Apis mellifera. Apicultural trade is arguably the main factor for the almost global distribution of most honey bee diseases, thereby increasing chances for multiple infestations/infections of regions, apiaries, colonies and even individual bees. This imposes difficulties to evaluate the effects of pathogens in isolation, thereby creating demand to survey remote areas. Here, we conducted the first comprehensive survey for 14 honey bee pathogens in Mongolia , where honey bee colonies depend on humans to overwinter. In Mongolia, honey bees, Apis spp., are not native and colonies of European A. mellifera subspecies have been introduced ~60 years ago. Despite the high detection power and large sample size across Mongolian regions with beekeeping, the mite Acarapis woodi, the bacteria Melissococcus plutonius and Paenibacillus larvae, the microsporidian Nosema apis, Acute bee paralysis virus, Kashmir bee virus, Israeli acute paralysis virus and Lake Sinai virus strain 2 were not detected, suggesting that they are either very rare or absent. The mite Varroa destructor, Nosema ceranae and four viruses  and Chronic bee paralysis virus) were found with different prevalence. Despite the positive correlation between the prevalence of V. destructor mites and DWV, some areas had only mites, but not DWV, which is most likely due to the exceptional isolation of apiaries (up to 600 km). Phylogenetic analyses of the detected viruses reveal their clustering and European origin, thereby supporting the role of trade for pathogen spread and the isolation of Mongolia from South-Asian countries. In conclusion, this survey reveals the distinctive honey bee pathosphere of Mongolia, which offers opportunities for exciting future research.Parasites and pathogens are apparent key factors for the detrimental health of managed European honey bee subspecies,  Apis mellifera, for pollination  –23 Acaraectively . PCR conAmplifications for the detection of viruses were performed with 2 μl diluted cDNA using MyTaq (Bioline) reagents and PCR conditions as describe above. Positive samples for Sacbrood virus (SBV), Black queen cell virus (BQCV), DWV, and Chronic bee paralysis virus (CBPV) were sequenced using different primers . PCR wasNosema spp. spore counts were performed in PCR-positive colonies (N = 14). Individual bees (N = 50 for each colony) were crushed with a mortar in 1000 μl water [μl water  and thenμl water  calculathttps://blast.ncbi.nlm.nih.gov). To infer the phylogenetic trees, the nucleotide sequences of the samples and of other annotated sequences retrieved from Genbank were aligned using the MUSCLE program and compared using the maximum likelihood method with the MEGA5.2 program under the Tamura 3-parameter [Phylogenetic analyses of the detected viruses were performed to confirm their species status and to detect their geographic affiliation. The identity of the detected species was confirmed using the NCBI Blast searches in Genbank  between the regions . However, mite prevalence was significantly different . Region B was less infested by mites, 14.3% (13 out 91 colonies); followed by region C  and the highest mite infestation was found in region A . Colonies from all four locations of region B were infested with V. destructor (4.5–66.7%); three locations out of four of the region C were infested at 33.3–100% and location 7 was free from the mite. At region A, screening of sealed drone brood exhibited infestation of all six colonies, with up to 6 mites from a single cell. Regarding the V. destructor haplotype identity, the blast comparison of the 797-nt sequences, corresponding to the cox1 mitochondrial gene from 13 mites analyzed, showed 100% identity with the haplotype K1.While  regions . There wNosema apis was not detected at all, PCR-based detection revealed N. ceranae only in region B, with significant differences in prevalence between locations . In the location 2, it was detected in 11.8% of the colonies (2/17), in the locations three, four and five 0% (0/3), 44.44% (12/27) and 11.36% (5/44), respectively. The number of spores in individual workers ranged from 5x104 up to 30.4x106 with significant differences between locations as determined by one-way ANOVA  = 6.571, p = 0.001).While All samples provided negative results for the casual agents of both European and American foulbrood by PCR-based detection.2 = 99.2, df = 3, p<0.001). SBV and BQCV were more prevalent (73.5% (111/151) and 49.6% (75/151), respectively) than DWV and CBPV (11.25% (17/151) and 5.96% (9/151), respectively).Acute bee paralysis virus (ABPV), Kashmir bee virus (KBV), Israeli acute paralysis virus (IAPV), and Lake Sinai virus strain 2 (LSV-2) were not detected in this survey. Prevalence differed significantly among the four detected RNA viruses . As shown in The SBV prevalence was significantly different between the regions . While BQCV was not detected in region A (0/15), all locations from region B tested positive with locations 3 and 4 being all positive (100% 3/3 and 100% 27/27). The samples of locations 2 and 5 show 47.1% (8/17) and 63.6% (28/44), respectively. Location 8 of the region C had 100% (9/9) infection with BQCV, but the other three locations were negative for this virus.The BQCV prevalence was significantly different between regions . In region B, all four locations were positive for DWV with significant variation between locations (location 2: 41.2% (7/17); location 3: 33.3% (1/3); location 4: 3.7% (1/27) and location 5: 2.3% (1/44); Pearson Chi Square test, χ2 = 22.3, df = 3, p<0.001). In region C, DWV was common in location 8 (77.7% (7/9)), but not found in locations 6, 7 and 9. Likewise, the samples of adult workers, sealed drone brood and mites of region A were also tested negative for DWV (N = 15 colonies).This virus was less frequent compared to SBV and BQCV . No sign2 = 3.4, df = 2, p = 0.19). Locations 3, 4 and 5 were positive 33.3% (1/3), 15% (4/27) and 7% (3/44), respectively; but location 2 was free of this virus. Three out of four locations of the region C were negative and only location 9 was positive with 8.3% (1/12). The region A was also free of this virus.This virus was the least prevalent one as it was only identified in 5.96% (9/151) of all tested colonies. The majority of the CBPV-positive workers were found in region B. However, no significant differences were found between regions  detected viruses of this survey . As showAltogether, the following combined virus infections were observed: single virus occurrence contained only SBV; in dual infection-SBV and DWV; in triple-BQCV, SBV and DWV; in quadruple all detected four viruses. Interestingly, those viruses were not observed in any other combinations. In addition, single hives contained a maximum of three viruses at the time in any of these combinations SBV-BQCV-DWV and SBV-BQCV-CBPV.The nucleotide sequences from SBV, BQCV, DWV and CBPV confirmed the identity of those viruses with 97–99% similarity.Phylogenetic trees were inferred for those viruses Figs –5. For tTo infer the BQCV phylogenetic tree the 511-nt sequence of the capsid protein coding region was used. Five BQCV Mongolian isolates from the North and Central regions as well as sequences from isolates from USA, Europe and Asia were used to construct the tree . In geneThe phylogeny of the Mongolian DWV isolates was inferred from 820-nt sequence of the capsid protein-coding region. Seven isolates from bees and mites from the North and Central regions as well as previous isolates retrieved from Genbank were used to construct the DWV phylogenetic tree . Two cluTo infer the CBPV phylogenetic tree the 335-nt sequence of the RdRp coding region was used. Two isolates derived from the North and Central regions together with isolates from other countries retrieved from the Genbank were used to construct the CBPV phylogenetic tree . Most boV. destructor mites and DWV  especially in region B and C . No other significant correlations were found.To reveal potential positive associations of pathogens in the samples from Mongolia, Pearson’s Chi-squared tests were performed (p<0.05). Overall locations, a positive correlation was found between the prevalence of Apis mellifera, pathosphere of Mongolia. The survey data show a distinctive pathosphere of colonies of European, A. mellifera, subspecies in this isolated country, where honey bees have been introduced as recent as ~60 years ago. We used standard methods to survey 151 colonies, distributed in three regions and 9 locations across the country, to register the presence of 14 parasites and pathogens of managed honey bees. Besides the mite V. destructor, the sampling scheme allowed for the detection of the tested pathogens, when only 5% of workers are infested with a probability of 0.92 [This is the first report addressing the honey bee,  of 0.92 . Therefo of 0.92 .P. larvae and M. plutonius, respectively) were not detected in any of the sampled colonies, thereby indicating their absence or most probably a very low prevalence at the time of sampling. AFB and EFB are widely distributed around the globe [A. woodi, was not detected. Although this mite is thought to have a global distribution, it has also not been found in Sweden, Norway, Denmark, New Zealand, and Australia, and in the US state of Hawaii [Despite this high detection power, the causative bacteria for American and European foulbrood , thereby suggesting that this region had some contact with bees or beekeeping material infested/contaminated with respective spores. The prevalence of N. ceranae in all surveyed regions in Mongolia was 12.58% (19/151). This is considerably high in light of a recent European survey that included 16 countries, and showed that only 3 countries  exceeded 10% of clinical prevalence [N. ceranae in regions A and C remain unclear, but the extreme isolation of some apiaries (up to 600 km) cannot be overruled. N. ceranae is expected to be affected by extreme cold climate [Since the first report of  in Asia  and its  in Asia , it seem[N. apis . To our is virus . N. ceraevalence . The und climate , but thi climate .V. destructor in the Mongolian apiaries varied between the geographical locations from none  to all  of the colonies infested. Those variations may be explained by differential mite treatments, e.g. in region B and location 7. In general, considerable differences in mite infestation levels between countries, regions or even apiaries are not uncommon. For example, samples collected at the same period of time (summer 2013) can show substantial differences ranging from 0% (Belgium) to 87% (Latvia) at the apiary level [The prevalence of ry level .V. destructor overall surveyed regions, DWV and V. destructor were not always found together at the location level. For example in locations 1, 6 and 9 only V. destructor was found but not DWV. In location 7, neither DWV nor V. destructor were found, possibly due to treatments by beekeepers. Mongolia hence offers a unique opportunity to address V. destructor and DWV in isolation. In sharp contrast to other areas [At present, there are no data about colony losses in Mongolia to link possible effect of mite infestation on the health of local colonies. Interestingly, despite a significant positive correlation between the prevalence of DWV and er areas ,41–45, DV. destructor mite, which is acting as a potent vector of this virus [Regarding viruses in general, SBV was the most prevalent one in our surveyed colonies (73.5%). This high prevalence of SBV is in line with other studies from European and Asian countries  can be governed by season [Since the prevalence of some viruses  and parasites  along the border with China impose natural geographic barriers, which seem to efficiently limit contact between honey bees from Mongolia and those from South-Asian countries. This may explain the distant phylogenetic relation between those geographically closed populations. The imports of honey bee colonies from Russia are the most plausible source of the detected European viruses, thereby supporting the view that global apicultural trade was and still is the most efficient way for long range transport of pathogens.A. mellifera, pathosphere of Mongolia, with the absence or low prevalence of otherwise globally distributed diseases, e.g. the presence of V. destructor in isolation from DWV. This creates an interesting case, which offers ample opportunity for exciting future research on honey bee health.In conclusion, this survey reveals to a certain level, the distinctive honey bee, S1 Table(S) indicates the primer sets used for sequencing.(DOCX)Click here for additional data file.


Compared to other pregnancy-related events, the full cost of stillbirth remains poorly described. In the UK one in every 200 births ends in stillbirth. As a follow-up to a recent study which explored the direct costs of stillbirth, this study aimed to explore the intangible costs of stillbirth in terms of their duration and economic implication.Systematic searches identified relevant papers on the psychological consequences of stillbirth. A narrative review of the quantitative studies was undertaken. This was followed by a qualitative synthesis using meta-ethnography to identify over-arching themes common to the papers. Finally, the themes were used to generate questions proposed for use in a questionnaire to capture the intangible costs of stillbirth.The narrative review revealed a higher level of anxiety and depression in couples with stillbirth compared to those without stillbirth. The qualitative synthesis identified a range of psychological effects common to families that have experienced stillbirth. Both methods revealed the persistent nature of these effects and the subsequent economic burden.The psychological effects of stillbirth adversely impacts on the daily functioning, relationships and employment of those affected with far-reaching economic implications. Knowledge of the intangible costs of stillbirth is therefore important to accurately estimate the size of the impact on families and health services and to inform policy and decision making.The online version of this article (doi:10.1186/s12884-015-0617-x) contains supplementary material, which is available to authorized users. The World Health Organisation (WHO) defines stillbirth as “the birth of a baby with no signs of life at, or after 28 completed weeks of pregnancy” . GloballStillbirth is a traumatic experience with reports of adverse psychosocial effects such as: anxiety, depression, shame, suicidal thoughts, post-traumatic stress disorder (PTSD) and guilt –7. The oHowever, stillbirth has been termed an ‘invisible death’ due to being neglected as a public health issue of importance to society and health policy makers . EffortsThe economic impact of an illness involves all costs and outcomes associated with its incidence. These include: direct costs - the monetary cost of all resources associated with the provision of an intervention e.g. health service use and medications , indirecDirect and indirect costs do not attempt to reflect the range of deterioration in quality of life from interventions/diseases. However, the term intangible costs encompasses the psychological dimensions of illness  but theyA recent paper by Mistry et al.  classifiAn exploration of the intangible costs of stillbirth will add to the evidence of the economic costs and consequences of stillbirth allowing a more complete appreciation of the burden of stillbirth. It is hypothesised that a realistic total cost will inform decision making on health resource allocation to prevent stillbirth or its adverse consequences.Building on the earlier quantitative study by Mistry et al.  we carriThe literature search followed the Centre for Review and Dissemination (CRD) guidelines  and was Papers were included if: the participants were mothers or/and fathers who had experienced stillbirth, the intervention/exposure was stillbirth and the outcome was the negative consequences of stillbirth. The study design included both qualitative and quantitative studies. The review was restricted to studies carried out in high-income countries (defined as the Organisation for Economic Co-operation and Development (OECD) member countries) and English language articles published in peer-reviewed journals. Papers published prior to 2000 were also excluded for pragmatic reasons as they may not reflect current experiences of parents.Four electronic databases: MEDLINE, PsycINFO, CINAHL and Web of Science (WoS) were comprehensively searched in June 2014  in order to determine the central concepts, and the details of the study participants, aims and methods were tabulated to serve as a context for interpreting and explaining each study. Next, the main ideas and quotes for each study were identified and tabulated. To explicitly show how the concepts correlated with each other, a grid was created and the concepts of each paper placed in it. Using ideas developed by Schutz , first aUsing reciprocal translation analysis (RTA) , 34, relThe themes from the qualitative synthesis were prioritised in terms of the frequency of their occurrence in the identified studies. Themes with similar ideas were merged into sections. In an attempt to move towards quantifying the evidence collated in the qualitative synthesis, questions are proposed, which could be developed for use in a large scale survey to provide this quantification. Finally, the questions developed from the sections were refined and related themes amalgamated to generate a proposed questionnaire.Six quantitative and 14 qualitative studies were selected for the review and synthesis.Among the six quantitative papers, “The months after were filled with therapy and medication for PTSD, anxious days and panicked nights” ([“Once I started to say I was home-birth (and) I didn’t have any scans, I always got the feeling ……well that’s why your baby died, because you didn’t have a scan” .nights” place…” “My husband went back to work quickly and seemed happy to do so…and I was left on my own”  of it” “having people acknowledge her life and death and her impact on my family, helped me to know that I’m not crazy” .hild….” .“My child’s death has changed me to be a more sensitive person to other’s feelings…..” .The merging of themes with similar ideas resulted in four sections and after refining and blending similar questions, 10 questions were proposed to cover the breadth of intangible costs and they focus on depression/profound grief, need for support, social isolation/return to normality and couples’ relationship/siblings' issues Fig. .Fig. 3Todepression’. This also came out strongly in the narrative review. This theme was followed closely by ‘need for support’. The next two most common themes were ‘profound grief’ and ‘social isolation’. Three themes: ‘couples’ relationship’, ‘siblings’ issues’ and ‘return to normality’ seemed to be of relatively equal importance. However, two of these themes (couples’ relationship and siblings’ issues) have the same connotation and seemed to point towards similar issues relating to impacts on the family. Less common was the theme of ‘recovery’ which is arguably an intangible benefit and therefore, is less relevant to the aim of the study.Among the eight themes from the meta-ethnography, the outstanding theme that emerged from nearly all the studies was ‘We aimed to find studies that identified the consequences of stillbirth, and their duration and impact so that they could be quantified and moved from intangible to tangible costs. A number of studies reported these negative feelings but most did not explicitly report the duration or their impact on quality of life. However, several studies , 49, 54 The overarching themes of profound shock and depression were consistent with results from the narrative review which showed that the high level of depression, anxiety and PTSD after a stillbirth, persisted for months  and up tThe synthesis also showed that stillbirth could cause relationship issues among couples leading to separation and divorce. Gender differences in grieving may lead to misunderstandings which inadvertently affect family functioning and relationships , 58. RelThe synthesis showed that men returned to work earlier than women with all fathers returning to work and most mothers either on full or part-time leave by the third month after a stillbirth . The conThe importance of formal and informal support cut across all aspects of the studies, with many parents feeling they needed expert support and reporting that support was limited. The Royal College of Obstetricians and Gynaecologists (RCOG) guidelines for care of families following a stillbirth, acknowledges the possibility of the need for psychological care but there is no practical advice on how healthcare professionals can support parents . The iso“…I spent so many years trying to find that ‘something’ that will give me peace” .The main strength of this paper is that it is the first to identify the probable main sources of the intangible costs of stillbirth. The only indirect evidence that presently exists is in studies that have described the psychosocial consequences of stillbirth. A second strength is the comprehensive and systematic attempt to identify relevant studies. The inclusion of both quantitative and qualitative studies conducted in the last 15 years allowed a deep assessment of the issues highlighted by these studies. The search strategy and the three-stage selection/exclusion process were wide and detailed. The use of meta-ethnography to interpret first-order constructs and produce new understandings resulted in a deeper insight and development of concepts than that obtained in a narrative literature review alone. Finally, the study addressed the research gap identified in a previous study on costs of stillbirth .As expected in any study, there were also limitations. Firstly, the study did not assess the relevance of the findings of the synthesis. A possible way of checking this would be to present it in a questionnaire form to population groups that have experienced stillbirth. However, by proposing questions that could be asked in prospective large scale surveys, the study developed a framework to guide future studies. A second limitation was the presentation of these questions in the words of the authors alone. Thus for a questionnaire, the questions will have to be refined by experts from this patient group to ensure the utmost sensitivity in the language used. Finally, there are controversies surrounding the inclusion of intangible costs in costing estimates , 65, 66,This study has important implications for health economists; maternity care providers and policy makers. Firstly, the findings justify the rationale for including intangible costs in economic evaluations of stillbirth by revealing their potentially significant contribution to the total costs. Thus, the common approach of estimating only direct and indirect costs is likely to miss relevant aspects of the total disease burden. It has been said that intangible costs are difficult to quantify so are not included in most studies. However, some studies have used recognised approaches such as “willingness-to-pay” (WTP) methods to place a monetary value on these costs. WTP is a complex method requiring specialized expertise in designing and implementing surveys and its use in this area is limited , 23.Secondly, the intangible costs, in addition to direct/indirect costs of stillbirth could be compared more realistically to the costs of other maternal or fetal outcomes such as live-births and premature births. This comparison will be useful for policy makers to decide on resource allocation to these sectors. Thirdly, after a stillbirth, supportive measures could be instituted at once; from the diagnosis, into and beyond the subsequent pregnancy to offset some of the intangible costs. Ultimately, evidence-based guidelines on how to practically support families after a stillbirth need to be developed. To address this need the policy emphasis needs to be directed to providing more funds towards development of interventions to reduce the adverse psychological effects of stillbirth on families. In addition, a true appreciation of the costs of stillbirth will allow an appropriate cost-benefit assessment of interventions to prevent stillbirth which are presently impossible.There is undoubtedly a need for more economic evaluation studies to be conducted on stillbirth. The area is still sparsely researched which has not allowed us to consider the impacts on health-service delivery or on health-care professionals. One suggestion for future studies would be to put a monetary value on these intangible costs. Furthermore, similar studies could also be carried out in low and middle income countries.The study has shown that stillbirths have a wide reaching and long-lasting impact with far-reaching economic implications. Considering the significant numbers of stillbirth worldwide, the large number of families affected and the long-lasting nature of its impact, it is surprising that stillbirths still attract relatively little attention from policy makers. Therefore, exploring the intangible costs associated with stillbirth is important to emphasize its burden and inform policy and decision making.


Poecile atricapillus) in Alaska. Subsequently, similar deformities have appeared in numerous species across continents. Despite the widespread distribution of this emerging pathology, the cause of AKD remains elusive. As a result, it is unknown whether suspected cases of AKD in the afflicted species are causally linked, and the impacts of this pathology at the population and community levels are difficult to evaluate. We applied unbiased, metagenomic next-generation sequencing to search for candidate pathogens in birds affected with AKD. We identified and sequenced the complete coding region of a novel picornavirus, which we are calling poecivirus. Subsequent screening of 19 AKD-affected black-capped chickadees and 9 control individuals for the presence of poecivirus revealed that 19/19 (100%) AKD-affected individuals were positive, while only 2/9 (22%) control individuals were infected with poecivirus. Two northwestern crows (Corvus caurinus) and two red-breasted nuthatches (Sitta canadensis) with AKD-consistent pathology also tested positive for poecivirus. We suggest that poecivirus is a candidate etiological agent of AKD.Avian keratin disorder (AKD), characterized by debilitating overgrowth of the avian beak, was first documented in black-capped chickadees ( Avian keratin disorder (AKD) is an increasingly common disease of wild birds. This disease, characterized by beak overgrowth, was first described in the late 1990s and has been spreading rapidly both geographically and in terms of host species affected. AKD decreases host fitness and can be fatal. However, the cause of the disease has remained elusive, and its impact on host populations is poorly understood. We found a novel and divergent picornavirus in 19/19 AKD-affected black-capped chickadees that we examined but in only 2/9 control cases. We also found this virus in 4 individuals of 2 other passerine species that exhibited symptoms consistent with AKD. Our data suggest that this novel picornavirus warrants further investigation as the causative agent of AKD. Poecile atricapillus]) in Alaska in the late 1990s . Birte 1990s , changeste 1990s , and higte 1990s  and aviate 1990s . As a rete 1990s . While dCyanopsitta spixii)  .Corvus caurinus]), red-breasted nuthatches (RBNH [Sitta canadensis]), and raptors . We then assembled the remaining sequences into contiguous sequences (contigs) using the Trinity de novo assembler  . CompariYP_009030047, AGS15016, and AGW95843).Poecivirus follows the typical picornavirus organization, with the single ORF coding for a polyprotein that includes the P1 region  and the P2 and P3 regions  . To furtADN94256)  and VP0 . AnalysiDN94256) , 20. AliDN94256)  support  and VP1 . The P1  and VP1 ; these a875/C. The poecivirus 2A protein does not exhibit sequence homology to any known picornavirus. The 2A protein does not contain the NPGP motif that facilitates C-terminal termination in aphthoviruses, avihepatoviruses, cardioviruses, erboviruses, senecaviruses, and teschoviruses  for NTP-binding sites but not the DDLxQ motif for putative helicase activity , which typically houses the active-site cysteine in picornaviruses   .P < 0.0001, likelihood-ratio chi-square test).Having characterized the sequence and genomic organization of poecivirus, we determined the infection status of 19 AKD-affected BCCH and 9 unaffected individuals using targeted PCR followed by Sanger sequencing . Of the AKD-affected BCCH, 19 of 19 (100%) tested positive for poecivirus via PCR, while 2 of 9 (22%) unaffected individuals tested positive; thus, individuals with AKD were significantly more likely to be infected with poecivirus than expected by chance  cells; however, we were unsuccessful. Viral RNA was initially detectable in the inoculum (prepared from virus-positive beak and other tissues) and supernatant but disappeared rapidly and remained undetectable for 72 days. This suggests that these cell lines or the culture conditions that we used were not permissive for viral replication, that the cell lines used might not express the receptor(s) used by poecivirus to gain entry into cells, or that the inoculum that we used did not contain replication-competent virus.To further characterize this virus, we attempted to develop The two NOCR and two RBNH with elongated beaks all tested positive for poecivirus via PCR using poecivirus-specific primers that target the 5′ untranslated region (UTR) and the P1 region. Sanger sequencing of the PCR products revealed virus with high nucleotide identity to the poecivirus detected in BCCH . The 5′ UTR regions displayed 92.4% (NOCR 674), 91.8% (NOCR 848), and 92.5% (RBNH 12 and 929) nucleotide identities with the BCCH poecivirus, while a 300-bp sequence in the P1 region displayed 92.0% , 91.0% (RBNH 12), and 91.3% (RBNH 929) nucleotide identities with the BCCH poecivirus. Across this region, virus from NOCR exhibited 24 mutations, of which 23/24 (95.8%) were synonymous with the virus detected in BCCH; meanwhile, the virus from RBNH exhibited 28 (RBNH 12) and 26 (RBNH 929) mutations, of which 27/28 (96.4%) and 25/26 (96.2%), respectively, were synonymous with the virus from BCCH. Furthermore, phylogenetic analysis of this region shows that the RBNH viruses were more closely related to each other than to viruses isolated from the other species and that the NOCR viruses were more closely related to each other than to viruses isolated from the other species. In addition, viruses isolated from RBNH and NOCR were more divergent from the BCCH-isolated poeciviruses than the BCCH viruses were from each other; this is consistent with the possibility of adaptation to particular host species.In this study, we used next-generation sequencing to investigate potential causes of avian keratin disorder (AKD), an emerging epizootic of beak deformities affecting numerous species across continents. Focusing on Alaskan BCCH, we found evidence of a single pathogen in the beaks of AKD-affected individuals: a novel picornavirus that we have named poecivirus. While we cannot rule out the presence of additional pathogens in other tissues, we did detect a strong, statistically significant correlation between poecivirus and AKD. Specifically, poecivirus was present in 19/19 (100%) AKD-affected BCCH and 2/9 (22%) control individuals. In addition to the correlation between poecivirus infection and AKD in BCCH, we detected poecivirus in two NOCR and two RBNH with elongated beaks. Importantly, poecivirus is not the first picornavirus associated with hyperkeratosis; Seneca Valley virus has been associated with hyperkeratosis in pigs .Complete polyprotein analyses of poecivirus reveal a novel virus whose closest relatives are all avian picornaviruses, including megriviruses, turkey hepatitis virus, and chicken picornaviruses 4 and 5. While the polyprotein exhibits characteristic picornaviral organization and motifs, including picornavirus-like capsid domains and typical motifs in the 2C, 3C, and 3D functional domains, it is distinct from known picornaviruses. According to the Picornaviridae Study Group of the International Committee on Taxonomy of Viruses (http://www.picornastudygroup.com/definitions/genus_definition.htm), novel picornavirus genera are defined by amino acid identities in the P1, P2, and P3 regions of <40%, <40%, and <50%, respectively. Poecivirus exhibits ≤34%, ≤25%, and ≤48% identity to any known picornaviruses in the P1, P2, and P3 regions, respectively. This, along with lack of homology in the 2A, 2B, and 3A regions between poecivirus and its closest relatives indicates that poecivirus likely represents a new genus of picornavirus.Picornaviridae family, although knowledge of the family taxonomy is changing rapidly, with 9 new genera approved in 2014 alone. While the family includes 22 fully sequenced picornavirus species isolated from avian species, only a few these are known from wild bird species, including turdivirus 1 and 2 . All 19 of these individuals tested positive for the presence of poecivirus. It is also possible that some individuals exhibit subclinical infection (as has been suggested for related viruses ) or thatin vitro system for growing wild-type virus, one possible alternative would be to use in vitro-transcribed RNA derived from a full-length cDNA of the poecivirus genome to transfect cultured cells; this would bypass a potential block to viral entry of cultured cells if, for example, cells used in culture lacked the appropriate receptor.AKD is an emerging disease of wild birds that has been increasing both in geographic scope and in the variety of species affected. The strong association between poecivirus and AKD in BCCH suggests that this is a promising candidate for the causative agent of AKD. It is noteworthy that AKD appears to affect an incredibly wide range of avian species. Several lines of evidence  lend support to the idea that multiple species are being afflicted by a disease with a common etiology. In keeping with this, we found poecivirus to be present in 4/4 individuals with overgrown beaks from 2 additional species (NOCR and RBNH). However, control individuals from these species were not available for testing at the time of this study; screening of additional species with AKD-like deformities for the presence of poecivirus and sequencing of poecivirus from a greater variety of species will be necessary to further test the association between poecivirus and AKD. In addition, next-generation sequencing would be required to obtain the full viral genome from each of these hosts to confirm that they are infected by poecivirus, as opposed to a related but distinct species of virus. Finally, viral challenge experiments will be required to demonstrate with certainty that poecivirus is the cause of AKD. In the absence of an in vitro culture system, and viral challenge of healthy individuals will provide the information necessary to determine with certainty the clinical contribution of poecivirus to AKD and to elucidate both the pathogenesis and transmission potential of this virus. Ultimately, identifying the causative agent of AKD is vital in order to monitor the impact of this disease on wild bird populations, understand the causes underlying current disease emergence, and determine the possibility of and necessity for interventions.In conclusion, the association between poecivirus and AKD warrants further investigation. The use of cloacal and/or buccal swabs to test individuals for infection with poecivirus provides a nonterminal method of sampling individuals for poecivirus infection that will make sampling large numbers of AKD-affected individuals substantially more feasible. The sampling of larger numbers of individuals, together with metagenomic characterization of other affected species, the development of an The majority of the research documenting, describing, and investigating AKD has been conducted in BCCH , 6, 14. Nineteen BCCH with AKD  were traAll work was conducted with the approval of the U.S. Geological Survey (USGS) Alaska Science Center Institutional Animal Care and Use Committee (assurance no. 2011-2) and under appropriate state and federal permits.Tissue samples from 8 BCCH with AKD were processed for the initial metagenomic deep sequencing . To extr−8 were filtered. Similarly, sequences that aligned with the Illumina adapter sequences or to ϕX174 control sequence were removed. The remaining sequences were assembled into contiguous sequences using the Trinity de novo assembler . We also used the more sensitive HHPRED hidden Markov model-based alignment and structure prediction software tool to search for more distant homologies  were removed . The firssembler . The resssembler . Analysissembler , and by mologies .The viral genome assembled from deep-sequencing reads was validated by Sanger sequencing  with free-end gaps; otherwise, default settings were used. Maximum-likelihood phylogenies were created using PhyML (PhyML plugin for Geneious version 2.2.0) with the LG model of amino acid substitutions, 100 bootstrap replicates, and otherwise default parameters .Quantitative PCR (qPCR) was used to determine viral tropism and monitor viral RNA levels in culture experiments. RNA was extracted with a Quick-RNA miniprep kit (tissue) or ZR viral RNA kit (tissue culture) (Zymo). RNA was reverse transcribed as described above; the qPCR mixtures contained 1× LC480 Sybr green master mix (Roche), 0.1 µM each primer , while chicken embryo cells were grown in Dulbecco’s modified Eagle’s medium (ATCC); both were supplemented with 10% fetal bovine serum, 50 units/ml penicillin, and 50 µg/ml streptomycin. QT6 cells were grown in Ham’s F-12K medium (ATCC) with 2 mM l-glutamine adjusted to contain 1.5 g/liter sodium bicarbonate, supplemented with 10% tryptose phosphate broth, 5% fetal bovine serum, 50 units/ml penicillin, and 50 µg/ml streptomycin.Duck embryo (CCL-141), chicken embryo (CRL-12203), and quail QT6 cells were obtained from the ATCC  and were grown at 37°C and 5% COg for 1 min, filtered through a 0.2-µm filter, diluted 1:60, and used to inoculate cell cultures. The cells were visually monitored for evidence of infection, and the supernatants were monitored for the presence of viral particles using quantitative real-time PCR (qRT-PCR).For inoculation experiments, portions of frozen, virus-positive beak, heart, brain, cloaca, kidney, liver, and ventriculus samples were minced with scalpels, suspended in serum-free DMEM containing 25 mM HEPES (pH 7.4), homogenized with a pellet pestle or Dounce homogenizer (beak), and placed on ice for 30 min. The homogenates were clarified by centrifugation at 10,000 × We tested for the presence of poecivirus in tissue samples from the 8 AKD-affected BCCH subjected to metagenomic deep sequencing, from 11 additional BCCH with AKD, and from 9 control BCCH. Half of a mandible from each black-capped chickadee was ground using a mortar and pestle on liquid nitrogen, and RNA was extracted using a Zymo Quick-RNA miniprep kit. In some cases, the cloaca was also tested; the cloaca was homogenized using a pellet pestle, and RNA was extracted using a Zymo Quick-RNA miniprep kit and reverse transcribed as described above. Samples were screened via PCR using poecivirus-specific primers see  and BCCHViral tropism was examined in four BCCH  via qPCR (as described above). We measured the levels of viral RNA relative to host RNA in the cloaca, beak, gastrointestinal tract, liver, lung, kidney, heart, and brain of all four individuals and in buccal and cloacal swabs collected from individuals 331 and 971. We also measured the levels of viral RNA relative to host RNA in the two BCCH that were unaffected by AKD but tested positive for poecivirus.Tissues were fixed in 10% formalin, embedded in paraffin, cut into 5-µm sections, mounted on glass slides, and stained with hematoxylin and eosin.The sequence of poecivirus has been deposited in GenBank under accession number KU977108. The sequences obtained from the NOCR and RBNH have been deposited under accession numbers KU986726, KU986727, KU986728, KU986729, KU986730, KU986731, KU986732, and KU986733.Table S1 Poecivirus-specific primers. Primer BCCHpic_1F and BCCHpic_9R, targeting the 5′ UTR and 3′ UTR, respectively, are not represented in the Sanger sequencing-validated poecivirus genome deposited in GenBank (accession number KU977108).Table S1, DOCX file, 0.01 MB


The recent outbreak of the Ebola virus in West Africa has highlighted the clear shortage of broad-spectrum antiviral drugs for emerging viruses. There are numerous FDA approved drugs and other small molecules described in the literature that could be further evaluated for their potential as antiviral compounds. These molecules are in addition to the few new antivirals that have been tested in Ebola patients but were not originally developed against the Ebola virus, and may play an important role as we await an effective vaccine. The balance between using FDA approved drugs versus novel antivirals with minimal safety and no efficacy data in humans should be considered. We have evaluated 55 molecules from the perspective of an experienced medicinal chemist as well as using simple molecular properties and have highlighted 16 compounds that have desirable qualities as well as those that may be less desirable. In addition we propose that a collaborative database for sharing such published and novel information on small molecules is needed for the research community studying the Ebola virus. The flavivirus that causes dengue fever infects up to 100 million people each year, leading to death in 2.5% of cases4. Other viral outbreaks including severe acute respiratory syndrome coronavirus (SARS-CoV) and the Middle East respiratory coronavirus (MERS-CoV), affect far fewer people but have high mortality rates and the potential to spread to epidemic size6. Thus, even with the development of vaccines and other treatments, viruses lead to a large burden on human health.Viruses remain a constant threat to global health, with new infections from Human Immunodeficiency Virus (HIV), Hepatitis B Virus (HBV) and Hepatitis C Virus (HCV) killing more than 3 million people annually8. However, a large number of virus types remain without any effective therapeutics, and there are few broad-spectrum anti-virals available. Thus, when viruses emerge that cause life-threatening infections, such as the recent Ebola virus epidemic in West Africa, there are no treatment options. Since it is likely that these and other types of infectious agents will emerge in the future, an important goal is to identify inhibitors to be available to contain such outbreaks.More than 30 small molecule drugs have been developed that have activity against individual viruses, including HIV, influenza, HBV, and more recently HCV3. Targets in this class fall into multiple categories: virus adsorption inhibitors, inhibitors of viral DNA or RNA synthesis, viral protease inhibitors required for virus maturation, and viral neuraminidase inhibitors required for virus elution7. In addition, cellular targets exist that are required for viral replication, including inosine monophosphate (IMP) dehydrogenase, which is required to supply the pool of guanosine triphosphate (GTP) that serves as a substrate for RNA and DNA, and S-adenosylhomocysteine (SAH) hydrolase, which is required for the methylation and hence maturation of viral DNA. While finding specific inhibitors to target each viral threat individually may be ideal, the cost-savings and feasibility of finding drugs that act as broad-spectrum antivirals, or those that target specific viral genus or family, is an important goal given the expense associated with developing any one drug for one disease3. There are important individual aspects of each virus, and viral family to consider, but nonetheless, many mechanisms of the viral life cycle are mirrored across families, and thus represent opportunities to learn from the many experimental studies that have already been performed.A large array of drug discovery efforts have proven that despite their small size, viral genomes represent suitable targets for drugs. Direct-acting antivirals, which target viral proteins rather than the host’s, have been the subject of extensive investigation9? Small molecules have several advantages over other therapeutic approaches including the ability to be produced at a large scale and stability necessary for broad distribution. We felt it was time to therefore focus more on small molecules while we await a vaccine. We have found that indeed there is much prior knowledge regarding small molecules that have been shown to be active against the Ebola virusin vitro or in animal models13, including a number of FDA-approved drugs16. A thorough literature search of PubMed, and CAS SciFinderTM  using terms including “Ebola” identified 55 molecules suggested to have activity against Ebola virusin vitro and/orin vivo  resulting from two high throughput screens against the Ebola virus15 and closely matched the receptor-ligand pharmacophores for the Ebola Viral protein 35 (VP35)10. Follow-up docking studies suggested that these compounds may have favorable inhibitory interactions with this receptor. VP35 is a cofactor in the RNA polymerase transcription complex, and helps the virus evade the immune response by blocking activation of the interferon regulatory factor 3, which is required for the induction of interferons alpha and beta. Thus, blocking VP35 should allow for an enhancement of the host immune response to the Ebola virus. It is proposed that similar compounds may be acting via a closely related mechanism, though there has been no experimental evidence to directly prove this yet.Recently, a pharmacophore16 has highlighted the ability of three clinically approved ion channel blockers to inhibit the Ebola virus cellular entry. The drugs amiodarone, dronedarone, and verapamil, were given at concentrations that are possible in human serum, and were effective against a number of filoviruses. The authors hypothesized that these drugs may act by disrupting late endosomal processing or by disrupting calcium signaling that is required for viral entry.Another recent study19. However, they are all orally bioavailable and generally safe for humans. Thus these repurposed drugs may represent a fast track to potential evaluation and approval as a feasible option for preventing the spread and mortality associated with the Ebola virus in a large population.Of course, none of these aforementioned FDA approved drugs were designed to target the Ebola virus. Amodiaquine and chloroquine are antimalarials, clomiphene and toremifene are selective estrogen receptor modulators. Amiodarone, dronedarone, and verapamil are anti-arrhythmics. Interestingly, all of these compounds have a common tertiary amine feature, which may suggest they could act through similar mechanism20. Faviparavir is thought to act by inhibiting the viral RNA-dependent RNA polymerase selectively and has demonstrated activity against a number of other viruses. At least one Ebola patient, who has since recovered, was given favipiravir21, and Japan offered to supply it to the World Health Organization.Several small molecules have actually been tested in very small numbers of humans for activity against the Ebola virus. For example there has been some press on favipiravir, which is undergoing phase 3 clinical trials in the US for influenza and is approved in Japan, as it has shown promising efficacy against the Ebola virus in mice22, in phase 3 clinical trials for treatment of cytomegalovirus and other DNA viruses has shown efficacy against the Ebola virusin vitro and animal studies are ongoing23. Brincidofovir is thought to mimic cytidine, a building block of DNA, and thereby inhibit viral DNA polymerases, and its mechanism of action against the Ebola virus, an RNA virus, is yet unknown. Brincidofovir, which has demonstrated safety in humans, has been given to at least two Ebola virus patients, one in Dallas and one in Nebraska. While unfortunately the Dallas patient died, the Nebraska patient survived24. It is of course too early to know the effect of this molecule on the progression of the disease. This compound is a pro-drug that is converted into the active antiviral, cidofovir diphosphate. Brincidofovir has higher oral bioavailability, intracellular concentrations of drug and increased antiviral potency22. This compound only appeared in the literature in 2014 and there is very little published information.A second experimental drug, brincidofovir9. While many are not ready for in human use, they may present an attractive starting point to be refined in a future drug discovery effort. For example, a novel nucleoside analog, BCX4430 demonstrated efficacy in mice and non-human primates against the Ebola virus25. This compound targets viral RNA polymerase activity by inducing early termination of transcription and thus blocking replication. BCX4430 is not only active against Ebola virus, but also targets other members of the Filovirus family as well as 8 other RNA virus families. Because of the potent and efficacious effects, BCX4430 is being fast-tracked for clinical trials in humans.Beyond these early stage drugs, there are a number of other compounds that have been identified as active against the Ebola virus as summarized by Erik De Clercq26 analysis of the over 320 NIH probe compounds using public and commercial sources of chemical structures and the issues related to doing this type of analysis27. The likely chemistry quality of these probes was scored based on a number of criteria including literature related to the probe and potential chemical reactivity. Through a series of machine learning models, we also computationally predicted the scores which were being identified through a painstaking manual process26. External validation and comparison with other measures of drug-likeness and filtering rules suggested a comparable level of accuracy26.We have recently described an expert’s medicinal chemistry29 31.In vivo a steady state plasma concentration could not be maintained at a therapeutic level. Rhodanines are known to be problematic PAINS compounds29. Interestingly amodiaquine was not scored favorably by PAINS or the medicinal chemist, yet this is a successful antimalarial drug.We have now carefully analyzed in a similar manner the 55 small molecules with activity against the Ebola virus identified from our literature search. The chemist’s (C.A.L.) decisions on compound quality are summarized in29 . PAINS at-test and ANOVA with JMP v. 8.0.1 . Significant differences were noted in LogP and Lipinski Rule of 5 violations between desirable and undesirable compounds although one molecule SARA-133 skewed the data with a molecular weight over 3000  Vault  using the Chemaxon toolkit ) of the molecules and compared these to their medicinal chemistry classification . The meaer 3000 . Removal32 . The compound representation in CDD Vault can be accessed via the text compound descriptors (as in theThe research described for small molecule inhibitors against the Ebola virus has occurred in a disconnected manner and there have been few efforts to summarize the total medicinal chemistry efforts to date. We think this research can learn from other areas in which there are currently efforts to improve collaboration and screening. In the area of Tuberculosis research, funding from the Bill and Melinda Gates Foundation and The European Commission have enabled the TB Drug Accelerator and the More Medicines For Tuberculosis, as large-scale collaborations between academia, research institutes and industry. These collaborations have promoted the selective sharing of related data in a secure environment between collaborators using the CDD Vault32 . The cenmpounds  freely a16 but with activity against Ebola virusin vitro orin vivo may represent useful starting points with the advantage that much is known regarding their ADME and Tox properties. However they may not be ideal in the opinion of a medicinal chemist. By bringing together all 55 compounds described in the literature with activity against the Ebola virus, this body of evidence may be more convincing than each study in isolation. In addition it allows us to consider structural features and molecular properties, which may provide insights into the target or mechanism of action. It is unclear whether any of the 55 compounds might also have activity when used as combination therapy as is the current standard of care for HIV, in order to overcome drug resistance, and this needs experimental evaluation. Clearly, we are a considerable distance from having an FDA approved drug for the Ebola virus, but this analysis illustrates that there are already drugs on the shelf that can be potentially repurposed in a shorter time period and at a lower cost and yet they do not appear to have been tested in patients with the Ebola virus. In addition there are at least 16 molecules which in the opinion of an experienced medicinal chemist are possibilities for further optimization. While novel compounds are likely more commercially viable they also will require considerable effort to assess safety. Resources will need to be allocated to this effort and administrators and scientists should perhaps consider some of the medicinal chemistry insights we have provided as well as using a collaborative database to share molecules that are active amongst all scientists. It is hoped these efforts could inspire further drug discovery efforts around small molecules.The compounds which are FDA approved drugs for other diseases37.To illustrate the level of interest in repurposing efforts for the Ebola virus, the following studies were identified upon submission that describe additional compounds as well as those already described hereinSupplemental Table 1. 55 compounds identified from the literature asin vitro orin vivo active against the Ebola virus. Molecule structures in various 2D formats are provided along with simple molecular descriptors, information relating to the target if known, indication, publication reference as well as medicinal chemistry insights and PAINS filter failures.http://dx.doi.org/10.5256/f1000research.6120.s42952 The authors have collected 55 small molecules reported to have activity against the ebola virus from the literature and organized them into a dataset that is easily searchable for those who may be interested in pursuing further research on the design of inhibitors of this virus.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. The authors have searched a collection of compounds for a common pharmacophore directed against Ebola proteins, and detected 55 hits. These were filtered by an experienced medicinal chemist using well-established techniques and intuition to provide 16 legitimate compounds that may serve the Ebola research community as a publicly available resource. This is an excellent example of the power of shared, collaborative research databases providing valuable resources to the research community.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


The aim of this study was to compare the health care costs for patients using additional homeopathic treatment (homeopathy group) with the costs for those receiving usual care (control group).Cost data provided by a large German statutory health insurance company were retrospectively analysed from the societal perspective (primary outcome) and from the statutory health insurance perspective. Patients in both groups were matched using a propensity score matching procedure based on socio-demographic variables as well as costs, number of hospital stays and sick leave days in the previous 12 months. Total cumulative costs over 18 months were compared between the groups with an analysis of covariance (adjusted for baseline costs) across diagnoses and for six specific diagnoses .Data from 44,550 patients  were available for analysis. From the societal perspective, total costs after 18 months were higher in the homeopathy group  than in the control group  with the largest differences between groups for productivity loss  and outpatient care costs . Group differences decreased over time. For all diagnoses, costs were higher in the homeopathy group than in the control group, although this difference was not always statistically significant.Compared with usual care, additional homeopathic treatment was associated with significantly higher costs. These analyses did not confirm previously observed cost savings resulting from the use of homeopathy in the health care system. Homeopathy is frequently used by patients in the German healthcare system. A cross-sectional survey representative of the population indicated that 60% of all 1503 persons interviewed had used homeopathy . HomeopaA recent systematic review that summarized the available literature on cost-effectiveness provided no distinct conclusion . HomeopaThe objective of this study was to compare the health care costs of insured persons who used additional homeopathic treatment within an integrated care contract framework with the costs for patients who used only usual care through one of the largest statutory health insurance companies in Germany.www.tk.de)). For this analysis, patients belonged to the homeopathy group if they subscribed to the integrated care contract in 2011 and if they were continuously insured through the TK for the observational period (12 months before and 18 months after subscription to the integrated care contract), regardless of whether they used homeopathy during the study period. The start date of the observation period for the homeopathy group was the start date of the integrated care contract subscription, which indicated the commencement of homeopathic treatment. The control group was drawn from a sample of all TK-insured persons who did not subscribe to the integrated care contract, who were continuously insured through the TK for the observational period, who contacted a physician during the first three months of the observation period, and who were successfully matched with a homeopathy user based on propensity scores. The start date of the observation period for the controls was determined as the start date of the integrated care contract subscription of the homeopathy users . In addition to comparing health care costs across diagnoses between groups, data were also analysed for specific confirmed diagnoses: depression (ICD-10 F32), migraine (G43), allergic rhinitis (J30), allergic asthma (J45), atopic dermatitis (L20), and tension headache (R51). We followed the guidelines for secondary data analyses. Patient claims data were pseudonymised by the statutory health insurance. We had no key for de-identifying the pseudonymous data. The study was approved by the Ethics Committee of the Charité—Universitätsmedizin Berlin (EA2/121/12).Health care cost data were compared for patients who used additional homeopathic treatment within the integrated care model homeopathy (homeopathy group) and those who used only usual care (control group) based on claims data from one of the largest German statutory health insurance company  with cumulative costs of month -12 until month 0 as the covariate was performed to compare the cumulative total costs after 18 months between both groups. Total costs included outpatient care costs (outpatient costs by homeopathic physicians and outpatient costs by other physicians), medication costs, productivity loss, costs of the integrated care contract, inpatient costs and other costs. Both total costs and single cost types were summarized for the period of 12 months before the study onset (month -12 to month 0) and for the subsequent 18 months, divided into three-month intervals  and compared between groups and between diagnoses using ANCOVAs with the respective baseline cost values as a covariate. Cost progression for the groups was compared from both the societal and statutory health insurance perspectives over the observation period. Data on the number of days of sick leave and number of hospital stays were compared between the groups and diagnoses. The test for the primary end point was two-sided with a significance level of 0.05. All other tests were two-sided with a significance level of 0.05, but were exploratory. The number of prescriptions as well as the number of the most common diagnoses  were analysed post-hoc and exploratory after discussing the results of the pre-planned analyses.Matching was performed with SAS software version 9.3 . We employed a pre-specified statistical analysis plan and used R version 3.1.0  for the Out of all those insured in this statutory health insurance company, 4,117,726 fulfilled the inclusion criteria. Of those 4,117,726, a sample of ten times more controls than homeopathy patients were drawn at random for each matching process. The matching process was then done in a 1:1 ratio. Data for 44550 patients were included in the analyses, and propensity score matching resulted in 22275 matched pairs. The mean age was 34 years (SD 20.1) and 67.3% of the patients were female . The propensity score matching resulted in a comparable sample that showed no relevant differences in baseline characteristics or costs .The adjusted cumulative total costs 18 months after integrated care contract onset were higher in the homeopathy group than in the control group. The adjusted mean difference of EUR 1350.16 [95% CI 1143.59–1556.73] was statistically significant . The higher costs in the homeopathy group were primarily driven by productivity loss  and outpatient care costs . Outpatient care costs in the homeopathic group were higher than those in the control group for both components  . ApproxiRegarding disease-specific costs, patients with depression accounted for most of these costs. Among the group suffering from depression, the adjusted cumulative total costs were higher in the homeopathy group than in the control group  . For theFrom both the societal and statutory health insurance perspectives, the cost progression was similar in both groups from month -12 to month 0 . The greatest cost difference could be observed in months 1–3 . This substantial difference largely resulted from the costs for initial homeopathic consultations. Costs progressed similarly from both the societal and statutory health insurance perspectives. From both perspectives, the costs for the homeopathy group were higher than those for the control group for the entire observation period. However, by the end of the observation period (month 18), the gap between the two groups had decreased . The numOur analysis showed that adjusted total health care costs after 18 months across diagnoses were higher in the homeopathy group compared to the control group. The cost difference was primarily attributable to productivity loss and outpatient care costs. Group differences decreased over time.This study used claims data from a large statutory health insurance company for secondary data analyses, resulting in a sample size of 44550 patients, including 22275 subscribers to the integrated care contract homeopathy. We used cumulative different unit costs for 12 months prior to the integrated care contract as a covariate for the propensity score. The nature of secondary data used in our study does not allow to draw conclusions about the severity of the patients’ diseases. However, health care costs can serve as a proxy for a medical condition or for the severity of a disease . TherefoA limitation of our study is that it was restricted to costs; therefore, we were unable to evaluate the effectiveness of the integrated care contract. Approximately 40% of the patients were matched according to a confirmed diagnosis. However, the patients’ reasons, primary or otherwise, for visiting a physician during the first three months of the observation period were unknown. For example, two patients could have been matched for the diagnosis of asthma although one of the matches might have also experienced and sought treatment for a costly car accident. However, such a situation could have occurred in both groups; because the matching also involved costs, we assume that we balanced the burden of disease in both groups. Another limitation of our study is the relatively short observation period of 18 months. Given that the costs for both groups appeared to converge at the end of the observation period, a longer observation period would have been desirable. Homeopathic treatment could be associated with initial higher investments that decline over longer courses of treatment. However, the data available for our study did not allow for testing this assumption. Regarding the generalizability of our results, approximately two-thirds of our sample comprised female participants, reflecting the current literature on predictions for complementary medicine usage . FurtherA recent systematic review by Viksveen on the cost-effectiveness of homeopathy showed that in eight out of fourteen studies, the homeopathic treatment was less cost-intensive than the conventional treatment; in four studies, the treatment costs were similar; and in two studies, the homeopathic treatment was more costly than conventional treatment . HoweverThe Viksveen review reported that the most relevant cost driver in homeopathic treatment are consultation costs and that overall medication costs are often reduced for homeopathic treatment compared with conventional practices . By contThe analyses of our present study did not confirm the previously observed cost savings resulting from the use of homeopathy in the health care system. In an 18-month integrated care program that offered and reimbursed homeopathy in addition to usual care, patients who used additional homeopathic treatment had significantly higher costs compared with patients who received only usual care.S1 Table(PDF)Click here for additional data file.


N-terminal domains (NTDs) that are closely related to those of feline and porcine strains. The increase in disease severity in dogs and the emergence of novel CCoVs can be attributed to the high level of recombination within the spike gene that can occur during infection by more than one CCoV type in the same host.Canine enteric coronavirus (CCoV) is an alphacoronavirus infecting dogs that is closely related to enteric coronaviruses of cats and pigs. While CCoV has traditionally caused mild gastro-intestinal clinical signs, there are increasing reports of lethal CCoV infections in dogs, with evidence of both gastrointestinal and systemic viral dissemination. Consequently, CCoV is now considered to be an emerging infectious disease of dogs. In addition to the two known serotypes of CCoV, novel recombinant variants of CCoV have been found containing spike protein  Coronaviridae, order Nidovirales, and was first recognized as a pathogen of dogs following virus isolation of the prototype 1-71 virus in 1971 during an outbreak of gastroenteritis in military dogs [Canine enteric coronavirus (CCoV) is a common infection of dogs, particularly those housed in large groups such as kennels, shelters, and breeding facilities. CCoV belongs to the family ary dogs . Howeverary dogs . Based oN protein with the genomic RNA forms the helical nucleocapsid that is surrounded by an icosahedral structure composed of the viral M protein. The virus enters cells upon receptor binding and membrane fusion  [Coronaviruses are enveloped, single-stranded, positive-sense RNA viruses that infect humans and a wide variety of animal species ,4. Coronprotein) , and, liprotein) . Coronavprotein) . While 3protein) , the corprotein) .Alphacoronavirus-1) from a taxonomic perspective. However, there are two distinct serotypes of CCoV: type I and type II [Coronaviruses are phylogenetically divided into several genera, termed alpha, beta, and gamma . The exi type II ,16. CCoV type II —A situat type II . It has  type II . A summa type II —with Elm type II ,21.More extensive genetic characterization led to such variant viruses being termed “FCoV-like” CCoVs, based on their similarity to the commonly circulating FCoV strains (FCoV-I) . Like otIn vivo, CCoV-I viruses are thought to co-circulate extensively with CCoV-II viruses, often occurring as co-infections [CCoV-I isolates are not culturable in cell culture systems, which has severely hampered the study of these viruses. fections ,26,27,28fections  . The NTD is an important determinant of intestinal tropism in closely related TGEV [The first identified CCoV strain 1-71, is now categorized as a type II virus (CCoV-II). Further classification of CCoV-IIs into distinct subtypes  has been proposed, based on the sequence of the first 300 amino acids of the spike protein, a region known as the ted TGEV ,32. The ted TGEV ,34,35. Tted TGEV ,34,36,37ted TGEV ,39. Liketed TGEV ,38 but aN-terminal domain. These viruses have been reported in both the United States and Sweden [Based on the finding of a novel NTD in A76-like viruses, we propose the creation of a new subgroup, CCoV-IIc, which consists of CCoV type II viruses with a CCoV-I- or FCoV-I-like d Sweden ,30. Othed Sweden . As CCoVd Sweden , this seCCoV-II viruses (such as 1-71) typically grow readily in cell culture, with A-72 cells (canine tumor fibroblast cells derived from unknown tissue type) widely used for virus propagation. 1-71 also grows well in a variety of feline cell lines , but, interestingly, not in many other canine cell lines .N (APN) has been shown to act as a common receptor for many alphacoronaviruses, including FCoV and TGEV [in vivo receptor for all CCoV-IIs, as shown for 1-71 [C-domain”, has resulted in the ability to use cAPN but not fAPN as a receptor. The relative lack of glycosylation on fAPN compared to cAPN may account for these differences [The coronavirus spike protein is a major antigenic determinant and is also responsible for host cell receptor binding and viral entry . Aminopeand TGEV . Althougand TGEV . This prand TGEV ,44. Presfor 1-71 . In the ferences .The receptor determinants for the serotype I FCoV/CCoV group are much less certain. While there is some evidence for fAPN as an FCoV type I receptor , other sIn addition to a specific proteinaceous receptor, there are indications that lectin-based interactions via sugar moieties, on either the virus or the host, may play a role in the receptor-binding complex for FCoV types I and II and TGEV ,50,51,52The coronavirus spike protein is activated by proteolytic cleavage at one or two sites (defined as S1/S2 and S2’) . In otheCCoV is transmitted by the fecal-oral route; however, it is unclear whether transmission by other routes including aerosols can occur. After ingestion, CCoV typically infects and replicates within the cytoplasm of mature epithelial cells along the sides and the tip of intestinal villi, while sparing the epithelium that lines the intestinal crypts . InfecteCCoV is generally thought of as a mild, but highly contagious, enteritis of young dogs, most often under 12 weeks of age ,61. In sIn recent years, an increasing number of reports of infections by highly virulent CCoVs have also been documented in puppies without apparent coinfections ,67,68,69It is clear that coronaviruses have high potential for emergence of novel variants with altered tropism and pathogenesis. In large part this can be explained by several factors: a combination of recombination and mutation within the viral genome and that many coronaviruses exist as quasi-species, the availability of natural reservoirs for the virus and the “modular” nature of the viral spike protein . Recent Definitive diagnosis of CCoV-induced disease is currently difficult; thus, it is not commonly done or available. The virus can be identified by visualization of viral particles in stool specimens following negative staining and examination by transmission electron microscopy; however, this is not a routinely available diagnostic tool. Virus isolation can be achieved for CCoV-II, but not for CCoV-I viruses, and again, this is not commonly available. The definitive test is post-mortem identification of viral antigen by immunofluorescence or immunohistochemical staining of tissue sections see . The mosA variety of inactivated and modified-live virus vaccines are commercially available and designed to prevent infection with CCoV . Current


Objective: We outline the cost of installing GeneXpert in district hospitals in Abuja, Nigeria.Since the endorsement of GeneXpert MTB/RIF by the WHO, many countries have embarked on implementing this technology. We prospectively documented costs related to the installation of GeneXpert at five sites. Costs were collected from receipts received from suppliers and normalized to USD 2012 values.Costs were often identified after initiating installation for many reasons. Installation varied widely between sites with sufficient space and power supply; sites with insufficient space or power supply and costs not directly associated with site installation. The basic cost for installation was USD 2,621.98 per machine. Sites that required additional space cost close to USD 7,000.00.Space and power requirements have a significant effect on installation costs. Countries need to carefully consider the placement of Xpert machines based on the quality and size of the available infrastructure. Since the World Health Organization WHO endorsement of Xpert MTB/RIF in December 2010 and its subsequent recommendation to health authorities to roll-out the technology in phases within the context of national plans, many high Tuberculosis (TB) burden countries have embarked on implementing the technology to improve the management of TB [In addition to the capital equipment expenditure and recurrent costs for the cartridges and calibrating machines , there aWHO in its 2012 global TB report ranked Nigeria as the country with the 10th highest burden of TB with characteristics of a low resource setting . The FedFive four-module Xpert instruments were received by the FCT-TBLCP in January 2012 and installed from April to July 2012 in district hospitals of the Local Area Councils (LAC) of the FCT. The equipment was part of a TB-REACH-funded project  to test a new approach to increase TB case detection amongst adults in the FCT and the hospital selected had to be located outside Abuja's metropolitan area. The equipment was purchased at the Foundation for Innovative New Diagnostic (FIND) price of 17,500 USD per instrument, computer and freight on board. The project developed a network of community health extension workers who conducted house-to-house visits to identify adults with symptoms of TB and motorbike riders who linked the teams to the laboratory facilities by transporting specimens. Patients were tested using smear-microscopy and smear-negative patients were further tested with Xpert using an interim diagnostic algorithm. The management of the patients was conducted by the programme using its treatment algorithms .During its planning phase, the project assessed the LAC hospital laboratories that routinely conducted smear microscopy to select those considered to have suitable facilities for the introduction of Xpert. Assessments were conducted by a team of laboratory staff, doctors and programme managers working with the Federal Ministry of Health, the FCT TBLCP and Zankli Medical Centre, a private TB research laboratory which supports the NTBLCP and acts as a reference diagnostic centre for the programme. The assessments documented the availability of space to accommodate the instrument and accessories, including a portable computer and bar reading stand, bench space, a backup system for electricity and the security of the premises. Laboratories also had to have at least two laboratory staff were willing to participate who had basic computer literacy for data entry, processing and printing of results.The costs incurred from the installation were prospectively documented. Costs were documented from receipts received from registered suppliers and normalized to United States Dollar (USD) 2012 values by using the mean exchange rate across the year (USD 1 = 156 Nigerian Naira). Costs were stratified in three categories: - Cost incurred to install the machines for laboratories with sufficient space and adequate electricity supply. These costs were considered the basic expense required for installation and were the same for all five laboratories. - Costs not directly associated with site infrastructure, including custom clearance and training. - Costs incurred into sites subsequently considered having inadequate electricity supply or insufficient space to accommodate additional equipment needs identified during the installation. Recurrent costs for running and maintaining the instruments are not included .Ethics: This study was approved by the Health Research Ethics Committee of Federal Capital Territory, Abuja-Nigeria and Liverpool School of Tropical Medicine United Kingdom.A wide range of costs were incurred during the installation of the instruments. Some of these problems were not foreseen before initiating the project and costs often snowballed due to variety of reasons. For example, despite the survey, it was soon recognized that the electricity backup system provided by the hospitals still allowed short electricity interruptions before a generator re-instated the supply. In turn, the extra equipment needed to secure uninterrupted electrical supply required additional laboratory space.The main basic costs incurred to install the machines in the laboratories are described in In two laboratories we purchased generators because the hospitals’ own generators were broken. The electrical equipment  was bulky and required more space than anticipated and was not possible to accommodate them within the laboratory premises. It became evident that the added equipment overwhelmed the laboratory and could not be accommodated into the existing premises.We therefore purchased and furnished cargo containers to provide additional bench, storage and office space. Refurbishment included air conditioners, as temperatures are usually >30°C. The costs for the sites requiring additional premises are shown in The WHO technical and operational guidance on Xpert  providesFinding the space required for the machines was a common problem, with the FCT health and human services having no space in two sites to build or expand the existing laboratories and competing priorities from other projects and programmes. Consequently, with the need to implement the project as quickly as possible, the procurement of cargo containers was the only option available to us. This added, on average, an additional USD 6,792.31 per site. Studies looking at the cost effectiveness of Xpert in India, South Africa and Uganda reported variations in the infrastructure cost . AlthougOur findings in Abuja demonstrate that countries need to carefully consider the placement of Xpert machines based on the quality and size of the laboratory infrastructure to ensure that additional placement costs are controlled. This echoes the concerns raised at the 2012 fourth Global Laboratory Initiative partners meeting, which observed that, although users were pleased with Xpert MTB/ RIF, there were concerns about the price of the technology; especially in terms of accelerated and sustained roll-out in both low- and middle-income countries.


Tomato mosaic virus was reported from jasmine in Florida. We present the first complete genome sequence of a tomato mosaic virus isolate from this woody perennial plant in the United States. Tobamovirus in the family Virgaviridae is composed of viruses with rigid rod-shaped virions that are easily transmitted by mechanical inoculation and plant-to-plant contact (Tobamovirus (http://www.ictvonline.org/virusTaxonomy.asp), which are distributed worldwide and include tobacco mosaic virus, tomato mottle mosaic virus, and hibiscus latent Fort Pierce virus. These tobamoviruses commonly infect herbaceous annual plant species, including tomato and pepper, and woody perennial plant species, including hibiscus (–Jasminum multiflorum) in Florida since 1999  from symptomatic jasmine was used initially to mechanically inoculate protocol . ToMV geprotocol , 8 were Comparative analysis showed that the Florida ToMV isolate sequence  was 99% identical to the reference ToMV sequence with 69 single-nucleotide differences. Of the 49 nucleotide differences within the predicted four tobamovirus open reading frames , 46 were silent, whereas 3 led to amino acid changes.KR537870.The complete genome sequence of the jasmine ToMV isolate was deposited in GenBank under the accession no.


A Task Force established by the Brazil Ministry of Health investigated the possible association of microcephaly with Zika virus infection during pregnancy and a registry for microcephaly cases among women suspected to have had Zika virus infection during pregnancy. A Task Force established by the Brazil Ministry of Health investigated the possible association of microcephaly with Zika virus infection during pregnancy and a registry for microcephaly cases among women suspected to have had Zika virus infection during pregnancy. In early 2015, an outbreak of Zika virus, a flavivirus transmitted by Aedes mosquitoes, was identified in northeastern Brazil. By September 2015 an increase in the number of infants born with microcephaly began to emerge, and Zika virus RNA was identified in the amniotic fluid of 2 women with fetuses having microcephaly on ultrasound. Among a cohort of 35 infants with microcephaly born during August-October 2015 in eight of Brazil’s 26 states and reported to the registry, the mothers of all 35 had lived in or visited Zika virus-affected areas during pregnancy, 25 (71%) infants had severe microcephaly , 17 (49%) had at least one neurologic abnormality , and among 27 infants who had neuroimaging studies, all had abnormalities . Tests for other congenital infections were negative. CSF tests for Zika virus infection are not yet available, and further studies are needed to confirm the association of microcephaly with Zika virus infection during pregnancy. [COMMENTARY. Microcephaly is defined as a head circumference -/= SD below the mean for sex and gestational age at birth . Except


Hyalomma marginatum. The geographical distribution of H. marginatum covers most of the Western Mediterranean basin. We aimed to investigate whether CCHF virus (CCHFv) is circulating in Tunisia. Samples from unexplained acute febrile patients (n = 181) and a high risk group of humans, mainly slaughter workers (n = 38), were collected in the summer of 2014 and analyzed for exposure to CCHFv using serological tests and real-time RT-PCR. Ticks were collected from Northern and Southern Tunisia during May–June 2014 and examined for the presence of CCHFv by real-time RT-PCR. Of the 181 febrile patients, 5 showed only high titers of IgM suggesting a recent exposure to CCHFv. Among 38 slaughter workers, 2 had IgG anti-CCHFv responses yielding a seroprevalence of 5.2%. No CCHFv was detected in ticks and sera. Our results provide evidence of human exposure to CCHFv in Tunisia.Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne disease associated with a high case fatality rate and transmitted mainly by  Nairovirus) is the etiologic agent of Crimean-Congo hemorrhagic fever (CCHF), a severe acute febrile illness characterized by fever and hemorrhage with case fatality rates of 9–50% [Crimean-Congo hemorrhagic fever virus (CCHFv) . Samples were also actively taken from abattoir workers (n = 38) in order to survey evidence of previous exposure among a group designated as high risk. Most of the participants originated from the governorates of Sousse, Sfax, and Mahdia . The study was conducted under ethical approval from the Ethics Committee HHS-IRB 00008931, University Hospital Farhat Hached, protocol reference date 08-04-2013.Ticks and human sera were examined for the presence of CCHFv by real-time RT-PCR . CollectBrucella, Rickettsia, and West Nile virus (WNV). All samples in this study were shown to be negative during the testing phase and were then analyzed for CCHF by serological testing. Of a total of 181 sera from febrile patients, 5 (2.7%) showed high titers of IgM anti-CCHFv activity with an absence of IgG  ; Greece Hyalomma scupense is the main vector of Theileria annulata, etiologic agent of theileriosis in North Africa [H. scupense completes its life cycle within the protection of walls and crevices of barns without any other host beside livestock, and subsequently, this tick species has not been implicated in the transmission of CCHFv [Hyalomma dromedarii were collected only from the vegetation and from resting sites of oryx and not from livestock , known to be the main amplifying hosts for CCHFv [Hyalomma marginatum, a main vector of CCHFv, is located mainly in the Northwestern part of Tunisia [H. marginatum are needed.In our study, no CCHFv was detected in the analyzed ticks. h Africa . H. scupof CCHFv . Hyalommor CCHFv . Hyalomm Tunisia , an areaH. marginatum in North Africa. To date, no clinical cases of CCHF (fever followed by hemorrhagic manifestations) have been observed in North Africa. It is of major epidemiological importance to point out that the detection of CCHFv in H. marginatum collected from birds in Morocco and in H. aegyptium collected from tortoises in Algeria provides strong evidence that the virus is transmitted in North Africa mainly by ticks belonging to the genus Hyalomma [H. aegyptium collected from tortoises in Algeria [H. marginatum covers significantly larger areas including Spain, Southern Italy, and North Africa. The absence of human clinical cases in Southern Europe could be explained by the lack of movements of livestock coupled with the apparent lack of movements of wild hosts for ticks between the Western Mediterranean basin and the Balkans, leading to a lack of connectivity between eastern and western populations of the vector and virus [Our results suggest circulation of CCHFv in Tunisia. The seroprevalence observed among the high risk population was not unexpected mainly because of the circulation of CCHFv among populations of Hyalomma , 24. The Algeria  could ex Algeria . This hy Algeria . The geond virus . Howevernd virus . During nd virus . Thus, pnd virus .In conclusion, our results provide strong evidence of circulation of CCHFv in Tunisia for the first time. Thus, further studies are recommended to be carried out on livestock, high risk groups of humans, birds, and ticks for a better understanding of the dynamic transmission of CCHFv in Tunisia and surrounding countries in North Africa.The authors declare no conflict of interest in relation with this paper.


Extracorporeal life support (ECLS) seems an efficient therapy for acute, potentially reversible cardiac or respiratory failure, when conventional therapy has been inadequate, or as bridge to transplant, but there is no evidence for cost-effectiveness. Our aim was to conduct an exploration of the hospital costs of ECLS therapy.Single-center retrospective exploratory cohort cost study. The study is performed from a hospital perspective with a time horizon of patients' complete hospital admission in which they received ECLS.All 67 consecutive adult patients who were admitted to the ICU of the University Medical Center Groningen (the Netherlands) in the period 2010 to 2013 and received ECLS treatment were included. The bottom-up micro costing method was used. Medical costs were estimated by multiplying every registered healthcare consumption with unit prices. Unit prices were largely based on Dutch reference prices. For each patient the personnel costs and material costs were assessed in detail. The costs of ECLS were differentiated in costs of procedures and costs of daily surcharge of therapy. Procedure related costs were subdivided in costs of devices and disposables, costs of additional human resources and surgery hours.In order to estimate costs of ECLS therapy based on indication all patients were categorized into six different subgroups: respiratory - bridge to recovery, respiratory - bridge to transplant, cardiac - bridge to recovery, cardiac - bridge to transplant, cardiac - post-cardiotomy, and ECPR.The mean age was 46 years, female sex 66%, APACHE II score 24, ECLS duration 135 ± 163 hours, ICU length of stay 18 ± 22, hospital length of stay 38 ± 45, and in hospital mortality 68%. The mean total hospital costs were €106.263 (€83.841 - €126.266) per patient. On average, 52% of the total costs arose from hospital nursing days and 11% of direct procedure related ECLS costs. Surgery and diagnostics represented a vast amount of the remaining costs.This large and detailed economic evaluation of hospital costs of ECLS therapy in the Netherlands showed that mean total hospital cost of ECLS treatment are €106.263 per patient. The majority of the costs are composed of hospital nursing days.