Astroviruses are enterically transmitted viruses that cause infections in mammalian and avian species. Astroviruses are nonenveloped, icosahedral viruses comprised of a capsid protein shell and a positive-sense, single-stranded RNA genome. The capsid protein undergoes dramatic proteolytic processing both inside and outside of the host cell, resulting in a coordinated maturation process that affects cellular localization, virus structure, and infectivity. After maturation, the capsid protein controls the initial phases of virus infection, including virus attachment, endocytosis, and genome release into the host cell. The astrovirus capsid is the target of host antibodies including virus-neutralizing antibodies. The capsid protein also mediates the binding of host complement proteins and inhibits complement activation. Here, we will review our knowledge on the astrovirus capsid protein (CP), with particular attention to the recent structural, biochemical, and virological studies that have advanced our understanding of the astrovirus life cycle. Astroviridae is divided into two genera: genus Mamastrovirus including viruses that infect mammals, and genus Avastrovirus including viruses that infect avian species. Astrovirus infections in birds cause diverse pathologies including enteritis, hepatitis, and nephritis. Astrovirus infections in mammals typically cause gastroenteritis and in rare cases cause neurological syndromes and encephalitis. Human astroviruses (HAstV) are recognized as a leading cause of childhood viral gastroenteritis, with eight canonical serotypes (HAstV-1 to HAstV-8) and several noncanonical human genogroups.The family Astroviruses are nonenveloped, T = 3 icosahedral viruses with a positive-sense, single-stranded RNA genome of 6–8 kb . The The astrovirus CP is synthesized from the viral subgenomic RNA (sgRNA) and is 72–90 kDa ,4,5,6 Fa. In genIn HAstV-infected Caco-2 cells , the 90 kDa HAstV capsid protein (VP90) is produced and assembles into viral particles ,9 Figur. AnalysiCaspase cleavage is required for HAstV release from cells, since the presence of caspase inhibitors drastically reduces the release of virus particles ,12 Figuc. While 5-fold [Once released outside of the host cell, immature HAstV particles comprised of VP70 are further processed by extracellular proteases to produce mature, infectious HAstV particles d. In cel5-fold ,15,16. F5-fold . Similar5-fold  . N-term5-fold . VP34 enastron means star in Greek). Infectious HAstV particles grown in cell culture and visualized by electron microscopy were observed to be ~41 nm in diameter and form spherical particles studded with spikes [In 1975, astroviruses were first visualized by negative-stain electron microscopy in stool samples of children with diarrhea and vomiting . Virus ph spikes .In the past five years, several exciting new structural studies have revealed high-resolution information about the astrovirus capsid protein and its assembly into immature and mature HAstV particles. First, the structures of immature (VP70) and mature, infectious (trypsin-digested) HAstV particles were resolved to moderate ~25 Å resolution by cryo-electron microscopy (cryo-EM) . Both imHigh-resolution information about the structural domains of the astrovirus capsid protein has been obtained by X-ray crystallography. The HAstV capsid core domain (HAstV-1 residues 80–411) forms the T = 3 icosahedral core of the virus particle. The structure of the HAstV capsid core reveals two linear subdomains: a typical jelly-role β-barrel found in many virus capsids (inner core) and a squashed β-barrel (outer core) ,27 Figud. MappinInterestingly, the structure of an avian (turkey) astrovirus capsid spike domain reveals a striking structural divergence compared to the structure of the human astrovirus capsid spike domain  Figure f. While Hepeviridae family. Like the astrovirus, HEV is an enterically transmitted, nonenveloped, positive-sense, single-stranded RNA virus. There are clear similarities in structural organization of the capsid proteins from the astrovirus and HEV, including positively charged N-termini, conserved inner and outer core domains, variable spike domains, and C-termini that are proteolytically cleaved. As a result, the immature HAstV particle shows a striking resemblance to the T = 3 icosahedral HEV virus-like particle [In all structural studies that have been carried out, there are notable similarities to the capsid of hepatitis E virus (HEV), a member of the particle   HAstV results in the generation of polyclonal antibodies that can be used to detect HAstV ,42,43. PThree studies have reported the generation of anti-HAstV mouse monoclonal antibodies (mAbs) ,23,41. TTwo studies reported the generation of anti-HAstV neutralizing mAbs that neutralize HAstV infectivity in cell culture ,23. In oFinally, the neutralizing mAb PL-2 (isotype IgG2a) was reported to selectively neutralize serotype HAstV-2 . mAb PL-Both human and avian astrovirus infection induces limited inflammation and cell death in intestinal tissues, suggesting that astroviruses may disrupt the innate immune system during infection ,47. One Analysis of the HAstV CP sequence revealed limited sequence homology between HAstV CP residues 79–139 (located in the CP inner core domain) and human neutrophil defensin-1 (HNP-1), a known inhibitor of C1 and MBL . A peptiThe early interactions of the astrovirus with its host cell have been poorly characterized. The first step in the virus replication cycle is the attachment of the virus capsid to a receptor on the cell surface f. HoweveThere is evidence that the HAstV CP spike contains a receptor-binding site. Initial evidence came from antigenic and sequence analyses, where neutralizing monoclonal antibodies that immunoprecipitate the CP spike domain (VP25 and VP27) were found to block HAstV attachment to host cells . These fThe largest and most surface-exposed putative receptor-binding site, the P site/Site 1, is comprised of several conserved polar and charged amino acids, which are characteristic for carbohydrate binding  Figure d. This sMature (trypsin-digested) HAstV-8 has been estimated to attach to cultured Caco-2 cells with a half-time of approximately 10 min . After bThe early interaction of HAstV with the host cell induces the activation of the extracellular signal-regulated kinase (ERK1/2) and the phosphoinositide 3-kinase (PI3K) pathways ,60 Figug. HAstVsOne of the hurdles to significant progress in our understanding of early interactions of the astrovirus with host cells has been the difficulty to obtain sufficient amounts of purified virus ,22, howeOverall, recent studies on the astrovirus CP have unveiled exciting new insights into the virus structure, proteolytic maturation and infectivity, interactions with host antibodies and complement, and entry into host cells. These studies have advanced our understanding of the astrovirus life cycle and have afforded new tools and methods to study the virus. These studies have major implications for the development of therapies to prevent and treat astrovirus infection. Nevertheless, there are still many significant questions that remain unanswered, including:What is the role of the acidic region at the C-terminus of the CP?What is the mechanism of astrovirus release from the host cell?Which extracellular protease(s) play a role in HAstV maturation in vivo?What is the mechanism by which protease maturation increases HAstV infectivity?Which of the protease cleavage sites are essential for HAstV infectivity?Capsid assembly and proteolytic processing:Are the spike dimers homodimeric for VP27 or heterodimeric for VP25 and VP27?What are the structures of CPs from other sequence-divergent astroviruses?What are the high-resolution structures of the immature and mature astrovirus particles?Capsid structure:Where do other neutralizing and non-neutralizing antibodies bind?What are the mechanisms by which neutralizing antibodies block infection?Do antibodies protect from astrovirus infection in vivo?What is the role of CP inhibition of complement activation in astrovirus pathogenesis?Capsid interactions with antibodies and complement:What is the astrovirus host cell receptor and do astroviruses use co-receptors?Do glycans play a role in virus cell entry?Where are the receptor binding site(s) on the CP surface?What viral factors trigger activation of the ERK1/2 and PI3K pathways and how do they facilitate virus entry and replication?How and where is the astrovirus genome released into the cytoplasm?How does the CP modulate tight junctions and intestinal barrier permeability?Virus entry into cells:


Implant-related infections, including those of peri-prosthetic joint (PJIs), osteosynthesis and other biomaterials, are biofilm-related. Pathogen identification is considered the diagnostic benchmark; however, the presence of bacterial biofilms makes pathogen detection with traditional microbiological techniques only partially effective. To improve microbiological diagnostic accuracy, some biofilm debonding techniques have been recently proposed. Aim of this health economics assessment study was to evaluate their economic impact on hospital costs.Direct and indirect hospital costs connected with the routine introduction of sonication and dithiothreitol treatment applied to hip and knee PJIs and of tissue cultures were examined. In particular the consequences of diagnostic inaccuracy, the opportunities, costs, and risks of each technique were calculated.Considering an average of five samples per patient, processed separately with traditional tissue culture with or without sonication of prosthetic components, or pooled together using the MicroDTTect device , the overall mean direct cost per patient was € 397 and € 393 for sonication or MicroDTTect, respectively, compared to € 308 for traditional tissue cultures. In terms of opportunity costs, MicroDTTect was the most effective technique, allowing for a 35% or 55% reduction in time required for sample treatment, compared to tissue cultures combined or not with sonication, respectively.Pooling together direct and indirect costs associated with false positive and negative results of the different diagnostic techniques, unnecessary medical treatments and possible medical claims, MicroDTTect or sonication become increasingly cost-effective when the extra-costs, generated by diagnostic inaccuracy of traditional tissue culture, took place, respectively, in 2% or 20% or more of the patients.This is the first study specifically focused on the economic impact of the routine clinical use of microbiological antibiofilm sampling and processing techniques in orthopaedics. Although our results may suffer from a potential country and hospital bias, as the data collection process for direct and indirect costs is specific to each institution and country, this analysis highlights the potential economic advantage to hospitals associated with the routine introduction of antibiofilm techniques for microbiological diagnosis of PJI. Implant-related infections, including those affecting joints (PJI), osteosynthesis and other biomaterials, are biofilm-related –3.Intra-operative clinical diagnosis and pathogen identification is considered the diagnostic benchmark , 5, howeComparison of DTT treatment with sonication has evidenced a similar rate in FP 5.88%) and a higher frequency of FN with sonication 28.6%) than with DTT (14.3%) . Superio% than wi8% and a To the best of our knowledge, only two contributions evaluate the economic impact of different diagnostic techniques for PJIs. Diaz Ledezma et al.  developeHence, this analysis aimed to fill this gap by assessing the total economic impact of introducing antibiofilm microbiological techniques to diagnose hip and knee PJIs, comparing their direct and indirect hospital costs with the current economic standard offered by routine microbiology testing. In our setting, the main source of additional costs is precisely a wrong diagnosis, thus ruling out all other possible complications that may emerge in the treatment of PJIs .The decision-analytic modelling approach used to conduct the cost-effectiveness analysis presented here was based upon the framework of Diaz-Ledezma et al. , who assOur analysis focused instead on three different intra-operative diagnostic approaches. We compared, in fact, the economic impact of tissue culture with that of two antibiofilm technique: sonication, and DTT, using MicroDTTect® system , a close system for intra-operative tissue and implant sampling, transport and antibiofilm processing.For each technique, we evaluated the consequences of diagnostic inaccuracy, relatively to routine PJI microbiological diagnosis. In a similar vein to Diaz-Ledezma et al. , we defiThe opportunities of each technique reflected the opportunity cost of each technique, as expressed by the Laboratory time-opportunity cost , which is the time needed by biologists and technicians to perform the diagnostic activity.With regards to the cost assessment, we adopted a broader definition which distinguished between direct and indirect costs. The former cost voice included both the material and other operational costs, whereas the latter referred to the additional costs  that might stem from a wrong diagnosis. Moreover, we distinguished between the indirect estimated medical costs resulting from diagnostic inaccuracy  and the indirect legal costs, that accounted for the incidence and costs related to medico legal claims following a wrong diagnosis. The resulting medical and legal costs were weighted by the probability of having a wrong diagnosis. Hence, our definition of risk had a double interpretation: if, on one side, it reflected the probability of a wrong diagnosis, just as in Diaz Ledezma et al. , on the PJIs were defined according to the criteria established by the International Consensus Meeting of Philadelphia: presence of a sinus tract communicating with the prosthesis, isolation of the same microorganisms from at least 2 samples or fulfilment of three of the following requisites: acute inflammation evidenced by histology of periprosthetic tissues, one positive culture, high synovial leukocyte count or a ++ result on the leukocyte esterase strip, high percentage of synovial neutrophils, elevated erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) . All theAs displayed in Fig. All direct costs were collected at the Galeazzi Orthopaedic Institute in Milan, Italy, on a case study of 20 patients, for a total of 100 tissue samples and 20 implants. These voice costs cover costs for materials (plastics and culture media) and laboratory staff activities . To this aim, we reviewed the impact of the L-TAT in our microbiology laboratory, which was defined as the time taken from sample arrival to the laboratory to the availability of a report to the clinician. This was calculated by prospectively documenting the dates and times when the sample was received in the laboratory, the report signed by the microbiologist, the report sorted by the laboratory clerical staff, and the final report received by the clinician . MoreoveThe additional medical costs that arise from microbiological diagnostic inaccuracy were considered as indirect costs and were extracted from the most recent literature. In fact, failure to identify the pathogen in a PJI (FN) may result in no or inadequate antibiotic treatment and higher risk of infection recurrence/persistence. Samples contamination and false pathogen identification (FP) may be associated with unnecessary antibiotic therapy and post-surgical surveillance and monitoring. Additionally, if the prosthesis was infected but the result was a wrong isolate, i.e. the isolate was not the real pathogen but a contaminant, the patient may have received inadequate or ineffective antibiotic treatment, with possible treatment failure and infection recurrence or persistence. In extreme circumstances, the patient may require further surgery to treat the misdiagnosed PJI.Before quantifying precisely these additional medical costs, we first had to assess the diagnostic accuracy of each technique, as prescribed by the algorithm displayed in Fig. We then proceeded with the assessment of the indirect additional medical costs that each wrong diagnosis generates  to 100% . In this analysis, we considered four different mitigation factors  to account for an increasingly share of wrong diagnosis actually generating additional costs.Since not all wrong diagnosis will actually generate additional and indirect costs, we applied to the total indirect cost of a wrong diagnosis a “The mitigation factor was then applied to the overall cost of intra-operative diagnostic inaccuracy for the treated patients, as derived in step 5. The resulting mitigated cost was then given by the following equation . On aggregate terms, MicroDTTect led to a 35% decrease of the time required for processing and analysing samples, relatively to tissue culture, or 55% if compared to sonication.The mean calculated direct costs of standard tissue culture, including all consumables and L-TAT, was € 61.5 per processed sample or € 307.5 per patient (5 processed samples) , 30. WheDiagnostic accuracy of the techniques under study was evaluated according to Drago et al.  and summFailure to identify the responsible for a PJI or a FN result have the following potential clinical consequences: possible inadequate antibiotic treatment and risk of infection recurrence/persistence. The costs resulting from inadequate and often prolonged antibiotic treatment were estimated on average as € 4500 (range € 2000 to € 8000), according to Hernández-Vaquero (2013) , while tSample contamination and FP results may also lead to prolonged antibiotic treatment; moreover, unnecessary post-surgical surveillance and monitoring was associated to average costs of € 4000 (range € 3000 to € 5000) , 27, 28,N = 3) or knee (N = 10) arthroplasty revision were settled at our Institute, due to post-surgical infections; the total amount paid over these three years was € 1,192,456 or € 298,114/year, with an average amount of € 91,727 paid to the recipient. Since 28% of these post-surgical infections were misdiagnosed PJIs, due to intra-operative FN results, the estimated medico-legal cost of diagnostic inaccuracy was € 333,887/year or € 650.85/patient . A reduction of FN results from 28% to 14%, as determined by the introduction of the MicroDTTect, would potentially result in a medico-legal cost per patient of € 325.42.Concerning medico-legal costs arising in the period considered (2010–2013), 1083 hip or knee revision surgeries were performed at our Institute; of these, 513 were considered as aseptic and 570 septic. During the same years, a total of 13 medico-legal claims following a hip . Sonication and MicroDTTect, by reducing the incidence of FP results from 23% to 6%, could abate the legal cost per patient to €125.52.We then weighted the medical and legal costs likely arising from a wrong diagnosis by the effective incidence of FN and FP results  used for our calculations are hospital and country specific. In fact, hourly labour cost and direct costs of materials may differ from one country to the other or among various institutions , 36. MorWith regards to the direct costs and opportunities collection process, we acknowledge the limited sample size (20 patients). It is worth stressing that the actual sample size is greater than 20, as it consists of 5 tissues samples and 1 prosthetic impact, for a total of 100 + 20 observations. Furthermore, we would recall that this is a pilot study, whose methodological framework can be rapidly and easily extended to a larger sample.Also, the indirect costs’ calculation can be prone to bias and errors. In particular, we have no data concerning the real-life impact of misdiagnosis and related costs. Hence, in the present study, to calculate the relative economic burden of wrong treatments, originating from inaccurate diagnosis, several assumptions have been made, based on previous papers and reports; these assumptions may not be completely accurate and reproducible for every institution and for every country.Similarly, concerning the quantification of the medico-legal risk, this has been calculated on the basis of our Institute database, which may not be assumed as necessarily representative of other hospitals. Furthermore, while wrong diagnosis and infection are a well-known potential trigger of medico-legal claims –41, the This cost analysis highlights the potential benefits of antibiofilm technologies that may offer a more accurate pathogen identification, leading to an improvement in the management of implant-related infections with a substantial economic balance or advantage.


Cobalamin deficiency  was found among 11%, whereas 24% of the infants had plasma cobalamin concentrations between 148–221 pmol/L. Elevated total homocysteine (>10 µmol/L) and methylmalonic acid (>0.28 µmol/L) indicating functional cobalamin deficiency were found among 53% and 75% of the infants, respectively. Based on a combined indicator of cobalamin status, 58% were found to have low cobalamin status. However, folate deficiency (<10 nmol/L) was not found as the lowest value of plasma folate was 20.7 nmol/L. It is important to examine the extent to which poor cobalamin status during infancy has immediate or long-term consequences.Cobalamin and folate are crucial micronutrients during infancy and they are required for growth and cognitive development. Due to the monotonous and predominantly vegetarian-based complementary feeding and poor maternal micronutrient status, infants from low- and middle-income countries are susceptible to cobalamin deficiency. However, data on plasma cobalamin and folate and the functional markers methylmalonic acid and total homocysteine from breastfed infants in Nepal are still needed. We collected plasma samples from 316 6–11-month-old breastfed infants with a length-for-age of less than minus one  Due to s system ,5,6,7,8.s system ,11,12,13s system ,15. Mores system , exists s system . This untem [12) .For infants of 7–12 months, 0.7 μg of cobalamin per day is set as the requirement, but achieving this amount from vegetarian-based complementary feeding is unlikely ,20. BreaPoor cobalamin status in combination with high serum folate levels may exacerbate neurological damage and could also worsen poor cognitive function in adults . In the 2) (NDHS 2016). The prevalence of anemia is also high affecting approximately 53% [In Nepal, stunting is a common form of malnutrition among children affecting approximately one third of children under the age of 5. Of women, 17% are undernourished . Written informed consent from one of the parents  was obtained. For illiterate parents, we took thumbprints in the presence of an impartial witness. In case of anemia (defined as hemoglobin <11 g/dL), iron supplementation was given according to national guidelines. Implementation of the study was as stated in the latest version of the Helsinki Declaration.The main study has obtained approval from the Nepal Health Research Council  in Nepal and from the Regional Committee for Medical and Health Research Ethics (REC #2014/1528) in Norway and registered at Lactobacillus leichmannii. The assay has been adapted to a microtiter plate format and is carried out by a robotic workstation. The between-day coefficient of variation ranged from 2–8% for MMA and tHcy and was 5% for both cobalamin and folate. All biochemical analyses were done at Bevital Laboratory, Bergen, Norway .Most blood samples (80%) were taken at the study clinic after 12 noon, and collected from one of the cubital veins into heparinized polypropylene tubes  which were protected from direct sunlight exposure. A total of 24% of the infants did not have any meal or snacks, during the day before blood sampling, whereas only 2% were not breastfed. The hemoglobin concentration was analyzed immediately following blood sampling with Hemocue , which was calibrated as per the guidelines defined by the manufacturer. The heparinized blood was centrifuged for 10 min at 700 g within 10 min after venipuncture and kept at −196 °C liquid nitrogen located at the field site clinic. Plasma was separated, transferred into polypropylene vials  and stored at −80 °C in Nepal until it was transferred to Norway on dry ice. Plasma tHcy and MMA were analyzed by gas chromatography-mass spectrometry (GC-MS) based on methylchloroformate derivatization . The pla12) is calculated based on the three biomarkers  as suggested by Fedosov et al. [z-scores below −2 z-score, respectively, as compared with WHO growth chart [The cut-off values for altitude adjusted 1400 m) anemia, low cobalamin, low folate, high MMA and high tHcy are as per the WHO/CDC guidelines: 11.3 g/dL, <148 pmol/L, <10 nmol/L, >0.28 μmol/L, and >10 µmol/L, respectively ,40. Func0 m anemiv et al. . A scoreth chart . Breastfth chart .12. To identify determinants for cobalamin and biomarker status, we used the 3cB12 and log-transformed tHcy, MMA and cobalamin concentrations as the dependent variables in multiple linear regression models. Candidate variables for these regression models are those listed in a priori to include age of the child (in months) in all regression models. Statistical analyses were undertaken using Stata®, version 15 .Descriptive statistics such as means, medians, percentiles, interquartile ranges, standard deviations and 95% confidence intervals (CI) were calculated for baseline characteristics and for plasma concentrations of cobalamin, folate, MMA, tHcy, and Hb, as well as the combined indicator 3cBThe mean age of the 316 enrolled infants was 8.3 months and 53% were male. One in every second family lived in a rented house and 50% were living in joint families. Most of the children were delivered at health centers (95%) with normal vaginal delivery (74%). At birth, 17% of the children had low birth weight (birth weight <2500 g) . Almost Janamghuti) was also given to 54% of infants, often within 3 months after delivery. One third of the infants were stunted (<−2 z-score), whereas the prevalence of underweight infants was 19% (Most of the infants (85%) received breast milk within 24 h of delivery (56% within one hour), but the prevalence of exclusive breastfeeding at 3 and 6 months was only 43% and 10%, respectively. Early introduction of homemade cereals (lito) or Cerelac (Nestlé) was a common practice as 40% of infants were given these foods by 3 months of age but this is recommended only after 6 months of age. An Ayurvedic liquid ( was 19% .12 was −0.70 (0.84). The percentile distributions of these bio-markers are presented in p = 0.002) in the fasting samples , none of other bio-markers were different according to the history of food/snack consumption before blood sampling. Based on plasma cobalamin concentration <148 pmol/L, only 11% of infants were cobalamin-deficient, whereas 24% had cobalamin concentration between 148–221 pmol/L. None of the infants were found to be folate-deficient when using the conventional cut-off value of folate concentration (<10 nmol/L). The folate status was found to be very good in this population ranging from 20.7 to 150.8 nmol/L. Three-fourths of the infants had elevated MMA levels (>0.28 μmol/L), while 53% had tHcy > 10 μmol/L indicating functional cobalamin deficiency  concentration of cobalamin, folate, tHcy and MMA were 271.6 (257.8–286.2) pmol/L, 61 (59–64) nmol/L, 10.8 (10.3–11.4) µmol/L, and 0.50 (0.46–0.55) µmol/L respectively. The mean (SD) 3cBficiency . However12, tHcy, MMA, and cobalamin derived from multiple linear regression analyses are presented in 12 increased with age whereas there were no significant association between age and MMA. Infants from a family staying on rent had lower 3cB12, and higher MMA and tHcy than those from families staying in their own property. Higher tHcy concentrations were also found among infants who were exclusively breastfed for three months or more. Stunted infants had higher MMA concentrations, whereas underweight infants had higher plasma cobalamin concentrations.Adjusted (and back-transformed when relevant) regression coefficient for 3cB12), 58% had low cobalamin status. One in every second infant had high tHcy or MMA suggesting that functional cobalamin deficiency is wide-spread. It should be noted that for cobalamin and the functional markers, there are no well-established cutoff values in infants. Various cutoffs and combinations have been suggested. Bjørke-Monsen et al used a cutoff of 6.5 µmol/L tHcy which was the 95th percentile in infants who had received an injection of vitamin B12 [In this cross-sectional analysis consisting of 6–11 months old breastfed infants included over a period of 12 months, we observed cobalamin deficiencies ranging from 11–75% depending upon the indicator that was used. None of the infants had folate deficiency. Based on the combined biomarker   or from z-scores of <−1, which may limit the generalizability of our findings. This was also reflected in the high prevalence of stunting (33%), in comparison to the 17 to 20% range in this age group in the last demographic survey of Nepal [We also recognize some limitations of our study. Due to our enrolment criteria in the main study, our samples are based on infants with length-for-age of Nepal . Howeverof Nepal .12) more than 50% of the infants with length for age <−1 z-score may be cobalamin-deficient, which is probably due to sub-optimal maternal nutritional status during pregnancy and lactation or predominantly vegetarian complementary feeding. It is important to identify immediate and long-term consequences of poor cobalamin status during infancy.Based on measurement of functional markers of cobalamin status, such as tHcy and MMA and the combined cobalamin indicator (3cB


Is Mayaro virus infection the latest in a series of new arbovirus diseases expanding across the Western Hemisphere?Since the launch of the 2000 Millennium Development Goals, we have seen an explosion of new arboviruses affecting the Americas. According to the Global Burden of Disease Study 2015, there has been a 143.1% increase in dengue fever cases between 2005 and 2015 . The facBut it does not look like the emergence and rapid expansion of new human arbovirus infections will abate anytime soon. In addition to Bourbon, Cache Valley, chikungunya, Heartland, Itaqui, Oropouche, Powassan, and Zika viruses , one of Like chikungunya virus, Mayaro virus is an alphavirus and a member of the Togaviridae family of enveloped RNA viruses  5, 6]. , 6. 5, 6Haemogogus mosquitoes and likely involves forest-dwelling nonhuman primates and possibly migratory birds as animal reservoirs . Of. Of19]. Could Mayaro virus infection become the “next chikungunya” in the Americas? Drs. Mario Luis Garcia de Figueiredo and Luiz Tadeu Moraes Figueiredo from Brazil’s prestigious University of Sao Paulo have suggested that both of these alphaviruses can “mutate and/or adapt to new zoonotic cycles and thus acquire a higher potential for emergency” to cause significant epidemics . This isThe Caribbean and tropical regions of Latin America have now become high-risk areas for the emergence of Mayaro virus infection epidemics. The countries at highest risk of emergence are also resource limited and lack diagnostic capacity at the local level; therefore, it is highly likely that any Mayaro virus infections would be presumed as chikungunya virus due to the similarity of clinical symptoms, possibly allowing rapid transmission and subsequent spread throughout Central and North America to occur under our global health radar.With locally acquired infections of both chikungunya and Zika virus now occurring in Texas and Florida in the US, we would presume these areas to also be at high risk for emergence of Mayaro virus. In May of 2016, the Texas Department of State Health Services reported on the first locally acquired case of chikungunya in Cameron County, Texas , while 2Emerging arbovirus infections have become a “new normal” for the Americas , includi


Andrena cineraria, Andrena fulva, Andrena haemorrhoa, Bombus terrestris, Bombus cryptarum, Bombus pascuorum, Osmia bicornis, and Osmia cornuta) sampled from four different localities in Belgium. Across the RNA sequencing libraries, 88–99% of the taxonomically informative reads were of the host transcriptome. Four viruses with homology to insect pathogens were found including two RNA viruses , a double stranded DNA virus (family Nudiviridae) and a single stranded DNA virus (family Parvoviridae). In addition, we found genomic sequences of 11 unclassified arthropod viruses , seven plant pathogenic viruses, and one fungal virus. Interestingly, nege-like viruses appear to be widespread, host-specific, and capable of attaining high copy numbers inside bees. Next to viruses, three novel parasite associations were discovered in wild bees, including Crithidia pragensis and a tubulinosematid and a neogregarine parasite. Yeasts of the genus Metschnikowia were identified in solitary bees. This study gives a glimpse of the microorganisms and viruses associated with social and solitary wild bees and demonstrates that their diversity exceeds by far the subset of species first discovered in honey bees.Bees are associated with a remarkable diversity of microorganisms, including unicellular parasites, bacteria, fungi, and viruses. The application of next-generation sequencing approaches enables the identification of this rich species composition as well as the discovery of previously unknown associations. Using high-throughput polyadenylated ribonucleic acid (RNA) sequencing, we investigated the metatranscriptome of eight wild bee species ( Transcriptomics is the study of all ribonucleic acid (RNA) molecules in a biological sample . In beesApis mellifera. These studies include investigations of the gut microbiome function organisms and viruses . This isfunction , host-pafunction  or both function .Bees interact with a remarkable diversity of microorganisms and viruses . These iIn this study, we aimed at exploring novel viruses and parasites associated with wild bees using high-throughput RNA sequencing. We adopted a polyadenylation enrichment strategy for two reasons: (1) to selectively enrich for RNA viruses with a polyadenylated genome (which are common in bees) or DNA virus transcripts and (2) to detect as well a significant fraction of the transcriptomes of eukaryote bee parasites and host. We increased the likelihood to encounter infected bees by pooling five individuals per sequencing library and sampling populations from different localities. Despite of the modest selection of only 8 of the nearly 400 wild bee species that occur in Belgium, the diversity of viruses and parasites associated with those species was high and comprised numerous novel associations to bees.Figure 1B); see also Bombus, Osmia, and Andrena were collected during the spring of 2015. In case of Bombus, only queens that fly early in the bumble bee life cycle could be sampled. Sampling authorization was obtained for protected areas in the southern provinces of the country. We only sampled on days with favorable weather conditions to ensure high bee activity. Bees were individually captured using a sweep net and directly euthanized by placing them on dry ice. At the end of the day, whole bees were stored in the lab at -80°C until further processing. The species determination was confirmed by sequencing of the COI barcode gene from a DNA extract of a single hind leg (see Supplementary File S1). Per locality, five individual bees of the same species were selected for RNA extraction. The species included were : Andrena cineraria (Acin), Andrena fulva (Aful), Andrena haemorrhoa (Ahae), Bombus cryptarum (Bcry), Bombus pascuorum (Bpas), Bombus terrestris (Bter), Osmia bicornis (Obic), and Osmia cornuta (Ocor). The species name abbreviation is extended by a number that refers to one of the four sampling localities, for example, Bter1 refers to B. terrestris sampled from locality 1 .Four sampling locations were selected in correspondence to historical collection sites in Belgium . We assumed that the head and abdomen tagmata consist of the predominant target tissues for pathogenic viruses and parasites including the alimentary tract, fat bodies, and hypopharyngeal glands. Hence, the thorax tagma was entirely (including wings and legs) left out in order to reduce sample complexity and host fraction. Accordingly, a single bee at a time was dissected on a -80°C surface and tagmata were directly submerged in QIAzol Lysis Reagent (Qiagen). In case of non-corbiculate Osmia and Andrena bees, external pollen was removed from the abdominal scopa using a sterile knife. Tissues were disrupted using a tissue homogenizer (Precellys) for 90 s at 5000 Hz in the presence of 250 mg 0.1 mm Ø zirconium beads and five 2.3 mm Ø stainless steel beads. RNA was extracted using the RNeasy Lipid Tissue Mini kit (Qiagen) following manufacturer’s instructions including a 15 min on-column DNase I (Qiagen) digestion step. RNA concentration was determined using an ND-1000 UV–Vis Spectrophotometer (Nanodrop Technologies). At this stage, the total RNA was pooled per 5 individuals (10 μg RNA each) per species group and per locality, resulting in 16 pooled total RNA samples. cDNA library construction and sequencing were performed at Omega Bioservices, Norcross, GA, United States (Supplementary File S1). The cDNA library preparation method included a polyA+ RNA selective enrichment step and preserved stranded information of the original RNA fragments. An average of 32.7 M (27.2–40.5 M) paired-end 100 bp reads per library was obtained.Prior to all tissue handlings, areas and pipettes were wiped with RNase Awayin silico protocols is given in Supplementary File S1. Briefly, after quality assessment, reads were trimmed and de novo assembled into contiguous sequences (contigs) using two independent algorithms, CLC Genomics Workbench 9 (Qiagen) and Trinity v2.2.0  of the non-redundant nucleotide and protein sequence databases using BLAST+ v2.2.31  was visualized as a heatmap using ggplot2 v2.2.1 , PCRs for sanger sequencing and GenBank submissions using proof-reading Platinum Pfx DNApol (Thermo Scientific), or in case of difficult templates using expand long template PCR system (Roche). Amplicons were cloned using the TOPO-TA cloning system (Life Technologies) and plasmids were extracted using the GeneJET Plasmid Miniprep Kit (Thermo Scientific). Purified plasmids were sequenced using M13 and custom primers at GATC Biotech .All primer sequences and corresponding polymerase chain reaction (PCR) parameters used in this study are included in Supplementary File S2. The viral replicase protein was used as a phylogenetic marker for RNA viruses, the NS1 protein for ssDNA viruses, and the DNApol, per os infectivity factor 2 (pif-2), and late expression factor 8 proteins for dsDNA viruses. Multiple sequence analyses were carried out using MAFFT v7 (Open reading frames (ORFs) were predicted using the online Expasy Translate tool. Conserved domains were predicted from amino acid (aa) sequences using the web-based Interproscan portal. Coverage graphs were exported from CLC Genomics Workbench v8.5 (Qiagen) after mapping paired-end reads to the contigs. Viral taxonomy is based on the International Committee on Taxonomy of Viruses website with the inclusion of the genus type species where possible. All virus abbreviations and accession numbers are summarized in MAFFT v7  and the MAFFT v7  and trimMAFFT v7 , a maximMAFFT v7  embeddedMAFFT v7 .PRJNA411946. Nucleotide sequences that were cloned and confirmed by Sanger sequencing have been submitted to GenBank under the following name and accessions: Tubulinosema sp. isolate Bpas2 (MF998087) and Apicystis sp. isolate Bpas1 (MF998086). Other GenBank submissions based on in silico assembled contigs are: A. haemorrhoa nege-like virus isolate Ahae2 (MF998082), Bee iflavirus 1 isolate Ahae2 (MF998083), Bee macula-like virus 2 isolate Ahae2 (MF998084), and B. cryptarum densovirus isolate bcry3 (MF998085). CLC de novo and Trinity contigs were stored at a freely accessible Figshare collection  and the average BUSCO notation was C:66.4% , F:15.3%, M:18.3%, n:4415. In full, out of the 4415 single-copy orthologs in the Hymenoptera lineage, 2930 (66.3%) were complete and single, 5 (0.1%) were complete and duplicated, 667 (15.1%) were fragmented, and 807 (18.3%) were missing. The transcriptome completeness was similar to an independent RNA-Seq study of B. terrestris queens  and nucleotide (nt) similarity to honey bee BQCV strains was low. Varroa destructor virus-1 (VDV-1) was detected in B. terrestris and O. cornuta. Read count was very low  but nt similarity to honey bee isolates was high. Intuitively, both BQCV and VDV-1 were present in very low copy number. Either the viruses were at an early infection stage in the bee host or they were associated with pollen  and Ganda bee virus (GABV). RT-PCR analysis confirmed SRBV in 66% individuals and GABV in 93% individuals . GABV is related to Apis bunyavirus 2 that was recently discovered in honey bees (Two honey bee RNA viruses were detected in wild bees. Black queen cell virus (BQCV) was found in sympatric h pollen . Two solh pollen  were alsney bees .Picornavirales), referred to as bee iflavirus 1 (BeeIV-1), was present in A. haemorrhoa. The virus is phylogenetically related to honey bee pathogenic viruses deformed wing virus, sacbrood virus, and slow bee paralysis virus . However, the partial polyprotein of BeeIV-1 had highest aa identity to Dinocampus coccinelae paralysis virus, a virus that induces behavioral changes in its parasitoid host , referred to as bee macula-like virus 2 (BeeMLV-2), was detected in multiple hosts B. pascuorum, O. cornuta, and A. haemorrhoa but was only present in high copy number in library Ahae2 . The recovered genome sequence of BeeMLV-2 was nearly complete and a similar organization was observed compared to related macula-like viruses . Based on RdRp protein phylogeny, BeeMLV-2 clustered close to members of Maculavirus in the family Tymoviridae and its closest relative was Bombyx mori macula-like virus . This virus was originally identified in cell cultures . The longest transcript had a similar genome organization to that of Ixodes scapularis associated virus-2 where the viral serine protease ORF1 overlaps the RdRp protein ORF2, a feature that is shared among sobemoviruses.Two contigs in it flies , mosquitit flies , ants, ait flies . PhylogeVirgaviridae were identified in all libraries except Aful1, Bter2, and Bpas2. Each bee species was associated with a particular nege-like virus. The genome sequence of viruses from different localities shared less than 1% sequence dissimilarity, which can be attributed to the natural variation in RNA viruses. The genome organization of four nege-like viruses are depicted in Figure 2A. Comparison of nege-like viruses from different bee species revealed strong analogy in genome organization and evolutionary relationship . Based on RdRp protein phylogeny, the A. haemorrhoa nege-like virus was related to the Sandewavirus phylogenetic group of negeviruses. The RdRp protein was most similar  to Tanay virus, a nege-like virus of mosquitoes . It is worth noting that the nege-viral sequences were not detected in nematode-associated B. terrestris and B. pascuorum libraries , despite of their relatively abundant presence in nematode-free libraries.Contigs related to insect-specific negeviruses and plant viruses of the family squitoes . The O. utumnata , dipterautumnata , and theuturalis . Bombus ematodes . The virO. cornuta libraries, one or two sets of contigs had homology to totiviruses. The most representative genome sequence was obtained in library Ocor2 with a length of 6.6 Kb . Genome organization is similar to that of Leptopilina boulardi toti-like virus (LbTV) where ORF1 overlaps ORF2 and translation of ORF2 is mediated by a -1 ribosomal frameshift. Sequences of libraries Ocor1 and Ocor2 are very similar (99% nt identity), but both are more dissimilar to the totivirus in library Ocor4 (93% nt identity) suggesting that different strains exist. Members of the family Totiviridae have a linear dsRNA genome of 4.6–6.7 Kb in size and are originally described from fungal hosts. However, several insect totiviruses have been discovered that currently fall apart in two groups, the one infecting Hymenoptera including parasitic wasps . In fact, Martinez et al.  and that infection positively correlates with developmental success  of O. bicornis consistently had around 80% aa identity to SRBV RdRp, nucleoprotein, and glycoprotein. Nine contigs (summed length 6394 nt) of A. cineraria had 36–42% aa identity to RdRp proteins of rhabdoviruses  including Wuhan ant virus and Apis rhabdovirus 1 and 2. Both rhabdoviruses were shown to elicit an anti-viral immune response in honey bees . OcNV is phylogenetically related to pathogenic nudiviruses in beetles . Because it is not possible to obtain the complete genome sequence of a DNA virus by RNA sequencing, we mapped the OcNV transcriptome to the complete genome of a related nudivirus  to estimate its completeness. Strikingly, OcNV homologs to all of the 20 core baculoviral genes shared among nudiviruses and baculoviruses  and one B. terrestris library (Bter1) were associated with viral sequences of ssDNA viruses of the family Parvoviridae. The closest relative (36% protein identity) was the ambidensovirus Diaphorina citri densovirus (DcDNV), although phylogenetic analysis of the replicase protein placed the B. cryptarum densovirus together with Lone star tick densovirus 1 (LstDNV-1) in a separate clade . A characteristic feature of ambidensoviruses is that structural (VP) and non-structural (NS) cassettes are transcribed from opposite DNA strands. In case of DcDNV, the VP and NS cassettes are separated by 21 nt , we anticipated a non-overlapping, ambisense genome organization similar to that of DcDNV. Indeed, gap-spanning PCR on a genomic DNA extract of an infected bee and Sanger sequencing of the amplicon confirmed that both transcripts originate from the same DNA template but are in opposite orientation and separated by 30 nt . Densoviral contigs were also found in library Bter1 with 70–75 and 88% nucleotide identity to the densoviral NS and VP transcripts in Bcry3, respectively, although the coverage was much lower. RT-PCR revealed that all (5) B. cryptarum queens and one B. terrestris queen were associated with a densovirus . Despite of the broad host range of densoviruses in insects, none so far have been reported in bees. The relation of densoviruses of the subfamily Densovirinae to their arthropod host ranges from mutualistic . Because the primary source of plant viruses in bees is likely to be dietary pollen, we anticipated a correlation between the abundance of plant virus and pollen-derived reads. Surprisingly, no plant viruses were detected in A. haemorrhoa and A. cineraria despite of the larger proportion of pollen-derived reads in these hosts. Four of the plant viruses detected in this study belong to the family Secoviridae  of which AMV and CLRV are nepoviruses. Recently, a plant pathogenic nepovirus (Secoviridae) was demonstrated to replicate in and negatively affect honey bees (Andrena spp.Seven well-characterized plant viruses were detected: cherry virus A (CVA), Prunus dwarf virus (PDV), cherry leaf roll virus (CLRV), Prunus virus F (PVF), strawberry latent ringspot virus (SLRSV), arabis mosaic virus (AMV), and crimson clover cryptic virus 2 (CCCV-2). Except from CCCV-2, all are +ssRNA viruses and some are serious pathogens of crops . Only thney bees . We coulAndrena libraries, low-abundant sequences were detected with up to 40% protein homology to narna-like viruses isolated from yeasts and crustaceans. Narnaviruses (Narnaviridae) have a monopartite, linear +ssRNA genome of 2.5–2.9 Kb in size and naturally infect fungi. Two fragmentary, narna-like viral contigs in A. fulva had a summed length of 2077 nt and a candidate partial genome sequence of 2.6 Kb was manually refined.In two Crithidia bombi was commonly detected in B. terrestris, B. pascuorum, and O. cornuta , and typically occupied less than 0.6% of sequencing libraries. A trypanosome nearly identical to Crithidia pragensis was identified in O. cornuta (Table 1). Crithidia pragensis is originally described from the fly Cordilura albipes in Central Europe . Either the microsporidium Nosema thomsoni or a very related species was associated with A. haemorrhoa . It was most identical to the Nosema associated with Andrena vaga , which, respectively, are low-virulent parasites recently described from Bombus atratus in S-America . An apicomplexan parasite equally similar to Apicystis bombi as Mattesia geminata (Table 3) was associated with two B. pascuorum libraries. However, microscopical examination of the infected thoraces revealed morphisms similar in shape as the navicular sporocysts of A. bombi .The trypanosome l Europe . The libena vaga . A third-America  and the -Siberia . The TubMetschnikowia pulcherima, Metschnikowia chrysoperlae, and Metschnikowia (Candida) pimensis. All are members of the M. pulcherima clade which is primarily associated with nectar and fruit-feeding insects , but was absent elsewhere . The nematode transcriptome fraction occupied 5.5 and 2.3% of the sequencing library, respectively. Sequence comparison of conserved house-keeping transcripts revealed that S. bombi of interspecific hosts was genetically distinct, which suggests a host-specific haplotype structuring of this parasite. Further, the tracheal mite Locustacarus buchneri was molecularly detected in B. pascuorum queens . Finally, the larvae of two conopid fly species, Myopa testacea and Myopa tesselatipennis, were detected as abdominal parasites. Myopa testacea was present in one O. cornuta female and three A. cineraria females whereas M. tesselatipennis was present in two A. haemorrhoa females . The fly transcriptome typically occupied less than 3% of the sequencing library.The parasitic nematode A. cineraria, A. haemorrhoa, A. fulva, B. cryptarum, and B. pascuorum) were not sequenced earlier. We identified over 20 associated viruses, 10 different species of bee parasites, and associated fungi. Two unknown RNA viruses in A. haemorrhoa (bee iflavirus 1 and bee macula-like virus 2) and two unknown DNA viruses in Osmia spp. and Bombus spp.  were related to other pathogenic viruses of insects. These viruses may be of pathological importance to the studied bees and other bee species, or in case of the DNA viruses to the rearing of the commercial pollinator species O. cornuta, O. bicornis, and B. terrestris. However, the potential pathological association of the viruses has to be validated by follow-up studies that include isolation of the virus particles and experimental inoculation in the bee host. We also discovered the widespread occurrence of nege-like viruses, negative-stranded RNA viruses, and other insect-specific viruses, which is in congruence with recent large-scale, viral metagenomics studies in arthropods. However, the role of these viruses and their significance with respect to insects in general is unknown. Further, next to typical bee parasites, we detected for the first time in Europe a Tubulinosema parasite in a bumble bee host, a fly trypanosome parasite that infected O. cornuta, and a neogregarine parasite that is related but molecularly dissimilar to A. bombi. We encourage future prevalence studies to include the novel associations of viruses and parasites reported here to learn more about their geographical distribution and impact on pollinator health.In this study, we combined a polyadenylated RNA enrichment with high-throughput RNA sequencing to explore the metatranscriptome of eight social and solitary wild bee species, of which the transcriptomes of five species (DdG, GS, and KS conceived the study. KS performed the sampling and practical work. DdG, FF, and KS wrote the manuscript. DdG, GS, FF, and KS revised 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.


Equus caballus) to equine practitioners and researchers cannot be ignored. An unevenly distributed population of equids harbors numerous diseases, which can affect horses of any age and breed. Among these, the affections of nervous system are potent reason for death and euthanasia in equids. Many episodes associated with the emergence of equine encephalitic conditions have also pose a threat to human population as well, which signifies their pathogenic zoonotic potential. Intensification of most of the arboviruses is associated with sophisticated interaction between vectors and hosts, which supports their transmission. The alphaviruses, bunyaviruses, and flaviviruses are the major implicated groups of viruses involved with equines/humans epizootic/epidemic. In recent years, many outbreaks of deadly zoonotic diseases such as Nipah virus, Hendra virus, and Japanese encephalitis in many parts of the globe addresses their alarming significance. The equine encephalitic viruses differ in their global distribution, transmission and main vector species involved, as discussed in this article. The current review summarizes the status, pathogenesis, pathology, and impact of equine neuro-invasive conditions of viral origin. A greater understanding of these aspects might be able to provide development of advances in neuro-protective strategies in equine population.The importance of horse ( Equus caballus) started approximately 5000 years ago . T,9. T6,9]st case was reported from human in year 1952 in Mumbai ..st reporst time reported VEEV is a zoonotic pathogen just like EEEV and WEEV in Venezuela. Ochlerotatus taeniorhynchus is a major vector, and cotton spiny rat is the most common rodent host responsible for the transmission of this virus [In 1938, Beck and Wyckoff 1is virus . During is virus ,91. EEE is virus ,93 as hais virus . The vacis virus -96. Theris virus . Prophylis virus ,99. DNA is virus ,101.EHV-1 belongs to subfamily alpha herpesvirinae, a member of genus varicellovirus and has been identified as pathogenic to domestic horses . Equids The clinical sign includes a high fever of 102-107°F for 1-7 days, nasal discharges, coughing, abortion, and other non-specific clinical signs . AbortioA number of diagnostic techniques were used for the diagnosis of EHV-1. The diagnosis of this virus by isolation, direct immunofluorescence and immunohistochemistry (IHC) is usually difficult, but PCR based assays for detection of the viral genes are quite reliable, fast and sensitive method for the detection of EHV-1 and EHV-4 -117. A rRabies is caused by Lyssavirus (negative strand ssRNA virus of family Rhabdoviridae). This disease often found to occur in three clinical forms including dumb, furious, and paralytic. The incubation period of this disease is approximately 15 days in experimental cases, while in clinical cases it is always fatal and leads to death within 5-10 days . The behThe diagnosis of encephalitic conditions in equines is based on many factors including season, vector and host involvement, type of etiology associated, clinical, serological, and molecular results. Laboratory diagnosis of most of arboviral infections is accomplished by testing of CSF or serum samples to detect virus-specific IgM and neutralizing antibodies. Serology is the key to ante-mortem diagnosis of alphavirus and flavivirus infection. No pathognomonic signs distinguish flavivirus infection from other CNS diseases in horses. IgM capture ELISA is the test of choice to detect recent exposure to alphavirus and flavivirus. CSF from horses with acute EEEV infection typically shows a neutrophilic pleocytosis. Although WNV-infected horses can have normal CSF, if the CSF is abnormal, there is mononuclear pleocytosis. Clinically, peripheral lymphopenia can be seen in EEEV and WNV. Laboratory diagnosis of NiV can be made by virus isolation, identification of the RNA by use of RT-PCR, IHC, histopathology, or serologic tests such as indirect ELISA and virus neutralization tests. Real-time PCR on samples obtained from CSF, nasal secretions, or neural tissue can be done to detect the neuropathic strain of EHV-1. PCR testing and IHC for EEEV and EHV-1 are relatively straightforward compared with that for WNV. In recent advance researchers, pattern recognition receptors  also found to have major role in detection of immune response against flaviviruses  Table-3Table-3.The nervous systems affections are ubiquitous in equines worldwide and often serve as a potent source of infection to other species. Recent emergence of many diseases of equine origin has pose a threat to human civilization. The nervous system associated outbreaks  have already been encountered globally in equines and often signifies the zoonotic potential as well. The different disease conditions associated with the nervous system shows their different spectrum of transmission and pathogenesis. Hence, nowadays, it becomes a continuous challenge to veterinarians to elucidate the different ways to establish diagnostics and therapeutic measures in more recent and advanced manner. For this, proper advances in neuroprotective strategies, proper vector control, and novel immunization strategies are highly recommended. The present review summarizes only the known viral encephalitic conditions, but still more studies are required to investigate some other unknown causes associated with the affections of the nervous system in equines.Collection, compilation and writing work along with construction of flow diagrams and tables done by RK. Editing and formatting work done by RDP. Both authors read and approved the final manuscript.


Exploring the genetic structure of influenza viruses attracts the attention in the field of molecular ecology and medical genetics, whose epidemics cause morbidity and mortality worldwide. The rapid variations in RNA strand and changes of protein structure of the virus result in low-accuracy subtyping identification and make it difficult to develop effective drugs and vaccine. This paper constructs the evolutionary structure of avian influenza virus system considering both hemagglutinin and neuraminidase protein fragments. An optimization model was established to determine the rational granularity of the virus system for exploring the intrinsic relationship among the subtypes based on the fuzzy hierarchical evaluation index. Thus, an algorithm was presented to extract the rational structure. Furthermore, to reduce the systematic and computational complexity, the granular signatures of virus system were identified based on the coarse-grained idea and then its performance was evaluated through a designed classifier. The results showed that the obtained virus signatures could approximate and reflect the whole avian influenza virus system, indicating that the proposed method could identify the effective virus signatures. Once a new molecular virus is detected, it is efficient to identify the homologous virus hierarchically. Exploring the genetic structure of biological population attracts the focus in the field of population biology, molecular ecology, and medical genetics . InfluenThe dramatic progress in sequencing technologies provides unprecedented prospects for the exploration of virus homologous and mutation trajectory in space and time. Understanding the evolution of influenza viruses has benefited from phylogenetic reconstructions of the hemagglutinin protein . In an aIn recent years, the granular computing (GrC) theory has become a hotspot in the field of artificial intelligence and machine learning, which comes from the idea that people solve the problems from different levels and views . ClusterIn this paper, our aim is to explore the evolutional relationships of the avian influenza viruses in the same subtype and among the subtypes considering both HA and NA fragments in the virus system. Moreover, the complex virus system should be reduced for further exploration, faced with thousands of samples in the dataset. Jointing the two protein sequences, the feature vectors are extracted from HA and NA proteins, respectively, for labeling the specific virus. Furthermore, the granular signatures in the viral granules are identified based on the obtained features to reduce the systematic and computational complexity and then its performance will be evaluated. This will provide the supports for the rationality of subtype identification. Once a new molecular virus is detected, it could be analyzed with obtained viral signatures and then the prevention and treatment measures can follow what were applied in the viral signature.http://www.ncbi.nlm.nih.gov/genomes/FLU/) , we define a relation Rλ :  ∈ Rλ↔R ≥ λ, where Rλ is a crisp proximity relation that satisfies the reflexivity and symmetry. Then, the equivalent classes of the transitive closure tr(Rλ) can be marked by [x]λ, which is derived by Rλ, and then X(λ) = {[x]λ∣x ∈ X} is a granularity corresponding to λ. The set {X(λ)∣λ ∈ } represents a fuzzy granular space on X, which is an ordered set, and satisfies that the bigger the threshold λ is, the finer the granularity is, denoted by ℵTR(X) [In , the gray ℵTR(X) .λ is marked as X(λ) = {a1, a2,…, acλ}, where ai = {xi1, xi2,…, xiJi} satisfying the conditions that |ai| = Ji (|·| stands for the number of the elements in a set) and ∑i=1cλJi = n. Some properties are explored, such as i = 1,2,…, cλ) is the center of granule ai and the center of X is X(λ) [2 stand for the 2-norm number in K-dimension space.The granularity derived by ion X(λ) , 20, defSamong(X(λ)) is monotone increasing, with the granularity changing from the coarse to the fine, while Swithin(X(λ)) is gradually decreasing. Notably, the total deviation (S(X(λ)) = Samong(X(λ)) + Swithin(X(λ))) is always constant Samong(X(0)) = Swithin(X(1)) = 0 and By analyzing the variance within and among the classes in statistics , Samong(X(λ)) reaches the minimum. There exists only one λ = λ0 to meet the optimization model, marked as Model Xλ0=aX. Compared with [Samong(X(λ)) > Swithin(X(λ)).Model  is a glored with , their mR on the finite set X = {x1, x2,…, xn} and D = {R∣x, y ∈ X} = {r0, r1,…, rN}, satisfying 1 = r0 > r1 > ⋯>rN, an algorithm is presented to detect the optimized hierarchical clustering and construct the hierarchy of complex system based on the fuzzy granular space [Given an FP relation (or SFP relation) ar space .  Input: an FP relation (or SFP relation). Output: the optimized hierarchical structure and the corresponding threshold.Step 1Step 2Step 3Step 4Step 5. For any aj ∈ A, B ⇐ B ∪ aj, A ⇐ A∖aj.Step 6. For ∀ak ∈ A, if ∃xj ∈ aj, yk ∈ ak satisfying R ≥ ri, B ⇐ B ∪ ak, A ⇐ A∖ak. Step 7Step 8. If A = ⌀, X(ri) = C; otherwise, go to Step 5.Step 9. If X(ri) ≠ X(ri−1),  S1 ⇐ |Samong(X(ri)) − Swithin(X(ri))|; otherwise, go to Step 2.Step 10. If S0 > S1, S0 ⇐ S1, go to Step 2. Step 11. Output ri−1, X(ri−1) and S0.O(n2). The concrete problems are decomposed hierarchically, which is consistent with the core idea of GrC. Given an FP (or SFP) relation on the finite set X, the optimization clustering structure constructed by Algorithm A is its first level structure. Furthermore, its second level structure is obtained if Algorithm A is repeatedly applied to all the equivalent classes in its first level structure. Therefore, Algorithm A can be used to construct multilevel structure in practical application.The computational complexity of Algorithm A is X∗ = {a1, a2,…, aJ} is constructed, where J = |X∗|. To reduce the complexity of the system, feature viruses (or signature viruses) could be extracted to approximately represent the equivalent class. According to the nearest-to-center principle, an objective function to select the signature is established, and it is formulated as follows:pi is the signature item of the granule ai and P = {p1, p2,…, pJ} is a signature set of the granularity X∗. In some way, the signature set P can be used to represent approximately the complex system X.Once the optimal granularity of the complex system is determined, it is of crucial importance to construct information granules for abstracting original samples. Generally, the granules are obtained according to the principle: the samples with the same features assemble in one granule. And the average of all samples in one class or the center of the class is efficacious to represent the core information. Suppose that a multilevel structure (or granularity) P, a classifier is designed for classifying the rest of the samples of the corresponding classes according to the principle of maximum similarity, marked as Model (qj (∈X∖P), the classifier is designed:pi ∈ P, i = 1,2,…, J, and Lj is the class the virus qj belongs to.To evaluate the performance of selected signature set as Model . Given aX∖P are assigned to |P| classes. All samples in X∖P are divided into |P| classes according to Model  labels the specific virus xi. Furthermore, the similarity between viruses xi and xj is measured:xi, xj) = ∑i=132xik · xjk stands for the inner product in 32-dimension space. Obviously, R is an SFP relation.Based on the feature vectors extracted from the viral HA and NA proteins, the 32-dimension vector Ω∗ is obtained. The FHEI is applied to virus system Ω∗ containing 909 avian influenza viruses, to obtain the reasonable partition and evolutionary structure.The virus dataset has redundant information as many viruses are labeled with the same host, the same occurrence time, and the same outbreak sites, which could pose the obstacle to explore the intrinsic relationship and difference among the subtypes. Thus, those with the same host, the same occurrence time, and the same location combine as one new point (a representative virus), which is the preliminary system simplification, and then a unique virus database Ω∗, the viral granular space (evolutionary structure) is constructed by using Algorithm A. On the first level, 3 equivalent classes were finally determined to partition the whole system, and the corresponding signature viruses are obtained, shown in bk∗ , and the virus signatures are extracted, shown in On the basis of the virus database k∗ k = 1,,…, 14, aP∗ on the second level structure is 76.57% by comparison, indicating that the second level structure of viruses system constructed by our model is effective.The virus signature could be used to approximate the whole system for they are selected from the classes as the granule information. Moreover, the classifier, designed based on the principle of maximum similarity, is applied to validate the performance of virus signature. The accuracy rate of the signature virus set P∗ containing 14 viruses can be used to approximate the whole system containing 909 viruses.Evaluating the performance of virus signature, the error rate is still 23.43%, which might be caused by the approximation process since all signature viruses are selected according to the nearest-to-center principle and they are not just on the center of each subclass, respectively. From the perspective of approximation, the signature set contains the most information of virus system according to the accuracy rate 76.57%. Therefore, the signature virus set P∗ can be constructed by applying the hierarchical clustering algorithm [The phylogenetic tree of the signature virus set lgorithm , shown ilgorithm .Among the 8 virus subtypes, 7 viruses are identified as the signature viruses except H7N9, for H7N9 viruses account for the minority of whole system . ExploriFrom the hierarchical structure of the feature viruses, A/blue-winged teal/LA/AI13-1225/2013(H7N7), A/duck/Korea/A349/2009(H7N2), and A/chicken/Abbottabad/NARC-2419/2005(H7N3) have similar evolution relationship (connect closely) for they equip the same HA type (H7). Besides, A/chicken/Cambodia/LC/2006(H5N1) and A/dog/Shandong/JT01/2009(H5N2) have the consistent conclusions. However, A/chicken/Italy/330/1997(H5N2) is far from them, which could be due to the fact that the outbreak time plays an important role in sequence mutation. If just considering the HA and NA proteins, the subtypes, such as H9N2 [The rapid variation of influenza viruses results in low-accuracy subtyping identification and makes it difficult to develop effective drugs. This article explored the homology of avian influenza virus system and identified the subtypes according to HA and NA protein fragments, which might provide the support for developing antiviral drugs and vaccines according to different subtypes. Phylogenetic reconstructions serve understanding the evolution of influenza viruses. However, the large amounts of virus dataset pose an obstacle for analyzing the evolutionary relationship and identifying the correct subtypes to predict the biological functions. Granular computing theory was applied to determine the partition of virus system based on the constructed granular space. A method and the corresponding algorithm were proposed for detecting the rational granularity. With the proposed algorithm applied repeatedly, a multilevel structure of whole system was constructed. To reduce the computational complexity, some key viruses were selected to approximate the whole system based on the coarse-grained idea. According to the nearest-center principle, virus signatures were identified and constructed the granular signature set of a multilevel structure of complex system. By designing a classifier, the performance of virus signatures was evaluated and the result showed that the virus signatures could reflect the most properties of virus system. Furthermore, hierarchical structure of virus signature was constructed by using hierarchical clustering algorithm. Both of the two structures have some consistent intrinsic relationship among the virus systems and between the different subtypes. Some viruses were detected as isolated points in the structure thought equipped with the same labels, which might be caused by the rapid variations in the RNA strands. The virus signatures have the potential use in new virus subtyping comparison and functional prediction.


P. aeruginosa have been linked to higher morbidity and mortality. To understand the impact of P. aeruginosa in NCFBE on health care costs and burden, we assessed healthcare costs and utilization before and after P. aeruginosa diagnosis. Using data from 2007 to 2013 PharMetrics Plus administrative claims, we included patients with ≥2 claims for bronchiectasis and >1 claim for P. aeruginosa; then excluded those with a claim for cystic fibrosis. Patients were indexed at first claim for P. aeruginosa and were required to have >12 months before and after the index P. aeruginosa. The mean differences in utilization and costs were assessed using paired Student’s t-tests for statistical significance. Mean total healthcare costs per patient were $36,213 pre-P. aeruginosa diagnosis versus $67,764 post-P. aeruginosa, an increase of 87% (p < 0.0001). Inpatient costs represented the largest proportion of total healthcare costs post-P. aeruginosa (54%) with an increase of four hospitalizations per patient (p < 0.0001). NCFBE patients with evidence of P. aeruginosa incur substantially greater healthcare costs and utilization after P. aeruginosa diagnosis. Future research should explore methods of earlier identification of NCFBE patients with P. aeruginosa, as this may lead to fewer severe exacerbations, thereby resulting in a reduction in hospitalizations and healthcare costs.Non-cystic fibrosis bronchiectasis (NCFBE) is a rare, chronic lung disease characterized by bronchial inflammation and permanent airway dilation. Chronic infections with  P. aeruginosa infection may experience even greater burden.Non-cystic fibrosis bronchiectasis (NCFBE) is a chronic disease characterized by a cyclical process of recurrent infections, pulmonary inflammation and structural damage to the airways. Prevalence of NCFBE in the U.S. has been estimated to be 139 cases per 100,000 . NCFBE pP. aeruginosa is the most prevalent cause of infection, which leads to more hospital admissions than other NCFBE-related infections [P. aeruginosa among NCFBE patients has been reported to be as high as 15–58%, however many of these estimates are based on single centre investigations and some studies include cystic fibrosis patients in their prevalence calculations [P. aeruginosa prevalence in the U.S. are not well understood. Between 50 and 80% of NCFBE cases are idiopathic and the remainder secondary to post-infection resulting from underlying disease, obstruction or other airway related injury ,5,6. Thefections ,9,10,11.ulations ,13,14,15P. aeruginosa infection and frequent exacerbations experience accelerated decreases in lung function, reduced quality of life, increased length of stay and risk of hospitalization, and both higher morbidity and mortality [P. aeruginosa infections have more frequent exacerbations and accelerated rapid disease progression than NCFBE patients without P. aeruginosa [Haemophilus influenzae, nontuberculous mycobacteria, and Moraxella catarrhalis, but these infections are not reported to be as prevalent as P. aeruginosa [NCFBE patients with ortality ,20,21,22ruginosa ,11,21,23ruginosa ,25. P. aeruginosa. Disease management requires obtaining sputum cultures and treatment options vary from inhaled antibiotics to dual anti-pseudomonal therapy [P. aeruginosa is among the most common pathogens responsible for nosocomial infections, a plethora of questions remain unanswered in determining the optimal therapeutic treatment in patients with NCFBE [P. aeruginosa may include the use of several antibiotics due to antibiotic resistance and treatment ineffectiveness, perhaps contributing to the higher costs seen following P. aeruginosa diagnosis in the present study [Management is complicated by the fact that no gold standard exists for the diagnosis, management, and treatment of  therapy ,26. Whilth NCFBE . A patient study ,27,28.P. aeruginosa on healthcare costs and utilization related to NCFBE in the U.S. Given the significant impact of P. aeruginosa on clinical outcomes in NCFBE, it is anticipated that P. aeruginosa diagnosis increases economic burden on the healthcare system versus NCFBE without P. aeruginosa. Therefore, the purpose of this study is to evaluate the healthcare costs and utilization pre- and post-P. aeruginosa diagnosis among commercially insured NCFBE patients in the U.S.Currently, no studies have effectively evaluated the impact of To complete this analysis, we used 2007–2013 claims data from the PharMetrics Plus database. This database provided pooling of adjudicated medical and pharmacy claims for over 100 million patient lives from more than 250 health plans across the U.S. As an administrative claims dataset, PharMetrics includes inpatient and outpatient diagnoses in International Classification of Diseases-Ninth Revision-Clinical Modification (ICD-9-CM and ICD-10) format, procedures in Current Procedural Terminology-Fourth Edition and the Healthcare Common Procedure Coding System and prescription records. Also included in the data are demographic variables, product and insurance type, provider specialty, and dates inclusive of plan enrollment .P. aeruginosa claim for the assessment of healthcare cost and resource utilization. Patients were stratified by P. aeruginosa diagnosis at any point during the study period using ICD-9-DM codes for 482.1 (pneumonia due to Pseudomonas) and 041.7 (pseudomonas). Patients were indexed at their first P. aeruginosa claim. NCFBE patients were originally identified by the presence of at least two claims with the ICD-9 code for bronchiectasis (494.XX) and the absence of any claims for cystic fibrosis (277.XX). To further ensure proper patient selection, the claims for bronchiectasis were required to be at least 90 days apart. Patients were required to have 12 months of claims experience before and after the index P. aeruginosa diagnosis on NCFBE patients [P. aeruginosa. Post diagnosis P. aeruginosa-related costs were those with a primary claim for P. aeruginosa. Costs outside these categories were classified as ‘other costs’, and included, but were not limited to: nursing home, home health, ambulance services, rehabilitation, durable medical equipment, and laboratory costs. Post diagnosis all-cause healthcare utilization measures included mean annual hospital, emergency room, physician office and pharmacy visits. All statistical analysis was conducted using SAS version 9.4 . All tests were conducted assuming a two-tailed test of significance and alpha level set a priori at 0.05. Demographic and medical characteristics for the cohort were measured using counts and percentages for categorical variables and measures of central tendency for continuous variables. Characteristics were presented by total sample. Negative binomial models were used to assess factors associated with counts of events to include inpatient hospitalizations, outpatient events, physician office visits, emergency room visits, and total pharmacy utilization while controlling for covariates. A paired Student’s t-test for statistical significance was conducted to determine the mean difference in utilization and costs. Model fit characteristics were assessed and assumptions of the model were upheld.Baseline characteristics of all patients were collected from the one-year pre-period and included Elixhauser comorbidity conditions, age, and gender. Mean and median total and disease-related 12-month post-index disease related healthcare utilization and costs were evaluated to measure the burden of patients . Post diP. aeruginosa. Females accounted for over half the NCFBE with P. aeruginosa population (60.47%). The majority of patients were 50+ years (88.83%), with 7.54% in the 18–49 and 3.07% in the 0–17 year age ranges. Chronic obstructive pulmonary disease (COPD), uncontrolled hypertension, and cardiac arrhythmias were the most prevalent comorbidities among NCFBE patients with P. aeruginosa   See . P. aeruginosa, mean total per patient costs pre- P. aeruginosa were $36,213 ± 132,270 on average compared to $67,764 ± 154,750 post-P. aeruginosa, equating to an 87% or $31,551 increase post-diagnosis (p < 0.0001). Mean hospital costs accounted for the largest proportion of healthcare costs both pre- and post-P. aeruginosa diagnosis , followed by other costs , pharmacy costs , office visit costs , and emergency room (ER) visit costs . Post-P. aeruginosa diagnosis pharmacy and other costs were over two times higher than pre-P. aeruginosa costs   See .P. aeruginosa-related healthcare costs were $18,407 , of which hospitalizations again accounted for the majority of costs . Mean other P. aeruginosa-related costs were $1206 (±3665), mean office visit costs were $256 (±1560), and mean ER visit costs amounted to $9 (±166). Total mean P. aeruginosa period . Mean ER visits increased by 82.51%, office visits by 66.04% and pharmacy visits by 56.50%   See .P. aeruginosa diagnosis, which is not surprising from a clinical perspective. However, this study is the first to describe an incremental cost post-P. aeruginosa diagnosis. Even though the cost differences found were significantly higher post-P. aeruginosa, it is possible that the true incremental cost may be substantially higher if P. aeruginosa was present but undetected in the pre-P. aeruginosa diagnosis period. Previous reports evaluating the economic burden of NCFBE have suggested that management of this disease is associated with high costs to the U.S. healthcare system, but have not evaluated the impact of P. aeruginosa diagnosis on these costs. One study estimated that in 2013 between 340,000 and 522,000 persons in the U.S. were receiving treatment for NCFBE during that year [P. aeruginosa contributes to the chronic cycle of infection and inflammation in NCFBE which is associated with exacerbations, increased symptoms, decline in lung function, reduced quality of life and ultimately, increased healthcare resource use [P. aeruginosa in the sputum, (2) frequency of severe exacerbations, and (3) systemic inflammation. All three of these factors are a major component of the increased economic burden of P. aeruginosa on NCFBE patients. However, whether the presence of P. aeruginosa among NCFBE patients resulted in increased costs was not addressed in these previous analyses [Healthcare cost and resource utilization increased substantially for NCFBE patients after urce use ,20,21,22P. aeruginosa patients increased by 115.63, 82.51, 66.04, and 56.60%, respectively, from the pre- to post-P. aeruginosa period. To put these findings in context, in a longitudinal retrospective observational cohort study by McDonnell et al. [P. aeruginosa experienced higher hospitalization rates than their non-P. aeruginosa infected counterparts. However, McDonnell et al. did not include the economic burden of PA in their analysis [P. aeruginosa. Costs beyond NCFBE were mostly attributed to acute healthcare services such as hospitalizations and emergency room visits. We found that resource utilization, including hospital, ER, office, and pharmacy visits, among P. aeruginosa is a component of two bronchiectasis severity scoring systems, the Bronchiectasis Severity Index (BSI) and the FACED (forced expiratory volume in one second (FEV1), age, chronic colonization, extension, and dyspnea) score. In a study by Goeminne et al., P. aeruginosa in addition to a variety of demographic and clinical factors, was associated with higher mortality in NCFBE [P. aeruginosa to be associated with higher mortality, as well as greater airflow obstruction, increased severity of disease and poorer quality of life among NCFBE patients [P. aeruginosa is accepted as a marker of disease severity, and assuming patients with more severe disease require more medical attention, it is not surprising that the current analysis found increased healthcare resource use and costs in the year following diagnosis. PA infection has been identified as a marker of more severe disease, which leads to higher costs. Furthermore, in NCFBE . This ispatients ,22. GiveP. aeruginosa diagnosis and treatment process of NCFBE patients with P. aeruginosa may also be responsible for increased healthcare utilization and costs found in the current analysis. The management of P. aeruginosa in NCFBE patients varies per patient and is largely dependent on patient history and clinician preference. Comprehensive outpatient follow-up care is required as treatment failures due to antibiotic resistance and exacerbations may occur. Patients with more severe exacerbations, such as hypoxemia or respiratory distress, are often hospitalized. Severe exacerbations that lead to hospitalizations are most troublesome and have the greatest impact on disease progression, healthcare resource utilization, and costs. Further, patients with severe exacerbations may require long-term antibiotic therapy. Clinicians suggest the optimal duration of long-term antibiotic therapy to range from three months to a year, leading to a further increase in healthcare utilization and cost [The delayed time to and cost ,31,32,33P. aeruginosa to healthcare use and costs, there needs to be an emphasis on increased rates of testing for NCFBE related infections. Establishing current rates of testing is difficult due to a lack of definitive guidelines. The potential cost of diagnostic testing is significant as there are a number of laboratory and imaging studies, tests, and procedures that may be helpful in confirming a P. aeruginosa infection. Testing methods may include: pulmonary function tests, sputum cultures, molecular tests, and antibiotic susceptibility tests [P. aeruginosa and thus, increasing the prospect of successful eradication [With the results of the current study linking ty tests ,34. Prevdication . FurtherP. aeruginosa are burdened with many other comorbidities may also account for higher resource utilization and cost found in this study [The current study found the majority of patients to be over the age of 50. This finding is consistent with a previous study that reported the majority of patients to be between the age of 45 and 64 years . The oldis study ,2,3,4.P. aeruginosa infection may not have necessarily coincided with the timing of testing and diagnosis of the infection. However, these patients appeared to consume greater healthcare services and incur increased costs following their P. aeruginosa diagnosis. Additionally, while claims data is useful in providing a large study population, the potential for misclassification exists. Claims data have the potential for errors and miscoding, which may potentially introduce misclassification bias. Patients may have been incorrectly identified due to this potential error. Another limitation of claims data is that data identifying specific treatments used or patient severity were not available. Therefore, it is not known if patients truly had a chronic infection of P. aeruginosa or the claim was for a single isolated incident.We acknowledge that the time at which the NCFBE patient acquired the P. aeruginosa on healthcare costs and resource utilization related to NCFBE, an area that had been previously unexplored. Management of P. aeruginosa may be complicated by multiple obstacles such as lengthy patient diagnostic experience, inconsistent or inappropriate treatment strategies, and more severely progressed disease, which lead to further increases in healthcare resource utilization and economic burden. Future economic analyses should investigate the prevalence, diagnosis, and treatment of P. aeruginosa and the implications for patients, payers, and other stakeholders.The findings from this study provided insight on the impact of


I wouldn’t be surprised to find my work on a politician-generated list of “ridiculous, wasteful” research projects: “Scientists examine how poop bacteria activate polio, an eradicated virus.” I have worked on poliovirus for 16 years. We work on other viruses too, but poliovirus has led to some of our most important discoveries. Many people only know polio as a paralytic disease that was eradicated from the United States decades ago. So why are we studying an eradicated virus?10 infectious viruses without even trying very hard. For perspective, if these 10,000,000,000 viruses were dollar bills, the stack would be 68 miles high. Working with poliovirus is safe due to vaccination. We can make targeted mutations in the genome and generate mutant polioviruses within days. There are mouse strains that can be infected. Most importantly, poliovirus has been studied for over 100 years. We know a lot about poliovirus and we have great tools in our toolbox. If you’re going to tackle a tough problem, it helps to have a great toolbox. For other fields, the ideal toolbox may be fruit flies, worms, or yeast. Collectively, these model systems have illuminated biology and have led to major advancements in human health. What have we learned using poliovirus?Poliovirus is an incredible model system. It grows like a weed. We can make virus stocks containing 10https://storify.com/HarmitMalik/on-one-of-the-enduring-friendships-in-virology). Marco’s lab has since shown that other viruses, including chikungunya, also rely on sloppy replication for optimal fitness. Several groups are exploring viruses with altered fidelity as vaccine candidates. In the end, our work with poliovirus was applicable to many viruses and may lead to new vaccine strategies.As a postdoctoral fellow in Karla Kirkegaard’s lab, I used poliovirus to show that RNA viruses benefit from a sloppy replication strategy. Many viruses, including Ebola, Zika, and influenza, have RNA as their genetic material. Replication of RNA by viral enzymes generates mutations, like a fast typist without spellchecker. Most of these mutations are damaging to the virus, but some can be beneficial. By isolating a mutant poliovirus that generated fewer mutations, I showed that a sloppy replication strategy benefits the virus by conferring adaptability during infection of animals. Marco Vignuzzi, then in Raul Andino’s lab, published similar results. Long story short, Marco and I began as competitors but are now great friends (see Over the past several years, my lab has shown that intestinal bacteria promote infection with poliovirus and other gut viruses. When we used antibiotics to deplete bacteria from mice, poliovirus replication and disease was reduced. We found that poliovirus sticks to bacteria, which aids viral infection and transmission. Tatyana Golovkina’s lab showed similar effects for another model virus, mouse mammary tumor virus, and our papers were published together. Since then, several groups have shown that a variety of gut viruses rely on intestinal bacteria for infection. One of my favorite examples is Stephanie Karst’s work with human norovirus. Norovirus infections are common and miserable, with explosive vomiting and diarrhea. However, we know relatively little about human norovirus. In spite of 40 years of effort, human norovirus could not be cultured in the lab, which severely limited progress. The only method to study viral replication was to infect human volunteers! Human stool samples were filtered to remove bacteria to generate “virus” preparations. Stephanie is a friend and knew about our poliovirus results. She added bacteria back to her virus samples, and then the virus could replicate in cultured cells. The ability to grow human norovirus in the lab is a huge advance that could lead to new treatment or prevention strategies. And it all started in small labs with curiosity-driven studies using poliovirus and mouse mammary tumor virus.The value of model systems and curiosity-driven basic science research is immense, but we often fail to convey this to the general public, to those in political office, and even to other scientists. Some scientists put a premium on clinical or translational relevance. Although a project need not have disease relevance to have high merit, basic science grant proposals can suffer a penalty from biased reviewers. When communicating their work, many scientists start with a description of the disease burden of their pathogen to highlight significance, rather than discussing why their biological question is interesting or important. It’s easy to understand why some nonscientists have trouble understanding the value of model systems when some scientists have the same problem. A diversified portfolio of research projects is needed—translational and basic science, model systems and disease models, and “big data” collaborative projects and investigator-initiated projects. But I hope that there will always be support for scientists that “geek out” over fruit flies, worms, yeast, or nearly eradicated viruses.


Workers’ Memorial Day, observed annually on April 28,Occupational injuries and illnesses also have economic costs. The societal cost of work-related fatalities, injuries, and illnesses was estimated at $250 billion in 2007, based on methods that focus on medical costs and productivity losses (MMWR (https://www.cdc.gov/niosh/topics/fishing/.New data on fatal falls overboard in the fishing industry, one of the nation’s most hazardous industries, are reported in this issue of


Filoviridae, such as ebola viruses and marburgviruses, cause hemorrhagic fevers associated with high mortality and are, therefore, of major public health importance worldwide1. The 2013–2016 West African Ebola virus (EBOV) disease outbreak further illustrated the epidemic potential and devastating effects associated with the members of Filoviridae. Bats have been identified as reservoirs for marburgviruses and are suspected to be involved in the transmission of ebola viruses to humans3. Since the discovery of Lloviu virus (LLOV) in Miniopterus schreibersii samples from Spain in 2002, European bats have also been considered as a possible host for filoviruses4. Subsequently, there have been no additional reports of LLOV detection or isolation, resulting in a paucity of knowledge on the biology and pathology of the virus. Moreover, no specific diagnostic test has been described, which further hampers surveillance studies5. Furthermore, the identification of genetically diverse filoviruses discovered in Chinese bat colonies has further highlighted the broad geographical distribution of filoviruses and their unexpected genetic diversity7.Members of the virus family M. schreibersii bat colony, causing the death of ~500 individuals in Northeast Hungary (Bükk mountain). After investigating the scene, only aged carcasses could be collected, which prevented virological investigation. Blood coagulate was detected on the nose of several individuals, referring to possible respiratory hemorrhages. Other known bat colonies in the area were also checked and were found to be unaffected at that time. Three years later, in 2016, five Schreiber’s bat individuals were reported with the gross pathologic symptom of respiratory bleeding . The bats inside the mine cavern system were unaffected at the time of sampling and were frequently monitored during 2017. An additional mortality event was detected in December 2017 that affected 25 individuals roosting separately. Unfortunately, the decaying condition of the carcasses did not permit virological examination; thus, a connection to LLOV infection cannot be made. On the basis of a previous visit to the cave site in July, we hypothesize that this latest M. schreibersii mortality event occurred between July and December 2017.In 2013, a mass-mortality incident occurred in a bleeding  in North6. The RT-PCR amplicon was purified from agarose gel, and then bi-directionally sequenced on an ABI Prism 310 Genetic Analyzer. On the basis of the preliminary sequence data for the Hungarian LLOV, a specific TaqMan probe-based real-time RT-PCR assay was designed . The One-Step RT-PCR (QIAGEN) conditions were as follows: 50 °C for 30 min, 95 °C for 15 min followed by 50 cycles of 94 °C for 30 s, 51 °C for 30 s and 72 °C for 1 min. To the best of our knowledge, this is the first published LLOV-specific diagnostic assay used on wildlife samples. To further support the presence of LLOV in our samples, we designed two additional nested primer sets specific to the nucleoprotein gene. The nucleoprotein-specific oligonucleotide sequences and nested RT-PCR reaction conditions are summarized in the Fresh carcasses from 2016 were collected and transported on dry ice to the biosafety level 4 (BSL-4) suit laboratory at the Szentágothai Research Centre, University of Pécs, for further examination. Briefly, after dissection, tissue samples were homogenized, and nucleic acid was extracted using a QIAamp Viral RNA Mini Kit (QAIGEN) according to the manufacturers’ recommendations. Nested RT-PCR specific for the RNA-dependent RNA polymerase gene was used to identify filovirus RNA as described previouslywww.ncbi.nlm.nih.gov) and further phylogenetic analyses showed that the Hungarian virus  was most closely related to LLOV isolate MS-Liver-86/2003 (JF828358) with 98% nucleic acid homology  search . Briefly, Vero E6 cells were prepared on a 24-well cell culture plate using Eagle’s minimum essential medium (MEM) with 10% fetal bovine serum (FBS) and antibiotics (streptomycin and ampicillin) as a growth medium. Plates with 80–90% confluent cell monolayers were washed with phosphate buffered saline, and then 200 μL from all examined tissues  homogenized in phosphate buffered saline solution was added. The inoculated plates were incubated at 37 °C in a CO8. Moreover, other studies involving recombinant LLOV proteins reported EBOV reminiscent receptor usage10. To assess the potential role of the virus in human or primate infections, further biological, molecular and in vitro investigations are needed.Although the human pathogenic potential of the virus is still unknown, the innate immune inhibitory function of LLOV-derived VP35 and VP24 proteins in human and bat cells has been reportedM. schreibersii bats in Spain (Cueva del Lloviu), LLOV has re-emerged in Europe, and we report its presence within the same bat species in Hungary. This incidence raises the question of whether it was a second introduction of LLOV in Europe or an unnoticed silent circulation proceeding among European bats. Our finding indicates a significant range expansion of the virus from the Iberian Peninsula of Spain, its currently only known geographic range. Due to the possible connection between LLOV infection and the mortality of M. schreibersii, it seems that this species is not the reservoir animal of LLOV. To clarify the exact source  and geographic range of LLOV, further investigations involving bat-connected ecological niches are needed without unnecessary disturbance. The potential ecological ramifications of the negative effects on local bat populations have been reported for White Nose Syndrome in NorthvAmerica11. M. schreibersii and closely related Miniopterus species are widely distributed across Eurasia and have been known to roost in huge colonies of up to several thousand individuals12. These bats represent a significant concern regarding conservation biology since they represent an important ecological resource and an integral part of European biodiversity. It seems that LLOV may cause severe disease in one species of bat, resulting in mass mortalities in colonies. Therefore, increased attention from the scientific community and conservation biologists is required. Although any direct causality between mortality events in M. schreibersii and LLOV infection has not been shown, our findings of LLOV range expansion demonstrate the need for further investigation.More than a decade after the mass mortality of supplemental Table and Figure