Koskinonodon perfectus and Apachesaurus gregorii, are known from Petrified Forest National Park (PEFO), AZ. Small, elongate intercentra are the single diagnostic postcranial characteristic of the smaller A. gregorii. However, a poor understanding of the earliest life stages of Koskinonodon perfectus and other large metoposaurids makes it unclear whether the proportions of the intercentra are a diagnostic feature for species discrimination or whether they are influenced by ontogeny. Previous work on metoposaurid intercentra has shown that ontogenetic information can be extrapolated from histological analyses. Here, we perform an analysis of the microanatomy and the histology of metoposaurid intercentra from PEFO to determine their ontogenetic maturity and in turn whether elongate intercentra are a reliable taxonomic feature for distinguishing North American metoposaurids. Our findings suggest that the elongate intercentra are the result of ontogenetic variation within a single large-bodied metoposaurid taxon rather than interspecific variation between two metoposaurids of vastly different adult sizes. These findings have significant implications for the taxonomy of North American metoposaurids and subsequently for interpretations of the Chinle Basin paleoenvironment. Furthermore, this study provides the first histological characterization of North American metoposaurid intercentra, thereby improving the understanding of vertebral ontogeny within Metoposauridae and offering new insights into the ecology of large metoposaurids.Metoposaurids are temnospondyl amphibians that are commonly recovered from the Chinle Formation deposits of North America. Two species,  Koskinonodon perfectus and Metoposaurus bakeri, and one of small size, Apachesaurus gregorii , AZ, USA. Koskinonodon perfectus is common in the lower units within the Chinle Formation (the Blue Mesa Member and lower part of the Sonsela Member) and is rare in the upper units (the upper part of the Sonsela Member and the Petrified Forest Member) , this is not a reliable metric given the role of ontogeny in changing body size  . A. gregribution . Althoughe taxon . The oriouazzoui . Furtherfeatures . Finallyze e.g., . This isn detail .A. gregorii has been interpreted to be a small yet mature metoposaurid taxon, it remains unclear whether the diagnostic features of the taxon, such as the shallow otic notches and the elongate intercentra, are the product of speciation or if they are merely a misinterpretation of features influenced by ontogeny in large metoposaurids. Such a possibility is rarely considered in determining whether small metoposaurid specimens are skeletally mature individuals of A. gregorii, as argued by Koskinonodon perfectus. Although A. gregorii represents a small yet mature taxon, many of the arguments rely heavily on external morphology and a scant sample of juvenile metoposaurid specimens, and its ontogenetic maturity has never been confirmed by more informative methods such as histological analyses. Accordingly, the diagnosis of A. gregorii, which relies heavily on elongate intercentra given their prevalence in depositional settings, is tentative in the absence of multiple specimens that can confirm more of the informative cranial features or that can clearly associate elongate intercentra with diagnostic cranial material. In this study, we focus on analyzing the histology and microanatomy of the single diagnostic postcranial trait of A. gregorii, elongate intercentra. The goal of the analysis is to elucidate additional ontogenetic information beyond that of the external morphology to better determine whether A. gregorii represents a small, mature metoposaurid or if it may in fact be an early ontogenetic stage of a large metoposaurid such as Koskinonodon perfectus. If A. gregorii were indeed a significantly smaller metoposaurid, we would expect to find histological and anatomical markers of relative maturity ) in the diagnostic intercentra.Although Metoposaurus and the Moroccan Dutuitosaurus   dissolved in acetone. All specimens were molded and casted according to National Park Service museum standards , with Caor axis) , but we or axis) . All speMetoposaurus krasiejowensis  that were created for Based on this classification system, we do not have any specimens referable to HOS 1 in the sample, as periosteal bone is clearly present in all intercentra. The majority of specimens belong to HOS 2 ; this designation is primarily made on the basis of relatively high vascularity within the external cortex. PEFO 38645 possesses a compact lamellar annulus, but no LAGs can be identified, rendering it referable to HOS 3 . SeveralA. gregorii are determined to belong to highly immature individuals. Several anatomical and histological features inform the ontogenetic assignment of these specimens: (1) a perforate notochordal channel; (2) a thick, more semi-circular periosteal region; (3) a greater abundance of calcified cartilage; (4) a minor degree of secondary remodeling; (5) relatively thin lamellar deposition within vascular canals; and (6) the absence of a compact lamellar annulus at the ventral margin  of a much larger size was analyzed by Metoposaurus krasiejowensis and D. ouazzoui  extreme thickening of the endochondral trabeculae at the articular surfaces; (2) the combination of a low periosteal apex with a compact lamellar annulus; (3) the absence of a clear periosteal cortex on the lateral margin in transverse profile; and (4) a thickened trabecular extension from the lateral margin to the inner endochondral region. This element was determined to be presacral, one of the posterior most elements sampled in this study, is of an intermediate size for the sample, and is of proportions typical of erfectus . It is nouazzoui . Given touazzoui . No abnoA. gregorii , we tentatively assign the PEFO material to Koskinonodon perfectus, with the understanding that future revision may be necessary as more diagnostic material is recovered  contains possible LAGs in the external cortex . This elnditions . As the nd e.g., , it is pKoskinonodon   and potentially form in different geologic settings than deposits that are skewed toward large metoposaurids. This in turn supports the hypothesis of natural ecological separation between life stages of metoposaurids. Additionally, taxa that are primarily associated with blue paleosol horizons may not be as stratigraphically restricted as previously thought, and a perceived faunal turnover may in fact be more closely linked to changes in the relative likelihood of preservation of different depositional settings.These findings, particularly with regard to the smallest intercentra, provide one line of evidence to support a hypothesis of niche partitioning between life stages of metoposaurids, which has been suggested in inonodon  and in Mposaurus . Such paposaurus . Furthern detail . A juvenity 485) . As a reth stage . Based ohorizons . PFV 040horizons . The assA. gregorii and Koskinonodon perfectus. Although fossils of A. gregorii are common in late Norian deposits, the vast majority of this material has consisted of isolated elongate intercentra, which we demonstrate here cannot be considered apomorphic. Limited fragmentary pectoral and pelvic material of A. gregorii has been described in the literature, but no justification for ascribing it to the taxon has ever been provided  may in fact be influenced by changing biomechanical demands throughout ontogeny, rather than being the result of speciation. The potential for intraspecific variation to exert an influence on metoposaurid morphology has also not been well studied in North American taxa even though studies of bonebeds of Metoposaurus krasiejowensis and Metoposaurus algarvensis have demonstrated a higher degree of variability in many cranial regions than previously thought (These findings reiterate the importance of evaluating the potential for morphological variation to be the result of ontogeny, especially when comparing two taxa of vastly different sizes, such as ed e.g., . Althouged e.g., . North Aed e.g., , which ced e.g., . Similarholotype . The potholotype . For exaholotype . Based o thought .A. gregorii is not in fact a distinct species, but rather that it is an early ontogenetic stage of a large metoposaurid, such as Koskinonodon perfectus. The stratigraphic distribution that is alleged to reflect ecological turnover could actually be controlled by taphonomic bias if niche partitioning characterized different life stages of Koskinonodon perfectus. The role of ontogeny and intraspecific variation in producing morphological variation in features such as cranial suture patterns, the basicranium, and the otic notch remain relatively unexplored in North American metoposaurids. Discovery and study of additional juvenile specimens is needed to establish a more robust ontogenetic characterization of the earliest stages of metoposaurid development, but our study has also demonstrated that underutilized methods of analysis such as paleohistology on existing specimens can shed new light on the paleobiology of extinct taxa with implications for taxonomy and ontogeny.Finally, we believe that our results provide one line of evidence that


DP-foams contain relatively small interconnected pores; PU-foams showed large pore size and interconnectivity; whereas PVA-foams displayed heterogeneous and poorly interconnected pores. PVA-foams swelled by 40 %, whereas DP- and PU-foams remained almost without swelling. Effective interconnectivity was investigated by submitting fluorescent beads of 3, 20 and 45 μm diameter through the foams. DP- and PU-foams removed 70-90 % of all beads within 4 h, independent of the bead diameter or bead pre-adsorption with serum albumin. For PVA-foams albumin pre-adsorbed beads circulated longer, where 20 % of 3 μm and 10 % of 20 μm diameter beads circulated after 96 h. The studies indicate that efficient bead perfusion does not only depend on pore size and swelling capacity, but effective interconnectivity might also depend on chemical composition of the foam itself. In addition due to the efficient sieve-effect of the foams uptake of wound components in vivo might occur only for short time suggesting other mechanisms being decisive for success of NPWT.Many wounds heal slowly and are difficult to manage. Therefore Negative Pressure Wound Therapy (NPWT) was developed where polymer foams are applied and a defined negative pressure removes wound fluid, reduces bacterial burden and increases the formation of granulation tissue. Although NPWT is used successfully, its mechanisms are not well understood. In particular, different NPWT dressings were never compared. Here a poly-ester urethane Degrapol Each year, approximately twenty million people around the world suffer from chronic wounds caused by diabetes (e.g. 7.0 million diabetic ulcers), circulatory problems and many other conditions such as surgical site infections that generate huge demands on the health care systems . Negativ® (DP)-foams [Here poly-ester urethane DegrapolP)-foams ,16,17 weP)-foams ,18,19, mP)-foams .Currently used NPWT materials consist mainly of polyurethane (PU)- or polyvinyl alcohol (PVA)-foams , thaPolyvinyl alcohol (PVA) is prepared by partial or complete hydrolysis of polyvinyl acetate to remove acetate groups and then polymerized. Polyvinyl alcohol has excellent film forming, emulsifying, and adhesive properties ,29,30. Iin vitro was performed. The aim of this study was to compare in an in vitro system DP-foams with commercially available PU- and PVA-foams  that® (DP)-foams were produced and their potential use as dressings for negative pressure wound therapy (NPWT) was explored. Morphology, swelling and effective interconnectivity of pores that is needed when negative pressure is applied through the foams to remove wound fluid, bacteria and cells during the therapy was compared between DP-foams and commercially available foams that are clinically used for NPWT in wound healing.In this study DegraPol® (DP-) with PU- and PVA-foams, cross sections of all materials were analyzed by scanning electron microscopy  this study aimed at investigating the effective interconnectivity of the pores under flow conditions to simulate uptake of vacuum-removed body fluids, bacteria or cell debris. Removal of fluorescent beads from solution was determined as a measure for the uptake of beads by the foams. As evident from the SEM images and from swelling experiments, differences in pore diameter and interconnectivity might indicate differential perfusion with fluorescence beads. Fluorescent beads of different diameters were used for perfusion as they correspond roughly to the size of bacteria (3 μm), single cells (20 μm) and small cell clumps (45 μm) that need to pass through the foams while applied in NPWT. To analyze bead perfusion, a flow device was used that could harvest polymer cylinders with a diameter of 5 mm and 2-5 mm in length. The flow device was connected to a pump, enabling continuous fluorescent bead supply and a reservoir chamber where beads were stirred by a magnetic stirring bar such that they did not precipitate. A schematic of the closed-loop system is shown in in vivo applications. These experiments revealed that although DP- and PU-foams differed considerably in their pore size the effective interconnectivity for fluorescent beads did not reflect the morphological appearance. Fluorescent beads were taken up by DP- and PU-foams very efficiently within short time and this finding was almost independent from the bead diameter as well as from pre-incubation of beads and scaffold with serum albumin.The bead perfusion experiments showed that BSA-adsorbed and native beads performed very similarly in DP- and PU-foams, as 90 and 70 %, respectively, of the 3 μm diameter beads were entrapped after 4 h. Essentially all beads of 3 μm diameter were removed from solution after 24 h. 80-90 % of 20 μm diameter beads were adsorbed after 4 h for DP- and PU-foams, respectively, and all 20 μm diameter beads were removed from solution after 24 h. In contrast, for PVA-foams, BSA-pre-adsorption showed a difference for 3 and for 20 μm diameter beads, in that 3 μm diameter BSA-pre-adsorbed beads circulated considerably longer over the entire testing period of 96 h than non-treated beads. 20 % of the 3 μm diameter beads remained circulating even after 96 h. For 20 μm diameter beads this effect was significant as 90 % of BSA-pre-adsorbed beads were entrapped within the foams only after 24 h, whereas non-treated beads were removed from the solution already after 4 h. For all further studies BSA-pre-adsorbed beads were used as the presence of serum albumin has to be considered for Bead perfusion through DP-, PU- and PVA-foams was visualized microscopically as strongly fluorescent beads could be observed by yellow depositions on black PU- and white DP- or PVA-foams, respectively b. This wWhen bead perfusion experiments were quantified and compared, it could clearly be shown that DP-foams behave very similar to PU-foams d. For 3 in vivo that often results in tight tissue ingrowth into the PU-foams during NPWT . This is found less when PVA-foams were used. Other groups have explored PVA’s chemical characteristics when using PVA-films or foams in applications where tissue ingrowth is not desired such as to prevent postoperative adhesions [The conclusion from these findings is that the pore size, swelling and interconnectivity of pores are not the only parameters decisive for effective bead penetration but also the chemistry of the foam, resulting in differential affinity between the beads and the polymer needs to be considered. Consistent with our findings would be that PU-foams induce very strong granulation tissue formation dhesions ,33.in vivo model for NPWT was very recently established [Our study tries to dissect NPWT-efficiency into different aspects that contribute to its successful use in wound healing. Here we analyzed purely materials related properties being effective foam interconnectivity, swelling and uptake of small particles. The length of the treatment varies depending the wound type and size being usually between 2 and 15 days or longer with foam changes every 2-3 days ,10. Duriablished . Therefo®) was produced according to the procedure described by Lendlein et al. [R)-hydroxybutyrate)-co-(ε-caprolactone)-diol Mn = 2,660 and 60 wt % poly(ε-caprolactone)-diol were dissolved in 1.4-dioxane and dried by heating and refluxing the solvent over molecular sieves (pore size 0.4 nm) situated in a Soxhlet apparatus mounted onto the reaction vessel, until the water content was below 20 ppm. The reaction mixture was cooled to 83 °C before the stochiometric amount, with respect to the two diols, of diisocyanate (TMDI) was added. After about one day of reaction, three portions of dibutyltin dilaurate (20 ppm) were added within one day in order to reach molecular weights between 60 and 110 kDa. The polymer was precipitated in dry ice cooled methanol and subsequently purified via dissolution in chloroform and filtration over a silicagel 60 (Fluka) column. A second precipitation in dry ice cooled methanol ended the process. DP-foams were produced by a freeze-immersion precipitation technique. A solution of DP in dioxane as solvent (5 wt %) was prepared and cooled to 10°C for 2 h. The cold solution was poured into a stainless steel mold having the shape of the desired porous body. The solution in the mold was then cooled to -25 °C in a laboratory freezer and kept at this temperature overnight. Afterwards the solidified structure was taken out of the mold and air dried at room temperature in vacuum until complete removal of the dioxane. Cylinders of the polymer were punched out for perfusion experiments after swelling to equilibrium.The polyester-urethane DP  and, polyvinyl-alcohol foam dressing  were purchased from the V.A.C. ® T.R.A.C. ™ Therapy systems, KCI Medical Products, Wimborne, UK). Cylinders of the polymer were punched out for perfusion experiments after swelling to equilibrium.Sterile black polyurethane foam dressings ; PO43- stock solution  or simulated body fluid (SFB), the foams were swollen to equilibrium. Swelling time and amount of swelling was determined for DP- and PU-foams of 5 mm in diameter and 5 mm length or 5 mm in diameter and 3 mm length. PVA-foams were analyzed using cylinders of 5 mm in diameter and 3 mm length or 5 mm in diameter and 2 mm in length. The samples were immersed in PBS  filled up to 1 L with double distilled water  or SFB. SBF was prepared by mixing a “Cax 3 H2O; ). Both sA custom-made flow device was used consisting of two parts A and B that could be connected through a screw thread leaving a sample container of 5 mm inner diameter and adjustable length (2-5 mm) by insertion of different numbers of O-rings between parts A and B . Each paTM YG microspheres , 20 µm FluoresbriteTM YG microspheres  and 45 µm FluoresbriteTM YG microspheres  were used for the experiments. The fluorescent beads were used either native in PBS or after pre-adsorption with bovine serum albumin. For this purpose 100 µL of the bead solution was pre-adsorbed in 1 mL 2 % bovine serum albumin  in PBS and incubated for 15 min, centrifuged 5 min at 10,000 rpm at room temperature. This procedure was repeated three times. The foams were pre-swollen in PBS at room temperature and cut into disks of 5 mm diameter and 5 mm length and inserted into the custom-made flow device that allowed perfusion with fluorescent beads. The flow rate was 8.1 mL/min using an Ismatec instruments pump (No. 7610-20). As the fluorescence of the circulating solution correlated with the number of fluorescent beads, two times 200 µL samples were extracted before and after 5, 20 min, 1 and 4 hours, and 1 and 3 days circulation time and transferred into white 96 well plates. The fluorescence was determined at 441 nm excitation and 486 nm emission wavelength both at 2.5 nm slit width in a PerkinElmer LS 55 Luminescence Spectrometer using the provided software Flwinlab. The numbers of the fluorescent beads in solution submitted through the foams were 8.4 x107 for the 3 µm diameter beads, the 20 µm bead solution contained 284,000 beads and the 45 µm bead solution 24,950 beads, respectively. The fluorescence at the beginning of the experiments corresponded to the number of beads (for the appropriate size of beads) and has been used as a reference. At the indicated time points the actual fluorescence was determined and relative fluorescence = To determine the interconnectivity of the foam pores fluorescent beads of different diameter were circulated through DP-, PU- and PVA-foams in a closed-loop system . The adsCylinders of DP-, PU- and PVA-foams (5 mm in diameter and 5 min in length) were swollen in PBS, perfused with 3 or 20 m diameter fluorescent beads for 24 h as indicated above or left in PBS only. The foams were fixed in 4 % paraformaldehyde dissolved in PBS for 10 min at room temperature. Then they were extensively rinsed with PBS and placed on a clean glass plate. The samples were analyzed by a Leica MZ 16A macroscope using upright light and the front lens No. 1.2  using a critical-point dryer . The samples were sputter-coated with 10 nm platinum and the images were recorded with a Zeiss SUPRA 50 VP at 1 kV and 5 kV using secondary electron signals.Cylinders of DP-, PU- and PVA-foams  were swollen in PBS and fixed in 4 % paraformaldehyde dissolved in PBS for 10 min and in 3 % glutaraldehyde in PBS for 30 min both at room temperature. The samples were dehydrated in a graded series of ethanol. Out of absolute ethanol the samples were dried over the critical point of COin vivo might occur only for short time suggesting biological mechanisms rather than materials characteristics being decisive for clinical success of NPWT in wound healing.This study demonstrates that the geometry of different NPWT dressings, even in their non-compressed form, is not the only parameter decisive for effective bead penetration but also the chemistry of the foam itself resulting in differential affinity between the beads and the polymers. In addition due to the efficient sieve-effect of the foams uptake of vacuum-removed wound components


Introduction. Costing evidence is essential for policy makers for priority setting and resource allocation. It is in this context that the clinical trials of ARVs and cotrimoxazole provided a costing component to provide evidence for budgeting and resource needs alongside the clinical efficacy studies. Methods. A micro based costing approach was adopted, using case record forms for maintaining patient records. Costs for fixed assets were allocated based on the paediatric space. Medication and other resource costs were costed using the WHO/MSH Drug Price Indicators as well as procurement data where these were available. Results. The costs for cotrimoxazole and ARVs are significantly different. The average costs for human resources were US$22 and US$71 for physician costs and $1.3 and $16 for nursing costs while in-patient costs were $257 and $15 for the cotrimoxazole and ARV cohorts, respectively. Mean or average costs were $870 for the cotrimoxazole cohort and $218 for the ARV. The causal factors for the significant cost differences are attributable to the higher human resource time, higher infections of opportunistic conditions, and longer and higher frequency of hospitalisations, among others. This is an increase from about 67,000 cases per annum in 2006. The increase has been occurring in spite of a decline in adult prevalence  from about 17 percent in 2000 to 14 percent in 2007. At the same time it has been estimated that of the 1,100,000 people living with HIV/AIDS (PLHA), a minimum of 490,000 are women while 120,000 are children. The annual AIDS-related mortality is in the region of 45,000 [The burden arising from the consequences of the spread of the human immune deficiency virus (HIV) and Acquired Immunodeficiency Syndrome (AIDS) epidemic in Zambia has been relatively high with respect to both morbidity and mortality effects. It has been estimated that the prevalence or progression of total infections of HIV increased from about 30,000 cases in 1993 to about 1,100,000 by 2010 while national population at this time was estimated to be 13 million [Apart from antiretroviral therapy, the World Health Organisation (WHO) has advocated the use of cotrimoxazole in low resource settings to enhance and complement ART administration. During the early part of 2000 decade, Medical Research Council (MRC) in collaboration with the University of Zambian Medical School undertook WHO supported clinical trials to evaluate clinical efficacy for initially cotrimoxazole and subsequently ARVs .Since the trials, the use of cotrimoxazole and ART for paediatrics has since been approved and is currently in use. However, the costs for treatment were not provided. Costing of the drugs, namely, cotrimoxazole and paediatric ARVs, became necessary in order to provide information on the cost-of-illness structure with respect to resource utilisation and programming budgets. Costing data plays an important aspect in the planning and budgeting processes of the Ministry of Health in Zambia which accounts for the provision of services to approximately 80% of the population . This isThis study augments the required cost data for children living with HIV/AIDS. This evidence is deemed essential or necessary in view of the lack of costing data on all major interventions available to the policy makers. For instance, it has been accepted that there needs to be a costed essential package of health care services that allows the health sector to have evidence of the cost of the interventions and their budgetary and resource allocation decisions . In the Research on costing of paediatric ARVs has been limited. A study by Marques et al.  focussedFor purposes of the RCT, Pedimune comprising fixed dose combinations (FDCs) of nevirapine (NVP), stavudine (d4T), and lamivudine (3TC) was manufactured exclusively for purposes of facilitating the trials. The data collection and analysis methods built on the clinical trial process. A brief outline of each of these is provided below.The Children with HIV Antibiotic Prophylaxis (CHAP) trial was a phase III placebo controlled trial design in nature. Evaluating the prophylactic properties of cotrimoxazole in assisting with reducing morbidity and mortality of children living with HIV/AIDS served as the main purpose of the trial .The primary endpoint designated was time to death or first hospital admission after randomisation. The number of children assessed for eligibility was 1,185, of which 851 were tested for HIV antibodies. 699 children were determined to be HIV-antibody positive and of this 541 consented to be enrolled into the trial. These were subsequently randomised comprising 265 in the cotrimoxazole arm and 269 in the placebo arm.The main purpose and objective of the CHAPAS trial was to determine the efficacy of newly formulated three in one paediatric ARVs. Additional objectives included assessing pharmacokinetic and adherence characteristics and practices, respectively, of each of the medications , 18.Eligibility for enrolment into the study was based on children with HIV aged between 3 months and 14 years, all inclusive, and who were eligible for ART, according to the WHO criteria.211 children were enrolled in a 1 : 1 ratio to start with Pedimune either at a full dose on a twice daily schedule or on a dose escalation schedule of once a day administration for 14 days which is subsequently scaled up to full dose, with the latter schedule having a 50 percent of normal dose of NVP. Enrolment took place over a 12-month period and follow-up continued until the last randomised child had completed a 48-week schedule. Streptococcus pneumoniae, frequency of clinical malaria, changes in weight and height, and the number of courses of cotrimoxazole required.Primary endpoints were defined as either time to death or grade 3 or 4 adverse reactions. Secondary endpoints were determined as time to death or hospitalisation, episodes of definitive and presumptive bacterial infections, resistance to cotrimoxazole in children developing bacterial infections, development ofThe data collection was made at the following points: (1) before trial commencement, (2) follow-up, that is, baseline and midline, and (3) end line of the trials. Data capture and recording used case report forms for daily capture of all resources used including care giving visits or facility visits by the patients and the carers. These forms captured both socioeconomic and clinical data. These data are summarised in Costing theory decomposes costs into different constituent parts. However, the application of cost theory seldom works in isolation as accounting methodologies and analytical tools are often, such as in the case of allocation of costs necessary. Allocation methodology, as shown in  International Indicator of Drug Pricing publication [Costs are differentiated depending on their related activity or functionality. Patients, communities and society in general, and providers and policy makers, that is, ministries of health, may all have a different conceptualisation of costs and costs may be evaluated from different perspectives that include patient, provider, or societal . Cost dalication , 21.Data analysis was done in Excel and Stata. The method for cost estimates and analysis in this study is based on the provider perspective.Cost structures are determined on the basis of fixed and variable costs. The fixed costs include the investment costs such as infrastructure and administrative costs. Variable costs include costs associated with providing specific services. These may include drugs, medical supplies, and nursing care.The cost estimates were adjusted to 2015 values using the United States consumer price indices for the period of 2006 and 2015. Administration costs such as the administration and management and security costs of the UTH were apportioned as a proportion of the share of the space of paediatric ward facilities of UTH. The laundry and catering costs were allocated based on the total inpatient load of the paediatric section relative to the total inpatient care admissions.Micro costing, alternatively termed as the “ingredients” approach, has been categorised as the most precise costing method and is associated as being the closest to a “gold standard” in costing terms . This isA problematic issue in handling costs often arises in the case of shared costs under a hospital environment in which several services are offered. While there are direct programme or service costs related to a particular condition and set of patients, for example, medications, there are however complications with respect to the question of how shared services, such as space, administration, medical, and nursing care, should be apportioned. It has been noted that none of the few costing studies available on paediatric ART other than the study by Marques et al.  have addA direct allocation method has been used to apportion costs in the study. The application of this method is justifiable in spite of the implied or actual exclusion of interactions between or among other departments. However, given the nature of RCTs and that the bulk of overhead costs arose in the cases of laboratory diagnosis and inpatient costs, it is appropriate to adopt this method. Cognisance is also taken of the options that involve stepdown allocation or partial adjustments for interaction of overhead costs; stepdown allocation with iterations or full adjustment for interaction of overhead departments; or simultaneous allocation in which there is full adjustment for interaction of overhead departments , 22, 23.As the costs were collected alongside the clinical trials, the case report forms were used to derive the resource consumption units and utilised in the costing substudy.Li is labour costs for each particular cadre involved such as the medical doctor, nurses, and laboratory technicians. Kt is capital items involved including buildings, laboratory equipment, and motor vehicles. Dd represents medicines and medical supplies including ARVs, cotrimoxazole, and antibiotics. Ll is laboratory diagnostic tests for haematology, biochemistry, and lymphocyte and virology test.The generic formula used in the costing exercise was as follows:Data on resource use was collected throughout the entire duration of the study. The resources consumed during the study were identified on the basis of the structure of sources and types of resource consumption in The fixed cost items in the study were identified as laboratory equipment for laboratory analysis and performance of activities such as CD4 counts, HIV/AIDS testing, malaria testing, motor vehicles, and infrastructure for the health facilities.The medications and medical supplies which were listed by individual patients were collected from the same sources as above.The building costs are estimated as the percentage share of the space. The use of equipment presents an option for determining the equipment cost. Building and equipment costs were annuitised at 3%.Laboratory costs were done for different tests. Among these were the following: stool culture, blood culture, sputum culture, urine microscopy HIV viral load, malaria parasite slide (MPS), cerebrospinal fluid (CSF) tuberculosis sputum, liver function tests, full blood count (FBC) differential tests, and T cell CD4 counts. These constituted some of the main and recurring tests which were captured for each individual and whose resource use was documented in terms of each test at a standard rate and unit costs were obtained from the WHO sources on pharmaceutical prices.The unit costs for physician consultation were based on the total number of visits made by the physician with respect to each patient. In the case of patients where death was the censoring event, the number of visits up to the time of death was computed. The data not captured as a result of death was treated as missing data. The missing data was averaged out .The total monthly wage was divided by the amount of time spent on consultation or nursing. This share of costs was allocated to the patient and multiplied by the total number of visits.The total consumption of medication was estimated. The unit costs of the medications used have been described above.Inpatient costs = direct + overhead costsCosts = fixed costs + variable costsDirect = costs of medicationsOverhead = laboratory costs + catering costs + administration costs + security costs + building/infrastructure costs + transport costs + other costsLaboratory costs = cost of sample tests per unitCatering costs (“hotel”) = costs of meals per hospitalisation visitAdministration costs = apportioned costs per total visitationBuilding costs = share of allocated costs to paediatric ARV careOverhead = building + admin + security + security An algorithm of hospitalisation costs is shown as follows:The hospitalisation data was captured for each individual patient who might have been hospitalised. The length of stay  was recorded for each patient along with all resource consumption. Cost estimates were computed on the basis of the unit costs and utilisation and estimated based on the total resource consumption and human resource costs. The inpatient fixed costs were based on the process of allocation previously mentioned. The hospital as a rule uses absorption costing for its management financial system. Consequently, shared costs are allocated using this approach which uses space and patient load as weighting criteria for allocation.The outpatient costs comprise mainly the consumption of resources such as human resource, medications, laboratory reagents, and fixed costs relating to shared costs (infrastructure and administration). This approach was used to determine average costs. Average costs are a derivative of the unit costs and quantities of resources consumed.The administration costs are disaggregated into hospital security, catering, laundry, infrastructure, and others. In each particular case, these are based on the space allocation of the paediatrics section as well as the clinical trials unit; the respective allocated costs were derived from the total paediatrics allocated costs of the University Teaching Hospital (UTH).Haematology: the samples were tested for haemoglobin, mean cell volume (MCV), platelets, white cell count, neutrophil, and lymphocyte counts.Biochemistry: creatine, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and bilirubin.Lymphocyte subsets: CD3, CD3+CD4, CD3+Cd8, and total lymphocyte count.These tests, haematology (lymphocytes), biochemistry, immunology (CD4 count), and microbiology (culture), were conducted at the minimum at each nursing visit and/or when the respondent reported not feeling well.Laboratory costs were made on the following key requirements as a matter of routine and for the initial selection purposes. The laboratory tests undertaken were as follows:In the CHAP trial, the mean age of respondents at initiation was 66 months. The mean CD4 count and CD percent were 585 and 12%, respectively, with mean weight of 17 kg. This is shown in The ARV cohort descriptive data shows mean CD4 count at initiation of 550, CD4% of 13, and weight of 15 kg. Mean data at 72–96 weeks of trial shows CD4 count of 1050, CD4% of 29, and weight of 14 kg. End of trial data show CD4 count of 808, CD% of 20, and mean weight of 16 kg. The mortality rate for the ARV cohort was just under 10% as 22 children died out of the 211 initially recruited.The costs for the two cohorts differ significantly. Total physician costs were US$6,425 and the nursing costs were US$390 for the cotrimoxazole cohort. Similar costs for the ARV cohort were $15,240 and $3,080, respectively. The mean costs for nursing and physician costs are $1.3 and $16 for cotrimoxazole, while costs for the ARV cohort are $22 and $71, respectively. Other costs are shown in The differences in the costs can be distinguished by the different sources comprising mainly inpatient costs and treatment for opportunistic infections. In terms of outcomes, the height, weight, and, most importantly, the CD4% are relatively higher in the ART cohort.The costs were determined based on the formulae provided earlier. Conceptually, these are summarised in The main aim of the costing was not to provide an identically distributed set of costs but to capture a detailed unit reflective composition of costs consistent with the data collection method which was based on the collection of patient level data that uses individual resources consumed and valuation of these unit ingredients of resource utilisation.The cost structure for the two therapies shows marked differences. The main drivers of the cost differences are hospitalisation frequency and occurrence of opportunistic infections. Hospitalisation in the case of the cotrimoxazole arm ranged up to 77 days as opposed to the ARV arm which had a maximum admission time of 37 days. Not only is the frequency much more in the case of cotrimoxazole, but also the seriousness of the illnesses and adverse events is much higher. This is again evident from the summary statistics presented in Tables The distribution of costs is skewed as may be expected with chronic conditions and relatively expensive therapies. Figures The skewness in the costs shown in Figures The structure of the costs for the two treatment arms shows quite diverse results. The costs for ARV are on the whole comparatively low. In particular the hospitalisation costs are markedly lower for ART than in the case of the costs for cotrimoxazole. The higher hospitalisation costs also reflect the frequency of hospitalisations and the higher costs related to the incidence of opportunistic infections. The cost structures ultimately have a key bearing on the final effectiveness results.The recorded opportunistic infections and related drug therapy provided are shown in Tables The causes of hospitalisation are also shown in Tables The cost structure shows a larger cost differential at the beginning in the first 1–5 years between the treatment care of comparator and intervention group. As the mortality rate is much higher for the cotrimoxazole group and much less for the ARV group, the cost differential narrows between the two groups as the surviving cases in the latter lead to higher relative treatment costs.The pricing applicable for medication cost is provided in Tables Data arising out of the need to meet the specific standards of the protocol different from routine generated data: protocols set out detailed and extensive monitoring and assessment criteria and data needs which may vary in practice; for example, the number of health care visits, nursing, physician care, and laboratory visits may be that much more extensive than under the normal environment. This may be further exacerbated by the rules and ethics concerning compliance, given the unknown consequences of the medication. These activities are most likely to lead to increased consumption use and increased costs.Censored data: this concerns not just the time of termination of the study but also the effect or consequences attributable to subjects that may be lost to follow-up or who simply drop out of the study prior to the attainment of the predetermined outcome. Again, the handling of these data gaps and subjects has to be decided regarding how it shall be managed in the analysis.Cost analysis asks questions about the following: what type of cost information is needed, why such cost information is needed, or alternatively in whose interests are costs being collected and analysed? Related questions to these are how costs are then imputed from market and nonmarket resources and how overhead as well as capital outlays ought to be considered or managed. The underlying process in this study was to adopt methods treating costs from a micro perspective (“the ingredients” approach) constituting hospital care  and community care comprising physician visits, nurse visits, and transport costs relating to clinicians' visits. Other costs related to direct patient care in terms of drugs/medications, laboratory costs, administrative costs (or other overhead costs), and equipment and building costs.While clinical trials are generally accepted as an ideal source of costing and cost evaluation data, there are nevertheless a number of concerns which are highlighted as possible areas of contention and caution. These concerns may be of relevance in the context of the two studies which are under consideration :Data ariIssues relating to internal validity and generalisability are particularly anchored in the nature of the clinical trial as a mechanism for data generation. The randomisation process of the patients in the RCT is the basis of the assumption of the internal validity. The generalisability in the study is particularly emphasised by the choice of the alternative therapy, namely, cotrimoxazole, which in itself is of high policy relevance in terms of both domestic and international health.The costs and cost analysis in the thesis are drawn from the resource use data from the randomised clinical trials. The total costs are computed as a function of the individual resource use from which the unit costs are derived. Allocations for the administrative costs were derived on the basis of the space use as well as time allocated to the care of the patients. Barber and Thompson  assert tThe distribution of costs and the range are consistent with the expected results. The results show almost a fourfold difference between the cotrimoxazole arm and the ARV arm. As mentioned earlier, although cotrimoxazole is cost-effective given low resource cost and being with no alternative, it is susceptible to exposure of the patients with more infections and a limited life expectancy.The data show that the differences in the cost are quite considerable. The outcomes are also clearly given in terms of the deaths averted through the use of ARVs in relation to cotrimoxazole. In addition, the results show the differences in both cost differences and other intermediate outcomes such as the rate of hospitalisation, the frequency and rate of opportunistic infections, and/or the frequency of health facility utilisation. These are different endpoints which are different forms of outcome or effects.The costing analysis is the first detailed analysis of costs for a paediatric ART programme based on clinical trials.Cases of missing data did not occur as the data was being collected alongside the clinical trials. However, there were cases of censored events due to death. Data was collected up to the time of the event. There was in this regard a complete cycle of data collection. The limitation of censored events forms part of the skewness of the data which was addressed through the bootstrapping process referred to earlier and which can be further addressed through linear regression analysis .Estimated cost results of cotrimoxazole and ART management show that there exist significant differences in using the two treatment interventions. Taking the key cost elements shows that the differences between cotrimoxazole and ART were as follows: physician costs were five times higher and nursing costs were almost 8 times higher while medications were almost twice as much for ART relative to cotrimoxazole, respectively. However, the hospitalisation costs and medications under hospitalisation times were almost four times as much for patients on cotrimoxazole patients on ART. The differences in total costs between the two therapies demonstrate the significance of the likelihood of the risk of misinterpretation of total cost alone as a determinant of cost savings in relation to HIV/AIDS. More fundamentally, the cost differences show that the costs will be higher due to the compounding or cumulative costs as patients fall increasingly susceptible to various illnesses with changes in CD4 counts over time due to decline in immunity. From both the provider and societal perspective, the resource demand due to these differences leads to loss of societal gains or benefits.


Rift Valley fever virus is an arbovirus that affects both livestock and humans throughout Africa and in the Middle East. Despite its endemicity throughout Africa, it is a rare event to identify an infected individual during the acute phase of the disease and an even rarer event to collect serial blood samples from the affected patient. Severely affected patients can present with hemorrhagic manifestations of disease. In this study we identified three Ugandan men with RVFV disease that was accompanied by hemorrhagic manifestations. Serial blood samples from these men were analyzed for a series of biomarkers specific for various aspects of human pathophysiology including inflammation, endothelial function and coagulopathy. There were significant differences between biomarker levels in controls and cases both early during the illness and after clearance of viremia. Positive correlation of viral load with markers of inflammation , markers of fibrinolysis (tPA and D-dimer), and markers of endothelial function (sICAM-1) were all noted. However, and perhaps most interesting given the fact that these individuals exhibited hemorrhagic manifestations of disease, was the finding of a negative correlation between viral load and P-selectin, ADAMTS13, and fibrinogen all of which are associated with coagulation pathways occurring on the endothelial surface. Rift Valley fever virus is an emerging virus of public health significance. In endemic areas up to 60% of the population are seropositive by adulthood. This implies that most of the individuals living in these endemic areas become infected with this virus at some point in their lifetime. This highly prevalent virus is poorly understood likely secondary to lack of diagnostic capacity in affected areas. In fact, the majority of cases are undocumented and spontaneously resolve. However, in some individuals the disease can be very severe and at times associated with hemorrhage or encephalitis. In this report we identify three men infected during the acute stage who also exhibited hemorrhagic manifestations. These men had elevated cytokine and chemokine levels consistent with immune activation. Our data show that biomarkers of fibrinolysis correlated positively with virus while biomarkers of coagulation correlated negatively with virus, suggesting that opposing hemostatic pathways are altered during disease, and that they correct with resolution of viremia. Phenuivirdae family [Phlebovirus [Rift Valley Fever Virus (RVFV) was recently re-classified in the e family , genus Pebovirus . Rift Vaebovirus . Transmiebovirus . Humans ebovirus . Outbreaebovirus , 5.Humans infected with RVFV can present with varying degrees of symptomatology ranging from asymptomatic or a mild influenza-like illness to severe disease with hepatitis, retinitis or encephalitis . LaboratIt is not well understood why there is such variation in the clinical presentation of RVF. The interaction of viral proteins with the host immune response may play a role in pathogenicity. RVFV is an enveloped negative strand RNA virus consisting of three RNA segments varying in size: large (L), medium (M) and small (S). The S segment encodes the N and NSs proteins; the M segment encodes the Gn, Gc, and NSm proteins; whereas the L segment encodes the polymerase . The NSsThe interaction between the immune system, coagulation, and the endothelium during hemorrhagic fever virus infection has been previously studied in human subjects with Lassa, Ebola, and dengue virus infections , 10–17. In March 2016, two human cases of RVF were identified in Uganda. A thirdThis study used de-identified pre-existing specimens and clinical records obtained during an outbreak and was determined to be exempt by the Centers for Disease Control and Prevention Human Research Protection Office. CDC Protocol Number: 6920.6 rads. In preparation for use in the assays, samples were thawed and centrifuged to remove membrane particles. The freeze thaw process and use of whole blood in these assays were previously demonstrated using 8 human blood samples from healthy controls obtained in the US [Blood samples obtained in EDTA tubes as part of routine clinical care for RVF patients were analyzed using commercial multiplex assays according to the manufacturer’s instructions. The samples were aliquoted, frozen, and gamma irradiated on dry ice with 5x10n the US . Eight h50)/mL, was generated from normal human whole blood spiked with a wild-type RVFV stock. The standard curve was used to convert raw Ct values to relative TCID50/mL.RNA was isolated from gamma-irradiated whole blood using MagMax Total RNA Isolation Kit . qRT-PCR of RNA was conducted with established primer and probe sets for the RVFV L segment . A standMaxisorp plates  were coated with RVFV lysate prepared from infected Vero-E6 cells as previously described , dilutedDescriptive analyses were performed using STATA 13.0  and Prism. Patient means were compared to those of the controls. P-values for all statistically significant comparisons are included in the supplementary table . PatientCase 1 was a middle aged male butcher who presented to a local hospital in Kabale District with a chief complaint of epistaxis. He had been well until a week prior to admission when he developed a high-grade fever associated with headache, neck pain, joint pain, and worsening fatigue. His admission labs were notable for an elevated white blood cell count of 16,100/μl, a decreased hemoglobin of 7.9 g/dl, and decreased platelets of 29,000/μl. Liver function tests revealed an elevated total bilirubin of 16.8 mg/dl and elevated transaminases. Creatinine and blood urea nitrogen were increased at 12.5 mg/dl and 240.5 mg/dl, respectively. Testing for malaria was negative. He was transferred to another hospital for transfusion and further management and recovered.Case 2 is a teenage student who presented to a local hospital in Kabale with a five day history of fever, myalgia, joint pain, headache and diarrhea as well as three days of epistaxis. He had cared for goats and sheep at home. On admission he was noted to have epistaxis and scleral icterus. Laboratory evaluation revealed an elevated unconjugated hyperbilirubinemia of 9.72 mg/dl, elevated AST of 470 U/L, decreased total protein of 3.43 g/dl, elevated amylase of 386 U/L, decreased hemoglobin of 7.3 g/dl, and decreased platelets of 45,000/μL. Renal function was normal. He was treated empirically with broad spectrum antibiotics and required blood transfusions. His hospital course was complicated by seizures and altered mental status, but gradually improved.Case 3 is a young adult builder who presented to a local hospital with fever, vomiting, abdominal pain, arthralgias, myalgias, and photosensitivity. He also had a history of epistaxis and hematemesis. Laboratory data were not available for this case.50/mL) in each of the three cases decreased over time  and ferritin , Case 2 (circle), Case 3 (triangle). The grey area represents the range of the biomarker concentration that was detected in 8 normal healthy individuals. The dotted line is the limit of detection of the assay.(PDF)Click here for additional data file.S1 TableMean analyte value from patients was compared to mean analyte value from controls. Any value in italics did not meet criteria for significance for the indicated disease period.(PDF)Click here for additional data file.


Solenopsis invicta Buren in its native range  with the ultimate goal of testing and releasing new viral pathogens into U.S. S. invicta populations to provide natural, sustainable control of this ant. RNA was purified from worker ants from 182 S. invicta colonies, which was pooled into 4 groups according to location. A library was created from each group and sequenced using Illumina Miseq technology. After a series of winnowing methods to remove S. invicta genes, known S. invicta virus genes, and all other non-virus gene sequences, 61,944 unique singletons were identified with virus identity. These were assembled de novo yielding 171 contiguous sequences with significant identity to non-plant virus genes. Fifteen contiguous sequences exhibited very high expression rates and were detected in all four gene libraries. One contig (Contig_29) exhibited the highest expression level overall and across all four gene libraries. Random amplification of cDNA ends analyses expanded this contiguous sequence yielding a complete virus genome, which we have provisionally named Solenopsis invicta virus 5 (SINV-5). SINV-5 is a positive-sense, single-stranded RNA virus with genome characteristics consistent with insect-infecting viruses from the family Dicistroviridae. Moreover, the replicative genome strand of SINV-5 was detected in worker ants indicating that S. invicta serves as host for the virus. Many additional sequences were identified that are likely of viral origin. These sequences await further investigation to determine their origins and relationship with S. invicta. This study expands knowledge of the RNA virome diversity found within S. invicta populations.Metagenomics and next generation sequencing were employed to discover new virus natural enemies of the fire ant,  Solenopsis invicta Buren is an invasive species native to southern South America [S. invicta likely escaped its natural enemies during U.S. founding events. Indeed, direct evaluations have shown a paucity of natural enemies in founding populations of S. invicta [The red imported fire ant,  America . The ant America , most li America . This an America , 4. Dama America . Populat America , 7. Thes invicta .S. invicta with the intention of providing sustainable control in the U.S. This effort led to the discovery of many pathogens and parasites of fire ants in their native and introduced ranges [S. invicta in South and North America [In the U.S., early eradication efforts were attempted , but eved ranges , some ofd ranges –13. Stil America , 15 warrS. invicta [S. invicta. The RNA viruses include Solenopsis invicta virus 1 [S. invicta. Solenopsis invicta queens infected with Solenopsis invicta virus 1 (SINV-1) have lower body weights that reduce the probability of successful colony founding [Despite the known usefulness of viruses to control insect pests , viruses invicta . To date virus 1 , Solenop virus 1 , Solenop virus 1 , and Sol virus 1 . All of founding . Solenopfounding . Solenopfounding  and altefounding . The impfounding .S. invicta from the native range , with the ultimate goal of their release into introduced U.S. populations as self-sustaining biocontrol agents. Metagenomics and next generation sequencing were employed to achieve this objective and resulted in the discovery of one new virus and multiple high-probability target sequences of likely viral origin providing future leads to pursue.The objective of this research was to discover new virus natural enemies of S. invicta colonies collected at 25 locations in the eastern portion of the Province of Formosa, Argentina , Gainesville, Florida collection and the Fundación Para el Estudio de Especies Invasivas (FuEDEI), Hurlingham, Buenos Aires, Argentina collection.Samples of workers, brood, and sexuals were obtained from a total of 182 rgentina . Collects region . Collecto latitude; SAL_2, collected south of 26.3o latitude; SAL_3, collected east of 58.3o longitude; and SAL_4, collected west of 58.5o longitude. The total number of colonies pooled in each group was 46 (SAL_1), 53 (SAL_2), 44 (SAL_3), and 39 (SAL_4). Total RNA (10 μg per group) was submitted to GE Healthcare  for mRNA purification, library preparation, and Illumina RNA sequencing (MiSeq).Total RNA was extracted from a pooled group of 15 worker ants from each colony fragment using the Trizol method followed by the PureLink RNA Mini Purification Kit according to the manufacturer’s instructions . RNA quality of each preparation was assessed by microfluidic analysis on an Agilent 2100 Bioanalyzer  using the RNA 6000 Nano kit according to the manufacturer’s instructions. RNA samples were pooled from ant colonies into four groups according geographic region ; Fig 1. Total RNA (200 ng) purified from each of the four pooled groups of worker ants  was used for mRNA purification with the Illumina TruSeq Stranded mRNA Library Preparation Kit . The low sample protocol was followed according to the manufacturer’s instructions. The RNA fragmentation step was omitted to maximize library insert length. Rather than fragmenting the RNA, the sealed plate was incubated at 80°C for 2 minutes to elute the primed mRNA from the RNA purification beads. This omission resulted in RNA fragmentation with an average final library size of 467 bp. Library sizes were determined empirically by microfluidic analysis on an Agilent 2100 Bioanalyzer and quantified using the Quant-iT dsDNA kit with broad range standards . Samples were pooled together at equimolar quantities and sequenced twice using the Illumina MiSeq (2x300) cycle kit with version 3 chemistry. Using the Illumina indices, the data were demultiplexed and the runs combined to assign the data to individual samples. All other procedures were followed according to the manufacturer’s instructions.Solenopsis invicta reference genome downloaded from http://www.ncbi.nlm.nih.gov/Traces/wgs/?val=AEAQ01#contigs using the Burrows-Wheeler Aligner (bwa-0.7.5a), a software package for mapping low-divergent sequences against a large reference genome [S. invicta unmapped reads were selected and converted to FASTA format using NextGENe-2.3.4 . Each read was then filtered and retained if the median score was ≥ 20 and base number ≥ 25. Unmapped and filtered individual MiSeq sequences were analyzed using BLASTX [http://www.uniProt.org; download date 11/14/2014). Sequences returning an expectation score less than 10−5 were tabulated.Sequences were aligned to the e genome . The S. g BLASTX  against Based on the BLASTX results, each sequence was annotated and assorted taxonomically. The sequences were binned into the following groups: Animal, Plant, Fungi, Bacteria, Archaea, Phage, and Non-phage virus. Also at this stage, sequences exhibiting identity to Enterobacteria phage phiX174, an internal control for Illumina processing  were rem−5 were tabulated.Non-phage virus sequences from each library identified from the BLASTX analysis were assembled using the CAP3 algorithm  in the VRaw sequence data from each library were deposited into the GenBank database as a Sequence Read Archive under accession number, SRP113235 (Bioproject PRJNA394996). Assembled sequences with viral identity  have beeContig_29 was unquestionably a near complete virus RNA genome. Therefore, this sequence was used as template for 5' and 3' RACE to acquire the entire genome sequence. For 3' RACE, cDNA was synthesized with the GeneRacer Oligo dT primer . PCR was subsequently conducted with the GeneRacer 3' primer and gene-specific primer, P1604 . For 5' S. invicta colonies (worker ants) field-collected from around Gainesville, Florida, (n = 27 from 3 locations) to determine whether the sequence was present in the U.S. field population and, if so, its prevalence. This experiment also provided additional confirmation of a potential viral origin. If amplification was observed in 100% of the samples, it was assumed that the sequence was of host origin and further experiments with the sequence were terminated. Viral infections rarely exhibit an incidence of 100% among field-collected arthropods [In order to provide further evidence of whether a contig was of viral origin, or not, oligonucleotide primers  were desthropods .S. invicta, strand-specific RT-PCR was conducted to detect the replicative genome strand by the modified method of Craggs et al. [2, 200 μM dNTP mix, 0.5 units of Platinum Taq DNA polymerase , 0.2 μM of each oligonucleotide primer p1601 and TAG  was actively replicating in s et al. . Pooled  and TAG , and 5 μS. invicta-unmatched sequences. Phage and non-phage virus categories comprised 50.2% of the total number of non-S. invicta sequences . The number of non-phage virus sequences was 292,499, or 33.4% of the total. Surprisingly, phage were well represented despite low numbers of bacterial sequences. However, those phage sequences detected exhibited high expression levels and were limited to the Microviridae (ssDNA). The remaining sequences were annotated to the following taxa: Animal (41.7%), Fungi (3.4%), Plant (2.1%), Protist (1.1%), Bacteria (1.0%), and Archaea (0.3%). The relative number of sequences across these broad taxonomic categories was fairly consistent among the four gene libraries . SINV-4  represented the largest fraction of S. invicta known virus sequences in the pooled libraries, followed by SINV-1 , SINV-2 , SINV-3 (n = 353), and SiDNV (n = 9)  . The smay/region .S. invicta virus-unmatched sequences  were assembled de novo yielding 171 contiguous sequences  with significant identity to non-plant virus genes  were also excluded from further analysis because they either did not match any sequence in the GenBank database and/or were less than 100 nucleotides in length.The us genes . An addin = 38) and using S. invicta as host. This assumption was based on the total sequence expression representation, representation across the libraries , and contig size. Expression representation has been used successfully to detect pathogens and is simply based on the fact that actively replicating genes  will be highly represented in non-normalized gene libraries [Dicistroviridae (n = 23). Fourteen of the sequences exhibited identity with unclassified viruses and one with a virus in the Nodaviridae .Among the 171 contiguous sequences with significant identity to non-plant virus genes, ibraries . Among tn = 15; high likelihood viral prospects). Therefore, we focused our effort on these fifteen contigs  separated and flanked by untranslated regions, and a short, overlapping ORF at the 5' end of ORF 2 (S. invicta (from Argentinean colonies). The presence of a replicative genome strand and a high expression level of SINV-5 sequences detected in all four gene libraries indicate that S. invicta likely serves as host for the virus  also exhibited high expression levels and were represented in all four libraries. These contigs also exhibited significant identity to dicistrovirus non-structural and structural proteins and likely represent new virus species. The remaining 11 contigs, Contig_70 to Contig_80 , had lowS. invicta colonies from Florida (n = 27) were also examined by RT-PCR to determine whether any of the fifteen high likelihood viral sequences were present in the U.S. S. invicta population .We also discovered what will likely be a second virus among the unidentified contigs in ulations  and likeS. invicta were left behind in South America when it was accidently introduced into the U.S. [S. invicta viruses discovered to date, plus the likely one just mentioned above, plus 5 of 15 likely viral contigs tested . By way of comparison, 31 viruses have been characterized from honey bees [In conclusion, our ongoing metagenomics/next generation sequencing efforts  have beeney bees . Future S1 Table(DOCX)Click here for additional data file.S2 TableContigs were first sorted in descending order based on the number of the sequences comprising it, followed by the libraries represented.(DOCX)Click here for additional data file.


Ebola virus is known to persist in semen of male survivors of Ebola virus disease (EVD). However, maximum duration of, or risk factors for, virus persistence are unknown. We report an EVD survivor with preexisting HIV infection, whose semen was positive for Ebola virus RNA 565 days after recovery from EVD. In March 2015 in Liberia, unprotected sexual intercourse was strongly suspected in the transmission of Ebola virus disease (EVD) from a male survivor of EVD to his female partner  of 32.39. While in the ETU, he continued his antiretroviral therapy. The patient was discharged on September 8, 2014, after showing a negative result for Ebola virus in blood by RT-PCR.On August 27, 2014, a 48-year-old man with a history of HIV infection who was receiving antiretroviral therapy was admitted to an Ebola treatment unit (ETU) in Monrovia, Liberia, with a 1-week history of fever, chills, and weakness and a 2-day history of vomiting and diarrhea. The next day, he had a positive result for Ebola virus in blood by real-time reverse transcription PCR (RT-PCR), with a cycle threshold (CWhen the patient was first given a diagnosis of infection with HIV-1 in October 2009 (CD4 cell count 46/μL) , he was t values <40, and indeterminate if only 1 gene target has a Ct <40. Since his enrollment in the program, the semen of the patient has been positive for Ebola virus RNA up to 565 days after he was discharged from the ETU. Ct values plateaued to indeterminate for samples up to 758 days after discharge (The patient enrolled in the Men’s Health Screening Program on October 21, 2015. Per program protocol, his semen was tested every 4 weeks for Ebola virus by RT-PCR using described methods (ischarge . Althoug–The prolonged period during which Ebola virus RNA was detected in this patient adds to evidence (Because HIV infection is treatable and testing is readily available in West Africa, semen testing programs for Ebola virus should consider offering HIV testing to male survivors of EVD with persistently detectable Ebola virus in semen. Furthermore, HIV care was interrupted during the Ebola outbreak in West Africa because of closure of clinics and interruption of ART distribution (


Genetic characterization of wild-type measles virus (MV) strains is a critical component of measles surveillance and molecular epidemiology. We have obtained complete genome sequences of six MV strains belonging to different genotypes, using random-primed next generation sequencing. Morbillivirus within the family Paramyxoviridae , is an enveloped virus with a negative-sense single-stranded RNA genome and is a member of the genus oviridae . MV genooviridae . The MV Mycoplasma spp. All virus isolates are available via the European Virus Archive (https://www.european-virus-archive.com).Viral isolates of circulating wild-type MV strains provide an important resource for virology and vaccine development. In this work, we have obtained complete genome sequences for the following six wild-type MV strains: MVi/Khartoum.SUD/34.97/2 [B3], a strain endemically circulating in Khartoum (Sudan) in 1997 ; MVi/Ams5 and 107 50% tissue culture infective dose [TCID50]/ml), using Bioke extraction reagents . Extracted RNA was reverse transcribed and second-strand synthesis was performed using random primers as previously described  package  had a 6-nucleotide insertion, giving rise to a full genome length of 15,900 nt and complying with the rule of six for replication competency in MV . These sMG912589 to MG912594.The MV sequences described in this study have been deposited in GenBank under the accession numbers


Epirrita autumnata) larva, as part of a search for the possible causes of the cyclical nature and mortality associated with geometrid moth dynamics and outbreaks in northern Fennoscandia. Abisko virus has a genome organization similar to that of the insect-infecting negeviruses, but phylogenetic and compositional bias analyses also reveal strong affiliations with plant-infecting viruses, such that both the primary host origin and taxonomic identity of the virus remain in doubt. In an extensive set of larval, pupal, and adult autumnal moth and winter moth (Operophtera brumata) outbreak samples, the virus was only detected in a few adult E. autumnata moths as well as the single larval transcriptome. The Abisko virus is therefore unlikely to be a factor in the Fennoscandia geometrid population dynamics.A novel, 10 kb RNA virus—tentatively named ‘Abisko virus’—was discovered in the transcriptome data of a diseased autumnal moth ( Epirrita autumnata) dominates while the winter moth (Operophtera brumata) prefers the warmer inland and coastal areas. However, the winter moth has started to appear at outbreak densities in locations where the autumnal moth was previously the sole outbreak species, probably due to climate change [Geometrid moth larvae are major defoliators of deciduous forests in central and northern Europe. In northern Fennoscandia, vast areas of mountain birch forests are recurrently defoliated by larvae of two geometrid species. In colder, continental areas the more cold-tolerant species, the autumnal moth , 86 pupae and 77 adults were collected for analysis, as well as 53 O. brumata pupae, from the six collection sites . The Abisko virus sequence was assembled from around 0.5 million end-paired ca. 100 nt sequences using the Trinity transcriptome assembly software [www.htslib.org, [16) for cDNA synthesis, followed by PCR with the EaNV-3RACE-F2 virus-specific forward primer  and an d primer , using tQuantitative qPCR was performed on a CFX Connect Real-Time PCR machine  using SSo Fast EvaGreen Supermix (Bio-Rad). The reaction volume was 20 μL containing 1 μL of the diluted cDNA template and 0.3 µM each of the forward and reverse primers . CyclingE. autumnata sample (25A), as a biological negative control. The cDNA reactions were incubated 60 min at 37 °C followed by 10 min at 70 °C. Excess cDNA primer was removed by adding 1u Exonuclease-I (Thermo Fischer 70073Z2500UN), incubating 30 min at 37 °C followed by 15 min at 70 °C, followed by diluting the cDNA 10-fold in sterile water [The Abiskovirus genomic (positive) and replicative (negative) RNA strands were quantified separately using four pairs of strand-specific assays; two located in open reading frame 1 (ORF-1) and two located in ORF-2/3. Strand-specific cDNA was generated in 10 μL reactions containing 1 μL RNA plus 2 μM tagged cDNA primer  heated tle water ,13. The www.ncbi.nlm.nih.gov, identifying key viral amino acid motifs and a close similarity with negeviruses, ciliviruses and members of the Virgaviridae. For each unique virus taxon identified by BLAST-P, a single accession (The predicted Abisko virus ORF1 and ORF2 amino acid sequences were queried with BLAST-P using default settings against the GenBank repository of non-redundant translated nucleotide sequences (nr database) at ccession  was extrwww.bioinformatics.org using the Sequence Manipulation Suite [www.R-project.org) using the ade4 package [Virus genomes often mimic the compositional biases of their hosts’ genomes , such thon Suite . The dinon Suite . These mon Suite ,21 to id package . The tra package  of the dAcinetobacter baumannii hypothetical protein (GenBank WP_071209019.1) and 7% of the reads to a single 10 kb RNA virus. Only 4% of the reads belonged to the E. autumnata transcriptome and the remaining reads were mostly of bacterial and fungal origin. Given its abundance, this 10 kb Abisko virus merited further investigation.The starting point of the current study was the 2013 autumnal moth outbreak in northern Fennoscandia. Among the samples collected at the Abisko sampling site were a number of larvae with obvious disease symptoms . The RNAThe Abisko virus sequence comprises a single-stranded RNA genome of about 10 kb, with a natural 3’ poly-A tail a relatively large 782 nt) 3′ untranslated region (3′UTR), a short 5′UTR, and three major ORFs, separated by an intergenic region (IGR) of about 229 nt and a 3 nt stretch following the end of the putative coat protein . ORF-1 c82 nt 3′ E. autumnata adult samples (78A) and by three of the assays in another adult (86A), with assay neg2(g) just missing the detection threshold (8971AUGUUA) is about as sub-optimal as can be and inferior to either of the possible ORF-2 start codons , neither of which are particular optimal themselves. Therefore, unless ORF-3 is translated through a dedicated IRES hidden in the CP gene , it mayVirgaviridae [Virgaviridae, which will have contributed to the uncertainty of its phylogenetic placement within the negeviridae. This is in itself not particularly unusual: RNA viruses in particular are well capable of acquiring gene cassettes from a variety of different origins, including related viruses [The phylogenetic analyses  confirm aviridae . It lies viruses  and even viruses . HoweverE. autumnata, with 0/86 pupae, 1/60 larvae, and 4/77 adults registering positive for the virus by RT-qPCR, as well as 0/52 O. brumata pupae  than in the larval sample . These data, indicating host status for the adults but uncertainty for larvae or pupae, present an interesting conundrum concerning the origin and transmission of Abisko virus. Since the adult moths do not feed, are only alive for a few weeks in late autumn and the females are largely immobile [E. autumnata adults is an accidental irrelevance, possibly involving acquisition during the larval phase and undetectable persistence through the pupal stage. Alternatively, E. autumnata is a primary host and transmission between individuals is through a mobile vector .The virus has a rather low prevalence in ta pupae . The ideimmobile , they reVirus genomes often mimic the compositional biases of their hosts’ genomes , such thE. autumnata larva from the 2012–2013 geometrid outbreak in northern Fennoscandia. Subsequent screening of an extensive set of natural samples revealed that the virus could not be detected in either larvae  or pupae of both E. autumnata and O. brumata, and that it could be detected in only four adult E. autumnata, collected from three different sites. The dispersed distribution, generous titers, and clear evidence for virus replicative-strand intermediates indicate that adult E. autumnata are a true host of the virus, while the absence of the virus or its replication intermediates from all pupae and all but one larva suggests that these are not hosts. Since the adults live only a few weeks, are not very mobile, and do not feed or defecate, the persistence of Abisko virus in the adult autumnal moth population is difficult to explain unless other transmission routes or hosts are involved. The genome organization and phylogenetic analyses identify Abisko virus most closely with the negeviruses, a newly described group of insect-infecting viruses [In this manuscript, we describe the discovery and a limited set of molecular features of a novel 10 kb RNA virus that was assembled from transcriptome data of a diseased  viruses ,7, but w


To the Editor: On April 9, 2016, a 32-year-old woman from Italy traveled to Santo Domingo in the Dominican Republic. She worked as a volunteer nurse in the outpatient clinic of a primary school of a nongovernmental organization based in Italy. She returned to Italy on April 17. She did not have sexual intercourse during her stay abroad. On April 26, she was referred to the travel clinic of the National Institute for Infectious Diseases Lazzaro Spallanzani in Rome for a febrile syndrome with rash, generalized headache, and weakness, which started on April 21. Approximately 24 hours later, she was admitted to the institute’s medical facility for a suspected neurologic involvement. At admission, she had abnormal gait, strong asthenia, and a disseminated pruritic rash on her face, abdomen, chest, and arms, but she did not have a fever.During physical examination, the patient was alert and fully oriented. Temperature was 36.9°C, pulse rate 90 beats/min, blood pressure 100/60 mm Hg, and respiratory rate 20 breaths/min. She had a diffuse erythematous macular rash and bilateral nonpurulent conjunctival hyperemia without meningeal signs. Findings of a neurologic examination of the upper limbs were within reference ranges.. Muscular strength was reduced in both legs (left > right), whereas tendon reflexes and all sensory modalities were within reference ranges. Results of a contrast-enhanced magnetic resonance imaging of the brain and spinal cord (on day 7), nerve conduction studies and electromyography (on day 8), and an electroencephalogram (on day 16) were within reference ranges. A lumbar puncture (on day 7) showed normal cell counts (<10 cells/mL), a normal glycorrachia/glycemia ratio (>0.5), and a slight increase in protein concentration (0.48 g/L [reference range 0.32−0.80 g/L]) in cerebrospinal fluid. Complete neuropsychologic examinations (on days 9 and 10) showed mild deficits in attention and mental processing speed and mental flexibility and moderate deficits in verbal and nonverbal memory tasks .Real-time reverse transcription PCR (rRT-PCR) results for dengue viruses 1–4 and chikungunya virus were negative in serum and cerebrospinal fluid (CSF), whereas Zika virus RNA was detected in serum (day 7), urine (up to day 27), CSF (day 6), saliva (up to day 13), and vaginal swab (up to day 13) . SpecifiStarting on day 7, intravenous polyvalent immunoglobulins were administered (0.4 g/kg/day for 5 days); no adverse events were observed. A second neuropsychologic examination was performed on day 16 and indicated persistent impairment in memory performances and an improvement in mental concentration and flexibility tasks .A second lumbar puncture (on day 17) showed an increased cell count , and CSF was negative for Zika virus RNA by rRT-PCR. The patient was discharged on day 20; she showed a progressive neurologic recovery starting on day 16. At 60-days follow-up visit, no neurologic deficits were reported.During the 2013–2014 outbreak of Zika virus in French Polynesia and in the context of the 2015–2016 Zika virus circulation (In our patient, Zika virus RNA was found in different systems, including the central nervous system and the genital tract. Recently, a mouse model of Zika virus infection by vaginal exposure demonstrated that Zika virus replicated within the genital mucosa, persisted postinfection, and was detected in the fetal brain of the mice (Our case highlights the potential for Zika virus neurotropism and the need for early identification of Zika virus–related neurologic symptoms. Moreover, the presence of Zika virus in the genital tract supports the recommendation of safe sex practice for women returning home from areas with ongoing Zika virus transmission.Methods for laboratory diagnosis and neuropsychologic examination.


During the 2016 Zika virus outbreak in Brazil, we detected Zika virus RNA in urine samples collected from Zika virus–positive pregnant women during different stages of pregnancy. Women had positive and negative intervals of viruria; 3 newborns had adverse outcomes. Further research is needed to clarify the relationship between viruria and outcomes for newborns. Zika virus is a reemerging flavivirus that was first isolated in Uganda in 1947 during the course of a yellow fever virus survey  was <38.5 and negative if the Ct was >38.5, as described previously (Pregnant women who were Zika virus positive by quantitative reverse transcription PCR (RT-qPCR) . The maximum period of detection was 7 months (patient no. 2). The number of collections varied depending on when during the pregnancy the patient started treatment at the facility and when the last urine sample was collected before delivery. Of the 12 newborns for which outcomes were known, 3 (25%) had adverse outcomes: 1 had subependymal cysts and 2 had unilateral abnormal otoacoustic emissions  was likely due to the sensitivity of the test. More studies are necessary to understand the dynamics of the replication of Zika virus in fetal tissues and its connection to viruria in pregnant women.We report findings of long-term viruria among Zika virus‒positive pregnant women, with Zika virus RNA being detectable in urine during different stages of pregnancy. Plasma and serum are considered the biologic fluids of choice for the molecular diagnosis of Zika virus for Urine, semen, and saliva have been described as specimens that can be used in the molecular detection of Zika virus after the clearance of viremia  and can be taken >5 days after the onset of clinical symptoms (Considering that some mothers had viruria just before delivery and newborns without adverse outcomes, the adverse outcomes of the 3 newborns might or might not have been related to the viruria. Because microcephaly and fetal abnormalities have been attributed to Zika virus, continuing to monitor women who had Zika virus infections during their pregnancies will be essential to manage adverse fetal outcomes. The monitoring of Zika virus viruria in pregnant women through the regular collection of urine samples over the course of the pregnancy proved to be a workable approach; however, the meaning of viruria and the consequences for newborns will need to be evaluated further. Whether virus replicates in fetal tissues and uses them as reservoirs remains to be determined.


Specifically, viruses belonging to the Sandfly fever Sicilian complex should be given careful consideration. This calls for implementation of direct and indirect diagnosis in National reference centers and in hospital microbiology laboratories and systematic testing of unelucidated febrile illness and central and peripheral nervous system febrile manifestations.Phleboviruses transmitted by sandflies are endemic in the Mediterranean area. The last decade has witnessed the description of an accumulating number of novel viruses. Although, the risk of exposure of vertebrates is globally assessed, detailed geographic knowledge is poor even in Greece and Cyprus where sandfly fever has been recognized for a long time and repeatedly. A total of 1,250 dogs from mainland Greece and Greek archipelago on one hand and 422 dogs from Cyprus on the other hand have been sampled and tested for neutralising antibodies against Toscana virus (TOSV), Sandfly fever Sicilian virus (SFSV), Arbia virus, and Adana virus i.e. four viruses belonging to the 3 sandfly-borne serocomplexes known to circulate actively in the Mediterranean area. Our results showed that (i) SFSV is highly prevalent with 71.9% (50.7–84.9% depending on the region) in Greece and 60.2% (40.0–72.6%) in Cyprus; (ii) TOSV ranked second with 4.4% (0–15.4%) in Greece and 8.4% (0–11.4%) in Cyprus; (iii) Salehabad viruses (Arbia and Adana) displayed also substantial prevalence rates in both countries with values ranging from 0–22.6% depending on the region and on the virus strain used in the test. These results demonstrate that circulation of viruses transmitted by sand flies can be estimated qualitatively using dog sera. As reported in other regions of the Mediterranean, these results indicate that it is time to shift these viruses from the " Phleboviruses transmitted by sandflies are endemic in the Mediterranean basin. An increased number of new viruses was described during the last decade. However, levels of exposure of human and animal populations are poorly known. A total of 1,250 dogs from Greece and 422 dogs from Cyprus were tested for the presence of neutralising antibodies signing previous infection with selected phleboviruses representing the 3 serological complexes known to be present in the Old World: Toscana virus (TOSV), Sandfly fever Sicilian virus (SFSV) and Salehabad viruses (Arbia and Adana viruses). Our data showed that (i) SFSV is largely predominant with infection rates higher than 50%, (ii) TOSV is widely distributed with 4.4% and 8.4% in Greece and Cyprus, respectively, and (iii) that viruses belonging to the Salehabad serocomplex should be further studied for their capacity to cause human disease in view of prevalence rates in dogs up to 22.6%. These findings confirm that dogs can be considered as excellent sentinels for sandfly-borne phleboviruses. The results also underline the importance to study the role of SFSV in humans and may lead to the set-up diagnostic tests for patients presenting unexplained febrile illness and neuroinvasive infections. Further studies are also needed to define whether these viruses cause diseases in dogs. Bunyaviridae family, Phlebovirus genus) transmitted by phlebotomines consist of three species or antigenic groups, namely Sandfly fever Naples, Salehabad, and Sandfly fever Sicilian serocomplexes. Each species contains several viruses among which Naples, Sicilian and Toscana virus cause 3-day fever, commonly called sandfly fever in humans; Toscana virus (TOSV) causes of neuroinvasive human infections such as meningitis and encephalitis [In the Old world, phleboviruses , closely related to Sandfly fever Sicilian virus (SFSV), was isolated during a large outbreak of sandfly fever in Swedish United Nations troops stationed in Cyprus; few cases were also caused by TOSV . In Gree the CSF , 13. To During the last decade, field-to-laboratory integrated studies associating virologists, parasitologists and entomologists have discovered several new phlebotomine-borne phleboviruses; thus there is an increased diversity in each of the three aforementioned species or serocomplexes . Althougdistricts of Cyprus and 32 prefectures belonging to 12 regions of Greece  was collected, after the written consent of the owner, and questionnaires with personal, epidemiological, and clinical data for each dog were completed.Only domestic dogs that were raised in the area were considered for the study. The domestic dogs were included after owners’ informed consent. Information regarding age, sex, was obtained after interviewing dog owners . Each doData on the region, gender, and age  were recorded. This study was ethically approved by the Institutional Animal Care and Use Committee of the University of Crete Medical School and conform with the European Union Directive 2010/63/EU regarding use of animals and biological specimens in research, as well as the relevant Hellenic legislation . Written informed consent was obtained from the dog owners, according to the aforementioned national legislations.Sandfly fever Naples virus species or complex) [Salehabad species or complex), and (iv) Adana virus strain 195  [Sera were tested by the virus microneutralisation assay (MN), described for phleboviruses  in paralcomplex) , (ii) SFcomplex) , (iii) Acomplex) .50 was added into each well except for the controls that consisted of PBS. A volume of 50 μL of EMEM medium enriched with 5% fetal bovine serum, 1% Penicilin Streptomycin, 1% L-Glutamine 200 mM, 1% Kanamycin, 3% Fungizone, was added to each well of the controls.Briefly, two-fold serial dilutions from 1:10 to 1:80 were prepared for each serum and a volume of 50μL was pipeted into 96-well plate. Viruses were titrated in Vero cells (ATCC CCL81). A volume of 50 μL containing 1000 TCID5 cells/mL of EMEM medium (as previously described) was added to each well, and incubated at 37C° in presence of 5% CO2.The first row of each plate contained control sera diluted 1:10 and Vero cells without virus.The plates were incubated at 37C° for one hour. Then, a 100μL suspension of Vero cells containing approximately 2 x10After 5 days (Toscana and Arbia virus) and 7 day (Sicilian and Adana virus), the microplates were read under an inverted microscope, and the presence  or absence  of cytopathic effect was noted. Cut-off value for positivity was set at titre ≥ 20 , 20, 21.Due to insufficient volume in Greek samples, ADAV was used for testing Cyprus specimens, only.Dog seroprevalence for each virus was estimated for each prefecture and mapped using the geographical information system software .The chi-square or Fisher’s exact tests were used to compare percentages of positivity among categories of the same independent variables and also the total prevalence of each virus. A p value < 0.05 was considered as statistically significant. Analyses were performed with StatLib and SPSS® 21 software for Windows.In Greece, a total of 1,250 sera  were collected. The median age was 36 months (range: 3–216). The sera were collected from 32 prefectures, but owing to the variability in the number of collected sera from each prefecture (range: 1–410), the sera were grouped into 12 regions. Of these 12 regions, Thessaly was not included in the analyses because it consisted of 1 serum only. For the other 11 regions, the number of sera ranged from 14 to 410.In Cyprus, a total of 442 sera  were collected. The median age was 36 (range: 3–144).They consisted of 67, 27, 97, 74, and 177 sera collected from the districts of Ammochostos, Larnaca, Limassol, Nicosia, and Paphos, respectively.vs 0.84).The two dog populations had the same median age (36 months) and a similar sex ratio  compared with 4.4% and 2.6% for TOSV and ARBV, respectively . SimilarThe distribution of TOSV positive sera is quite homogenous within the studied regions . There is no significant difference according to the sex of the dogs. In contrast, it appears that the prevalence increases with the age, although it is not statistically significant even when the results of dogs from Greece and Cyprus are merged .In both countries, a statistic association was found between SFSV prevalence and geographic area.In Cyprus, none of the sera containing ARBV-NT-Abs were also positive for ADAV-NT-Abs and vice versa; this demonstrated that there is no cross-reactivity through MN assay between these two viruses despite the fact that they belong to the same serocomplex. Exposure to ARBV and ADAV is significantly associated with the age with higher rates observed in older dogs. In contrast, the high rates of SFSV-NT-Abs were observed in the 6-11-month age class in Cyprus and Greece.Salehabad species, was also identified in the CSF of a patient with meningitis [At the outset of our study, the following data were available for Greece: (i) there was no serological data in domestic animals  for any phlebovirus transmitted by sand flies, (ii) in the 1970's, 13.1% of 38 adults living in Crete had NT-Abs against Naples virus, and 24.7% and 8.5% of 632 human sera from Athens inhabitants had NT-Abs against Naples virus and Sicilian virus, respectively ; (iii) mningitis , 13. To At the outset of our study, the following data were available for Cyprus: (i) there was no serological data in domestic animals  for any phlebovirus transmitted by sand flies; (ii) first evidence of the presence of TOSV, Naples and Sicilian viruses were observed in Swedish soldiers of the United Nations force  through The recent discovery of several new viruses belonging to the three species associated with phlebotomines in the Old World has raised questions about the viral strain currently circulating in the two regions. Since broadly cross-reactive techniques such as ELISA and IIF are not capable to distinguish between viruses belonging to the same serocomplex, we decided to use microneutralisation assay using viral strains or surrogates which presence had been assessed in Greece and Cyprus. Indeed, we consider it valid to use SFSV as a surrogate for SFCV (isolated in Cyprus) and other SFSV-related viruses because amino acid distances observed between the proteins that elicit neutralizing antibodies (Gn and Gc) are well within the acceptable range, ie <5% different for SFSV and SFSV-related viruses, 31. ThuSince collecting human sera displaying a large geographic distribution was challenging, we decided to use dog sera; indeed, dog sera can be good surrogates for the following reasons: (i) dogs are readily infected with phlebotomine-borne phleboviruses which are human pathogens , 31, 32;The largest amount of data available on dogs, at the outset of this study, concerned TOSV, which observed rates (4.4% in Greece and 8.4% in Cyprus) are in the same order of magnitude as those recently reported in dogs in Tunisia , in Algvs 51.7%) [Sandfly fever Naples serocomplex in which 6 new viruses were described during the last decade  [In the present study, dogs living in the Ionian island of Corfu showed a much lower seroprevalence compared to the human population living in the same island 3.9% s 51.7%) ; howeverZerdali) , 33–37 iZerdali) . The sam% s 51.7%Zerdali) , 39 and Zerdali) . It is wZerdali) , 20. TheSalehabad species is 21.7%. This is congruent with the results observed in Adana, southern Anatolia, Turkey where domestic animals were presenting high rates of NT-Ab against viruses belonging to the Salehabad serocomplex [Salehabad complex can infect humans and other vertebrates. Interestingly, Adria virus RNA has been detected in the CSF of a Greek patient presenting with meningitis [Salehabad virus complex. Isolation of Adria virus is now a priority in order to pursue the studies using neutralization-based serological studies in humans and animals.The absence of cross-protection between ARBV-NT-Abs and ADAV-NT-Abs confirm previous data from Turkey . Accordiocomplex . Tesh etocomplex  did not ocomplex . This suningitis  but was Very high rates of SFSV-NT-Abs were observed in Cyprus and Greece. In the latter, rates were consistently above 50% (range 50.7–84.9%); in Cyprus, rates were above 40% (range 40.0–72.6%) except in Ammochostos (26.3%). The extremely high prevalence rates observed with SFSV in young dogs show that this virus continues to circulate very actively in these regions, and beyond as recently described in dogs from Tunisia  and in Phlebotomus neglectus [The massive prevalence of SFSV-NT-Ab observed in our study is not unexpected and is congruent with entomological and human data in the literature: (i) isolation of Corfu virus on the eponymous island from eglectus ; (ii) SFeglectus –5; (v) aeglectus ; (vi) a eglectus . In conteglectus , 16; theeglectus , 40, SFSIn conclusion, this study indicates that (i) sandfly-borne phleboviruses belonging to 3 distinct genetic and antigenic groups are widely spread and co-circulate; (ii) dogs represent excellent qualitative sentinels for virus transmitted by sandflies and further studies must be done to estimate the role of dogs in the dynamics of transmission, and whether they play a role as reservoir hosts in the natural cycle of these viruses.Since several of these viruses are proven human pathogens, our results plead for performing similar studies using human sera to identify geographic hot spots. The increasing number of sequence data for these phlebotomine-borne phleboviruses now enables to design and develop real-time molecular assays. The improved diagnostic toolbox will allow to investgate the medical impact of these viruses in patients presenting unexplained febrile illness and neuroinvasive infections.


Ourmiavirus are characterized by having non-enveloped bacilliform virions with a series of discrete lengths from 30 to 62 nm composed of a single coat protein (CP). The genome consists of three positive-sense single-stranded RNAs, each encoding a single protein. The RNA-dependent RNA polymerase (RdRp) has closest similarity to that of viruses from the family Narnaviridae; the movement protein (MP) is similar to the MPs of tombusviruses; the CP shows limited similarity to the CPs of several plant and animal viruses. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the genus Ourmiavirus, which is available at www.ictv.global/report/ourmiavirus.Members of the plant virus genus  The bacilliform virions of Ourmiaviruses constitute a series of particles with conical ends (apparently hemi-icosahedra) and cylindrical bodies, 18 nm in diameter. The bodies of the particles are composed of a series of double discs, the most common particle having two discs (particle length 30 nm), a second common particle having three discs (particle length 37 nm), with rarer particles having four discs (particle length 45.5 nm) and six discs (particle length 62 nm). There is no envelope .The genome is composed of three positive-sense single-stranded RNAs. In Ourmia melon virus, the RNAs are 2814, 1064 and 974 nt in length  [1]. TheNarnaviridae, but is distinct from the RdRP of yeast viruses classified in the genus Narnavirus [The putative RdRp carries the conserved GDD motif and has closest affinity with the RdRp of a number of viruses related to members of the family rnavirus . A proternavirus  but therrnavirus . Synthesrnavirus . The MP rnavirus . Detailsrnavirus .Ourmiavirus: Ourmia melon virus, members of which were initially isolated from melon plants in Iran [Epirus cherry virus, with members isolated from cherry trees in Greece [Cassava virus C, members of which were isolated from cassava from equatorial Africa [To date, three species of plant viruses are classified in the genus  in Iran , Epirus n Greece , and Casl Africa .www.ictv.global/report/ourmiavirus.Full ICTV Online (10th) Report: