidentified arthropod . Soon after, a small, papular lesion developed. During his return flight to Italy, ≈48 hours after the bite, the patient experienced high fever, shaking chills, anorexia, malaise, nausea and vomiting, and blurred vision. During the next 5 days, these signs and symptoms worsened, and the man was admitted to the “Ospedali Riuniti di Bergamo” in northern Italy. His medical history was unremarkable, but he frequently traveled abroad and had been vaccinated against yellow fever in 1998.3), thrombocytopenia , and increased liver enzymes . The patient was given acetaminophen, and fever and general malaise progressively decreased over the next 5 days. He was discharged 11 days later, on May 17, in good general condition despite persistence of asthenia.Laboratory test results showed leukopenia  5 gene of flaviviruses (www.ncbi.nlm.nih.gov/blast/Blast.cgi] submission showed 97% identity with AF331718). This unexpected result called for further investigations to confirm the diagnosis of an ALKV infection. Thus, an ALKV-specific nested reverse transcription–PCR selective for a wider region of a different gene (E) was designed by using the following primers: outer forward 5′-TGGAACCCCACACGGGTGACT-3′; outer reverse 5′-ATGCCCACTGTCGGTTGGCG-3′; inner forward 5′-CCCACAGCAATCGAAAAACGGCATC-3′; inner reverse 5′- GCCCACATCACAGGTGACATGACC-3′.Acute-phase and convalescent-phase serum samples  were sent to the virology laboratory of the “Lazzaro Spallanzani” National Institute for Infectious Diseases in Rome to be tested for dengue and West Nile viruses. Immunoglobulins (Ig) G and M for both viruses were detected by immunofluorescence of both samples; for each virus, IgG titer was All residual biological samples collected during the patient’s hospital stay were sent to the virology laboratory of the Spallanzani Hospital  in compliance with biosafety procedures. The new ALKV PCR result was positive, and the sequence of the amplicon (GenBank accession no. HM629508) showed high homology with ALKV (99% identity with AF331718). The phylogenetic trees based on partial sequences of NS5  and E F genes coAfter submitting this article, we detected ALKV infection in a second patient. This patient had traveled to the same area ≈1 month later, visited the same camel market, and was affected by a milder disease. NS5 (HQ218942) and E (HQ218941) gene sequences obtained from this patient have been included in the phylogenetic tree, showing that they cluster together with those from the first patient .The 2 patients had traveled to an area of the world where ALKV had not been previously reported. Although viremia was demonstrated 10 days after symptom onset, and we can reliably suppose that it started when fever and chills appeared, the probability of a susceptible vector in Europe is small, and the infection seems not to be transmissible from human to human.Flaviviridae and because of the absence of commercially available serologic tests and reference biologic materials for their development. However, surveillance of travelers returning from areas where highly dangerous infectious diseases are endemic should be improved and should include ALKV. The finding that the distribution of this virus is wider than previously thought and that it includes the African continent is in line with the hypothesis that tick-borne flaviviruses originated in Africa (Laboratory diagnosis of this infection is not easy to obtain and requires a specialized laboratory because of antibody cross-reactivity with other members of the family The detection of 2 independent infection events for travelers who visited the same area in a restricted period strongly supports the hypothesis of sustained local ALKV circulation. Further veterinary and entomologic investigations are needed to expand understanding of the geographic distribution of ALKV and to assess the danger for local populations and visitors. It would be advisable to inform travelers about the danger of coming into contact with infected animals in areas where the virus has been reported. Avoidance of or minimization of exposure to infected ticks should be recommended as the most effective prevention measure.


A West Nile virus (WNV) disease outbreak occurred in 2010 in northern Greece with a total of 262 laboratory-confirmed human cases and 35 deaths. A serological and molecular surveillance was conducted on samples of hunter-harvested wild birds prior to and during the outbreak.Serum and tissue samples from 295 resident and migratory wild birds, hunter-harvested during the 2009–2010 and 2010–2011 hunting seasons at the epicenter of the outbreak in northern Greece, were tested for the presence of WNV-specific antibodies by immunofluorescence assay and virus neutralization test. WNV neutralizing antibodies were detected in 53 avian samples. Fourteen positive sera were obtained from birds hunter-harvested up to 8 months prior to the human outbreak. Specific genetic determinants of virulence  were recognized in a WNV lineage 2 strain isolated from a hunter-harvested Eurasian magpie and a nucleotide mismatch was revealed between this strain and a mosquito WNV strain isolated one month earlier in the same area.This is the first report regarding exposure of wild birds to WNV prior to the 2010 outbreak, in Greece. Results provide evidence of the implication of wild birds in a local enzootic cycle that could allow maintenance and amplification of the virus before and during the outbreak. Findings of past exposure of migratory birds to WNV upon their arrival in Greece during autumn migration, suggest avian species with similar migration traits as candidates for the introduction of WNV into Greece. The possibility that an endemic circulation of WNV could have caused the outbreak, after an amplification cycle due to favorable conditions cannot be excluded. West Nile virus (WNV) is a flavivirus of major public health concern for the last 2 decades, as associated disease outbreaks are increasing worldwide. The main route of infection is through the bite of infected mosquitoes; humans and horses develop viremia levels of low magnitude and short duration, insufficient to re-infect mosquitoes, and thus do not serve as amplifying hosts for WNV in nature .Culex species, the main vectors. Moreover, local movements of resident birds and long-range travel of migratory birds may both contribute to the spread of WNV [On the contrary, various avian species develop viremia levels sufficient to infect mosquitoes and even bird to bird transmission of the virus by direct contact has been reported . Hence, d of WNV ,4.In 2010, a major outbreak of WNV human infections occurred in northern Greece, with 262 laboratory-confirmed cases and 35 deaths .For the purposes of our participation in an FP7 EU research project (“WildTech”), wild birds samples that have been collected by the Hunting Federation of Macedonia and Thrace since 2009 were used for serological and molecular surveillance regarding exposure to various pathogens. The objective of this study was to detect possible exposure of wild birds to WNV prior to and during the outbreak. In addition we further investigated the detected WNV magpie strain for important virulence markers. These markers have been recognized to be a prerequisite for the development of viremia levels in wild birds necessary for them to be considered amplifying hosts.Corvidae family, like Eurasian magpies (Pica pica) and hooded crows (Corvus cornix) were prioritized; these corvid species are resident, with a wide daily dispersal range of up to 20 km, social, roosting in large colonies and abundant in both wetlands and urban areas [Streptopelia turtur) were also targeted; they are suspected to be a principal introductory host of WNV via their migration routes, as the virus has been isolated from actively migrating turtle doves [Anas platyrhynchos) have been found to carry WNV antibodies and recent experimental studies have also proven that Anseriformes may be able to function as carriers of WNV [Our team conducted a serological and molecular surveillance in serum and tissue samples of wild birds hunter-harvested in 2009–2010 and 2010–2011 official hunting seasons (from 20 August until 28 February of the following year). Samples were collected at the epicenter of the 2010 outbreak  test kit (EUROIMMUN®) with slight modifications of manufacturers’ instructions, to detect avian WNV antibodies, as previously described; the determined cut-off value of 1:30 was used and a (FITC)-labeled goat anti-bird antibody was applied (Bethyl Laboratories Inc.) . PositivSerological results are summarized in Table Seven sera of turtle doves hunter-harvested upon the days of their arrival in resting areas  was performed as described previously . A posit5 PFU/ml) that allow efficient transmission of WNV Lineage 1 from avian hosts to mosquitoes [The N-linked glycosylation motif (N-Y-T/S) at residues 154–156 of the E protein is present in the magpie strain; E protein glycosylation is considered a prerequisite for the development of the necessary viremic levels in avian blood  upon their arrival in resting areas of Greece during their autumn migration from breeding areas of central Europe to wintering areas of Africa, indicates exposure probably in the areas of their origin. This finding in addition to the molecular similarity of WNV strains isolated in Greece with strains isolated in previous years in Austria and Hungary ,14 suggeResults of this study strengthen the need for a continuous active serological and molecular surveillance system regarding WNV and other flaviviruses, which may provide timely information regarding virus introduction and circulation, further dispersion or introduction of new strains.The authors declare that they have no competing interests.PB and AG collected the samples, and created the map figure. ZD and CI prepared serum and tissue samples and performed the RNA extraction. GV, AT and LA carried out virological and molecular work. GV, CB, VS and LP conceived the study, selected and organized the employed protocols, carried out the sequencing studies and phylogenetic analysis and drafted the manuscript. All authors read and approved the final manuscript.