User fees, transportation costs, and time costs impair access to healthcare by rural communities in low and middle income countries. However, effects of time costs on demand for healthcare are less understood than effects of user fees for health providers. In addition, prospective patients might not know about all health services available. This study aims to investigate how the family caregivers of febrile children respond to the pecuniary costs and time costs in their choice of health providers in rural Papua New Guinea.Using an original questionnaire, we surveyed households in the catchment area surrounding Dagua Health Center in East Sepik Province, Papua New Guinea, during February–March 2015. We estimated the probability of choosing one among four categories of providers  via a mixed logit model in which we restrict alternatives to those for which family caregivers knew cost information.Of 1173 family caregivers, 96% sought treatment for febrile children from four categories of providers. Almost all knew the location of the health center and a health volunteer, but only 50% knew the location of aid posts. Analysis by discrete choice model showed that pecuniary costs and time costs were inversely associated with the probability of choosing any type of provider. We then changed pecuniary costs and time costs counterfactually to calculate and compare the probability of choosing each provider. Time costs affected the choice more than pecuniary costs, and individual heterogeneity appeared among caregivers with respect to pecuniary costs. When pecuniary or time costs of VHVs are altered, substitution between VHVs and home-treatment appeared.Our findings suggest that policies to increase awareness of aid posts and reduce time costs in addition to treatment fees for each category of healthcare provider could help developing economies to improve access to essential healthcare services. Access to essential healthcare services is an important public health issue in low and middle income countries (LMIC). Though health policy causing change of supply curve  has been promoted, the importance of intervention overcoming demand side barriers was also emphasized to improve healthcare service utilization . Among dOn the other hand, Acton  pointed The population living in rural parts of LMIC countries, however, confront long distances, undependable transportation, and steep travel costs to access healthcare. Numerous studies examine how distance traveled affects choice of healthcare providers there –16. In rIn Papua New Guinea (PNG), as the mortality rate for children under 5 years old is the third worst among 23 developing East Asia and Pacific Region economies , policieThis study examines how the family caregivers of febrile children respond to the pecuniary costs and time costs in their choice of health providers. We used discrete choice models to estimate the probability of an alternative being chosen. We calculated own- and cross-cost responsiveness to predicted choice probability for each provider by counterfactual analysis and examined substitutability between alternatives. We also performed a sub-group analysis for severity of minor patients’ symptoms.The study covered 23 villages in the catchment area surrounding the Dagua Health Center, located 56 km west of Wewak, the capital of East Sepik Province of PNG. In the lowland swamp along the main coastal road malaria is endemic year-round. The Dagua Health Center is operated 24 h a day by the Catholic Church. Eleven health professionals provide medical/public health services, including ambulatory care, hospitalization for general internal medicine , and outreach services (immunization and emergency care). It treated an average of 20.1 outpatients per day in 2016, a figure not considered to constitute excessive demand .With only one health professional assigned, each aid post provides general outpatient care and normal delivery services. Its staff is nationally certified by completing a two-year program at a medical college or a university and is allowed to perform the same clinical works as nurses. Our surveyed population had access to four such aid posts. Although records on the number of their patients were unavailable, we observed that two or fewer patients awaited treatment at any given time, suggesting the demand for their services was not excessive. While the traditional practitioners of herbal or spiritual remedies were commonplace, the area had only one distributor of Western pharmaceuticals. The provincial capital city of Wewak, has only one general hospital and two public clinics. The hospital provides general medicine, surgery, pediatric, obstetric, psychiatric, and physical therapy in emergency outpatient and inpatient departments besides overseeing provincial public health. Staffed by five or so professionals, public clinics provide general outpatient and normal delivery services.Outpatient fees at these formal healthcare facilities are capitation payments, covering consultation, clinical exams, laboratory tests, medications, and follow-up visits. The fees vary from one facility to another, but the costs are less than the nation’s minimum hourly wage. Public transportation, in the form of buses or public motor vehicles (PMVs), are available to villagers beyond walking distance. PMVs operate daily, except on Sundays, from villages to Wewak, once in the morning, and from Wewak to villages, once in the afternoon. The government regulates transportation charges on the basis of distance traveled. Neither public nor community-based health insurance had been introduced in the area studied.To improve access to basic healthcare, lay workers called “village health volunteers” (VHVs) have served in area studied since 2007 . The VHVDuring February–March 2015 using an original questionnaire, we conducted a cross-sectional survey of households in which parents or parent surrogates were taking care of children under age 15. Trained field interviewers collected data about episodes of fever among their children and the respondent’s choices of health providers during the 2 weeks preceding the interview. The lead researcher double-checked questionnaires completed by field interviewers. Missing and erroneous values were corrected by revisiting respondents.Questions asked about instances of fever, home-treatment at its onset, initial choices of health provider, the second, and the third or subsequent choices of health provider. The survey also asked the caregivers to give information on all health providers where they may take their children when they are sick: namely,  locationWe define the pecuniary costs as “caregiver’s out-of-pocket payments for a health provider plus round-trip transportation fees to get there”. We use survey respondents’ self-reported estimate of pecuniary costs for each healthcare provider respondents had named, including the ones they had not chosen. As we described above, out-of-pocket payment for a health provider was equal to a fixed sum covering consultation, clinical exams, laboratory tests, treatments, drugs, and follow-up visits for VHVs, the aid posts, or the health center.Time cost is defined as “the caregiver’s opportunity cost for his/her self-reported time spent in seeking and obtaining healthcare away from home or work”. To calculate time costs, first we calculate time required for care at each health provider for each caregiver. When caregivers would travel to a health provider only on foot, total time required for its care was calculated as round-trip walking time plus its wait/treatment time. When caregivers would use public transportation to a health provider, total time required for its care was calculated as time difference between clock time of returning home and clock time of leaving home. We then calculate the individual time costs by multiplying the time required for care at each health provider by the caregiver’s wage rate.To check the self-reported travel time, we calculated individual travel distances for each provider. We first recorded the locations of each house and each health provider with global positioning system (GPS) devices . We then measured individual distances, using a digital map of the area  and Quantum GIS 2.14, as [We calculated the probability for a caregiver to choose a particular type of healthcare provider using a discrete choice model in which the individual tries to maximize utility by their choice. However, our respondents were not necessarily aware of all the providers available in the area. Providers they were unaware of could not have been placed in the set of eligible alternatives. Also, conceivably, respondents would not choose providers they believe offer low utility even if they were aware of them. For these reasons, we postulated that to be an eligible alternative,  a healthThe model’s explained variable was “a healthcare provider chosen for a febrile child in the two weeks before the interview date.” We defined home-treatment as all the treatment given within a household at any time during one fever episode without incurring any pecuniary costs. Home-treatment, for instance, included monitoring the sick child without treatment in a family. Following the previous studies, time costs of home-treatment were normalized to zero , 8.When a caregiver did not have cost information of the health center, any aid post, or any VHV, we excluded those alternatives from his/her choice set. We assumed all caregivers could have chosen home-treatment. Therefore, the number of alternatives in a choice set varied for each caregiver between two to four, and its average was 3.4. A small number of respondents visited multiple healthcare providers for the same episode. In such cases, we set the first provider visited as the explained variable.j by respondent i is given asxij is a vector of alternative-specific variables. zi is a vector of respondent-specific variables. εij is the error term, which mixed logit models assume follows an extreme value distribution. In Eq.  takes a value of 1 if a respondent knows cost-related information of alternative j and 0 otherwise. Probability of choice is the integral of the logit probability over density function vi. Assuming f(v) is a probability density function of vi with a normal distribution, the probability of choosing alternative j selected by respondent i is described asIf alternative-specific variables  alone have a random component, utility for the choice of alternative asmixlogit. Simulation methods with 500 Halton draws approximate the maximum log likelihood.We used Stata15  and the command Individual-specific variables were the minor child’s gender, age, perceived severity of illness, caregiver’s education, presence of Western drugs in the household, household size (number of persons), and an index of household assets. Assets were selected to proxy long-term wealth by constructing a linear index of asset ownership and housing characteristics using principle component analysis . Seven dOur results reveal that almost all (98%) of caregivers had the information on the location of Dagua Health Center and VHVs, while 65% of them on the locations of public clinics in Wewak, 56% of them on traditional health practitioners, 54% of them on aid posts, and 12% of them on the dispenser of Western pharmaceuticals , but self-reported time varied widely among respondents with identical travel distances, reflecting differences in respondents’ life-styles and faulty recollections. Large measurement errors in variables create unreliable coefficients in empirical models, and the effect of travel time on choice of treatment provider can be underestimated .Therefore, we used ordinary least squares (OLS) regression to adjust self-reported round-trip time on walking distance, transportation distance, and wait/examination time as explanatory variables Table . The coeAlmost all caregivers (99%) were females . Among females, almost all (> 99%) were non-wage workers and spent time for domestic work and farming. Therefore, difference of wage rate seemed little among the caregivers. As we did not get the information on wage rate of domestic work or agricultural work in PNG, we adopted formal minimum wage rate for estimating wage rate of study caregivers. Using these adjusted time spent for taking their children to the healthcare providers, we calculated time costs of the visits by multiplying them by the hourly minimum wage in Papua New Guinea Kina  .Table As travel time using public transit is the longest to the health center for most caregivers, the average time costs are the highest for the health center, followed by the aid posts and then by the VHVs.Also wait/treatment time was the longest at the health center. At the center, practitioners work 24 h in shifts and absenteeism is not a problem; lengthy waits were observed in the morning with the simultaneous arrival of new patients by the public transportation services. For the aid posts or for VHVs, however, our observation suggested that it was not the concentration of patients that caused lengthy waits. Within the survey area, a single health worker was assigned as a VHV or to each aid post, and many worked in their homes as farmers. Lengthy waits occurred when they had not reported for work, primarily because they were farming in their distant fields, and caregivers had to wait their return.Time costs evaluated at minimum wage exceed pecuniary costs for all providers because public transportation and treatment fees at formal facilities are relatively inexpensive, but it takes substantial time to reach them in rural PNG.Table Results show that 89% of households own mobile phones, 46% have access to safe drinking water, 41% own radios, 14% have metal or concrete roofs, 13% have generators, 6.4% have brick or metal walls, and 4.0% own a vehicle or outboard motorboat. The median and mode for the number of assets is 2, the average is 2.1, and 88% of households own three or fewer of the assets surveyed.We employed a mixed logit model with home-treatment as the base alternative to estimate the choice probability among 439 observations . We estimated four models; in Model 1, alternative-specific explanatory variables consist of only the pecuniary costs, in Model 2, they consist of only the time costs, in Model 3, they consist of the pecuniary costs and time costs, and in Model 4, they consist of only the total costs, or the sums of the pecuniary costs and the time costs. The coefficient of any cost in any model is significantly negative Table . The ran2 statistics of the four models reveals Model 3 is the best specification of the four. Consequently, we have adopted Model 3 as the basis of our counterfactual analysis regarding the pecuniary costs of a health provider without changing values of the other explanatory variables. The mean choice probability for each type of provider, given a hypothetical change in its pecuniary costs, is its average predicted choice probability. The mean choice probability curve for the alternative is downwardly convex and barely changes when the costs exceed PGK 10  and time costs as explanatory variables, and we estimated our model using stated cost information, resulting in that a treatment choice set with individual heterogeneity . Compared to reducing pecuniary costs, reducing time costs significantly affected the probability that caregivers would choose treatment by VHVs, an aid post, or a health center. We also observed individual heterogeneity in effects of pecuniary costs on choice of healthcare provider. Although IMIC have restricted health and financial resources, they need to develop policies to reduce time costs and pecuniary costs to improve access to essential healthcare.


Banana streak virus (BSV) belongs to the members of the genus Badnavirus, family Caulimoviridae. At present, BSV contains nine species in the International Committee on Taxonomy of Viruses (ICTV) classification report (2018b release). Previous study indicated that the viral particles of Banana streak virus Acuminata Yunnan (BSV-Acum) were purified from banana (Cavendish Musa AAA group) leaves in Yunnan Province, China, and its complete genome was obtained. To further determine whether this sample infecting with Banana streak GF virus (BSGFV), the polymerase chain reaction (PCR) cloning and complete genome analysis of the Banana streak GF virus Yunnan isolate (BSGFV-YN) isolate were carried out in this study. The result showed that BSGFV-YN infecting Cavendish Musa AAA group was co-infecting this sample. Its genome contains a total of 7,325 bp in length with 42% GC content. This complete genome sequence was deposited in GenBank under accession number MN296502. Sequence analysis showed that the complete genome of BSGFV-YN was 98.14% sequence similarity to BSGFV Goldfinger, while it was 49.10–57.09% to other BSV species. Two phylogenetic trees based on the complete genome and ORFIII polyprotein indicated that BSGFV-YN and other BSV species clustered into a group, while it was the highest homology with BSGFV Goldfinger. Although BSGFV-YN and BSGFV Goldfinger were highly homologous, their cultivating bananas are different. The former cultivating banana was from Cavendish Musa AAA group, while the latter cultivating banana was from Goldfinger Musa AAAB group. Compared with BSGFV Goldfinger, the genome of BSGFV-YN has an extra multiple repetitive sequences in the intergenetic region between ORFIII and ORFI, suggesting that this region might be related to host selection. In summary, a BSGFV-YN distant from BSV-Acum was identified from the same sample, and its complete genome sequence was determined and analyzed. The study extends the polymorphism of BSVs in China and provides scientific clue for the evolutionary relationship with host selection of badnaviruses. Musa spp.), a perennial monocotyledonous herb, is the fourth largest food crops and the third largest tropical fruit in the world. Bananas are susceptible to variety of viruses (Banana streak virus (BSV) (Banana bunchy top virus (BBTV) (Cucumber mosaic virus (CMV)  , Banana s (BBTV)  and Cucuus (CMV) . Banana oviridae . BSV is oviridae . Moreoveoviridae . BSV is oviridae . BSV posoviridae .ORFI and ORFII encode proteins of unkown function. ORFIII, the largest ORF, encodes a ∼200 kDa polyprotein which is hydrolyzed by protease to produce a variety of functional proteins related to the viral life cycle, containing movement protein (MP), coat protein (CP), aspartic protease (AP), reverse transcriptase (RT) and ribonuclease H (RNaseH) , the nucleotide sequence similarity less than 80% or the amino acid sequence similarity less than 89% is considered as a new species (Banana streak GF virus (BSGFV), Banana streak IM virus (BSIMV), Banana streak MY virus (BSMYV), Banana streak OL virus (BSOLV), Banana streak UA virus (BSUAV), Banana streak UI virus (BSUIV), Banana streak UL virus (BSULV), Banana streak UM virus (BSUMV) and Banana streak VN virus (BSVNV) are identified by ICTV. In addition, three other BSV species of Banana streak CA virus (BSCAV) (Banana streak virus-isolate GD (BSV-GD) (Banana streak virus Acuminata Yunnan (BSV-Acum)  in the positive strand . ORFI an(RNaseH) . Bioinfo species . At pres (BSCAV) , Banana (BSV-GD)  and BanaSV-Acum)  have notBanana streak virus (BSV) in China are under investigation. Isolation and sequencing of the BSGFV and other new BSVs are greatly abundant the diversity of the badnavirus in China and provides an important data for disease resistance breeding. Based on the characteristics of circle double-stranded DNA molecule of the BSV genome, the complete genome sequence of BSGFV-YN was obtained by segmental PCR amplification, and the genomic structure and evolutionary relationship were further analyzed. The study will extend the polymorphism of BSV in China and provide scientific clue for the evolutionary relationship with host selection of badnaviruses.The diversities of complete genome sequences of more than nine BSV species indicated that the virus is highly variable and polymorphic . FurtherMusa AAA group. Total DNA was extracted and stored at −80 °C. Previous study showed that the sample was infected with Banana streak virus Acuminata Yunnan (BSV-Acum), and its complete genome was obtained and analyzed in our laboratory (Banana leaves showing streak symptoms were collected by permissions of a farmer (Shao-cheng Shen) from Yunnan, China in 2009. The banana belongs to the Cavendish boratory . In ordeNC_007002.1), seven specific pairs primers were designed by Primer Premier 5.0 , 2.5 mM dNTPs 4 µL, 10 ×EasyTaq buffer 5 µL, 5 µM forward and reverse primers 4 µL, total DNA template 2 µL, and added ddH2O to 50 µL. PCR reaction condition was pre-denaturation at 94 °C for 3 min; followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 90 s; and a final extension step at 72 °C for 10 min. The 6 µL PCR product was analyzed by electrophoresis in 1.5% agarose gel, and the remaining PCR product was purified by an OMEGA gel recovery kit . The purified PCR product of each DNA fragment was cloned into the pMD18-T vector , and then transformed into E. coli DH5a competent cells . Three positive clones were randomly selected for Sanger sequencing at Invitrogen .According to the complete genome sequence of BSGFV deposited in NCBI (GenBank accession number mier 5.0 . The prihttp://blast.ncbi.nlm.nih.gov/Blast.cgi), and the possible species or strain of BSV was preliminarily identified. Based on the classification of ICTV (2018b release), 37 badnaviruses were downloaded from GenBank for further comparison and analysis (https://www.ncbi.nlm.nih.gov/orffinder/) and bioinformatic analysis. Subsequently, the sequence similarities of complete genomes, ORFI∼III and their putative proteins between BSGFV-YN with other 15 badnaviruses were analyzed by Sequence Identity Matrix Program in BioEdit software. The movement protein (MP), coat protein (CP), aspartic protease (AP), reverse transcriptase (RT), ribonuclease H (RNaseH) and two cysteine-rich domains in BSGFV-YN ORFIII were predicted according to the report by ORFIII and ORFI of BSGFV-YN and BSGFV goldfinger (NC_007002.1) were also analyzed by GeneDoc software.The seven overlapping fragments were edited by ChromasPro software  and were used to assemble into the complete genome of BSGFV-YN by BioEdit software . The genanalysis . The opeIn order to further determine the homology and evolutionary relationships of BSGFV-YN with other badnaviruses, two phylogenetic trees based on the complete genomes and ORFIII polyproteins of BSGFV-YN and other 37 badnaviruses were constructed by using MEGA6.0 software . The evoMN296502.To obtain the complete genome sequence of BSGFV-YN, 7 specific pair primers were used for segmental PCR amplification . The PCRORFI) was found at 484–1,011 bp while the second ORF (ORFII) was located at 1,008–1,346 bp, both of which were predicted to encode proteins with unknown function. However, the third ORF (ORFIII) located at 1,343-6,841 bp encoded a polyprotein with the largest in size (ORFI has a 4-base overlap (ATGA) with the start codon region of ORFII; similarly, the termination codon region of ORFII and the start codon region of ORFIII are also overlap of a 4-base, with ATGA as well.Sequence analysis showed that the characteristic of BSGFV-YN complete genome was similar to the typical badnavirus. In detail, the BSGFV-YN contains three open reading frames (ORFs) on the positive strand of viral genome. The first ORF ( in size . FurtherCanna yellow mottle associated virus (CaYMV), Kalanchoe top-spotting virus (KTSV), Pineapple bacilliform CO virus (PBCOV), or Sugarcane bacilliform MO virus (SBMOV) was 53.58%, 52.92%, 51.49% and 49.16%, respectively. Further, the BSGFV-YN ORFs (I∼III) were 99.43%, 99.11% and 98.91% nt sequence similarities with those of BSGFV Goldfinger, while they shared 99.62%, 98.82% and 99.02% at the amino acid levels. The sequence similarity between BSGFV-YN ORFs (I∼III) and other badnaviruses ORFs (I∼III) were even more lower , of tRNAmet. This binding site is a common feature among all badnaviruses. The first nucleotide “T” is a highly conserved base required for replication and is set as the starting base of the sequence. The non-coding region also contains a TATA box domain (TATATA), which is located at 7,162–7,168 bp. In addition, the non-coding region has a 63 bp sequence that longer than BSGFV Goldfinger, and two kinds of repeating sequences were found in the 63 bp and adjacent region. A short repetition “Repeat S” and relative long repetition “Repeat L” have been found  group was clustered into Group I, which is different from the result of phylogenetic tree based on the complete genome. This phylogenetic tree also showed that BSGFV-YN had the highest homology with BSGFV Goldfinger , but the BSGFV-YN was cloned from another banana line (Musa AAA), indicating the BSGFV can infect different banana varieties.As shown in Badnavirus of the family Caulimoviridae, including nine species of BSV, suggesting the high diversity and polymorphism of BSV species. The badnaviruses, one of the important plant viral pathogens, endanger global agriculture. They are widely infecting the economic crops, such as bananas, sugarcane, citrus, cocoa, taro, black pepper, yam and other tropical and subtropical crops resulting in severe diseases  was obtained by seven segmental PCR cloning strategies. Previous study has been showed that the sample was infected with Banana streak virus Acuminata Yunnan (BSV-Acum), and its complete genome was obtained from the purified viral particle  was 441 bp longer than BSOLV-IN1/IN2 between the regions of the ORFIII and ORFI, but their host derived from the different banana varieties , and then reverse transcribed to viral DNA by RT to complete viral DNA replication. Therefore, the RT enzyme is conserved in all badnaviruses, which is consistent with its protein function . AP, as Banana streak GF virus (BSGFV), was cloned from the banana sample of Cavendish Musa AAA group that has been reported as being infected with BSV-Acum. Bioinformatics analysis showed that BSGFV-YN is highly homologous to the BSGFV Goldfinger isolated from Musa AAAB group, indicating that BSGFV can infect different banana varieties. BSGFV-YN may be a novel isolate of BSGFV in the new host, which provides an important clue for revealing the polymorphism of BSV genome.In summary, BSGFV-YN, a new isolate of the 10.7717/peerj.8459/supp-1Table S1Click here for additional data file.10.7717/peerj.8459/supp-2Table S2Click here for additional data file.


New network technologies are framed as eliminating ‘transaction costs’, a notion first developed in economic theory that now drives the design of market systems. However, the actual promise of the elimination of transaction costs seems unfeasible, because of a cyclical pattern in which network technologies that make that promise create processes of institutionalization that create new forms transaction costs. Nonetheless, the promises legitimize the exemption of innovations of network technologies from critical scrutiny. We aim to show that the focus on the eradication of transaction costs, first, allows technological development to be exempted from public scrutiny, and, second, narrows the scope of societal and economic concerns to an undesirable extent as new societal and economic asymmetries emerge. Its normative claim is that promises about the reduction of transaction costs should be not be taken at face value, but they should be made into subject of public reflection and deliberation.Promises about the capacities of new technologies are an important driver for technological development, helping to mobilize resource bases . In the performative for economic markets , an ideologically driven, open-source project – though it should be noted that OpenBazaar is being developed by the for profit company OB1 , IPFS is a content-centric approach similar to BitTorrent, where content can be fractured and distributed in the peer network while remaining searchable through the hash pointers. Accordingly, all the listings on the OpenBazaar are stored in the distributed database without central servers. This brings about two critical consequences: First, there is no central database provider that can claim intermediary fees, and, second, the marketplace is potentially censor-resistant. Furthermore, there is no single centralized search engine , and any provider can freely index the marketplace data base. This means that fees associated with the information asymmetry of market participants can also be lower than in in the case of competitors like Amazon.Probably the so-far most well-documented project that involves distributed ledger technology is OpenBazaar  is basically a system that allows a contractual payment to be frozen until the agreement is achieved between majority of contracting parties . In order to avoid the collusion of interests, contracting parties can employ an extended number of independent notaries. Other potential options include double  escrows, where both parties freeze funds so that a defaulting party inevitably loses, or even smart contracts that ensure automatic implementation of contractual obligations. Still, the crucial point is that no single authority that could charge rent for the mitigation of contractual issues is present here, thus creating potential for lowered transaction costs, on litigations and contract enforcements. Currently in development is an additional layer built on Ethereum blockchain that can provide decentralized advertising on the platform, using smart contracts for the implementation of auctions for ad content spaces.6 Finally, the implementation of cryptocurrency payments and anonymity (TOR) network aims to eliminate transaction costs associated with privacy and cybersecurity of market participants. Using cryptocurrencies for payment, market participants can enjoy lower intermediary fees for the transfer of value operations in the absence of centralized financial intermediaries such as banks and payment systems like PayPal.Another important feature of the OpenBazaar platform concerns the resolution of contracts and litigations by a number of mechanisms. At this moment, this is arguably the least novel aspect, since, as with many other existing platforms, market participants are encouraged to rely on the reputation of their counteragents, and use ‘Ricardian contracts’ rather than blockchain based smart contracts, for arbitrage.7 Moreover, these tools and instruments can bring about new forms of negative market externalities, such as the propagation of markets for illegal goods and services following the so-called darknet markets’ paradigm (hosted on the TOR network). The social impact of such markets is too broad an issue for univocal assessment, but there is little doubt that they can carry new and unexpected social costs for the non-participants of such markets.The promises made about decentralized marketplaces such as OpenBazaar need to be qualified. A starting observation here is that, following Thus it can be argued that while these technologies may enhance the privacy of market participants , they can also impose new cybersecurity and privacy risks when such platforms contribute to the propagation of trade in identity data and malware, affecting an even wider number of stakeholders. OpenBazaar developers appear to acknowledge these issues, but address them in an idealistic interpretation of the Coasean framework by expecting that various third-party providers of search engines for the marketplace will implement the appropriate policing practices . Whether8 already suggest that established market participants, rather than small startups aiming to disrupt existing markets, may be better positioned to benefit from blockchain-based crowdfunding to support their business models s models . InformaTransaction costs do not evaporate because of the rise of decentralized markets or other forms of fintech. Instead, these technologies create new rules and practices that induce new costs. Though it might be the case that, on the whole, the new transaction costs are lower, they also contain some rather worrying aspects. Consumers have not become more ‘rational’ in cyberspace, but increasingly appear to assume heuristics for trust, as seen in many examples of ICO funding schemes prompting crackdowns from government regulators e.g. . One of The expanding scope of motivations to tackle innovation costs also strongly influences the conceptual framework of innovation. By promoting technological development in terms of promises of ‘reducing transaction costs’ or ‘creating more trust’, the capacity to express other considerations needing to be taken into account is drastically reduced. These promises come to define the future technology, but they also come to define those who will (have to) use this technology. As this technology-imposed definition of affected actors becomes mixed with the technical complexity of new forms of information and communication technologies, a skewed distribution of knowledge and resources may further undermine the overall ethical and societal acceptability of these technologies.In this article, we have explored an intertwining of promises and technological development in the development of network technologies. This development can be sketched out as the following circular pattern:Innovators promise that their new technology will lead to the reduction (or even eradication) of transaction costs.A resource base, consisting of investments, institutional support and legitimacy, is created so the new technology can be further developed.The societal uptake of the new technology creates new processes of institutionalization, and new forms of professional, cognitive and technical specialization emerge as responses to the development of new sociotechnical systems.New forms of specialization rules set off new transaction costs and the loop will start of again.This cyclical patterns seemingly gives rise to a ‘perpetuum mobile’ of conquering and creating transactions costs. Indeed, increases in calculative powers and interconnectedness give rise to new promises that are more far-reaching than ever before. The development of blockchain-based solutions is based on promises to eliminate transaction costs by eliminating market fees of intermediation carried out by centralized authorities. Such tempting visions of markets with zero transaction costs obscure questions about the overall desirability of the technologies and institutions involved. Added to that is the observation that, in the absence of appropriate regulations, existing market monopolies should be able to successfully colonize new distributed ledger-enabled ecosystems and institutions, so that market asymmetries are further reinforced. In those cases, the promises of decentralization and consequent social benefits may fail to materialize.The pull of reducing transaction costs is part of a sociotechnical imaginary that is not reflected upon and that allows technology developers to evade responsibility for the moral implications of their work. As we have shown, new information technologies may come with new asymmetries of power, which necessitate not only new behavioral rules, but also new moral codes that have to address these asymmetries. Moreover, the exclusive focus on information and accompanying transaction costs might hinder a full grasp of associated societal and political problems. Indeed, digital networks are effectuations of specific social theories that in many cases have strong ideological connotations cf. . At timeThe reluctance to intervene and scrutinize also has to do with a reliance on policies that only target negative side-effects, such as cybercrime and privacy protection, of these technologies. Policies, however, can go much further than just remedying side-effects; they can actively guide the innovation process so as to develop new technologies that are broadly desirable – especially if they are as radical as fintech and have potential to affect fundamental human rights.Given the constitutive role of promises, it seems constructive from a democratic and ethical point of view to think about the possibilities of turning the articulation of promises and expectations in to an inclusive and deliberative process. Promises about the reduction of transaction costs should be not be taken at face value, but they should be made into subject of reflection and deliberation. In this, it is of the utmost importance not to see promises as unavoidable futures, but as strategic activities to help innovators and other stakeholders build the resource base they require to further their goals and interests. This take on the role of promises in technology development implies that new responsibilities have to be taken up by technology developers, regulators and researchers .Making innovation processes more responsive implies that limited sociotechnical imaginaries and one-sided problem definitions need to be opened up for wider examination, by attending a plurality of future pathways . This deA final point that needs to be raised here pertains to responsibilities of researchers in a domain that is characterized by performativity. As has been shown here, insights developed in research tend to spill over into the ‘real world’, as they become internalized in the mental models of actors involved in institutional and technological innovation. The notion of transaction costs is not only a descriptive label, but is also an explicit point of reference for technology development. An awareness about the interconnection between theory and practice necessitates the reconsideration of the epistemological and moral starting points of institutional economics, and should prompt theorists to develop new concepts and new frameworks to understand the challenges provided by technologies currently implemented and being implemented. The seamlessness, speed of development, and unavoidability of these new technologies make it necessary to reflect and act with much more caution than is practiced now.


Chicken anemia virus (CAV), avian reovirus (ARV), infectious bursal disease virus (IBDV), Marek’s disease virus (MDV) and reticuloendotheliosis virus (REV) all cause immunosuppressive disease in birds through vertical or horizontal transmission. Mixed infections with these immunosuppressive pathogens lead to atypical clinical signs and obstruct accurate diagnoses and epidemiological investigations. Therefore, it is essential to develop a high-throughput assay for the simultaneous detection of these immunosuppressive viruses with high specificity and sensitivity. The aim of this study was to establish a novel method using a RT-PCR assay combined with fluorescence labeled polystyrene bead microarray (multiplex xTAG assay) to detect single or mixed viral infections.Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS). The limit of detection was 1.0 × 103 copies/μL for IBDV and 1.0 × 102copies/μL for the other four viruses. Ninety field samples were tested and the results were confirmed using conventional RT-PCR methods. The detection results of these two methods were 100% consistent. The established multiplex xTAG assay allows a high throughput and simultaneous detection of five chicken immunosuppressive viruses.The results showed that the established xTAG assay had no nonspecific reactions with avian influenza virus (AIV), infectious bronchitis virus (IBV), newcastle disease virus (NDV), infectious laryngotracheitis virus (ILTV), The multiplex xTAG assay has been showed to be an additional tool for molecular epidemiology studies of five chicken immunosuppressive viruses in the poultry industry.The online version of this article (10.1186/s12917-018-1663-1) contains supplementary material, which is available to authorized users. Immunosuppression caused by viral infections is a common syndrome in the poultry industry. The result is temporary or permanent immune suppression, leaving the flock highly susceptible to other pathogenic agents. Young bird flocks are susceptible to five main immunosuppressive viruses, such as avian reovirus (ARV), chicken anemia virus (CAV), infectious bursal disease virus (IBDV), Marek’s disease virus (MDV) and reticuloendotheliosis virus (REV) –11. ImmuVirus isolation and electron microscopy is the current standard for virus identification. However, these processes are time and labor intensive and require technical proficiency in these methods. Serological tests are sufficient to separate infected birds and are widely used on poultry production farms although immunosuppressed animals may test as falsely negative. Molecular biology techniques are now in use for characterization of viral pathogens. Conventional PCR and RT-PCR tests detect specific genes from target viral genomes. Agarose gel electrophoresis-based detection is insensitive compared with real-time and multiplex PCR/RT-PCR assays –19. ThesAn alternative to these procedures is the commercial MagPlex-TAG system that enables high-throughput detection of multiple analytes in a single reaction. This allows simultaneous detection of viral target in a mixed infection , 21. TheMycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) , infectious bronchitis virus (IBV), newcastle disease virus (NDV), infectious laryngotracheitis virus (ILTV), 3copies/μL RNA for IBDV and 1 × 102copies/μL for ARV, CAV, MDV and REV. The sensitivity of multiplex xTAG assay for CAV was ten-fold higher than conventional PCR and had similar sensitivity for the other four viruses  of the replicates above the cut-off value from the highest plasmid or RNA dilution. The limit of detection was 1.0 × 105 and 1 × 107 copies/μL were prepared for reproducibility test. The inter-assay variation fell between 1.4 to 4.6%. The intra-assay variations were blind tested in duplicates by another user using three parallel reactions at 1 × 105 and 1 × 107 copies/μL. These variations ranged from 1.2 to 3.3%  and Mycoplasma synoviae (MS) were purchased from the China Veterinary Culture Collection.The virus strains IBDV B87, REV C15 and the vaccine strain MDV 814 were purchased from Guangdong Animal Epidemic Prevention and Material Reserve Center. Cell culture adapted strains CAV GD-2014, ARV GD-2, Avian influenza virus (AIV) H7N2 subtype, newcastle disease virus (NDV) F48E9 strain, infectious bronchitis virus (IBV) Massachusetts 41, infectious laryngotracheitis virus (ILTV) N-71851 strain (ATCC VR-783) were maintained in our laboratories. 90 chicken samples  from a poultry farm in Guangdong province were submitted to our laboratory at Guangdong Laboratory Animals Monitoring Institute for routine animal health surveillance.2O and stored at − 80 °C.Frozen chicken organ samples were homogenized for 15–30s before use. Cultured cell harvests were frozen and thawed several times followed by low speed centrifugation. The supernatants were stored at − 80 °C until use. Lyophilized vaccine samples were suspended in phosphate buffered saline following the manufacturer’s protocol. Viral genomic DNA and RNA were extracted from homogenates and lysed cell culture supernatants using TIANamp Virus DNA/RNA Kit following the company’s protocol . The samples after extraction were eluted in 70 μL RNase-free ddHhttps://www.ncbi.nlm.nih.gov/) and each primer pair was designed based on multiple sequence alignment from Clustal W implemented in Geneious version 5.4.3 . Primer oligomers were evaluated using Primer Premier 5.0 software (www.premierbiosoft.com).Full-length viral genome sequences were retrieved from the National Center for Biotechnology Information (NCBI) GenBank database (VP1 gene), MDV (MEQ gene), REV (ENV gene), ARV (S1 gene) and IBDV (VP2 gene) (Table xTAG assay uses target-specific primers which have linker overhangs (TAG sequences) on one primer which allow the PCR products to attach to a differently colored bead sets  and biotin on the other primer so that the PCR products contain biotin. Attached to each differently colored bead is an anti-TAG sequence which only binds to the complementary TAG sequence on the primer. After PCR amplification, color-coded beads were hybridized with the target PCR products via the TAG and anti-TAG complementarity. And then streptavidin-R-phycoertyhrin was added to bind to the biotin on the PCR amplicons. The intensity of fluorescence of the beads and R-phycoerythrin was determined by Luminex® 200 instrument. All primer sets were synthesized and purified by polyacrylamide gel electrophoresis (PAGE) . The gene targets were CAV (e) Table .Table 4PThe nucleic acid amplifications were performed using the OneStep RT-PCR Kit . Each 20 μL reaction contained 1 μL nucleic acid template, 0.8 μL OneStep RT-PCR Enzyme Mix, 250 nM of each primer, 0.8 μL dNTP mixes and 4 μL OneStep RT-PCR buffer. Cycling conditions for the multiplex PCR were 50 °C for 30 min, 95 °C for 15 min, 40 cycles of 94 °C for 25 s, 55 °C for 25 s, and 72 °C for 20 s and 72 °C for 10 min. After reaction completion, 5 μl of the reaction mix was added to 75 μl of streptavidin-R-phycoerythrin  and 20 μl beads (approximately 2500 beads). The hybridization process was carried out at 37 °C for 30 min in a thermal cycler. After the hybridization reaction, the products were analyzed on the Luminex® 200 instrument.The median fluorescent intensity (MFI) was calculated based on conditions suggested by the manufacturer and at least 50 beads were counted for each bead set in a single experiment. The data was analyzed using the Luminex xPONENT software 3.1. Negative controls containing all the hybridization components except target nucleic acid were used in each experiment to reduce background interference. The cut-off value was defined as the mean of the net MFI from negative PCR controls plus three times of standard deviation (MFI + 3SD).Sensitivity testing was determined by using 10-fold serial dilutions of DNA or RNA standards as assay templates. PCR products of the five viral PCR products were cloned in plasmid vector pGEM T Easy . The constructs of IBDV, REV and ARV were in vitro transcribed using a RiboMax Large Scale RNA production system T7 (Promega) according to the manufacturer’s instructions. RNase-free DNase (40 U) (Promega) was used for processing. Trizol LS reagent  was used for RNA isolation to eliminate DNA interference. RNA concentrations were estimated by UV spectrophotometry from the average of five repeats. The copy numbers of DNA or RNA standards were calculated based on the nucleic acid concentration and molecular weight. Assay sensitivity was compared to conventional PCR using 10-fold serial dilutions of DNA or RNA standards. All the samples were performed in triplicate.Assay specificity was tested using samples of non-target viruses in the reactions, such as AIV, IBV, NDV, ILTV, MG and MS. PCR products from specificity experiments were excised from 1.0% agarose gels using an AxyPrep DNA gel extraction kit  and sequenced .Homogenates of organ and swab samples were used for RNA extraction and simultaneously tested for the presence of CAV, MDV, IBDV, REV and ARV using the multiplex xTAG assay and conventional PCR Table  30]. PC. PC30]. Additional file 1:Table S1. The average of the MFI values with SD of specificity test by the xTAG and conventional PCR/RT-PCR assay. (DOCX 15 kb)Additional file 2:Table S2. Results for clinical samples examined by the xTAG multiplex RT-PCR and conventional PCR/RT-PCR assay. (DOC 17 kb)


Tymovirales. The genome consists of 7106 nucleotides excluding the poly(A) tail. Five open reading frames were identified, with the first encoding a putative viral replicase with methyl transferase (MTR), AlkB, helicase, and RNA dependent RNA polymerase (RdRp) domains. The genome organization downstream of the replicase resembles that of members of the order Tymovirales with an unconventional triple gene block (TGB) movement protein arrangement with none of the other four putative proteins exhibiting significant homology to viral proteins. Phylogenetic analysis using replicase conserved motifs loosely placed RAVA within the Betaflexiviridae. Data strongly suggest that RAVA is a novel virus that should be classified as a species in a new genus in the Betaflexiviridae or a new family within the order Tymovirales.A novel virus with distinct genome features was discovered by high throughput sequencing in a symptomatic blackcurrant plant. The virus, tentatively named Ribes americanum virus A (RAVA), has distinct genome organization and molecular features bridging genera in the order  Ribes spp.), a berry known for its potential health effects [Ribes americanum; PI 617879) showed virus-like symptoms  have resulted in the rapid discovery and characterization of several novel plant viruses ,2,3,4. T effects . This stsymptoms . The plaTymovirales includes five families, namely Alphaflexiviridae, Betaflexiviridae, Gammaflexiviridae, Deltaflexiviridae, and Tymoviridae [Tymovirales indicated that RAVA is a virus with unique genome features, possibly representing a new genus in the Betaflexiviridae family, or being the type representative of a yet to be described family. Discovery of this novel virus adds information to the current knowledge on this diverse group of viruses, and expands the number of virus species reported in the order.The order oviridae . Membersoviridae . This ma® RLM-RACE Kit  was used to obtain the 5′ terminus. The 5′ and 3′ RACE-RT-PCRs were performed using virus-specific primers and programs described in Double-stranded RNAs were extracted from 20 g of symptomatic leaves from currant accession PI 617879  and subjTymovirales were obtained from GenBank  for 10 min each and infiltrated with Araldite resin. Ultrathin sections were done using an RMC PowerTome PC microtome. Grids were stained with either uranyl acetate or phosphotungstic acid and examined in an FEI Titan ChemiSTEM TEM.1–5. The genome encodes five ORFs with untranslated regions (UTRs) of 140 and 172 nt for the 5′ and 3′ termini, respectively (The genome of RAVA is 7106 nucleotides (nt), excluding the poly-A tail and begins with the pentanucleotide motif GAAAAectively . 1436-44 and four bipartite signals; FAKCRQLDPENLLLSEALLVNDLIKWLRE328-65, KIRLGSKDRVIGSCKDWTTKVIKISKG913-39, GCGKTKPLMDLILKSNDNILILVPRKRLGDS1034-64, and PRKRLGDSWTSKMGHKKNVRV1057-78. The entire protein shares low aa identities with replicases of other members of Tymovirales showing the highest identity (23%) to the orthologs in members of the genus Chordovirus . The sequence identity of the conserved domains of RdRp ranges from 21–57%; the highest being with members of Betaflexiviridae  and the lowest with members of the Tymoviridae, Alpha-, Gamma- and Deltaflexiviridae (21–32%) (data not shown). The MTR domain also showed higher identity to members of the Betaflexiviridae (24–33%) compared to the other families in Tymovirales.ORF 1 encodes a putative protein of 207 K , presumably involved in virus replication as it contains domains of methyl transferase , AlkB homologue of 2OG-Fe(II) oxygenase , helicase , and RNA dependent RNA polymerase . The polyprotein contains five nuclear localization signals; one monopartite signal, AVRKRLRFA13-35). ORF 3 partially overlaps ORF 2 and encodes a peptide of 60 aa (p3) with a calculated molecular mass of 7 K with two predicted transmembrane domains at the N and C termini . The secondary structures of the transmembrane regions contain one and two α-helixes respectively  . This pu280,283) predicted to be involved in ligand binding with calcium (Ca)  protein of unknown function speculated to be the capsid protein (CP) of the virus. This protein contains several α-helixes and coils and a single β-sheet . Among s-TASSER; ).Betaflexiviridae  looselyiviridae A,B, withTissue tested positive for the virus by RT-PCR was used in electron microscopy. No virus-like particles were identified, nor were there any structures that could belong to an endophyte, a potential host of the virus (data not shown).Alphaflexiviridae and Betaflexiviridae families. Because of the unique genome organization, we employed electron microscopy on tissue that was tested positive for the virus to determine whether a bacterial or fungal endophyte may be the cryptic host of the virus. In the absence of any structures other than those associated with the plant cell, we determined that RAVA is a plant virus. The genome initiated with the pentanucleotide sequence (GAAAA1-5) that is conserved in Alpha- and Betaflexiviridae [Potexvirus, Allexivirus, Lolavirus, Mandarivirus  and Foveavirus, Robigovirus, Carlavirus, and the unassigned members (in the Betaflexiviridae)  . The repAlphaflexiviridae and Betaflexiviridae , and ‘potex-like’ , and are usually overlapping [Robigovirus and an unassigned member, sugarcane striate mosaic-associated virus, in which only two, TGBp 2 and TGBp 3, are overlapping. Despite the similarity in the 5′ part of the genome coding for the replicase, the RAVA genome differed significantly from genomes of members of the iviridae . First arlapping , with thThe organization of TGB ORFs is highly conserved among plant viruses ,28. PoteTymoviridae [AUGG . In somin bold; . The KozAlpha- and Betaflexiviridae members is the CP  suggestive of possible recombination events in which the ancestral viruses had a common 5′ part while acquiring the 3′ genes from different origins, which further diversified over time [Betaflexiviridae, whereas the remaining proteins are phylogenetically distant. Changes in phylogenetic relationships for different regions of the genome of a virus is an indication of recombination [Trivirinae subfamily, however the rest of the genome is Quinvirinae-like  and the distant phylogenetic relationship with other members distinguishes it from both existing genera and unassigned members of the family, suggesting a tentative classification. Therefore, we propose that RAVA represents a new, monotypic genus in the family Betaflexiviridae or a family (given the low replicase aa identities with existing viruses) for which the names Ravavirus/Ravaviridae are proposed. RAVA is a unique, previously undescribed virus of tymo-like lineage which cannot be assigned to any of the currently recognized virus taxa. With regard to its molecular features such as the number of genes encoded in the genome, organization, and the presence of conserved pentanucleotide at 5′ termini, RAVA resembles members of the order


As not much is known about the prevalence and predictors of nutritional deficiencies among vegans in the Czech Republic, we evaluated whether supplement use and duration of adherence to the vegan diet are associated with the risk of cobalamin and iron deficiencies. Associations between self-reported supplementation and duration of vegan diet with biomarkers of cobalamin  and iron status  were assessed by cross-sectional analyses of medical data from a clinical nutrition center. Data from 151  adult vegans (age 18–67 years), who were free of major chronic diseases and 85  healthy non-vegans (age 21–47 years) were analyzed. Overall, vegans had significantly lower cobalamin, hemoglobin and ferritin levels, but higher folate and MCV values compared to non-vegans. Vegans not using cobalamin supplements were at higher risk of low plasma cobalamin than regularly supplementing vegans , whereas no significant differences in cobalamin status related to duration of the vegan diet were observed. Regularly supplementing vegans had similar levels of cobalamin/holotranscobalamin as non-vegans. Despite lower ferritin and hemoglobin levels, there was no indication of a higher risk of iron-deficiency among vegans. To conclude cobalamin deficiency risk depends on supplementation status and not on the duration of an exclusive vegan diet, which underlines the need to integrate cobalamin status monitoring and counselling on supplement use in routine clinical care in the Czech Republic. Veganism is defined as a philosophy and way of living, which seeks to exclude, as far as possible and practicable, all forms of exploitation of, and cruelty to, animals for food, clothing and any other purpose . While aBased on the concept of the so-called rational vegan diet, promoted by vegan organizations and by some scientific societies, meat and meat products, dairy, eggs and honey can be replaced by legumes and grains as the main sources of protein and nutrients. A rational vegan diet should theoretically meet all of the requirements for nutrients except of cobalamin, which should be supplemented ,6. HowevMedical records of the out-patient department at a tertiary clinical nutrition center  were used for the present study. Data of vegan adults, who attended medical check-ups between 2012 and 2019, and volunteers from previous studies  of the dPeripheral blood samples were obtained after an over-night fast. The biomarkers of interest were chosen and serum analyses were performed immediately in the ISO certified institutional laboratory by validated routine methods. For cobalamin and folate chemiluminescence immunoassay, for holotranscobalamin electrochemiluminiscent Elecsys active B12 assay and for iron FerroZine colorometry was used, all analyzed automatically on a Cobas 8000 system . Homocystein, ferritin (chemiluminescence immunoassay) and transferrin (immunoturbidimetry) using Advia Centaur XP . Saturation of transferrin was calculated as TS = 3.98 × ([iron] μmol/L)/([transferrin] g/L) and total iron binding capacity was calculated as TIBC = [unbound iron capacity] μmol/L + [iron] μmol/L. As per hemoglobin (HGB) in whole blood, it was measured using photometry, hematocrit using direct impedance measurement and mean corpuscular volume MCV was calculated as hematocrit/red blood cell count (RBC). For cobalamin, holotranscobalamin, folate, ferritin and homocysteine reference values provided by manufacturer were used. Other reference intervals used are based on Czech population data .https://www.r-project.org/, using the tidyverse package . Tidyverse: Easily Install and Load the ‘Tidyverse’. R package version 1.2.1. https://CRAN.R-project.org/package=tidyverse) and ggpubr package (Alboukadel Kassambara (2018) ggpubr: ‘ggplot2’ Based Publication Ready Plots. R package version 0.1.7 https://CRAN.R-project.org/package=ggpubr). A two-sided p-value of 0.05 was used to denote statistically significant associations.For our statistical analyses we divided the study participants into groups based on the durations that they reported to have followed an exclusive vegan diet , as well as self-reported supplement use (non-vegans vs. “non-supplementing” vegans vs “irregularly supplementing” vegans vs. “supplementing” vegans). Kruskal-Wallis tests were carried to test for differences in blood biomarkers among vegans and non-vegans, and across the above-mentioned strata of supplement use and duration of the vegan diet. ANCOVA models were then used to evaluate differences in biomarkers between non-vegans and vegans stratified by the mentioned categories of supplement use and vegan diet duration adjusting for age, sex, and mutually adjusting for supplementation and duration, respectively. In addition, multivariable logistic regression models with the odds ratios  of low plasma cobalamin levels , increased homocysteine (defined as homocysteine levels > 15 µmol/L), decreased ferritin (ferritin < 30 µg/L) and other clinically relevant combinations of biomarkers were carried out to assess differences in biomarker levels by strata of cobalamin supplement use and duration of the vegan diet, again with mutual adjustment for these variables in addition to adjustment for age and sex. R  was used for all analyses and plots , despite the fact that there was no significant difference in levels of holotranscobalamin between vegans and non-vegans .Vegans have significantly higher risk of cobalamin levels below 190 ng/L: OR 5.49 (95% CI 1.6–18.87). When defining deficiency based on the levels of cobalamin and homocysteine together, irregularly supplementing vegans have 3.89 (95% CI 1.13–13.45) higher and non-supplementing vegans have 21.48 (95% CI 5.49–84.02) higher risk for deficiency than regularly supplementing. Using combination of markers for assessment the risk of laboratory signs of cobalamin deficiency , 16.9% of the vegan study population was cobalamin deficient; 5.56% among the supplement users, 17.9% among the irregular supplement users, and 52.9% among the non-users, while only 3 non-vegans out of 85 could be classified as cobalamin deficient. Compared to non-vegans, vegans have OR 7.05 (1.6–31.06) for having been classified as deficient.With regard to duration of vegan diet, the highest prevalence of lowered cobalamin levels was found among short term vegans (28.2%) compared to mid-term (4.55%) and long-term (17.1%) while only 3.7% in non-vegans. In multivariable logistic regression analyses , the lower prevalence of low levels of cobalamin among medium-term vegans in comparison to short-term vegans was significant. Of note, there were only two vegans with folate values below 4.6 µg/L . For details in the differences among vegan subgroup see Based on duration of vegan diet (compared to non-vegans), ORs were for short-term vegans: 15.07 (3.13–72.56), medium-term vegans: 1.96 (0.32–12.22) and for long-term vegans: 9.28 (1.76–48.9). Based on supplementation habits, there were significantly higher risks for not-supplementing and irregularly supplementing vegans: OR 18.89 (3.34–106.71) and 8.61 (1.81–49.93), respectively. The difference among supplementing vegans and non-vegans was not significant .Anemia, defined as hemoglobin levels below 130 g/L for males or 120 g/L for females, was recorded in 12.87% of participants, 13.6% of the vegan participants . In non-vegans, it was in 4.94% . In the logistic regression model taking into account the age and sex the difference in the risk of lower hemoglobin among groups for vegans compared to non-vegans OR: 3.25, 95% CI 1.04–10.16). The lower values of MCV, defined as MCV < 82 fL, were found in 7.53% of vegans and 7.4% of non-vegans. In the logistic regression model, the difference was not significant (vegans compared to non-vegans OR: 1.05 (0.38–2.98). Ferritin values under 30 µg/L were found in 44.76% of study participants, 33.3% of non-vegans and 46.2% of vegans. In logistic regression, the difference in risk of the values of ferritin under 30 µg/L was for vegans . When looking on the combination of markers suggesting the ongoing iron deficiency, the combination of low hemoglobin and low ferritin values , 7.41% of non-vegans and 11.6% of vegans could be classified a possibly deficient. The difference in risk was not significant in logistic regression model adjusting for age and sex: OR 1.71 (0.62–4.66). Similar results were obtained when looking on the combination of low ferritin, low iron and low iron binding capacity  µmol/L and iron binding capacity < 45 µmol/L), in which case 51% 4 of non-vegans and 50.1% of vegans had at least one pathological values, while the number was 40.5% for non-vegans, again the difference was not significant in logistic regression. For selected markers, more detailed analysis investigating the difference in the risk of values suggesting iron deficiency based on the duration of veganism in vegan participants was prepared see .In the current study involving 151 Czech vegans and 85 non-vegan controls, we showed that vegans have significantly lower levels of cobalamin, hemoglobin and ferritin, but higher folate and MCV. The risk of laboratory signs of cobalamin deficiency among vegans was strongly related to the non-use of dietary supplements, and even irregular users of supplements were at higher risk for low levels of cobalamin and holotranscobalamin compared with regular users or non-vegans. By contrast, our study does not indicate that the duration of adherence to the vegan diet alone is related to the risk of cobalamin deficiency. Despite lower ferritin values among vegans (not related to the duration of vegan diet), the overall presence of the typical laboratory markers of iron deficiency anemia was low in both among vegans and non-vegans, and there were no statistically significant differences in the prevalence of such signs. Ferritin and hemoglobin values were significantly lower among vegan vs. non-vegan men, while no such difference was observed among women.While evidence from the past years on homocysteine as a biomarker of cardiovascular disease risk is not consistent ,13, our The observed tendency for higher MCV values among vegans was no longer statistically significant after adjustment for supplement use. Also, almost all of the participants in the study were in the normal range of MCV, despite having low cobalamin or showing a tendency for lower ferritin. Based on this finding, we assume that MCV may not be a valid marker for cobalamin or iron deficiency anemias in vegans, even though it is traditionally recommended for this purpose. Possibly, our observation can be either explained by higher levels of folate in vegans  or by lower ferritin levels .While the duration of adherence to the vegan diet was not associated with cobalamin, holotranscobalamin or homocysteine levels in our study, we observed a trend towards decreased iron load and storage with a longer vegan diet duration, ferritin being significantly lower in vegans than in non-vegans (not related to the duration of the vegan diet). Interestingly, many vegans in our study showed iron status parameters that clustered around the threshold value for iron deficiency, but only a minority of them had levels indicating iron deficiency. Although such borderline values could be interpreted as a risk factor for sideropenic anemia, low iron stores were also suggested to be partially responsible beneficial health effects of a vegan diet ,15. HighGiven the increasing prevalence of alternative diets, guidelines for cobalamin deficiency assessment by blood markers according to which health professionals can be trained are needed. In this regard, it would have been of interest to evaluate other reliable markers of cobalamin deficiency used in practice, namely methylmalonic acid MMA ,20 in ouFurther limitations of the present study have to be considered. First, our vegan study population was a convenience sample of adults, who voluntarily attended healthcare checkups at the outpatient department of Clinical Nutrition. It is conceivable that this sample may have a higher degree of health consciousness together with greater trust in Western medicine and practicing physicians compared to other Czech vegans, who may be more prone to utilizing alternative medicine. Thus, it can be speculated that the overall prevalence of cobalamin deficiency among vegan adults in the Czech Republic may be higher than in the current sample of vegans. Our study did not include children, pregnant females, or lactating females, who may be particularly vulnerable to consequences of cobalamin deficiency. Indeed, borderline cobalamin status in pregnant women can be associated with increased risk of acquired newborn B12 deficiency and cobalamin alone may not be sufficient to diagnose deficiency among them, which is why MMA tests are required . Future Our data may stimulate future research, and foster a more evidence-based debate about medical guidelines and recommendations targeting populations with alternative lifestyles in eastern European countries. There is clear indirect evidence that the popularity of alternative diets is increasing even in regions, in which the traditional cuisine is heavily based on animal products and where the availability of plant-based alternatives is not as high as in countries with a longer tradition of veganism. While we showed there is a substantial proportion of vegans, who do not supplement B12 sufficiently, the current shift in eating habits towards plant-based diets is not reflected by current public health recommendations and guidelines.To conclude, we observed that Czech vegans are at higher risk of cobalamin/holotranscobalamin and ferritin deficiency overall. This difference was driven by irregular or non-use of cobalamin supplements in subgroups of the vegan study population, while regular supplement users were not at a higher risk of cobalamin deficiency. The duration of adherence to the vegan diet was not associated with risk for low cobalamin values. Public health services and healthcare professionals in the Czech Republic and other countries, where the vegan diet is becoming more popular, should meet the needs of vegans, especially regarding cobalamin status monitoring to prevent cobalamin deficiency.


Miguel A. ArandaKristiina MäkinenJeanmarie VerchotWe are very pleased to present in this Special Issue of Molecular Plant Pathology a series of short reviews on the topic “Extracellular and intracellular perception of plant viruses”. The issue summarizes important breakthroughs in plant defence against viruses. Significant advances made in recent years provide new insights into the mechanisms of antiviral immunity after virus recognition by host cells. Plant viruses constitute an important cause of major crop losses worldwide and threaten food security. The advances in the field of plant–virus interactions will expand the potential strategies for breeding and engineering resistance in crops. The articles in this issue discuss the co‐evolving virulence and plant resistance strategies exemplifying the evolutionary arms race between viruses and their hosts.nucleotide‐binding leucine‐rich repeat (NLR) receptors leading to effector‐triggered immunity (ETI), have been seen as the most critical contributions to antiviral defence in plants. As discussed in the review entitled “Perspectives on intracellular perception of plant viruses” by Meier et al. in this issue, a considerable body of knowledge on how viruses are perceived by NLRs, and how NLRs are activated and use downstream signalling pathways to initiate defence responses, has developed during these years. In spite of that, our understanding of the underlying phenomena is still far from complete. After their concise but comprehensive review, the authors draw future lines for research, and point out structural analyses of NLRs complexed with their partners as one source of critical information to understand intracellular perception of plant virus and defences activation.For the past twenty years, the endogenous RNA silencing pathway acting to degrade viral RNAs, and intracellular perception of viral proteins by et al. also discuss these aspects in their review entitled “Virus perception at the cell surface: revisiting the roles of receptor‐like kinases as viral pattern recognition receptors”. Importantly, they make a case of the NUCLEAR SHUTTLE PROTEIN (NSP)‐INTERACTING KINASE 1 (NIK1), an RLK identified in an interaction screening with NSP, which is a begomoviral protein. Activation of NIK1 leads to repression of ribosomal protein genes, suppression of global translation, including begomoviral mRNAs, and confers resistance to begomoviruses. Their data nicely fits in a model in which begomovirus‐derived nucleic acids could act as PAMPs to activate NIK1, and NSP acts as a viral effector that suppresses NIK1. Whether or not this model could be extended outside of begomoviruses is a matter of future studies.In a first layer of the plant immune system, extracellular perception of pathogen‐associated molecular patterns (PAMPs) leads to pathogen‐triggered immunity (PTI). Recent findings strongly suggest the existence of PTI‐like responses against viral infection. The newly uncovered layer of antiviral defence initiates at the plasma membrane and leads to a broad range of early responses to plant viruses. As discussed in “Molecular dialogues between viruses and receptor‐like kinases in plants” by Macho and Lozano‐Duran, activation of receptor‐like kinases (RLKs) on the cell surface during virus infection leads to activation of PTI‐related genes and multiple outcomes of plant's antiviral defence. Effects on the level of developmental rearrangements and cell‐to‐cell movement of RNAi are also possible. It is under debate whether plant viral proteins can directly act as PAMPs or whether the infection induces production of danger‐associated molecular patterns (DAMPs) for RLK activation. An increasing body of evidence is revealing that RKLs are targets of viral proteins. Teixeira Both RNA silencing and, as recently found, antiviral PTI, recognize double‐stranded (ds) RNAs as defence signals. During virus infection, replication and expression of viral genomes leads to accumulation of virus‐derived transcripts and dsRNAs. Since viruses are intracellular pathogens, we have well accepted that the cellular RNA silencing machinery recognizes viral RNAs. Antimicrobial PTI, however, involves plasma membrane‐localized pattern‐recognition receptors (PRRs). In the insightful review entitled “Perception of double‐stranded RNA in plant antiviral immunity”, Annette Niehl and Manfred Heinlein discuss several different scenarios on how perception of dsRNA by PRRs could happen during dsRNA‐mediated antiviral PTI.The series of papers closes with a review on autophagy and plant viruses. During the recent years, autophagy has turned out to have an important role in plant virus infection and antiviral immunity as highlighted by Kushwaha, Hafren and Hoffius in their review entitled “Autophagy‐virus interplay in plants: from antiviral recognition to pro‐viral manipulation”. The authors discuss a wide variety of examples of autophagy in antiviral recognition and degradation, pro‐viral counterdefence and manipulation, disease‐related cell death, immunity‐related hypersensitive reaction and other viral functions.The coming years will inevitably deepen our still limited knowledge on the mechanisms and functions of extracellular and intracellular recognition of viruses, and the contribution of autophagy in plant virus infection. We are sincerely convinced that this series of reviews can contribute to identify new important research avenues, particularly for young researchers joining the exciting field of plant virology.


Infection and transmission of honey bee viruses pose a serious threat to the pollination services of crops and wild plants, which plays a vital role in agricultural economy and ecology. RNA interference (RNAi) is an effective defense mechanism against commonly occurring viral infections of animals and plants. However, recent studies indicate that the effects of RNAi on the honey bee can induce additional impacts and might not always be effective in suppressing the virus. Moreover, the RNAi responses differed in relation to the developmental stage of the insect and the target tissue used, even though the same method of delivery was used. These results indicate that further analysis and field experiments should be performed to characterize the varying effectiveness of RNAi-based methods for treating honey bee viral infections. In this review, we provide an overview of the current knowledge and the recent progress in RNAi-based anti-viral treatments for honey bees, focusing in particular highlight the role of the dsRNA-delivery method used and its effect on RNAi efficiency and demonstrate the potential practical value of this tool for controlling the virus. We conclude studying the gene function and disease control of honey bee by RNAi technology requires a complex consideration from physiology, genetics to environment. Varroa destructor , chronic bee paralysis virus (CBPV), black queen cell virus (BQCV), Israeli acute paralysis virus (IAPV), and sacbrood virus (SBV) can make honey bee display the visible symptoms such as deformed wing, paralyzed, black cell and pupae sacbrood. In addition, the viruses establish acute infections such as the infection caused by acute bee paralysis virus (ABPV), which produces apparent symptoms . Thence,Discitrovirus family. The viruses from Discitrovirus family have been shown to readily establish persistent infections and cause large economic losses in the apicultural industry because these viruses are able to replicate efficiently by using internal ribosome entry sites (IRES)-mediated translation mechanism, which is different from the cap-dependent replication mechanism used by most other viruses , c-Jun N-terminal kinase (JNK) and Janus kinase/Signal Transducer and Activator of Transcription (Jak-STAT) pathways have been confirmed that play a vital role in resistance against virus infection . In addiFigure 1A)  . RNAi cagure 1A) . Direct nogaster . Genome nogaster . Experimilencing . RNAi hailencing , immunitilencing , and genilencing  as well ilencing . In addi ceranae , ectoparstructor  and smalstructor . More imstructor . With thstructor . Recentld by SBV .Table 1), there are more challenges associated with dsRNA delivery in honey bees than in other insects due to the lack of bee cell cultivation system  can induce the anticipated responses, which could then activate cellular or humoral actions related to physiology and survival . In addivitellogenin gene will cause the honey bee workers into extremely earlier forages and leading to behavior maturation (vg) and ultraspiracle (usp), and found that vg plays a key role among the vg, usp and juvenile hormone (JH) during the process of behavioral maturation. However, A study has shown that silencing of turation . To undeD. melanogaster and Manduca sexta have only been achieved in hemocytes compared to other tissues  was higher than that of short dsRNA (31 bp) . In addiV was 0% . TherefoNosema apis , was shown to bind to Ago-2 to inhibit slicing of mRNA in vitro , or among IAPV, Kashmir bee virus (KBV) and Kakugo virus (KV). Even if purified virus was from experimental infection honey bee samples, it still might host several viruses . Since injection of dsRNA into honey bees is not a convenient and practical method, future research should focus on developing methods that enable efficient uptake of dsRNA by the target tissues and also enable the dsRNA to persist in vivo after oral application. In addition, study is required to address several questions, including the role and interactions of siRNA from other pathogens with the host RNAi machinery. Taken together, we conclude that much works have to be done to make the RNAi-based treatment strategy become reliably effective tool to study gene functions and gene mechanisms of honey bees.For systematic RNAi application, the size and quantity of dsRNA used should be considered. Based on the findings of the previous studies, we speculate that there may have been two factors that may have influenced the results of RNAi treatment in some studies. The first factor may have been inefficient dsRNA uptake or no response of intracellular RNAi machinery . The secCH and QD conceived this manuscript. DY, XX, HZ, SY, XW, and DZ participated in the writing, reviewing, and critical analysis of this manuscript. CH and QD coordinated the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling Editor declared a shared affiliation, though no other collaboration, with several of the authors .


Vector-borne viruses are becoming increasingly important from a public health standpoint with the emergence or reemergence of viruses and extension of the areas at risk. Here, we report the whole-genome sequences of two alphaviruses, namely, one Igbo-Ora virus and one Babanki virus, that were isolated several decades ago in Africa from human serum. Vector-borne viruses are becoming increasingly important from a public health standpoint with the emergence or reemergence of viruses and extension of the areas at risk. Here, we report the whole-genome sequences of two alphaviruses, namely, one Igbo-Ora virus and one Babanki virus, that were isolated several decades ago in Africa from human serum. Alphavirus genus and Togaviridae family and cause diseases in humans. Both of them are arthropod-borne viruses. Igbo-Ora virus was first isolated in Nigeria  were used as targeted sequences. Finally, 697,876 and 1,412,595 viral sequences, covering overall the target sequences from their 5′ to 3′ ends, were obtained and used for whole-genome assembly with SPAdes version 3.1.0 , which were both isolated from human serum specimens collected in the Central African Republic in 1966 and 1975, respectively. These viruses were isolated and amplified by serial passages in brains of newborn mice (unknown passage numbers). Brains were homogenized in Hanks’ solution and centrifuged. Supernatants were lyophilized and stored in sealed glass vials at room temperature until 2012. Viral genomic material was extracted from lyophilizates that were resuspended in phosphate-buffered saline using a QIAamp viral RNA minikit (Qiagen). The extracted RNA was retrotranscribed into cDNA using SuperScript III enzyme and random hexamers (Life Technologies). The cDNA fragments were ligated and then amplified using the phi29 DNA polymerase and random hexamers .For HB67652 and HB741570, the lengths of the whole-genome sequences were 11,818 and 11,714 nucleotides, with average coverages of 3,221× and 1,933× and GC contents of 48.1% and 51.6, respectively. The coding sequence lengths of HB67652 were 7,542 and 3,744 nucleotides for the 1st and 2nd open reading frame (ORF), respectively. The coding sequence lengths of HB741570 were 7,548 and 3,738 nucleotides for the 1st and 2nd ORF, respectively. Of note, a leaky stop codon near the nsP3 gene 3′ end is present in HB741570, unlike in HB67652. HB67652 and HB741570 share >99% nucleic acid identity with the only 2 other complete genomes of Igbo-Ora and Babanki viruses currently available in GenBank (accession numbers While Igbo-Ora virus and Sindbis viruses have confirmed potential to cause widespread epidemics in humans, the epidemic potential of Babanki virus remains unclear , 10. FurMF409176 and MF409178. The raw sequencing data have been submitted to the SRA database under the accession numbers SRR9712540 and SRR9712544.These whole-genome sequences are available in GenBank under accession numbers


Zika and chikungunya viruses were first detected in Fiji in 2015. Examining surveillance and phylogenetic and serologic data, we found evidence of low-level transmission of Zika and chikungunya viruses during 2013–2017, in contrast to the major outbreaks caused by closely related virus strains in other Pacific Island countries. Zika virus and chikungunya virus (CHIKV) have caused outbreaks in several tropical regions, including the Pacific  in Fiji (We retrieved surveillance data for patients with prolonged fever (PF), defined as any fever lasting  in Fiji , as well in Fiji  Table 1. in Fiji  Figure 1 in Fiji . Analysi in Fiji  Table 4.Surveillance data recorded during 2013–2017 indicated cyclical increases in AFR and PF each year, concurrent with the hot and rainy season occurring in December–April . MoleculWe aligned the envelope (E) gene sequences of Zika virus strains collected in Fiji during 2015–2016  Table 2 We aligned the E1 gene sequences of Fiji CHIKV strains collected during 2015–2016  Table 2 <0.0368) and higher in urban than in rural areas (p = 0.0136). During 2013–2015, we observed a significant increase in DENV seroprevalence  among persons 0–19 years of age (p = 0.0013), women and girls (p = 0.0002), and participants living in the Central Division (p = 0.0018) and urban areas (p = 0.0048). Seroprevalence in 2015 remained lower in persons 0–19 years of age than in other age groups (p<0.0137) but was significantly higher in women and girls compared with men and boys (p = 0.039) and in urban compared with rural areas (p = 0.0053).Zika virus seroprevalence in 2013 was 7.8% (95% CI 6.1%–10%); we observed no significant differences between age groups, sexes, residential divisions, or areas . In 2015Analysis of paired samples collected in 2013 and 2015 from the same participants supported previous serologic findings on all samples collected  Table 5.Aedes aegypti and Ae. albopictus, are abundant.We found evidence of low-level transmission of Zika virus and CHIKV in Fiji for multiple years after their initial introduction into a population that probably was immunologically naive, despite an ecologic environment subject to large and recurrent DENV outbreaks. Similar evidence of low-level Zika virus circulation has been observed in other settings (Our study highlights the difficulties in detecting and anticipating outbreaks of Zika virus and CHIKV and the value of having multiple data sources available. Stronger clinical and laboratory surveillance capacities are needed to ensure the early detection of these and future infectious disease threats.Additional information for sustained low-level transmission of Zika and chikungunya viruses after emergence in the Fiji Islands.


Eidolon helvum) in Ghana, West Africa. To investigate this further, longitudinal blood sampling was undertaken quarterly between 2012 and 2014 on wild E. helvum at two sites in Ghana. Serum samples were collected and tested for LBV-neutralizing antibodies using a modified flourescent antibody virus neutralisation (FAVN) assay (n = 294) and brains from moribund or dead bats were tested for antigen and viral RNA (n = 55). Overall, 44.7% of the 304 bats sampled had LBV-neutralising antibodies. None of the brain samples from bats contained lyssavirus antigen or RNA. Together with the results of an earlier serological study, our findings demonstrate that LBV is endemic and circulates within E. helvum in Ghana even though the detection of viral infection in dead bats was unsuccessful. Confirmation that LBV infection is endemic in E. helvum in Ghana is an important finding and indicates that the potential public health threats from LBV warrant further investigation.Bats are key species for ecological function, but they are also reservoirs of zoonotic agents, such as lyssaviruses that cause rabies. Little is known about the maintenance and transmission of lyssaviruses in bats, although the observation of clinically sick bats, both in experimental studies and wild bats, has at least demonstrated that lyssaviruses are capable of causing clinical disease in bat species. Despite this, extensive surveillance for diseased bats has not yielded lyssaviruses, whilst serological surveys demonstrate that bats must be exposed to lyssavirus without developing clinical disease. We hypothesize that there is endemic circulation of Lagos bat virus (LBV) in the straw-coloured fruit bat ( Chiroptera) represent a large group of wildlife, comprising ~20% of the 4600 mammalian species recognized to date [Wildlife populations constitute a large and largely undefined reservoir of infectious agents, some of which are zoonotic ,2,3. Inf to date . HoweverRhabdoviridae, genus Lyssavirus) are the aetiological agents that cause the disease rabies—an acute, progressive, viral encephalitis that is almost invariably fatal [Lyssaviruses  in Ghana [E. helvum, were made through post-mortem sampling of moribund and dead bats as well as through serological assessment of samples for LBV neutralizing antibodies.One bat lyssavirus that has been associated with the infection of terrestrial carnivores is Lagos bat virus (LBV), a virus most commonly associated with infection of African fruit bats. Fruit bats of several species have been identified as reservoir hosts for LBV, with spill-over infections reported in dogs, cats and a mongoose ,12,13,14in Ghana . While tin Ghana , knowledThe study was approved by the Institutional Review Board of the Nuguchi Memorial Institute for Medical Research of the University of Ghana (NMIMR-ITB CPN 002/13-14 IORG0000908) and the Ethics Committee of the Zoological Society of London (WLE/0638).E. helvum roost sites in Ghana, West Africa. The roosts comprised between approximately 3000 to 1,000,000 bats each, depending on year and season. One roost was located in Greater Accra , within the grounds of a large general infirmary  in the center of the city. The other roost was in Tanoboase in the Brong-Ahafo region , within a protected forest area  to enable identification in case of recapture. Bats were released at the site of capture immediately following sampling, after ensuring haemostasis at the site of venipuncture.Wild bats were captured using mist nets from 2012 to 2014 inclusive, as detailed in Members of the public who frequented the areas under the roosts were asked to report any dead or sick bats they found. Also, during each sampling visit, searches for dead or sick bats were undertaken in the immediate vicinity of the roosts. Sick bats were euthanised using intramuscular ketamine and medetomidine, followed by exsanguination via cardiac puncture. Bat carcasses were stored in a freezer at −70 °C until necropsy. At necropsy, a range of tissues, including brain, was collected and stored fixed or frozen for later investigation.Fluorescein isothiocyanate (FITC)-conjugated anti-rabies nucleocapsid polyclonal antibody conjugate  was used diluted 1:40 in 0.1 M phosphate buffered saline( PBS) pH 7.2 to stain virus antigen in acetone-fixed impression smears taken from the hippocampus, cerebellum and brain stem of bats found dead or sick (following euthanasia), as described previously . Slides 50/50 μL. Both negative sera from an unvaccinated dog and positive anti-LBVNig56 rabbit serum samples were used as serological controls for the assay. Each test serum sample was diluted three-fold (starting with a 1:9 dilution) in minimum essential medium (MEM), and incubated with the viral inoculum for 1 h at 37 °C before being added to BHK-21 cells in suspension in a 96-well plate. Following incubation for 48 h at 37 °C, the plates were fixed in 80% chilled acetone for 20 min, and stained with FITC-labelled anti-rabies polyclonal antibody . Fluorescent foci present in cells infected with LBV were visualized at 488 nm using a fluorescence UV microscope. The lack of accurately-titred control sera meant that all neutralizing antibody levels were expressed as D50 values. The D50 for each sample is the dilution endpoint at which 50% of the wells showed the presence of virus. D50 values were calculated using the Spearman-Karber method. A serological cut-off was assigned according to neutralization of control sera and a back titration of input virus. The presence of antibodies capable of neutralizing LBV in collected bat sera was assessed using a modified version of the fluorescent antibody virus neutralization assay (mFAVN), as described previously ,15. BrieTotal RNA was extracted from brain tissue samples using Trizol (Invitrogen) following the manufacturer’s protocol. A pan-lyssavirus SYBR Green real-time (q) RT-PCR  and a paThe chi-squared test was used to investigate if there were significant differences in seroprevalence according to the age, sex and roost location of bats.n = 136) were considered seropositive . Of those bats that tested serologically positive, 45.2% were known to be male (n = 95/210) while 61.9% were identified as female (n = 52/84). Results from samples collected from bats of undetermined sex or age were not included in the statistical analyses. There was no significant difference in the different age groups that tested positive  (n = 236) was slightly higher than in the sub-adult and juvenile bats combined .Samples taken and data regarding age, sex and serology are detailed in positive . This copositive . Seropre = 0.78) . SeropreE. helvum dams (ID: 900 and ID: 905) were caught with their pups  at the 37-Military Hospital roost site. All four animals were apparently healthy with a good body condition and no outward signs of disease. Following serological assessment, the two mothers were both seronegative with a D50 of 5.20. In contrast, both pups were seropositive with detectable anti-LBV neutralizing antibody D50 of 46.77 and 81.00 for animals 901 and 904, respectively.In June 2013, two nursing n = 94/213) were seropositive (n = 42/91) of bats at Tanoboase (p = 0.84).Overall, there was no statistically significant difference in seroprevalence between the two sites . At 37-Mpositive a compareanoboase b (chi-sqp = 0.83)  and the rainy season (April–November) ( = 0.83) .n = 11) had traumatic injuries of unknown origin. All brain samples were negative for lyssavirus antigen and RNA using FAT and the RT-PCRs, respectively.Forty-six bats were examined post-mortem, of which 38 bats had been collected from the 37-Military Hospital roost and eight bats collected from Tanoboase. Thirty-three bats were found dead, while 13 bats were found moribund and were euthanized, following clinical assessment, on welfare grounds . Most mon = 62; 2013—42.7%, n = 164; 2014—47.4% n = 78). These results, taken together with similar LBV serology results reported previously [E. helvum population. Previous studies have reported serological evidence of exposure to LBV in different E. helvum populations, including in Nigeria , Ghana , and Kenya  [A total of 304 bat sera was collected and tested for LBV neutralizing antibodies from 2012 to 2014. Of the 304 sera tested, 136 tested positive  . Seropren = 102) ,17,18. TE. helvum populations and possibly also those of other bat species.The size of populations involved within the roosts studied and the movement between defined roosts and other roosting sites that remain undefined during migratory periods may influence virus exposure and hence seropositivity. There was no difference in seroprevalence between the two field sites when assessing data comparing sex and age of sampled bats. However, this species is migratory and individuals may travel long distances to feed  perhaps Lasiurus cinereus) [Of the 294 sera from wild-caught bats tested of known age class, 134 (45.5%) were serologically positive for LBV neutralizing antibodies. There was no significant difference in the seroprevalence detected between the different age groups tested. The slightly higher proportion of seropositive adult bats (0.46) over juvenile bats (0.43) are similar to levels reported in studies addressing serological positivity to RABV in hoary bats (inereus) ,23. The The effect of migration and coloniality on infection dynamics is not known. Reduced migration and population connectivity has been suggested as a mechanism that could increase the amplitude of seasonal outbreaks and increase the probability for spillover of pathogens to other species, such as human beings . The absThe current understanding of lyssavirus serological positivity within bat populations is poor. Persistence of neutralizing antibodies to lyssaviruses for longer than 12 months has been described previously ,26. The The distribution of LBV lineages across Africa is diverse (reviewed in ). In GhaWhere higher titres are seen, it may indicate repeated exposure to antigenically-similar viruses that drive an anamnestic response. A multiple exposure infection study with rabies virus in bats  demonstrE. helvum is widely hunted in Ghana [n = 567) succeeded in isolating LBV in one E. helvum bat [In an attempt to detect live virus in bats at each study site, diseased and dead bats were assessed for the presence of virus antigen and nucleic acid in brain material. Clinical signs observed in moribund bats included paralyses, ataxia, weakness, vocalization and self-urination. The inability to detect LBV antigen or RNA in the brain samples of moribund or dead bats supports previous findings  that thein Ghana . Many balvum bat .E. helvum and other bat species was previously demonstrated in Ghana [E. helvum in Ghana [E. helvum colonies provides evidence of long-term maintenance and circulation of LBV in this bat population. Demonstrating the maintenance of antibody titres within bat populations is important as it indicates that individual bats are being re-exposed to virus, which in turn suggests that bats are shedding virus and are exposing conspecifics. Whilst this perhaps is not surprising, demonstrating what appears to be a stable level of seroprevalence in a population is important. The mechanisms of transmission of bat lyssaviruses remain ill-defined but minimally, the data presented here reiterate that virus continues to circulate within the bat population, which is important in our understanding of the relationship between bats and lyssaviruses. A high prevalence of antibodies against Lagos bat virus (LBV) in in Ghana ,30. Furtin Ghana . The curOngoing studies have indicated the occurrence of close interactions between chiropteran, domestic animal, and human populations. Current methods used to confirm rabies virus infection in people and terrestrial carnivores are unable to differentiate between different lyssavirus species. As human consumption of bats is practiced in many parts of Ghana , the pot