key: cord-274620-6ebl319q authors: ceballos, nidia aréchiga; morón, sonia vázquez; berciano, josé m.; nicolás, olga; lópez, carolina aznar; juste, javier; nevado, cristina rodríguez; setién, álvaro aguilar; echevarría, juan e. title: novel lyssavirus in bat, spain date: 2013-05-17 journal: emerg infect dis doi: 10.3201/eid1905.121071 sha: doc_id: 274620 cord_uid: 6ebl319q a new tentative lyssavirus, lleida bat lyssavirus, was found in a bent-winged bat (miniopterus schreibersii) in spain. it does not belong to phylogroups i or ii, and it seems to be more closely related to the west causasian bat virus, and especially to the ikoma lyssavirus. b ats have been considered natural hosts of a wide diversity of viruses, including human pathogens such as lyssaviruses, severe acute respiratory syndrome coronavirus, henipavirus, and filoviruses (1) . within the genus lyssavirus, 12 (2) and ikoma lyssavirus (ikov) (3) . bats are the natural reservoirs for most lyssaviruses, and to our knowledge, only mokv and ikov have never been detected in bats. rabv is the only virus known to establish epidemiologic cycles in bats and carnivores, and it is responsible for most human infections, mainly transmitted by dogs. the genus lyssavirus comprises at least 2 phylogroups: phylogroup i (rabv, duvv, eblv1-2, ablv, arav, irkv, bblv, khuv) and phylogroup ii (lbv, mokv, and shibv). phylogroup iii consists of wcbv (4). according to a recent phylogenetic reconstruction that included the novel ikov and was based on a fragment of 405 nt from the nucleoprotein gene, ikov has proven to be highly divergent (3) and probably also forms part of phylogroup iii. during 1977-2011 in europe, 988 cases of bat rabies were reported to the rabies bulletin europe. bats of the species eptesicus serotinus and e. isabellinus, which account for >95% of cases, are considered the major natural reservoirs of eblv-1. several bat species within the genus myotis are reservoirs for eblv-2, bblv, and the central asian lyssaviruses arav and khuv (5) . wcbv has been isolated in the common bent-winged bat miniopterus schreibersii (6) . other bat species might act as eventual hosts, although in spain, bat rabies has been declared only in e. isabellinus bats (7) . the possibility of a wider host range has been suggested by some surveys on natural bat colonies of other bat species describing neutralizing antibodies and genomic fragments related to eblv-1 (8) . in july 2011, a bat was found in the city of lleida and taken to the wildlife care center of vallcalent (lleida, catalonia). the bat arrived lethargic and dehydrated, died soon after admission, and its carcass was frozen at -20°c. on march 12, 2012, as part of the rabies surveillance program in spain, the bat carcass was received by the national center of microbiology, where rabies testing was conducted by 2 generic reverse transcription pcr (rt-pcr) methods for lyssavirus detection (9,10) and 2 commercial rabies antiserum assays (bio-rad laboratories, marnes la coquette, france; and fujirebio, inc., tokyo, japan) for antigen detection by fluorescent antibody testing. brain smears were positive for lyssavirus by rt-pcr and fluorescent antibody testing, and an oropharyngeal swab sample was positive by rt-pcr. further attempts to isolate the virus by tissue cultures were unsuccessful after 2 blind passages in bhk-21 and murine neuroblastoma cells. the negative results could be explained by the fact that the sample had been stored at -20°c for 8 months and had been frozen and thawed twice before cell culture testing; however, the possibility of the cell lines not being permissive for the virus cannot be excluded. the bat was morphologically identified as a bentwinged bat (m. schreibersii) and genetically identified by cytochrome b sequencing (11) . the genomic sequence of the corresponding fragment of the diagnostic rt-pcr on the conserved region of the nucleoprotein gene, determined by blast (http://blast.ncbi.nlm.nih.gov/), showed no substantial sequence similarity to previously known lyssaviruses. to determine the identity of the lyssavirus, we sequenced a larger fragment (565 bp), including the variable codifying region of the nucleoprotein gene (genbank accession number submitted). we reconstructed an overall the best-fit nucleotide model, gtr + i + g, available in mrbayes,was selected according to the corrected akaike information criterion. the phylogenetic reconstruction was based on a dataset representative of all known lyssaviruses, including the recently described ikov. the topology obtained showed that this sequence is more closely related to ikov and wcbv than to the lyssaviruses in phylogroups i and ii (figure) . these results suggest that this sequence tentatively belongs to a new lyssavirus species named after the location of collection, lleida bat lyssavirus (llebv). the highest nucleotide identity was with ikov (71. 7%-64%) . the lowest nucleotide identity was with the only lyssavirus found in bats of the iberian peninsula, eblv-1. the nucleotide identity among the previously known lyssaviruses was 63.5%-80.0% in this particular fragment, and the lowest identities among strains belonging to the same lyssavirus were 80.4% for ablv and 79.9% for lbv (the most distant lbv strain has been suggested to be a different lyssavirus) (online technical appendix, wwwnc.cdc.gov/eid/article/19/5/12-1071-techapp1.pdf). the lyssavirus-specific antigen reactivity and association with a genomic sequence found in a bent-winged bat in northeastern spain could be derived from the tentative new virus llebv. according to our phylogenetic reconstruction, the virus does not seem to belong to phylogroup i, which comprises most bat lyssaviruses, or to the african phylogroup ii. the evolutionary relationships between the llebv sequence with wcbv and ikov sequences need to be clarified before it can be determined whether they form >1 different phylogroups. of note, the new llebv was detected in m. schreibersii bats, as was wcbv, the other european lyssavirus outside phylogroup i. the genus miniopterus has traditionally been considered to belong to the family vespertilionidae as do other bat genera linked to lyssaviruses in eurasia (eptesicus, myotis, and murina). however, recent molecular analyses have confirmed that the genus miniopterus belongs to the family miniopteridae (12) . m. schreibersii bats are migratory, widely distributed across southern europe and eurasia. large numbers (thousands) of these bats overwinter in caves and move in the spring to different and sometimes distant summer roosts for reproduction (13) . these ecologic features make it relatively easy for an infectious agent to quickly spread out within and among the populations. consequently, it is difficult to imagine that wcbv or llebv are locally restricted; both could be located far from where they were found. neutralizing antibodies against wcbv have been found in bats in africa (14) . the cumulative description of new bat lyssaviruses in recent years shows the convenience of always using generic amplification primers for rabies diagnosis based on rt-pcr to complement antigen detection. no human exposure to the new virus has been reported. however, because of the divergence exhibited by lle-bv and ikov, and the growing evidence of inadequate protection/cross-neutralization against viruses outside phylogroup i, the effectiveness of current rabies vaccines for these viruses remains a concern (15). bats: important reservoir hosts of emerging viruses novel lyssavirus in natterer's bat ikoma lyssavirus, highly divergent novel lyssavirus in african civet complete genomes of aravan, khujand, irkut and west caucasian bat viruses, with special attention to the polymerase gene and non-coding regions current state of bat rabies surveillance in europe novel lyssaviruses isolated from bats in russia phylogeny of european bat lyssavirus 1 in eptesicus isabellinus bats in spain european bat lyssavirus infection in spanish bat populations screening of active lyssavirus infection in wild bat populations by viral rna detection on oropharyngeal swabs rt-pcr for detection of all seven genotypes of lyssavirus genus the iberian contribution to cryptic diversity in european bats molecular phylogenetics of the chiropteran family vespertilionidae migratory movements of miniopterus schreibersii in the north-east of spain possible emergence of west caucasian bat virus in africa passive immunity in the prevention of rabies: a neglected tool for a neglected disease we thank the genomics unit of the instituto de salud carlos iii for the analyses of the genomic sequences.this research was financially supported by project no. saf 2009-09172 of the general research program of the spanish ministry of science and education. c.r.n. was supported by a research fellowship from the universidad de alcalá de henares.dr aréchiga ceballos is postdoctoral fellow in the unidad de aislamiento y deteccion viral at the centro nacional de microbiologia, instituto de salud carlos iii (spain), supported by the consejo nacional de ciencia y tecnologia, mexico. her research interests are focused on zoonoses, particularly those related to lyssavirus, bats, and procionids. sign up to receive emailed announcements when new podcasts or articles on topics you select are posted on our website. www.cdc.gov/ncidod/eid/subscribe.htm key: cord-017407-nc9cyu2u authors: mehlhorn, heinz title: introduction: the world of bats date: 2013-09-03 journal: bats (chiroptera) as vectors of diseases and parasites doi: 10.1007/978-3-642-39333-4_1 sha: doc_id: 17407 cord_uid: nc9cyu2u bats are unique flying mammalians that occur worldwide feeding either on insects, fruits or even on blood depending on the species. since they are active during the night, many myths have been launched during the last 500 years. all these facets are shortly considered here and prepare for a hopefully intense look into their hidden life. bats-their english name points to their fluttering up and down wing movementsare a peculiar group of the so-called mammalia (¼ animals with breast nipples) in the zoological system, where they are described as chiroptera (greek: cheir ¼ hand, pteron ¼ wing) (neuweiler 1993; niethammer and krapp 2004, claus et al. 1932) . this term refers to their morphological peculiarity that thin portions of leathery skin (plagiopatagium, propatagium, dactylopatagium and uropatagium) are stretched between the body and all fingers reaching from there to the knuckles of both feet (claus et al. 1932 ; fig. 1.1) . these skin portions can be folded, when the bats rest in their typical position: head down under (figs. 1.2 and 1.3), fastholding by help of the claws and their five toes at each foot. their size may vary considerably between the different species. there exist rather large species (e.g. macroderma gigas in australia), which reach a body length of up to 20 cm, show a "wingspan" of up to 60 cm when stretched and weigh nearly 200-250 g. the smallest species (e.g. craseonycteris thonglongyai) have a body length of about 3 cm at the maximum and a body weight of only 2 g thus belonging to the smallest mammalians at all. the body of all bats is covered with very fine, shiny (often brownish) hair. bats are active at night seeking their food, which consistsdepending on the species-of insects, fruits or even of blood. since their rather quick flight is absolutely silent, so that they may approach and pass humans like a dark flash, many fairy tales developed in different human cultures (chaps. 7, 8). thus, their hidden way of life caused fear, especially in those cases, when many bats fly simultaneously around houses or around heads of persons, since they live together in groups of often more than 100-150 animals hiding themselves during daytime in the treetops (e.g. plant eating flying foxes e.g. in australia) or in tunnels of railways, galleries of closed mines, in old, rottening empty buildings on practically all continents except for antarctica. this peculiar way of life and their strange behaviour at night led to the fact, that for a long time the knowledge on their claus et al. 1932 . note that at the thumb and the index finger of each hand claws are present as well as at each of the five toes. cl ¼ clavicula; australian "flying fox" hanging head down at a branch of a tree in the botanical garden of sydney-happily sleeping during daytime morphology and their mode of reproduction was scarce. thus it was a big surprise when it was discovered, that these animals give birth to their offspring and do not lay eggs at hidden places. in general, they bear only one "baby" per year, although they possess two nipples at their breast. during the flight of the mother, the young bats are transported being attached to the hair of mother's belly often biting into one of the nipples for further stabilisation during the flights. absolutely surprising was also the discovery that the bats support their nightly flights not only by excellent eyes, which can catch even remnants of light, but also that they have developed a system of expelling ultrasound waves (holland et al. 2006; skiba 2009 ), a system of perception of the repelled waves and in addition they possess a nerve system, which allows them to react and to steer their muscles in milliseconds in order to avoid crashes with invisible obstacles in the night. these inventions are not unique, however, extremely scarce in the animal kingdom. it is less than 100 years that humans detected these waves, which reach far beyond the border of human hearing ranging up to 200 khz. thus the bats are extremely interesting with respect to their abilities and activities. while bats in warm countries are active all year around, the european species hibernate. on one side most of the bats-especially the insect feeders-are very useful for humans and thus are strictly protected, since their breeding places are endangered, fig. 1.3 a "flying fox" being disturbed during sleeping showing his pointed teeth to announce its "annoyance" especially in the surroundings of the growing industrial cities. on the other side, some of the species damage fruit plantations by eating fruits or just by biting into fruits in monocultures in australia and southeast asia (figs. 1.2 and 1.3) . a few bat species in south and central america (chap. 7) even suck blood of animals staying on meadows at night. these hosts are injured by the sharp and pointed teeth of the vampire bats, suffer from blood loss and even may become infected by pathogens such as those of true rabies (lyssa virus). indeed there exist several more of such bat transmitted viruses. therefore, also the harmless and very useful species that feed on insects have recently been set in the focus of scientific interest, since they are now known also as possible true or mechanical vectors of severe agents of diseases (especially of viruses) that may hit humans and animals (see chap. 3), if they get in contact with bats or their infected faeces (drosten 2012). especially drosten's laboratory and the microbiological institute of the university of hong kong showed that numerous bat species were carriers of the human-associated corona virus (sars-cov). in total, eight variations had been characterised (http:// covdb.microbiology.hku.hk:8080/cov-newpages/index.html). thus, this book is designed to present some interesting and important features on the biology, morphology, reproduction and the potential vectorship of bats on one side and to throw a glimpse on the mysterious beliefs that are still whispered today when bats are flying ( fig. 1.4) . thus this book comprises several chapters of different length that deal with some of the most interesting and most important findings in bat research. however, it is evident that there remain many more surprising news to be detected in the future. unbelievable reservoir of viruses bat navigation: bat orientation using earth's magnetic field handbook of the mammalia in fledermäuse: kennzeichen, echoortung und detektoranwendung key: cord-103460-5thh6syt authors: carlson, colin j.; albery, gregory f.; merow, cory; trisos, christopher h.; zipfel, casey m.; eskew, evan a.; olival, kevin j.; ross, noam; bansal, shweta title: climate change will drive novel cross-species viral transmission date: 2020-07-14 journal: biorxiv doi: 10.1101/2020.01.24.918755 sha: doc_id: 103460 cord_uid: 5thh6syt at least 10,000 species of mammal virus are estimated to have the potential to spread in human populations, but the vast majority are currently circulating in wildlife, largely undescribed and undetected by disease outbreak surveillance1,2,3. in addition, changing climate and land use are already driving geographic range shifts in wildlife, producing novel species assemblages and opportunities for viral sharing between previously isolated species4,5. in some cases, this will inevitably facilitate spillover into humans6,7—a possible mechanistic link between global environmental change and emerging zoonotic disease8. here, we map potential hotspots of viral sharing, using a phylogeographic model of the mammal-virus network, and projections of geographic range shifts for 3,870 mammal species under climate change and land use scenarios for the year 2070. range-shifting mammal species are predicted to aggregate at high elevations, in biodiversity hotspots, and in areas of high human population density in asia and africa, driving the cross-species transmission of novel viruses at least 4,000 times. counter to expectations, holding warming under 2°c within the century does not reduce new viral sharing, due to greater range expansions—highlighting the need to invest in surveillance even in a low-warming future. most projected viral sharing is driven by diverse hyperreservoirs (rodents and bats) and large-bodied predators (carnivores). because of their unique dispersal capacity, bats account for the majority of novel viral sharing, and are likely to share viruses along evolutionary pathways that could facilitate future emergence in humans. our findings highlight the urgent need to pair viral surveillance and discovery efforts with biodiversity surveys tracking range shifts, especially in tropical countries that harbor the most emerging zoonoses. dispersal limits). even with dispersal limits, these first encounters are predicted to produce al-207 most one hundred new viral sharing events (rcp 2.6: 96 ± 2.3; rcp 8.5: 86 ± 3.9) that might 208 include zebov, and which cover a much broader part of africa than the current zoonotic niche 209 of ebola 68 . human spillover risk aside, this could expose several new wildlife species to a 210 deadly virus historically responsible for sizable primate die-offs 69 . moreover, for zoonoses like emerging threats like ranavirus causing conservation concern, pathogen exchange among am-271 phibians may be especially important for conservation practitioners to understand 82 . finally, 272 marine mammals are an important target given their exclusion here, especially after a recent 273 study implicating reduced arctic sea ice in novel viral transmission between pinnipeds and sea 274 otters-a result that may be the first proof of concept for our proposed climate-disease link 83 . because hotspots of cross-species transmission are predictable, our study provides the first 276 template for how surveillance could target future hotspots of viral emergence in wildlife. in the 277 next decade alone, billions could be spent on virological work trying to identify and counteract 278 zoonotic threats before they spread from wildlife reservoirs into human populations 2 . these to implement the grubb outlier tests for a given species we defined a distance matrix between 322 each record and the centroid of all records (in both environmental or geographic space, respec-323 tively) and determined whether the record with the largest distance was an outlier with respect 324 to all other distances, at a given statistical significance (p = 1e − 3, in order to exclude only 325 extreme outliers). if an outlier was detected it was removed and the test was repeated until no 326 additional outliers were detected. the worldclim dataset is widely used in ecology, biodiversity, and agricultural projections 331 of potential climate change impacts. worldclim makes data available for current and future for herbivores and omnivores, the maximum is estimated as d = 3.31m 0.65 . 514 we used mammalian diet data from the eltontraits database 113 , and used the same cutoff as 515 schloss to identify carnivores as any species with 10% or less plants in their diet. we used body 516 mass data from eltontraits in the schloss formula to estimate maximum generational dispersal, 517 and converted estimates to annual maximum dispersal rates by dividing by generation length, formula performs notably poorly for bats: for example, it would assign the largest bat in our 527 study, the indian flying fox (pteropus giganteus), a dispersal capacity lower than that of the gray 528 dwarf hamster (cricetulus migratorius). bats were instead given full dispersal in all scenarios: 529 14 given significant evidence that some bat species regularly cover continental distances 46,47 , and 530 that isolation by distance is uncommon within many bats' ranges 49 , we felt this was a defensible 531 assumption for modeling purposes. moving forward, the rapid range shifts already observed 532 in many bat species (see main text) could provide an empirical reference point to fit a new allo-533 metric scaling curve (after standardizing those results for the studies' many different method-534 ologies). a different set of functional traits likely govern the scaling of bat dispersal, chiefly the 535 aspect ratio (length:width) of wings, which is a strong predictor of population genetic differ-536 entiation 49 . migratory status would also be important to include as a predictor although here, 537 we exclude information on long-distance migration for all species (due to a lack of any real 538 framework for adding that information to species distribution models in the literature). . using a linear model, we show that elevation (c), species richness (d), and land use (e) influence the number of new overlaps for bats and non-bats. slopes for the elevation effect were generally steeply positive: a log 10 -increase in elevation was associated with between a 0.4-1.41 log 10 -increase in first encounters. results are averaged across nine global climate models. legends refer to scenarios: cl gives climate and land use change, while cld add adds dispersal limits. 20 a. b. extended data figure 8 : projected viral sharing from suspected ebola reservoirs is dominated by bats. node size is proportional to (left) the number of suspected ebola host species in each order, which connect to (middle) first encounters with potentially naive host species; and (right) the number of projected viral sharing events in each receiving group. (node size denotes proportions out of 100% within each column total.) while ebola hosts will encounter a much wider taxonomic range of mammal groups than current reservoirs, the vast majority of viral sharing will occur disproportionately in bats. extended data figure 9 : data processing workflow. summary of species inclusion across the modeling pipeline for species distributions and viral sharing models. the final analyses in the main text use 3,139 species of placental mammals across all scenarios. extended data figure 10 : species distribution modeling workflow for a single species. a focal species (the sand cat, felis margarita) is displayed as an illustrative example. the present day climate prediction (top left) was clipped to the same continent according to the iucn distribution (top right). this was then clipped according to cervus elaphus land use (second row, left). the known dispersal distance of the red deer was used to buffer the climate distribution (second row, right). the future distribution predictions (rcp 2.6 shown as an example) are displayed in the bottom four panels, for each of the four pipelines: only climate (third row, left); climate + dispersal clip (third row, right); climate + land use clip (bottom row, left) and climate + land use + dispersal clip (bottom row, right). the four distributions clearly display the limiting effect of the dispersal filter (bottom right panels) in reducing the probability of novel species interactions (bottom left panels). the land use clip had little effect on this species as the entire distribution area was habitable for the red deer. bats as 'special' reservoirs for emerging zoonotic 805 pathogens a comparison of bats and 808 rodents as reservoirs of zoonotic viruses: are bats special? are bats really 'special' as viral reservoirs? 811 what we know and need to know mass extinctions, biodiversity and mitochon-813 drial function: are bats 'special' as reservoirs for emerging viruses? current opinion in 814 virology viral zoonotic risk is homogenous among taxonomic 816 orders of mammalian and avian reservoir hosts virological factors 819 that increase the transmissibility of emerging human viruses transmissibility of emerging viral 822 zoonoses origins of hiv and the aids pandemic. cold spring harbor 824 perspectives in medicine 1 origin and evolution of pathogenic coronaviruses key: cord-261547-8tfbhmzo authors: góes, luiz gustavo bentim; campos, angélica cristine de almeida; carvalho, cristiano de; ambar, guilherme; queiroz, luzia helena; cruz-neto, ariovaldo pereira; munir, muhammad; durigon, edison luiz title: genetic diversity of bats coronaviruses in the atlantic forest hotspot biome, brazil date: 2016-07-26 journal: infect genet evol doi: 10.1016/j.meegid.2016.07.034 sha: doc_id: 261547 cord_uid: 8tfbhmzo bats are notorious reservoirs of genetically-diverse and high-profile pathogens, and are playing crucial roles in the emergence and re-emergence of viruses, both in human and in animals. in this report, we identified and characterized previously unknown and diverse genetic clusters of bat coronaviruses in the atlantic forest biome, brazil. these results highlight the virus richness of bats and their possible roles in the public health. brazil harbours 15% of the world's bat diversity and carries 178 distinct bats species (nogueira et al., 2014) . out of these, a total of 113 species exist only in the atlantic forest biome (afb), which is the second largest rain forest of the south america, and is one of the unique regions with highest biodiversity in the world (paglia et al., 2012) . bats are historically unique and widespread mammals playing essential roles in the emergence and re-emergence of viruses of both veterinary and public health importance. although viruses of diverse genetic backgrounds can co-exist asymptomatically in bats, majority of these viruses are single stranded rna viruses (calisher et al., 2006) . coronaviruses (covs) are enveloped, positive-sense, single-stranded rna viruses in the family coronaviridae, and are usually associated with respiratory, enteric, hepatic and neurological pathologies of varying severity (woo et al., 2012) . coronaviruses are classified into four genera; alphacoronavirus (α-cov) and betacoronavirus (β-cov) have been exclusively identified in mammals, whereas gammacoronavirus and deltacoronavirus are mainly detected in avian species (woo et al., 2012; ictv, 2015) . based on the genetic relatedness, the β-covs can further be subdivided into four clades: a to d (drexler et al., 2014) . all covs that can potentially infect human were originated from animal reservoirs, and four of such covs are believe to be transmitted to human through bats including α-covs (229e and nl63) and highly pathogenic β-cov (severe acute respiratory syndrome and middle east respiratory syndrome) (bolles et al., 2011; chan et al., 2015; corman et al., 2013; huynh et al., 2012) . recently, a number of novel bats covs have been identified, primarily from african, asian and european bats (calisher et al., 2006; chu et al., 2006; drexler et al., 2014) , as well as from south american countries including costa rica, panama, ecuador, mexico and brazil (corman et al., 2013; goes et al., 2013) . collectively, these studies indicate the co-existence of bats and viruses at the interface of viral evolution and bats ecology. a limited number of studies have been conducted in brazil to map the nature and breath of bats in harbouring viral populations. in previous studies, a total of five distinct cov lineages have been detected in just 10% of local bats (15 species), and most of these are belonging to α-cov (brandao et al., 2008; corman et al., 2013; goes et al., 2013) . these attributes, and the existence of a large number of human beings (120 million) in the atlantic forest biome, brazil, clearly highlight the potential of bats in not only carrying zoonotic viruses but also in possible transmission of viruses to human beings. to ascertain the diversity of bats covs circulating in the brazilian bats, a total of 401 intestine tissues from 17 bat species were collected from 2010 to 2014 encompassing bats with distinct diet habits (table 1 , fig. 1a , b). bats from urban (n = 192) and rural (n = 10) areas were received from 14 municipalities of the northwestern state of são paulo by the rabies laboratoryof universidade estadual paulista (unesp), araçatuba-sp. additionally, 99 and 100 samples were collected from iguaçu national park, and from two distinct disturbed landscape sites, respectively, and provided by our collaborators of the zoology department at unesp, rio claro-sp. all activities were infection, genetics and evolution 44 (2016) infection, genetics and evolution j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / m e e g i d authorized and approved by the ethic committee of institute of biomedical research from university of são paulo (56-18-03/2014) and bats species were identified based on the morphological characteristics including head-boby, forearm size, and dental arch as described by vizotto and tadei (1973) ; gregorin and taddei (2002) and miranda et al. (2011) . nucleic acids extracted from 30 mg of intestine tissue of individual bat using nuclisens® easymag® were subjected to first strand cdna synthesis with random hexamers and rt-pcr high-capacity cdna archive kit (applied biosystems). the cdna preparations were screened by pancoronavirus nested pcr assay targeting the rna-dependent rna polymerase (rdrp) gene (chu et al., 2011) . positive samples were sequenced and final sequences were submitted to the genbank (kt717380-kt717394) (supplementary table) . a dataset, consisting of sequence generated in this study and all publically available bats covs, was phylogenetically analysed by neighbour-joining method in mega6 software using kimura's two-parameter correction and 10,000 bootstrap values (tamura et al., 2013) . coronaviruses were detected in 15 bat intestines samples from eight bat species with distinct diet habit, demonstrating a marked potential of covs distribution among bat species in afb that harbours 9% of world's bat diversity. all covs-positive bat species were geographical distributed in the neotropics and in anthropogenic area apparently affected by fragmented forests, and bats-abundance urbanized areas. the detection of covs varied by bat species; undisturbed forest remnant (4%), fragmented forests (4.5%) and urban areas (3.1%) ( table 1) . phylodynamics analyses indicated the circulation of 13 α-cov and 2 β-cov in afb bats (fig. 1c) . this is of primary interest as majority of bat covs surveillance studies conducted in the new world bats detected only α-covs (corman et al., 2013; drexler et al., 2014; osborne et al., 2011) . a higher resolution analysis indicated a distinct distribution and diversity of α-cov and β-cov lineages (fig. 1d) . α-cov sequences obtained from bats of same genus presented high nucleotide sequence similarity (e.g. artibeus, glossophaga, carollia, molossus, myotis and sturnira) (fig. 1d and supplementary table), even with sequences detected in other studies from bats of geographically distant regions. this relation can be exemplified by the high similarity of the cov rna partial sequences (91,9%) detected in carollia perspicillata species from fenix-pr, brazil (kt717385), and fyzabad, trinidad and tobago (eu769557) (carrington et al., 2008) , located at a distance of at least 3900 km. similar results were previously reported for a variety of bat covs and are taken as evidence of co-evolution of cov genotypes and specific host genera (drexler et al., 2014; corman et al., 2013; anthony et al., 2013) . moreover, two previously uncharacterized α-cov from myotis nigricans and myotis riparius and one β-cov cluster were identified in studied bats ( fig. 1c and d) . cov sequences from myotis bats genera presented high nt sequence similarity (98.9%) and were most closely related to bat covs (kc110771) collected from brazilian molossus molossus and tadarida brasiliensis. a distinct cluster of α-covs were also detected in sturnira bat that grouped with α-covs lineage 1, a group with an evolutionary history of recombination and cross-species transmission between domestic and livestock animals, such as feline, canine and swine α-covs (lorusso et al., 2008) . this clade presents nt similarity between 73.9% and 74.4% with transmissible gastroenteritis virus (dq811789) and feline coronavirus (af124987) depending on the sturnira lineage (supplementary table) . this branching pattern may possibly explain the common-ancestral origin of α-covs lineage 1 species from bats and other animal species. however, extensive evolutionary studies on complete genome sequences of these isolates are required to provide information on the virus origin and divergence. out of two β-covs, the virus from eumops glaucinus clustered within mers-cov containing lineage c. the second β-cov detected in artibeus lituratus shown highest similarity (96.4%) with cov reported from costa rica (corman et al., 2013) and lower similarity (69.3%) with mers-cov (supplementary table) . these sequences showed sequence similarities with β-cov detected in pipistrellus bats that poses high homology with human mers-cov (jx869059). notably to observe that the eumops bat positive for cov was found on an urban area and was predated by a domesticated cat. it was not possible to rule out any event of virus transmission to human, however, this highlight the potential of domesticated animals in virus transmission and disease dynamics at the virus-animalhuman interface. this is of special importance due to the established role of animals in the transmission of viruses to human being (johnson et al., 2015) . taken together, these results represent the first detection of lineage c β-cov in south american bats. despite of close relationship between lineage c β-covs in asia, africa and europe, cumulative data indicate that this lineage showed a more diversified host family distribution in americas in mormoopidae, phyllostomidae and molossidae bat species (anthony et al., 2013; corman et al., 2013; goes et al., 2013) . we present a great diversity of cov genotypes and clusters in brazilian bats, highlighting a biogeographic distribution of bats covs in the region. it is indispensable in future to investigate the evolutionary events in genetically diverse bats covs using complete genome sequences, and their possible transmission potentials to human being. although it is not possible to calculate the risk of "spill over" events of brazilian bats covs to humans, our results reinforce the need for expanded and continuing surveillance of covs in bat fauna, including those in the afb regions of brazil. supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.meegid.2016.07.034. coronaviruses in bats from mexico sars-cov and emergent coronaviruses: viral determinants of interspecies transmission a coronavirus detected in the vampire bat desmodus rotundus bats: important reservoir hosts of emerging viruses detection and phylogenetic analysis of group 1 coronaviruses in south american bats middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease coronaviruses in bent-winged bats (miniopterus spp avian coronavirus in wild aquatic birds ecology, evolution and classification of bat coronaviruses in the aftermath of sars novel bat coronaviruses chave artificial para a identificação de molossídeos brasileiros (mammalia, chiroptera) evidence supporting a zoonotic origin of human coronavirus strain nl63 virus taxonomy spillover and pandemic properties of zoonotic viruses with high host plasticity chave ilustrada para determinação dos morcegos da região sul do brasil checklist of brazilian bats alphacoronaviruses in new world bats: prevalence, persistence, phylogeny, and potential for interaction with humans lista anotada dos mamíferos do brasil/annotated checklist of brazilian mammals evolution. an eocene big bang for bats mega6: molecular evolutionary genetics analysis version 6.0 chave para determinação de quirópteros brasileiros discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus a: brazil's map and sites for bats capture/collection. bats were collected from 17 sites (a-q) in the atlantic forest biome from two adjacent paraná and são paulo states, brazil. b: based on morphological characteristics, bats were classified into different families (positive families were bold and underlined) this study was supported by fapesp (são paulo research foundation) process number 2013/11006-0 and british council grant number 172710323. cruz-neto a.p was supported by fapesp grant process number 2008/57687-0. we thank luiz aurélio de campos crispin and mariana cristine pereira de souza for their contributions to this study. key: cord-281956-obwtd33y authors: mayer, fabiana quoos; dos reis, emily marques; bezerra, andré vinícius andrade; cerva, cristine; rosa, júlio; cibulski, samuel paulo; lima, francisco esmaile sales; pacheco, susi missel; rodrigues, rogério oliveira title: pathogenic leptospira spp. in bats: molecular investigation in southern brazil date: 2017-05-12 journal: comp immunol microbiol infect dis doi: 10.1016/j.cimid.2017.05.003 sha: doc_id: 281956 cord_uid: obwtd33y the present study aimed to investigate the frequency of pathogenic leptospira spp. in brazilian bats and to determine possible risk factors associated to it. ninety two bats of 12 species were evaluated. whole genomic dna from kidneys was extracted and real-time pcr specific to pathogenic leptospira spp. was applied. association between the frequency of specimens positive for leptospira spp. and sex, age, bat species or family, season of collection, geographic localization and feeding habits was evaluated. the results showed that 39.13% of analyzed bats were found positive for leptospira spp. nine bat species had at least one positive result. there was no association among the evaluated variables and frequency of pathogenic leptospira spp. although the limitations due to lack of leptospira spp. isolation, leptospiral carriage was demonstrated in bats of different species from southern brazil, which reinforces the need for surveillance of infectious agents in wild animals. leptospirosis is a zoonotic disease caused by pathogenic strains of leptospira spp., which colonize host kidneys and are eliminated in urine. the transmission occurs by direct contact with contaminated urine or indirectly through contaminated water or soil [1] . leptospirosis is worldwide distributed, and its incidence varies with climate, animal reservoirs and surveillance [2] . rainy periods are associated to higher frequencies of disease, especially in large cities, since there is an increased chance of population contact with contaminated water in inadequate sanitation areas [3] . in brazil, more than 60,000 human leptospirosis cases were confirmed between 2000 and 2016 and the regions with higher frequency of confirmed cases are the southern and southeastern regions [3] . the higher incidences occur in low-income populations of most populous cities, such as são paulo or in regions with poor sanitation system [4] . besides public health issues, leptospirosis is responsible for economic losses for the animal production sector. this is caused by costs with vaccination and decrease of production due to abortion, lower milk production and animal death [1] . an important feature of leptospirosis is the wide host range that are susceptible to disease or can serve as bacteria reservoirs [5] , which hinders its control and epidemiologic understanding. several studies have demonstrated that rodents are the main leptospirosis reservoirs [6] . however, there is a growing knowledge about the role of different wild animal species on disease cycle, which may be important due to their abundance and increasing contact with domestic animals [7, 8] . thus, identifying wild reservoirs is a key factor in leptospirosis epidemiology knowledge [9] . among leptospira spp. wild reservoirs, bats are being evaluated in different parts of the world with different results according to region [10] . bats have been implicated in epidemiological cycles of several emerging and re-emerging zoonosis, such as rabies [11] , severe acute respiratory syndrome (sars) [12] , leptospirosis [8, 13] and recently ebola in africa [14] , which points them as important key players in the epidemiology of infectious diseases. bats are found on every continent http except antarctica [15] . in brazil, 178 bat species have been recorded [16] , of which 40 are settled in rio grande do sul state, the brazilian southern region [17] . despite this wide range of bat species and high rates of leptospirosis, there are few brazilian studies on this subject showing low bat leptospiral infection rates [4, 18] ; however, they were performed in urban areas of southeast region. thus, in the present study, we sought to investigate the frequency of pathogenic leptospira strains in bats from different areas of rio grande do sul, the southern brazilian state, which has a different ecosystem. permission for this work on bats was granted by ethical commission on animal experimentation of the "instituto de pesquisas veterinárias desidério finamor" (ceua/ipvdf) (process number 03/2012). the study did not involve any direct manipulations of live bats and relied entirely on collection of tissue samples from dead bats. all experiments were performed in compliance with the european convention for the protection of vertebrate animals used for experimental and other scientific purposes (european treaty series-no. 170 revised 2005) and the procedures of the brazilian college of animal experimentation (cobea). bat samples (n = 92) from 31 and 2 municipalities of rio grande do sul and santa catarina states, respectively ( fig. 1) were included in the study. the samples were sent to instituto de pesquisas veterinárias desidério finamor for rabies diagnosis between december 2010 and december 2012. species identification was performed based on pre-vious studies and age determination was made through epiphysis fusion and dentition evaluation [19, 20] . for detection of pathogenic leptospira spp. in bat kidneys, total dna was extracted as previously described [21] and quantified using a spectrophotometer (l-quant, loccus biotechnology, brazil). a conventional pcr for glyceraldehyde-3-phosphate dehydrogenase (gapdh) gene was performed for dna quality confirmation [22] . about 25 ng of dna were used as template for taqman ® real-time pcr with primers and probe targeting lipl32 as previously published [23] . all reactions were carried out in triplicate and positive results were considered if at least two reactions had detectable ct. positive and negative controls were included in each run. to determine the real-time pcr analytical sensitivity, a conventional pcr using the reference isolate pomona dna was performed and the resulting amplicon was cloned in a pcr2.1 vector (topo ta cloning ® kit, invitrogen, usa). after escherichia coli dh5-α transformation, plasmid dna was recovered [24] and quantified with qubit (invitrogen, usa). the number of molecules was calculated as follows: [6.02 × 10 23 molecules × cloned vector quantity (g)]/molecular weight of cloned vector (g). standard curve was made with amounts ranging from 10 9 to 10 −1 leptospiral dna molecules as templates for real-time pcr. in order to evaluate an association between the positivity frequency for pathogenic leptospira spp. and the dependent variables, chi-square (sex variable) or fisher exact test (age, bat species or family, season of collection, geographic localization and feeding habits variables) were ninety two bat specimens were analyzed. they were classified into 12 different species, from phyllostomidae, molossidae and vespertilionidae families. most of the samples were from free tailed bats (tadarida brasiliensis) ( table 1 ). the majority of animals were adults (82.61%). sample distribution by sex was homogeneous and the higher frequency of collection occurred during spring and winter ( table 1 ). the number of analyzed bats by municipality is indicated in fig. 1 . the real-time pcr was able to detect 10 genome copies of leptospira spp. with 103.42% efficiency (r 2 = 0.99) (fig. 2.) , which was the same detection capacity than the previous study [23] . all dna amplified the gapdh gene, showing that the samples were in appropriate conditions for molecular analysis. of these, 36 (39.13%) had positive results for pathogenic leptospira (table 2 ) and 9 species had at least one positive bat ( table 2) . among the species with higher number of analyzed specimens, tadarida brasiliensis had almost half of the animals positive for leptospiral genome carriage (21/46; 45.65%; table 2 ) and molossus molossus had a lower positivity rate (4/19; 21.05%; table 2 ). a low number of other bat species has also been surveyed and their infection rates varied from 0% (eptesicus furinalis, eumops patagonicus and sturnira lilium) to 100% (histiotus velatus (2/2), lasiurus ega (1/1) and myotis nigricans (1/1)) ( table 2 ). in 33 municipalities where the samples were collected, 20 had at least, one positive bat (fig. 1.) . factors such bat species, leptospira spp. serovar, age and breeding season may influence bat leptospiral infection rates. in the present study, there was no association among age (p = 0.502), sex (p = 0.867), species (p = 0.139), season of collection (p = 0.838), location (p = 0.477), feeding habits (p = 1.000) or family (p = 0.09) and the frequency of positive results for pathogenic leptospira. in the present study, bats from southern brazil were shown to be leptospira spp. carriers. the data showed higher frequency than previous brazilian studies -39.1% vs 7.8% and 1.75% in botucatu and são paulo, respectively [25, 26] . this may be related to the environment in which the bats live, as in the present study bats from south brazilian areas, most of them distant from urban centers, were evaluated. a variable number of bats from the surveyed species were analyzed; however, it was possible to observe an equal distribution of positive and negative results for tadarida brasiliensis, the species presenting a higher number of analyzed specimens; and a lower rate of positive results for molossus molossus, the second species with more samples. considering the family, there seems to be higher frequency of positive results within vespertilionidae family, although there was no statistical significance; thus, further studies are needed to confirm that different bat families have different degrees of susceptibility to leptospira spp. infection. the leptospiral host specificity could not be accessed, since there was failure to amplify the secy gene fragment. this may have been occurred due to low bacterial loads or poor dna quality, as the bats arrived in the laboratory days after death. previous studies also report difficulty to amplify larger amplicons from kidney samples positive for leptospiral real-time pcr, even though the bat specimens were captured avoiding dna quality issues [27, 28] . regarding the feed behavior, the presence of leptospiral dna was detected in insectivorous species, which refutes the hypothesis that sharing food with rodents increases the likelihood of bats to carry leptospira spp. [29] . as the bats' habitats were not accessed, it was not possible to determine the routes by which the animals were probably infected. taking into account the leptospira spp. transmission routes, the contact with contaminated soil or water could be considered. no statistical difference was observed regarding leptospira spp. positivity frequency when analyzing sex, age, local or season of collection. some of these variables have been described to influence leptospiral carriage in rodents [30] ; however, the observed results may be related to low sample number and the way of selection, which was by convenience. the majority of bats evaluated in the present study were found distant from large urban centers and many of them in unusual situations, such as in daylight, sick, or dead. this can indicate increased risk of co-infection with other infectious agents; however, to test this hypothesis, new studies with animals randomly selected should be performed. currently, urban sprawl has been responsible for the closer contact of humans and wild animals. bats often live at roofs, gaps between buildings and urban vegetation, which may have impact on disease transmission to humans and domesticated animals. as the number of analyzed specimens by municipality varied and in general was low, it was not possible to associate bat leptospiral carriage and human cases of leptospirosis; moreover, it was not possible to access the leptospiral serovars infecting the bats. these limitations precluded an analysis about the role of these animals on human leptospirosis transmission, which needs to be further investigated. there are differences associated with social, health and environmental conditions regarding infectious diseases transmissibility, which are still of major concerns worldwide. since in the last decades some important infectious diseases have emerged or re-emerged, challenging the global public health security, a better knowledge on the ecology of different infectious agents becomes important. the present study, even though presenting limitations due to lack of leptospira spp. isolation and genotyping, brings new knowledge on leptospiral carriage in brazilian bats. the data reinforces the need for surveillance of infectious agents, especially the zoonotic ones which are hosted by wild animals. evaluation of a modified taqman assay detecting pathogenic leptospira spp. against culture and leptospira-specific igm enzyme-linked immunosorbent assay in a clinical environment the contribution of bats to leptospirosis transmission in são paulo city, brazil, am pathogenesis of leptospirosis: cellular and molecular aspects leptospirosis: a zoonotic disease of global importance animal leptospirosis in latin america and the caribbean countries: reported outbreaks and literature review diversity of bat-associated leptospira in the peruvian amazon inferred by bayesian phylogenetic analysis of 16s ribosomal dna sequences maximizing the chances of detecting pathogenic leptospires in mammals: the evaluation of field samples and a multi-sample-per-mammal, multi-test approach leptospira and paramyxovirus infection dynamics in a bat maternity enlightens pathogen maintenance in wildlife geographical distribution of vampire bat-related cattle rabies in brazil bats are natural reservoirs of sars-like coronaviruses leptospiral antibodies in flying foxes in australia investigating the zoonotic origin of the west african ebola epidemic bats: important reservoir hosts of emerging viruses checklist of brazilian bats biogeografia de quirópteros da região sul leptospira spp. and toxoplasma gondii antibodies in vampire bats (desmodus rotundus) in botucatu region, sp brazil chave artificial para determinação de molossídeos brasileiros (mammalia: chiroptera) los murciélagos de argentina: clave de identificación comparison of in house polymerase chain reaction with conventional techniques for the detection of mycobacterium tuberculosis dna in granulomatous lymphadenopathy food safety in raw milk production: risk factors associated to bacterial dna contamination detection of pathogenic leptospira spp. through taqman polymerase chain reaction targeting the lipl32 gene preparation and transformation of competent e. coli using calcium chloride the contribution of bats to leptospirosis transmission in sao paulo city, brazil, am leptospira spp. and toxoplasma gondii antibodies in vampire bats (desmodus rotundus) in botucatu region, sp, brazil, j. venomous anim malagasy bats shelter a considerable genetic diversity of pathogenic leptospira suggesting notable host-specificity patterns pathogenic leptospira spp. in bats, madagascar and union of the comoros use of pcr to identify leptospira in kidneys of big brown bats (eptesicus fuscus) in kansas and nebraska, usa household characteristics associated with rodent presence and leptospira infection in rural and urban communities from southern chile emily marques dos reis was recipient of finep/cnpq scholarship and andré vinícius andrade bezerra was recipient of fapergs scholarship. this project was financially supported by finep (grant number sanimars 01100783-00). the sponsors had no role in the study design, in the collection, analysis and interpretation of data, in the writing of the manuscript and in the decision to submit the manuscript for publication. key: cord-254543-gxwtqqdu authors: suwannarong, kanokwan; schuler, sidney title: bat consumption in thailand date: 2016-01-22 journal: infect ecol epidemiol doi: 10.3402/iee.v6.29941 sha: doc_id: 254543 cord_uid: gxwtqqdu background: human consumption of bats poses an increasing public health threat globally. communities in which bat guano is mined from caves have extensive exposure to bat excreta, often harvest bats for consumption, and are at risk for bat-borne diseases. methods: this rapid ethnographic study was conducted in four provinces of thailand (ratchaburi, sakaeo, nakorn sawan, and phitsanulok), where bat guano was mined and sold during the period april–august 2014. the aim of this study was to understand behaviors and risk perceptions associated with bat conservation, exposure to bats and their excreta, and bat consumption. sixty-seven respondents playing various roles in bat guano mining, packaging, sale, and use as fertilizer participated in the study. data were collected through interviews and/or focus group discussions. results: in spite of a bat conservation program dating back to the 1980s, the benefits of conserving bats and the risks associated with bat consumption were not clear and infrequently articulated by study respondents. discussion: since bat consumption continues, albeit covertly, the risk of bat-borne diseases remains high. there is an opportunity to reduce the risk of bat-borne diseases in guano-mining communities by strengthening bat conservation efforts and raising awareness of the health risks of bat consumption. further research is suggested to test behavior change strategies for reducing bat consumption. z oonotic pathogens cause an estimated 70% of emerging and re-emerging infectious diseases in humans (1á3). bats in particular are important wild animal reservoirs for zoonotic viruses, as they host more viruses per species than other mammals (4) . several emerging diseases, such as severe acute respiratory syndrome (sars) (5) , ebola virus (6, 7) , nipah virus (8) , and middle eastern respiratory syndrome (mers-cov) (9) , have been linked to bats. there is nevertheless little research on bat and human interaction, the human perceptions about bats and the associated disease risk, and the populations that are the most at risk (10) . globally, there are two main types of bats: megabats (megachiroptera) and microbats (microchiroptera). megabats are found in the subtropics and tropics of asia and africa, and feed on nectar and pollen, petals, bracts, fruit, leaves, and water (11) . in thailand, where the study was undertaken, there are several species of flying foxes and fruit bats. microbats are geographically more widespread than the megabats. they are sometimes referred to as insectivorous bats because insects are their primary food. bats are hunted, eaten, and used for medicine and natural insect control. their excrement (guano) is used as fertilizer (10, 12á14) . bat guano mining is common globally, especially in countries in africa, latin america, and the caribbean and east asian countries (including thailand). guano mining can be very lucrative: the guano produced by a large cavedwelling population of bats might be worth several million dollars each year (13, 15) . bat guano is a very rich fertilizer, primarily due to its high nitrogen, potassium, and phosphorus content (16) . in thailand, guano produced by the microbat tadarida plicata, the wrinkle-lipped freetailed bat, is favored for its rich organic content. it is the most abundant bat in thailand, lives in 17 caves across the country, and is insectivorous (12) . exposure to bats, bat guano, and bat excreta through bat consumption puts people at risk of direct contact with bat-borne viruses (14, 17) . because of the potential for human contact with infected bats, the prevent project, a part of the us agency for international development's (usaid) emerging pandemic threats (ept) program, decided to conduct this exploratory, rapid ethnographic study. of the study was to understand the various ways by which people are exposed to bats and bat excreta in the context of guano mining, and its packaging and sale as fertilizer in thailand; the perceived health risks associated with such exposure; and the measures that may be used to mitigate risk among people involved in the guano-mining business. this paper focuses on bat consumption only indirectly, given the current legal prohibitions against capturing and selling bats. the study was conducted in four sites in thailand where bat guano is mined and sold. the sites, located in four provinces (ratchaburi, sakaeo, nakorn sawan, and phitsanulok), were selected to represent several different ways in which bat guano mining is organized and regulated. observing the different ways guano mining is handled was intended to capture possible variations in the age groups and numbers of people who are exposed to bats and bat excreta, the extent of their exposure, and the measures that are taken to reduce exposure. preference was given to sites where other usaid ept program activities were taking place, especially other studies bat-borne and other zoonotic viruses. a map of the selected provinces is shown in fig. 1 . a rapid ethnography approach was utilized to understand perceptions and behaviors regarding exposure to bats and bat excreta. although the research conducted examined different aspects of exposure to bat and bat excreta, this paper reports only our findings on bat consumption. the research was implemented from april to august 2014. six types of respondents (aged 20 years and above) were included in the study, for a total 67 interviews: 1. mine managers. the mine manager is the person who runs day-to-day operations at the guano mine. there were only one or two managers at the selected sites. the willingness of the mine manager(s) to be interviewed was one of our criteria for site selection. 2. male and female miners. the selection of the miners was purported to capture the range of different ages, gender, and other characteristics noted by the mine managers. the eligible miners were required to have been engaged in the guano-mining work for a minimum of 6 months. the researchers approached the miners at the cave where they worked; obtained their preliminary consent, home addresses, and phone numbers; and made appointments to interview them at a convenient time at their homes or another location of their choice. 3. adults who dry, package, and/or purchase the guano at the mine site. we included respondents who were willing to respond to questions and also lived near the mines. 4. spouses or other adult family members of miners who wash miners' soiled clothing or otherwise come into direct or indirect contact with bats or their excreta. these respondents were referred by the miners. 5. owners or managers of shops that sell guano. these respondents were referred by the guano buyers at the mine. they were interviewed in their shops or another location of their choice. 6. users of guano for fertilizer. these respondents were referred either by shopkeepers, miners, or miners' spouses. preference was given to users residing in the communities of the miners. they were interviewed in their homes or another location of their choice. numbers of respondents' by province and type are shown in table 1 . the five interviewers (two male and three female) used semi-structured interview guides to conduct one-onone, open-ended interviews in thai with each type of respondent. the interviewers were trained to probe and spontaneously add questions based on the discussion and information provided by the respondents. a note-taker accompanied each interviewer to record observations related to body language, the presence of other individuals during the interview, and details of the environment in which the interview took place. written informed consent was obtained prior to each interview. the interviews were audio-recorded with permission from the respondents. in two sites, we conducted focus group discussions, inviting all respondents (except mine managers), who wished to join. in these discussions, we summarized what we learned from the individual interviews about exposure to bats and bat excreta and asked for verification and corrections, discussed risk mitigation strategies and barriers to their use, and elicited opinions about the feasibility and acceptability of potential strategies to minimize exposure to guano, such as wearing goggles and masks. the interviews were transcribed and translated into english. the english translations were thematically coded using the qualitative research software, nvivo 10. data on specific topics of interest were systematically compared across interviews and variations by gender and site noted. guano miners have an appreciation and respect for bats that live in their communities or are seen existing community caves or fruit orchards. in recent years, it has become illegal to trap or eat bats. in spite of this, most respondents knew people in the community who had trapped bats, usually at the mouths of caves or in fruit orchards, and/or had eaten bats, although they said this was less common since the regulations went into effect. it is legal for community members to collect and eat the occasional bat that falls to the ground after colliding with electric power lines. respondents who either had eaten bats or knew people who had eaten bats said that they were usually prepared by frying or stir-frying with garlic, chili and/or basil; or boiling in curry after discarding the intestines and wings. some people also ferment bat meat as they would pork, and serve it with rice; or prepare a spicy bat soup. in addition to cooking bats, the practice of drinking bat blood (e.g. as a health supplement or a few drops mixed with whiskey) was reported as a rumor in three of the four sites. below is information on the consumption of bats from respondents in each province (identified as a, b, c, and d to protect confidentiality). historically, until about 10 years ago, bats were caught in nets at the top of the mountain at the cave mouth and sold in batches of up to 100 at the food stall. since the arrival of the governmental wildlife conservation office, however, this practice has all but stopped. the royal forest department prohibits trapping bats for consumption and levies a fine of 500 baht on those who trap or this respondent was the same person (the mine manager and owner of the shop); b in practice, the categories of miners and spouses were not mutually exclusive. during interviews with spouses, we learned that both husbands and wives often work in guano mines. in these cases, we interviewed the spouses both as miners and as spouses of miners, but classified them as spouses in the table above to avoid double-counting. citation: infection ecology and epidemiology 2016, 6: 29941http://dx.doi.org/10.3402/iee.v6.29941 eat bats. as in the other provinces, bats are sometimes trapped, and eaten or sold secretly. the bats that are eaten in the community are the flying fox and lotus bats, which tend to be comparatively large, fatty bats. it was also noted that lesser dog-faced fruit bat (khang kao na ma) and the shortridge's rousette bat (khang kao bua) were occasionally consumed in the community. some of the illegally trapped bats are sold to restaurants for 4á5 baht per bat. the restaurants sell bats as a gourmet item that is likely not found on the published restaurant menu. bat blood is used as a health supplement in the province. fresh bat blood mixed with alcohol is sometimes used to relieve back, leg, and waist pain or to nourish the body and prevent 'wasting disease'. bat blood can be purchased in the community for 5á10 baht per bottle. the royal forest department did not seem to be active in this province at the time of the study. the villagers were knowledgeable about the need to conserve bats, and there did not appear to be a fine for catching or consuming bats in this community. in the past, people in this community ate the large lye's flying fox bats that were brought home by the miners. reportedly, restaurants still place private orders for bats with a few people who occasionally set up nets at the mouth of the cave to catch them. the bats are purchased for 1 baht per bat. at the time of the study, there was a 500-baht fine for trapping and/or eating bats in this province. bats that died a natural death could be consumed with no fine. mid-sized bats are illegally trapped and sold in the community. nonspecific cave bats, bats caught in nets at the mango or red cotton flower fields, and the flying fox bat were among the bat types secretly caught as an illegal food source. in addition to bat meat, respondents reported that bat blood is occasionally consumed as a health supplement in the community, as it is said to be useful for treating asthma in children (especially if the blood is taken from a bat's ear or wing), curing other unnamed illnesses, or increasing strength. province d although trapping and consuming bats has been regulated for some time in this province with a fine that is rumored to be very high (50,000á60,000 baht), numerous respondents spoke of their knowledge or experience in trapping and consuming bats. the most common bats consumed are fruit bats that become caught in nets placed over fruit trees or sugar palms. small bats are also occasionally eaten. bats that fall from after flying into power lines can be legally consumed and are often made into a spicy bat soup or stir fry. bat blood is occasionally consumed and is considered to be an aphrodisiac. efforts to protect bat populations in thailand's caves began in the 1980s and have continued until the present time. recent legislation makes it illegal to trap or eat bats in thailand. currently, the royal forest department discourages the trapping and eating of bats by levying steep fines. the fines were noted in all of the communities we studied. the benefits of conserving bats and the risks associated with bat consumption, however, were infrequently articulated by the study respondents, indicating that illegality was the greatest deterrent to trapping and eating bats, not concerns about animal conservation or health risk reasons. however, several studies presented relevant evidences that bat consumption might be risky for bat-borne diseases (10, 14, 17) . aside from direct consumption, the transmission of disease from bats to people in bangladesh is through the contamination of raw date palm sap by bats. presumably, the transmission to humans occurs through the consumption of food contaminated with bat saliva or urine, and the infection of domestic animals (cattle, pigs, and goats) (18) . further community-level research should be conducted on the perceived economic and other benefits related to bat catching and consumption, as well as community knowledge and perceptions of the risks associated with bats. findings from this research may illuminate barriers to bat conservation and lead to the development of strategies and interventions for eliminating bat consumption. this has the potential to mitigate the risk of transmission of zoonotic diseases from bats to humans in thailand, as well as in other countries where bats are eaten. american people through the united states agency for international development (usaid). beyond bushmeat: animal contact, injury, and zoonotic disease risk in western uganda global trends in emerging infectious diseases bats as a continuing source of emerging infections in humans a comparison of bats and rodents as reservoirs of zoonotic viruses: are bats special? bats and emerging zoonoses: henipaviruses and sars fruit bats as reservoirs of ebola virus long-term survival of an urban fruit bat seropositive for ebola and lagos bat viruses adaptive evolution of bat dipeptidyl peptidase 4 (dpp4): implications for the origin and emergence of middle east respiratory syndrome coronavirus characteristics and risk perceptions of ghanaians potentially exposed to bat-borne zoonoses through bushmeat old world phytophagous bats (megachiroptera) and their food plants: a survey diet of wrinkle-lipped free-tailed bat (tadarida plicata buchannan, 1800) in central thailand: insectivorous bats potentially act as biological pest control agents investigating the role of bats in emerging zoonoses: balancing ecology, conservation and public health interests. fao animal production and health manual analysis of patterns of bushmeat consumption reveals extensive exploitation of protected species in eastern madagascar corporate-update-ecoland-international-inc-an-organic-fertilizer-company.html?print01 corporate update: ecoland international, inc. á an organic fertilizer company bats as bushmeat in madagascar transmission of human infection with nipah virus this is noted that the first author, dr. suwannarong, conducted this study while worked for the prevent project/fhi360 with dr. schuler and fhi360 team. we thank our health and local authorities and communities in saraburi, ratchaburi, nakorn sawan, and phitsanulok provinces, who supported and participated in this study. we also thank our local researchers and the operational staff, who assisted all data collection and logistics management at fields. we thank helen bristow for her kind support with the qualitative data coding and analysis for this study. finally, we also thank kara tureski and tula michaelides for their reviews of this manuscript. this study was funded by prevent project which is implemented by fhi 360 with funds from usaid cooperative agreement ghn-a-00-09-00002-00; this study was made possible by the generous support of the key: cord-003775-1axsebya authors: lelli, davide; lavazza, antonio; prosperi, alice; sozzi, enrica; faccin, francesca; baioni, laura; trogu, tiziana; cavallari, gian luca; mauri, matteo; gibellini, anna maria; chiapponi, chiara; moreno, ana title: hypsugopoxvirus: a novel poxvirus isolated from hypsugo savii in italy date: 2019-06-19 journal: viruses doi: 10.3390/v11060568 sha: doc_id: 3775 cord_uid: 1axsebya interest in bat-related viruses has increased considerably during the last decade, leading to the discovery of a rising number of new viruses in several bat species. poxviridae are a large, diverse family of dna viruses that can infect a wide range of vertebrates and invertebrates. to date, only a few documented detections of poxviruses have been described in bat populations on three different continents (america, africa, and australia). these viruses are phylogenetically dissimilar and have diverse clinical impacts on their hosts. herein, we report the isolation, nearly complete genome sequencing, and annotation of a novel poxvirus detected from an insectivorous bat (hypsugo savii) in northern italy. the virus is tentatively named hypsugopoxvirus (hypv) after the bat species from which it was isolated. the nearly complete genome size is 166,600 nt and it encodes 161 genes. genome analyses suggest that hypv belongs to the chordopoxvirinae subfamily, with the highest nucleotide identity (85%) to eptesipoxvirus (eptv) detected from a microbat eptesicus fuscus in wa, usa, in 2011. to date, hypv represents the first poxvirus detected in bats in europe; thus, its viral ecology and disease associations should be investigated further. poxviruses are dsdna viruses with large genomes (130 to 360 kb) that belong to the family poxviridae. the family is divided into the entomopoxvirinae and the chordopoxvirinae subfamilies of viruses, which infect insects and vertebrates, respectively. according to the international committee on taxonomy of viruses (ictv) 2017 release [1] , 11 genera have been created to classify chordopoxviruses (avipoxvirus, capripoxvirus, centapoxvirus, cervidpoxvirus, crocodylidpoxvirus, leporipoxvirus, molluscipoxvirus, orthopoxvirus, parapoxvirus, suipoxvirus, and yatapoxvirus), but other viruses remain unclassified and new genera are likely to be recognized in the future. poxviruses show a diverse host range, with some viruses having wide host tropism (e.g., orthopoxviruses) and thus being consequently associated with greater zoonotic risks [2] , and others having strict host specificity. in recent decades, bats have been increasingly recognized as reservoirs of emerging viral infections, which has important ramifications for animal and public health [3] . however, the majority of bat-borne viruses that can cause severe diseases in humans and other mammals, do not cause apparent clinical signs in bats. consequently, it has been assumed that bats may have a "special" relationship with viruses based on physiological, ecological, evolutionary, and/or immunological aspects, which allow them to act as special viral reservoirs with exaggerated viral richness [4] [5] [6] [7] . currently, four poxviruses from the microchiroptera and macrochiroptera suborders have been detected in bat populations on three continents (america, africa, and australia) [8] . specifically, eptesipoxvirus (eptv) was isolated in north america in 2011 from eptesicus fuscus [9, 10] ; eidolon helvum poxvirus 1 (ehpv1) was detected in west africa in 2009 from eidolon helvum [11] ; the pteropox virus (ptpv) was identified in northwestern australia in 2015 from pteropus scapulatus [12] ; and a fourth poxvirus was also identified in south australia from miniopterus schreibersii bassanii in 2009 [13] . it is remarkable that these viruses are phylogenetically divergent and are associated with variable clinical manifestations. virological investigations focused on poxviruses in bat populations may have a positive impact for future ecological studies of bat-pathogen interactions. moreover, from the perspective of the one health approach, bats could benefit from these studies, since european bat populations are currently undergoing a global decline that could be linked with so far overlooked viral infections. in this study, we report the isolation, nearly complete genomic sequencing, and annotation of a novel poxvirus detected from an insectivorous bat (hypsugo savii) in northern italy. the virus was tentatively named hypsugopoxvirus (hypv), according to the bat species from which it was isolated. phylogenetic analyses suggest that hypv belongs to the chordopoxvirinae subfamily, revealing the highest similarity (85%) with eptesipoxvirus (eptv) detected from the microbat eptesicus fuscus in wa, usa in 2011, which is associated with bat necrosuppurative osteomyelitis in multiple joints. hypv is the first poxvirus detected in bats in europe and its viral ecology and disease associations should be investigated further. dead bats from different species were collected for virological investigations from wild animal rescue/rehabilitation centers in the context of a general surveillance project that has been implemented in northern italy since 2009-2010, which focuses on the detection of emerging bat viruses [14] [15] [16] . the bats were taxonomically identified based on their morphologic characteristics, according to the european bat identification keys [15] . the carcasses were necropsied, and tissue samples were collected for further laboratory exams, particularly for viral detection and isolation. after necropsy, organ samples (lungs, heart, kidney, brain, and intestines) were mechanically homogenized in minimal essential medium (1 g/10 ml), which contained antibiotics. they were then centrifuged at 3000 g for 15 min. samples were inoculated in confluent monolayers of vero and marc 145 cells (african green monkey), incubated at 37 • c with 5% co 2 and observed daily for seven days to assess their cytopathic effects (cpes). in the absence of cpes, the cryolysates were sub-cultured twice onto fresh monolayers. cell culture supernatants showing cpe were partially purified by ultracentrifugation at 35,000 rpm for 2 h (rotor tst41 kontron) through a 25% (w/w) sucrose cushion, and the pellet was re-suspended in pbs. this antigen was kept at −70 • c and then submitted for viral identification with the ngs approach and negative-staining electron microscopy (nsem) by using the airfuge (beckman instruments, palo alto, ca, usa) method [17] . viral dna was extracted from 200 µl of positive cell culture supernatants using a biosprint 96 one-for-all vet kit (qiagen s.p.a., milan, italy). sequencing libraries were made with a nextera flex kit (illumina inc. san diego, ca, usa) in accordance with the manufacturer's instructions. libraries were sequenced on a miseq instrument (illumina inc. san diego, ca, usa) by using a miseq reagent kit v2 in a 250 cycle paired-end run. data were assembled de novo by the clc genomic workbench v.11 (qiagen s.p.a., milan, italy). genome annotation and analysis was performed with tools from the bioinformatics suite developed at the viral bioinformatics resource centre [18] . the genome annotation transfer utility (gatu) [19] uses a reference genome to automatically annotate poxvirus genes with clear orthologs in the reference. other possible genes were presented to the annotator for further characterization and to make final annotation decisions. the case specifically concerned a juvenile hypsugo savii male that spontaneously died in a wildlife recovery center in valpredina, cenate sopra (bg), northern italy after several weeks of hospitalization. the sick bat was originally found alive on july 17, 2017 in telgate (bergamo province, northern italy) by a private citizen who brought it to the center. clinically, the bat had a humerus fracture, sensory depression and a lack of appetite but normal body mass. the death occurred 54 days after admission to the center on september 9, 2017; then, the carcass was sent to the lab for necroscopy and further analyses. pathological lesions in the internal organs indicative of infectious diseases were not observed, but a soft bone callus due to pathological healing of the humerus fracture associated with osteomalacia and calcium deficiency was detected. a virus was isolated on marc 145 cells inoculated with the organ pool composed of the bat's heart and lungs. the cpe occurred on the third day post-inoculation during the second passage and was characterized by a diffused degeneration of a monolayer with rounded cells floating in the culture medium ( figure 1a ,b). the cell culture supernatant showing cpe was submitted to the ngs in order to identify and characterize the unknown isolate. furthermore, nsem performed on the purified and concentrated antigen revealed the presence of viral particles that unequivocally morphologically resembled those belonging to the genus orthopoxvirus ( figure 1c ). the virus was tentatively named hypsugopoxvirus (hypv), according to the bat species from which it was isolated. table 1 summarizes the basic information on the hypv identified in this study in comparison with all known poxviruses detected to date in bats worldwide. after ngs sequencing, the nearly complete viral genome of a poxvirus was obtained from one contig of 166,600 nucleotides originating from 85,678 reads with an average coverage of 118.53. the nearly full genome sequence of the viral strain was determined and compared with those of other members of the poxviridae family available on genbank. for the nearly complete viral genome sequencing, blast analysis revealed the highest nucleotide identity (85%) to the eptesipoxvirus (eptv) strain "washington", a member of the chordopoxvirinae subfamily identified in microbats in the usa ( table 2 ). the nearly complete genome sequence for hypv was submitted to genbank under accession number mk860688. [3, 4] a conservative approach was taken for genome annotation to avoid over-annotating open reading frames (orfs) that were unlikely to represent functional genes. orfs less than 50 codons or overlapping by more than 25% with well-characterized genes were not considered for annotation unless supported by other evidence. a total of 161 genes were annotated for hypv, showing a percentage value of nt identity with its closest related virus eptv ranging from 42.5% for the hypv-2 gene (serpin 2) to 100% for the hypv-90 gene (vltf-3) ( table 3) . when the seven conserved genes-rpo147, rap94, mrna capping enzyme large subunit, p4a precursor, rpo132, vetf-l, and dna primase-were considered individually, the value of nt similarity with eptv ranged from 90.5% to 98.5%. the above conserved genes that have been used for phylogenetic analysis in previous studies [10, 12] are presented in bold in table 3 . hypv showed nucleotide divergence from its closest relative, eptv. the smaller genome size with 166,600 nt encoding 161 genes for hypv in comparison to 176,688 nt and 191 genes for eptv, is likely due to the omission of the itrs from the analysis and therefore, is not possible to establish the exact length of its the viral genome. two orfs (hypv-24 and hypv-25, table 3 ), whose function is still unknown, appear to be unique to hypv. table 3 . hypv genome annotation and nucleotide identities for each gene to the most similar strain eptesipoxvirus (eptv). the seven conserved genes used for phylogenetic analysis in previous studies [10, 12] are presented in bold. (5 -3 ) or "−" (3 -5 ) ). the potential zoonotic risks associated with bats and their fascinating and special relationship with viruses have attracted the attention of many researchers worldwide. consequently, general and target surveillance on bat populations has increased in the last decade with the purpose of clarifying the genetic diversity of bat-associated viruses as well as acquiring comprehensive information on bat-pathogen interactions. in fact, viral disease prevention and biological conservation issues could both benefit from such research. virological surveillance of bat populations in italy is a relative novelty and has only recently been extensively applied, but almost immediately, a great heterogeneity of virus identifications has been observed. viruses belonging to several viral families, such as reoviridae [14] , coronaviridae [15, [20] [21] [22] [23] [24] , paramyxoviridae [24] , rhabdoviridae [16, 25] , and astroviridae [26] , have been detected, allowing the identification of some novel/previously unknown viral agents. the results of the general surveillance of bats, which have been randomly applied so far as pilot virus discovery studies, may drive future activity to more specific longitudinal and target studies aimed at understanding the epidemiology of potential new pathogens. in this study, a novel poxvirus, hypv, was detected from the microbat hypsugo savii in italy. this likely represents the first poxvirus detection in bats in europe. in fact, only four poxviruses have been documented to date in bat populations worldwide, and these and these have diverse and somehow incomplete descriptions, with just some common aspects. firstly, ehpv1 was detected in 2009 with a high-prevalence in throat swabs from apparently healthy african megabats (eidolon helvum), and metagenomic analysis identified poxvirus sequences that were most closely related with molluscum contagiosum (mocv), a human-only pathogen [11] . in the same year of 2009, another bat poxvirus was incidentally detected in south australia during the investigation of an outbreak of parasitic skin disease in a population of the microbat species, miniopterus schreibersii bassanii. in one of the twenty-one bats examined, an independent (non-nematode-associated) lesion containing intracytoplasmic inclusion bodies indicative of poxvirus infection was observed, and this was confirmed with electron microscopy [13] . between 2009 and 2011, eptv was detected in adult big brown bats (eptesicus fuscus) with severe joint disease (tenosynovitis and osteoarthritis) at a wildlife center in northwestern united states. phylogenetic analysis revealed that eptesipoxvirus is most closely related to the cotia virus, a virus detected in sentinel suckling mice in sao paulo, brazil in 1961 [27, 28] . ptpv was detected from an australian little red flying fox (pteropus scapulatus) that died following entrapment on a fence. post-mortem examination revealed multiple nodules on the wing membranes. phylogenetic analysis indicated that ptpv is not closely related to any other poxvirus isolated from bats or other species, and that it likely should be placed in a new genus [12] . it is noteworthy that ptpv and ehpv were isolated from megabat hosts (pteropus scapulatus and eidolon helvum, respectively), whereas eptv and hypv were isolated from microbats (eptesicus fuscus and hypsugo savii, respectively). while ehpv was detected in apparently healthy bats, the other viruses were identified in sick bats and their association with the pathological condition was assumed. specifically, clinical symptoms of eptv in eptesicus fuscus manifested in the form of joint swelling and increased lethargy [10] . ptpv-infected pteropus scapulatus presented vesicular to nodular skin lesions on the wing membranes that are typical of poxvirus infections [13] . hypv was detected in a bat showing pathological healing of the humerus fracture associated with osteomalacia and calcium deficiency. neither symptom was directly linked to fatality and thus the capability of these viruses still needs to be ascertained, including the role of hypv in causing deadly disease in bats. the results of our study indicate that hypv presents the typical morphology of the orthopoxvirus genus and that it could be isolated in cell culture. indeed, its final identification was obtained by genomic characterization. the nearly complete genomic sequencing clearly demonstrated that hypv is a new virus that is distantly related to its closest known relative eptv (wa, usa, 2011) with a nucleotide identity of 85% (almost whole genome). indeed, the percentage value of the nt identity of hypv with eptv ranged from 42.5% for the hypv-2 gene (serpin 2) to 100% for the hypv-90 gene (vltf-3). regarding orfs annotation the hypv was shown to be defective in particular in the itr genes i.e., 12 out of 13 described in eptv, but this should be not a real structural defect but more likely due to the omission of the itrs from the analysis. on the contrary, two orfs, whose function is still unknown, appear to be unique to hypv. to conclude, a new poxvirus, hypv, was detected in bats in europe and its viral ecology and disease associations should be investigated further. author contributions: d.l. designed the study and wrote the manuscript; a.l. performed electron microscopy, participated in study coordination, and helped to draft the manuscript; c.c. and l.b. performed the next-generation sequencing and data analysis; a.m.g. and m.m. performed the sampling and data collection; g.l.c. performed the clinical investigations; a.p. and f.f. performed the necropsies and molecular tests; e.s. and t.t. were involved in the virological analysis and interpretation of the results; a.m. performed the molecular genetic studies and helped to draft the manuscript. all of the authors have read and approved the final manuscript. an increasing danger of zoonotic orthopoxvirus infections bats: important reservoir 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insectivorous bats from central-southern italy cotia virus: a new agent isolated from sentinel mice in sao paulo biological characterization and next-generation genome sequencing of the unclassified cotia virus span232 (poxviridae) this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license acknowledgments: special thanks to anna tirelli, loredana zingarello, giovanni bozzoni and all technicians in the izsler virology section for their valuable technical work and support in virological analysis. the authors declare no conflict of interest. key: cord-276052-gk6n8slx authors: yadav, pragya; sarkale, prasad; patil, deepak; shete, anita; kokate, prasad; kumar, vimal; jain, rajlaxmi; jadhav, santosh; basu, atanu; pawar, shailesh; sudeep, anakkathil; gokhale, mangesh; lakra, rajen; mourya, devendra title: isolation of tioman virus from pteropus giganteus bat in north-east region of india date: 2016-09-09 journal: infect genet evol doi: 10.1016/j.meegid.2016.09.010 sha: doc_id: 276052 cord_uid: gk6n8slx bat-borne viral diseases are a major public health concern among newly emerging infectious diseases which includes severe acute respiratory syndrome, nipah, marburg and ebola virus disease. during the survey for nipah virus among bats at north-east region of india; tioman virus (tiov), a new member of the paramyxoviridae family was isolated from tissues of pteropus giganteus bats for the first time in india. this isolate was identified and confirmed by rt-pcr, sequence analysis and electron microscopy. a range of vertebrate cell lines were shown to be susceptible to tioman virus. negative electron microscopy study revealed the “herringbone” morphology of the nucleocapsid filaments and enveloped particles with distinct envelope projections a characteristic of the paramyxoviridae family. sequence analysis of nucleocapsid gene of tiov demonstrated sequence identity of 99.87% and 99.99% nucleotide and amino acid respectively with of tiov strain isolated in malaysia, 2001. this report demonstrates the first isolation of tioman virus from a region where nipah virus activity has been noticed in the past and recent years. bat-borne viruses have become serious concern world-wide. a survey of bats for novel viruses in this region would help in recognizing emerging viruses and combating diseases caused by them. bat-borne viruses are considered to be important emerging viruses, as they can pose a serious threat to human and animal health. henipaviruses, coronaviruses, filoviruses and rabies-causing lyssaviruses are all transmissible from bats to humans. bats are primary reservoir host and often the resulting human disease is fatal. they are known to harbor more zoonotic viruses per species than rodents and recognized as a significant source of zoonotic agents (newman et al., 2011; calisher et al., 2006; mackenzie et al., 2003; pavri et al., 1971; mourya et al., 2014; raut et al., 2012; wynne and wang, 2013) . old world fruit bats of the family pteropodidae, particularly species belonging to the genus pteropus, have been considered as natural hosts for a large number of emerging viruses, especially of the family paramyxoviridae (calisher et al., 2006) . due to special characteristics, pteropus bats are the perfect reservoir for most of the recently emerging zoonotic pathogens. they often live in large colonies or roosts and travel long distances; thus they are very effective in transmitting viruses among colony members and disseminating them over a considerable distance. interactions among bats, humans and livestock are constantly increasing due to anthropogenic activities, thereby increasing the potential for transmission of viruses. deforestation in tropical areas destroyed the natural habitats of these fruit bat species thus forcing them to live in the vicinity of human settlements. the resulting close contact is responsible for the emergence of highly pathogenic paramyxoviruses, like hendra and nipah virus (niv) in human populations in southeast asia and australia (mackenzie et al., 2003) . paramyxoviridae is a family of viruses that comprises important pathogens like nipah virus, measles virus, human parainfluenza virus type 3 and human respiratory syncytial virus (aguilar and lee, 2011) . while investigating niv in urine samples of giant fruit bats of the pteropus genus on tioman island, malaysia, in 2001, researchers isolated a novel virus which was placed in the rubulavirus genus of the paramyxoviridae family. the virus was named as tioman virus (tiov) after the place of isolation from malaysia (chua et al., 2001) . in this communication, we report the isolation and confirmation of a tioman virus isolated from pteropus species of bats from north-east region of india. the scientific advisory committee, institutional biosafety committee, and institutional animal ethical committee of national institute of infection, genetics and evolution 45 (2016) to determine the presence of niv in pteropus bats, a survey was conducted in two states of north-east region, india i.e. west bengal and assam states that share boundaries with bangladesh. the criteria for selection of the study areas were based on earlier reports of a niv seropositive bat from the myanaguri area in west bengal and confirmed human cases from siliguri and nadia districts of west bengal and roosting areas of pteropus bats (yadav et al., 2012; chadha et al., 2006) . sixty-eight pteropus bats were collected from jalpaiguri (n = 8) and cooch behar (n = 39) districts of west bengal and dhubri district (n = 21) of assam on two occasions from march to may 2015. mist nets were used to capture the bats. after capturing the bats, species identification and morphometry was done. further, the bats were euthanized and necropsies were performed in the field following proper biosafety measures. blood, organs (kidney, liver, and spleen), throat swabs, rectal swabs and urine samples were collected from bats. waste disposal was done following guidelines and proper precautionary measures. organ specimens were frozen in liquid nitrogen immediately after necropsy, while blood samples were kept at room temperature for 30 min and centrifuged for 10 min at approximately 1000 g. separated serum was aliquoted into labeled cryovials. vials of serum were transported at + 4°c in styrofoam box to national institute of virology (niv), pune, for further investigation. liver/spleen and kidney tissues of 68 bats were homogenized in sterile minimum essential medium (mem; gibco) using a homogenizer (genogrinder 2000; bt&c inc., lebanon, nj, usa). further, tissue homogenates were centrifuged at 5000 rpm for 10 min, and 0.1 ml of the supernatants was inoculated on to monolayers of vero ccl-81 cells grown in 24-well cell culture plates after removing the growth medium. the cells were incubated for 1 h at 37°c to allow virus adsorption, with rocking every 10 min for uniform virus distribution. after the incubation, the inoculum was removed and the cells were washed with 1 × phosphate buffer saline (pbs). finally mem supplemented with 2% fetal bovine serum (fbs) was added to each well. the cultures were incubated further in 5% co 2 incubator at 37°c and observed daily for cytopathic effects (cpe) under an inverted microscope. cultures that showed cpe were harvested and the suspension was centrifuged at 5000 rpm for 30 min at 4°c; the supernatants were processed immediately or stored at −86°c in 1 ml aliquots. viral rna was extracted using tripure reagent and rna extraction kit (qiagen, valencia, ca, usa) as per the manufacturer's instruction. virus isolations were also attempted with other specimens of bats (throat and rectal swabs, urine), following the same protocol (chua et al., 2002) . cell culture supernatants and pellets of vero ccl-81 infected cells showing distinct cpe were examined by negative-stain transmission electron microscopy (tem) as described previously (brenner and horne, 1959; gangodkar et al., 2010) . to identify the virus isolate, various diagnostic tests were undertaken targeting niv and genus paramyxovirus using specific primers by rt-pcr (guillaume et al., 2004; tong et al., 2008) . further, the isolates were screened by rt-pcr using primers targeting the nucleocapsid gene and phosphoprotein gene of paramyxoviruses, as described earlier (chua et al., 2001) . amplified products were further sequenced targeting nucleocapsid and phosphoprotein gene. the sequences obtained by sequencing were curated using sequencher 5.0 (gene codes corporation, ann arbor, mi, usa) version software. the curated sequences were aligned using clustal w (embl-ebi, cambridgeshire, uk), and a phylogenetic tree was constructed using the neighbor-joining algorithm (kimura 2parameter model) with 500-bootstrap replicates as implemented by mega v 6.0 software (tamura et al., 2007) . in order to study susceptibility of different vertebrate cells to tiov, the infectious virus titer was determined by estimating 50% tissue culture infective dose (tcid 50 ) using reed and muench method (reed and muench, 1938) . four vertebrate cell lines vero e-6 cells, pipistrellus ceylonicus bat embryo cells, baby hamster kidney-21 (bhk-21) and madine darbey canine kidney (mdck) cells were used for virus infection. the 70-80% confluent monolayer of the cells was infected with 10 multiplicity of infection (m.o.i) virus and observed for seven post-infection days. the methodology of cell infection by the virus was similar as mentioned above in section 2.4. two passages of the virus were made in all cell lines in order to confirm the susceptibility of the cells. all the cells were studied for susceptibility based on cpe and further confirmed using real-time rt-pcr. to explore the possibility of propagation in embryonated chicken eggs, 0.2 ml of vero ccl-81 grown tiov was inoculated in the allantoic cavities of 10-day-old embryonated white-leghorn chicken eggs. the eggs were incubated at 37°c for 5 days and were observed for sluggishness and mortality after every 24 h. allantoic fluids from infected eggs were harvested after 5 days of incubation and stored at −86°c. first blind passage was performed and allantoic fluid was tested for tioman virus by real-time rt-pcr. cytopathic effect was (cpe) observed in vero ccl-81cells inoculated with a kidney tissue homogenate of p. giganteus bat (nivan159672). the characteristics of cpe included cell fusion and formation of syncytium with aggregation of the nucleolus. cpe was prominent on post-infection-day 3 and cell detachment was observed on days post-infection-4 (dpi) (isolation dated 21st april 2015) (fig. 1 ). the supernatant was tested by pcr, sequencing and electron microscopy for identification of the suspected virus isolate. negative contrast electron microscopy of the cell supernatant of vero ccl-81 infected with virus isolate showed the presence of virus particles with the typical paramyxovirus morphology. the "herringbone" morphology of the nucleocapsid filaments, a characteristic of the paramyxoviridae family, was clearly visible (fig. 2) . distinct enveloped paramyxovirus particles with envelope projections of approximately 9 nm in length were also visualized. out of 68 bat sample processed, kidney samples from two bats (nivan159672, nivan151165) were found to be positive by rt-pcr. pcr products of 473 bp were observed for nucleocapsid gene of tiov. amplified products were further confirmed by sequencing partial nucleocapsid and phosphoprotein genes of tiov (gene bank accession no. kt601212, kt601213, kt601214. kt601215). sequence analysis showed 99.87% nucleotide sequence identity with both nucleoprotein gene and phosphoprotein gene sequences of the malaysian tiov isolate respectively (bat/2002/genbank af298895) (fig. 3) . partial sequences of tiov phosphoprotein and nucleocapsid gene revealed that tiov strains from india and malaysia are from one lineage, it also makes up a clade with menagle virus while tuhoko, achimota and sosuga viruses make up a separate clade. tiov isolated from kidney tissue homogenate of bat showed a titer of 10 4.61 /100 μl by tcid 50 in vero ccl-81 cell line. cpe-based susceptibility studies showed that all the studied vertebrate cell lines were susceptible to tiov with varying productivity. cpe in vero ccl-81 cell line became evident by 2nd dpi and there was total degeneration of cells by 4th dpi. vero e-6 cells, pipistrellus ceylonicus bat embryo cells and bhk-21 cell line showed cpe by 5th dpi. ps (porcine stable cell line) cells did not show cpe in first passage; however it showed distinct cpe at 7th dpi in the second passage. mdck cells showed growth of tiov with rounding and detachment of cells within 72h post-infection. however, susceptibility study by cpe showed that tiov grows faster in vero ccl-81 cells in comparison with other vertebrate cells. the study of susceptibility of different vertebrate cells to tiov indicated that vero ccl-81 cell lines are best suited for propagation of tiov. this may be useful for viral replication studies in future. embryonated eggs did not show any sluggishness or mortality in the initial passage and first blind passage. real-time rt-pcr using tiov specific primers and probe on the allantoic fluid of both the passages did not show any virus amplification. this showed that tiov did not grow in embryonated eggs. the present study reports the isolation of tiov from pteropus giganteus bat from dhubri, assam, india (fig. 4) ; this is the second report of tiov isolation besides malaysia (chua et al., 2001) . tiov is antigenically related to menangle virus (bowden and boyle, 2005) which is also harbored by pteropid fruit bats; the menangle virus caused an outbreak of fetal deformities in pigs in australia in 1997 (philbey et al., 1998) . all the above-named pteropus-borne viruses group in a single clade, which separates them from other paramyxoviruses. the bats and flying foxes belonging to the order chiroptera are ecologically remarkable. they are among the most abundant, diverse and geographically dispersed vertebrates and are natural reservoirs for a number of highly pathogenic zoonotic viruses. bats are known to have persistent viral infections at a rate higher than other mammals, possibly due to shorter antibody half-life in these animals (calisher et al., 2006) . detailed studies are needed on their importance as reservoirs of viruses and their potential to harbor important pathogen causing human and animal diseases. there is scanty information available regarding the hosts, reservoirs and transmission of tiov, though direct transmission via ingestion of fruit by humans has been suggested (lehle et al., 2007) . however, batto-human transmission of tiov has not yet been reported. neutralizing antibodies against tiov have been detected in human serum samples from tioman island in malaysia, from where the virus was first isolated (yaiw et al., 2007) . tiov's estimated prevalence of 1.8% is suggestive of its potential to cause subclinical infection in humans. experimental studies have shown that tiov is capable of infecting and replicating in pigs and its main cellular targets are lymphocytes, thymic epithelioreticular cells and the tonsillar epithelium in these animals (yaiw et al., 2008) . hence, pigs could act as an intermediate or amplifying host for human transmission, as has happened during menangle virus and niv outbreaks (parashar et al., 2000) . during niv outbreaks in malaysia, pigs played a critical role in transmitting the disease to pig handlers by direct contact. pig farms are a source of daily livelihood for a large number of populations in assam and other states like nagaland. the nagaland pig production and marketing project is funded by the national agricultural innovation project with a contribution from the international fund for agricultural development and aims to develop sustainable solutions to livelihood improvement in one of the poorest districts in india. pig farming was rampant during the year 2008 and being a good reservoir of many diseases in recent past the number of japanese encephalitis cases and outbreaks were increased in these areas. undetected mild tiov infection could occur in naturally infected pigs and this could facilitate viral transmission to humans via contact with oral secretions; this transmission could cause serious illness by crossing the species barrier. therefore, the role of bats and pigs in transmitting viruses to humans in asia needs to be determined. although no evidence of tiov illness in humans or animals exists, tiov's close relationship to other disease-causing bat paramyxoviruses, including niv, suggests the possibility that it too may cross the species barrier (bowden and boyle, 2005) . our study has shown the presence of tiov by highlighting its isolation from pteropus bat from dhubri district, assam india. the presence of large colonies of pteropus bats in close proximity of human settlements warrants implementation of necessary steps for detection and identification of emerging bat-borne viruses circulating in north-east region of india. emerging paramyxoviruses: molecular mechanisms and antiviral strategies completion of the full-length genome sequence of menangle virus: characterization of the polymerase gene and genomic 5-trailer region a negative staining method for high resolution electron microscopy of viruses bats: important reservoir hosts of emerging viruses nipah virus-associated encephalitis outbreak tioman virus, a novel paramyxovirus isolated from fruit bats in malaysia isolation of nipah virus from malaysian island flying-foxes. microbes infect dengue virus induced autophagosomes and changes in endomembrane ultrastructure imaged by electron tomography and whole-mount-grid cell culture techniques specific detection of nipah virus using real-time rt-pcr (taq man) henipavirus and tioman virus antibodies in pteropodid bats, madagascar managing emerging diseases borne by fruit bats (flying foxes), with particular reference to henipaviruses and australian bat lyssavirus malsoor virus, a novel bat phlebovirus, is closely related to severe fever with thrombocytopenia syndrome virus and heartland virus investigating the role of bats in emerging zoonoses: balancing ecology, conservation and public health interest. fao animal production and health manual no. 12 case-control study of risk factors for human infection with a new zoonotic paramyxovirus, nipah virus, during a 1998-1999 outbreak of severe encephalitis in malaysia isolation of a new parainfluenza virus from a frugivorous bat, rousettus leschenaulti, collected at poona, india. am an apparently new virus (family paramyxoviridae) infectious for pigs, humans, and fruit bats isolation of a novel adenovirus from rousettus leschenaultii bats from india a simple method of estimating fifty percent endpoints mega4: molecular evolutionary genetics analysis (mega) software version 4.0 sensitive and broadly reactive reverse transcription-pcr assays to detect novel paramyxoviruses bats and viruses: friend or foe? detection of nipah virus rna in fruit bat (pteropus giganteus) from india serological evidence of possible human infection with tioman virus, a newly described paramyxovirus of bat origin tioman virus, a paramyxovirus of bat origin, causes mild disease in pigs and has a predilection for lymphoid tissues authors express their sincere gratitude to the secretary and director general, indian council of medical research, new delhi for her continuous support. we would like to acknowledge icmr for funding extramural project 'multi-site epidemiological and virological survey of nipah virus: special emphasis on north-east region of india' (grant number 2013:1445). authors are grateful to dr. ms chadha (scientist 'f'& head of department), influenza department for continuous guidance and support and dr. r laxminarayanan, senior administrative officer, niv, pune, for rendering logistic support. technical assistance rendered by divya bhattad, kumar bagmare, amita bargat, shital melag and uk shende (laboratory) is gratefully acknowledged. the authors declare that they have no competing interests. key: cord-270335-8vqi9c68 authors: seifert, stephanie n; letko, michael c; bushmaker, trenton; laing, eric d; saturday, greg; meade-white, kimberly; van doremalen, neeltje; broder, christopher c; munster, vincent j title: rousettus aegyptiacus bats do not support productive nipah virus replication date: 2019-11-04 journal: j infect dis doi: 10.1093/infdis/jiz429 sha: doc_id: 270335 cord_uid: 8vqi9c68 nipah virus (niv) is a bat-borne zoonotic pathogen that can cause severe respiratory distress and encephalitis upon spillover into humans. nipah virus is capable of infecting a broad range of hosts including humans, pigs, ferrets, dogs, cats, hamsters, and at least 2 genera of bats. little is known about the biology of niv in the bat reservoir. in this study, we evaluate the potential for the egyptian fruit bat (efb), rousettus aegyptiacus, to serve as a model organism for studying niv in bats. our data suggest that niv does not efficiently replicate in efbs in vivo. furthermore, we show a lack of seroconversion against niv glycoprotein and a lack of viral replication in primary and immortalized efb-derived cell lines. our data show that despite using a conserved target for viral entry, niv replication is limited in some bat species. we conclude that efbs are not an appropriate organism to model niv infection or transmission in bats. nipah virus (niv) is a zoonotic pathogen that can cause acute respiratory illness and fatal encephalitis upon spillover into human populations. since its discovery in malaysia in 1998, niv has emerged as a persistent public health problem in southeast asia warranting inclusion on the world health organization blueprint list of priority diseases for which research toward effective countermeasures is urgently needed [1] . unlike other paramyxoviruses, niv has a broad host tropism with multiple taxa supporting viral replication including ferrets, hamsters, cats, dogs, african green monkeys, squirrel monkeys, and pigs [2] [3] [4] [5] . nipah virus uses 2 major envelope glycoproteins to enter the host cell: the receptorbinding protein (g) and the fusion protein (f) [6] . after attachment of the niv g to the host cell receptor, ephrin-b2 or ephrin-b3, niv f fusogenic activity is triggered leading to the merger of the virion and host cell membranes and subsequent virus infection [7] [8] [9] [10] [11] . ephrins are highly conserved across mammalian taxa given their key role in development of the central nervous system [12] , likely contributing to the unusually broad host range of niv. fruit bats in the genus pteropus have been identified as the primary reservoir hosts for niv [13] . spillover of niv from bats to humans is thought to occur through ingestion of food or liquids contaminated with infected bat urine [14] . pteropus spp bats are known to feed from the spigots of date palm sap collection jars, often urinating into and contaminating the collection jars before human consumption [14, 15] . although pteropus spp bats are implicated as the primary reservoir for niv, they comprise only a fraction of the fruit bat visitation to date palm sap collection jars in bangladesh [15] . several other fruit bat species overlap with the distribution of pteropus spp bats and visit date palm sap collection jars, suggesting that if other bats are potential secondary reservoirs for the virus, then they may contribute to nipah virus spillover. less is known about the infection and enzootic transmission dynamics of niv in reservoir populations or between coroosting bat species. nipah virus has been isolated from the urine of both wild caught [16] [17] [18] and experimentally challenged [19] pteropus spp bats. nipah virus ribonucleic acid (rna) has also been detected in an insectivorous bat, hipposideros larvatus [20] , and serological evidence of niv infection has been detected in several other species of frugivorous and insectivorous bats, including rousettus leschenaultia [21, 22] . despite efforts to understand enzootic transmission dynamics, many questions remain regarding the biology of niv infection in bats. studies of wild caught pteropus spp suggest potential for viral recrudescence [16, 23] ; however, the hypothesis that niv may persist in an individual bat and re-emerge under times of stress has yet to be confirmed experimentally. pteropus spp bats are suboptimal model organisms for studying niv due to size and availability. in contrast, the egyptian fruit bat (efb), rousettus aegyptiacus, belongs to the same taxonomic family as pteropus spp, pteropodidae, and has been successfully used to model marburg virus transmission [24, 25] and serological cross-reactivity after filovirus challenge [26] . egyptian fruit bats are common in zoological settings because they are small, amenable to handling, and reproduce readily in captivity. the efb transcriptome is well annotated [27] , and there have been recent efforts to analyze the genome in context of antiviral immunity [28] . in this study, we evaluate efbs as a model system for niv infection in bats. all work with niv was conducted in the biosafety level (bsl) 4 facility at the rocky mountain laboratories, division of intramural research, national institute of allergy and infectious diseases, national institutes of health following standard operating procedures as approved by the institutional biosafety committee. egyptian fruit bats were sourced from a us-based zoological facility. all animal experiments were approved by the rocky mountain laboratories institutional animal care and use committee (asp no. 2018-042e) and performed following the guidelines of the association for assessment and accreditation of laboratory animal care, international (aaalac) by certified staff in an aaalac-approved facility. nipah virus was obtained through the special pathogens branch of the centers for disease control and prevention (atlanta, ga). nipah virus bangladesh was isolated from a throat swab from a human patient in 2004 (genbank accession number ay988601); the virus was propagated on vero-e6 cells and passaged a total of 2 times. the virus stock was deep sequenced at the rocky mountain laboratories genomics core unit before the start of this study to confirm that no fungal or bacterial contaminants were present. primary efb cell lines were generated as previously described [29] , with modifications, from kidney (raksm) and lung (ralu) tissue samples obtained from an efb euthanized under bsl2 conditions. in brief, tissue homogenates were washed in phosphate-buffered saline and resuspended in primary cell culture (d12) media containing dulbecco's modified eagle's medium (dmem)/f-12 supplemented with nonessential amino acids, 12% fetal bovine serum (fbs), 1 mm l-glutamine, 50 u/ml penicillin, 50 μg/ml streptomycin, 1 μg/ml amphotericin, and 1 mm sodium pyruvate. after 1 passage, amphotericin was excluded from the d12 media. cells were not maintained after 3 passages. jordan at probiogen ag, berlin, germany) were obtained and grown in d12 media without amphotericin. vero-e6 cells were used as a positive control. each cell line was seeded in triplicate in 12-well plates and inoculated with niv at a multiplicity of infection of 0.1 in dmem supplemented with 2% fbs, 1 mmol/l l-glutamine, 50 u/ml penicillin, and 50 μg/ml streptomycin. supernatants were collected at 0, 24, 48, and 72 hours postinoculation and stored in avl buffer (qiagen) at −80°c until inactivation and rna extraction. after inactivation of the virus with avl and ethanol as described in [30] , rna extraction was performed on the qiacube (qiagen) with the machery-nagel nucleospin virus core kit (machery-nagel). we then performed quantitative real-time reverse-transcription polymerase chain reaction (qrtpcr) as described in [31] on the quantstudio 5 real-time pcr system (thermo fisher scientific) with the inclusion of a serially diluted known concentration of niv on each plate to calculate tissue culture infectious dose (tcid 50 /ml) equivalent for each sample. sequences for the niv receptors, ephrin-b2 and ephrin-b3, that have been experimentally demonstrated to support niv entry [32] were downloaded from national center for biotechnology information (ncbi) in addition to the efb ephrin-b2 and ephrin-b3 sequences. the sequences for each ephrin were translated before alignment using the mafft v7.388 [33] plugin implemented in geneious prime 2019.0.4 (biomatters ltd). nipah virus was diluted in sterile dmem, and 10 5 tcid 50 /ml was administered to each of 12 adult r aegyptiacus bats via the intraperitoneal route of inoculation in a final volume of 200 μl. oronasal, urogenital, and rectal swabs were collected daily in addition to swabbing the excreta pan of each cage for the first 14 days followed by twice-weekly sampling through 28 days postinoculation (dpi). temperature and weight for each bat was taken at the time of sampling. blood was drawn before inoculation, then at 7 dpi, 14 dpi, and 21 dpi for survivors in addition to terminal blood draws at 3 dpi, 7 dpi, and 28 dpi for serological analyses. tissue samples were taken at necropsy and either stored at −80°c until rna extraction or placed in 10% formalin for histopathology and immunohistochemistry analysis. the rna extraction and qrtpcr were conducted as described in [31] and performed on a quantstudio 5 real-time pcr system (thermo fisher scientific). sera were analyzed for presence of immunoglobulins (ig) specific to the niv-g using a luminex xmap-based multiplex bead assay adapted from [34] . in brief, blood was collected into serum-separating tubes before centrifugation at 1000 ×g for 10 minutes; serum was then collected and frozen at −80°c. each sample received a dose of 8 mrads irradiation while on dry ice before heat-inactivation at 56°c for 30 minutes. soluble niv-g (niv-sg) was produced in a freestyle 293-f stable cell-line expression system before purification as described [35] and coupling to bio-plex pro magnetic cooh beads (bio-rad). we diluted each serum sample 1:250, and each serum sample was run in duplicate with the bio-plex 200 system (bio-rad) with purified rabbit igg against niv-sg diluted to 1:1000 as the positive control. in vitro replication kinetics showed no appreciable increase in niv titer over a 72-hour period on the 4 efb cell lines tested including the 2 primary efb cell lines and the 2 immortal efb cell lines (figure 1 ). the hammer-headed fruit bat cell line supported moderate niv replication relative to the vero-e6 cell line (figure 1 ). an alignment of the niv host receptors, ephrin-b2 and ephrin-b3, show no unique amino acid changes in the critical g protein-binding (g-h) loop between the efb sequences and the sequences of species that have been confirmed to facilitate niv entry (figure 2) . the efbs showed no significant change in temperature or weight throughout the study period, although variability was high for both metrics ( figure 3a and b) . we did not detect niv rna in any of the tissue samples or swab samples tested by qrtpcr, with no samples amplifying within 40 thermal cycles ( the naive mean fluorescence intensity; however, 1 bat showed a slight increase in mean fluorescence intensity relative to the naive bat serum at 21 and 28 dpi (figure 4) . we confirmed that the inoculum contained 10 5 tcid 50 /ml through back-titrations of the diluted viral stock (data not shown). none of the sectioned tissue samples showed pathology associated with niv infection, and no niv antigen was detected via immunohistochemistry staining ( figure 5 ). our data show a lack of productive niv replication in efbs. the lack of detectable viral rna in both the swabs and the tissues across all time points suggests that the bats did not shed virus nor was viral replication detected in any of the tested tissue types (table 1) ; viral replication and shedding are qualities associated with natural hosts [36, 37] . back-titrations of the viral inoculum confirmed that the efbs received a 10 5 tcid 50 dose niv, which is higher than the 5 × 10 4 tcid 50 niv challenge that resulted in productive viral replication in guinea pigs and pteropus bats [19] . a lack of detectable virus in any efb tissue samples 3 days postinoculation suggests that the virus was rapidly cleared; halpin et al [13] report that henipavirus inoculum is cleared within 48 hours. the bats did not seroconvert against niv g in the given timeframe of 28 days, although 1 individual had a slight increase in mean fluorescence intensity at 21 and 28 dpi, which may have increased above our cutoff if given more time (figure 4 ). previous studies have demonstrated that efb cells are permissive to ebola virus, but experimentally challenged bats did not shed virus or support productive replication [38, 39] despite compatibility between the ebola virus glycoprotein and the host receptor, npc1 [40] . these data suggest that productive viral replication is blocked by a mechanism other than compatibility with the host receptor. likewise, van doremalen et al [41] reported a lack of efficient viral replication in efbs when challenged with bat severe acute respiratory syndrome-like coronavirus wiv1 despite in vitro receptor compatibility. given the lack of unique variation in either efb ephrin-b2 or ephrin-b3 relative to compatible host ephrin sequences (figure 2) , it is likely that niv virus replication is not inhibited by a lack of binding between niv g and the efb ephrin-b2 or ephrin-b3, nor the subsequent f-mediated activation and membrane fusion process. indeed, niv f and g can mediate productive cell-cell fusion in a variety of different mammalian species including several that are negative or refractory to productive infection, such as rabbit and mouse [32, 42] . because we did not detect viral replication or shedding, we conclude that efbs are not a suitable model system for modeling niv transmission dynamics in bats. however, follow-up studies to determine the mechanism of inhibition of viral replication in efbs would be valuable in elucidating the evolution of niv in its natural reservoirs. few controlled studies have been conducted using bats as a model organism, and, as such, there are few reagents commercially available for studying the immunobiology of bats in response to viral infection. further research is urgently needed to expand upon the current capacity to conduct research in bats, particularly when considering that 5 of 9 viruses associated with the world health organization's blueprint list of priority diseases [1] likely originated as batborne zoonoses. recent studies have applied machine learning algorithms to prioritize surveillance for high-impact pathogens such as niv and ebola virus using data on ecological traits, life history, demographic traits, and species distributions [43, 44] . although it is important to consider broader ecological characteristics in determining potential for a host species to contribute to virus spillover and maintenance, understanding the limitations to reservoir potential on a mechanistic level for high-impact pathogens such as niv would further improve predictive modeling work. testing viral entry and replication through in vitro assays are important first steps in determining host potential, but this should be followed by in vivo experiments when possible. supplementary materials are available at the journal of infectious diseases online. consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. the who r&d blueprint: 2018 review of emerging infectious diseases requiring urgent research and development efforts a neutralizing human monoclonal antibody protects against lethal disease in a new ferret model of acute nipah virus infection a golden hamster model for human acute nipah virus infection development of an acute and highly pathogenic nonhuman primate model of nipah virus infection experimental nipah virus infection in pigs and cats functional properties of the fusion and attachment glycoproteins of nipah virus role of endocytosis and cathepsin-mediated activation in nipah virus entry ephrin-b2 ligand is a functional receptor for hendra virus and nipah virus ephrinb2 is the entry receptor for nipah virus, an emergent deadly paramyxovirus two key residues in ephrinb3 are critical for its use as an alternative receptor for nipah virus activation of paramyxovirus membrane fusion and virus entry ephrins and their receptors: binding versus biology henipavirus ecology research group. pteropid bats are confirmed as the reservoir hosts of henipaviruses: a comprehensive experimental study of virus transmission nipah virus transmission from bats to humans associated with drinking traditional liquor made from date palm sap use of infrared camera to understand bats' access to date palm sap: implications for preventing nipah virus transmission henipavirus ecology research group. characterization of nipah virus from naturally infected pteropus vampyrus bats isolation of nipah virus from malaysian island flying-foxes nipah virus in lyle's flying foxes experimental nipah virus infection in pteropid bats (pteropus poliocephalus) rousettus aegyptiacus bats do not support productive nipah virus replication • jid 2019:xx xx xxxx • 7 bat nipah virus nipah virus infection in bats (order chiroptera) in peninsular malaysia antibodies to nipah or nipahlike viruses in bats henipavirus ecology research group. evidence for nipah virus recrudescence and serological patterns of captive pteropus vampyrus oral shedding of marburg virus in experimentally infected egyptian fruit bats (rousettus aegyptiacus) modelling filovirus maintenance in nature by experimental transmission of marburg virus between egyptian rousette bats comparative analysis of serologic cross-reactivity using convalescent sera from filovirusexperimentally infected fruit bats de novo transcriptome reconstruction and annotation of the egyptian rousette bat the egyptian rousette genome reveals unexpected features of bat antiviral immunity bat airway epithelial cells: a novel tool for the study of zoonotic viruses effective chemical inactivation of ebola virus comparison of the pathogenicity of nipah virus isolates from bangladesh and malaysia in the syrian hamster functional studies of host-specific ephrin-b ligands as henipavirus receptors mafft multiple sequence alignment software version 7: improvements in performance and usability serologic evidence of fruit bat exposure to filoviruses expression system for recombinant henipavirus glycoproteins replication and shedding of mers-cov in jamaican fruit bats (artibeus jamaicensis) bats: important reservoir hosts of emerging viruses experimental inoculation of egyptian rousette bats (rousettus aegyptiacus) with viruses of the ebolavirus and marburgvirus genera experimental inoculation of egyptian fruit bats (rousettus aegyptiacus) with ebola virus establishment of fruit bat cells (rousettus aegyptiacus) as a model system for the investigation of filoviral infection sarslike coronavirus wiv1-cov does not replicate in egyptian fruit bats (rousettus aegyptiacus) membrane fusion tropism and heterotypic functional activities of the nipah virus and hendra virus envelope glycoproteins undiscovered bat hosts of filoviruses prioritizing surveillance of nipah virus in india we thank marcel a. müller (institute of virology, charite -universitätsmedizin berlin, germany) and ingo jordan (probiogen ag, berlin, germany) for providing the immortalized bat cell lines used in this study, the centers for disease control and prevention for providing the initial nipah virus (niv) stock, and friederike feldmann (laboratory of virology, national institute of allergy and infectious diseases [niaid]) for preparing and maintaining the niv stocks. we thank spencer l. sterling and lianying yan (uniformed services university) for invaluable technical assistance with soluble niv-sg expression and purification. we thank emmie de wit (laboratory of virology, niaid) for advice and laboratory support. finally, we thank drs. patrick hanley, jamie lovaglio, and dana scott and the animal caretakers of the rocky mountain veterinary branch, niaid for support.disclaimer. the content of this publication does not necessarily reflect the views or policies of the department of health and human services, nor does the mention of trade names, commercial products, or organizations imply endorsement by the u.s. government. the views expressed in the manuscript are solely those of the authors, and they do not represent official views or opinions of the department of defense or the uniformed services university of the health sciences.financial support. this research was funded by the intramural research program of niaid, national institutes of health.potential conflicts of interest. all authors: no reported conflicts of interest. all authors have submitted the icmje form for disclosure of potential conflicts of interest. key: cord-257321-l1swyr6g authors: chen, lihong; liu, bo; wu, zhiqiang; jin, qi; yang, jian title: drodvir: a resource for exploring the virome diversity in rodents date: 2017-05-20 journal: j genet genomics doi: 10.1016/j.jgg.2017.04.004 sha: doc_id: 257321 cord_uid: l1swyr6g emerging zoonotic diseases have received tremendous interests in recent years, as they pose a significant threat to human health, animal welfare, and economic stability. a high proportion of zoonoses originate from wildlife reservoirs. rodents are the most numerous, widespread, and diverse group of mammals on the earth and are reservoirs for many zoonotic viruses responsible for significant morbidity and mortality. a better understanding of virome diversity in rodents would be of importance for researchers and professionals in the field. therefore, we developed the drodvir database (http://www.mgc.ac.cn/drodvir/), a comprehensive, up-to-date, and well-curated repository of rodent-associated animal viruses. the database currently covers 7690 sequences from 5491 rodent-associated mammal viruses of 26 viral families detected from 194 rodent species in 93 countries worldwide. in addition to virus sequences, the database provides detailed information on related samples and host rodents, as well as a set of online analytical tools for text query, blast search and phylogenetic reconstruction. the drodvir database will help virologists better understand the virome diversity of rodents. moreover, it will be a valuable tool for epidemiologists and zoologists for easy monitoring and tracking of the current and future zoonotic diseases. as a data application example, we further compared the current status of rodent-associated viruses with bat-associated viruses to highlight the necessity for including additional host species and geographic regions in future investigations, which will help us achieve a better understanding of the virome diversities in the two major reservoirs of emerging zoonotic infectious diseases. zoonotic diseases comprise a significant and increasing proportion of all emerging human infectious diseases, and most of them originate from wildlife (kruse et al., 2004; jones et al., 2008) . two mammalian orders, rodentia (rodents) and chiroptera (bats), represent the most relevant potential sources of new zoonoses (calisher et al., 2006; meerburg et al., 2009; smith and wang, 2013) . the rodentia is the single largest order of mammals. there are more than 2000 living species of rodents, which comprise~40% of all mammalian species, including mice, rats, hamsters, guinea pigs, voles, chinchillas, chipmunks, gophers, muskrats, gerbils, woodchucks, and many others (huchon et al., 2002) . despite their great species diversity, all rodents share a common feature e a single pair of continuously growing incisors, which are used to gnaw food, excavate burrows, and defend themselves. rodents are native on all continents except antarctica, and they show a wide range of lifestyles, ranging from terrestrial, subterranean, arboreal, to aquatic habitats (wolff and sherman, 2007) . rodents are common hosts for pathogens that transmit diseases to humans and domestic animals because of their properties of close association with humans, large social group size, intense social interaction, high population density and widespread geographic distribution. since the middle ages, rodents have been known to contribute to human diseases, as black rats were associated with the spread of the plague (perry and fetherston, 1997) . however, rodents are also a threat to public health in modern times. they have been implicated as reservoir hosts of zoonotic pathogens, such as viral hemorrhagic fever viruses, including arenaviruses (junin, machupo, and lassa) and hantaviruses (hantaan, dobrava, and sin nombre) (enria and pinheiro, 2000; goeijenbier et al., 2013) . also, global climate change and continued urbanization have led to increased problems with rodent-associated zoonoses (mills et al., 2010) . recent metagenomic studies have identified a wide diversity of novel viruses in rodents from families/ genera that contain important human pathogens, including new species/variants of picornaviruses, hepaciviruses and sapoviruses (firth et al., 2014) . therefore, it is necessary to enhance our knowledge of the virome diversity in rodents to facilitate future prevention and control of emerging zoonotic diseases. bats are the second most diverse mammalian orders on the earth, with more than 1200 living species identified worldwide (altringham, 2011) . the importance of bats as natural hosts for several important viral agents, including ebola virus, marburg virus, hendra virus, nipah virus, severe acute respiratory syndrome (sars) coronavirus, and middle east respiratory syndrome (mers) coronavirus, has been established (calisher et al., 2006; wong et al., 2007; ge et al., 2013; smith and wang, 2013; munster et al., 2016) . due to the wide-scale microbial surveillance programs conducted and novel high-throughput detection methods developed in recent years, there are a plethora of newly identified viruses in both rodents and bats. our group already constructed the first database of bat-associated viruses (dbatvir) in 2014 (chen et al., 2014) . in this study, we present a newly established drodvir database for a thorough and modern understanding of rodent-associated viral zoonoses (http://www.mgc.ac.cn/drodvir/). moreover, we used the information from two sister databases to perform a comparative analysis on the virome diversities of the two major reservoirs of emerging zoonotic infectious diseases. drodvir is a sequence-centric database, since molecular methods are now commonly used in virus detection and functional analysis. to retrieve all available sequences of rodent-associated viruses from the public domain, we first performed exhaustive searches in both the pubmed and nucleotide databases of ncbi using a group of keywords pertaining to rodents. then, the retrieved genbank records were downloaded to a local system and parsed by an in-house bioperl script to generate human-readable tables of the metadata, including the sampling time, location, rodent species, specimen type (e.g., feces, blood, or tissues), and viral detection method (e.g., pcr or metagenomics), for further review. for published sequences, additional meta information was extracted from the related literature using manual curation. as the drodvir database focuses on natural rodent-associated mammal viruses that are most relevant to emerging zoonotic infectious diseases, the following filter criteria are employed to exclude unrelated records: i) samples derived from animals other than rodents; ii) sequences of phages; iii) insect or plant viruses; iv) laboratory-cultured viruses in model rodents. taxonomic information of all viruses and rodents was derived from the taxonomy database of ncbi. in addition, we collected information concerning the viruses (e.g., genome architecture, average size and virus graphics) and the rodents (e.g., common names and known geographic ranges) from the viralzone databases (masson et al., 2013) and the icun red list (www.iucnredlist.org), respectively. moreover, the established phylogenetic relationships between different rodent families from a previous study were carefully incorporated into the database (meredith et al., 2011) . the drodvir database reused the background data schema and the majority of the foreground perl cgi and javascript codes of the dbatvir database because they are efficient to provide versatile and user-friendly interface (chen et al., 2014) . given that many users of drodvir and dbatvir databases may overlap, the similar architecture makes the new database instantly familiar with previous users. in addition, the two sister databases with similar web design and analytical functions will make it very easy to perform any comparative analysis on bat-and rodent-associated viruses for all users. the ncbi blast and muscle programs were integrated into the database to allow users to conduct sequence similarity searches and multiple sequence alignment on the web page (altschul et al., 1997; edgar, 2004) . the fasttree program and jsphylosvg library were used for online phylogenetic tree construction and visualization, respectively (price et al., 2010; smits and ouverney, 2010) . the main page of drodvir database provides a highly responsive and intuitive user interface, with a look similar to a desktop application rather than a traditional website. on the left side is a multifunctional menu panel for easy navigation, and on the right side is a tabbed content panel for presenting tables, figures, trees, blast outputs, and other results. to maximize the visible section of the content panel for users with limited screen size, the menu panel can be collapsed into a clickable vertical bar automatically (depends on visitors' screen resolution setting) or manually (by single click the icon on the top right corner) (fig. 1a) . features that were previously available only in standalone applications, including collapsible menus, expandable trees, sortable grids, tabbed panels and live statistical pie charts, provide high performance and an improved user experience. drodvir inherits the major features from the dbatvir database (chen et al., 2014) . users can either browse or search the database contents. in browse mode, the menu panel offers three submenus in accordion style for users to browse the database by categories of viruses/rodents or geographic regions. users who are familiar with certain viruses can use the 'browse by virus' submenu to explore the current host range in rodents and their geographic distributions (fig. 1b) , whereas those who are interested in given types of rodents may try the 'browse by rodent' submenu to investigate the known virome diversity in different rodentia hosts (fig. 1c ). in addition, the 'browse by region' submenu allows the users to easily summarize the current research efforts conducted on rodentassociated viruses for any countries or continents (fig. 1d ). all three submenus are organized as hierarchical and expandable trees and can be easily switched on/off by single click on the title bar. each branch of the trees in the submenus provides a direct link to individual tab in the main content panel, which presents a uniform sortable grid that includes brief information of the viruses, specimens, associated rodents, determined sequences and related literature (fig. 1a) . each line of the table can be further expanded by double click to show additional information, such as virus detection method, genbank accession number(s), sequence submission date, submitters and their affiliations. a clickable linear map is available for each full-length viral sequence to highlight the genome architecture of the virus (fig. 1a) . for brevity, the table provides up to 100 records by default. handy toolbar is available for users to page up/down if more records exist, or alternatively, turn off paging to force to display all records within a single table. in addition, the main table offers various manipulating functions, i.e., sorting/ filtering the table by column contents, reordering the columns, adjusting column width or hiding some columns. in search mode, two parallel query forms are provided to the users for text information search and blast sequence similarity search, respectively ( fig. 2a) . the text search engine allows the extraction of virus/rodent/specimen/sequence information from the database using either a simple query for a quick start or combined search patterns for advanced usages. the search results are organized into an individual high-performance grid in the content panel as aforementioned. to facilitate online data analysis, two visualization tools are integrated into the result table: i) a statistical pie chart is available with a single click on the column title of virus family, rodent species/family, sample type and sampling country (fig. 2b) ; ii) a global map with indicative markers is provided for the column of sampling country to better illustrate the geographic distribution of the rodent-associated viruses (http://www.mgc.ac. cn/cgi-bin/drodvir/main.cgi?func¼map). in addition, the result table and the related sequences can be easily downloaded as excel and fasta files respectively for further offline analyses. to bridge the gap between virologists and zoologists, the information concerning the rodents including common names, known distribution and phylogenetic relationships of rodents is also available and the hierarchical taxonomic tree shows virus families (bolded branches) by default for brevity. c: the second layer of the main menu for browsing the database by rodent category. several branches are expended to show lower taxonomic levels (i.e., subfamily, genus and species). d: the third layer of the main menu for browsing the database by geographic regions. the geographic tree is organized by continents and countries. searchable in the drodvir database (fig. 2c) . the blast sequence similarity search form enables the users to submit their own sequences for homology comparison among all known rodentassociated viruses. moreover, an automatic pipeline for multiple sequence alignment and online phylogenetic tree construction is offered to facilitate follow-up sequence analysis. detailed step-bystep instruction of all aforementioned usages of the drodvir database is available in the online help page (http://www.mgc.ac. cn/drodvir/howto.htm). the genomic studies on rodent-associated viruses were initiated earlier than on bat-associated viruses. both have been boosted by the genomic era during the past two decades. in addition, metagenomic approaches or high-throughput sequencing (hts)-based methods have played a major role in the discovery of novel viruses in both rodents and bats in recent years (fig. 3a) . however, many genbank records provide insufficient meta information about the detection methods applied, so the contributions of metagenomics or hts-based approaches are systematically underestimated in fig. 3a . currently,~25% sequences of rodent-or bat-associated viruses are deposited in public domains without related literature. to date (november 2016), 5491 viruses from 26 families and 6268 viruses from 24 families were identified from rodents and bats, respectively. however, only 21 viral families are shared by rodent-and bat-associated viruses. viruses from families arenaviridae, arteriviridae, and picobirnaviridae, carried by rodents, have not yet been detected in bats. similarly, viruses from filoviridae, identified in bats, have not been found in rodents so far (fig. 3b) . we avoided the use of lower taxonomic concepts such as genus or species because of the inconsistent criteria used for such definitions as described previously (anthony et al., 2013) and because there are many newly identified viruses grouped into unclassified genera or species in public data. our current data show that the virome diversities in rodents and bats are similar at the family level, though a previous study suggests that bats harbor more zoonotic viruses per species than rodents (luis et al., 2013) . remarkably, the aforementioned viruses were identified from 194 rodent species and 246 bat species. given that there is nearly twice the number of living rodent species as bat species, the current investigation efforts cover onlỹ 9% of the rodent species but~21% of the bat species. thus, the virome diversity in rodents has probably been underestimated compared with that of bats due to the limitation of the currently available data. future studies to screen additional rodent species are required to achieve a better estimation of the virome diversity in rodents. the geographic distribution of the hosts being investigated is also an important factor to affect the estimation of virome diversity in both rodents and bats, since some viruses carried by the hosts are likely regional even for the same host species. for example, the ebola virus and marburg virus were exclusively identified from bats in africa. therefore, a better understanding of the virome diversity in either host requires the investigation efforts to cover as many countries as possible. indeed, the current studies already include countries from all six continents. thus far, the coverage of countries in most continents is far from comprehensive (fig. 3c) . although the species diversity of rodents/bats in different countries is different and the current efforts in most countries could not cover all resident host species, further investigations dedicated to those unexplored regions of each continent are essential for our understanding of virome diversity in both rodents and bats. the current data are disproportionally distributed in different viral families for both rodent-and bat-associated viruses. this could be a reflection of particular public concerns on some wellknown zoonotic viruses carried by rodents or bats. for example, 52% of the rodent-associated viruses currently identified were from family bunyaviridae (mostly are hantaviruses), whereas~41% of the bat-associated viruses reported so far were from family rhabdoviridae (mostly are lyssaviruses). nevertheless, the biased data may also be a helpful measure for the potential distribution range of each viral family in the hosts. for instance, viruses from the family bunyaviridae are the most widely distributed viruses among rodent-associated viruses, which have been detected in 109 rodent species from 64 countries worldwide (fig. 3b) . however, although the current data of viruses from the family coronaviridae (i.e., coronavirus) in bats are less than half of those from family rhabdoviridae, coronaviruses were surprisingly identified in 121 bat species from 39 countries, which makes them the most widely distributed bat-associated viruses thus far (fig. 3b) . because the causative agents of both sars and mers pandemics are coronaviruses, their widespread occurrence in bats further highlights their potential threats to public health. also, the current data show that viruses from some other families such as rhabdoviridae, paramyxoviridae, flaviviridae, herpesviridae, arenaviridae, and picornaviridae are broadly distributed in terms of host species (rodents and/or bats) and geography (fig. 3b) . therefore, these viral families should be the hotspots for future studies dedicated to the prevention and control of zoonotic viral diseases. as of november 2016, the drodvir database has collected information on 5491 rodent-associated animal viruses of 26 virus families detected from 194 rodent species in 93 countries worldwide. these data will give us an overview and snapshot of the current research regarding rodent-associated viruses and provide a substantial source for future attempts to assess and predict epidemic risks. moreover, this work provides the scientific community with a framework and platform for further exploring the virome diversity of rodents. mammals are supposed to harbor over 320,000 undiscovered viruses (anthony et al., 2013) . rodents and bats are important reservoirs for an increasing number of zoonotic infectious diseases with a significant impact on public health. the two most diverse and geographically widespread mammalian orders play vital roles in the maintenance, evolution, and spread of many emerging infectious disease agents. our comparative analysis primarily explored the virome diversities in rodents and bats based on the summary of previous studies and highlighted the necessity to cover additional host species and geographic regions in future investigations. for easy comparative analysis on rodent-and bat-associated viruses in further studies, we plan to combine the two sister databases, dbatvir and drodvir, to form an integrated resource for the two major reservoirs of emerging zoonoses in the future. bats: from evolution to conservation gapped blast and psi-blast: a new generation of protein database search programs a strategy to estimate unknown viral diversity in mammals bats: important reservoir hosts of emerging viruses dbatvir: the database of bat-associated viruses. database (oxford) muscle: multiple sequence alignment with high accuracy and high throughput rodent-borne emerging viral zoonosis: hemorrhagic fevers and hantavirus infections in south america detection of zoonotic pathogens and characterization of novel viruses carried by commensal rattus norvegicus isolation and characterization of a bat sars-like coronavirus that uses the ace2 receptor rodent-borne hemorrhagic fevers: under-recognized, widely spread and preventableepidemiology, diagnostics and treatment rodent phylogeny and a timescale for the evolution of glires: evidence from an extensive taxon sampling using three nuclear genes global trends in emerging infectious diseases wildlife as source of zoonotic infections a comparison of bats and rodents as reservoirs of zoonotic viruses: are bats special? viralzone: recent updates to the virus knowledge resource rodent-borne diseases and their risks for public health impacts of the cretaceous terrestrial revolution and kpg extinction on mammal diversification potential influence of climate change on vector-borne and zoonotic diseases: a review and proposed research plan replication and shedding of mers-cov in jamaican fruit bats (artibeus jamaicensis) yersinia pestiseetiologic agent of plague fasttree 2eapproximately maximumlikelihood trees for large alignments bats and their virome: an important source of emerging viruses capable of infecting humans jsphylosvg: a javascript library for visualizing interactive and vector-based phylogenetic trees on the web rodent societies: an ecological and evolutionary perspective bats as a continuing source of emerging infections in humans key: cord-262434-q4tk96tq authors: baker, kate s.; murcia, pablo r. title: poxviruses in bats … so what? date: 2014-04-03 journal: viruses doi: 10.3390/v6041564 sha: doc_id: 262434 cord_uid: q4tk96tq poxviruses are important pathogens of man and numerous domestic and wild animal species. cross species (including zoonotic) poxvirus infections can have drastic consequences for the recipient host. bats are a diverse order of mammals known to carry lethal viral zoonoses such as rabies, hendra, nipah, and sars. consequent targeted research is revealing bats to be infected with a rich diversity of novel viruses. poxviruses were recently identified in bats and the settings in which they were found were dramatically different. here, we review the natural history of poxviruses in bats and highlight the relationship of the viruses to each other and their context in the poxviridae family. in addition to considering the zoonotic potential of these viruses, we reflect on the broader implications of these findings. specifically, the potential to explore and exploit this newfound relationship to study coevolution and cross species transmission together with fundamental aspects of poxvirus host tropism as well as bat virology and immunology. been identified in bat populations for which the zoonotic potential is unknown, including novel influenza types and hepadnaviruses [26, 27] . as a result, there has been well-grounded speculation that owing perhaps to physiological, ecological, evolutionary, and/or immunological reasons, bats may have a "special" relationship with viruses [15, 28, 29] and be particularly good viral reservoirs with exaggerated viral richness [30] . indeed, a recent intensive study found that a single bat species likely carries ≥58 different viral species from only nine viral families [31] . as well as the obvious first step of considering the zoonotic potential of newly identified bat viruses, further exploring the impacts of these findings and the opportunities they present for multiple research fields is necessary to capitalize on these discoveries. poxvirus infections have recently been identified in bats, comprising part of the increase in viral families newly identified in this taxonomic order. here, we review the current evidence of poxvirus infections in bats, present the phylogenetic context of the viruses within the poxviridae, and consider their zoonotic potential. finally, we speculate on the possible consequences and potential research avenues opened following this marrying of a pathogen of great historical and contemporary importance with an ancient host that has an apparently peculiar relationship with viruses; a fascinating and likely fruitful meeting whose study will be facilitated by recent technological advances and a heightened interest in bat virology. there are three documented detections of poxviruses in bat populations under distinct circumstances (summarized in table 1 ). the viruses were detected in animals from both bat suborders on three different continents. they had varied clinical impacts on their hosts and were phylogenetically dissimilar. genetic sequence of one bat poxvirus was detected at high prevalence during active surveillance on apparently-healthy african straw-colored fruit bats (eidolon helvum) [32] . metagenomic analysis of pooled throat swabs collected from e. helvum in ghana in 2009 contained poxvirus sequences most closely related with molluscum contagiosum (mocv) a human-only pathogen ( figure 2 ). detected sequences were distributed across the mocv genome and reconstructed sequences relating to 23 viral genes were deposited in genbank as being derived from eidolon helvum poxvirus 1 [32] . retrospective analysis of throat swabs from individual bats revealed a high prevalence of this virus in the apparently healthy study population with 13% (n = 5/40) of swabs containing poxviral dna. notably, the detection of true poxvirus sequences in this metagenomic study, in which sequences related to multiple genes distributed throughout the genome were found and reconfirmed in individual throat swab samples, is distinct from the detection of poxvirus-like sequences described in other metagenomic studies performed on pooled bat feces, whose presence was ultimately attributed to the presence of other (non-pox) viruses or viral elements integrated into host genomes [33, 34] . between 2009 and 2011, a poxvirus associated with pathology (tenosynovitis and osteoarthritis) was detected in six adult big brown bats (eptescicus fuscus, a microbat) sampled at a wildlife center in the north western united states [35] . the clinical illness of the bats was progressive and ultimately led to their euthanasia. histopathological examination of the joint lesions was indicative of poxvirus infection, which was confirmed by electron microscopy. the virus was successfully isolated on an african green monkey cell line (bsc40) and the genome was partially characterized (seven full protein coding sequences). phylogenetic analysis revealed that the novel eptesipox virus was most closely related with cotia virus, a virus detected in sentinel suckling mice in sao paulo, brazil in 1961 ( figure 2 ) [36, 37] . finally, a bat poxvirus was again detected in a clinical setting, in south australia in 2009. the virus was identified as an incidental infection during investigation of an outbreak of parasitic skin disease in a population of southern bentwing bats (miniopterus schreibersii bassanii, a critically-endangered microbat species) [38] . bats presented with white nodular skin lesions that contained encysted nematodes. however, in one of the twenty-one bats examined, an independent (non-nematode associated) lesion contained intracytoplasmic inclusion bodies indicative of poxvirus infection, which was confirmed with electron microscopy [38] . no further confirmation or characterization of the virus was reported, and both the epidemiology and consequent conservation implications of poxviral disease for this species remain unknown. the three detections of poxviruses in bat populations are distinct and inherently incomplete stories with very few common threads; high-prevalence detection in throat swabs from apparently healthy african megabats, severe joint disease in several north american microbats and, negligible though comorbid skin disease in an endangered australasian microbat. further to their varied clinical impact, the partial genetic characterization of the former two viruses shows that these viruses are genetically diverse. the two viruses are most closely related with the very distinct poxviruses, molluscum contagiosum virus and cotia virus respectively (figure 2 ), and although only partially genetically characterized, a small (100 amino acids) region of overlap in their rap94 proteins has only 62% amino acid identity (please see table s1 in the supplementary files). that this is as far as these new viruses can be contrasted demonstrates the dearth of information currently available for further investigation of poxviruses in bats. the finding of poxviruses in bats is not unique among wildlife taxa (in fact it would have been more surprising had they not been found to carry poxviruses) and there is no reason to believe they would have greater zoonotic potential than other animal poxviruses. poxviruses with varying zoonotic potentials have been found in a broad range of wildlife taxa including hundreds of bird species, reptiles, marine mammals, macropods, marsupials, monotremes, ungulates, equids, and primates [1, 2, 5, [39] [40] [41] [42] and there is currently insufficient evidence available to determine what the zoonotic potential of bat poxviruses might be on this spectrum. for example, although eidolon helvum poxvirus 1 is closely related to mocv, a human-only contagion, poxvirus-associated lesions mirroring mocv-disease have also been found in horses, donkeys and a red kangaroo [41] [42] [43] . similarly, the discovery of eptesipox virus in north american brown bats is analogous to the discovery of the other north american poxviruses found in voles, skunks, raccoons and squirrels, which are also detected at high prevalence in their reservoir hosts [44, 45] . notably however, in the initial eptesipox virus report, the authors comment that poxvirus infection manifesting as musculoskeletal disease (osteomyelitis) has also been reported in human varv and vaccinia virus (vacv) infections [35] . however, given that no bat poxviruses identified to date are orthopoxviruses, and the little information available, it is clear that much more detail is needed before the potential threat of bat poxviruses to man can be commented on. notably however, the two hosts in which poxviruses have been identified are widely distributed across their respective continents (africa and north america) and both habit urban areas, so have ample opportunities for contact with potential spillover hosts (i.e., humans and domestic animal species). to determine the zoonotic risk posed by bat poxviruses there are, as for other novel viruses, a number of obvious and relatively straightforward investigations that can be done. full genomic characterization of these viruses to identify known and putative poxvirus host range genes (discussed further below) would be an obvious step. similarly, testing the in vitro host range of isolated viruses such as eptesipox virus would help inform whether human and further animal cell lines are permissive for infection (i.e., that they contain the necessary host factors to support infection and do not contain antiviral components that restrict infection). serological and clinical surveillance of human populations for poxvirus infections in geographical regions near detection sites, and/or overlapping with bat home ranges would be a direct approach that would provide samples useful for evaluating multiple candidate zoonoses. whether bat poxviruses pose a zoonotic threat will likely comprise part of the future research agenda as these investigations are prudent for the discovery of all novel viruses. however, our current knowledge on bat poxviruses does not allow us to make firm predictions about their ability to infect humans. irrespective of their potential role as zoonotic agents however, the study of poxviruses in bats opens unique avenues of highly relevant research for multiple research fields beyond the individual host-pathogen relationships. further field (in situ), in vitro and in silico studies could elucidate the possible coevolution, cross species infections and mechanisms of host range restriction of bat poxviruses, the implications of which are relevant for bat ecologists, virologists and emerging infectious disease specialists (including those with a specific interest in bats) alike. it is likely that comparative phylogenetics of bats and poxviruses would inform and deepen our understanding of origins and evolution of both elements. bats and poxviruses are diverse host and pathogen taxa respectively and given their 0.5 million years of likely co-existence [46] , there is surely a vast amount of knowledge to be gained by studying the phylogenetic relationships between bats and poxviruses. further sampling of bat populations for poxviruses would undoubtedly dramatically expand the poxvirus phylogeny, as has occurred subsequent to the study of other viral taxa in bat populations [47] [48] [49] [50] [51] [52] [53] . comparative phylogenetics of bats and their poxviruses could differentiate between ancient co-speciation, or a more recent introduction and dissemination, of poxviruses among bat species. the two thus far partially characterized bat poxviruses are quite distinct from each other and are both relatively basal (i.e., have older most recent common ancestors with other extant viruses) in the poxvirus phylogeny when compared with other mammalian-infecting poxviruses. it is possible that if evidence of coevolution between bats and poxviruses were present, as has been suggested for the north american poxviruses [44] , this could inform the phylogenies of both bats and poxviruses which are complicated by convergent evolution and horizontal gene transfer respectively [54] [55] [56] . in addition to allowing the study of co-evolution, such studies provide the context for the identification of cross-species infections. with concerted research effort to identify reservoir species of bat poxviruses and cross species infections of poxviruses in bats could be identified and would have important implications for both bat and zoonotic-disease specialists. continued serological and molecular studies of naturally infected bat populations would allow the clinical effect and ecological impact of cross species poxvirus infections in bats to be assessed. we already noted that poxvirus infections across species barriers can devastate wildlife populations (e.g., squirrelpox, see introduction), an effect so severe that it was used to control introduced rabbit species in australia in the 1950s [57] . white nose syndrome, a fungal pathogen causing massive die offs in north american bat populations, is an unfortunate contemporary example of the severe ecological impacts that emerging pathogens can have on bat populations [58, 59] . hence, from an ecological perspective if a bat poxvirus, e.g., eptesipox virus with its severe disease manifestations, were an emerging cross-species infection it would be useful to identify this rapidly, especially in already endangered species as is the case of the southern bentwing bat in which a poxvirus was reported. further to the conservation implications of such research, combining data regarding cross species infection and ecological aspects of host taxa (e.g., behavior, habitat, range overlap, host relatedness) will likely inform key concepts of virus sharing among bat species, as has been done with lyssaviruses [60, 61] . given the heightened interest in bat virology, further analysis of bat poxviral isolates from both within-and cross-species infections will allow for a deeper understanding of the extent and mechanisms of poxvirus host restriction. many bat cell lines have now been developed [62] [63] [64] [65] [66] , and at least one of these allows productive poxvirus infection [62] . such tools will allow the in vitro refinement of host range definitions beyond detection in the field. furthermore, full genome sequencing information of poxviruses (now a comparatively easy and cost effective task) would facilitate the in silico identification of poxvirus host range gene orthologues, as recently done by bratke and colleagues who performed a systematic survey for the presence of known poxviral host range genes on among chordopoxviruses [3] . furthermore, applying new bioinformatics tools to genomic sequence information and host range data could facilitate the identification of novel host-range determinants, perhaps even unique to bat poxviruses [12, 67] . in addition, with the aforementioned in vitro tools in place, hypothetical host range genes can be validated, advancing our fundamental knowledge of poxvirus host range restriction. finally, and most speculatively, the identification of genes involved in poxvirus host range restriction in bats may represent a unique opportunity to study bat immunology, which may have broader implications for their confirmed roles as zoonotic reservoirs. since genes that interplay with the host innate immune system, not those involved with cell entry, are typically responsible for host range determination in poxviruses [8, 9] , the identification of bat-unique poxvirus host range genes could facilitate the cognate identification of (possibly novel) host immune factors. this is particularly important for bats as they potentially have antiviral immunity distinct from our own, which seemingly allows them to harbor numerous human pathogens viruses asymptomatically [29] . some preliminary evidence of this distinction existing for poxviruses is that in the single described report of infection of bat cell lines with poxviruses, bat cells were found to behave very differently from other mammalian cell lines, being susceptible to a highly attenuated strain of vaccinia virus [62] . with several bat genomes recently sequenced [68] and the capabilities of newer proteomic approaches, it is realistic that novel non-orthologous innate immune factors of bats (if they exist) could be identified. that these novel immune factors might then 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animal, plant and ecosystem health host phylogeny constrains cross-species emergence and establishment of rabies virus in bats rates of viral evolution are linked to host geography in bat rabies cell lines from the egyptian fruit bat are permissive for modified vaccinia ankara establishment, immortalisation and characterisation of pteropid bat cell lines authentication of the r06e fruit bat cell line establishment of cell line from embryonic tissue of pipistrellus ceylonicus bat species from india & its susceptibility to different viruses type i interferon reaction to viral infection in interferon-competent, immortalized cell lines from the african fruit bat eidolon helvum genome-wide association study identifies vitamin b5 biosynthesis as a host specificity factor in campylobacter comparative analysis of bat genomes provides insight into the evolution of flight and immunity the authors thank daniel streicker and gustavo delhon for their helpful comments on the manuscript. both authors reviewed the literature, wrote, and edited the manuscript. the authors declare no conflict of interest. key: cord-001455-n7quwr4s authors: rapin, noreen; johns, kirk; martin, lauren; warnecke, lisa; turner, james m.; bollinger, trent k.; willis, craig k. r.; voyles, jamie; misra, vikram title: activation of innate immune-response genes in little brown bats (myotis lucifugus) infected with the fungus pseudogymnoascus destructans date: 2014-11-12 journal: plos one doi: 10.1371/journal.pone.0112285 sha: doc_id: 1455 cord_uid: n7quwr4s recently bats have been associated with the emergence of diseases, both as reservoirs for several new viral diseases in humans and other animals and, in the northern americas, as hosts for a devastating fungal disease that threatens to drive several bat species to regional extinction. however, despite these catastrophic events little information is available on bat defences or how they interact with their pathogens. even less is known about the response of bats to infection during torpor or long-term hibernation. using tissue samples collected at the termination of an experiment to explore the pathogenesis of white nose syndrome in little brown bats, we determined if hibernating bats infected with the fungus pseudogymnoascus destructans could respond to infection by activating genes responsible for innate immune and stress responses. lesions due to fungal infection and, in some cases, secondary bacterial infections, were restricted to the skin. however, we were unable to obtain sufficient amounts of rna from these sites. we therefore examined lungs for response at an epithelial surface not linked to the primary site of infection. we found that bats responded to infection with a significant increase in lungs of transcripts for cathelicidin (an anti-microbial peptide) as well as the immune modulators tumor necrosis factor alpha and interleukins 10 and 23. in conclusion, hibernating bats can respond to experimental p. destructans infection by activating expression of innate immune response genes. bats, members of the mammalian order chiroptera, have evolved a range of characteristics that allow them to adapt to changing environmental conditions. they are the only mammals capable of powered flight, most bat species undergo torpor to conserve energy and species that inhabit high northern latitudes hibernate for up to eight months with body temperatures below 10uc [1] . bats are extremely diverse, making up a fifth of all known mammalian species. they occupy a variety of niches across most of the world where they contribute in many ways to ecological balance [2] . bats have been implicated as reservoirs for human bacterial [3] [4] [5] and viral pathogens [6] . in recent years bats have also been associated with the emergence of several devastating viral diseases in humans and other animals [7] [8] [9] [10] [11] [12] [13] [14] [15] . circumstantial evidence even suggests that most human and domestic animal pathogens of at least four viral families -coronaviridae, paramyxoviridae, lyssaviridae and filoviridae may have arisen in bats [15] [16] [17] and bats are more likely than rodents to host zoonotic viruses [18] . interestingly, with the exception of rabies and other lyssaviruses, viruses do not appear to cause overt pathology in bats, suggesting the evolution of benign relationships between bats and their pathogens [14, 19] . despite the growing realization of the importance of bats in environmental and human health and disease, we know relatively little about how bats interact with their pathogens and commensal microbes. data on their immune responses to infection during torpor or long-term hibernation are particularly scarce because individuals of some species spend as long as 8 months a year in hibernation (norquay and willis, in press) and are therefore difficult to sample. the few studies on bats and other mammals suggest that the energy conserving benefits of torpor and hibernation may be enhanced by a state of immunosuppression [20] . the fungus pseudogymnoascus destructans (formerly known as geomyces destructans) is a cold-adapted saprophytic fungus that targets hibernating bats. it appears to be a recent introduction to the northern americas [21] and in recent years has been responsible for the death of an estimated 6 million bats and the potential regional extinction of some species [22, 23] . this disease is called white nose syndrome (wns) for the white fungal mycelia on the faces and wings of affected bats. although damage caused by the fungus is restricted to the superficial skin, infected bats clearly show signs of systemic physiological perturbation such as dehydration, hypovolemia and metabolic acidosis [24] . infected bats arouse from torpor more frequently than uninfected bats [25, 26] possibly leading to emaciation. recently, we investigated the pathogenesis of p. destructans [24, 25] . we used samples collected during the experiment to address the question: can hibernating bats respond to infection by activating genes responsible for innate immune and stress responses? while the site of primary site of fungal infection and occasional secondary bacterial infection was the skin of the wings [25] , most of this tissue was needed for histopathology and fungal detection and we were unable to obtain sufficient amounts of rna from these sites. to test the hypothesis that bats mount a systemic response to infection we examined lungs as an example of an epithelial surface which could have come in contact with aerosolized fungal spores despite being remote from the site of infection or may have responded to infection of peripheral areas [27, 28] . fifty four male m. lucifugus bats were collected from a wnsfree cave in manitoba, canada. protocols for collecting and transporting bats, infection with p. destructans, maintenance of bats in hibernation and sample collection have been described previously [24, 25] . briefly, bats in groups of 18 were either sham inoculated or inoculated with n. american or european isolates of p. destructans. bats were housed at 7uc and greater than 97% humidity with ad libitum water. all bats were equipped with data loggers to monitor skin temperatures. bats were euthanized during the experiment when humanely required or at the termination of the experiment 120 days after inoculation. immediately following euthanasia samples from segments of wing as well as various tissues were preserved in rnalater (life technologies, am7021) or in formalin. samples in rnalater were kept at 220uc until they were processed. we did not find p. destructans isolate-specific differences and treated the bats as either infected or sham-infected (control). during necropsy we collected representative samples for histopathology from all major organ systems. in addition, representative samples were taken from all areas of the wing and rolled on dental wax before placing in 10% neutral buffered formalin. tissues were processed routinely for histology, 5 mm sections cut and stained with periodic acid-schiff stain to highlight fungal hyphae. liver and other tissues were processed routinely and stained with hematoxylin and eosin. wings were subjectively scored on a scale of 0 to 5 with 5 being very severe with .50% of wing covered in fungal hyphae. bacterial score was from 0 to 5 with 5 indicating wide-spread and abundant bacteria being present in many areas within the dermis and underlying connective tissues. interstitial lung neutrophil assessment was similarly evaluated on a scale of 0 to 5, with 5 being very severe. tissues were homogenized in 2 ml sealed vials with a 5 mm steel bead, 0.1 g of 0.1 mm zirconium silica beads, 350 ml of rlt buffer (with b-mercaptoethanol) (rneasy plus kit, qiagen, 74136) using a retsch mm400 tissue homogenizer at 30 hz twice for 2 minutes each. rna from tissues was extracted using the procedure provided with the rneasy plus kit. cdna synthesis cdna synthesis was performed with 1 mg of rna per reaction using the quantitect reverse transcription kit (qiagen, 205313). cdna samples were either stored at 4uc if proceeding to use in qrt-pcr, or stored at 280uc until they were needed. to identify myotis genes for various cytokines and proteins involved in immune responses we scanned the m. lucifugus genome for regions with sequences similar to transcripts of proteins from human, mouse and rat. for genome sequences that were not annotated, m. lucifugus exons detected were compared with known exon-intron junctions in corresponding genes in human, mouse and rat to ensure that the sequences did indeed represent homologous genes. for mrnas where qrt-pcr primers had been described for other species, m. lucifugus primers were designed from the sequence of corresponding m. lucifugus genes. for mrnas where such information was not available we designed primers to optimize for annealing temperature and specificity, and to minimize dimer formation. in all cases the veracity of the pcr products was determined by electrophoresis and confirmed by sequencing. details of primers are in table 1 . stratagene's mx3005p pcr cycler was used in conjunction with quantifast sybr green pcr kit (qiagen, 204056). in addition to the primer sets mentioned, gapdh and cytochrome b were both used for every sample as normalizers. for each sample tested, 2 ml (1 mm, final concentration) of diluted forward primer, 2 ml (1 mm) of diluted reverse primer, 12.5 ml of sybr green master mix, and 8.5 ml of cdna was used. samples were tested in duplicate in each run and values used in the analysis were averages of the duplicates. the thermal profile used was as follows: 95uc for 5 minutes for initiation, then 40 cycles of 95uc for denaturing for 10 seconds, and 60uc for annealing and extension for 30 seconds with readings at the end of every cycle, then 1 cycle of 95uc for 1 minute, 55uc for 30 seconds, and 95uc for 30 seconds. the denaturation characteristics of the products were determined by incremental increase from 55uc to 95uc. a no template control (ntc) was used in every sample tested. reactions that generated primer dimers or products with spurious denaturation profiles were not considered. in all cases the identity of the pcr products was confirmed by determining their nucleotide sequences (macrogen). only results from reactions that yielded unambiguous results (products of the expected size and sequence, homogenous denaturation profiles, no products for no-template controls) were used for analysis. data from qrt-pcr and histopathological scores were analysed with spss statistics version 21. for qrt-pcr the relative levels of a transcript for each bat were calculated as cycle threshold (ct) normalized separately (dct) for levels of transcripts for two ''house-keeping'' genes -glycerol 6 phosphate dehydro-genase (gapdh) and cytochrome b. a lower ct (or dct) of one (1) indicates approximately a two-fold higher concentration of rna. the significance of differences of mean values of dct between infected and mock-infected (control) bats were determined using a student t-test. pearson's coefficients were calculated for the dct levels for cytokine transcripts for bats in each treatment class and lung interstitial neutrophil scores and mean bacterial and hyphae scores for 5 wing sections for each bat. methods were approved by the university of saskatchewan committees on animal care (protocol #20100120) and biosafety (permit #vmb03). recently we demonstrated that the european and north american strains of p. destructans were equally pathogenic for hibernating m. lucifugus [24, 25] . during these experiments histological sections from wings and other tissues were examined and graded for the presence of hyphae, edema, necrosis, bacteria (epidermal or invasive), neutrophil infiltration and inflammation. we also preserved samples which were then used in this study. we determined levels of transcripts for several immune and stress response genes (table 1) in lungs from infected and control bats. only those qrt-pcr reactions that yielded unambiguous results were considered. there were significant differences for transcripts for the interleukins (il) 10 and 23, the pro-inflammatory cytokine tumour necrosis factor alpha (tnfa), and the anti microbial and immunoregulatory peptide cathelicidin (figure 1 ). in figure 1a lower dct of one indicates approximately a two-fold higher concentration of rna. infected bats as a group had lower dct values (on an average 8 to 14 fold higher concentrations of rna, histograms in figure 1 ) for the four genes. while the group of infected hibernating bats had higher levels of transcripts for il10, il23, cathelicidin and tnfa than hibernating uninfected bats, the levels varied over a wide range with some infected bats having levels comparable to (or lower than) those of uninfected animals. since the level of fungal infection and pathology varied considerably among infected bats, we attempted to correlate transcript levels with scores for fungal infection, fungal hyphae and bacterial infection in wings and neutrophil accumumyotis homologues for innate response genes were identified by scanning the myotis genome for sequences that matched those of well-characterized reference organisms. where qrt-pcr primers for reference organisms d had been validated in the literature b,c , we used the corresponding myotis sequences as primers. myotisspecific substitutions in these primers are in bold a . for other genes (hsp70, tnfa, nos2, il-10, grp78) myotis-specific primers were designed from potential myotis exon sequences using parameters for optimal use in pcr reactions. in all cases the identity of pcr products were confirmed by sequencing. primers were used in qrt-pcr at a final concentration of 1 mm. doi:10.1371/journal.pone.0112285.t001 lation in lungs (table 2 ). il23 and cathlicidin levels correlated with all four parameters of disease and inflammation. il10 correlated with level of infection, and hyphae and bacterial scores in the wing, and tnfa correlated with level of infection and wing hyphae score. there was an inverse relationship between transcript levels and dct. the correlation coefficients are therefore negative. our results suggest that hibernating m. lucifugus are capable of inducing the expression of genes for cytokines in response to infection. the bats we examined were kept under conditions that closely mimicked temperature and humidity encountered in their natural hibernacula (i.e., 7uc and .97% relative humidity) [24, 25] . during hibernation m. lucifugus body temperature is maintained within 1-2uc of ambient temperature for weeks at a time with return to normothermic temperatures during periodic arousal [24, 25] . it is difficult to imagine mammalian gene expression at temperatures this low and it is possible that the genes were transcribed during the arousal periods. our previous study indicated that p. destructans-infected bats aroused more frequently than uninfected bats [25] , a pattern which has also been observed in the wild [26] and this may also have contributed to higher basal levels of gene expression in their cells. however, our observation that the levels for il 23, cathelicidin, il 10 and tnfa and not the other transcripts examined (dectin 1, defensin b1, hepcidin, il 17, cytchrome b, heat shock protein 70, nitrogen oxide synthetase) were higher in the lungs of infected bats, suggest that the response was a specific to infection. we observed mild to moderate levels of interstitial neutrophils in the lung of infected bats. on average, the lungs contained 8 to 20 times more transcripts for the anti-microbial peptide cathelicidin as well as for the immune modulators tnfa and il 10 and 23. the increase in levels of il 23, cathelicidin and il10 correlated with the lung interstitial scores ( table 2) . little is known about the role in bats of these bioactive proteins, information from other species indicates that they are intimately involved in the response to infection. for instance: cathelicidins belong to a family of antimicrobial peptides with an intrinsic ability to kill bacteria and fungi but they also function as chemoattractants for inflammatory cell recruitment and cytokine release. they are found in the lysosomes of neutrophils, macrophages and epithelial cells after activation or infection with a wide variety of pathogens [29] . humans, mice and dogs have a single gene for cathelicidin while several related genes have been discovered in pigs, horses, cattle and chickens. m. lucifugus has at least two genes with similarity to the human gene. our primers were designed to amplify transcripts for the gene most similar to that for human cathelicidin. interleukin 23 is a multifunctional pro-inflammatory cytokine involved in both innate and adaptive responses [30] . it is an important mediator of dectin-dependent response of the protective neutrophils to lung infections by aspergillus [31] . it is also important in the maturation of t helper cells. in addition to being protective il23 may also play a negative role by inducing chronic inflammation and exacerbating the effects of aspergillus and candida infections [32] . interleukin 10 is also known as cytokine synthesis inhibitory factor (csif) because of its role in modulating the inflammatory response. its effect is to suppress th1 responses and stimulate th2 responses leading to b-cell survival and proliferation and antibody synthesis. il10 is produced by a number of cell types including monocytes, and activated subsets of regulatory and helper t-cells b-cells. its synthesis is tightly regulated and is mediated by the nfkb and ap1 pathways after stimulation by commensal and pathogenic microorganisms [33] . if the role of il10 in wns is indeed to direct the immune system to a th2 response, our results are in contrast with those of moore et. al. [34] who found increased levels of il4 in the serum of wns-affected bats. work in other species suggests that il4 directs the immune system to a th1 response. tumour necrosis factor alpha (tnfa) is a monocyte derived cytotoxin implicated in tumour regression, septic shock and cachexia. tnfa binding to its receptor stimulates the activation of the transcription factor, nfkb, which activates gene expression required for several defensive responses [35] . the presence of neutrophilia in the lung interstitia of infected bats, despite a lack of obvious pathology or the presence of microorganisms, is puzzling. however, these observations may be explained by: a) injury in the skin can lead to complement-induced tnfa expression in the lungs followed by inflammation and neutrophil infiltration [27] . b) immune responses at mucosal surfaces are coordinated, with stimulation at one surface leading to responses at other mucosae [36] . the pulmonary response may have been due to stimulation of the upper respiratory tract by fungal spores. c) there have been suggestions that hibernating mammals show marked leukopenia [20] raising the possibility that circulating leukocytes are sequestered in some organ, possibly the lungs. the infected animals may have responded to inflammation in the wings with an increase in neutrophils, which then migrated to the lungs. d) lung neutrophils in infected bats, and the accompanying increase in the transcripts of the immune modulators may represent the end of the infectious process in the lungs and the removal of fungal spores and hyphae. this suggests that bats are capable of suppressing a lung or systemic infection by p. destructans and may explain why wns, the disease, occurs only after bat-to-bat contact rather than by infection through inhalation. while relatively little is known about the response of bats to infections, work with other species indicates that the host's response to pathogens is complex involving the interconnected innate, relatively non-specific mechanisms as well as the more specific adaptive systems of humoral and cellular immunity. the increased transcripts we detected in infected bats represent but a few components of this complex network. while our study shows that hibernating bats can respond to an infection, it does have some limitations and our results should be interpreted with caution. for instance, we have used increase in the stable levels of transcripts for various cytokine genes as a surrogate for increases in their respective proteins. however mrna and protein levels do not always correlate, largely because of complex regulatory processes that separate transcription of mrnas from their translation into protein [37] [38] [39] . in addition, only one of the transcripts, for cathelicidin, is directly linked to an anti-fungal response. the remainder, il 23, il 10 and tnfa may be indirectly related or may represent a non-specific response to the stress of infection. the importance of temporal 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immune responses of bats: a review hibernation: the immune system at rest? bat white-nose syndrome: an emerging fungal pathogen an emerging disease causes regional population collapse of a common north american bat species experimental infection of bats with geomyces destructans causes white-nose syndrome pathophysiology of white-nose syndrome in bats: a mechanistic model linking wing damage to mortality inoculation of bats with european geomyces destructans supports the novel pathogen hypothesis for the origin of white-nose syndrome frequent arousal from hibernation linked to severity of infection and mortality in bats with white-nose syndrome chemotactic mediator requirements in lung injury following skin burns in rats lung injury secondary to chemotactic factor-induced leukocyte activation innate immune defense system of the skin the il23/th17 pathway as a therapeutic target in chronic inflammatory diseases neutrophils produce interleukin 17a (il-17a) in a dectin-1-and il-23-dependent manner during invasive fungal infection th17 cells in the setting of aspergillus infection and pathology the regulation of il-10 production by immune cells hibernating little brown myotis (myotis lucifugus) show variable immunological responses to white-nose syndrome molecular basis of nf-kappab signaling role of inducible bronchus associated lymphoid tissue (ibalt) in respiratory immunity aebersold r (1999) correlation between protein and mrna abundance in yeast correlation of mrna and protein in complex biological samples insights into the regulation of protein abundance from proteomic and transcriptomic analyses regulation of cathelicidin antimicrobial peptide expression by an endoplasmic reticulum (er) stress signaling, vitamin d receptor-independent pathway dectin-1 expression at early period of aspergillus fumigatus infection in rat's corneal epithelium expression of human beta-defensins 1 and 2 in kidneys with chronic bacterial infection the small molecule, genistein, increases hepcidin expression in human hepatocytes expression of interleukin-17 associated with disease progression and liver fibrosis with hepatitis b virus infection: il-17 in hbv infection the immunomodulatory effect of bone marrow stromal cells (bmscs) on interleukin (il)-23/il-17-mediated ischemic stroke in mice discovery of herpesviruses in bats key: cord-003232-nquw7qga authors: kuchipudi, suresh v.; nissly, ruth h. title: novel flu viruses in bats and cattle: “pushing the envelope” of influenza infection date: 2018-08-06 journal: vet sci doi: 10.3390/vetsci5030071 sha: doc_id: 3232 cord_uid: nquw7qga influenza viruses are among the major infectious disease threats of animal and human health. this review examines the recent discovery of novel influenza viruses in bats and cattle, the evolving complexity of influenza virus host range including the ability to cross species barriers and geographic boundaries, and implications to animal and human health. influenza viruses are known to constantly evolve and cross species barriers. the genetic diversity of influenza viruses is ever increasing with more novel influenza subtypes being discovered periodically. the purpose of this review is to provide an up-to-date overview of ecology and evolution of influenza viruses including the novel influenza viruses in bats and cattle. in addition, we discussed the growing complexity of influenza virus-host interactions and highlighted the key research questions that need to be answered for a better understanding of the emergence of pandemic influenza viruses. influenza is among the major infectious disease problems affecting animal and human health globally. several human influenza pandemics have been recorded since 1590 ad [1] , with the most significant of those being the "spanish flu" of 1918, often referred to as the "mother of all pandemics" [2] . spanish flu pandemic is believed to have affected approximately 25-30 percent of the world's population and caused more than 50-60 million human deaths globally [3] . influenza infections in humans occur either as epidemic (seasonal or interpandemic) influenza caused by influenza a and b viruses, or as sporadic pandemic influenza caused by influenza a viruses [4] . study of influenza pandemics has been of great interest to epidemiologists. influenza epidemics and pandemics have been repeatedly occurring for centuries, but to date the ability to predict a pandemic has not been achieved [5] . influenza viruses belong to the family orthomyxoviridae (from the greek orthos, meaning "standard, correct", and myxa, meaning "mucus"). members of orthomyxoviridae are characterized by virions that are either spherical or pleomorphic measuring 80-120 nm in diameter. genomes are comprised of single-stranded negative-sense rna that is arranged in either eight, seven or six segments depending on the genus. classification of the historically described a, b and c influenza viruses based on antigenic differences in nucleocapsid (np) and matrix (m1) proteins. influenza a viruses (iavs) are further classified into subtypes based on the antigenic properties of the external glycoproteins namely iavs are known to infect humans and a wide variety of animals including pigs, horses, minks, seals, whales and birds [19] . avian influenza viruses (aivs) continue to spread, evolve and cause significant economic losses to poultry industries globally. unlike low pathogenic avian influenza viruses (lpaivs) that cause mild clinical signs in domestic poultry, infection of gallinaceous poultry with highly pathogenic avian influenza viruses (hpaivs) results in severe fatal disease often causing up to 100% mortality within two-three days [20] . aivs have also been implicated in the generation of influenza viruses that have caused all human influenza pandemics. in particular, human infection with certain contemporary eurasian lineage hpai h5n1 viruses frequently caused severe disease with a case fatality rate of up to 50% [21] . ability to infect a wide range hosts is a key contributing factor to the complex and seemingly expanding genetic diversity of iavs. it is now well established that iavs infect domestic pets such as dogs and cats, adding to the list of host species that could potentially expose humans to influenza viruses. an h3n8 equine influenza virus in 1999 and an avian virus-like h3n2 strain around 2005 or 2006 were transmitted to dogs, and these canine influenza viruses have been circulating in the u.s. dog population ever since [22] . after the first report of pandemic h1n1 influenza a virus (ph1n1) infection of cats in italy in 2011 [23] , more than 500 cats became infected with influenza subtype h7n2 in animal shelters in new york, ny, usa during 2016-2017 [24] . while transmission of these animal viruses to humans has not yet been documented, the close contact of many humans with pet dogs and cats presents an increased risk of opportunity for such a host jump. transmission of iavs between animals and humans is bidirectional such that humans are infected by animal influenza viruses and human influenza viruses have been shown to transmit to animals. in particular, studies established that domestic pets are susceptible to infection by human influenza viruses. for example, dogs experimentally inoculated with a human seasonal h3n2 or pandemic (pdm) h1n1 (2009) showed nasal shedding and seroconversion. however, dogs inoculated with influenza b virus did not exhibit virus shedding or seroconversion highlighting that interspecies transmissibility is a key feature of iavs and not ibvs [25] . aquatic birds such as ducks, gulls, and shorebirds have historically been identified as the primary reservoir of all the known iav subtypes [26] . recently, northwest atlantic gray seals were suggested as an endemically infected wild reservoir population for diverse influenza viruses [27] . arguably, influenza virus host range could be much broader than currently known, with additional reservoirs that are yet to be revealed. prior to 2011, 16 antigenically different ha (h1-h16) and nine different na (n1-n9) types had been described, all of which were found in the aquatic bird reservoir. however, the number of ha and na types have now expanded after the identification of two novel influenza-like viruses in fruit bats. the first was in a frugivorous yellow-shouldered bat (sturnira lilium) in guatemala [28] . soon after, a second virus was found in the flat-faced fruit bat (artibeus planirostris) in peru [29] . the newly identified bat flu viruses are genetically distinct from all previously known iavs and hence are designated as novel subtypes namely h17n10 and h18n11 [29] . serological surveys showed wide spread prevalence of h17/h18-specific antibodies among bat populations in central and south america [29] . finding novel bat-influenza viruses is not surprising as bats represent 24% of all known mammalian species [30] and have been known to be natural reservoirs of many deadly zoonotic rna viruses including ebola virus [31] [32] [33] . it is believed that bats have the capacity to harbor more influenza virus genetic diversity than all the other mammalian and avian species combined [29] . notably, little yellow-shouldered bats in central america have been proposed as a potential sylvatic mammalian reservoir of influenza [28] . discovery of novel bat iavs raises numerous questions including the host range evolution of iavs and the role of bats in evolution of iavs. the potential for the newly discovered bat iavs to reassort with previously known iav types is being explored and providing novel information about the iav life cycle. bat flu viruses have been reconstructed by reverse genetics using synthetic dna which has allowed for more functional understanding of these viruses [34] . multiple experimental studies have shown that reassortment of bat iavs and conventional iavs does not occur under experimental conditions [35] [36] [37] . this is partly explained by functional differences between bat iavs and conventional iavs. iav nonstructural protein ns1 is a multifunctional protein that plays a crucial role in evading host immune responses and serves as a key virulence factor [38, 39] . unlike the ns1 of conventional iavs, bat ns1 fails to bind to the host p85β, a regulatory subunit of the cellular metabolism-regulating enzyme phosphoinositide 3-kinase (pi3k) [40] . in addition, the surface glycoprotein ha and na genes of bat iavs only show low nucleotide sequence identity with those of the conventional iavs [28, 29] . notably, it has been found that the bat iav hemagglutinin (ha) and neuraminidase (na) proteins lack the receptor binding activity that are characteristic of the conventional iav [28, 41] . ha of bat iavs does not bind to the classical avian (saα2,3-gal) or human (saα2,6-gal) iav receptors highlighting the possibility of unique entry mechanisms that are yet to be identified [42] . consequently, it was found that bat iavs initiate infection at the basolateral membrane of cells unlike conventional iavs which preferential initiate infection on the apical surface of cells [34] . these findings highlight that there are major evolutionary constraints to bat iavs to effectively reassert with conventional iavs and/or infect other species including humans. while the zoonotic potential of bat iavs is not yet fully established and continues to be a focus of ongoing research, bats have been found to be susceptible to infection by conventional iavs. a recent study found serological evidence of iav h9 subtype infection in 30% of frugivorous bats tested in africa [43] . a key source of iav genetic diversity could come from the replication of iavs in a non-native host species that initiate evolution of new virus variants [44] . the receptors for previously known iavs are sialic acids (sa) on host cells [45] . consequently, the expression of these receptors on host cells is a key determinant of the ability of iavs to infect a host [46] . aivs preferentially bind to sa receptors that are linked to galactose by an α 2,3 linkage (saα2,3-gal), while human and classical swine influenza viruses show preference to α2,6 linked sas (saα2,6-gal). it is widely believed that hosts that co-express both saα2,3-gal and saα2,6-gal receptors could support reassortment of iavs and hence play a major role in the evolution of iavs [45, 47] . we have demonstrated abundant co-expression of both saα2,3-gal and saα2,6-gal receptors that are compatible with avian and human iav binding in respiratory and digestive tracts of little brown bats, the most widespread bat species in north america [48] . the potential for bats to support infection by multiple conventional iav types and thus serve as a source of novel genetic variants remains to be fully explored. although influenza viruses infect humans and a wide range of animals and birds, cattle were never considered to be susceptible to influenza virus infection. however, a novel influenza virus has recently been identified in several animals including swine, cattle, sheep, and goats. the virus was first isolated as an influenza c-like virus from pigs with respiratory illness in oklahoma, usa, in 2011 [49, 50] . the virus was subsequently classified as influenza d virus (idv), and the virus has now been reported from many countries including united states, france [51] , italy [52] , china [53] , japan [54] ireland [55] and countries in north and west africa [56] . the international committee on taxonomy of viruses (ictv), which is responsible for developing, refining, and maintaining a universal virus taxonomy, has recently released revised classification of orthomyxoviridae [57] . in earlier classification, orthomyxoviruses belonged to any one of the five genera: influenzavirus a, influenzavirus b, influenzavirus c, thogotovirus and isavirus [58] . however, the most recent classification of orthomyxoviridae includes eight genera and nine species (table 1) . genus alphainfluenzavirus comprises the species influenza a virus, betainfluenzavirus comprises the species influenza b virus, gammainfluenzavirus comprises the species influenza c virus, and delatinfluenzavirus comprises the species idv [58] . like other influenza viruses, idvs possess a negative-sense single-stranded rna genome. however unlike iavs that contain 8 segments, idvs comprise seven genomic segments that are predicted to encode nine proteins, including glycoprotein hemagglutinin-esterase fusion (he), polymerases pb2, pb1, and p3, nucleoprotein, matrix protein (m1 and cm2), and nonstructural proteins (ns1 and nep) [59] . idvs use 9-o-acetylated sialic acid as their cellular receptor on host cells such as icvs [60] . it is well established based on several epidemiological studies that cattle are the primary reservoirs of idvs [50, 59] . in addition, idvs have also been isolated from a range of animals including pigs, sheep, goat, horses and camelids. while the precise role of idvs in clinical disease in animals is not yet fully investigated, their role in causing respiratory infections in cattle has been implied. two recent studies carried out metagenomic characterizations of the virome associated with bovine respiratory disease in feedlot cattle and found correlation of idv presence with bovine respiratory disease (brd) clinical signs, raising exciting new prospects for understanding and combatting this complicated disease [61, 62] . brd complex is one of the major diseases affecting the cattle industry in the usa and around the world. productivity losses due to brd are estimated to be $23.60 per calf with an annual economic impact of more than one billion dollars to the u.s. cattle industry [61] . brd is associated by multiple pathogens and accounts for approximately 70-80% of the morbidity in the usa [63] and 84.5-99.9% of the morbidity in mexican feedlot cattle. brd results in the use of widespread therapeutics and antibiotics in feedlots, which increasingly raises public health concerns of promoting antibiotic resistance [64, 65] . pathophysiology of brd involves complex interactions between host, pathogen, environment and management factors. in feedlot cattle, brd is initiated by viral infection followed by stress due to travel which is typically followed by a secondary infection by resident bacteria [66] . viral infection can cause increased susceptibility to secondary bacterial infections by either immunosuppression or by damaging the epithelium of upper airways and injuring lung parenchyma which facilitates the migration of bacterial pathogens and colonization of the lower respiratory tract. depending on several factors, the clinical outcome of brd can be variable; however, higher morbidity and mortality are observed in the event of mixed viral and bacterial infections [67] . many viral pathogens have been implicated in brd, which include bovine viral diarrhea virus, bovine herpesvirus 1, bovine respiratory syncytial virus and bovine parainfluenza 3. experimental studies in calves with idv showed damage that results in the induction of inflammation in the trachea. idv could be a significant player in brd and could facilitate coinfections with other bovine pathogens [59] . in addition to the animal health implications of idvs, a recent study found idv antibodies in 34 out of 35 persons that had contact with cattle and only 2 out of 11 that did not have any exposure to cattle [68] . the results of this study raised the possibility that idv could be relevant from a public health stand point and that it could pose a zoonotic risk to cattle-exposed workers. with much still to be characterized about the new idv species, exploring its impact on human an animal health through epidemiological studies will be vital to understanding spread of this virus. emerging and novel zoonotic infections often result from pathogens jumping from their original host into novel host species [69] . the host range evolution of mammalian viruses typically involves more closely related hosts [70, 71] . in particular, rna viruses with broad host range are more likely to jump between distantly related species [70, 72, 73] . a key determinant in the host range evolution of a virus is the mechanism of viral entry used by the viruses. analysis of 64 human viruses revealed that the viruses that use receptors that are highly conserved in their amino acid sequence across species have the broadest host range [74] . the ability of viruses to bind to an alternative receptor is sometimes key in species jumping. aivs need to change to preferentially bind to saα-2,6-gal receptor to efficiently transmit between humans. in experimental studies, it was shown that the shift from saα-2,3-gal to saα-2,6-gal binding requires four mutations for a ha of hpai h5n1 viruses [75] . however, several newly emerged h5n1 aivs in egypt have been found to have acquired the human receptor saα-2,6-gal binding ability during their emergence in birds [76] . influenza a, b, c and d viruses have varying susceptible host range ( figure 1) . notably, the newly discovered idvs have the widest host range after iavs. further, humans and pigs are susceptible to infection by all four types of influenza viruses. with the extensive host range that continues to grow and the zoonotic potential, influenza viruses remain a major challenge to epidemiologists. owing to their tremendous potential to affect animal and human health, there is a need to carry out in-depth and comprehensive studies to unravel the ecological complexity of influenza virus host range evolution. in particular, we feel that researchers should focus on answering the key questions, "what is the role of bats in the ecology and evolution of iavs?", "are idvs involved in the epidemic influenza infections in people?", and "are birds susceptible to infection by idvs?" vet. sci. 2018, 5, x for peer review 6 of 10 need to carry out in-depth and comprehensive studies to unravel the ecological complexity of influenza virus host range evolution. in particular, we feel that researchers should focus on answering the key questions, "what is the role of bats in the ecology and evolution of iavs?", "are idvs involved in the epidemic influenza infections in people?", and "are birds susceptible to infection by idvs?" a history of influenza influenza: the mother of all pandemics the origin and virulence of the 1918 "spanish" influenza virus reviewing the history of pandemic influenza: understanding patterns of emergence and transmission a history of influenza influenza: the mother of all pandemics the origin and virulence of the 1918 "spanish" influenza virus reviewing the history of pandemic influenza: understanding patterns of emergence and transmission non coding extremities of the seven influenza virus type c vrna segments: effect on transcription and replication by the type c and type a polymerase complexes domestic pigs are susceptible to infection with influenza b viruses epidemiology and pathogenesis of influenza selection and analysis of antigenic variants of the neuraminidase of n2 influenza viruses with monoclonal antibodies antigenic and genetic characteristics of swine-origin 2009 a(h1n1) influenza viruses circulating in humans avian-to-human transmission of the pb1 gene of influenza a viruses in the 1957 and 1968 pandemics compatibility among polymerase subunit proteins is a restricting factor in reassortment between equine h7n7 and human h3n2 influenza viruses selective packaging of the influenza a genome and consequences for genetic reassortment the genome-packaging signal of the influenza a virus genome comprises a genome incorporation signal and a genome-bundling signal interaction network linking the human h3n2 influenza a virus genomic rna segments a supramolecular assembly formed by influenza a virus genomic rna segments an in vitro network of intermolecular interactions between viral rna 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restriction conformational plasticity of the influenza a virus ns1 protein unexpected functional divergence of bat influenza virus ns1 proteins structural and functional characterization of neuraminidase-like molecule n10 derived from bat influenza a virus bat-derived influenza hemagglutinin h17 does not bind canonical avian or human receptors and most likely uses a unique entry mechanism serological evidence of influenza a viruses in frugivorous bats from africa influenza a virus polymerase is a site for adaptive changes during experimental evolution in bat cells differences in influenza virus receptors in chickens and ducks: implications for interspecies transmission endocytosis of influenza viruses. microbes infect comparative distribution of human and avian type sialic acid influenza receptors in the pig avian and human influenza virus compatible sialic acid receptors in little brown bats isolation of a novel swine influenza virus from oklahoma in 2011 which is distantly related to human influenza c viruses characterization of a novel influenza virus in cattle and swine: proposal for a new genus in the orthomyxoviridae family influenza d virus in cattle detection of influenza d virus among swine and cattle influenza d virus infection in herd of cattle influenza d virus in cattle serologic evidence for influenza c and d virus among ruminants and camelids pathogenesis of influenza d virus in cattle an open receptor-binding cavity of hemagglutinin-esterase-fusion glycoprotein from newly-identified influenza d virus: basis for its broad cell tropism metagenomic characterization of the virome associated with bovine respiratory disease in feedlot cattle identified novel viruses and suggests an etiologic role for influenza d virus a metagenomics and case-control study to identify viruses associated with bovine respiratory disease where have we been, where are we now, and where do we want to go? anim a ten-year (2000-2009) study of antimicrobial susceptibility of bacteria that cause bovine respiratory disease complex-mannheimia haemolytica, pasteurella multocida, and histophilus somni-in the united states and canada metaphylactic antimicrobial therapy for bovine respiratory disease in stocker and feedlot cattle review of brd pathogenesis: the old and the new board-invited review: recent advances in management of highly stressed, newly received feedlot cattle serologic evidence of exposure to influenza d virus among persons with occupational contact with cattle the evolution and genetics of virus host shifts phylogeny and geography predict pathogen community similarity in wild primates and humans a tale of two phylogenies: comparative analyses of ecological interactions emerging pathogens: the epidemiology and evolution of species jumps host range and emerging and reemerging pathogens human viruses: discovery and emergence experimental adaptation of an influenza h5 ha confers respiratory droplet transmission to a reassortant h5 ha/h1n1 virus in ferrets acquisition of human-type receptor binding specificity by new h5n1 influenza virus sublineages during their emergence in birds in egypt acknowledgments: authors acknowledge the support of penn state animal diagnostic laboratory for providing resources and access needed for the preparation of this review. the authors declare no conflict of interest.acknowledgments: authors acknowledge the support of penn state animal diagnostic laboratory for providing resources and access needed for the preparation of this review. the authors declare no conflict of interest. key: cord-021805-2j07zw6q authors: epstein, jonathan h. title: emerging diseases in bats date: 2018-09-28 journal: fowler's zoo and wild animal medicine current therapy, volume 9 doi: 10.1016/b978-0-323-55228-8.00040-0 sha: doc_id: 21805 cord_uid: 2j07zw6q nan the majority of emerging infectious diseases are zoonotic, and most originate in wild animals. 1 the rate of emerging disease has increased significantly over the past few decades, and the majority of emerging pathogens are rna viruses. these are distinctive in that they have the ability to mutate rapidly compared with dna viruses and bacteria, allowing them to adapt to new hosts and spread more effectively. 2 little is understood about the dynamics of zoonotic viruses in their natural reservoirs, yet it is becoming clear that anthropogenic environmental change is driving the spillover of pathogens from wildlife populations into domestic livestock and humans. activities such as urbanization, agricultural intensification, and global travel and trade are expanding the interface between people, livestock, and wildlife, providing continuous opportunities for pathogens to spill over into human populations and then move around the world. 3 viral spillover between wildlife and domestic animals or humans probably occurs more frequently than is recognized, owing to limited or poor surveillance systems. 4 zoonotic disease emergence is most likely to occur in regions where biodiversity and human population density are high and where human activities that alter the environment-such as urbanization, agricultural expansion, and deforestation-are most intensive. 2, 5 among mammalian taxa, bats (order chiroptera) carry more zoonotic viruses than other mammalian groups. 6 why bats are special is not completely understood, but there appears to be a combination of ecologic, genetic, and immunologic factors, the last two of which have only recently begun to be explored. 6, 7 bats have been associated with several zoonotic viruses that have recently been discovered and linked to significant human and animal disease, including severe acute respiratory syndrome (sars), middle east respiratory syndrome (mers), ebola and marburg viruses, and nipah virus (niv) 8 (see also chapters 19, 34, and 42 ). there are more than 1200 species of bats in the world, forming the order chiroptera, which makes them the second most speciose taxonomic group of mammals after rodents, representing 20% of mammalian diversity. 9 emerging diseases in bats bats are found on every continent and in every environment in which humans live. they successfully exploit human dwellings, constructs, and food resources, which creates opportunity for direct and indirect contact with people and domestic animals. although bats typically avoid direct contact with people, indirect exposure to excreta created by bats roosting within households, buildings, mines, and caves may lead to human infection with bat-borne pathogens. 10 frugivorous bats are often found roosting in trees in rural and even urban environments. 11, 12 they will eat cultivated fruit such as mangos, rambutan, and guava, as well as and other human-provided food resources, which, when contaminated with bat excreta, may also serve as route of infection for people or animals. despite their potential to carry zoonotic viruses, bats are overwhelmingly beneficial to people and plants, performing vital ecosystem services in the form of agricultural pest control 13, 14 and seed dispersal and pollination. 15 in many parts of the world, bats are hunted for food, sport, or traditional medicine. 16 the butchering and consumption of bats provides an opportunity for the transmission of blood-borne pathogens. 8 many novel viruses or viral sequences have been identified in bats, but in most cases their ability to infect other species remains unknown. one of the major challenges to predicting zoonotic disease emergence is our inability to translate viral genotype into phenotype (clinical presentation and pathogenicity of a virus). viral discovery has, however, significantly expanded our understanding of the phylogenetic breadth of important viral families such as filoviruses (e.g., ebola virus), paramyxoviruses (e.g., niv), and coronaviruses (e.g., sars coronavirus [cov]), which is necessary for both better understanding what makes viruses pathogenic and also for recognizing wildlife reservoirs of viral pathogens, once they do emerge, more rapidly. 17 the following is a review of recently emerging zoonotic viruses that have bats as a natural reservoir. these examples highlight viruses whose emergence has been linked to human behaviors and that have caused significant morbidity and mortality in people, but have also involved other species in the transmission chain between bats and people, making them relevant to both human and animal health. the indian subcontinent. [27] [28] [29] [30] in addition to niv, rna sequences from closely related paramyxoviruses have been identified in this bat. 31 nonneutralizing antibodies reactive to niv have been found in domestic animals in bangladesh, including pigs, goats, and cattle, suggesting that spillover of nipah-like viruses has occurred, although no human cases have been linked to domestic animal infections in bangladesh. 32 hendra virus has continued to cause outbreaks in horses across queensland and the adjoining state of new south wales since 1994, and evidence of infection has been detected in each of the four species of flying fox present in this range. 33 although the definitive mode of transmission between bats and horses remains uncertain, it is hypothesized that infected bats feeding or roosting in trees within horse enclosures contaminate the area beneath, and horses are exposed either by direct exposure to excreta or by ingesting contaminated feed or water. 33 infected horses are then able to transmit the virus to other horses and to humans. outbreaks in horses are sporadic; however, since 2006 there has been a marked increase in the frequency and number of equine cases identified. pteropus species are the primary natural reservoirs for henipaviruses throughout asia and australia 24,34 ; however, the full geographic extent and host diversity for henipaviruses is still being studied. antibodies against a nipah-like virus were recently detected in the straw-colored fruit bat (eidolon helvum), a migratory pteropodid bat, and in hunters in cameroon, suggesting that related viruses may be circulating in central africa. 35,36 antibodies against nipah-like viruses have also been detected in insectivorous bat species in china. 37 to date, human infections have been identified in relatively few countries compared with the distribution of henipaviruses in bats (india, bangladesh, malaysia, the philippines, singapore, and australia). 8 although no treatment or vaccine for niv currently exists, the advent and commercial production of a hendra virus vaccine for horses in 2014 have offered an effective tool for limiting hev cases in australia. 38 currently niv is listed as a priority by the world health organization (www.who.int/blueprint/ priority-diseases/en/) and the coalition for epidemic preparedness innovations (cepi; www.cepi.net) for the development of a vaccine. experimental nipah vaccines that utilize soluble g proteins, like the hendra vaccine, have been found to be effective in nonhuman primate models. 39 niv's broad geographic host range, its ability to infect multiple domestic animal species and humans, its repeated spillover in populous areas and ability to spread among people, and its association with high mortality rates make niv a significant threat to human and animal health. 8, 40 because of the potential severity of henipavirus infection in people and livestock, improved surveillance systems are needed to both ensure rapid detection and response to outbreaks as well as to identify high-risk areas where host, virus, and an interface that promotes spillover exist so that effective interventions can be implemented. niv is a zoonotic paramyxovirus (genus henipavirus) first recognized in malaysia in 1998 as a respiratory and neurologic disease in domestic pigs; it subsequently infected farm workers. 18 the initial spillover occurred because mango orchards were planted next to pig enclosures. the mangos attracted frugivorous bats that carried niv; the proximity to the pig enclosures allowed contaminated fruit to be dropped and consumed by pigs. the size of the farm created an environment that could support a sustained niv outbreak in pigs over the course of a year, which fueled the broader epidemic. 19 the outbreak in malaysia spread via the movement of infected pigs from farm to farm, ultimately leading to the depopulation and closure of thousands of farms and the infection of 265 people in malaysia and singapore, of whom 105 died. 18 after niv was stamped out by the systematic depopulation of pig farms, policies were put into place that required a buffer zone between orchards and livestock enclosures on commercial farms. this solution has proven effective in removing the key interface that led to niv spillover and emergence on the index farm, and there has not been an outbreak since, despite the continued presence of the two pteropid host species and continued livestock production. four years prior to the discovery of niv in malaysia, hendra virus was discovered as the cause of an outbreak of severe respiratory and neurologic disease in horses in racing stables in hendra, a suburb of brisbane in the eastern australian state of queensland. fourteen horses were affected with respiratory and neurologic signs, and the horses' trainer became sick and died after being exposed to the horses. hendra virus was ultimately traced back to flying foxes (pteropus spp., of which there are four in australia) as the natural reservoir. 20 niv's genetic relationship to hendra led to the investigation and confirmation of the two endemic pteropid bat species in malaysia as reservoirs for niv. 21 in bangladesh, outbreaks of niv encephalitis in people have been reported on a near annual basis since 2001, with some causing case fatality rates of 100%. 22,23 outbreaks in bangladesh are seasonal and spatially clustered within the western half of the country. 22 the consumption of raw date palm sap has been the primary exposure associated with infection, and the timing of date palm sap harvesting (november-april) aligns with human niv encephalitis outbreaks. 22 niv, like hendra virus, is excreted by pteropus bats in saliva, urine, and feces; in experimental infections it does not cause visible clinical signs or severe pathology despite widespread viral infection of endothelial tissue. 24,25 contamination of date palm sap likely occurs when the indian flying fox (pteropus medius) feeds from the sap flow or from sap collection pots. 26 in bangladesh and india, antibodies against niv as well as viral rna have been detected in p. medius, which is the only pteropid bat on ebola viruses. 51 as with cov and henipaviruses, filoviruses appear to be geographically widespread in bat hosts in both africa and asia. although some ebola viruses and marburg virus have been associated with high mortality rates in people, ebola reston virus illustrates how genetic diversity within a viral group can influence pathogenicity in humans or domestic animals. until there is an a priori method for determining pathogenicity from genetics, filovirus surveillance and ecologic research in bats and other wildlife-including work done at the international centre for medical research, franceville (cirmf) 42,47 ; the us centers for disease control 44, 45, 52 ; and the us agency for international development (usaid) under its emerging pandemic threats: predict program 53 -will help to provide a better understanding of filovirus host ecology and viral genomics and inform strategies to reduce the risk of ebola virus disease and outbreaks of marburg virus disease (see also chapter 19) . covs comprise a large viral family known to infect a wide variety of animals, including humans. prior to the emergence of sars and mers, only four covs were known to infect humans. 54 the sars pandemic of 2002-2003 infected more than 8000 people in 27 countries and had a case fatality rate of ~9%. 55,56 mers-cov (as of june 2017) has infected more than 2000 people in 27 countries and had a 35% case fatality rate. 57 these two epidemics solidified covs as a viral family of concern for human health. sars-cov emerged from bats through the live animal markets of southern china in 2003. 55 the close caging of various mammalian species, including bats, and the general lack of effective biosecurity practices in handling and butchering animals in live animal markets facilitated the infection of multiple species, including civets (paguma larvata), raccoon dogs (nyctereutes procyonoides), and ferret badgers (melogale spp.), all of which were initially suspected as being the primary source of the virus in early investigations. 58 initially, civets were implicated as the source of sars-cov, and markets and farms were depopulated of civets as a control measure. importantly, farmed civets outside the marketplace did not have evidence of sars-cov infection, suggesting an alternate reservoir. 59, 60 the eventual discovery of sarslike covs in bats was an important step in understanding the natural reservoir, although early bat viral isolates did not use the same cell entry mechanism as sars-cov and therefore were not able to cause sars in animal models. in 2012, nearly 10 years after the initial discovery of bat sarslike covs, a cov much more closely related to sars-cov and capable of directly infecting humans was identified among chinese horseshoe bats (rhinolophus sinicus) in yunnan, china. 61 although bats are no longer legally sold in live animal markets in china, there are still communities, ebola virus was first discovered in 1976; since then there have been more than 26 outbreaks of ebola virus disease. 41 over the past 40 years, the natural reservoir for ebola virus has remained a mystery. although some of the outbreaks were epidemiologically linked to contact with wild animals, few had evidence directly linking cases to contact with bats. 8 human infections in central africa have been associated with such contact and with the consumption of infected animals such as gorilla (gorilla gorilla), chimpanzee (pan troglodytes), or duiker (cephalophus spp.) carcasses. 42 in december 2013, an outbreak of ebola zaire virus, of unprecedented magnitude in west africa, began in guéckedou, guinea, following a single introduction from an unknown animal reservoir (hypothesized to be a bat) into the human population. 43 importantly, human social dynamics, rather than repeated introductions from an animal reservoir, were responsible for the rapid and uncontrolled spread of ebola virus disease through guinea, sierra leone, and liberia, underscoring the importance of human-wildlife interaction in spillover and the triggering of epidemics and pandemics. however, over the past decade there has been a growing body of evidence suggesting that multiple bat species carry ebola viruses, whereas marburg virus appears to be primarily carried by the egyptian fruit bat (rousettus aegyptiacus), a common frugivorous bat found throughout the african continent and in the middle east. 44 marburg virus infection occurs seasonally in r. aegyptiacus, with peak infection rates occurring during the birthing season. 45 as with henipaviruses, experimental infections with marburg virus in r. aegyptiacus suggest that there is minimal pathology and no visible signs of disease in these bats when infected and that they may shed virus for up to 19 days postinoculation. 46 ebola virus zaire has been detected in several different bat species in central africa, including the hammer-headed fruit bat (hypsignathus monstrosus), franquet's epauletted fruit bat (epomops franquetti), and the little collared fruit bat (myonycteris torquata). 47 ebola virus has not yet been isolated from bats; however, viral rna and antibodies have been detected in several species. ebola reston virus, a species causing disease in macaques but not humans or pigs, was detected in the common bent-wing bat (miniopterus schreibersi), a common insectivorous bat, in the philippines. 48 antibodies reactive to ebola zaire antigen have been found in leishenault's fruit bat (rousettus leishenaulti) in bangladesh, and although a filovirus has not yet been identified, these findings suggest that an immunogenically related filovirus is circulating in these bats. 49 novel filoviruses, yet to be characterized, have been found in the cave nectar bat (eonycterus spelea) and r. leishenaulti in china. 50 these viruses may be closely related to those causing the immune response detected in the same species in bangladesh. the npc-1 receptor, used by filoviruses for cell entry, is conserved across several bat species, which further supports a broad bat species range for henipaviruses may provide insight into where surveillance should be targeted based on viral diversity hot spots. currently there are geographic regions such as latin america where there is a disproportionately low number of zoonotic viruses that have been characterized in bats, relative to bat species richness, making this a region where surveillance efforts could potentially bring a high yield. 6 ultimately the integration of host ecology with viral discovery will be important for understanding the risk of viral spillover. niv is an important reminder that simply the presence of a host and virus is not sufficient for zoonotic transmission to occur. a viable "interface" or mechanism of transmission is also needed for spillover (provided that people or domestic animals are susceptible to infection). experimental studies will also be vital to clarify the pathogenesis and transmissibility of novel viruses. reverse engineering has made it much easier to "rescue" or recreate viruses in the laboratory from sequence data. genetically modified mice provide an important model for the study of susceptibility to infection and pathogenesis in human physiology. the recent deluge of new viruses found in bats globally warrants a degree of caution against overstating their threat to human or animal health when communicating findings to the public or policy makers. in the majority of instances, there is no evidence that any newly discovered virus in bats has infected any other animal or person, thereby making it simply a bat virus until proven otherwise. when newly identified viruses are related to known zoonoses, they are often presented as potential threats to human or animal health, but there is the potential to cause undue public alarm when reporting these findings. given the potential for negative and scientifically unsupported actions against bats that include extermination, messaging to the public should provide appropriate context where there is a lack of evidence for human or animal health impact and emphasize that bats are ecologically invaluable animals. extermination of bats should not be considered an effective response to an outbreak of a bat-borne pathogen or a control measure to prevent outbreaks. this approach actually enhanced the local transmission of marburg virus among bat populations following the extirpation of bats from a mine in uganda. 72 there will continue to be a large research and surveillance focus on bats as hosts for zoonoses. data are mounting to support bats as important reservoirs compared with other mammals, and large-scale surveillance efforts like predict and the recently launched global virome project, a 10-year effort to identify the majority of viruses in key wildlife species in emerging disease hot spots, 73 will shed more light on the total diversity of viruses in bat species and the types of human-animal interfaces that exist in different geographic and cultural contexts. understanding specific human behaviors that promote contact with bats and developing strategies that limit bat-human-domestic animal contact without harming bats is key to reducing the risk of viral spillover while also preserving bats and the ecologic services they provide. including some in yunnan, that hunt and eat rhinolophus bats, raising the possibility that sars could reemerge. in 2012, another novel cov was discovered in people in saudi arabia. 62 ultimately named mers-cov, its genetic relationship to sars-cov and other beta covs found in bats in hong kong led early investigations to focus on bats as a potential reservoir. a short rna fragment matching mers-cov was found in an egyptian tomb bat (taphozous perforatus) in saudi arabia, although epidemiologic studies have not confirmed this species as a reservoir. 63 mersrelated covs have been found in other bat species in asia and africa 64 ; however, dromedary camels are the most likely animal source of infection for people. 65 juvenile camels shed mers-cov more frequently than adults, and infection is associated with a mild respiratory disease. 65 nosocomial transmission has also been a significant risk factor for human mers-cov infection. 66 in 2015, an outbreak involving 81 people occurred in south korea and was linked to hospitalbased transmission. 67 the discovery of sars-like covs in bats fueled further investigation and the discovery of a large diversity of covs in bats and the hypothesis that all human covs originated in bats. 64, 68 it is estimated that 1200-6000 covs are carried by bats worldwide, some of which will also have the potential to emerge in human or domestic animal populations. 64 porcine epidemic diarrhea virus (pedv) is an alphacoronavirus that in 2013 emerged in the united states and reemerged in asia, causing economically significant disease outbreaks in domestic pigs. 69 although pedv has not been directly linked to bats, it does cluster phylogenetically with other alphacoronaviruses that have been found in bats. 64, 70 cov diversity and richness correlate with bat species richness, but as with all categories of novel viruses, it is not currently possible to determine which viruses have zoonotic potential. 64 hospital-or community-based severe acute respiratory infection (sari) surveillance in regions with high bat biodiversity and high-risk bat-human interfaces (e.g., guano mining 71 ) should consider cov screening as part of a diagnostic approach to investigating respiratory disease clusters in people or diarrheal disease in animals. identification of novel covs as etiologic agents in humans or domestic animals will provide additional insights into genetic determinants of pathogenicity and their relationship with bat covs. within each of the groups of viruses discussed, it is likely that there are still many as yet undiscovered species, strains, and genetic variants comprised by the genetic diversity of nature. in addition, the high mutation rate of rna viruses-and in the case of covs the ability to recombine-means that new genotypes are continuously being created. this presents a serious challenge to cataloging viral diversity, but doing so may ultimately pay off by allowing for the identification of genetic determinants of pathogenesis. also, having a library of sequences from all bat covs, filoviruses, or risk factors for human disease emergence zoonoses 3 prediction and prevention of the next pandemic zoonosis anthropogenic change, biodiversity loss, and a new agenda for emerging diseases origins of major human infectious diseases anthropogenic environmental change and the emergence of infectious diseases in wildlife host and viral traits predict zoonotic spillover from mammals immunology of bats and their viruses: challenges 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resurgence in kitaka mine bat population after extermination attempts the global virome project seasonal pulses of marburg virus circulation in juvenile rousettus aegyptiacus bats coincide with periods of increased risk of human infection modelling filovirus maintenance in nature by experimental transmission of marburg virus between egyptian rousette bats fruit bats as reservoirs of ebola virus molecular evidence of ebola reston virus infection in philippine bats ebola virus antibodies in fruit bats genetically diverse filoviruses in rousettus and eonycteris spp filovirus receptor npc1 contributes to species-specific patterns of ebolavirus susceptibility in bats oral shedding of marburg virus in experimentally infected egyptian fruit bats (rousettus aegyptiacus) human coronaviruses: what do they cause? a novel coronavirus associated with severe acute respiratory syndrome who: summary of probable sars cases with onset of illness from 1 isolation and characterization of viruses related to the sars coronavirus from animals in southern china antibodies to sars coronavirus in civets molecular evolution analysis and geographic investigation of severe acute respiratory syndrome key: cord-254713-ghcwfcx2 authors: razanajatovo, norosoa h; nomenjanahary, lalaina a; wilkinson, david a; razafimanahaka, julie h; goodman, steven m; jenkins, richard k; jones, julia pg; heraud, jean-michel title: detection of new genetic variants of betacoronaviruses in endemic frugivorous bats of madagascar date: 2015-03-12 journal: virol j doi: 10.1186/s12985-015-0271-y sha: doc_id: 254713 cord_uid: ghcwfcx2 background: bats are amongst the natural reservoirs of many coronaviruses (covs) of which some can lead to severe infection in human. african bats are known to harbor a range of pathogens (e.g., ebola and marburg viruses) that can infect humans and cause disease outbreaks. a recent study in south africa isolated a genetic variant closely related to mers-cov from an insectivorous bat. though madagascar is home to 44 bat species (41 insectivorous and 3 frugivorous) of which 34 are endemic, no data exists concerning the circulation of covs in the island’s chiropteran fauna. certain malagasy bats can be frequently found in close contact with humans and frugivorous bats feed in the same trees where people collect and consume fruits and are hunted and consumed as bush meat. the purpose of our study is to detect and identify covs from frugivorous bats in madagascar to evaluate the risk of human infection from infected bats. methods: frugivorous bats belonging to three species were captured in four different regions of madagascar. we analyzed fecal and throat swabs to detect the presence of virus through amplification of the rna-dependent rna polymerase (rdrp) gene, which is highly conserved in all known coronaviruses. phylogenetic analyses were performed from positive specimens. results: from 351 frugivorous bats, we detected 14 coronaviruses from two endemic bats species, of which 13 viruses were identified from pteropus rufus and one from eidolon dupreanum, giving an overall prevalence of 4.5%. phylogenetic analysis revealed that the malagasy strains belong to the genus betacoronavirus but form three distinct clusters, which seem to represent previously undescribed genetic lineages. conclusions: our findings suggest that covs circulate in frugivorous bats of madagascar, demonstrating the needs to evaluate spillover risk to human populations especially for individuals that hunt and consume infected bats. possible dispersal mechanisms as to how coronaviruses arrived on madagascar are discussed. coronaviruses (covs) are enveloped viruses with singlestranded positive-sense rna belonging to the subfamily coronavirinae in the family coronaviridae (order nidovirales). genomes of covs range from 25 to 32 kb and show high genetic diversity [1] . covs are classified into four genera: alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus [2] . in mammals and birds, covs are associated with upper and lower respiratory illnesses or gastroenteritis. in humans, covs infections are commonly caused by hcov-229e and hcov-oc43 which generally cause mild respiratory illnesses [3] . a new cov that causes severe acute respiratory syndrome (sars-cov) emerged in humans in 2002-2003 and infected more than 8,000 individuals with mortality rates estimated at around 10% [4] . the emergence of sars-cov and its mortality rate have raised the risk of a new pandemic that could threaten public health. for this reason, the scientific community invested considerable interest in the identification and characterization of covs especially within mammal reservoirs. subsequently, two novel human covs were discovered: hcov-nl63 in 2004 [5] and hcov-hku1 in 2005 [6] . in june 2012, a third novel coronavirus named hcov-emc/2012 (renamed mers-cov) was isolated from patients presenting with acute respiratory distress and pulmonary inflammation [7, 8] . studies which aimed to identify potential reservoirs of emerging human covs have revealed that the betacoronavirus sars-cov was closely related to covs detected in bats, specifically members of the genus (rhinolophus), which brought the hypothesis of a spillover of this virus to several animal species (including civet cats and raccoons) sold in chinese markets as bushmeat for human consumption [9] [10] [11] . bats have since become a particular focus and a number of alphacoronavirus and betacoronaviruses have been identified in many frugivorous and insectivorous bat species and in many countries worldwide in asia, the americas and europe (see review from drexler et al. 2014) [12] . genomic characterization of the recently discovered mers-cov showed that this virus belongs to the genus betacoronavirus and seems to be closely related to bat coronaviruses hku4 and hku5 isolated from bats (tylonycteris and pipistrellus) [13] . african bats are known to harbor a range of pathogens (e.g., ebola and marburg viruses) that can infect humans and cause disease outbreaks [14] [15] [16] . some authors have reported the detection of bat covs from mainland africa [17] [18] [19] [20] [21] . a recent study in south africa detected a genetic variant closely related to mers-cov from neoromicia zuluensis, an insectivorous bat [22] . the authors hypothesize that mers-cov may have a common ancestors with covs borne by bats from africa. though madagascar is home to 44 bat species (41 insectivorous and 3 frugivorous) of which 34 are endemic [23] [24] [25] , no data exists concerning the circulation of covs in malagasy bats. certain bat species on the island can be frequently found in close contact with humans, particularly members of the family molossidae; these synanthropic species roost in human-occupied buildings, like houses, schools or hospitals [26] , whilst frugivorous bats feed in the same fruit trees where people collect and consume fruits. moreover, hunting and consumption of bat bush meat, especially the larger frugivorous species of the family pteropodidae, is widespread on the island, bringing hunters, purveyors and consumers into contact with bats [27] . in this study, we report the detection of covs amongst frugivorous bats in madagascar. our results demonstrated for the first time that covs belonging to the genus betacoronavirus are circulating amongst two endemic frugivorous bats species in madagascar. a total of 351 bats belonging to 3 endemic bat species of the family pteropodidae were captured and sampled: rousettus madagascariensis (n = 179), pteropus rufus (n = 76) and eidolon dupreanum (n = 96) ( table 1) . none of the throat swabs from any bat species (n = 265) tested positive for cov, but 4.5% (14/313) of fecal specimens tested positive for cov. prevalence within p. rufus, e. dupreanum and r. madagascariensis was respectively 17.1% (13/76), 1.0% (1/96) and 0% (0/141). all positive specimens originated from bats captured in the menabe region ( figure 1 ). short amplicon sequences of 329 bp in length of the rdrp gene were obtained for all pcr-positive animals, whereas larger fragment of 993 bp sequences could only be obtained from seven of the 14 pcr-positive animals. all amplicon sequences were aligned in-frame with a compilation of reference sequences from genbank for which collection-date data was available [28] , giving final alignments containing 51 different sequences of 993 bp in length and 64 different sequences of 329 bp in length. gtr + i + g was identified as the optimal substitution model using jmodeltest v2.1.2 [29] . multiple phylogenies were generated in beast using different combinations of model parameters, and the best models were selected using the tracer [30] . bayes factor analysis employing marginal likelihoods, as detailed in [12] . all parameter combinations produced identical, strongly supported tree topologies (data not shown). as has elsewhere been determined by lau et al. [12] , bayesian skyline using a relaxed, exponentially distributed clock model was found to be the best fitting model for rdrp dated-tip phylogenies. the final phylogenetic analyses ( figure 2 ) revealed that strains from madagascar are members of the betacoronavirus genus, rooting with hong kong strain btcov-hku9 (hm211100) and kenyan strain ky77 (gu065421), with posterior probabilities of 1. these lineages could be described as sars-like, and were uniquely affiliated with frugivorous bat hosts of the family pteripodidae. malagasy strains were sub-divided into three distinct clusters: two of which were closely related (clusters 1 and 2) and originating from p. rufus, and one more distantly related (cluster 3) containing a strain detected from e. dupreanum and sequences previously detected from e. helvum in kenya [21] . the malagasy covs were detected from bats captured in three different sites of the menabe region (west of madagascar). within cluster 1, strains were originated from bemanonga, mahabo and ankiliabo. within cluster 2, strains were originated from bemanonga and ankiliabo. the only virus detected belonging to cluster 3 was detected in one bat captured in mahabo. overall, identities among malagasy covs ranged from 65 to 100% at the nucleotide level and 70.9 to 100% at the amino acid level (data not shown). molecular dating estimates based on the 993 bp fragment of the rdrp gene estimated the timescale of evolution of the coronavirus family to be thousands to tens of thousands of years, however dating estimates proved inaccurate, with broadly spanning hpd values at individual node positions. in the context of this study, we detected 14 coronaviruses forming nine genetically distinct strains in two endemic malagasy frugivorous bat species. the overall prevalence (4.5%) is consistent with those identified in studies elsewhere [31] [32] [33] . thirteen viruses were detected from pteropus rufus and 1 virus from eidolon dupreanum. we did not detect covs among the sampled r. madagascariensis. the detection of novel bat covs supports the observation that these viruses are diverse and have a nearly worldwide distribution [17, 32, 34, 35] . we observed that all malagasy bat covs detected in the present study belong to a sars-related subgroup of the betacoronaviruses, with relatively close homology to btcov-hku9 [36] . our strains displayed 3 distinct clusters: 2 clusters associated with p. rufus and 1 cluster associated with e. dupreanum. it can be inferred from the results that multiple clusters of covs occurring within malagasy bat populations co-circulate and possibly in a syntopic manner. the high nucleotide and amino acid divergence between clusters and compared to the reference virus btcov-hku9 suggests previously undescribed genetic lineages. given the mobility of bats, and the especially long distances that can be travelled by colonies of fruit bats [37] , these coronaviruses may be spread over a large region. however, host-genus-specific phylogenetic clustering (figure 2 , inset) suggests likely host-specificity which may limit viral spill-over. thus, further molecular epidemiology studies would be required to fully understand the dispersal potential of covs amongst malagasy bats species. it is important to remember that, although all three of madagascar's fruit bat species were sampled, nothing is known of cov dynamics in tropical fruit bats, and many factors such as seasonality, bioclimate and the presence of other host species may have important influences on cov prevalence in these populations. more studies are needed in different locations including different species, particularly those with an insectivorous diet, to reveal a more comprehensive view of the diversity of these viruses in madagascar. since the strains of betacoronavirus identified from madagascar are closely related to those known from africa, some preliminary biogeographic considerations are in order. all three bat species analyzed herein are endemic to madagascar. the genus pteropus has a broad distribution from the australia-new guinea region west across the indian ocean to offshore islands of tanzania; it is unknown from the african continent. the genus eidolon is composed of two species: e. helvum occurring on the african mainland and offshore islands and e. dupreanum restricted to madagascar. based on a phylogeographical study, both species show broad panmictic population structure [38] . further, these two taxa are estimated to have diverged from one another sometime in the late miocene or early pliocene [39] . the genus rousettus is broadly distributed across africa and asia and the ancestral origin of the malagasy species, r. madagascariensis, is unresolved [40] . as with the other two pteropodidae species occurring on madagascar, r. madagascariensis shows little genetic population structure and presumably broadly disperses across the island, which in turn has important epidemiological implications for these bats transmitting different zoonoses. although estimates of the most recent common ancestors (mrcas) proved inaccurate in our study, most likely as a result of a limited sequence availability from the identified viral strains, standard evolutionary analyses have estimated cov origins to date to somewhere between 500-10,000 yrs. in the past [41] [42] [43] . nevertheless, further investigations into the relevance of mrca prediction methodologies are required and a great level of caution must be employed in the interpretation of mrca data. . alternatively, viral lineages may have been imported to madagascar in recent history: while the vast majority of the island's bat fauna is endemic, a few species apparently disperse across the mozambique channel. probably the best example is the molossidae species mops midas, for which, based on genetic data, southern african animals are nested within malagasy populations [44] . this bat makes its day roosts in rock crevices and may broadly occur synoptically at such sites with e. dupreanum and r. madagascariensis. these two fruit bat species are known to feed in the same fruiting trees with p. rufus [45] , which would complete the cycle of how covs originated from africa mainland could be carried to madagascar and transmitted to different species of pteropodid bats. although we were not able to evaluate risk of human infection, the strains detected here may be considered as potential human pathogens, as bats are natural reservoirs of some pathogenic covs. isolation of malagasy covs using cell culture and molecular analysis of spike (s) gene could better evaluate risk of human infection. also, a longitudinal study amongst people who frequently handle live bats (e.g. bat hunters, bat bushmeat purveyors, and scientists), and who represent populations at higher risk of infection, would be interesting to establish possible cases of transmission to humans and public health risks. in our study, we confirm that covs are circulating in two species of endemic bats in madagascar. further work on cov diversity amongst the island's bat species, as well as aspects of the ecology and behavior of susceptible taxa, are needed to understand the origin, evolution and dispersal of these viruses across the island. to conclude, the results of our study demonstrate the need to develop research programs that aim at surveying viruses in the wild, especially in bats, in order to address possible emergence of zoonotic viruses within human populations. we sampled frugivorous bats in four different areas of madagascar: anjohibe, anosibe an' ala, menabe and toliara (table 1/ figure 1 ) based on known accessible colonies of roosting bats and sites where bats are frequently hunted and eaten by people. in the region of menabe, we selected 5 different sites situated at a mean distance of 28 km around mahabo to capture and collect specimens, while in the three other regions, sampling occurred at a single site. sampling was carried out under protocols approved and permitted by ministry of environment and forest (authorization # 301/08, 101/09, 163/10, 032/ 11 and 261/11). fruit bats were captured by the use of mist-nets set near roosting sites (trees or caves) and with help of professional hunters [46] . rectal and throat swabs were collected from each individual bat. all bats were identified according species specific morphological features well known by our field trained team (ecologist and veterinarian) and subsequently released. swabs were placed in viral transport media, almost immediately conserved in liquid nitrogen in the field and stored at −80°c upon arrival at the laboratory in antananarivo. rna was extracted from specimens using the qiaamp viral rna minikit (qiagen, courtaboeuf, france) according to the manufacturer's protocol. briefly, total rna was extracted from 140 μl of each sample and eluted in 65 μl of qiagen ave elution buffer. the extracted rna was either immediately analyzed or stored at −80°c until use. extracted rna was reverse transcribed to generate cdna by using the m-mlv reverse transriptase (invitrogen, california, usa) into a 2 step reactions. first, 2 μl of rna were mixed in a solution containing 0.5 μm of random hexamer primers (roche diagnostics, mannheim, germany), 1 u/μl of rnase inhibitor (10 000 units) and 8.5 μl of water, at 80°c for 5 min, 50°c for 5 min and 4°c for 15 min. then, 8 μl of rna issued from the first step was added to a mixture of 0.5 mm of each dntp (deoxynucleotide triphosphates), 10 u/μl of reverse transcriptase m-mlv, 1 x of buffer and 0.01 m of dtt (dithiothreitol), and incubated at 42°c for 50 min and 95°c for 5 min. pcr assay was performed to amplify the rna-dependent rna polymerase (rdrp) gene which is highly conserved in all known coronaviruses [47] . the primers pair (forward 5′-ggttgggactatcctaagtgtga-3′; reverse 5′-c catcatcagatagaatcatcata-3′) was designed to amplify a 440 bp fragment as described previously [47] . reaction mixture was carried out using the gotaq/dntp mix, custom kit (promega corporation, madison, usa). briefly, 5 μl of cdna was mixed with 1 x of green gotaq flexi buffer, 1.5 mm of mgcl 2 , 0.2 mm of each dntp, 0.2 μm of each primer, 0.025 u/μl of gotaq dna polymerase and 28.75 μl of water nuclease-free giving a final volume of 50 μl. the thermocycling was performed under the following conditions: activation at 95°c for 10 min and 40 cycles of denaturation at 95°c for 1 min, annealing at 58°c for 1 min, extension at 72°c for 1 min, and a final extension at 72°c for 5 min. all negative samples were tested in a semi-nested pcr with the same pcr program and using the following pair of primers (forward 5′-ggttg ggactatcctaagtgtga-3′; reverse 5′-atcagata gaatcatcatagaga-3′). amplicons products were subsequently electrophorezed on a 2.0% agarose gel and visualized using ethidium bromide under uv light. all specimens that showed a positive band at the expected size (440 bp) were sequenced on both strands by beckman coulter genomics (essex, uk). from the sequences obtained from the 440 bp, fragment, we designed new primers (reverse) that were strain specific for malagasy batcov (5′-gatgacc tgtatattccca-3′ and 5′-atgacctatacatacc catg-3). we then amplified a large fragment of the rdrp gene by using the consensus forward primer 5′-gtgtacgctgctgatcctgctatgca-3′ [48] . the following conditions were performed: 95°c for 10 min and 40 cycles of denaturation at 95°c for 1 min, annealing 56°c for 1 min, extension at 72°c for 1 min, and a final extension at 72°c for 5 min. the final size of sequences used for molecular dating was 1086 bp. sequences from the 440 bp or 1086 bp fragments of the rdrp gene were cleaned and aligned with reference sequences collected from a literature search. alignment was performed using the translation alignment tool in geneious pro™ v.6.3.2, created by biomatters (available from http:// www.geneious.com/), and the default clustalw cost matrix. the final alignment was respectively 329 bp and 993 bp in length for fragments 440 bp and 1086 bp, and contained no free-end or internal gaps. from these alignments, the appropriate substitution model was identified in jmodeltest v.2.1.2 [29, 49] . using the appropriate substitution model, 2*10^8 iterations were run with or without the use of a 3base codon model, using different clock models, alternating between constant and bayesian skyline population size models, and seeding with uncorrelated log-normally distributed priors. trees were sampled every 2x10^5 iterations, and analysis convergence was assessed in tracer v.1.4.0 [50] (available from http://beast.bio.ed.ac.uk/tracer). all analyses converged after a 10% burn-in to give effective sample size values for all parameters superior to 200. bayes factor analyses were performed in tracer v1.4.0, with 1000 bootstrap replicates to assess the relative performance of model selections on the generated phylogenies. after 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immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution this study was conducted in collaboration with the association madagasikara voakajy and the bangor university (darwin initiative project), a project examining aspects of emerging viruses in small wild mammals. the work in the toliara region was part of action concertée inter-pasteurienne (acip) research program. we would like to thank felicien herbert randrianandrianina of madagasikara voakajy and local hunters for their help in capturing bats. david a. wilkinson's post-doctoral fellowship was funded by "run-emerge", a european project funded by european commission under fp7 program. the authors declare that they have no competing interest.authors' contributions nhr and daw carried out the molecular genetic studies, participated in the sequence alignment and drafted the manuscript. lan coordinated the fieldwork, participated to the sampling collection, participated to molecular testing and drafted the manuscript. jhr participated in the design of the study and coordinated the fieldwork smg and rkj helped to draft the manuscript. jpgj participated in the design of the study and helped to draft the manuscript jmh: conceived, designed and coordinated the study and helped to draft the manuscript. all authors read and approved the final manuscript key: cord-003166-k3jxvzfi authors: noh, ji yeong; jeong, dae gwin; yoon, sun-woo; kim, ji hyung; choi, yong gun; kang, shien-young; kim, hye kwon title: isolation and characterization of novel bat paramyxovirus b16-40 potentially belonging to the proposed genus shaanvirus date: 2018-08-22 journal: sci rep doi: 10.1038/s41598-018-30319-7 sha: doc_id: 3166 cord_uid: k3jxvzfi the bat paramyxovirus b16-40 was first isolated in korea in this study. using the isolated virus, we could obtain not only genomic information, but also several biological characteristics of the virus. in the phylogenetic analysis, the virus was found to belong to the recently proposed genus shaanvirus. through sequence analyses and in vitro testing, the isolated virus was also found to have haemagglutinin-neuraminidase (hn) protein as one of the structural proteins. when mouse antiserum was generated against the isolated virus and tested, it was cross-reactive to human parainfluenza virus 1 in an indirect immunofluorescence assay but could not cross-neutralize human parainfluenza virus 1. in addition, the bat paramyxovirus b16-40 was not infectious in the mouse model. collectively, this study provided basic information on further classification of the bat paramyxovirus b16-40 and related viruses in the proposed genus shaanvirus. bats are considered a reservoir of severe emerging infectious diseases. severe acute respiratory syndrome coronavirus (sars-cov), middle east respiratory syndrome coronavirus (mers-cov), nipah virus, hendra virus, and ebola virus are all thought to be bat-borne viruses 1, 2 . notably, bats also host major mammalian paramyxoviruses from the family paramyxoviridae, order monone-gavirales 3, 4 . while henipaviruses (nipah and hendra viruses) in south east asia and australia are associated with fruit bats 5 , other paramyxoviruses have been detected not only in fruit bats but in insectivorous bats worldwide [6] [7] [8] [9] . a potential pathway for nipah virus transmission from bats to humans was found to be associated with a human-bat interface, specifically date palm sap shared by bats and humans 10 . in addition, serological evidence of possible human infection with a bat-originated paramyxovirus, tioman virus 11 , reinforces the epidemiological role of bats in the emergence of pathogens such as paramyxoviruses in humans. in addition to these bat paramyxoviruses with zoonotic potential, other new paramyxoviruses have been reported. these include several new mammalian paramyxoviruses such as beilong virus and j virus, which remain unassigned under the family paramyxoviridae 12 . recent bat-associated paramyxoviruses were proposed to be grouped in a separate phylogenetic clade within a potentially separate genus such as shaanvirus 13 which was distantly related to jeilongvirus 14 . in addition, novel strains of bat paramyxoviruses in diverse genera have been reported continuously [15] [16] [17] . based on the recent papers, bat paramyxoviruses found worldwide to date have belonged to the genera rubulavirus, morbillivirus, henipavirus and the unclassified proposed genera shaanvirus. expanded classifications for grouping newly identified viruses in bats can be accomplished by further studying the biological characteristics of novel paramyxoviruses as well as genome characterization 18 . in this study, active surveillance was performed to reveal paramyxoviruses circulating in korean bats. a total of 232 bat samples were collected at 48 sites in natural bat habitats and tested for the possible existence of paramyxoviruses. identification of novel bat paramyxovirus b16-40. in this study, 232 bat samples were collected at 48 sites from natural bat habitats and tested for the possible existence of paramyxoviruses (supplementary table s1 ). based on the rt-semi-nested pcr using the consensus paramyxovirus primers targeting the rdrp region 19 , five samples were positive and confirmed to be bat paramyxoviruses by sequencing. b16-6 was from a urine sample of miniopterus schreibersii at bt cave in hapcheon in march 2016, and b16-40 was from a feces sample of miniopterus schreibersii at g cave in danyang in april 2016. b16-148 and b16-154 were from feces samples of murina ussuriensis at oj cave in youngwol and at s cave in pyungchang in july 2016, respectively. b16-230 was from a urine sample of miniopterus schreibersii at blr cave in seogwipo in december 2016. among these, one paramyxovirus (b16-40) was successfully isolated. the isolated virus, bat paramyxovirus b16-40, was isolated from the korean bat miniopterus schreibersii. the virus was successfully cultivated after three blind passages in the marc-145 cell line (fig. 1) , which is a clone of the ma-104 cell line derived from kidney epithelial cells of an african green monkey 20 . viral particles were observed in a pellet of infected marc-145 cells under a transmission electron microscope (fig. 1b) . in terms of viral growth, infectious viruses were increased in the culture supernatant and infected cells from 24 hours post inoculation (hpi) and were consistently released for 144 hpi, as measured using tissue culture infectious dose 50 (tcid 50 ) method (fig. 1c ). infectivity in c57bl/6 mice. when 6-week-old female c57bl/6 mice were inoculated via the intranasal and intragastric routes with 10 5 tissue culture infectious dose (tcid) 50 /ml or 10 2.5 tcid 50 /ml of bat paramyxovirus b16-40, we found no evidence of infection, i.e. no viral shedding, histopathological findings, or seroconversion. characterization of the bat paramyxovirus b16-40. using high throughput sequencing (hiseq. 2000 sequencing system based on the transcriptome de novo sequencing platform), 23,336 contigs totaling 8,010,635 base pairs (bps) with an average length of 343 bps were obtained. in the taxonomy annotation with mg-rast, 12,522 contigs were annotated. while most of the annotated sequences associated with eukaryotes, 13 sequences were associated with family paramyxoviridae. the 17,771 bases of viral genomic sequences were obtained by rt-pcr using primers designed based on the paramyxoviridae-associated contigs and race sequencing. the genomic sequence of the virus encodes 8 open reading frames (orfs) ( fig. 2a) , which is consistent with bat paramyxovirus strain ms-parav/anhui2011 (kc154054) 13 . in addition, maximum-likelihood trees showed that the bat paramyxovirus b16-40 strain was closely related to the bat paramyxovirus ms-parav/anhui2011 (kc154054) and btmi-parav/qh2013 (kj641657) strains rather than beilong virus, j-virus, and tailam virus (figs 2b and s1). even though the hn amino acids sequences were similar to those from viruses in the proposed genera shaanvirus, it was also related to that of sendai virus and human parainfluenza virus 1, which belong to a different genus, respirovirus showing (table 1) . based on the partial rna-dependent rna polymerase (rdrp) gene, the bat paramyxovirus isolates and other korean bat paramyxoviruses belong to the genus shaanvirus, with 68.8-79.0% nucleotide similarities (fig. 2c ). this suggests a single, closely related group of paramyxoviruses circulating in korea. consistent with the genetic findings, the bat paramyxovirus b16-40 was found to have haemagglutinin and neuraminidase activities in the haemadsorption assay (0.5% chicken red blood cells in pbs (ph 7.4)) and neuraminidase assay, respectively (fig. 3 ). in the preliminary studies of mouse antisera (balb/ ca mice) against human respiratory syncytial virus a (kbpv-vr-41), human mumps virus (kbpv-vr-51) and human parainfluenza virus 1 (kbpv-vr-44), the antisera against human parainfluenza virus 1 (kbpv-vr-44) was only cross-reactive with the bat paramyxovirus b16-40 as determined using the immunofluorescence assay. therefore, further cross-reactivity and cross-neutralizing tests between human parainfluenza virus 1 (kbpv-vr-44) and the bat paramyxovirus b16-40 were performed. as shown in table 2 , partial cross-reactivities were observed between the two viruses in the indirect immunofluorescence assay, even though they belong to different genera. among the six mouse sera against the bat paramyxovirus b16-40, two sera were cross-reactive to human parainfluenza virus 1 (kbpv-vr-44) with 10 and 40 as the end-point titers for the fluorescent signals. in addition, among three pooled sera (two mouse sera each) against human parainfluenza virus 1 (kbpv-vr-44), one pooled serum was cross-reactive to the bat paramyxovirus b16-40 with 40 as the end-point titer for the fluorescent signal ( table 2 , fig. 4 ). while the mouse antisera neutralized the homologous virus with 20-160 serum neutralizing titer, they did not neutralize the heterologous virus (table 2 ). the bat paramyxovirus b16-40 in this study was similar to viruses in the recently proposed genera shaanvirus and jeilongvirus. the genus jeilongvirus includes beilong virus, which was first isolated from human kidney mesangial cells 21 , and j virus, first isolated from mice 22 . these two viruses were only recently proposed to form jeilongvirus as a distinct genus 14 . in addition, the newly discovered bat paramyxoviruses, ms-parav/ anhui2011 and btml-parav/qh2013, were recently proposed to form the additional genus shaanvirus due to their genomic length and sequence identity differences 13 . however, their taxonomy has not yet been confirmed by the international committee on taxonomy of viruses (ictv, https://talk.ictvonline.org). the proposed genera shaanvirus and jeilongvirus have two novel genes putatively encoding sh (small hydrophobic) and tm (transmembrane) proteins 12, 13 . two viral genes were also located between the fusion and haemagglutinin-neuraminidase genes of bat paramyxovirus b16-40. further, their nucleotide and amino acids reference viruses belonging to paramyxoviridae were included and aligned by mafft. the phylogenetic tree was generated by the maximum-likelihood method with 1,000 replicates of bootstrap sampling and the kimura 2-parameter model using mega 6. bat paramyxovirus b16-40 isolated in this study is indicated with the blue box. (c) phylogenetic analysis based on partial rna-dependent rna polymerase (rdrp) nucleotide sequences of bat paramyxoviruses detected in the korean bats and other reference viruses belonging to paramyxoviridae. the phylogenetic tree was generated by the maximum-likelihood method with 1,000 replicates of bootstrap sampling and the kimura 2-parameter model using mega 6. korean bat paramyxoviruses are indicated by blue boxes. sequences were similar to the sh and tm genes identified from bat paramyxovirus strain ms-parav/anhui 2011. therefore, the isolated bat paramyxovirus b16-40 might belong to the proposed genus shaanvirus rather than jeilongvirus. the bat paramyxovirus b16-40 in this study was predicted to have a haemagglutinin-neuraminidase (hn) protein as an attachment glycoprotein in the sequence analysis. consistent with the genetic findings, the bat paramyxovirus b16-40 was found to have haemagglutinin and neuraminidase activities in the haemadsorption assay (0.5% chicken red blood cells in pbs (ph 7.4)) and neuraminidase assay, respectively ( fig. 2a,b) . notably, even though the hn amino acids sequences were similar to those from viruses in the proposed genera shaanvirus, it was also related to that of sendai virus and human parainfluenza virus 1, which belong to a different genus, respirovirus (table 1) . maximum-likelihood tree analysis based on the full hn amino acid sequences also showed that the bat paramyxovirus b16-40 was related to viruses in the genus respirovirus ( supplementary fig. s1 ). therefore, the b16-40 hn amino acid sequence was compared to that of human parainfluenza virus 1 (kbpv-vr-44), and six regions that have at least six conserved amino acids were found. these regions were located on the sialidase superfamily domain and were structurally gathered together ( supplementary fig. s2 ). among the seven neuraminidase active sites (r, d, e, r, r, y, and e 23 ), two of these (e at the third site and y) were located within the conserved amino acid sequences. these bioinformatics findings might indicate that the hn proteins of bat paramyxovirus b16-40 and human parainfluenza virus 1 (kbpv-vr-44) are genetically related, even though they are in different genera. further study on their antigenic and evolutionary relationship should be followed up. notably, in a previous study, bat serum (diluted 1/40) from african wild bat, micropteropus pusillus was reactive with human parainfluenza virus 1 in an indirect immunofluorescence assay 3 . there may be two possibilities to explain this. first, the african bat might have been infected with a human parainfluenza virus 1-related virus in the genus respirovirus that has not yet been found. however, as there has been no evidence of bat paramyxoviruses related to the genus respirovirus in recent studies 13, 15, 16 , the first possibility seems low. second, the bat might have been infected by other bat paramyxoviruses which were cross-reactive with human parainfluenza virus 1. in fact, in this study, when mouse antisera were made and tested against bat paramyxovirus b16-40 and human parainfluenza virus 1 (kbpv-vr-44), the two viruses were partially cross-reactive to each other in an indirect immunofluorescence assay. this result provided an evidence that there could be cross-reactivity between viruses belonging to different genera. therefore, the bat paramyxovirus b16-40-related viruses circulating in bats might be one of the reasons why the wild bat sera was reactive with human parainfluenza virus 1 in the previous study. however, we could not find the cross-neutralization between bat paramyxovirus b16-40 and human parainfluenza virus 1. based on the recent studies, cross-neutralization was found between the viruses in the same genus of paramyxoviruses: sendai virus and human parainfluenza virus 1, which both belong to the genus respirovirus 24 , and human mumps virus and african bat mumps virus, which both belong to the genus rubulavirus 25 . further, the engaged proteins in the cross-reactivity between bat paramyxovirus b16-40 and human parainfluenza virus 1 had not been revealed. therefore, although bat paramyxovirus b16-40 in this study was cross-reactive with human parainfluenza virus 1 in indirect immunofluorescence assay, it is difficult to say that the bat paramyxovirus b16-40 was antigenically related with human parainfluenza virus 1. since a number of paramyxoviruses have been newly found around world, their classification has been difficult due to the limited criteria, such as sequence alone 18 . additional input from the biological context may be helpful. in this study, a bat paramyxovirus b16-40 belonging to the proposed genus shaanvirus was first isolated. through virus isolation, we could obtain not only nucleotide sequence information, but also several biological characteristics of the virus. its putative hn protein was demonstrated to have haemagglutinin and neuraminidase activities. mouse antisera against the bat paramyxovirus b16-40 was also cross-reactive to human parainfluenza virus 1 in the indirect immunofluorescence assay, but could not cross-neutralize human parainfluenza virus 1. additionally, the bat paramyxovirus b16-40 was not infectious in 6-week-old female c57bl/6 mice. therefore, this study provided basic information on further classification of the bat paramyxovirus b16-40 and related viruses in the proposed genus shaanvirus. samples. in 2016, a total of 232 samples were collected at 48 sites in natural bat habitats. most samples were collected as fresh guano under the colony of bats. in some cases, bats were captured by a fine mesh net to collect fresh feces as well as oropharyngeal and urine swabs, and the animals were then immediately released. the collected samples were placed into transport medium (noble bio. co. ltd) in a 10% suspension and were transported to the lab (bsl2) for further analysis. the major bat species at the collection sites were determined based on morphology and on previous data from bats' roosting sites 26 table s1 ). rna extracted from bat samples was tested by rt-semi-nested pcr using consensus paramyxovirus primers targeting the rdrp region 19 . the nucleotide sequences of the positive samples were determined by cosmogenetech co. ltd. five of the 232 bat samples were confirmed positive to the bat paramyxoviruses by sequencing. virus isolation was performed from the rt-semi-nested pcr-positive samples including feces, urine, and oropharyngeal swabs from bats. each bat sample was diluted 10-fold with fresh dulbecco modified eagle medium (dmem) and filtered with a 0.2-î¼m filter before being inoculated into the marc 145 cell line, a clone from the ma-104 cell line from kidney epithelial cells of an african green monkey 20 ; blind passaging was performed up to three times. the cytopathic effect (cpe) was confirmed in cells infected with one sample (b16-40) of five pcr-positive samples. next, a monolayer of marc 145 was infected with bat paramyxovirus (b16-40) at a multiplicity of infection (moi) of 0.1. two days post-infection, the infected cells were harvested and pelleted. the infected cell pellet was fixed with 2.5% glutaraldehyde in pbs (ph 7.4) for 2 hours and sent for transmission electron microscopy (tem) to the advanced analysis center in the korean institute of science and technology in korea. briefly, after washing with pbs the cells were post-fixed in 2% osmium tetroxide and dehydrated in an ethanol series (70%, 80%, 85%, 90%, 95%, and 100% (two times)). then, the sample was transited with 100% propylene oxide and embedded in epoxy resin. the embedded sample was sectioned into 70-80 nm sections with an ultramicrotome (ultra cut c; leica), stained with 2% uranyl acetate and lead citrate on a copper grid, and examined using tem mode on a cryotecnai f20 (fei ltd., usa). culture supernatant and freeze-thawed cell lysate were centrifuged at 3,000 ã� g for 10 minutes to remove cell pellets and then used for viral titration. the infectious viruses were quantified from the prepared samples using the tissue culture infectious dose 50(tcid 50 ) method. briefly, the prepared samples were 10-fold serially diluted and inoculated to monolayers of marc-145 cells in 4 wells. the highest dilution showing 50% of viral infection was obtained by observing cytopathic effects with a light inverted microscope (ix71s1f-3, olympus corporation). the tcid 50 was calculated using the spearman-karber method. genomic sequencing and analyses. a virus isolate showing cytopathic effects (cpes) was filtered through a 0.2 î¼m filter and ultra-centrifuged at 250,000 ã� g for 1 hour. each pellet was suspended in 500 âµl of 1x digestion buffer (turbo dna free kit; ambion) and treated with turbo dnase; the suspension was incubated at 37 â°c for 30 min. the rna from the suspension was isolated using the trizol ls reagent according to the manufacturer's instructions (ambion). the extracted rna was submitted to macrogen (seoul, korea) for high throughput sequencing in a hiseq. 2000 sequencing system based on the transcriptome de novo sequencing platform. the obtained viral contigs were analysed and annotated by the mg-rast server 27 . the cut-off for the annotation was 10 â��5 maximum e-value, 60% minimum percentage identity, and 15 for minimum alignment length. the viral genomic sequences were obtained by rt-pcr using primers based on the paramyxoviridae-associated contigs obtained by high throughput sequencing. additionally, 3â�² and 5â�² end sequences were obtained by race sequencing based on pcr using the adapter-oligo dtvn primer from bionics co. ltd. for human parainfluenza virus 1 (kbpv-vr-44), the 1,992 bases of the hn gene were obtained by rt-nested pcr following the previously published method 28 . the obtained genomic rna sequences and rt-pcr-positive amplicons were further analysed with related sequences in genbank using mafft 29 and mega version 6 30 . genomic sequence data generated in this study have been deposited in genbank under accession number mg230624. the full hn sequence of human parainfluenza virus 1 (kbpv-vr-44) has also been deposited in genbank under accession number mg255129. the hn amino acid sequences of human parainfluenza virus 1 (kbpv-vr-44) and bat paramyxovirus were further compared with the bioedit program 31 . putative hn protein structures were predicted using the phyre2 website 32 and visualized with the ucsf chimera 1.11.2 program 33 . haemadsorption and neuraminidase assays. to investigate the activity of the hn protein, the haemadsorption assay was used. monolayer cultures of marc 145 cells in 96-well plates were inoculated with the bat paramyxovirus b16-40 at an moi of 0.1. after 48 hours, the cells were gently washed with pbs and incubated for 1 hour with 0.5% chicken red blood cells (rbcs) in pbs (ph 7.4) at room temperature. then, the cells were washed with pbs twice to remove unbound rbcs and observed under a microscope. in addition, the neuraminidase assay was performed with the viral isolate using the na-fluor influenza neuraminidase assay kit (thermo) according to the manufacturer's protocol. the virus was diluted 2-fold with the na-fluor 2ã� assay buffer in a black 96-well plate and incubated for 1 hour at 37 â°c with the na-fluor substrate. after adding the stop solution, the plate was read at the excitation and emission wavelengths of 355 nm and 460 nm, respectively, using a perkinelmer victor2 fluorometer. a total of 44 6-week-old female c57bl/6 mice maintained under specific-pathogen-free (spf) conditions were divided into two groups of negative controls (n = 4 each) and four inoculation groups (n = 9 each). the inoculation groups were inoculated with 400 âµl of 10 5 tcid 50 or 10 2.5 tcid 50 of the bat paramyxovirus b16-40 via intra-gastric administration routes, and 30 âµl of 10 5 tcid 50 or 10 2.5 tcid 50 of the bat paramyxovirus b16-40 via the intranasal administration routes. each inoculation was conducted twice with 1 week in between. weight loss was monitored daily for up to 2 weeks. the lungs, liver, brain, and intestine were harvested at 7 or 14 days post-challenge, fixed with 4% paraformaldehyde in pbs, ph = 7.2, and submitted to biolead inc., korea, for histopathological examination. additionally, rna was extracted from each tissue and from oral swabs and fecal samples collected daily for 7 days post-challenge. the extracted rna was tested using rt-semi-nested pcr with the consensus paramyxovirus primers 19 . in addition, real time pcr was performed with newly designed primers and probes targeting regions of the membrane and fusion protein. briefly, the designed primers were as follows: forward primer: pvm-f5â�²-cccaggagtatggttatcaagtgagg-3â�²; reverse primer: pvm-r 5â�²-tcca ttgggctctctttgtttgc-3â�²; taqman probe: pvm-p 5â�²-fam-cccatcccagaccagccacca gaccc-tamra-3â�² real time rt-pcr was performed as follows: reverse transcription at 45 â°c for 10 min, followed by pcr â��95 â°c for 5 min, cycling 40 times at 95 â°c for 10 sec, and 60 â°c for 30 sec. using the 10-fold diluted virus (bat paramyxovirus b16-40), standard curves were performed for every reaction. generation of mouse antisera against bat paramyxovirus b16-40 and human paramyxoviruses. we next observed preliminary cross-reactivity in indirect immunofluorescence assay between several human viruses belonging to the family paramyxoviridae and the bat paramyxovirus b16-40. 6-week-old female balb/ca mice maintained under spf conditions were intramuscularly inoculated twice, with 2 weeks in between, with a 1:1 ratio mixture of addavax adjuvant and live virus stock (bat paramyxovirus b16-40, human respiratory syncytial virus a (kbpv-vr-41), human mumps virus (kbpv-vr-51), and human parainfluenza virus 1 (kbpv-vr-44), respectively, from the korea bank for pathogenic viruses. two weeks after the last immunization, antisera against the virus were obtained by cardiac puncture for further studies related to cross-reactivity in indirect immunofluorescence assay. mouse antisera against the bat paramyxovirus b16-40 (10 5.75 tcid 50 /ml) and human parainfluenza virus 1 (kbpv-vr-44, 10 5.75 tcid 50 /ml) were further compared through serum titration using the indirect immunofluorescence assay and serum neutralization test. the washed cells were fixed with 100% ethanol for 1 hour at â��20 â°c and rehydrated with pbs (ph 7.4) for 10 min at room temperature. primary antibodies used in the experiments were the mouse antisera against bat paramyxovirus b16-40 and human parainfluenza virus 1 (kbpv-vr-44). the sera were diluted with 50% culture supernatants from the cell lines in fresh dmem; the corresponding mock-infected cells were treated in the same way as the target virus culture. then, the sera were serially diluted 2-fold from an initial dilution of 1:10 and added to the fixed infected or mock-infected cells and incubated for 1 hour at room temperature. the plates were washed three times with pbs containing 0 with the same serum diluent and was added to the plates and incubated for 1 hour at room temperature. after three washes with pbs-t, the plates were viewed with a fluorescence microscope they were then diluted 2-fold from an initial dilution of 1:10 in 96-well plates and mixed with 200 tcid 50 of each virus. the plates were then incubated at 37 â°c for 1 hour. the virus-serum mixtures for the bat paramyxovirus b16-40 and human parainfluenza virus 1 (kbpv-vr-44) were added to monolayer cultures of marc 145 and llc-mk2 cells, respectively, and incubated at 37 â°c for 1 hour. the marc 145 cells and llc-mk2 cells were then washed once with pbs bats as reservoirs of severe emerging infectious diseases detection of severe acute respiratory syndrome-like, middle east respiratory syndrome-like bat coronaviruses and group h rotavirus in faeces of korean bats bats host major mammalian paramyxoviruses taxonomy of the order mononegavirales: update 2017 hendra and nipah infection: emerging paramyxoviruses isolation of multiple novel paramyxoviruses from pteropid bat urine novel paramyxoviruses in free-ranging european bats novel paramyxoviruses in bats from sub-saharan africa identification of novel paramyxoviruses in insectivorous bats of the southwest indian ocean nipah virus transmission from bats to humans associated with drinking traditional liquor made from date palm sap serological evidence of possible human infection with tioman virus, a newly described paramyxovirus of bat origin the complete genome sequence of j virus reveals a unique genome structure in the family paramyxoviridae deciphering the bat virome catalog to better understand the ecological diversity of bat viruses and the bat origin of emerging infectious diseases the immune evasion function of j and beilong virus v proteins is distinct from that of other paramyxoviruses, consistent with their inclusion in the proposed genus jeilongvirus coronavirus and paramyxovirus in bats from northwest italy circulation of alphacoronavirus, betacoronavirus and paramyxovirus in hipposideros bat species in zimbabwe. infection novel alphacoronaviruses and paramyxoviruses cocirculate with type 1 and severe acute respiratory system (sars)-related betacoronaviruses in synanthropic bats of luxembourg problems of classification in the family paramyxoviridae sensitive and broadly reactive reverse transcription-pcr assays to detect novel paramyxoviruses enhanced replication of porcine reproductive and respiratory syndrome (prrs) virus in a homogeneous subpopulation of ma-104 cell line beilong virus, a novel paramyxovirus with the largest genome of non-segmented negative-stranded rna viruses a new mouse paramyxovirus (j virus) structural studies of the parainfluenza virus 5 hemagglutinin-neuraminidase tetramer in complex with its receptor, sialyllactose safety and immunogenicity of an intranasal sendai virus-based human parainfluenza virus type 1 vaccine in 3-to 6-year-old children cross-neutralization between human and african bat mumps viruses sounds of the bats in korea the metagenomics rast server-a public resource for the automatic phylogenetic and functional analysis of metagenomes a study of genetic variability of human parainfluenza virus type 1 in croatia mafft multiple sequence alignment software version 7: improvements in performance and usability mega6: molecular evolutionary genetics analysis version 6.0. molecular biology and evolution in nucleic acids symposium series the phyre2 web portal for protein modeling, prediction and analysis ucsf chimera-a visualization system for exploratory research and analysis data availability. the datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-30319-7.competing interests: the authors declare no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. key: cord-272250-asuxx1ln authors: robertson, kis; lumlertdacha, boonlert; franka, richard; petersen, brett; bhengsri, saithip; henchaichon, sununta; peruski, leonard f.; baggett, henry c.; maloney, susan a.; rupprecht, charles e. title: rabies-related knowledge and practices among persons at risk of bat exposures in thailand date: 2011-06-28 journal: plos negl trop dis doi: 10.1371/journal.pntd.0001054 sha: doc_id: 272250 cord_uid: asuxx1ln background: rabies is a fatal encephalitis caused by lyssaviruses. evidence of lyssavirus circulation has recently emerged in southeast asian bats. a cross-sectional study was conducted in thailand to assess rabies-related knowledge and practices among persons regularly exposed to bats and bat habitats. the objectives were to identify deficiencies in rabies awareness, describe the occurrence of bat exposures, and explore factors associated with transdermal bat exposures. methods: a survey was administered to a convenience sample of adult guano miners, bat hunters, game wardens, and residents/personnel at buddhist temples where mass bat roosting occurs. the questionnaire elicited information on demographics, experience with bat exposures, and rabies knowledge. participants were also asked to describe actions they would take in response to a bat bite as well as actions for a bite from a potentially rabid animal. bivariate analysis was used to compare responses between groups and multivariable logistic regression was used to explore factors independently associated with being bitten or scratched by a bat. findings: of 106 people interviewed, 11 (10%) identified bats as a potential source of rabies. a history of a bat bite or scratch was reported by 29 (27%), and 38 (36%) stated either that they would do nothing or that they did not know what they would do in response to a bat bite. guano miners were less likely than other groups to indicate animal bites as a mechanism of rabies transmission (68% vs. 90%, p = 0.03) and were less likely to say they would respond appropriately to a bat bite or scratch (61% vs. 27%, p = 0.003). guano mining, bat hunting, and being in a bat cave or roost area more than 5 times a year were associated with history of a bat bite or scratch. conclusions: these findings indicate the need for educational outreach to raise awareness of bat rabies, promote exposure prevention, and ensure appropriate health-seeking behaviors for bat-inflicted wounds, particularly among at-risk groups in thailand. rabies is an exceptionally fatal encephalitis caused by rhabdoviruses in the lyssavirus genus. transmission typically occurs when broken skin is contaminated with saliva from an infected mammal-usually in association with a bite but in rare instances by scratches. the most well-known and ubiquitous lyssavirus is the rabies virus (rabv), which circulates in new world bats and both old and new world terrestrial mammals. the vast majority of human rabies cases worldwide are transmitted by dogs infected with rabv. a lesser known member of the genus is the mokola virus, which has been isolated from a number of terrestrial mammals in africa (most notably shrews) and has caused at least two human cases [1] , [2] . reservoirs for the remaining nine members of the lyssavirus genus appear to be exclusively old world bats [3] . rabies is a major public health problem in asia. of the estimated 55,000 human cases that occur annually worldwide, more than half occur in asian countries [4] . in recent decades, initiatives aimed at raising rabies awareness (e.g. the world rabies day campaign) and lowering human exposure risk through mass vaccination of leading reservoir species have been implemented globally, coinciding with the development of highly potent human rabies vaccines [4] , [5] , [6] . notable trends have subsequently followed. of all asian countries, thailand has experienced the steadiest decline in human rabies cases, with a near 10-fold decrease in reported cases during the last 20 years [7] . much of this decline is attributable to the country's very extensive use of rabies vaccine in the treatment of persons bitten by dogs. in 2003, for instance, more than 400,000 people in thailand were vaccinated against rabies following potential rabies exposures [8] . historically in southeast (se) asia, animal-based prevention efforts for rabies have almost exclusively been centered on dogs. most reported human cases in the region are traced to these animals either through an exposure history or through molecular or antigenic subtyping of variants from rabid human patients [9] , [10] . the canine-associated rabies variant has been the only one linked to terrestrial wildlife and domestic animals in thailand [8] , further evidence that dogs are the main lyssavirus reservoir in the region. to date, no human cases of rabies linked to lyssaviruses other than canine-associated rabv have been reported in thailand or the rest of se asia. like other dog-rabies endemic countries, the majority of human rabies victims in thailand are children under the age of 15 years [11] . within the last ten years, however, interest in bats and their role in lyssavirus transmission has increased in the region. the discovery of the australian bat lyssavirus (ablv) in australian flying fox bats (pteropus spp.) in the mid-1990's and the isolation of new bat lyssaviruses in the former soviet union [12] , [13] were pivotal in turning scientific interest towards the study of potential asian bat reservoirs. in the last 10 years, evidence of lyssavirus maintenance in se asian chiropterans has emerged from surveillance in cambodia, thailand, bangladesh, and the philippines [14] , [15] , [16] , [17] . although lyssaviruses have not yet been isolated from these mammals, neutralizing antibodies associated with lyssaviruses have been detected in sera from both regional mega and microbats. these findings strongly suggest that asian bats maintain lyssaviruses like their counterparts in europe, africa, australia, and the americas. human deaths due to bat-borne rabies infection have been well documented in these continents [12] , [18] , [19] , [20] . in particular, rabid vampire bats are a major cause of human mortality in south america's amazon region [21] . routine surveillance for bat rabies is lacking in asia and as a consequence, understanding is limited regarding the extent of lyssavirus circulation among se asian bats and the impact on animal and public health. however, evidence thus far raises pressing questions about human health risks. the potential implications of bat rabies are particularly salient in se asia because human-bat interaction occurs routinely in many locales. bat guano is regularly mined from caves for use as a fertilizer. hunting of bats for sale and personal consumption occurs as well, despite laws to stop this practice. the presence of large numbers of bats at many buddhist temples also promotes exposures, as these sites are focal points for commerce, tourism, and religious expression. because the severity of skin trauma inflicted by bats is usually minor and unlikely to prompt a medical visit on the basis of physical injury alone, public knowledge of appropriate healthseeking behaviors following a bat exposure is especially important in preventing cases of bat-borne rabies. rabies postexposure prophylaxis (pep) for bat bites and scratches, as recommended by both the world health organization (who) and the u.s. advisory committee on immunization practices (acip), includes thorough wound washing and the administration of rabies immune globulin (in non-immunized individuals) and rabies vaccine administered in a series of doses [4] , [22] . when administered promptly and properly, rabies pep is highly effective in preventing the disease. to date, however, no intervention has proven effectiveness in stopping the clinical course after symptom onset, a fact which further underscores the importance of early care following a possible lyssavirus exposure. little is known about the extent of bat-specific rabies awareness in se asia. to assess the knowledge and practices of individuals who are most at-risk of bat exposures in thailand, we surveyed persons who regularly come in contact with bats or bat dwellings through occupational activities and other practices. we sought to elucidate gaps in knowledge that potentially have bearing on rabies prevention, describe the occurrence of bat-associated exposures in this population, and explore factors associated with bat exposures that are of potential consequence to lyssavirus transmission. the desired sample size was 200 individuals. surveyed individuals were a convenience sample of adults who collect guano from caves, engage in bat hunting, work or reside at temples that serve as sites for mass bat roosting, or work as game wardens responsible for monitoring and protecting bat caves. engagement in at least one of these activities within the last 5 years and being 18 years or older were inclusion criteria for participation. individuals were recruited from eight provinces as shown by figure 1 . recruitment areas were selected based on proximity to bat caves and/or mass bat roosting sites and certain community characteristics known to promote batassociated activities (e.g., an agrarian-economy that benefits from guano fertilizer use). participants were classified based on the activity in which they most frequently engaged, and were primarily recruited from rural and semi-rural localities. in farming communities near bat caves, village leaders and other local contacts provided assistance in locating individuals known to engage in guano mining. such individuals were also found through referral from existing participants. a similar method was employed to locate bat hunters/trappers in communities where fruit farms are known to attract flying fox bats. to recruit temple workers/residents and game wardens, permission from supervisory officials at buddhist temples and national parks was obtained before individuals under their management were approached for participation. recruits were not offered or given incentives for participating. the study design and consent process was approved by the institutional review board (irb) at cdc (protocol# 5709). all participants were verbally informed of the study's purpose and assured that their responses would be kept anonymous, even if they engaged in illegal activities. oral consent was obtained to ensure rabies is a fatal encephalitis caused by lyssaviruses. evidence of lyssavirus circulation has recently emerged in southeast asian bats. we surveyed persons regularly exposed to bats and bat habitats in thailand to assess rabies-related knowledge and practices. targeted groups included guano miners, bat hunters, game wardens, and residents/personnel at buddhist temples where mass bat roosting occurs. of the 106 people interviewed, 11 (10%) identified bats as a source of rabies. history of a bat bite/ scratch was reported by 29 (27%), and 38 (36%) expressed either that they would do nothing or that they did not know what they would do in response to a bat bite. guano miners were less likely than other groups to indicate animal bites as a mechanism of transmission (68% vs. 90%, p = 0.03) and were less likely to say they would respond appropriately to a bat bite or scratch (61% vs. 27%, p = 0.003). these findings indicate a need for educational outreach in thailand to raise awareness of bat rabies, promote exposure prevention, and ensure health-seeking behaviors for bat-inflicted wounds, particularly among atrisk groups. anonymity and accommodate illiterate participants, and was documented by the interviewer electronically via personal digital assistants (pda) prior to administering the survey. this method of obtaining informed consent was approved by cdc's irb. a 41-item structured questionnaire was developed in english and translated and reviewed by native thai speakers employed by the office of the u.s. cdc, international emerging infections program in bangkok. the questionnaire was designed to be administered in thai via face-to-face interviews, with responses entered in pdas using geoage fast software. not all questions provided data used in this study. the questionnaire was developed based on socio-ecological reasoning about gaps in rabies knowledge that potentially translate into failed prevention on the individual level. data were collected on demographics; primary bat-associated activity and years of experience; history of rabies vaccination; and type and frequency of bat exposures such as cave entry, direct contact with bats, bites and scratches from bats, and bat consumption. individuals who reported receiving rabies vaccination were asked to indicate whether it was in direct response to an animal exposure (i.e. pep) or for pre-exposure immunization (prep), which is a vaccination series most often administered to people who have a relatively high likelihood of rabies virus exposure due to occupational risks or other factors. those who reported receiving prep were asked to describe its administration and only those who indicated receiving a series of injections spaced over multiple days were counted as having prep. to assess rabies-related knowledge, participants were asked to rate their understanding of the disease as either ''little or none'', ''basic'', or ''extensive''; explain how humans acquire the disease, and identify animal sources of the disease. each knowledge question was evaluated independently, and the validity of a participant's self-reported knowledge level was not verified using other responses. participants were also asked to describe the severity of rabies. only responses that emphasized death or profound suffering with no suggestion that recovery was likely were considered evidence that the participant recognized rabies as being severe. awareness of other diseases that humans can get from bats was also elicited. to assess health-seeking practices following transdermal bat exposures, participants were asked about actions they would take if they were bitten or scratched by a bat. responses to this openended question were compared to a similar question later asked about actions a person should take following a bite from a potentially rabid animal, based on the participant's own understanding of what constitutes a potentially rabid animal. questions that were specifically asked about bats preceded all questions asked about rabies to minimize reporting bias, and whenever feasible, participants were asked open-ended questions to minimize the interviewer's influence on responses. participants were also interviewed away from other people. interviewers were instructed to not ask questions in a leading manner and to allow as much time as necessary for participants to answer. survey responses were transferred to a computer, exported into microsoft excel, and then imported into sas version 9.2 for analysis. data were summarized using descriptive statistics and comparisons by bat-associated activity group were made using chi-square or fisher's exact test. multivariable logistic regression analysis was used to explore factors independently associated with being bitten or scratched by a bat. variables related to the bats & rabies kp www.plosntds.org outcome at p-values#0. 25 were included in the model. crude and adjusted odds ratios (or) with 95% confidence intervals (ci) were calculated. associations were statistically significant at p-values less than 0.05. the study was conducted during august 3-18, 2009. a total of 106 people were interviewed. interviews lasted an average of about 10 minutes. demographic characteristics, history of rabies vaccination, and primary bat-associated activity of the participants are described in table 1 . all temple workers/residents and game wardens were involved in their activity at the time of interview; 71% of guano miners and 53% of bat hunters reported that their most recent engagement had occurred within the previous 12 months. of all groups, guano miners had fewer years of schooling, with 89% educated at the primary level or less versus 55% of nonguano miners (p = 0.001). temple workers/residents were more likely to have greater than 15 years of experience in their activity than other groups (59% vs. 26%, p = 0.001). thirty-one percent of participants reported a history of receiving either rabies prep (7.5%) or pep (23.5%) within their lifetimes. of those who had received rabies pep, 96% reported that they had been vaccinated in response to a dog exposure and 4% for a cat exposure. no participants reported receiving pep for a bat exposure and none reported receiving both prep and pep. there were no statistically significant differences between activity groups with respect to rabies vaccination. table 2 describes participant's responses to rabies-related knowledge questions by activity group. a majority of participants (54%) reported having little or no knowledge of rabies. proportionately more temple workers/residents reported basic or extensive knowledge than non-temple workers/residents (p = 0.03). self-assessed rabies knowledge appeared to be lowest among guano miners, but not to a statistically significant degree (p = 0.06). although most (85%) participants seemed to be aware that animal bites cause rabies, significant differences were observed between activity groups. only 68% of guano miners indicated animal bites as a mechanism of transmission compared to 90% of non-guano miners (p = 0.03). when asked to identify which animals are sources of rabies, only 11 (10%) participants named bats. in contrast, dogs were named by 80 (76%), cats were named by 41 (39%), and other mammals (including rodents and large domestic animals) were named by 24 (23%). fourteen participants (13%) were unable to name any animals as rabies sources. differences between activity groups with respect to bat attribution were not statistically significant, and individuals who attributed rabies to dogs were no more likely to also attribute rabies in bats than those who did not attribute rabies to dogs (11% vs 8%, p = 1.0) (not shown in table 2 ). when asked whether they were aware of any other diseases (besides rabies) that humans can get from bats, 18 (17%) answered yes; all were temple workers/residents (not shown in table 2 ). table 3 shows how participants responded when asked ''what actions would you take if you were bitten or scratched by a bat?'' and ''if someone has been bitten by an animal potentially infected with rabies what should that person do?'' twenty-eight (26%) participants expressed that they would seek medical care or rabies pep for a bat bite or scratch, while a significantly higher proportion (95%) advocated these actions if the bite came from a potentially rabid animal (p = 0.0001). the proportion of participants who either said they would do nothing or that they didn't know what they would do if bitten or scratched by a bat was significantly higher than the proportion answering similarly when asked about an exposure to a potentially rabid animal (36% vs. 2%, p = 0.0001). guano miners were more likely than non-guano miners to give this response for bat exposures (61% vs. 27%, p = 0.003). an incidental finding (not shown in the table) was that previous recipients of prep or pep were more likely than nonrecipients to advocate a health-seeking behavior for bat bites and scratches but not to a stasticially significant extent (82% vs 56%; p = 0.15). table 4 describes bat-related exposures by the number and proportion of participants who reported experiencing it at least once in their lifetimes, along with those who reported experiencing it more than five times a year. a history of transdermal bat exposure (bite or scratch) was reported by 29 (27%) participants. table 5 shows factors independently associated with a bat bite or scratch history. variables considered for inclusion in the multivariable model on the basis of biological plausibility included age, sex, years of experience, education, knowledge self-assessment, frequency in bat caves/roost areas, and bat-associated activity. all except the latter three variables were removed from the final model due to unadjusted p-values.0.25. no two variables were so strongly associated with one another or the outcome as to suggest the presence of colinearity. in the final model, self-assessed rabies knowledge of ''little or none'' was not significantly predictive of a bat bite or scratch history, although a strong association was observed prior to adjustment for other variables. individuals who engaged in guano mining, bat hunting, or visiting a bat cave or roost area more than 5 times a year were more likely to report a history of bat bites or scratches. in this survey among persons at risk for bat exposure in thailand, we found that although general awareness of rabies transmission and severity were relatively high, awareness of bat rabies in particular was low, with only 10% of participants identifying bats as a potential source of rabies and 36% failing to say they would take any specific action if bitten or scratched by a bat. bat exposures conducive to potential lyssavirus transmission were also common in this population and were reported by members of all four activity groups, supporting more than just a theoretical risk for these types of incidents. we found that guano miners reported the highest frequency of transdermal bat exposures, were the least knowledgeable about rabies, and were the least likely to say they would respond to bat exposures in a manner that would ensure rabies prevention. based on these findings we conclude that of the groups we surveyed, bat rabies has the greatest potential impact on guano miners. the potential risk associated with guano mining is even more stark given that moribund bats (i.e., those most likely to be rabid) normally fall to the floor of caves, where they can readily come in contact with someone collecting bat droppings by hand. the effectiveness of any bat-borne rabies prevention strategy may hinge upon how well it diffuses into communities where guano mining regularly occurs. education at the community level is an important strategy in the prevention of human rabies [4] . although the decreasing incidence of human rabies in thailand points to the effectiveness of past and present rabies education efforts, our findings demonstrate a need to raise public awareness of the potential risk of rabies associated with bat exposures. special attention should be placed on communities where bats or bat guano are commonly utilized, and if school-based, programs should include primary level students to ensure that they reach those who do not progress past this level of schooling. in addition to emphasizing the importance of exposure avoidance and countering attitudes that inappropriately lower risk perception towards bats, wound washing and healthcare utilization following bat bites and scratches are practices that should be promoted. similarly, if the awareness we observed in the public is indicative of awareness in the medical community, outreach to healthcare professionals might also be needed to ensure that patients presenting with bat exposures are treated in accordance with who guidelines [4] . studies aimed at assessing knowledge and practices in the thai medical community should be explored to ensure that such outreach is well-informed. education at temples and national parks is also recommended to ensure personnel at these sites know to avoid unnecessary bat contact and respond appropriately to bat-inflicted injury. a a strategy that integrates community outreach with law enforcement should be considered as well. comparing the proportion of participants who advocated a given action for a bat exposure with the proportion who advocated the same action for an exposure to a potential rabid animal. b includes cleaning the wound with water, soap, and/or a common antiseptic agent (e.g. betadine). c includes prayer or visiting a traditional healer. d includes bleeding the wound, bandaging, or using topical home remedies (e.g. local herbs). doi:pntd.0001054.t003 table 4 . bat-associated exposure histories reported by participants. to date, there have been no reported cases of human rabies cases associated with bats in thailand. one plausible explanation is that the prevalence of bat lyssaviruses in se asia is so low that humans are rarely if ever exposed to these pathogens. it is also possible that the prevailing assumption about dogs as the usual source of rabies leads patients and their family members to overlook relevant bat encounters when recounting animal exposures, resulting in misdiagnosis or misattribution. additionally, the rate of human rabies vaccination in the population may be high enough to protect many people against bat rabies. in our study, we found that almost a third of all participants reported a history of rabies vaccination, mostly as a result of dog-associated pep. this suggests that the percentage of individuals in our study population with at least some lyssavirus immunity is relatively high and may help account for why bats have yet to be linked to any human rabies cases in the country. however, immunity levels could change if pep use becomes more conservative in the future. currently, funds annually spent on the purchase of human rabies biologics by the thai government are quite substantial [8] , and this financial burden may be difficult to sustain indefinitely. there are some limitations to our study that should be noted. first, it is unlikely that our relatively small convenience sample is representative of all persons engaged in bat-related activities in thailand. our findings may have also been subject to reporting bias, since guano miners and bat hunters may have been less willing than others to answer questions truthfully due to the illegal nature of their work. this potential bias may have led participants to understate their years of experience, which could explain why this variable was not found to be associated with a history of transdermal bat exposures. estimated participation rates for these two groups were also much lower than the other two groups (participation rates were hard to definitively ascertain because participation was ultimately premised on self-identification). additionally, we classified individuals based on their self-reported primary batassociated activity; however, a few participants indicated involvement with other activities (e.g,. guano miners that also hunt bats) either presently or in the past. having such a history was not accounted for this study, although it potentially could be associated with an increased lifetime risk of transdermal bat exposures. the desired sample size of 200 persons was somewhat arbitrarily determined given the lack of reliable estimates for the study population size. failure to meet this number was largely due to the difficulty in finding willing participants who engaged in bat hunting and guano mining, and the limited availability of personnel and funds that could be used to extend the study period. as a consequence of our small sample size and low statistical power, truly significant associations may have gone undetected in this study. however, by recruiting from several provinces, we minimized the influence that geography might have imparted on the associations we observed. another limitation is that the validity and reliability of the questionnaire may have been suboptimal because the survey instrument was not subject to very rigorous in-field testing. our findings have relevance to zoonotic diseases other than rabies. se asian bats have been linked to the encephalitis-causing nipah virus and hendra virus [23] , [24] , [25] , and the corona virus associated with severe acute respiratory syndrome (sars) [26] , [27] . less novel diseases associated with bats also include histoplasmosis, an invasive fungal respiratory disease linked to bat guano exposure [28] . additionally, evidence suggests that bat ectoparasites may transmit pathogens such as bartonella and rickettsia [29] , [30] . in this study, we found that exposures that could potentially facilitate transmission of these diseases appear to occur relatively frequently, with 36% of surveyed participants reporting that they experience direct contact with bats at least twice a year. bat consumption-an activity that in and of itself may be low risk (assuming the bat is well cooked) but could be associated with increased disease risk through contact with bat carcasses-was reported by more than half the participants. exposure to toxic or infectious aerosols is another potential hazard for this population as well, since almost all participants reported regularly being in bat caves and roosting areas. more epidemiological studies are needed to better assess the risks associated with bat-related exposures, particularly in regions of the world where outbreaks of severe zoonoses have occurred and questions remain regarding animal reservoirs for such diseases. parnkaew rattanasilpkancharn from the thailand ministry of health; dr. sumalee boonmar, benchawan pongurgsorn, pongpun sawatwong, and manoon hirunsalee from cdc-thailand ieip; and dr. amy turmelle, felix jackson, and dustyn palmer, of the poxvirus and rabies branch, cdc. disclaimer statement: the findings and conclusions in this report are those of the author(s) and do not necessarily represent the official position of the centers for disease control and prevention. isolation of a rabies related virus from the cerebrospinal fluid of a child with aseptic meningitis a fatal human infection with mokola virus shimoni bat virus, a new representative of the lyssavirus genus world rabies day: focusing attention on a neglected disease oral vaccination of wildlife against rabies: opportunities and challenges in prevention and control the rabies situation in far east asia transmission dynamics of rabies virus in thailand: implications for disease control complex genetic structure of the rabies virus in bangkok and its surrounding provinces, thailand: implications for canine rabies control rabies situation in cambodia moving towards the elimination of rabies in thailand australian bat lyssavirus: a recently discovered new rhabdovirus rabies surveillance in the former soviet union serologic evidence of lyssavirus infection in bats survey for bat lyssaviruses lyssavirus surveillance in bats serologic evidence of lyssavirus infections among bats, the philippines rabies surveillance in the united states during epidemiology and pathogenicity of african bat lyssaviruses first encounter of european bat lyssavirus type 2 (eblv-2) in a bat in finland bat-transmitted human rabies outbreaks, brazilian amazon human rabies prevention-2008: recommendations of the advisory committee for immunization practices nipah virus infection in bats (order chiroptera) in peninsular malaysia duplex nested rt-pcr for detection of nipah virus rna from urine specimens of bats bat nipah virus review of bats and sars identification of a novel coronavirus in bats fungal infections among returning travelers association of baronella with the fleas (siphonaptera) of rodents and bats using molecular techniques dectection of rickettsia, borrelia, and bartonella in carios kelleyi (acari: argasidae) the authors thank dr. robert gibbons and the us department of defense's armed forces research institute of medical sciences (afrims); dr. rattiya naksuwan from the thailand ministry of agriculture; key: cord-102977-yci9kq6x authors: liu, haiming; luo, jiaohua; guillory, bobby; chen, ji-an; zang, pu; yoeli, jordan k.; hernandez, yamileth; lee, ian (in-gi); anderson, barbara; storie, mackenzie; tewnion, alison; garcia, jose m. title: ghsr-1a is not required for ghrelin’s anti-inflammatory and fat-sparing effects in cancer cachexia date: 2019-12-06 journal: biorxiv doi: 10.1101/866376 sha: doc_id: 102977 cord_uid: yci9kq6x adipose tissue (at) atrophy is a hallmark of cancer cachexia contributing to increased morbidity/mortality. ghrelin has been proposed as a treatment for cancer cachexia partly by preventing at atrophy. however, the mechanisms mediating ghrelin’s effects are incompletely understood, including the extent to which its only known receptor, ghsr-1a, is required for these effects. this study characterizes the pathways involved in at atrophy in the lewis lung carcinoma (llc)-induced cachexia model and those mediating the effects of ghrelin in ghsr+/+ and ghsr−/− mice. we show that llc causes at atrophy by inducing anorexia, and increasing at inflammation, thermogenesis and energy expenditure. these changes were greater in ghsr−/−. ghrelin administration prevented llc-induced anorexia only in ghsr+/+, but prevented wat inflammation and atrophy in both genotypes, although its effects were greater in ghsr+/+. llc-induced increases in bat inflammation, wat and bat thermogenesis, and energy expenditure were not affected by ghrelin. in conclusion, ghrelin ameliorates wat inflammation, fat atrophy and anorexia in llc-induced cachexia. ghsr-1a is required for ghrelin’s orexigenic effect but not for its anti-inflammatory or fat-sparing effects. every year, over 1,500,000 individuals in the us are diagnosed with cancer. cachexia (involuntary 63 loss of muscle and adipose tissue) is present in up to 80% of cancer patients, is strongly associated 64 with higher morbidity and mortality, and is reported as the direct cause of death in 20-40% of these 65 patients (dewys, begg et al., 1980 , fearon, strasser et al., 2011 . adipose tissue, once considered 66 only a high-energy fuel reserve, has emerged recently as an active metabolic organ modulating 67 inflammation, energy expenditure and food intake in non-cancer settings (you & nicklas, 2006) . 68 accelerated loss of adipose tissue plays an important role in cancer cachexia contributing 69 significantly to the increased morbidity and mortality seen in this setting (fouladiun, korner et al., 70 2005) . 71 72 increased inflammation is common in the setting of cancer (garcia, garcia-touza et al., 2005) and 73 is associated with adipose tissue wasting in human studies (lerner, hayes et al., 2015) . white 74 adipose tissue (wat) is a significant source of inflammatory cytokines accounting for more than 30% 75 of circulating interleukin (il)-6 (michaud, boulet et al., 2014) and this and other inflammatory 76 cytokines have been linked to wat atrophy in the setting of cancer (petruzzelli, schweiger et al., 77 2014, tsoli & robertson, 2013 , tsoli, swarbrick et al., 2016 . also, a phenotypic switch from wat to 78 brown adipose tissue (bat) known as "browning" is thought to contribute to the overall increase in 79 energy expenditure and wat atrophy seen in cancer cachexia (petruzzelli et al., 2014) . 80 nevertheless, the mechanisms regulating adipose tissue atrophy and dysfunction in this setting are 81 incompletely understood. 82 9 ee levels in response to llc tumor implantation when compared to ghsr +/+ . animals 174 co-administered ghrelin were not statistically different from vehicle-treated, tumor-bearing animals. 175 tumor implantation also decreased spontaneous locomotor activity in both genotypes and ghrelin 176 administration did not prevent these changes . the respiratory quotient (rq), was 177 significantly decreased by tumor implantation and was not affected by genotype or ghrelin 178 administration (fig 5 g-i) . adipose tissue atrophy is a central component of the cancer anorexia and cachexia syndrome 182 (cacs) leading to increased morbidity and mortality (das, eder et al., 2011) . recently, emerging 183 roles for inflammation, wat browning and increased bat thermogenesis have been demonstrated 184 in this setting (daas, rizeq et al., 2018 , dalal, 2019 , han, meng et al., 2018 , kir, white et al., 2014 kliewer, ke et al., 2015 , petruzzelli et al., 2014 , rohm, schafer et al., 2016 2019, wang, zhu et al., 2019) ; however, the pathways involved and their potential as therapeutic 187 targets are not well-known. ghrelin and agonists of its only known receptor, show 188 potential to ameliorate cacs at least in part by preventing fat atrophy, but the specific mechanisms 189 mediating these effects have not been fully characterized. given that there are no treatments for cancer cachexia and that several clinical trials targeting this pathway have failed to 191 meet their primary endpoints (garcia et al., 2015 , temel, abernethy et al., 2016 , there is a pressing 192 need to improve our understanding of the mechanisms of action of ghrelin in this setting. in this 193 study we show that ghrelin prevents llc tumor-induced weight loss, fat atrophy and wat 194 inflammation without affecting tumor-induced bat inflammation, wat browning, and increased bat 195 uncoupling and whole-body energy expenditure. we confirmed that its orexigenic effects are 196 ghsr-1a-dependent, and also show that other novel ghsr-1a-independent mechanisms are 197 involved given the partial improvements in fat atrophy and wat inflammation seen in ghrelin-treated, 198 ghsr -/animals. also, this is the first report of macrophages as the source of wat and bat in the setting of cacs. weight loss and survival rates are correlated with il-6 levels in cancer patients (garcia et al., 2005, 202 moses, maingay et al., 2009 , scott, mcmillan et al., 1996 . these observations and several 203 mechanistic studies support the premise that inflammation plays a central role in cacs. increases 204 11 in il-1β and tnf contribute to anorexia (baracos, martin et al., 2018 , braun, zhu et al., 2011 , khatib, 205 gaidhane et al., 2018 , and tnf and il-6 promote lipolysis and inhibit lipogenesis in wat leading to 206 weight loss (fearon, glass et al., 2012 , han et al., 2018 , jeanson, carriere et al., 2015 2014, ruan, hacohen et al., 2002) . in non-cancer settings, one third of the circulating il-6 is 208 produced by wat (mohamed-ali, goodrick et al., 1997) and most of this wat-derived il-6 comes 209 from the stroma-vascular fraction composed of endothelial cells, monocytes/macrophages, 210 myocytes, and fibroblasts (fain, madan et al., 2004) , although it can also be derived from 211 adipocytes (fain, 2006) . macrophages in wat are known to be the source of proinflammatory 212 cytokines in conditions leading to at hypertrophy including obesity (di gregorio, yao-borengasser 213 et al., 2005 , divoux, tordjman et al., 2010 , lumeng, deyoung et al., 2007 ) but this has not been 214 previously shown in cacs. here we show that llc tumor implantation induces an increase in 215 inflammatory cytokines in circulation as well as in bat and wat. moreover, these at cytokines 216 appear to be derived exclusively from macrophages residing in these tissues. adipose tissue 217 atrophy in cancer patients with cacs has been associated with an increase in subcutaneous at 218 macrophages (batista, henriques et al., 2016 , de matos-neto, lima et al., 2015 , henriques, sertie 219 et al., 2017 and tissue inflammation (batista, olivan et al., 2013 , de matos-neto et al., 2015 henriques et al., 2017). although, macrophage infiltration has also been described in wat from 221 tumor-bearing rodents (henriques et al., 2017 , machado, costa rosa et al., 2004 2014), to our knowledge this is the first report of macrophages as the source of pro-inflammatory 223 cytokines in adipose tissue in cacs. these findings may explain why at remains an important 224 source of pro-inflammatory cytokines even when the adipocyte mass is significantly reduced in this 225 setting. also, this may be clinically relevant to cancer patients since knowing the source of 226 inflammation may allow us to target these pathways more effectively (henriques, lopes et al., 227 2018). previously, we have shown that activation of ghsr-1a by ghrelin or ghsr-1a agonists (ghs) 230 increases food intake and body weight (13, 39, 40) . our group and others also have shown that 231 ghrelin reduces fat oxidation and lipolysis and increases lipogenesis and adiposity in a rodent model 232 of cisplatin-induced cachexia by a combination of food intake-dependent and independent 233 mechanisms (chen et al., 2015 , garcia et al., 2013b , porporato, filigheddu et al., 2013 . ghrelin is 234 thought to have anti-inflammatory effects in other settings (deboer, zhu et al., 2008 , dixit, schaffer 235 et al., 2004 , tsubouchi, yanagi et al., 2014 but this is not yet clear in cacs. some reports suggest 236 an anti-inflammatory effect of native ghrelin administration, but this was not confirmed in other 237 studies using ghsr-1a agonists (chen et al., 2015 , garcia, friend et al., 2013a . in the current 238 study, we report that ghrelin modulates inflammation in a tissue-specific manner. ghrelin did not 239 prevent tumor-induced increases in circulating inflammatory cytokines or in bat il-1β or mcp-1 240 protein levels. however, it mitigated llc-induced inflammation in wat. this effect was seen in both 241 genotypes although it was clearer in wild type animals partly because ghsr -/mice appear to be 242 resistant to tumor-induced inflammation. ghsr-1a is not expressed in adipocytes (sun, garcia et 243 al., 2007) but is present in macrophages (ma, lin et al., 2013) and our findings are consistent with a 244 previous report showing that old, non-tumor-bearing ghsr -/mice have reduced macrophage 245 infiltration, a shift on macrophage differentiation towards a more anti-inflammatory phenotype, and 246 decreased inflammation in adipose tissue (lin, lee et al., 2016) . however, a ghsr-1a-independent 247 effect of ghrelin on macrophages is also possible as it has been proposed in other settings (avallone, 248 demers et al., 2006 , bulgarelli, tamiazzo et al., 2009 , lucchi, costa et al., 2017 . taken together, 249 our data is consistent with a wat-specific, anti-inflammatory effect of ghrelin that is partly ghsr-1a 250 dependent. this is clinically relevant as ghsr-1a agonists are in clinical development for cacs 251 and their effect on these ghsr-1a independent pathways is not known (garcia et al., 2015) . also, 252 13 the differences we report between serum, wat and bat levels underscore the limitations of relying 253 exclusively on circulating cytokine levels when trying to determine the potential role of inflammation 254 in other tissues. 255 256 energy expenditure is an important mechanism in the regulation of body weight and is increased in 257 cacs (garcia et al., 2013a , kir, komaba et al., 2016 , rohm et al., 2019 . factors contributing to ee 258 include physical activity and resting ee (ree) (silver, dietrich et al., 2007, vazeille, jouinot et al., 259 2017) and adipose tissue can lead to an increase in ree by uncoupling oxidative phosphorylation in 260 mitochondria thereby releasing heat through activation of a proton leak (nicholls, 1976, 261 okamatsu-ogura, kitao et al., 2007) . in wat, browning has been noted in multiple cancer cachexia 262 models with adipocytes showing an upregulation of the main regulator of thermogenesis, ucp1 263 (dong, lin et al., 2018 , vaitkus & celi, 2017 . in bat, increased thermogenesis has been reported in 264 cachectic animals (kir et al., 2014) independently of decreased food intake or their ability to 265 maintain their body temperature (tsoli, moore et al., 2012) . proinflammatory cytokines have been 266 suggested as key drivers of wat browning (han et al., 2018 , petruzzelli et al., 2014 and of bat 267 thermogenesis through activation of sympathetic nervous system or targeting bat directly (arruda, 268 milanski et al., 2010 , dascombe, rothwell et al., 1989 , li, klein et al., 2002 , tsoli et al., 2012 . here 269 we show that llc-tumor implantation led to an increase in total ee in spite of a significant decrease 270 in physical activity, suggesting an increase in ree. this was associated with an increase in ucp-1 271 expression in wat (browning) and in bat. moreover, these effects were more marked in ghsr -/mice 272 suggesting a protective role of ghsr-1a in this setting. these results agree with previous reports in 273 aged, non-tumor-bearing ghsr -/showing higher levels of thermogenesis and energy expenditure 274 when compared to aged-matched, wild-type mice (lin, saha et al., 2011) . the effect of ghrelin or 275 ghsr1a agonists on energy expenditure is unclear with some studies showing a decrease in ee 276 14 (borner, loi et al., 2016 , villars, pietra et al., 2017 while others showed no effect (adachi, takiguchi 277 et al., 2010 , tschop, smiley et al., 2000 , vestergaard, djurhuus et al., 2008 . in this study, we did 278 not see a significant effect of ghrelin on preventing llc-induced fat browning, bat thermogenesis, 279 increased ree or decreased physical activity in the setting of cacs despite the fact that ghrelin 280 prevented fat and weight loss and anorexia. we hypothesize that differences in the models, route of 281 administration and treatment regimen and agents used (llc mice vs. c26 mice or hepatoma model 282 in rats, administration via s.q. vs. oral gavage vs. osmotic mini pump, ghrelin vs. ghsr1a agonists) 283 could account for these discrepancies. more studies will be needed to test this hypothesis. calderon-dominguez, mir et al., 2016) . in cacs the aforementioned tumor-induced inflammation is 289 thought to play an important role in bat thermogenesis (petruzzelli et al., 2014 , tsoli et al., 2012 ; 290 however, the source of inflammation in bat is not known. similar to wat, we found that bat il-6 291 and tnf come exclusively from macrophages in the setting of cachexia. however, their expression 292 in bat were lower than in wat and no significant changes were found in response to tumor 293 implantation or ghrelin. we found a significant tumor-effect on increasing il-1β levels in bat 294 although ghrelin did not prevent this increase, suggesting tissue-specific differences in inflammation 295 between bat and wat in response to tumor and ghrelin. taken together, these results are 296 important because they show that tumor-induced wat browning and bat thermogenesis are 297 associated with significant increases in ree and appear to be independent of inflammation given 298 that downregulating inflammation does not prevent uncoupling in wat and that bat il6 and tnf 299 levels were not upregulated upon tumor implantation. in addition, our data suggests that wat is a 300 15 significant source of inflammatory cytokines, which express the highest levels of il-1β, il-6, and 301 tnf when compared to bat and circulating levels. 302 303 there were limitations to our approach. this study was not set up to establish the safety of ghrelin 304 administration in the setting of cancer. nevertheless, none of the studies published to date using 305 ghrelin or ghsr-1a agonists in mice or humans have shown an increase in tumor progression 306 (sever, white et al., 2016) . also, the experiments were not designed to characterize other 307 mechanisms contributing to the protective role of ghsr-1a in this setting. lastly, our data suggest 308 that there is an alternative receptor for ghrelin although identification of this receptor remains elusive 309 and is the focus of other studies. 310 in summary, ghrelin prevents llc tumor-induced body weight and fat loss by a combination of 312 ghsr-1a-dependent mechanisms including preventing anorexia, and other mechanisms that are 313 partly ghsr-1a-independent. the increase in inflammation in at induced by tumor implantation is 314 prevented by ghrelin only in wat; however, tumor-induced wat browning, and increased bat 315 inflammation, uncoupling and whole body energy expenditure are not prevented by ghrelin even 316 when the presence of ghsr-1a appears to contribute to maintaining energy balance in this setting. 317 tumor-induced wat browning and bat thermogenesis are associated with significant increases in 318 ree and these seem to be independent of inflammation given that downregulating it does not 319 prevent these changes. these results are clinically relevant because they show that ghrelin five to seven-month-old male c57bl/6j growth hormone (gh) secretagogue receptor wild type 329 (ghsr +/+ ) and knockout (ghsr -/-) congenic mice were used for all experiments. briefly the ghsr +/+ and 330 ghsr -/mice were originally from dr. roy g. smith ph.d's laboratory (sun, butte et al., 2008) the procedures of tumor implantation (ti) and ghrelin intervention were described previously (chen 342 et al., 2015) . in brief, mice were injected subcutaneously (s.q.) with lewis lung carcinoma (llc) 343 cells (1 × 10 6 cells, crl1642, american type culture collection, manassas, va) into the right flank 344 or with equal volume and number of heat-killed llc cells (hk). approximately 7 days after tumor 345 implantation (ti), when the tumor was palpable (~1cm in diameter), the tumor-bearing mice were 346 treated with either acylated ghrelin (as-24160, anaspect, fremont, ca) at a dose of 0.8 mg/kg or 347 vehicle (0.9% sodium chloride, 8881570121, covidien, dublin, ireland), s.q., twice daily, while 348 mice in hk group received vehicle (saline, same volume), s.q., twice daily for two weeks. the comprehensive laboratory animal monitoring system (clams™, columbus instruments, 363 columbus, oh) was used to identify metabolic parameters of the animals as we previously 364 described (guillory, chen et al., 2017) . ghsr +/+ and ghsr -/mice were individually housed in clams 365 cages for 96 hours before ti as well as at the endpoint (see the supplemental fig. 5 , timeline for the 366 study). the first 12 hours of clams was considered as the acclimation phase and the data for the 367 next 72 hours were analyzed. oxygen consumption (vo 2 ) (ml/h), carbon dioxide production (vco 2 ) 368 (ml/h), and locomotor activity (infrared beam-break counts) were recorded automatically by the 369 clams system every 20 min. the respiratory exchange ratio (rq) and energy expenditure (ee, or 370 heat generation) were calculated from vo 2 and vco 2 gas exchange data as follows: rq = 371 vco 2 /vo 2 and ee = (3.815 + 1.232 × rq) × vo 2 , respectively. energy expenditure was then 372 18 normalized to lbm for statistical analysis using two-way analysis of variance (anova). alternatively, 373 we also analyzed ee value by ancova with lbm as a covariate. locomotor activity was measured 374 on x-and z-axes by the counts of beam-breaks during the recording period. the data shown in the 375 results was summarized as the mean of every 6 hours in a 72-hour-period. for iwat and bat samples, 150ug of the protein lysate was diluted with diluent 41 and loaded onto 385 each well. the plate was incubated at room temperature (rt) with shaking for 2h followed by 3 386 times of wash in phosphate buffered saline with .05% tween 20 (pbs/t). sulfo-tag labeled 387 detection antibody was then added to plates and incubated for 2.5h. after another 3 washes in 388 pbs/t, read buffer t(2x) was added and the plate was read on msd sector imager (msd). 389 390 immunohistochemistry 391 the iwat and bat were mounted with oct (vwr 25608-930, vwr, radnor, pa) and flash frozen 392 in liquid nitrogen-chilled isopentane immediately after tissue collection. the oct-mounted iwat 393 and bat blocks were sliced at 14μm using a cryostat (leica cm3050s, nussloch, germany) at 394 -40 o c. before the process of staining, slides were dehydrated at rt for 30 minutes followed by 395 incubating in methanol for 15 minutes at -20 o c. to identify the colocalization of f4/80 and il-6 or 396 tnfα in iwat and bat, slides were blocked with 10% donkey serum for 1 hour at rt and followed 397 by incubating in primary antibodies (f4/80 monoclonal antibody 1:100, mf48000, thermo fisher 398 scientific; anti-il-6 antibody 1:100, ab6672, abcam; tnf alpha monoclonal antibody, fitc, 399 ebioscience™ 1: 200, thermo fisher scientific) signaling). the stained slides were dehydrated by 70%, 90%, 100% ethanol, and 100% xylene 411 sequentially and mounted with coverslips by using permount (sp15-100, thermo fisher scientific). 412 all stained slides were imaged by nikon nie microscope at 20x (iwat) or 40x (bat). the positive 413 cells (immunofluorescence) or positive area (dab stain) in the section were quantified and 414 normalized to the total area of the section (mm 2 ) using imagej analysis software (national institutes 415 of health, http://rsb.info.nih.gov/ij/). two-way anova was performed to identify differences between genotypes (ghsr +/+ vs. ghsr -/-) 419 across treatments (hk, tv, and tg) followed by fisher's lsd post hoc test. for inflammatory 420 20 cytokines, kruskal-wallis test was performed to identify the differences between groups. for energy 421 expenditure, ancova was also used for analysis in addition to anova with lbm as a covariate to 422 identify differences between genotypes across treatments followed by fisher's lsd post hoc test. 423 values are presented in mean ± sem. all statistical testing was performed using ibm spss version 424 adachi s, takiguchi s, okada k, yamamoto k, yamasaki m, miyata h, nakajima k, fujiwara y, 446 hosoda h, kangawa k, mori m, doki y (2010) effects of ghrelin administration after total 447 gastrectomy: a prospective, randomized, placebo-controlled phase ii study. and thermogenic responses to il-1beta in mice ghrelin-induced adiposity is independent of orexigenic effects a switch from white to brown 617 fat increases energy expenditure in cancer-associated cachexia acylated and unacylated ghrelin impair skeletal muscle atrophy in 621 mice coactivator of nuclear receptors linked to adaptive thermogenesis diet-induced obesity causes insulin resistance in 625 mouse brown adipose tissue an amp-activated protein kinase-stabilizing peptide 629 ameliorates adipose tissue wasting in cancer cachexia in mice energy metabolism in cachexia what we talk about when we talk about fat tumor necrosis factor-alpha 633 suppresses adipocyte-specific genes and activates expression of preadipocyte genes in 3t3-l1 634 adipocytes: nuclear factor-kappab activation by tnf-alpha is obligatory the relationship between weight loss 636 31 and interleukin 6 in non-small-cell lung cancer is there an effect of ghrelin/ghrelin analogs on cancer? a 638 systematic review changes in body mass, energy balance, physical function, 640 and inflammatory state in patients with locally advanced head and neck cancer treated with 641 concurrent chemoradiation after low-dose induction chemotherapy peptidomimetic regulation of growth hormone secretion characterization of adult ghrelin and ghrelin receptor 646 knockout mice under positive and negative energy balance ghrelin and growth hormone secretagogue receptor expression 648 in mice during aging anamorelin in 650 patients with non-small-cell lung cancer and cachexia (romana 1 and romana 2): results from 651 two randomised, double-blind, phase 3 trials ghrelin induces adiposity in rodents a guide to analysis of mouse energy 656 metabolism activation of thermogenesis in brown adipose tissue and 659 dysregulated lipid metabolism associated with cancer cachexia in mice cancer cachexia: malignant inflammation, tumorkines, and metabolic 661 mayhem lipolytic and thermogenic depletion of adipose tissue 663 in cancer cachexia ghrelin relieves 665 cancer cachexia associated with the development of lung adenocarcinoma in mice the role of adipose tissue in cancer-associated cachexia immune modulation 670 of brown(ing) adipose tissue in obesity relation between hypermetabolism, cachexia, and survival in cancer 673 patients: a prospective study in 390 cancer patients before initiation of anticancer therapy acute effects of ghrelin administration on glucose and lipid metabolism receptor agonist hm01 attenuates cachexia in mice bearing colon-26 (c26) tumors multiple roles of adipose tissue in cancer formation and progression beige adipocytes are a distinct type of thermogenic fat cell in mouse and 686 human chronic inflammation: role of adipose tissue and modulation by weight loss hk: 692 heat-killed + vehicle; tv: tumor + vehicle; tg: tumor + ghrelin. changes in (a) body weight (carcass 693 weight, n = 8-10) and (b) fat body mass by nmr expressed as % change from baseline average cumulative 695 food intake (fi) normalized to baseline fi (g/g, black areas represent food intake in the nighttime, 696 and the bottom areas in the bars represent food intake in the daytime, n = 4-6). * p < 0.05 compared 697 to hk within the same genotype. # p <0.05 compared to tv within the same genotype hk: 703 heat-killed + vehicle; tv: tumor + vehicle; tg: tumor + ghrelin. protein levels of inflammatory 704 markers (a)il-1β, (b) il-6, and (c) tnf; and (d) macrophage marker mcp-1 in iwat (pg/mg) 01 compared to hk within the same genotype. # p < 0.05 compared to tv within the 706 same genotype. no genotype difference was detected. data are shown as mean ± se. n = 707 6-7/group. (e-f) colocalization of inflammation and macrophages in iwat. (e) representative 708 images of colocalization of inflammatory marker il-6 and macrophage marker f4 f4/80 in fitc green; nuclei in dapi blue). (f) representative images of colocalization of 710 inflammatory marker tnf and macrophage marker f4/80 in iwat (tnf in fitc green positively stained inflammatory markers and colocalizations with 712 macrophages are indicated by the white arrows. scale bars hk: 715 heat-killed + vehicle; tv: tumor + vehicle; tg: tumor + ghrelin. protein levels of inflammatory 716 markers (a)il-1β, (b) il-6, and (c) tnf; and (d) macrophage marker mcp-1 in iwat (pg/mg) 001 compared to hk within the same genotype. # p < 0.05; ### p < 0.001 718 compared to tv within the same genotype. no genotype difference was detected. data are shown 719 as mean ± se. n = 6-7/group. (e-f) colocalization of inflammation and macrophages in bat representative images of colocalization of inflammatory marker il-6 and macrophage marker f4/80 f4/80 in fitc green; nuclei in dapi blue). (f) representative images of 722 colocalization of inflammatory marker tnf and macrophage marker f4/80 in bat positively stained inflammatory markers and 724 colocalizations with macrophages are indicated by the white arrows. scale bars hk: heat-killed + vehicle; tv: tumor + vehicle; tg: 727 tumor + ghrelin. (a) representative ihc images of ucp-1 in iwat. (b) ucp-1 positive area is 728 expressed as % of the total analyzed area in iwat ucp-1 in bat. (d) ucp-1 positive area is expressed as % of the total analyzed area in bat 001 compared to hk within the same genotype. genotype effects 731 are shown as p-values above the corresponding figures (p < .05). data are shown as mean ± se. 732 scale bars hk: heat-killed + vehicle; tv: tumor + 735 vehicle; tg: tumor + ghrelin. (a-c) energy expenditure adjusted by lbm is expressed (a) compared 736 to the baseline; (b) every 6 hours; and (c) average of every 6 hours. (d-f) ambulatory activity is 737 expressed (d) compared to baseline g-i) respiratory quotient (rq) is expressed (g) compared to baseline every 6 hours; and (i) average of every 6 hours. *p<0.05 compared to hk within the same genotype genotype effects are shown in p-values above the corresponding figures (p < 0.05). n = 4 for hk 741 groups and n = 6 for the rest of the groups. data are shown as mean ± se data 744 is expressed as box-and-whisker plot showing the median (middle line), mean (middle cross), upper 745 and lower quartiles (box), maximum and minimum (whiskers) supplemental fig. 2. high resolution images of immunohistochemistry staining in iwat representative images of colocalization of inflammatory marker il-6 and macrophage marker f4/80 f4/80 in fitc green; nuclei in dapi blue). (b) representative images of 752 colocalization of inflammatory marker tnf and macrophage marker f4/80 in iwat positively stained inflammatory markers and 754 colocalizations with macrophages are indicated by the white arrows. scale bars supplemental fig. 3. high resolution images of immunohistochemistry staining in bat representative images of colocalization of inflammatory marker il-6 and macrophage marker f4/80 f4/80 in fitc green; nuclei in dapi blue). (b) representative images of 759 colocalization of inflammatory marker tnf and macrophage marker f4/80 in bat positively stained inflammatory markers and 761 colocalizations with macrophages are indicated by the white arrows. scale bars effects of ghrelin on llc-induced protein-level changes in inflammation (il-1β mcp-1) in plasma (pg/mg, n = 11-14). *, **: different than hk 765 within the same genotype (*: p < .05; **: p < .01). genotype effects are shown in p-values above the 766 corresponding figures timeline of current study. ghsr +/+ and -/-mice were injected with llc (t, 1 × 769 106 cells, s.q.) into the right flank or with equal volume and number of heat-killed llc cells (hk) the 771 tumor-bearing mice were treated with either acylated ghrelin, 0.8 mg/kg (tg) or vehicle (0.9% 772 sodium chloride, tv), s.q., twice daily, while mice in hk group received vehicle (saline, same 773 volume), s.q., twice daily for two weeks days before tumor noted, baseline) and weekly till the endpoint. all the mice were 775 individually housed in clams cages for 96 hours before ti (11-7 days before tumor noted, baseline) 776 as well as at the endpoint borner t, loi l, pietra c, giuliano c, lutz ta, riediger t (2016) the ghrelin receptor agonist hm01 469 mimics the neuronal effects of ghrelin in the arcuate nucleus and attenuates anorexia-cachexia 470 syndrome in tumor-bearing rats. am j physiol regul integr comp physiol 311: r89-96 471 braun tp, zhu x, szumowski m, scott gd, grossberg aj, levasseur pr, graham k, khan s, 472 damaraju s, colmers wf, baracos key: cord-256370-cz88t29n authors: jansen van vuren, petrus; wiley, michael; palacios, gustavo; storm, nadia; mcculloch, stewart; markotter, wanda; birkhead, monica; kemp, alan; paweska, janusz t. title: isolation of a novel fusogenic orthoreovirus from eucampsipoda africana bat flies in south africa date: 2016-02-29 journal: viruses doi: 10.3390/v8030065 sha: doc_id: 256370 cord_uid: cz88t29n we report on the isolation of a novel fusogenic orthoreovirus from bat flies (eucampsipoda africana) associated with egyptian fruit bats (rousettus aegyptiacus) collected in south africa. complete sequences of the ten dsrna genome segments of the virus, tentatively named mahlapitsi virus (mahlv), were determined. phylogenetic analysis places this virus into a distinct clade with baboon orthoreovirus, bush viper reovirus and the bat-associated broome virus. all genome segments of mahlv contain a 5' terminal sequence (5'-gguca) that is unique to all currently described viruses of the genus. the smallest genome segment is bicistronic encoding for a 14 kda protein similar to p14 membrane fusion protein of bush viper reovirus and an 18 kda protein similar to p16 non-structural protein of baboon orthoreovirus. this is the first report on isolation of an orthoreovirus from an arthropod host associated with bats, and phylogenetic and sequence data suggests that mahlv constitutes a new species within the orthoreovirus genus. bats have been increasingly associated with emerging and re-emerging viruses. the likelihood of possible transmission of these pathogens to humans is ever increasing as a result of human encroachment on animal habitats, climate change and change of human behaviour. pathogens of particular public health importance are filoviruses [1, 2] , coronaviruses [3, 4] , paramyxoviruses [5, 6] and lyssaviruses [7, 8] . other viruses, without a known human disease link, have also been detected recently [9] [10] [11] . some human pathogens, such as rift valley fever virus, that have been detected in bats were likely a result of coincidental infection and do not constitute proof that bats play a role as reservoirs [12] . bats are parasitized by a number of ectoparasites, including mites, bat flies, ticks and fleas, and often by some or all of these simultaneously [13] . the bat flies are members of two families in the diptera order, namely, the streblidae and nycteribiidae, and are highly host-specific obligate ectoparasites of bats [13] [14] [15] . both bat fly families are hematophagous and potentially capable of (qiagen). cellular debris was removed by centrifugation at 14,000ˆg for 3 min, and the supernatant used for subsequent virus isolation and nucleic acid extraction procedures. viruses 2016, 8, x 5mm stainless steel beads (qiagen). cellular debris was removed by centrifugation at 14,000 × g for 3 min, and the supernatant used for subsequent virus isolation and nucleic acid extraction procedures. the wells of 24-well tissue culture plates (nunc) were seeded with vero e6 cells and grown to 80%-90% confluency in eagle's minimum essential medium (emem, lonza) supplemented with antibiotics (penicillin/streptomycin/amphotericinb, lonza) and 10% foetal calf serum at 37 °c and 5% co2. culture medium was removed and the monolayers in individual wells inoculated with 200 μl of ectoparasite pool homogenates (one pool representing parasites from one bat). after one hour adsorption at 37 °c, the inoculum was removed and fresh emem containing antibiotics and 2% foetal calf serum added. the 24-well plates were incubated for 14 days and cytopathic effects (cpe) monitored. a second and third blind passage was performed for all samples by inoculating and incubating monolayers as described above with 200 μl of undiluted supernatant from the preceding passage. supernatants were collected from all wells displaying cpe after three blind passages, a 1/10 dilution prepared in emem, and 1 ml of this used to inoculate a 25 cm 2 tissue culture flask. if the same cpe was noted in the sub-cultured 25 cm 2 flask, a 1/100 dilution of this supernatant was prepared and used to inoculate a 75 cm 2 tissue culture flask for preparation of stock virus. stock virus titres were determined by standard tissue culture infectious dose 50 (tcid50) titrations on 96-well microtitre plates as described previously [36] . the wells of 24-well tissue culture plates (nunc) were seeded with vero e6 cells and grown to 80%-90% confluency in eagle's minimum essential medium (emem, lonza) supplemented with antibiotics (penicillin/streptomycin/amphotericinb, lonza) and 10% foetal calf serum at 37˝c and 5% co 2 . culture medium was removed and the monolayers in individual wells inoculated with 200 µl of ectoparasite pool homogenates (one pool representing parasites from one bat). after one hour adsorption at 37˝c, the inoculum was removed and fresh emem containing antibiotics and 2% foetal calf serum added. the 24-well plates were incubated for 14 days and cytopathic effects (cpe) monitored. a second and third blind passage was performed for all samples by inoculating and incubating monolayers as described above with 200 µl of undiluted supernatant from the preceding passage. supernatants were collected from all wells displaying cpe after three blind passages, a 1/10 dilution prepared in emem, and 1 ml of this used to inoculate a 25 cm 2 tissue culture flask. if the same cpe was noted in the sub-cultured 25 cm 2 flask, a 1/100 dilution of this supernatant was prepared and used to inoculate a 75 cm 2 tissue culture flask for preparation of stock virus. stock virus titres were determined by standard tissue culture infectious dose 50 (tcid 50 ) titrations on 96-well microtitre plates as described previously [36] . for electron microscopy specimen preparation, 80%-90% confluent vero e6 monolayers in 25 cm 2 flasks were inoculated with stock virus and monitored for cpe. at the first sign of cpe, culture supernatant was collected, cleared of cellular content by centrifugation (3000ˆg for 5 min), and subsequently fixed in an equal volume of 2.5% glutaraldehyde in 0.1 m hepes buffer (ph 6.9) for visualization of virus particles by negative staining. a beckman airfuge ® (beckman coulter, brea, ca, usa) was used to concentrate all samples (10 min at 207 kpa), after which droplets of sample were adsorbed to 0.25% formar-coated copper grids for a minimum of 10 min, rinsed twice in deionised, distilled water and stained briefly in 2% phosphotungstic acid (ph 6.9). for ultramicrotomy, the remaining infected monolayers were flooded with the same fixative overnight, then routinely processed (postfixation in 1% buffered osmium tetroxide, graded ethanol dehydration, infiltration with a low viscosity resin (agar scientific, stansted, uk) and overnight polymerisation at 70˝c). seventy nm sections were cut on a leica em-uc6, double stained with saturated uranyl acetate and lead citrate, and viewed at 80 kv on a biotwin spirit (fei company, hillsboro, or, usa). imaging was done with an olympus quemesa ccd camera (olympus, tokyo, japan). single-primer amplification (sispa), rapid amplification of cdna ends (race), next-generation sequencing (ngs) and bioinformatics stock virus culture supernatant was added to trizol-ls (life technologies, waltham, ma, usa) at a ratio of 100 µl supernatant to 300 µl trizol-ls. rna was extracted using a column based kit (direct-zol rna kit, zymo research, irvine, ca, usa). to increase sensitivity, rrna was depleted using the same method as described previously [37] . rnas were converted to cdna and amplified using sispa as described previously with modifications [38] . to enhance coverage of the terminal ends, an oligo containing three rgtp at the 3' end (gccggagctctgcagatatcggccattat ggccrgrgrg) was added during first-strand cdna synthesis and the reverse transcriptase was changed to maxima h minus (thermo scientific, waltham, ma, usa), which has terminal transferase activity that enables addition of the rgtp containing oligo to the 5' end during cdna synthesis. amplicons were sheared and libraries prepared using the illumina truseq dna library preparation kit (illumina, san diego, ca, usa). sequencing was performed either on an illumina miseq (illumina) or nextseq 500 (illumina) using either a 2ˆ150 or 2ˆ250 version2 kit. illumina and sispa adapter sequences were trimmed from the sequencing reads using cutadapt-1.2.1 [39] , quality filtering was conducted with prinseq-lite (-min len 50-derep 14-lc method dust-lc threshold 3-trim ns left 1-trim ns right 1-trim qual right 15) [40] and reads were assembled into contigs using ray meta with kmer length = 25 [41] . resultant contigs were aligned to the ncbi sequence database using blast. the mega (version 6) program was used to prepare alignments (clustalw) of nucleic acid segment sequences, deduced amino acid sequences, phylogenetic trees and pairwise distance calculations [42] . the publicly available reovirus sequences used in the analysis were obtained from ncbi-nucleotide (genbank). nucleotide sequences from a small number of viruses from each genus in the reoviridae family were used to prepare a maximum likelihood tree showing the placement of mahlv in the family based on the full rna-dependent rna polymerase (rdrp) encoding segment. maximum likelihood trees were prepared using amino acid sequences of all open reading frames from all segments, showing the placement of mahlapitsi virus (mahlv) in the orthoreovirus genus relative to other viruses in this genus for which sequence is available on genbank. virus sequence accession numbers are summarised in table 1 . the evolutionary history was inferred by using the maximum likelihood method based on the jtt matrix-based model [43] . the tree with the highest log likelihood is shown. the percentage of trees in which the associated taxa clustered together is shown next to the branches (1000 bootstrap iterations). initial tree(s) for the heuristic search were obtained by applying the neighbor-joining method to a matrix of pairwise distances estimated using a jtt model. the tree is drawn to scale, with branch lengths measured in the number of substitutions per site all positions containing gaps and missing data were eliminated. evolutionary analyses were conducted in mega6 [42] . open reading frames were located and deduced protein amino acid sequences prepared by using the clc genomics workbench (qiagen). putative functions of the new virus deduced proteins were determined by blastx similarity searches to sequences available on genbank. the ectoparasite pool homogenate used for virus isolation was used as dna source for phylogenetic confirmation of species. dna was extracted using trizol (invitrogen, waltham, ma, usa) and the method as described by the manufacturer. amplification of the cytochrome c oxidase subunit i (coi) gene was performed with barcoding primers as described by tortosa et al.: lco1490 and hco2198 [13] . polymerase chain reaction was carried out in 50 µl reactions containing 25 µl mytaq red mix 2ˆ(bioline, london, uk), 2 µl of forward and reverse primer (10 µm), dna template (10 µl) and nuclease free water (11 µl). amplification steps were 94˝c for 5 min, 25 cycles of 94˝c for 60 s, 48˝c for 60 s and 72˝c for 90 s, and 72˝c for 10 min. pcr product was purified using the minelute kit (qiagen). purified pcr amplicon products were then sequenced at the nicd core sequencing facility (nicd, sandringham, south africa). replication of mahlv was evaluated in two cell lines: vero e6 (source african green monkey kidney) and c6-36 (source aedes albopictus mosquitoes). cells were grown to 50%-70% confluency in 25 cm 2 flasks, supernatant removed and respective flasks inoculated with 1 ml of 10´1, 10´2, 10´3 and 10´5 dilutions from stock virus (1ˆ10 6 tcid 50 /ml) in emem. after 1 h adsorption at 37˝c (veroe6) or 28˝c (c6-36), the inoculum was removed, cells washed with 5 ml phosphate buffered saline (pbs) and fresh emem, antibiotics and 2% foetal calf serum (hyclone, logan, ut, usa) added. cultures were incubated for 13 days at 37˝c (veroe6) or 28˝c (c6-36) while 0.5 ml aliquots of supernatant were collected from each flask directly after inoculation and addition of fresh medium (day 0), followed by day 4, 7 and 13. rna was extracted from 140 µl of the serial supernatant collections (qiamp viral rna kit, qiagen) and subjected to taqman real-time rt-pcr. a taqman real-time rt-pcr was developed to detect the rdrp gene of mahlv. primers and probe sequences are: forward morv_796f (5'-tagtggttcgtatgcgtggt-3'), reverse morv_893r (5'-aacagccattcaatctcagg-3') and probe morv_875p (fam-ggcacatatccctcaactgg-bhq), with the number in the oligonucleotide name indicating the nucleic acid position in the segment encoding rdrp. real-time rt-pcr was performed on the extracted rna using the qiagen one-step rt-pcr kit (qiagen) on a smartcycler (cepheid, sunnyvale, ca, usa) with the following program: reverse transcription (50˝c for 30 min), hot-start taq activation (95˝c for 15 min) and 50 cycles of amplification (95˝c for 15 s; 52˝c for 25 s plus signal acquisition; 72˝c for 20 s). rna extracted from diluted stock mahlv (final 1ˆ10 5 tcid 50 /ml) was used as a qualitative positive control in each run. from a total of 273 bat ectoparasite pools subjected to virus isolation by three blind passages, two yielded an agent that caused obvious cytopathic effects in the form of syncytia (giant cell) formation by three or four days post inoculation (d.p.i.) (figure 2 ). the parasite pool that yielded mahlv isolate 2511 was collected from an apparently healthy adult female rousettus aegyptiacus bat captured at mahune cave in may 2013. cpe in vero cells were noted after two blind passages, and the supernatant collected on day five from passage four in a 75 cm 2 flask containing infected vero cells yielded 1ˆ10 6.25 tcid 50 /ml of the unknown virus. the second parasite pool that yielded mahlv isolate 06-24 was collected from an apparently healthy juvenile male rousettus aegyptiacus bat captured at mahune cave in june 2013. cpe in vero cells were noted after three blind passages, and the supernatant collected on day five from passage five in a 75 cm 2 flask containing infected vero cells yielded 1ˆ10 6 tcid 50 /ml of the virus. these supernatants, passage four of 2511 and passage five of 06-24, were used for subsequent identification by tem and ngs. the ectoparasites from which the viruses were isolated were morphologically identified as bat flies, eucampsipoda africana theodor (diptera: nycteribiidae) ( figure 3 ) [44] . sequencing of the cytochrome c oxidase subunit i gene (coi) and alignment to sequences available on genbank, followed by phylogenetic analysis (figure 4 ) confirms that the bat flies in this study are closest related to eucampsipoda spp. identified before. initial screening of negatively-stained culture supernatants revealed the presence of rounded icosahedrons lacking envelopes, which resembled non-rotavirus-like virions of the reoviridae ( figure 5 ). two-layered capsids with an outer diameter of 70-75 nm (n = 30) and an inner core of 42-45 nm, possessed clearly defined solvent channels radiating outwards through the clustered capsomers of the outer capsid layer ( figure 5 ). although the dimensions of the negatively-stained, inner capsid layer were comparable to those recorded after processing for ultramicrotomy, the outer layer was slightly larger, occasionally measuring up to 81 nm in diameter. the coi partial sequence from the bat fly sequenced from this study is indicated by the red star. initial screening of negatively-stained culture supernatants revealed the presence of rounded icosahedrons lacking envelopes, which resembled non-rotavirus-like virions of the reoviridae ( figure 5 ). two-layered capsids with an outer diameter of 70-75 nm (n = 30) and an inner core of 42-45 nm, possessed clearly defined solvent channels radiating outwards through the clustered capsomers of the outer capsid layer ( figure 5 ). although the dimensions of the negatively-stained, inner capsid layer were comparable to those recorded after processing for ultramicrotomy, the outer layer was slightly larger, occasionally measuring up to 81 nm in diameter. (a) negatively-stained particle with two distinct layers; (b) icosahedral negatively-stained particle; (c) resin-embedded, sectioned viral particle in cytoplasm of infected vero e6 cell. white arrows indicate some of the characteristic spaces between the finger-like, capsomeric projections surrounding the solvent channels through the outer capsid layer. evident in ultrathin sections of infected vero e6 cells were multinucleate cells with extensive nuclear lobing, and cytoplasmic inclusion bodies associated with developing, double-shelled virus particles ( figure 6 ). tem observations therefore suggested that the isolated virus belonged to the reoviridae, sub-family spinareovirinae. (a) negatively-stained particle with two distinct layers; (b) icosahedral negatively-stained particle; (c) resin-embedded, sectioned viral particle in cytoplasm of infected vero e6 cell. white arrows indicate some of the characteristic spaces between the finger-like, capsomeric projections surrounding the solvent channels through the outer capsid layer. evident in ultrathin sections of infected vero e6 cells were multinucleate cells with extensive nuclear lobing, and cytoplasmic inclusion bodies associated with developing, double-shelled virus particles ( figure 6 ). tem observations therefore suggested that the isolated virus belonged to the reoviridae, sub-family spinareovirinae. evident in ultrathin sections of infected vero e6 cells were multinucleate cells with extensive nuclear lobing, and cytoplasmic inclusion bodies associated with developing, double-shelled virus particles ( figure 6 ). tem observations therefore suggested that the isolated virus belonged to the reoviridae, sub-family spinareovirinae. an unbiased next-generation sequencing approach using sispa amplification confirmed the presence of a novel orthoreovirus. initial sequencing results of both isolates yielded enough sequence coverage to identify all 10 segments. a polyetheleneglycol (peg) precipitated preparation of isolate 06-24 yielded the most viral specific reads and formed 11 contigs aligning to othoreoviruses using blastn and blastx. both the 5' and 3' ends were missing for all the segments, so to obtain complete genomes for each isolate, rrna depletion and a combination of sispa and rapid amplification of cdna ends (sispa-race) was done. read numbers were also increased by running samples on an illumina nextseq 500. both an increase in the percentage of viral reads aligning to the genome segments and an increase in coverage of the ends were observed. presence of mahlv in the original homogenates from which the isolates were obtained was confirmed by sispa amplification and ngs directly from the homogenates. expectedly, only a low number of reads from both homogenates mapped to the mahlv sequence due to the high amount of host sequence obscuring viral specific sequences combined with likely low viral load in the homogenates and the relatively low sensitivity of the sispa method. a maximum likelihood tree, constructed with nucleic acid sequence data for the rna-dependent rna polymerase (rdrp) encoding segments of representative viruses from the different genera within reoviridae (figure 7) shows the placement of both isolates amongst other orthoreoviruses in the family. maximum likelihood trees were prepared using the deduced amino acid sequences from the open reading frames (orf's) of all the virus' segments and those of other viruses in the orthoreovirus genus (figures 8-10) . a distinct clade is formed by mahlv, bush viper reovirus, baboon orthoreovirus and broome virus within the genus. the above-mentioned clade is visibly distinct from others composed of bat-associated viruses; the nelson bay orthoreovirus and bat-derived mammalian orthoreoviruses. the closest relative of mahlv, based on sequence homology of a conserved core protein, is bush viper reovirus (lambda b nucleic acid identity-63.7%; rdrp amino acid identity-66.3%) while the closest bat-associated virus is broome virus (lambdab nucleic acid identity-60.7%; rdrp amino acid identity-58.0%) ( table 2) . homology of the divergent major outer capsid protein of mahlv to known orthoreoviruses is much lower: sigma b nucleic acid identity-28.7%-41.9%; amino acid identity-5.6%-24.3% (table 3) . the genome segments of mahlv were named according to the nucleotide length, which is consistent with the nomenclature of other orthoreoviruses [26] . a summary of the mahlv genome is given in table 4 . the total genome size is 23,200 nucleotides and predicted to encode eleven proteins, seven of which are structural. all ten genome segments of mahlv contain an identical 3' terminal sequence, ucauc-3', which is conserved between all known species of orthoreovirus, and an identical 5' terminal sequence, 5'-gguca which is unique to mahlv. non-coding regions (ncrs) are present at both ends of the genome segments, with the 5' ncrs being shorter in nucleotide length than 3' ncrs. the nucleotide sequences of the two isolates of mahlv, 2511 and 06-24, are not identical. nucleotide homology of the rdrp encoding segment between the two isolates is 93.5% (99.8% deduced amino acid sequence), and 80.4% (89.0% deduced amino acid sequence) for the sigma b encoding segment. putative protein functions were determined by blastx similarity searches to sequences available on genbank, revealing putative functions known for other orthoreoviruses. the segments l2, l3, m1, m3, s1, s2 and s3 each contain a single start aug codon in close proximity to the 5' end. the l1 segment of mahlv contains two aug start codons in close proximity to the 5' end, at positions 14 and 19. agcaugg) . the open reading frame initiating at position 29 is 2031 nucleotides in length and putatively encodes for an outer capsid protein involved in membrane penetration during infection (mub). the second aug initiates a 492 nucleotide open reading frame but the deduced amino acid sequence does not match any viral protein of note on genbank. the deduced sigma a protein of mahlv (segment s1) contains the fusogenic orthoreovirus-wide conserved arginine amino acid at position 273. the s4 segment is bicistronic and encodes for a 14 kda protein similar to p14 membrane fusion protein of bush viper reovirus and a non-overlapping 18 kda protein similar to p16 non-structural protein of baboon orthoreovirus without a known function ( table 4 ). the isoelectric point of mahlv p18 is acidic (5.02), similar to that of p16 of broome virus and baboon orthoreovirus and contrary to that of other orthoreoviruses. the first ten amino acids in the putative 14 kda protein of mahlv are identical to the first ten amino acids in the p13 fusion protein of broome virus and represent the myristoylation consensus sequence required for fusion activity of the protein. the s4 segment does not encode a cell attachment protein, an observation also characteristic of broome virus and baboon orthoreovirus. 1 63.0 54.3 53.9 63.1 63.8 63.3 63.6 64.7 mahlv replicated efficiently in vero cell culture, with the inoculum containing a high dose of virus (10 5 tcid 50 /ml) leading to rapid monolayer destruction after inoculation, with a peak in virus rna (measured by real-time rt-pcr) by day 7, followed by a decrease on day 13. the inoculums containing a lower virus dose (10 4 and 10 3 tcid 50 /ml) resulted in a peak of rna detection on day 13. all three above-mentioned inoculum doses yielded detectable virus rna by day 4 after inoculation. the inoculum containing 10 1 tcid 50 /ml virus did not generate detectable viral rna until day 7, and was still showing an upward trend on day 13 (last sampling day). the virus did not replicate in the insect cells (c6-36) up to day 13, but adaptation to these cells through serial passaging was not attempted. the role of bats in harbouring pathogens of public health and veterinary importance is becoming an increasingly popular topic of research within the field of emerging and zoonotic diseases. the most notable viruses in which natural transmission from bats have been implicated include filoviruses, coronaviruses, paramyxoviruses, herpesviruses, lyssaviruses and bunyaviruses. the implication of bats in transmission or maintenance of some of these viruses is very circumstantial and often based only on serological evidence. more convincing evidence for others is based on detection of viral nucleic acid and isolation of live virus, although this does not conclusively prove that a vertebrate host is a reservoir. finding pathogens in bats leads to the questions of how they are transmitted between bats, and from bats to incidental hosts such as humans. one possible transmission mechanism could be bat-associated hematophagous arthropods, such as the bat flies, but migration of parasites between bats is not well understood and would require further entomological investigation to better understand [13] . two isolates of a novel fusogenic orthoreovirus were cultured and we determined their full genome sequences, which were compared to currently known viruses in the genus. the two isolates are not identical but similar enough to suggest that they are merely two isolates of the same virus. this suggests that there are multiple variants of the virus present in the host population. members of a species within the orthoreovirus genus are usually identified by a number of characteristics: amino acid and nucleotide sequence identity, organization of the polycistronic genome segment and host species [19] . for conserved core proteins, an amino acid identity >85% for homologous proteins indicates that two viruses belong to the same species, while identity <65% indicates a possible new species. when comparing the amino acid sequence of more divergent outer capsid proteins, >55% identity indicates one species and <35% indicates different species. nucleic acid sequence identity of homologous segments of >75% indicates the same species and <60% a new species. the nature of conserved genome segment termini sequences of orthoreoviruses is also useful for virus classification [45] . the divergence of mahlv sequence from other known orthoreoviruses combined with a unique conserved 5' genome segment end and a unique host species, suggests that this is a new virus species in this genus. along with broome virus and baboon orthoreovirus, mahlv is the third orthoreovirus that lacks an identified cell attachment protein in the s4 segment. this unique characteristic further strengthens the phylogenetic classification which places these viruses in a separate clade and suggests that entry of these viruses into cells is mediated differently than for other orthoreoviruses. taking the abovementioned criteria and the sequence characteristics of the novel virus described here into consideration, we propose that mahlapitsi virus constitutes a new species within the orthoreovirus genus. to our knowledge this is the first description of an orthoreovirus in africa with an indirect link to bats. considering the rich diversity of bat species found on the continent and increased scientific interest in this field, this is unlikely to be the only such virus to be isolated from bat ectoparasites in years to come. however, to our knowledge this is the first orthoreovirus to be isolated from an arthropod host, since all currently known viruses in this genus are associated with vertebrates. mahlv did not replicate on c6-36 cells in this study but aedes albopictus, from which the c6-36 cell line is derived, is classified in a completely different dipteran family, the culicidae, likely pointing to a cell receptor incompatibility. another possible explanation could be the temperature at which insect cells are cultured compared to mammalian cells, which might be incompatible with this virus. the arthropod-borne nature of mahlv transmission needs further investigation, especially to establish whether nycteribiid flies are only involved in mechanical or possibly biological transmission, and if the virus is even transmitted to bats. various other genera in the reoviridae family contain vector-borne viruses, including banna virus (seadornavirus), colorado tick fever virus (coltivirus) and bluetongue virus (orbivirus). our isolation of mahlv from arthropods might direct some attention to the possible role of insects in the transmission of currently known orthoreoviruses, or possibly the presence of other yet unknown viruses in various arthropods. we have no information on the geographical range of mahlv, but the wide distribution of rousettus aegyptiacus in africa and the middle east [46] , and the strict host preference and specificity of bat flies [13] [14] [15] , dictate that their ranges will overlap. we have no data to suggest that mahlv has any human health implication, but this warrants further investigation. the virus grows to high titers in vero e6 cells which suggests that it may infect vertebrates, although growth in in vitro systems cannot be translated directly into replication in a vertebrate host. it is important to note, also, that this was after blind passage and cell culture adaptation (cpe noted after two-three blind passages). any risk of human infection for now, however, is only likely in individuals who come into close contact with wild egyptian fruit bats and their ectoparasites. although highly host-dependent, the bat flies have been noted to leave their bat hosts and crawl on bat researchers (personal observation). respiratory disease has been noted in humans infected with melaka, kampar and nelson bay orthoreovirus, including limited human-to-human transmission [22, 23, 32, 33] . thus the potential for mahlv to infect humans, and spread between humans, cannot be excluded until further investigation is done, especially considering that the virus grows very efficiently on a monkey-derived cell line. in conclusion, we have identified a novel orthoreovirus which we propose should constitute a new species within the genus. two virus strains were isolated from ectoparasitic bat flies collected from egyptian fruit bats from a south african cave roost. this represents the first isolation of an orthoreovirus from arthropods and the first african virus in this genus with an indirect link to bats. fruit bats as reservoirs of ebola virus marburg virus infection detected in a common african bat bats are natural reservoirs of sars-like coronaviruses middle east respiratory syndrome coronavirus in bats, saudi arabia nipah virus: a recently emergent deadly paramyxovirus isolation of hendra virus from pteropid bats: a natural reservoir of hendra virus isolation of rabies virus 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orthoreovirus in the aborted fetus of a stellar sea lion (eumetopias jubatus) high similarity of novel orthoreovirus detected in a child hospitalized with acute gastroenteritis to mammalian orthoreoviruses found in bats in europe reptilian reovirus: a new fusogenic orthoreovirus species investigation of a potential zoonotic transmission of orthoreovirus associated with acute influenza-like illness in an adult patient imported case of acute respiratory tract infection associated with a member of species nelson bay orthoreovirus standards for sequencing viral genomes in the era of high-throughput sequencing isolation of genetically diverse marburg viruses from egyptian fruit bats virological and serological findings in rousettus aegyptiacus experimentally inoculated with vero cells-adapted hogan strain of marburg virus selective depletion of rrna enables whole transcriptome profiling of archival fixed tissue viral genome sequencing by random priming methods cutadapt removes adapter sequences from high-throughput sequencing reads quality control and preprocessing of metagenomics datasets scalable de novo metagenome assembly and profiling molecular evolutionary genetics analysis version 6.0 the rapid generation of mutation data matrices from protein sequences an illustrated catalogue of the rothschild collection of nycteribiidae (diptera) in the british museum (natural history), with keys and short descriptions for the identification of subfamilies, genera, species and subspecies sequence at both termini of the 10 genes of reovirus serotype 3 (strain dearing) mapping the zoonotic niche of marburg virus disease in africa the authors would like to thank the following individuals for their contributions towards fieldwork and technical assistance: busi mogodi, justice kgatitsoe, antoinette grobbelaar, terence scott, joe kgaladi, marinda mortlock, marike geldenhuys, jessica coetzer, andre coetzer. we would like to thank dorothy southern and alfred musekiwa for proofreading the manuscript.the project is jointly funded by the following grants awarded to: janusz t. paweska the grant holders acknowledge that opinions, findings and conclusions or recommendations expressed in any publication generated by gdd and nrf-supported research are those of the authors and that the gdd and nrf accept no liability whatsoever in this regard.author contributions: petrus jansen van vuren and janusz paweska conceived and designed the experiments; all authors were involved in some aspect of performing the experiments and interpretation of the data; petrus jansen van vuren, janusz paweska, monica birkhead, michael wiley and gustavo palacios analysed the data; petrus jansen van vuren wrote the paper with inputs from all other authors; janusz paweska, petrus jansen van vuren, wanda markotter and gustavo palacios contributed funding. all authors read and approved the final manuscript. the authors declare no conflict of interest.viruses 2016, 8, 65 key: cord-256452-77xij0fc authors: allen, louise c.; turmelle, amy s.; mendonça, mary t.; navara, kristen j.; kunz, thomas h.; mccracken, gary f. title: roosting ecology and variation in adaptive and innate immune system function in the brazilian free-tailed bat (tadarida brasiliensis) date: 2008-11-11 journal: j comp physiol b doi: 10.1007/s00360-008-0315-3 sha: doc_id: 256452 cord_uid: 77xij0fc bats have recently been implicated as reservoirs of important emerging diseases. however, few studies have examined immune responses in bats, and even fewer have evaluated these responses in an ecological context. we examined aspects of both innate and adaptive immune response in adult female brazilian free-tailed bats (tadarida brasiliensis) at four maternity roosts (two natural caves and two human-made bridges) in south-central texas. immune measurements included in vitro bactericidal ability of whole blood and in vivo t cell mediated response to mitogenic challenge. bactericidal activity in t. brasiliensis varied with roosting ecology, but appears to be sensitive to colony-level effects. blood from females living at one cave had significantly lower bactericidal ability than blood from females at three other sites. t cell mediated response in this species was associated with variation in roost ecology, with females from two caves having greater responses than females from two bridges. t cell mediated response and bactericidal activity were negatively correlated with one another within individuals that were tested for both. variation in immunological response of t. brasiliensis is important for understanding the influence of the environment on the frequency and distribution of immunologically competent individuals and for understanding disease-host dynamics in this and other colonial species. the emergence in wildlife of infectious diseases, such as aids, ebola, west nile virus, sars, and hantaviruses, highlight the need to understand the ecology of reservoir hosts to disease, and the interactions of immunological response and ecological variation. recently, bats have become a focus of attention as possible reservoirs of several emerging pathogens, including sars-like viruses (li et al. 2005) , and other corona viruses (dominguez et al. 2007; gloza-rausch et al. 2008) , nipah and hendra viruses (joharra et al. 2001; halpin et al. 2000) , ebola virus (leroy et al. 2005) , herpes virus (wibbelt et al. 2007) , and others (reviewed in calisher et al. 2006) . bats also have long been recognized as reservoir hosts, and vectors to humans and domestic animals, of rabies virus (messenger et al. 2003; constantine 1967) and related lyssaviruses (fraser et al. 1996; botvinkin et al. 2003) . however, little is known about variation in immune function and susceptibility to disease in freeranging bats (calisher et al. 2006; dobson 2005 ; but see christe et al. 2000) . improving our understanding of variation in immune function among individuals and populations of bats is important for understanding the role of bats as potential vectors of emerging diseases (messenger et al. 2003; dobson 2005) . bats possess a variety of life-history traits that are likely to affect their immune function and the role of bats as reservoirs and vectors of disease (calisher et al. 2006 ). many species are colonial, although considerable variation in colony size exists (kunz 1982; o'shea and bogan 2003) . the brazilian free-tailed bat (tadarida brasiliensis), for example, roosts in some of the largest aggregations of mammals on earth, with several thousand to several million individual bats estimated to form maternity colonies in caves and under highway bridges (davis et al. 1962; mccracken 2003; keeley and keeley 2004; betke et al. 2008) . colonial living brings the possibility of increased exposure to infectious pathogens, with direct links to immune defense and protection. relationships have been shown between coloniality and immune responsiveness in several avian species, although evidence is conflicting. for example, tella et al. (2001) found that t cell mediated immune response in fledglings of the magellanic penguin (spheniscus magellanicus) was negatively correlated with colony size, whereas møller et al. (2001) , examining immunity at the interspecific level, found that highly colonial swallows and martins, have higher levels of t and b cell response compared to less social songbirds. bats sometimes roost in proximity to urban and agricultural landscapes , and occasionally within human dwellings. in south-central texas, t. brasiliensis uses both natural and man-made structures as roosts, including caves, bridges, buildings, and bat houses (kunz and reynolds 2003; mccracken 2003; sgro and wilkins 2003) . roosts can differ in structure, capacity and possible quality (lausen and barclay 2006; neubaum et al. 2006) . given the recognized role of bats in disease transmission, it is important to understand variation in their immune condition in relation to ecological variables, particularly for those species roosting in close proximity to humans. innate immunity establishes an early line of defense against invading pathogens and inhibits the progression of infection. in contrast, the adaptive immune responses are induced upon antigen processing and have greater importance for clearing infections (goldsby et al. 2003) . the innate and adaptive arms of the immune response interact to protect individuals, although their competence may vary among individuals (blount et al. 2003) . most previous research on the effects of ecological conditions on immune function has focused primarily on the adaptive immune response (but see tieleman et al. 2005 and blount et al. 2003) , by estimating t cell proliferation in response to mitogenic challenge (tella et al. 2001; tella et al. 2002) . evaluation of multiple components of immune response is needed to develop a more complete understanding of immune competence in free-ranging animals. in the present study, we evaluate aspects of both the innate and adaptive arms of the immune system in t. brasiliensis over a period of 5 months at four maternity colonies (two natural caves, two man-made bridges) in south-central texas. bactericidal activity of whole blood in culture, one aspect of the innate immune response, primarily measures complement-mediated cytotoxicity (merchant et al. 2003) , which is a major factor in defense against viruses (blue et al. 2004) . t cell mediated response, which contains aspects of both innate and adaptive immune components, is also important in clearance of viruses, including rabies virus (hooper et al. 1998 ). this study was designed to characterize responses of t. brasiliensis to two immune challenges. we postulated that variation in both the innate and adaptive arms of the immune system would be affected by differences in roosting ecology as it relates to roost environment and colony size. this study was conducted in south-central texas from may to september of 2005. bactericidal activity and t cell infiltration were assessed in free-ranging adult female brazilian free-tailed bats (t. brasiliensis), captured at two natural caves (frio cave and davis cave) and two large pre-caste concrete highway bridges (seco creek bridge and east elm creek bridge). periods of data collection corresponded to female reproductive stages: pregnancy (may-june); lactation (june-july); post-lactation/nonreproductive (august-september). earlier studies indicated that mating in t. brasiliensis occurs prior to spring migration (davis et al. 1962; cockrum 1969) , though recent evidence suggests that mating also occurs in texas in march and april (keeley and keeley 2004) . outside of the mating season, however, many of these roosts are occupied by non-territorial maternity colonies, comprised mostly of females. thus, we have focused on adult females in the present study. approximately 15 bats were captured on a given night at each site, most of which were sampled within 2-4 days in each sampling period, for a total of 327 bats. both immunological assays were not always performed on each bat, due to temporal constraints of the specific protocols. body mass, reproductive condition (non-reproductive, pregnant and lactating) and age class (adult or juvenile; anthony 1988) were determined for each individual. juveniles were not included in the analyses, due to low sample sizes (n = 20) over the entire study period. bats were held for up to 14 h for the immunological assays described below and then released at the site of capture. all individuals received a site-specific tattoo on their wings to prevent re-sampling. tattoos on wings provide effective markings on these bats (lollar and schmidt-french 1998) that can last up to 3 years. all capture, handling and experimental procedures were approved by the university of tennessee animal care and use committee (#890) to g.f.m. and boston university animal care and use committee (05-012) to t.h.k., and under texas parks and wildlife department permit spr-0305-058 to t.h.k. bacterial killing ability of the bat's whole blood against escherichia coli was measured to represent one aspect of the innate immune response. this technique has been used successfully as an in vitro assay of innate immune function in free-ranging animals (tieleman et al. 2005) . a small amount of whole blood (6 ll) was collected in sterile heparinized capillary tubes via venopuncture (kunz and nagy 1988) within 2 h of capture from each bat. although matson et al. (2006) found a decrease in bactericidal ability of the blood of several species of birds at this length of time post-capture, preliminary data on t. brasiliensis shows no reduction in bactericidal ability up to 2 h postcapture. the blood was diluted to 1:50 in rpmi-1640 media (roswell park memorial institute), supplemented with 5% fetal bovine serum (fbs). the e. coli (atcc 8739; microbiologics, usa) solution was diluted to 1:1,000 using sterile phosphate buffered saline (pbs). a total of 140 ll of diluted blood was mixed with 10 ll of diluted bacteria. once mixed, 50 ll of the combined blood and bacterial dilution was spread onto labeled trypticase soy agar plates (bd diagnostic systems, usa) at both 0 and 60 min post-mixing. two control plates consisted of 5% fbs in rpmi with equivalently diluted bacteria and no blood. all plates were incubated at 37°c for 12 h, after which colonies of e. coli were visually counted and recorded. the unit-less index used to calculate the bactericidal ability of the blood was standardized for both controls of a given assay: this index assigned large positive values when bats had blood with high cytotoxic activity and negative values when blood had little or no cytotoxic activity against the bacteria. the index method controls for bacterial die-off occurring within an assay that is unrelated to the blood's bactericidal ability. only data from assays where there was less than a 15% difference between the 0 and 60 min controls were used. three individuals whose bactericidal indices were over three standard deviations lower than the mean of the remaining individuals were excluded from the data set, because these values deviated from the normal distribution of bactericidal ability found in our samples. in the final analyses, bactericidal indices were derived from 89 individual adult female bats. a subcutaneous injection of phytohaemagglutinin (pha-p #l8754; sigma, usa) was administered to assess t cell infiltration in a total of 163 individual bats. injections were administered in the interfemoral membrane (uropatagium) below the knee at the point of contact with the leg. prior to injection, the area was measured with a digital micrometer (mitutuyo #293-230, japan). the experimental area on each bat was injected with 0.05 ml of 3 mg/ml pha in pbs. as a control, the contralateral side was then injected with 0.05 ml of pbs. swelling at the sites of injection was subsequently measured at 12 hours post-challenge. measurements of pha swellings in the uropatagium of bats are very similar to the more common measurements made in the wing web of birds. the swelling produces thickened cellular infiltrate between the two layers of skin adjacent to the leg, thus our measurements are comparable in execution to those conducted in birds. although bactericidal ability of blood was not tested in every bat, all bats were bled within 3 min of capture, for a companion study on stress hormones, therefore all bats in our study were bled once prior to pha challenge. to ensure consistency in assessing the pha test, one of us (l.c.a.) performed all injections, and another (a.s.t.) made all measurements. care was taken to conduct repeatable, blind measurements with each swelling measured twice and averaged. following navara et al. (2005) , the unit-less index below was used to determine the swelling response to the mitogenic challenge standardized against the control response: indices greater than one designate bats with marked t cell infiltration to the injection area, whereas smaller index values indicate that little or no response was observed. one individual with an index value that was over three standard deviations beyond the normal range of indices was excluded from the data set, resulting in analyses of t cell infiltration for 162 individual adult female bats. while this technique has been widely used in avian immunology to assess t cell mediated response (bonforte et al. 1972; tella et al. 2001) , recent studies have cautioned that the proper identification of cellular components present in the swelling is necessary to accurately interpret the response (kennedy and nager 2006; martin et al. 2006a) . in a separate group of t. brasiliensis (n = 18), we analyzed pha treated and control tissues with biopsies of the swellings taken 6-21 h post-challenge. we documented significantly larger numbers of lymphocytes (mean cell count = 6), as well as heterophils (mean cell count = 35), in pha-challenged tissues that were not observed in any numbers in control tissues, demonstrating that specific cellular infiltration had occurred in response to the pha challenge. we found that measurements of the swellings at 12-h postinjection capture the maximal swelling response of individual bats, and represented when increased lymphocyte and heterophil infiltration occurred (turmelle and mendonça, unpublished data) . the immune response to pha challenge in bats suggests that both innate and adaptive components are responsible for local swelling at the injection site, as was found in birds by martin et al. (2006a) . because pha elicits t-lymphocyte response at the site of injection, this method is a good overall measure of cell-mediated immune function in this species. the bactericidal indices among all individuals included in the final data set were normally distributed. however, t cell responses among individuals were not normally distributed, and thus the data were log-transformed to fit a normal distribution prior to statistical analysis. non-transformed values, means ± standard error, of bactericidal and t cell indices are presented. indices for either assay did not differ statistically between reproductive classes (bka, p = 0.374; pha, p = 0.961). thus, all reproductive classes were combined prior to analyses of the data sets. for both immune indices, a nested mixed anova model, with one covariate (general linear model; using jmp 5.0.1), was used for comparison of roost type. by roost type we refer to the structure of the roost (bridge or cave), whereas by colony we refer to the location at which the individual bats were sampled (frio cave, davis cave, seco creek bridge and east elm creek bridge). because we were only able to sample four colonies, a nested design was used (comparing roost types, with colony nested within roost type) to examine if there is significant variation in roost types beyond variation due to differences among colonies alone. roost type, and colony nested within roost type were independent variables and body condition, a ratio of body mass to right forearm length, was a covariate in the overall model. we also treated colony as a random effect (i.e., we are interested in generalizing to other colonies of brazilian free-tailed bats); roost type (bridge or cave), a fixed effect, was tested over colony nested within roost type. because colony size varies with roost type (the majority of cave colonies, including those studied here, have more bats living in them than bridge colonies), we were unable to directly test for the effects of population size on immune function, but we will explore this hypothesis in our discussion below. lastly, we tested for a linear association between bactericidal ability and the log-transformed t cell response data, within individuals subjected to both assays (n = 74), using pearson's productmoment correlation (a = 0.05). the overall model of roost type, colony within roost type and body condition significantly explained variation in bacterial killing ability (p = 0.03, f = 2.88 (3, 88) , r 2 = 0.12). within the model, colony (within roost type) was the only significant predictor of the variation in bactericidal ability (p \ 0.05; fig. 1a ). blood from females at davis cave had significantly lower killing ability (20.19 ± 4.32) than the three other colonies; frio cave (35.46 ± 4.60), seco creek bridge (44.31 ± 7.59), and east elm creek bridge (38.68 ± 6.73). other variables including body condition (p = 0.77) and roost type [bridge (41.15 ± 5.15) and cave (27.35 ± 3.22); p = 0.06] were not significant predictors of bactericidal ability in female bats, although roost type borders on significance. there were no interactions between roost type or colony nested within roost type and body condition. the overall model of roost type, colony within roost type and body condition significantly explained variation in pha response (p \ 0.01, f = 8.34 (3, 161) , r 2 = 0.18). within the model roost type and colony (within roost type) were both significant predictors of pha response (p \ 0.001 and p = 0.02, respectively). females from caves (1.68 ± 0.07) exhibited a greater t cell response than those living in bridges (1.05 ± 0.12). females from davis cave (1.54 ± 0.08) and frio cave (1.90 ± 0.10) had larger responses than those at the bridge colonies, east elm creek (1.14 ± 0.21) and seco creek (1.01 ± 0.15; fig. 1b) . variation in t cell response was not explained by body condition (p = 0.33). again, there were no interactions between roost type or colony nested within roost type and body condition. within individual females, there was a weak but significant negative correlation between bactericidal ability and t cell response to pha (pearson's q = -0.190, p = 0.05). females that produced larger swellings in response to pha had blood with lower bactericidal ability (fig. 2) . this study demonstrates that individual t. brasiliensis differ in their ability to mount functional immunological responses, and that roosting ecology significantly impacts immune function. we also provide evidence of a negative association between innate and adaptive immune responses within individuals. blood from female bats at davis cave had lower bacterial killing ability compared to females at frio cave and the two bridge sites (fig. 1a) . this trend is also present when roost types are compared; suggesting that there are inherent differences between bridges and caves that likely affect immunity. although this result is not linked to body condition, there may be additional factors that affect innate immunity which are not included in our model. one possible factor could be differences in ectoparasitism among the colonies surveyed. high parasite loads affect immune function adversely in a variety of vertebrates (sheldon and verhulst 1996; møller et al. 1999) . examination of infestations of ectoparasitic mites on t. brasiliensis at these same sites show that individuals at davis cave, the site with the lowest mean bka index, had the highest degree of mite cover, followed by individuals at frio cave and then at bridge sites (turmelle 2005) . although these types of immune responses are not directly related, we suggest that fig. 1 a bka index (bactericidal ability of blood; mean ± se) at the four colonies sampled, blood from bats at davis cave had significantly lower bactericidal ability than blood from bats at the other colonies (p \ 0.05). caves versus bridges (p = 0.06). sample sizes for frio and davis caves and seco creek and east elm creek bridges are n = 30, 34, 11, 14, respectively. b pha index (response to pha injection at 12 h; mean ± se) at the four colonies sampled. letters denote statistical differences between colonies, estimated using post-hoc analysis. bats sampled at two cave colonies had significantly greater response to pha challenge than those at the two bridges (colony, p = 0.02; roost type, p = 0.001). sample sizes for frio and davis caves and seco creek and east elm creek bridges are n = 50, 76, 24, 12, respectively higher parasitism of bats in the colony at davis cave may reflect a weakened immune system overall; however, the exact causative mechanisms are currently unknown. research on the relationships between ectoparasitism and immune responses in t. brasiliensis is in progress. the lower t cell responses in females occupying bridges compared to their cave-dwelling counterparts indicates that adaptive immune response is also influenced by roost type (fig. 1b) . this supports our hypothesis that environmental differences may exist between roost types. bats living in bridges may experience physiological stress associated with living in man-made roosts that negatively impact adaptive immune responses. as reviewed by nelson et al. (2000) immune suppression associated with stress hormones has been demonstrated in several species of vertebrates. notwithstanding, evidence linking stress hormone levels and immune system function in the brazilian free-tailed bat is currently lacking. alternatively, this pattern may also reflect differences in colony size between the roost types. while our model does not directly test for the effects of colony size on swelling response to pha, a posthoc test shows a positive correlation (p \ 0.0001, f = 19.79 (3, 161) , r 2 = 0.11; fig. 3 ) between mean pha response and the best current estimates of colony size (betke et al. 2008) . because colony size varied with roost type, and the effects of both variables may be working in combination, additional research will be needed to tease apart these effects. similarly, in an interspecific study møller et al. (2001) found that highly colonial swallows and martins had more robust t cell responses compared to less social songbirds, and they suggested that higher levels of parasitism observed in larger colonies may cause individuals to allocate more resources to combat infection. tella et al. (2001) , found the opposite relationship, examining colonies of the magellanic penguin (spheniscus magellanicus), with larger colonies having reduced t cell responses compared to smaller ones. the authors point out that density dependent food limitations and crowding negatively affected body condition and immunocompetence in fledglings. since we found pha response to increase with colony size, it is likely that these bats may not be particularly sensitive to crowding and food limitations. because the adaptive arm of the immune system responds to chronic infection (klasing 2004) , greater investment in immunity is expected in organisms threatened by a larger number of pathogens (read and allen 2000) . evidence suggests that bats in caves have higher ectoparasite loads than those roosting beneath bridges (turmelle 2005) . differences in exposures to other pathogens may also vary by roost type. for example, once the spores of histoplasma capsulatum and other fungi that grow in the accumulated guano below bats become airborne they can infect lungs and mucosal membranes (mcmurray and russel 1982) . resistance to h. capsulatum infection in mammals is dependent on a cellular immune response primarily mediated by t cells (cain and deepe 1998; deepe 1994) . one critical determinant of the course of infection is the inflammatory response evoked in response to host-pathogen interactions. the inability to evoke the appropriate inflammatory response can lead to disease progression. compared to bats roosting in caves, bats roosting under bridges may be less exposed to fungi due to smaller accumulations of guano and greater air circulation between the guano and the roosting bats. thus, we expect that higher pressures from parasites and fungi in caves may result in cave-roosting bats investing more energy in mounting memory-related, adaptive, resistance to the pathogens they commonly face. these findings are in contrast with those of christe et al. (2000) who found that reproductive females of myotis myotis had lower t cell responsiveness and higher mite loads than non-reproductive females, and that during lactation immunocompetence was positively correlated with body mass. in the current study, we found no evidence for differences in t cell response due to body condition or reproductive stage. moreover, we found evidence for a positive relationship between ectoparasitic mite loads and t cell response, although this pattern may be indirect owing to differences in roosting condition in bridge and cave-roosting bats. results from our study have shown a negative correlation between pha response and bactericidal ability within individual bats (fig. 2) . forsman et al. (2008) found similar results, and showed that humoral bactericidal activity was negatively related to cutaneous immune activity (pha martin et al. (2006b) found that some immune responses can negatively affect other recent immunological activity in female white-footed mice, when examining simultaneous wound healing and cutaneous immune response. our data suggests that individuals may not be able to maximally activate all aspects of immunity owing to competing costs and the variety of strategies associated with a multiple-component immune system (klasing 2004) . t cell mediated inflammation aids in the clearance of viruses (hooper et al. 1998) ; therefore individuals at greater risk of coming in contact with viruses, such as rabies in bats, may be selected to invest more into adaptive immune defenses, potentially at the expense of reduced innate immune responsiveness. in addition to rabies and other lyssaviruses, brazilian free-tailed bats may be incidental hosts to various arboviruses, including the flaviviruses (family flaviviridae, genus flavivirus) rio bravo virus (rbv; constantine and woodall 1964) , st louis encephalitis (sle; allen et al. 1970; herbold et al. 1983) , and west nile virus (wnv; davis et al. 2005; pilipski et al. 2004) , and the alphaviruses (family togaviridae, genus alphavirus) eastern equine encephalitis (eee), and western equine encephalitis (wee) (constantine 1970) . the infection cycle for these arboviruses occurs primarily between birds and arthropods, with incidental infection of mammals generally not producing significant viremia to act as amplifying hosts or facilitate transmission. enzootic foci for arboviruses are generally swampy areas, drainages, or irrigated agricultural land, with infection occurring primarily during the summer months. both risk factors are relevant for roosting aggregations of brazilian free-tailed bats, particularly colonies that roost over standing water (bridges) or near irrigated agricultural lands (both caves and bridges). infection with flaviviruses or alphaviruses should induce a viral neutralizing antibody response that confers lifelong immunity against the specific virus responsible for infection. in the context of our data, we would predict that bats which are unable to mount strong t cell mediated antibody responses would be more likely to succumb to viral infection. in subclinical cases, bats with lower t cell mediated response may develop limited protection against future or related exposures. determining the ecological factors that predict variation in the ability of individual brazilian free-tailed bats to exhibit functional immunological responses is important to understand disease dynamics and population health in this and other species of colonial animals. a number of variables can affect the ability of organisms to combat pathogens and viral infections, and this study is a first step toward understanding susceptibility related to immunocompetence. a companion paper investigating links between roosting ecology and disease exposure (rabies virus) is currently in review. evidence from this current study suggests that immune responsiveness and presumably disease susceptibility are linked to the roosting ecology of the host. we predict that the immunotypic composition of a colony will influence pathogen transmission and persistence in free-ranging bats (dimitrov et al. 2006 (dimitrov et al. , 2007 . energetically costly aspects of immunological defense may also impose tradeoffs and energetic limitations for other seasonal behaviors and life-history functions that are important for survival. from this perspective the emerging field of ecoimmunology has contributed to our deeper understanding of the role of immunological competence in population regulation and the evolution of coloniality (lochmiller 1996) . the implications of such studies are further enhanced by examining species that are known to play a role in infectious disease transmission. studies of arthropod-borne virus infections in chiroptera: viii. evidence of natural st. louis encephalitis virus infection in bats age determination in bats 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birds estimates of population sizes in summer colonies of brazilian free-tailed bats (tadarida brasiliensis) contribution of bats to the maintenance of histoplasma capsulatum in a cave microfocus antibacterial properties of serum from the american alligator (alligator mississippiensis) bats, emerging virus infections, and the rabies paradigm parasitism, host immune function, and sexual selection immune defense and host sociality: a comparative study of swallows and martins variable effects of yolk androgens on growth, survival, and immunity in eastern bluebird nestlings autumn migration and selection of rock crevices as hibernacula by big brown bats in colorado monitoring trends in bat populations of the united states and territories: problems and prospects. us geological survey, biological resources discipline west nile virus antibodies in bats from new jersey and new york the economics of immunity roosting behavior of the mexican freetailed bat (tadarida brasiliensis) in a highway overpass ecological immunology: costly parasite defenses and trade-offs in evolutionary ecology offspring body condition and immunocompetence are negatively affected by high breeding densities in a colonial seabird: a multiscale approach is cell-mediated immunity related to the evolution of life-history strategies in birds? constitutive innate immunity is a component of the pace-oflife syndrome in tropical birds ecology of parasitism on brazilian free-tailed bats (tadarida brasiliensis) discovery of herpesviruses in bats acknowledgments we thank nickolay hristov, jonathan reichard, sarah duncan, cynthia schmaeman, and laura borrelli for countless hours of assistance with fieldwork. we thank irving marbach, dwayne davis, and bill cofer for allowing access to their property. special thanks to pat morton and meg goodman (texas parks and wildlife), mark bloschock (texas department of transportation), bain walker (hill country adventure tours), and barbara french (bat conservation international) for support in the field, and to margaret and j. david bamberger (selah-bamberger ranch) for hospitality. additionally, we thank christopher richardson and james sullivan for help with statistical analysis. funding was provided by grant eid 0426082 from nsf/nih program for the ecology of infectious disease to g.f.m. and t.h.k. all experimental procedures comply with the current laws of the united states of america. key: cord-005012-bgo0uwob authors: hiller, thomas; rasche, andrea; brändel, stefan dominik; könig, alexander; jeworowski, lara; teague o’mara, m.; cottontail, veronika; page, rachel a.; glebe, dieter; drexler, jan felix; tschapka, marco title: host biology and anthropogenic factors affect hepadnavirus infection in a neotropical bat date: 2018-12-18 journal: ecohealth doi: 10.1007/s10393-018-1387-5 sha: doc_id: 5012 cord_uid: bgo0uwob the tent-making bat hepatitis b virus (tbhbv) is a hepadnavirus closely related to human hepatitis b virus. the ecology of tbhbv is unclear. we show that it is widespread and highly diversified in peters’ tent-making bats (uroderma bilobatum) within panama, while local prevalence varied significantly between sample sites, ranging from 0 to 14.3%. females showed significantly higher prevalence than males, and pregnant females were more often acutely infected than non-reproductive ones. the distribution of tbhbv in bats was significantly affected by forest cover, with higher infection rates in areas with lower forest cover. our data indicate that loss of natural habitat may lead to positive feedback on the biotic factors driving infection possibility. these results underline the necessity of multidisciplinary studies for a better understanding of mechanisms in pathogen–host relationships and for predictions in disease ecology. electronic supplementary material: the online version of this article (10.1007/s10393-018-1387-5) contains supplementary material, which is available to authorized users. over the last century, the majority of the world's biosphere has been vastly transformed. many forests are lost or fragmented due to plantations and farmland and traversed by road networks (ellis et al. 2010; ellis 2011) . the loss of natural habitat directly results in loss of biodiversity and drives many specialist species to extinction. in contrast, some generalist species can persist and occasionally even profit from changes in the environment, with some showing inflated population densities in disturbed areas (fahrig 2003; suzán et al. 2008) . fragmentation of habitats has been shown to decrease health and fitness of animals and to promote diseases, parasitism, and virus occurrence (keesing et al. 2006; acevedo-whitehouse and duffus 2009; cottontail et al. 2009; brearley et al. 2013) . highly diverse tropical forests, in particular, are especially affected by habitat loss, as the functioning of these ecosystems depends on intact animal assemblages (laurance et al. 2011; aide et al. 2013) . bats are a good indicator group for habitat health as they are highly diverse and play major roles in pollination, pest control and seed dispersal (jones et al. 2009; cunto and bernard 2012) . in addition to their importance for ecosystem functioning, bats also are long-lived (seim et al. 2013) and can have large group sizes and high mobility (luis et al. 2013) , all of which make them suitable model organisms for epidemiological studies (drexler et al. 2012; brook and dobson 2015) . bats were identified as hosts to a great variety of viruses, some of them causing zoonotic diseases (e.g., ebola, sars, rabies), but research has focused mainly on rna viruses, leaving dna viruses largely unexplored (calisher et al. 2006; jones et al. 2008; brook and dobson 2015) . of particular interest among the dna viruses are orthohepadnaviruses that infect mammals (rasche et al. 2016) . hepatitis b, a representative of these orthohepadnaviruses, is one of the most common and serious viral infectious diseases in humans, causing acute and chronic infections of the liver, resulting in an estimated 900,000 deaths each year (who global hepatitis report 2017). transmission occurs chiefly by direct contact with infected blood (who fact sheet #204, updated july 2016). in humans, the course of infection depends on the time of virus acquisition. infection with hepatitis b virus (hbv) during birth results in 90% of chronic courses, while infection during early childhood leads to chronicity in 30% of all cases. in contrast, over 95% of infections acquired by immune-competent adults result in acute and self-limiting course (seeger and mason 2000; gish et al. 2015) . chronic courses of infection may be a common feature of orthohepadnaviruses. in woodchucks, an animal model of hbv, perinatal infection with the woodchuck hepatitis virus (whv) results in 100% chronic courses infection (seeger and mason 2000) . orthohepadnaviruses are known from north american rodents, non-human apes, and humans (locarnini et al. 2013 ) but were recently also found in bats, both in the old world and in the new world (drexler et al. 2013; he et al. 2013 he et al. , 2015 . the tent-making bat hepatitis b virus (tbhbv), isolated from peters' tent-making bats (uroderma bilobatum) in panama, is antigenically closely related to primate hbv (drexler et al. 2013) . drexler et al. (2013) showed that tbhbv surface proteins allowed specific viral binding and in vitro entry into human hepatocytes via the human hbv high-affinity receptor ntcp (natrium taurocholate co-transporting protein), providing an interesting viral model organism. uroderma bilobatum is a widespread, highly mobile frugivorous bat in the neotropics that is specialized on eating figs. it commonly occurs in secondary forests and constructs its own roosts by modifying leaves of seven different plant families, often palms, into tent-like shelters (baker and clark 1987; rodríguez-herrera et al. 2007 ). it adapts well to anthropogenically modified habitats and readily roosts in the eaves of human houses, as well as in the leaves of non-native palms such as cocos nucifera, coccothrinax barbadensis, or livistona chinensis (timm and lewis 1991; rodríguez-herrera et al. 2007 ) that are often planted as garden ornamentals (sagot et al. 2013) . its potentially close contact with humans, the relatedness of tbhbv to primate hbv, and the ability of tbhbv to use the human ntcp are factors supporting the importance of virus ecology studies in u. bilobatum. we investigated environmental and host traits favoring tbhbv infection as well as patterns of transmission and distribution in u. bilobatum. between september 2012 and august 2015, we collected blood samples of peters' tent-making bat, u. bilobatum (phyllostomidae: stenodermatinae) in panama. the samples were mainly obtained in the panama canal zone (around barro colorado island (bci) and gamboa) as well as from the regions bocas del toro, los santos, and veraguas ( fig. 1) , representing a total of 46 capture locations. capturing and handling of bats were approved by the smithsonian tropical research institute (iacuc protocols 100316-1001 (iacuc protocols 100316-18 and 2013 (iacuc protocols 100316-0401-2016 and by the panamanian government (ministerio de ambiente, research permits: se/a-68-11, se/a-75-13, se/a-69-14; export permits: sex/a-37-14, sex/a-22-15, and sex/a-60-15). bats were captured using mist nets (ecotone, 6 9 2.5 m, 16 mm mesh and 2 9 70 denier) set in the forest or with hand nets directly at the roost. individual bats were kept separately in freshly cleaned, soft cloth bags until processing. sex, age [juvenile, subadult, and adult; based on grade of ossification of the epiphyseal plates (sharifi et al. 2008) ], and reproductive status (scrotal and non-reproductive in males; pregnant, lactating, post lactating and non-reproductive in females) were determined, and adult bats were individually marked with necklaces following handley and gardner (1991) or pit tags (id100; euro id, weilerswist, germany). up to 70 ll of blood was drawn with edta coated capillaries (75 ll, kabe, germany) by puncturing the cephalic vein with a 27 g needle (sterican, braun, germany) (cottontail et al. 2009 ). after processing, all bats were offered sugar water and released at the capture site. blood samples were stored on ice in the field and thereon at -80°c until processing in the laboratory. dna was purified from blood specimens using the magna pure 96 dna and viral na small volume kit (roche, mannheim, germany). following protocols described in drexler et al. (2013) , purified samples were screened for hepadnavirus dna using a broadly reactive nested-pcr leading to amplification of a 354 bp fragment encoding the surface and polymerase proteins. near-full genome amplifications were conducted using strain-specific primers (primer sequences available upon request). viral loads were quantified by specific real-time pcr as described before (drexler et al. 2013) . to detect specific antibodies against tbhbv proteins in bat serum, immunofluorescence assays were conducted using huh7 cells transfected with 1.1 overlength expression plasmids of tbhbv as described (drexler et al. 2013 ). sequences were aligned with geneious 9.1.3 using the mafft package (version 7.222). dnasp 5.10.01 was used to detect point mutations within a near-complete genome alignment of 15 novel tbhbv sequences and 4 published sequences (nc_024445, kc790379, kc790380, kc790381) (drexler et al. 2013) . the antigenic loop within the s-orf was predicted in tmhmm (v.2.0). neighbor-joining phylogenetic analysis of a 2503 nucleotide fragment was conducted in mega7 (kumar et al. 2016 ) using the complete deletion option and a percentage distance substitution model including a bootstrap analysis with 1000 replicates. all statistics were performed in r v3.3.1 (r development core team 2016). we tested for virus genetic isolation by geographic distance using a mantel test (mantel(), r package vegan, oksanen et al. 2016 ). tip-randomization tests for capture year and capture region were conducted using bats (parker et al. 2008) . therefore, we used the pcr screening fragments available from all positive samples, including a total of 24 sequences and a sequence length of 354 nucleotides. we used mcmc trees created in beast v.1.8.3 (drummond and rambaut 2007) using a hky substitution model under a strict clock and 10 million generations (burn-in 1 million). as a basis for further analyses, we extracted data for forest cover and forest cover loss using arcmap (esri 2011) and maps based on landsat images provided by the university of maryland (hansen et al. 2013 , http://eartheng inepartners.appspot.com/science-2013-global-forest). we used a 1 km radius around each capture site (resulting in 314 ha), based on recent telemetry studies estimating the home range of u. bilobatum at around 23 ha (villalobos-chaves et al. 2016) . forest cover was calculated based on a map of tree canopy cover density (0-100%) from 2000, setting the threshold at 85% density, which resulted in 99% of the tree canopy cover density values for each of the protected study sites (bohio, gigante, and peña blanca). forest cover loss was extracted from a map representing the loss of forested areas during the period 2000-2014, encoded as percental loss of forested area in a 1 km radius around each capture site. using generalized linear mixed effects models (glmer(), r package lme4, bates et al. 2007 ), we identified the variables best predicting tbhbv infections. initial data exploration was conducted following zuur et al. (2010). our full model consisted of the infection status (infected vs. non-infected, binomial distribution) as a response variable and the variables sex, reproductive status, percent forest cover, and percent forest cover loss in a 1 km radius around each capture site as fixed effects. we also included the year of capture as well as the capture location nested in capture region as random effects. we scaled the fixed variables and tested for multi colinearity using variance inflation factors (vifs) implemented in the r library aed (zuur 2010). further, we tested for spatial autocorrelation of infected bats in our data set as well as in model residuals of our candidate models using moran's i test (moran.i(), r package ape, paradis et al. 2004) . the best models were selected based on corrected akaike's information criterion (aic c ) and the 'all-subset approach' as described in (symonds and moussalli 2011) (dredge(), r package mumin, barton 2016) . parameter estimates were obtained using model averaging of set of top models, based on d2 aic c as cutoff (burnham and anderson 2003; symonds and moussalli 2011) . for all models included in the top-model set, we calculated pseudo-r 2 values to estimate model fit by accounting for the variation explained by both fixed and random effects (rsquared.glmm(), r packaged mumin). pregnant and lactating females were grouped together with scrotal males as reproductive, to obtain a single reproductive status variable for both females and males. to identify biotic and abiotic factors influencing tbhbv infections, we included in our statistical analysis 54 u. bilobatum blood samples from a former study (drexler et al. 2013) , as well as 7 additional individuals of the same sampling period that had not been previously analyzed, all collected in the bci canal zone. we collected a total of 474 individual blood samples, from which 20 (4.2%) tested positive for tbhbv dna (table 1, supplementary table 1 ). immunofluorescence analyses revealed the presence of antibodies against tbhbv in samples from 12 additional tbhbv pcr-negative individuals and showed positive reactions in 4 of the pcrpositive samples. the detection rate of antibody-positive bats (3.4%) was comparable to pcr-positive bats (4.2%). ratios of ift-positive to pcr-positive varied between 0 and 4.5 and differed significantly between the sampling sites bci and gamboa (pairwise fisher's test with bonferroni correction, p < 0.01). the viral loads were high with up to 3.20 9 10 12 copies per ml serum and an average of 1.96 9 10 11 cp/ml serum (see table 1 for details). near-complete tbhbv genomes (2503 nt, whole genome comprises 3149 nt (drexler et al. 2013) were sequenced from 15 individual samples and shorter fragments for the five remaining samples. all sequences were uploaded to genbank and are accessible under the following numbers: mg231914-mg231918 and mg252537-mg252551. to detect hints for immune pressure, nonsynonymous substitutions were analyzed among the genome of 19 sequences, including 4 formerly published tbhbv sequences (drexler et al. 2013) (fig. 2) . accumulation of mutations was detected in presumably antigenically active sites within the surface protein-coding open reading frame (orf), including c-terminal amino acids within the pres1 domain and the predicted antigenic loop. in a phylogenetic analysis, viruses clustered with previously described tbhbv strains from panama (drexler et al. 2013) (fig. 3) . virus strains did not show any clustering according to year of sampling using a tip-random-ization test including all available tbhbv fragments (354 nt). further, clustering according to sampling sites was only significant for two regions, namely gamboa and veraguas (table 2) . some virus strains sampled among large geographic ranges showed low genetic distance (fig. 3) , for example, b404 from los santos, which is closely related to sequences from the bci canal zone, approximately 200 km away. indeed, using a mantel test, no support for isolation by distance was detected (mantel test, r = 0.023, p = 0.166). combining results of pcr-and ift-screening females exhibited a higher prevalence (9.7%) than males (3.8%) (chi-squared, x 2 = 5.55, df = 1, p = 0.019). local prevalence of pcr-positive bats varied between 0 and 14.3%. all variables used in our mixed models to predict tbhbv infections had variance inflation factors (vifs) below 3, indicating the absence of colinearity among those variables (zuur et al. 2010) . we further did not detect spatial colinearity between our observations (moran's i, i = 0.00, p = 0.24). to ascertain that no regional effect is driving our modeling outcome, we excluded during our preliminary model selection process individual sample regions, however, we obtained throughout consistent results. of the 16 models created by the all-subset model approach (supplementary table 3 ), only two entered in the d2 aic c model set (table 3) . both top models shared three variables, the extrinsic factor forest cover as well as the intrinsic variables sex and reproductive status, indicating a high importance of these variables. for decreasing forest cover the local tbhbv prevalence was increasing; additionally, females as well as reproductive active individuals tended to have a higher prevalence compared to males or non-reproductive active individuals, respectively (table 4 ). the variable forest cover loss was slightly positively correlated with higher tbhbv prevalence, but it was weighted fairly low when averaged over all subset models (table 4 ). model residuals of both top models did not show spatial autocorrelation (moran's i, i = -0.00, p = 0.91; i = -0.00, p = 0.99). the conditional pseudo-r 2 values estimating model fit for the two top models were 0.33 and 0.34, respectively (table 3) . based on these two models incorporating 535 individual observations, we plotted the predicted probabilities of a tbhbv infection showing all three significantly explaining variables, whereas forest cover loss was kept constant in the graph for the second model (ggeffect(), r package ggeffects, lüdecke 2017). the graphs show probabilities at least ten times higher in areas without forest cover than in fully forested areas (fig. 4) and fit well the distribution of the raw data used (fig. 5) . our model implicates therefore a strong influence of the available forest cover throughout all combinations of sex and reproductive status on the infection probability with tbhbv. despite the high similarity of tbhbv to human hepatitis b virus, no cross-species infections between the natural reservoir species u. bilobatum and humans are reported, nor expected (drexler et al. 2013; rasche et al. 2016) . in fact, this close relatedness offers an exceptional possibility to study disease transmission among host bats in their natural environment, as mechanisms and restrictions might be directly adapted from well-studied host-virus interaction in humans and animals (seeger and mason 2000; menne and cote 2007; wang et al. 2011 ). our study shows that tbhbv infections in u. bilobatum occur not only in the panama canal zone where they were initially discovered (drexler et al. 2013 ), but are actually widespread in panama (more than 10.000 km 2 ). detected viral loads in infected bats were very high (mean 1.96 9 10 11 ge/ml serum) and are comparable to highest levels reached in chronically infected woodchucks and untreated chronic human patients (seeger and mason 2000) . since high viral load is a predictor for hbv-related liver cirrhosis, hepatocellular carcinoma, and increased mortality in chronic human patients (chen et al. 2009 ), similar consequences on the health of chronically tbhbvinfected bats are imaginable, but further studies are necessary for confirmation. near-complete genome sequencing of 15 new virus strains suggests that the currently observed tbhbv infections did not originate from a single outbreak in the canal zone, but are widely distributed across panama. since closely related tbhbv sequences are found in distant sample sites and the diversity at individual sample sites reflects the overall tbhbv diversity combined with the relatively slow evolution of hepadnaviruses, a long-term relationship between the bat host u. bilobatum and tbhbv in panama can be assumed (littlejohn et al. 2016 ). all samples cluster together with sequences previously published (drexler et al. 2013) , confirming the assignment as tbhbv. we did not find significant clustering of samples within years of collection, coinciding with the relatively slow virus evolution. however, samples collected in veraguas as well as in gamboa clustered more often together than expected by chance. these samples were frequently obtained from bats sharing the same roost or were roosting in close vicinity to each other, suggesting a largely unobstructed gene flow of virus strains within a given population. clearly, we found no indication of genetic isolation by distance, suggesting a largely unobstructed gene flow of the virus strains within the country. this concurs with the wide distribution, high mobility, and abundance of this common bat species (baker and clark 1987; meyer et al. 2009; mantilla-meluk 2014) . notably, tbhbv strains from the gamboa sampling site are dispersed among the phylogenetic tree, embracing the complete tbhbv diversity at this particular site. here, in contrast to the other sampling sites, u. bilobatum were frequently observed roosting in the village of gamboa closely together under overhanging roofs (o'mara et al. 2017) . suitable roosting conditions are known to attract bats and favor the formation of larger groups (campbell et al. 2006) . a recent study has shown that resident bats are attracted to unknown, new individuals, possibly to obtain information about new food sources, which might lead to close contact between roost members and unfamiliar individuals (ramakers et al. 2016) . together this might increase the genetic diversity of tbhbv, especially in areas with limited high-quality roosts. the analysis of non-synonymous mutations among the tbhbv genome revealed a clustering of amino acid substitutions within the c-terminus of the pres1 coding region and within the predicted antigenic loop of the surface protein. equivalent sites are exposed to immunological pressure in the human hbv (sloan et al. 2008; glebe and bremer 2013) , therefore hinting at similarities within the immunological response in tent-making bats and humans infected with hepadnaviruses. in addition, the pres1 region represents a favored aim for non-synonymous mutations since the overlapping polymerase orf does not code for any functional elements in this region, but rather serves as a spacer between the terminal protein and the reverse transcriptase . the combination of pcr and immunofluorescence analyses enabled us to distinguish between different stages of infections, if we assume a similar etiopathology of tbhbv as observed in hbv infections of humans. the occurrence of individuals with only an antibody response without the actual presence of viral dna (n = 12) suggests that u. bilobatum may have overcome an infection with tbhbv (seeger and mason 2000) . we identified also acute infections, in which the tbhbv dna was present, while no antibody reaction could be detected (n = 16), indicating recently obtained infections (seeger and mason 2000; liang 2009 ). most of these individuals were caught within 3 months after the main mating period in february-march and september-october (fleming et al. 1972) . bats tested positive for both tbhbv dna-and tbhbv-specific antibodies (n = 4) may represent acute infections preceding seroclearance or chronic infections (seeger and mason 2000; liang 2009 ). to actually confirm chronic infections, consecutive sampling of tbhbv dna positive individuals over at least two time points is necessary, but difficult to achieve in the field. the prevalence of current tbhbv infections detected in the bci canal zone area (0.8%, n = 262) was lower than the prevalence previously reported for the same study area (9.3%, n = 54; drexler et al. 2013) . the original description of tbhbv (drexler et al. 2013 ) was based on a much smaller sample size (54 vs. 262 individuals), and the high prevalence might have been influenced by the fact that four of the five positive bats were caught in the same night at the same fruiting fig tree (cottontail, pers. comm.) and might represent roost mates. o'mara et al. (2014) showed that u. bilobatum roost mates may pass on the information of a promising food source and might therefore share foraging sites and show group foraging behavior. the social organization of u. bilobatum might favor the transmission of tbhbv within a roosting group. groups may consist of polygynous harems of 5-50 (kunz 1982; chaverri et al. 2010 ) individuals, with one male per 5-10 females, but the animals are also found to roost in multi-male or multifemale groups (chaverri et al. 2010) . assuming a similar possibility for sexual transmission of tbhbv as in hbv, there are two general ways of transmission within a harem group: (1) the male is a chronic carrier, transmitting tbhbv onto the females, or (2) an infected female joins the harem, and passes the virus to the male, which thereafter infects the remaining females. both hypotheses lead to a female-biased infection prevalence typical for polygynous mating systems (ashby and gupta 2015) as it was observed in this study, with females showing a higher prevalence (9.7%) than males (3.8%). furthermore, chronically infected pregnant females with high viral load will most likely transmit the virus to their newborns during birth, which is a hallmark for all orthohepadnaviruses, including human hbv. perinatal transmission will induce chronic infections in 90% of offspring (gish et al. 2015) . in conclusion, sex and reproductive state were significant factors affecting the probability of an infection with tbhbv. in addition to these intrinsic factors, we identified forest cover as a significant external factor (fig. 3) . the loss of habitat and therefore also of natural roosting options changes the roosting behavior of u. bilobatum (sagot et al. 2013) . their high flexibility, adaptability, and ability to roost in ornamental palms in close vicinity to humans (rodríguez-herrera et al. 2007; sagot et al. 2013 ) and occasionally even under the eaves of houses (o'mara et al. 2017 ) allow them to persist in areas with relatively low densities of natural roosting options. roosting under house eaves could offer energetic benefits by eliminating the necessity of building a new tent every few months (chaverri et al. 2010) . loss of natural habitat and the corresponding native roost plants may therefore lead to higher densities of bats in areas with coconut palms, which are most often cultivated in and around villages (chaverri et al. 2007; sagot and stevens 2012; sagot et al. 2013) . higher densities may also lead to larger social groups, in which females might be more receptive to stress than males as they show higher cortisol levels when restrained (o'mara et al. 2017) , possibly weakening the immune system (rønsholt et al. 1998 ; van der poel et al. 2000) , which coincides well with our observed female-biased infection prevalence. lewis (1992) showed that groups in anthropogenic influenced areas are more stable due to this scarceness of possible roosts compared to natural habitats, favoring virus transmission within the group. roosting in gardens and houses bring bats in close contact to humans, who, if noticing the bats, often attempt to expulse them by destroying their tents (sagot and stevens 2012; streicker et al. 2012 ). however, this might result in raising infection levels among bats, as evicted bats subsequently try to enter other colonies, causing distress and raising fight frequency. through enforced roost site changes, already infected bats may directly introduce tbhbv into healthy colonies (kerth et al. 2002; streicker et al. 2012 ) and may cause more rapid spreading in anthropogenic areas than in natural habitats. as tbhbv is closely related to human hepatitis b, the close contact to humans of a possible reservoir species might sound alarming, but current studies reveal no imminent risk of any cross-species infections between bats and humans in their natural habitat. further, most bats pose no risk to humans at all and often go unnoticed, but contribute critically important ecosystem services such as pollination, seed dispersal, and pest control (kunz et al. 2011 ). nevertheless, this study emphasizes the necessity of a better understanding of host-virus systems in the wild. many aspects, host sociality for example, remain unexplored, but could prove highly important in the propensity for disease transmission luis et al. 2013) . although the number of sampled bats in our study was extensive, the low infection prevalence and therefore the low number of positive bats entering our models may limit the precision of our predictions. nevertheless, our results are significant and we are confident that our interpretation is well supported. we demonstrate that the loss of natural habitat can promote the spreading of tbhbv and can increase the probability of u. bilobatum becoming infected with tbhbv. anthropogenically altered habitats provide ideal circumstances for the spread of infection, for example, offering conditions promoting numerous, large roosting groups in close vicinity to each other, roosting conditions that typically avoided in pristine habitats lacking human disturbance. as demonstrated by estrada-peña et al. 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and control epidemiological considerations of rodent community composition in fragmented landscapes in panama a brief guide to model selection, multimodel inference and model averaging in behavioural ecology using akaike's information criterion tent construction and use by uroderma bilobatum in coconut palms (cocos nucifera) in costa rica characterisation of a recently isolated lyssavirus in frugivorous zoo bats the influence of sex and reproductive status on foraging behavior and seed dispersal by uroderma convexum (chiroptera: phyllostomidae). in: sociality in bats establishing a new animal model for hepadnaviral infection: susceptibility of chinese marmota-species to woodchuck hepatitis virus infection hepatitis b fact sheet aed: data files used in mixed effects models and extensions in ecology with r zuur af, ieno en, elphick cs (2010) a protocol for data exploration to avoid common statistical problems we want to thank the smithsonian tropical research institute, especially oris acevedo and belkys jimenez, for providing the infrastructure and logistics for field work. we are grateful to all people of the bci and gamboa batlabs for help and assistance in the field. we further want to thank nina schwensow for a crash course in statistical analysis of molecular data and two anonymous reviewers who helped to improve the manuscript. this study was funded by german research foundation (dfg) spp 1596 grants dr 810/1-1, gl 595/4-1, and ts 81/6-1 (to j.f.d., m.t., and d.g.), which had no influence in study design or interpretation of the results. conflict of interest the authors declare that they have no conflict of interest. key: cord-010307-sxh5mq1q authors: milne, d. j.; armstrong, m.; fisher, a.; flores, t.; pavey, c. r. title: structure and environmental relationships of insectivorous bat assemblages in tropical australian savannas date: 2005-11-23 journal: austral ecol doi: 10.1111/j.1442-9993.2005.01535.x sha: doc_id: 10307 cord_uid: sxh5mq1q abstract patterns in the composition of assemblages of microbat species sampled during the late dry season (the ‘build‐up’) in north australian savannas were assessed against a range of environmental factors as well as four a priori defined habitat types (riparian, escarpments, coastal and woodlands). distinct species assemblages were most strongly associated with topographic and climatic variables. there were also limited associations with vegetation structure, fire and local roost potential but no associations with insects or water availability. total species diversity at sample sites was associated with distance to rivers and rainfall. in general, species assemblages were not clearly defined and the number of significant environmental associations was relatively few. we compare these associations with those reported for bat assemblages elsewhere in australia. understanding of the diversity and evolutionary ecology of australia's mammal fauna has not been uniform across orders. in particular, most detailed tests of evolutionary hypotheses (e.g. johnson 1998; fisher et al . 2001 ) omit bats (order chiroptera). assessments of population trends and extinction proneness have also excluded chiroptera (e.g. johnson 2002) . this is a significant shortcoming as bats represent over 30% of australia's mammal species, many of which are endemic. although australian mammal diversity peaks in the tropical forests of eastern queensland including cape york peninsula, significant diversity also occurs in the savannas of north-western australia where 94 species are known ). an assessment of the response of mammals within this region to 23 environmental variables revealed that a single environmental gradient (of substrate and disturbance) described the distribution of all species, excluding bats . rock-inhabiting mammals are a significant component of this fauna, however, diversity of this assemblage decreases with decreasing outcrop size and increasing isolation. identified three other trends. first, that the mammal fauna of eucalypt open forest/woodland habitats of north-western australia is characterized by extensive distributions of its component species. sec-ond, that monsoon forests support a depauperate mammal fauna. last, that the mammal fauna of this region undergoes substantial latitudinal change associated with a steep north-south rainfall gradient. did not include systematic sampling of bats, preventing a rigorous examination of the response of the bat fauna to environmental measures. however, data from captures (mist netting, harp trapping, roost searches) indicated that most bat species were present across the environmental range sampled ). here we revisit the issue of the response of bats to environmental variables in the tropical savanna of the northern territory and north-west queensland using a more rigorous data set. our data collection incorporated the use of ultrasonic detectors to sample bats and geographic information system (gis) derived variables to represent environmental conditions. the study region supports a rich microbat fauna (26 of australia's 65 species, 15 of australia's 20 genera), including one endemic species ( taphozous kapalgensis ), and both of australia's monotypic genera ( rhinonicteris , macroderma ). we assessed environmental factors at two levels, first at the landscape scale, using data available from a gis, and second at a local scale where information was collected on the physical environment and food resource availability (insects) at individual sampling sites. we predicted that the high vagility of bats would result in species responding broadly to environmental variables. however, specific responses to a number of environmental variables were expected. in particular, we predicted that the distribution, composition and segregation of bat assemblages would respond to geographical patterns in annual rainfall, presence of rocky escarpments, water bodies and canopy cover. although a relationship with insect abundance and composition was examined, we predicted that this relationship would not be significant given the generalist feeding ecology of most insect-eating bats (fenton 1990) . the bat assemblages of tropical australian savannas are also compared with assemblages elsewhere in australia. specifically, we compared our results with community composition and environmental association studies in north queensland rainforest (crome & richards 1988) , mangroves in north-western western australia (mckenzie & muir 2000) , and open forest/ woodland in victoria (kutt 1995; lumsden & bennett 1995; herr 1998) , south-east new south wales (nsw) (law et al . 1999 ) and tasmania (taylor & o'neill 1988) . the study area, called the top end of australia, included the tropical savanna of the northern terri-tory and north-west queensland, north of 18 ° s, but excluding offshore islands (fig. 1) . across this area, maximum mean weekly temperature ranges between 32 ° c and 39 ° c and mean annual rainfall between 360 mm and 1720 mm (houlder 2000 ; fig. 1 ). rainfall is highly seasonal with almost all precipitation occurring from november to april. topographic relief is relatively low. the maximum elevation is 553 m on the arnhem land plateau, with the main areas of topographic relief being the kakadu escarpment and the eastern edge of the kimberley region in the southwest of the study area. eucalypt woodlands and forests dominate 78% of the study area (fox et al . 2001) . other notable environments include monsoon rainforests and floodplains dominated by sedgelands and grasslands. on average, over half (52%) of the top end is burnt every year (a. edwards, pers. comm. 2004) . a total of 39 sampling sites were located across the top end (fig. 1) 1 . map of the study area showing the location of sampling sites and site labels as well as average annual rainfall isohyets (in millimetres). sites are symbolized according to bat assemblage (diamond = group 1, square = group 2, cross = group 3, triangle = group 4, circle = group 5). sonal variation on species composition. each site was a circular plot of 100 m radius. plots were primarily selected to cover a large geographical area and to sample four broad habitat types: 1. riparian -adjacent to perennial rivers, creeks or permanent waterholes (10 sites). 2. escarpments -sandstone cliffs (11 sites). 3. coastal -coastal and near coastal environments (excluding estuaries and mangroves) (eight sites). 4. woodlands -continuous areas of eucalypt woodlands or open forests not associated with the other habitats types (10 sites). habitat types were chosen a priori and were based on information gleaned from species' distribution maps and descriptions of microbat habitat preferences (strahan 1995; churchill 1998 ) that suggested these habitats may contain distinctive species assemblages. two sites were usually sampled at a time on the same nights. with one exception, no two sites within a sampling pair sampled the same habitat type. distances between sampling pairs ranged between 2 km and 30 km (mean 10 km). at each site we used a range of sampling techniques to maximize the likelihood of obtaining a full inventory of bat species (kuenzi & morrison 1998; murray et al . 1999; o'farrell & gannon 1999) . bats were sampled using two (18 sites) or three (21 sites) harp traps over two consecutive nights as well as one night of shot sampling for a 3-h period after dusk. harp traps were usually placed across 'flyways' (tracks, streams or other gaps within the vegetation where bats are more likely to be trapped) and were either positioned side by side or spaced between 20 and 30 m apart. we also conducted active searches of caves, road culverts and any other features potentially used as diurnal roosts by bats within 400 m of the centre of the sampling site. in addition, bat calls were recorded at every site with ultrasonic bat-detectors (anabat ii, titley electronics, ballina, australia) using two methods. the first method involved placing a detector on the ground, elevated to approximately 40 ° , and operated from dusk for a cumulative total of at least six recording hours over two consecutive nights (maximum 22 h, mean 20 h). this time period has been shown to sample 90% of species calls at a given site (milne et al . 2004) . detectors were connected to either an anabat ii delay switch with output recorded to 90-min cassette tape (sony chrome ux, tokyo, japan) via tape recorder (optimus ctr-115 d, fort worth, usa) (18 sites) or an anabat v zcaim and computer (toshiba portégé 3440ct or toshiba tecra 700ct d, tokyo, japan) running anabat6 software in monitor mode (21 sites). there are no differences in the species detected between these two recording techniques (milne et al . 2004) . for the second method, an anabat detector was held in the hand and manually activated on detection of a bat-call and actively pointed in the direction of the call. calls were recorded via tape-recorder and cassette tape, for 3 h after dusk for one night. all recorded calls were identified according to milne (2002) . at several sites (14), shot sampling was not permitted. instead we trapped bats using mist-nets at these sites. it is likely therefore, that some 'high-flying' bat species that are readily detected using shot sampling, may not have been trapped at these sites. however, we expect this will have a negligible effect on our results as shot sampling at all other sites, used in conjunction with anabat detectors, enabled us to collect an extensive reference call library for 'high-flying' bat species for the entire study area (milne 2002) . anabat detectors were systematically used at all sites and will normally detect 'high-flying' species that are not readily trapped (o'farrell & gannon 1999) . we collated environmental data for each site from field habitat measurements, analysis of spatial data and insect sampling. at the centre of each site we measured tree basal area, canopy cover and stem count, 10 m either side of a 100-m transect (0.2 ha) in an area of undisturbed vegetation usually adjacent and parallel to flyways where harp traps were set. on escarpment sites, the transects either traversed the escarpment or were situated at the base of the escarpment. basal area and stem counts were derived by measuring diameter at breast height (d.b.h.) of every tree along the transect, whereas canopy cover was measured using a spherical densiometer at 0 m, 50 m and 100 m along the transect. for the entire site (3.1 ha), we measured slope, maximum canopy height, crown cover, rock cover, distance to water and local roost potential. crown cover in three height classes (1-3 m, 3-10 m and > 10 m) was estimated using crown separation ratios (mcdonald et al . 1998 4 -mostly large trees and rock outcrop with large cracks and holes we chose to assess whole trees and rock outcrops rather than count individual hollows because small microbats ( < 10 g) can roost in hollows equivalent to their own body diameter (pers. obs. 2004). entrances to these hollows are very small and would regularly be overlooked if we attempted to count hollows directly. large trees have been shown to contain more tree hollows than smaller trees (whitford 2002) and are preferred roost sites for many bat species (lunney et al . 1988; herr & klomp 1999; law & anderson 2000; lumsden et al . 2002) . several variables were derived using gis from a 3 s ( c . 100 m) digital elevation model (dem, provided by the department of defence) including elevation, ruggedness index (the range in cell values of the dem within a 3 × 3 cell neighbourhood), and distance to 25 and 100 m 'escarpments' (defined here as any adjacent dem cells having an altitude difference of 25 or 100 m). climate variables (annual mean temperature, minimum monthly temperature and annual rainfall) were derived using bioclim (houlder 2000) . other gis data included fire frequency (number of years in which the site was burnt over the preceding 7 years) and years since last fire (data sets provided by the bushfires council of the northern territory), distance to perennial rivers, and ndvi (normalized difference vegetation index, which is a measure of vegetation 'greenness' derived from satellite imagery) and projective foliage cover (meakin et al . 2001) . at each site we trapped flying nocturnal insects for one night concurrently with bat sampling. the insect trap was constructed from a white cotton sheet (1.5 m × 2.5 m), suspended off the ground by strings tied to the corners to form a funnel, one end higher than the other. at the bottom of the funnel a hole was cut in the sheet and a plastic jar (65 mm diameter × 130 mm depth) partially filled with 70% ethanol was attached to hang underneath. a 12-v fluorescent light ('col-light' brand col-lite, maleny, australia) was hung from the higher end of the sheet to attract insects. the trap was positioned approximately 100 m from the anabat detector so as not to disturb bats from natural flight habits in the vicinity of the detector, and left unattended for the entire night. insects that fell into the jar were collected the following morning. in the laboratory, insect samples were filtered through a 2-mm sieve to remove the smallest insects (mostly < 3 mm in length) and then identified to order and assigned to four size (head-body length) classes: < 5 mm, 5-10 mm, 10-15 mm and > 15 mm. the choice of size classes was based on the range of body sizes found to be prey items of bats in tasmania (o'neill & taylor 1989) . analysis of bat communities was based on species presence-absence at each site derived from the combination of all sampling methods. anabat calls for the following combinations cannot be reliably separated in the top end: (i) chalinolobus nigrogriseus , scotorepens greyii and scotorepens sanborni ; (ii) miniopterus schreibersii and pipistrellus westralis ; and (iii) nyctophilus arnhemensis , nyctophilus bifax and nyctophilus geoffroyi (milne 2002) . anabat call sequences that were attributed to these species combinations were therefore excluded from the analysis, although species within these combinations were included if identified using one of the physical sampling methods. the one exception was s. greyii and s. sanborni which cannot be readily separated in the field (churchill 1998 ) and were treated here as a single species although in some areas their distributions are allopatric (mckenzie & muir 2000) . species assemblages were assessed using patn software (belbin 1994) . similarities in species composition between sites were calculated using the bray-curtis association measure. cluster analysis (unweighted pair group mean) was used to define assemblages (groups of sites) following visual inspection of the dendrogram. anosim (clarke & green 1988 ) was used to test whether bat species composition differed significantly between the defined assemblages as well as the four a priori habitat types. the relationship between sites was also portrayed by ordination (multidimensional scaling) of sites by their bat species composition. in both analyses, only sites with at least three species were included. all environmental variables (table 1) were continuous or rank ordered. variables were initially compared using the spearman rank correlation test. where pairs of variables had a correlation coefficient greater than 0.8, one of the pair was excluded from further analysis. the mean of each environmental variable was calculated for each group of sites derived from the cluster analysis and the significance of differences between bat assemblage groups was tested using kruskal-wallis anova . the relationship between environmental variables and the arrangement of sites in the ordination space was also tested using vector fitting (kantvilas & minchin 1989) . finally, generalized linear modelling (glm; crawley 1993) was used to develop a predictive habitat model for total site species richness. a poisson error distribution and log link function was used and a backward stepwise procedure was adopted to generate the minimum adequate model with only those variables having a significant correlation in the vector fitting included in the model development. a total of 23 microbat species were identified from the 39 sites, representing over 80% of the species recorded from north-western australia . two species known to occur in the top end , macroderma gigas (ghost bat) and saccolaimus saccolaimus were not detected in this study. we identified five groups from the classification of all sites by their species composition (fig. 2) . the initial classification divided the sites into four groups. we subdivided the largest of these groups into two and assigned two outlying sites to group 1 based on the relative position of these sites in the ordination. anosim analysis confirmed that the groups differed significantly in composition ( r = 0.70, p < 0.001) and that there was a significant difference between each pair of groups ( p < 0.01 or better). the occurrence of bat species within the derived groups and habitat types is summarized in table 2 and the geographical distribution of sites (classified according to group) is shown in fig. 1 . four species were ubiquitous throughout the groups and habitats ( chaerephon jobensis , pipistrellus adamsi, mormopterus loriae and saccolaimus flaviventris ) while three species were each detected at single sites only ( hipposideros diadema , hipposideros stenotis and m. schreibersii ). the distribution of sites in ordination space and the relationship with environmental vectors is shown in fig. 3 . a total of 14 environmental variables were significantly correlated with variation in species composition between sites (table 3) . a summary of mean values for these variables for each group is provided in table 4 . a description of species composition and the environmental characteristics for each group is provided below. species that were detected most often in this group include chalinolobus gouldii (present in all sites), c. jobensis, s. flaviventris, s. greyii/s. sanborni and p. adamsi. chalinolobus gouldii was strongly associated with this group (i.e. tended to occur in group 1 more than the other four groups). group 1 had the highest total species richness and mean site species richness of all groups. sites were characterized by high percent-age canopy cover, frequent burning and high annual rainfall and were located in the north and west of the top end. the greatest number of sites occurred in this group (14) . species that were detected most often include vespadelus caurinus (present at all sites), s. flaviventris , taphozous georgianus and c. jobensis . v. caurinus was strongly associated with this group. sites were characterized by rugged, steep rocky slopes, high elevations and short distances to escarpments and rivers. minimum temperatures were cool and annual rainfall low. sites were widely distributed across the top end, except the coastal zone. species detected most often in this group include s. flaviventris, n. arnhemensis, myotis macropus and s. greyii/s. sanborni. n. arnhemensis was strongly associated with this group. group 3 was not clearly associated with any of the environmental variables measured and occupied an intermediate value on most environmental gradients. however, this group displayed the highest mean values for minimum temperature. figures represent percentage of sites within each group or habitat in which each species was detected. differences in proportions were tested using χ 2 statistic and differences in mean species richness were tested using kruskal-wallis anova (ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001). this group had equal fewest sites (five) and had the lowest total and mean site species richness. species detected most often include c. jobensis (present at all sites) s. flaviventris, m. loriae and c. nigrogriseus. there were no strong species associations, although c. nigrogriseus occurred at proportionately more sites in this group than any other. sites were characterized by lower mean annual temperatures, long distances to rivers and no rock cover. species detected most often include s. flaviventris, p. westralis (both present at all sites), p. adamsi, m. loriae and t. kapalgensis. p. westralis and t. kapalgensis were strongly associated with this group. group 5 also had relatively few sites and low species richness, but was associated with the minima or maxima of several environmental variables including long distances to escarpments, flat terrain at low elevations with no rock, low local roost potential, high annual temperatures and low fire frequency. all five sites were located near the coast (fig. 1) . there was a significant difference in species composition between habitat types (anosim, r = 0.35, p < 0.001) as well as between all pairwise combinations of habitats except between 'woodland' and 'riverine' (r = 0.037, p = 0.27). v. caurinus and t. georgianus were detected most often in 'escarpment' habitat. both of these species, as well as rhinonicteris aurantius and c. nigrogriseus, were absent from 'coastal' habitat. p. westralis was strongly associated with 'coastal' habitat and absent from both 'escarpment' and 'riparian' habitat. n. arnhemensis was also absent from 'escarpment' habitat. all habitats had similar total and site species richness, with slightly lower species richness in the 'coastal' habitat. the relationship between groups and habitats is summarized in table 5 . the habitat type of each site was not independent of group classification (χ 2 = 32.54, p < 0.01). most of the 'escarpment' sites occurred in group 2 (steep, rocky, rugged sites) with two further sites in group 1. 'coastal' sites mainly occurred in group 3 (few environmental correlates) and group 5 (flat, low elevation), whereas 'riparian' sites occurred across four of the groups and 'woodland' sites were evenly represented across all five groups. we found no significant associations between bat species assemblages and various measures of insect availability including total number of insects, total number of insect orders, total number of insects in various size classes, proportion of insects in various size classes or total number of insects in each order. values are the mean for sites in each group, h-values refer to the kruskal-wallis statistic (ns; not significant; *p < 0.05; **p < 0.01; ***p < 0.001). habitat modelling identified distance to perennial rivers and annual rainfall as the major predictors for site species richness (table 6 ). the minimum adequate model was only moderately robust with 40% of the deviance captured. this suggests that there was considerable 'noise' in the data or that some important explanatory variables were not quantified. as predicted, the insectivorous bat fauna of northwestern australia responded broadly to most environmental variables. the main environmental feature associated with the distribution of microbat assemblages in the study area was topography (variation in elevation, slope, topographic ruggedness and distance to escarpments). not surprisingly, therefore, species considered to be obligate cave roosters (hipposideros ater, h. diadema, h. stenotis, m. macropus, m. schreibersii, r. aurantius, t. georgianus, v. caurinus, vespadelus finlaysoni) , mainly occurred in (but were not restricted to) groups 1 and 2 which were associated with high values for the topographic variables. although we expected a relationship between microbat assemblages and distance to escarpments, the significant effect of elevation was not predicted. elevation generally increased away from the coast and was auto-correlated with 'distance to coastlines', making it unclear which of these features was most important in influencing bat composition. the second factor influencing bat composition was climate, specifically annual rainfall (corrolated with maximum temperature and latitude), mean temperature and minimum temperature (corrolated with temperature range; refer table 1 ). the influence of annual rainfall was expected given the relationship between rainfall and species composition exhibited by the entire mammal fauna of north-western australia . a similar pattern was shown by the vegetation (bowman et al. 1988 ) and birds (whitehead et al. 1992 ) of north-western australia. in contrast to the significant relationship identified between bat assemblages and mean climatic variables, there was no significant relationship between ambient temperature (measured at 10 pm each sampling night) at each site and species composition. at the time of year that we sampled, temperature was unlikely to limit the number of bat species that were active. however, during the dry season, low inland temperatures may reduce insect activity, restrict bat activity to the earlier, warmer times of the night and/or induce some species to enter torpor. therefore, restricting our sampling to one period of the year may have affected our results but sampling at different times of the year would have required a much greater sampling effort. between years, there was no observable difference in general weather patterns during each sampling period, therefore inter-year variations were unlikely to have affected our results. there were significant associations between bat species assemblages and fire frequency (corrolated with time since fire). the effects of fire on landscapes in northern australia can depend on the number of times an area is burnt and on the time since last fire (assessed here), fire intensity, seasonal timing of fires and spatial extent of burning (dyer et al. 2001; andersen et al. 2003) . the link between fire and bat species composition is likely to be an indirect one. it is also possible that characteristics of the landscape such as fuel loads, geography and habitat type are actually the primary influence for species assemblages and fire frequency is a secondary consequence of these landscape characteristics. therefore, our results should be viewed with caution and further investigation into the effects of fire on bat species assemblages is required before conclusions can be drawn. we assessed several variables involving insect availability at each site. none of the variables showed any significant relationship with microbat assemblages. this suggests that, in the top end at least, available food resources do not influence the composition of bat communities. this conclusion was consistent with previous research on insect-eating bats that indicated most species capture prey opportunistically (fenton minimum adequate models and explanatory power (per cent of deviance captured) are shown. probability levels *p < 0.05, **p < 0.01. 1990). specific research on tasmanian bats also concluded that bat assemblages were generally opportunistic foragers (o'neill & taylor 1989) . four aspects of our sampling strategy may have influenced our analysis. first, we did not sample non-volant insects and other arthropods that are eaten by some bat species in the top end (e.g. spiders, c.r. pavey, unpubl. data 2004) . second, high flying insects that are preyed on by bats such as taphozous spp. were probably not attracted to our light trap. third, bats may only show a response to insects at certain times of the year. it is likely that at the time of sampling (late dry season), insects were abundant and food resources did not affect the activity of bats. fourth, insect sampling was limited to one night per site, which may not have been sufficient to provide an adequate representation of overall insect availability. therefore, we suggest that a combination of insect sampling methods should be used in future assessments of prey availability and bat assemblages, particularly when the diversity of bats is high. these methods should aim to sample volant and non-volant invertebrates. did our study adequately sample a cross section of the major environmental gradients in the top end? compared with much of australia, the environment of the top end is relatively uniform. landscape relief is low, woodlands dominate most of the landscape, temperature varies little throughout the year and the climatic gradients are gradual. therefore, environmental variation is relatively small, and fewer sampling sites should be required compared with areas with greater topographic, climatic and vegetative variation. however, there may have been two significant deficiencies in our sampling. first, the highly seasonal rainfall in the monsoon tropics results in starkly contrasting 'wet' and 'dry' seasons. from our study, we were unable to say how bat composition may vary seasonally and there are no data available to assess seasonal patterns. second, the chosen study area was huge (530 000 km 2 ) and most of the north-east of the top end (arnhem land) was unsampled. therefore, clearer patterns of bat assemblages may have emerged if we had sampled more comprehensively, both spatially and temporally. most sites (34) were sampled in pairs and the minimum distance between any two sites was 2 km (mean 10 km). bats can travel long distances during the night. foraging distances for a selection of species range between 1 km and 10 km (herr & klomp 1999; law & anderson 2000; lumsden et al. 2002) , therefore some of our results were potentially autocorrelated due to the same bats being sampled at both sites within a pair. therefore, we assessed the similarity in site species composition using the bray-curtis index. this index was calculated by dividing the number of shared species between pairs of sites by the total number of species of both sites. the resulting value was plotted against the distance between each pair of sites (fig. 4) . the scatter of points was highly variable, however, the slope of the regression line was shallow. this pattern indicated that the relative change in species composition as a result of geographical separation was small. one of the environments largely neglected during sampling was monsoon rainforest, although 'riverine' sites did sample components of monsoon rainforest environments. we considered this had little effect on our results because monsoon rainforests occupy just 0.5% of the landscape (based on mapping by fox et al. 2001 ) and usually occur in patches less than 5 ha (russell-smith 1991) . in addition, menkhorst and woinarski (1992) found no bat species that were tightly associated with monsoon rainforests in the top end and these forests support a depauperate mammal fauna in general . some of the environmental variables that we found to be significantly correlated with bat assemblages in the top end differed from those related to bat community variation in other areas of australia. waterbodies have been found to support high species diversity and some species are strictly associated with them (law et al. 1998 ; south-west slopes of nsw.; young & ford 2000; central western queensland) . in the top end, glm analysis suggested bat species richness increases with decreasing distance to perennial rivers. however, species richness was not exceptionally high at our 'riparian' sites. further, group 2, which was on average closest to rivers, did not have the highest species diversity. also, we found no significant difference in species assemblages between 'riverine' and 'woodfig. 4 . similarity in bat species composition between pairs of sites (using the bray curtis similarity index, refer text) plotted against the distance between each pair. graph also displays the fitted linear regression line. land' habitats and there was no relationship with distance to available surface water. given that sampling was carried out during the driest time of the year (late dry season september-november) the likelihood of detecting significant associations with waterbodies was maximized. a relationship between vegetation structural complexity and microbat diversity has been established in studies in western australia (mckenzie & muir 2000) and nsw (law et al. 1998) . by contrast, we found significant correlations of bat species diversity with canopy cover but no associations with structural complexity. compared with the vegetation in the areas sampled by mckenzie and muir (2000) and law et al. (1998) , the vegetation of the top end is usually shorter and contains fewer understorey layers (d. lewis pers. comm. 2004 ). this limits the degree of vegetation structural complexity in the top end that likely accounts for the lack of correlation between structural complexity and bat communities. although we identified significant differences between the species assemblages within classification groups and habitat types, the assemblages were not clearly defined. most species occurred in more than one group and some were present in all groups. in addition, there were no associations between insect variables and bat assemblages and relatively few associations with environmental variables. this pattern is not restricted to microbats. birds, reptiles and nonvolant mammals also exhibit 'loose' patterns of species composition (woinarski & fisher 1995; woinarski et al. 2000) and limited associations with particular environments and environmental gradients woinarski et al. 1999) in the top end. woinarski et al. (1999) suggested this trend was a consequence of the homogeneity of eucalypt woodlands and forests that dominate the top end landscape. this relatively uniform environment militates against highly specialized and habitat-specific faunas. however, there were exceptions. specifically, some microbat species had a clear association with rugged rocky areas, particularly escarpments and adjacent areas. these areas provided a complex mix of habitats that contained foraging and roosting sites suitable for both cave and tree roosting species. this pattern extended to other vertebrate species as well. rocky escarpment regions in the top end support high species diversity as well as a number of endemic or habitat restricted species (woinarski & gambold 1992; . vegetation corridors beside rivers and surrounding areas (but not the waterbodies themselves) appeared to be important environments as they supported high bat species richness. bats are regularly characterized by the foraging strategy they employ within their immediate environment (mckenzie & rolfe 1986; neuweiler 1989; schnitzler & kalko 1998) . rivers are often associated with environments with tall dense vegetation. these areas do not appear to be of conservation significance because we did not observe high species richness at our 'riparian' sample sites. however, riverine environments usually have a distinct outer 'edge' and vegetation surrounding these areas is usually shorter and relatively open. we propose that these areas had greater species richness as they provide a diversity of environments for bats that employ different foraging strategies. we recommend that further research be conducted to examine the relationship between rivers and bats in the top end. our study did not take into account longer-term bat population dynamics. bats in the top end are poorly surveyed and, with few exceptions, surveys have been unstructured and unsystematic. therefore, attempting to identify and compare historical trends in bat populations is very difficult. given the top end environment is (currently) relatively unmodified, it could be assumed that mammal populations will remain stable and secure over the short to medium term. unfortunately, this is not the case. woinarski et al. 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biogeographic patterns, environmental correlates and conservation of avifauna in the northern territory hollows in jarrah (eucalyptus marginata) and marri (corymbia calophilla) trees. i. hollow sizes, tree attributes and ages gradient analysis of a tropical herpetofauna: distribution patterns of terrestrial reptiles and amphibians in stage iii of kakadu national park wildlife of lancewood, acacia shirleyi thickets and woodlands in northern australia. 1. variation in vertebrate species composition across the environmental range occupied by lancewood vegetation in the northern territory gradient analysis of the distribution of mammals in stage iii of kakadu national park, with a review of the distribution patterns of mammals across north-western australia distribution patterns of vertebrates in relation to an extensive rainfall gradient and variation in soil texture in the tropical savannas of the northern territory bird distribution in riparian vegetation in the extensive natural landscape of australia's tropical savanna: a broad-scale survey and analysis of a distributional data base changes in mammal populations in relatively intact landscapes of kakadu national park bat fauna of a semi-arid environment in central western queensland our thanks go to bryan baker, chaz delacoeur, chris grant, david holland, marieke lettink, tony mitchell, steve owen, terry reardon, nathalia velez and felicity watt for their help with field work. greg connors, andrew edwards and peter brocklehurst assisted with various aspects of the analysis. john sheppard tracked down many of the references. thanks to irene rainey and peta jones who did a wonderful job with data entry. many parks and wildlife rangers and landholders assisted in planning surveys and locating suitable sampling sites. john woinarski, chris johnson, brad law, michael bull and an anonymous referee provided valuable comments on the manuscript. d.m. also thanks john woinarski for his support throughout this project. the tropical savannas crc provided funding for this project. animal sampling procedures were approved by the n.t. animal ethics committee (ref: a02001). key: cord-268645-5op2m7pu authors: wu, zhiqiang; yang, li; ren, xianwen; he, guimei; zhang, junpeng; yang, jian; qian, zhaohui; dong, jie; sun, lilian; zhu, yafang; du, jiang; yang, fan; zhang, shuyi; jin, qi title: deciphering the bat virome catalog to better understand the ecological diversity of bat viruses and the bat origin of emerging infectious diseases date: 2015-08-11 journal: the isme journal doi: 10.1038/ismej.2015.138 sha: doc_id: 268645 cord_uid: 5op2m7pu studies have demonstrated that ~60%–80% of emerging infectious diseases (eids) in humans originated from wild life. bats are natural reservoirs of a large variety of viruses, including many important zoonotic viruses that cause severe diseases in humans and domestic animals. however, the understanding of the viral population and the ecological diversity residing in bat populations is unclear, which complicates the determination of the origins of certain eids. here, using bats as a typical wildlife reservoir model, virome analysis was conducted based on pharyngeal and anal swab samples of 4440 bat individuals of 40 major bat species throughout china. the purpose of this study was to survey the ecological and biological diversities of viruses residing in these bat species, to investigate the presence of potential bat-borne zoonotic viruses and to evaluate the impacts of these viruses on public health. the data obtained in this study revealed an overview of the viral community present in these bat samples. many novel bat viruses were reported for the first time and some bat viruses closely related to known human or animal pathogens were identified. this genetic evidence provides new clues in the search for the origin or evolution pattern of certain viruses, such as coronaviruses and noroviruses. these data offer meaningful ecological information for predicting and tracing wildlife-originated eids. emerging infectious diseases (eids) pose a great threat to global public health. approximately 60%-80% of human eids originate from wildlife, as shown by the typical examples of hemorrhagic fever, avian influenza and henipavirus-related lethal neurologic and respiratory diseases that originated from rodents, wild birds and bats (jones et al., 2008; wolfe et al., 2007; lloyd-smith et al., 2009; marsh and wang, 2012; smith and wang, 2013) . the primary issues associated with the prevention and control of eids are how to quickly identify the pathogen, determine where it originated and control the chain of transmission. these issues, along with the limited knowledge of the viral population and ecological diversity of wildlife, complicate the study of eids. therefore, meaningful information afforded by the understanding of the viral community present in wildlife, as well as the prevalence, genetic diversity and geographical distribution of these viruses, is very important for the prevention and control of wildlife-borne eids daszak et al., 2000) . bats are mammals with a wide geographical distribution, extensive species diversity, unique behaviors (characteristic flight patterns, long life spans and gregarious roosting and mobility behaviors) and intimate interactions with humans and livestock (calisher et al., 2006) . they are natural reservoirs of a large variety of viruses, including many important zoonotic viruses that cause severe diseases in humans and domestic animals, including henipaviruses, marburg virus and ebola virus (luis et al., 2013; quan et al., 2013; o'shea et al., 2014) . the severe acute respiratory syndrome (sars) outbreak in 2003, which resulted in nearly 8000 cases and 800 deaths worldwide, was suspected to have originated in bats and then spread to humans (li et al., 2005) . all of these examples reveal that zoonotic viruses carried by bats can be transmitted directly or via certain intermediate hosts from bats to humans or domestic animals with high virulence. as most bat-borne pathogens are transmitted by four routes (airborne, droplet, oral-fecal and contact transmission) from the respiratory tracts, oral cavities or enteric canals of bats to other species, it is particularly important to determine the viral communities present at these locations. in this study, bat individuals of 40 representative species across china were sampled by pharyngeal and anal swabbing, to assess the variety of viruses residing in bat species. metagenomic analysis was then conducted to screen the viromes of these samples. here we outline the viral spectrum within these bat samples and the basic ecological and genetic characteristics of these novel bat viruses. the identification of novel bat viruses in this study also provides genetic evidence for cross-species transmission between bats or between bats and other mammals. these data offer new clues for tracing the sources of important viral pathogens such as sars coronavirus (sars-cov) and middle east respiratory syndrome cov (mers-cov). pharyngeal and anal swab samples were collected separately and then immersed in virus sampling tubes (yocon, beijing, china) containing maintenance medium and were temporarily stored at − 20°c. the samples were then transported to the laboratory and stored at − 80°c. viral nucleic acid library construction, next-generation sequencing and taxonomic assignments a tube with either a pharyngeal or anal swab sample in maintenance medium was vigorously vortexed to re-suspend the sample in solution. samples from the same bat species and from the same site were then pooled. the pooled samples were processed with a viral particle-protected nucleic acid purification method and then amplified by sequenceindependent reverse transcriptase-pcr as described previously wu et al., 2014) . briefly, the samples were centrifuged at 10 000 g for 10 min at 4°c. supernatant from each sample was filtered through a 0.45-μm polyvinylidene difluoride filter (millipore, darmstadt, germany), to remove eukaryotic and bacterial-sized particles. the filtered samples were then centrifuged at 100 000 g for 3 h at 4°c. the pellets were re-suspended in hank's balanced salt solution. to remove naked dna and rna, the re-suspended pellet was digested in a cocktail of dnase and rnase enzymes, including 14 u of turbo dnase (ambion, austin, tx, usa), 20 u of benzonase (novagen, darmstadt, germany) , and 20 u of rnase one (promega, madison, wi, usa) at 37°c for 2 h in 1 × dnase buffer (ambion). the viral nucleic acids were then isolated using a qiamp minelute virus spin kit (qiagen, valencia, ca, usa). viral first-strand cdna was synthesized using the primer k-8n (5′-gaccatctagcgacct ccac-nnnnnnnn-3′) and a superscript iii system (invitrogen, carlsbad, ca, usa). to convert firststrand cdna into double-stranded dna, the cdna was incubated at 37°c for 1 h in the presence of 5 u of klenow fragment (neb, ipswich, ma, usa) in 1 × neb buffer 2 (final volume of 25 μl). sequenceindependent pcr amplification was conducted using 1 μm primer k (5′-gaccatctagcgacct ccac-3′) and 0.5 u phusion dna polymerase (neb). the pcr products were analyzed by agarose gel electrophoresis. a dna smear of larger than 500 bp was excised and extracted with a minelute gel extraction kit (qiagen). the amplified viral nucleic acid libraries were then analyzed using an illumina ga ii sequencer (illumina, sandiego, ca, usa) for a single read of 81 bp in length. the raw sequence reads were filtered using previously described criteria , to obtain valid sequences. each read was evaluated for viral origin by conducting alignments with the ncbi nonredundant nucleotide (nt) database (nt) and protein database (nr) using blastn and blastx (with parameters -e 1e-5 -f t). reads with no hits in nt or nr were further assembled using the velvet software (v1.2.10, pittsburgh supercomputing center, pittsburgh, pa, usa) and the contigs were again aligned with nt and nr to identify any viruses that were present. the taxonomies of the aligned reads with the best blast scores (e-value o10 − 5 ) from all lanes were parsed and exported with megan 4-metagenome analyzer . besides the alignment-first analytical strategy, we also tested assembly-first strategy to analyze the sequence data. the reads were firstly assembled by metagenomics assemblers (for example, metavelvet and idba-ud) (namiki et al., 2012; peng et al., 2012) and the output contigs were then aligned to nt and nr. as the percentage of viral reads was small in the whole data sets and most of the assembled contigs were thus of bacterial and host origins, the assembly-first strategy generally had lower sensitivity than the alignmentfirst strategy in this study. identification of the prevalence and positive rate of each virus according to the molecular clues provided by metagenomic analyses, the sequence reads classified into the same virus family or genus by megan 4 were extracted and then assembled with seqman program (lasergene, dnastar, madison, wi, usa). a draft genome with several or a large number of single-nt polymorphisms of each virus was obtained. based on the partial genomic sequences of the viruses obtained by the assembly, we designed specific nested primers for pcr or reverse trancriptase-pcr to screen for each virus in individual samples from each bat species (the primer sequences for each virus are available in supplementary table s2 ). genome sequencing of each virus in positive samples by pcr the accurate locations of the reads and the relative distances between reads of the same virus were determined based on the alignment results exported using megan 4. representative positive samples for each virus were selected for genome sequencing as viral quasi-species. the reads with accurate genomic locations were then used for reads-based pcr to identify partial genomes. based on the partial genomic sequences obtained by specific nested pcr, the remaining genomic sequences were determined using inverse pcr, genome walking, and 5′-and 3′ rapid amplification of cdna ends. genome sequences and phylogenetic and evolutionary analyses all genome sequences have been submitted to genbank. the accession numbers for all sequences are listed in supplementary table s2 . the ga ii sequence data have been deposited into the ncbi sequence reads archive under the accession number sra051252. routine sequence alignments were performed using megalign program (lasergene) or t-coffee with manual curation. mega5.0 (phoenix, az, usa) was used to align the nt and amino acid (aa) sequences using muscle package with the default parameters. the best substitution model was then evaluated using model selection package. finally, a maximum-likelihood method with an appropriate model was used to conduct phylogenetic analyses with 1000 bootstrap replicates. supplementary table s1 . bat viromes in china z wu et al main bat species, the 17 most common genera and 6 families of both frugivorous and insectivorous bats that reside in urban, rural and wild areas throughout china. all pharyngeal and anal swab samples were classified and combined into 84 pools and then subjected to virome analysis . finally, a total of 179 gb of nt data (2 241 761 959 valid reads, 81 bp in length) was obtained. in total, 20 632 698 reads were best matched with viral proteins available in the ncbi nr database (~0.8% of the total sequence reads). the number of virusassociated reads in each lane varied between 8283 and 2 919 423. in total, 79 families of phages, insect viruses, fungal viruses, mammalian viruses and plant viruses were parsed. after excluding bat habit-related non-mammalian viruses, including insect viruses (mainly of the families baculoviridae, iflaviridae, dicistroviridae and tetraviridae; subfamily densovirinae), fungal viruses (mainly of the families chrysoviridae, hypoviridae, partitiviridae and totiviridae), phages (the order caudovirales and families inoviridae and microviridae) and plant viruses (matched reads of the 60 non-mammalian virus families are provided in supplementary table s23 ), overview of the reads of 19 families of mammalian viruses in each pooled sample is shown in figure 2a and supplementary table s24 . in addition, an overview of the classification from family to genus of the identified bat viruses is shown in figure 2b . the reads related to the family parvoviridae comprised the largest proportion of viruses as shown in figure 2 , most of which were classified into the subfamily densovirinae. the dominating abundance of insect densoviruses was associated with the insectivorous habits of bats. the existence and prevalence of virus strains in 17 of the 19 families were confirmed. in total, 692 positive results were confirmed by pcr screening (positive rates are shown in supplementary table s2 ) and 144 viruses of representative positive samples were selected for genomic sequencing as quasi-species of these viruses (supplementary table s2 ; the 94 full-length sequenced viruses are labeled in red). the most widely distributed families of mammalian viruses were herpesviridae, papillomaviridae, retroviridae, adenoviridae and astroviridae. the diverse reads related to these families occupied~61% of the total viral sequence reads (supplementary table s24 ). the assessment of diverse reads of bat herpesviruses, bat papillomaviruses, bat retroviruses, bat astroviruses and bat adenoviruses (supplementary figure s2) , and full-length sequenced representative strains of these viruses revealed that most of these viruses were distinct from each other within each family (30%-65% nt identities). in addition to the above families, many reads related to the families circoviridae, paramyxoviridae, coronaviridae, caliciviridae, polyomaviridae, rhabdoviridae, hepeviridae, bunyaviridae, reoviridae, flaviviridae and picornaviridae, and the subfamily parvovirinae, exhibited low nt or aa sequence identities with known viruses. although sequence reads related to the families orthomyxoviridae and hepadnaviridae were occasionally present in some of the samples, we failed to amplify any sequences of viruses in these families, which may have been a result of very low viral loads. although samples from 307 frugivorous bats of two species (rousettus leschenaultia and cynopterus sphinx) were collected for virome analysis, only a few reads related to herpesvirus, papillomavirus and retrovirus were found. this finding revealed that the virome of frugivorous bats is far less abundant than that of insectivorous bats in this study. bat main single-stranded rna viruses (coronaviridae, paramyxoviridae and picornaviridae) the family coronaviridae contains two subfamilies, coronavirinae and torovirinae. the subfamily coronavirinae includes four approved genera, alphacoronavirus, betacoronavirus, deltacoronavirus and gammacoronavirus. it is a group of large enveloped viruses with a positive single-stranded rna genome (~26 to 32 kb in length). six αand β-covs (hku1, oc43, nl63, 229e, sars-cov and mers-cov) are human pathogens that cause mild-tosevere disease (corman et al., 2014b; li et al., 2005; cui et al., 2007; o'shea et al., 2014) . here, 30 novel bat covs (btcovs) were identified separately in 12 bat species of 9 genera from 15 provinces (supplementary table s2 and supplementary figure s1 ). covs from seven bat species are reported for the first time. phylogenetic trees were constructed based on the deduced rna-dependent rna polymerase (nsp12) (figure 3a spike (s) and figure 3b proteins). the sequence identities of these btcovs are shown in supplementary table s4 . eleven btcovs were assigned to a group with lineage-b beta-covs and three btcovs were assigned to a group with lineage-c beta-covs. the btvs-betacov/sc2013 identified in vespertilio superans bats had a closer genetic relationship with mers-cov than with other btcovs (supplementary figure s3) , as well as with neocov identified in african neoromicia capensis bats (corman et al., 2014a) . one btcov, bthp-betacov/zj2013, represented a separate clade related to lineage b. the overall nt identity of this cov genome with lineage-b covs was only 58% and the rna-dependent rna polymerase and s proteins shared only 78% and 41% aa identities with those of lineage-b covs. this cov contained an unusual putative s-related orf2 between orf1ab and the s gene (supplementary figure s4 and supplementary table s3 ) (quan et al., 2010) . a signal peptide (1-13 aa) and a transmembrane region (413-435 aa) were identified in the s-related orf2, suggesting that this protein might be a surface protein. the remaining 15 btcovs were all assigned to the genus alphacoronavirus and formed many novel separate clades. for btmr-alphacov/sax2011 and btnv-alphacov/sc2013, although the phylogenetic tree constructed based on rna-dependent rna polymerase indicated that these two viruses were clustered with hku10, the phylogenetic tree based on the s protein indicated that these two viruses represented two separate clades far from other covs, suggesting that recombination may occur in their genomes. although the orf1ab, e, m and n genes of btrf-alphacov/hub2013 and btms-alphacov/ z wu et al gs2013 shared very high sequence identities (higher than 98%), the s genes of these two viruses shared only 85% nt identity. a similar phenomenon was observed between btrf-alphacov/yn2012 and hku2. the results of sample-by-sample screening of bat samples from the same gathering place revealed that btcov strains of the same species identified in the same cave had significantly diverse features. some key gene segments, such as the s and orf8 genes, presented great diversity with low sequence identities (supplementary figure s5) , indicating that these two genes are hypervariable regions within the btcov genome. similar to recently reported sars-like covs (sl-covs) (wiv1, rs3367 and lyra11) (ge et al., 2013; he et al., 2014) , two btcovs, btrs-betacov/ yn2013 and btrs-betacov/gx2013, identified in rhinolophus sinicus from the yunnan and guangxi provinces shared the highest similarities with sars-covs in the backbone (including orf1ab, e, m and n genes), orf6, orf7a, orf7b and orf8 genes compared with other bat lineage-b β-covs (supplementary tables s11-s18 and supplementary figures s6a and b and s7) . furthermore, the s genes of lineage-b β-covs from r. sinicus had much higher genetic diversity and were scattered among the phylogenetic clades of sars-covs and lineage-b covs from other bat species (supplementary figure s6c) . co-infections of cov strains of sublineages 1 and 2 of group 1 in miniopterus fuliginosus were detected in two anal specimens collected in guangdong and henan. the covs of sublineage 1 with highly similar backbone sequences presented differing degrees of variation in the s region. recombination was confirmed by similarity plots, bootscan analysis and detection of putative breakpoints around the s regions in the genomes of lineage 1 covs in m. fuliginosus and m. pusillus. (supplementary table s21 and supplementary figure s8 ). the family paramyxoviridae is a group of large enveloped viruses with negative-sense singlestranded rna genomes (~15 to 19 kb in length) that are responsible for a variety of mild-to-severe human and animal diseases (mayo, 2002; smith and wang, 2013) . bat paramyxoviruses (btparavs) were separately identified in 9 bat species from 10 provinces of china (supplementary table s2 and supplementary figure s1 ). the full-length sequence of 3 viruses (btmf-parav/ah2011, btml-parav/ qh2013 and btha-parav/gd2012) were almost completely determined (supplementary figure s9 and supplementary table s6 ) and 11 other viruses were partially or completely sequenced in the l gene. twelve of the 14 novel btparavs identified in the different bat species could be clustered together and formed a separate phylogenetic clade. alternatively, these btparavs could be classified into a potential separate genus, shaanvirus, with btmf-parav/ah2011 and btml-parav/qh2013 as prototypes. the genomic organizations of these two viruses were similar to those of three previously reported members of the genus jeilongvirus. however, the genomes of btms-parav/anhui2011 and btml-parav/qh2013 were shorter in length than those of the three rodent viruses and the sequence identities were low (supplementary table s5 ). the remaining two novel btparavs could be clustered together with rubulaviruses as new species (figure 3c . the central domain of the n protein contained three conserved motifs common to all paramyxoviruses, and the six conserved domains within the l proteins of the order mononegavirales (lau et al., 2010) could be found in all three fulllength sequenced viruses. picornaviruses (picovs) of the family picornaviridae are small, non-enveloped, positive single-stranded rna viruses with a genome of 7-9 kb in size. the members of the family picornaviridae cause mucocutaneous, encephalic, cardiac, hepatic, neurological and respiratory diseases in a wide variety of vertebrate hosts (tracy et al., 2006; wang et al., 2015) . nineteen bat picovs (btpicovs) were identified in 11 bat species from 9 provinces (supplementary table s2 and supplementary figure s1 ). phylogenetic analysis of the rnadependent rna polymerase genes was conducted (figure 3d . seven btpicovs could be clustered with three previously reported btpicovs (clade 1) and could then be divided into separate sub-clades according to their host genera. clade 3 contained two sub-clades formed by four btpicovs of two bat genera. clade 2 was formed by two btpicovs identified in different bat genera and clustered in a sister relationship with the genus sapelovirus. clade 4 contained two btpicovs identified in the same bat genus that were closely related to the genus kobuvirus. the previously reported miniopterus schreibersii picov-1 was closely related to the genera cardiovirus and senecavirus. two btpicovs, btrf-picov-2/yn2012 and btmf-picov-1/sax2011, showed lower aa identities with other known picovs. different btpicovs identified from the same bat genus, such as rhinolophus or miniopterus, in different locations showed very close genetic relationships. the aa identities of the predicted rnadependent rna polymerase proteins of these novel btpicovs were low compared with known picovs (supplementary table s7 ). the predicted p1, p2 and p3 regions and cleavage sites of these viruses showed typical features of picovs (supplementary table s22 ). the family circoviridae is a group of viruses with small, non-enveloped, circular single-strand dna genomes of 1.7-3 kb in length (fauquet and fargette, 2005) . porcine circovirus (cv)-2 is the main swine pathogen (chae, 2005) . thirty-four novel bat cvs (btcvs) were identified in 13 bat species table s2 and supplementary figure s1 ). the genome sizes of these viruses varied from 1643 to 2979 nts. seven new clades of btcvs in the genera circovirus and cyclovirus were found. clade 2 was formed by two viruses clustered in a sister relationship with two pathogenic viruses, porcine and dog cvs (chae, 2005; li et al., 2013) , at the same root with a short branch length. three btcvs were closely related to human cycloviruses. seven btcvs were assigned to the proposed genus cyclovirus and formed four separate clades, three of which were closely related to human cycloviruses. a separate clade, clade 7, was constructed from two btcvs and was closely related to the genus cyclovirus. in addition to these viruses, 17 novel btcvs branched out of the root of cvs and cycloviruses revealed the presence of new genera different from the known genera (figure 4a) . viruses of the family parvoviridae comprise a group of small, non-enveloped viruses with linear positive-sense single-stranded dna (~5 kb genomes) that infect vertebrate animals and cause mild-tosevere diseases (brown, 2010) . bat parvoviruses (btpvs) and bat bocaviruses were identified in nine bat species from eight provinces (supplementary table s2 and supplementary figure s1 ). in addition to viruses in the genera bocavirus, parvovirus and amdovirus, four btpvs were most similar to the recently reported human bufavirus members and the bufavirus-related wuharv parvovirus, with similar ns1 and vp1 proteins (higher than 60% aa identities) (phan et al., 2012; yahiro et al., 2014) . bthp-pv/gd2012, in the genus parvovirus, was very closely related to a recently identified rat pv, with 89% aa identity (figure 4b) . the aa identities among the predicted vp1 proteins of these btpvs and bat bocaviruses, and other known members of the subfamily parvovirinae, are shown in supplementary table s8 . bat adeno-associated viruses have been described previously (li et al., 2010b) ; however, considering the nonpathogenic nature of adeno-associated viruses, we did not perform further verification of these viruses. other rare bat viruses six bat caliciviruses (btcalvs), four bat polyomaviruses, one bat hepatitis e virus, one bat rhabdovirus, one bat bunyavirus, one bat orthoreovirus and one bat rotavirus were identified. phylogenetic in previous studies, zoonotic viruses in more than 15 virus families have been identified in bats around the world (chen et al., 2014; o'shea et al., 2014) . two bat virome analyses conducted by li et al. (2010a) and donaldson et al. (2010) have revealed the presence of covs, herpesviruses, picovs, cvs, adenoviruses, adeno-associated viruses and astroviruses in some bat species of north america. one bat virome analysis conducted by ge et al. (2012) mainly described insect viruses in some bat species of china. one bat virome analysis conducted by ng et al. (2013) has revealed the presence of a novel rhabdovirus in big brown bats, and one bat virome analysis conducted by he et al. (2013) has described the spectrum of viruses harbored by several bat species in myanmar. different from these previous reports, this study was the first to characterize the pharyngeal and anal virome of representative bat samples in china. we did not perform additional verification of non-mammalian viruses because of the association of the abundance of these viruses (not initially harbored in bats) with their life habits. this report suggests that bats harbor a large spectrum of mammalian viruses. except for a few viruses, such as btcalvs, btcvs and btpvs, which are closely related to known viruses, most of the bat viruses identified here that were widely distributed . these findings reveal that these three bat genera may act as major reservoirs for diverse mammalian viruses in china. notably, all bats collected in this study were considered to be apparently healthy and showed no overt signs of disease, further confirming that bats can tolerate diverse viruses through their unique metabolic and immune systems (o'shea et al., 2014) . this study extends the host range for members of each viral family and reveals unique ecological and evolutionary characteristics of bat-borne viruses. the diverse btcovs were grouped into several novel evolutionary clades that significantly differed from those of all known αand β-covs, providing additional evidence to support investigations of the evolution of bat-originated covs. with regard to btparavs, a previous study has revealed that bats host major mammalian paravs in the genera rubulavirus, morbillivirus, henipavirus and the subfamily pneumovirinae (drexler et al., 2012) . however, in this study, except for 2 viruses assigned to the genus rubulavirus, the remaining 12 viruses formed a new genus distant from the known genera and the 14 identified btparavs showed no direct relationship with the known human or animal pathogens of the family paramyxoviridae. these results suggest an entirely different distribution of btparavs in china than previously reported. although the classifications of bat herpesviruses, bat papillomaviruses, bat retroviruses, bat astroviruses, bat adenoviruses, btpicovs, btcvs, btpvs and bat bocaviruses were extended according to the current virus taxonomy file released by the international committee on taxonomy of viruses, the large number of novel viruses grouped into the various evolutionary clades identified in this study further expand the taxa to include many new viral genera and species. many new clades formed by btcvs distinct from all known members of the genera circovirus and cyclovirus could be candidates for many new genera. viruses related to henipaviruses, ebola virus, rabies virus and pathogenic bunyaviruses were not detected in the chinese bat species examined in this study. diverse herpesviruses and papillomaviruses identified countrywide support the hypothesis that these dna viruses from different bat species are located in different phylogenetic positions within each family without strict host or geographic specificity (garcia-perez et al., 2014) ; however, many other dna or rna viruses, such as btcovs, btparavs, btpicovs and btpvs or bat bocaviruses, identified from the same or different bat species from different locations shared high sequence identities and close genetic relationships. these phenomena indicate that certain bat-originated dna and rna viruses have the potential for intra-or cross-species transmission concomitant with the migration, co-roosting and intra-or inter-species contact of their bat hosts. the identification of some viruses, such as certain rat pv-related btpv, norovirus-related btcalvs, human or swine cv-related btcvs and bat rotavirus in the rotavirus a group, also provides a new understanding of the evolution of these viruses in different mammalian hosts and possible transmission events that occur between bats and other hosts. furthermore, btcovs had more distinctive features than the other bat viruses. highly diverse s genes or orf8s were present in particular covs carried by bats of the same species from different locations or even the same gathering place. considering the diversity of covs, co-infections may create opportunities for recombination and the emergence of new covs that are able to adapt to new hosts. these findings may explain why tracing the potential cov-related eids in insectivorous bats is often complicated by the presence of diverse key genomic segments and no virus with an identical genome sequence related to the pathogens causing human or animal eids has been identified in insectivorous bats. instead, the origin of the ebola virus and henipaviruses could be relatively easily confirmed by the identification of identical viruses in frugivorous bats (calisher et al., 2006; leroy et al., 2005; smith and wang, 2013) . only lineage-b β-covs of six bat species (r. ferrumequinum, r. sinicus, r. pusillus, r. macrotis, r. affinis and chaerephon plicata) from china (sl-covs) are closely related to sars-covs, as similar orf1ab, e, m and n genes, and the presence of a unique structural orfs (including orf6, 7a, 7b and 8) have been identified (supplementary figure s10 ) (holmes and enjuanes, 2003; li et al., 2005; woo et al., 2009; quan et al., 2010; woo et al., 2012; yang et al., 2013) . recently, a functionally similar s gene has been identified in sl-covs of r. sinicus and r. affinis (wiv1, rs3367 and lyra11) with less sequence identity to the s gene of sars-cov, but which is capable of using the human ace2 as a receptor for virus entry (ge et al., 2013; he et al., 2014) . however, knowledge gaps exist between bat sl-covs and sars-covs with regard to the s gene and the unique structural orf that prevent the determination of which bat virus species is the direct ancestor of sars-cov. in this study, two btcovs (btrs-beta-cov/yn2013 and btrs-betacov/gx2013) in chinese horseshoe bats (r. sinicus) provided bat-originated unique structural orfs that were nearly identical to the original sars-cov isolated during the earliest phase of the sars pandemic (after transmission to humans, this region of sars-cov experiences ongoing adaptive evolution in humans, with gradual deletion (2004)), providing some information to fill the knowledge gap with regard to the origin of human sars-covs. in addition, the higher similarities of the backbones of these two btcovs to sarsa strategy to estimate unknown viral diversity in mammals evidence for camel-to-human transmission of mers coronavirus the expanding range of parvoviruses which infect humans bats: important reservoir hosts of emerging virusesclin a review of porcine circovirus 2-associated syndromes and diseases dbatvir: the database of bat-associated viruses molecular evolution of the sars coronavirus during the course of the sars epidemic in china mers coronaviruses in dromedary camels rooting the phylogenetic tree of mers-coronavirus by characterization of a conspecific virus from an african bat characterization of a novel betacoronavirus related to middle east respiratory syndrome coronavirus in european hedgehogs evolutionary relationships between bat coronaviruses and their hosts emerging infectious diseases of wildlife-threats to biodiversity and human health metagenomic analysis of the viromes of three north american bat species: viral diversity among different bat species that share a common habitat bats host major mammalian paramyxoviruses international committee on taxonomy of viruses and the 3,142 unassigned species novel papillomaviruses in free-ranging iberian bats: no virus-host co-evolution, no strict host specificity, and hints for recombination metagenomic analysis of viruses from the bat fecal samples reveals many novel viruses in insectivorous bats in china isolation and characterization of a bat sars-like coronavirus that uses the ace2 receptor virome profiling of bats from myanmar by metagenomic analysis of tissue samples reveals more novel mammalian viruses identification of diverse alphacoronaviruses and genomic characterization of a novel severe acute respiratory syndrome-like coronavirus from bats in china lack of middle east respiratory syndrome coronavirus transmission from infected camels virology. the sars coronavirus: a postgenomic era global trends in emerging infectious diseases identification and complete genome analysis of three novel paramyxoviruses, tuhoko virus 1, 2 and 3, in fruit bats from china fruit bats as reservoirs of ebola virus bat guano virome: predominance of dietary viruses from insects and plants plus novel mammalian viruses circovirus in tissues of dogs with vasculitis and hemorrhage bats are natural reservoirs of sars-like coronaviruses prevalence and genetic diversity of adenoassociated viruses in bats from china epidemic dynamics at the human-animal interface a comparison of bats and rodents as reservoirs of zoonotic viruses: are bats special? hendra and nipah viruses: why are they so deadly? a summary of taxonomic changes recently approved by ictv antibodies against mers coronavirus in dromedary camels metavelvet: an extension of velvet assembler to de novo metagenome assembly from short sequence reads distinct lineage of vesiculovirus from big brown bats, united states bat flight and zoonotic viruses idba-ud: a de novo assembler for single-cell and metagenomic sequencing data with highly uneven depth acute diarrhea in west african children: diverse enteric viruses and a novel parvovirus genus identification of a severe acute respiratory syndrome coronavirus-like virus in a leaf-nosed bat in nigeria bats are a major natural reservoir for hepaciviruses and pegiviruses bats and their virome: an important source of emerging viruses capable of infecting humans evolution of virulence in picornaviruses hepatitis a virus and the origins of picornaviruses origins of major human infectious diseases coronavirus diversity, phylogeny and interspecies jumping discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus virome analysis for identification of novel mammalian viruses in bat species from chinese provinces novel henipa-like virus, mojiang paramyxovirus, in rats novel human bufavirus genotype 3 in children with severe diarrhea, bhutan unbiased parallel detection of viral pathogens in clinical samples by use of a metagenomic approach novel sars-like betacoronaviruses in bats mers-related betacoronavirus in vespertilio superans bats receptor usage and cell entry of bat coronavirus hku4 provide insight into bat-to-human transmission of mers coronavirus covs, and the highly diverse s genes present in only r. sinicus imply that frequent recombination events occur among sl-covs of r. sinicus and other hosts, suggesting that the transmission of sl-covs from r. sinicus to other mammals is a result of the viruses obtaining novel s genes. these data indicate that human sars-covs most likely originated through zoonotic transfer, either directly or indirectly, from chinese horseshoe bats via a complicated adaptation process and a series of rare recombination events.although the transmission of mers-cov has been confirmed by the detection of identical mers-cov sequences in dromedary camels and humans (azhar et al., 2014; chu et al., 2014; meyer et al., 2014) , the zoonotic transmission of this virus from dromedaries to humans is still considered to be rare (hemida et al., 2015) and the wildlife source of mers-covs remains unknown. the data obtained here and a recently reported lineage-c btcov, neocov, identified by another group (corman et al., 2014a; yang et al., 2014a) provide new clues about the sources and pathways of human-and camel-derived mers-covs in bats of the family vespertilionidae. bat species of this family may have important roles in mers-cov evolution (yang et al., 2014b) . furthermore, the diverse s genes of these mersrelated covs may provide an opportunity for their recombination to ultimately generate new covs.in conclusion, the understanding of the viral community characteristics, genetics and ecological distribution of bat viruses could enable the rapid identification of novel viruses with variant genomes and could thus facilitate the tracing of eids in bats. furthermore, this strategy could be extended to other wildlife or livestock worldwide, ultimately increasing knowledge of the viral population and ecological community, thus minimizing the impact of potential wildlife-originated eids on public health by providing meaningful basic data. the authors declare no conflict of interest. key: cord-004510-cbutpjre authors: seetahal, janine f. r.; greenberg, lauren; satheshkumar, panayampalli subbian; sanchez-vazquez, manuel j.; legall, george; singh, shamjeet; ramkissoon, vernie; schountz, tony; munster, vincent; oura, christopher a. l.; carrington, christine v. f. title: the serological prevalence of rabies virus-neutralizing antibodies in the bat population on the caribbean island of trinidad date: 2020-02-05 journal: viruses doi: 10.3390/v12020178 sha: doc_id: 4510 cord_uid: cbutpjre rabies virus (rabv) is the only lyssavirus known to be present within the caribbean. the island of trinidad, is richly diverse in chiropteran fauna and endemic for bat-transmitted rabies with low rabv isolation rates observed in this population. we aimed to determine the seroprevalence of rabies virus neutralizing antibodies (rvna) in light of spatio-temporal and bat demographic factors to infer the extent of natural exposure to rabv in the trinidadian bat population. rvna titers were determined by the rabv micro-neutralization test on 383 bat samples representing 21 species, comprising 30.9% of local bat diversity, from 31 locations across the island over 5 years. rvna was positively detected in 33 samples (8.6%) representing 6 bat species (mainly frugivorous) with titers ranging from 0.1 to 19 iu/ml (mean 1.66 iu/ml). the analyses based on a multivariable binomial generalised linear mixed-effects model showed that bat age and year of capture were significant predictors of seropositivity. thus, juvenile bats were more likely to be seropositive when compared to adults (estimate 1.13; p = 0.04) which may suggest early exposure to the rabv with possible implications for viral amplification in this population. temporal variation in rabies seropositivity, 2012–2014 versus 2015–2017 (estimate 1.07; p = 0.03) may have been related to the prevailing rabies epizootic situation. regarding other factors investigated, rvna was found in bats from both rural and non-rural areas, as well as in both hematophagous and non-hematophagous bat species. the most common seropositive species, artibeus jamaicensis planirostris is ubiquitous throughout the island which may potentially facilitate human exposure. the findings of this study should be factored into public health assessments on the potential for rabies transmission by non-hematophagous bats in trinidad. rabies is a highly fatal but preventable zoonotic disease of major public health significance [1, 2] . the causative agent rabies virus (rabv) is the type species and most ubiquitous of the lyssavirus genus [3] . it is the only lyssavirus known to circulate in the americas [4] . while, the major global burden of rabies is attributed to dog-mediated transmission [5] , sylvatic-transmission is becoming increasing important in the epidemiology of rabies [6] [7] [8] [9] . this is particularly relevant in the americas with the decline of canine-transmitted cases [10] [11] [12] and the recognition of distinct rabv variants in numerous bat species [9, 13, 14] . due to the aerial nature of their reservoir, these variants are more defined by species than by geographical boundaries [15] and in the americas, nearly 30 distinct bat rabv variants have been found thus far [16] . although these variants can be transmitted between bat species and to other mammals [14, 17] , in latin america and the caribbean the vampire bat is the bat species most implicated as a reservoir in this region [18] . the caribbean island of trinidad, located just 12 km away from the northeast coast of south america, is richly diverse in chiropteran fauna with 68 identified species, including two species of hematophagous bats [19] . the island is enzootic for bat rabv which has been so far isolated from nine bat species [19, 20] . of these, the hematophagous desmodus rotundus species is considered the most effective vector on account of its feeding practices [21] and to date, is the only bat directly implicated in transmission of the virus on the island [22] . some earlier unconfirmed studies have suggested that apparently healthy bats could harbour and transmit rabv for extended periods [23] [24] [25] [26] , however to date this has not been conclusively proven by modern diagnostic methods. in trinidad viral isolation from bats has been rare with a rabies positivity proportion of 0.05% (two positive of 4399 tested between 1971 and 2015) obtained from samples acquired mainly through active surveillance in the bat population [27] . despite the bias towards healthy bats inherent to this type of sampling, it has been suggested that this low proportion is a consequence of virus only being periodically imported from the south american mainland (as bats fly from mainland to island) causing vampire bat epizootics with occasional viral spill-over to the livestock population [22] . nonetheless, over the last 50 years, despite the apparent low levels of rabv circulation in the bat population, five significant epizootic events have occurred on the island [28] . in light of the apparently low prevalence of virus among bats, rabies antibody levels may be used as an indicator of virus exposure to gauge the risk of virus transmission. few studies on rabies antibody prevalence have been conducted within the caribbean [29] [30] [31] [32] and the only report from trinidad (conducted in 1974 during a small epizootic event) demonstrated a seropositive proportion of 12.8% [29] . we therefore sought to determine the current seroprevalence of rabies virus neutralizing antibodies (rvna) in the trinidadian bat population over a period of five years in order to infer the extent of natural exposure to rabvs and the spatio-temporal dynamics of rabv infection in the bat population. we also aimed to determine whether seroprevalence varied with factors related to bat demographics and habitat, with a view to identifying potential risk factors for transmission to susceptible animal and human populations. bats were caught mainly using mist nets set at ground level at dusk and night from february 2012 to april 2017 on the island of trinidad (see table s1 ). bat trapping and specimen collection were carried out under special game licenses issued annually by the wildlife section, forestry division, ministry of agriculture, land and fisheries, trinidad and tobago in accordance with the government of trinidad and tobago conservation of wildlife act (1958) chapter 67:01 (section 10). field and laboratory protocols were approved by the ethics committee, faculty of medical sciences, university of the west indies, st. augustine campus (14th february 2012). bats were transported to the laboratory in individual mesh bags for processing. all bats were apparently healthy at the time of sampling and were sampled only once. biometrics (i.e., forearm length, weight and head and body length) and the appearance of other external characteristics (e.g., tail, chin warts and nose leaf) were recorded for each animal to aid in their morphological species identification using locally developed field identification keys [19] . juvenile bats were distinguished from adult bats by the lack of ossification in the epiphyseal joint between the metacarpal and proximal phalanx bones demonstrated upon trans-illumination of the wing. bats were humanely euthanized by anaesthetic overdose using isoflurane and organ tissues were collected by dissection and stored in-house for other parallel studies and future studies including species identification using genetic methods. blood was sampled by cardiac puncture and sera was separated by centrifugation and stored at −80 • c until further processing. the micro-neutralization test was developed to test smaller volumes of test serum making it an ideal comparison to the traditional rapid fluorescent focus inhibition test (rffit) [33] . the microneutralization test was conducted following the protocol outlined in smith et al. [33] using the cvs-11 rabv variant. all serum samples were screened at 1:10 dilution and positive sera were run to end-point. a cut off titer of 1:10 (0.1 iu/ml) was chosen based on the value for 50% neutralization of the challenge virus in accordance with previous studies [33, 34] . the procedure for performing the micro-neutralization test involves using four-well teflon coated slides to perform serial dilutions while employing a humidity chamber throughout the procedure to avoid any evaporation. minimum essential medium (mem) (12 µl) was added to each well of each slide and 3 µl of each test serum was serially diluted. standard rabies immunoglobulin (srig) was used as positive control serum in preparing the positive control slide [35] . cvs-11 was prepared for the working dilution at 50 ffd 50 (50% fluorescent focus forming doses) per ml and 12 µl of this working dilution of virus, was added to each well of each test slides as well as to the positive control slide and the back-titration slide. the slides were incubated in the humidity chamber at 37 • c with 0.5% co 2 for 90 min. after completion of this incubation period, 24 µl of mouse neuroblastoma cells (mna) were added to each well, equivalent to 1.4 × 10 4 cells per well. slides were again incubated at 37 • c for 20 h with 0.5% co 2 then fixed with acetone and stained with fitc-anti-rabies immunoglobulin (fujirebio diagnostics, malvern, pa, usa) before being observed for the presence of fluorescent foci with a fluorescence microscope. the reed-muench method [36] was used to calculate the endpoint titer, which was converted to international units per millilitre (iu/ml) based on comparison to srig diluted at 2 iu/ml. rvna titer values were recorded originally as a continuous variable and they were further categorized as a binary variable, either as positive (≥0.1 iu/ml) or negative (<0.1 iu/ml) serological status. the variables investigated in the study were related to the bat captured, i.e., sex, age and dietary habits; or to external factors such as season of capture (dry vs wet season; i.e., january-may vs june-december), year of capture, urbanization level at capture location, and district of capture. this investigation aimed to study the factors associated with the rabies serological status of the captured bats. the response variable in the analyses was positive (≥0.1 iu/ml) or negative (<0.1 iu/ml) serological status according to the rvna titer obtained. initially, the analysis comprised univariable explorations to investigate the associations between the binary status (i.e., positive or negative) of each bat and the different factors investigated in the study, utilizing a binomial generalized linear model (glm). the proportion of seropositive bats and the 95% confidence intervals (ci) were also computed. the variables significantly associated (p < 0.05) in the univariable model were included in a multivariable binomial glm and a backward elimination process was followed to build the final model. then, the model evolved, allowing for random effects at the county level, utilizing a binomial generalised linear mixed-effects model (glmm). this approach allowed accounting for the potential over-dispersion present in our data due to clustering at the level of county. the goodness of fit measurement akaike's information criterion (aic), was used for comparison between nested models. additionally, substantial changes in the estimated coefficients in the models and increases in standard errors during the model building process were investigated. the wald tests were used to examine and to present the significance (p value < 0.05) of the variables retained in the final model. all the analyses and graphs were performed using the minitab (version 18) [37] and r statistical software environment [38] using the libraries stats, epicalc and lme4. in this study, 409 sera samples from bats in trinidad were collected for rvna testing indicative of exposure to rabv. of these, 383 samples representing 31 geographical locations on the island (see figure 1 and table s1 ) were of suitable quality and quantity for determination of rvna titers. twenty-one (21) species of bats representing four of the nine families known to be present on the island, were tested from these locations. the most common species in the sample set was the desmodus rotundus (n = 107; 27.9%). after this artibeus jamaicensis planirostris (n = 89; 23.2%), carollia perspicillata (n = 70; 18.3%) followed by molossus molossus (n = 11; 2.9%) bats made up the majority of tested samples. rvna was positively detected in the sera of 33 bats representing 8.6% of the sample population (95% ci 5.81, 11.43) with seropositive titers ranging from 0.1 to 19 iu/ml (mean 1.66 iu/ml; sd ± 4.18), as illustrated in table s2 . bats from all counties except county nariva/mayaro tested positive for rabies antibodies. as detailed in table 1 table s2 ). six species of bats accounted for the 33 seropositive samples (table s2 ) and were distributed as follows: sixteen (48.48%) from the species a. jamicensis planirostris, six (18.18%) each from artibeus lituratus and d. rotundus, three (9.09%) from c. perspicillata and one (3.03%) each from phyllostomus hastatus and glossophaga soricina. most of the seropositive samples (n = 33) were from frugivorous (25 of 33; 75.8%) and hematophagous (6 of 33; 18.2%) bat species ( figure s1 ). lower numbers of bats with mixed dietary preference and nectarivores were positive when compared to frugivorous and hematophagous species and no insectivorous bats were seropositive for rvna. non-hematophagous bats (n = 275) demonstrated a seropositivity proportion of 9.8% (95% ci (6.8-13.9); 27 of 275) compared to 5.6% (95% ci (2.6-11.6); 6 of 108) for hematophagous bats (n = 108), as illustrated in table 2 . however, there was no significant association between bat diet and seropositivity for rabies antibody (p > 0.05) according to the results of the univariable glm. overall from the entire test population (n = 383), more female bats (n = 237) were tested than males (n = 146). females accounted for 57.6% (19 of 33) of all positives bats (n = 33) as opposed to 42.4% (14 of 33) for males ( figure s1 ). the seropositivity proportion for males was 9.6% (95% ci (5.3, 15.6)) versus 8.0% (95% ci (5.0, 12.2)) for females. however, there was no significant association between sex and seropositivity for rabies antibody (p-value > 0.05) according to the results of the univariable glm. although juvenile bats comprised only 6% (23 of 383) of the test population (n = 383) they accounted for 24.2% (8 of 33) of all seropositive samples (see figure s1 ). their seropositivity proportion was 34.8% (95% ci (18.8, 55.1)) significantly higher (p ≤ 0.001) than the 6.9% (95% ci (4.8, 10.1)) noted in adult bats. likewise, there was a statistically significant difference between the mean rvna titer status in juveniles compared to adults, with juveniles having greater risk (estimate 1.97; p < 0.01) according to the results of the univariable glm). individual seropositivity status for artibeus juvenile and dam pairs were not consistently similar. for example, in one instance, both the juvenile (t15) and dam (t14) a. jamaicensis planirostris were rvna positive with identical titer values of 0.13 iu/ml, whereas another juvenile bat (t25) of the same species with a similar titer value was associated with a seronegative dam (t24). this dam (t24) was also associated with two other pups (t26 and t27) both of which were seronegative. for the remaining juvenile bats, the dam-pup association was only established for one seronegative adult and juvenile a. lituratus pair, which were t37 and t38 respectively. all other juveniles were not affiliated with a dam. about two-thirds of the samples tested (257 of 383) were from bats sampled during the dry season and these accounted for 75.8% (25 of 33) of rvna positive samples ( figure s1 ). the prevalence of rvna positive samples during the dry season (9.7%; 95% ci (6.7, 14.0); 25 of 257) was similar with that for the wet season (6.4%; 95% ci (3.33, 12.0); 8 of 126) with no significant difference in the seropositivity between the seasons (p > 0.05) according to the results of the univariable glm. the majority of tested samples (72.8%; 279 of 383) were from bats trapped in rural areas where there were low human population densities (figure 1 ). the prevalence of rvna positive samples in these areas was 6.5% (95% ci (3.9, 10.0); 18 of 33) compared to 14.4% (95% ci (8.3, 22.7); 15 of 33) in non-rural areas. seropositive bats from non-rural areas were sampled from both residential and non-residential areas, with 93% (95% ci (70.1-98.9)) of the seropositive samples from non-rural areas sampled at non-residential locations. the association between seropositivity and urbanization level at the location of capture was statistically significant, with urban areas showing a greater risk in comparison to rural areas (estimate 1.42, p < 0.01) according to the glm. seropositive bats were mainly from two counties, st. george east and st. patrick, with seropositivity rates of 18.2% (95% ci (11.8, 26. 2)) and 6.9% (95% ci (2.6, 14.4)) respectively ( figure s1 ). other districts had lower seropositivity rates (2.5-5.9%) and county nariva/mayaro had no positives samples (see table 1 ). the most seropositive samples per sampled district originated in champs fleurs (table s2) . however, there was no significant difference in the seropositivity across the counties (p-value > 0.05) according to the results of the univariable glm. the seropositivity of bats captured by roost extraction was 9.9% (95% ci (6.2, 14.7); 21 of 213) versus 7.1% (95% ci (3.7, 12.0); 12 of 170) for those captured in the field at the feeding grounds but this difference was not found to be significant (p > 0.05) in the glm. the results of the final multivariable glmm retained two variables as significantly associated with the rabies serological status (see table 3 ). regarding the bat age, the juveniles appeared to have a greater risk that adults and the samples taken during the first period of sampling (2012-2014) also showed a greater risk than those sampled within the second period (2015-2017). in comparison to viral investigative studies for rabv in bats, there have been limited investigations into the serological prevalence of rabies antibodies in bat populations. in latin america and the caribbean, the previous serological studies revealed significantly variable rates for the prevalence of rabies antibodies in bats [29] [30] [31] [39] [40] [41] [42] [43] [44] which may be attributed to differences in viral population dynamics influenced by spatio-temporal and bat demographic factors as discussed below. in an earlier trinidadian rabies seroprevalence study [29] , only four of 68 bat species documented on the island (i.e., 5.9% of local bat species diversity) [19] were tested, with only the artibeus species found to be seropositive, compared to 30.9% (21 species) in the present study. in line with studies that show a correlation between the number of bat species from which rabv has been isolated and research effort [13] , we found six seropositive bat species and by extension more seropositive bat species and possible isolations of rabv can be expected with more extensive studies covering more bat species. in trinidad, all except phyllostomus hastatus have previously been reported to be rabv positive [20] , while this species was found to be rabies positive in south america [45] . it is important to note that bat taxonomic identification on the basis of morphological characteristics does not distinguish cryptic species so future work will include genetic characterization to clarify species designations. comparison of the seropositivity rate observed in the current study (8.6% for the period 2012-2017) with the rate of 12.8% reported in the 1974 study [29] suggests that there is temporal variation in rabies seropositivity in trinidad. rabies serological studies conducted several years apart in french guiana and grenada, also showed proportion variations over time, with 6.6% and 10% respectively in earlier years and 10.7% and 7.2% more recently [29] [30] [31] [32] . in our study, year of capture was statistically significantly associated to serological status with, a bat sampled in 2012-2014 having a greater risk of being seropositive than one sampled during 2015-2017. in terms of the rabies epizootic situation at these time, there is evidence of viral exposure during both periods. in 2012, the year in which 79% (26 of 33) of rvna positive bat samples in our study were sampled (see table s2 ), one vampire bat (n = 253) was confirmed to be rabies positive [46, 47] . on the other hand, in 1974, although no bats were found to be rabid from the small number tested (n = 6), 12 bat-transmitted rabies cases were diagnosed in the livestock population clearly indicating virus was circulating in at least the vampire bat population [28] . although not reported herein, 65 brain samples from bats specimens sampled during the period of this study were tested for rabv and other lyssaviruses by real-time rt-pcr [48] , however none were positive for rabv [49] . the majority of seropositive samples in this study were <2.0 iu/ml, consistent with natural primary exposure [50] . some studies have shown that rabies antibody levels decline to undetectable levels between 5 and 6 months post-primary exposure, and up to one year after secondary exposure [50, 51] . similar waning of passive immunity is thought to occur in juvenile bats [50] . so seropositive bats found in this study may have been naturally exposed to rabv up to a year prior to capture which would mean that rabv circulated in the trinidadian bat population at the very least during the period 2011 to 2016. only 15% of seropositive bats had rvna titers higher than 2.0 iu/ml, which could be attributed to immunological priming from past exposures with subsequent anamnestic responses [50, 52] . so relatively high rabies antibody titers may occur without active viral infection, because of pre-existing acquired immunity from past exposures [50, 53] . this phenomenon may also explain the absence of mass mortalities among bats during epizootic events. longitudinal temporal monitoring of vampire roosts in peru and french guiana [31, 41] have provided insights into non-lethal rabv infection by demonstrating seroconversion with highly fluctuating individual seroprevalence rates over time, which perhaps reflects viral persistence in the roost with periodic reactivation. on the contrary, rather than single colony perpetuation of rabv, one study suggests enzootic viral persistence may occur due to movement of infectious bats among colonies, with a high frequency of immunizing non-lethal exposures [54] . assessment of natural variations in antibody levels for individual bats can be further investigated by capture (mark) and release studies with bat recapture and successive sampling over a period of time to identify changes in rvna titer levels and determine the maintenance of immunity. this study demonstrates variation in rabies seroprevalence by district in trinidad, with st. george east and st. patrick accounting for 85% of all rvna positive samples, consistent with localisation of rabies livestock epizootics in these regions [27, 28] . however, more structured sampling across the island, which would account for sample site variation, is necessary to confirm the observed pattern. differences between regions may be related to the influence of bat population densities on viral dynamics. in theory, large bat roosts provide ideal conditions for viral spread amongst roost mates resulting in larger numbers of bats exposed to higher amounts of virus and thus higher rates of seroconversion [55] . nevertheless, streicker et al. [41] found limited evidence for a relationship between vampire bat colony size and exposure to rabv. affected bats may also be more likely to forgo normal foraging behaviour in preference to staying in roost [56] . although our results showed no relationship between seropositivity and roost versus field capture, more targeted sampling may reveal underlying associations. the fruit bat, a. jamaicensis planirostris was the species most commonly found to be seropositive on the island. this species is highly adaptable and is widespread throughout the island roosting in crevices of both homes and non-residential buildings [19] and therefore has a high potential for human contact. in light of this, public health officials and wildlife biologists should collaborate to address potential risks for human virus exposure with minimum ecological disruption. in general, frugivorous bats had the highest rvna seroprevalence for trinidadian bats, which is similar to the situation in grenada [29, 30] . as with other studies, [31, 57] rvna levels varied among bat species (see table s2 ) within the same area, suggesting variable rabies exposure and infection dynamics. the wide range of rvna titer values observed for the hematophagous species (see table 2 ) may suggest greater natural exposure to rabv, which may more likely result in abortive infections when compared with other species [53, 58] . in the caribbean (including trinidad) despite isolation of rabv from 16 bat species, only the desmodus viral variant has thus far been definitely identified [18] . however, the prevalence of rvna in non-haematophagous bats (particularly artibeus species), in both past and present studies may suggest the presence of other rabies variants or may indicate transmission of the desmodus variant to these species during roost co-habitation [20] . along these lines, and similar to the situation in other areas [59] non-hematophagous bats may play a significant role in rabv transmission within the caribbean. the multivariable glmm analysis found that juvenile bats were more likely to be seropositive than adults. this is consistent with previous studies which suggest that bats may be exposed to rabv soon after birth [60] , and that virus amplification within the susceptible young population can facilitate an increase in rvna seroprevalence after a birth pulse, through active immunological responses [61, 62] . this in turn can facilitate long-term maintenance of the virus [63] . the role of pre-natal exposure is debatable, as contradictory evidence has thus far been provided for in-utero rabv transfer [61, 64] . alternatively, passive transfer of maternal antibodies may have resulted in seropositive juvenile bats, which may not have been naturally exposed to the virus [65] . this may have been the case with the seropositive juvenile and dam pair in this study (t14 and t15). although, all attempts were made to maintain appropriate identification records for dam-offspring pairs by observational analysis of dam-pup attachment and nursing prior to and after bat capture, alloparental nursing cooperative behaviour [66, 67] cannot be ruled out as an explanation for the seropositive juvenile bat paired with a seronegative dam in this study (t25 and t24). genetic maternity analysis, to confirm the maternal relationships between these adult-juvenile pairs [68] , was beyond the scope of the current study, but should be employed in future studies into the relationship between maternal and juvenile rvna titers. in another study where age was noted to be a significant predictor of rvna, higher seropositivity rates in juvenile bats compared to adults were thought to reflect active infection rather than the presence of maternally derived antibodies, as titers were much higher in juveniles than the adult females and early antibody decline consistent with passive immunity was not observed [41] . single timepoint sampling employed in the current study did not allow for assessment of antibody decline, but titer levels were noted to be comparable between the seropositive dam-pup pair. twelve out of the 33 (36%) seropositive bats identified in this study were from one maternity roost. this finding supports the evidence for a higher viral prevalence in pregnant and lactating female bats [69, 70] . the estimated prevalence of rabv in wild bat populations within endemic regions is typically <1% [56, 71] . over the last century, since the isolation of rabv in trinidadian bats, the rabv prevalence proportion in this population, mainly from active surveillance, has gradually decreased from 3.3% (1930s) to 0.4% (1950-60s) to the most recent estimate of 0.05% [27, [72] [73] [74] . this apparent decline may reflect lower levels of viral circulation in the bat population and may be responsible for the lower seroprevalence proportion demonstrated in this contemporary study (8.6%), as compared the previous study conducted in the 1970s (12.8%) [29] . conversely, studies conducted in south america demonstrate an inverse relationship between rabv prevalence and rvna seroprevalence [43, 44, 75] . this may be attributed to the protective effect of herd immunity and death of virus-infected individuals, with the virus less able to establish infection and sustain bat to bat transmission due to increasing numbers of immune individuals. hence it has been suggested that rabv epizootics in bat populations are unidirectionally migratory with a periodicity of at least four years between events, which allows the population to regenerate a threshold of susceptible individuals [76] . in trinidad, bat surveillance has traditionally targeted hematophagous bats with desmodus species comprising the vast majority of tested bats [22, 27] . however, the prevalence of rvna in non-hematophagous bats found in this study suggests an expansion of rabies diagnostic testing to non-hematophagous species may be warranted to obtain a comprehensive picture of rabies host diversity and viral dynamics and ultimately determine the risk of transmission [31] . in general the trinidadian population is well aware of the risks associated with hematophagous bats due to the history of rabies on the island [27] . however, the potential for rabies transmission by non-hematophagous bats may be overlooked and persons may not seek medical care after exposures to these bats. although the potential risk for exposure to an actively infected rabv bat is relatively low, our results indicate that the virus can be associated with non-hematophagous bats so adequate precautions must be taken when handling all bats and appropriate post exposure prophylaxis is recommended in accordance with international guidelines regardless of the species of bat [5] . rabies serological rates from trinidad and other countries in which rabies is enzootic [40] [41] [42] were comparable to those found in grenada [29, 30] , which is surprising because despite the single historical isolation of rabv from an artibeus bat [29] , the virus is not known to be enzootic in the grenadian bat population. this along with the high rvna prevalence among the artibeus bats [29, 30] , which are known to have long distance flight ranges over open water [77] may imply translocation of the virus from endemic areas such as trinidad by bat movement. taken together with the ubiquity of the artibeus species throughout the caribbean [78] the presence or introduction of rabv into other caribbean islands previously thought to be free of bat rabies is plausible. this has direct implications for the island of tobago which is only 42 km from trinidad, compared to 160 km from trinidad to grenada. in general, the lack of rabid clinical syndromes in bats may not necessarily reflect the spatial range of lyssaviruses due to exposures resulting in seroconversion rather than overt disease or latent infection [23, 79] . reports of serological evidence of other lyssaviruses in the old world within areas previously thought to be 'free' of these viruses [6, 80] have highlighted the value of serological surveillance to determine public health risk. consequently, serological surveys for anti-rabv antibodies in bat populations within the caribbean thought to be historically "rabies-free", especially those islands closer to the american continent, may be worthwhile to establish the true geographical range of rabv in the americas. such wide scale investigations might consider prioritizing species demonstrated to have flight ranges over large distances, particularly over open water such as artibeus jamaicensis [77] and tadarida brasilensis [81] species. with respect to sampling effort, surveillance for antibodies may be more efficient than viral detection in the long-term, however, the presence of these antibodies do not necessarily demonstrate active infection, but reflects past exposure to a phylogroup i lyssavirus [82] . therefore viral isolation and typing would be necessary to confirm the lyssavirus circulating in these bat populations. similarly in trinidad, although no other lyssavirus has been detected here or within the americas, the detection of rvna does not necessarily mean that rabv is circulating in the bat population as the existence of a closely related lyssavirus that elicits a cross-reactive immune response cannot be ruled out [82] furthermore, seroprevalence levels may vary over time at any given location based on bat population dynamics, infection kinetics and seasonal influences [80] , thus caution must be exercised in the extrapolation of prevalence data. table s1 : geographic locations and sampled years for the bat serological sample set utilized in the study, table s2 : demographic and habitat information for rvna positive bat samples. travel vaccination for rabies human monoclonal antibody and vaccine approaches to prevent human rabies taxonomy of the order mononegavirales: update the global phylogeography of lyssaviruses-challenging the 'out of africa' hypothesis world health organization. who expert consultation on rabies serologic evidence of lyssavirus infections among bats, the philippines notes from the field: wildlife rabies on an island free from canine rabies for 52 years-taiwan pathology and molecular detection of rabies virus in ferret badgers associated with a rabies outbreak in taiwan rabies transmitted by vampire bats to humans: an emerging zoonotic disease in latin america? progress towards eliminating canine rabies: policies and perspectives from latin america and the caribbean rabies update for latin america and the caribbean overview of rabies in the americas bat borne rabies in latin america host phylogeny constrains cross-species emergence and establishment of rabies virus in bats the evolutionary history and dynamics of bat rabies virus rabies virus vectors and reservoir species lyssaviruses and rabies: current conundrums, concerns, contradictions and controversies. f1000 res rabies in the caribbean: a situational analysis and historical review bats of trinidad and tobago: a field guide and natural history a review of the bats of trinidad and tobago: descriptions, rabies infection and ecology the biting and feeding habits of the vampire bat, desmodus rotundus evolutionary history and phylogeography of rabies viruses associated with outbreaks in trinidad rabies in the vampire bat of trinidad with special reference to the clinical course and the latency of infection the transmission of paralytic rabies in trinidad by the vampire bat (desmodus rotundus murinus wagner 1840) fruit-eating bats and paralytic rabies in trinidad a transmissao da raiva dos herbivoros pelos morcegos da familia desmodontidae the history of rabies in trinidad: epidemiology and control measures of bats and livestock: the epidemiology of rabies in trinidad, west indies rabies virus and antibody in bats in grenada and trinidad natural exposure of bats in grenada to rabies virus bioecological drivers of rabies virus 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deciphering serology to understand the ecology of infectious diseases in wildlife communal nursing in the evening bat, nycticeius humeralis nursing behavior in mexican free-tailed bat maternity colonies mate fidelity and intra-lineage polygyny in greater horseshoe bats detection and prevalence patterns of group i coronaviruses in bats reproduction and nutritional stress are risk factors for hendra virus infection in little red flying foxes the study and control of paralytic rabies transmitted by bats in trinidad extracts from the administration report of the surgeon general 1925-1941 bat transmitted paralytic rabies in trinidad virological and serological diagnosis of rabies in bats from an urban area in the brazilian amazaon an ecological strategy for controlling rabies through the elimination of vampire bats mitochondrial dna polymorphism in three antillean island populations of the fruit bats, artibeus jamaicensis phylogenetics and phylogeography of the artibeus jamaicensis complex based on cytochrome b dna sequences rabies: virus and disease serological evidence of lyssaviruses among bats on southwestern indian ocean islands population struture of a widespread bat (tadarida brasiliensis) in an island system evidence of two lyssavirus phylogroups with distinct pathogenicity and immunogenicity this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors would like to thank the anti-rabies unit of the ministry of agriculture, land and fisheries and the trinidad and tobago bat conservation and research unit for their support in the field. we appreciate the assistance of jasmin camacho with field sample collection and we are grateful to ron mahabir for his assistance with the production of the map for this study. we also acknowledge the department of geomatics, engineering and land management, university of the west indies, st. augustine campus and the geographic information systems unit, ministry of agriculture, land and fisheries for the provision of digital data layers for the map. the findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the centers for disease control and prevention. the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. key: cord-003482-f1uvohf0 authors: malmlov, ashley; bantle, collin; aboellail, tawfik; wagner, kaitlyn; campbell, corey l.; eckley, miles; chotiwan, nunya; gullberg, rebekah c.; perera, rushika; tjalkens, ronald; schountz, tony title: experimental zika virus infection of jamaican fruit bats (artibeus jamaicensis) and possible entry of virus into brain via activated microglial cells date: 2019-02-04 journal: plos negl trop dis doi: 10.1371/journal.pntd.0007071 sha: doc_id: 3482 cord_uid: f1uvohf0 the emergence of zika virus (zikv) in the new world has led to more than 200,000 human infections. perinatal infection can cause severe neurological complications, including fetal and neonatal microcephaly, and in adults there is an association with guillain-barré syndrome (gbs). zikv is transmitted to humans by aedes sp. mosquitoes, yet little is known about its enzootic cycle in which transmission is thought to occur between arboreal aedes sp. mosquitos and non-human primates. in the 1950s and ‘60s, several bat species were shown to be naturally and experimentally susceptible to zikv with acute viremia and seroconversion, and some developed neurological disease with viral antigen detected in the brain. because of zikv emergence in the americas, we sought to determine susceptibility of jamaican fruit bats (artibeus jamaicensis), one of the most common bats in the new world. bats were inoculated with zikv prvabc59 but did not show signs of disease. bats held to 28 days post-inoculation (pi) had detectable antibody by elisa and viral rna was detected by qrt-pcr in the brain, saliva and urine in some of the bats. immunoreactivity using polyclonal anti-zikv antibody was detected in testes, brain, lung and salivary glands plus scrotal skin. tropism for mononuclear cells, including macrophages/microglia and fibroblasts, was seen in the aforementioned organs in addition to testicular leydig cells. the virus likely localized to the brain via infection of iba1(+) macrophage/microglial cells. jamaican fruit bats, therefore, may be a useful animal model for the study of zikv infection. this work also raises the possibility that bats may have a role in zika virus ecology in endemic regions, and that zikv may pose a wildlife disease threat to bat populations. introduction zika virus (zikv) was first isolated from a sentinel rhesus macaque in uganda in 1947 and subsequently from aedes africanus mosquitoes in the same location [1] . the first human cases were identified in 1954 in nigeria and serosurveys found evidence of a broad geographic distribution for zikv throughout africa and asia with sporadic cases in humans [2, 3] . the first recognized zikv epidemic occurred in yap state, federated state of micronesia in 2007. an estimated 73% of residents were infected, and of those 18% presented with clinical disease [4] . in 2013, a second epidemic occurred in french polynesia with 28,000 cases reported. during the latter outbreak, the incidence rate of guillain-barré syndrome (gbs) increased 20-fold and first indication of a connection between zikv infection and gbs was established [5] . the virus spread to brazil in 2015 [6, 7] and has since disseminated throughout much of tropical south america, central america, the caribbean, and the southern united states, with more than 200,000 confirmed cases [8] . zikv can also cause congenital zika syndrome (czs) in naïve populations and is therefore a virus of high concern [3] . zika virus is maintained in an urban cycle, transmitted between an aedes mosquito vector and humans thereby maintaining endemicity [9] . it is generally accepted that the virus transmits between non-human primates and vectors in a sylvatic cycle; however, the sylvatic cycle has not been well characterized in the old world and little is known about a new world sylvatic cycle [9, 10] . molecular analysis of zikv to better understand viral phylogenetics suggests that animal hosts affected viral evolution and therefore may play an important role in viral ecology [11] . in the 1950s and '60s, the susceptibility of bats to zikv was investigated. shepherd and williams [12] screened 172 wild bats from 12 different species in uganda for antibodies against zikv and found 16/44 little free-tail bats (tadarida pumila) and 26/36 angolan freetail bats (t. condylura) were seropositive by hemagglutination inhibition assay. additionally, two angolan free-tail bats were experimentally inoculated with zikv and serially bled to test for viremia. both animals were viremic on days 2, 4 and 6 as determined by paralysis in mice inoculated with the sera from those two bats [12] . simpson and o'sullivan [13] experimentally inoculated three straw-colored fruit bats (eidolon helvum), three egyptian fruit bats (rousettus aegyptiacusi), and five angolan free-tail bats. two of the straw-colored fruit bats were viremic and had seroconverted. one of the egyptian fruit bats was viremic and two had seroconverted. the angolan free-tail bats were euthanized on days 1, 3, 5, 7 and 10 days post inoculation and screened for viral tropism. at one day post infection, a kidney was trace positive [13] . finally, reagan et al. [14] inoculated 20 new world little brown bats (myotis lucifigus) by 5 different routes: intracranial, intraperitoneal, intradermal, intrarectal and intranasal. bats in all groups, with the exception of the intranasal group, developed fatal neurological disease 4-7 days post inoculation. brain tissue was virus-positive in all animals with clinical disease, determined by inoculation of mice with brain homogenate suspension [14] . considering the evidence that african bats are naturally susceptible to zikv and that little brown bats develop disease, the question emerged: could bats serve as a natural reservoir host for zikv in the new world? to test this hypothesis, we inoculated jamaican fruit bats (artibeus jamaicensis), among the most abundant bats in the caribbean, central america and mexico, with zikv to examine virology, immunology and pathology of the infection. although virus was detected in several organs, including the testes and brains, no overt clinical signs were detected, and substantial viremia or viruria was not evident. these results suggest that jamaican fruit bats are unlikely to serve as amplification hosts but that zikv infection may constitute a wildlife disease threat to bats. bats for this project were obtained from the colorado state university breeding colony approved by the institutional animal care and use committee (protocol 16-6512a). two experimental infections were conducted; a pilot study and a time course study. in the pilotstudy, three male bats (aj-z7, aj-z8, aj-z9) were intradermally inoculated with 7.5x10 5 plaque forming units (pfu) zikv, strain prvabc59; a high dose to assess susceptibility. no signs of disease were apparent during this 28 day experiment; however, all three bats had antibody titers of 3200 on day 28 (table 1) . after demonstration of susceptibility in the pilot study, a time course study was conducted. six male bats (aj-z1 through aj-z6) were identically inoculated and two were euthanized at 2, 5 and 10 days post inoculation (dpi). no conspicuous signs of disease were observed in any of the inoculated bats. necropsies immediately followed euthanasia and no significant gross pathology was evident. quantitative probe-based reverse transcription pcr (qrt-pcr) was performed on seruminoculated vero cell supernatants, serum, brain, lung, liver, spleen, kidney, urinary bladder, prostate and testes from bats from both studies. in addition, urine collected during the time course study was similarly assayed. urine from bats aj-z6 at 3 dpi and aj-z7 at 5 dpi had low levels of vrna whereas bat aj-z1, euthanized at 2 dpi, had low levels of vrna in its brain ( fig 1) . all other samples were negative. sera from aj-z2 at 2 dpi, and aj-z3 and aj-z4 at 5 dpi were negative by elisa. sera were blind passaged on vero e6 cells in an attempt to isolate zikv and all were negative for cpe and pcr. hematoxylin and eosin stain (h&e). heart, lung, liver, kidney, testes, prostate, urinary bladder, and brain were collected from all 9 animals as well as salivary glands from 3/9 bats. all samples were blindly read by one pathologist. a summary of the consistent histopathology findings is listed in table 2 . for the time course study, aj-z1 at 2 dpi showed mild pulmonary congestion with multifocal areas of interstitial pneumonia, mild intra-alveolar hemorrhage and mild atelectasis. terminal airways had slightly increased amounts of mucus. kidneys had multifocal interstitial infiltrates of small numbers of lymphocytes. all other tissues were within normal limits. in ajz2 at 2 dpi, lungs showed milder pathology than aj-z1 with minimal interstitial to perivascular infiltrates predominately lymphocytes and macrophages with a band of collapsed air spaces subjacent to the pleural surface. there were focal lesions in the left ventricle of the heart where there was individual cell loss or else fragmentation of the sarcoplasm of scattered cardiomyocytes. degenerate/necrotic cardiomyocytes were accompanied by infiltrations of small numbers of macrophages, lymphocytes and satellite cells. all other tissues were within normal limits. lungs from aj-z3 at 5 dpi had minimal focal interstitial histiocytic pneumonia with atelectasis. kidneys showed multifocal chronic lymphohistiocytic pyelitis with a few degenerate and detached epithelial cells accumulating in the renal pelvis and infiltration of pelvic stroma by small numbers of mixed inflammatory cells. mandibular salivary gland showed focal moderate cellular infiltrates of periductular lymphocytes and macrophages. affected salivary ducts contained detached and degenerate epithelial cells and leukocytes. occasional ducts were encircled by granulation tissue and a few heterophils. rare apoptosis was evident in the lining epithelium of such ducts. all other tissues were within normal limits. aj-z4 at 5 dpi had lungs with minimal alveolar septal infiltrates scattered within collapsed lung parenchyma along with multifocal microscopic hemorrhages. kidneys had multifocal areas of mineralization. in the outer medulla and at the cortico-medullary junction were rare perivascular infiltrates of lymphoplasmacytes. esophagus and lymphoid tissue associated with palatine salivary gland showed focal mild lymphoplasmacytic inflammation. moderate numbers of lymphocytes and plasma cells were arranged in columns parallel to the respiratory mucosal epithelium of the nasophayrnx. the lumen contained increased amounts of mucus and a few inflammatory cells, mainly heterophils and lymphocytes. in the testicles, there was focal testicular degeneration manifested by presence of giant spermatids in the lumina of affected seminiferous tubules and accumulation of a small numbers of interstitial lymphocytes and macrophages. all other tissues were within normal limits. lungs from aj-z5 at 10 dpi had minimal interstitial to perivascular infiltrates with multifocal atelectasis and microscopic hemorrhages. the left papillary muscle of the heart showed rare multifocal cardiomyocyte necrosis characterized by rounding up of individual cardiomyocytes. necrotic cardiomyocytes appeared with hypereosinophilic cytoplasm, devoid of cross striations or fragmented and rarely vacuolated. minimal interstitial hypercellularity due to increased activity of satellite cells and infiltration of small numbers of lymphocytes was observed in the vicinity of degenerate/necrotic cardiac muscle fibers. kidneys had an area of focal lymphoplasmactyic pyelitis. additionally, there was a focal area of mineralization and inflammation in the inner medulla. all other tissues were within normal limits. aj-z6 at 10 dpi had occasional focal inflammation and cardiomyocyte degeneration in the left ventricle and interventricular septum. area ca3 of the hippocampus in the brain showed focal pyrimidal neuronal necrosis with a focal area of mineralization around a vessel in the cerebral cortex along with focal gliosis and individual neuronal necrosis (fig 2) . all other tissues were within normal limits. testicular, neural and salivary glands' lesions are believed to be associated with zikv infection as they were not seen with other viral infections. in the pilot study bats, aj-z7 at 28 dpi had more prominent interstitial pneumonia with congestion of the lungs compared to earlier time points. the heart had minimal cardiomyocyte degeneration and necrosis with hypercellular interstitium and increased amounts of mature fibrous connective tissue. the kidney had focal interstitial infiltrates of the cortical and outer medullary interstitium. the brain showed degenerate neurons in area a3 of the hippocampus. all other tissues were within normal limits. aj-z8 had minimal focal testicular degeneration (fig 3) . all other tissues were normal. aj-z9 had perivascular lymphocyte pulmonary infiltrates and atelectasis. heart demonstrated locally extensive lymphocytic and histiocytic pericarditis. kidneys showed multifocal interstitial lymphocytic infiltrates. brain had focal, perivascular infiltrates of small numbers of lymphocytes at the subfornical commissure. the reticular formation showed multifocal neuronal degeneration/necrosis. immunohistochemistry and immunofluorescence. tissues were stained with a polyclonal antibody for zikv (cdc, fort collins). ajz-3 at 5 dpi with inflammation of the mandibular salivary gland had moderate immunoreactivity in the lumen of affected ducts (fig 4) . aj-z5 at 10 dpi had immunoreactive cells in the brain and mononuclear cell immunoreactivity in the testes ( fig 5) . additionally, aj-z5 demonstrated immunoreactivity in purkinje cells of the cerebellum (fig 6a) . aj-z8 at 28 dpi had immunoreactive cells around the pulmonary arteries in the lungs ( fig 7a) . aj-z8 also had immunoreactivity perivascullarly in the tunica albuginea of the testes (fig 8a) . scrotal skin had focal lymphocytic dermatitis with immunoreactive mononuclear cells ( fig 8d) . cell morphology consistently identified mononuclear cells compatible with macrophages and fibroblasts as the primary cell types showing immunoreactivity against zikv antigen. brain and testicular tissues stained with both goat polyclonal goat anti-iba1 (green) and monoclonal 4g-2 flavivirus e specific antibodies (red) showed co-localization (yellow) of zikv antigen in cytoplasm of activated microglial cells with their characteristic morphology in the cerebral cortex of infected bats 10 dpi in the time course study and 28 day dpi in the pilot study (fig 9) . increased microgliosis was noted in the vicinity of co-localization sites. the gliosis was also prominent in the cerebellum and hippocampus especially around dead neurons. in the testicles, occasional macrophages showed similar co-localization similar to that noted in the brain in the testicular interstitium, inner layer of tunica albuginea and scrotum. cells consistent in morphology with leydig cells were similarly highlighted by zika viral antigen only showing strong immunoreactivity using polyclonal anti-zikv antibody. two bat infection experiments were conducted in this investigation; 1) a pilot study to determine susceptibility of jamaican fruit bats to zikv infection, and 2) a time course study to better understand pathophysiology and chronology of events pertaining to the dynamics of viremia, viral tropism, replication and shedding of the virus in a new world bat species. the goal was to determine whether bats can be used as an animal model for zikv pathogenesis and to assess the possible role of bats in zikv ecology in the new world. in the pilot experiment, no signs of disease were apparent during the 28-day study. sera collected at euthanasia indicated modest antibody titers of 3200 for each bat by elisa (table 1) , whereas the human -convalescent control serum titer was �12,800. bats typically have low to modest antibody titers, perhaps due to limited somatic hypermutation and affinity maturation [15] [16] [17] [18] [19] [20] [21] [22] . concerning viremia, cell-serum supernatants, blind passage supernatants, and neat serum results were all negative. although serum is routinely used for zikv diagnostics in humans, it may not be the most suitable sample [23] [24] [25] [26] [27] . in one investigation zikv patient had negative serum sample for the duration of the study, whereas whole blood yielded positive qrt-pcr results from days 9 to 101 [27] . one possible explanation for the phenomenon of negative serum in human patients is that the virus during acute infection disseminates via a cell-associated viremia or as novel findings suggest that the virus gets phagocytized in neutrophils and therefore whole blood is a more sensitive diagnostic sample than serum. viruria is commonly detected in zikv-infected humans [26] ; therefore, urine may be an equally important diagnostic sample with higher viral load in early infection when compared to blood in humans and other primates [23] [24] [25] [26] . although urine collection from bats was challenging, we collected urine from some of the inoculated bats in the time course study. aj-z6 exhibited viruria only at 3 dpi, and aj-z7 was equivocal only at 5 dpi, corroborating the findings in other mammals that urine may be a route of viral shedding early in infection. urine from one human patient was positive from the first time point (6 dpi) through 14 dpi and again on day 56. similarly, saliva from that same patient was positive from day nine through day 14 and again on day 49 [27] . another investigation compared diagnostic samples of 80 infected patients and showed that urine was positive in 50 of them, whereas serum was only positive in 19 patients by qrt-pcr. the study concluded that viral loads in urine were tenfold higher compared to serum and that uremia lasted longer [25] . these data corroborated the first study that identified zikv shed in urine in which there was a higher viral load in urine for longer duration compared to serum [26] . zikv rna in plasma was detected in the bats by qrt-pcr between 2 and 6 dpi, but between 2 to 17 dpi in urine [26] . the lack of detectable viremia in the serum of bats is congruent with some of the human and nhp investigations in that viremia is low and short-lived. detached renal pelvic urothelial cells and degenerate salivary gland ductular epithelium as seen in the current study will make urine and saliva equally important fluids to collect in order to maximize detection of zikv in the acute and established stages of infections. for this experiment, all male bats were used because female bats are prioritized for colony expansion. zikv exhibited tropism for the testes with strong immunoreactivity in reproductive organs (figs 4 & 7) . histologically, minimal focal testicular degeneration in two bats ( fig 2) suggests viral related pathology may be minimal. in humans it has yet to be completely elucidated what reproductive organs harbor zikv, it has been determined that semen contains zikv both in both vasectomized and unvasectomized men [27, 28] . this suggests that zikv is sequestered in the testes and/or accessory sex glands. mouse models have demonstrated zikv infection and associated pathology in the testes [29] [30] [31] of humanized blt mouse model with infection primarily targeting macrophages and leydig cells [32] . limited investigation has been done relating to infection of accessory sex glands in mouse models, but one study that assessed the prostate found no virus, possibly due to differential expression of the receptor candidate in the testes but not in the prostate [29] . for this experiment the finding of viral antigen and viral rna in the testes but not in the prostate is consistent with published animal models and may suggest the potential for bats to serve as another animal model. three bats had histopathological alterations in the hippocampus at later time points and one bat had viral nucleic acid present in the brain as determined by qrt-pcr demonstrating tropism for the cns, a tissue predilection also documented in humans and animal models. zikv has a predilection for nervous tissue in animal studies and disease manifestation in humans. as a neurological teratogen, zikv has been detected in the brain mononuclear cells in human newborns with fatal microcephaly and fetal miscarriages. histological lesions are varied but may include parenchymal calcification, microglial nodules, gliosis, cell degeneration, mononuclear infiltration and necrosis [33] [34] [35] . in non-human animal models, evidence for viral tropism has been found in brain and/or peripheral nervous tissue [36] [37] [38] [39] . in immunocompromised mouse models, the virus has a predilection for the brain but with the mice engineered for specific immune traits it is difficult to know to what extent this recapitulates natural zikv pathophysiology [40] . in the bats used in this experiment, evidence of zikvinduced pathology in the brain is consistent with what has been seen in human newborns and fetuses. the novel finding of co-localizing zikv antigen in bat iba1 + microglial/macrophage cells lends support to the earlier evidence of microglial cell infection via axl ligand bridging zikv particles to glial cells [41] . iba1 (aka, allograft inflammatory factor 1, aif1) is a microglia/macrophage-specific calcium-binding protein, which has actin-bundling activity that participates in membrane ruffling and phagocytic activity of activated microglia. activated microglial cells appeared with increased ability of cell migration and phagocytosis, which is controlled by remodeling of membrane cytoskeleton [42] . the morphology of cells with co-localization in the brain of infected bats is consistent with activated microglia depicting prominent branched processes. recent primate models in rhesus and cynomolgus macaques demonstrated similar viral distribution of zikv antigen to that in bats, described herein. high-level of zikv was evident in cerebellar neurons and the same studies documented involvement of iba1 positive microglial cells in cns infections. in primate models there is increasing evidence that zikv antigen was detected in individuals with the highest peak plasma viremia, which in part implies that zikv may initially seed the cns by a passive spillover from circulating monocytes to resident microglial cells. this is further substantiated in all of human and animal studies, which did not show any evidence of disruption to bbb or viral distribution reminiscent of circumventricular distribution seen in alphavirus animal models [43] . in addition to brain and testes immunoreactivity, scrotal skin and mandibular salivary gland also harbored viral antigen. distribution of viral antigen in bat tissues suggests that infection in this species recapitulates human infection, which is thought to start with infection of epidermal and dermal cells with subsequent dissemination to multiple organs including salivary glands as viral rna can be detected in human saliva [44, 45] . the histopathology for ajzika virus infects new world bats z5, 5 dpi showed sialoadenitis and the presence zikv antigen by ihc (fig 3) . this suggests zikv may be shed in the saliva, although additional animal experiments need to be performed to confirm such a route of shedding. the results presented here suggest that jamaican fruit bats may be a suitable animal model for examining zikv infection to elucidate its pathogenesis. jamaican fruit bats may also serve as a model to ascertain sexual transmission, in utero transmission, teratogenesis and neurological pathophysiology. it may be that zikv is a wildlife disease threat for bats that could lead to infertility in some males, which could impact bat populations. zikv is thought to be maintained in two different distinct cycles: sylvatic-cycling between non-human primates (nhp) and mosquito species, and urban-cycling between humans and mosquito species [3] . while there are limited data on what mosquito species feed on jamaican fruit bats, evidence for natural flavivirus infection has been identified in wild new world bats. dengue virus (denv) rna and antibodies to denv were detected in multiple species of bats, including jamaican fruit bats, in mexico [46] . additionally, antibodies to denv were detected in multiple bat species including those of the artibeus genus in costa rica and ecuador [47, 48] . these data indirectly provide evidence for mosquito-bat interactions in the wild; either through consumption of bat-blood meals taken by mosquitoes or bat consumption of infected mosquitoes. as it pertains to a wildlife reservoir, wild nhps have antibody to zikv including several monkey species trapped near ziika forest [10] , and wild and semi-captive orangutans in borneo [49] . not only have nhp been found to be seropositive, but also many other mammals, including rodents, horses, cows, and goats [50, 51] . furthermore, experimental inoculation of various north american species resulted in seroconversion (cottontail rabbits, boar goats, pigs, and leopard frogs) and demonstrated viremia (nine-banded armadillo and leopard frogs) [52] . molecular epidemiology suggests animals play an important role in an enzootic cycle [11] . much about the enzootic cycle of zikv has yet to be understood but it stands to reason that bats may be capable of maintaining the virus in nature. jamaican fruit bats are found in northern south america, central america, and the caribbean-areas that now have zikv potentially exposing bat populations to the virus [8, 53] . however, the data presented here suggest it is unlikely that jamaican fruit bats can serve as amplification hosts of zikv, unless virus sequesters in some as-yet unidentified way that could lead to periodic shedding of virus. it may also be that some bats become persistently infected and can transmit sexually to maintain virus within populations of bats. further experimental and field studies will be necessary to fully understand the ecological role of bats in zikv maintenance. all animal procedures were approved by the colorado state university (csu) institutional animal care and use committee (protocol 16-6512a) and were in compliance with u.s. animal welfare act. bats csu has a captive colony of jamaican fruit bats (artibeus jamaicensis), a neotropical fruit bat indigenous to much of south america, central america and the caribbean [53] . colony bats are kept in a free flight room measuring 19'w x 10'l x 9'h. roosting baskets are hung from the ceiling throughout the room and drapes of different cloth material are positioned for hanging and roosting. ambient temperature is maintained between 20˚c and 25˚c, with humidity between 50% and 70%, and a 12 hour light/12 hour dark light cycle via a computer-controlled system. diets consist of a combination of fruits (shamrock foods, fort collins, co), tekald primate diet (envigo, huntington, uk), molasses, nonfat dry milk and cherry gelatin that are placed in multiple feeding trays around the room once a day. fresh water is provided. in addition, fruit is hung around the room to stimulate foraging behavior and serve as enrichment. for infection experiments, bats were trapped using a butterfly net and placed in an 20"d x 12"w x 18"h cage for 24 hours prior to inoculations to allow for acclimation. hanging clothes were provided for roosting and coverage. food and water are placed in open trays in the bottom of the cage and changed daily. tray liners were changed every two days, and cages and hanging clothes are changed every two weeks. due to the social nature of these bats, minimums of two bats were kept in cages at all times to mitigate potential stress. two sets of experiments were performed; a pilot study and a time course study. zika virus strain prvabc59. prvabc59 was isolated in 2015 by centers for disease control and prevention (fort collins, co) from an infected individual who traveled to puerto rico (genbank accession no. hq234499). the virus stock titer is 3x10 7 plaque forming units (pfu) per ml of media, and the fourth passage was used for both studies. for the pilot study, three male bats were anesthetized with 1% to 3% isoflurane to effect with an oxygen flow rate of 1.5 l/min, administered with a gas mask. animals were placed on a heating pad to maintain body temperature and respirations continuously monitored. the dorsum of each animal was disinfected with 70% ethanol and 25ul containing 7.5x10 5 p.f.u of virus was administered subcutaneously (sc) at the level of the scapula with a sterile hypodermic 25 gauge needle in a biosafety cabinet. when procedures were finished, bats were removed from isoflurane and placed back in the cage in ventral recumbency. respirations were monitored until animal was fully awake and ambulated normally. bats were identified as aj-z7, aj-z8 and aj-z9. animals were euthanized at 28 days post-inoculation (dpi). for the time course study, six male bats were anesthetized under the same protocol as the pilot study. animals were placed in ventral recumbency. after disinfecting the dorsum of each animal with 70% ethanol, 0.15mls of 1% lidocaine was administered sc at the level of the last rib with a 25 gauge sterile hypodermic needle as a local anesthetic. iptt300 transponders (biomedic data systems, inc., seaford, de) were inserted sc at the level of the caudal edge of the scapula. twenty-five microliters containing 7.5x10 5 p.f.u of virus was administered sc at the level of the cranial edge of the scapula. recovery followed the same protocol as for the pilot study bats. animals were identified as aj-z1 through aj-z6. aj-z1 and aj-z2 were euthanized at two dpi. aj-z3 and aj-z4 were euthanized at 5 dpi. aj-z5 and aj-z6 were euthanized at 10 dpi. female bats were excluded from the study because they are prioritized for breeding to sustain and expand upon the colony. for the pilot study, bats were visually monitored twice daily for fourteen days, and then monitored once a day for an additional fourteen days. for the time course study, bats were monitored twice a day throughout the experiment. for both studies, energy levels, behavior, ability to ambulate, respirations, presence of oral or nasal discharge, and fecal consistency were all assessed. during the time course study urine was collected at 2, 3, 5 and 10 dpi from as many bats as possible. urine was collected by allowing bats to grasp screen cloth with their feet and then the bat was placed in a clear solo cup (dart container, lake forest, il) with the screen covering the top of the cup as a lid, and kept in place with a rubber band. this allowed the bats to hang in a clear container. bats were monitored for 45 minutes. if they urinated, bats were removed from the collection contraption and placed back in the cage without disrupting the urine. urine collection was attempted on all remaining bats at each time point, but not all bats would urinate at each collection attempt. urine was successfully collected as follows: two dpi from aj-z3 and aj-z4; three dpi from aj-z3, aj-z5 and aj-z6; five dpi from aj-z3, aj-z4, aj-z5 and aj-z6; and ten dpi from aj-z5 and aj-z6. urine was pipetted off the surface of the cup with a sterile pipette tip and put in a 1.5 ml microcentrifuge tube and stored at -80˚c for future use. urine volume ranged between 5 ul and 15 ul. bats were deeply anesthetized and maintained with 3% isoflurane and an oxygen flow rate of 1.5 l/min. deep pain was assessed by firmly pinching skin and toes with forceps and assessed for any response. a thoracotomy was then performed with sterile standard scissors to puncture through the skin, muscle and diaphragm just caudal to the sternum and cut through the wall of the chest cavity caudally to cranially-removing and preventing negative pressure from building in the thorax. cardiac blood was collected with a 21 gauge sterile needle inserted into the apex of the heart. a maximum blood volume of between 1 and 1.5mls is collected in a syringe and transferred to a red top tube (rtt). rtts sat at room temperature for one hour to allow a clot to form and then centrifuged at 1000 x g for 10 min at room temperature. serum was removed from the clot, placed in a new microcentrifuge tube and stored at -20˚c. serum from bats at 2 and 5 dpi were used to assess for viremia. serum from 10 dpi and the 28 dpi pilot study bats were used to determine antibody titers. because blood draws yield a small volume of blood (50 μl whole blood for a non-terminal blood draw, 500 μl whole blood for terminal blood draw) it was necessary to prioritize samples to optimize data retrieved. in order to assay the serum for viral rna and perform serology, earlier time points were used to assess for viremia and later time points for seroconversion. along with sample partitioning for data maximization, the small blood volume led to concerns that there would be an undetectably small viral load. to circumvent this issue, neat serum and 1:10 diluted serum were inoculated onto vero cells to amplify any virus that may have been present at low levels. one blind passage on vero cells was done and cell supernatants assayed by qrt-pcr. the remaining serum from three of the four bats was assayed directly for zikv rna. necropsies were performed immediately after euthanasia. bats were assessed for gross pathology. the following tissues were collected for both experiments: heart, lung, liver, spleen, kidney, urinary bladder, prostate, testes, and brain. a portion of tissues were collected and kept at -80˚c for rna extraction, and a portion placed in 10% buffered formalin for histology at a 1:10 weight to volume ratio for histology. for a negative control animal a male bat was trapped from the colony and euthanized under the same protocol as the experimental infection bats. vero e6 cells (atcc) were propagated to 60% confluency in a 96-well tissue culture plate and infected with zikv strain prvabc at an m.o.i. of 0.1. after a one hour incubation period, unbound virus was removed and replaced with 2% fbs-dmem and incubated for a maximum of three days. media was then replaced with 85% acetone for 20 minutes at -20˚c to fix virusinfected cells to plate and serve as an antigen for enzyme-linked-immunosorbent assay (elisa). plates were stored at 4˚c until use and used within two weeks. plates were washed 5x with 0.05% tween 20-pbs and blocked with superblock t20 (tbs) blocking buffer (thermo fisher scientific, waltham, ma) for one hour at room temperature. serum from an uninfected bat was used for a negative control. a convalescent human serum sample (kindly provided by b. foy, csu) was used as a positive control. a two-fold serial dilution was used starting at 1:100 to 1:12800. diluted serum was placed in wells and incubated for two hours at room temperature. serum was removed and plates washed. hrp-conjugated protein a/g (thermo fisher scientific, waltham, ma) was added at a concentration of 2 μg/ml to each well, and incubated for 30 minutes at room temperature. hrp-conjugated protein a/g was used in place of a secondary antibody as it targets the fc portion of an antibody, which is highly conserved and therefore can be used for multiple animal species [54] . plates were washed and 150 μl of abts peroxidase substrate (2 component) (kpl, gaithersburg, md) added according to manufacturers' instructions, incubated at room temperature for 30 minutes, and then 150 μl of abts peroxidase stop solution (kpl, gaithersburg, md) added. plates were read on an emax plus microplate reader (cambridge scientific, watertown, ma). absorbance was measured at 405 nm and the limit of detectable response was set at three standard deviation values above mean negative control serum. trizol reagent was used for rna extraction from serum-cell supernatants, serum, urine and tissues according to ambion, life technologies protocol. for tissues, approximately 50 mg of tissue was homogenized with one ml of trizol reagent. a 5mm stainless steel bead (qiagen, valencia, ca) was used with a tissuelyser lt (qiagen, valencia, ca) at 50 hz for 5 minutes. one ml of trizol was added to urine to 5 to 15 μl of urine. one ml of trizol was added to 160 μl of serum from aj-z2, aj-z3, and aj-z4. two-hundred microliters of serum-cell supernatants were added to one ml of trizol. samples were then incubated at room temperature for 5 minutes. chloroform (thermo fisher scientific, waltham, ma) was added, samples were mixed, incubated for 3 minutes at room temperature and centrifuged at 12,000 x g for 15 minutes at 4˚c. the aqueous phase was removed, 4 μg of glycogen (thermo fisher scientific, waltham, ma) and 100% molecular grade isopropanol added (thermo fisher scientific, waltham, ma). samples were incubated at room temperature for 10 minutes and then centrifuged at 12,000 x g for 10 minutes at 4˚c. supernatant was removed and 75% molecular grade ethanol (thermo fisher scientific, waltham, ma) was added to rna pellet. samples were vortexed and centrifuged at 7500 x g for 5 minutes at 4˚c. wash was removed and air-dried. rna was resuspended in rnase-free water and stored at -80˚c for future use. vero cells were grown to 70 to 80% confluency in a 48-well tissue culture plate with 10% fbs-dmem. media was removed and 100 ul of bat serum from 2 dpi bats and 5 dpi bats was inoculated onto cells. additionally, serum from each bat was diluted 10-fold in 2% fbs (millipore sigma) pbs supplemented with 1% calcium and magnesium, and inoculated onto cells. samples were incubated for one hour at 37˚c. inoculum was removed and cells washed twice in sterile pbs. two-percent fbs-dmem was added to wells and plates were incubated at 37˚c, 5% co 2 . cells were assessed daily for cytopathology (cpe) through day 7 but none was observed. two-hundred microliters of the supernatant was removed on day 7 and used for rna extractions. an additional 100 μl of supernatant was blind passaged onto vero cells at 70 to 80% confluency. cells were incubated for one hour at 37˚c, washed twice with sterile pbs and 2% fbs-dmem added. on day seven, supernatant was removed and trizol extractions performed for rna recovery. serum was treated as such in an attempt to amplify viral load and increase assay sensitivity serum may not be the most sensitive diagnostic sample [23] [24] [25] [26] . if any serum was remaining it was directly used for trizol rna extractions. serum samples remained from aj-z2 at 2 dpi, and aj-z3 and aj-z4 at 5 dpi. no serum remained from aj-z1. roche real time ready rna virus master kit (roche, indianapolis, in) was used on rna extracted from serum-cell supernatants, serum, urine and tissue to assay for zikv rna according to manufacturers' instructions. primers used were zikv 1086 (ccgctgcccaa cacaag) and zikv 1162c (ccactaacgttcttttgcagacat). probe was zikv 1107-fam (agcctaccttgacaagcagtcagacactcaa) [55] . two-hundred nanograms of sample rna was added to each reaction. reactions were performed in duplicate. standards were a non-infectious clone of full length zikv strain prvabc59 by which concentration was determined through optical density. molecular weight of the genome sequence was used to calculate copy number [56] . a log 10 dilution series of the standard was made and linear regression used to determine copy number equivalents of positive samples. amplification was performed according to manufacturers' protocol for roche real time ready rna virus master kit (roche diagnostics corporation, indianapolis, in) with pcr conditions as follows: 8 min at 50˚c, 30 s at 95˚c, and 45 cycles of 10 s at 95˚c, 20 s at 60˚c and 10 s at 72˚c. tissues fixed in 10%-buffered formalin were cut in and submitted to colorado state university veterinary diagnostic laboratory (csu vdl, fort collins, co) for paraffin embedding, sectioning and staining with hematoxylin and eosin, as well as immunohistochemistry (ihc). tissues cut in on bats to assess for histology included: heart, lung, liver, kidney, testes, prostate, urinary bladder and brain. additionally, for aj-z3 and aj-z5 mandibular salivary gland was cut in. aj-z4 had esophagus and lymphoid tissue that included palatine salivary gland cut in. antibody for ihc was a polyclonal rabbit antibody that targets prem and e proteins of zikv and was provided by csu vdl's pathology department. the bond-iii automated instrument (leica biosystems, wetzlar, germany) was used for ihc staining. all slides were blindly read by a diplomat of the american college of veterinary pathologists. brain tissues was prepared for immunohistochemical and immunofluorescence staining as previously reported [57] . tissue was dehydrated by using a graded ethanol series of 70% ethanol for 2 h, 80% overnight, 90% for 2 h and 100% for 2 h. brain tissues were then post-fixed in dimethylbenzene for 30 min and embedded in dimethylbenzene-paraffin at 60˚c for 2 h, after which samples were embedded in a metal frame. sagittal sections were collected at 5um thick. all dewaxing, antigen retrieval and immunofluorescence staining was automated using a leica bond rxm. in short, sections were dewaxed using ethanol and then boiled in antigen retrieval solution for 10 minutes. the cooled sections were incubated in 3% h2o2 for 15 min at room temperature and then blocked with 2% donkey and goat serum (millipore sigma) for 1 hour. rabbit anti-iba1 (wako chemicals usa, irvine, ca) and 4g-2 flavivirus e specific monoclonal antibodies (cdc, fort collins) were diluted in tbs to final concentrations of 1:250 and 1:50, respectively. sections were incubated in primary antibodies concurrently at room temperature for one hour. following removal of unbound primary antibodies by washing, goat anti-rabbit secondary (alexafluor-555) and donkey anti-mouse secondary (alexafluor-647) was added and incubated for 1 hour at room temperature. finally, dapi counterstain (vector laboratories, burlingame, ca) was applied and sections were washed with tbs prior to cover slipping for imaging. stained sections were imaged on a ziess lsm 800 with airyscan laser-scanning confocal microscope (ziess, oberkochen, germany) using a 63× oil immersion objective. each field of view was imaged as a z-stack (8-10 planes, .5-μm step size) transformed into a single maximum projection image using the ziess zen (blue) imaging software. zika virus. i. isolations and serological specificity zika virus: a report on three cases of human infection during an epidemic of jaundice in nigeria zika virus: history, emergence, biology, and prospects for control zika virus outbreak on yap island, federated states of micronesia rapid spread of emerging zika virus in the pacific area zika virus outbreak, bahia, brazil. emerg infect dis first report of autochonous transmission of zika virus in brazil zika cases and congenital syndrome associated with zika virus reported by countries and territories in the americas potential for zika virus to establish a sylvatic 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rapid and specific detection of asian-and african-lineage zika viruses fluoxetine prevents lps-induced degeneration of nigral dopaminergic neurons by inhibiting microglia-mediated oxidative stress the authors would like to than brent davis, cdc, fort collins, co for supplying anti-zikv polyclonal and specific monoclonal antibodies. key: cord-003757-uwbpbai7 authors: chionh, yok teng; cui, jie; koh, javier; mendenhall, ian h.; ng, justin h. j.; low, dolyce; itahana, koji; irving, aaron t.; wang, lin-fa title: high basal heat-shock protein expression in bats confers resistance to cellular heat/oxidative stress date: 2019-06-22 journal: cell stress and chaperones doi: 10.1007/s12192-019-01013-y sha: doc_id: 3757 cord_uid: uwbpbai7 bats, unique among mammals with powered flight, have many species with the longest size-proportionate lifespan of all mammals. evolutionary adaptations would have been required to survive the elevated body temperatures during flight. heat shock protein (hsp), highly conserved master regulators of cell stress, expression was examined across tissues and various cell lines in bats. basal expression level of major hsps (hsp70 and hsp90) is significantly higher in two different bat species compared to other mammals. this hsp expression could be a bat-unique, key factor to modulate cellular stress and death. consequently, bat cells survive prolonged heat treatment, along with other stress stimuli, in a hsp-dependent manner, whereas other mammalian cells succumbed. this suggests hsp expression in bats could be an important adaption to intrinsic metabolic stresses like flight and therefore an important model to study stress resilience and longevity in general. electronic supplementary material: the online version of this article (10.1007/s12192-019-01013-y) contains supplementary material, which is available to authorized users. bats are the only mammal that have mastered true flight, and consequently, they must endure elevated body temperatures throughout flight. additionally, most have longer lifespans than terrestrial mammals of similar body sizes, despite their high metabolic rate and its predicted consequences on oxidative metabolism. as such, they have one of the greatest disparities between body mass and longevity (ball et al. 2018; bozek et al. 2017; davies et al. 2014; hughes et al. 2018; munshi-south and wilkinson 2010; wilkinson and adams 2019) . their longevity has been attributed, at least in part, to enhanced oxidative stress resistance and protein homeostasis yin et al. 2016) . the body temperature of bats raises drastically during flight (o'shea et al. 2014) , and there is an excessive increase in metabolic rate and presumably oxidative stress, with heart rates up to 1200 bpm (o'mara et al. 2017; podlutsky et al. 2005) . anecdotal evidence from carers suggests that fruit bats can tolerate prolonged heat stress in the wild if they remain well hydrated. heat shock proteins (hsps), as evident from their namesake, are produced in response to high temperature though also from other stress responses. there are five general classes: hsp30/40 (dnajs), hsp60 (hspd), hsp70 (hspa1/4/ 5/8/9), hsp90 and small hsps. heat shock protein 70 (hsp70) and 90 (hsp90) are among the better characterized hsps. a key function of hsps is to react to environmental and physiological stress, bind and repair damaged proteins, and reestablish protein homeostasis. high levels of hsps have been observed in several long-lived animals (morley and morimoto 2004; salway et al. 2011; singh et al. 2006 ). this could be due to the enhanced protein repair mechanisms facilitated by increased hsp levels, limiting prolonged cellular damage (galbadage and hartman 2008; heidler et al. 2010; minois et al. 1999; salmon et al. 2009; swindell 2009; walker and lithgow 2003; zhao et al. 2005) . both hsp70 (beere et al. 2000) and 90 (pandey et al. 2000) have been shown to inhibit apoptosis by interfering with the formation of the apoptosome, thereby reducing cell death during heat shock (mosser et al. 1997) . hsps, often neglected by immunologists, interact with vital components of both the innate and adaptive immune systems (srivastava 2002) . hsp70 and 90 are also up-regulated during viral infection (geller et al. 2012; glotzer et al. 2000; nagy et al. 2011) . it is now well-established that bats are reservoirs for several viruses capable of crossing the species barriers, evident by recent outbreaks of related viruses including ebola virus (leroy et al. 2005) , sars coronavirus (li et al. 2005) , mers coronavirus (ithete et al. 2013) , and directly with nipah virus (chua et al. 2002) and hendra virus (halpin et al. 2000; young et al. 1996) . these viruses often cause severe morbidity and mortality in incidental mammals, while bats remain clinically asymptomatic upon infection (storm, 2018 #74; schuh, 2018 #73; schuh, 2017 #75) . throughout evolution, bats may have gained novel functions allowing them to coexist with these viruses, thus evading the onset of severe pathology (wynne et al 2014 , zhang et al. 2017 zhou et al 2016) . hsp70 has been shown to inhibit 'cytokine storm', induced by viruses like sars (huang et al. 2005) , through binding/degrading the p65 subunit of nfκb, thus reducing overall inflammation and pathology (tanaka et al. 2014) . here, we show that bats have elevated expression of heat shock proteins (hsps) in various tissue and cell lines from two species, comparative sequence analysis of hsps from ten bat species and that increased hsp expression in vitro confers survival of cells during prolonged heat stress. this evolutionary advantage aids in bat physiology, though, and may have considerable impacts on other unique aspects of bat's longevity and zoonotic potential. the two species characterized include the australian black flying fox (pteropus alecto) and the cave nectar bat (eonycteris spelaea). both animals are frugivorous in nature, feeding mostly on nectar, flowers, seeds and fruit and are considered long-lived at 19.7 years (670.1 g) and 22 years (58.7 g), respectively (wilkinson and adams 2019 #59). these bats are largely data deficient with regard to maximum age, and closely related species are documented with much longer lifespans. both are in the order yinpterochiroptera and are considered adept at flying with single-day flight ranges documented of over 40 km (nowak 1999 #78; hall 2000 #77) . both species have similar blood glucose levels and respiratory capacity (/body size), suggesting similar metabolism between the two (maina 1991 #82; hill 1984 #81; peng 2017 #79) . we hypothesise that higher levels of hsps allow bats to endure the metabolic stresses induced by high temperatures, presumably that generated by sustained flight, and this alters the ability of bats to survive at these temperatures. this in turn may impact the bat's lifespan (podlutsky et al. 2005) and pathogen-host equilibrium as hsps are involved in both processes ). the evolutionary conserved function of hsp's does not appear to be altered, merely the regulation of expression and intrinsic availability. healthy eyonicteris spelaea, rattus tanezumi, chalcophaps indica and treron vernans were caught in singapore at the beginning of an activity cycle and rested prior to processing. all work was done with the ethics approval of national university of singapore (iacuc permit # b01/12), and the national parks permits np/rp11-011-3a and np/rp12-004-2. all c57bl/6 mice were healthy, male, 8-15 weeks old. healthy resting, adult, bats were used for e. spelaea tissue, and samples were obtained from 1 female and 2 males, with an average weight of 58 g. adult pteropus alecto bats, physically damaged but otherwise healthy, were collected from bat carers around south-east queensland (australia), transiently housed and processed at the resting state. three males and 1 female were used for ngs with an average body weight of 692 g. these weights are close to the expected weights for these species (wilkinson and adams 2019 #59) . all experiments were performed in accordance with relevant guidelines and regulations. the generation of palut02 (rrid:cvcl_dr91) and pakit03 (rrid:cvcl_dr89) cell lines has been described previously (crameri et al. 2009 ). eonycteris spelaea lung epithelia (eslut02) cell line was generated following our previously established method (crameri et al. 2009 ) and selected for based on optimum culturing conditions matching those of most mammalian cells. this cell line exhibits a typical doubling time of 2-3 days, expresses no detectable hif1α, minimal cellular/ mitochondrial ros production, and minimal uptake of trypan blue or pi and has been culture up to at least 70 passages, indicating suitable culturing conditions. paki, eslu, bhk-21 (mesocricetus auratus, rrid:cvcl_1915), mdck (canis lupus familiaris, rrid:cvcl_b033), a549 (human, rrid:cvcl_0023) and wi-38 (human, rrid:cvcl_0579) cell lines were all cultured in dmem (gibco) with 10% (v/v) fbs (bioind #04-001-1a) (well established for long-term culture). duck embryo cell-line ccl-141 from anas platyrhynchus domesticus (rrid:cvcl_t281) was purchased from atcc and cultured in eagle's minimum essential medium (emem) (gibco) with 10% fbs, as recommended. all tissue was preserved in rnalater except for muscle, which was snap frozen in liquid nitrogen then processed with trizol directly to preserve the limited rna amounts. all the tissue samples tested are performed in biological replicates unless otherwise stated. cell-line studies were performed across multiple passages in separate experiments. paki, bhk and mdck cells were all initially grown and adhered overnight to 96-well black-wall tc-treated plates (nunc) at 37°c and heat-treated at 40°c for 4-24 h. prior to treatment, cells were loaded at 37°c with vybrant cell metabolic assay kit with c12-resazurin (thermo fisher scientific), according to the manufacturer's protocol (1:2000), washed twice in pbs and fresh phenol-red free dmem was added (gibco, thermoscientific). briefly, the c12-resazurin is converted to a fluorescent by-product by cellular esterases in an atp-dependent manner, and the fluorescence signal is proportional to the amount of atp. c12-resazurin by-product was then measured with an excitation/emission maxima of 563/ 587 nm. enough un-converted dye is loaded for 24 h of constant imaging accounting for minor bleaching. fluorescent signal of the converted resorufin control was the same at 37/ 40°c. knockdown of hsp90 and hsp70 by sirnas was performed using rnaimax (thermo fisher scientific) with oligos purchased from idt (table s4 ) according to the manufacturer's protocol. for sirna knockdown of hsp90, a combination of hsp90aa1and ab1 was used at a ratio of 1:1. cells were washed twice with pbs to remove excess dye and cultured in dmem with 10% fbs at 37°c and 40°c in a tecan plate reader and detected using ex/em at 560 nm/590 nm wavelength. cell viability was calculated by normalizing against the 2-h time point after the dye had completely stabilized. the cell viability was plotted over time using graphpad prism software and a growth/survival (kaplan-meier) curve constructed. the significant difference between the different cell growth curves over time was calculated using two-way anova, bonferroni multiple comparisons. snapped frozen tissues were placed in trizol® reagent (invitrogen) and homogenized using ceramic beads in tissue digester (fastprep-24™, m.p. biomedical, llc, santa ana california, usa). rna and protein were extracted according to the manufacturer's protocol. proteins were solubilized in 1% sds with proteinase inhibitors cocktail (roche) and separated on 10% or 15% sds-page gels and transferred onto to pvdf membranes (milipore). membranes were blocked with 5% skim milk and probed with anti-hsp90 (ac88 #ab13492; abcam), anti-hsp70 (3a3, #ab5439; abcam) or anti-gapdh (pierce) overnight. after washing, the membrane is incubated with goat anti-mouse igg-hrp (santa cruz biotechnology) for 2 h. all antibodies are diluted at 1:5000. membranes were visualized using ecl prime chemiluminescence reagent (ge healthcare) and a myecl imager (thermo fisher scientific), with densitometry performed using myimageanalysis software (thermo fisher scientific). abcam anti-hsp70 and hsp90 antibodies have been tested against several animal species including human mouse, chicken and fish and is known to bind to the conserved region of hsp70 and hsp90 (mapped in supplementary table s1b, c). complementary dna (cdna) from tissue and cells was made using quantitect® reverse transcription kit (qiagen). quantitative pcr (qpcr) was performed to determine mrna levels of different hsp genes. reactions were setup using the sensifast™ sybr no-rox kit (bioline) and assays were run on the cfx96 touch™ real-time pcr detection system (bio-rad) under the following cycling condition: 95°c for 5 min, followed by 40 cycles of 95°c for 10 s and 55°c for 30 s, and ending with a melt profile analysis. relative expression of the targeted gene was determined by rest formula (pfaffl 2001) , relative to housekeeping gene gapdh. qpcr primers for hsp90aa1, hsp90ab, hsp70 and gapdh genes are listed in table s1 . gapdh has previously been shown to be an appropriate housekeeping gene in multiple species and across tissue (kosir, 2010 #85; foss 1998 #84; grant 2009 #83) . the complete coding regions of hsp genes, hsp90aa1, hsp70 and hsp90ab1, across different species were collected from genbank (http://www.ncbi.nlm.nih.gov/genome) and aligned using clustalw in strap (gille and frommel 2001) . three gene data sets were created and each data set contained 18 mammalian species, belonging to seven groups: bats (eptesicus fuscus, pteropus alecto and myotis davidii), cetartiodactyla (bos taurus, tursiops truncatus and sus scrofa) , odd-toed ungulates (equus caballus and ceratotherium simum simum), carnivores (felis catus and canis lupus familiaris), eulipotyphla (sorex araneus and erinaceus europaeus), rodents (mus musculus, rattus norvegicus and spermophilus tridecemlineatus) and primates (homo sapiens, pan troglodytes and gorilla gorilla). a non-bootstrapped maximum likelihood phylogenetic reconstruction was performed by using phyml 3.0 (guindon et al. 2010) , under gtr + i + g model. tests for selection on ancestral hsps of different taxa were inferred by using paml 4.8 (yang 2007) , under a free ratio model, which allows each branch having different dn/ds (ratio of nonsynonymous to synonymous changes) values. promoter analysis was performed using the 1500 bp directly upstream of the atg for bat (xm_006917965.3) and human (nm_005347.5) hspa5 gene. the sequence was passed through genepromoterminer (http://gpminer.mbc.nctu.edu. tw/) and analysed using transfac binding sites (core score 1.0, matrix threshold 0.95) (lee et al. 2012 ). viability of cells was measured at a single timepoint using propidium iodide (pi) exclusion dye assay. briefly, pakit03 cells were transfected with either sirna control, sirna for hsp70 or sirna hsp90 and incubated for 24 h before treatment with spermine nonoate for 6 h at concentrations of 0.1, 0.25, 0.5 and 1 mm, respectively. the media was then removed, replaced with fresh dmem and incubated for 72 h. supernatants were collected, floating cells spun down and added to adherent cells followed by incubation with propidium iodide (pi) at a final concentration of 5 μg/ml for 15 min prior to flow cytometry analysis. spermine nonoate was purchased from cayman chemical in powder form. flow cytometry was conducted using macsquant analyser 10 (miltenyi biotec) and laser excitation wavelength was set at 488 nm and filtered emission at 615/50 nm. a total number of 10,000 live cells were analysed per analysis. the percentage of viable cells were calculated using the equation: viable cells (%) = number of cells (pi-negative) / number of cells (pi-negative) at 0 mm spermine nonoate treatment from 10,000 total rna was checked using the rna 6000 labchip kit on the agilent bioanalyzer (agilent technologies, palo alto, ca). rnaseq libraries were prepared using illumina tru-seq stranded total rna with ribo-zero gold kit following the manufacturer's instructions (illumina, san diego, ca, usa). libraries were validated with an agilent bioanalyzer (agilent technologies, palo alto, ca), diluted and applied to an illumina flow cell using the illumina cbot system. sequencing was performed on an illumina hiseq 3000 sequencer at the duke-nus genome biology facility with the paired-end 150-bp read option. after trimming and cleaning for quality assurance (including distribution of reads), all reads were mapped to the p.alecto reference genome (ncbi genome database: asm32557v1, 1.01) with bowtie and rsem abundance estimation was performed (li et al., 2005 #86) . e. spelaea reads were de novo assembled with tophat/cufflinks (ghosh 2016 #87) , and the fpkm for both bowtie/rsem mapped data sets was calculated using cufflinks. normalized fpkm values in liver tissue for p.a (n = 4 individuals, gse129377), e.s (n = 2 individuals, gse129199), and public datasets for h.s (n = 16 individuals, gse94660) and m.m. (n = 9 individuals, gse95135) were compared. unless specifically mentioned in the figure legend, all cell experiments were performed in a biological (n) triplicate with multiple experiments. animal tissue studies involved three individuals for qpcr/western blot and multiple individuals (as indicated) for rnaseq. graphs for qpcr are combined for technical duplicates × three biological replicates (six values used in each graph), and western blot quantification graphs are combined for three separate western blots. cell viability assays are a representative experiment out of three separate experiments (in biological triplicate each time with 12 readings per well/replicate). error is represented as sd unless otherwise stated and p* < 0.05, **p < 0.02, ***p < 0.01 for a one-way anova relative to the matched control (bat vs mouse etc. unless indicated in the figure) or students unpaired, two-tailed, t test, as indicated. graphs were generated using graphpad prism, and heatmaps and boxplots were generated in morpheus (broad institute). all data used in this manuscript is available in open source databases (ncbi sra bioprojects, h.s gse94660 m.m. gse95135 p.a gse129377 e.s gse129199) or available to authors upon request. to confirm heat tolerance at the cellular level, as predicted by elevated core body temperature during flight, bat cells were subjected to prolonged heat stress. bat kidney cells (pakit03) (crameri et al. 2009 ) and lung cells palu and eslu were cultured alongside kidney cells from other species (mdck and bhk) and human lung cells (a549) cells at 37°c and 40°c for 24 h of culture. while all six cell lines were able to grow at 37°c, as indicated by c12-resazurin conversion by atp (fig. 1a, b) , only bat cells pakit03, palu and eslu were able to survive, and even proliferate, for 24 h at 40°c (fig. 1c, d) . the atp availability assay, c12-resazurin, documents not just cell survival but continuous metabolic activity by the esterase required to generate fluorescence. this highlights a unique ability to tolerate heat stress by bat cells in culture. fig. 1 survival of bat cells at 40°c and expression of hsps. a kidney cell lines, paki (bat), mdck (canine) and bhk (hamster) were cultured at 37°c along with b lung cell lines palu (p.a. bat), eslu (e.s. bat), a549 (human) and viability was measured each hr for 24 h with c12-resazurin atp-conversion. c kidney cells were subjected to 40°c heat treatment for 24 h along with d lung cell lines. statistical significance was calculated using two-way anova (graphpad prism software) comparing the cell growth rate over time; ***p < 0.0001. f expression levels of hsp90aa1, g hsp90ab1 and h hsp70 in various tissue of c57bl/6 mice, wild caught rat (r. tanezumi) and bats (e. spelaea and p. alecto) (n = 3) were measured using sybr-green qpcr (tissues as indicated, relative to gapdh). i hsp levels in each tissue were combined into an average value for hsp90aa1, hsp90ab1 and hsp70. the expression of hsps in bats (e. spelaea and p. alecto) was compared against mouse and rat. error bar denotes standard derivation (sd) to determine the level of hsp70 and hsp90 expression in bats, real-time pcr (qpcr) was used to quantify the relative amounts of hsp rna. as hsp90 involves two different genes (hsp90aa1 and hsp90ab1), we used two qpcr primer sets along with hsp70 (hspa5) to determine the rna expression in two bat species, the singaporean cave nectar bat (eonycteris spelaea) and the australian back flying fox (pteropus alecto). expression was compared relative to gapdh, used previously for cross-species/tissue comparison (ahn 2019 #90; kosir 2010 #85; foss 1998 #84; grant 2009 #83) . inbred (c57bl/ 6) laboratory mice and wild-captured rats (asian house rat, rattus tanezumi) were added for comparison. the additional of a field-captured rodent was to control for any stress response to collection of wild animals and minimize discrepancies between laboratory and wild-caught animals. tissues tested included heart, lung, spleen, kidney, muscle, brain, liver, intestine, wing and lymph node, collected during the resting state, depending on availability in each species. as shown in fig. 1f , g, we found that e. spelaea, in most tissues, has a significantly higher level of hsp90aa1, hsp90ab and hsp70 compared to rodents. p. alecto also has significantly higher levels of hsp90aa1 and hsp70, with a trend for hsp90ab1 that did not reach significance (p = 0.06). figure 1i shows a cumulative average of the tissues of each species for each gene. all three hsps are highly expressed in the lung tissues of both e. spelaea and p. alecto (and liver for e. spelaea) compared to those in mice and rats (fig. 1f-h) . moreover, we further validated this high hsp expression at the protein level with conserved antibodies known to detect multiple species. this shows that the amount of hsp70 and hsp90 (complex) in both the e. spelaea and p. alecto muscle (at the resting, unstimulated state) is significantly higher compared to mouse, where minimal hsp expression is expected at the resting state (fig. 2a , antibody binding sites, table s1 ). to confirm in mammalian cell lines (from two different organs, kidney and lung, respectively), we analysed cells by qpcr and western blot. for kidney, we found that pakit03 (bat) cells expressed significantly higher amounts of hsp70 and hsp90a/b both at the mrna (fig. 2b-d) and protein levels (fig. 2e) at 37°c compared to the mdck (dog) and bhk (hamster) cells under normal conditions. under heat treatment at 40°c for 3 h, all kidney-derived cell lines increased the hsp expression at the rna level ( fig. 2b-d) . interestingly, the relative amounts of hsp70, hsp90aa1 and hsp90ab expressed by pakit03 are still significantly higher than those of the other two mammalian cell lines. similar observations were found in lung-derived cells, as shown in fig. 2f -i. with cells maintained basally at 37°c, bat cells (both p. alecto and e. spelaea) produced significantly higher amounts of hsp70 and hsp90 at both mrna and protein levels than the a549 and wi-38 cells of human origin (fig. 2f-i) . with heat treatment, eslu and palu cells also produced significantly higher amounts of hsp70, hsp90aa1 and hsp90ab compared to a549 and wi-38 cells by qpcr. while maintaining a high basal expression of hsp's, bat cells still retain the ability for further heat induction of hsps to levels far higher than other mammalian cells. proteins with critical functions exhibit high conservation (castello et al. 2016; garg et al. 2015; ng et al. 2016) . to determine if this was true for bat hsps, we analysed the available bat genomic sequences and found a 99.9% hsp sequence identity between bats and other mammals (fig. s1 ). next, we examined the amino acid selection pressure and found that all three bat hsp genes exhibited slightly higher selection pressure values compared to non-bat mammalian groups (fig. 3ac) ; however, this was not statistically significant with the low dn/ds ratio inferring that the conservation of genes in this pathway is critical for their function. to examine expression of hsp 40, 60, 70, 90 and known hsp regulators, de novo transcriptome assembly from liver of wild bats for p. alecto (n = 4) and e. spelaea (n = 2) was compared with publicly available sequence data of human (n = 16) and mouse (n = 9) from the same illumine hiseq3000 platform/pipeline. the average normalized fpkm is shown (fig. 3d) with high expression of some hsp70 (particularly hspa5/8/9), hsp90 (hsp90aa1/hsp90b1), hsp40 (dnaja1) and hsp60 (hspd1) genes in bats compared to fig. 2 expression at the protein level and correlation to cell line expression. a protein was extracted from the muscle of c57bl/6 mice, e. spelaea and p. alecto (n = 3 individuals each) using trizol extraction method and probed with anti-hsp70, anti-hsp90 and anti-gapdh antibodies (left panel), and levels were quantified using myimageanalysis software (thermo fisher scientific inc) and normalized against gapdh (right panel). statistical significance was calculated using one-way anova (graphpad prism software) comparing either e. spelaea or p. alecto to the mouse or rat; ***p < 0.0001, **p < 0.001, and *p < 0.05. error bar denotes standard derivation (sd). kidney (mdck, bhk and paki) cell lines were used to determine the expression of b hsp90aa1, c hsp90ab1 and d hsp70 by qpcr as previously at 37°c and 40°c, respectively. e protein expression of hsp70 and hsp90 in the cell lines at 37°c was probed with anti-hsp70, anti-hsp90 and anti-gapdh antibodies. similarly, for lung cell lines (a549, w138, palu and eslu) for qpcr (f-h) and protein (i). statistical significance was calculated using two-way anova (graphpad prism software) comparing bat cell lines (pakit03, palu and eslu) to other cell lines; **p < 0.001, and *p < 0.05. error bar denotes standard derivation (sd) mouse and human (with these significant gene families displayed in the adjacent boxplot). interestingly, this higher rna expression does not correlate with the common regulators of hsp expression such as hsf-1/2, prdm1/4, irf2, fos and tp53 (hayashida et al. 2010; prasad et al. 2007; zhang et al. 2011) . while there are slightly higher levels of other implicated regulators smad1, nfatc1, bach2, stat1, mzf1 and nfya in bat liver tissue, the overall fpkm is still minimal for all regulatory genes except possibly stat1/mzf1/nfya (table s2) . to identify if genetic elements in the promoter are responsible for differential expression, the promoter for human (hs) hspa5 (the highest expressed) was analysed (transfac 8.3.4 ) and displayed in fig. 3e . while several elements are missing, or exhibiting lower sequence identity, in the p. alecto (pa) hspa5 (fig. 3f ) promoter compared to human, the majority of elements are conserved. there are no obvious additions with new promoter elements/transcription factor (tf) binding sites visible in the pahspa5 promoter. of note, the identity/frequency increased for nfya/caat and mzf1binding sites. taken together with higher expression of these tfs in the liver, this correlates with higher expression of hspa5 in bat liver. it is worth to note that in the paki and palu cell lines, the expression of most known hsp promoter tfs does not correlate with the high expression of hsp70/90, particularly in comparison to hek93, which expresses high levels of hsp's concurrent with high levels of most hsf tfs. table s3 includes ngs fpkm values of hsp genes, tfs and housekeeping genes from pakit03, palu and hek293 for comparison. there is a slightly elevated level of tp53, mzf1 and nfya in the bat cell lines that may partially explain the expression status. as high basal hsp expression may be responsible for the survival of bat cells at 40°c, we used sirna to knockdown hsp90 and hsp70 in bat pakt03 and palu cells. the knockdown efficiency was determined to be approximately 90% (fig. s2) . while crispr-cas9 can be used in pakit03 cells, knockdown of hsp90 and hsp70 families via crispr proved non-viable, likely due to the essential nature of hsps. however, pakit03 and palu cells with hsp90 and hsp70 knocked down by sirna showed minimal signs of abnormal growth (fig. 4a, b) or morphological change at 37°c compared to non-silencing (ns) control sirna. in contrast, when treated for 24 h at 40°c, neither hsp knockdown treatments survived with pakit03 or palu cells. the control-treatment cells survived and continued to proliferate (fig. 4c, d) . not only do these findings support the predicted role of hsps during heat stress, but they also strongly suggest that expression of both hsp70 and hsp90 is essential for the survival of bat cells during this prolonged heat stress. to further understand the role of hsps in flying animals, we compared hsp levels of bats with two flying birds, pigeon and dove. muscle from two avian species were compared against p. alecto (fig. 4e) . p. alecto expressed higher levels of hsp70 and 90 compared to chalcophaps indica (emerald dove) and treron vernans (pink-necked green pigeon) (fig. 4f) . the protein levels of hsp70 and 90 in an avian cell line (ccl-141, anas platyrhynchos domesticus) were also significantly lower compared to pakit03 (fig. 4g, h) , though albeit than previously examined cells. as we believe high basal hsp expression is a bat-specific adaptation to heat stress, ccl-141 was subsequently cultured at 37°c and 40°c to examine its resistance to heat stress. while ccl-141 continued to grow at 37°c, the duck cells stopped growing or dividing at 40°c and maintained a constant viability, possibly senescence (fig. 4i ). as flight of bats is metabolically demanding and high hsp expression is imprinted even in cultured bat cells, we evaluated the protective role of hsps to oxidative stress that can occur from high metabolic demands. parental pakit03 cells and hsp70 or hsp90 sirna-treated cells were subjected to increasing concentrations of spermine nonoate for 6 h, followed by incubation for 72 h and then examined for cell viability. knockdown of either hsp70 or hsp90 in pakit03 cells demonstrated a reduction in cell confluency as assessed via phase-contrast microscopy 72 h after 1 mm spermine nonoate removal (fig. s2 ). in addition, using pi exclusion for viability, we observed significant reduction in the percentage of viable pakit03 cells in the presence of hsp70 knockdown for all spermine nonoate concentrations and with 0.1 and 0.25 mm concentrations for hsp90 treatment (fig. 4j) . high doses of spermine nonoate decreased the number of e viable cells even in control cells. these observations suggest that hsps are essential for survivability of bat cells during a type of oxidative stress, nitric oxide, which is potentially generated from flight. it is conceivable and generally accepted that major evolutionary adaptation is required for bats to sustain its ability to fly. recent comparative genomics studies have confirmed that bats are different to all other mammalian groups (ahn et al. 2016; ng et al. 2016; zhang et al. 2013; zhou et al. 2014) . however, it is extremely difficult to pinpoint major genetic adaptations to explain why bats have a relatively longer lifespan or to their apparent reservoir host potential. the adaptations required to sustain a high rate of metabolism and high body temperature during daily flight may provide insight. while proving high basal hsp expression of bats is an adaptive evolution from flight is not possible without a non-flying bat species for comparison, we speculate that this high expression pattern of hsps in bats is likely an adaptation to cope with both high heat and metabolic stress. here, we report the first evidence that bats of two different species, p. alecto and e. spelaea, have elevated basal level expression of both major hsps, hsp70 and hsp90, in live animal-derived tissues, and this is imprinted in laboratoryderived cell lines. additionally, the liver of animals exhibited increased expression of other hsp families including hsp40 (dnaja1) and hsp60 (hspd1). salway et al. (salway et al. 2011) previously reported that there is a positive correlation between lifespan and the level of hsp expression in animals. in our studies, we have demonstrated that the expression levels of hsp90 and hsp70 are significantly higher than those in the tissue of pigeon, duck, mouse, rat and human and in cell lines of human, dog, hamster and duck origin. this is especially true for hsp70 (hspa5), which has a 10fold higher level of expression in bats compared to rodents. a comparison to smaller bats with exceptional longevity quotients would be required for a correlation of hsp expression to lifespan in bats. in comparison with other flying animals, pigeon and duck have lower levels of hsp70 and 90 compared to p. alecto. this suggests that while birds still increase their core body temperature (mcnab 1966) during flight, hsp expression is not be the main protective effector in birds. this suggests that high basal hsp expression is a bat-specific phenomenon. the levels we observed in pigeon and duck are comparable to the study of finches from salway et al. (salway et al. 2011) . this was partially confirmed by the hsp expression in ccl-141 duck cells failing to confer the resistance to heat stress observed in multiple bat cells. other primary avian cells have been reported to survive at 40°c, presumably through hsp-independent mechanisms (harper, 2011 #88) . to determine if bat hsps could functionally differ, we examined genetic evolution and dn/ds selection pressure. as bat hsps are highly homologous to other mammalian hsps, with low dn/ds variation, this seems unlikely and it emphasizes their critical function in the biology of the host. it has previously been shown that there is a disconnection between cross-species sequence homology and expression levels in the oriental fruit moth . this highlights that altering expression of hsps rather than altering the function of hsps still confers a phenotypic advantage. this may be the case in other species, such as the bat. further annotation and sequencing data from available bat genomes will allow a full phylogeny-independent characterization of all hsp genes from bats, though our preliminary indicates that hsp function is not altered. analysis of the highly expressed hsp70 gene revealed only minor differences in the promoter region with no particularly enhanced elements compared to the human promoter. while caution must be used when using algorithms designed against human on bat transcription factors (tfs), there was an increased number of binding sites above the threshold for the nfya/caat dna-binding elements and also mzf1. both of which show higher expression in the liver tissue of both bat species compared to mouse and human. additionally, there is a 150-bp gap in the genome for the p. alecto promoter, which may mask other binding elements. intriguingly, hsp expression in bats has been linked to hibernation with increased hsp70 and hsp90 expression observed during torpor (zhang et al. 2013) . while the species investigated here are not known for hibernation, partial torpor is recorded in most fruit bat species along with resting bradycardia during flight (o'mara et al. 2017 ) that resembles partial torpor. this may highlight evolutionary conserved pathways for thermos fig. 4 stress survival in bat and avian cells. a the expression of hsp70 and hsp90 in paki cells was reduced using sirna knockdown and cultured at 37°c while tracking cell viability as previous. b as per a but for palu cells. c heat treatment was applied to sirna-treated paki cells at 40°c for 24 h together or to d palu cells as per a. statistical significance was calculated using two-way anova (graphpad prism software) comparing the cell growth rate over time; ***p < 0.0001. experiment was performed in triplicate on three separate occasions, and one representative experiment is shown. e example western blot for protein expression for bat, duck and pigeon muscle tissue (species as indicated,) and quantified as previous. f quantification of expression data for e normalized against gapdh. three individuals per species were tested. g duck cell line ccl-141 was compared to paki cells by western blot as per e) (h). quantification of data in g relative to gapdh as previous, three repeats from continuous passages are shown. i heat-treated viability curves, as described previously, are presented for 37°c and 40°c for 24 h of culture relative to the 0 h timepoint. j sirnatreated paki cells subjected to na-nonoate treatment for 6 h, followed by incubation for 72 h total, dose as indicated. cell survival was measured by pi uptake. error bar denotes standard derivation (sd) regulation in bats leading to higher expression of hsps. indeed, nfya, a known regulator of hsp 70/90 and a tf expressed higher in bats (fig. 4d) , has also been implicated in torpor (han et al. 2015) . other additional elements compared to human included gata, nfat, elk1/ets and myb binding sites, though these had lower binding scores. a full functional analysis of the promoter elements for each hsp gene in better annotated genomes and the binding capacity of each tf would be required to clarify this alternative, hsf-independent, expression of hsps in bats. as hsp expression can be further induced two-fold with heat treatment, similar to non-bat mammalian cells, it suggests that baseline hsp expression in bats is important while retaining endogenous mechanisms for induction with heat shock and likely other stress inducers such as the unfolded protein response. as this is the case, we do not believe before and after flight to be the most significant contribution to hsp expression but rather an imprinted basal expression. that bat cells were no longer able to grow at 40°c when either hsp70 or hsp90 was abolished by sirna and highlights their role in endurance of heat-induced stress in bat cells, whether related to flight or independent. this high base level in vitro confers basal thermo-tolerance. this confirms the known classical function of hsp for resistance to heat-induced apoptosis or cell death (mosser et al. 1997) , largely due to hsp70's ability to bind apf-1 and prevent recruitment of caspases for the formation of the apoptosome complex (beere et al. 2000) . moreover, flight itself is metabolically demanding and could in turn increase cellular oxidative stress, affecting cell survivability. our data with spermine nonoate treatment, in the absence of heat shock, demonstrated the pro-survival feature of hsp70 and hsp90 in bat cells, although the definitive mechanism remains to be elucidated. the little brown bat myotis lucifugus, with an estimated lifespan of 34 years (anage, http://genomics.senescence.info/species/), can reduce free radicals in their bodies (brunet-rossinni 2004) . due to the wide-ranging tissue expression, the role of hsps in the general protein homeostasis in bats must be considered, along with any possible implication for reduced ageing. this notion is also supported by two recently published studies on the long-lived naked mole rat. while hsps were highly conserved to other mammals, they observed reduced protein degradation and unfolding despite increased oxidative stress (perez et al. 2009 ). another recent study observed a higher level of hsp70 and hsp25 in the naked mole rat (rodriguez et al. 2016 ) possibly enhancing protein repair and reducing cellular damage. there may be other implications for high hsp expression in explaining why bats are effective hosts to zoonotic viruses (hu et al. 2014; miller et al. 2016; plowright et al. 2015; zhu et al. 2009 ). hsp70 can promote ebola virus replication (shurtleff et al. 2014) . nipah virus has been shown to utilize hsp90 for chaperoning viral l polymerase (bloyet et al. 2016) , and dengue virus (denv) uses hsp70 and hsp90 as alternative receptors for viral entry (reyes-del valle et al. 2005) . hsp expression also affected the mutation rate/genetic diversity of influenza viruses (phillips et al. 2017) . the basal expression level of several hsp genes is genetically imprinted in vivo in live animal-derived tissues from two species of bat, with cultured cell lines displaying the same high expression. this is required for bat cells to sustain prolonged heat stress and affects their response to oxidative stress. together with published literature linking high hsp expression level with long lifespan and promotion of virus entry and/or replication, this speculatively links the two most fascinating features of bat biology, i.e. longer lifespan and an exceptional ability as a zoonotic host. unique loss of the pyhin gene family in bats amongst mammals: implications for inflammasome sensing dampened nlrp3-mediated inflammation in bats and implications for a special viral reservoir host comparative metabolomics of aging in a long-lived bat: insights into the physiology of extreme longevity heat-shock protein 70 inhibits apoptosis by preventing recruitment of procaspase-9 to the apaf-1 apoptosome requirement of hsp90 chaperoning in addition to 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drosophila caused by histone deacetylase inhibitors irf7 in the australian black flying fox, pteropus alecto: evidence for a unique expression pattern and functional conservation contraction of the type i ifn locus and unusual constitutive expression of ifn-alpha in bats detection of diverse astroviruses from bats in china publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgements we wish to acknowledge gary crameri and dr. michelle baker at csiro/aahl for access to pteropus cell lines.authors' contributions ytc designed and performed the experiments with jk and ati. ytc and ati drafted the manuscript. jc and ati performed the bioinformatics analysis. ihm and dl helped in the e. spelaea sampling and the establishment of e. spelaea cell lines. jn, ytc and ati helped in the collection of the p. alecto bat sampling and jn established the p. alecto and e. spelaea tissue cdna libraries. ati designed the heat treatment assay. ki provided mentorship and funding for jk. jn, ati and lfw revised the paper and provided scientific insight. all authors read and approved the final manuscript. we acknowledge tan jun hao for his help in amino acid sequence alignment of the hsps. we thank gary crameri and the queensland animal science precinct team led by hume field for their advice and help in catching and handling p. alecto. conflict of interest the authors declare that they have no competing interests. key: cord-272009-yxjhfg7m authors: cui, jie; han, naijian; streicker, daniel; li, gang; tang, xianchun; shi, zhengli; hu, zhihong; zhao, guoping; fontanet, arnaud; guan, yi; wang, linfa; jones, gareth; field, hume e.; daszak, peter; zhang, shuyi title: evolutionary relationships between bat coronaviruses and their hosts date: 2007-10-17 journal: emerg infect dis doi: 10.3201/eid1310.070448 sha: doc_id: 272009 cord_uid: yxjhfg7m recent studies have suggested that bats are the natural reservoir of a range of coronaviruses (covs), and that rhinolophid bats harbor viruses closely related to the severe acute respiratory syndrome (sars) cov, which caused an outbreak of respiratory illness in humans during 2002–2003. we examined the evolutionary relationships between bat covs and their hosts by using sequence data of the virus rna-dependent rna polymerase gene and the bat cytochrome b gene. phylogenetic analyses showed multiple incongruent associations between the phylogenies of rhinolophid bats and their covs, which suggested that host shifts have occurred in the recent evolutionary history of this group. these shifts may be due to either virus biologic traits or host behavioral traits. this finding has implications for the emergence of sars and for the potential future emergence of sars-covs or related viruses. recent studies have suggested that bats are the natural reservoir of a range of coronaviruses (covs), and that rhinolophid bats harbor viruses closely related to the severe acute respiratory syndrome (sars) cov, which caused an outbreak of respiratory illness in humans during [2002] [2003] . we examined the evolutionary relationships between bat covs and their hosts by using sequence data of the virus rna-dependent rna polymerase gene and the bat cytochrome b gene. phylogenetic analyses showed multiple incongruent associations between the phylogenies of rhinolophid bats and their covs, which suggested that host shifts have occurred in the recent evolutionary history of this group. these shifts may be due to either virus biologic traits or host behavioral traits. this fi nding has implications for the emergence of sars and for the potential future emergence of sars-covs or related viruses. s evere acute respiratory syndrome (sars) emerged in november 2002 in southern people's republic of china (1) , and a sars coronavirus (sars-cov) was identifi ed as the etiologic agent (2) . these events and the identifi ca-tion of sars-cov in animals associated with the wildlife trade in southern china (3) have led to a rapid resurgence of interest in covs of different origins. this resurgence led to discovery of 2 novel human covs (4, 5) ; identifi cation of sars-like covs in horseshoe bats (rhinolophus macrotis, r. ferrumequinum, r. pearsoni, and r. sinicus) (6, 7) ; and identifi cation of other covs in bat species (r. sinicus, r. ferrumequinum, miniopterus magnater [m. magnater has been misidentifi ed as m. schreibersi (8) in reports on sars-like cov], pipistrellus abramus, p. pipistrellus, tylonycteris pachypus, myotis ricketti, and scotophilus kuhlii) (7, (9) (10) (11) (12) . however, evolutionary relationships among these covs and their bat hosts have not been examined. studies in species other than bats have examined hostvirus phylogeny and identifi ed coevolutionary relationships (13) (14) (15) (16) or incongruous phylogenetic patterns (17) . these fi ndings suggest recent pathogen host shifts (defi ned as interspecies transmission followed by establishment and long-term persistence in the new host species [18] ). other studies have demonstrated that the relationship between viral phylogeny and geographic location and identifi cation hosts (viral phylogeography [19] ) can yield information on the origin of emerging zoonoses (19, 20) . knowing the high genetic diversity of bat covs, we carried out a systematic phylogenetic study of the viruses and their hosts to examine evolutionary relationships between bat covs and bats. the aim was to further investigate the origin of sars-like covs and sars. our results suggest host-pathogen divergence and host shifts in the recent evolutionary history of these viruses and their hosts. we discuss host behavioral traits and viral traits that might have given rise to these patterns and comment on the implications of our fi ndings for the emergence of sars-cov. only covs from bats were included in this study. we used gene sequences that tang et al. obtained from 10 bat species (r. sinicus, r. ferrumequinum, r. macrotis, r. pearsoni, m. magnater, p. abramus, p. pipistrellus, t. pachypus, s. kuhlii, and myotis ricketti) (10) . an additional 57 bat cov sequences available in genbank were also included in this analysis. tissue samples were obtained from 3-mm wing membrane biopsy specimens from wild bats, which had been caught in 9 provinces of china, that had been preserved in 99% ethanol. genomic dna was extracted by using the dneasy tissue kit (qiagen, valencia, ca, usa) and stored at -20°c. we used complete cyt b sequences of r. ferrumequinum, p. abramus, and p. pipistrellus, which have recently been published and are available in genbank. we generated cyt b sequences from m. magnater (n = 4), t. pachypus (n = 3), r. macrotis (n = 2), r. pearsoni (n = 2), r. sinicus (n = 2), s. kuhlii (n = 1), and myotis ricketti (n = 1). pcr mixtures were prepared in 50-μl volumes containing 25 μl 2× extaq dna polymerase (takara, kyoto, japan). two pairs of primers, bat_cytb_1 (5′-tag aat atc agc ttt ggg tg-3′) (21) with bat_cytb_2 (5′-aaa tca ccg ttg tac ttc aac-3′) (21) , and bat_cytb_2 with bat15r (5′-tca gct ttg ggt gtt gat gg-3′) (22) , were used because of amplifi cation specifi city of certain primers in some species. amplifi cation was conducted at an initial denaturing temperature at 94°c for 30 s; 34 cycles of denaturation at 94°c for 30 s, annealing at 55°c for 30 s, and extension at 72°c for 90s; and a fi nal extension at 72°c for 10 min. the pcr samples were then stored at 4°c. the complete mitochondrial cyt b gene (1,140 bp) was amplifi ed and sequenced. these sequences have been submitted to genbank and accession numbers are shown in the table. for virus phylogeny studies, sequences from a 440bp fragment of the rna-dependent rna polymerase (rdrp) gene, which is highly conserved among different covs, were obtained and analyzed (10) . multiple alignments of the 440-bp rdrp partial sequence of bat covs were conducted in clustalx version 1.81 (23) . bayesian analyses were conducted with mrbayes version 3.1.2 (24) . four consecutive metropolis-coupled markov chain monte carlo computations were run for 2 million generations, with trees sampled every 100 generations. initial trees were random. on the basis of stabilization of preliminary runs, the fi rst 3,000 trees were discarded before generation of the consensus tree. the bayesian consensus tree was rooted to breda virus (ay427798), a related cov ( figure 1 ). for bat phylogeny, we used the complete mitochondrial cyt b gene to construct maximum likelihood (ml) and bayesian phylograms. the cyt b sequence data were aligned by using clustalx version 1.81 as above. ml analysis was performed by using paup* version 4.0b (27) . the most appropriate substitution model (generalized time reversible + γ + i) with the parameters matrix = 0.4835 × 9.6665 × 0.3815 × 0.2973 × 7.1418, base frequency = 0.3576 × 0.3420 × 0.0748, rates = gamma, shape = 0.6008, and proportion of invariable sites unable to accept substitutions = 0.4078 for ml and subsequent bayesian analysis was calculated by using the program modeltest 3.7 (26) . we used heuristic searches (10 replicates, random addition of taxa, with tree bisection and reconnection branch swapping), followed by 100 bootstrap iterations for robustness of the ml tree. bayesian analysis was also used to construct a tree with 4 simultaneous markov chains for 1 million generations. trees were sampled every 20 generations, and the fi rst 5,000 trees were discarded before the consensus tree was made (on the basis of practical values of stabilizing likelihood). we compared the genetic diversity of covs isolated from rhinolophids and vespertilionids and the corresponding diversity among bat taxa by using the index of nucleotide diversity (π) described by nei (28) in arlequin version 3.1 (29) . analyses were performed on uncorrected pairwise genetic distances between sequences. by combining information derived from the phylogram of bat covs, together with data on the geographic origin of viruses, we were able to describe the phylogeographic distributions for known covs from bats in china (figures 1, 2 ; table) . bat sars-like covs formed a monophyletic clade. species-specifi c host restriction was found for covs in 4 of 7 bats species (myotis ricketti, m. magnater, p. abramus, and t. pachypus) sampled from >1 geographic location, and these clustered with high bayesian posterior probability. overall phylogenetic relationships between virus lineages were similar across our analyses, and well-supported genetic structure was observed within some cov lineages. for example, covs isolated from m. magnater were monophyletic but formed 3 well-supported clades with no evidence of geographic structure (bayesian posterior probability [pp] = 1.0 for each). a similar pattern was apparent in covs from myotis ricketti, which formed 2 geographically overlapping independent clades (pp = 0.99 and 1.0, respectively). one t. pachypus was infected by a virus that clustered with moderate statistical support (pp = 0.91) within the larger clade associated with p. abramus, which indicated a potential interspecies transmission event or recent evolutionary host shift (defi ned as interspecies transmission followed by establishment and long-term persistence in the new host species [18] ) (figure 1) . phylograms of host sequences were also constructed and were essentially of the same topology with high support whether derived by using mrbayes version 3.1.2 or mega3 (data not shown). when we mapped host phylogram to virus, virus phylogeny did not always track host phylogeny (figure 3 ). when separate host-virus phylograms were constructed for the 2 bat families (verspertilionidae and rhinolophidae), different corresponding relationships were evident. verspertilionids and their covs showed phylogenetic congruence, and rhinolophids and their covs showed incongruous phylogenies (figure 4) . we found evidence for evolutionarily divergent relationships for some vespertilionid viruses and their hosts when analyzed at the family scale (figure 4, panel a) . for example, divergence between viruses harbored by p. pipistrellus and p. abramus is congruent with their hosts. the divergence among other viruses was incongruent with divergence of host species, e.g., those from s. kuhlii and myotis ricketti. rhinolophid bats and their viruses were analyzed at a different taxonomic scale (within genus). in this co-phylogeny, viral host shifts were the evident virus-host feature (figure 4, panel b) . except for r. macrotis, all rhinolophidae bats had 2 distinct lineages of covs, and host shifts were found among viruses carried by r. ferrumequinum, r. pearsoni, and r. sinicus. genetic diversity of covs harbored by rhinolophids and vespertilionids was similar (vespertilionids π = 0.27 ± 0.13; rhinolophids π = 0.25 ± 0.13). in contrast, genetic diversity of cyt b sequences from bats was much higher among the vespertilonids (π = 0.17 ± 0.007) than among the rhinolophids (π = 0.09 ± 0.006). covs sequenced from different bats of the same species clustered together, even when bats were collected in locations 1,000-2,000 km apart. this pattern was found for covs from p. abramus, t. pachypus, myotis ricketti, and m. magnater. bats of the genus miniopterus are known to migrate long distances (30) , which explains why the phylogeny of viruses isolated from m. magnater sampled in distant places (guangxi, anhui, fujian, and hong kong) although some of these values are low, our analysis demonstrated a pathway for future study (28) . lines between the 2 trees were added to help visualize virus and host sequence congruence or incongruence. lacks geographic structure. in nonmigrating species such as bats of the genera pipistrellus and tylonycteris, intimate physical contact of bats in same cave or the same bamboo roost site, as well as periodic exchange of bats among neighboring colonies, may facilitate virus transmission among populations. despite the co-roosting of many bats species, we found little evidence of host shifts for some viruses. for example, covs from m. magnater and myotis ricketti sampled in the same cave in guangxi were divergent, although sample size was limited. although myotis ricketti has a closer phylogenetic relationship with t. pachypus, p. pipistrellus, and p. abramus than with m. magnater and s. kuhlii, its behavior and habits are closer to those of the last group. for example, myotis ricketti and s. kuhlii bats roosts in caves (although s. kuhlii also roosts under palm leaves), whereas t. pachypus roosts inside bamboo and p. abramus roosts almost entirely in old buildings. thus, it seems plausible that the close phylogenetic relationship between viruses harbored by myotis ricketti and s. kuhlii refl ects the similar behavior and ecology of their hosts. the phylogenetic and phylogeographic associations we found suggest that there may be a coevolutionary relationship between some bat covs and their hosts. for example, sister taxa within the genus pipistrellus independently maintained 2 distinct viruses that share a most recent common ancestor. a similar relationship was apparent among the viruses of some closely related genera (e.g., pipistrellus and tylonycteris), whereby divergence of each genus was mirrored by divergence in viral phylogeny. however, viruses are usually thought to have evolved more recently than their hosts (31) . thus, apparent coevolutionary patterns may refl ect either a high frequency of host shift among closely related bat species or simultaneous lineage splitting of hosts and viruses. host shifts among related bats might be favored by a variety of mechanisms, including preadaptation to overcome immune defenses or greater rates of interspecifi c contact relative to distantly related bat species. phylograms with better resolution would enable statistical comparison of phylogenetic congruence and estimation of divergence times. in the vespertilionids, close phylogenetic concordance between host and virus suggests a close, possibly evolutionarily divergent relationship. however, there are different scales of comparison between the vespertillionidae, in which all but 1 cov came from separate genera, and the rhinolophidae, in which we examined a co-phylogeny of multiple species within 1 genus. genetic diversity in the vespertilionids sampled was nearly double that of the rhinolophids, which was probably due to the greater number of species sampled and their broader taxonomic range. despite this greater genetic diversity among vespertilionid bat hosts, the genetic diversity of covs did not differ between vespertilionids and rhinopholids. this diversity suggests that vespertilionids may maintain undiscovered covs or that rhinolophids might harbor disproportionate cov diversity relative to diversity of their genus. we propose that future work may identify more vespertilionid bat covs, which would enable an accurate comparison of propensity for host shifts within this group. in the rhinolophids, the host phylogram demonstrated genetic divergence between r. ferrumequinum and other species, as shown by the division of rhinolophus bats into 2 groups. each of these groups harbors covs from 2 clusters (sars-like covs and other covs), which suggests multiple introductions of covs into these species. lack of concordance between phylogenies of rhinolophid bats and their covs can be interpreted as evidence for host shifts between bats of the genus rhinolophus. different species of rhinolophus are often observed roosting rm, r. macrotis. boldface branches in panel b contain severe acute respiratory syndrome-like covs reported. lines between bat and virus trees were added to help visualize congruence or incongruence. although this fi gure implies differences in propensity for host shifts between these families, all but 1 of the vespertilionid covs are from different genera, whereas all rhinolophid covs are from the same genera, which make meaningful comparisons diffi cult. overall mean genetic differences are much greater between vespertilionid species than between rhinolophid species. inside the same cave, which facilitates virus transmission between species. however, the degree of host shifting of rhinolophid bat covs may not be particularly high relative to other genera of bats. this observation will be clarifi ed when a greater diversity of covs from other bat genera is reported and the sequences are analyzed. these requirements support the need for further research on bat viruses (32, 33) . host-shifting within the genus rhinolophus would likely be promoted if these bats shed covs in a way that makes them more available to other rhinolophus spp.; had behavioral traits that lead to increased contact with other rhinolophus spp.; or if covs harbored by these bats have structural, biologic, or other traits that make them more readily transmitted to other rhinolophus spp. two lines of evidence suggest that host traits are the most parsimonious explanation for host shifts within the genus rhinolophus. first, sars-like covs and other rhinolophid covs (rfv1 and rpv1) show evidence of interspecies transmission. second, covs from other bat groups that are phylogenetically much closer to rfv 1 and rpv 1 than to the sars-like covs do not show evidence of successful host shifts. thus, the ability to jump hosts is unlikely to be a strictly viral trait. the phylogeography of bat covs suggests that the bat sars-like covs form a monophyletic clade that is both phylogenetically distinct from other bat covs and geographically isolated. although we acknowledge that this interpretation may be limited by sample size, it may also indicate that rhinolophid bats, the hosts of a cluster of sars-like covs within which human and civet sars cov nestle phylogenetically (6, 7) , are more likely to foster the host shifts of covs than are other bat species. the potential for close contact between bats, civets, and humans in the wildlife trade in southern china, coupled with a possible propensity of these bats to foster cov host-shifts, could explain sars-like covs as the source of sars-cov. this potential supports molecular results on bat covs that suggest a recent host shift from bats to civets or other animals and humans (34) . such host shifts may indicate a risk posed by other bat covs for novel disease emergence. finally, the ability of sars-like covs to be transmitted between and establish in new species (i.e., to undergo host shifts) is consistent with other covs. this has been shown for several covs of livestock species (35) and has been used to support their inclusion as 1 of the groups of viruses most likely to be responsible for emerging zoonoses, even before the emergence of sars (36) . the total diversity of covs (including sars-like covs) in bats has likely not been fully described. the genus rhinolophus (8) contains 77 bat species distributed in asia, europe, and africa. the recent discoveries of bat covs in the united states (37) and sars-covs in african bats (38) support the hypothesis that covs are diverse and widespread in bat species. given the diversity of covs in this group, and their propensity for host shifts, further viral discovery in rhinolophids may assist in understanding and ultimately controlling the emergence of zoonotic viruses. bats are increasingly recognized as reservoirs of many highly lethal zoonotic agents (32) . understanding their diversity, behavior, and mechanisms of virus transmission may play a key role in preventing future outbreaks of both known and unknown zoonotic diseases of bat origin. identifi cation of a novel coronavirus in patients with severe acute respiratory syndrome newly discovered coronavirus as the primary cause of severe acute respiratory syndrome isolation and characterization of viruses related to the sars coronavirus from animals in 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hostparasite assemblages the ecology and genetics of a host shift: microbotryum as a model system the phylogeography of human viruses establishment of multiple sublineages of h5n1 infl uenza virus in asia: implications for pandemic control phylogenetics of small horseshoe bats from east asia based on mitochondrial dna sequence variation evolution of the cytochrome b gene of mammals the clustal_x windows interface: fl exible strategies for multiple sequence alignment aided by quality analysis tools mrbayes: bayesian inference of phylogenetic trees mega3: integrated software for molecular evolutionary genetics analysis and sequence alignment modeltest: testing the model of dna substitution paup* beta version: phylogenetic analysis using parsimony (*and other methods). version 4. sunderland (ma): sinauer associates molecular evolutionary genetics arlequin ver. 3.0: an integrated software package for population genetics data analysis. evolutionary strong population substructure is correlated with morphology and ecology in a migratory bat error thresholds and the constraints to rna virus evolution what links bats to emerging infectious diseases? science bats: important reservoir hosts of emerging viruses evolutionary insights into the ecology of coronaviruses animal coronaviruses: what can they teach us about the severe acute respiratory syndrome? the evolvability of emerging viruses detection of group 1 coronaviruses in bats in north america coronavirus antibodies in african bat species people's republic of china key: cord-271687-sxl8g85p authors: mathews, fiona title: chapter 8 zoonoses in wildlife: integrating ecology into management date: 2009-03-14 journal: adv parasitol doi: 10.1016/s0065-308x(08)00608-8 sha: doc_id: 271687 cord_uid: sxl8g85p zoonoses in wildlife not only play an important ecological role, but pose significant threats to the health of humans, domestic animals and some endangered species. more than two‐thirds of emerging, or re‐emerging, infectious diseases are thought to originate in wildlife. despite this, co‐ordinated surveillance schemes are rare, and most efforts at disease control operate at the level of crisis management. this review examines the pathways linking zoonoses in wildlife with infection in other hosts, using examples from a range of key zoonoses, including european bat lyssaviruses and bovine tuberculosis. ecologically based control, including the management of conditions leading to spill‐overs into target host populations, is likely to be more effective and sustainable than simple reductions in wildlife populations alone. bovine tuberculosis. ecologically based control, including the management of conditions leading to spill-overs into target host populations, is likely to be more effective and sustainable than simple reductions in wildlife populations alone. parasites and pathogens in wildlife are a natural part of biodiversity. an abundance of theoretical studies indicate that they have key roles in ecological and processes, including the regulation of population size may and anderson, 1979) and the maintenance of genetic diversity (may and anderson, 1983; read et al., 1995) . occasional field experiments have also demonstrated impacts of sublethal infections on reproductive rates (munger and karasov, 1991) and susceptibility of wildlife to predation (hudson et al., 1992) . yet the vast majority of empirical studies consider wildlife pathogens and parasites only if they threaten the health of humans or their domestic animals, and often overlook their natural history. partly this reflects the priorities of funding agencies; and their concerns are not trivial. more than 70% of emerging (or re-emerging) pathogens of humans are thought to have wild animals as their natural reservoirs (taylor et al., 2001) . examples include sars-cov (severe acute respiratory syndrome coronavirus), avian influenza a (h5n1) virus (bird flu), nipah virus, hantavirus and west nile virus. the economic imperative for controlling zoonoses that affect domestic stock can be very strong. for example, bovine tuberculosis (btb) in the united kingdom undoubtedly has a reservoir in wild badger populations, and the direct cost of the disease to agriculture is projected to reach £1 billion by 2011 (department for environment, food and rural affairs (defra), 2004). it has been recognised relatively recently that disease can also pose a serious threat to the survival of endangered wildlife (lyles and dobson, 1993; may, 1988; smith, 1982) . this can either be through direct mortality, where losses are greater than the birth rate, or through effects on birth rate, longevity and survival, which suppress the population size to a level that renders it susceptible to extinction by stochastic effects (table 8 .1). generalist pathogens with a wide host range are particularly problematic, since even virulent species can persist in alternative hosts while driving the rare host to extinction (begon and bowers, 1995) . although remaining low on the list of priorities compared with other threats such as habitat loss, efforts are therefore sometimes now made to control infectious diseases for conservation reasons. examples of recent successes include the control of canine distemper virus in black-footed ferrets (mustela nigripes) (williams et al., 1988) , and rabies in african wild dogs (lycaon pictus) (hofmayer et al., 2004) and ethiopian wolves (canis simensis) (haydon et al., 2004) . in europe alone, there are at least 35 zoonotic parasites and pathogens in wildlife that are known to be important either to public health or the agricultural economy (artois et al., 2001) . for many other infectious agents, such as cryptosporidium parvum, the epidemiological role of wildlife is unknown. despite the many attempts to control zoonoses in wildlife, the success rate is poor. typically, measures are adopted as crisis management (usually in the form of culling) following an outbreak, with little understanding of the ecology of species or its relationship to the pathogen. crisis management also means that proper scientific designs with appropriate controls are often lacking; it is therefore difficult to evaluate the effectiveness of a given intervention. even where there is a long history of attempts to control a disease through the management of a wildlife reservoir, the results have not been encouraging. for example, efforts over the last 30 years to control btb in cattle in the united kingdom by culling of badgers has failed to yield significant benefits, with analyses of the recent randomised controlled trial of badger culling concluding that culling could not contribute meaningfully to future control strategies for btb (donnelly et al., 2005) . similarly, the culling of foxes has been discounted as a means of rabies control in western europe (blancou et al., 1991) . however, success has been achieved through the use of widespread vaccination (administered via bait) (aubert, 1995) . rabies is currently the only example of a widespread strategy of vaccination being favoured over the control of the host species (artois et al., 2001) . this review considers the ecology of zoonoses in wildlife and the links between infection in wildlife and humans or livestock. it proposes that a shift to ecologically based control, explicitly considering the natural history of wildlife hosts and their pathogens, is crucial in minimising the risk presented to humans, domestic animals and endangered species from zoonoses. this approach will also yield benefits for the conservation and welfare status of wild animals. notwithstanding the complexities of specific relationships, the probability of a zoonosis being passed from wildlife into another host population, be it humans, domestic stock or an endangered wild species, is always influenced by several key parameters. these are the intensity of infection in the reservoir hosts; the size, or depending on the case, the density, of the infected host population; the degree and nature of the contact between infected individuals (or infectious particles in the environment such as infected faeces); and the susceptibility of the in-contact animal. (for indirectly transmitted parasites and pathogens, the role of vectors and/or intermediate hosts must also be considered.) whether the zoonosis persists after initial invasion is also determined by the new host's population size. a great variety of models has been developed to describe the transmission dynamics of macro-and microparasites, taking into account the nuances of particular host and parasite population structures (diekmann and heesterbeek, 2000; heesterbeek and roberts, 1995; scott, 1988; scott and smith, 1994 ). yet empirical research has lagged behind the theoretical advances. the legacy of researchers like elton and chitty (chitty, 1952 (chitty, , 1954 elton, 1931; elton et al., 1931 elton et al., , 1935 , who sought not only to describe pathogens but to understand their ecological role, has not been sustained. (there are a few notable exceptions, including the long-term studies of small mammals in the north of england (beldomenico et al., 2008a,b; feore et al., 1997) ; rodent reservoirs of hantavirus in the united states (calisher et al., 2007; mills et al., 1997) and macroparasite infections in laboratory models (ehman and scott, 2004; scott, 1988; scott and anderson, 1984; scott and smith, 1987) .) this deficiency was noted in a key text in the field in 1995 (grenfell and dobson, 1995) and again in the follow-up publication in 2002 (hudson et al., 2002) . we lack even species lists of parasites and pathogens for most, if not all, wild animals. while pathogens that affect international trade are reported to the world organization for animal health (oie), and many of these affect wildlife (see artois et al., 2001) for the lengthy list of those likely to affect wildlife in europe), there is no agreed systematic programme of surveillance (kulken et al., 2005) . even where programmes exist, they lack integration with surveillance in humans and domestic animals at both local and international scales. disease surveillance in wildlife is usually driven by outbreaks in humans or domestic animals (childs, 2004) . virulent pathogens are, therefore, more likely to be detected than more benign ones (williamson et al., 1986) . such studies are also, by their nature, not designed to screen for a range of pathogens, so opportunities to investigate the epidemiology and ecology of coinfections are often lost. systematic surveillance of representative samples of the population is difficult and time consuming. yet prevalence estimates can be seriously skewed if the only data available are derived from passive surveillance of carcasses. not only are estimates likely to be too high if they are based on samples of wildlife found dead or sick by the public, but even road kills and game bags are likely to over-represent certain population classes (such as dispersing juvenile male mammals) and animals in compromised health. disease-responsive surveillance also offers little information on the frequency with which transfer events are likely to occur. for example, many of the 'spectacular' epidemics derived from bat viruses, such as hendra virus, nipah virus, sars-cov-like virus, have been observed only a small number of times. we do not know why this should be the case. is transfer of zoonoses from bats to terrestrial vertebrates generally rare due to a lack of appropriate contact? or is there regular inter-specific transmission of other viruses but these go undetected because they lack the extreme pathogenicity of hendra and nipah viruses to stimulate screening efforts? pro-active surveillance of wildlife and of apparently healthy human or livestock populations could help answer these questions. structuring and species specificity of a pathogen in wildlife hosts? screening for european bat lyssaviruses in europe is an exemplary case of research stimulated by public health concerns. the first recorded european bat rabies case was in hamburg in 1954 and several other cases were identified subsequently (king et al., 2004 ). yet surveillance of bats was not really pursued until a woman in denmark was bitten by a serotine bat (eptesicus serotinus) infected with european bat lyssavirus 1 (eblv1). since then more than 800 rabies-positive bats have been identified across europe; the vast majority being serotine bats infected with eblv-1 (harris et al., 2006) . in the united kingdom, screening efforts were intensified following the death of a man in scotland from eblv-2 in 2002, after apparent contact with many bats in the united kingdom and europe (fooks et al., 2003) . in contrast with classical rabies (rabv) there is now good evidence that at least some bats (and possibly other animals) can produce neutralising antibodies and survive eblv infection for at least 6 years (serra-cobo et al., 2002; van der poel et al., 2000) , and experimental models suggest that eblv-2 might be inherently less virulent than eblv-1 (vos et al., 2004) . eblv-2 also appears to have a much more restricted geographical range than eblv-1, and small numbers of positive bats have been identified in the united kingdom, switzerland, the netherlands, denmark and germany (department for environment, food and rural affairs (defra), 2008; racey et al., 2004; vos, 2007; vos et al., 2007) . these cases have all been in the closely related daubenton's (myotis daubentoni) and pond bats (m. dasycneme). structuring of eblvs therefore is apparent from these data both across geographical areas and across species. the serotine bat occurs over most of europe, extending north to 55 latitude (england south of the wash estuary, denmark and southern sweden); daubenton's bats are common across europe; and the pond bat is present in a wide band across central and eastern europe (between 48 and 60 latitude; absent from the united kingdom (schober and grimmberger, 1997) ). yet neither eblv-1 nor eblv-2 appears throughout their hosts' ranges. some suggest that in the case of eblv-1, this may be because long-distance travel is uncommon in serotines, the primary host (vos et al., 2007) . yet the species is widely distributed, and it is unlikely that there are gaps between populations that could not be travelled with relative ease; dispersing movements of up to 300 km have also been recorded (hutterer et al., 2005) . interestingly, eblv-2 also appears to have a patchy distribution, despite its host species, at least in continental europe, being migratory over long distances (vos, 2007) . whether the geographical distribution is, in reality, less patchy than it currently appears requires co-ordinated surveillance effort and a willingness by statutory authorities to publish test results even if they are negative. it is clear that active surveillance (systematic screening of bats in the wild) has been undertaken in a few countries only, and passive surveillance (submission of dead bats by members of the public for screening) has involved few, if any, animals in a number of european countries, including portugal, ireland, greece, the czech republic and slovakia (racey et al., 2004) . a range of european bats, most of which are common and widespread, has been identified as having active eblv infections in addition to the key hosts (table 8 .2). it is striking then that the vast majority of reported cases come from just three species. undoubtedly, the numbers of bats of each species submitted by the public does not match their abundance in the wild, but is influenced by the closeness of their contact with humans (and their cats, which are a major cause of bat mortality). for example, few woodland specialists have been submitted, whereas bats that frequently roost in houses, particularly pipistrelles, long-eared bats and possibly serotines, are over-represented (harris et al., 2006) . even active surveillance does not attempt comprehensive surveys of all species in proportion to their abundance: instead, it focuses on the three species already identified as being important sources of eblvs, potentially failing to estimate properly the prevalence in others. despite these limitations, the data clearly suggest that species partitioning occurs. the common pipistrelle bat is known to be susceptible to experimental infection with eblv-1 (kuzmin and botvinkin, 1996) . yet none of the more than 10,000 pipistrelles (p. pipistrellus and also p. pygmaeus, which is cryptic with p. pipistrellus) surveyed in the netherlands, france and the united kingdom (harris et al., 2006; picard-meyer et al., 2006; van der poel et al., 2005) has proved positive for the virus. whether structuring across bat species driven by differing immunoresponsiveness to particular eblv types, by a lack of transmission opportunities or by other mechanisms, is unclear. multi-species summer, and particularly hibernation, roosts are known, though the amount of inter-specific direct contact appears to vary by season and species. for example, bats in houses and trees tend to use single-species roosts, even if more than one species is present at the site (park et al., 1996) . there may be more potential for inter-specific contact at key underground sites used by bats. in a survey of more than 76,000 bats of 13 different species roosting in caves in turkey, it was noted that multi-species clusters frequently occurred in the post-hibernation season, but not during hibernation; and the horseshoe bats (rhinolophus spp.) only ever formed single-species clusters (furman and ö zgü l, 2004) . many bat species also use swarming sites-enclosed areas often in and around caves-for display purposes. at these sites, hundreds or thousands of bats of mixed species congregate (glover and altringham, 2008; parsons et al., 2003) . the amount of contact, for example, via urine or aerosol droplets, between species at these events is unknown. more field research is needed to investigate the opportunities for disease transmission across bat species, and across geographical barriers. interestingly, in the united kingdom, only a single case of exposure to eblv-1 has been found (the test was able to detect exposure rather than live virus), whereas in other european countries with eblvs, the apparently more pathogenic eblv-1 is more common (defra, 2008; racey et al., 2004; vos, 2007) . to date, seven daubenton' bats (myotis daubentoni) in the united kingdom have been found to have eblv-2 infection (defra, 2008) , the latest case being diagnosed in may 2008. it is notable that although a low prevalence (around 2%) of seropositivity was detected during active surveillance of daubenton's bats in scotland, live virus was not isolated from any of them (brookes et al., 2005) . similar results were found in an active-surveillance study in spain, which found that up to 60% of individuals in some colonies were seropositive for eblv-1, but the prevalence of active infection was less than 1.1% (serra-cobo et al., 2002) . it is currently difficult to interpret these results, but the vertical transmission of antibodies, as well as acquired immunity, is a possibility. while it is clear that eblv-1 and eblv-2 can cause deaths in unvaccinated humans, whether natural immune responses and cryptic recovery (i.e., without the virus having invaded the central nervous system and become symptomatic) are possible remains unknown. it is unfortunate that there was been no serological testing of bat workers in the united kingdom to establish the natural prevalence of neutralising antibodies to eblvs prior to 2002. since that date, following the fatality in scotland from eblv-2, it has been officially recommended that bat handlers be vaccinated against rabies. the take-up rate of vaccination has been very high. this understandable management of the public-health crisis means that it is now not possible to gather information that would have helped indicate the pathogenicity of eblv-2 to humans, and also whether exposure was more widespread than the single fatal case. it certainly appears that despite other species being susceptible in experiments, natural spillover into other non-bat hosts to produce clinical symptoms is rare, with the only known case for eblv-1 being a single stone marten . there are no reports of spill-over for eblv-2. the apparently simple task of establishing the prevalence of a pathogen in wild animals can be fraught with difficulty. even assuming that a reasonably random cohort can be sampled, there is usually no opportunity to repeat 'live-tests' in cases of diagnostic uncertainty. establishing values for other key parameters is equally problematic. fundamental data on the sex-and age-distributions of infection are often not recorded. sometimes this is because the surveys (particularly for 'crisis management') were not designed with research in mind. sometimes it is practically difficult for the data to be acquired. bats, for example, can live more than 30 years, yet it is impossible in the absence of long-term banding studies on the particular population being surveyed, to judge the age of animals with much greater accuracy than 'juvenile', 'young of the year', and 'adult'. weight is frequently used as a surrogate for age or maturity, particularly in studies of rodents, but there can be difficulties in distinguishing age from dominance effects, since both are correlated with body mass. the size of the population (or its density) is also often estimated with huge margins of error, as surveyors simply lack the time to undertake detailed ecological studies in addition to collecting clinical samples. distinguishing between different burdens of infection (particularly for macro-parasites) and stages of infection (particularly for micro-parasites) is frequently overlooked. this makes it difficult to use the data to parameterise epidemiological models. for example, animals infected with btb but in which the bacilli are encysted present no risk of transmission at that particular time point, yet these groups are often combined when data from post-mortem examinations are used. the fact that the disease may reactivate at some future time (measured by the overall prevalence) is not relevant to the calculation of the basic reproductive rate of the disease r 0 . by conducting large-scale surveys of representative populations of wildlife on british farms, workers were able to build deterministic models to investigate the likelihood of the disease persisting in each host species. initially they assumed that no between-species transmission was present. using the prevalence of infectious individuals, together with field data on population structure and density derived from the same sites, they computed the basic reproductive number r 0 for each of the species. the analyses showed that even when the maximum likely prevalence was assumed (based on the upper 95% confidence limit), the r 0 (the basic reproductive rate of the disease) ranged from just 1.003 in wood mice to 1.05 in rats. (the lower confidence intervals for prevalence always gave r 0 values that were <1.0; mathews et al., 2006b .) it is therefore unlikely that the disease would persist within single-host systems in the wild: the animals are unable to pass on the infection to their conspecifics at a rate high enough to maintain the disease. the findings are robust to underdiagnosis of infection: to affect the r 0 materially, the prevalences would need to be have been underestimated very substantially. if, instead of single-host models, we assumed multiple-host systems, then higher prevalences should have been observed in the field than those recorded. alternatively, to achieve the prevalences seen in reality, the withinspecies transmission rate would have to be even lower than the very low value calculated. they have therefore been able to conclude that multi-species transmission of btb within farmland wildlife communities appears unlikely. perhaps the best example of long-term epidemiological studies in wildlife leading to epidemiological models of value to human health comes from studies of hantavirus infection in the united states. large-scale studies of several thousand rodents were conducted by four separate research teams, but were co-ordinated by common methodologies (calisher et al., 2007; easterbrook et al., 2007b; glass et al., 2007; mills et al., 1997) . using longterm datasets, with repeated trapping at set grids, the teams were able to explore key components of the transmission pathway. seropositivity was higher in males and in heavier animals, suggesting horizontal transmission among adult males. decreasing prevalence with age among the youngest deer mice suggests that infected dams confer passive immunity to pups. in the main host of sin nombre virus, the deer mouse (peromyscus maniculatus), gender, age, wounding, season and local relative population densities were linked with the period prevalence of antibody (used as a marker of infection). nevertheless, antibody prevalence and some of the risk factors associated with antibody prevalence, such as relative population density, gender bias and prevalence of wounding, varied significantly among sites and even between nearby trapping arrays at a single site. this suggests that local micro-site-specific differences play an important role in determining relative risk of infection in rodents and, consequently, in humans. these data are now being used in spatially explicit models of the risk of human disease outbreaks (eisen et al., 2007) . as described for bat lyssaviruses, the contact rates between infectious and susceptible individuals (or a vector and a susceptible) is a critical step in the transmission pathway. yet compared with the effort that goes into improving, for example, the accuracy of a diagnostic test, very little attention is paid to measuring it in the field. this failure may offer some explanation for the difficulties faced in attempted disease control programmes. for example, disease is a primary threat to the survival of the critically endangered ethiopian wolf (canis simensis). since the early 1990s, outbreaks of rabies and canine distemper virus (cdv) have had significant impacts on wolf population dynamics randall et al., 2004 randall et al., , 2006 . these diseases are maintained in local domestic dog populations, and a programme of dog vaccination was therefore introduced in 1996, with the aim of reducing the population of susceptible dogs and hence the risk of transmission to wolves. attempts were made to achieve coverage of more than 70% of susceptible dogs during annual vaccination campaigns. this was not an easy task since dogs in ethiopia are used for guarding cattle and are not tame. rabies vaccines have high efficacy, and in theory, this level of coverage should prevent rabies outbreaks 95% of the time (coleman and dye, 1996; world health organization, 2004) . over 30,000 vaccinations have taken place, and at least initially, the number of rabies cases in dogs declined (randall et al., 2006) . nevertheless, a rabies outbreak occurred in wolves in 2003, and could be linked with more than 35 sympatric dogs with clinical symptoms consistent with rabies . mathews has, therefore, been analysing the reasons for the apparent failure of the vaccination strategy, focusing on the population dynamics of the domestic dogs, using data collected by the ethiopian wolf conservation programme. the key factor appears to be the growth of the dog population, which, as in other african countries, is keeping pace with, or even outstripping, human populations (cleaveland et al., 2000; rhodes et al., 1998) . eighty-six percent of all households owned dogs, rising to 93.5% in rural areas. virtually nothing is known about the true contact rate between domestic dogs and ethopian wolves. it is clear that interactions do occur as wolf-dog hybrids are seen. we might speculate that diseased dogs, and aggressive dogs that are difficult to vaccinate, might be even more likely to interact with wolves than would healthy ones. some data are available on the demography of the dogs surrounding the bale mountains national park-one of the strongholds of the remaining ethiopian wolf population-as a result of a questionnaire survey administered by the ethiopian wolf conservation programme. the rate of increase in the dog population size appears to be around 5% per annum, and the turnover rate is also high. this creates a constant influx of new susceptibles into the dog population. it is difficult to keep pace with these, given the financial and logistical constraints on the numbers of visits veterinarians can make to each village. there also appears to be some geographical clustering of vaccination effort, and the implications of pockets of unvaccinated dogs on the probability of rabies transmission to wolves is currently being explored. the vast majority of efforts to control zoonoses in wildlife hosts, rather than in domestic animals, rely on culling strategies. in simple terms, the idea is to depress the population of the reservoir host to a level at which the disease can no longer be sustained, because the density of infected and susceptible hosts is too low. few of these culling programmes have systematically examined either the total population size or the level of population reduction likely to be required to achieve the desired endpoint. even where this has been done, it can be difficult practically, as with vaccination, to achieve the level of coverage desired. the strategy to control btb in badgers and cattle in the united kingdom has had the culling of badgers as its cornerstone for more than 30 years. when it became evident that gradual badger culling was having little or no impact on the incidence of the disease in cattle, a formal review of the programme was introduced, culminating in a large-scale randomised trial of badger culling. this has demonstrated that there is no clear-cut reduction in btb in cattle. on the contrary, whereas the incidence of btb in farms at the centre of 100 km 2 badger culling zones fell by around 19%, the incidence in farms up to 2 km away from the borders of these zones increased by around 29% (donnelly, 2005) . similar results were found in comparable areas where clusters of badgers were removed reactively, following nearby outbreaks of btb in cattle (donnelly et al., 2003) . at least part of the explanation for the failure to achieve effective btb control is likely to be the alteration in contact rates between infected and susceptible badgers, and also between infected badgers and cattle, as a result of the culling. there has been only one detailed study of m. bovis epizootiology in undisturbed badgers (culling having been suspended at the site, woodchester park in gloucestershire, england in 1978; delahay et al., 2000) . this study showed that btb does not spread rapidly at high incidence through badger populations, but rather is distributed patchily among a minority of individuals. social groups are relatively stable, and long-term dispersal movements are uncommon, though shorter movements do occur more regularly (rogers et al., 1998; vicente et al., 2007) . there is a correlation between rates of inter-group movement and the incidence of new infections (rogers et al., 1998; vicente et al., 2007) . while spatial clusters of infection exist, there is no strong synchrony between neighbouring groups, suggesting that there is only limited transmission between adjacent social groups (delahay et al., 2000) . both individuals and groups are more likely to be incident cases where the social group was diminishing in size, although there is no apparent relationship with group size itself, suggesting that it is the change in group size, and possibly the associated social dynamics, that influences disease risk (vicente et al., 2007) . badger culling operations have clear impacts on the behavioural ecology of the survivors. woodroffe et al. (2006) found that badger social group ranges increased among survivors within reactive and pro-active culling areas and along the perimeters of pro-active culling areas. their finding, at a large scale, accords closely with the observations of of individual and group behaviour in two zones of badger removal in england, as well as those of o' corry-crowe et al. (1996) in ireland. in all cases, the spatial organisation of social groups was considerably altered following the culls, with a large increase in the extent of overlaps between social groups (e.g., fig. 8.1) . the numbers of ranges with which each group overlapped also increased. there was a rather chaotic alteration in population densities (e.g., fig. 8.2) . in the examples shown, culling was conducted in 1995, largely targeting areas of highest badger density (in effect, the largest social groups). one year later badger density was, unsurprisingly, lower in the culled areas, whereas there had been some increases elsewhere. in 1997, although the population as a whole had not grown, the density remained low, or even fell further, in two removal areas, but increased elsewhere. by 1998 and 1999, the distribution of badgers in the study area was radically different from that at the outset, with some previously high density, but culled, areas remaining depauperate . thus, while the population density recovered as a whole, the badgers built up in a different place. this sort of radical redistribution has not been reported in undisturbed populations. the changes have not only implications for absolute contact rates, but also the nature of contacts. for example, bite-wounds-an important route of btb transmission-were more common in the macdonald et al. ( ) study following social perturbation. 1995 1996 (post-cull) figure 8.1 badger social territories before and after the selective removal of social groups following btb incidents in local farms. social structure also plays an important role in hantavirus transmission. in deer mice, both wounding and sin nombre virus antibody prevalence increased with mass. although it occurred in both sexes, the a 1995 c 1997 e 1999 b 1996 d 1998 badgers removed in bro increase was much more pronounced in males. wounding was more frequent in adult males than in adult females, and adults had more wounds than juveniles. the highest rate of infection was seen in individuals with the most wounds. similarly, in rats (rattus norvegicus) hantavirus infection (seoul virus) was associated with both wounding and elevated testosterone levels (easterbrook and klein, 2008; easterbrook et al., 2007a) . it is therefore evident that changes to social structure-for example, by the removal of a dominant male-could have important implications for the epidemiology of a disease. rather than attempt to control disease by vaccination or culling, an alternative approach is to understand the factors leading to disease outbreaks in the first place and to manage these (dobson, 2005) . habitat changes that lead to alterations in population structure or migratory patterns, for example, are likely to affect the risk of zoonotic disease transmission (dobson and may, 1986) . the effect of habitat fragmentation on disease processes has rarely been investigated, but it has recently been shown that trypanosoma cruzi infection rates are higher in fragmented than continuous atlantic forest (vaz et al., 2007) ; and the risk of lyme disease in new york is also apparently increased by fragmentation (allan et al., 2003) . interestingly, the division of endangered ethiopian wolf population into small sub-populations, joined by habitat corridors, has been shown to allow rabies control to be achieved using a low-coverage vaccination strategy (haydon et al., 2004) . the strategy operates by eliminating the largest outbreaks of disease, and so enhances meta-population persistence, rather than by the conventional objective of reducing the reproductive number of the disease to less than one (haydon et al., 2004) . human activities that artificially increase, rather than decrease, animal densities also influence disease processes. these increases can be the result of losses of absolute habitat area, or from the provision of supplementary food or water. in the united states, the practice of supplementary feeding of house finches and white-tailed deer has lead to an increase in the incidence of mycoplasmal diseases and btb, respectively (hartup et al., 1998; schmitt et al., 1997) , presumably because of greater opportunities for disease transmission, and possibly also immunosuppressive effects of aggression at the feeding sites. btb has also been an intractable problem in the british cattle herd, with the incidence rising inexorably since 1979. with a cull of badgers recently being ruled out, somewhat controversially, as offering no meaningful contribution to the long-term control of the disease (donnelly et al., 2005) , it is worth asking whether consideration of the ecology of the badgers and cattle might help generate workable solutions. over the past 30 years, along with the increase of btb, there has also been an increase in badger densities, and it is likely that this contributes at least in part to the disease in cattle. so why have badger populations risen? might the answer lie in changes in land use? macdonald and newman (2006) speculate on a possible role for climate change, with milder winters and hence greater earthworm availability improving survival rates. changes could also have occurred in the susceptibility of badgers to btb and/or of cattle to btb. for example, the average milk production of a dairy cow rose from 3,750 l in 1970 to 5,395 l in 1995 (farm animal welfare council, 1997) , possibly to the detriment of the animal's immune status. similarly, stress resulting from cull-associated social perturbations, or from other changes to habitat, food availability or population density, may have influenced the innate immune response of badgers. little is known of the physiological responses of free-living wild mammals to poor environmental quality or other potential stressors. an argument has been made for polychlorinated biphenyls (pcbs) and other pollutants contributing to phocine distemper outbreaks (ross et al., 1996 (ross et al., , 2000 , but this is has been questioned (o'hea, 2000) . while the role of toxicants is not clear, recent work indicates that high population densities in wild field voles is associated with compromise in haematological and immunological indices. poor body condition appeared to affect the inflammatory response (as indicated by lower neutrophil and monocyte peaks) and lower immunological investment (as indicated by lower lymphocyte counts (beldomenico et al., 2008a,b) . i have found, with co-worker jon blount, preliminary evidence of increased oxidative stress (measured by serum malondialdehyde concentration) among non-infected badgers from farms with recent btb in cattle, compared with those at sites free of btb ( fig. 8.3 ). there is also a considerable literature from farm (moberg and mench, 2000) , laboratory (galloway and handy, 2003) , and free-living aquatic animals (liney et al., 2006) showing that environment has a strong impact on stress responses, and that these can lead to pathological and pre-pathological alterations in immune function and overall health status. while it might be difficult to intervene directly to reduce the causes of stress in animals, ecologically based interventions that reduce both disease susceptibility and the opportunities for transmission may be possible. for example, in a study of 120 british farms it has been shown that habitat management and cattle herd size were strongly associated with the risk of btb in dairy cattle (mathews et al., 2006a) . reduced risk of btb was associated with the management of farmland in ways favourable to wildlife, including greater hedgerow availability, a lack of gaps in hedgerows, increasing hedgerow width and the presence of ungrazed wildlife strips adjacent to hedgerows. all of these measures are encouraged by recent european common agricultural policy reforms (2005) . broadly, habitat could influence cattle contact rates or be associated with agricultural management practices in ways relevant to btb transmission (such as reduced herd size). favourable habitat may lower the susceptibility of badgers to btb or reduce the number of inter-group excursions; alternatively, cattle on hedgerow-rich farms may be at reduced risk of ingesting contaminated soil. taking for simplicity just one of the parameters contributing to the effects-the total length of hedgerow-an increase of 1 km/100 ha was associated with a decrease in the odds of btb by about 12.5% (95% confidence interval 0.3% increase to 26.3% decrease) in univariate analysis. in absolute terms, this equates to the annual risk of btb changing from the current rate of 9.2% (2,152 confirmed incidents in 23,471 herds in 2004) to 8.1% (1,901 incidents) for herds in the west of england if a policy of moderate hedgerow density increase were adopted. this would mean a reduction of 251 infected herds per year. by comparison, systematic badger culling appears able to reduce the odds of btb by a maximum of about 19% and may even increase the prevalence in neighbouring areas (donnelly et al., 2005) . change in land use has also been linked to the emergence of two henipaviruses, nipah virus and hendra virus in the 1990s, and land use management may therefore offer part of the solution. both viruses appear to be asymptomatic in their natural hosts, fruit bats (genus pteropus). they are amplified in domestic animals, pigs and horses, respectively, where they cause mortality, and can then be passed on to humans (chua et al., 2000; halpin et al., 2000) . the closeness of rna sequence match between pteropus sp., livestock and human isolates of each virus suggests that a sudden change in virulence is a less likely explanation of their rapid emergence into domestic animals and humans than is the ecological change that have affected the habitat of their natural hosts. many flying fox species are in decline, with roosting and feeding sites being deforested, and converted to agricultural or urban use. a number of hypotheses have been proposed to explain exactly how nipah virus emerged (see breed et al., 2006) , all of which involve the establishment of piggeries in previously forested regions still used by fruit bats (chua et al., 2002) . increased contact rates are also the likely explanation for the emergence of hendra virus in australia, with many pteropus populations having relocated into urban areas (hall and richards, 2000) . with no vaccine available, and pteropus in need of conservation, ecologically based strategies to limit contact rates between bats and livestock offer the best prospects of controlling the disease (field and mackenzie, 2004) . managing the risks from zoonoses to the health of humans and domestic animals is complex. it is also fundamentally important: virtually all emerging infectious diseases have originated in wildlife. superficially, the simplest method of control is via a reduction in reservoir host-disease prevalence, this being achieved by culls of host populations. however, effective reductions in population densities can be difficult to achieve in practice and may be undesirable where the target is of conservation concern. for example, most bat species are threatened, and yet they appear to be particularly important sources of emerging viruses (calisher et al., 2006; dobson, 2005) ; and despite not being endangered, badgers in the united kingdom are legally protected. an alternative, and possibly complementary, strategy is to manage the ecological conditions leading to disease spill-overs. this will not only benefit the health of humans and their domestic stock, but must surely also lead to benefits for the conservation and welfare of wild animals. effects of forest fragmentation on lyme disease risk population biology of infectious diseases: part i control of infectious diseases of wildlife in europe epidemiologie et lutte contre la rage en france et en ecology of infectious diseases in natural populations the dynamics of health in wild field vole populations: a haematological perspective poor condition and infection: a vicious circle in natural populations fox rabies. in ''the natural history of rabies.'' 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who effects of culling on badger meles meles spartial organisation: implications for the control of bovine tuberculosis key: cord-295554-0pzjyrdf authors: lima, francisco esmaile de sales; campos, fabrício souza; kunert filho, hiran castagnino; batista, helena beatriz de carvalho ruthner; carnielli júnior, pedro; cibulski, samuel paulo; spilki, fernando rosado; roehe, paulo michel; franco, ana cláudia title: detection of alphacoronavirus in velvety free-tailed bats (molossus molossus) and brazilian free-tailed bats (tadarida brasiliensis) from urban area of southern brazil date: 2013-03-16 journal: virus genes doi: 10.1007/s11262-013-0899-x sha: doc_id: 295554 cord_uid: 0pzjyrdf a survey was carried out in search for bat coronaviruses in an urban maternity roost of about 500 specimens of two species of insectivorous bats, molossus molossus and tadarida brasiliensis, in southern brazil. twenty-nine out of 150 pooled fecal samples tested positive by reverse transcription-pcr contained fragments of the rna-dependent rna polymerase gene of coronavirus-related viruses. the sequences clustered along with bat alphacoronaviruses, forming a subcluster within this group. our findings point to the need for risk assessment and continued surveillance of coronavirus infections of bats in brazil. electronic supplementary material: the online version of this article (doi:10.1007/s11262-013-0899-x) contains supplementary material, which is available to authorized users. bats (order chiroptera, suborders megachiroptera and microchiroptera) are one of the most diverse and widely distributed groups of mammals, representing *20 % of all known mammalian species [1] . about a 100 different viruses have been identified in bats of different species in asia, europe, north america and africa. therefore, such species may be natural reservoirs for a large variety of potentially zoonotic rna viruses, such as lyssaviruses, paramyxoviruses, ebola and marburg viruses as well as the recently emerged severe acute respiratory syndrome coronavirus (sars-cov) [2] [3] [4] [5] [6] . a variety of other coronaviruses have been detected in many bat species from asia, including specimens of the genus rhinolophus, which were found to be infected with sars-like cov. phylogenetic analyses of such viruses revealed that those form a large clade within betacoronavirus genus, along with sars coronaviruses from palm civets and the sars coronaviruses recovered from humans during the 2003 outbreak [7, 8] . these data suggested that the agent responsible for the 2002-2003 pandemic might have originated from bats. in addition, in 2012, a new human coronavirus (hcov-emc), which has been associated to clinical disease that resembles sars, emerged in the middle east. this new virus appears to have originated from bats, raising the possibility that hcov-emc jumped species directly from bats to humans [9] . in brazil, most studies looking for associations between bats and viruses have focused on the role for those species as reservoirs for rabies virus [10] . however, to date, more than 160 bat species have been detected in brazil, comprising members of the families phyllostomidae, vespertilionidae, and molossidae. it is estimated that at least 40 bat species live in the state of rio grande do sul, southern brazil, where the predominantly sub-tropical climate seems to favor the settlement of such species [11] . in view of the potential role that bats may play in the transmission of new viral infections to humans and other species, this study was set up in search for coronavirus genomes in bats from the urban area of porto alegre (30°01 0 59 00 s; 51°13 0 48 00 w), a town with about 1.5 million inhabitants and capital of the state of rio grande do sul, brazil. with that purpose, coronavirus rna was searched in feces of two species of synanthropic insectivorous bats collected in a maternity roost within the urban area of the city. a maternity roost of bats known to have direct contact with people and domestic animals was identified in the summer of 2012 in the attic of a residence in the central area of porto alegre, southern brazil. the colony was estimated to harbor about 500 bat specimens of insectivorous bats of two species, velvety free-tailed bats (molossus molossus) and brazilian free-tailed bats (tadarida brasiliensis). speciation was confirmed by amplification and sequencing of the mitochondrial cytochrome b (cytb) gene as described [12] . one hundred and fifty fecal samples were collected from the attic floor as follows: a plastic film was spread on the ground of the attic compartment and fresh droppings were collected with clean disposable forks in the following night. each sample consisted of five fecal droppings, which were immediately sent to the laboratory and stored at -80°c. the samples were then submitted to total rna extraction with trizol (invitrogen tm ). cov rna screening was performed by reverse transcription-polymerase chain reaction (rt-pcr) in a total volume of 25 ll reaction using conserved primers for the rna-dependent rna polymerase gene (forward: 5 0 -ggttgggactatc ctaagtgtga-3 0 and reverse: 5 0 -ccatcatcagatag aatcatcata-3 0 ). this pair of primers is expected to give rise to amplicons of 440 bp [7] . the cycling conditions were: 5 min at 94°c followed by 35 cycles of 1 min at 94°c, 1 min at 49°c and 1 min at 72°c, followed by a final extension time of 5 min at 72°c. bovine coronavirus (bcov) rna was used as a positive control to optimize the assay. standard precautions were taken to avoid pcr contamination; blank controls without template were included in every set of five rt-pcr assays. five microliters of the pcr products were electrophoresed in 1.5 % agarose gels and the products visualized on uv light after staining with ethidium bromide. the amplicons obtained were cloned into pcr ò 2.1-topo ò cloning kit (invitrogen) before being submitted to nucleic acid sequencing. sequencing was performed with the big dye terminator cycle sequencing ready reaction (applied biosystems, uk) in an abi-prism 3100 genetic analyzer (abi, foster city, ca), following the manufacturer's protocol. sequence analyses were performed with the blast software [13] . nucleotide sequences were aligned and compared to human and animal cov sequences available at genbank database with the program clustalx 2.0 [14] . alignments were optimized with the bioedit sequence alignment editor program version 7.0.9 [15] . the protocol to generate the phylogenetic trees was selected with the program modeltest 3.7 [16] . phylogenetic analysis was carried out using mega 5.0; pairwise genetic distances were calculated by the tamura 3-parameter model and phylogenetic trees were constructed using the neighbourjoining method. bootstrap values were determined by 1,000 replicates to assess the confidence level of each branch pattern. pcr amplicons with the expected size of the targeted region were obtained from 29 out of the 150 (19.33 %) pools of bat fecal samples. the nucleotide sequences of sixteen randomly selected amplicons were determined and submitted to genbank (accession numbers kc 110770 to kc 110785). genetic analyses provided evidence that the viruses circulating in these two species of insectivorous bats belong to the genus alphacoronavirus. when compared with each other, all the obtained sequences showed a high nucleotide and amino acid identity (90.6 to 100 % and 98 to 100 %, respectively) (supplemental material). the rdrp sequences examined here were distantly related (\75 % nt identity) to other known alphacoronaviruses. the closest bat coronaviruses rdrp sequences found in genbank were the asian (btcov/a633/2005) and north american (rm-btcov 6 and rm-batcov 11) bat coronaviruses (fig. 1) . the percentage of nucleotide similarity between the sequences described here and those of asian and north american coronaviruses ranged from 72.4 to 76 %, whereas at the amino acid level, the similarity ranged from 74 to 81 % (data not shown). during the last two decades, several studies have shown that various important human and animal pathogens are of bat origin; these species have become targets for several surveillance studies aiming the detection of other potentially pathogenic viruses for humans and other animals. the association of these pathogens and possible disease outbreaks caused by direct or indirect contact of humans with bats stimulated the development of research activities on bat-borne viruses. in addition, the advances of molecular techniques offer opportunities for the discovery of novel dna and rna bat viruses without the need for virus isolation and bat pooled fecal samples being used as source for viruses, preventing animal manipulations [17, 18] . in our study, we detected rdrp sequences of bat cov at a frequency of 19.33 % in the examined samples; such frequency is comparable to previous results obtained in similar studies from different bat species in other countries (ranging figure) . the tree was generated based on the neighbor joining method in the mega program. the nucleotide sequence of the equivalent genome fragment of sars-cov was included as outgroup (fig. 1) . these results show that similar coronaviruses are found in different bat species that are distributed in geographically distant regions, suggesting a low degree of host restriction for coronavirus in those bat populations. in contrast to the enormous diversity of cov genomes found in old world bats [12, 25] , in this study and in several others concerning the cov detection in new world bats, only alphacoronaviruses were detected [2, 3, 17, 18] . based on these results, it has been hypothesized that covs found in new world bats are less diverse than those detected among old world bats [26] . in this initial study, samples were restricted in location and variety of bat species, and we found only alphacoronaviruses. such findings do not reflect data on incidence or prevalence of such infections in bat populations. however, one cannot exclude the possibility that a greater diversity may become apparent in brazilian bats as long as larger numbers of samples from a wider spectrum of species are examined. to our knowledge, this is the first report of cov detection in feces from presumably healthy insectivorous bats in brazil. however, it is very likely that other bat species might also be infected with similar viruses. additional studies with larger numbers of bats and bat species, as well as the continued vigilance on the occurrence of viral infections in bats over the years is required to follow the evolution of bat coronaviruses in its interactions with the different bat host species. in addition, the detection of covs in brazilian bat populations in close proximity to human inhabitants may represent a risk to human health. our findings point to the need to identify the prevalence of covs in brazilian bats, to perform risk assessment studies and continued surveillance of coronavirus infections from both urban and rural environments. mammal species of the world: a taxonomic reference vector borne zoonotic dis proc. natl. acad. sci. usa bioedit: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/nt acknowledgments we would like to thank the government agencies finep, cnpq, and capes by the financial support. p.m. roehe, a.c. franco, and f.r. spilki are cnpq research fellows. key: cord-287748-co9j3uig authors: kobayashi, tomoya; murakami, shin; yamamoto, terumasa; mineshita, ko; sakuyama, muneki; sasaki, reiko; maeda, ken; horimoto, taisuke title: detection of bat hepatitis e virus rna in microbats in japan date: 2018-05-29 journal: virus genes doi: 10.1007/s11262-018-1577-9 sha: doc_id: 287748 cord_uid: co9j3uig several recent studies have reported that various bat species harbor bat hepatitis e viruses (bathev) belonging to the family hepeviridae, which also contains human hepatitis e virus (hev). the distribution and ecology of bathev are not well known. here, we collected and screened 81 bat fecal samples from nine bat species in japan to detect bathev rna by rt-pcr using hev-specific primers, and detected three positive samples. sequence and phylogenetic analyses indicated that these three viruses were bathevs belonging to genus orthohepevirus d like other bathev strains reported earlier in various countries. these data support the first detection of bathevs in japanese microbats, indicating their wide geographical distribution among multiple bat species. bats are known to be natural reservoirs of various zoonotic viruses such as rabies virus, nipah virus, and severe acute respiratory syndrome (sars) coronavirus [1] [2] [3] . in addition, hepatitis e virus (hev)-like viruses have been detected in bats in several countries [4, 5] . bat hev (bathev) belongs to family hepeviridae, genus orthohepevirus and is a non-enveloped, positive-sense, single-stranded rna virus. orthohepevirus is divided into four species, orthohepevirus a-d [6, 7] . orthohepevirus a contains hev, which is a causative agent for human acute hepatitis. orthohepevirus b includes avian-hev associated with hepatitis-splenomegaly syndrome. orthohepevirus c is divided into c1 (rodent-hev) and c2 (carnivore-associated hev) genotypes. orthohepevirus d comprises bathev, which has 57.4-64.8% identity with hev [8] . recently, bathevs were detected from macrobats and microbats in a variety of countries [4, 5] . however, limited information about the distribution of bathevs in other regions and their ecology is available. here, we surveyed several japanese microbat species to detect bathevs. to examine whether bathevs exist in japanese bats, we collected 81 bat fecal samples from nine bat species captured in five different prefectures of japan in 2015, with permission from the ministry of the environment, japan and the respective local government (fig. 1a ). bats were caught using a harp trap and kept in a pouch for an hour to check the signs of disease as well as to obtain fresh feces. all captured bats did not show any obvious symptoms. the feces were added in a medium containing antibiotics, and frozen in dry ice. we extracted rna from the fecal samples and performed rt-pcr using a primer set ( table 1) that was designed specifically against the conserved region of the rna-dependent rna polymerase (rdrp) of hev, to screen the hev genomes. two samples (bthev-ej1 and bthev-ej2) from the japanese short-tailed bat (eptesicus japonensis) and 1 sample (bthev-ps1) from the brown long-eared bat (plecotus sacrimontis) were found to be positive. although we attempted to sequence the entire genome of the bathevs, we failed to amplify the whole genome using rt-pcr. therefore, we amplified partial orf1, which encodes non-structural protein including rdrp, and entire capsid coding region of bthev-ej1, -ej2, and -ps1 (corresponding to nucleotides (nt) 2171-6690, nt 3585-6690, and nt 3847-6690 of bathev/bs7, respectively) using specific primers ( table 1 ). the sequences of bthev-ej1 and bthev-ej2, which were collected in the same site on different days, were 99% identical (nt 2904 out of 2915). the identity between bthev-ej1/-ej2 and bthev-ps1 using a part of the rdrp was about 75% at the nucleotide level, suggesting the presence of multiple bathevs in japan. blast analysis indicated that bthev-ej1/-ej2 showed the highest sequence identities to bathev/bs7, a german strain detected from the serotine bat (eptesicus serotinus), among strains previously reported in other countries. on the other hand, bthev-ps1 showed the highest sequence identity to bthev/nms098b, a german strain detected from the daubenton's bat (myotis daubentonii). in particular, 82% identity observed between bthev-ej1/-ej2 and bathev/bs7 or 77% identity observed between bthev-ps1 and bthev/nms098b were greater than that observed between japanese strains. these data suggest that similar viruses exist in geographically distant regions. we then phylogenetically analyzed the sequences by maximum-likelihood analysis using clustalw and mega version 7.0 [9] . a phylogenetic tree constructed using the partial amino acid sequences of rdrp indicated that the japanese viruses were included in orthohepevirus d (fig. 1b) , demonstrating that all bathevs are classified in this species. we also amplified the full-length capsid (orf2) sequences by rt-pcr and analyzed them phylogenetically. the resulting tree confirmed that the novel japanese viruses were included in orthohepevirus d (fig. 1c) . the sequences of bthev-ej1/-ej2 were placed in a position neighboring the german bathev/bs7 strain, confirming the phylogenetic similarity between these strains. all bats captured in this study were insectivores and hibernate in winter. although there is no information about migration of e. japonensis and p. sacrimontis, bat species closely related to them were reported to migrate only a few kilometers from their colonies per night [10, 11] . they had different ecology in the terms of the habitat. bthev-ej1 and -ej2 were detected on different days from two e. japonensis bats, both of which used eaves of the same house as night roost in nagano. although the e. japonensis bats formed mixed colony at the roost with myotis ikkonikovi and rhinolophus ferrumequinum bats, bathevs were only detected in e. japonensis, implying bthev-ej1/-ej2 might have a narrow host range. p. sacrimontis bats usually form a small colony without other species of bats. indeed, p. sacrimontis bats, from which bthev-ps1 was detected in this study, were captured near such small colony in a ruin in aomori. thus, bthev-ps1 is likely circulating in the p. sacrimontis since the bats have low opportunity to come in contact with other species of bats. the closely related bathevs (bthev-ej1/-ej2 and bat hev/bs7) have been detected in different species of eptesicus bats (e. serotinus and e. japonensis). since the distribution areas of these bats are not overlapping, viruses ancestral to bthev-ej1/-ej2 and bathev/bs7 might have infected ancestral eptesicus and might have branched into different species in the process of evolution. for virus isolation, we inoculated the rt-pcr-positive fecal samples not only into several bat cells (bkt, fbkt, and demkt1 cells) but also into other mammalian cell lines (madin-darby canine kidney (mdck), african green monkey veroe6, human a549, madin-darby bovine kidney (mdbk), and swine pk15 cells) since we suspected that the bat fecal samples may contain several pathogens other than bathevs. all inoculated cells were incubated for 12-15 days with media changes at 2-3 days interval. after the incubation, cells were blindly passaged three times. however, we could neither recover any infectious viruses nor detect bathev rna in the inoculated cells by rt-pcr. in conclusion, the present study showed the presence of several bathev strains, which were independently classified to obtain fresh feces, bats were kept in a pouch for an hour, and the feces were then collected by a sterilized cotton bud and transferred to 1 ml of dulbecco's modified medium eagle's minimum essential medium (dmem) supplemented with 100 u/ml of penicillin, 1 mg/ml of streptomycin, 100 µg/ml of gentamycin, and 2 µg/ml of amphotericin. the feces were suspended well and then centrifuged at 10,000×g for 15 min at 4 °c. the supernatants were used for rna extraction with isogen ls reagent (nippon gene). cdna was synthesized using prime script rt reagent kit (takara bio) with a mixture of random hexamer and oligo dt primers. pcr amplifications were performed using the kod fx neo (toyobo) with consensus hev primer sets (panhev f and r), which were designed in this study to amplify a 191-bp fragment of the rna-dependent into orthohepevirus d, in japanese bats, suggesting wide geographical distribution of bathev among multiple bat species. although these data suggest limited transmissibility of bathev to other animals, further studies are needed to determine its zoonotic potential. fields virology members of the international committee on the taxonomy of viruses hepeviridae study group acknowledgements we thank mr. mitsuru mukohyama for helping us key: cord-289584-rbp7p8s9 authors: zhou, ling; sun, yuan; lan, tian; wu, ruiting; chen, junwei; wu, zixian; xie, qingmei; zhang, xiangbin; ma, jingyun title: retrospective detection and phylogenetic analysis of swine acute diarrhoea syndrome coronavirus in pigs in southern china date: 2019-01-09 journal: transbound emerg dis doi: 10.1111/tbed.13008 sha: doc_id: 289584 cord_uid: rbp7p8s9 swine acute diarrhoea syndrome coronavirus (sads‐cov), a novel coronavirus, was first discovered in southern china in january 2017 and caused a large scale outbreak of fatal diarrheal disease in piglets. here, we conducted a retrospective investigation of 236 samples from 45 swine farms with a clinical history of diarrheal disease to evaluate the emergence and the distribution of sads‐cov in pigs in china. our results suggest that sads‐cov has emerged in china at least since august 2016. meanwhile, we detected a prevalence of sads‐cov (43.53%), porcine deltacoronavirus (8.83%), porcine epidemic diarrhoea virus (pedv) (78.25%), rotavirus (21.77%), and transmissible gastroenteritis virus (0%), and we also found the co‐infection of sads‐cov and pedv occurred most frequently with the rate of 17.65%. we screened and obtained two new complete genomes, five n and five s genes of sads‐cov. phylogenetic analysis based on these sequences revealed that all sads‐cov sequences in this study clustered with previously reported sads‐cov strains to form a well defined branch that grouped with the bat coronavirus hku2 strains. this study is the first retrospective investigation for sads‐cov and provides the epidemiological information of this new virus in china, which highlights the urgency to develop effective measures to control sads‐cov. swine acute diarrhoea syndrome coronavirus (sads-cov) is a newly discovered coronavirus which is an enveloped, positive and singlestranded sense rna virus with a genome size of approximately 27 kb (gong et al., 2017; pan et al., 2017; zhou et al., 2018) . sads-cov belongs to the family coronaviridae which contains four genera, alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus (woo, huang, lau, & yuen, 2010; woo et al., 2012) . so far, six coronaviruses have been identified from pigs, which include porcine epidemic diarrhoea virus (pedv), porcine respiratory coronavirus (prcv), sads-cov and transmissible gastroenteritis virus (tgev) that all belong to the alphacoronavirus genus, as well as one betacoronavirus, porcine hemagglutinating encephalomyelitis virus (phev) and one deltacoronavirus, porcine deltacoronavirus (pdcov) (lin, saif, marthaler, & wang, 2016; wesley, woods, & cheung, 1991; woo et al., 2010) . among these viruses, sads-cov is the most newly discovered coronavirus, which has been first reported in 2017 in china and is considered to be an hku2-related coronavirus with a bat-origin (gong et al., 2017; zhou et al., 2018) . in january 2017, sads-cov was detected in a swine farm and subsequently spread rapidly to three other farms in *these authors contributed equally to this work. guangdong province and caused the fatal swine acute diarrhoea syndrome (sads) characterized by the clinical signs with severe, acute diarrhoea and rapid weight loss of piglets. the symptoms of sads-cov are similar to those that caused by other swine enteric coronaviruses such as pdcov and pedv, but sads-cov is more harmful than these viruses because it has led to the death of almost 25,000 piglets in a short time and resulted in more significant economic losses (dong et al., 2015; sun, wang, wei, chen, & feng, 2016; zhou et al., 2018) . so, it is urgent to investigate the molecular epidemiology and transmission patterns of sads-cov for establishing effective controls for this new coronavirus. in the present study, we performed the retrospective pcr testing on diarrheal samples from 45 swine farms in guangdong province to evaluate the emergence and the distribution of sads-cov in pigs in china. the prevalence and co-infection information of sads-cov from eleven sads-cov-positive farms was provided. the sequences of sads-cov, including two complete genomes, five nucleocapsid protein (n) genes and five spike protein (s) genes, were also identified and characterized to investigate the phylogenetic relationships of sads-cov. samples were homogenized in phosphate-buffered saline (pbs) (20% w/v), frozen and thawed three times, then centrifuged for 10 min at 10,000 g. viral nucleic acid was extracted following the manufacturer's recommendations of axyprep tm body fluid viral dna/rna miniprep kit (axygen scientific, inc). the virus nucleic acid was stored at −80°c until pcr was performed. a pair of primers (forward primer 5′-ggtccctgtgaccgaagttttag-3′, reverse primer 5′-gcgttctgcgataaagcttaaaactatta-3′) was designed to detected sads-cov based on the conserved n gene of this virus. one step rt-pcr using primescript ™ one step rt-pcr kit ver.2 with dye plus (takara, biotechnology, dalian, china) was carried out to amplify the target fragments by the following thermal profile of 50°c for 30 min, 94°c for 3 min, 35 cycles of denaturation at 94°c for 30 s, annealing at 55°c for 30 s, an extension at 72°c for 30 s, and a final step of 72°c for 5 min. four other diarrheal pathogens including pedv, pdcov, rotavirus (rv), and tgev from sads-covpositive farms were also tested by rt-pcr according to the previously described methods (liu, zhu, liao, xu, & zhou, 2015; mai et al., 2017; stevenson et al., 2013) . specific primer pairs based on reported sads-cov strains (genbank accession numbers: mf094681-mf094684) were designed for s genes, n genes and complete genome amplifications, respectively (table s1 ). pcr assays were performed with the following thermal profile: 95°c for 5 min, 35 cycles of 95°c for 30 s, 50°c for 30 s, and 72°c for 1 min 15 s, followed by a final 10 min extension at 72°c. the products were purified following the manufacturer's the nucleotide sequences were assembled and aligned using the dnastar program (dnastar v7.1, madison, wi, usa). phylogenetic trees were constructed using the neighbour-joining method in mega 7.0 software with bootstrap analysis of 1,000 replicates. percentages of replicate trees in which the associated taxa clustered are shown as nearby branches (chenna et al., 2003; kumar, stecher, & tamura, 2016; tamura, nei, & kumar, 2004 ). the two complete genomes (accession number mg605090 and f i g u r e 4 phylogenetic analysis of the s genes of sads-cov and reference coronavirus species. the tree was constructed as per figure 2 above. five new sequences of s genes studied in this work were indicated with "black solid circles" diarrhoea samples. our results showed that the first sads-cov positive sample was collected in august 2016 from the farm ls with a history of diarrhoea, as well as from other two farms tp and zw, which indicates that sads-cov has emerged in pigs in china at least since august 2016. and this time point is 5 months earlier than the first discovered time reported by our previous study (zhou et al., 2018) . as the same time, clinical signs of sads-cov during the retrospective investigation included sever and acute vomiting and diarrhoea, leading to death in piglets that were less than 5 days of age with a mortality rate of around 50%. these clinical presentations were similar to those signs in the large scale outbreak of sads-cov reported by zhou et al. (2018) , except the mortality rate in piglets later increased to 90%. based on the rates of infection documented in our work, it revealed that pedv (78.25%) was still the primary cause of the porcine diarrhoea, which is consistent with previous studies that pedv has been considered to be the major pathogen responsible for the porcine diarrhoea epidemic in china since 2010 (ge et al., 2013; sun et al., 2012; zhao et al., 2016) . the phylogenetic relationships of sads-cov sequences were also identified in this study. the results showed that all sads-cov sequences clustered together to form an independent branch and separated from other viral sequences in the genus alphacoronavirus. our results also indicated that both the complete genomes, n genes and s genes of all sads-cov strains shared the highest nucleotides identifies with those corresponding sequences of four bat coronavirus hku2 strains. in this work, the phylogenetic trees of full length genomes and s genes of sads-cov sequences showed that the sads-cov branch clustered with these four hku2 strains, which is same to previous results (gong et al., 2017; pan et al., 2017; zhou et al., 2018) . besides the genomes and s genes, the tree of n genes in our study revealed the identical result too. so far, a total of eight full-length genomes of sads-cov have been reported in guangdong province of china (gong et al., 2017; pan et al., 2017; zhou et al., 2018 ; this study). the two new genomes of sads-cov sequences in this work shared 100% nucleotides identities with the sequence mf167434 published by gong et al. (2017) and our four previously reported sequences (zhou et al., 2018) , and shared 99.8% nucleotides identities with the sequence mf370205 studied by pan et al. (2017) . the results suggest that these eight sads-cov sequences may come from the same origin. only the phylogenetic tree of s genes in our work showed that sequences of the alphacov were divided into two sublineages, alphacov1 which contained all sads-cov sequences and alphacov2, clustering together with sequences of the beltacov and the delatcov, respectively. and this result was consistent with the study of pan et al. (2017) . as a newly discovered coronavirus, the availability of sads-cov sequences data is limited which prevents better understandings of the molecular epidemiology of this virus. meanwhile, being a rna virus, sads-cov may mutate rapidly and exhibit high genetic differences (drummond, pybus, rambaut, forsberg, & rodrigo, 2003; kühnert, wu, & drummond, 2011 the authors declare no conflict of interests with any organization. ma https://orcid.org/0000-0001-6285-312x infectious diseases, 23, 1607-1609. https://doi.org/10.3201/eid2309. porcine enteric alphacoronavirus gds04 | mf167434 rhinolophus bat coronavirus hku2 ch/gd-01/2017/p2 | mf370205 bat coronavirus hku2 | nc009988 bat coronavirus hku2 strain hku2/hk/46 bat coronavirus hku2 strain hku2/hk/33 human coronavirus 229e | nc002645 camel alphacoronavirus isolate riyadh/ry141/2015 | nc028752 229e-related bat coronavirus strain btky229e-1 | ky073747 porcine enteric alphacoronavirus gds04 | mf167434 rhinolophus bat coronavirus hku2 ch/gd-01/2017/p2 | mf370205 bat coronavirus hku2 strain hku2/hk/33 bat coronavirus hku2 strain hku2/hk/46 bat coronavirus 1a | nc010437 bat coronavirus hku8 strain afcd77 | eu420139 rousettus bat coronavirus hku10 | nc01887 porcine epidemic diarrhoea virus strain gds01 | km089829 porcine epidemic diarrhoea virus strain cv777 | kt323979 porcine epidemic diarrhoea virus| nc003436 human coronavirus nl63 | nc005831.2 nl63-related bat coronavirus strain btkynl63-9a | nc032107 229e-related bat coronavirus strain btky229e-1 | ky073747 camel alphacoronavirus isolate riyadh/ry141/2015 | nc028752 human coronavirus 229e | nc002645 porcine hemagglutinating encephalomyelitis virus | nc007732 human coronavirus oc43 strain sc2481 | ky983583 mouse hepatitis virus strain mhv-a59 c12 mutant | nc001846 murine hepatitis virus strain jhm complete genome | ac000192 middle east respiratory syndrome coronavirus | nc019843 bat sars coronavirus hku3-1 | dq022305 bat sars-like coronavirus rsshc014 | kc881005 bat sars-like coronavirus wiv1 | kf367457 european turkey coronavirus 080385d | kr822424 bulbul coronavirus hku11-796 | fj376620 porcine deltacoronavirus isolate pdcov/chjxni2/2015 | kr131621.1 porcine coronavirus hku15 strain hku15-44 | nc016990 rhinolophus bat coronavirus hku2 ch/gd-01/2017/p2 | mf370205 bat coronavirus hku2 strain hku2/hk/46 bat coronavirus hku2 strain hku2/hk/33 human coronavirus oc43 strain sc2481 | ky983583 porcine hemagglutinating encephalomyelitis virus | nc007732 mouse hepatitis virus strain mhv-a59 c12 mutant | nc001846 murine hepatitis virus strain jhm complete genome | ac000192 middle east respiratory syndrome coronavirus | nc019843 bat sars coronavirus hku3-1 | dq022305.2 sars coronavirus | nc004718 bat sars-like coronavirus rsshc014 | kc881005 bat sars-like coronavirus wiv1 | kf367457 european turkey coronavirus 080385d | kr822424 porcine deltacoronavirus isolate pdcov/chjxni2/2015 | kr131621.1 porcine coronavirus hku15 strain hku15-44 | nc016990 bulbul coronavirus hku11-796 | fj376620 bat coronavirus hku8 strain afcd77 | eu420139 bat coronavirus 1a | nc010437 camel alphacoronavirus isolate riyadh/ry141/2015 | nc028752 multiple sequence alignmentwith the clustal series of programs porcine deltacoronavirus in mainland china measurably evolving populations epidemiological survey of porcine epidemic diarrhea virus in swine farms in a new bat-hku2-like coronavirus in swine phylogenetic and epidemic modeling of rapidly evolving infectious diseases mega7: molecular evolutionary genetics analysis version 7.0 for bigger datasets evolution, antigenicity and pathogenicity of global porcine epidemic diarrhea virus strains the porcine microrna transcriptome response to transmissible gastroenteritis virus infection the detection and phylogenetic analysis of porcine deltacoronavirus from guangdong province in southern china discovery of a novel swine enteric alphacoronavirus (sea-cov) in southern china emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences outbreak of porcine epidemic diarrhea in suckling piglets epidemiology and vaccine of porcine epidemic diarrhea virus in china: a mini-review prospects for inferring very large phylogenies by using the neighbor-joining method genetic analysis of porcine respiratory coronavirus, an attenuated variant of transmissible gastroenteritis virus coronavirus genomics and bioinformatics analysis discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus the rate of co-infection for piglet diarrhea viruses in china and the genetic characterization of porcine epidemic diarrhea virus and porcine kobuvirus fatal swine acute diarrhea syndrome caused by an hku2-related coronavirus of bat origin key: cord-021152-6znmkvy9 authors: montecino-latorre, diego; goldstein, tracey; gilardi, kirsten; wolking, david; van wormer, elizabeth; kazwala, rudovick; ssebide, benard; nziza, julius; sijali, zikankuba; cranfield, michael; mazet, jonna a. k. title: reproduction of east-african bats may guide risk mitigation for coronavirus spillover date: 2020-02-07 journal: nan doi: 10.1186/s42522-019-0008-8 sha: doc_id: 21152 cord_uid: 6znmkvy9 background: bats provide important ecosystem services; however, current evidence supports that they host several zoonotic viruses, including species of the coronaviridae family. if bats in close interaction with humans host and shed coronaviruses with zoonotic potential, such as the severe acute respiratory syndrome virus, spillover may occur. therefore, strategies aiming to mitigate potential spillover and disease emergence, while supporting the conservation of bats and their important ecological roles are needed. past research suggests that coronavirus shedding in bats varies seasonally following their reproductive cycle; however, shedding dynamics have been assessed in only a few species, which does not allow for generalization of findings across bat taxa and geographic regions. methods: to assess the generalizability of coronavirus shedding seasonality, we sampled hundreds of bats belonging to several species with different life history traits across east africa at different times of the year. we assessed, via bayesian modeling, the hypothesis that chiropterans, across species and spatial domains, experience seasonal trends in coronavirus shedding as a function of the reproductive cycle. results: we found that, beyond spatial, taxonomic, and life history differences, coronavirus shedding is more expected when pups are becoming independent from the dam and that juvenile bats are prone to shed these viruses. conclusions: these findings could guide policy aimed at the prevention of spillover in limited-resource settings, where longitudinal surveillance is not feasible, by identifying high-risk periods for coronavirus shedding. in these periods, contact with bats should be avoided (for example, by impeding or forbidding people access to caves). our proposed strategy provides an alternative to culling – an ethically questionable practice that may result in higher pathogen levels – and supports the conservation of bats and the delivery of their key ecosystem services. the order chiroptera is the second largest order of mammals with more than 1000 identified species [1] . the members of this order, bats, provide important ecosystem services (reviewed in [2, 3] ). for example, insectivorous bats can reduce arthropod herbivory [4] [5] [6] , increase agricultural yields [7] , reduce the need for insecticides [8] , and prevent large financial losses in agriculture [9] [10] [11] . plant-visiting chiropterans provide pollination and seeddispersing services (reviewed in [3] ), certain nectivorous bats are pollinators of economically important plants [12] , and frugivorous bats can be important for reforestation [13] . finally, cave-roosting bats produce guano, the main energy source in many cave ecosystems [14, 15] , and the mining of this product is an income source in poor communities [16] . however, current evidence supports that bats are a natural host of several disease-causing viruses across the globe, including zoonotic viruses, such as rabies virus (rhabdoviridae, genus lyssavirus); hendra and nipah viruses (paramyxoviridae, genus henipavirus); and marburg and ebolaviruses (filoviridae, genus marburgvirus and ebolavirus, respectively; [17, 18] ). bats are also hosts of several viruses of the coronaviridae family [19] [20] [21] [22] . molecular evidence suggests that the severe acute respiratory syndrome betacoronavirus (sars-cov beta-cov) and the middle-east respiratory syndrome betacov (mers-cov) originated from bats [23] [24] [25] [26] [27] [28] [29] [30] [31] . both viruses emerged in humans during the past two decades, specifically in china (2002) and saudi arabia (2012). the sars-cov pandemic included 8096 cases in 27 countries with a~10% case fatality [32] , while mers-cov has affected 2279 people in 27 countries with a case fatality of 35% [33] . incidental cases of mers-cov are still detected mainly in saudi arabia [32] , and it is thought that camels are important for human infection [34] [35] [36] . today it is known that: i) a high genetic diversity of coronaviruses (covs) is present in more than 100 bat species, including viruses related to sars and mers cov [37] ; ii) covs are prone to move and adapt to new host species [38] ; iii) plausibly, all mammalian-adapted covs may have originated in bats [20, 21, 38] , including a recently emerged highly fatal alphacoronavirus in piglets [39] and the 229e human cov [22, 40, 41] ; and iv) covs found in bats can use human receptors for cell entry [21, 25, 42, 43] . these lines of evidence suggest that future spillover of coronaviruses humans is feasible. because covs are found in bat species that have adapted to be in close contact with humans, such as the straw-colored fruit bat (eidolon helvum) and the brazilian free-tailed bat (tadarida brasiliensis [44, 45] ), high contact "bat-human" interfaces currently exist around the world. if the bats in these interfaces shed covs with the ability to infect humans, then opportunities for spillover through direct exposure to feces [37] or the contamination of food are created, as these viruses can remain infectious in the environment for days [46] . therefore, strategies aiming to mitigate human exposure to covs, and thus, the risk of spillover and disease emergence are needed, while supporting the conservation of bats and their important ecological roles. longitudinal sampling with specific species has shown that the proportion of bats shedding covs varies seasonally [47] [48] [49] [50] and that fecal cov-rna loads can also be heterogeneous over time [51, 52] . if exposure through contact with bat feces is a main pathway for zoonotic cov spillover to humans but shedding of these pathogens is not uniform over time, then mitigation strategies aiming to prevent bat cov-shed exposure could be targeted temporally, directed especially at highrisk seasons. such a strategy could guide policy in limitedresource settings where sampling bats for cov testing is not feasible and it could support an ethically acceptable management to mitigate spillover risk. however, the few species and locations tested to date do not allow for identification of a potential seasonal shedding pattern to responsibly suggest temporal spillover risk management across species and geography. therefore, assessment of the cov dynamics in a broader range of bat species that show different life history traits, as well as in diverse geographic and ecological circumstances, could be extremely useful. to this end, we evaluated the dynamics of cov shedding in different bat species sampled in several locations in east africa at different times of the year. this geographical region has been identified as a hotspot of pathogen emergence [53] , where cov host switching events seem to be higher compared to other areas [22] , but, to our knowledge, no study on cov dynamics in bats has been conducted. specifically, we hypothesized that bat species exhibit seasonal trends in cov shedding that are associated with the reproductive season. we assessed this hypothesis by fitting bayesian statistical multivariable models to evaluate whether cov shedding in bats is positively associated with the time period when pups are becoming independent from the dam. beyond the inclusion of several species sampled in different countries at different times, we explicitly identified the reproductive events for each species at the time of sampling and also included other traits, such as the aggregation of individuals at the roost, that may be involved in cov dynamics. samples (rectal swabs and fresh feces) were collected from bats captured in uganda, rwanda, and tanzania ( fig. 1) , between september 2011 and april 2014 with permission from local authorities and under the institutional animal care and use committee at the university of california, davis (protocol number: 16048). bats were captured in 36 unique locations between latitudes − 0.9739 and − 10.7506 (fig. 1 ). these locations were selected because they represented potentially high-risk interfaces for contact between bats and humans, such as areas of land-use change, human-dwellings, ecotourism sites, markets, and places with potential for occupational exposure [54] . locations in close proximity (euclidean distance < 20 km) in which sampling was conducted within the same week were considered a single sampling event. the remaining sampling events that occurred in the same location or spatially close to others but conducted in different weeks were considered independent sampling events. as result, we collected samples from 30 unique sampling events. all captures were conducted using mist nets set in the early morning or at dawn. individuals were released after sample collection. samples were handled as previously described [22, 55] . each sample was immediately transferred to vials containing viral transport media and nuclisens® lysis buffer (biomérieux, inc., marcy-i'étoile, france), which were maintained in liquid nitrogen until storage in a -80c freezer in each country. rna was extracted from all samples, and cdna was prepared as previously described [22, 55] . two broadly reactive consensus pcr assays targeting non-overlapping fragments of the orf1ab were used to detect both known and novel covs [56, 57] . amplified products of the expected size were cloned and sequenced as described in [22] . sequences were analyzed and edited using geneious prime 2019.1.3 [58] . a sample was considered positive when at least one pcr assay yielded a sequence that matched coronaviruses in genbank. coronavirus sequences were classified as belonging to a specific taxa following previously described methodologies [22] . bats were categorized as adults and juveniles based on size, and morphological and behavioral characteristics were observed at sample collection. the sex of the bats was also recorded. identification of some bat species can be challenging in the field. for this reason, field-identified species were confirmed by dna barcoding using the cytochrome b or cytochrome oxidase subunit 1 mitochondrial genes [59] . obtained sequences were compared against sequences in the genbank and barcode of life databases [60, 61] . when possible or necessary, sequences from both genes were used for species identification. a threshold of 97% nucleotide identity was used to confirm the species; sequences with 95-97% nucleotide identity were assigned a "conferre" (cf.) species status, and sequences below 95% nucleotide identity were either classified to the genus level or as unidentified. sequences with > 97% nucleotide identity to more than one species for either gene, were classified to the genus level unless they clearly clustered with sequences from other animals in the same geographic area. if barcoding results for all of the first ten bats tested per species were in agreement with the field identification, we assumed that the field identification for the remaining bats of that species in each country was correct. otherwise, all of the remaining samples were barcoded to ensure correct speciation. we recorded when sampled females were pregnant based on abdominal palpation, had attached pups (indicating recent parturition), and were lactating, as well as when juveniles were captured. therefore, we were able to track pregnancy, lactation, and recent birth pulses. moreover, we accessed the data in the pantheria [62] and amniote [63] databases, and we thoroughly reviewed the literature on the biology of the bats species we sampled for latitudes similar to our sampling locations. with the gathering of these information sources we established the timing of the birth pulses, lactation periods, and the weaning of pups for each species. for the details justifying the dates inferred for these three life history events for each species and the corresponding bibliographical references see additional file 1. once the timing of these events was confirmed or inferred, we were able to establish 2 seasons that occur at least once during the year across all observed bat species: i) when juveniles are being weaned and female-pup contact decreases ("recent weaning" [rw]) and ii) the rest of the year (hereafter "n-rw" for "not recent weaning"). we chose to evaluate risk of cov shedding for the first period because past longitudinal studies with microchiropterans in germany and china found higher cov-rna loads approximately 1 month after parturition [51, 52] . similarly, peaks were found 2 months after the formation of a maternity colony of myotis macropus [49] , which would match a post weaning period for that species. here, we defined the end of the rw period as 1 month after the last pups were weaned. we assumed that 1 month would provide a reasonable time window for the colony to "clear" the cov susceptibility status of this period and acquire the susceptibility corresponding to the season(s) when weaning does not occur (n-rw period), if differences actually exist. finally, once we determined these two seasons, we categorized each bat sample into one of them depending on the week of the year in which they were taken. because some species had more than one litter per year, there could be more than one rw period during the year. it is worth noting that we were able to define these periods for those species in our dataset that have synchronized reproduction, whose biology was properly described, and whose taxonomy is generally accepted. when we could not assign a reproductive period to specific bats, this season was imputed (see methods: statistical analysis). we characterized specific traits of each bat species studied based on previous scientific literature on pathogen dynamics in bats [51, [64] [65] [66] [67] [68] [69] [70] [71] : colony size (small, medium, or large if a typical colony contains one to dozens, hundreds to thousands, or thousands to millions of individuals, respectively); roost type ("closed" if the species has been reported to use caves, mines, roofs, or other confined spaces; or "open" if the bats have been typically reported roosting in the foliage of trees); the aggregation of bats in clusters while roosting (no, yes); and the number of litters per year of the species at equatorial latitudes. references are provided in additional file 2. we also considered data from pantheria and amniote [62, 63] . we could not include other species traits, such as the mating strategy (harem or another) and the segregation of females in maternity colonies, because available studies were incomplete or contradictory. we did not include factors, such as multi-species occupancy of the roost, because we did not observe all of the roosts. further, we did not assess postpartum estrus, as within the study area, it is only known to occur in some molossid bats [72] , of which we only sampled a small number. to statistically estimate the association between the rw and cov detection we used a bayesian inference to model the detection of covs as a bernoulli process of the form: where cov i , the detection of cov in rectal swabs (1 if detected, 0 otherwise) from the i th bat (sample), is assumed to follow a bernoulli process parameterized by p i , the probability of cov detection on the i th bat. this parameter was related to a set of candidate covariates as: with s j~n ormal (0, σ s ) and sp k~n ormal (0, σ sp ). here x and y are binary covariates representing the rw season and juvenile age category. we specifically included these two terms to separate the potential association of the season with cov detection from the seasonal presence of juveniles. because it was not feasible to allocate all species in the rw or n-rw seasons based on previous research, we assumed that these unknown reproductive seasons were "missing at random" and they were imputed as a function of the latitude at sampling, the day of the year of the sampling event, the number of litters per year of the corresponding species (one litter per year versus more than one litter per year), and the historical precipitation of the month at the sampling event location. the description of the imputation model is provided in additional file 3. the terms s j and sp k represent the sampling eventand the species-specific intercepts, respectively, because we assumed that bats sampled in the same event and bats belonging to the same species were not independent with respect to cov detection. no sampling event involved the same bat colony in successive rw and n-rw seasons, therefore, we assumed that cov detection was not temporally correlated within sampling locations. we constructed the model by adding other covariates one-at-a-time to this working model: the c 1...l categorical variables; and they remained in the model if they were judged to confound the relationship between cov detection and the reproductive seasons or between age and cov detection (i.e., their inclusion caused meaningful changes in the posterior probability distributions [ppds] of the specific reproductive season or age coefficients). finally, c 1...l categorical variables could be retained as well if they were marginally associated with cov detection (the corresponding coefficient ppd did not include zero). to assess the goodness-of-fit of the models, we evaluated the congruence between cov detection in the data and in the posterior predictive distributions yielded by the models by: i) ages and seasons, ii) age, iii) per season, iv) per age and season, and v) per sampling event. all models were constructed using "stan" v. 2.17.0 [73] which was run from "r" v. 3.6.0 [74] through the package "rstan" v. 2.17.3 [75] . weakly informative priors were assigned for all coefficients: normal(0, 1.5) for the estimates of α 0 , β's, γ, and ρ's. the σ s and σ sp had a prior half-cauchy (0,5) following previous suggestions [76, 77] . the ppds were estimated by sampling in parallel from 4 mcmc chains for 4000 iterations following 3000 iteration warm-up for a total of 4000 saved samples for each parameter ppd. convergence was assessed by the gelman-rubin statistic [78] and graphically using trace plots. the code to fit the models is available at https://github.com/ dmontecino/east-african-bats-and-cov-shedding. we sampled 753 individuals, all of them aged and successfully identified to belonging to 15 species. nycteris thebaica, pipistrellus hesperidus, and rhinolophus clivosus were assigned the "conferre" status. hipposiderids were assigned only to the genus level because barcoding did not provide certainty on species identification in line with previously recognized taxonomic difficulties [79] [80] [81] ; however, the biology of the candidate hipposiderid species is similar ( fig. 2 ; [82, 83] ), and we were able to used them for estimation purposes. we excluded scotophilus viridis (n = 6) from the analysis because their reproductive traits are unknown, and this species also has taxonomic difficulties for species identification [84] . therefore, these six individuals were removed, and 747 bats were included in the study. we were able to infer the reproductive season for all bats except for lissonycteris angolensis, rhinolophus cf. clivosus, and pipistrellus cf. hesperidus (n = 117, 104 adults and 13 juveniles) that had limited available knowledge on biology and reproductive season. these reproductive seasons were imputed as explained above. this imputation process did not substantively affect the proportion of bats in each reproductive period or the crude cov detection per age (additional file 3: figure s3 .2). moreover, we had partial data for neoromicia nana and triaenops persicus lactation period and we assigned one that is likely longer than it would be in reality based on the other species. even under this overextended period, none of these bats (n. nana and t. persicus) could have being sampled during the rw season, so this knowledge gap did not risk a misclassification (fig. 2) . in the end, 274 and 356 bats were allocated in the n-rw and rw periods, respectively. the distribution of bats across the reproductive periods per age and sampling event was heterogeneous as expected due to the opportunistically nature of sampling. indeed, 233 and 273 adult bats were in the n-rw and rw periods, respectively, while 41 and 83 juvenile bats were sampled in these seasons, respectively. in total, 30.79% bats were positive for covs (n = 230). within the subset of adult bats, 26.22% were positive (n = 160) while 51.09% of the juvenile bats were positive (n = 70). the detection of cov shedding was variable across seasons and bat species, as well as across sampling events (0, 100, 9.69, and 25.84% for the minimum, maximum, median, and mean detection, respectively). the covs found per species are shown in additional file 4: table s4 . a summary of the roosting and reproductive traits of the bat species sampled is provided in table 1 . all bats except n. nana (n = 9) and e. helvum (n = 315) roosted in "closed" structures, such as caves, abandoned mines, and roofs. within the group of bats using "open" structures, e. helvum was the only species with cov positive individuals. therefore, we did not use this covariate to assess a potential association with cov shedding. the models showed adequate sampling. the 4 markov chain monte carlo chains converged graphically, whilst all gelman-rubin statistics were < 1.004. the selected model had a number of effective samples for each coefficient of at least 1636. the data were properly fitted, as well (additional file 5: figure s5 .1), although some predictions lacked precision. the ppds of the fixed coefficients are shown in additional file 5: figure s5. 2. the selected model to assess periodic differences in cov shedding included season and age, species-specific intercepts, and sampling event-specific intercepts. beyond the species-specific terms, we included a binary categorical covariate equal to 1 for e. helvum and t. persicus and 0 otherwise. we incorporated this term because cov detection in these species was remarkably higher than the other species. as expected, this fixed effect was correlated with the corresponding species-specific intercepts (the remaining correlations were all low); however, we decided to keep it to assess if the main findings hold even when accounting for the bat species with highest detection. the corresponding means, standard deviations, and 90% hpdi are shown in table 2 . the coefficients' ppds from the selected model indicate an association between age and cov shedding, with juveniles presenting 1.26-2.94 times higher odds to shed compared to adult bats (90% hpdi). the coefficients' ppds also point to an association between the reproductive season and cov shedding as well, with an estimated odds 1.71-16.00 times higher to shed during the period when pups are being compared to other seasons (90% hpdi). the proportions of cov shedders estimated by reversing the 90% hpdi of the logits were: 0.02-0.22, 0.09-0.59, 0.01-0.13, and 0.05-0.42 for juveniles during the "n-rw" and "rw" periods, and adults during the "n-rw" and "rw" periods, respectively (90% hpdi). these values refer to bats not belonging to the species e. helvum or t. persicus. finally, the predicted cov detections, based on the posterior predictive distributions, were 0.01-0.18, 0.06-0.54, 0.00-0.05, and 0.04-0.36, for these same groups (90% hpdi; fig. 3, left) . in practical terms, these last values imply that juveniles during the "rw" period are, on average, 3.34 times more likely to be detected shedding covs compared to juveniles in the "n-rw" period. adults during the "rw" period are, on average, 3.93 times more likely to be detected shedding covs compared to adults in the "n-rw" period. in both seasons, juveniles are, on average, more likely to shed covs, than adults. the selected model suggests a higher odds of cov detection in e. helvum and t. persicus compared to other species. the species-specific intercept terms, once the e. helvum -t. persicus effect is included, suggest no further differences in terms of cov (fig. 3, right) ; however, the estimates are not precise. the sampling event-specific random intercepts suggest that a few specific locations could show differential cov shedding but that most of them do not explain further variation (additional file 6). if coronavirus shedding by bats follows temporal patterns that are generalizable across species and locations, then mitigation strategies targeting the prevention of human exposure and potential spillover could be directed toward high-risk periods, through mechanisms that can also support bat-human coexistence and the provision of bat ecosystem services. previous research has focused on viral identification in specific locations and in few species [47] [48] [49] [50] , resulting in a limited representation of viral dynamics in association with few ecological settings, biological traits, and reproductive strategies. additionally, few studies of coronavirus shedding patterns have employed statistical models and, in consequence, the potential complex web of factors and causal relationships that may determine this process has not been fully explored. here, we aimed to address these issues by statistically modeling coronavirus rna detection in several bat species, captured at different times and locations in east africa and involving different ecological contexts and life histories. using data from several hundreds of bats, we found that, beyond spatial, taxonomic, and life history differences; i) the odds of coronavirus shedding is higher during the period when pups are being weaned (up to a month after the lactation period is over), and ii) juvenile bats have higher odds to shed these viruses. moreover, the ratios of predicted detections per bat category (age and reproductive season) suggest that juvenile bats during the recent weaning period have relatively higher shedding compared to bats out of this period no matter their age. caution must be taken with these ratios because we used a logit link and our data had high proportion of cov shedding in specific groups. however, our results are consistent, and they are in agreement with previous research conducted in a restricted number of species and locations. similar seasonality of coronavirus shedding has been observed in germany, australia, thailand, china, and ghana (west africa). in the specific species involved in these previous studies, higher coronavirus shedding and viral loads were detected weeks after the birth pulse [47] [48] [49] [50] [51] [52] 85] . further, and consistent with our results, detection of higher levels of coronavirus in juveniles has been reported in micro-as well megabats from africa, asia, europe, and north and south america [22, 48, 50, [86] [87] [88] [89] . it has been proposed that the increased detection of coronaviruses after the birth pulse is attributable to the waning of passively-received maternal antibodies in juveniles [51] . this idea has been frequently cited; however, we are not aware of any longitudinal age-specific sd standard deviation and 80%, hpdi = 90% high posterior density interval coronavirus seroprevalence study in bats. such studies are important to understand the drivers of pathogen persistence and spillover risk, and in consequence, to ethically manage and prevent bat pathogen exposure. nevertheless, this kind of research is difficult to conduct due to logistical challenges, our questionable ability to obtain statisticallyrepresentative samples across age groups, cross-reactivity of serological assays, and the difficulties to differentiate serodynamics derived from closed-population processes from those caused by migratory movements. although extrapolations for antibody dynamics across viruses and species are not simple [90] , bat serodynamics for hendra virus are congruent with the increased detection of coronaviruses after the birth pulse. pups passively receive maternal hendra virus antibodies which decline after the first month of age up to 6 months of age [91] [92] [93] [94] . this decline would lead to a period in which young bats tend to be more susceptible to infection, become infected, and then shed virus. consistently, coronavirus shedding peaked weeks after the birth pulse in a german and a chinese species [51, 52] and immunologically naïve bats shed higher coronavirus loads [30] . over time, as young bats clear hendra virus infection, they become seropositive again [91] [92] [93] [94] . concordantly, capture-mark-recapture studies support the clearance of coronaviruses in infected bats [47, 95] , which would become seropositive. however, young bats may not reach adult seroprevalence levels until they are older than a year, as occurs with hendra virus [91, 92, 94] . therefore, the population of juvenile bats would remain comparatively more susceptible to viral infection and shedding beyond the period immediately after weaning. age-specific henipavirus seroprevalence in african e. helvum is in agreement with the serodynamics described for hendra virus [90] . additionally, coronavirus transmission may be favored by high colony density created by the birth pulse, as previously proposed [85, 95] , and then the seasonal influx of susceptible juveniles could accelerate viral spread across the entire colony, including adult bats. indeed, adult myotis macropus in an australian colony showed a peak of coronavirus detection after the birth pulse [85] . the peak of coronavirus detection for two hipposideros species and nycteris cf. gambiensis sampled in ghana occurred during the months that encompassed the birth pulse and nursing after accounting for the age of the sampled individuals [50] . higher coronavirus infection has also been reported in lactating females [85, 88] , which overlap with the period of pup weaning and decay of maternally-derived immunity; however, the opposite has also been found [48, 50, 85] . in practical terms, public health managers could anticipate high risk periods for coronavirus shedding to target interventions. assuming that higher spillover risk is a function of higher viral shedding [67] and that all coronaviruses with zoonotic potential behave ecologically similarly to coronaviruses detected in this study, managers could target the prevention of human-bat direct (consumption) or indirect (bat droppings) contact specifically during the high-risk season: around and just after weaning, the timing of observable juveniles or individuals smaller than adults. for the species and interfaces defined herein, those management periods have now been determined (fig. 2) . for others, direct observation of bats at high-risk transmission interfaces could be used to identify time periods when non-adult sized bats are present. however, observation of dependent pups is not always easy [96] . of course, for specific species, birth pulses and lactation seasons could also be used to more precisely establish high risk periods similar to the methods we used here, including a combination of direct observation, reports from previous literature, and consultation with knowledgeable bat biologists. our proposed risk-driven strategy i) is evidence-based, as it builds upon coronavirus shedding patterns observed across several chiropteran species present around the world; ii) does not require the advanced laboratory capacity often lacking in resource-restricted settings where intense bat-human interfaces usually occur; iii) is a good alternative to the ideal but expensive and resourceintensive longitudinal surveys; and iv) it may prevent the exposure to viruses belonging to other taxa whose observed bat shedding dynamics resemble our findings for coronaviruses (e.g. paramyxoviruses [97] ), the lower coronavirus detections in african emballonurids (c. afra and t. mauritianus) and the higher detections in e. helvum, african hipposiderids (hipposideros sp. and t. persicus), and r. cf. clivosus that we found are consistent with previous reports [22, 44, 50, 98] and should be considered by managers when providing risk-based spillover prevention strategies. moreover, sars-like coronaviruses in africa have been found in hipposiderid, rhinolophid, and molossid bats [22, 57, 98] , and mers-related coronaviruses have been found in vespertillionid bats [22, 28, 29] . therefore, it seems reasonable to prioritize the identification of birth pulses and lactation seasons, and thus determine high-risk periods of coronavirus shedding, for these bat families. interestingly, e. helvum roost in tight clusters that can contain hundreds of individuals [99] , similar to t. persicus. on the other hand, emballonurids, showing the lowest crude detection levels across families (represented by coleura afra and taphozous mauritanus here) tend not to cluster while roosting [100, 101] . we did find an association between coronavirus shedding and whether the species typically aggregate in clusters while roosting when the variable "e. helvum -t. persicus" was not considered, but we chose a different model not including this term because we did not directly assess bat roosts and our categorization may oversimplify the continuum from mostly solitary roosting (e.g., neromocia nana) to common tight aggregations of bats (e.g., mops condylurus). using this categorization could be misleading, as some species differentially cluster while roosting depending on temperature, colony size, colony type (e.g., maternity colony versus not a maternity colony), and season [72] . future studies should consider the roosting habits of bats, as this trait could further support riskbased management to prevent or reduce human exposure. the risk-driven strategy we propose provides a contactreduction alternative that is ethically favorable compared to often-employed measures, such as culling or other reactive measures, that ensue when the public becomes aware of a health threat without a suggested practical option to reduce their risk for exposure. in addition to ethical concerns and being logistically difficult and expensive, culling has failed to reduce disease in wild populations and can result in even higher pathogen levels. for example, "badger culling can make no meaningful contribution to cattle tuberculosis control in britain" [102] . a culling program to reduce echinococcus multilocularis prevalence in red foxes (vulpes vulpes) resulted in an increase of infection [103] . this strategy has also failed to control rabies in canids around the globe [104] . similar results have been observed in bats. culling failed to reduce rabies seroprevalence in desmodus rotundus in perú and could have increased the levels of exposure to the virus [105, 106] . in argentina, the extermination of bats changed the direction of spread of rabies in livestock but did not prevent its advancement [107] . in uganda, miners exterminated a colony of rousettus aegyptiacus bats after an outbreak of marburg virus in 2007 that involved 4 miners in close contact with these bats. five years later, a new outbreak occurred in miners from the same mine. the second time, marburg virus rna was detected in a higher proportion in the r. aegyptiacus that recolonized the mine (13.3%, n = 400; [108] ) compared to rna detection before culling in this cave (5.1%, n = 611; [109] ) and other caves in uganda (2.5%, n = 1622; [67] ) and gabon (4.8%, n = 187; [110] ) where culling has never been reported. culling can also cause demographic changes, leading to a higher proportion of juvenile individuals. this change may occur because of a disproportionate cull of older individuals; the potential increase in survival of pups at lower population densities, followed by higher recruitment of juvenile females into reproductive age [111] ; the hypothetical increase of young dispersers immigrating from neighboring colonies into culled, less dense, and better resourced colonies [105, 112] ; or by causing compensatory reproduction [113] . this last possibility may have not been studied in bats but seems unlikely due to their high conception rates and usual litter size of one. examples of younger populations after culling have been reported in the red deer (cervus elaphus), racoon (procyon lotor), american mink (mustela vison), and australian brushtail possums (trichosurus vulpecula), among others [114] [115] [116] [117] . as our results and past research consistently show higher viral shedding and detection in young individuals, activities leading to a younger bat population are not advisable for viral spillover management. similar results are expected when fruit bats are culled based on being categorized as "agricultural pests"; therefore, this kind of management may create higher risk of viral exposure to the human population. virological, ecological, and epidemiological research on bats over the last 15 years has helped to identify chiropterans as hosts of zoonotic viruses and to document that human-driven environmental change, human behavior, and human-to-human transmission are the key drivers for the creation of bat-human interfaces, spillover, and epidemics of emergent viruses, respectively [118] [119] [120] . in the context of the current biodiversity and bat conservation crisis [121, 122] , we must not omit these facts when attempting to effectively, and responsibly frame and communicate disease risks associated with bats. realistic, data-based risk communication is of paramount importance to avoid framing bats as a threat to humans and to support bat conservation given their important ecological roles [123, 124] . with this background, it seems a proper time for the scientific community studying "bat-associated" viruses to move the conversation from bat spillover risk assessments to the planning of pro-biodiversity and subsequently pro-ecosystem strategies aiming to mitigate spillover risk. science is valued not only for the diagnosis of problems but because it finds solutions to them. here, we have attempted to aid the progress of scientific and management dialogue by proposing, not only a management strategy to limit potential coronavirus spillover, but one that is context-and logistically-grounded and pro-conservation, promoting the delivery of the key ecosystem services provided by bats. data from hundreds of bats collected in east-africa show that coronavirus shedding is expected to be more frequent when pups are becoming independent from the dam, independently of the age of the bats, their species, their location, and their life histories; however, the odds of shedding do differ by species. these results can guide temporal-based mitigation strategies to prevent bat-associated coronavirus exposure using non-lethal methods in limited-resource settings, where longitudinal surveillance is not feasible, by identifying high-risk periods for coronavirus shedding when contact with bats should be avoided. supplementary information accompanies this paper at https://doi.org/10. 1186/s42522-019-0008-8. additional file 1: summary of the inferred start date of the birth pulse, the end of the lactation period, and the start date of the mating period per microchiropteran species. additional file 2: bibliographic references for the traits of the bat species included in the study. additional file 3: imputation of the reproductive season to those bats whose biology is insufficiently known. model methods, results, and literature cited. table s3 . summary of the coefficients' posterior probability distributions of the selected model for the imputation of the reproductive seasons of bats missing this data. figure s3 carlo iteration. right: the distribution of the crude coronavirus detection per reproductive season across the 5,000 markov chain monte carlo sampling iterations after imputing the periods when un-inferred. the black boxplots show the distribution of the coronavirus detection per period, while the light and dark colored boxes above and below show the interquartile detection in non-adults and adult bats, respectively, per period. additional file 4: table s4 . summary of the alpha-and betacoronaviruses (alphacov and betacov, respectively) found in the microbats tested. additional file 5: results of the model to assess the association between cov shedding and the "recent weaning" season in eidolon helvum and microbats. figure s5 authors' contributions dml = designed the project; collected, analyzed, and interpreted data; and drafted, edited, and prepared the final manuscript and figures. tg = guided laboratory analysis and generated data, especially barcoding and sample testing, and supported manuscript writing. kg, dw, evw, rk = organized data collection and supported manuscript writing. bs, jn, zs, mc = data collection and supported manuscript writing. the predict consortium = provided support in the design and implementation of surveillance and interpretation of data. jkm = designed and supervised the project, including sampling design and data collection; supported data analysis and interpretation; and drafted, edited, and prepared the final manuscript. all authors read and approved the final manuscript. this study was supported by the generous support of the american people through the united states agency for international development (cooperative agreement numbers ghn-a-oo-09-00010-00 and aid-oaa-a − 14 -00102). the contents of this paper are the responsibility of the authors and do not necessarily reflect the views of the us agency for 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vampire bats control of bovine rabies through vampire bat control marburgvirus resurgence in kitaka mine bat population after extermination attempts isolation of genetically diverse marburg viruses from egyptian fruit bats is marburg virus enzootic in gabon? impact of human disturbance, density, and environmental conditions on the survival probabilities of pipistrelle bat (pipistrellus pipistrellus) bats and emerging zoonoses: henipaviruses and sars compensatory reproduction in feral horses consequences of harvesting on age structure, sex ratio and population dynamics of red deer cervus elaphus in central norway effects of culling on mesopredator population dynamics demography of three populations of american mink mustela vison in europe effects of intensive trapping on breeding and age structure of brushtail possums, trichosurus vulpecula the consequences of human actions on risks for infectious diseases: a review factors in the emergence of infectious diseases human ecology in pathogenic landscapes: two hypotheses on how land use change drives viral emergence accelerated modern human-induced species losses: entering the sixth mass extinction bats in the anthropocene the role of one health in wildlife conservation: a challenge and opportunity the role of protected areas in supporting human health: a call to broaden the assessment of conservation outcomes publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we appreciate the support of the american people through the united states agency for international development (usaid) emerging pandemic threats predict project (cooperative agreement number ghn-a-oo-09-00010-00). we thank the governments of rwanda, tanzania, and uganda for permission to conduct this study, and the country predict teams that led and conducted the field activities. we are mostly grateful of all the technicians involved in the processing, testing, and barcoding of the samples, especially ruth maganga (sokoine university of agriculture) and brett smith (one health institute, university of california -davis). finally, we appreciate the help of dr. evan eskew for supporting with stan coding and dr. wes johnson for guidance with bayesian inference. key: cord-258160-v08cs51n authors: wang, lin-fa; shi, zhengli; zhang, shuyi; field, hume; daszak, peter; eaton, bryan t. title: review of bats and sars date: 2006-12-17 journal: emerg infect dis doi: 10.3201/eid1212.060401 sha: doc_id: 258160 cord_uid: v08cs51n bats have been identified as a natural reservoir for an increasing number of emerging zoonotic viruses, including henipaviruses and variants of rabies viruses. recently, we and another group independently identified several horseshoe bat species (genus rhinolophus) as the reservoir host for a large number of viruses that have a close genetic relationship with the coronavirus associated with severe acute respiratory syndrome (sars). our current research focused on the identification of the reservoir species for the progenitor virus of the sars coronaviruses responsible for outbreaks during 2002–2003 and 2003–2004. in addition to sars-like coronaviruses, many other novel bat coronaviruses, which belong to groups 1 and 2 of the 3 existing coronavirus groups, have been detected by pcr. the discovery of bat sars-like coronaviruses and the great genetic diversity of coronaviruses in bats have shed new light on the origin and transmission of sars coronaviruses. bats have been identified as a natural reservoir for an increasing number of emerging zoonotic viruses, including henipaviruses and variants of rabies viruses. recently, we and another group independently identified several horseshoe bat species (genus rhinolophus) as the reservoir host for a large number of viruses that have a close genetic relationship with the coronavirus associated with severe acute respiratory syndrome (sars). our current research focused on the identification of the reservoir species for the progenitor virus of the sars coronaviruses responsible for outbreaks during 2002-2003 and 2003-2004 . in addition to sars-like coronaviruses, many other novel bat coronaviruses, which belong to groups 1 and 2 of the 3 existing coronavirus groups, have been detected by pcr. the discovery of bat sars-like coronaviruses and the great genetic diversity of coronaviruses in bats have shed new light on the origin and transmission of sars coronaviruses. s evere acute respiratory syndrome (sars) represents the 21st century's first pandemic of a transmissible disease with a previously unknown cause. the pandemic started in november 2002 and was brought under control in july 2003, after it had spread to 33 countries on 5 continents, resulting in >8,000 infections and >700 deaths (1) . the outbreaks were caused by a newly emerged coronavirus, now known as the sars coronavirus (sars-cov). in (3) . these findings indicate independent species-crossing events. they also indicate that a sars epidemic may recur in the future and that sars-like coronaviruses (sarslike-covs) that originate from different reservoir host populations may lead to epidemics at different times or in different regions, depending on the distribution of the reservoirs and transmitting hosts. the recent discovery of a group of diverse sars-like-covs in bats supports the possibility of these events and further highlights the need to understand reservoir distribution and transmission to prevent future outbreaks. because of the sudden and unpredictable nature of the sars outbreaks that started in november 2002 in southern people's republic of china, structured and reliable epidemiologic studies to conclusively trace the origin of sars-cov were not conducted. however, accumulated studies from different groups, which used a variety of approaches, indicated an animal origin on the basis of the following findings. 1) genome sequencing indicated that sars-cov is a new virus with no genetic relatedness to any known human coronaviruses (4, 5) . 2) retrospective serologic studies found no evidence of seroprevalence to sars-cov or related viruses in the human population (6) . 3) serologic surveys among market traders during the 2002-2003 outbreaks showed that antibodies against sars-cov or related viruses were present at a higher ratio in animal traders than control populations (7-9). 4) epidemiologic studies indicated that early case-patients were more likely than later case-patients to report living near a produce market but not near a farm, and almost half of them were food handlers with probable animal contact (7). 5) sars-covs isolated from animals in markets were almost identical to human isolates (9). 6) molecular epidemiologic analyses indicated that human sars-cov isolates could be divided into 3 groups from the early, middle, and late phases of the outbreaks and that early-phase isolates were more closely related to the animal isolates (10). 7) human sars-covs isolates from the 2003-2004 outbreaks had higher sequence identity to animal isolates of the same period than to human isolates from the 2002-2003 outbreaks (3). the first evidence of sars-cov infection in animals came from a study conducted in a live animal market in early 2003 (9) . from the 25 animals sampled, viruses closely related to sars-cov were detected in 3 masked palm civets (paguma larvata) and 1 raccoon dog (nyctereutes procyonoides). in addition, neutralizing antibodies against sars-cov were detected in 2 chinese ferret badgers (melogale moschata). this initial study indicated that at least 3 different animal species in the shenzhen market were infected by coronaviruses that are closely related to sars-cov. given the vast number of live animals being traded in animal markets in southern people's republic of china, knowing which other animals are also susceptible to these viruses is crucial. unfortunately, for a variety of reasons no systematic studies were conducted on traded animals during the outbreak period. experimental infection of different animals therefore became a component of the sars-cov investigation. currently, >10 mammalian species have been proven to be susceptible to infection by sars-cov or related viruses (table 1) . rats were also implicated as potentially susceptible animals that may have played a role in the transmission and spread of sars-cov in the well-publicized sars outbreaks in the amoy gardens apartment block in hong kong special administrative region, people's republic of china (23) . in guangdong in 2004, the first human with a confirmed case of sars was reported to have had no contact with any animals except rats (2) . experimentally, we have obtained serologic evidence that sars-cov replicates asymptomatically in rats (b.t. eaton et al., unpub. data). further studies are needed to clarify the potential role of rats in the transmission of sars-cov. studies by 2 independent groups suggested that avian species were not susceptible to sars-cov infection and that, hence, domestic poultry were unlikely to be the reservoir or associated with the dissemination of sars-cov in the animal markets of southern people's republic of china (22, 24) . although in 1 live animal market, 3 species were found to be infected by viruses related to sars-cov (9), all subsequent studies have focused mainly on palm civets, possibly because the rate of detection was higher in civets or because the number of civets traded in southern people's republic of china exceeds that of other wildlife groups. the isolation of closely related sars-cov in civets during the 2002-2003 and 2003-2004 outbreaks and the close match of virus sequences between the human and civet isolates from each outbreak (3, 9, 25) strongly suggest that civets are a direct source of human infection. however, these studies did not clarify whether animals other than civets were involved in transmission of sars-cov to humans or whether civets were an intermediate host or the natural reservoir host of sars-covs. during the 2002-2003 outbreaks, none of the animal traders surveyed in the markets, who supposedly had very close contact with live civets, displayed sars symptoms (7) (8) (9) . during the 2003-2004 outbreaks, at least 1 human sars patient had had no contact with civets (2). these observations seem to indicate that ≥1 other animal species may play a role in transmission of sars-cov to humans. most, if not all, civets traded in the markets are not truly wildlife animals; rather, they are farmed animals. civet farming is relatively new in people's republic of china and has rapidly expanded during the past 15 years or so. tu although not universally true, natural reservoir hosts tend to have coevolved with their viruses and usually do not display clinical signs of infection (29) . however, when palm civets were experimentally infected with 2 strains of human sars-cov, all developed clinical signs of fever, lethargy, and loss of aggressiveness (11) . civits' high susceptibility to sars-cov infection and wide presence in markets and restaurants strongly indicates an important role for civets in the 2002-2003 and 2003-2004 sars outbreaks. however, the lack of widespread infection in wild or farmed palm civets makes them unlikely to have been the natural reservoir host. the presence of sars-like-covs in different species of horseshoe bats in the genus rhinolophus has recently been reported. we found, in a study of horseshoe bat species in different regions of mainland people's republic of china in 2004 (30) , that each of the 4 species surveyed had evidence of infection by a sars-like-cov: 2 species (r. pearsoni and r. macrotis) had positive results by both serologic and pcr tests, and 2 (r. pussilus and r. ferrumequinum) had positive results by either serologic or pcr tests, respectively. bats with positive results were detected in the provinces of hubei and guangxi, which are >1,000 km apart. a group in hong kong (31) found that, when analyzed by pcr, 23 (39%) of 59 anal swabs of wild chinese horseshoe bats (r. sinicus) contained genetic material closely related to sars-cov. they also found that as many as 84% of the horseshoe bats examined contained antibodies to a recombinant n protein of sars-cov. a previous study indicated a certain level of antigenic cross-reactivity between sars-cov and some group 1 coronaviruses (6) and that several group 1 coronaviruses had recently been found in bats. therefore, the actual seropositive proportion of r. sinicus might be <84%. nevertheless, the relatively high seroprevalence and wide distribution of seropositive bats is consistent with the serologic pattern expected from a pathogen's natural reservoir host (29) . genome sequencing showed that the genome organization of all bat sars-like-covs is almost identical to that of the sars-covs isolated from humans or civets. they shared an overall sequence identity of 88% to 92%. the most variable regions were located in the 5′ end of the s gene, which codes for the s1 domain responsible for receptor binding, and in open reading frame 10 (orf10 or orf8, depending on the nomenclature used) region immediately upstream from the n gene ( figure, panel a, region b) , which is known to be also prone to deletions of various sizes (3, 9) . most human sars-covs isolated during the late phase of the 2002-2003 outbreaks have a 29-nt deletion in this region; this deletion is absent in the civet isolates or human isolates from the early phase of the outbreaks (3, 9) . the bat viruses also lack the 29-nt deletion, indicating that sars-covs and sars-like-covs share a common ancestor. furthermore, sequence analyses indicated the existence of a much greater genetic diversity of sars-like-covs in bats than of sars-covs in civets or humans, which supports the notion that sars-cov is a member of this novel coronavirus group and that bats are a natural reservoir for it. the overall genome sequence identities between the human/civit sars-covs and the bat viruses rp3 (isolated from r. pearsoni) and hku3-1 (isolated from r. sinicus) were 92% and 88%, respectively. the sequence identity between the bat isolates rp3 and hku3-1 is 89%, which indicates that the genetic divergence among the bat isolates is as great as the divergence between each of the bat viruses and the human/civet isolates. in addition, phylogenetic trees based on different protein sequences display different tree topologies, as shown in the figure (panel b) , which indicates the existence of multiple evolutionary pathways for different regions of the genome. the discovery of sars-cov has boosted the search for novel coronaviruses of human and animal origin. bats synopsis have been chosen as the main target because of their species diversity, large population size, broad geographic distribution, ability for long-distance migration, and habit of roosting in large groups. in addition to the sarslike-covs described above, many other coronaviruses have been detected by pcr among diverse bat populations in hong kong ( (32) sampled 309 individual bats representing 13 species from 20 different locations in rural hong kong. they detected coronavirus-related viral genomic rna in 37 bats, representing a prevalence of 12%. partial sequencing of rdrp and helicase genes identified 8 coronavirus genome types, 2 of which were the same as those reported previously (28, 30, 31) . the other 6 novel types of coronaviruses were obtained from 6 different bat species and phylogenetically positioned in 2 of the existing 3 coronavirus groups. four were in group 1, derived from bat species m. magnater, m. pusillus, myotis ricketti, and r. sinicus; the other 2 were in group 2, from bat species pipistrellus abramus and tylonycteris pachypus. to accommodate the newly discovered genetic diversity of group 2 coronaviruses, the authors proposed the following subdivisions: group 2a (coronaviruses existing before the discovery of sars-cov), group 2b (sars-cov and sars-like-covs), and group 2c (novel bat coronaviruses discovered in this study). attempts to isolate virus in veroe6, mrc-5, llc-mk2, frhk-4, huh-7.5, and hrt-18 were unsuccessful. in were clustered with known bat sars-like-covs (or group 2b), 40 belonged to group 1, and the remaining 22 formed a separate cluster in group 2, most likely clustering with the group 2c viruses reported by woo et al. (32) . attempts to isolate virus in veroe6, frhk4, and cv1 were unsuccessful. in addition to the diversity of coronaviruses in bats, 3 more observations can be drawn from these studies. first, none of the bat coronaviruses discovered so far belonged to group 3. second, with very few exceptions, most bat coronaviruses seem to be species-specific; i.e., different bat species from a similar location harbor different coronaviruses, whereas the same bat species from different geographic locations carry coronaviruses of the same genetic lineage (32, 33) . third, among the 5 published studies involving bat coronaviruses (28, (30) (31) (32) (33) , no researchers were able to isolate live virus from any of the swab samples collected despite the use of many different cell lines and the presence of high levels of viral genetic materials shown by quantitative pcr. emergence of zoonotic viruses from a wildlife reservoir requires 4 events: 1) interspecies contact, 2) cross-species virus transmission (i.e., spillover), 3) sustained transmission, and 4) virus adaptation within the spillover species (34) . these 4 transition events occurred during the sars outbreaks and contributed to the rapid spread of the disease around the world. the role of civets in directly transmitting sars-cov to humans has been well established. the most convincing case was the infection of a waitress and a customer in a restaurant where sars-cov-positive civets were housed in cages (25) . two key questions remain: what is the natural reservoir host for the outbreak sars-cov strains, and how were the viruses transmitted to civets or other intermediate hosts? although not conclusive, the data obtained so far strongly suggest that bats (horseshoe bats in particular) are most likely the reservoir host of sars-cov. as indicated above, bat coronaviruses seem to be species-specific and sars-like-covs discovered so far are exclusively associated with horseshoe bats. we hope that continued field study will eventually identify the direct progenitor of sars-cov among the 69 different known horseshoe species. the facts that the cross-species transmission of sars-cov seems to be a relatively rare event and that legal and illegal trading of wildlife animals between people's republic of china and other countries occurs raise the possibility that the natural reservoir species may not be native to people's republic of china. thus, we should expand our search into regions other than hong kong and mainland people's republic of china. another approach to search for the natural reservoir of sars-cov is to conduct infection experiments in different bat species. if we assume that the progenitor viruses come from bats, chances are high that the human/civet sars-covs are still capable of infecting the original reservoir species. without knowing the natural reservoir of sars-cov, predicting the exact mechanism of transmission from reservoir host to intermediate host is difficult. however, the fecal-oral route represents the main mode of transmission among animals. although mixing of live reservoir hosts (e.g., bats) and intermediate hosts (e.g., civets) would be an efficient means of transmission, the main source of cross-species transmission in the animal trading chain (including warehouses, transportation vehicles, markets) may come from contaminated feces, urine, blood, or aerosols. this may also be true for civet-to-human transmission. as shown in the case of the infected restaurant customer in 2004, the customer had no direct contact with civets and was sitting at a table ≈5 m from the civet cages (25) . although at this stage we cannot rule out the possibility of direct transmission from the natural reservoir host to humans, molecular epidemiologic studies (2,10) and studies of the receptor-s protein interaction (35) indicate that the progenitor viruses are unlikely to be able to infect humans and that a rapid viral evolution in an intermediate host (such as civets) seems to be necessary to adapt the virus for human infection. ability to efficiently use the receptor molecules (ace2 for human and civet) seems to be a major limiting factor for animal-to-human and human-to-human transmission (35) . this also explains why the sars-cov was able to cause the human pandemic but the closely related bat sars-like-covs were not. for the sars-like-covs to infect humans, substantial genetic changes in the s1 receptor-binding domain will be necessary. these changes may be achieved in 1 of 2 possible ways. they could be achieved by genetic recombination, as coronaviruses are known to be able to recombine. for example, bat sars-like-covs and another yet unknown coronavirus could coinfect an intermediate host, and the bat viruses would gain the ace2 binding site in the s1 domain by recombination. the alternative is continuous evolution independent of recombination. coronaviruses in bats could have a spectrum sufficiently diverse to encompass the progenitor virus for sars-covs. the progenitor virus's ability to bind human ace2 may be acquired or improved by adaptation (i.e., point mutations) in >1 intermediate host before it could efficiently infect humans. the existence of at least 3 discontinuous highly variable genomic regions between sars-cov and sars-like-cov indicates that the second mechanism is more likely. in conclusion, the discovery of bat sars-like-covs and the great genetic diversity of coronaviruses in bats have shed new light on the origin and transmission of sars-cov. although the exact natural reservoir host for the progenitor virus of sars-cov is still unknown, we believe that a continued search in different bat populations in people's republic of china and neighboring countries, combined with experimental infection of different bat species with sars-cov, will eventually identify the native reservoir species. a positive outcome of these investigations will greatly enhance our understanding of spillover mechanisms, which will in turn facilitate development and implementation of effective prevention strategies. the discovery of sars-like-covs in bats highlights the increasingly recognized importance of bats as reservoirs of emerging viruses (36) . moreover, the recent emergence of sars-covs and other bat-associated viruses such as henipaviruses (37, 38) , menangle, and tioman viruses (36) , and variants of rabies viruses and bat lyssaviruses (38, 39) also supports the contention that viruses, especially rna viruses, possess more risk than other pathogens for disease emergence in human and domestic mammals because of their higher mutation rates (40) . severe acute respiratory syndrome laboratory diagnosis of four recent sporadic cases of communityacquired sars cross-host evolution of severe acute respiratory syndrome coronavirus in palm civet and human characterization of a novel coronavirus associated with severe acute respiratory syndrome the genome sequence of the sars-associated coronavirus a novel 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coronavirus infection of golden syrian hamsters pathology of guinea pigs experimentally infected with a novel reovirus and coronavirus isolated from sars patients aged balb/c mice as a model for increased severity of severe acute respiratory syndrome in elderly humans prior infection and passive transfer of neutralizing antibody prevent replication of severe acute respiratory syndrome coronavirus in the respiratory tract of mice sars-associated coronavirus transmitted from human to pig susceptibility of pigs and chickens to sars coronavirus possible role of an animal vector in the sars outbreak at amoy gardens domestic poultry and sars coronavirus, southern china sars-cov infection in a restaurant from palm civet antibodies to sars coronavirus in civets molecular evolution analysis and geographic investigation of severe acute respiratory syndrome coronavirus-like virus in palm civets at an animal market and on farms identification of a novel coronavirus in bats the ecology of wildlife diseases bats are natural reservoirs of sars-like coronaviruses severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats molecular diversity of coronaviruses in bats prevalence and genetic diversity of coronaviruses in bats from china zoonotic viruses of wildlife: hither from yon animal origins of the severe acute respiratory syndrome coronavirus: insight from ace2-s-protein interactions bats: important reservoir hosts of emerging viruses emerging henipaviruses and flying foxes-conservation and management perspectives emerging encephalitogenic viruses: lyssaviruses and henipaviruses transmitted by frugivorous bats european bat lyssaviruses: distribution, prevalence and implications for conservation diseases of humans and their domestic mammals: pathogen characteristics, host range and the risk of emergence the work conducted by our multination collaborative team was jointly funded by a special key: cord-277039-yo5ojr0s authors: mendenhall, ian h.; kerimbayev, aslan a.; strochkov, vitaliy m.; sultankulova, kulyaisan t.; kopeyev, syrym k.; su, yvonne c.f.; smith, gavin j.d.; orynbayev, mukhit b. title: discovery and characterization of novel bat coronavirus lineages from kazakhstan date: 2019-04-17 journal: viruses doi: 10.3390/v11040356 sha: doc_id: 277039 cord_uid: yo5ojr0s coronaviruses are positive-stranded rna viruses that infect a variety of hosts, resulting in a range of symptoms from gastrointestinal illness to respiratory distress. bats are reservoirs for a high diversity of coronaviruses, and focused surveillance detected several strains genetically similar to mers-coronavirus, sars-coronavirus, and the human coronaviruses 229e and nl63. the bat fauna of central asia, which link china to eastern europe, are relatively less studied than other regions of the world. kazakhstan is the world’s ninth largest country; however, little is understood about the prevalence and diversity of bat-borne viruses. in this study, bat guano was collected from bat caves in three different sites of southern kazakhstan that tested positive for coronaviruses. our phylogenetic reconstruction indicates these are novel bat coronaviruses that belong to the genus alphacoronavirus. in addition, two distinct lineages of kazakhstan bat coronaviruses were detected. both lineages are closely related to bat coronaviruses from china, france, spain, and south africa, suggesting that co-circulation of coronaviruses is common in multiple bat species with overlapping geographical distributions. our study highlights the need for collaborative efforts in understudied countries to increase integrated surveillance capabilities toward better monitoring and detection of infectious diseases. bats are mammals in the order chiroptera that possess a range of unique ecological, immunological, and behavioral attributes. bats are exceptionally speciose, comprising 20% of all mammalian species, and they are the only mammals that are capable of true flight [1] . most bat species are gregarious and roost in large colonies, which can number over one million individuals [2] . they are relatively long-lived for their body size, and temperate species often undergo torpor or hibernation [3] . bats also act as rich reservoirs of virus diversity with at least 23 families of viruses detected, including double-stranded dna viruses, single-stranded dna viruses, and positive-and negative-sense single-stranded rna viruses [4] . bats are incriminated as the source of several medically important virus families, including filoviruses, coronaviruses, paramyxoviruses, and reoviruses [5, 6] . several recent zoonotic spillover events and outbreaks directly or indirectly originated from bats [1] . coronaviruses are positive-sense rna viruses in the order nidovirales and the family coronaviridae. these viruses have the largest genome of any single-stranded rna viruses that infect vertebrates, and they are capable of recombining to create new strains. they infect a wide variety of mammals and birds, including infectious bronchitis virus in birds and transmissible gastroenteritis virus in pigs [7] . in humans, seasonal coronaviruses can cause both upper and lower respiratory tract infections, with increased disease severity in the elderly, children, and immunocompromised patients [8] . the zoonotic sars-coronavirus (sars-cov) outbreak originated in southern china from horseshoe bats, where wet markets permitted atypical contact between species, including subsequent spillover to humans [9] . recent work showed that all genetic components of sars-cov co-circulate among different bat species sharing the same cave, underlying the opportunity for its re-emergence [10] . on the other hand, camels are the putative natural reservoir for mers-coronavirus, although recent phylogenetic analysis indicated that bats harbor coronaviruses that are ancestral to the mers-cov lineage [11] . more recently, an hku2-cov outbreak caused by transmission from bats to pigs in china killed nearly 25,000 piglets [12] . central asia is one of the largest grassland and steppe habitats in the world, although little is known about its resident bat fauna. this habitat type is primarily located in russia, mongolia, and kazakhstan. kazakhstan is the largest land-locked country in the world; it is relatively arid (<300mm rainfall), and comprises plains and hills, with forested areas primarily restricted to the mountains in the south (tien shan) and the east (alatul and altai). described to date, there are 27 species of bats in kazakhstan, with 15 of these resident in turkestan oblast, and the most common species are vespertilio murinus (linnaeus, 1758) and myotis mystacinus (kuhl, 1817) [13, 14] . while there is substantive bat research in russia and mongolia, there is little work on the kazakhstan bats and less on associated virus communities [15, 16] . in this study, we collected fresh bat guano from three caves at different locations in kazakhstan and conducted molecular screening for coronaviruses. our objective was to explore and understand the diversity of bat coronaviruses in of kazakhstan. here, we identified and sequenced novel bat coronaviruses and determined the evolutionary relatedness of the viruses. to the best of our knowledge, this study represents the first detection of bat coronaviruses from kazakhstan. bat guano was collected from three sites in turkestan oblast from 11 april to 16 may 2017. these sites were the kepterkhan tunnel and qaraungir cave in tulkibas rayon district, with additional guano collected in the ungirli cave in altyntau, sozak rayon ( figure 1 ). bat feces were collected from plastic sheets placed underneath bat roosts. multiple fecal pellets were placed into cryovials with viral transport media using polyester swabs, which were subsequently placed into a liquid-nitrogen dry shipper and then transferred back to the research institute for biological safety problems (ribsp) in gvardeiskiy, kazakhstan, where all samples were stored at −80 • c. bat guano was vortexed for 30 s and rna was extracted using a qiaamp viral rna mini kit (qiagen, hilden, germany), performed following the manufacturer's instructions. coronavirus nucleic acid was amplified using pan-coronavirus primers that amplified a 440-bp region of the rna-dependent reverse polymerase (rdrp) (pancor in-6: ggttgggactatcctaagtgtga and pancor in-7: ccatcatcagatagaatcatcata). a pcr was run using a superscript iii one-step rt-pcr system with platinum taq dna polymerase (invitrogen, carlsbad, ca, usa) in a rotorgene thermocycler (qiagen, hilden, germany). the reaction consisted of 2 µl of rna, 12.5 µl of 2× reaction buffer, 1 µl of reverse transcriptase, 1 µm of forward and reverse primer, and water to a total of 25 µl. the thermocycler protocol followed a previously described protocol with the reverse transcription (rt) step held at 56 • c for 20 min, followed by a denaturation step at 94 • c for 2 min, followed by 45 cycles of 94 • c for 15 s, 56 • c for 30 s, and 68 • c for 1 min, with a 5-min extension period at 68 • c [17] . pcr products were visualized on a 2% agarose gel stained with ethidium bromide. a positive control, an rdrp sequence from human coronavirus 229e in a p-gem t-easy plasmid (promega, madison, wi, usa), was run with each set of reactions. positive samples with the appropriate amplicon size were purified and sequenced at ribsp using the genetic analyzer 3130xl (thermofisher, waltham, ma, usa) with a big dye terminator cycle sequencing kit, v. 3.1 (thermofisher). electropherograms were inspected in geneious v11 [18] . a total of 12 rdrp sequences were newly generated from this study. to further understand the evolutionary relationships of these viruses, we analyzed novel bat coronavirus sequences in combination with 2811 rdrp sequences of coronavirus from different host species worldwide, representing the three genera: alpha-, beta-, and gamma-coronaviruses. global rdrp sequences were downloaded from national center for biotechnology information sequence database (genbank) and aligned using transalign [19] . this large dataset was manually aligned and further down-sampled to 248 sequences to reduce redundant and similar sequences. maximum-likelihood phylogeny of the partial rdrp gene (460 bp) was reconstructed by raxml; gtr + gamma was selected for the model of nucleotide substitution as it allows rate heterogeneity among sites, as implemented in geneious v11 [20] . branch support was assessed using 1000 bootstrap replicates; bootstrap values greater than 50% were indicated at major nodes. a total of 200 bat guano samples were collected from three sites: kepterkhan tunnel (n = 101), qaraungir cave (n = 50), and ungirli cave (n = 49). each cave was occupied by two bat species, the dominant species being myotis blythii (lesser mouse-eared bat) and the more infrequent species being hypsugo savii (savi's pipistrelle). overall, 25 (12.5%) of all guano samples screened were positive for coronaviruses: qaraungir cave with the highest percent positive (24%) and kepterkham tunnel with the lowest (6.9%) ( table 1) . sequence data were successfully generated for 12 of the 25 pcr positive samples. our rdrp phylogeny demonstrates that all 12 new cov sequences (genbank accession mk603150-mk603161) from kazakhstan bats belong to the genus alpha-cov (figure 2 ; figure s1 , supplementary materials). the alpha-cov genus comprises a large number of coronaviruses from diverse hosts, including bats, shrews, dogs, cats, ferrets, pigs, and humans. in kazakhstan bats, the new cov sequences were found to be segregated into two different groups. the majority (11 sequences) of the kazakhstan covs formed a strongly monophyletic single clade (bootstrap (bs) = 95%), referred to here as "kz3", with the nucleotide sequence identities ranging from 94.7% to 100%. the kz3 clade was a sister group to three bat cov sequences from spain (miniopterus schreibersii and myotis blythii) and china (myotis ricketti). within the kz3 clade, two smaller co-circulating lineages (kz-3a and kz-3b) were formed that are also strongly monophyletic (bs = 82% and 98%, respectively). the kz-3b sub-lineage contained seven bat sequences (ribsp-7, 1, 13, 18, 62, 65, and 66) with 100% nucleotide similarity; however, the samples were collected from two different sites. bat kz-3b sequences (ribsp-7, 11, 13, and 18) were collected from the qaraungir cave, whereas bat kz-3b sequences (ribsp-62, 65, and 66) were collected from the altyntau cave. this suggests the kazakhstan bats residing in two different caves appear to harbor highly similar cov strains. the kz43 group contains only one kazakhstan sequence (ribsp-43) from the altyntau cave, which is markedly divergent (nucleotide identities: 70.2-72.9%) from the kz3 sequences. the ribsp-43 sequence is most closely related with pipistrellus pipistrellus cov (nucleotide identity: 88.1%) described from france in 2014. these sequences in turn grouped with diverse bat species from broad geographical regions including spain (nyctalus lasiopterus and myotis myotis), china (pipistrellus pipistrellus), and south africa (neoromicia cf capensis). taken together, our results indicate that kazakhstan bats may harbor a wider diversity of alpha cov, possibly by means of the regional and intercontinental spread of the virus. although the pooled feces prevented attribution to a singular species, the two lineages of alpha cov may be derived from the different species of bats, myotis blythii and hypsugo savii. kz3 is similar to myotis coronavirus sequences, while the kz43 is most similar to a pipistrellus sequence and h. savii is a pipistrelle bat. our approach was opportunistic sampling in three sites, and the total sample size was 200, limiting the conclusions we can make about bat-borne coronaviruses in central asia; however, this study provides a baseline for future studies. intense coronavirus surveillance is ongoing in china since the sars-cov outbreak, and it was demonstrated that co-roosting can maintain all strains with the necessary components to make sars [10] . there is a paucity of bat-borne virus surveillance efforts across central asia, even though this region is one of the largest grassland biomes in the world. this area is also of interest because it is where the eurasian and asian bat populations may be panmictic, as seen in other bat species with large distributions [21, 22] . the range of m. blythii and h. savii is extensive, with both species found from china and northern india through to spain, and these populations may be co-roosting at multiple sites due to the paucity of roosting areas in the steppe [23, 24] (figure 3 ). myotis blythii is considered an occasional migrant, with movement of greater than 450 km recorded. the migratory behavior of hypsugo savii is not known [25] . the potential panmixia and co-roosting of these bat species may lead to a similar mixing of viruses. these results provide a foundation to study bat-borne coronaviruses in kazakhstan and highlight the need for collaborative efforts in understudied countries to increase integrated surveillance capabilities toward better monitoring and detection of infectious diseases. the following are available online at http://www.mdpi.com/1999-4915/11/4/356/ s1: figure s1 . phylogenetic relationships of the rdrp gene sequences of coronavirus, inferred using the maximum-likelihood method with the gtr + gamma model in raxml. representative virus isolates from alpha-, beta-, and gamma-coronavirus were included in the analysis. colored branches and symbols denote viruses collected from different hosts. new cov sequences generated from this study are marked by red branches. bootstrap support values greater than 50% are displayed at major nodes. the scale bar indicates the number of nucleotide substitutions per site. bats: important reservoir hosts of emerging viruses microchiropteran bats: global status survey and conservation action plan bat ecology bats as viral reservoirs bats and their virome: an important source of emerging viruses capable of infecting humans host and viral traits predict zoonotic spillover from mammals evolutionary insights into the ecology of coronaviruses human coronaviruses: what do they cause? bats are natural reservoirs of sars-like coronaviruses discovery of a rich gene pool of bat sars-related coronaviruses provides new insights into the origin of sars coronavirus rooting the phylogenetic tree of middle east respiratory syndrome coronavirus by characterization of a conspecific virus from an african bat fatal swine acute diarrhoea syndrome caused by an hku2-related coronavirus of bat origin catalog of the mammals of ussr bat study in the kharaa region bats of the russian far east. dalnauka vladivostok bat coronaviruses and experimental infection of bats, the philippines geneious basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data using amino acids to facilitate the multiple alignment of protein-coding dna sequences raxml version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies the conservation genetics of three cave-dwelling bat species in southeastern europe and anatolia continent-wide panmixia of an african fruit bat facilitates transmission of potentially zoonotic viruses the iucn red list of threatened species the iucn red list of threatened species bat migrations in europe: a review of banding data and literature; federal agency for nature conservation we wish to thank kairat tabynov and sulushash zhumabayeva at jacobs. we also thank s. nurabayev, e. burashev, n. rametov, and b. burabayev for their participation in the fieldwork. we also wish to thank jayanthi jayakumar at duke nus for technical assistance. the authors declare no conflict of interest. key: cord-287131-svtdfeop authors: campos, angélica cristine almeida; góes, luiz gustavo bentim; moreira-soto, andres; de carvalho, cristiano; ambar, guilherme; sander, anna-lena; fischer, carlo; ruckert da rosa, adriana; cardoso de oliveira, debora; kataoka, ana paula g.; pedro, wagner andré; martorelli, luzia fátima a.; queiroz, luzia helena; cruz-neto, ariovaldo p.; durigon, edison luiz; drexler, jan felix title: bat influenza a(hl18nl11) virus in fruit bats, brazil date: 2019-02-17 journal: emerg infect dis doi: 10.3201/eid2502.181246 sha: doc_id: 287131 cord_uid: svtdfeop screening of 533 bats for influenza a viruses showed subtype hl18nl11 in intestines of 2 great fruit-eating bats (artibeus lituratus). high concentrations suggested fecal shedding. genomic characterizations revealed conservation of viral genes across different host species, countries, and sampling years, suggesting a conserved cellular receptor and wide-ranging occurrence of bat influenza a viruses. i nfluenza a viruses are major causes of human disease and are predominantly maintained in avian reservoirs (1) . the segmented influenza a genome facilitates reassortment events in birds or intermediate hosts, such as swine and horses, leading to emergence of new variants potentially capable of causing zoonotic infections (2) . bats are major sources of zoonotic pathogens (3) . in pioneering studies from 2012 and 2013, the first bat influenza a viruses, termed h17n10 and h18n11, were discovered in 2 bat species, sturnira lilium (little yellow-shouldered bat) and artibeus planirostris (flat-faced fruit-eating bat) (4, 5) . bat-associated influenza a viruses are phylogenetically highly divergent from avian-associated influenza a viruses in their hemagglutinin (ha) and neuraminidase (na) genes, suggesting these viruses represent ancient influenza a strains (2) . consistent with their genetic divergence, batassociated influenza a surface proteins lack typical hemagglutination and neuraminidase activities (6) , leading to the terminology ha-like (hl) and neuraminidase-like (nl) for bat-associated influenza surface proteins. so far, only 4 individual bat specimens yielded influenza a genomic sequences during the pivotal investigations (4, 5) . hl18nl11 has only been found in 1 a. planirostris bat captured in peru in 2010 (5) , challenging definite host assessments. to investigate bat influenza a virus epidemiology, we investigated bats in southern brazil during 2010-2014. for this study, we sampled 533 individual bats representing 26 species and 3 families across 28 sampling sites (table 1) . bats were captured using mist nets, euthanized, and necropsied and were identified on the basis of morphological criteria by trained field biologists as described previously (7) . only intestine samples were available for virological analyses. the instituto brasileiro do meio ambiente e dos recursos naturais (21748-1), instituto ambiental do paraná (235/10), and the ethics committee of the institute of biomedical science from the university of são paulo (56-18-03/2014) authorized sampling. we tested intestine specimens from all bats using 2 highly sensitive, broadly reactive nested reverse transcription pcrs targeting different regions of the influenza a polymerase basic (pb) 1 gene (5, 8 a. lituratus bats were the most abundantly sampled species ( table 1) . the low overall influenza virus detection rate in this study (0.4%, 95% ci 0.0%-1.5%) was not significantly different by fisher exact test from the previous 2 studies (1/110 bats for hl18nl11 [0.9%, 95% ci 0.0%-5.5%; p = 0.43]; 3/316 bats for hl17nl10 [1.0%, 95% ci 0.0%-2.9%; p = 0.37]). apparently low rates of acute influenza a virus infection in bats are not consistent with high seroprevalence of 72% in different bat species according to a preliminary investigation (5) and may hint at seasonal variation in bat influenza virus infections, comparable to other batborne rna viruses (9) . sanger sequencing of the screening pcr amplicons suggested close genetic relatedness of the strains circulating in brazil with the hl18nl11 strain circulating in peru. virus concentrations in the positive intestine specimens as determined by strain-specific quantitative real-time reverse transcription rt-pcr (appendix table 1 , https://wwwnc. cdc.gov/eid/article/25/2/18-1246-app.pdf) were high (1.5 × 10 9 and 4.9 × 10 10 rna copies/g of tissue). high hl18nl11 concentrations in intestinal specimens are consistent with qualitative data from the pioneering study on hl18nl11 (5) and may suggest intestinal tropism and potential fecal shedding into the environment. we determined the full coding sequence of all 8 segments of the viral genomes using primers aiming at amplifying overlapping regions of bat influenza a virus genomes (genbank accession nos. mh682200-15) (appendix table 1 ). the 2 hl18nl11 variants in brazil differed by 15 nt from each other across the combined 8 genomic segments. four of those substitutions were nonsynonymous, causing amino acid exchanges in the pb2 (v203i), pb1 (r334k), nucleoprotein (g484s), and na (v191i) genes (table 2; figure 1 , panel b). this finding suggests recent common ancestry of the hl18nl11 variants identified in the 2 positive bats and was consistent with their detection in the same site 5 days apart. comparison of the full coding sequence of the novel hl18nl11 variants revealed high sequence identity between the peru and the brazil strains, 93.5%-96.9% nucleotide identity across all 8 genomic segments ( table 2 ). the genomic relatedness of peru and brazil hl18nl11 strains was surprising given a time span of 2 years, a geographic distance exceeding 2,000 km, and 2 different bat species that tested positive in our study and the previous study (5) . all critical amino acid residues associated with influenza a virus replication and entry (4,5) were conserved between the brazil and the peru hl18nl11 strains, including the ha monobasic cleavage site motif piketr/glf (5) . thermodynamic modeling revealed that the amino acid exchanges observed between the brazil and peru hl18nl11 strains did not alter the tridimensional structure of the hl and nl proteins, and neither mapped to the putative receptor binding site of the hl protein ( figure 1 , panel c), nor to the putative active site of the nl protein ( figure 1 , panel d) (6) . this result suggests preservation of the biologic activity of these glycoproteins in different bat species and supported a broadly conserved cellular receptor of bat influenza a viruses that differs from sialic acid receptors used by avian-associated influenza a viruses (10) . significantly fewer amino acid exchanges were observed between the hl proteins of brazil and peru bat influenza virus than between the respective nl proteins (p = 0.007 by fisher exact test) ( n20t, k350r, l357m, i380l, i387v n20t, k350r, l357m, i380l, i387v, g484s nl 93.5% i11v, i15l, v82i, v200i, l254i, a264t, v284i, d332e, v378i, g382e i11v, i15l, v82i, v191i, v200i, l254i, a264t, v284i, low rate of nonsynonymous substitutions in the hlencoding genes of bat influenza a virus variants was reminiscent of strong purifying selection acting on the hemagglutinin genes in avian-specific influenza a virus strains (11) . this finding may suggest comparable evolutionary dynamics between chiropteran and avian reservoirs. definite assessments will require considerably larger datasets of bat influenza a virus strains. a. lituratus bats and a. planirostris bats, in which hl18nl11 was originally detected in peru, represent closely related, yet genetically and morphologically clearly distinct bat species (12) . the distribution of these bat species overlaps (figure 1, panel a) , potentially facilitating virus exchange across the populations. phylogenetic analyses confirmed the close genetic relationship between peru and brazil hl18nl11 variants across all 8 segments (figure 2 ; appendix table 2 ), suggesting lack of reassortment events according to the available data. our data thus suggest host associations of hl18nl11 beyond the species level, comparable to genus-level host associations of other batborne rna viruses such as coronaviruses (13) . the zoonotic potential of hl18nl11 is unclear, yet humanderived cell lines were susceptible to infection by chimeric vesicular stomatitis virus pseudotyped with hl18 (14) . the abundance of a. lituratus bats within latin america (figure 1 bats are sources of high viral diversity and high-profile zoonotic viruses worldwide. although apparently not pathogenic in their reservoir hosts, some viruses from bats severely affect other mammals, including humans. examples include severe acute respiratory syndrome coronaviruses, ebola and marburg viruses, and nipah and hendra viruses. factors underlying high viral diversity in bats are the subject of speculation. the hypothesis is that flight, a factor common to all bats but to no other mammals, provides an intensive selective force for coexistence with viral parasites through a daily cycle that elevates metabolism and body temperature analogous to the febrile response in other mammals. visit our website to listen: http://www2c.cdc.gov/podcasts/player.asp?f=8632573 global patterns of influenza a virus in wild birds chiropteran influenza viruses: flu from bats or a relic from the past? host and viral traits predict zoonotic spillover from mammals a distinct lineage of influenza a virus from bats new world bats harbor diverse influenza a viruses the neuraminidase of bat influenza viruses is not a neuraminidase genetic diversity of bats coronaviruses in the atlantic forest hotspot biome, brazil non-random patterns in viral diversity amplification of emerging viruses in a bat colony hemagglutinin homologue from h17n10 bat influenza virus exhibits divergent receptor-binding and ph-dependent fusion activities evolutionary dynamics and global diversity of influenza a virus speciation dynamics of the fruit-eating bats (genus artibeus): with evidence of ecological divergence in central american populations ecology, evolution, and classification of bat coronaviruses in the aftermath of sars synthetically derived bat influenza a-like viruses reveal a cell type-but not species-specific tropism replication and shedding of mers-cov in jamaican fruit bats (artibeus jamaicensis) we thank mariana cristine pereira de souza, cairo monteiro de oliveira, and luciano matsumiya thomazelli for laboratory support. dr. campos is a postdoctorate researcher affiliated with the university of sao paulo and charité-universitätsmedizin berlin. her research focuses on emerging viruses from bats. key: cord-004502-3mkv3bal authors: wei, gang; sun, honglin; liu, jun-li; dong, kai; liu, junli; zhang, min title: indirubin, a small molecular deriving from connectivity map (cmap) screening, ameliorates obesity-induced metabolic dysfunction by enhancing brown adipose thermogenesis and white adipose browning date: 2020-03-16 journal: nutr metab (lond) doi: 10.1186/s12986-020-00440-4 sha: doc_id: 4502 cord_uid: 3mkv3bal background: obesity occurs when the body’s energy intake is constantly greater than its energy consumption and the pharmacological enhancing the activity of brown adipose tissue (bat) and (or) browning of white adipose tissue (wat) has been considered promising strategies to treat obesity. methods: in this study, we took a multi-pronged approach to screen ucp1 activators, including in silico predictions, in vitro assays, as well as in vivo experiments. results: base on connectivity map (cmap) screening, we obtained multiple drugs that possess a remarkably correlating gene expression pattern to that of enhancing activity in bat and (or) swat signature. particularly, we focused on a previously unreported drug-indirubin, a compound obtained from the indigo plant, which is now mainly used for the treatment of chronic myelogenous leukemia (cml). in the current study, our results shown that indirubin could enhance the bat activity, as evidenced by up-regulated ucp1 expression and enhanced mitochondrial respiratory function in vitro cellular model. furthermore, indirubin treatment restrained high-fat diet (hfd)-induced body weight gain, improved glucose homeostasis and ameliorated hepatic steatosis which were associated with the increase of energy expenditure in the mice model. moreover, we revealed that indirubin treatment increased bat activity by promoting thermogenesis and mitochondrial biogenesis in bat and induced browning of subcutaneous inguinal white adipose tissue (swat) of mice under hfd. besides, our results indicated that indirubin induced ucp1 expression in brown adipocytes, at least in part, via activation of pka and p38mapk signaling pathways. conclusions: our results clearly show that as an effective bat (as well as beige cells) activator, indirubin may have a protective effect on the prevention and treatment of obesity and its complications. the prevalence of obesity has been progressively rising worldwide over the past two decades, reaching pandemic levels [1] . according to the world health organization (who), in 2030, over one billion people in the world will be affected by obesity [2] . obesity is one of important risk factors for metabolic diseases such as type2 diabetes mellitus, non-alcoholic steatohepatitis, cancers, etc., all of which contributes to a decline in both life quality and lifespan [3] [4] [5] . obesity occurs when the body's energy intake constantly exceeds its energy consumption. at present, the available drugs to treat obesity are mainly through limiting energy intake, including inhibiting intestinal lipid absorption (such as orlistat) or inhibiting appetite (such as phenylalanine) [6] . though these medications are effective, the adverse side effects (such as steatorrhea or depression) due to long-term use limit drug adherence of patients. therefore, there is an urgent need for safer and more effective pharmacological approaches to weight loss. obesity is defined as a state of excessive or abnormal fat accumulation of sufficient magnitude which may pose a threat to the health of people [4, 7] . however, mammals, in fact, possess two kinds of adipose tissue with distinct physiological functions: white adipose tissue (wat) and brown adipose tissue (bat). wat stores excess energy in the form of triglycerides. in contrast, bat increases energy expenditure by dissipating chemical energy as heat (thermogenesis), potentially counteracting obesity and related disorders [8] . indeed, recent researches have shown that bat transplantation could reduce the body weight gain and ameliorate glucose homeostasis in leptin deficient (ob/ob) obese mice and high-fat diet (hfd) -induced obese mice [9, 10] . importantly, bat transplantation in polycystic ovary syndrome (pcos) rats also exhibited a significantly improvement in the key features of pcos by increasing energy expenditure [11] . recently, a number of studies have proved that adult humans possess functional bat and its activity is negatively correlated with body mass index [12] [13] [14] . in humans, activation of bat alleviates obesity, and decreases elevated plasma triglyceride concentrations [5] . an important feature of bat is the expression of uncoupled protein 1 (ucp1), which is located in the inner membrane of the mitochondria [15] [16] [17] . in response to external stimuli (such as cold), ucp1 can increase the permeability of the inner mitochondrial membrane, during which ucp1 increases heat production instead of atp synthesis [18] [19] [20] . as compared to other ucp proteins (e.g., ucp2 or ucp3), ucp1 is thought to be the only gene responsible for adaptation to non-shivering heat production [21, 22] . ucp1-null mice display intolerant to cold [23] [24] [25] and develop obesity housed at a thermoneutral temperature [26, 27] . by contrast, transgenic expression of ucp1 in adipose tissues reduces fat deposition and improves energy metabolism in rodents or pigs (in which a functional ucp1 gene is absent) [28] [29] [30] . recently, a group of brown-like fat cells have been identified in white adipose tissue exposed to various stimulation (e.g., the cold), which are called "brite (brown inwhite) or beige" adipocytes [31] . this process is called the "browning" of wat. similar to adipocytes in bat, beige cells also express ucp1 protein (though at a lower level), resulting in increased mitochondrial respiration and energy expenditure. correspondly, a pharmacological approach to increase ucp1 expression and activates of bat thermogenesis and (or) recruits brown-like brite/beige cells in wat may be a safer avenue to enhance wholebody energy expenditure, one complementary and alternative medicine for anti-obesity therapy [32, 33] . the connectivity map (cmap) is developed by the broad institute of mit and harvard university which is a web-based tool and allows users to screen of molecules for a physiological or disease gene signature [34] [35] [36] . both drugs and diseases have so-called genetic signatures which consist of sets of genes known to be turned up or down, on or off in cells exposed to a particular drug or in patients with a particular disease. this screening is obtained by comparing microarray gene expression data of small compounds in cmap database with the gene signature of the phenotype based on the user's interest using a patternmatching algorithm. by this way, a list of compounds exhibits in the results, which shown significantly correlating gene expression patterns as compare to that within the interest phenotype, thus facilitating potential treatments for disease in a specialized search engine. since its publication, cmap has been widely used and become one of the most effective tools for drug repositioning (also known as drug repurposing) and compounds screening to treat associated diseases. in recently studies published in nature med and cell, ozcan's group of harvard medical school employed the cmap database to identify candidate drugs to reduce er stress in obesity. in this context, they found that withaferin a and celastrol as leptin sensitizers, can reduce the body weight and mitigate the metabolic abnormalities of diet-induced obesity mice, including hepatic steatosis [37, 38] . besides, there were some other examples of cmap used for identifying chemicals and (or) in combination with other therapies in treating t2d diabetes, muscular atrophy, inflammatory bowel disease and cancer [39] [40] [41] [42] [43] [44] [45] . recently, as part of the nih lincs consortium, more than a 1000-fold scale-up of the cmap (termed l1000) was made [46, 47] . the expanded cmap will play a greater role in exploring the relationships among drugs, genes, and diseases. in our current study, we have created a gene expression signatures by utilizing the genes data obtained from cit-genedb. citgenedb is a comprehensive database including genes of enhancing or suppressing cold-induced thermogenesis (cit) in human and mouse, which are validated by in vivo or ex vivo experiments in mice [48] . cit-genedb has been integrated into gene ontology (go) and mgi mammalian phenotype ontology and can facilitate the research of cit with systems biology perspectives. based on cmap database, we then used the signature to identify small compounds which could upregulate the ucp1 expression and enhanced activity in adipose (bat and wat), and thereby offer a treatment for obesity. in the current study, we paid particular attention to indirubin, a compound obtained from the indigo plant a compound obtained from the indigo plant, which is now mainly used for treating chronic myelogenous leukemia (cml). our results indicate that as an effective bat (as well as beige cell) activator, indirubin may have a protective effect on the prevention and treatment of obesity and related diseases, which involved in the up-regulation of ucp1 expression and enhancing the bat activity and (or) inducing browning of swat, at least in part, via activation of pka and p38mapk signaling pathways. the chemical compounds derived from cmap screening were purchased from chemical biology technology platform of chinese academy of sciences (shanghai, china) and applied in a dose of 10 μm (final concentration) for each drug within cell experiments. indirubin used in animal treatment was purchased from medchemexpress (catalog no.: hy-n117; cas no.: 479-41-4; purity: >98%, monmouth junction, nj). indirubin was first dissolved in dimethyl sulfoxide (dmso) and stored in the dark at − 20°c. during our experiments, indirubin was diluted into corresponding concentration with the corn oil (sigma-aldrich). all the other chemicals were purchased from sigma chemical co. (st. louis, mo, usa) unless otherwise specified. the primary antibodies included anti-ucp1 (abcam, ab10983 and ab23841), anti-pgc1α (santa cruze biotechnology, sc-13,067), anti-oxphos (abcam, ab110413), anti-phospho-(ser/thr) pka substrate (cell signaling technology, #9621), anti-phospho-creb (cell signaling technology, #4276s, san antonio, tx, usa), anti-vdac1 (cell signaling technology, #4661), anti-β-tubulin (cell signaling technology, #2146) . cell culture, brown adipocytes differentiation, and function analysis c3h10t1/2 cells were purchased from shanghai institute of biochemistry and cell biology (shanghai, china) and routinely maintained in high glucose dmem containing 10% fetal bovine serum (v/v), 100 u/ml penicillin, and 100 μg/ ml streptomycin at 37°c in a humidified atmosphere of 5% co 2 in air. for induction to brown adipocytes, the cells were incubated with medium that contained 20 nm insulin, 1 nm 3, 3, 5-triiodo-l-thyronine (t3), 1 μm dexamethasone, 0.5 mm isobutylmethylxanthine, 125 nm indomethacin, and 1 μm rosiglitazone for two days and were subsequently cultured in medium containing with only insulin and t3 for an additional 4 days. for the assessment of cellular respiration, o 2 consumption of mature adipocytes were measured at day 6 using an xf24-3 extracellular flux analyzer according to the manufacturer's instructions (agilent technologies, santa clara, ca, usa). cells treated with dmso were used as control. for the next-generation cmap (https://clue.io) analyses, a gene expression signature (termed enhancing activity in bat and (or) swat signature) was created by using the genes profile obtained from citgenedb [48] . the online tool of 'query' was then used to directly explore the similarity between gene signatures of drugs and query samples with the query parameters of 'gene expression (l1000)', 'touchstone' and 'individual query'. for the prediction of novel drugs that enhance the activity of adipose in bat and (or) browning of wat, the genes of enhancing or suppressing cold-induced thermogenesis in human and mouse were used as the up-regulated genes or downregulated genes, respectively. drugs similarity was ranked according to the cmap connectivity score (ranging from − 100 to 100) and displayed as a form of heat map, followed the 'perturbation type' were set as 'compound'. the cell count kit-8 (cck-8) assay was used for evaluating cell viability as described previously [49] . in brief, c3h10t1/2 cells were treated with indirubin or other compounds at the indicated concentrations either for 48 h in the undifferentiated state or for the entire differentiation process (until day 6). the absorbance was measured at a wavelength of 450 nm on a plate reader (perkin elmer, waltham, ma, usa). four-week-old c57bl/6 male mice were obtained from shanghai slac laboratory animal company and were housed four to five per cage under constant environmental conditions (12 h/12 h light-dark cycle, temperature 22 ± 2°c, relative humidity at 50 ± 15%) in an office of laboratory animal welfare-certified animal facility. after acclimatization for one week, the mice were randomly divided into different groups fed with either normal chow diet (ncd) or high fat diet (hfd, 60% of energy from fat, research diets inc., cat.no. d12492) for six weeks. then, mice were treated with indirubin (3.5 mg/kg) or vehicle (corn oil, sigma-aldrich) as control by intraperitoneal injection (once every two days) for another 8 weeks while simultaneously fed ncd or hfd, respectively. water and food were provided ad libitum unless otherwise specified. the body weight was assessed every wednesday morning, and the food intake (mean daily food consumption) was measured at the tenth week by calculating the amount of food consumed at 24-h intervals for 6 days. after a 7-week treatment with either indirubin or vehicle, the total fat and lean masses of mice were tested with minispec nmr instrument (bca-body composition analyzer, bruker corporation, billerica, ma, usa). at the end of the experiments, the blood plasma samples were collected and the weights of liver tussues and adipose tissues including bat, subcutaneous inguinal white adipose tissue (swat) and epididymay white adipose tissue (ewat) were measured. tissues were preserved for gene expression, western blot analyses, histology, and immunohistochemistry experiments. all animal procedures in this study were performed in accordance with guideline from the animal care committee of shanghai jiao tong university. whole-body oxygen consumption was assesed with tse lab master system (tse systems, bad homburg, germany), as described previously [50] . briefly, mice were maintained in respiration chambers (22 ± 2°c, a 12-h light/dark cycle) with free access to food and water. mice were adapted in the metabolic chambers for 24 h, and then vo 2 , vco 2 , and physical activity were monitored during the next 24 h. heat production and respiratory exchange ratio (rer) were then calculated [51] . for glucose tolerance test (gtt), mice had free access to drinking water and were fasted overnight (16 h, 5:00 pm to 9:00 am). the fasting blood glucose and body weight of each mouse was recorded followed by an intraperitoneally (i.p.) injection of d-glucose (sigma-aldrich, 10% in saline, 1.5 g/kg body weight). insulin tolerance tests were conducted in mice fasted 5 h (9:00 am to 2:00 pm) by i.p. injection of insulin (0.75 iu/kg body weight, novolin, usa). blood glucose levels were detected with an accu-chek glucose monitor (roche diagnostics, indianapolis, in, usa) at 0, 15, 30, 60, 90, and 120 min. alanine aminotransferase (alt), aspartate aminotransferase (ast), total triglyceride (tg), high-density lipoprotein (hdl-c), low-density lipoprotein (ldl-c), total cholesterol (tc) in serum were determined using commercial standard enzymatic assay kits (nanjing jiancheng bioengineering institute, nanjing, jiangsu, china). liver tg and tc were measured by using commercially available assay kits (applygen technologies inc., beijing, china) and further normalized to protein concentration. levels of insulin in the serum were measured using high sensitive mouse insulin immunoassay kit (ais, cat.no. 32270) following the manufacturer's instructions. for histological analysis, tissues were fixed with 4% paraformaldehyde and embedded in paraffin. the sections were then prepared and stained with hematoxylin and eosin (sigma) to observe the general morphological features. adipocyte area was determined using a microscope (olym-pus bx51, japan). at least 5 fields from random sections of each mouse sample were quantified, and the mean value was calculated. immunohistochemistry staining was performed using rabbit anti-ucp1 antibody (at 1:500 dilution, abcam, ab10983) as described previously [52] . total rna was extracted from tissues or cells with trizol reagent (invitrogen, carlsbad, ca, usa). reverse transcription of total rna (500 ng) and rt-qpcr analysis were conducted with commercial kits (vazyme biotech, nanjing, jiangsu, china). rt-qpcr analysis was performed in duplicate for each sample and repeated three times utilizing a roche lightcycler 480 system (roche diagnostics, mannheim, germany). cyclophilin a was used as a control. primer sequences used are listed in supplymentary table 1 . for quantification of mtdna, total dna was isolated from bat, swat and differentiated c3h10t1/2 cells with tianamp genomic dna kit (tiangen biotech co., ltd., beijing, china). quantitative pcr was performed in duplicate with mtdna specific primer (cox ii) and nuclear-specific pcr (β-globin). primers used in qpcr analysis were designed from the published primer sequences by yuan et al. [50] . the results were shown as abundance ratio of mtdna target gene coxii to βglobin, which correspondingly represented mitochondrial dna and genomic dna, respectively. protein from tissues or cells was extracted by ripa buffer (50 mm tris, 150 mm sodium chloride, 0.1% sds, 1.0% triton x-100, 0.5% sodium deoxycholate) containing protease and phosphatase inhibitor cocktail (roche diagnostics, rotkreuz, switzerland). extracts were spun down, and the cell debris were removed before analysis by western blot. equivalent samples (40-60 μg of protein in each well) were separated by sds-page and transferred to pvdf membrane (millipore, burlington, ma, usa). membranes were blocked with 5% (w/v) fat-free milk in tbst buffer for 1 h and incubated with different antibodies overnight at 4°c, followed by incubation with secondary antibodies for 1 h at room temperature. blots were conducted using enhanced chemiluminescence reagents (thermo fisher scientific) and detected in a luminescent image analyzer according to the manufacturer's protocols (las-4000, fujifilm, tokyo, japan). data were expressed as mean ± sd. the statistical significance of differences was determined using either the student's unpaired t test (2-tailed) or one-way anova followed by bonferroni's multiple comparison post hoc tests. a value of p < 0.05 was considered statistically significant. the cmap links drugs with diseases or physiological phenotypes by using a pattern-matching algorithm and measuring similarities in gene expression [35, 46] . in this context, both drugs and diseases (and physiological phenotypes) have so-called genetic signatures which are sets of genes known to be turned up or down, on or off in a particular disease (or physiological phenotype) and in cells treatment by a particular drug. we hypothesized that perturbagen signatures of compounds in cmap with corresponding signatures similar to that genetic signature of enhanced activity in bat and (or) swat, will have good effects on improving obesity and obesityrelated diseases. to test this hypothesis, we first created a gene expression signature (termed enhancing activity in bat and (or) swat signature) from the gene profiles obtained from citgenedb (fig. 1a , supplymentary table 2 ). importantly, we chose the enhancing cit genes as up-regulated genes and suppressive cit genes as down-regulated genes. we then employed the query of cmap for drugs which have a gene expression pattern positively correlating to the enhancing activity in bat and (or) swat signature. during screening, multiple drugs in the cmap were identified that had a significantly correlating gene expression pattern to that of enhancing activity in bat and (or) swat signature, including rutin and myricetin (fig. 1b) . these results demonstrate the validity of the cmap, because both rutin and myricetin can induce the ucp1expression in differentiated adipocytes, and enhance the thermogenesis of bat and browning of swat in previous publications [50, 52] . we next set up a phenotype screen platform in vitro by examining the ucp1 expression and further oxygen consumption of differentiated c3h10t1/2 cells exposure to compounds derived from the cmap. herein, we focus on one drug of natural compounds derived from this model, which is named indirubin (fig. 1c ) and used as a chinese medicine for the treatment of chronic myelogenous leukemia (cml) [53] . prior to testing the effects of indirubin in adipocytes, we performed a cck-8 assay in c3h10t1/2 cells to select optimal doses of indirubin. indirubin has no significant cytotoxicity in concentrations up to 40 μm against the c3h10t1/2 cells for treatment of 48 h (supplymentary figure 1a) . moreover, indirubin did not show significant cytotoxicity when treated chronically (6 days) during differentiation (supplymentary figure 1b) . to assess the effect of indirubin on ucp1 expression, we differentiated c3h10t1/2 cells into adipocytes in the presence of several doses of indirubin for 6 days. at a dose of 5 μm and higher, indirubin markedly upregulated the expression of ucp1 mrna (fig. 1d) . consistently, indirubin (10 μm) treatment also increased the mrna expression levels of thermogenic-related genes (pgc1α, dio2, cideα, prdm16, and fgf21), fao (fat acid oxidation) -related genes (pparα cpt1α, cpt1β, lcad), lipolysis-related genes (hsl, atgl) and mitochondrial biogenesis-related genes (nrf1/2, tfam) (fig. 1e) . however, indirubin treatment did not significantly affect the mrna expression of common adipogenic marker genes pparg2 and ap2 (also known as fabp4) (fig. 1e) . to determine the impact of indirubin on mitochondrial activities, oxygen consumption rate (ocr) was determined by the seahorse system [54] . in agreement with gene expression data, indirubin increased the mitochondrial ocr, especially basal oxygen consumption as well as maximal respiration capacity ( fig. 1f-g) . therefore, these data show that indirubin may be a potential drug for combating obesity and obesity-related discords, in regard to its potential in inducing ucp1 expression and enhancing mitochondrial respiratory function in vitro. next, we further assesed the effects of indirubin treatment in vivo. mice were fed with normal chow diet (ncd) or high-fat diet (hfd) for six weeks, and then simultaneously treated with indirubin (or vehicle) at a dose of 3.5 mg/kg through intraperitoneal injection (once every two days) for another 8 weeks. when mice fed under ncd conditions, indirubin treatment had no obvious effects on body weight gain as well as body composition (fig. 2a-c) . however, indirubin treatment significantly inhibited the body weight gain in mice fed on a hfd (fig. 2a) , which was generally owing to the decreased fat mass (fig. 2b-c) . in addition, indirubin treatment did not remarkably affect the food intake in both ncd and hfd groups (fig. 2i) . to examine whether indirubin-mediated decline in adiposity and body weight gain is associated with increased whole-body energy expenditure, mice were then housed in metabolic cages for monitoring o 2 consumption after 6 weeks' treatment of indirubin. on the normal chow diet, indirubin-treated mice exhibited comparable o 2 consumption ( fig. 2d-e) . when placed on hfd, indirubintreated mice displayed significantly increased o 2 consumption without much difference in physical activity ( fig. 2d-e, h) , which suggested that the reduced body weights were not due to alteration in physical activity. moreover, indirubin-treated mice displayed a higher reliance on fat oxidation, which was reflected by the relative lower-rer values (fig. 2f) . because body weights were significantly different between vehicle-and indirubin-treated mice under hfd conditions, energy expenditure (ee) was statistically adjusted for body weight using analysis of covariance (ancova). in line with increased o 2 consumption, indirubin-treated mice exhibited greater ee than that of hfd group (fig. 2g) . these data suggest that indirubin treatment is resistant to hfd-induced obesity by increasing whole-body energy consumption. a flow chart depicting the process of cmap screen using citgenedb to identify potential small-molecule for inducing ucp1. b, c the chemical structure and the cas number of indirubin. d dose-dependent effect of indirubin on ucp1 expression in differentiated c3h10t1/2 cells on day 6 of brown adipogenesis. e rt-qpcr analysis of genes related to thermogenesis, fatty acid oxidation, mitochondrial biogenesis and common adipogenic marks in differentiated c3h10t1/2 cells (6 days of differentiation) after indirubin (10 μm) treatment. f oxygen consumption rates (ocr) was measured in differentiated c3h10t1/2 cells (6 days of differentiation) in the presence or absence of indirubin using a seahorse xf24 analyzer. g basal oxygen consumption and maximum respiratory capacity from seahorse assay were determined. data in d-g are presented as mean ± sd of six independent experiments performed in duplicate. * p < 0.05, ** p < 0.01 compared with vehicle indirubin improves whole-body glucose homeostasis, reduces lipid accumulation in adipose, and ameliorates fatty liver as reduced adiposity is usually related to better glucose homeostasis, we next investigated if reduced fat mass in indirubin-treated mice under hfd would result in improved glucose disposal ability and insulin sensitivity. as our expected, fasting blood glucose levels and insulin levels were obviously lower in indirubin-treated mice under hfd (fig. 3a-b) , whereas corresponding fasting glucose levels and insulin levels were not significantly different between ncd groups (fig. 3a-b) . consistent with these results, indirubin treated mice under hfd also displayed improved glucose handling as determined by glucose and insulin tolerance tests (at weeks 10 and 12, respectively) ( fig. 3c-f ). mice were then dissected for further investigation. in line with the lean phenotype, total organ weights of bat (− 26.09%), swat (inguinal wat, − 24.80%) and ewat (epididymal wat, − 22.20%) were decreased in indirubin-treated mice of hfd groups but not in mice of ncd groups (fig. 4a-b) . moreover, indirubin-treated mice under hfd exhibited remarkably decreased lipid content and adipocyte size in bat, swat and ewat as evidenced by histological analysis (fig. 4c-f ), which were correlated with body weight loss (fig. 2a) . in addition, the symptom of fatty liver induced during hfd was also alleviated by indirubin treatment, as assessed by morphological examination and histological analysis ( fig. 5a-b) . after 14 weeks of hfd, indirubin-treated mice exhibited a significantly reduction in liver weights (fig. 5c) . consistent with this result, indirubin treatment (b-c) the total fat mass b and lean mass c were determined using minispec nmr instrument after 7-week treatment with either vehicle or indirubin. d-h metabolic cage analyses of mice after 7-week treatment with either vehicle or indirubin (n = 6 in each group). the vo 2 consumption during a 12-h light: 12-h dark cycle d, energy expenditure (ee), mean vo 2 consumption levels e, the respiratory exchange ratio (rer; vco 2 /vo 2 ) f, and physical avtivity h, were measured simultaneously. g energy expenditure values adjusted for body weight using ancova. i food intake (mean daily food consumption) was measured after 7-week treatment with either vehicle or indirubin by calculating the amount of food consumed at 24-h intervals for 6 days. data are presented as mean ± sd (n = 6 in each group). # p < 0.05, ## p < 0.01, ### p < 0.001 compared with ncd + vehicle group; * p < 0.05, ** p < 0.01 compared with hfd + vehicle group significantly ameliorated neutral lipid accumulation in livers of hfd-induced mice, as indicated both by means of h&e staining and oil red o analysis (fig. 5a ). in addition, indirubin-treated mice also displayed reduced levels of liver triglyceride (− 54.75%) and liver cholesterol content (− 10.66%) in (fig. 5d-e) in livers of hfdinduced mice. as a higher degree of obesity often leads to lipid metabolism abnormalities, we next evaluated the effects of indirubin on hfd-fed mice by measuring serum lipid metabolism-related biochemical parameters. as shown in table 1 , indirubin treatment prevented the hfdinduced elevation in the serum levels of tg, tc and ldl-c, whereas indirubin-treated mice exhibited considerably increase in the serum levels of hdl-c under hfd conditions. in line with these results, as shown in fig. 5f , rt-qpcr assays also confirmed that indirubin treatment could reduce the mrna expression levels of lipogenesisassociated genes (fans, acc1). meanwhile, we found less expression of mcp1, tnf-α and il-6 cytokines in liver tissues of mice after indirubin treatment under hfd (fig. 5f) , suggest an improved chronic inflammation state. of note, we also analyzed serum levels of ast and alt to determine whether indirubin treatment causes liver injury. as shown in fig. 5g -h, compared with ncd groups, liver damage was verified by obviously elevated levels of serum ast and alt in hfd-fed mice, whereas indirubin treatment protected against the increase in ast and alt levels ( fig. 5g-h) . these results indicated that indirubin treatment, at least in the concentration applied in this study, had no side effects on liver function. taken together, these data show that indirubin treatment can improve glucose metabolism, reduce lipid accumulation in adipose and adipocyte size, as well as decrease hepatic fat deposition in hfd-fed mice. in order to investigate the close relationship of enhanced energy consumption to bat activation, we examined the molecular biological features of bat in mice after indirubin treatment. consistent with enhanced wholebody energy consumption, the bat marker ucp1 was upregulated significantly in indirubin-treated mice under hfd by immunohistochemical analysis (fig. 6g) . furthermore, rt-qpcr analysis of bat showed that indirubin treatment also led to upregulation of expression levels of bat-specific genes related to heat generation, including ucp1, pgc1α, prdm16, dio2, elovl3, cideα (fig. 6a) . meanwhile, the mrna expression levels of genes controlling fatty acid oxidation (pparα, cpt1α, cpt1β, mcad) were significantly increased by indirubin treatment in bat (fig. 6b ). in addition, the mrna expression level of hormone-sensitive lipase (hsl), which is closely related to triglyceride hydrolysis showed a slight but significant increase in indirubin-treated mice under hfd (fig. 6c) . however, as shown in fig. 6d , common adipocytes makers (pparγ2, ap2) were not significantly affected after indirubin treatment, though the expression level of the insulin sensitizing adipokine adiponectin (also known as adipoq) was significantly higher relative to control hfd-fed mice. in addition, the bat-enriched genes mentioned above showed no change or a slight increase in expression levels between ncd groups (fig. 6a-d) . these cumulative evidences indicated that indirubin had potential to increase the thermogenic capacity in bat of hfd-fed mice. since the higher activity of bat is often positive correlating to the number and activity of mitochondria. therefore, we next measured the mrna expression levels of mitochondriogenesis-related factors. as expected, nrf1/2 and tfam expression levels were also upregulated (fig. 6e) . consist with this, the number of mitochondria was increased in bat of indirubin-treated mice under hfd as quantified by mitochondrial dna (mtdna) copy number (fig. 6f) . furthermore, the protein abundance of voltage-dependent anion channel 1 (vdac1), which is a major isoform highly and predominantly expressed on the mitochondrial outer membrane, was obviously upregulated after indirubin treatment in bat of mice under hfd (fig. 6i ). in addition, the abundance of proteins (ucp1, pgc1α, and oxphos) related to thermogenesis and β-oxidative phosphorylation were markedly unregulated in bat of indirubin-treated mice under hfd (fig. 6i) . however, bat-enriched genes and (or) proteins mentioned above and mtdna copy number showed no change or a slight increase in expression levels between ncd groups (fig. 6a-h) . taken together, these results indicate that indirubin can promote thermogenesis and mitochondrial biogenesis in bat, thereby enhancing endogenous bat activity and burning of fat. recent researches have proved that browning of swat can also increase energy metabolism and exhibit beneficial effects on anti-obesity [55] [56] [57] [58] . to further assesed the reason of swat mass loss in indirubin-treated mice under hfd conditions, molecular biological characteristics of swat were researched. interestingly, immunohistochemistry results indicated that ucp1 was strikingly stimulated in swat of mice under hfd in response to indirubin treatment. besides, we next examined the bat-enriched genes in swat (fig. 7g) . our results showed that the mrna expression levels of genes related to thermogenesis (ucp1, pgc1α, prdm16, elovl3, fgf21) and fatty acid oxidation (pparα, lcad) were significantly upregulated in swat of mice treated with indirubin under hfd. lipid droplets lipolysis in swat is essential for regulating bat fuel sources and wat thermogenesis during metabolic adaption [59, 60] . in our study, as shown in fig. 7c , the mrna expression levels of genes involved in lipolysis such as adipose triacylglycerol lipase (atgl) and hsl were obviously reduced in swat of hfd-fed control mice relative to ncd groups, whereas the mrna expression levels of these two genes were considerably upregulated in swat of indirubintreated mice under hfd, leading to the activation of lipolysis and fueling thermogenesis during bat activation and swat browning. more importantly, beige cell marker genes (cd137, tmem26, tbx1) [61] were also markedly increased in swat of indirubin-treated mice as compared to hfd-fed control mice (fig. 7e) . however, there was no significant change in common adipose markers (pparγ2, ap2, adiponectin) at mrna expression level under both diets (fig. 7d) . as beige cells has the characteristics similar to bat, we also investigated the mitochondrial biogenesis in swat. the mrna expression levels of tfam and nrf1/2 in swat were notably upregulated after indirubin treatment under hfd (fig. 7f) . in parallel, indirubin treatment increased the number of mitochondria in swat, as was further manifest by increased mtdna copy number (fig. 7g) and mitochondrial outer membrane protein vdac1 (fig. 7j) . consistently, western blot analysis indicated that the abundance of proteins (ucp1, pgc1α, and oxphos) related to thermogenesis and β-oxidative phosphorylation were significantly unregulated in swat of indirubintreated mice under hfd (fig. 7j) . these results indicate that increased browning of swat in response to indirubin can act synergistically with bat activation on anti-obesity. data are presented as mean ± sd (n = 6 in each group). # p < 0.05, ## p < 0.01, ### p < 0.001 compared with ncd + vehicle group; * p < 0.05, ** p < 0.01, *** p < 0.01 compared with hfd + vehicle group indirubin induces ucp1 expression and enhance bat activity relying on pka and p38mapk signaling pathways to identify the possible mechanism underlying the effects caused by indirubin treatment, we determined expression levels of key signaling molecules involved in the regulation of brown adipose function. we investigated campdependent protein kinase a (pka) and p38 mapk signal pathways, both of which play important roles in the mobilization of fat to be hydrolyzed, oxidized, and uncoupled. firstly, we used anti-phosphorylated pka substrate antibody to evaluate pka activity after indirubin treatment in differentiated adipocytes. as shown in fig. 8a , indirubin treatment increased pka activity as observed by increased phosphorylation of pka substrate. creb and p38 mapk are important downstream targets of pka, both of which have been proved as key elements in inducing ucp1 expression [19, 62] . in our current study, the increased protein abundance of phosphorylated forms of creb and p38 mapk were also observed by western blot analysis after treatment with indirubin compared with vehicle-treated cells (fig. 8a-c) . more importantly, we separately treated cells with pka inhibitor (h89) and p38 mapk inhibitor (sb203580) in the presence or obscene of indirubin at 6 days of differentiation. as shown in fig. 8a , inhibition of pka with h89 abolished indirubin-induced expression of bat-enriched genes, including ucp1, pgc1α. similarly, elevated expression of bat-enriched genes (ucp1 and pgc1α) were eliminated after p38 mapk inhibitor treatment (fig. 8b) . these results indicate that indirubin treatment can promote thermogenesis via activated pka and p38 mapk pathways. collectively, these results suggest that indirubin may be a useful as herbal medicine for inducing ucp1 expression and enhance bat activity (as well as browning of swat) via activation of pka and p38mapk signaling pathways, thereby potentially in preventing and treating obesity and obesity-associated metabolic diseases. obesity occurs when the body's energy intake is constantly greater than its energy consumption, which has become a growing public health concern in modern society [4] . since the recent discovery of functional bat or batlike tissue in adult humans, the development and function fig. 4 indirubin treatment decreases adipose tissue mass and adipocyte cell size in hfd-induced obese mice. a mean tissue weights of bat, iwat, and ewat of mice fed ncd or hfd after 8-week treatment with either vehicle or indirubin. b representive photographs of bat, iwat, and ewat in hfd-induced obese mice after 8-week treatment with either vehicle or indirubin. c representative hematoxylin and eosin (h&e) staining images of bat, iwat, and ewat of mice fed ncd or hfd after 8-week treatment with either vehicle or indirubin. scale bar = 50 μm. d mean adipocyte size of adipose tissues quantified from h&e-stained section in c, six fields per mouse, using metaxpress software. data in a, d, e and f are presented as mean ± sd (n = 6 in each group). # p < 0.05, ### p < 0.001 compared with ncd + vehicle group; * p < 0.05, ** p < 0.01, compared with hfd + vehicle group of brown or beige adipocyte has been widely and deeply researched for its potential as a target of anti-obesity therapeutics [63] . although many screening approaches (in vitro and in vivo) have been adopted for the discovery of chemicals to activate ucp1 expression [64] [65] [66] [67] , to identify safe and effective drugs for upregulating the activity of brown and (or) beige adipocytes is still a challenge, especially given the high-cost and time-consuming traditional methods for screening ucp1 activators. in order to address this challenge, we took a multi-pronged approach to screen ucp1 activators (through drug repurposing), including in silico predictions, in vitro assays, as well as in vivo experiments. in the in silico predictions, the cmap database was employed to select appropriate drug candidates for further in vitro and in vivo validation. our results indicate that indirubin is a promising natural c mean tissue weights of liver were measured in mice fed ncd or hfd after 8-week treatment with either vehicle or indirubin. d-e liver tg d and tc e content, serum alt g and ast h were measured in mice fed ncd or hfd after 8-week treatment with either vehicle or indirubin. f rt-qpcr analysis of genes related to adipogenesis and inflammation in livers of mice fed ncd or hfd after 8-week treatment with either vehicle or indirubin. g-h serum alt g and ast h were measured in mice fed ncd or hfd after 8-week treatment with either vehicle or indirubin. data in c-h are presented as mean ± sd (n = 6 in each group). ## p < 0.01, ### p < 0.001 compared with ncd + vehicle group; * p < 0.05, ** p < 0.01, *** p < 0.01 compared with hfd + vehicle group plasma profiles of mice with or without indirubin treatment. data are presented as mean ± sd (n = 6 in each group). #p < 0.05 compared with ncd + vehicle group;*p < 0.05, **p < 0.01 compared with hfd + vehicle group product for protecting obesity and associated diseases through enhancing bat activity and inducing browning of swat, at least in part, by activating pka and p38 mapk signal pathways. in this work, we firstly used a gene profile obtained from citgenedb to construct the gene expression signature as enhancing activity in bat and (or) swat signature. because the genes of enhancing or suppressing cit in citgenedb were all validated via perturbation experiments in mice, it will be more effective to highlight the role of core genes in regulation of the activity of brown or beige adipocytes. for example, the complete list of coldmodulated genes in bat after microarray or rna-seq data analysis is easily obtained from gene expression omnibus fig. 6 indirubin increases bat activity by promoting thermogenesis and mitochondrial biogenesis in hfd-induced obese mice. a-e rt-qpcr analysis of genes related to thermogenesis a, fatty acid oxidation b, lipolysis c, common adipogenic marks d and mitochondrial biogenesis in bat of mice fed ncd or hfd after 8-week treatment with either vehicle or indirubin. f measurement of mtdma copy number in bat of mice fed ncd or hfd after 8-week treatment with either vehicle or indirubin. g immunohistochemistry (ihc) staining with a ucp1-specific antibody in bat of mice fed ncd or hfd after 8-week treatment with either vehicle or indirubin. scale bar = 50 μm. h-i western blot analysis of proteins levels of ucp1, pgc1α, vdac1, and oxphos in bat of mice fed ncd h or hfd i after 8-week treatment with either vehicle or indirubin. β-tubulin serves as a loading control. data in a-f are presented as mean ± sd (n = 6 in each group). * p < 0.05, ** p < 0.01 compared with hfd + vehicle group (geo) [68] [69] [70] , but the more highly expression genes are usually not consistent with the positive effects of enhancing cold-induced thermogenesis (or vice versa), possibly involving the negative feedback control or compensation effect. in this way, we identified a group of drugs in cmap database that have higher scores of similarity (connectivity score) of gene-expression profiling as compared to the established enhancing activity in bat and (or) swat signature, thereby potentially activating the ucp1 expression and inducing energy expenditure to combating obesity and associated diseases. interestingly, rutin [50] and myricetin [52] , both of which have been proved to have the capacity to elevate of ucp1 expression and enhance the thermogenesis in bat and browning of swat, were also present in our results, indicating the feasibility of utilizing this fig. 7 indirubin promotes browning of swat by increasing bat-specific markers and mitochondrial biogenesis in hfd-induced obese mice. a-f rt-qpcr analysis of genes related to thermogenesis a, fatty acid oxidation b, lipolysis c, common adipogenic marks d, beige cell markers e and mitochondrial biogenesis f in swat of mice fed ncd or hfd after 8-week treatment with either vehicle or indirubin. g measurement of mtdma copy number in swat of mice fed ncd or hfd after 8-week treatment with either vehicle or indirubin. h immunohistochemistry (ihc) staining with a ucp1-specific antibody in bat of mice fed ncd or hfd after 8-week treatment with either vehicle or indirubin. scale bar = 50 μm. i-j western blot analysis of proteins levels of ucp1, pgc1α, vdac1, and oxphos in bat of mice fed ncd (h) or hfd i after 8-week treatment with either vehicle or indirubin. β-tubulin serves as a loading control. data in a-f are presented as mean ± sd (n = 6 in each group). # p < 0.05 compared with ncd + vehicle group; * p < 0.05, ** p < 0.01 compared with hfd + vehicle group system to screen drugs for stimulating ucp1 expression and elevating activity in bat and (or) swat. further in vitro studies we focused on a previously unreported compound-indirubin, revealed that indirubin can enhance ucp1 expression as well as other thermogenic genes expression in differentiated brown adipocyte (fig. 1e) . these results demonstrated that indirubin could directly activate brown adipocytes in a cellautonomous way. two important characteristics of bat or beige adipocytes are high mitochondrial dna copy number and high rates of oxygen consumption. our data showed that indirubin treatment significantly enhanced the mitochondrial ocr (fig. 1f-e) in differentiated brown adipocytes, suggesting an elevated of the mitochondrial activities. collected, our results suggest that indirubin can increase activity of the brown adipocytes by upregulating the bat-enriched gene expression and by increasing mitochondrial activity. we next focused on indirubin for further in vivo research. in the present study, our results showed that indirubin treatment remarkably reduced body weight gain and adiposity and improved whole-body metabolism in hfd mice, but there was no significant difference on normal chow diet conditions (fig. 2) . at the molecular level, we found that indirubin treatment in vivo resulted in higher ucp1expression levels (both in mrna and protein levels) in bat and swat ( fig. 6 and fig. 7, respectively) , which was in accord with the in vitro screening results (fig. 1) . additionally, indirubin treatment also strongly upregulated the expression levels of bat-enriched thermogenic genes involving in lipid metabolism and mitochondrial biogenesis, as well as the oxphos proteins in bat and swat ( fig. 6 and fig. 7) . importantly, indirubin treatment also caused browning effect on swat, as reflected by the increased expression of beige specific markers (tbx1, cd137, and tmem26) in hfd-fed mice (fig. 7e) . these results suggest that indirubin normalized body weight and fat mass, which was likely involved in bat activation and browning of swat, accompanied by increased energy expenditure and thermogenic gene expression in bat and swat. bat also have a secretory role and bat-derived endocrine factors (the so-called brown adipokines or batokines) have been thought to contribute to the systemic consequences of bat activity. in particular, adiponectin plays a key role in mediating the benefits of bat transplantation in rodents, including improvements in wholebody energy expenditure and glucose homeostasis [9] [10] [11] . besides, previous research also reveals that patients with obesity and nafld exhibited a reduced adiponectin transcription in adipose tissue and decreased adiponectin concentration in plasma [71] . in addition, other moleculars, such as fgf21 and bmp, have also been suggested as exercise mimetics [72] [73] [74] . interestingly, our present findings showed that indirubin treatment did not lead to obvious alteration in the expression levels of common adipogenic maker genes (pparγ2, ap2) in bat and swat, but not adiponectin in bat, which was increased significantly (fig. 6d) . in consist with this, the expression level of fgf21was also significant upregulated in differentiated brown adipocytes (fig. 1e) and swat of mice under hfd after treatment with indirubin (fig. 6d) . moreover, in our current work, we also found that indirubin improved systemic glucose and lipid homeostasis, ameliorated hepatic steatosis and obviously decreased the expression of inflammation-related genes in liver tissues induced by hfd (fig. 5) . the precise mechanism is not yet clear, even so, increased secretion of batokines after bat activation and browning of swat, such as adiponectin and fgf21, may also exert beneficial effects on treating type 2 diabetes and associated several metabolic comorbidities. finally, we further explored the underlying pathway by which indirubin upregulates ucp1 in adipocytes, which further enhances the activity of activity in bat and (or) swat. it has been proved that increased pka activity specifically in adipose tissue could upregulate ucp1 expression and ameliorate metabolism discords. pka activates p38 mapk and increases the expression of ucp1, thereby promoting thermogenesis in bat [75, 76] . moreover, increased pka activity has also been linked to induction of browning in swat [77, 78] . in adipocytes, activated pka increases the thermogenesis-related genes such as pgc1α and ucp1 expression through phosphorylating the transcription factor creb [22, 61] . pgc1α plays a key role in thermogenesis and oxidative metabolism. it has been reported that pgc1α promotes the expression of nuclear-encoded mitochondrial genes, thereby subsequently enhancing the mitochondrial biogenesis [61, 79] . up-regulation of pgc1α expression in adipose tissues leads to a robust resistance to obesity and related diseases [80] . the activation of pka triggers p38 mapk phosphorylation, which also phosphorylates and activates of downstream effector pgc1α [75, 81, 82] . pgc1α then modulates the expression of ucp1, thereby promoting thermogenesis and browning in adipocytes [83] [84] [85] . given that the pka and p38 mapk signaling pathway is critical for bat activation and browning of (s) wat, we next to evaluate whether indirubin treatment had any effect on pka activity in adipocytes. our study in vitro showed that indirubin significantly increased phosphorylation of pka substrate (fig. 8a) . in particular, the present study showed that indirubin treatment could significantly increase the abundance of active phosphorylated forms of creb and p38 mapk in adipocytes. we also treated adipocytes with pka inhibitor (h89) and p38 mapk inhibitor (sb208503), respectively. pka inhibitor or p38 mapk inhibitor treatment clearly blocked the level of brown fat enriched mark proteins (ucp1 and pgc1α) (fig. 8a-b) . these observations could result from the increased activity of pka and p38 mapk in adipocytes, suggesting that indirubin activated bat (as well as beige cell) via pka and p38 mapk dependent pathways. it is interesting to notice that different compounds with even modest structure changes may lead to quite different physiological effects. one recent research has suggested that indirubin-3′-oxime (i3o), which is a synthesized analog of indirubin, has potential to prevent obesity and metabolic syndrome by inhibiting the differentiation of preadipocytes into mature adipocytes [86] . in our study, we noted that treatment of brown adipocytes with i3o in vitro can only displayed a slight but no significant increase in the expression of bat marker gene ucp1 ( supplymentary figure 2a) , suggest different mechanism involved in the regulation of ucp1 expression compared with indirubin. besides, our results showed that i3o markedly reduced the mrna expression of genes related to activity and function of brown adipocytes (mcad, lcad, hsl, pgc1α) in the differentiation of c3h10t1/2 cells, though i3o slightly but not significantly inhibited common adipogenic marker pparγ (supplymentary figure 2b) . likewise, it should be considered that other closely related compounds, such as sb216763 and sb415286 (two gsk3 inhibitors), may also regulate one or both pathways controlled by indirubin in fat. recent reports have indicated that small-molecule inhibitors of gsk3 have favorable metabolic effects in rodents, which exhibit some of the same metabolic effects as fgf21 administration, including improved glucose tolerance and prevention of diet-induced obesity [87] [88] [89] [90] . one potential mechanism by which these agents could induce thermogenenic activation is by inhibitory phosphorylation of gsk3 in a pka-dependent manner, which in turn result in enhanced activity of p38 mapk signaling module [91] . in our study, we found treatment of differentiated brown adipocytes with indirubin (10 μm) indeed showed a modest inhibition of gsk3β by enhanced the phosphorylation of ser-9 (p-gsk3β) (ser9) in vitro (supplymentary figure 3) . however, from our results, we are unable to conclude on the relative contribution of gsk3β or its paralogs to bat activation. a precise mechanism by which indirubin treatment in adipocytes lead to increased thermogenesis remains to be further determined, but it is clear that pka and p38 mapk activation is very important for the effects. nonetheless, the fact that indirubin treatment has a cell autonomous effect on bat activation and browning of swat in vivo makes indirubin in particular a very promising drug candidate to protect against obesity and its related complications (fig. 8d) . this study we offer new clues for the indirubin, a purified compound obtained from the indigo plant that decreased whole-body weight and adiposity, and improves glucose homeostasis and insulin sensitivity in hfd-fed mice. specifically, screening potential ucp1 activators with the method of cmap, which further enhance bat activity and induce browning of wat, could be a feasible therapeutic strategy for obesity and associated diseases. supplementary information accompanies this paper at https://doi.org/10. 1186/s12986-020-00440-4. additional file 1 supplymentary figure 1 (a-b) cytotoxicity of indirubin upon induction in c3h10t1/2 cells before (48 h) (a) or after differentiation on day 6 (b). figure 2 rt-qpcr analysis mrna expression of ucp1 and bat-enriched genes in differentiated c3h10t1/2 cells on day 6. data are presented as mean ± sd of six independent experiments performed in duplicate. * p < 0.05, ** p < 0.01 compared with vehicle. figure 3 western blot analysis performed with the indicated antibodies (p-gck3β, gck3β) in differentiated c3h10t1/2 cells on day 6. table 1 primer sequences used in this study. table 2 the enhancing cit genes used as up-regulated genes and suppressive cit genes from citgenedb. will all americans become overweight or obese? estimating the progression and cost of the us obesity epidemic global burden of obesity in 2005 and projections to 2030 epidemiology of obesity and diabetes and their cardiovascular complications 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expenditure and improve metabolic health selective activation of mitogen-activated protein (map) kinase kinase 3 and p38alpha map kinase is essential for cyclic amp-dependent ucp1 expression in adipocytes a pgc1-alpha-dependent myokine that drives brown-fat-like development of white fat and thermogenesis peroxisome proliferator-activated receptorgamma coactivator 1 alpha (pgc-1 alpha): transcriptional coactivator and metabolic regulator pgc-1alpha is required for exercise-and exercise training-induced ucp1 upregulation in mouse white adipose tissue the small molecule indirubin-3′-oxime activates wnt/beta-catenin signaling and inhibits adipocyte differentiation and obesity effects of a novel glycogen synthase kinase-3 inhibitor on insulin-stimulated glucose metabolism in zucker diabetic fatty (fa/fa) rats long-term treatment with novel glycogen synthase kinase-3 inhibitor improves glucose homeostasis in ob/ ob mice: molecular characterization in liver and muscle anti-obesity effects of 3-hydroxychromone derivative, a novel small-molecule inhibitor of glycogen synthase kinase-3 anti-diabetic efficacy of kicg1338, a novel glycogen synthase kinase-3beta inhibitor, and its molecular characterization in animal models of type 2 diabetes and insulin resistance gsk3 is a negative regulator of the thermogenic program in brown adipocytes springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we express our gratitude to all of the participants who consented to participate in this study.authors' contributions gw was a major contributor in writing the manuscript. gw, jll, mz conceived and designed, supervised, and led the study. gw, hls, kd, j-ll collected samples. gw, jll contributed to discussion and revision of the manuscript. gw, jll analyzed the data and wrote the manuscript. all authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord-262815-fg76s168 authors: anthony, s. j.; ojeda-flores, r.; rico-chávez, o.; navarrete-macias, i.; zambrana-torrelio, c. m.; rostal, m. k.; epstein, j. h.; tipps, t.; liang, e.; sanchez-leon, m.; sotomayor-bonilla, j.; aguirre, a. a.; ávila-flores, r.; medellín, r. a.; goldstein, t.; suzán, g.; daszak, p.; lipkin, w. i. title: coronaviruses in bats from mexico date: 2013-05-01 journal: journal of general virology doi: 10.1099/vir.0.049759-0 sha: doc_id: 262815 cord_uid: fg76s168 bats are reservoirs for a wide range of human pathogens including nipah, hendra, rabies, ebola, marburg and severe acute respiratory syndrome coronavirus (cov). the recent implication of a novel beta (β)-cov as the cause of fatal respiratory disease in the middle east emphasizes the importance of surveillance for covs that have potential to move from bats into the human population. in a screen of 606 bats from 42 different species in campeche, chiapas and mexico city we identified 13 distinct covs. nine were alpha (α)-covs; four were β-covs. twelve were novel. analyses of these viruses in the context of their hosts and ecological habitat indicated that host species is a strong selective driver in cov evolution, even in allopatric populations separated by significant geographical distance; and that a single species/genus of bat can contain multiple covs. a β-cov with 96.5 % amino acid identity to the β-cov associated with human disease in the middle east was found in a nyctinomops laticaudatus bat, suggesting that efforts to identify the viral reservoir should include surveillance of the bat families molossidae/vespertilionidae, or the closely related nycteridae/emballonuridae. while it is important to investigate unknown viral diversity in bats, it is also important to remember that the majority of viruses they carry will not pose any clinical risk, and bats should not be stigmatized ubiquitously as significant threats to public health. coronaviruses (covs), in the subfamily coronavirinae, are enveloped, single-stranded positive-sense rna viruses with spherical virions of 120-160 nm (king et al., 2012) . they are among the largest rna viruses, with complex polyadenylated genomes of 26-32 kb, and are divided into four genera: alphacoronavirus (a-cov) and betacoronavirus (b-cov) (infecting mainly mammals), and gammacoronavirus (c-cov) and deltacoronavirus (d-cov) (infecting mainly birds) (king et al., 2012; woo et al., 2012) . infection with covs is often asymptomatic, however, they can be responsible for a range of respiratory and enteric diseases of medical and veterinary importance. chief among these is the severe acute respiratory syndrome (sars)-cov, which caused a pandemic in [2002] [2003] . this outbreak lasted for 8 months, infected 8096 people and resulted in 774 deaths (who, 2004) . since then, a renewed public health interest in these viruses has been stimulated by the emergence of a novel b-cov in nine people from the middle east. in these cases the patients suffered from acute, serious respiratory illness, presenting with fever, cough, shortness of breath and difficulty breathing (zaki et al., 2012) . five cases later died. 3these authors contributed equally to this work as joint first authors. 4these authors contributed equally to this work as joint last authors. it is currently unclear where this particular virus came from, though genomic analyses have shown similarity to bat covs (zaki et al., 2012) . given that the majority of emerging pathogens are known to originate in animals (jones et al., 2008) , the concern is that this current outbreak may represent a further example of zoonotic transmission from wildlife to people, though further ecological, immunological and evolutionary information is still required to confirm this. knowledge about cov diversity has increased significantly since the sars pandemic, with the description of several novel viruses from a wide range of mammalian and avian hosts (cavanagh, 2005; chu et al., 2011; dong et al., 2007; felippe et al., 2010; guan et al., 2003; jackwood et al., 2012; lau et al., 2012b; woo et al., 2009a woo et al., , b, 2012 . bats in particular seem to be important reservoirs for covs, and discovery efforts have been increasingly focused on them since the recognition of sars-like covs in rhinolophid species (lau et al., 2005; li et al., 2005) and because bats appear to be reservoirs for a large number of other viruses (calisher et al., 2006; drexler et al., 2012; jia et al., 2003; leroy et al., 2005; rahman et al., 2010; towner et al., 2007) . several of the novel covs described in the last decade were identified in bats of various species and demonstrate a strong association between bats and covs (august et al., 2012; carrington et al., 2008; chu et al., 2009; dominguez et al., 2007; drexler et al., 2011 drexler et al., , 2010 falcó n et al., 2011; ge et al., 2012b; gloza-rausch et al., 2008; li et al., 2005; misra et al., 2009; osborne et al., 2011; pfefferle et al., 2009; quan et al., 2010; reusken et al., 2010; tong et al., 2009b; woo et al., 2006; yuan et al., 2010) . the large number of covs that continue to be described in bats suggest that many (if not most) bat species might be associated with at least one cov. given that there arẽ 1200 extant bat species known, the existence of an equally large diversity of covs must be considered likely. initially, most discovery effort was targeted towards bats from china (ge et al., 2012a; lau et al., 2010b; li et al., 2005; tang et al., 2006; woo et al., 2006) , followed by limited surveillance in other south-east asian countries including japan, the philippines and thailand (gouilh et al., 2011; shirato et al., 2012; watanabe et al., 2010) . in the old world, novel covs have been found in both europe and africa (august et al., 2012; drexler et al., 2011 drexler et al., , 2010 gloza-rausch et al., 2008; pfefferle et al., 2009; quan et al., 2010; reusken et al., 2010; rihtaric et al., 2010; tong et al., 2009b) . in contrast, very few investigations have been conducted in the new world and little is known about the diversity of covs found here. dominguez et al. (2007) were the first to test bats in the new world for cov, followed by donaldson et al. (2010) and osborne et al. (2011) . these groups tested bats captured in colorado and maryland and found a-covs from five different species of evening bats (eptesicus fuscus, myotis evotis, myotis lucifugus, myotis occultus and myotis volans). all were unique compared with covs found in asia. misra et al. (2009) then tested m. lucifugus samples from canada and detected a similar a-cov to those found in myotis bats from colorado. in south america, carrington et al. (2008) identified an a-cov in two species of leaf-nosed bats, carollia perspicillata and glossophaga soricina, which clustered most closely with covs from north american and european bats. nothing is known about the diversity of covs in mexico. many of the bats studied in canada and the usa are also found in mexico, yet it is unknown whether similar viruses are found here. it is also unknown whether b-covs exist in the americas, or whether the a-covs predominate. this is a substantive gap in our knowledge of cov ecology because one-third of all bat species (and 75 % of all known bat genera) are found in the neotropics, which includes southern mexico (osborne et al., 2011; wilson & reeder, 2005) . it seems probable that the high ecological, trophic and taxonomic diversity found in neotropical and nearctic bats in mexico (arita & ortega, 1998) would be matched by an equally diverse population of novel covs. in this study we examined 42 species of bats using broadly reactive consensus pcr for the discovery of novel covs, and found an additional 13 viral lineages/clades, clustering in both the a-cov and the b-cov genera. phylogenetic analysis of these new viruses has provided insight into the molecular epidemiology of covs, and shows that host speciation is a significant driver in cov evolution. the goal of this study was to increase our knowledge of cov diversity in bats from southern mexico. three sites were included in the study: campeche, chiapas and mexico city (mexico distrito federal; d.f.) ( fig. 1 ). at two of the sites (chiapas and campeche) bats were captured in disturbed and undisturbed habitat to investigate how anthropogenic activity may affect host and viral diversity. such habitat gradients do not exist in d.f., which is a highly urbanized site. a total of 1046 samples were collected from 606 individuals, of 42 different bat species (table 1) . host diversity was examined at all sites. in chiapas, a species richness of 32 was recorded, and the calculated shannon-wiener diversity index (h9) was 2.81 (table 2) . a comparison of undisturbed and disturbed habitat in chiapas (shannon t-test) revealed no significant difference in richness and diversity (p50.11). in campeche the overall species richness was 16 and the diversity index h9 was 2.167 (table 2) . again, no significant difference in host diversity was seen between the undisturbed and disturbed habitats (p50.44). previous work has shown that bat diversity often reflects the level of disturbance for a given habitat, with lower diversity recorded in disturbed areas (medellín et al., 2000) . no such distinction was observed here between disturbed and undisturbed sites. this may reflect the dominance of bats from the genera artibeus and carollia (table 1) , both of which contain species that are known to be more adaptable and resistant to the effects of habitat fragmentation (medellín et al., 2000) . an increased sampling effort including larger spatial and temporal scales will be needed to assess whether the abundance and (fig. 1b) . richness of less-well represented species alter the overall bat diversity in each fragment. in d.f. (mexico city), eight species were captured and the diversity index h9 was 1.69 (table 2) . sampling effort was not consistent among the three sites, precluding any direct comparisons of diversity between chiapas, campeche and d.f. broadly reactive consensus pcr revealed cov sequences in 32/606 (5.3 %) bats (table 1) . sequence analyses indicated high phylogenetic diversity and the presence of 13 distinct clades at the nucleotide level (fig. 2) . clades 5a/5b and 11a/ 11b had high nucleotide sequence identity and collapsed into a single group when analysed at the amino acid level (data not shown). nine of the viruses clustered with known a-covs, and four clustered with b-covs (fig. 2) . one of the a-covs (mex_cov-6) was closely related to a virus identified previously in an eptesicus fuscus bat, sampled on the appalachian trail in maryland, usa (donaldson et al., 2010) . we therefore extend the known geographical range of this virus to south-eastern mexico and present the discovery of a further 12 novel covs. prior to this study, very little was known about the diversity of covs in the neotropics, despite the high diversity of bat species found here (wilson & reeder, 2005) . here, we demonstrate that several additional viruses from both the genus alphacoronavirus and the genus betacoronavirus exist in mexico. this particular study was limited to the analysis of a 329 bp fragment of the rna-dependent rna polymerase (rdrp), however, it was sufficient for the identification of these novel strains and therefore satisfied our primary goal of discovery. cov-positive sample types included 27 rectal swabs, four oral swabs and one blood sample (annotated on fig. 2) . the high number of positive rectal samples agrees with previous studies, which showed cov detection in bats to be almost exclusively restricted to faeces (lau et al., 2005; li et al., 2005; pfefferle et al., 2009; tang et al., 2006) . detection in oral swabs has also been demonstrated, but much less frequently (carrington et al., 2008) . phylogenetic analyses of this short fragment show that covs cluster based on the relatedness of host species. fig. 2 shows that all of the a-covs detected in phyllostomid bats cluster together; as do all a-covs discovered in miniopterid bats. the families vespertilionidae and molossidae are closely related (agnarsson et al., 2011; teeling et al., 2005) , and viruses from these bats also cluster together, though the additional presence of cov hku2 (from a rhinolophid bat; woo et al., 2006) in this group is currently unexplained. in the b-cov genus a similar pattern is observed. all viruses identified in rhinolophid bats cluster together, as do viruses from the vespertilionid/molossid group, and equally so in the related mormoopid/phyllostomid group. these results suggest purifying selection, which is apparently effective at the level of host species or genus. for example, the a-cov mex_cov-1 was only found in carollia spp. bats, but could be present variably in the species carollia sowelli or carollia perspicillata. the same is true of the b-covs mex_cov-11a and mex_cov-11b, both of which were only found in artibeus spp. bats, but which could be present in either artibeus lituratus or artibeus phaeotis. mex_cov-6 was found in an eptesicus sp. bat and clustered very closely with the previously identified eptesicus-associated cov (genbank accession no. hq585086). finally, the close association of mex-cov-7 and -8 with myotis velifer and tadarida brasiliensis, respectively, also suggest strong host specificity. these results agree with previous studies that show individual covs are associated with a single species or genus, even among co-roosting species -including miniopterus, rousettus, rhinolophus and hipposideros bats (chu et al., 2006; drexler et al., 2010; gouilh et al., 2011; pfefferle et al., 2009; quan et al., 2010; tang et al., 2006) . and the same appears to be true in myotis ricketti in asia (tang et al., 2006) , in chaerephon and rousettus in africa (tong et al., 2009b) , and in nyctalus and myotis spp. bats in europe (august et al., 2012; drexler et al., 2010; gloza-rausch et al., 2008) . in all cases it was concluded that even if populations of these species were thousands of kilometres apart, highly similar covs could be detected. it is important to qualify that our results are based on a short sequence, and additional studies will be required to assess whether our observations are consistent when additional sequences from other genes/proteins are considered. mex_cov-5b was the only virus to be found in two distinct (but related) genera, having been detected in both artibeus and carollia bats (fig. 2) . such findings have been reported previously, albeit rarely (lau et al., 2012a; osborne et al., 2011; tong et al., 2009a) , and demonstrate that covs can infect individuals from different genera/suborders. it is interesting to note that this particular bat (carollia sowelli, pmx-1232) was captured in a disturbed habitat. increased efforts for viral discovery in this region will be required to investigate whether disturbed habitats provide increased risk or opportunity for viruses to spillover into new species, as previously suggested (cottontail et al., 2009; keesing et al., 2010; suzán et al., 2012) . that said, the health risk to people probably remains low, and bats should not be viewed as a liability, especially given the vital ecosystem functions they serve (medellín, 2009 ). strong associations of covs with host species/genus could prove to be extremely useful in identifying potential eu769557 btcov ify2ba carollia perspicillata (phyllostomidae) eu769558 btcov 1co7ba glossophaga soricina (phyllostomidae) ) ) ) ) ) ) ) ) ) ) ) e) ) ) ) ) ) ) ) reservoirs for viruses that do spillover into other species, assuming that an emergent virus still shares sufficient similarity to those circulating in the original host. a phylogenetic analysis of the new human b-cov that recently emerged in saudi arabia showed that the virus clusters with viruses from bats in the vespertilionid/ molossid families, and that the closest relative is the mex_cov-9 virus that was identified in a nyctinomops laticaudatus bat from this study (fig. 3) . sequence identity between these two viruses is 86.5 % at the nucleotide level, but 96.5 % at the amino acid level. when only the first and second nucleotide positions are considered, nucleotide identity jumps to 97.1 %, demonstrating both that there is strong purifying selection acting on these viruses, and that mex_cov-9 and human b-cov have probably been evolving separately for quite some time. these results do not mean the saudi arabian cov originates in nyctinomops spp. bat, but do suggest that any search for the original reservoir of this virus should perhaps focus on bats in the molossid/vespertilionid families, or the related nycterid/emballonurid families. artibeus was the only genus shown to contain more than one cov, with the detection of the a-covs mex_cov-5a and 5b, mex_cov-4, and the b-covs mex_cov-11a/b. however, bats in this genus were also the most frequently sampled (table 1) , which probably explains why more viruses were identified. other studies have also identified multiple covs within a single species, including rhinolophus sinicus (yuan et al., 2010) and rousettus leschenaulti in china (lau et al., 2010a) , and miniopterus spp. and rhinolophus spp. from europe ; all of which were shown to be doubly and triply infected with different covs. further studies focusing on rarely represented species would be needed to investigate whether population size determines the ability for bats to carry more than one cov, or whether all genera are capable of supporting multiple covs independently of population size. in this study, when multiple covs were discovered in a given bat species/genus they were often closely related, for example a-covs mex_cov-5a/b and b-covs mex_cov-11a/b. when examined at the amino acid level, these clades collapsed into single groups, yet they maintained separate clades at the nucleotide level, suggesting the contemporary evolution of new strains in these bats. together, these results highlight the importance of screening sufficient numbers of individuals per species when attempting to describe viral diversity in a given population/region. methods used to assess the diversity of host species were also used to measure cov diversity at undisturbed and disturbed sites in chiapas and campeche. no significant difference was seen in cov diversity across gradients (chiapas, p50.10; campeche, p50.47), mirroring the non-significant difference also observed in the host (described above). sites and sampling. bats were captured at three different sites in mexico, the reserva de la biosfera montes azules (chiapas), the reserva de la biosfera calakmul (campeche), and mexico city (d.f., fig. 1 ). the first two sites, located in south-eastern mexico represent regions of high species diversity, and are characterized by large tracts of continuous primary vegetation, while mexico city represents a highly urbanized site. in chiapas and campeche bats were collected in two landscape gradients, assigned as: (1) 'undisturbed' forest (ud), where any sign of human impact is largely absent; and (2) 'disturbed' (d), defined as the transition zone between areas of primary vegetation and agriculture/livestock, and by the presence of urban areas. landscape units were separated by at least 10 km. capture effort included two nights of trapping using 569 m mist nets by roosts or foraging sites. nets were opened at dusk and remained open for 4 h consecutively. identification of animals was made using field guides (medellín et al., 2008) . oral and rectal swabs, and blood were collected (when possible) from each animal. for blood samples ,10 % of the blood volume was collected and for small bats blood was taken using protocols previously described (smith et al., 2010) . a veterinarian was present for all sampling and all animals were released safely at the site of capture. samples were collected directly into lysis buffer and preserved at 280 uc until transfer to the center for infection and immunity for cov screening. capture and sample collection was approved by the institutional animal care and use committee at the university of california, davis (protocol number: 16048). laboratory testing. total nucleic acid was extracted from all samples using the easymag (biomérieux) platform, and cdna synthesis performed using superscript iii first strand synthesis supermix (invitrogen), all according the manufacturer's instructions. cov discovery was performed using broadly reactive consensus pcr primers, targeting the rdrp (quan et al., 2010) . pcr products of the expected size were cloned into strataclone pcr cloning vector and sequenced using standard m13r primers. if an individual tested positive for cov, the species of the bat was secondarily confirmed with genetic barcoding, targeting both cytochrome oxidase subunit i and cyt-b mitochondrial genes, as described previously (townzen et al., 2008) . analysis. sequences were edited using geneious pro (5.6.4). alignments were constructed using clustal w, executed through geneious, and refined manually. neighbour-joining and maximumlikelihood trees were built in mega (5.0), and bootstrapped using 1000 repetitions. nucleotide trees that represent a consensus of both methods are presented. evaluations of host and viral diversity at each site/habitat were made using the shannon-wiener diversity index (h9) using the past 1.81 software. this index takes into account the number of individuals as well as the number of taxa. a 0 value means that the community has only a single taxon. comparison of the shannon-wiener diversities (entropies) were calculated between habitat types for each region using the shannon t-test, described by poole (1974) . viruses discovered in this study are indicated by 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mitochondrial cytochrome oxidase subunit i and cytochrome b gene sequences bat coronaviruses and experimental infection of bats, the philippines summary of probable sars cases with onset of illness from 1 mammal species of the world molecular diversity of coronaviruses in bats more and more coronaviruses: human coronavirus hku1 discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus intraspecies diversity of sars-like coronaviruses in rhinolophus sinicus and its implications for the origin of sars coronaviruses in humans isolation of a novel coronavirus from a man with pneumonia in saudi arabia we acknowledge funding from the united states agency for international development (usaid) emerging pandemic threats predict, nih-ai57158 (nbc-lipkin), nih niaid r01 a1079231 (non-biodefense eid), dtra and conacyt ( # 290674). we thank angélica menchaca, ana cecilia ibarra, adriana fernández, william karesh, jonna mazet, karen moreno, stephen morse, monica quijada, ivan olivera and ashley case. we further thank dr nicole arrigo for editing and comments on the manuscript. the contents of this paper are the responsibility of the authors and do not necessarily reflect the views of usaid or the us government. key: cord-288451-npefpo3t authors: yinda, claude kwe; zeller, mark; conceição-neto, nádia; maes, piet; deboutte, ward; beller, leen; heylen, elisabeth; ghogomu, stephen mbigha; van ranst, marc; matthijnssens, jelle title: novel highly divergent reassortant bat rotaviruses in cameroon, without evidence of zoonosis date: 2016-09-26 journal: sci rep doi: 10.1038/srep34209 sha: doc_id: 288451 cord_uid: npefpo3t bats are an important reservoir for zoonotic viruses. to date, only three rva strains have been reported in bats in kenya and china. in the current study we investigated the genetic diversity of rvas in fecal samples from 87 straw-colored fruit bats living in close contact with humans in cameroon using viral metagenomics. five (near) complete rva genomes were obtained. a single rva strain showed a partial relationship with the kenyan bat rva strain, whereas the other strains were completely novel. only the vp7 and vp4 genes showed significant variability, indicating the occurrence of frequent reassortment events. comparing these bat rva strains with currently used human rva screening primers indicated that most of the novel vp7 and vp4 segments would not be detected in routine epidemiological screening studies. therefore, novel consensus screening primers were developed and used to screen samples from infants with gastroenteritis living in close proximity with the studied bat population. although rva infections were identified in 36% of the infants, there was no evidence of zoonosis. this study identified multiple novel bat rva strains, but further epidemiological studies in humans will have to assess if these viruses have the potential to cause gastroenteritis in humans. rva/bat-wt/ken/ke4852/2007/g25p [6] , and possesses the following genotype constellation: g25-p[6]-i15-rx-c8-mx-ax-n8-t11-e2-h10 17 . two other bat rvas were found in china in a lesser horseshoe bat (rhinolophus hipposideros) and a stoliczka's trident bat (aselliscus stoliczkanus) named rva/bat-tc/chn/mslh14/2012/ g3p [3] and rva/bat-tc/chn/myas33/2013/g3p [10] , respectively 18, 19 . phylogenetic analysis showed that strains mslh14 and myas33, although sampling sites were more than 400 km apart, shared the same genotype constellation (g3-p[x]-i8-r3-c3-m3-a9-n3-t3-e3-h6) except for the p genotype which was p [3] for mslh14 and p [10] for myas33. to further study the genomics of rva in bats and their zoonotic potential in humans, we screened stool samples of straw-colored fruit bats (eidolon helvum) living in close proximity with humans in the south west region of cameroon (fig. 1) , as well as samples from infants with gastroenteritis. our choice of this region is due to the fact that bats are considered a delicacy and the species sampled are the most commonly eaten bat species in these localities. sample characterization. a total of 24 pools of 3-5 bat fecal samples were constituted, enriched for viral particles and sequenced. illumina sequencing yielded between 1.1 and 7.8 million reads per pool, and diamond classification 20 of the obtained contigs indicated that five pools contained a significant amount of rva sequence reads. the percentage reads mapping to rva in each pool ranged from 0.1-2.4% (table 1) . partial segments were completed by regular pcr and sanger sequencing, to obtain at least the entire orf for each of the obtained variants. obtained sequences were used for phylogenetic comparison with a selection of representative members of each genotype. the rva strains discovered in this study were named rva/bat-wt/cmr/batli08/2014/ g31p [42] , rva/bat-wt/cmr/batli09/2014/g30p [42] , rva/bat-wt/cmr/batli10/2014/g30p [42] , rva/bat-wt/ cmr/batly03/2014/g25p [43] and rva/bat-wt/cmr/batly17/2014/g30p[47] hereafter referred to as batli08, batli09, batli10, batly03 and batly17, respectively. all the obtained sequences were highly divergent from established genotypes and were therefore submitted to the rotavirus classification working group (rcwg) for novel genotype assignments (see below) and to genbank (accession numbers: kx268743-kx268797). phylogenetic analysis. the vp7 gene of batly03 was 96% identical (on the nucleotide (nt) level, supplementary data s1) to the kenyan bat rva strain ke4852 counterpart, which had been previously classified as a g25 genotype (fig. 2 ). batli10 and batli09 were 100% identical and also clustered closely with strain batly17 (92% similar). this cluster was only distantly related to all other known vp7 rva sequences as well as to strain batli08, which also formed a unique long branch in the phylogenetic tree. both clusters only show similarities below 74% with established genotypes (fig. 2) . the vp7 of these 4 strains (batli10, batli09, batly17 and batli08) did not belong to any of the established rva g-genotypes, according to the established criteria 6 , and were assigned genotypes g30 (batli09, batli10 and batly17) and g31 (batli08) by the rcwg. for vp4, vp1 and vp3, all five cameroonian bat rvas strains were distantly related to other known rva strains, including the kenyan and chinese rva strains and were therefore assigned to novel genotypes according to the rcwg classification criteria (fig. 3) . the vp4 gene of strains batli08, batli09 and batli10 (representatives of the novel genotype p[42]) were almost 99-100% identical to each other and only 56-75% identical to any other p-genotype. that of strains batly03 and batly17 had 29% nt dissimilarity to each other and their nt identity ranged from 60-76% with other p-genotypes and therefore were assigned the genotypes p[43] and p[47], respectively. the vp1 and vp3 genes of batli08, batli09, batli10 and batly17 were nearly identical (nt identity range 99-100%) and clustered together but distinct from other established r and m-genotypes, thereby representing the new genotypes r15 and m14, respectively (fig. 3) . the vp1 and vp3 genes of batly03 were only distantly related to the other four cameroonian bat rvas (68-77% nt identity) and are the sole member of the newly assigned genotypes r16 and m15, respectively (fig. 3) . the vp6, vp2, nsp2, nsp3 and nsp5 gene segments of 4 of our strains (batli08, batli09, batli10 and batly17) were distantly related to their counterparts of other mammalian and avian rvas fig. 4) . for all the 4 strains, these gene segments clustered together and were 99-100% identical to each other and consequently they constitute new genotypes for the different gene segments (i22, c15 n15, t17 and h17, respectively). the vp6, vp2, nsp2, nsp3 and nsp5 gene segments of batly03 phylogenetically clustered together with the kenyan bat rva strain ke4852 in the previously established i15, c8, n8, t11 and h10 genotypes, respectively (fig. 4) . for nsp1, the cameroonian bat strains batli08, batli09, batli10 and batly17 clustered closely together (98-100% nucleotide sequence identity) in the novel genotype a25, and showed only 67% nucleotide similarity to strain batly03 (a26). these 5 new nsp1 gene segments were only 42-45% identical to that of the most closely related established nsp1 genotype a13 and a14 (from cow) (fig. 5) . the nsp4 gene segments of all the 5 rvas discovered in this study were quite divergent to those of other known bat rotaviruses (at most 69% nucleotide sequence identity) and other rvas (approximately 45-68% nucleotide similarity) forming two distinct clusters. the nsp4 gene segments of strains batli08, batli09, batli10 and batly03 (genotype e22) were 100% identical but all were 37-38% divergent from that of batly03 (e23, fig. 5 ). batli10 (p10) batlyp03 (p03) batli08 (p08) batli09 (p09) batlyp17(p17) bat rotaviruses in humans? several different primer pairs are currently being used to detect human rva vp7 and vp4 gene segments, to determine the g-and p-genotypes using sequencing or multiplex pcr assays [21] [22] [23] [24] . in order to find out if the currently used human rva screening primers would detect the bat rva strain from this study in case of zoonosis, we compared these primers with their corresponding sequences in the respective gene segments (table 2 and supplementary data s2). overall, the similarity percentages for the vp7 forward and reverse primer between the bat rva sequences and the human primers were 57.1-100% and 63.2-95%, respectively. for vp4, the percentage similarity ranged from 63.6-94.4% and 76.2-85.7% for the forward and reverse primers, respectively. vp7 forward primers beg9, sbeg9 and 9con1-l showed a (near) perfect match with batly03-g25, whereas strain batli08-g31 and batly17-g30 (first 6 nt are missing for this strain), showed up to 10 and 4 nucleotide mismatches at the 3′ end of the primers, respectively. vp7 forward primer 9con1-l showed a perfect match with all the genotypes (g25, g30 and g31). considering the vp7 reverse primers enda, vp7-rdeg, end9 and rvg9, batly03-g25 did not show a perfect match as there were 4, 1, 4 and 2 mutations, respectively. the mismatches with enda, vp7-rdeg and rvg9 were near the middle or at the 5′ end of the primer, whereas 2 of those of end9 were close to the 3′ end. comparing the same vp7 reverse primers with strain batli08-g31 and batli09-g30 also showed mismatches. for enda and vp7-rdeg maximum 2 mismatches are located in the middle or near the 5′ -end, whereas for end9 and rvg, there were multiple mismatches of which 2 and 7 mismatches, respectively were right at the 3′ -end. for vp4 forward primer vp4additionally, to determine if any of these bat rvas could cross species and infect humans, we designed primers (rva-vp6_40f and rva-vp6_1063r) from an alignment of both human and bat rva vp6 segments to screen 25 diarrheic infant samples (infants living around the same region where the bat samples were collected). thirty-six percent of human samples were positive for rva, however, none of them was of bat rva origin. they all possessed the typical human genotype i1 and were 99% identical to the gambian, senegalese, belgian and brazilian wa-like g1p [8] (table 3) . screening human samples for these bat rvas indicated no interspecies transmissions and primer comparison showed that not all the strains can be picked up with the currently used screening primers. bats have been proven to harbor several human pathogenic viruses including sars, mers-related coronaviruses, as well as filoviruses, such as marburgvirus, or henipaviruses, such as nipah and hendra virus [13] [14] [15] , but bat rvas [10] , were isolated from a lesser horseshoe bat, and a stoliczka's trident bat in china, respectively 18, 19 . to better understand the spread and diversity of rva in bats, we performed an rva screening in cameroonian bats, after trapping both male and female, young and adult bats close to human dwellings in muyuka, limbe and lysoka localities of the south west region of cameroon (fig. 1) . using an unbiased viral metagenomics approach, we identified 5 divergent novel bat rva strains, 4 of which were genetically similar to each other. the fifth strain was related to the kenyan bat strain. interestingly, all these rvas were identified in adult (both female and male) straw-colored fruit bats (eidolon helvum) which is in contrast to human and other animals whereby rva (symptomatic) infections occur mostly in juveniles 1 . also, diarrhea or other obvious signs of sickness were not noticed in these bats. this may suggest that bats may undergo active virus replication and shedding without obvious clinical signs 25 , which potentially could increase human exposure. even though there exists a considerable genetic divergence between bat rva and human rva, suggestions have been made about potential interspecies transmission of chinese and kenyan bat rva strains. the two table 2 . nucleotide comparison between the sequence of human rva screening primers for vp7 (beg9, sbeg9, 9con1-l, enda, vp7-rdeg, end9 and rvg9) and vp4 (vp4-1-17f, con3 and con2) with their corresponding region of the new bat rva genotype. black shaded nucleotides indicate dissimilar nucleotides between a strain segment sequence and primer and bold numbers at the beginning and end of sequence indicate nucleotide positions for different strains. for clarity the reverse complement sequence of the reverse primers is used for comparison with bat rva sequences. chinese rva strains are genetically quite conserved (all segments of both strains have the same genotype except for their vp4 gene). based on genome comparisons of chinese bat and partial human rva strains from thailand (cmh079 and cmh222) and india (69 m, 57 m and mcs60), xia and colleagues speculated that asian bat rvas may have crossed the host species barrier to humans on a number of occasions 19 . in addition, the unusual equine strain e3198 26 shares the same genotype constellation with either myas33 and/or mslh14 in all segments except vp6. this data therefore suggests that this equine rva strain most likely share a common ancestor with asian bat rvas. furthermore, the genotype constellations of these asian bat rva (table 3 ) are reminiscent to the au-1-like genotype backbone of feline/canine-like rva strains, as well as to the genotype constellation of two unusual simian rvas (rrv and tuch) 27 , suggesting that interspecies transmissions might have also occurred in the distant past. moreover, an unusual ecuadorean human rva, ecu534 28 is closely related to bat sequences from brazil recently submitted to genbank. similarly, possible interspecies transmission trends were also suggested by he and colleagues 18 between bovine strain rva/cow-wt/ind/rubv3/2005/g3p [3] and the bat strain mslh14. given the novelty of the bat rva strains described here, it is questionable if the currently used human rva screening primers (for vp7 and vp4) will pick up these divergent strains in case an interspecies transmission from bats to humans would occur. comparisons (table 2 and supplementary data s2) of these primers with the corresponding sequences showed that the primer combination 9con1-l and vp7-rdeg 21, 22 , would most likely detect both g25 and g31 rva strains. also, the combination of either beg9 or sbeg9 with end9 or rvg9 24 might be successful in amplifying g25, but the same combinations might not be able to pick up the novel g31 and g30 genotypes in pcr screening assays. furthermore, the primer combination beg9/sbeg9 and enda 29 are likely to detect g25, but will be unsuccessful in case of g30 and g31 especially if the forward primer beg9 is used. considering vp4, both forward primers, vp4-1-17f 30 and con3 23 in combination with the reverse primer con2 will be sub-optimal in detecting any of the p genotypes. generally, with the exception of strain g25, detection of most of the bat strains will be sub-optimal or not successful at all for the different available primer combinations. therefore, zoonotic events of bat rva strains could easily be missed with the current screening primers depending on the primer combinations, pcr conditions and/or circulating zoonotic strains. in order to investigate the possibility of bat rva infecting humans who are living in close contact with bats, we used novel primers (rva-vp6_40f and rva-vp6_1063r) designed from an alignment of both human and bat vp6 rva segment to screen 25 infant samples from patients with gastroenteritis, living around the same region where the bat samples were collected. interestingly, 36% of human samples were positive, however, none of these were positive for bat rvas. all were of the typical human rva genotype i1 and therefore there is no evidence for interspecies transmissions of bat rva to humans. however, this result is not conclusive as only a small sample size was considered here. sampling a larger number of subjects and from different localities around the region might result in more conclusive answers with respect to the zoonotic potential of these bat rva strains. the genotype constellations of the two chinese bat rvas showed clear indication of recent reassortment event(s) because they possessed different p genotypes (p [10] for myas33 and p [3] for mslh14), and some gene segments were nearly identical whereas others were not 18, 19 . their genotype constellation differs markedly from the kenyan straw-colored fruit bat strain (ke4852). although this strain showed a unique genotype backbone, some of its segments were similar to some human and other animal rvas 17 . moreover, ke4852 share the same genotypes in several gene segments (vp2, vp6, vp7, nsp2, nsp3 and nsp5) with our bat rva strain batly03 indicating possible reassortment events between different bat rva strains, as well as a large geographical spread of this virus. furthermore, batli08, batli09, batli10 and batly17 had conserved genotype constellations (in vp6, vp1-vp3, nsp1-nsp5) with 98-100% nucleotide sequence similarity except for the vp7 of batli08 and vp4 of batly17, again confirming reassortment events within bat rvas. the high genetic divergence and partial relatedness of most of the segments of the different bat rva strains and the ones identified in this study indicate the frequent occurrence of reassortment events in the general bat population and those of cameroon in particular. also, with the current knowledge of the genetic diversity, there seems to exist several true bat rva genotype constellations, as has been previously described for humans, and cats/dogs 10, 31 . however, this needs to be further confirmed by identification of a larger number of rvas from bats from different age groups and different geographical locations. ethical authorization. ethical authorization for the use of human samples was obtained from the cameroon national ethics committee, yaoundé. all human experiments were performed in accordance with the ministry's national ethics committee guidelines. ethical authorization for the protocol and the use of animal samples was also obtained from the cameroon national ethics committee, yaoundé. all animal experiments were performed in accordance with the ministry's national ethics committee guidelines. all experimental protocols used in this study were approved by cameroon national ethics committee. administrative authorization was obtained from the delegation of public health for south west region, cameroon. informed consent was obtained from human subjects or their parents or guardians. bat sample collection. bat samples were collected between december 2013 and may 2014 using a previously described method 32 . briefly, bats were captured in 3 different regions (lysoka, muyuka and limbe) of the south west region of cameroon (fig. 1) using mist nets around fruit trees and around human dwellings. captured bats were retrieved from the traps and held in paper sacks for 10-15 min, allowing enough time for the excretion of fresh fecal boluses. sterile disposable spatulas were used to retrieve feces from the paper sacks, and placed into tubes containing 1 ml of universal transport medium (utm, copan diagnostics, brescia, italy). labeled samples were put on ice and then transferred to the molecular and cell biology laboratory, biotechnology unit, university of buea, cameroon and stored at − 20 °c, until they were shipped to the laboratory of viral metagenomics, leuven, belgium where they were stored at − 80 °c. each captured bat was assessed to determine species, weight (g), forearm length (mm), sex, reproductive state, and age. all captured bats were then marked by hair clipping to facilitate identification of recaptures, and released afterwards. trained zoologists used morphological characteristics to determine the species of the bats before they were released. no clinical signs of disease were noticed in any of these bats. sample preparation for ngs. eighty-seven fecal samples were grouped into 25 pools each containing three to five samples and treated to enrich viral particles as follows: fecal suspensions were homogenized for 1 min at 3000 rpm with a minilys homogenizer (bertin technologies, montigny-le-bretonneux, france) and filtered consecutively through 100 μ m, 10 μ m and 0.8 μ m membrane filters (merck millipore, massachusetts, usa) for 30 s at 1250 g. the filtrate was then treated with a cocktail of benzonase (novagen, madison, usa) and micrococcal nuclease (new england biolabs, massachusetts, usa) at 37 °c for 2 h to digest free-floating nucleic acids. nucleic acids were extracted using the qiaamp viral rna mini kit (qiagen, hilden, germany) according to the manufacturer's instructions but without addition of carrier rna to the lysis buffer. first and second strand cdna synthesis was performed and random pcr amplification for 17 cycles were performed using a whole transcriptome amplification (wta) kit procedure (sigma-aldrich), with a denaturation temperature of 95 °c instead of 72 °c to allow the denaturation of dsdna and dsrna. wta products were purified with msb spin pcrapace spin columns (stratec, berlin, germany) and the libraries were prepared for illumina sequencing using the nexteraxt library preparation kit (illumina, san diego, usa). a cleanup after library synthesis was performed using a 1.8 ratio of agencourt ampure xp beads (beckman coulter, inc., nyon, switzerland) 33 . sequencing of the samples was performed on a hiseq 2500 platform (illumina) for 300 cycles (2 × 150 bp paired ends). partial sequences were completed using rt-pcrs with specific primers (supplementary s4). for gene segments lacking the 5′ and/or 3′ ends of the orf the single primer amplification method (primers in supplementary s4) was used as described previously 34 . sanger sequencing was done on an abi prism 3130 genetic analyzer (applied biosystems, massachusetts, usa). lysoka local clinic and kumba district hospital of the south west region of cameroon ( fig. 1 ) after informed consent was obtained from patients or their parents or guardians. the patients were either diarrheic or came into contact with bats directly (by eating, hunting or handling) or indirectly (if family member is directly exposed to bats). the samples were put in utm containing tubes and stored the same way like the bat samples. screening primers (supplementary data s4) were designed from a consensus sequences of human and bat vp6 rvas and a total of 25 samples from infants (0-3 years) who had diarrhea were screened by reverse transcriptase polymerase chain reaction (rt-pcr) using the onestep rt-pcr kit (qiagen). the products of positive samples were sequenced using sanger sequencing method. genomic and phylogenetic analysis. raw illumina reads were trimmed for quality and adapters using trimmomatic 35 , and were de novo assembled into scaffold using spades 36 . scaffolds were classified using diamond in sensitive mode 20 . contigs assigned to rva were used to map the trimmed reads using the burrows-wheeler alignment tool (bwa) 37 . open reading frames (orf) were identified with orf finder analysis tool (http://www.ncbi.nlm.nih.gov/gorf/orfig.cgi) and the conserved motifs in the amino acid sequences were identified with hmmer 38 . amino acid alignments of the viral sequences and maximum likelihood phylogenetic trees were constructed in mega6.06 39 , using the gtr + g (vp1, vp6, nsp2 and nsp3), gtr + g + i (vp2-vp4, vp7 and nsp1), hky (nsp4) and t92 (nsp5) substitution models (after testing for the best dna/protein model), with 500 bootstrap replicates. nucleotide similarities were also computed in mega by pairwise distance using p-distance model. phylogenetic analyses were performed using appropriate reference strains in addition to the rva discovered in this study. determinants of rotavirus host range restriction-a heterologous bovine nsp1 gene does not affect replication kinetics in the pig virus taxonomy: ninth report of the ictv candidate new rotavirus species in sheltered dogs exotic rotaviruses in animals and rotaviruses in exotic animals full genome-based classification of rotaviruses reveals a common origin between human wa-like and porcine rotavirus strains and human ds-1-like and bovine rotavirus strains recommendations for the classification of group a rotaviruses using all 11 genomic rna segments are human p[14] rotavirus strains the result of interspecies transmissions from sheep or other ungulates that belong to the mammalian order artiodactyla full-length genomic analysis of porcine g9p[23] and g9p[7] rotavirus strains isolated from pigs with diarrhea in south korea. infection, genetics and evolution: journal of molecular epidemiology and 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divergent gene constellations that originated from interspecies transmission and reassortment characterization of novel vp7, vp4, and vp6 genotypes of a previously untypeable group a rotavirus distribution of human rotavirus g types circulating in paris, france, during the 1997-1998 epidemic: high prevalence of type g4 genetic analyses reveal differences in the vp7 and vp4 antigenic epitopes between human rotaviruses circulating in belgium and rotaviruses in rotarix and rotateq genotype constellation and evolution of group a rotaviruses infecting humans metagenomic analysis of the viromes of three north american bat species: viral diversity among different bat species that share a common habitat modular approach to customise sample preparation procedures for viral metagenomics: a reproducible protocol for virome analysis g8 rotavirus strains isolated in the democratic republic of congo belong to the ds-1-like genogroup trimmomatic: a flexible trimmer for illumina sequence data spades: a new genome assembly algorithm and its applications to single-cell sequencing fast and accurate short read alignment with burrows-wheeler transform hmmer web server: interactive sequence similarity searching molecular evolutionary genetics analysis version 6.0. molecular biology and evolution r: a language and environment for statistical computing. r foundation for statistical computing geographic analysis and modeling with raster data this work is licensed under a creative commons attribution 4.0 international license. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in the credit line; if the material is not included under the creative commons license, users will need to obtain permission from the license holder to reproduce the material. to view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ key: cord-270803-jtv5jmkn authors: wang, lin-fa; walker, peter j.; poon, leo l.m. title: mass extinctions, biodiversity and mitochondrial function: are bats ‘special’ as reservoirs for emerging viruses? date: 2011-11-09 journal: curr opin virol doi: 10.1016/j.coviro.2011.10.013 sha: doc_id: 270803 cord_uid: jtv5jmkn for the past 10–15 years, bats have attracted growing attention as reservoirs of emerging zoonotic viruses. this has been due to a combination of factors including the emergence of highly virulent zoonotic pathogens, such as hendra, nipah, sars and ebola viruses, and the high rate of detection of a large number of previously unknown viral sequences in bat specimens. as bats have ancient evolutionary origins and are the only flying mammals, it has been hypothesized that some of their unique biological features may have made them especially suitable hosts for different viruses. so the question ‘are bats different, special or exceptional?’ has become a focal point in the field of virology, bat biology and virus-host co-evolution. in this brief review, we examine the topic in a relatively unconventional way, that is, our discussion will be based on both scientific discoveries and theoretical predictions. this approach was chosen partially because the data in this field are so limited that it is impossible to conduct a useful review based on published results only and also because we believe it is important to provoke original, speculative or even controversial ideas or theories in this important field of research. lin-fa wang 1 , peter j walker 1 and leo l m poon 2 for the past 10-15 years, bats have attracted growing attention as reservoirs of emerging zoonotic viruses. this has been due to a combination of factors including the emergence of highly virulent zoonotic pathogens, such as hendra, nipah, sars and ebola viruses, and the high rate of detection of a large number of previously unknown viral sequences in bat specimens. as bats have ancient evolutionary origins and are the only flying mammals, it has been hypothesized that some of their unique biological features may have made them especially suitable hosts for different viruses. so the question 'are bats different, special or exceptional?' has become a focal point in the field of virology, bat biology and virus-host co-evolution. in this brief review, we examine the topic in a relatively unconventional way, that is, our discussion will be based on both scientific discoveries and theoretical predictions. this approach was chosen partially because the data in this field are so limited that it is impossible to conduct a useful review based on published results only and also because we believe it is important to provoke original, speculative or even controversial ideas or theories in this important field of research. bats (order chiroptera), one of the most abundant, diverse and geographically dispersed vertebrates on earth, have recently been shown to be reservoir hosts of a number of emerging viruses responsible for severe disease outbreaks in humans and livestock [1 ,2,3]. the first recognition that bats are involved in the ecology of human disease came during the 1920s when rabies virus was identified in bats in south and central america [4] . however, the discovery of henipaviruses in the mid-1990s and the subsequent recognition that bats may be a natural host of sars-like coronaviruses and filoviruses marked a new era of fresh research into the role of bats as an important reservoir host of viruses which have the potential to cause disease in humans and livestock [3, [5] [6] [7] [8] [9] . the recent surge of interest in bats as a reservoir of viruses was driven by two factors. first, in less than 20 years, several high profile viral pathogens have been proven or hypothesized to have a bat origin. since hendra virus was first discovered in 1994, there have been at least 17 known spillover events in australia with a mortality rate in humans of approximately 60% [10] . the closely related nipah virus has been responsible for devastating disease outbreaks in malaysia, bangladesh and india with mortality rates ranging from 40% to 90%, resulting in the deaths of approximately 200 humans [11] . filoviruses (ebola and marburg viruses) have caused outbreaks in africa with associated human mortality rates as high as 90%, and have been linked to mass gorilla die-offs, making them both a public health and conservation concern [12] [13] [14] . the outbreak of severe acute respiratory syndrome (sars) in 2002-2003, due to a previously unknown coronavirus, resulted in more than 8000 human infections with a mortality rate close to 10% and an estimated cost of $50 billion in lost tourism and trade [15] [16] [17] . the association of these high profile pathogens and disease outbreaks with bats has led to an increase in public interest, funding and research activities on these and many other bat-borne viruses. however, it should be emphasized that, although closely related sars-like coronaviruses have been detected in horseshoe bats, the exact natural reservoir of the coronavirus responsible for the sars outbreaks is still unknown [8, 9] . the true natural reservoir of ebola virus is also still being debated as rodents, insectivores and bats have all been identified as potential sources of infection in primates [18 ,19] . the second driver for the recent surge in bat virus research has been advances in modern molecular techniques which have presented opportunities for discovery of novel bat viruses, that were considered impossible or nonpractical just a decade ago. using pan-virus-specific primers and next-generation sequencing, it is now possible to detect and characterize novel viral sequences without the need for virus isolation by cell culture or the identification of virions by electron microscopy. numerous publications in the past few years have reinforced the observation, first made by sulkin and allen in 1974 [20], that bats carry a wide range of novel rna and dna viruses. these results also provide support to the notion, as first observed during the investigations of bat coronaviruses (see below), that bats within a geographic location and/or taxonomic group have an unusual ability to harbor a large number of genetically diverse viruses. more recently, two metagenomic studies on bat fecal samples have revealed a great number of novel bat viruses, some of which have moderate sequence identity to previously known mammalian viruses, including members of the parvoviridae, circoviridae, picornaviridae, adenoviridae, poxviridae, astroviridae, herpesviridae and coronaviridae [21 ,22 ] . further systematic surveillance will be required to determine whether bats are the natural hosts of these novel viruses, but these results clearly indicate that there are many bat viruses yet to be identified. interestingly, similar to other metagenomic analyses of human or other animal fecal samples, these studies have also identified numerous sequences derived from viruses infecting insects, plants and bacteria. although these viruses are unlikely to infect bats, one might hypothesize that they could play an important role in facilitating the dispersal of these viruses to different geographical locations and different hosts. the high detection rate and great genetic diversity of viral sequences from bats have not only propelled further scientific and public interest in this field, but also led to debate on the importance of bats as reservoirs of zoonotic viruses. the question 'are bats different?' has been raised at many international conferences and has been the topic of several previous reviews [1 ,2,20,23 ]. while the currently available data are too limited to provide a conclusive answer, this review aims to examine different hypotheses which may eventually allow us to resolve this intriguing and fundamentally important question. it should be noted, however, that some of the discussions presented in this review are largely speculative or even controversial. this has been done intentionally by the authors to invigorate discussion and further research on this topic. bats have several features that might help to explain the seemingly high rate of virus detection. bats constitute the second largest order of mammals. there are about 1240 bat species worldwide, which represents more than 20% of all mammalian species [24] . bats are classified in the order chiroptera in which there are two suborders: the yinpterochiroptera (also known as megachiroptera), which contains the megabats, and the yangochiroptera (microchiroptera), which includes the majority of microbat families [25] . the wide range of bat species could provide a large 'breeding ground' for viruses. the earliest known bat fossil dates to 52.5 million years ago (mya) [26, 27] . extrapolation of fossil records and genetic data has suggested that the basal split from other placental mammals in the superorder laurasiatheria occurred during the late cretaceous period approximately 80-90 mya, with extensive diversification of extant bat families commencing approximately 62 mya [28] [29] [30] . bat viruses may therefore have co-evolved with or adapted to bats over many millions of years. besides, bats are the only mammalian species that can fly and some bat species can migrate hundreds of miles to their overwintering or hibernation sites [1 ]. thus, bats have more opportunities than terrestrial mammals to have direct or indirect contact with other animal species at different geographical locations, thereby enhancing the opportunity for interspecies virus transmission. in addition, some insectivorous bats exhibit exceptionally long life-spans of 25-35 years and live in panmictic populations comprising of millions of individuals. the long life-span of bats may facilitate the transmission of chronic persistent infections, whereas the unusually large and complex structure of bat populations may ensure a sufficient number of immunologically naive juveniles for bat viruses to persist in bat colonies. some bat species also have a capacity for hibernation over winter or to enter into daily torpor to conserve energy. the reduced body temperature and metabolic rate may suppress robust immune responses and reduce the rate of virus replication, thereby delaying virus clearance from bat populations [20,31 ]. purely a numbers game: more bat species = more viruses? in one of the most comprehensive reviews on bat viruses, calisher et al. [1 ] listed 66 different bat viruses that have either been isolated or detected. since then, many more novel bat viruses, as well as variants of previously known bat viruses, have been reported. in total, 15 virus families -10 families of rna viruses and five families of dna virus -are known to infect 75 bat genera [23 ] . the detection rate of novel viruses or viral sequences appears to have been higher in bats than that in any other mammalian species for the past two decades or more. it could be argued that, as bats represent the second largest group of mammals (comprising 20% of all mammalian species), it is not entirely surprising that there are many bat viruses. however, some of our recent indirect evidence suggests that bats may be atypical hosts of at least some viruses. firstly, the genetic diversity and prevalence of infection of some rna viruses in bats is unusually high. we previously reported the detection of genetically highly diverse astroviruses and coronaviruses in bat fecal samples, with the prevalence of infection of these novel bat viruses in the range of 10-50% [32, 33] . however, similar surveillance studies for astrovirus and coronavirus in rodents sampled at the same geographic location indicated that none of the samples (n = 441) were positive for coronavirus, whereas only 1.6% of the tested brown rat (rattus norvegicus) samples (n = 371) were positive for astroviruses [34 ] . at least at this location, bats appear to harbor many more coronaviruses and astroviruses than rodents. secondly, phylogenetic analysis of viral sequences has revealed that a large number of coronaviruses recognized in other mammalian species share a common ancestor with various other bat coronaviruses ( figure 1 ). these findings suggest that bats are likely to be the natural reservoir from which all presently known mammalian coronavirus lineages have evolved [35] . the high prevalence of viral infection in bats, together with some of the unusual characteristics of bats discussed above, may have facilitated the transmission of bat viruses to other mammals. as surveillance data for viruses in wildlife are currently scarce, it may be premature to conclude that bats host a greater diversity of viruses than other animals. for example, more than 45 hantaviruses have been identified in rodents -the largest group of mammals on earth (40% of all mammalian species) -and each hantavirus appears to have co-evolved with a specific rodent [36, 37] . rodents are also considered to be the natural reservoir of arenaviruses with which they appear to have co-evolved [38] and waterfowls are known to be the natural reservoir of influenza viruses [39] . it is possible that bats, rodents, birds and other wildlife may be ancient reservoirs of different sets of virus taxa. further systematic surveillance for viruses in different wildlife populations using metagenomics or other molecular approaches is required to determine if the large number of viruses identified in bats is just simply numbers game. nevertheless, the prevalence of infection of certain bat virus families appears to be much higher than has been reported for the viral families co-evolved with rodent and avian species, suggesting that bats may have some intrinsic properties which make them more suited as a reservoir host. the five great mass extinctions that have punctuated the history of life on earth have played a major role in shaping the modern biosphere [40] and it is reasonable to assume that mass extinctions will also have impacted profoundly on the evolutionary history of viruses. the most recent mass extinction, the k-t extinction, occurred 65 million years ago. it followed the earth impact of the large bolide that created the 180-300-km-wide chicxulub crater in northern yucatan, mexico [41, 42] and resulted in 70-80% reduction in marine diversity at the species level, 50% at the genus level, and the loss of 70% of all species worldwide [43, 44] . the k-t extinction will also have impacted on viral diversity. indeed, as the survival of virus populations is inextricably linked to the survival of their host species, the rate of virus extinction during precipitous mass extinctions is likely to have been far greater than that of their hosts. virus extinction will have occurred not only as a consequence of host extinction but also through decreases in host population size and host isolation to a level that could not sustain ongoing virus transmission (table 1) . even temporary host species decline or isolation, followed by recovery and survival, will have had potential for virus extinction. survival will have favored those viruses that could persist either in the environment or in the host, those that caused no disease or mortalities, those that were transmitted vertically, those with a broad host range, and those for which the host survived with little impact on population size. surviving host species that were largely unaffected by such a devastating mass extinction event are likely therefore to have been important sources of extant viral biodiversity. because of the exceptional paucity of the fossil record, the evolutionary history of bats is not as well documented as many other vertebrate lineages. however, as discussed above, bats are known to have origins in the late cretaceous period and appear to have diversified rapidly during the period immediately after the k-t extinction [24, 45, 46] . it has been argued that the short intense heat pulse caused by the ballistic atmospheric re-entry of ejecta following the bolide impact created a catastrophe that set the stage for later evolutionary events [47] . indeed, ancestral bats, rodents, insectivores and some birds are likely to have had the characteristics of animals mass extinctions, biodiversity and mitochondrial function: wang, walker and poon 651 table 1 effects of the k-t bolide impact on host populations and likely consequences on the contemporary virosphere. characteristics required for survival of the nuclear winter and food chain collapse that is predicted to have followed the initial impact [51] . bats are now second only to rodents as the most ecologically and morphologically diverse mammalian clades, adapting to almost every terrestrial environment and accounting for over 20% of extant mammalian diversity [24] . it follows that bats may also be one of the most important sources of extant mammalian virus diversity and supports the view that bat viruses may have ancient origins and a long history of co-evolution with their hosts. as described above, bats do appear to host a strikingly wide range of viruses and are likely the natural reservoir from which all presently known mammalian coronavirus lineages have evolved [35] . it has also been suggested that the ubiquity, wide genetic diversity and deeply rooted phylogeny of bat lyssaviruses and paramyxoviruses indicate that bats may be their natural ancestral hosts [52, 53] and bats, rodents and shrew have been found to contain integrated filovirus-like genome elements that suggest a very ancient relationship [18 ] . although there may not be a direct link between host diversity and virus diversity, the long evolutionary history and the ecological diversity of bats will also have presented a myriad of opportunities for cross-species transmission of viruses to and from many other host species, further enhancing their role as amplifiers of viral biodiversity. the discovery of endogenous viral elements (eves) integrated into animal genomes appears to provide the long-sought opportunity to trace the deep evolution of viruses and the role bats may have played in shaping the modern virosphere [54] . as discussed above, persistence in the absence of pathology or disease appears to be a common characteristic of bat viruses in their natural host population and this is also indicative of a highly evolved relationship [1 ,18 ,32,55 ] . the ecological balance that maintains infection and transmission in the absence of disease favors both pathogen and host and it can be argued that each may have contributed to its evolution [1 ]. the host aims to detect and contain or eliminate the pathogen through an effective immune response to avoid disease or mortality. the virus needs only to maintain replication and transmission beyond the extinction threshold (ro > 1) and the long-term survival of the virus may be improved if this can be achieved in the absence of disease or mortality [56] . however, the high replication rate, mutation frequency and potential for recombination of viruses, particularly rna viruses, provide a potential for continual adaptation and refinement that far exceeds that of their hosts [57] . it could be argued, therefore, that the most significant characteristic of viral infections in bats may not be the effectiveness of a highly evolved host immune response, but rather the absence of pathology as the result of an ancient and highly evolved viral survival strategy. for many rna viruses such as those commonly infecting bats, accessory proteins and evolved secondary functions of other viral proteins play a key role in infection by blocking host innate immune defences, modulating cellular signaling pathways and re-directing normal cellular functions [58] [59] [60] . the refinement of these functions during a long evolutionary history in bats may well have defined a successful strategy for long-term survival, even through the periods of catastrophic environmental disruption and diminished biodiversity. conversely, the severe pathology and disease that often occurs as a result of spill-over of bat viruses into other vertebrate hosts may result not from an inherently less effective immune response but from the disturbance of this finely tuned interaction of viral proteins with their targets in host cells. it can also be argued that there are several ways in which the harboring of well-adapted viruses might also bring a biological advantage to bats. one possibility is through symbiotic enhancement of innate immunity. although innate immunity has long been considered a broad, nonspecific and nonanamnestic first line of host defence, recent studies have demonstrated that persistent infection with one pathogen may prime host innate immunity to provide cross-protection from others. this has been best illustrated by a study in mice demonstrating that herpesvirus latency confers protection from bacterial in-mass extinctions, biodiversity and mitochondrial function: wang, walker and poon 653 table 2 favorable characteristics for survival and proliferation following the k-t mass extinction of bats and other potential sources of extant viral biodiversity. survival fection [61] . in a paper by roossinck, examples of 'good viruses' and virus-host symbiosis have also been reviewed for viruses infecting human, wasps, plants, fungi, aphids and bacteria [62 ] . highly adapted viruses persistently infecting bat populations might also serve to protect bats at the species or population level from predators (e.g., tree roosting animals such as raccoons and opossums, owls and hawks, and primates) in a sense acting as defensive 'biological weapons'. the best defensive weapons are those that do no harm to the host species and are released only when there is an imminent threat of danger and the emerging bat viruses (e.g., henipaviruses and filoviruses) satisfy these requirements. henipaviruses are believed to persist in bat populations at a very low viral load and are totally harmless to their natural host. however, under stress, the viral load increases, facilitating transmission to other animals [63, 64] . they have a very broad range of susceptible hosts and are highly lethal in many different vertebrate species [65] . such a mechanism might not be able to protect every individual animal in a population, but it would be an effective way to preserve the species. in principle, such a symbiotic relationship with viruses would benefit any animal species and there is evidence that such relationships do exist in very different hosts including humans, mice to fungi and bacteria [62 ] . it is perhaps the long period of co-evolution and some unique selective pressures that have driven its emergence and dominance in bats. bats have a relatively low reproductive rate (usually one birth with one pup per year) compared to other animals such as rodents and, as discussed above, bats tend to live in very large and dense populations. these biological and behavioral characteristics may demand far more robust mechanisms to fight infection and predation in order to avoid extinction. flight capability, longevity and innate immunity -are they linked? as discussed above, some of the 'unique' biological characteristics of bats are believed to contribute to the observation that they appear to harbor a large number of viruses without clinical signs of disease. while the scientific data are not sufficient to make any conclusive link, it is tempting to speculate on the interplay for some of these factors. in table 3 , three key aspects of the biology of bats are analyzed in the context of their impact on cellular metabolism and infectious agents. flight ability is the most distinguishing feature of bats amongst mammals. flight consumes a large amount of energy, demanding a much higher rate of metabolism. in general, it is believed that a high metabolic rate, such as that in bats, is likely to generate more metabolic byproducts, which, in turn, will increase the rate of oxidative damage to mitochondrial dna and other cellular structures [66] . according to the 'rate of living' theories, animals with a high metabolic rate are likely to be short-lived [67 ,68] . although the combination of small body size, high metabolic rate and long lifespan in bats does not seem to be compatible with this view, recent studies on mitochondrial dna and cellular processes have indicated that multiple mechanisms exist in bats (and other long lifespan animals such as birds) to allow them to be more efficient in resisting oxidative damages than short lifespan animals [67 ] . oxidative damage to dna is also an important mechanism of tumorigenesis [69] . it is therefore interesting that unpublished anecdotal observations suggest that bats have a lower rate of tumorigenesis than most other animals. an extensive literature search revealed only a few recent papers describing tumors in egyptian fruit bats [70] [71] [72] . in one case, a sarcomatoid carcinoma was diagnosed in the lung of a 10-year-old male captive bat, and in the other case a gastrointestinal leiomyosarcoma was found in a 10-yearold female bat. during our own study to establish bat cell lines, a wide international collaborative effort examining bats from australia, asia and africa failed to identify any tumors from a large number of individual bats representing more than ten different bat species [73] (g. crameri, l.-f. wang, unpublished observations). although the jury is still out, it is not impossible that efficient mechanisms for countering oxidative damage in bats result in a lower rate of tumorigenesis. on the other hand, it is also possible that the low reporting rate of bat tumors results from a lack of appropriate detection/diagnostic methods for bat tumors or general interest in this area of research. mitochondria are key organelles in controlling cellular metabolism. for bats, the efficient function of mitochondria 654 emerging viruses table 3 potential association of unique bat biological features with a symbiotic relationship with viruses. unique biological feature impact on metabolism impact on infectious agents true ability to fly requiring more energy efficient metabolism greater chance of inter-species and long distance transmission rapid change of body temperature highly efficient sensing and regulation of temperature effect on immune system favoring persistence long lifespan relative to body size more efficient mechanism to prevent oxidative damage to dna is likely to be essential for key biological characteristics such as flight, body temperature changes and lifespan, all of which could impact on the ecology of viral infection (table 3) . until very recently, it was not recognized that mitochondria also act as a center of signaling pathways for apoptosis, inflammation and innate immune responses [74 ,75,76] . this is a very new and rapidly evolving field of research but it is clear that mitochondria are involved in signaling for antiviral and antibacterial immunity [76] . all published studies to date have been conducted in human or mouse cell lines so it will be extremely interesting and important to conduct parallel studies in bat cells to determine whether mitochondria have similar functions in controlling innate immune responses in bats. in summary, we speculate that the key unique biological features of bats, that is, ability to fly, high metabolic rate and longevity, are functionally interconnected and mitochondria are the key cellular organelles that link all of these processes. these features, in turn, all have an impact on the bat's ability to control tumors and infection. this fundamental and common innate ability of bats may help explain their seemingly super anti-ageing, antitumor and anti-infection characteristics. multiple hypotheses are presented in this review in an attempt to address the question as to whether bats are special as reservoir hosts of viruses. while we are not able to provide a definitive answer to the question, we hope that the range of new ideas and angles presented here will stimulate those who work in the field to explore further in the future. it is possible that all of the aspects discussed here, although some of them seem to be mutually exclusive, may play a part in the overall picture of high-rate detection of viruses and infection with no diseases in bats. if bat's innate ability to counter biological imbalance proves to be different from or more robust than other mammals in whatever way or shape, it will provide a tremendous opportunity for us to 'learn from bats' and apply some of these principles to human and animal health, either via therapeutic intervention in humans or transgenic modification in livestock animals. however, one must recognize that despite the great interest in bat viruses in recent years, bat biology research is in its infancy compared with existing knowledge of infection in humans and other animals such as rodents. there is a total lack of research tools and reagents to address any of the hypotheses in depth. thus, there is an urgent need to advance the basic study of bat biology and bat immunology to help remove the road blocks. taylor dj, leach rw, bruenn j: filoviruses are ancient and integrated into mammalian genomes. bmc evol biol 2010, 10:193. this is the first report of endogenization in the mammalian genome of nonretroviral rna viruses with extranuclear replication. the endogenous viral elements were most commonly detected in bats, rodents and insectivores. phylogenetic analysis suggested an ancient association between filoviruses and mammals that was dated to tens of millions of years ago. olival kj, epstein jh, wang l-f, field he, daszak p: are bats unique virus reservoirs? in conservation medicine, edn 2. edited by aquirre aa, ostfeld rs, daszak p. oxford university press (in press). a recent review on the same topic as this review, but focusing on different aspects. it is worth to read this book chapter in conjunction with the current review for a more complete appreciation of the subject. bats and viruses: a brief review sur une grande epizootie de rage swanepoel r: fruit bats as reservoirs of ebola virus a morbillivirus that caused fatal disease in horses and humans nipah virus: a recently emergent deadly paramyxovirus bats are natural reservoirs of sars-like coronaviruses severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats henipaviruses: emerging paramyxoviruses associated with fruit bats henipavirus vaccine development. j bioterrorism biodefense ebola outbreak killed 5000 gorillas the ecology of ebola virus bats, clocks, and rocks: diversification patterns in chiroptera order chiroptera early eocene bat from wyoming linking the wasatchian/bridgerian boundary to the cenzoic global climate optimum: new magnetostratiographic and isotopic results from south pass a highresolution genetic signature of demographic and spatial expansion in epizootic rabies virus placental mammal diversification and the cretaceous-tertiary boundary a molecular phylogeny for bats illuminates biogeography and the fossil record host and viral ecology determine bat rabies seasonality and maintenance an example of using mathematical modeling to identify factors that are important for viral infection dynamics in bat populations novel astroviruses in insectivorous bats identification of a novel coronavirus in bats detection of novel astroviruses in urban brown rats and previously known astroviruses in humans the most recent paper out of a series studies on astroviruses conducted by the hong kong group, which demonstrated that the genetic diversity of astroviruses in bats is greater than those in other mammals evolutionary insights into the ecology of coronaviruses emergence and persistence of hantaviruses virus evolution and genetic diversity of hantaviruses and their rodent hosts evolution of the old world arenaviridae and their rodent hosts: generalised host-transfer or association by descent? influenza: emergence and control mass extinctions in the marine fossil record new links between the chicxulub impact structure and the cretaceous/tertiary boundary extraterrestrial cause for the cretaceous-tertiary extinction. experimental results and theoretical interpretation extinctions in the fossil record selectivity of end-cretaceous marine bivalve extinctions the delayed rise of present-day mammals a phylogenetic supertree of the bats (mammalia: chiroptera) survival in the first hours of the cenozoic evolutionary biology -a first for bats primitive early eocene bat from wyoming and the evolution of flight and echolocation a bony connection signals laryngeal echolocation in bats energy, volatile production, and climatic effects of the chicxulub cretaceous/ tertiary impact reassessing conflicting evolutionary histories of the paramyxoviridae and the origins of respiroviruses with bayesian multigene phylogenies genomic diversity and evolution of the lyssaviruses endogenous viral elements in animal genomes this paper reports the use of a systematic screening in-silico to detect the common occurrence of endogenous elements derived from a diverse array of dna and rna viruses in animal genomes. analysis of the sequences of the endogenous viral elements (eves) with respect to extant viruses indicated ancient origins infectious disease modeling and the dynamics of transmission the evolution and emergence of rna viruses mechanisms of severe acute respiratory syndrome pathogenesis and innate immunomodulation understanding the accessory viral proteins unique to the severe acute respiratory syndrome (sars) coronavirus inhibition of interferon induction and signaling by paramyxoviruses virgin hwt: herpesvirus latency confers symbiotic protection from bacterial infection the good viruses: viral mutualistic symbioses an updated review on virus-host symbiosis. it is an excellent paper to read for those wishing to learn a bit more about the importance and progress in this area of research anthropogenic deforestation, el nino and the emergence of nipah virus in malaysia nipah virus outbreak in malaysia hendra and nipah viruses: different and dangerous oxidative damage to dna: relation to species metabolic rate and life span bats and birds. exceptional longevity despite high metabolic rates an excellent review for anyone who is interested in the subjects of aging, longevity and flying ability of animals in the rate of living viral oncogene-induced dna damage response is activated in kaposi sarcoma tumorigenesis sarcomatoid carcinoma in the lung of an egyptian fruit bat (rousettus aegyptiacus) gastrointestinal leiomyosarcoma in an egyptian fruit bat (rousettus aegyptiacus) microchip-associated leiomyosarcoma in an egyptian fruit bat (rousettus aegyptiacus) establishment, immortalisation and characterisation of pteropid bat cell lines emerging role of damageassociated molecular patterns derived from mitochondria in inflammation this paper describes the establishment of bat primary and immortalized cell lines which are becoming increasingly important for isolation of bat viruses and for basic study on virus-bat interaction recent advances in apoptosis, mitochondria and drug resistance in cancer cells mitochondria in innate immune responses mass survival of birds across the cretaceous-tertiary boundary: molecular evidence the role of mitochondria in innate immunity is a relative new topic in immunology and this updated review provides an excellent starting point to review the recent discoveries and future research directions bat mating systems impact winter and the cretaceous-tertiary extinctions -results of a chicxulub asteroid impact model a theoretical exercise in the modeling of ground-level ozone resulting from the k-t asteroid impact: its possible link with the extinction selectivity of terrestrial vertebrates evidence for echolocation in the oldest known bats impact of the terminal cretaceous event on plant-insect associations we thank e.c. holmes for useful discussions on the evolution of bat viruses. key: cord-018821-e9oxvgar authors: webber, quinn m. r.; willis, craig k. r. title: sociality, parasites, and pathogens in bats date: 2016-04-27 journal: sociality in bats doi: 10.1007/978-3-319-38953-0_5 sha: doc_id: 18821 cord_uid: e9oxvgar little is known about the ecology of many of the parasites and pathogens affecting bats, but host social behavior almost certainly plays an important role in bat-parasite dynamics. understanding parasite dynamics for bats is important from a human public health perspective because of their role as natural reservoirs for recent high-profile emerging zoonotic pathogens (e.g. ebola, hendra) and from a bat conservation perspective because of the recent emergence of white-nose syndrome (wns) in north america highlighting the potential population impacts of parasites and pathogens. although some bat species are among the most gregarious of mammals, species vary widely in terms of their social behavior and this variation could influence pathogen transmission and impacts. here, we review the literature on links between bat social behavior and parasite dynamics. using standardized search terms in web of science, we identified articles that explicitly tested or discussed links between some aspect of bat sociality and parasite transmission or host population impacts. we identified social network analysis, epidemiological modeling, and interspecific comparative analyses as the most commonly used methods to quantify relationships between social behavior and parasite-risk in bats while wns, hendra virus, and arthropod ectoparasites were the most commonly studied host-parasite systems. we summarize known host-parasite relationships in these three systems and propose testable hypotheses that could improve our understanding of links between host sociality and parasite-dynamics in bats. parasitism is ubiquitous in nature. parasites affect fitness of their hosts and thus can shape host population dynamics. defined broadly, a parasite is any organism that grows, feeds, and/or is sheltered at the expense of another organism (i.e. the host). parasites can be categorized into different ways but one common approach is to differentiate microparasites, which are typically unicellular or multicellular microbes (e.g. bacteria, prions, viruses, protozoans, fungi) with short generation times and a life cycle that occurs entirely on/in the host, from macroparasites which are multicellular parasites with longer generation times and more complex life cycles that may include multiple host species (table 5 .1; anderson and may 1979; hudson et al. 2002) . many microparasites can be pathogenic and cause identifiable disease in their hosts with recognizable physiological or behavioral signs or symptoms (table 5 .1). for example, batrachochytrium dendrobatiidis is a fungal microparasite of amphibians that infects keratinized tissues, thickens the epidermis, and eventually causes mortality, all of which are identifiable signs of the associated disease, chytridiomycosis (voyles et al. 2009; rosenblum et al. 2010) . macroparasites tend to result in chronic infections of their definitive hosts, decrease host fecundity, and usually cause morbidity rather than mortality (hudson et al. 2002) . experimental infection of great tits (parus major) with the hen flea (ceratophyllus gallinae) resulted in reduced reproductive success via increased nest failure during incubation and the nestling period (fitze et al. 2004) . parasite dynamics and impacts are often quantified using two metrics: intensity (i.e. load) and prevalence. intensity quantifies the number of infections per individual host within a given population, while prevalence is measured as a proportion of infected individuals within a sample from a given host population (table 5 .1). understanding variation in parasite prevalence and intensity can thus be important for making inferences about the evolution of social behavior and the potential fitness consequences associated with parasite infection. although empirical data from wild host populations are surprisingly scarce, host behavior is considered an important predictor of parasite intensity, prevalence, and impacts (moore 2002) . a longstanding hypothesis in parasite ecology predicts an influence of the host social system, and the duration and frequency of social contacts within host populations, on parasite prevalence or intensity (loehle 1995; altizer et al. 2003) . social systems, defined as groups of conspecifics that regularly interact more frequently with one another than with members of other groups, represent the highest level of sociality, while social organization and social structure describe the size, composition, and spatiotemporal distribution and cohesion of social systems (table 5 .2; whitehead 2008) . social systems can be further subdivided into two categories: colonies and aggregations, and this distinction is important for understanding host-parasite dynamics in bats. colonies are groups of individuals that may or may not be genetically related but which exhibit non-random patterns of association, and frequent close contact with each other (kerth 2008) . it is often assumed that groups of bats roosting in a common structure represent a colony but often such groups may not meet the colony definition and, instead, represent aggregations. aggregations are defined as assemblages of individuals that happen to occur in a shared environment at the same time, perhaps due to an attraction to that environment rather than social bonds with other individuals (table 5 .2). variation in social dynamics within and between colonies and aggregations can mediate host-parasite dynamics (webber et al. 2016 ). host an animal or plant on which a parasite lives. martin (2010) reservoir host (ecological definition) hosts that do not exhibit clinical disease as a result of infection. nunn and altizer (2006) reservoir host (medical definition) hosts that serve as a source of infection and potential reinfection of people and sustain parasite populations when humans are not available. martin (2010) parasite (ecological definition) any organism that lives on and draws nutrients from another living organism (the host), usually to the host's detriment. nunn and alitzer (2006) parasite (medical definition) an organism that grows, feeds, and is sheltered on (ectoparasite) or in (endoparasite) a different organism while contributing nothing to survival of its host. martin (2010) microparasite pathogens, or disease-causing microbes (viruses, bacteria, protozoa, fungi). anderson and may (1979) macroparasite multicellular parasites (helminthes, arthropods, most ectoparasites) may and anderson (1979) disease (ecological definition) pathology caused by infection, including outward physical signs and internal or behavioral changes. nunn and alitzer (2006) disease (medical definition) a disorder with a specific cause (may or may not be known) and recognizable signs and symptoms. martin (2010) pathogen disease-causing agent. nunn and alitzer (2006) virulence (ecological definition) disease-induced host mortality and/or reductions in fecundity nunn and alitzer (2006) virulence (medical definition) the disease-producing (pathogenic) ability of a microorganism. martin (2010) infection invasion of the body by harmful organisms (pathogens), such as bacteria, fungi, protozoa, or viruses. martin (2010) prevalence a measure of morbidity based on current levels of disease in a population; measured as a proportion (i.e. number of infections divided by number of individuals). martin (2010) intensity a measure of morbidity based on current levels of disease in a population; measured as an absolute number (i.e. the total number of infections). martin (2010) epidemiology the science concerned with the study of the factors determining and influencing the frequency and distribution of disease in a defined human or animal population. martin (2010) relationships between host social systems and parasite dynamics are complex, but several mechanisms are predicted to influence these patterns and are likely important for bats. for example, hosts that occur in large, high-density colonies are predicted to have more frequent interactions resulting in more opportunities for parasite transmission (stanko et al. 2002; tompkins et al. 2011) . dense aggregations may result in contacts that are fewer and shorter in duration than those occurring in colonies, but are still likely to provide more opportunities for parasite transmission than might occur for solitary bats or those in very small colonies. this variation in host density also has the potential to influence one of the most fundamental parameters of disease ecology, the basic reproduction number or basic reproductive ratio (r 0 ). r 0 is an important metric of parasite fitness typically defined, for microparasites, as the number of secondary infections caused by an infectious individual in an entirely susceptible population or, for macroparasites, the number of female larvae established from a single female worm (hudson et al. 2002) . when r 0 > 1 infection persists within the host population and when r 0 < 1 infection cannot become established (perkins et al. 2003) . variation in social behavior can impact r 0 if certain individual hosts disproportionately infect a large an individual's reaction to a novel object or situation. réale et al. (2007) number of conspecifics, and thus inflate r 0 above the persistence threshold of one (lloyd-smith et al. 2005) . although empirical data from numerous vertebrates (e.g. ungulates: ezenwa 2004) and invertebrates (e.g. bees: otterstatter and thomson 2007 ) support theoretical relationships between parasitism and sociality, there are few data for most host-pathogen systems involving wild mammals, including bats. bats are among the most ecologically diverse of mammals with an enormous range of social systems (kunz and lumsden 2003; kerth 2008; johnson et al. 2013) . for example, colony or aggregation sizes of bats range from completely solitary to millions of individuals, while social systems range from small, closed societies with potentially long-term social bonds (e.g. thyroptera tricolor: chaverri 2010) to enormous, likely passive aggregations of individuals attracted to high-quality habitats (e.g. myotis lucifugus during autumn swarming : fenton 1969) . social behavior in bats presumably evolved in response to the costs and benefits associated with close conspecific contact. benefits of social roosting may be numerous and include social thermoregulation, cooperative behavior, and information transfer. many temperate bats rely on social thermoregulation to decrease energy expenditure during periods of energy limitation, such as pregnancy and lactation (e.g. eptesicus fuscus: willis and brigham 2007) , while some tropical species also appear to exploit social thermoregulation (e.g. uroderma bilobatum: lewis 1992) . the evolution of cooperative behavior in bats was likely facilitated by strong female philopatry and stable group structure (emlen 1994) . vampire bats (desmodus rotundus) are well known for their cooperative behavior (i.e. reciprocal altruism) and females rarely transfer between groups (wilkinson 1987; carter and wilkinson 2013) . this combination suggests an evolutionary scenario, where stable group structure ultimately led to selection favoring cooperative behavior. information transfer about predation risk (e.g. kalcounis and brigham 1994) and high-quality foraging sites (e.g. mccracken and bradbury 1981) are also commonly cited as potential benefits of sociality for bats. despite these potential benefits of social behavior, risk of infection with microand macroparasites is thought to represent a potentially pronounced fitness cost of being social (côté and poulin 1995) . this cost is illustrated most obviously by the recently emerged infectious disease white-nose syndrome (wns), which is caused by the fungal microparasite pseudogymnoascus destructans (blehert et al. 2009 ). wns has resulted in catastrophic declines of temperate hibernating bats in north america (frick et al. 2010 ) and prompted urgent conservation and management attention fenton 2012) . p. destructans is an invasive pathogen that appears to have evolved with bats from the old world, where it does not cause mortality of infected hosts, and to date, is known to occur on at least 15 hibernating bat species (puechmaille et al. 2011; zukal et al. 2016) . p. destructans grows in exposed skin of the muzzle, ears, and wing membranes of bats during hibernation (blehert et al. 2009; warnecke et al. 2012) . for north american species, infection with p. destructans causes an increase in energy expenditure (verant et al. 2014) and arousal frequency (boyles and willis 2010; reeder et al. 2012; warnecke et al. 2012 ) which lead to premature depletion of fat stores during hibernation. although the mechanism inducing increased energy expenditure and arousals by infected bats is still not fully understood (for review see willis 2015) , variation in social behavior could mediate fungal transmission and growth, especially since affected species tend to hibernate in large colonies or aggregations in caves or mines. understanding host-parasite dynamics in the context of social behavior for wns is therefore important from a conservation perspective. in addition to serious conservation threats for some species, bats also appear to be reservoir hosts for a number of emerging infectious diseases (eids) of public health concern (luis et al. 2013; plowright et al. 2015) . reservoir hosts tend not to exhibit clinical disease as a result of infection (baker et al. 2013 ) and reservoir host populations may therefore provide large pools of infected hosts that could facilitate spillover events to heterospecifics, including humans, livestock, or pets (luis et al. 2013; plowright et al. 2015) . interestingly, their apparent ability to tolerate infection with a wide diversity of viral parasites is one factor supporting the recent hypothesis that bats are 'special' with respect to their propensity to host zoonotic microparasites (luis et al. 2013; brook and dobson 2015) . the recent identification of a number of viral, protozoan, and bacterial microparasites in bats supports this hypothesis and has prompted significant analysis and discussion about bats as natural hosts to microparasites associated with eids of humans or livestock luis et al. 2013; olival and hayman 2014; veikkolainen et al. 2014) . for example, recent evidence suggests that bats host more zoonotic viruses per species compared to rodents, and human encroachment into bat habitats, particularly in the tropics, could facilitate spillover events (daszak et al. 2000; luis et al. 2013) . several hypotheses have been proposed to explain the apparent zoonotic potential of bats. enormous variation in body temperature (t b ) and metabolic rate (mr) between rest and sustained flight in many heterothermic bats could reduce fitness or pathogenicity for many viral parasites (o'shea et al. 2014) . interestingly, widespread torpor expression in bats appears to reduce the likelihood of hosting zoonotic viruses, possibly because viral replication decreases as a result of reduced t b and mr during torpor (luis et al. 2013; stawski et al. 2014 ). this hypothesis suggests physiological tolerance as a mechanism allowing bats to serve as reservoir hosts, but evidence linking host sociality as an additional mechanism is limited . despite the fundamental evolutionary importance, and conservation and public health significance of bat-parasite interactions, studies of the influence of bat sociality on parasite transmission and acquisition are limited, especially for microparasites. here, we review the literature on relationships between social behavior of bats and their associated parasites. first, we provide an overview of the tools, techniques, and methodologies that have been used to quantify relationships between sociality and parasitism in bats, as well as relevant techniques that have been used for other vertebrates and which could be applied to bats. second, we summarize the role of sociality in three relatively well-studied bat-parasite systems: wns, hendra virus (hev), and arthropod macroparasites. finally, we propose testable hypotheses and observational and experimental studies important for understanding the influence on parasite dynamics of two important concepts in behavioral ecology of bats: fission-fusion social organization and individual behavioral tendencies (i.e. personality). we conducted an extensive search of the literature and compiled a list of articles based on combinations of key word searches on web of science. we searched the term "chiroptera" and "bat" with every possible combination of social*, gregarious*, colony, aggregation, fission-fusion (i.e. a common type of bat social system, see below), viral, pathogen, disease, ectoparasite*, endoparasite*, parasite*, infection* and epidemiology*. our initial search yielded 223 unique articles but we eliminated articles that did not explicitly quantify or discuss a link between some aspect of sociality and parasite risk. this left only 35 articles that fully satisfied our search criteria. four of these used comparative analyses to examine effects of species-specific socioecological traits on parasite risk (table 5 .4), 15 addressed sociality in the context of microparasites (table 5 .4), and 16 addressed sociality in the context of macroparasites (table 5 .5). the studies we identified employed a range of methodologies to quantify links between sociality and parasite risk (tables 5.3, 5.4 and 5.5). methods included social network analysis (n = 3), epidemiological modeling (n = 5), and interspecific comparative analyses (n = 4). details about the underlying theory and the implementation of these methods is available elsewhere (e.g. social network analysis: croft et al. 2008; epidemiological modeling: may 2006 ; comparative analyses: garland et al. 2005) , and here we focus on the progress that has been made to date in identifying knowledge gaps where future research on bat-parasite dynamics could be focused. social network analysis is based in mathematical graph theory and has been widely applied in human sociobiology, since the 1950s (e.g. cartwright and harary 1956; wasserman and faust 1994) . for studies of wildlife, network analysis was first applied in primate sociobiology (e.g. sade and dow 1994) , and has recently been applied more broadly to many other taxa (e.g. fewell 2003; hamede et al. 2009; drewe 2010) , including bats (for review see johnson et al. 2013) . networks consist of nodes (individuals or locations) and edges (interactions between nodes) through which a variety of individual and group level metrics can be quantified (wey et al. 2008) . the roles and importance of individuals or locations can then be assessed based on these metrics (see croft et al. 2008 for review). for example, individuals with certain combinations of traits can influence network dynamics by potentially acting as intermediaries connecting smaller subgroups within larger groups (wey et al. 2008; krause et al. 2010 ). in addition, bipartite networks (or two-mode networks) can be constructed to assess associations between individuals and ecologically relevant locations (e.g. a population of organisms and their nesting sites). network metrics are especially useful because they can be used as predictor variables for relevant-dependent variables (e.g. infection status or parasite intensity) in standard statistical models (e.g. general linear models). network analysis has allowed disease ecologists and parasitologists to make important strides quantifying how non-random social interactions affect parasite transmission and dynamics (for review see godfrey 2013). network analyses have become increasingly popular for studies of bats (johnson et al. 2013) . for instance, network analyses have been applied in a bat-habitat management context by identifying critical roosting locations that serve as 'hubs' within a roost network and simulating the consequences of removing those key sites for stability of the social group (rhodes et al. 2006; silvis et al. 2014) . network analyses have also been used to identify and quantify fission-fusion behavior within bat colonies (fortuna et al. 2009; patriquin et al. 2010; kerth et al. 2011; johnson et al. 2012 ). in the context of disease ecology, so far only two studies have connected social networks with epidemiological models to make inferences about host-pathogen dynamics (e.g. fortuna et al. 2009; webber et al. 2016) . fortuna et al. (2009) showed that giant noctule bats (nyctalus lasiopterus) form highly modular, fission-fusion colonies, and used an epidemiological model, parameterized using network metrics, to show that this arrangement reduces the spread of information or disease within the population because the colony was effectively segregated into modules divided among many roost trees (n = 73 trees for 25 bats: fortuna et al. 2009 ). for big brown bats (eptesicus fuscus), webber et al. (2016) recently showed that network structure depends on the habitat context. although tree-roosting e. fuscus reuse tree hollows between years (willis et al. 2003) , they frequently switch roosts within years (on average every 1.7 days: willis and brigham 2004) and rarely return to the same roost within a given summer. in building roosts, however, e. fuscus switch much less frequently and commonly return to the same roost repeatedly within the same year (ellison et al. 2007; webber et al. 2016) . webber et al. (2016) applied epidemiological models to social networks constructed based on these patterns of roosting behavior in forests versus buildings and found that parasite dynamics should vary between these habitat contexts with more rapid pathogen dissemination within building roosting colonies. constructing epidemiological models from empirical data undoubtedly improves inference for predicting epidemic outcomes, but as yet there are no studies that quantify real-world relationships between the social behavior of bat hosts and their parasites to test predictions of epidemiological models (table 5 .3). although this form of data collection can be labor-intensive and potentially expensive (craft and caillaud 2011) , there are numerous examples of network analysis being used to integrate information on host-contact patterns and parasitism in systems with highly gregarious hosts. for example, network analysis elucidated the role of meerkat (suricatta suricata) allogrooming as a predictor for the prevalence of myobacterium tuberculosis, the microparasite that causes tuberculosis (drewe 2010) . quantifying association patterns of meerkats was largely conducted via focal animal observation, a type of data collection that is nearly impossible for free-ranging bats. thus, advances in technology, such as passive transponders (pit tags), data-logging telemetry, and/or proximity data loggers may be useful for quantifying association patterns and constructing social networks for bats with implications for epidemiology and disease ecology (e.g. willis and brigham 2004; patriquin et al. 2010; kerth et al. 2011; johnson et al. 2012 ). although empirical data are sparse, host-parasite dynamics, and the impacts of parasites on host populations have a rich theoretical history founded on anderson and may's (1979) seminal models. their classic microparasite epidemiological model conceptualizes host population dynamics in terms of susceptible (s), exposed (e), infected (i), and resistant/recovered (r) population pools, and these pools can be combined in a range of ways depending on the nature of host-pathogen interactions in the wild (e.g. si, sis, sir, seir models). in a standard sir model, individuals transition from s to i as a function of transmission rate (β) and from i to r as a function of survival of infection (v), while birth (a), and death rates (b, a + b for infected hosts) of each pool drive overall population dynamics (fig. 5.1 ; anderson and may 1979) . since being developed, sir epidemiological models have been widely used to infer microparasite impacts on host populations for humans (e.g. anderson and may 1982) , wildlife (e.g. mccallum et al. 2009 ) and plants (e.g. gilligan et al. 1997) . although appropriate for modeling the impacts of many infectious diseases (e.g. measles, whooping cough: anderson and may 1992) , classic sir models compartmentalize individuals into broad categories which may fail to capture variation in aspects of host biology that could influence parasite dynamics and impacts (keeling and eames 2005) . for example, classic sir models often assume that individual hosts in the population associate at random but, particularly for highly social species, non-random association patterns are far more likely in nature. incorporating network structure into epidemiological models eliminates random-mixing assumption of classic sir models by assigning each individual a finite number of permanent or temporary contacts between which transmission can occur (keeling and eames 2005; may 2006) . implications for network epidemiological modeling include the ability to identify highly connected individuals that may be involved in a disproportionate number of transmission events (e.g. super-spreaders: lloyd-smith et al. 2005) and weakly connected individuals that may benefit from lower risk of infection. epidemiological models informed by variation in social connectedness can be highly effective for predicting pathogen dynamics (lloyd-smith et al. 2005) but, as noted above, so far only two studies have applied these methods specifically to bats (fortuna et al. 2009; webber et al. 2016) . however, to our knowledge no study has explicitly tested model predictions on a natural bat-parasite system in the wild. an alternative to network epidemiological models informed by short-term (i.e. one season) association data, is the use of long-term population data for model parameterization. these data ideally include information on changes in population or colony sizes and changes in population demographic structure and/or vital rates. most often for bats, long-term field data on colony size and population connectivity, in combination with laboratory data on immune physiology have been used (table 5 .4; george et al. 2011; plowright et al. 2011) . for example, field and laboratory data were combined to model ecological factors driving hev spillover events occurring in australia from flying foxes (pteropus sp.) to horses and predicted that decreased migratory behavior of the host increased host density in urban areas, which contributed to the duration and intensity of hev outbreaks (plowright et al. 2011, see below) . similarly, mark-recapture data, combined with birth and death rates of different demographics, have been used to parameterize models of rabies dynamics (george et al. 2011) . rabies in e. fuscus is highly seasonal, with peaks in prevalence at maternity colonies (i.e. during june and july) after parturition when pups provide a supply of immunologically naive hosts (george et al. 2011 ). thus, the chance of spillover is greatest during the post-parturition period when females roost in large colonies and the influx of juveniles in the population increases overall prevalence. these studies highlight the value of predictive models incorporating the behavior of bats to make predictions about parasite dynamics, with potential implications for human public health. however, even models which incorporate host ecology and behavior (e.g. habitat selection) do not typically account for variation in social behavior which could also play an important role (moore 2002) . for microparasites, the two most likely parameters influenced by variation in social behavior are transmission rate (β) and the survival and subsequent reproduction of infected individuals (fig. 5.1 ). for example, recent appreciation of individual differences in host behavior has led to the realization that homogenous mixing or so-called 'mass action' transmission may not reflect the dynamics of most host-parasite systems (mccallum et al. 2001; moore 2002; barber and dingemanse 2010) . despite this realization, though, for many host-pathogen systems, estimating β can be difficult and modeling studies tend to rely on some plausible range of values for β (e.g. griffin and nunn 2012) . for some microparasites, however, it may be possible to experimentally manipulate transmission and determine β empirically. for example, a basic pairwise experiment, where pairs of individuals (one infected, one susceptible) are housed together could help to determine the proportion of contacts that result in transmission, and narrow the range of plausible β values. incorporating empirically derived data on association patterns of individual bats into epidemic models could help to improve our predictions of potential epidemic scenarios. these analyses (i.e. informed by empirical data) are still relatively scarce for bats (table 5 .4), but recent interest in host-parasite interactions and social network analysis provides an excellent opportunity to connect real data with model predictions. comparative analyses provide insight into potential species-specific variation in evolutionary adaption (i.e. changes in response to natural selection) within a group of organisms (garland et al. 2005) . from the perspective of parasites, a host is effectively a habitat, which means principles of community ecology, and biotic and abiotic characteristics of the "host ecosystem," can be used to explain patterns of parasite community composition (morand and poulin 1998) . thus, in contrast to intraspecific analyses which typically assess variation in prevalence or intensity of infection among individuals, comparative studies typically examine interspecific variation in parasite species richness among host species because intensity and prevalence are unlikely to be comparable in terms of their impacts, across different host and parasite species. in most cases, comparative studies of parasite species richness "correct for" host phylogeny. two closely related species are more likely to share similar functional traits compared to two more distantly related species because of their more recent evolutionary history (ives and garland 2010) . more parasites are also likely to have been identified for host species that have been well-studied so, in addition, most comparative studies also account for publication bias by including the total number of publications on a given host species as a covariate in statistical models, or by using residuals of a linear regression between number of publications and parasite species richness as the response variable (e.g. nunn et al. 2003; lindenfors et al. 2007; turmelle and olival 2009 ). to date, relatively few studies have used comparative analyses to test effects of social behavior on parasite species richness in bats and, so far, most have focused on viruses with one quantifying macroparasites (table 5 .3). in contrast, studies of primates, the best-studied mammalian taxon in terms of relationships between social behavior and parasite richness, focus on a wider breadth of parasites (i.e. ecto and endoparasites as well as bacterial and viral pathogens: nunn et al. 2003; vitone et al. 2004; griffin and nunn 2012) . for bats, colony size appears to be one of the most important predictors of viral richness, and epidemiological and evolutionary theory predict that highly colonial bat species should harbor greater parasite diversity, because of increased opportunities for parasite reproduction and evolution . however, observed relationships have not always followed this pattern for bats (turmelle and olival 2009; gay et al. 2014) . turmelle and olival (2009) found no effect of colony size on viral richness in bats but, rather, identified species' conservation status and global f st (i.e. population genetic structure) as the most important predictors. on the other hand, among southeast asian bats, gay et al. (2014) found a negative relationship between colony size and viral richness, which contradicts epidemiological theory (loehle 1995) . one potential limitation of these results could be that the authors used a categorical metric of population size (i.e. small, medium or large), which likely underrepresents the complexity of social behavior (gay et al. 2014) . alternatively, in the case of gay et al.'s (2014) results, it could be that bats in large colonies express immune or behavioral traits that provide protection from increased parasite risk in that social context. although they have still not been widely used, in our view comparative studies have great potential to help understand how bat social behavior influences parasite diversity and co-evolves with parasites, in part because bats are so diverse and exhibit a wide range of social and mating systems. for example, promiscuous bat species should host a greater number of parasite species compared to harem-breeding and monogamous bats because, independent of colony size or density, polygynandrous species presumably come in contact with a greater number of conspecifics during mating compared to polygynous species, which likely has implications for host-parasite coevolution. however, it is important to note that mating systems vary considerably among bats (mccracken and wilkinson 2000) and other aspects of bat social systems (e.g. degree of fission-fusion or roost fidelity: see below) likely co-vary with mating system and are equally important as predictors of parasite richness. thus, we recommend that future studies attempt to include more detailed information on bat social systems in comparative analyses. for example, a meta-analysis of primate social structure showed that differences in social network modularity (i.e. sub-grouping within a larger group) in 19 species led to differences in disease dynamics among species (griffin and nunn 2012) . although it may not be possible to include such detailed social association data for bats at present, as more social network studies accumulate, we recommend the use of network metrics as predictor variables of parasite richness in comparative analyses. species-specific data on typical contact rates among individuals and demographics, and between sympatric species, as well as data on dispersal patterns and group stability within bat species (e.g. fission-fusion) would also be valuable as predictor variables to better understand how host social traits in bats influence their parasite ecology and evolution. although very different in terms of their ecology and population impacts, based on our literature review, the relationship between bat sociality and parasite impacts and dynamics has been relatively well-studied for three sets of parasites: p. destructans, the cold-tolerant fungal pathogen of bats that causes wns; hev, which can lead to potentially fatal zoonotic disease in humans and horses; and the many species of arthropod ectoparasites which have been relatively well-studied because they can be easily sampled from bats captured in the field. we also identified four articles which addressed the relationship between rabies, or european lyssavirus, and bat social behavior (table 5 .4), but rabies in bats has been the focus of a number of reviews (e.g. messenger et al. 2003; kuzmin and rupprecht 2006; rupprecht et al. 2011; o'shea et al. 2011 ) so here we focus on less well-characterized bat-parasite interactions. wns is an eid of urgent conservation concern because it is causing staggering rates of mortality among hibernating bats in eastern north america (blehert et al. 2009; frick et al. 2010; wilder et al. 2011; langwig et al. 2012; frick et al. 2015) . despite its recent emergence , however, wns is among the most well-studied microparasites of bats in the context of social behavior. interestingly, one of the hallmark behaviors observed for p. destructans infected bats, both in the wild and in the laboratory, is a reduction in clustering behavior as wns infection progresses over the course of hibernation (langwig et al. 2012; wilcox et al. 2014 ). this phenomenon could lead to increased energy expenditure or evaporative water loss which has implications for survival and potentially future reproduction (willis et al. 2011; boratyński et al. 2015) . reduced clustering could be part of a stereotyped behavioral response by animals to infection, known as "sickness behavior" (hart 1988) , which could either reduce the likelihood of becoming infected, or reinfected, by sick individuals in the hibernaculum or reduce the likelihood of infecting susceptible individuals, which could be important for inclusive fitness if bats cluster with kin during hibernation (wilcox et al. 2014; bohn et al., in review) . in the laboratory, transmission occurs via direct physical contact and there is no evidence of airborne transmission (lorch et al. 2011) , while environmental transmission in the wild likely contributes strongly to infection dynamics because p. destructans can survive in hibernacula in the absence of bats (verant et al. 2012; hoyt et al. 2015) . although the most adversely affected bat species tend to associate with large numbers of conspecifics throughout the year, transmission appears to occur almost exclusively within hibernacula during autumn and throughout the winter . variation in sociality within and between bat species appears to influence host-parasite dynamics in wns. in the context of host density, there are two theories relevant to the relationship between transmission and host-parasite dynamics. first, density-dependent transmission predicts that host infection scales as a function of host density so that, at low host density, pathogen transmission declines, and a given pathogen fades out (greer et al. 2008) . second, frequency-dependent transmission predicts that host infection is driven by the total number of interactions among infected and susceptible individuals in the population, regardless of population density, so that when host density is low, a given pathogen can persist (greer et al. 2008) . interestingly, the relationship between wns transmission and host density during hibernation appears to be species-specific. among relatively solitary species (i.e. perimyotis subflavus and myotis septentrionalis) that hibernate individually or in small clusters, aggregation size (i.e. the numbers of bats in the hibernaculum) best predicted the number of surviving bats within a given hibernaculum, with larger aggregations experiencing faster apparent declines (langwig et al. 2012 ). this observation is consistent with density-dependent transmission models. in contrast, for species that vary more widely in aggregation size (i.e. m. lucifugus and m. sodalis), mortality was equally severe across a range of aggregation sizes (langwig et al. 2012) , which is consistent with frequency-dependent transmission models. this suggests that differences in species-specific social behavior during hibernation can modulate wns transmission and infection. unlike other infectious diseases of bats (e.g. rabies: george et al. 2011 ) demographic structure in the active season does not appear to drive wns dynamics. healthy m. lucifugus emerge from hibernation over an approximately eight-week period in spring (norquay and willis 2014; czenze and willis 2015) . females tend to emerge prior to males, with the females in the best condition emerging first. this is presumably because larger fat reserves allow females to cope with potentially inclement weather, but also initiate reproduction earlier by exploiting warmer, passively heated maternity roosts, and occasional warm nights with flying insects available (norquay and willis 2014) . however, bats infected with p. destructans tend to emerge from hibernation much earlier than healthy bats presumably because their fat stores are depleted. moreover, bats that survive hibernation with wns often have severe wing damage in spring and individuals with the most damaged wings tend to have the smallest energy reserves (fuller et al. 2011 ). for many diseases, an influx of immunologically naive hosts (i.e. juveniles) leads to a dramatic increase in prevalence immediately following reproduction due to vertical transmission from mothers to offspring. in wns, however, seasonal dynamics of the disease are driven by dramatic seasonal changes in host physiology, specifically sustained low body temperature during hibernation (warnecke et al. 2012; langwig et al. 2012 langwig et al. , 2015 . for p. destructans-infected bats in spring and early summer, an increase in body temperature limits infection and transmission despite the fact that females tend to aggregate in potentially large maternity colonies . although transmission likely does not occur readily for females at maternity colonies, high mortality rates during hibernation may decrease the number of potentially reproductive females that form maternity colonies . therefore, normal benefits incurred from colonial roosting, such as social thermoregulation, may be dramatically reduced for wns-surviving bats, which could have additional negative impacts on survivors. for example, the energetic costs of wound healing may be significant for infected individuals upon emergence from hibernation (fuller et al. 2011 ) and the decreased availability of social thermoregulation and increased likelihood of roosting solitarily may further increase energetic expenditure (wilcox and willis 2016) . therefore, for species affected by wns, the lack of potential colony members, and thus potential for social thermoregulation, during spring and early summer could ultimately result in decreased survival and reproduction . in addition to influencing transmission of p. destructans, theory predicts that population size influences risk of extinction (de castro and bolker 2005) . using counts of bats during winter hibernaculum surveys as a proxy for population size, frick et al. (2015) showed that, for five of six wns-affected species, probability of local extinction from a given hibernaculum decreased as population size increased. this suggests that host aggregation and social behavior could influence risk of extinction from wns. one possible mechanism is that larger populations have greater flexibility to cope with decreases in population numbers, and are thus less likely to face declines below population thresholds where extinction becomes inevitable. interestingly, social behavior may also help to explain differences in the consequences of p. destructans infection for european versus north american bats. prior to the emergence of wns, hibernating aggregations of affected north american species were approximately tenfold higher, on average, than colony sizes of ecologically similar european species . however, after wns emergence, colonies of affected north american species have declined precipitously and appear to be stabilizing at sizes similar to those of european bats . this suggests the possibility that european colony sizes could have been larger in the past and similar to pre-wns colony sizes in north america (frick et al. 2016) . taken together, empirical data from north america and inferences from europe suggest that wns could select against larger colony sizes (frick et al. 2016 ) resulting in remnant colonies which are, on average, smaller and less social than pre-wns colonies. we suggest that future studies attempt to quantify social tendencies at the individual and population levels for pre-and post-wns infected populations to assess the impact of this conservation pathogen for social evolution in bats. in australia, bats from the genus pteropus are reservoir hosts for hev, a recently emerged zoonotic virus from the family paramyxoviridae. flying foxes are only briefly infectious with no clinical disease but shed hev in urine, saliva, feces, and placental fluids (halpin et al. 2011) . hev is lethal to horses and humans, and transmission presumably occurs when horses ingest food or water contaminated by bats (plowright et al. 2008) . horses appear to act as amplifier hosts (daszak et al. 2006) , as all human cases have been directly traced back to an infected horse (field et al. 2012) . although, spillover events from flying foxes to horses are complex and few data exist linking individual social tendencies of the bats to hev transmission, host socioecology likely mediates host-pathogen dynamics for this zoonotic disease. colony/aggregation sizes of pteropus species can range from as few as 10,000 individuals in p. alecto maternity colonies to millions of individuals for p. scapulatus. based on experimental data and closely related human paramyxoviruses (e.g. measles), hev may require large host population sizes to provide enough susceptible individuals for persistence because of a relatively short infectious phase and life-long host immunity (daszak et al. 2006) . historically, naturally occurring nectar resources in native forests supported large, seasonally-migrating flying fox populations (eby 1991; plowright et al. 2015) but human-mediated habitat alterations have resulted in patchily distributed natural food resources, which were already ephemeral in nature (i.e. seasonally produced nectar). therefore, flying foxes have begun to colonize urban and periurban areas to exploit highly abundant, consistently available anthropogenic food resources, such as fruiting trees planted in gardens or horse paddocks (eby et al. 1999) . in many cases, urban bats do not migrate because food resources are consistently available, which increases the likelihood of hev-positive bats coming in contact with horses (plowright et al. 2011) . therefore, recent emergence and spillover of hev in australia appears directly linked to increases in the size and abundance of urban bat colonies as well as reduced migratory behavior (plowright et al. 2015) . although urban bat aggregations appear to remain relatively large throughout the year, temporal dynamics of hev are at least partially mediated by host socioecological traits such as colony size and migration (plowright et al. 2011). as noted above, social group size has been identified as a potentially important predictor of parasitism, with larger groups generally hosting more parasites (côté and poulin 1995; rifkin et al. 2012; patterson and ruckstuhl 2013) . among temperate bats, sexual segregation and variation in social group size between sexes appears to be among the most important mediators of ectoparasitism. adult males tend to roost individually or in small groups, while females form maternity colonies (kunz and lumsden 2003) . for males, parasitism tends to remain low-throughout spring and early summer and as the mating season progresses ectoparasite prevalence and intensity increases (webber et al. 2015a) . most studies investigating the link between colony size and ectoparasitism in bats have found the predicted positive relationship (e.g. luçan 2006; reckardt and kerth 2009; encarnaçao et al. 2012) , although this is not always the case (e.g. zhang et al. 2010; postawa and szubert-kruszynska 2014) . one explanation for lack of correlation between group size and ectoparasitism at female colonies in some studies may be linked to variation in host association patterns and roost selection/ephemerality. for instance, bamboo shoots used as roosts by flat-headed bats (tylonycteris pachypus and t. robustula) are highly ephemeral and likely contribute to frequent roost-switching (lewis 1995) as well as variation in group size and composition (zhang et al. 2010) . frequent roost switching breaks up social contacts that could favor direct host-host transmission of ectoparasites. meanwhile, because some ectoparasites rely on stable roost structures for a portion of their reproductive cycles, bats that use ephemeral roosts rarely come in contact with ectoparasites that rely on a stable roost for reproduction (lewis 1995) , which could supersede the theoretical relationship between colony size and parasite risk. although, social contacts among females help to explain observed patterns of ectoparasitism at maternity colonies, social organization (i.e. composition of groups) can also mediate ectoparasitism. most females present at maternity colonies are either pregnant or lactating and links between temporal variation in social organization (i.e. transition from pregnancy to lactation) and ectoparasitism are supported by co-evolution of some host-ectoparasite reproductive cycles (christe et al. 2000) . for example, reproductive cycles of some mite species respond to pregnancy hormones of their female hosts (lourenço and palmeirim 2007) . once pups are born there is also a pulse of mites, which results in significant vertical transmission from adult females to juveniles (christe et al. 2000; luçan 2006) . lactating females may also tolerate significant ectoparasite burdens. during lactation, females may have less energy available to mount behavioral (i.e. grooming) or immunological responses to infection (zuk and mckean 1996; christe et al. 2000) . thus, while females are lactating and pups are non-volant, ectoparasite prevalence, and intensity can be exceptionally high. however, once pups have been weaned and are volant, they tend to be more heavily parasitized than females until the colony disperses because they presumably have a naive immunoresponse and less-experience grooming (christie et al. 2000; czenze and broders 2011) . although ectoparasitism tends to increase with colony size, juveniles are usually the most infected hosts at maternity colonies. adult females still face significant ectoparasite burdens, while ectoparasitism for adult males generally increases throughout swarming as conspecific contact rates increase (webber et al. 2015a) . many bat ectoparasites are also vectors for bacterial pathogens (e.g. bartonella sp., veikkolainen et al. 2014 ). transmission of bacterial or viral pathogens via ectoparasite vectors among bat hosts could be influenced by sociality, especially for highly gregarious females at maternity colonies. in theory, the same principles which regulate host-pathogen dynamics should also apply to host-vector-pathogen dynamics, where the duration and frequency of host-host contact facilitates ectoparasite transmission, which in turn facilitates vector-based pathogen transmission. for instance, recent empirical evidence suggests that wing mites (spinturnix sp.) can transmit p. destructans propagules among hibernating bats in europe, a chain of transmission which is facilitated by direct host-host contact (luçan et al. 2016) . we suggest that more future studies quantify links between host sociality, vector ectoparasites, and vector-borne bacterial, viral, and fungal pathogens. progress has been made in identifying relationships between some aspects of sociality and parasitism in bats, but much more empirical data from wild and captive bats of more species is certainly needed. we propose a series of testable hypotheses and possible experimental and observational studies about the role of bat sociality in parasite dynamics. we focus on two aspects of sociality which have been relatively little-studied for bats in the context of parasitism: fission-fusion dynamics and consistent individual differences in behavior or personality. fission-fusion is the temporary splitting and reforming of colonies, where lack of consensus, or even conflict, result in temporary fission, but the cost of remaining apart is greater than subsequent fusion (sueur et al. 2011) . within maternity colonies, females exhibit fission-fusion sociality (e.g. m. bechsteinii: kerth and könig 1999; nyctalus lasiopterus: popa-lisseanu et al. 2008 ) and change roosts every few days but not all members of the group will move together, resulting in variable group size and composition (e.g. patriquin et al. 2010) . fission-fusion sociality and frequent roost switching by forest-living bats may have evolved as a mechanism to avoid parasites (reckardt and kerth 2007; bartonička and gaisler 2007) , although a trade-off between the costs of parasitism and benefits of remaining loyal to high-quality roosts likely exists. empirical data have linked fission-fusion behavior to ectoparasite avoidance (e.g. reckardt and kerth 2007) and some theoretical modeling also supports this hypothesis (kashima et al. 2013) . however, there are currently no studies investigating microparasite transmission within fission-fusion societies. we hypothesize that the relationship between fission-fusion dynamics and parasite risk and impacts will vary depending on the mode of transmission for a given type of parasite. we expect that for most microparasites and some ectoparasites (e.g. basilia nana: reckardt and kerth 2007) that require direct contact between hosts for transmission, fission-fusion dynamics could serve to disrupt parasite transmission. bats that display fission-fusion behavior typically switch roosts every 1-2 days (willis and brigham 2004) and data from m. bechsteinii suggest that bats are able to detect roosts that have recently been occupied by other bats (reckardt and kerth 2007) . thus, when bats switch roosts they appear to select sites that have not been occupied recently which could help them avoid infested roosts. in contrast, we expect that for macroparasites with alternative transmission strategies, such as mobile ectoparasites (e.g. crawling or flying), fission-fusion dynamics are likely to be less effective as a parasiteavoidance strategy. in fact, theory predicts that bats would be more likely to avoid mobile ectoparasites if they remained in a single, large group because of encounter-dilution effects (côté and poulin 1995; rifkin et al. 2012) . we suggest studies employing social network analysis of wild bats, combined with estimates of micro-and macroparasite prevalence, and intensity to disentangle relationships between host social behavior, including fission-fusion dynamics, and the ecology of parasite transmission (for review see godfrey 2013). animal personality refers to consistent individual differences in behavior that are stable within an individual across time and situations (sih et al. 2004; wolf and weissing 2012) . recently, personality has become more widely appreciated as a possible explanatory variable for a number of ecologically relevant traits including juvenile development (stamps and groothuis 2010) , energy expenditure (careau et al. 2008 ) and social structure (krause et al. 2010; wolf and krause 2014) . because personality influences non-random association patterns observed within social groups (krause et al. 2010) , between-individual variation in personality traits are expected to influence parasite transmission patterns within and between groups (barber and dingemanse 2010; kortet et al. 2010) . in general, the most active, exploratory or sociable (see table 5 .2 for definitions) individuals are expected to face greater risk of transmitting and/or acquiring parasites (barber and dingemanse 2010; kortet et al. 2010) . despite enormous recent interest in animal personality among behavioral ecologists, evolutionary physiologists, and conservationists (for review see sih et al. 2004; réale et al. 2007; careau et al. 2008; wolf and weissing 2012) , there are currently only three published studies examining effects of personality on any ecological trait in bats (menzies et al. 2013; kilgour and brigham 2013; webber et al. 2015b ). these studies highlight that individual bats, like many other vertebrates exhibit differences in personality but there is clearly room for much more research on personality in bats, especially in the context of parasite dynamics. we propose a series of hypotheses that would help to understand relationships between personality and parasite prevalence/intensity and transmission in the context of sociality. as for the implications of fission-fusion dynamics described above, these hypotheses vary for contact-mediated micro-and macroparasites versus mobile macroparasites with host-seeking behavior. first, we hypothesize a relationship between individual sociability and parasitism for contact-mediated parasites. theory predicts a positive relationship between host sociability and parasite risk, because the most sociable individuals are the most highly connected group-members within the social network and may therefore be exposed to, and themselves transmit, a disproportionate number of infections (barber and dingemanse 2010) . by contrast, for parasites with active host-seeking behavior, individual activity and exploration may be most important because less active or exploratory individuals may be easier targets for mobile macroparasites. webber et al. (2015b) identified the expected negative relationship between the prevalence and intensity of fleas (a relatively mobile ectoparasite) and the activity component of personality for female, but not male m. lucifugus, which highlights the potential complexity of these relationships. personality may have had a larger effect on females because females may be selective with their mating partners, so if less active females mate with fewer males they may retain a higher proportion of fleas, which would otherwise be transmitted during mating (webber et al. 2015b ). we suggest that future studies attempt to disentangle relationships between contact versus mobile parasites and sociability, activity, and exploration components of personality in bats. studies of host-parasite dynamics in bats are important from both a conservation and human public health perspective and, as we describe above, the implications of bat social behavior for parasite risk can be dramatic. based on our review of the literature, we identified social network analysis, epidemiological modeling (often, though not always, parameterized using empirically-derived data), and phylogenetically-informed comparative analyses as the methods most commonly used for quantifying links between sociality and parasitism in bats. in general, these studies suggest complex relationships and studies aimed at fully elucidating these links may require integration of techniques and data collection from multiple disciplines (e.g. behavior, ecology, epidemiology, parasitology, and physiology/immunology). we also found that, despite its recent emergence, wns is already among the best-studied pathogens of bats in the context of sociality perhaps, in part because researchers who focus on bats have brought behavioral and ecological expertise to bear on this important conservation problem. the link between host social aggregation and hev is also well established, again likely because of perceived societal importance, in this case 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white-nose syndrome without borders: pseudogymnoascus destructans infection tolerated in europe and palearctic asia but not in acknowledgments we thank jorge ortega for inviting us to contribute to this interesting book project and an anonymous reviewer for comments on an earlier version of this chapter. qmrw was funded through a manitoba graduate scholarship (mgs). funding for ckrw's research on host-parasite dynamics in bats is provided by a discovery grant and a discovery accelerator supplement from the natural sciences and engineering research council (nserc, canada). key: cord-303941-3lg1bzsi authors: han, hui-ju; wen, hong-ling; zhou, chuan-min; chen, fang-fang; luo, li-mei; liu, jian-wei; yu, xue-jie title: bats as reservoirs of severe emerging infectious diseases date: 2015-07-02 journal: virus research doi: 10.1016/j.virusres.2015.05.006 sha: doc_id: 303941 cord_uid: 3lg1bzsi abstract in recent years severe infectious diseases have been constantly emerging, causing panic in the world. now we know that many of these terrible diseases are caused by viruses originated from bats (table 1), such as ebola virus, marburg, sars coronavirus (sars-cov), mers coronavirus (mers-cov), nipah virus (niv) and hendra virus (hev). these viruses have co-evolved with bats due to bats’ special social, biological and immunological features. although bats are not in close contact with humans, spillover of viruses from bats to intermediate animal hosts, such as horses, pigs, civets, or non-human primates, is thought to be the most likely mode to cause human infection. humans may also become infected with viruses through aerosol by intruding into bat roosting caves or via direct contact with bats, such as catching bats or been bitten by bats. bats have been known to be reservoirs of rabies virus for a long time (pawan, 1959a,b) , and bats have also been considered as natural hosts of many common human and animal viruses, such as measles, mumps, parainfluenza, canine distemper and hepatitis c viruses (drexler et al., 2012; quan et al., 2013) . however, bats have caught our attention very recently, due to their association with several severe emerging infectious diseases. currently, bats have been considered to be natural reservoirs of sars-cov, mers-cov, niv, hev, ebola virus, and marburg viruses. these viruses have attracted global attention in recent years for their severity and/or easy transmission. this article reviews special features of bats in viral transmission and maintenance, bat's role as the reservoirs of some important viruses, the methods for bat-borne viruses' transmission to humans, and ecological drivers of bat-borne emerging infectious diseases. bats belong to the order chiroptera (greek means hand-wing) and can be further classified into two suborders: the yinpterochiroptera (megachiroptera, commonly known as megabats) and the yangchiroptera (microchiroptera, commonly known as microbats) (simmons, 2005) . megabats eat fruit and microbats live on insects, small mammals, fish or blood. megabats have no echolocation and microbats possess echolocation (except for rousettes and its relatives) ( table 1) . bats have many features that enable them to carry a diversity of viruses. with approximately 1240 recognized species worldwide, bats account for about 25% of all mammalian species, making them the second largest order of mammals (jones et al., 2005; turmelle and olival, 2009 ). the huge diversity of bat species can provide a large breeding ground for viruses. in addition, bats are ancient species and can be traced back to 52.5 million years ago (clyde et al., 2001; jepsen, 1966) . the long evolutionary history provides long period for a variety of viruses to co-evolve with bats to make bats their natural reservoirs. in order to facilitate flying, bats developed hollow bones to reduce body mass; as a result, they do not have bone marrow as other mammals to produce b cells. this special immunological characteristics of bats may enable bats to carry a large number of viruses without obvious illness (dobson, 2005) . during winter time, some bats hibernate to conserve energy. reduced body temperature and metabolic rate may suppress immune responses and delay viral clearance from bats (george et al., 2011; sulkin and allen, 1974) . some features of bats may keep viruses circulating among the bat population. bats are social animals, millions of individuals can be found in a single cave. the large number of bats in a colony with physical proximity facilitates viral transmission among bats, maintaining viruses circulating stably among bats. a study showed that several emerging viruses could be amplified in a bat colony during the breeding season (drexler et al., 2011) . microbats developed echolocation for navigation. apart from ultrasonic waves, the vibration of the larynx can also generate aerosols, which may also play an important role in viral transmission among bats (calisher et al., 2006) . some features of bats can contribute to viral dispersal. with a large number of species, bats can be found in all continents except the antarctica and inhabit various ecological niches, including trees, caves, and man-made structures, such as tunnels, deserted houses, even occupied houses in rural areas. the worldwide distribution and various habitats of bats pose the public to a general risk of infection with bat-borne viruses. bats are special as the only mammals that can actually fly. bats fly in their daily quest for food and seasonal migration, some of which can fly up to nearly 2000 km (fleming and eby, 2006) . the ability to fly enables bats to carry viruses to distant areas. the eating behavior of bats can also be linked to viral transmission. fruit bats cannot take a large amount of food, and to meet their demand for energy, instead of swallowing, they just chew to absorb sugars and higher energy components, and spit out the partially digested fruits. discarded fruits contaminated by viruses in bat saliva may be eaten by other animals and infect them (dobson, 2005) . in addition, despite their small size, bats have a relatively long life span, most of the species live for 10-20 years and some can live up to 30 years (brunet-rossinni and austad, 2004) . the longevity of bats also increases the possibility of viral dispersal. hev (formerly known as equine morbillivirus) was first recognized in australia in 1994, when it caused severe respiratory or neurological diseases in horses and humans (murray et al., 1995) . many investigations were done in search for the natural reservoir of this new highly pathogenic virus. initial screening of 2411 horses for hev-specific antibodies showed negative results (ward et al., 1996) , and a later extensive serosurvey of 5264 sera from 46 animal species, including both domesticated and wild animals, showed no evidence of infection. an epidemiological investigation pointed to fruit bats as a candidate natural reservoir, and serosurvey of 224 serum samples of fruit bats showed that 20 contained hevspecific antibodies (young et al., 1996) . three virus isolates were later obtained from urine and lung tissue from two of 460 individual fruit bats, and serological testing and sequencing of 200 nucleotides in the matrix gene indicated that the three isolates were identical to hev (halpin et al., 2000) . isolation of hev, together with serological and epidemiological evidence indicates that fruit bats are natural reservoirs of hev in australia. niv first emerged in malaysia in 1998, when it caused an outbreak of respiratory illness in pigs and encephalitis in humans. niv and hev were classified into a new genus, henipavirus, within the family paramyxoviridae. studies also tend to link niv to bats. serological evaluation in outbreak areas of malaysia showed that domesticated animals, such as dogs, cats and ponies had nivspecific antibodies (chua et al., 2000) , while wild boar, hunting dogs and rodents were all negative (yob et al., 2001 ). an extended survey of 14 species of bats highlighted that two fruit bat species had relatively high seropositivity, 31% and 17% for small flying fox (pteropus hypomelanus) and large flying fox (p. vampyrus), respectively. niv was isolated from p. hypomelanus urine and their partially eaten fruits, which was nearly identical to isolates from pigs and humans (56 nt changes) (abubakar et al., 2004; chua et al., 2002b) . recently, niv was isolated from p. vampyrus, which was about twice the difference between the human and p. hypomelanus isolates (98 nt changes) (rahman et al., 2010) . serological findings and viral isolation indicate that fruit bats are natural reservoirs of niv. sars-cov is the causative agent of sars (severe acute respiratory syndrome), which emerged as a new clinical severe human disease in guangdong province of china in 2002. initially, palm civets sold in live animal markets were demonstrated to carry sars-cov and were considered as the reservoirs of sars-cov (guan et al., 2003) , but later studies pointed to bats as the source of sars-cov. one group of researchers took bat samples from the natural environment, with 408 bats representing nine species. three species of horseshoe bats (genus rhinolophus) showed relatively high seroprevalence of sars-neutralizing antibodies, ranging from 28% to 71%. in addition, bat-cov rna was demonstrated 92% identity with human sars-cov isolates . another group of researchers screened nasopharyngeal and anal swabs of 120 bats and found that bat-cov sequences detected in 23 chinese horseshoe bats (rhinolophus sinicus) manifested 88% identity with the sars-cov, with great differences in spike genes (lau et al., 2005) . these bat sars-covs may be the progenitors of sars-cov, but cannot be closely related, since they were phylogeneticlly disparate from sars-cov and were unable to use the sars-cov cellular receptor molecule, human angiotensin converting enzyme ii (ace2), to enter cells (ren et al., 2008) . later, a study reported the whole-genome sequences of two novel bat-covs detected in chinese horseshoe bats, which were far more closely related to sars-cov than any previously identified ones, especially in the receptor binding domain of the spike protein, and they also isolated live sars-like cov from bat fecal samples. in vitro testing showed that this virus had a broad species tropism and used ace2 from humans, civets and chinese horseshoe bats for cell entry (ge et al., 2013) . thus chinese horseshoe bats are considered to be the natural reservoirs of sars-cov. mers-cov is a novel coronavirus, which was isolated from a man with acute pneumonia and subsequent renal failure in saudi arabia in 2012 . full genome sequence analysis of mers-cov and serological data provided evidence for transmission of mers-cov from camels to humans (azhar et al., 2014) , and mers-cov specific antibodies and rna have also been detected in camels during mers-cov outbreak investigations . however, phylogenetic analysis showed that mers-cov belonged to lineage c of the genus betacoronavirus, along with the bat-cov hku4 and hku5 . hence, bats are supposed to be the natural reservoirs of mers-cov, while camels may just act as intermediate animal hosts that facilitate the spillover from bats to humans. recent studies have revealed a diversity of mers-related covs in bats from saudi arabia , africa (ithete et al., 2013) , europe (annan et al., 2013) , and asia . studies on receptor usage by mers-cov can also give a clue of bat origins. initial study found that the receptor use by mers-cov was different from the one used by sars-cov. the receptor may be a conserved one, since mers-cov can replicate in both bat and human cells . subsequently, dipeptidyl peptidase 4 (dpp4 or cd26) was identified as the cellular receptor for mers-cov . bat coronavirus hku4, which is closely related to mers-cov, can also use dpp4 as receptor to initiate cellular entry, and this discovery indicated that bats might be the natural reservoirs of mers-cov (wang et al., 2014) . ebola virus first came into our knowledge in the democratic republic of congo in 1976 (johnson et al., 1977; report of an international, 1978) . since then, this notorious virus has been decimating gorilla, chimpanzee and human populations in africa with high mortality. during the 2013-2014 epidemic, ebola virus has caused 21,121 human infections and 8304 deaths (as of january 9th, 2015, http://www.cdc.gov/vhf/ebola/outbreaks/2014-west-africa/ index.html). during ebola outbreaks in humans and great apes between 2001 and 2003 in gabon and the republic of congo, to investigate the natural reservoirs, more than 1000 small vertebrates were captured. serum antibodies specific for ebola virus were found in three different bat species with prevalence as high as 25% (leroy et al., 2005) . given the lack of overt disease after being infected, bats are considered as likely natural reservoirs of ebola virus. the viruses discussed above tend to be restricted to certain geographic regions with a particular bat reservoir, such as hev and niv associated with flying foxes in australia and southeast asia and ebola virus associated with egyptian fruit bats in africa. most bat population roost in tropical forests or caves and they do not make frequent contact with people. how bats transmit viruses to humans is a mystery until now. below are the hypotheses of how bat-borne viruses are transmitted to humans. a popular theory for bats to transmit viruses to humans is through intermediate hosts, which are in close contact with humans and can amplify viruses. when fruit bats eat fruit, they contaminate the fruit with viruses in their saliva and urine; discarded fruit fall to the ground and is consumed by intermediate hosts, such as pigs, horses, and nonhuman primates; humans become infected by contact with or consuming products of the intermediate animal hosts. flying foxes are thought to transmit niv/hev in southeast asia and tropical australia. during the 1998 niv outbreak in malaysia, it is thought that pigs were infected by niv by consuming mangoes contaminated by flying foxes. mangoes were a food source for flying foxes, and discarded mangoes contaminated by the saliva and urine of the bats fell into the pigsties and were consumed by pigs, resulting in the cross-species infection of pigs and subsequently humans (chua et al., 2002a) . hev is endemic in flying-foxes in australia. hev spills over from bats to domestic animals, primarily horses, which amplify the virus and subsequently infect humans (murray et al., 1995) . bats contaminate a drip zone around trees where they feed or roost by excreting urine, feces and saliva (with partially eaten fruit). horses may be exposed to hev when consuming contaminated grass, fruit, feed or water; or when browsing or sniffing contaminated surfaces within this drip zone (plowright et al., 2015) . fruit contaminated by bats is also thought to transmit ebola virus to apes in central africa (leroy et al., 2005) . in the case of the 2003 sars outbreak in china, the transmission of sars-cov from bats to humans was made possible by the special taste for wildlife cuisine, including civets, which acted as intermediate hosts for sars-cov. in south china, civets are thought to strengthen health in winter, and the demand was so high that farming of this wildlife species was widespread. trade of civets in live wet markets exposed other susceptible animals. therefore, bat sars-cov was likely introduced into humans through civets and other animals (liu, 2003) . as for mers in middle east, dromedary camels are hypothesized as intermediate animals to transmit mers-covs from bats to humans (memish et al., 2014) . camels are popular in middle east countries both for entertainment and transportation. people may be infected through direct contact with infected camels, which can shed virus in respiratory secretions (azhar et al., 2014) . mers-covs were also detected in camel milk samples from mares infected with the virus (reusken et al., 2014) . whether the viruses is excreted in the milk or is introduced as contaminant during the milking process, it poses a risk for people to become infected by consumption of unpasteurized camel milk. although bats are rarely in contact with people, people may become infected with bat-borne viruses by consuming bat meat. wild animal meat or 'bush meat', including bats, are taken as delicacies in some regions of africa. the capture and selling of wild animals increases the risk of being infected by zoonotic viruses. consumption of infected bats may transmit such bat-borne viruses as ebola virus to humans. in 2007 ebola hemorrhagic fever reemerged in the democratic republic of congo (drc) causing 186 deaths. epidemiological investigation showed that the outbreak was due to consumption of fruit bats, which were migrating towards the southeast and were massively hunted as a major source of protein by local villagers (leroy et al., 2009 ). consumption of well-cooked bat meat may not be a problem, but the handling of bats may increase the risk of infection. in addition, accidental bite by a bat may result in rabies. one example is the case of an elderly man in south africa who died of duvenhage virus (duvv) infection after being scratched by a bat (adjemian et al., 2011) . some bats with a large population roost in caves. bat-borne viruses may contaminate air in caves where bats live and people may become infected by inhaling viruses in the air when they enter caves. research showed that cynomolgus macaques could suffer a lethal viral hemorrhagic fever after exposure to small-particle aerosols containing marv-angola (alves et al., 2010) . there was a report that humans were infected by marburg virus after visiting or working in caves in africa, and transmission by aerosol could not be excluded as a possible mode of infection (timen and koopmans, 2008) . for a spillover to occur, a range of conditions and events must be met. bats must be present and be infected, in most cases shedding viruses. viruses excreted by bats must survive in the environment (if transmitted indirectly), with access to intermediate animals. human or intermediate animals must be exposed to a sufficient dose of virus for an infection to be established, and humans or intermediate animals must be susceptible to the virus (plowright et al., 2015) . however, of all these conditions required for a spillover, it is of utmost importance for bats and humans or animals in close contact with humans to be present in the same habitat, which can be a result of intrusion into each other's living environment. once infected, some factors can accelerate transmission in human society. below we describe ecological factors that increase interaction between bats and humans and factors that accelerate transmission in humans. in human history, the emergence of new pathogens has been accompanied by increased density of humans and other animals. with human population explosion, more space and resources are needed to meet ever-increasing demand for food and housing. factors that contribute to the intrusion of bats into human living environment can be summarized into a 'push' and a 'pull' (brüssow, 2012) . a 'push' refers to the enormous demand for more space and resources brought by the human population explosion, which leads to the destruction of bat habitats and shortage of food. natural environmental changes, such as typhoons and droughts, can also place stresses on bats. a 'pull' involves the living environments built by humans, characterized by urbanization, intensive agriculture and food animal breeding, which attracts bats into human living environments for an abundant of food supply. the 1998 niv outbreak in malaysia was thought to be a combined result of anthropogenic deforestation and drought caused by el nino, which pushed flying fox bats from their natural habitats into farms (chua et al., 2002a) . the rapid development of modern transportation, and the mobility of people, animals and goods lead to the rapid spread of emerging infectious diseases. local transportation is important in sustaining endemics, while international air travel may facilitate cross-continent spread (bobashev et al., 2008) . during the 1998 niv outbreak in malaysia, the transport of infected pigs led to the expansion of outbreak areas in malaysia (lam and chua, 2002) , and the follow-up niv outbreak in singapore among abattoir workers (paton et al., 1999) . due to air transit, the sars outbreak that initially emerged in guangdong, china in november of 2002, quickly spread to 25 countries as far away as canada by the end of march of 2003 (cdc, 2003 . another example is the 2014 ebola hemorrhagic fever outbreak, the largest ebola virus outbreak to date, which quickly spread in west africa and resulted in massively affected areas including guinea, liberia, sierra leone and nigeria. ebola virus also spread to the usa by infected patients who traveled from west africa (von drehle, 2014) . cultural practices can also play an important role in the transmission of emerging infectious diseases. during the 2001 niv outbreak in bangladesh, person-to-person transmission was attributed to the social norms that require family members to maintain close physical contact with the sick one (luby et al., 2009) . in africa, traditional burial practices require the living ones to make contact with the deceased, which also facilitates transmission during ebola virus outbreaks (dowell et al., 1999) . socioeconomic factors may also contribute to transmission, associated with poor health care. one example is the 2014 west africa ebola virus outbreak, first-line health care workers were infected, because of lack of knowledge as well as the unavailability of proper personal protective equipment (kilmarx et al., 2014) . person-to-person transmission during the 2001 bangladesh niv outbreak was also thought to be a result of poverty-induced sharing of eating utensils and food with the infected person (brüssow, 2012) . with ever-increasing interest in bat-borne viruses, and the availability of molecular biotechnology, novel viruses are continually detected or isolated from bats all around the world. in recent years, viral metagenomics may provide a glimpse of bat viral diversity (dacheux et al., 2014; donaldson et al., 2010; ge et al., 2012; he et al., 2013) . so far, viruses detected in bats can be classified into as many as 22 families and many are novel viruses (http://www.mgc. ac.cn/dbatvir/). the pathogenicity of many of these novel viruses for humans remains unknown, and further efforts are needed to determine their potential threats to humans. in addition, previously known bat-borne viruses are also being detected in more bat species, and the geographic distribution of these viruses is also expanding. with human activity increasingly overlapping the habitats of bats, emerging infectious diseases from bat-borne viruses will undoubtedly increase. transmission modes discussed above are just current hypothesis that could theoretically explain some of the spillover events. however, the spillover process is still something of a black box that is scarcely understood and much more research is needed to expand our understanding of the spillover events. there are many questions that remain to be answered about the ecology of bat-borne infections: (1) are bats the natural reservoirs or just transient carriers of these novel viruses? (2) how long can bats harbor these viruses? (3) what are the routes of virus shedding? (4) how long can viruses excreted by bats survive in the environment? 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mers-related betacoronavirus in vespertilio superans bats nipah virus infection in bats (order chiroptera) in peninsular malaysia serologic evidence for the presence in pteropus bats of a paramyxovirus related to equine morbillivirus isolation of a novel coronavirus from a man with pneumonia in saudi arabia the authors are grateful to dr. david h. walker (department of pathology, university of texas medical branch at galveston, texas 77555-0609) for reviewing out manuscript. this research was supported by grants from shandong province science and technology development program (2014gsf121004) and supported by the shandong university. key: cord-255137-utg8k7qs authors: yinda, claude kwe; vanhulle, emiel; conceição-neto, nádia; beller, leen; deboutte, ward; shi, chenyan; ghogomu, stephen mbigha; maes, piet; van ranst, marc; matthijnssens, jelle title: gut virome analysis of cameroonians reveals high diversity of enteric viruses, including potential interspecies transmitted viruses date: 2019-01-23 journal: msphere doi: 10.1128/msphere.00585-18 sha: doc_id: 255137 cord_uid: utg8k7qs diarrhea remains one of the most common causes of deaths in children. a limited number of studies have investigated the prevalence of enteric pathogens in cameroon, and as in many other african countries, the cause of many diarrheal episodes remains unexplained. a proportion of these unknown cases of diarrhea are likely caused by yet-unidentified viral agents, some of which could be the result of (recent) interspecies transmission from animal reservoirs, like bats. using viral metagenomics, we screened fecal samples of 221 humans (almost all with gastroenteritis symptoms) between 0 and 89 years of age with different degrees of bat contact. we identified viruses belonging to families that are known to cause gastroenteritis such as adenoviridae, astroviridae, caliciviridae, picornaviridae, and reoviridae. interestingly, a mammalian orthoreovirus, picobirnaviruses, a smacovirus, and a pecovirus were also found. although there was no evidence of interspecies transmission of the most common human gastroenteritis-related viruses (astroviridae, caliciviridae, and reoviridae), the phylogenies of the identified orthoreovirus, picobirnavirus, and smacovirus indicate a genetic relatedness of these viruses identified in stools of humans and those of bats and/or other animals. these findings points out the possibility of interspecies transmission or simply a shared host of these viruses (bacterial, fungal, parasitic, …) present in both animals (bats) and humans. further screening of bat viruses in humans or vice versa will elucidate the epidemiological potential threats of animal viruses to human health. furthermore, this study showed a huge diversity of highly divergent novel phages, thereby expanding the existing phageome considerably. importance despite the availability of diagnostic tools for different enteric viral pathogens, a large fraction of human cases of gastroenteritis remains unexplained. this could be due to pathogens not tested for or novel divergent viruses of potential animal origin. fecal virome analyses of cameroonians showed a very diverse group of viruses, some of which are genetically related to those identified in animals. this is the first attempt to describe the gut virome of humans from cameroon. therefore, the data represent a baseline for future studies on enteric viral pathogens in this area and contribute to our knowledge of the world’s virome. the studies also highlight the fact that more viruses may be associated with diarrhea than the typical known ones. hence, it provides meaningful epidemiological information on diarrhea-related viruses in this area. d iarrhea is the second most common cause of death worldwide and accounts for about 8 to 9% of the 5.9 million yearly deaths in children under the age of 5 (1, 2) . most of these deaths occur in southeast asia and sub-saharan africa (3, 4) . the chances of infection with enteric viruses are higher in developing countries than developed countries, probably due to suboptimal sanitation and hygienic conditions and low quality of drinking water, especially in rural areas (5) . in cameroon, a limited number of studies have investigated the prevalence of enteric pathogens as the cause of gastroenteritis in humans. these studies mainly focused on the epidemiology of a limited number of pathogens such as rotavirus, norovirus, and enteroviruses, revealing significant differences in the prevalence of these viruses in different settings and time periods (4, 6, 7) . in parts of cameroon, a high prevalence of several enteric viruses such as enterovirus, norovirus, rotavirus, and adenovirus was found in children and adults (8) . generally in africa, many episodes of gastroenteritis remain unexplained as no etiological agent is determined (9, 10) . a proportion of the unexplained gastroenteritis cases are likely due to other known viruses, for which no tests were performed. however, a part of these gastroenteritis cases could also be caused by novel viral agents. transmission of these enteric viruses is predominantly fecal-oral, and humans are constantly exposed to these viruses through various routes (11) . one of these routes is zoonosis from reservoirs in wild or domestic animals, either by insect vectors or by exposure to animal droppings or tissues. one rich but, until recently, underappreciated reservoir of emergent viruses is bats. of the ϳ5,500 known terrestrial species of mammals, about 20% are bats (12) . several viruses pathogenic to humans are believed to have originated in bats over the last several years, including severe acute respiratory syndrome (sars)-and middle east respiratory syndrome (mers)-related coronaviruses, as well as filoviruses, such as ebola and marburg viruses, or henipaviruses, such as nipah and hendra viruses (13) (14) (15) (16) (17) (18) . in the southwest region of cameroon, bats are hunted and eaten. such close interactions provide ample opportunity for zoonotic events to occur (19) . previously, we identified a plethora of known and novel eukaryotic viruses in cameroonian fruit bats using a viral metagenomics approach, including viruses known to cause gastroenteritis in humans (sapovirus, sapelovirus, and rotaviruses a and h) and those not yet associated with gastroenteritis (bastrovirus and picobirna-like viruses) (20) (21) (22) (23) . in the current study, we metagenomically screened 221 human fecal samples collected in the same region (where bats are hunted and eaten), to assess (i) if any viruses of animal origin could be identified and (ii) which known human gastrointestinal viruses were present. these fecal samples were collected from children less than a year old to adults of more than 60 years who had gastroenteritis and/or were in contact with bats. additionally, since the gut virome typically contains both eukaryotic and prokaryotic viruses (phages), of which the latter usually represents the largest fraction of the gut virome, we also analyzed the phageome of these samples. sample characterization. a total of 221 human fecal samples (131 from kumba and 90 from lysoka) were collected from two hospitals in the southwest region of cameroon, for viral metagenomics screening. from these fecal samples, a total of 63 pools were constituted in categories based on age, bat contact status, and location (see table s1 in the supplemental material). illumina sequencing of all the 63 human pools generated in total approximately 708 million raw paired-end (pe) reads (between 4.3 and 53.4 million reads per pool). after trimming, 67% of the reads (471 million) were retained and 86% of these retained trimmed reads (405 million) were annotated using diamond. of these, 18% (74 million) could be attributed as viral. ngs viral read distribution/abundances. in each of the categories of pools, phages make up at least 84% of the total number of viral reads while the maximum proportion of eukaryotic viral reads is 16%. a similar annotation profile was observed for pools of patients in different age groups, different locations, and different bat contact statuses (fig. s1 ). further analysis of eukaryotic viral reads revealed that at least 70% of the reads mapped to viruses of the families astroviridae, reoviridae, and anelloviridae (fig. 1a) . other viruses were also present, particularly those that are known to cause gastroenteritis belonging to the families adenoviridae, caliciviridae (sapovirus and norovirus), and picornaviridae (of which about 60% were enteroviruses [fig. 1b and fig. s2] ). also, reads from viruses known to cause other human diseases (parvoviridae) or other animal diseases (circoviridae) or not associated with any diseases at all (picobirnaviridae) were present in variable numbers in the different groups ( fig. 1b to e) . the rest of the viral families were either plant-or insect-associated viruses. notably, in age groups a to d, the percentage of pools in which picobirnaviridae viruses were present increased with age with low percentages in age groups a and b (fig. 1c) . also, the percentages of pools positive for anelloviruses differed with respect to age, with higher percentages in young children and the elderly. further, there were no observable trends in the percentage of eukaryotic viral presence with respect to bat contact status or location ( fig. 1d and e) . figure 1f shows a heat map of the percentage of pools in which eukaryotic viral families were present in human and bat pools, while fig. s3 presence in human and bats at the genus level (23) . astroviridae (mamastrovirus), calciviridae (sapovirus), picornaviridae (parechovirus), and reoviridae (rotavirus), viral families known to cause gastroenteritis in humans, were identified in both bat and human pools from the same region. also, mammalian viruses not yet established to cause gastroenteritis (picobirnaviridae, circoviridae, and parvoviridae [bocaparvovirus]) were also common in both bats and humans from the same regions ( fig. 1f and fig. s3 ). phylogeny of eukaryotic viruses. in this study, we focused on viruses from which near-complete genomes were obtained, particularly those that are known to cause viral gastroenteritis (belonging to the astroviridae, caliciviridae [norovirus and sapovirus] , picornaviridae [enterovirus, parechovirus, cosavirus] , parvoviridae, reoviridae, and adenoviridae [human mastadenovirus]). furthermore, we also looked at other viruses not fully proven to cause gastroenteritis in humans but which have only sporadically been associated with gastroenteritis, like picobirnaviridae and small circular singlestranded dna viruses. phylogenetic analysis was done for each of the selected viruses using the protein or nucleotide sequences of suitable conserved regions and representative members of their viral family, genus, or species. reoviridae. reoviridae is a large viral family of segmented dsrna viruses with a wide host range. they are further divided into two subfamilies and 15 genera. genomes of viruses belonging to the reoviridae contain 9 to 12 segments (24) . in total, reoviridae reads were found in 6 pools, and (nearly) complete genomes of 2 viruses of the family reoviridae were obtained from pool hp55. samples in this pool were from two diarrheic children (less than 5 years), originating from kumba and without contact with bats. mammalian orthoreovirus. mammalian orthoreoviruses (morvs) contain 10 segments, l1 to l3, m1 to m3, and s1 to s4, coding for 12 to 13 proteins (24, 25) . a morv strain was identified represented by 16,913 reads (0.4% of all viral reads of the pool). phylogenetic analysis based on the nucleotide sequences of each of the 10 segments of this morv ( fig. 2 and fig. s4 ) showed topological incongruence with four distinctive patterns. based on segments l2 and s1, this strain clustered with bat strains wiv3 and wiv5 from china with 86% and 70% nucleotide (nt) identity, respectively ( fig. 2a and b ). for the l1 and s2 segments, the human strain clustered with the ndelle murine strain, also from cameroon, with 95% and 92% nt identity, respectively (fig. 2c and d) . on the other hand, segment s3 of the cameroonian morv strain clustered with a human strain and a civet morv strain from china (88% and 89% nt identity, respectively [fig. 2e] ). the rest of the segments (l3, m1 to m3, and s4) did not cluster together clearly with any of the abovementioned strains (fig. s4) . rotavirus a. rotavirus a (rva) contains 11 segments coding for 11 or 12 proteins: vp1 to vp4, vp6, vp7, and nsp1 to nsp6 (26, 27) . we identified a near-complete rva sequence which made up 99% (4.3 million) of the eukaryotic viral reads of that pool. the nsp3 segment was not identified in the sample. the vp7 gene of this strain was genetically most related to rva/human-tc/usa/wa/1974/g1p1a [8] and rva/human-tc/usa/rotarix/2009/g1p [8] (nt identity of 92 and 97%, respectively) while the vp4 gene was 90% identical to the same strains. the phylogenetic trees of the remaining segments shared the same clustering pattern (fig. 3a and b and fig. s5 ). according to the rotavirus classification scheme, this strain is a typical wa-like g1p [8] named rva/ human-wt/cmr/cmrhp55/2014/g1p [8] . cmrhp55 was distantly related to bat rva strains identified from the same regions (only 69 to 71% nt identity). picornaviridae. the picornaviridae represent a large family of small, cytoplasmic, nonenveloped icosahedral ssrna viruses consisting of 80 species, grouped into 35 genera. they have a genome of 7.1 to 8.9 kb in size and are most often composed of a single orf encoding a polyprotein flanked by a 5= and 3= utr (28) . the members of the family picornaviridae can cause gastroenteritis, meningitis, encephalitis, paralysis (nonpolio and polio-type), myocarditis, hepatitis, upper respiratory tract infections, and diabetes (29, 30) . out of the 63 pools, 41 contained picornaviridae reads, making the picornaviridae the eukaryotic viral family of which reads could be identified in the highest number of pools. enterovirus. the genus enterovirus (ev) consists of 15 species: enterovirus a to l and rhinovirus a to c. ev a, b, c, and d are found in humans; e and f in cattle; g in pigs; h, j, and l in monkeys; k in rodents; and species i in dromedary camels (http://www .picornaviridae.com). in this study, eighteen (nearly) complete genomes of evs were obtained. the strains were named ev/human/cmrhpxx/cmr/2014, here referred to as ev-cmrhpxx. all eighteen genomes were found in pools of age groups a and b (ͻ3 and 3 to 20 years, respectively). eight of these were identified in age group a, three (ev-cmrhp1, 5a, and 5b) of which were pools consisting of samples of infants who had indirect contact with bats while the rest (ev-cmrhp14, 45, 52a, 52b, and 55) were those that had no contact with bats. the ten other strains were identified in pools belonging to age group b, three of which had direct contact with bats (ev-cmrhp8a, 8b, and 9), 5 indirect contact 4, 35a, 35b, and 39) and two with no contact . based on the phylogenetic analysis of the vp1 nucleotide sequences, the evs found in this study were quite divergent from each other, belonging to three different species of enterovirus, a, b, and c (fig. 4a ). most of the strains belonged to the enterovirus c clade (ev-cmrhp1, 3, 4, 8a, 8b, 9, 14, 18, 35a, 52a, and 55) , while ev-cmrhp35b, 39, and 45 clustered within the enterovirus b genotype, and evcmrhp5a, 5b, 52b, and 58 in the genogroup enterovirus a. some pools had multiple strains of ev present, and some of these clustered together (cmrhp8a and 8b: vaccine type pv-3), whereas other pools contained distinct ev species (ev-cmrhp35a and 35b; 52a and 52b). the presence of vaccine strains in pool hp8 probably indicates recent vaccination events of the infants in this pool. apart from ev-cmrhp39 (which clustered with 11c52_cmr), all the ev strains identified here were distantly related to those previously identified in the far north region of cameroon (31) . furthermore, none of the human strains from cameroon were related to any of the animal ev strains (from chimp or gorilla). a summary of the detailed classification of these evs using an online typing tool (32) is shown in table 1 . parechovirus. the genus parechovirus is comprised of two species, parechovirus a (human parechovirus [hpev] ) and parechovirus b (ljungan virus, isolated from bank voles) (33) . hpev is subdivided into 19 types (hpev1 to -19) . hpev is associated with mild gastrointestinal or respiratory illness; however, severe disease conditions, such as meningitis/encephalitis, acute flaccid paralysis, and neonatal sepsis, may occur (34) (35) (36) . here, three (nearly) complete hpevs were identified in pools hp2, hp46, and hp48 with sequence lengths of 7,142 bp, 7,202 bp, and 7 ,219 bp, respectively, collected from children less than 3 years old (age group a). in terms of bat contact status, they were in pools of those either in indirect contact with bats (hp2 and hp48) or without contact (hp46). they were all distantly related to each other, with hpev-cmrhp46 and hpev-cmrhp48 having the highest identity (76% and 86% nt and aa identity, respectively). phylogenetically, hpevs in hp46 and in hp48 fell into a clade of type 1 hpevs (fig. 4b ). the hpev in hp46 clustered together with hpev1/harris strain with 76% nt identity, while cmrhp48 clustered closely with japanese and norwegian strains a1086-99 and no-3694 (84 to 90% nt identity) . furthermore, hpev-cmrhp2 clustered distantly with type 16 hpevs from china and bangladesh with only 70 to 71% nt identity. considering the 75% identity demarcation for hpev types (37, 38) , this strain potentially represents a novel type. cosavirus. the genus cosavirus consists of five species (cosavirus a, b, and d to f), which have been associated with gastroenteritis in children (39) . six near-complete [8] strains. red, cameroonian human rva strain identified in this study; blue, cameroonian bat rva strains. trees were constructed human cosavirus (hcosv) genomes were identified: 1 from children less than 3 years old (hp49), 3 from those between 3 and ͻ20 years old (hp6a and hp6b, hp57), and 2 from pools of individuals between 20 and ͻ60 years old (hp44, hp24). some of these pools had direct or indirect contact with bats (hp6, hp24, and hp44), while others had no contact with bats (hp49 and hp57). phylogenetic analysis (fig. 4c ) showed that cosaviruses from hp6b, hp49, and hp57 formed a clade with two other strains from australia and nigeria (hcosv/e1/aus and hcosv/ng385/nga) in species hcosv e. meanwhile the strains in hp6a, hp24, and hp44 clustered with hcosv in species a, d, and b, respectively. therefore, it seems that humans in cameroon host a diverse range of cosaviruses. cardiovirus. the genus cardiovirus consists of three species, cardiovirus a to c. species b includes saffold virus (safv) infecting humans. it has been found in cases with acute flaccid paralysis, respiratory tract infections, and diarrhea in china (40) (41) (42) . here, we found a near-complete genome of a safv in one pool (hp35) belonging to the age group between 3 and ͻ20 years old who had indirect contact with bats. the vp1 phylogenetic trees were based on the nucleotide sequences of the vp1-p2a region for the species hepatovirus a and the vp1 region for the rest of the genera. all the trees were constructed using the gtrϩgϩi nucleotide substitution model using raxml, with the automre flag, which enables a posteriori bootstrapping analysis. only bootstrap values greater than 70% are shown. bars indicate nucleotide substitutions per site. red, novel strains from this study; blue, human cameroonian enterovirus strains from other studies; green, animal enterovirus strains from cameroon. segment of the identified safv was 72 to 74% and 78 to 80% identical (on nt level) to safv strains in types 5 and 6, respectively. phylogenetic analysis based on the vp1 region confirmed the clustering of the novel strain between types 5 and 6 with more phylogenetic relatedness to type 6 ( fig. 4d) . hence, this novel safv strain may be a distant member of type 6 or represent a new type. hepatovirus a. hepatitis a virus (hav), now hepatovirus a, belongs to the genus hepatovirus, which consists of nine species (hepatovirus a to i). the hepatovirus a species is comprised of a single serotype, hav, subdivided into human and simian viruses (43) . it causes acute hepatitis throughout the world (44) . there were three (nearly) complete hav genomes in pools hp2, hp4, and hp6, all of which were pools from those in direct (hp6) or indirect (hp2 and hp4) contact with bats. these strains were either from infants less than 3 years old (hp2) or from children between 3 and ͻ20 years old (hp4 and hp6). based on the vp1-p2a region, the nt identity between these strains was 98 to 99%. strains in hp4 and hp6 were 99% identical to brab13, isolated from a patient from the netherlands in 2001, who was staying in a hippie community with visitors from all over the world and under primitive living conditions (45) . on the other hand, the hav strain in hp2 was closely related to strain g2b1-vp from france (98% nt identity). therefore, all strains identified here are genotype iia (fig. 4e) , increasing the number of completely sequenced genotype ii strains to five (the other two strains are ba/ita/2012 and cf53/berne). astroviridae. astroviridae is a family of nonenveloped, spherical viruses with a linear ssrna(ϩ) genome of 6.8 to 7kb, containing three overlapping orfs. the family is divided into two genera: genus mamastrovirus (mastvs) and genus avastrovirus (aastvs). the genera are further divided into 33 and 7 species, respectively (46) . fourteen out of the sixty-three human pools contained astroviridae reads, and we were able to obtain eight near-complete genomes of mastvs (hp2, 3, 6, 34, 35, 43, 45, and 46) . additionally, these pools were either from children less than 3 years old (hp2, hp45, and hp46), age group 3 to ͻ20 (hp3, hp6, and hp35), or between 20 and ͻ60 (hp34 and hp43). phylogenetic analysis of the rdrp and capsid regions ( fig. 5a and b) depicted clustering of the novel mastvs in species 1 (cmrhp2, 3, 34, 35d, 43, and 46) , 6 (cmrhp45), and 9 (cmrhp6). in the mastvs1 clade, there seems to be topological inconsistency in the different phylogenetic trees. strain astv8_yuc8 (af260508) clustered with the novel strains cmrhp2, 3, and 35d in the capsid tree, while in the rdrp tree it clustered with the chinese strain v4-guangzhou, suggesting a recombination event between these strains in the past. bat astrovirus identified in cameroon (23) phylogenetic trees based on the nucleotide sequences of the rdrp (a) and capsid (b) genes of the astvs identified in this study and representative strains from genbank. trees were constructed using the gtrϩgϩi nucleotide substitution model using raxml, with the automre flag, which enables a (continued on next page) formed a clade (in the rdrp tree) with other bat astroviruses from guangxi but was distantly related to the human astvs from the same region. caliciviridae. caliciviridae are a family of nonenveloped viruses with a linear ss-rna(ϩ) genome of 7.3 to 8.3 kb, containing two or three orfs. the family contains five genera (47, 48) . in total, caliciviridae reads were found in 16 pools belonging to either the norovirus or sapovirus genus. norovirus. this genus consists of a single species, norwalk virus (nv), divided into 5 genogroups. genogroups i, ii, and iv infect humans, whereas genogroup iii infects bovine species and genogroup v has been isolated from mice (49) . three nearcomplete nvs were present in the 16 pools that contained caliciviridae reads (hp1, hp18, and hp59), from people who had indirect (hp1 and hp59) or no (hp18) contact with bats, and from age group a (hp1), b (hp18), or c (hp59). the phylogenetic tree (fig. 6a) showed that the four nvs belonged to two genogroups: i (nv_cmrhp18, genotype i.3) and ii (nv_cmrhp1 and nv_cmrhp59, genotypes ii. 12 and ii.13, respectively) . the novel strain nv_cmrhp18 was more than 98% similar to strain c13/ 2009cmr_gi.3 (a partial sequence [jf802509]) isolated from the littoral region of cameroon in 2009, whereas strains of genogroup ii from the same study (ii.4, ii.8, ii.17) were distantly related to those identified here (ii.12 and ii.13) (7) . strains from this previous study were not included in the phylogenetic analysis because only 200 to 300 nt of the capsid region was available in databases. sapovirus. the genus sapovirus (sav) consists of a single species, sapporo virus. it has been detected in humans, pigs, minks, dogs, sea lions, bats, chimpanzees, rodents, and carnivores (50, 51) . three near-complete sav genomes were present in pools hp4 (age group b), hp15 (age group a), and hp22 (age group d) from people who were in indirect contact, were not in contact, and were in direct contact with bats, respectively. phylogenetic analysis (fig. 6b) showed that sav from hp22 could be classified as a giv genotype, and the savs hp4, hp53, hp46, hp56, and hp15 belonged to genotype gii. the phylogenetic tree showed that the bat savs found in cameroon (in blue) (22) clustered together and formed a clade with other bat savs from china and hong kong but divergent from these human savs, indicating no evidence of interspecies transmission of savs in this region. picobirnaviridae. picobirnaviruses (pbvs) belong to the family picobirnaviridae, genus picobirnavirus, and are small bisegmented dsrna viruses with a total genome size of about 4 kb. segment one encodes a polyprotein, containing the capsid protein, and segment two encodes the rdrp. based on the rdrp gene, pbvs are classified into two genogroups. although pbv is genetically highly diverse and has been found in stool samples of a broad range of mammals, its true host(s) remain(s) enigmatic. the disease association is unclear, but pbv infection has been associated with gastroenteritis in both animals and humans (52, 53) . up to 28 out of the 63 pools contained reads annotated as picobirnaviridae with most of the positive pools from individuals in age groups above 20. we could obtain 37 (near-complete) rdrp sequences of pbvs from these 28 pools. phylogenetic analysis based on rdrp (fig. 7) revealed the clustering of the novel strains in four different clades: in genogroup i (26 strains) , in genogroup ii (9 strains), and in 2 clades (3 strains) of uncharacterized picobirna-like viruses that use an alternative mitochondrial invertebrate genetic code (lysoka picobirna-like virus cmrhp9 and cmrhp10b and kumba picobirna-like virus cmrhp21a). interestingly, a wolf pbv strain from portugal (ans53886) from genogroup i clustered together with human strains from cameroon with an aa identity of 76% with strains cmrhp26a and cmrhp35. likewise, in genogroup ii, strains cmrhp34a, cmrhp63b, and cmrhp26c clustered closely (75 to 76% aa identity) with a portuguese feline strain (agz93689). intriguingly, the cameroonian human picobirna-like viruses cmrhp9 and cmrhp10b were 99% identical to a cameroonian bat strain picobirna-like virus, p11-300, suggesting a possible interspecies transmission. however, their true host has not yet been determined. it could be that the true hosts of pbvs are found in both humans and bats and that this therefore explains their presence in both. small circular, rep-encoding, ssdna (cress-dna) genomes. (i) smacovirus. smacovirus (scv) is a relatively recently described virus with a small circular dna genome with a size of about 2,529 bp. it belongs to the smacovirus group and is an unclassified eukaryotic virus of unknown origin (54) . in this study, we identified two scv sequences, one complete genome (huscv-cmrhp10) and a near-complete genome (huscv-cmrhp03). they were identified in pools of patients belonging to age group b, coming from lysoka and having direct (hp3) or indirect (hp10) contact with bats. these strains shared 99% amino acid identity. their replicase genes were 94% and 95% identical to chimpanzee (kp233190) and human (huscv3, kt600069) strains from the united states, respectively. based on the capsid region, these novel cameroonian strains were 98 to 99% identical to the chimp strain and only 85% identical to the human strain huscv3. the close genetic relatedness of human strains to a strain found in a chimpanzee sample suggest that these viruses infect a host shared between chimps and humans, and if indeed smacovirus infects mammals, this could be a case of interspecies transmission (55) . phylogenies of the replicase (fig. 8a ) and the capsid genes ( fig. 8b) indeed showed a cluster of these cameroonian strains with a human genogroup i and pecovirus (c and d) of the replicase and capsid genes, respectively. the trees were constructed using the lgϩgϩi substitution model using raxml, with the automre flag, which enables a posteriori bootstrapping analysis. only bootstrap values greater than 70% are shown. bars indicate amino acid substitutions per site. red, cameroonian human strains; blue, previously known human smacoviruses or pecovirus. and a chimpanzee strain from the united states. however, the topological inconsistency in the replicase and capsid trees may suggest a recombination event between these strains in the (distant) past. (ii) pecovirus. pecoviruses (peruvian stool-associated circo-like viruses [pecvs] ) are cress-dna genomes that were first identified in the feces of a patient during an outbreak of acute gastroenteritis in the netherlands and later in samples of peruvian children (55, 56) . subsequently, they were identified in other humans, pigs, a dromedary camel, and a seal (55) (56) (57) (58) . here we identified a genome sequence (hupecv-cmrhp60) of 2,937 bases made up of two orfs that code for a capsid (372 aa) and a replicase protein (336 aa). unlike other human pecvs, the cameroonian strain shared the same canonical nonamer (nantattac) atop the predicted stem-loop structure with seal, dromedary, and porcine pecv strains. the rep showed 31 to 42% aa sequence identity to all other rep genes, and a rep-based phylogenetic analysis (fig. 8c) showed that hupecv-cmrhp60 clustered together with pecovirus genomes from a seal and 3 human strains. based on the cap protein (fig. 8d) , the cameroonian strain was only 22 to 42% identical to all other pecoviruses and clustered only distantly from the seal strain, a porcine strain, and the human strains. this demonstrates the existence of a high level of genetic diversity within this group of circular dna genomes, pointing to the possible existence of multiple species in this clade. furthermore, we identified 2 incomplete sequences related to sewage-associated circular dna molecules recovered from a sewage treatment oxidation pond in new zealand (59), with only 38% aa identity on the rep protein, further expanding the great diversity of cress-dna genomes in the cameroonian population. bacteriophages. bacteriophages are viruses that infect and replicate within bacteria. their presence therefore reflects the gut microbiota of the patients. because most of the obtained viral reads were classified as bacteriophages, we further investigated the bacteriophage composition of the human samples. with virsorter (60), a tool developed to identify highly divergent dsdna phages from metagenomics data, 5,905 of the contigs in our data set were identified as phages. from these, the tool diamond (61) annotated 2,647 as bacterial, 21 as metazoan, and only 606 (ϳ10%) as viral, while 1,309 contigs remained unannotated. from the contigs annotated as viral by diamond, most were phages belonging to the myoviridae (236 contigs), podoviridae (95 contigs), siphoviridae (145 contigs), and microviridae (36 contigs) families. to get insight into the differences in the bacteriophage communities, we compared the virsorter-identified bacteriophage richness between the different age groups (fig. s6a) , locations (fig. s6b, ) and bat contact status (fig. s6c ), all of which showed no significant differences. to identify the potential bacterial hosts of these phages, we searched for bacterial crispr spacer sequences in the phage contigs, to identify its potential host. the search revealed that the most likely hosts of these phages are bacteria of the families bacteroidaceae, bifidobacteriaceae, enterobacteriaceae, enterococcaceae, erysipelotrichaceae, eubacteriaceae, lactobacillaceae, odoribacteraceae, streptococcaceae, and veillonellaceae (table s2) . network analysis of human and bat phageomes. in order to visualize the genetic relatedness between the human and bat gut phageome, a recently developed bioinformatics tool (vcontact) was used. it groups phages based on their genome sequences into viral clusters which correlate rather well with viral genera as defined by the international committee of taxonomy of viruses (ictv) (62) . a total of 30,875 protein clusters were predicted using the prokaryotic and archaeal refseq combined with the proteins predicted from the phage contigs identified from the human and bats pools using virsorter. using a network analysis approach (fig. 9) , 792 viral genome clusters were predicted of which 173 contained reference phages together with bat or human phage contigs, whereas the rest contained only bat, only human, or bat-human clusters. figure 9 shows that both cameroonian human and bat phage contigs identified in our studies are spread across the known phage sequence space. however, several of the phage contigs constituted completely new clusters (indicated by filled gray ovals), completely unconnected to phages in the reference database. also, the genetic diversity of several previously known phage subclusters was significantly expanded (as indicated by open ovals) while some clusters (in brown ovals) were made up of only bat and human phages identified in this study. recently, we thoroughly investigated the gut virome of fruit bats from cameroon (20) (21) (22) (23) 63) and showed the presence of many novel and divergent eukaryotic viral families, including viruses known to cause gastroenteritis in humans. the aim of the current study was to investigate the gut virome of humans (n ϭ 221) from cameroon and to further determine if bat viruses are possible causative agents of gastrointestinal infections in humans. twenty-four percent of the 471 million generated trimmed reads were assigned as viral. most of these reads were bacteriophages, which is in accordance with previous studies (64) . the eukaryotic viral reads include those that belong to viral families that are commonly associated with gastroenteritis in humans (adenoviridae, astroviridae, caliciviridae, picornaviridae, and reoviridae), viruses that are uncommon causes of gastroenteritis (orthoreovirus), or those that have been identified in humans but not associated with disease (anellovirus, smacovirus, and picrobirna-like viruses). virsorter_node_210_length_3873_cov_4_79189-cat_1 virsorter_node_388_length_2881_cov_7_17939-cat_1 virsorter_node_5_length_42493_cov_21_423-circular-cat_1 virsorter_node_8_length_45832_cov_51_2672-cat_1 virsorter_node_4_length_43684_cov_82_015-circular-cat_2 virsorter_node_15_length_28369_cov_37_8786-cat_2 virsorter_node_16_length_34096_cov_10_5837-cat_1 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virsorter_node_119_length_3091_cov_3_65196-cat_2 virsorter_node_71_length_5867_cov_6_41969-cat_2 virsorter_node_3_length_41933_cov_233_011-cat_2 virsorter_node_258_length_3503_cov_8_13689-cat_2 virsorter_node_321_length_1425_cov_3_60831-cat_1 virsorter_node_3_length_47484_cov_53_1945-cat_2 virsorter_node_6_length_18914_cov_22_0195-cat_2 virsorter_node_58_length_6232_cov_20_7313-cat_2 family were identified most frequently (fig. 1b) . this is partly because it is one of the largest viral families and is made up of at least 29 genera, many of which are transmitted through the fecal-oral or respiratory route (28) . most of these infections were in pools of individuals less than 20 years of age (fig. 1c) . this finding is consistent with previous findings from cameroon, where a high prevalence of ev in children was reported using pcr-based approaches (65) . furthermore, most of the evs here were of genotype c, also supporting a recent study that identified a high rate of ev cs in the northern regions of cameroon (31) . therefore, the high prevalence of ev c is probably national. however, the absence of genetic relatedness between the cameroonian human ev strains and animal strains (from chimp and gorilla [66] ) does not indicate interspecies transmission of evs from animals. additionally, we report for the first time (nearly) complete genomes of picornaviruses of the genera parechovirus, cardiovirus, hepatovirus a, and cosavirus from cameroonian patients. this broadens the range of picornaviruses found in the cameroonian population, indicating that picornaviruses might be playing a vital role in gastroenteric viral infection in the cameroonian population, especially given that most of these were from samples of sick children. rotavirus a (rva), a common viral gastroenteritis-causing agent, was identified only in a limited number of pools. this was previously observed in cameroon, and possible reasons for the low prevalence could include the acute nature of rotavirus infections or seasonal changes in rotavirus infections (6) . of note, rotavirus vaccination was introduced in cameroon in april 2014, coinciding with the period of sample collection of this study (february to september 2014); however, the vaccination campaign had not started in the sampling locations within this period, and therefore, the result represents a prevaccination rotavirus prevalence status. the identified rotavirus strain showed 3% nt differences with the vaccine strain, further suggesting that this was a wild-type rva strain, rather than a vaccine-derived strain. uncommon human gastroenteric virus: mammalian orthoreovirus (morv). this first morv strain from cameroon showed topological incongruence in its phylogeny, thereby pointing to possible reassortment events in the past. the phylogenetic clustering of some segments to strains from animals (rodents and bats) could be an indication of a zoonotic event or could also be due to the absence of related strains in databases from an unknown host. given that this strain was from a pool of samples from two children suffering from severe diarrhea, it is not unlikely that this strain might have contributed to the disease. therefore, morv might be playing a greater role in diarrheal diseases in this region than was previously known. hence, extensive epidemiological studies in different regions and in different hosts are required to fully delineate the prevalence, genomics, and interspecies transmissibility of morv. viruses not (yet) associated with gastroenteritis: picobirnaviridae, smacovirus, and anelloviridae. apart from the above-mentioned gastroenteritis-related viruses, several other viruses with unelucidated gastroenteric roles were also identified in this study. first, we observed fewer reads of picobirnaviruses (pbvs) in pools from children than in adults. previous studies also detected a relatively low percentage of children with pbvs (67, 68) . this therefore adds up to the notion that pbvs are likely to be absent in infants and young children and only start to increase with age and potentially a changing diet, though this needs to be further proven (69, 70) . interestingly, the genetic relatedness of a human picobirna-like virus with one that was found in a bat pool from the same region suggests an interspecies transmission. however, these picobirna-like sequences are translated using an alternative mitochondrial codon, indicating that their hosts may not be mammals. a principal component analysis of the codon usage bias of different known mitochondrial genome sequences, mitoviruses, and pbvs seems to suggest that they may have the same lifestyle as mitoviruses known to infect fungal mitochondria (71) . however, the recent identification of a bacterial ribosomal binding site in pbv genomes suggests prokaryotes as a potential host (72) . given that the mitochondria have descended from ancient eubacterial endosymbionts (73) , this may explain the clustering of these pbvs with mitoviruses. therefore, the question about the true host of pbvs remains controversial. second, for the first time, two strains of african smacovirus (scv) were identified in cameroonian samples. their genetic relatedness to a chimpanzee strain (isolated from a captive chimp in a zoo in san francisco) and a strain from a child from the united states (54, 55) indicates either an interspecies transmission event or the presence of a shared viral host in both humans and chimps. although the role of smacovirus in gastroenteritis has not been elucidated, their presence in cases of unexplained diarrhea in french patients seems to indicate a potential role in gastroenteritis (54) ; hence, these could be instances of interspecies transmissions. the percentages of pools positive for anelloviruses were higher in age categories of children and the old and lower for the middle-aged groups. given the well-established notion that infants and the elderly have reduced immunity (74, 75) , this could be in line with previous studies that suggest a link between the burden of anelloviruses and host immune competence (76) (77) (78) . despite their ubiquity, anelloviridae have an undefined implication in hosts' health and are thought to be probably asymptomatic (harmless) or even beneficial. however, they have been associated with hepatitis, pulmonary diseases, hematologic disorders, myopathy, and lupus, but it is not clear if their presence is the cause or the result of disease progression (79) (80) (81) (82) . human viruses and interspecies transmission from bats. in bats from the same area, we were able to identify gastroenteritis-related and nonrelated viruses. here, the corresponding viruses identified from the families astroviridae (astrovirus), caliciviridae (sapovirus), and reoviridae (rva) are genetically diverse from those identified in bats from the same region, indicating no evidence of recent interspecies transmissions between bats and humans (63) . however, genetic relatedness of human morv to animal strains showed the possibility of zoonosis between humans and not only bats but animals in general. additionally, the presence of some cameroonian strains of scv and pbv in bats or other animals would indicate interspecies transmissions if their infectivity in these animals is fully elucidated. human and bat phageome. in this study, we detected a huge phage community with a great diversity beyond the range of known bacteriophages in reference databases, potentially representing the gut microbiome diversity in the patients (83, 84) . overall, this further supports the idea that the full phageome richness is still to be completely elucidated (85) . furthermore, network analysis indicates the presence of completely novel phage groups and that phage genera in the gut microbiota might be shared between humans and bats. conclusion. several diverse viruses were discovered in the gut virome of cameroonians. some of these were already known to be the causative agent of gastroenteritis, whereas others are likely to be the cause of gastroenteric problems in the patients. further screening of patients for these viruses will be needed to establish their prevalence in the population, allowing for more appropriate measures and treatment and prevention of viral gastroenteritis. also, to be able to completely elucidate the role of the novel viruses like pecovirus and smacovirus, more studies are required. further attention should also be given to newly identified viruses (for example, morv) and their potential as emerging pathogens in the human population. ethical authorization. ethical authorization for the use of human samples was obtained from the cameroon national ethics committee, yaoundé. all human experiments were performed in accordance with the ministry's national ethics committee guidelines. sample collection and preparation. human fecal samples were collected between february and september 2014, after informed consent was obtained from patients in two different hospitals (lysoka health district and kumba district hospital of the southwest region of cameroon). this region was chosen because here bats are hunted, sold, and eaten. diarrheic patients and/or people who came into contact with bats directly (by eating, hunting, or handling) or indirectly (if a family member was directly exposed to bats) were eligible for sampling. a total of 221 samples were collected from subjects between age 0 and ͻ3 years (age group a, 80 samples), 3 and ͻ20 (age group b, 63 samples), 20 and ͻ60 (age group c, 65 samples), and 60 and older (age group d, 13 samples). all the samples were from people who had symptoms of gastroenteritis, except 2 from age group c who had contact with bats. samples were then placed into labeled tubes containing universal transport medium (utm), placed on dry ice, and stored at ϫ20°c, until being shipped to the laboratory of viral metagenomics, leuven, belgium. the samples were stored at ϫ80°c until used (63) . fecal samples were first diluted using utm, and equal volumes of the dilutions were pooled based on the location, age, and bat contact status (direct, indirect, or none). each pool contained two to five samples, and for the different age groups (a to d) we had 22, 17, 20, and 4 pools, respectively . the pools were then treated according to the netovir protocol (86) . briefly, the pools (10% [wt/vol] fecal suspensions) were homogenized for 1 min at 3,000 rpm with a minilys homogenizer (bertin technologies) and filtered using an 0.8-m pes filter (sartorius). the filtrate was then treated with a cocktail of benzonase (novagen) and micrococcal nuclease (new england biolabs) at 37°c for 2 h to digest free-floating nucleic acids. total nucleic acids (both rna and dna) were extracted using the qiaamp viral rna minikit (qiagen) according to the manufacturer's instructions but without addition of carrier rna to the lysis buffer. first-and second-strand synthesis and random pcr amplification for 17 cycles were performed using a slightly modified whole-transcriptome amplification (wta2) kit procedure (sigma-aldrich). wta2 products were purified with msb spin pcrapace spin columns (stratec), and the libraries were prepared for illumina sequencing using a slightly modified version of the nextera xt library preparation kit (illumina), which is described in detail in reference 86. samples were pooled in an attempt to obtain an average of approximately 10 million paired-end reads per pool. sequencing was performed on a nextseq 500 high-output platform (illumina) for 300 cycles (2 ϫ 150-bp paired ends). genomic and phylogenetic analysis. ngs reads were analyzed as described in the work of yinda et al. (20, 63) . briefly, raw reads were trimmed using trimmomatic (parameters: headcrop: and fastuniq to remove identical reads. the de novo assembly or reads and annotation of reads were performed using spades (with the meta flag) and diamond (with the sensitive option using the genbank nonredundant database), respectively (61, 87, 88) . open reading frames (orfs) of contigs of interest were identified and further analyzed for conserved motifs in the amino acid sequences using ncbi's conserved domain database (cdd) (89) . nucleotide and amino acid alignments of viral sequences were done with muscle implemented in mega7 (90) or mafft (91) . substitution models were determined using modelgenerator (92) , and phylogenetic trees were constructed using raxml (93) , with the automre flag, which enables a posteriori bootstrapping analysis. all trees were visualized in figtree (http://tree.bio.ed.ac.uk/software/figtree/) and midpoint rooted for purposes of clarity. phageome analysis. contig annotation with diamond is dependent on the accuracy of the database used, and in most databases, phages are poorly annotated. however, virsorter uses a manually curated database of virus reference genomes augmented with metagenomic viral sequences sampled from freshwater, seawater, and human gut, lung, and saliva. hence, for further identification of bacteriophages, scaffolds ͼ1 kb were classified using virsorter (decontamination mode [60] ). only scaffolds assigned to categories 1 and 2 were considered bacteriophage contigs and were filtered for redundancy at 95% nucleotide identity over 70% of the length using cluster genomes (94) . then, trimmed reads from each pool were mapped using bowtie 2 (95) to the bacteriophage contigs, and the generated bam files were filtered to remove reads that aligned at ͻ95% identity using bamm (http://ecogenomics.github .io/bamm/). abundance tables were obtained and normalized for total number of reads of each sample. for the richness comparison, mann-whitney tests were used, and for the clustering, an adonis test was performed. all downstream analyses were done in r (96) using the vegan package (97) . furthermore, to identify the potential corresponding bacterial host, a database of these contigs was made to which a nucleotide blastn search (100% identity without gaps) was performed using a fasta file of crispr sequences (98) as query. these sequences correspond to different bacterial hosts, and their presence in the phage genome highlight the potential host of the phage. to see if the phage community of these humans is related to those of the bats from the same locality, a visualization of the network of both human and bat phageomes was performed using vcontact (62) . initially, proteins were predicted using prodigal (99) , and combined with the viral refseq of archaeal and prokaryotic predicted proteins. a database was generated from the contigs of bat pools, human pools, and viral refseq proteins, and blastp was performed against the combined proteins. the output of blast was used to run vcontact, and the output network was visualized in cytoscape (100). data availability. all sequences were deposited in genbank under the following accession numbers: mh608285 to mh608287 and mh933752 to mh933860 (details in table s3 ). raw reads were submitted to the ncbi's short read archive (sra) under the project id prjna491626. supplemental material for this article may be found at https://doi.org/10.1128/ msphere.00585-18. ef051629_hpev-1/bni-788st/deu/xx jn867764/hcosv/a13-np6/npl gu985458_mink_astv-sms ay720892_humanastv5-goiania/go/12/94/brazil fj890351_sea_lionastv1 human-astv_cmrhp35d hq889774_pigeonanv-sh10 human-astv_cmrhp6 z25771_humanastv-newcastle human-astv_cmrhp46 human-astv_cmrhp3 fj973620_humanastv-va1 ita fj402983_humanastv-mlb1-wd0016 jx857869_humanastv-va4/human/nepal/s5363 hq916317_bovineastv/b76-2/hk fj890352_sea_lionastv2 z25771_humanastv-newcastle hq916313_bovineastv/b18/hk 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assumptions for choice of matrix are not justified raxml version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies clustering viral genomes in ivirus fast gapped-read alignment with bowtie 2 r: a language and environment for statistical computing. r foundation for statistical computing community ecology package the crisprdb database and tools to display crisprs and to generate dictionaries of spacers and repeats prodigal: prokaryotic gene recognition and translation initiation site identification integration of biological networks and gene expression data using cytoscape g31_rva/bat-wt/cmr/batli08/2014/g31p [42] g30_rva/bat-wt/cmr/batli10/2014/g30p [42] g3_rva/bat-wt/chn/myas33/2013_g3p [10] g25_rva/bat-wt/ken/ke4852/07/2007/g25p [6] g21_rva/cow-wt/jpn/azuk-1/2006/g21p [29] g13_rva/horse-tc/gbr/l338/1991/g13p [18] g3_rva/human-tc/jpn/au-1/1982/g3p [39] g15_rva/cow-tc/ind/hg18/1995/g15p [21] g30_rva/bat-wt/cmr/batli09/2014/g30p [42] g22_rva/turkey-tc/deu/03v0002e10/2003/g22p [35] g8_rva/human-tc/ind/69m/1980/g8p [4] 10g18_rva/pigeon-tc/jpn/po-13/1983/g18p [17] g20_rva/human-wt/ecu/ecu534/2006/g20p [28] g26_rva/pig-wt/jpn/tj4-1/2010/g26p [x] g14_rva/horse-tc/usa/fi23/1981/g14p [12] g1_rva/human-wt/cmr/cmrhp55/2014/g1p [8] g7_rva/turkey-tc/irl/ty-3/1979/g7p [17] g3_rva/bat-tc/mslh14/2012/g3p [3] g6_cow g25_rva/bat-wt/cmr/batly03/2014/g25p [43] g19_rva/chicken-tc/gbr/ch-1/197x/g19p [17] g1_rva/human-tc/usa/wa/1974/g1p[1]a8 g10_rva/human-tc/gbr/a64/1987/g10p [14] g9_rva/human-tc/usa/wi61/1983/g9p[1]a8 g30_rva/bat-wt/cmr/batly17/2014/g30p [47] g27_rva/sugarglider-tc/jpn/sg385/2012/g27p [36] g24_rva/cow-tc/jpn/dai-10/2007/g24p [33] g11_rva/pig-tc/mex/ym/1983/g11p[9]7 g16_rva/mouse-tc/usa/ew/xxxx/g16p [16] acknowledgments c.k.y. was supported by the interfaculty council for development cooperation (iro) from the ku leuven. n.c.-n. and l.b. were supported by the flanders innovation & entrepreneurship (vlaio). this work was supported by ku leuven grant ejx-c9928-stg/15/020bf. the funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.c the authors declare no competing financial interests. key: cord-309512-d8n9711b authors: bacus, michael g.; dayap, stephen adrian h.; tampon, nikki vanesa t.; udarbe, marielle m.; puentespina, roberto p.; villanueva, sharon yvette angelina m.; de cadiz, aleyla e.; achondo, marion john michael m.; murao, lyre anni e. title: global genetic patterns reveal host tropism versus cross-taxon transmission of bat betacoronaviruses date: 2020-05-05 journal: biorxiv doi: 10.1101/2020.05.04.076281 sha: doc_id: 309512 cord_uid: d8n9711b emerging infectious diseases due to coronavirus (cov) infections have received significant global attention in the past decade and have been linked to bats as the original source. the diversity, distribution, and host associations of bat covs were investigated to assess their potential for zoonotic transmission. phylogenetic, network, and principal coordinate analysis confirmed the classification of betacoronaviruses (betacovs) into five groups (2a to 2e) and a potentially novel group, with further division of 2d into five subgroups. the genetic co-clustering of betacovs among closely related bats reflects host taxon-specificity with each bat family as the host for a specific betacov group, potentially a natural barrier against random transmission. the divergent pathway of betacov and host evolution suggests that the viruses were introduced just prior to bat dispersal and speciation. as such, deviant patterns were observed such as for 2d-iv, wherein cross-taxon transmission due to overlap in bat habitats and geographic range among genetically divergent african bat hosts could have played a strong role on their shared cov lineages. in fact, a few bat taxa especially the subfamily pteropodinae were shown to host diverse groups of betacovs. therefore, ecological imbalances that disturb bat distribution may lead to loss of host specificity through cross-taxon transmission and multi-cov infection. hence, initiatives that minimize the destruction of wildlife habitats and limit wildlife-livestock-human interfaces are encouraged to help maintain the natural state of bat betacovs in the wild. importance bat betacoronaviruses (betacovs) pose a significant threat to global public health and have been implicated in several epidemics such as the recent pandemic by severe acute respiratory syndrome coronavirus 2. here, we show that bat betacovs are predominantly host-specific, which could be a natural barrier against infection of other host types. however, a strong overlap in bat habitat and geographic range may facilitate viral transmission to unrelated hosts, and a few bat families have already been shown to host multi-cov variants. we predict that continued disturbances on the ecological balance may eventually lead to loss of host specificity. when combined with enhanced wildlife-livestock-human interfaces, spillover to humans may be further facilitated. we should therefore start to define the ecological mechanisms surrounding zoonotic events. global surveillance should be expanded and strengthened to assess the complete picture of bat coronavirus diversity and distribution and their potential to cause spillover infections. emerging and re-emerging infectious diseases greatly affect public health and global economies (1). these diseases involve pathogenic strains that recently evolved, pathogens that infect human 52 population for the first time, and pathogens that re-occur at higher frequency (2). majority of these 53 emerging infectious diseases are caused by microorganisms from non-human source or zoonotic 54 pathogens from wild animals (3). in particular, emerging infectious diseases due to coronavirus (cov) however, some deviations were also noted. for example, bat hosts that belong to genetically 212 unrelated taxa were mixed in some betacov groups. the mollosidae bat eumops glaucinus was found in world leaf-nosed bat trianeops persicus in 2d-iv of epomophorinae bats. looking at the host, certain bat 217 families were observed to harbor betacovs that belong to various lineages. the rousettinae bats were 218 found to carry both 2d-i/2d-iii and 2d-iv betacovs, and the old world fruit bats 2b, 2d-iv, and 2e. the the divergence of the bats and their betacovs were compared to evaluate common evolutionary 223 pathways (fig. 5b) . the vesper microbats diverged as a separate group from the rest of the bats. the in contrast, our findings support a previously proposed hypothesis that covs limit cross-species although bat betacovs are host taxon-specific, their evolutionary pathways are different from evolution with its host. instead, this is indicative that the currently circulating viruses may have been introduced relatively recently, i.e. to the most recent common ancestors of each bat taxon but prior to 283 global dispersion and speciation, during which the virus acquired adaptation to its host. the recent 284 introduction of betacovs in bats implies that other factors may have had the opportunity to influence 285 virus-host dynamics. in the succeeding discussions, we will present two deviant phenomena that 286 exemplify this: cross-taxon transmission of covs and bat hosts with multi-cov lineages. we provide genetic evidence for cross-taxon transmission as indicated by genetically unrelated microbial threats to health: taxonomy-history?taxnode_id=20186105 a new virus isolated from the human respiratory 480 tract identification of a new human coronavirus molecular pathology of emerging coronavirus 485 infections dobsonia moluccensis. the iucn red list of threatened species notes on the dry season roosting and foraging behaviour 51 food availability and annual migration of the 57 replication and shedding of mers-cov in jamaican fruit bats 597 (artibeus jamaicensis) further evidence for bats as the evolutionary source of middle east 600 respiratory syndrome coronavirus coronaviruses: important emerging human 602 pathogens urbanization and disease 604 emergence: dynamics at the wildlife-livestock-human interface mechanisms of zoonotic severe acute respiratory syndrome coronavirus host range expansion in 608 human airway epithelium wuhan municipal commission of health and health on 64 detection and characterization of a novel bat-borne coronavirus in singapore using multiple 626 molecular approaches preliminary assessment of activity 628 pattern and diet of the lesser dog faced fruit bat cynopterus brachyotis in a dipterocarp forest development of a one-step rt-632 pcr assay for detection of pancoronaviruses (α-, β-, γ-, and δ-coronaviruses) using newly 633 designed degenerate primers for porcine and avianfecal samples jmodeltest: phylogenetic model averaging. molecular biology and 639 evolution bayesian phylogenetic and phylodynamic data integration using beast 1.10. virus 642 evolution posterior 644 summarization in bayesian phylogenetics using tracer 1.7. systematic biology past: paleontological statistics software 646 package for education and data analysis key: cord-270143-muxrxvyo authors: markotter, wanda; geldenhuys, marike; jansen van vuren, petrus; kemp, alan; mortlock, marinda; mudakikwa, antoine; nel, louis; nziza, julius; paweska, janusz; weyer, jacqueline title: paramyxoand coronaviruses in rwandan bats date: 2019-07-02 journal: trop med infect dis doi: 10.3390/tropicalmed4030099 sha: doc_id: 270143 cord_uid: muxrxvyo a high diversity of coronaand paramyxoviruses have been detected in different bat species at study sites worldwide, including africa, however no biosurveillance studies from rwanda have been reported. in this study, samples from bats collected from caves in ruhengeri, rwanda, were tested for the presence of coronaand paramyxoviral rna using reverse transcription pcr assays. positive results were further characterized by dna sequencing and phylogenetic analysis. in addition to morphological identification of bat species, we also did molecular confirmation of species identities, contributing to the known genetic database available for african bat species. we detected a novel betacoronavirus in two geoffroy’s horseshoe bats (rhinolophus clivosus) bats. we also detected several different paramyxoviral species from various insectivorous bats. one of these viral species was found to be homologous to the genomes of viruses belonging to the jeilongvirus genus. additionally, a henipavirus-related sequence was detected in an egyptian rousette fruit bat (rousettus aegyptiacus). these results expand on the known diversity of coronaand paramyxoviruses and their geographical distribution in africa. bats (order chiroptera) account for 20% of all mammalian species and are distributed worldwide. with the advancement in detection techniques and increased surveillance, bats are being increasingly recognized as hosts for many zoonotic viruses [1] , including filo-, paramyxo-, corona-and lyssaviruses [2] [3] [4] [5] . regions in africa are considered a hotspot for emerging infectious diseases with more than 50% of recently emerging diseases originating from wildlife species on this continent [6, 7] . although several surveillance studies have been implemented to detect potential zoonotic viruses in bats, including from countries in the congo basin and east africa, limited information is available for rwanda. importantly, in the bordering democratic republic of congo and uganda, marburg and ebola disease outbreaks in humans have occurred [8] , and corona-and paramyxoviruses have been reported to circulate in bats [3, [9] [10] [11] . coronaviruses are positive-sense rna viruses with the potential to cause respiratory, gastrointestinal, hepatic, and neurological diseases in their hosts [12] and are divided into four genera namely alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus [5] . bats host a large diversity of coronaviruses and the expanding research can be largely attributed to the emergence of novel coronaviruses of public health and veterinary importance. three such viruses emerged in the last 17 years, including the severe acute respiratory syndrome (sars) coronavirus in 2002, middle east respiratory syndrome (mers) coronavirus in 2012, and the swine acute diarrhoea syndrome (sads) coronavirus in 2017 [13, 14] . bat coronaviruses have been shown to be associated with particular bat genera and similar viruses have been identified throughout the geographical distribution of their hosts [11, 12, 15] . diverse bat coronaviruses related to sars coronavirus (now termed the sarbecovirus subgenus) have been identified from the rhinolophus bat genus (horseshoe bats) in asia, europe and africa [11, 16, 17] . continued surveillance within these bats species in china identified lineages of recombinant sars-related coronaviruses nearly identical to human sars coronaviruses, capable of using the same receptor molecules [17] [18] [19] . as a result, these viruses have therefore been postulated to be capable of direct human infection [18, 19] . bats from various african countries, including kenya, ghana, nigeria, tanzania, uganda and south africa, have yielded a large diversity of novel coronaviruses [11, [20] [21] [22] [23] [24] [25] [26] [27] . some of the bat coronaviruses identified have been shown to be genetically related to known human coronaviruses such as hcov-229e, hcov-nl63, and mers coronavirus [11, 21, [24] [25] [26] . paramyxoviruses are negative-sense single-stranded rna viruses capable of infecting a diverse host range including mammals, birds, reptiles and fishes [28] . the taxonomic classification of viruses in the paramyxoviridae family has recently undergone several changes [29] . in an attempt to accommodate the rapidly growing number of paramyxoviruses described, the previously known avulavirus and rubulavirus genera were elevated to the sub-family level (avula-and rubulavirinae) each with two new genera. in addition, several unclassified rodent-borne viruses were classified to newly established genera (narmovirus and jeilongvirus) in the sub-family orthoparamyxovirinae to which the henipa-, morbilliand respirovirus genera belong. several zoonotic paramyxoviruses have emerged as important public health threats in the past three decades. the emergence of hendra and nipah viruses (henipavirus genus) during the 1990s in australia and southeast asia respectively, marked the first report of zoonotic paramyxoviruses of considerable public health importance [30, 31] . these viruses are characterized by high morbidity and mortality rates and outbreaks have been reported on a near annual basis. in addition, another paramyxovirus, sosuga virus (pararubulavirus genus), emerged as the etiological agent of a single non-fatal human infection contracted in uganda, africa [32] . the natural wildlife reservoir for these zoonotic viruses was determined to be the fruit bat species occurring in these areas, i.e., flying foxes from the pteropus genus for the henipaviruses [33, 34] , and the egyptian rousette bat (rousettus aegyptiacus) for the rubulavirus [9] . viruses related to the henipaand orthorubulaand pararubulavirus genera as well as a number of unclassified viruses have been described from countries bordering rwanda as well as other african countries [3, 9, 10, [35] [36] [37] . r. aegyptiacus, hipposideros spp. and miniopterus inflatus have tested positive for paramyxoviral rna that is closely related to known human pathogens including the henipaviruses, human mumps virus, human parainfluenza virus 2 and human parainfluenza virus 4 [3, 37] . in this study, we report the detection of a novel betacoronavirus in rhinolophus clivosus sampled in the ruhengeri cave system in rwanda. in addition, we report on the detection of jeilongvirus and related sequences in hipposideros spp. as well as a henipavirus-related sequence in the fruit bat species r. aegyptiacus. these results expand on the known diversity of these virus groups and their geographical distribution in africa. in december 2008, a team from the university of pretoria, national institute for communicable diseases, and rwanda tourism board and national park authority visited two cave sites in ruhengeri, rwanda (gps coordinates: 1 • 30 14.2" s 29 • 37 59.9" e; figure 1 ) where bats were caught at night using mist nets in the surrounding areas, and two bank g4 forest strainer harp traps (bat conservation and management, inc., usa, australia) at the cave entrances. when collecting samples in the field, personal protective equipment used included tyvek suits (dupont tm , wilmington, de, usa), disposable over gowns (stylianou medisupplies ltd, middle east), 3m full powered air purifying respirators (3m, maplewood, mn, usa), gumboots (bata industries ® , pinetown, south africa), double layer nitrile gloves (lasec, cape town, south africa) and leather gloves (evrigard, johannesburg, south africa). in december 2008, a team from the university of pretoria, national institute for communicable diseases, and rwanda tourism board and national park authority visited two cave sites in ruhengeri, rwanda (gps coordinates: 1°30'14.2"s 29°37'59.9"e; figure 1 ) where bats were caught at night using mist nets in the surrounding areas, and two bank g4 forest strainer harp traps (bat conservation and management, inc., usa, australia) at the cave entrances. when collecting samples in the field, personal protective equipment used included tyvek suits (dupont tm , wilmington, de, usa), disposable over gowns (stylianou medisupplies ltd, middle east), 3m full powered air purifying respirators (3m, maplewood, mn, usa), gumboots (bata industries®, pinetown, south africa), double layer nitrile gloves (lasec, cape town, south africa) and leather gloves (evrigard, johannesburg, south africa). bats were placed in individual cotton bags before processing. bats were morphologically identified [38] and data including sex, reproductive status, forearm length and weight were also recorded. samples collected from bats included fecal and oral swabs, wing biopsies in 70% ethanol and blood (serum) for use in viral surveillance studies. oral swabs were collected by gently swabbing the inside of the mouth (cheeks and tongue) with a sterile swab (vwr critical swab, atlanta, ga, usa). fecal material or swabs (vwr critical swab, atlanta, ga, usa) were collected from the bat or the cotton bag, when it was available. sterile foreceps were used to collect fecal pellet(s) and place them in 2 ml microcentrifuge tubes (sarstedt, nümbrecht, germany). urine was collected with a sterile swab (vwr critical swab, atlanta, ga, usa) from individual bats as was available. in instances where bats died during processing, necropsies were performed and various organs and tissues, including kidney, spleen, heart, pectoral muscles, liver, lung, stomach, bladder, tongue, brain and lymph nodes, were collected and placed in 2 ml cryotubes (sarstedt, nümbrecht, germany). all samples were collected in rnalater preservative inactivation solution (qiagen, hilden, germany), stored at 4 °c, then transported to and tested in south africa at the national institute for communicable diseases (nicd) and university of pretoria. permits were obtained from the rwanda development board/tourism & conservation and animal ethics was obtained from the animal ethics committee, university of pretoria. for viral rna detection, rna was extracted from kidney, spleen, urine, fecal, rectal and intestinal samples (table s1 ). rna from kidney (n = 6), spleen (n = 5) and urine (n = 13), were extracted using the bats were placed in individual cotton bags before processing. bats were morphologically identified [38] and data including sex, reproductive status, forearm length and weight were also recorded. samples collected from bats included fecal and oral swabs, wing biopsies in 70% ethanol and blood (serum) for use in viral surveillance studies. oral swabs were collected by gently swabbing the inside of the mouth (cheeks and tongue) with a sterile swab (vwr critical swab, atlanta, ga, usa). fecal material or swabs (vwr critical swab, atlanta, ga, usa) were collected from the bat or the cotton bag, when it was available. sterile foreceps were used to collect fecal pellet(s) and place them in 2 ml microcentrifuge tubes (sarstedt, nümbrecht, germany). urine was collected with a sterile swab (vwr critical swab, atlanta, ga, usa) from individual bats as was available. in instances where bats died during processing, necropsies were performed and various organs and tissues, including kidney, spleen, heart, pectoral muscles, liver, lung, stomach, bladder, tongue, brain and lymph nodes, were collected and placed in 2 ml cryotubes (sarstedt, nümbrecht, germany). all samples were collected in rnalater preservative inactivation solution (qiagen, hilden, germany), stored at 4 • c, then transported to and tested in south africa at the national institute for communicable diseases (nicd) and university of pretoria. permits were obtained from the rwanda development board/tourism & conservation and animal ethics was obtained from the animal ethics committee, university of pretoria. for viral rna detection, rna was extracted from kidney, spleen, urine, fecal, rectal and intestinal samples (table s1 ). rna from kidney (n = 6), spleen (n = 5) and urine (n = 13), were extracted using the trizol reagent (invitrogen, carlsbad, ca, usa), and fecal material/swabs (n = 99), rectal and/or intestinal samples (n = 8) (table s1 ) were extracted using the duet rna/dna extraction kit (zymoresearch, ca, usa) from samples homogenized in 300 µl of phosphate buffered saline (lonza, basel, switzerland). both extraction methods were performed according to the manufacturer's instructions without deviations. dna was extracted using the dneasy blood & tissue kit (qiagen, hilden, germany) from heart tissues. confirmation of species identification of bats, in which viral rna was detected, was performed by amplifying the cytochrome b (cyt b) or cytochrome oxidase one (coi) gene region and determining the dna sequence. selection of cytochrome region for amplification was based on availability of credible comparative sequences in public databases (ncbi genbank and bold). previously reported pcr primers targeting these two regions were used or modified [39] [40] [41] . following the pcr analysis, reactions were subjected to agarose gel electrophoresis on a 1.5% agarose gel (lonza, basel, switzerland) and pcr amplicons were gel-purified using the wizard ® sv gel dna clean-up system (promega, madison, wi, usa) according to the manufacturer's instructions and without deviation. all amplicons were subjected to sanger sequencing for both the forward and reverse reactions on an abi 3100 dna sequencer (ae applied biosystems) at the sequencing facility of the university of pretoria. host gene sequences were subsequently compared to bat sequences available in the public domain (on the ncbi genbank and bold databases), results were interpreted and compared with the respective morphological field identifications. fecal, rectal and/or intestinal samples from 101 bats (table s1 ) were extracted and analysed for coronavirus rna. complementary dna (cdna) was prepared using 100 ng random hexamers (ie hplc purified, integrated dna technologies, coralville, ia, usa) with 200 u superscript iv reverse transcriptase (thermo scientific, waltham, ma, usa). additionally, cdna was treated with 2 u rnase h (thermo fisher scientific) incubation at 37 • c for 20 min and inactivated at 65 • c for 10 min. presence of coronavirus rna was detected with a coronavirus genus-specific hemi-nested rt-pcr assay which targets the rna dependent rna polymerase (rdrp) gene for amplification as described in geldenhuys et al. [27] . as the hemi-nested rt-pcr assay produced only short amplicons (approximately 260 bp), the rdrp-grouping unit (rgu) assay was used to extend the sequenced region of the identified betacoronaviruses to 820 bp [16] . a hemi-nested rt-pcr assay was performed using the randomly primed cdna prepared as well as forward primers from drexler et al. [16] (sp3080 5'-ctt ctt ctt tgc tca gga tgg caa tgc tgc-3' and sp3195 5'-ata ctt tga ttg tta cga tggt ggc tg-3') in combination with a reverse primer (p1beta_rev2016 5'-cat crt cas dia rda tca tcat-'3) from the geldenhuys et al. [27] assay. assay conditions from geldenhuys et al. [27] were used with modifications to cycling conditions including longer annealing and extension cycles (45 cycles of 94 • c for 30 s, 42 • c for 60 s and 72 • c for 80 s). agarose gel electrophoresis and purification of all pcr products were performed as previously described for molecular host species identification. kidney from insectivorous bats, spleen from frugivorous bats and urine from both groups (n = 24; table s1 ) were tested with the use of two broadly-reactive assays targeting the avula-rubulavirinae (ar) sub-families, and the respiro-morbilli-henipvirus (rmh) genera. for both assays, published primers targeting the conserved polymerase (l) gene [42] were used in combination with adapted two-step hemi-nested rt-pcr protocols. samples were tested with the ar assay as previously described [37] . for the rmh assay, the samples were tested as previously described [37] , with minor variations in the protocol. for the first-round pcr, 25 mm mgcl 2 (thermofisher scientific, waltham, ma, usa) was added and the nuclease-free water (ambion, foster city, ca, usa) was adapted for a final reaction volume of 50 µl. all cycling conditions and the protocol for the hemi-nested pcr remained the same as for ar. agarose gel electrophoresis and purification of all pcr products were performed as previously described for molecular host species identification. sequencing was performed as previously described for molecular host species identification. sequences were viewed, edited and a consensus generated using the bioedit sequence alignment editor software version 7.2.5 [43] . cipres was used for clustalx alignments, determining the best dna substitution model for nucleotide sequence analysis using the jmodeltest software and for constructing bayesian phylogenies using the beast version 1.8 software [44] [45] [46] . bayesian mcmc chains were set to 20 million iterations, sampling every 2000 steps for optimal ess scores. output files were visually inspected to check for convergence using the tracer software version 1.7 [47] . the final phylogenies were constructed in treeannotator with a burn-in value of 10%. for visualization and manipulation of the phylogenetic tree, the figtree version 1.4.2 software was used. pairwise similarities between sequences were analysed in mega x with complete deletion [48] . in total, samples from 101 bats constituting five genera were tested for coronaviruses (table 1 and table s1 ). of these, two samples contained coronavirus rna, originating from two individuals of the rhinolophus genus. barcoding and molecular identification confirmed the host species to be rhinolophus clivosus (table s1 ). coronavirus sequences were extended to 820 bp with rgu assay primer sets [16] . the two sequences (rh-btcov/441/rwanda/08 and rh-btcov/445/rwanda/08) share 99.9% nucleotide identity; pairwise similarities and phylogenetic analysis group these sequences with other lineage b betacoronaviruses (figure 2 ). the closest relative to the rwandan betacoronavirus was reported from kenya, btcovky72 (tao et al. unpublished; genbank accession number ky352407.1), though the rhinolophus species is not specified. the sequences share very close sequence similarities (95.5% nucleotide identity and 100% amino acid identity), suggesting that similar betacoronaviruses may be harbored by both kenyan and rwandan rhinolophus bats. full genome comparisons will be able to determine if kenyan and rwandan rhinolophus bats are infected by the same betacoronavirus species. within the analyzed conserved rdrp gene segment, this rwandan rhinolophus betacoronavirus also shares pairwise similarities of 88% nucleotide identity (98.9% amino acid identity) to the bulgarian betacoronavirus rh-btcov/bm48-31/bgr/08 [16] , as well as close similarities (86.1-86.4% nucleic acid similarity and 98.9-99.2% amino acid identity) to asian rhinolophus sars-related coronaviruses such as sarsr-rh-btcov/rp3 and sarsr-rh-btcovwiv16 [18, 49] . other coronavirus surveillance in rwanda, and surrounding countries such as uganda and tanzania also report sars-related coronaviruses from the rhinolophus genus [11] . unfortunately, the sequences cannot be compared as an assay targeting a different conserved peptide of the rdrp gene was used [22] . boldface indicates positive samples. twenty-four samples from 23 bats were tested for paramyxovirus rna (table s1 ), none of which tested positive with the avula-rubulavirinae (ar) assay. an overall percentage positivity for paramyxovirus rna, detected using the respiro-morbilli-henipavirus (rmh) assay, was found to be 16.6% (n = 4). three of the viral sequences were detected in the insectivorous bat species hipposideros ruber and otomops martiensseni, while one other sequence was detected in the frugivorous bat species rousettus twenty-four samples from 23 bats were tested for paramyxovirus rna (table s1 ), none of which tested positive with the avula-rubulavirinae (ar) assay. an overall percentage positivity for paramyxovirus rna, detected using the respiro-morbilli-henipavirus (rmh) assay, was found to be 16.6% (n = 4). three of the viral sequences were detected in the insectivorous bat species hipposideros ruber and otomops martiensseni, while one other sequence was detected in the frugivorous bat species rousettus aegyptiacus. host identification of positive samples was confirmed using molecular analysis (table s1 ). phylogenetic analysis of the sequences indicated that the insectivorous bat-borne viral sequences grouped with the jeilongvirus genus as well as in a jeilongvirus-related clade (figure 3 ). one of the h. ruber sequences (batpv/hip_rub/up401 /rwa/2008) described from this study potentially groups within the jeilongvirus genus. the second h. ruber-derived viral sequence (batpv/hip_rub/up450/rwa/2008) grouped with a paramyxoviral sequence detected in a bat from the same genus sampled in cameroon in 2010, however, was not identical. the detection of two diverse viruses from bats of the same species and same population has previously also been reported in insectivorous bats sampled in other african countries [36] . these observations can in part be explained by the generation of viral quasi-species populations due to the high mutation rate of rna viruses as a consequence of rna proofreading deficiency of the rna dependent rna polymerase [50] . a larger pool of diverse viruses within a bat population and the co-roosting of several cave-dwelling bat species may facilitate viral sharing between different bat species [51] . however, ongoing biosurveillance in these cave-dwelling bat species will be required before active viral sharing can be shown. the paramyxoviral sequence detected in the o. martiensseni bat (batpv/oto_mar/up535/rwa/2008) was near identical to the viral sequences previously described from several individuals of the same species sampled in kenya in 2011 [36] . these sequences shared a 99.3% similarity on both nucleotide and amino acid level. the r. aegyptiacus-derived viral sequence (batpv/rou_aeg/up438/rwa/2008) grouped within a henipavirus-related clade and was near identical to a paramyxoviral sequence detected in the same host species previously reported from kenya [36] . sequence similarity shared between these two sequences was found to be 98.1% and 98.7% on nucleotide and amino acid level, respectively. to our knowledge, this study reports on the first evidence of paramyxovirus rna in bats from rwanda. two of the four viral sequences detected in h. ruber, were not closely related to the paramyxovirus sequences previously reported (sharing nucleotide and amino acid similarities of less than 80% and 83.5%, respectively) and might represent novel viral species. however, a more rigorous analysis with variable genes such as the fusion and hemagglutinin gene will be required before putative species can be inferred. as observed in previous studies, viral sequences from frugivorous bats were mostly found to belong to the henipavirus genus or a related clade, while insectivorous bat-associated viral sequences have been linked to other genera including morbilliand jeilongvirus [36] . this observation was again reflected in the current study. the detection of highly similar viral sequences from bats in rwanda and kenya, which are more than 1000 km apart, can be explained by either the phenomena of metapopulations or the hypothesis of co-evolution of paramyxoviruses with their bat hosts [3, 52, 53] . rna viruses have an exceptionally high mutation rate commonly associated with quasi-species populations and with the potential to cross the species barrier, among others. this is evident in the high diversity of paramyxoviruses described to date and the wide host range associated with these viruses [3, 28, 36, 54] . additionally, due to the emergence of sars, mers and sads, it is widely accepted that coronaviruses are capable of readily adapting to new hosts [5] . the rwandan caves are considered an ecotourism site and guano is also mined on a small scale, providing an ideal bat-human interface. several insectivorous bat species co-roost with the egyptian fruit bats in these caves and future studies should investigate viral sharing. the egyptian fruit bat also uses these caves as a maternity roost and studies have shown that increased viral shedding is linked to reproductive cycles [37] . longitudinal biosurveillance studies can therefore identify high risk periods in the future. as such, the detection of a sars-related bat coronavirus potentially circulating within the rhinolophus population and a henipavirus-related paramyxovirus in r. aegyptiacus in the ruhengeri region may merit further investigation to determine exposure, and the potential for spill-over events to occur. to date, emphasis of paramyxovirus surveillance has mostly been placed on fruit bats, the henipavirus genus and related viruses due to the association of other henipavirus species with zoonotic events [30, 31] . however, research regarding the zoonotic potential of the insectivorous bat-associated viruses is still lacking. one major aim regarding surveillance of wildlife populations is to identify potential zoonotic agents and to evaluate any threat to the public as well as domestic animal health. though these bat-borne viruses are unlikely to pose a significant threat, it still merits continued monitoring of the chiropteran species within these caves as well as mammalian species that inhabit the surrounding area. 2010, however, was not identical. the detection of two diverse viruses from bats of the same species and same population has previously also been reported in insectivorous bats sampled in other african countries [36] . these observations can in part be explained by the generation of viral quasi-species populations due to the high mutation rate of rna viruses as a consequence of rna proofreading deficiency of the rna dependent rna polymerase [50] . a larger pool of diverse viruses within a bat population and the coroosting of several cave-dwelling bat species may facilitate viral sharing between different bat species [51] . however, ongoing biosurveillance in these cave-dwelling bat species will be required before active viral sharing can be shown. the paramyxoviral sequence detected in the o. martiensseni bat (batpv/oto_mar/up535/rwa/2008) was near identical to the viral sequences previously described from several individuals of the same species sampled in kenya in 2011 [36] . these sequences shared a 99.3% similarity on both nucleotide and amino acid level. the r. aegyptiacus-derived viral sequence (batpv/rou_aeg/up438/rwa/2008) grouped within a henipavirus-related clade and was near identical to a paramyxoviral sequence detected in the same host species previously reported from kenya [36] . sequence similarity shared between these two sequences was found to be 98.1% and 98.7% on nucleotide and amino acid level, respectively. for countries where the bat-human interface is more pronounced, as a result of ecotourism, guano mining or bat hunting and consumption, surveillance is key to identify the diversity of viruses present and their potential host species. longitudinal and well-structured surveillance programmes would better characterize the circulation and shedding periods of the paramyxo-and betacoronaviruses within the ruhengeri cave system. such information would be most valuable towards well-considered zoonotic disease risk assessments and mitigation strategies. supplementary materials: the following are available online at http://www.mdpi.com/2414-6366/4/3/99/s1, table s1 : bats collected and tested in this study. host and viral traits predict zoonotic spillover from mammals filoviruses in bats: current knowledge and future directions bats host major mammalian paramyxoviruses bat lyssaviruses global epidemiology of bat coronaviruses global capacity for emerging infectious disease detection emerging infectious diseases of 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meals dna primers for amplification of mitochondrial cytochrome c oxidase subunit i from diverse metazoan invertebrates. mol molecular identification of bloodmeals from biting midges (diptera: ceratopogonidae: culicoides latreille) in denmark sensitive and broadly reactive reverse transcription-pcr assays to detect novel paramyxoviruses bioedit: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/nt jmodeltest 2: more models, new heuristics and highperformance computing creating the cipres science gateway for inference of large phylogenetic trees posterior summarization in bayesian phylogenetics using tracer 1.7 molecular evolutionary genetics analysis across computing platforms bats are natural reservoirs of sars-like coronaviruses an eco-epidemiological study of morbilli-related paramyxovirus infection in madagascar bats reveals host-switching as the dominant macro-evolutionary mechanism a comparative analysis of viral richness and viral sharing in cave-roosting bats novel paramyxoviruses in free-ranging european bats novel paramyxoviruses in australian flying-fox populations support host-virus coevolution identification of novel paramyxoviruses in insectivorous bats of the southwest indian ocean we would also like to thank the staff of the rwanda tourism board and national parks and wendy white from the kwazulu-natal bat interest group for assisting in the logistics and fieldwork. the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results. key: cord-317244-4su5on6s authors: maganga, gael d.; bourgarel, mathieu; obame nkoghe, judicael; n'dilimabaka, nadine; drosten, christian; paupy, christophe; morand, serge; drexler, jan felix; leroy, eric m. title: identification of an unclassified paramyxovirus in coleura afra: a potential case of host specificity date: 2014-12-31 journal: plos one doi: 10.1371/journal.pone.0115588 sha: doc_id: 317244 cord_uid: 4su5on6s bats are known to harbor multiple paramyxoviruses. despite the creation of two new genera, aquaparamyxovirus and ferlavirus, to accommodate this increasing diversity, several recently isolated or characterized viruses remain unclassified beyond the subfamily level. in the present study, among 985 bats belonging to 6 species sampled in the belinga caves of gabon, rna of an unclassified paramyxovirus (belinga bat virus, belpv) was discovered in 14 african sheath-tailed bats (coleura afra), one of which exhibited several hemorrhagic lesions at necropsy, and viral sequence was obtained in two animals. phylogenetically, belpv is related to j virus and beilong virus (beipv), two other unclassified paramyxoviruses isolated from rodents. in the diseased belpv-infected c. afra individual, high viral load was detected in the heart, and the lesions were consistent with those reported in wild rodents and mice experimentally infected by j virus. belpv was not detected in other tested bat species sharing the same roosting sites and living in very close proximity with c. afra in the two caves sampled, suggesting that this virus may be host-specific for c. afra. the mode of transmission of this paramyxovirus in bat populations remains to be discovered. members of the paramyxoviridae family are pleomorphic enveloped viruses [1] divided into two subfamilies, paramyxovirinae and pneumovirinae. paramyxovirinae has recently been subdivided into seven genera: aquaparamyxovirus, avulavirus, ferlavirus, henipavirus, morbillivirus, respirovirus, and rubulavirus (http://ictvonline.org/virustaxonomy. asp?version52012). viruses of this family affect a wide range of animals, including primates, birds, carnivores, ungulates, snakes, cetaceans and humans, and cause a wide variety of infections, such as measles, mumps, pneumonia and encephalitis in humans, and distemper, peste des petits ruminants, newcastle disease and respiratory tract infections in animals. however, several paramyxoviruses (pvs) have not been classified into any of these seven genera, including nariva virus (narpv) [2] , mossman virus (mospv) [3] , beilong virus (beipv) [4] , j virus (jpv) [5, 6] , tupaia paramyxovirus (tuppv) [7] and tailam virus [8] , all of which belong to a group of novel paramyxoviruses isolated from wild animals, as well as salem virus isolated from horses [9] . among them, only jpv has been shown to be pathogenic, causing extensive haemorrhagic lesions in rodents [6] . horizontal transmission is the principal mode of intraspecies pv infection, suggesting that contaminated faeces, urine or saliva may be responsible for spillover to other species [10] . bats have a close evolutionary relationship with several genera of mammalian paramyxoviruses [11] . otherwise, bat-borne paramyxoviruses are in close relationship to known paramyxoviruses of mammalian. these small mammals are known to harbour a broad diversity of pvs, including emergent henipaviruses (nipah virus and hendra virus) and rubulaviruses [menangle virus, tioman virus, mapuera virus, and tuhoko virus 1, 2 and 3 (thkpv-1, thkpv-2 and thkpv-3)]. a very broad diversity of paramyxoviruses, including henipa-, rubula-, pneumo-and morbilli-related viruses, have been detected in six of ten tested bat families [11] . whereas most of the viruses identified in bats do not seem to cause clinical disease in these animals, there have been reports of rabid bats [12, 13] and of unusually large numbers of animals succumbing to infection by rabies virus [14] . as part of a large-scale investigation of viral diversity in bats and of associated zoonotic risks, we have previously detected a bat paramyxovirus in one insectivorous african sheath-tailed bat (coleura afra) [11] , exhibiting several hemorrhagic lesions at necropsy. we therefore examined occurrence of this bat paraymxovirus in other bats. the study was conducted in the belinga mountains (northeast gabon), where ebola outbreaks occurred in 1994-1996 and 2001-2002 . all the capture events, animal handling, euthanasia and transfer of samples across country borders were gabon. there are no patents, products in development, or marketed products to declare. this does not alter the authors' adherence to all the plos one policies on sharing data and materials, as detailed in the online guide for authors. performed in accordance with the guidelines of the american society of mammalogists (http://www.mammalsociety.org/committees/animal-care-anduse) [15] : bats were captured following recommendations by kunz and parsons [16] and identified by trained field biologist. captured bats were removed carefully from nets as soon as possible to minimize injury, drowning, strangulation, or stress. safe and humane euthanasia was achieved through the use of inhalant anaesthetic (halothane) prior to autopsy. all work (capture, euthanasia and autopsy) was carried out with authorization from the gabonese ministry of water and forestry (département de la samples of liver, spleen, kidney, lung, heart, gut, brain and salivary glands were collected, stored in liquid nitrogen and transferred to the cirmf laboratory (centre international de recherches médicales de franceville, gabon), where they were stored at 280˚c until analysis. blood samples were also collected, except from the smallest individuals (body mass ,12 g). a total of 985 bats (table 1) interestingly, at autopsy, only one c. afra out of the 26 individuals, from batouala cave, exhibited diarrhea and severe hemorrhagic lesions in both thoracic and abdominal organs, along with lung congestion and pleurisy. virological screening of the diseased bat was performed to identify the cause of these lesions. the screening process included the whole paramyxoviridae family and also filoviruses [marburg virus (marv) and zaire ebola virus (ebov)], given the nature of the lesions and the known filovirus tropism for bats. briefly, approximately 100 mg each of this animal's liver and spleen were pooled and crushed in 600 ml of cold pbs in a ball-mill tissue grinder (genogrinder 2000, spex centripep). total rna was extracted using a biorobot ez1 and the ez1 rna tissue mini kit (qiagen) according to the manufacturer's guidelines. the rna was then tested for paramyxoviruses, using three heminested reverse transcription-pcr (hnrt-pcr) assays targeting the polymerase gene [17] , and also for marburg virus [18] and zaire ebola virus [19] . however, the screening was extended to arenaviruses, flaviviruses, alphaviruses, polyomaviruses, orthopoxviruses, parapoxviruses, influenza viruses, lyssaviruses and rhabdoviridae family. to further investigate the presence of the virus in bat populations, a strain-specific real-time rt-pcr assay (primers: gb09-478-f, 59-ggcggctcttaaaagt-gaatg-39; gb09-478-r, 59-gcggggtcaaattggtcat-39; probe: gb09-478-p, 59-tccagcacaaacatatccgagaaggctag-39) was designed within the initial pcr fragment and was used to test total rna extracted from mixed liver and spleen samples from each of all the other bat species. the amplification was performed in a final volume of 25 ml, containing, 12.5 ml taqman r 2x pcr master mix (applied biosystems), 0.5 ml each primer and probe (10 mm), 1 ml bovine serum albumin (1 mg/ml) (invitrogen), 5 ml cdna and rnase-free water (invitrogen). amplification generally involved 2 min at 55˚c, 10 min at 95˚c followed by 45 cycles of 15s at 95˚c and 1 min at 58˚c. in order to determine the organ distribution of this virus in infected bats, total rna was extracted from heart, liver, spleen, kidney, lung, intestine and brain samples from all 14 real-time rt-pcr-positive bats, as described previously, and screened, using the same strain-specific real-time rt-pcr assay shown above. transmission by hematophagous arthropods was studied. hematophagous arthropods were collected inside faucon and zadié caves. mosquitoes were sampled using cdc light traps whereas bat-flies were manually collected on bats (coleura afra, miniopterus inflatus, hipposideros cf. ruber and rousettus aegyptiacus) trapped in the faucon and zadié caves between november and december 2009, june 2010, and january, march and april 2011. after the morphologic species determination, insects were crushed by monospecific pools (up to 10 specimens for mosquitoes, and between 1 and 5 for bat-flies) in 300 ml pbs. total rna was extracted using 100 ml of the supernatant from each pool by using the rneasy mini kit (qiagen) and then was tested with the specific realtime rt-pcr assay described above. the viral sequences obtained were first compared to those available in the public database using the algorithm "blastn" from blast program [20] and then aligned with homologous sequences of paramyxoviridae reference strains from genbank, using the mega program version 5 [21] . bayesian inference of phylogeny was done using mrbayes v.3.2 software and the gtr+g+i nucleotide substitution model [22] for two million generations with a burn-in of 25%. one of three hnrt-pcr, respiro-, morbilli-and henipaviruses pcr (rmh-pcr) assays yielded an amplicon of 559 base pairs (bp) from diseased bat, in collaborative work with the bonn institute of virology bonn (germany). the pcr product was sequenced with dye terminator chemistry (applied biosystems). no other virus was detected in the diseased bat. blast and phylogenetic analysis confirmed that the sequence from diseased bat, designated batpv gb09 478 (genbank accession number hq660155), belonged to the paramyxoviridae family. it showed 65% and 66% nucleotide identity, respectively, with jpv and beipv, and had pairwise nucleotide identities of 36-42% with the henipavirus group and 37-40% with the morbillivirus group. phylogenetic analysis indicated that this bat pv, that we named belinga bat virus (belpv), clustered with unclassified paramyxoviruses and was located between the genera morbillivirus and henipavirus (fig. 1) . in this study, among all animals tested by the strain-specific real-time rt-pcr assay only 14 c. afra bats, including the diseased animal, were positive. bat samples positive by specific real-time rt-pcr were then tested with rmh-hemi nested-pcr for molecular characterization. another 439-bp pcr product was amplified from one liver-spleen pool from a second c. afra specimen, and was sequenced. these two phylogenetically related sequences (fig. 1) , designated batpv gb09 478 and batpv gb09 450, displayed 100% of nucleotide identity. belpv-specific rna was detected only in c. afra, with a prevalence of 14.9% (14/ 94 of the c. afra individuals sampled from the faucon and batouala caves) ( table 1) . interestingly, no bats belonging to the other five species tested positive. belpv rna was detected in the heart (8/12) and liver (5/14) , at low and variable loads ( table 2 and fig. 2 ). no significant difference in the belpv rna detection rate was found between the two organs (x 2 52.476, df51, p.0.20). in the bat exhibiting severe hemorrhagic lesions (gb09 478), both the liver and heart were belpv-positive, with a higher viral load in the heart (ct value 528). in total, 432 arthropods were collected inside faucon and zadié caves, including culicidae (10 uranotaenia nigromaculata and 320 culex wigglesworthi), nycteribiidae (51 eucampsipoda africana, 26 nycteribia schmidlii scotti and 21 penicillidia fulvida) and streblidea (brachytarsina allaudi and 1 raymondia huberi huberi) ( table 1) . no arthropods tested were positive for belinga bat virus (table 1 ). the belpv nucleotide sequence obtained showed similarity with the jpv and beipv sequences. belpv has previously been reported to hold a phylogenetic position between the genera henipavirus and morbillivirus. the same phylogenetic position had been observed with mospv and j-v [4] . in this study, organs with high belpv concentrations are different from those found with high paramyxovirus concentrations in pteropodids and microchiroptera bats. in microchiroptera bats from brazil, spleen has been found more positive than the others organs with highest viral load, as in eidolon helvum (megachiroptera bat) in africa [11] . however, in our study majority of spleen were not available. the within-host belpv distribution tended to be organ-specific. belpv seemed to be restricted to the heart and liver. in contrast, jpv has been isolated from blood, lung, liver, kidney and spleen of experimentally infected laboratory mice [6] but not in heart. the belpv distribution for the heart and liver, together with the high viral load in heart tissue, could suggest that this virus is likely to be present in the bloodstream and might thus be transmitted during aggressive contacts between bats, or by blood-sucking vectors. nethertheless, viremia was not proven. belpv rna was not searched from blood because in these small species of bats blood was difficult to collect in the field. we detected belpv only in coleura afra and not in other bat species sharing the same roosting sites and living in very close proximity in the two caves sampled. however, it has been shown that bats of different species occupying the same roosting sites can share the same viruses. marburg virus had been detected in rousettus aegyptiacus and hipposideros sp. bats living in kitaka cave in uganda [23] and miniopterus inflatus and rousettus aegyptiacus bats caught in goroumbwa mine in the democratic republic of the congo [24] . these bat species are known to live in close proximity. thus, virus transmission between different bat species is possible [25] . thus, we can speculate that the failure to detect belpv in other bat species sharing the same caves would suggest that this virus has strong host specificity for c. afra, as well as restricted intraspecies transmission. henipaviruses occur naturally in fruit bats belonging to the genus pteropus [26] , and this also appears to be true of severe acute respiratory syndrome-like coronaviruses in rhinolophus bats [27, 11] . in view of our data we can assume that belpv might have pathogenic potential for its host c. afra. indeed, high viral load was detected in the heart of the diseased bat, and the lesions were consistent with those reported in wild rodents and mice experimentally infected by jpv [6] . although belpv rna was also detected in asymptomatic bats, pathogenicity may appear in long term under some immunological and/or ecological conditions. indeed, virus must not induce pathology to persist or adapt within its reservoir host. many authors suggested that persistence in the absence of pathology or disease appears to be a common characteristic of bat viruses in their natural host population [28, 29] . however, a severe immunodepression for instance, may increase the risk of infection with opportunistic pathogens. under some environmental conditions (cool environments for example), some avirulent pathogens, such as geomyces destructans, causative agent of white-nose syndrome, may become pathogenic in hibernating bats in north america [30, 31] . nevertheless, infection by belpv may be mild for bats and thus the pathology observed not directly related. otherwise, it may also be that this animal had an underlying disease or infection with a different pathogen. even in this case, we might not draw any conclusions neither establish a link with lesions seen. therefore, the pathogenicity of the belpv should be demonstrated by experimental animal infection. otherwise, viral antigens or rnas should be detected histologically in the lesions of naturally-infected bats. however, the unavailability of biological tissues from the diseased bat failed to perform these analyzes. consequently, other captures of coleura afra species are considered in order to find belpv again for further studies (pathogenicity to its host, isolation and complete genome characterization). however, coleura afra is a migratory species living in colonies of several hundred individuals. in gabon, this species, which has been recently described, is not present all year round in the caves of the north-east of the country, making the studies on this species difficult and thereof partly explaining the lack of virological studies. some viruses appear to cause clinical disease in wild-living bats; these include lyssaviruses and an ebola-like filovirus named lloviu virus [32, 33, 34] . bats are the natural reservoirs for many viruses, including emerging zoonotic viruses such as sars-cov [27] , hendra and nipah viruses [26, 35] , ebola virus [36] , marburg virus [23, 37] , rabies virus and other lyssaviruses [11] . in general, humans are infected through an intermediate amplifying host such as palm civets for sars-cov, horses for hendra virus and pigs for nipah virus [38] . however, in humans nipah virus outbreaks linked to bats exposure have been reported [39] . it remains to be shown whether the belpv reported here presents a zoonotic risk. nonetheless, like most rna viruses, for example coronaviruses, characterized by high mutation and/or recombination rates [40] , pvs may adapt to novel hosts, including humans. a serological test capable of detecting antibodies to this virus in human populations living in the vicinity of these animals is needed to assess zoonotic potential. all the blood-sucking arthropods collected from bats, as well as mosquitoes collected in the caves where bat sampling took place, were negative for belpv, in keeping with the lack of known pv vectors [41] . however, belpv transmission by blood-sucking vectors within the gabonese population of c. afra cannot be ruled out. indeed, a haemosporidian parasite (polychromophilus) was found in a blood parasite vector (penicillidia fulvida) in faucon cave in gabon in 1977 [42] and also in its host m. inflatus (greater long-fingered bat) from the same cave in 2010 and 2011 [43] . in addition, the methodology used to collect flying hematophagous insects (based on light traps) possibly introduced a bias by selecting only those attracted by light. therefore, we can not exclude that additional sampling techniques could increase the number of mosquitoes species or groups known to colonize caves such as sandflies or biting midges. hence, the natural mode of transmission of this unclassified paramyxovirus in bat populations, through batbat aggression for example, remains to be determined. this association between c. afra and belpv could serve as an interesting model, (i) to evaluate modes of transmission within host populations, (ii) to study hostvirus interactions (pathogenesis and host specificity), and (iii) to evaluate the zoonotic risk of a newly identified virus. further studies of c. afra populations and a broader diversity of arthropod vectors, spanning larger areas and time scales, are needed to confirm this apparent host-virus specificity, and to determine the modes of belpv transmission. further studies are needed to characterize complete belpv genome and demonstrate the pathogenicity of this virus for its host coleura afra. conceived and designed the experiments: gdm mb eml. performed the experiments: gdm jon nn. analyzed the data: gdm jfd. contributed reagents/ materials/analysis tools: gdm mb cd cp jfd eml. wrote the paper: gdm cp eml sm. paramyxoviridae: the viruses and their replication nariva virus, a hitherto undescribed agent isolated from the trinidadian rat, zygodontomys b. brevicauda (j.a. allen and chapman) mossman virus, a paramyxovirus of rodents isolated in queensland beilong virus, a novel paramyxovirus with the largest genome of non-segmented negative-stranded rna viruses studies on a virus isolated from wild mice (mus musculus) a new mouse paramyxovirus (j virus) isolation and molecular characterization of a novel cytopathogenic paramyxovirus from tree shrews complete genome sequence of a novel paramyxovirus, tailam virus, discovered in sikkim rats identification and phylogenetic comparison of salem virus, a novel paramyxovirus of horses reproduction and nutritional stress are risk factors for hendra virus infection in little red flying foxes bats host major mammalian paramyxoviruses encephalitis caused by a lyssavirus in fruit bats in australia airborne transmission of lyssaviruses rabies in nonhematophagous bats guidelines of the american society of mammalogists for the use of wild mammals in research ecological and behavioral methods for the study of bats sensitive and broadly reactive reverse transcription-pcr assays to detect novel paramyxoviruses marburg virus infection detected in a common african bat newly discovered ebola virus associated with hemorrhagic fever outbreak in uganda basic local alignment search tool mega5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance and maximum parsimony methods mrbayes: bayesian inference of phylogenetic trees isolation of genetically diverse marburg viruses from egyptian fruit bats studies of reservoir hosts for marburg virus genomic characterizations of bat coronaviruses (1a, 1b and hku8) and evidence for co-infections in miniopterus bats isolation of hendra virus from pteropid bats: a natural reservoir of hendra virus bats are natural reservoirs of sars-like coronaviruses bats: important reservoir hosts of emerging viruses novel astroviruses in insectivorous bats bat white-nose syndrome: an emerging fungal pathogen? inoculation of bats with european geomyces destructans supports the novel pathogen hypothesis for the origin of white-nose syndrome pathogenesis studies with australian bat lyssavirus in grey-headed flying foxes (pteropus poliocephalus) bats and lyssaviruses discovery of an ebolavirus-like filovirus in europe nipah virus infection in bats (order chiroptera) in peninsular malaysia fruit bats as reservoirs of ebola virus is marburg virus enzootic in gabon? bats as a continuing source of emerging infections in humans risk factor for nipah virus encephalitis in bangladesh severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats paramyxovirus and pneumovirus diseases of animals and birds: comparatives aspects and diagnosis dionisia bunoi n. g. n. sp., haemoproteidae parasite du microchiroptè re hipposideros cyclops au gabon the chiropteran haemosporidian polychromophilus melanipherus: a worldwide species complex restricted to the family miniopteridae we thank all the persons involved in sample collections, and especially andré délicat, peggy motsch, dieudonné nkogué, tabea binger, peter vallo, and nil rahola. we acknowledge heïdi lançon for improving the english. cirmf is supported by the government of gabon, total-fina-elf gabon, and ministère de la coopération française. key: cord-308614-gsgntf4c authors: eshar, david; weinberg, maya title: venipuncture in bats date: 2010 journal: lab anim (ny) doi: 10.1038/laban0610-175 sha: doc_id: 308614 cord_uid: gsgntf4c though not as common as small rodents in laboratory settings, bats are being increasingly used in research studies. knowledge of proper blood sampling techniques is essential for care and management of bats. to minimize handling and to avoid sample failure, all needed equipment should be prearranged and organized in advance. equipment needed includes a 1-to 3-ml syringe or 0.5-ml insulin syringe; 25-or 27-gauge needles; microtainer collection tubes with heparin and calcium-edta; microhematocrit capillary tubes and sealing clay; glass slides; chlorhexidine-based scrub equivalent to 1% of the total body weight at each draw. several bat species can act as reservoir hosts of zoonotic pathogens including lyssaviruses, severe acute respiratory syndrome (sars) coronavirus, ebola virus, henipaviruses, west nile virus, st. louis encephalitis virus and leptospira spp. 2, 5 . all personnel working with bats should be vaccinated against rabies 2 . coats and gloves should be used to minimize contact with body secretions, such as blood or saliva, that may contain infectious organisms 2 . manual restraint is usually not a problem in bats, but healthy bats can wriggle excessively unless properly restrained. all restrained bats can deliver a defensive bite, and the digits and associated claws of large bats can scratch an unprotected handler or pull fingers in for a bite 2 . bats should be restrained using either protective thick gloves or a towel to hold the head and the limbs (fig. 3) . the duration of manual restraint should be minimized to reduce stress and prevent hyperthermia and marked elevations in cortisol and glucose 6 . stressed bats may require chemical restraint, either by inhalation or by injection, although chemical restraint may have complicating side effects. bats should be anesthetized for procedures requiring close contact to reduce risk of biting and transmission of secretions 2 . bats are homeotherms, maintaining their body temperature between 35 °c and 39 °c (ref. 2). additionally, large lungs and naked flight membranes result in greater heat loss (6 times greater) and thermal conductance in recent years, interest in research using bats has grown. these small mammals have many unique features that attract great attention and can now be more commonly found in many research centers 1 . second to rodents, bats are the most abundant and diverse mammalian group in nature. the order chiroptera is divided into suborders microchiroptera and megachiroptera, with further subdivision into 18 families and 1,116 recognized species. bats used in studies are either wildcaught or originate from captive breeding colonies 1 . blood testing in bats may be done as part of a clinical evaluation or for hematological research. sites for blood collection from bats include the median (brachial) vein, cranial vena cava, jugular vein, orbital sinus and heart [2] [3] [4] . venipuncture of the cephalic and the saphenous (interfemoral) veins are two of the best techniques for quick and safe collection of blood in bats. these nonterminal techniques can be used to collect small to medium volumes of blood with good visualization of the blood vessels and can be done on anesthetized or manually restrained, unsedated bats. the cephalic vein is located along the leading edge of the patagium or the antebrachial wing membrane (fig. 1) . the saphenous (interfemoral) vein is found in the uropatagium (tail membrane) parallel to the femur (fig. 2) . the blood volume of bats comprises ~10% of the total body weight (9.0-11.0 ml per 100 g) 2 . as in other species, it is considered safe to remove a blood sample david eshar, dvm 1 though not as common as small rodents in laboratory settings, bats are being increasingly used in research studies. knowledge of proper blood sampling techniques is essential for care and management of bats. can also be centrifuged so that plasma can be removed and frozen until later analysis 7, 8 . cephalic and saphenous (interfemoral) venipuncture is a non-terminal technique that allows simple, safe and efficient blood sampling in bats of various sizes. be used for the venipuncture using a chlorhexidine-based scrub solution. for most cases, a 25-gauge needle with a 1-ml syringe or a 0.5-ml insulin syringe with attached 27-gauge needle is used (fig. 2) . once the needle is inserted into the vessel and blood is identified in the needle hub, a gentle negative pressure (suction) is intermittently applied until the desired sample volume is obtained. care should be taken not to use excessive pressures that can cause collapse of the vein or hemolysis of the blood sample. alternatively, microhematocrit capillary tubes can be used for sample collection. a 25-gauge needle can be used to puncture the vessel in small bats, and the tube can be inserted into the hub of the needle to collect the sample, or if a vein is accidently punctured, blood can be collected into a microhematocrit capillary tube directly from the incision site 2 (fig. 1) . bats' wing membranes are very thin and lack subcutaneous fat to aid in hemostasis of the punctured blood vessels. the venipuncture site should be gently pressed using a piece of cotton gauze until bleeding completely stops. special attention should be given to small bats because large hematomas and ongoing bleeding can result in severe and even life-threatening blood loss. once back in its cage, the bat should be closely monitored for renewed bleeding resulting from wing flapping. bat blood can altered by inappropriate collection, handling and storage 2 . moderate hemolysis during sample collection can cause test results to show hyperkalemia, hyponatremia, hypochloremia and elevated levels of aspartate transaminases 8 . the sample should be processed within 6 hours of sampling, as prolonged contact between the resting plasma and the red cells causes elevation in phosphorus and decreases in chloride and glucose concentrations 8 . blood samples treated with heparin can be analyzed immediately after collection and solution; leather and non-sterile gloves; and chemical restraint agents (as needed). some bat species weigh less than 100 g, and in many cases, only a small sample is obtained. hence, we prefer to use blood analyzers (such as the abaxis vetscan chemistry analyzer) that require only small blood sample volumes (0.l ml of whole blood or plasma). to avoid clotting and to reduce sample collection time, syringes should be prepared with heparin in advance 7 . venipuncture of the cephalic and saphenous veins without anesthesia usually requires one phlebotomist and one or two individuals to restraint the bat. one assistant will hold the bat's head and body while a second assistant gently spreads the wing and holds the vein at its most proximal end, to allow the vessel to engorge with blood and dilate. for a right-handed phlebotomist, the right wing of the bat is extended while the left wing is securely held by the first assistant. the left hand of the phlebotomist is used to either compress the vein or add stability while the right hand holds the syringe. the venipuncture site can be warmpacked prior to the procedure to facilitate blood flow. aseptic techniques should figure 2 | venipuncture of the right saphenous (interfemoral) vein in the uropatagium of a male egyptian fruit bat (rousettus aegyptiacus) using a 1-ml syringe with a 27-gauge needle. the restrainer manually holds the bat's neck and feet using thick gloves to protect against bites. science and the conservation of bats chiropterans (bats). in zoo animal and wildlife immobilization and anesthesia a method of bleeding small bats a new method of bleeding small and infant bats public health awareness of emerging zoonotic viruses of bats: a european perspective basal, diurnal, and stress-induced levels of glucose and glucocorticoids in captive bats comparison of serum and plasma for determination of blood biochemical values in malaysian flying foxes (pteropus vampyrus) the effect of time at which plasma separation occurs on biochemical values in small island flying foxes (pteropus hypomelanus) key: cord-277306-r8jki3x4 authors: osborne, christina; cryan, paul m.; o'shea, thomas j.; oko, lauren m.; ndaluka, christina; calisher, charles h.; berglund, andrew d.; klavetter, mead l.; bowen, richard a.; holmes, kathryn v.; dominguez, samuel r. title: alphacoronaviruses in new world bats: prevalence, persistence, phylogeny, and potential for interaction with humans date: 2011-05-12 journal: plos one doi: 10.1371/journal.pone.0019156 sha: doc_id: 277306 cord_uid: r8jki3x4 bats are reservoirs for many different coronaviruses (covs) as well as many other important zoonotic viruses. we sampled feces and/or anal swabs of 1,044 insectivorous bats of 2 families and 17 species from 21 different locations within colorado from 2007 to 2009. we detected alphacoronavirus rna in bats of 4 species: big brown bats (eptesicus fuscus), 10% prevalence; long-legged bats (myotis volans), 8% prevalence; little brown bats (myotis lucifugus), 3% prevalence; and western long-eared bats (myotis evotis), 2% prevalence. overall, juvenile bats were twice as likely to be positive for cov rna as adult bats. at two of the rural sampling sites, cov rnas were detected in big brown and long-legged bats during the three sequential summers of this study. cov rna was detected in big brown bats in all five of the urban maternity roosts sampled throughout each of the periods tested. individually tagged big brown bats that were positive for cov rna and later sampled again all became cov rna negative. nucleotide sequences in the rdrp gene fell into 3 main clusters, all distinct from those of old world bats. similar nucleotide sequences were found in amplicons from gene 1b and the spike gene in both a big-brown and a long-legged bat, indicating that a cov may be capable of infecting bats of different genera. these data suggest that ongoing evolution of covs in bats creates the possibility of a continued threat for emergence into hosts of other species. alphacoronavirus rna was detected at a high prevalence in big brown bats in roosts in close proximity to human habitations (10%) and known to have direct contact with people (19%), suggesting that significant potential opportunities exist for cross-species transmission of these viruses. further cov surveillance studies in bats throughout the americas are warranted. bats play important roles in maintaining and transmitting zoonotic viruses [1, 2, 3] . more than 99 different viruses have been detected in and/or isolated from bats of diverse species [2] (and c. calisher, personal communication). rabies virus and other lyssaviruses infect bats of many species, and old world fruit bats (family pteropodidae) are reservoirs for both hendra and nipah viruses [4, 5, 6] . two newly discovered human reoviruses, melaka virus and kampar virus, associated with influenza-like illnesses in humans, may be transmitted from small flying foxes (fruit bats; pteropus hypomelanus) based on the close phylogenetic relationships of these viruses to pulau virus, a bat reovirus [7, 8] . egyptian fruit bats (rousetttus aegyptiacus) are known reservoirs of marburg and certain ebolaviruses [9, 10] . in humans, domestic animals, and birds, coronaviruses are common respiratory and enteric pathogens, and several covs cause systemic disease. among the 5 known human coronaviruses, hcov-229e and hcov-nl63 are alphacoronaviruses (formerly called group 1 covs), hcov-oc43 and hcov-hku1 are betacoronaviruses (formerly group 2a), and the severe acute respiratory syndrome-related coronavirus (sars-cov) and sarslike covs are also betacoronaviruses (formerly group 2b). the sars pandemic of 2002-03 was caused by sars-cov, a zoonotic coronavirus recently emerged from horseshoe bats (suborder microchiroptera, family rhinolophidae, genus rhinolophus) from different locations in southeastern china [11, 12] . extensive worldwide surveillance of bats showed that bats carry an enormous diversity of covs [13, 14, 15, 16, 17, 18, 19, 20, 21, 22] . phylogenetic analysis of complete genome sequences of coronaviruses from bats, humans, birds, and other vertebrates suggests that bats may be the reservoir hosts from which all coronavirus lineages originated [23, 24] . the potential for emergence of zoonotic viruses into the human population depends on the prevalence of the virus in its host species, host range mutations within viral quasispecies, and the degree to which the reservoir host interacts with humans. in 2006, we reported the first detection of alphacoronavirus rna in feces of north american bats sampled in the rocky mountain region of colorado [17] . here we describe a much larger and more comprehensive study of coronavirus prevalence, epizootiology, geographic distribution, and persistence, as well as preliminary phylogenetic analysis of cov genome sequences in bats in colorado. capture, marking, and sampling of bats followed guidelines of the american society of mammalogists [25] and animal protocols were approved by the institutional animal care and use committee of the u.s. geological survey, fort collins science center ('standard operating procedure 01-01 for the capture, handling, marking, tagging, biopsy sampling, and collection of bats') and colorado state university (csu iacuc number 03-096a). bats were captured under authority of a scientific collecting license (permit numbers: 07tr738a3, 08tr2010, and 09tr2010) issued by the colorado division of wildlife. insectivorous bats of the families vespertilionidae (16 species) and molossidae (1 species) were sampled at 16 rural sites (sites #1-16, fig. 1 ) in the rocky mountain region during the summer of 2007. bats were identified to species based on external morphological characteristics as described in regional faunal manuals [26, 27] adopting revised taxonomy for myotis occultus [28] and parastrellus hesperus [29] . to determine whether covs persist in bat populations over the course of several years, additional bat fecal samples were collected during the summers of 2008 and 2009 at two rural sites in north central and southeastern colorado. in addition, big brown bats (eptesicus fuscus) were sampled at 5 different sites (sites #17-21) within a single urban municipality in northern colorado (fort collins) during the summers of 2007 and 2008. these sites were chosen because they were in close proximity to humans [30] . site #17 was in a vintage farmhouse that is currently being used as a family visitation center; site #18 was a natural creek surrounded by suburban neighborhoods; site #19 was in the recreation center of a church; site #20 was within an education building, and site #21 was within a picnic pavilion at a public park. several of these sites had been previously used in rabies ecology studies, and some bats had been tagged with passive integrated transponders (pit tags) for host demographic analysis [31, 32] . this allowed for repeated capture and sampling of known individual bats. all bats were either captured in mist nets during the night as they drank or foraged near open water, or were caught in mist nets or harp traps as they emerged from roosts. whenever possible, the species, sex, reproductive status, age (adult or juvenile), date, and location of capture were recorded for each bat sampled. bats were sampled as previously described [17] , typically within 5-10 min of capture, and then released. anal/rectal swabs or fecal pellets were taken using sterile calcium alginate swabs and stored in rnalater (ambion, austin, tx) and/or m4 viral transport medium (vtm, remel; lenexa, ks). all samples were stored at 270uc prior to analysis. based on sample type and medium results were pooled for analysis of prevalence surveys. in a post hoc analysis we identified differences in the efficacy of different sampling methods (text s1) such that the data represent minimal estimates of the prevalence of cov infection in bats. bat carcasses submitted to the colorado department of public health and environment (cdphe) that were negative for rabies viruses were sent to our laboratory for detection of cov rna. these bats had been submitted from counties throughout colorado for rabies testing to rule out the need for post-exposure rabies prophylaxis of humans who had had close contact with these animals [30, 33] . intestines were removed from the bats and stored at 270uc prior to analysis. extracted rna was eluted in 60 ml of rnase-free water and stored at 280uc. before rt-pcr, 50 microliters of rna was treated with zymo onestep pcr inhibitor removal kit (zymo research, orange, ca) following the manufacturer's instructions. cdna was generated by superscript iii reverse transcriptase (invitrogen, carlsbad, ca) with random hexamers in a 20 ml reaction using 11 ml of rna as a template according to the manufacturer's instructions. all samples were analyzed in duplicate. reverse-transcription products were stored at 220uc. all cdna samples collected from bats at rural sites or during 2007 were screened for cov rna by pcr with a pair of pan-cov consensus primers [13] that amplify a highly conserved region (400 nucleotide amplicon) of the coronavirus rnadependent rna polymerase (rdrp) gene as previously described [17] except that we used 2.0 mmol/l of primers and 1 ml of cdna or pcr product (for hemi-nested reactions). to increase the sensitivity of rna detection, based on our previously published bat cov sequences [17] and new data from this study, we designed specific primers within the amplicons of alphacoronaviruses from bats of several species in the genus myotis and big brown bats (table s1 ). all of the specimens collected from longlegged and big brown bats were also tested with these primers. to obtain longer nucleotide sequences, rt-pcr was performed using consensus degenerate primers from several regions within the rdrp gene in a superscript iii one-step rt-pcr system with platinum taq high fidelity kit (invitrogen, san diego, ca, usa). similarly, we designed consensus primers that targeted a highly conserved region of the s2 region of the alphacoronavirus spike gene, and made primers from an exact s2 sequence obtained from a big brown bat (table s1) . to minimize the possibility of contamination, all rt and pcr reactions were prepared in an enclosed acrylic nucleic acid workstation equipped with a uv light (clone zone, usa scientific, ocala, fl) in a room separate from the main laboratory. water controls without template included in every rt and pcr experiment gave no false-positive results. amplicons were analyzed by agarose gel electrophoresis and sequenced on an abi 3730 dna sequencer (applied biosystems technologies, carlsbad, ca) at the university of colorado school of medicine cancer center dna sequencing and analysis core. samples were scored as positive if cov rna was detected on two pcr runs. statistical significance was determined using fisher's exact test. phylogenetic analyses were conducted using mega version 4, and phylogenetic trees were constructed using the neighbor-joining method [34] . the nucleotide sequences from this study were deposited in genbank under accession numbers hq336973-hq336976 and jf414933-jf414936. table 1) . at site #4, a high-elevation meadow in a mountainous area of north-central colorado, 76 long-legged bats were sampled during three consecutive summers (2007) (2008) (2009) . although the sampled bats were not individually marked, the consistent capture of large numbers of females soon after sunset at the site indicated that most of the sampled bats likely came from a nearby maternity roost. female bats often show year-to-year fidelity to maternity roosts [35] . the percentage of long-legged bats that tested positive for cov rna at site #4 varied by year from 6% to 31% (table 2) . at site #5, an arid grassland bisected by canyons in southeastern colorado [36] , 56 bats of eight different species were sampled during two consecutive summers (2008 and 2009). only big brown bats at site #5 were positive for cov rna. although the number of big brown bats sampled at site #5 was small (4 in 2008 and 14 in 2009), the prevalence of cov rna in these bats during these two summers was high (29% to 100%) ( table 2) . in the five different urban locations (sites #17-21), 465 samples were collected from big brown bats during the summers of 2007 all of the urban bat sampling sites were part of a previous study of the ecology of rabies in big brown bats that emphasized host demography [31, 32] , and 113 (24%) of the 465 bats from these sites sampled for this study had been previously individually tagged. sixteen (14%) of these tagged bats were captured and sampled more than once (14 captured twice, and 2 captured three times). five (31%) of the 16 repeatedly sampled tagged bats captured in 2008 were positive for cov rna, but no cov rna could be detected in subsequent samples ( table 3 ). four of the 5 bats became negative for cov rna within 6 weeks after they tested positive for cov rna. (the fifth bat was not recaptured after turning positive). thus, in this small group of serially sampled bats, individual bats were not continually shedding detectable amounts of cov rna, so did not appear to be persistently infected. the age and sex distributions of the 999 (94%) bats sampled for which these data were available and the subset of big brown bats in the urban maternity roosts sampled are shown in table 4 . juvenile bats were two times more likely to be positive for cov rna than adults bats (13% vs. 6%, p = 0.008). in the urban maternity roosts, as expected, the majority of the big brown bats sampled were adult females, but juvenile bats (10 of 52 tested, 19%) were also more than twice as likely to be positive for cov rna than adult bats (36 of 413 tested, 9%, p = 0.03). from the samples positive for cov rna, we obtained nucleotide sequences of amplicons ranging in length from 93-356 nt from the rdrp region of gene 1b. these formed three clusters (.90% nt identity within each cluster). the first cluster (a) included cov rnas of big brown bats from sites #5 and #17-21, the one big brown bat from site #4, and two long-legged bats from site #4 that were collected in 2007 and 2009. the sequence of the a cluster (representative bat: rm-bt-cov 453/2007 ef) was 96% identical to the same region from a big brown bat (rm-bt-cov 65) reported in our previous study [17] . the second cluster (b) (representative bat: rm-bt-cov 09-07/2009 mv) was found in 2 long-legged bats (one sampled in 2008 and one in 2009) and one western long-eared bat sampled at site #4. these sequences had .97% identity in this region to cov rna obtained from several occult bats (m. occultus; rm-bt-cov 6 and 11) reported previously [17] . the third cluster (c) of cov amplicons (representative bat: rm-bt-cov 429/2007 mv) were from other long-legged bats sampled at site #4. these sequences were 96% identical to that from an occult bat (rm-bt-cov 3) reported previously (table 5 and figure s1 ). cluster a had ,65% identity with clusters b and c, whereas clusters b and c had 83% identity to one another. an 1100 nt sequence encoding the s2 domain of the spike glycoprotein was obtained from a big brown bat collected at site #4 in 2007 (rocky mountain bat-cov 453/2007 ef). we compared this sequence to s2 sequences of other known coronaviruses (table 5 and figure 2 ) and found that this genome was distantly related to other known alphacoronaviruses in group 1a, with ,67% nucleotide identity to covs. we also obtained a 700 nucleotide sequence in the same region of s2 from the long-legged bat (rm-bat-cov 433/2007 mv) that had a similar sequence to this big brown bat in the rdrp gene (both in rdrp cluster a). these s2 amplicons had .98% nt sequence identity. the closest bat coronavirus spike sequence to rm-bt-cov 453/2007 found in genbank, was bt-cov a701, from an old world species, rickett's big-footed bat (myotis ricketti) sampled in southeast china in 2005 [14] (65% nucleotide identity, 65% amino acid identity). an approximately 4000 nt sequence in 2 segments of the rdrp gene was obtained from one of the little brown (rm-bt-cov-15/ 2006/ml) and one of the big brown bats (rm-bt-cov-61/2007/ ef) that were submitted to the cdphe. these nt sequences were only 62% identical, indicating that they represented two unique viruses in bats of these two species. these sequences were distantly related (,75% nt identity) to other known alphacoronaviruses, with ,75% nt identity to covs in this group, including all currently available old world bat covs (table 5 and figure 3 ). this is the first multiyear surveillance project of covs in wild bats in north america. cov rna was detected in approximately 7% of all bats sampled (likely an underestimate of prevalence, text s1), comparable to the prevalence of cov rna detected in various species of bats reported in other parts of the world (ranging from 2-55%) [14, 18, 19, 21, 22, 37, 38, 39] . in our study no cov rna was detected in bats in 13 of the 17 species we sampled (also likely biased negatively). failure to detect covs in bats of these species could be related to the smaller numbers sampled. however, a relatively high prevalence of cov rna was detected in bats of 2 species collected at several different sites: 12% for big brown bats and 8% for long-legged bats, and at lower prevalence, 3% in little brown bats and 2% in western long-eared bats. in marked contrast to the enormous diversity of cov genomes found in old world bats [14, 24, 40] , in this and several other cov surveillance studies of new world bats [17, 18, 22] , all covs detected were alphacoronaviruses. our data indicate that nucleotide sequences of alphacoronaviruses harbored by colorado bats are distinct from those found in old world bats. two recent studies of the bat guano virome using next generation sequence technology also only detected alphacoronaviruses in the new world bats of the species tested, as well as a diverse array of other types of viruses [41, 42] . thus, so far there appears to be much more limited cov diversity in new world bats of the species tested than in old world bats. betacoronaviruses have only been detected in old world bat species belonging to the families pteropodidae (rousettus spp) and rhinolophidae (rhinolophus spp.) which belong to the chiropteran suborder yinpterochiroptera. based on available evidence, betacoronaviruses could be restricted to hosts in the suborder yinpterochiroptera (families pteropodidae, rhinolophidae, megadermatidae, craseonycteridae, rhinopomatidae). no bat families of the suborder yinpterochiroptera occur in the new world. [43] . the finding of only alphacoronaviruses in our study may be because bats of these species are resistant to other covs and/or bats from different parts of the new world have yet to be tested for cov infection, as we sampled bats from only a subset of the hundreds of species that reside in the new world. these observations also support the hypothesis that coronaviruses may have co-evolved with their bat hosts, as no species of bat is found both in the new world and old world [44] . to date, however, only a small subset of new world species of bats has been tested for coronavirus infection. as 75% of living genera of all bats worldwide are found in the new worlds tropics alone, further cov surveillance in bats of additional species from different regions in the western hemisphere may reveal hitherto undetected varieties of coronaviruses. the seasonal epidemiology and persistence of new world cov infections in individual bats and within bat populations has not been elucidated. the most comprehensive epidemiological investigation of covs to date in old world bat populations showed that the prevalence of sars-rh-batcovs in rhinolophid bats over a four-year period at collection sites in hong kong sar and china peaked in the spring and varied from year to year. we found similar results in new world bats. at site #4 long-legged bats had an alphacoronavirus rna prevalence of 31% in 2007, 19% in 2009, but only 6% in 2008. in all five of the urban maternity roosts sampled, covs persisted in bat roosts throughout the course of the non-hibernating part of the year (spring/summer) and persisted from year to year. we also found that the prevalence of cov infection in these bat roosts tended to peak in late spring/ early summer. the prevalence of infection with human covs also shows significant annual variations [45] , possibly depending on environmental conditions and/or fluctuating cov antibody levels in the population. possible seasonal variation in cov infection rates may explain why in our initial 2006 study we found a high prevalence (50%) of alphacoronavirus rna in occult bats [17] , but in 2007 we did not detect any positive individuals (22 tested in the same region). the majority of the bats sampled in our study were adult females because they were primarily captured from maternity roosts. the highest prevalence of infection was noted in juvenile bats. in germany, cov infection was also found to be associated with young age and was more common in female bats from maternity roosts compared to female bats found at foraging or swarming sites [19] . these findings support the hypothesis that younger bats may be more susceptible to cov infection and may serve to propagate and maintain these viruses within bat colonies. no overt clinical manifestations of disease were observed in any of the captured bats, including those that were infected with covs. in the small subset of bats that were tagged and recaptured, no individual bat remained persistently positive for cov rna after 6 weeks. similar findings were made in rhinolophid bats in asia that harbor sars-like-bat-covs [37] and in fruit bats experimentally infected with bat covs which showed no signs of illness [39] . these data suggest that although covs persist within bat populations, individual bats may experience only self-limited infections with covs without apparent illness. phylogenetic studies of cov genomes in old world bats in asia and europe have suggested that some bat covs may infect bats of only one species or several closely related species. in asia and germany, different species of bats roosting in the same cave were found to host different covs, whereas bats of the same species in different locations harbored similar covs [14, 19] . in europe, strict associations were found between bat cov deduced amino acid sequences in an 816 bp fragment of the rdrp gene and their specific bat hosts [40] . in africa, covs found in one species of bat were not detected in bats of different species co-roosting in the same cave [38] . similarly, our study showed that new world bats of the same species in geographically distinct locations and over the course of several years harbor similar covs. in contrast to these findings, in kenya some covs appear to be able to infect old world bats of several different species [21] . our preliminary nucleotide sequence data also suggests that we found very closely related cov nucleotide sequences in new world bats from three different species of myotis (m. volans, m. evotis, and m. occultus). furthermore, in site #4, we found similar nucleotide sequences in the spike and replicase genes in cov rnas from both a big-brown bat and a long-legged bat, suggesting that at least some new world bat covs may be able to infect bats of different genera. these findings are notable, as recent phylogenetic studies of rabies viruses in bats suggest that host species barriers play a key role in cross species transmission of viruses [46] . to assess the potential for zoonotic transmission of bat covs, we focused part of this present work on north american bats that have the closest contact with humans and sampled roosts where big brown bats had histories of contact or potential for contact with people [30] . big brown bats are common inhabitants of buildings in cities and towns in colorado and across the united states, and are the primary species encountered by humans in terms of potential exposure to disease agents [30, 33, 47] these bats had a high prevalence of cov infection, ranging from 0-67% (overall 10%) depending on the site and time of year. big brown bats submitted to the cdphe for rabies testing because of known direct contact with humans also had a very high prevalence (19%) of cov infection. because bats which have known or potential contact with humans have such a high prevalence of cov infection, opportunities exist for potential transmission of these viruses to humans. following the sars epidemic, intensive surveillance detected a great diversity of covs throughout the animal kingdom. covs can undergo a high frequency of rna recombination, both in vitro and in vivo, which may play an important role in their evolution and virulence [48] . old world bat covs of several different genotypes were found to co-exist in a single bat [49] . thus recombination between different bat covs could potentially occur in vivo, giving rise to new cov genomes. two strains of hcov-hku1 have recombined to yield a novel hcov-hku1 genotype [16] , and recombination between different strains of sars-cov-like viruses in bats may have given rise to civet sars-cov [37] . the great diversity of covs, their high frequency of rna recombination, their ability to persist in bat populations, and the finding that some covs can apparently infect bats of divergent genera, suggest that ongoing evolution of covs in bats may pose a continuing threat for emergence of novel covs into new hosts. table s1 primers and rt-pcr programs. a. consensus primers targeted a highly conserved region of the s2 region of the spike gene and from an exact sequence obtained from one of the big brown bats. pcr was performed under the following conditions: one ml of cdna was amplified in a 50-ml reaction containing, 0.2 mmol/l deoxynucleoside triphosphates, 1 u of phusiontaq high-fidelity dna polymerase (finnzymes, espoo, finland), and 2.0 mmol/l primers by the following pcr program: 30 sec at 98uc; 40 cycles for 10 sec at 98uc, 15 sec at 50-52uc (depending on the primer set), and 15 sec at 72uc; and then 10 min at 72uc. b. primers used for detection of cov sequence in bat samples. one microliter of cdna was amplified in a 50-ml reaction containing 1.5 mmol/l mgcl 2 , 0.2 mmol/l deoxynucleoside triphosphates, 2.5 u of hotstartaq (qiagen), and 2.0 mmol/l primers using the following pcr program: 15 min at 95uc; 45 cycles for 1 min at 95uc, 1 min at 48uc for my-f and my-r and 50uc for ef-f and ef-r, and 1 min at 72uc; and 10 min at 72uc. c. to obtain additional sequences for phylogenetic analysis, for two of the cdphe intestinal samples, rt-pcr was performed using consensus degenerate primers from several areas within the rdrp gene in a superscript iii one-step rt-pcr system with platinum taq high fidelity kit (invitrogen, san diego, ca, usa). primers and protocols were kindly provided by suxiang tong, phd and ying tao, phd of the centers for disease control and prevention, atlanta, georgia, usa. text s1 influence of different sampling and analysis techniques on cov rna detection. 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syndrome coronavirus and close relatives of human coronavirus 229e in bats bat coronaviruses and experimental infection of bats, the philippines genomic characterization of sars-related coronavirus in european bats and classification of coronaviruses based on partial rna-dependent rna polymerase gene sequences metagenomic analysis of the virome of three north american bat species: viral diversity between different bat species that share a common habitat bat guano virome: predominance of dietary viruses from insects and plants plus novel mammalian viruses a molecular phylogeny for bats illuminates biogeography and the fossil record a phylogenetic supertree of the bats (mammalia: chiroptera) host phylogeny constrains cross-species emergence and establishment of rabies virus in bats trends in national surveillance for rabies among bats in the united states in vivo rna-rna recombination of coronavirus in mouse brain co-existence of different genotypes in the same bat and serological characterization of rousettus bat coronavirus hku9 belonging to a novel betacoronavirus subgroup the authors are grateful to suxiang tong, phd and ying tao, phd of the centers for disease control and prevention, atlanta, georgia, usa, for sharing their primers and protocols for long range bat cov rt-pcr in advance of publication. field assistance in capturing bats was provided by j. bleak, r. choi, l. ellison, a. englert, a. gann, l. gayton, d. neubaum, m. neubaum, b. smart, e. snider, e. tuttle, and e. valdez. we thank c. willis and w. iko for helpful comments on an earlier draft of this manuscript. any use of trade product, or firm names is for descriptive purposes only and does not imply endorsement by the u.s. government. key: cord-295433-olmein3q authors: banerjee, arinjay; kulcsar, kirsten; misra, vikram; frieman, matthew; mossman, karen title: bats and coronaviruses date: 2019-01-09 journal: viruses doi: 10.3390/v11010041 sha: doc_id: 295433 cord_uid: olmein3q bats are speculated to be reservoirs of several emerging viruses including coronaviruses (covs) that cause serious disease in humans and agricultural animals. these include covs that cause severe acute respiratory syndrome (sars), middle east respiratory syndrome (mers), porcine epidemic diarrhea (ped) and severe acute diarrhea syndrome (sads). bats that are naturally infected or experimentally infected do not demonstrate clinical signs of disease. these observations have allowed researchers to speculate that bats are the likely reservoirs or ancestral hosts for several covs. in this review, we follow the cov outbreaks that are speculated to have originated in bats. we review studies that have allowed researchers to identify unique adaptation in bats that may allow them to harbor covs without severe disease. we speculate about future studies that are critical to identify how bats can harbor multiple strains of covs and factors that enable these viruses to “jump” from bats to other mammals. we hope that this review will enable readers to identify gaps in knowledge that currently exist and initiate a dialogue amongst bat researchers to share resources to overcome present limitations. bats are an ancient and diverse group of ecologically important mammals, constituting almost a quarter of all mammalian diversity and inhabiting every continent except antarctica. more than 1300 species of bats belong to the order chiroptera and are further classified into two suborders, yinpterochiroptera and yangochiroptera [1] [2] [3] . the yinpterochiroptera suborder includes the non-echolocating pteropodidae family and the echolocating rhinolophoidea superfamily. yangochiroptera contains the remaining echolocating microbat families. the two suborders diverged over 50 million years ago [4] [5] [6] . in addition to the important role that bats play in preservation of ecological balance, they have also been speculated to harbor a wide variety of viruses. many of the viruses in bats can cause disease in humans and agriculturally important animal species. these viruses include lyssaviruses, filoviruses, henipaviruses and coronaviruses [1, [7] [8] [9] . in this mini-review, we focus on the role of bats as reservoir hosts for important human and animal coronaviruses. we discuss the evidence of coronavirus spillover from bats, how bat ecological niches may contribute to spillover and the need to further explore bat-coronavirus interactions using viruses that have been naturally detected in bats. sars-cov emerged in humans in 2002 and efficient human to human transmission resulted in a global sars epidemic which lasted 8 months [15] . initial studies investigating animal sources of the virus from "wet markets" in the guangdong province of china suggested that himalayan palm civets and raccoon dogs were the most likely hosts responsible for human transmission [22] ; however, the role of bats as the original animal reservoir hosts of sars-cov was speculated as similar viruses were detected in them [27, 28] . years later, during an ecological surveillance of bats in the same region, a sars-like cov that closely matched the human sars-cov was isolated from the chinese horseshoe bat. bat sars-like cov could replicate in hela cells expressing angiotensin-converting enzyme 2 (ace2) receptor from human, civet and bat. the virus replicated in cells derived from human, bat and pig. no civet cells were tested [29] . these data suggest that sars-cov could have spilled over into humans directly from the chinese horseshoe bat while the palm civets in the "wet market" were incidental hosts [30] . however, the exact mechanism by which the zoonotic transmission event to humans occurred is still not clear. retrospective studies have found low levels of seroprevalence of sars-like cov in healthy individuals in hong kong dating back to 2001. interestingly, in 13 of 17 of these seropositive patients, the antibodies responded more strongly against the sars-like-cov isolated from a himalayan palm civet compared to the human sars-cov isolate [31] . these data suggest that low levels of human exposure to zoonotic sars-like covs occurred prior to the sars-cov epidemic that began in 2002, but went unidentified. mers-cov emerged in saudi arabia in 2012 and continues to cause human disease with a case fatality rate of 35% [16] . dromedary camels are a natural reservoir host for mers-cov. in the arabian peninsula and across northern africa, the seroprevalence rate for mers-cov in dromedary camels ranges from 70% to nearly 100% [32] [33] [34] [35] [36] [37] . live mers-cov has been successfully isolated and cultured from camel specimens [38] . approximately 55% of primary mers-cov cases are a result of direct contact with dromedary camels or camel products [39] ; however, the remainder of primary mers-cov cases have no history of contact with camels or infected individuals and thus, where they came into contact with the virus is unknown. a recent study found that 16 out of 30 camel workers surveyed in saudi arabia show evidence of prior mers-cov infection via seroconversion and/or virus-specific cd8+ t cell responses without any history of significant respiratory disease. this study suggests that camel workers with asymptomatic or mild disease may serve as another route of exposure [40] . although camels are thought to be the primary zoonotic reservoir for human transmission, there is strong evidence that bats are the ancestral reservoir host for mers-cov [24, [41] [42] [43] . mers-cov is a group c betacoronavirus and is phylogenetically related to batcovs identified in various bat species that belong to the vespertilionidae family. this includes batcov hku4, batcov hku5, neocov, and pdf-2180 [41, 44, 45] . furthermore, the spike protein from hku4 and mers-cov are highly similar and both use human dipeptidyl-peptidase 4 (dpp4) for virus entry [25, 46, 47] . it is not clear when mers-cov spread from bats to camels, but widespread exposure to the virus in the middle east and north and east africa dates back as early as the 1980s, suggesting that camels have served as a zoonotic reservoir for mers-cov for at least 30 years [33, 34, 37, 40, 48] . in addition to emerging highly pathogenic coronaviruses, human coronaviruses that cause the common cold are also thought to have their origins in bats. hcov-nl63 was first identified in a pediatric patient with bronchiolitis in 2004, but since then it has come to be appreciated that the virus causes approximately 1-9% of the common colds each year and has most likely circulated in humans for centuries with worldwide distribution [49] [50] [51] . a survey of bats in the u.s. found novel alphacoronaviruses, one of which was isolated from the north american tricoloured bat, perimyotis subflavus and was found to be a recent common ancestor of hcov-nl63 with an estimated divergence of~550 years ago [52] . hcov-nl63-like sequences were also identified in bats in africa [53] , further supporting a bat origin for hcov-nl63. although hcov-nl63-like viruses have been identified in bats, these viruses have sequences quite distant from the hcov-nl63 sequences, suggesting a possible intermediate host. hcov-229e also appears to have its origins in bat species. hcov-229e, another cause of the common cold, was first identified in 1967 and has been circulating in the human population for some time [54] . hcov-229e-related viruses have been found in hipposiderid bats during surveillance studies in kenya and ghana [53, 55] . in 2007, a novel alphacoronavirus was identified in an outbreak of respiratory disease in alpacas in the us, which is geographically separated from the bat species that harbor hcov-229e-like viruses in africa [56] . full genome sequencing and phylogenetic analysis of the alpaca cov placed it as an intermediate between the bat hcov-229e-related viruses and hcov-229e from humans [56] . by analyzing more bat, alpaca and human hcov-229e and hcov-229e-related sequences, evidence of genomic changes that occurred between bat and alpaca hcov-229e evolution and subsequently between alpaca and human evolution were identified [57] . interestingly, during tests of dromedary camels for mers-cov, about 6% of the camels studied were positive for hcov-229e [58] . seropositive camels were more prevalent in the arabian peninsula compared to africa and the earliest seropositive sample was from 1997 in a study that looked at samples from 1983 to 2014 [58] . these data all support the notion that hcov-229e has its ancestral origins in bat species while camelids serve as a more recent zoonotic reservoir for human transmission. a recent study has shown that hcov-229e (human strain) is incapable of infecting and replicating in cell lines from multiple bat species [59] . thus it is critical to isolate bat and camel strains of hcov-229e-related viruses to dissect the role of these mammals in the evolution of hcov-229e. porcine epidemic diarrhea (ped) was recognized as an enteric disease in pigs in the united kingdom as early as 1971. pedv was detected in belgium in 1978 [60] . the full-length genomic sequence of the prototype belgian pedv cv777 strain was determined in 2001 [61] . pedv cv777 is more closely related to a scotophilus bat coronavirus (btcov) 512/2005 than to other known alphacoronaviruses, such as transmissible gastroenteritis coronavirus (tgev) and hcov-229e and hcov-nl63, in phylogeny as well as genome organization [21] . this finding suggests that pedv and btcov/512/2005 have a common evolutionary precursor and that cross-species transmission of coronavirus may have occurred between bats and pigs. pedv has since emerged in north america and continues to cause periodic outbreaks that significantly affect producers [18, 62] . multiple pedv vaccine candidates have been shown to provide varying levels of protection in pigs [63, 64] . an effective vaccine may enable control of future pedv outbreaks along with strict biosecurity practices. although pedv propagates in human embryonic kidney cells [65] , no clinical cases of pedv have been reported in humans so far. we (banerjee and misra et al.) have also shown that pedv can infect kidney cells from big brown bats (eptesicus fuscus) [66] . pedv replication in bat cells has not been extensively studied. efforts are focused on designing therapeutics and vaccines to prevent ped in pigs. more recently, a novel hku2-related bat coronavirus, sads-cov has been shown to cause fatal enteric disease in pigs. zhou et al. identified sads-cov as the causative agent for a large-scale outbreak of fatal disease in pigs in china that caused the death of 24,693 piglets across four farms. sads-cov-like viruses with 96-98% similarity to sads-cov were detected in 9.8% of the bats that were sampled in this region [9] . none of the human serum samples that were collected from farm workers were positive for antibodies against sads-cov [9] . thus, sads-cov does not pose a risk for human transmission yet. further studies will be required to confirm the ability of sads-cov to infect and propagate in human cells. understanding how bats maintain a virus within a population is important for predicting spillover transmission events. for many viruses with known or suspected bat reservoirs, spillover transmission events typically occur within a defined time frame and location, which corresponds with higher than normal virus levels in the bat reservoir host. the reason for these "pulses" of virus within the reservoir host population are not clear, but proposed theories and evidence supporting these theories have been reviewed by plowright et al. [67] . in the case of marburg virus (marv), for example, ecological surveillance data shows a clear biannual spike in the prevalence of marv positive bats within the kitaka cave population, which correlates with an increase in the number of juvenile rousettus aegyptiacus bats due to the biannual birthing cycle. this pulse of virus positive bats correlates with an increased incidence of human spillover events [68] . horizontal transmission of marv between r. aegyptiacus bats was confirmed in a controlled experimental setting [69] . furthermore, recent experimental data has shown that bats infected with marv clear infection and maintain long-term immunity. this finding suggests that susceptible naïve juvenile bats are critical for maintaining marv within the population [69] . studies with hendra virus have shown that reproductive and nutritional stress can increase the levels of virus in little red flying foxes (pteropus scapulatus) [70] . the increase in virus replication may enhance the chances of a virus spillover. similar ecological studies need to be undertaken for bats and covs. other stressors, such as secondary infections, may also affect the relationship between bats and their viruses. a recent study by one of our laboratories (misra et al.) suggest that infection of little brown bats (myotis lucifugus) with white-nose syndrome causing fungus (pseudogymnoascus destructans) leads to an increase in replication of a persistently infecting coronavirus in these bats [71] . a recent study by anthony et al. evaluated the global diversity of coronaviruses in almost 20,000 animals and humans. during the course of this study, they found that the diversity of coronaviruses was highly associated with the diversity of bat species and this diversity separated into 3 distinct geographical regions, which mirrored the distribution of different species of bats. the authors report particular associations between bat families and viral sub-clades that suggest co-evolution [72] . a survey of coronaviruses isolated from bats in kenya found a high prevalence of coronaviruses in cardioderma cor, ediolon helvum, epomophorus labiatuc, hipposideros sp., miniopterus minor, otomops martiensseni, rhinolophus hildebrandtii, rhinolophus sp., and triaenops afer. the phylogenetic analysis of these novel covs found a number of cross-species transmission events, although the majority of these events appeared to be transient spillover events [53] . the recombination frequency of coronaviruses, which can be as high as 25% for the entire genome [73] , could lead to bats being an important reservoir for coronavirus recombination and virus evolution, much like birds and pigs are for influenza virus. indeed, there is strong evidence to suggest that a recombination event occurred between hcov-229e-like viruses found in hipposideros bats and hcov-nl63-like viruses found in triaenops afer bats, where the gene encoding for the spike protein is more closely related to the hcov-229e virus [53] . furthermore, the majority of recombination events identified in coronaviruses isolated from bats suggest recombination hotspots around the spike gene [23, 53] . in theory, bats could serve as an important reservoir for coronaviruses and coronaviruses with altered host tropism may very well evolve in bats. although bats are known to harbor a wide variety of coronaviruses, the mechanisms for virus spillover into humans or livestock are widely unknown. there is evidence that there are seasonal fluctuations in virus replication [74, 75] , however, the interconnectedness of virus replication rates and virus spillover have not been explored for bats. typically, coronaviruses found in bats have or require an intermediate host before spilling over into humans, like what is observed with mers-cov and camels. unlike the amount of information available from studies of other bat viruses such as nipah, hendra, ebola, and marburg viruses, we know very little, if anything about how coronaviruses are transmitted directly to humans or if direct human transmission does not occur and spillover via an intermediate host is required. bats are known to harbor a wide range of viruses including many that are highly pathogenic in humans. research to determine the mechanisms by which bats limit disease following virus infection is a relatively new field and can be difficult due to a lack of reagents and the need to develop appropriate in vitro and in vivo systems. even with these limitations, a variety of studies have been performed that evaluate the bat immune response to virus infection at the genomics level, in vitro using cell culture systems, and performing experimental infections in vivo. of note, very few of these studies are focused on coronavirus infections in bats and are rather centered around henipavirus and filovirus infections. future studies evaluating the virus-host interactions of bats and coronaviruses, particularly with bat cov isolates are important in determining why bats serve as important reservoirs for covs and how they control infection to limit severe pathological consequences. multiple studies have elucidated unique adaptations in the antiviral responses of bat cells. the primary bat species being used to study the bat immune response to virus infections in vitro and in vivo are pteropus alecto (black flying fox), rousettus aegyptiacus (egyptian rousette), and artibeus jamaicensis (jamaican fruit bat). papenfuss et al. were the first to sequence the p. alecto transcriptome and identified approximately 500 genes (3.5% of p. alecto transcribed genes) that encode immune-related proteins [76] . a similar number of immune genes were also identified in the transcriptomes of r. aegyptiacus and a. jamaicensis [77, 78] . this included the expression of canonical pattern recognition receptors including toll-like receptors (tlrs) 1-10, retinoic acid-inducible gene i (rig-i), and melanoma differentiation associated protein 5 (mda5) [76, 77] . furthermore, genes for different immune cell subsets, t-cell receptors (tcrs), cytokines and chemokines, and interferon-related genes were detected, while genes encoding for natural killer (nk) cell receptors were largely absent. work has been done to characterize many of these genes in cell lines derived from various bat species including p. alecto [79] [80] [81] . a large amount of interest in bat immune responses has focused specifically on the interferon response. genomic analysis of the interferon loci has shown species-specific evolution in which p. alecto has a contracted type i ifn locus [82] whereas p. vampyrus, m. lucifugus, and r. aegyptiacus have expanded the number of type i ifn genes [83, 84] . it has been observed that there may also be species specific differences in the baseline expression of type i ifns. p. alecto cells constitutively express three different ifnα genes [82] whereas cells generated from r. aegyptiacus do not show constitutive expression of ifnα [84] ; however, baseline expression of interferon alpha/beta receptors, ifnar1 and ifnar2, as well as a variety of interferon-stimulated genes are upregulated in these bat cells compared to human cells [85] . the molecular mechanisms that enable the differential expression pattern of ifns in bats are not known. thus, it is important to acknowledge that different species of bats may have evolved specific strategies to control viruses that they co-evolved with. although it appears that bats have many of the genes that are important for responding to virus infection, how this response compares between human and bat cells is just beginning to be examined. rna sensing and subsequent antiviral responses in bat cells have been studied using viruses known to induce an interferon response, such as sendai virus or newcastle disease virus or by transfecting a synthetic surrogate of viral double stranded rna (poly(i:c)) [80, [86] [87] [88] [89] [90] . these studies show that bat cells respond to rna and induce an antiviral response. many viruses encode proteins that antagonize the host response to infection and dampen the innate antiviral response. it has previously been shown that the v and w proteins of nipah and hendra viruses can inhibit antiviral responses in bat cells, similar to what is observed in human cells [90] . a more recent study showed that marv can inhibit the antiviral response in a r. aegyptiacus bat cell line and that this inhibition is dependent on the viral protein vp35 [85] . coronavirus accessory proteins are dispensable for replication but they play an important role in pathogenesis and virus fitness under the natural environment of a host [13, 91] . multiple studies with pedv, sars-and mers-covs have identified accessory proteins that can effectively inhibit an ifn response in mammalian cells [12] [13] [14] [91] [92] [93] [94] [95] . however, to date, there have been no published studies looking at the role of these accessory proteins in modulating antiviral responses in bat cells. in addition to studying the role of cov proteins in antagonizing the antiviral response in bat cells compared to other mammalian cell lines, it is also important to determine how covs isolated from bats compare to those isolated from humans. coronavirus accessory genes have co-evolved with their natural host for optimum functionality [91] and thus it is important to identify the role of accessory proteins in both their natural and spillover hosts. many of the covs that have been reported in bats, with the exception of few, such as a sars-like cov (bat sl-cov-wiv1) [29] , have been detected by molecular techniques that detect trace amounts of viral nucleic acids. to overcome this limitation, reverse genetics systems using the whole genome sequence from covs isolated from bats could be generated, propagated and evaluated in both bat and human cell lines [96] . this would allow researchers to better understand the role of viral proteins in a species-specific context. the vast majority of studies evaluating the bat host response to virus infection has been performed in cell lines. however, there is a great need to understand what happens during a virus infection in bats in vivo. the ability to study these questions is a daunting task and requires specialized facilities and staff, appropriate species selection especially for covs, and generating the necessary reagents. because of these limitations, only a handful of studies have been performed looking at the in vivo response of bats to virus infection. in fact, there are only two published studies in which experimental infections in bats using covs was performed. the first study was performed in an attempt to rescue a bat cov isolate. watanabe et al. detected covs in 57.1% of insectivorous bats and 55.6% of frugivorous bats; however, they were unable to culture the virus in vitro. to propagate covs detected in a lesser dog-faced fruit bat (cynopterus brachyotis), they administered intestinal samples orally to leschenault rousette bats (rousettus leschenaulti). virus could be detected by quantitative real-time pcr (qpcr) on 2 to 5 days after infection and there was an increase in viral rna while no clinical disease was observed. based on these data, the authors reported that this bat cov replicates in leschenault rousette bats; however, they were not able to isolate live virus [97] . this study emphasizes the importance of bat species selection for studying covs in bats. ideally, we would want to study a bat cov in the same species that it was detected in. the second study aimed to determine if bats could be infected with mers-cov and, if so, what the host response looks like. munster et al. infected ten jamaican fruit bats (artibeus jamaicensis) with mers-cov/emc2012. the authors detected virus shedding in the respiratory and intestinal tracts for 9 days. although the bats showed evidence of virus replication, no overt signs of disease were observed. a moderate and transient induction of the innate immune response was seen, but there were no signs of inflammation. based on their observations, the authors reported that jamaican fruit bats support the replication of mers-cov and thus, bats could be potential ancestral hosts of mers-cov [98] . this study has not been repeated in an insectivorous bat species. although several mers-like viruses have been detected in bats since the study in jamaican fruit bats, none have been successfully isolated [25, 41, 47, 99] . another study focused on looking at species-specific tropism. in this study, the authors focused on the mers-cov receptor dipeptidyl peptidase 4 (dpp4). widagdo et al. mapped the tissue distribution of dpp4 in multiple bat species to identify the differences in tissue tropism of mers-cov. in their study, the authors report that dpp4 in insectivorous bats is primarily detected in the gastro-intestinal (gi) tract and kidneys, whereas frugivorous bats express dpp4 in the respiratory and gi tracts [43] . other studies determined that dpp4 expression in camels is primarily in the upper respiratory tract [100] whereas dpp4 expression in humans is highest in the lower respiratory tract [101] . these data suggest that the tissue tropism in bats may be different than that in other mammalian species and that this may dictate the course of disease and disease severity. the ability of bats to harbor several different coronaviruses may seem like a mystery, but the same is true for rodents. although bats harbor more zoonotic viruses per species, rodents harbor a larger total number of zoonotic viruses [102] . after the sars outbreak, bats have been extensively sampled for coronaviruses and other viruses alike. we may be looking too hard and one may argue that we could find a similar diversity of viruses in other animals if we looked as robustly. metagenomics has enabled us to identify the broad range of viruses in bats and with time, we will expand this to other hosts of zoonotic viruses. for now, we know that bats are major evolutionary reservoirs and ecological drivers of cov diversity [72] . we can leverage this knowledge to design studies that will allow us to identify factors that cause covs to spillover from bats to other hosts. a recent study demonstrated that secondary infection with the white-nose syndrome fungus (pseudogymnoascus destructans) increases cov replication in m. lucifugus [71] . this study opens up a new avenue of investigation in infection dynamics. considering bats harbor multiple viruses, it is necessary to identify the impact these viruses have on each other. how do these viruses modulate the numerous host responses in bats and how does that affect virus replication? several such questions remain and studies are currently delayed due to the inability to isolate bat-covs similar to sars-cov, mers-cov, pedv and sads-cov. ecological and epidemiological studies to identify landscape changes and human practices that 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protein antagonizes beta interferon production by sequestering the interaction between irf3 and tbk1 reverse genetics with a full-length infectious cdna of the middle east respiratory syndrome coronavirus bat coronaviruses and experimental infection of bats, the philippines replication and shedding of mers-cov in jamaican fruit bats (artibeus jamaicensis) rapid detection of mers coronavirus-like viruses in bats: pote1ntial for tracking mers coronavirus transmission and animal origin differential expression of the middle east respiratory syndrome coronavirus receptor in the upper respiratory tracts of humans and dromedary camels dipeptidyl peptidase 4 distribution in the human respiratory tract: implications for the middle east respiratory syndrome a comparison of bats and rodents as reservoirs of zoonotic viruses: are bats special? ecological dynamics of emerging bat virus spillover community intervention to prevent nipah spillover key: cord-333317-oai67igl authors: efremova, agrafena; colleluori, georgia; thomsky, mikhail; perugini, jessica; protasoni, marina; reguzzoni, marcella; faragalli, andrea; carle, flavia; giordano, antonio; cinti, saverio title: biomarkers of browning in cold exposed siberian adults date: 2020-07-22 journal: nutrients doi: 10.3390/nu12082162 sha: doc_id: 333317 cord_uid: oai67igl cold-exposure promotes energy expenditure by inducing brown adipose tissue (bat) thermogenesis, which over time, is also sustained by browning, the appearance, or increase, of brown-like cells into white fat depots. identification of circulating markers reflecting bat activity and browning is crucial to study this phenomenon and its triggers, also holding possible implications for the therapy of obesity and metabolic diseases. using rt-qpcr, we evaluated the peripheral blood mononuclear cells (pbmc) expression profile of regulators of bat activity (cidea, prdm16), white adipocytes browning (hoxc9 and slc27a1), and fatty acid β-oxidation (cpt1a) in 150 siberian healthy miners living at extremely cold temperatures compared to 29 healthy subjects living in thermoneutral conditions. anthropometric parameters, glucose, and lipid profiles were also assessed. the cold-exposed group showed significantly lower weight, bmi, hip circumference, and pbmc expression of cidea, but higher expression of hoxc9 and higher circulating glucose compared to controls. within the cold-exposed group, bmi, total cholesterol, and the atherogenic coefficient were lower in individuals exposed to low temperatures for a longer time. in conclusion, human pbmc expresses the brown adipocytes marker cidea and the browning marker hoxc9, which, varying according to cold-exposure, possibly reflect changes in bat activation and white fat browning. obesity is a multifactorial chronic disease whose prevalence is~20% and~40% in europe [1] and united states [2] , respectively. during the last decades, the worldwide increase in obesity incidence made the need for effective therapeutic strategies extremely urgent, especially in consideration of its comorbidities and associated mortality [1] [2] [3] [4] . obesity is characterized by an aberrantly elevated amount of white adipose tissue (wat) resulting from a dysfunctional regulation of the energy balance [5] . the modulation of energy intake and expenditure is extremely complex and results from the integration of numerous neuroendocrine and environmental signals [6, 7] . cold-exposure is one of the environmental stimuli promoting energy expenditure through the activation of thermogenic pathways, a crucial response to ensure survival to hostile temperature conditions [6, 7] . specifically, cold promotes β-adrenergic stimulation via the sympathetic nervous system (sns), which in turn induces thermogenesis by activating brown adipose tissue (bat) [6] [7] [8] . bat burns fat to produce heat given the expression of ucp1 [6] . interestingly, upon cold stimulation, white adipocytes can transdifferentiate into beige (also known as "brite") adipocytes (brown-like phenotype with elevated expression of ucp1) in a process known as "browning," leading to heat production [9, 10] . importantly, during the phenomenon of browning, proliferation and differentiation of brown adipocyte precursors also occurs, contributing to the expansion of the heat-producing cell population [5, 11] . in mice, bat activation was shown to protect against obesity, type 2 diabetes and atherosclerosis [12, 13] . hence, the study of bat regulation has been particularly attractive as a potential target for obesity treatment [5, 14] . human adults have varying amounts of bat which decreases with advancing age and bmi [15, 16] . the study of bat activation and browning in humans is not simple due to several limitations. the most used procedure available for this purpose is the study of (18)f-fdg (2-deoxy-2-[ 18 f]fluoro-d-glucose) uptake by positron-emission tomography-computed tomography (pet-ct) which, besides different technical limitations, is costly and complex [15, 17, 18] . alternative techniques to assess bat activation and wat browning are needed. studies by palou and colleagues conducted on female rats demonstrated that the expression of regulators of bat activity (cidea, prdm16), wat browning (hoxc9 and slc27a1), and fatty acid β-oxidation (cpt1a) in both tissues, correlates with the expression of the same modulators in the peripheral blood mononuclear cells (pbmc) upon cold stimulation [19] . the authors concluded that these genes could be considered suitable brown/beige markers to be assessed in pbmc, avoiding the use of invasive procedures [19] . however, it is unknown whether brown/beige markers' expression in human pbmc is detectable and whether it varies depending on cold-exposure. we recently showed that cold-exposed siberian adults (living outdoor) display greater β-adrenergic activation and browning of visceral adipose depots compared to individuals living in thermoneutral conditions [20] . the main objective of our study was to evaluate whether pbmc of cold-exposed siberian miners expresses different levels of the brown/beige and fatty acid utilization markers compared to the pbmc of siberian adults living in thermoneutral conditions. our secondary aim was to evaluate differences in the metabolic status between our groups under investigation. this was an observational, cross-sectional study. the study was conducted in 2013 in the verkhoyansky and anabrasky districts of yakutia, following the guidelines in the declaration of helsinki for the ethical treatment of human subjects. the protocol was approved by the local committee review board (supplemental file s1). in this study, we recruited 150 healthy male diamond miners engaged in open-pit diamond mining at anabrasky district of yakutia (polar zone, cold-exposed group) and healthy control subjects living in the town-center of verkhoyansky, district of yakutia (urban area) in thermoneutral conditions. subjects enrolled in the cold-exposed group spent an average time of 8 h per day working in the mine for 3 months (december to february) when the average temperature is −45/−52 • c [21] . cold-exposed individuals lived close to the mining camp in dedicated accommodations in the countryside. at the time of recruitment, the study coordinator conducted a questionnaire-based interview to estimate the amount of time spent in cold conditions based on professional responsibilities. for the cold-exposed group, all blood sampling and anthropometric evaluations were performed in the camp or miners' dwellings. twenty-nine healthy male subjects from the same region were enrolled in the control group during summer (months of august) when the average temperature is +16 • c to +18 • c. similarly, at the time of recruitment, the study coordinator conducted a questionnaire-based interview to make sure that none of the individuals belonging to the control group was exposed to cold during the last three months before enrollment. for this purpose, a questionnaire formulated ad hoc by the research team was used. control subjects were living in the urban area, where the impact of cold during the winter months is minimal due to the presence of heating systems in houses and in means of transportation (+20-25 • c). based on the data collected during the interview, winter months cold exposure for the control group was estimated to be 20-30 min per day maximum and was mainly due to brief walks in open areas. the study coordinator contacted the manager of the miners' company "anabar diamonds" and got permission to access the mining camp for cold-exposed subjects' recruitment. healthy control subjects were recruited through the walk-in clinic of the scientific center for complex medical problem of yakut. subjects were eligible to be enrolled in the control group if they did not suffer from any chronic disease and if they were not taking any medication or undergoing any kind of medical treatment. medical information of study subjects belonging to the cold-exposed group was provided by the medical doctor of the working camp, whose role was to take care of workers' health. individuals with a documented diagnosis of any chronic or metabolic disease such as metabolic syndrome, type 2 diabetes, dyslipidemia or taking any medication that could affect glucose or lipid metabolism were excluded from this study. before enrollment in the study, all participants signed a written consent form. at the time of recruitment, body weight and height were measured by the standard weighing scale and stadiometer, respectively. bmi (kg/m 2 ) was calculated by dividing the weight (in kilograms) by height (in meters) squared. waist circumference (cm) was measured in a standing position midway from the lower edge of the costal arch to the iliac crest of the ilium bone. hip circumference (cm) was measured in the standing position at the level of the greater trochanters of the femurs. blood samples were collected in the morning between 8:00 and 11:00 am from study subjects who were asked to fast overnight. samples collection was performed during february for the cold-exposed group and in august for the control group, after 3 months of exposure to cold and thermoneutral conditions, respectively. after collection, samples were immediately frozen at −60 • c and transported to the laboratory of the yakut scientific center of complex medical problems, yakutsk russian federation. samples collection for the control group was performed at the clinical laboratories of the scientific center for complex medical problem of yakut. control glucose, triglycerides, total cholesterol and high-density lipoprotein (hdl) cholesterol measurements were determined using an automated biochemical analyzer labio 200 (mindray medical international limited, nanshan, shenzhen 518057, china) using biocon kits (biocon, electronic city, 560100 bangalore india). low-density lipoprotein (ldl) and very-low-density lipoprotein (vldl) cholesterols were assessed using the following formulas: ldl = total cholesterol-vldl-hdl; vldl = (tg)/2,2. the atherogenic coefficient ka was calculated using the formula ka = (total cholesterol-hdl)/hdl [22] . the optimal coefficient of atherogenity is considered to be between the value 2 and 3; where values higher than 3, predispose individuals to elevated risk for atherosclerosis and cardiovascular events. whole blood samples were collected into edta coated vacutainers and immediately transported to the laboratory for further processing. pbmcs were isolated by gradient separation using optiprep™ medium (d1556, sigma-aldrich, st. louis, mo, usa), according to manufacturer's instructions, with modifications previously described by paolu and colleagues [19] . briefly, blood was filled up to 6 ml with solution c (146 mm nacl and 1 mm hepes). blood was then layered to form a density barrier by mixing 2.7 ml of optiprep medium with 9.3 ml of optiprep diluent without intermixing (3 ml of density barrier per 2 ml of blood-solution c mixture) in a centrifuge tube. afterward, the tube was centrifuged at 700× g for 20 min at 20 • c with acceleration and deceleration adjusted at zero. the layer containing pbmcs and platelets was collected from the interface between plasma layer and optiprep medium. to wash pbmcs and to remove the platelets, the collected material was centrifuged in solution c at 400× g for 10 min at 20 • c. samples were kept at −70 • c before rna isolation. total rna extraction was performed using the rneasy plus minikit (qiagen, hilden, germany, cat.#74134) following the manufacturer instructions. in brief, 8 ml of human peripheral blood were added with rbc lysis buffer to a final volume of 45 ml and incubated at room temperature for 10 min. cells were pelleted by centrifugation at 600× g for 10 min. the supernatant was removed, and the pellet was resuspended in 1 ml of rbc lysis buffer. afterward, cells were pelleted and subsequently resuspended in 1 ml dpbs and pelleted again by centrifugation. the pellet was resuspended in 1200 µl of trizol and 0.2 ml of chloroform and vortexed for 15 s. samples were centrifuged at 13,000 rpm for 10 min at 4 • c. the upper phase was transferred to clean microcentrifuge tubes and an equal volume of cold isopropanol was added to the mixture. the tubes were inverted several times and placed in a −20 • c freezer for precipitation. samples were centrifuged at 13,000 rpm for 10 min at 4 • c. the supernatant was then carefully removed, and the pellet was rinsed with 0.5 ml of ice-cold ethanol (75%). samples were then centrifuged at 13,000 rpm for 10 min at 4 • c, the supernatant was removed, and samples were let dry at room temperature for 10 min. the rna pellet was dissolved in 20 µl of rnase-free water. rna quality was assessed with an implen p-300 nanophotometer. after quantitation, the rna samples were stored at −80 • c before proceeding with retrotranscription. fifty nanograms of total rna from pbmcs were reverse transcribed into cdna using the iscript cdna synthesis kit (bio-rad laboratories, hercules, ca, usa, cat.#1708891) following the manufacturer's instructions. the reaction was performed using a t-100 thermal cycler (bio-rad), and the conditions were: 25 • c for 5 min, 42 • c for 30 min, and 85 • c for 5 min. gene expression was assessed by rt-qpcr using the sfx96 real-time system. target genes were chosen based on the study by palou et al., who identified markers detectable in rats pbmc [19] . gene functions and selected primers are described in table 1 . each pcr reaction mix included diluted (1:5) cdna template, forward and reverse primers (1µm), sybr green pcr master mix (bio-rad, cat.#1725272) and nuclease-free water to a total volume of 20 µl. pcr reaction conditions were as follows: 15 s at 95 • c, 1 min at 60 • c, and 15 s at 95 • c. gene expression data for each target are expressed as relative quantification (∆∆ct) adjusted for the housekeeping gene gapdh (forward primer gtcggagtcaacggatttggt; reverse primer agtgatggca tggactgt). the normality of the variables was assessed through the kolmogorov-smirnov test. a nonparametric approach was followed due to the small sample size. median and interquartile ranges (iqr) were used to summarize the variables. the wilcoxon sum-rank test was used to evaluate differences between the two groups. in order to investigate differences between groups, the non-parametric ancova with smoothed regression and young and bowman test was applied. markers and biochemical variables were the dependent variables, and age and bmi were the covariates. one model for each dependent variable was performed. the non-parametric ancova was also applied to evaluate the effect of the number of cold-exposure hours on the distribution of each marker and biochemical variables in cold exposed subjects, using bmi as covariate. based on the cold-exposure time distribution, four classes were considered: 1 or 2 h, 4 or 4.5 h, 8 or 10, 11 h. benjamini-hochberg p-value adjustment method was applied. for the biochemical variables, the interaction between classes of cold exposure and bmi was also considered. a total of 179 subjects were enrolled in this study: 29 of them belonged to the control group, while 150 were miners belonging to the cold-exposed group. the distributions of each variable were asymmetric; hence, a non-parametric statistical approach was chosen. all subjects were male with median age equal to 32 years (iqr: 28; 38). among the cold-exposed individuals, 35% were exposed for less than 5 h, with 21% of subjects exposed for less than 2 h; 65% were exposed to cold for more than 5 h, with 55% exposed for 11 h. table 2 shows the characteristics of the enrolled subjects. in brief, no significant differences were found for age, waist circumference (wc), and waist to hip circumferences ratio (w/h) between the two groups. meanwhile, weight, height, bmi, and hip circumference were significantly lower in the cold-exposed group compared to the controls ( table 2 ). the pbmc gene expression study revealed significantly lower cidea and higher hoxc9 expression levels in the cold-exposed group compared to the control group, while no significant differences in other markers' expression were detected (table 3) . after adjustment for age (and with and without adjustment for bmi), circulating total cholesterol, ldl, hdl, vldl, and triglycerides, as well as the atherogenic coefficient were comparable between the two groups in analyses. however, the cold-exposed group had significantly higher circulating glucose levels compared to the control group (table 3) . table 4 shows the result of the analysis conducted on the cold exposed group, and due to the number of markers' missing values, we compared the subjects exposed to cold for less than 11 h and the subjects exposed to cold for 11 h. no clear trend in the adjusted medians and no statistically significant differences in the distribution of markers were observed. this could be attributed to the high variability of markers distribution in each cold-exposure group. a statistically significant difference among cold-exposure groups was observed for bmi, total cholesterol, and the atherogenic coefficient, for which the adjusted medians decreased when the number of cold-exposure hours increased. table 4 . pbmc markers and biochemical variables comparison within the cold exposed group based on time of exposure to cold. ci 95% refers to 95% confidence interval; p refers to non-parametric ancova test via smoothing regression with benjamini-hochberg's p-value adjustment; each model was adjusted for and bmi. ka: atherogenic coefficient. no adjusted median and ci 95% were reported because only 1 subject was in the subgroup. † comparison between group of subjects exposed to cold for less than 11 h and subject exposed to cold for 11 h. this is the first study investigating the expression of browning, beige, and fatty acids utilization regulators in the pbmc of human subjects chronically exposed to extremely cold temperatures compared to controls living in thermoneutral conditions. cold-exposed subjects expressed lower levels of the brown adipocytes' marker cidea and higher levels of the beige adipocytes' marker hoxc9 compared to controls, while the expression of the other investigated genes did not differ significantly between groups. interestingly, cold-exposed individuals from this study had higher circulating glucose, but lower body weight, bmi and hip circumference compared to controls, possibly reflecting a healthier metabolic status. our data proved that certain brown and beige adipocytes markers are detectable in human pbmc and vary according to cold exposure, potentially reflecting changes in bat activation, wat browning, and related metabolic status. the increased energy expenditure associated with cold-induced bat activation and wat browning has attracted enormous interests for its potentials in the treatment of obesity and metabolic diseases [5, 16, 23, 24] . during the last decades, in fact, numerous efforts have been made to identify browning regulators and alternative stimuli responsible for bat activation [5, 14, 16, 23, [25] [26] [27] . however, since only invasive techniques are available for the assessment of browning [17] , the study of this phenomenon in humans presents important limitations. hence, the identification of circulating markers of browning or of browning-induced metabolic changes would be extremely useful. several circulating "batokines" have been discovered [28] , although none of them has been recognized as a valid marker of bat activation. interestingly, paolu et al. demonstrated that cold-induced changes in the expression of few genes regulating browning, beiging, and fatty acid oxidation in rats' bat and wat are reflected by changes in the expression of the same regulators in the pbmc, pointing the attention to new potential analytical candidates [19] . nevertheless, this finding has never been explored and validated in humans. we recently demonstrated that cold-exposed siberian adults (living outdoor) display higher browning of visceral adipose depots compared to individuals living in thermoneutral conditions [20] . the aim of this study was to explore whether siberian subjects belonging to the same population also exhibit a differential pbmc expression of the markers identified by palou and colleagues. cold-exposed subjects enrolled in our study expressed higher levels of the beiging's marker hoxc9 and lower amounts of the brown adipocytes' marker cidea compared to controls. this finding is in some ways consistent with the ones of palou and colleagues whose study was conducted on female rats of different ages (1, 2, 4, and 6 months) exposed to cold for one week [19] . consistently to our results, in fact, cold exposure lead to a significant increase in the pbmc hoxc9 expression of adult rats (4 and 6 months) [19] . this marker is considered to be specific to the beige adipose depots, and its expression is known to increase upon browning stimulation (rosiglitazone administration) [29] . the increase of hoxc9 in the pbmc of our cold-exposed human subjects could thereby reflect the expression changes in their adipose depots, making it a potential circulating candidate to be used as a browning marker. our data are consistent with the findings from rna sequencing studies showing that human bat has a gene expression signature resembling the one of beige adipocytes [30] . on the other hand, palou and colleagues did not detect significant changes in adult rats pbmc expression of cidea exposed to cold, but they only revealed an evident reduction in the mrna levels of the marker in the bat [19] . cidea is widely expressed on the surface of lipid droplets of brown adipocytes and is responsible for the formation of large lipid droplets through the promotion of lipid exchange between them [31, 32] . adipocytes' cidea expression increases in conditions that favor triglycerides deposition [32] , an opposite phenomenon compared to what happens during bat activation. according to some studies, in fact, it antagonizes ucp1 expression [33] . cidea lower expression in the pbmc of our cold-exposed group compared to controls could reflect similar differences in this marker's bat expression, whose levels reduced following exposure to low temperatures in animal models [19, 34] . the evaluation of pbmc expression of cidea has the potential of great clinical relevance in the study of human bat activation that needs further exploration. in our study, we could not detect differences in the pbmc expression of cpt1a4, scl27, and prdm16 comparing cold exposed individuals to controls. this finding is in contrast with the study of palou and colleagues who observed an increased expression in cpt1a4 and scl27 in female adult rats pbmc upon cold exposure [19] . the differences in our findings and the one of palou could be attributed to several elements, for example, the diverse experimental models, gender, age, conditions under investigation, and the variability of these markers in the studied groups. the cold-exposed group of the present study also had lower body weight, bmi, and hip circumference, possibly reflecting a healthier metabolic status [35] . although we did not detect significant differences in cholesterol levels between our two groups, our data revealed lower levels of bmi, total cholesterol, and atherogenic coefficient with increasing daily time of exposure to cold. this finding is consistent with evidence reporting increased lipid utilization and improved lipid profile induced by bat activation produced by cold stimulation [13, [36] [37] [38] . on the other side, other reports revealed a u-shaped relationship between environmental temperature and cardiovascular risk, with the last one increasing for temperatures lower than −1 • c and higher than 20 • c [39] . however, most of these studies analyzed temperatures ranging from~−15 to 30 • c, different from our study, in which cold exposed individuals were exposed to temperatures lower than −30 • c. furthermore, our cold-exposed group had higher fasting glucose levels compared to controls. although this finding may seem counterintuitive, considering that acute bat activation increases glucose uptake [37, 38] , plasma glucose levels do not change upon acute cold-exposure in humans [37, 38] . thus, it is possible that in individuals with normal glycemia, bat chronic stimulation requires higher basal glucose levels for its usage, without leading to metabolic abnormalities (impaired fasting glucose or insulin resistance). importantly, the fasting glucose levels of both of our groups were within the range of normality. our study has several limitations. although we studied a unique population exposed to extremely cold temperature, the cross-sectional nature of our investigation does not allow us to establish a cause-effect relationship between the variables under analysis. furthermore, since we investigated siberian, male adults adapted to live in very cold temperatures, we do not know if our findings can be extended to different ethnicities, gender, or ages. our data need to be validated by additional long-term studies with a larger sample size, evaluating in parallel bat and pbmc gene expression and bat activation through multiple techniques. furthermore, data regarding the metabolic status of this population, such as insulin sensitivity and the existing correlation with the metabolic profile, should be investigated more in-depth. in conclusion, this is the first study demonstrating that human pbmc expresses markers of brown adipocytes and browning and that cidea and hoxc9 mrna levels vary according to cold-exposure. based on our results, we believe that cidea expression in human pbmc could mirror its expression in bat in a condition of chronic activation, while hoxc9 expression could mirror the one of white adipocytes undergoing white-to brown transdifferentiation, making both markers potentially useful circulating indexes of bat and browning activation deserving further investigation and validation. supplementary materials: the following are available online at http://www.mdpi.com/2072-6643/12/8/2162/s1, file s1: protocol. author contributions: conceptualization: a.e., m.t., and s.c.; methodology and investigation: a.e., g.c., m.t., j.p., m.p., m.r., g.c., a.g., and s.c.; data analyses and interpretation: g.c., a.f., f.c., a.g., and s.c.; writing, original draft: g.c., a.g., and s.c.; revising manuscript content and approving final version, all take responsibility for the manuscript content, the integrity of the data analysis and approval of the final version of the manuscript: a.e., g.c., m.t., j.p., m.p., m.r., a.f., f.c., a.g., and s.c.; a.e. and g.c. contributed equally to this manuscript and are considered as co-first authors. all authors have read and agreed to the published version of the manuscript. funding: this work was supported by grants from the italian ministry of university (prin 2017, #2017l8z2em) . the authors declare no conflict of interest. european society of endocrinology clinical practice guideline: endocrine work-up in obesity prevalence of obesity among adults and youth: united states obesity report; world health organization website covid-19 and fat embolism: a hypothesis to explain the severe clinical outcome in people with obesity convertible visceral fat as a therapeutic target to curb obesity anatomy and physiology of the nutritional system brown adipose tissue: function and physiological significance the emergence of cold-induced brown adipocytes in mouse white fat depots is determined predominantly by white to brown adipocyte transdifferentiation bi-directional interconversion of brite and white adipocytes tracking adipogenesis during white adipose tissue development, expansion and regeneration betaar signaling required for diet-induced thermogenesis and obesity resistance brown fat activation reduces hypercholesterolaemia and protects from atherosclerosis development transcriptional and epigenetic control of brown and beige adipose cell fate and function the presence of ucp1 demonstrates that metabolically active adipose tissue in the neck of adult humans truly represents brown adipose tissue identification and importance of brown adipose tissue in adult humans brown adipose tissue development and metabolism cold-activated brown adipose tissue in healthy men gene expression of peripheral blood mononuclear cells is affected by cold exposure a large proportion of mediastinal and perirenal visceral fat of siberian adult people is formed by ucp1 immunoreactive multilocular and paucilocular adipocytes russian federation, jakutsk alteration of plasma lipid profiles and atherogenic indices by stachytarpheta jamaicensis l. (vahl) a prdm16-driven metabolic signal from adipocytes regulates precursor cell fate the changed metabolic world with human brown adipose tissue: therapeutic visions dietary proteins, brown fat, and adiposity recruited brown adipose tissue as an antiobesity agent in humans micrornas in brown and beige fat brown adipose tissue as a secretory organ recruited vs. nonrecruited molecular signatures of brown, "brite," and white adipose tissues genetic and functional characterization of clonally derived adult human brown adipocytes the brown adipocyte protein cidea promotes lipid droplet fusion via a phosphatidic acid-binding amphipathic helix cidea is associated with lipid droplets and insulin sensitivity in humans acute cold exposure-induced down-regulation of cidea, cell death-inducing dna fragmentation factor-alpha-like effector a, in rat interscapular brown adipose tissue by sympathetically activated beta3-adrenoreceptors the fto gene is associated with a paradoxically favorable cardiometabolic risk profile in frail, obese older adults brown adipose tissue activity controls triglyceride clearance brown adipose tissue improves whole-body glucose homeostasis and insulin sensitivity in humans brown adipose tissue oxidative metabolism contributes to energy expenditure during acute cold exposure in humans the nonlinear association between outdoor temperature and cholesterol levels, with modifying effect of individual characteristics and behaviors this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord-305024-343l2ha7 authors: sonntag, michael; mühldorfer, kristin; speck, stephanie; wibbelt, gudrun; kurth, andreas title: new adenovirus in bats, germany date: 2009-12-17 journal: emerg infect dis doi: 10.3201/eid1512.090646 sha: doc_id: 305024 cord_uid: 343l2ha7 we tested 55 deceased vespertilionid bats of 12 species from southern germany for virus infections. a new adenovirus was isolated from tissue samples of 2 pipistrellus pipistrellus bats, which represents the only chiropteran virus isolate found in europe besides lyssavirus (rabies virus). evidence was found for adenovirus transmission between bats. s ince the recent discoveries of ebola virus, henipavirus, and severe acute respiratory syndrome-associated coronavirus infections, interest in the role of bats as hosts for pathogens has markedly increased (1) . with the exception of worldwide studies on bat lyssaviruses (2) , most virologic investigations in bats have been limited to a particular zoonotic agent implicated in a geographically localized disease outbreak (3) (4) (5) . in the remaining studies, various medically less important viruses have been discovered in bats in the americas, africa, asia, and australia (1, 6) . as a result of increasing research efforts regarding bats and infectious diseases in europe, 2 new virus groups were recently detected, namely beta-and gammaherpesviruses in organ tissue (7) and group i coronaviruses in feces of european vespertilionid bats (8) . however, because all bat species in europe/germany are protected by strict regulations, the acquisition of suitable samples for virus isolation is rather challenging in comparison to most other parts of the world. we performed an extensive search for unknown viruses in 55 german vespertilionid bats based on both generic pcr assays and virus isolation techniques, as part of a broader study investigating histopathologic changes in german bats in association with infectious pathogens. dead or moribund bats of 12 species (barbastella barbastellus, eptesicus nilssoni, e. serotinus, myotis daubentonii, m. mystacinus, nyctalus leisleri, n. noctula, pipistrellus kuhli, p. nathusii, p. pipistrellus, plecotus auritus, and vespertilio murinus) were collected at certifi ed bat rehabilitation centers in southern germany and were investigated macroscopically, bacteriologically, and histologically. for virologic examination, homogenized organ tissue was inoculated onto veroe6/7 cells and monitored daily for cytopathic effects. remaining tissue material was used for rna/dna extraction and further molecular analysis by generic pcr assays to detect members of several virus families including fl aviviruses, hantaviruses, coronaviruses, orthomyxoviruses, and paramyxoviruses. the species of bat involved was determined by amplifi cation and sequencing of the cytochrome b (cytb) gene, a standard technique for species identifi cation (9) . of the tested samples from 55 bats, virus was initially detected in only 2 adult common pipistrelles (p. pipistrellus, nos. 198/07 and 199/07). a cytopathic effect was detected in vero e6/7 cells after the second passage, indicating the presence of virus in the cell culture. purifi ed supernatant of these cell cultures was subjected to negative-staining electron microscopy, which showed numerous adenovirus-like particles ( figure 1, . genomic fragments were generated by generic adenovirus-specifi c pcr (10) and a virus discovery based on cdna-amplifi ed fragment length polymorphism pcr method (11) . partial sequence of the dna polymerase gene was generated from longrange pcr product. purifi ed pcr products were directly sequenced by using the bigdye terminator cycle sequencing ready reaction kit (applied biosystems, foster city, ca, usa) and analyzed on an abi 3770 automatic sequencer (applied biosytems). c, clone; p, ≈550-bp nested pcr product. verifi ed by the fi rst reaction of a generic adenovirus-specific nested pcr (10) . the obtained sequence of a fragment of the dna polymerase gene (≈550 bp) indicated that the viruses were a novel virus type within the genus mastadenovirus and was tentatively named bat adenovirus 2 (bat adv-2) strain p. pipistrellus virus 1 (ppv1). to obtain additional sequence information of bat adv-2, a random pcr method (virus discovery based on cdna-amplifi ed fragment length polymorphism) (11) was applied, which showed >20 adenovirus sequences distributed over the genome (figure 1, panel b) . the partial sequence of the bat adv-2 dna polymerase (3,408 bp; genbank accession no. fj983127) was obtained after lon-grange pcr by using the expand long range dntpack (roche, mannheim, germany) according to the manufacturer's directions and the following 2 primers: abs f1-b (5′-aaaagaggcaaagcaagacagtgg-3′) and abs r2-b (5′-ggcgggcaacaaagacctca-3′). after repeated sequence analysis of the partial dna polymerase gene for validation, we found that the identities of bat adv-2 ppv1 to closely related adenoviruses ranged from 68% to 74% on nucleic acid level (table 1) , with the closest relationship found to canine adenoviruses 1 and 2. so far, the only other adenovirus in bats has been accidentally isolated from primary bat kidney cells of a healthy ryukyu fl ying fox (pteropus dasymallus yayeyamae) (12) , which proved to have a rather distant phylogenetic relationship to bat adv-2 ( figure 2) . on the basis of newly acquired sequence information, we designed a specifi c real-time taqman pcr to detect bat adv-2 (abs forward 5′-cacaagtgg tgtctttgagagca-3′, abs reverse 5′-agagggatac aaactgatggaaaca-3′, abs tm 6fam-ctaactt-ggctggtggagtgcgaaac-q). cycler conditions were as follows: predenaturation (95°c for 10 min), 45 amplifi cation cycles (95°c for 30 s, 61°c for 30 s, 72°c for 30 s), and fi nal extension (72°c for 10 min). after screening all 55 bats from germany of 12 species, comprising an additional 11 common pipistrelles, an identical adenovirus was detected in 1 additional common pipistrelle. moreover, the tissue tropism of bat adv-2 was investigated in all 3 infected bats ( table 2 ). of all tested organs, bat adv-2 was detected in high dna copy numbers in the intestine of all 3 bats with lesser dna copy numbers in liver and kidneys, whereas the other organs contained little or no adenovirus dna. unfortunately, due to advanced tissue decomposition in most of the organs, including liver, kidneys, and intestines, thorough histopathologic examination of the 3 bats was markedly impaired. in contrast to maeda et al. (12) , who postulated the necessity of primary bat cells to isolate dna virus from chiroptera, our isolation of a dna virus from an european bat in a permanent cell line (monkey kidney cells) proved the opposite. we believe that the rare detection and isolation of viruses might be attributed to the fast natural degradation of bats of the suborder microchiroptera in comparison to that of other animal carcasses, most likely due to their extremely low weight (2-10 g). although viruses were not detected by various generic pcr assays from homogenized frozen tissue samples, we isolated a novel virus from a hibernating insectivorous bat species. this virus was detected in high dna copy numbers in the intestine of 3 bats that died of natural causes. the fact that no other viral or bacterial agents were detected in these animals suggests a clinical correlation to the isolated adenovirus. moreover, all 3 bats belonged to the same species and were of similar age. several days before their death, they were found moribund and subsequently admitted together to the rehabilitation center, which highlights the strong likelihood of infection in the colony of origin. cross-contamination during tissue preparation can be excluded because sterilized instruments were used for each animal, and after every incision, instruments were cleaned with 70% ethanol and a bunsen burner fl ame to destroy adhering tissue remnants. various adenovirus types of the genus mastdenovirus infect a range of different mammals and cause respiratory, ocular, and gastrointestinal diseases. here, in all 3 infected bats, the highest copy number of adenovirus dna was detected in the intestine, which suggests a correlation with a gastrointestinal disease. the host range of mastadenoviruses is known to be limited to a single (or a few closely related) mammalian species (13) with a probable co-evolution between virus and their hosts (14). the acquired partial sequence of the bat adv-2 dna polymerase with the closest relation to canine adenovirus (only 74% at the nucleic acid level) and the isolation from a new animal host suggests that this virus is a new adenovirus species within the genus mastadenovirus. a comparison to the only other adenovirus found in a bat (fl ying fox, order megachiroptera) with the available sequence information of a ≈550-bp fragment of the dna polymerase gene showed their distant relationship. this strict separation refl ects either the co-evolutionary development between the 2 adenoviruses (bat adv-1 fbv1, bat adv-2 ppv1) and their host families pteropodidae and vespertilionidae or a host switch of the virus originating from a yet-undetermined vertebrate host. to elucidate this problem, further research will be necessary. in conclusion, we isolated a new virus from freeranging vespertilionid bats, which represents the only chiropteran virus isolate besides lyssavirus (rabies) found in europe. moreover, the detection of this chiropteran virus can be connected with its transmission between individual bats living in close proximity to other bats. ecological and behavioral methods for the study of bats european bat lyssavirus type 2 rna in myotis daubentonii isolation of hendra virus from pteropid bats: a natural reservoir of hendra virus isolation of nipah virus from malaysian island fl ying-foxes severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats bats: important reservoir hosts of emerging viruses discovery of herpesviruses in bats detection and prevalence patterns of group i coronaviruses in bats, northern germany species determination: the role and use of the cytochrome b gene detection and analysis of six lizard adenoviruses by consensus primer pcr provides further evidence of a reptilian origin for the atadenoviruses identifi cation of a new human coronavirus isolation of novel adenovirus from fruit bat (pteropus dasymallus yayeyamae) characterization of a new species of adenovirus in falcons adenoviruses: general features we are grateful to bat protectionists for bat collection; jung-won sim-brandenburg, julia tesch, and angelina kus for excellent technical assistance; andreas nitsche for design of the realtime pcr assay for bat adv-2; and ursula erikli and alan curry for their dedicated editorial help.mr sonntag is a master's degree student at the centre for biological safety, robert koch institute, berlin. his primary research interest is the isolation and molecular characterization of human and animal pathogens, with a special focus on emerging viral infections. key: cord-006502-6ajms947 authors: cheng, chak kwong; bakar, hamidah abu; gollasch, maik; huang, yu title: perivascular adipose tissue: the sixth man of the cardiovascular system date: 2018-08-31 journal: cardiovasc drugs ther doi: 10.1007/s10557-018-6820-z sha: doc_id: 6502 cord_uid: 6ajms947 perivascular adipose tissue (pvat) refers to the local aggregate of adipose tissue surrounding the vascular tree, exhibiting phenotypes from white to brown and beige adipocytes. although pvat has long been regarded as simply a structural unit providing mechanical support to vasculature, it is now gaining reputation as an integral endocrine/paracrine component, in addition to the well-established modulator endothelium, in regulating vascular tone. since the discovery of anti-contractile effect of pvat in 1991, the use of multiple rodent models of reduced amounts of pvat has revealed its regulatory role in vascular remodeling and cardiovascular implications, including atherosclerosis. pvat does not only release pvat-derived relaxing factors (pvrfs) to activate multiple subsets of endothelial and vascular smooth muscle potassium channels and anti-inflammatory signals in the vasculature, but it does also provide an interface for neuron-adipocyte interactions in the vascular wall to regulate arterial vascular tone. in this review, we outline our current understanding towards pvat and attempt to provide hints about future studies that can sharpen the therapeutic potential of pvat against cardiovascular diseases and their complications. wingless-related mmtv integration site 5a wt wild-type xe991 4-pyridinylmethyl-9(10h)-anthracenone the vascular bed is a composite system enriched by various types of blood vessels, each specialized to adapt to the metabolic and hemodynamic requirements of a specific organ. most of the blood vessels within the vascular tree are surrounded by a functionally unique and specialized adipose tissue aggregate differing from general fat depots, termed perivascular adipose tissue (pvat) [1] . staying in close proximity to the tunica adventitia of large-, medium-, and smalldiametered arteries, pvat is a crucial paracrine/endocrine organ for cardiometabolic regulation [2] . except the cerebral vasculature, pvat widely distributes around large arteries and veins, small and resistance vessels, skeletal muscle microvessels, and within organs, including kidney in the renal sinus around both renal artery and vein [3] . during tumor expansion, adipose tissue around tumor vasculature plays a pivotal role in secreting angiogenic factors [(as fibroblast growth factor 2 (fgf2) and vascular endothelial growth factor a (vegfa)), adipokines (as adiponectin and leptin), and cytokines (as interleukin-6 (il-6))], all of which contribute to a pro-angiogenic microenvironment [4] , though whether those adipose tissue belongs to pvat remains to be discussed. under the stress of exogenous risk (as smoking and obesity), pvat might act as a negative modulator in the progression of type 2 diabetes and cardiovascular diseases (cvds), including hypertension, atherosclerosis, and coronary heart diseases [5, 6] . in a genome-wide expression investigation of in vitro differentiated adipocytes derived from paired subcutaneous and coronary perivascular adipose tissues, which were isolated from patients, a signature of 307 differentially expressed genes mainly for the regulation of vascular morphology, angiogenesis, blood clotting, and inflammation was identified [7] . depending on the regions in the vascular bed where pvat is located, pvat displays phenotypic and functional heterogeneity [3, 8] . pvat majorly exhibits several defining features of brown adipose tissue (bat), including the presence of multiple lipid droplets, high mitochondrial content, and expression of thermogenic genes (like ucp-1) [9] . however, certain pvat (like mesenteric pvat) has been characterized as white adipose tissue (wat)-like, due to the presence of relatively large lipid droplets, low mitochondrial content, and the relatively low expression levels of uncoupling protein 1 (ucp-1) [10] . in contrast, certain pvat (like coronary pvat) is more beige adipose tissue-like since the expression levels of brown adipocyte-related genes, including cell deathinducing dna fragmentation factor-α-like effector a (cidea), ucp-1, and carnitine palmitoyltransferase 1 b (cpt 1 b), are apparently different from those of typical bat in studies using fat deposits collected from 129sve mice and human [11, 12] . hence, with features that could hardly fit into the predefined fat tissue, it is reasonable to categorize pvat as the fourth type of adipose tissue [9, 13] . pvat plays active paracrine/endocrine roles in regulating arterial vascular tone of enclosed blood vessels and its microenvironment consists of various cell types and molecules ( fig. 1) . above tunica adventitia, adipocytes within pvat (either brown-like, white-like, or beige-like) are responsible for the production of many biologically active molecules, with possible autocrine and/or paracrine effects, modulating vascular function and cardiometabolic complications [13, 14] . in brief, these molecules can be categorized into putative vasoactivators, including both perivascular contraction factors (pvcfs) and perivascular relaxing factors (pvrfs) [15, 16] , adipokines like adiponectin, leptin, and resistin, cytokines such as tumor necrosis factor-α (tnf-α) and interleukins, and certain gaseous molecules like nitric oxide (no) and hydrogen sulfide (h 2 s) [17, 18] . through secretion of cytokines during the pathogenesis of atherosclerosis, pvat was confirmed, in both rodents and human atherosclerotic aortae [19] , to promote the recruitment and infiltration of immune cells, including macrophages and t cells, which in terms produce additional chemokines initiating a feed-forward loop for inflammatory response [19, 20] . furthermore, resident multipotent stem cells within pvat, named mesenchymal stem cells (mscs), show multiple differentiation capacity towards adipocytes, endothelial cells, smooth muscle cells (smcs) and osteoblasts for repair and replacement [21] [22] [23] . for instance, isolated human perivascular stem cells were able to undergo in vitro osteogenic differentiation and form bone in vivo after intramuscular implantation in male severe combined immunodeficiency (scid) mice [24] . pvat also provides an interface for interactions between perivascular adipocytes and autonomic perivascular nerve [25] . in brief, perivascular adipocytes express different receptors, including adrenoceptors (ars), receptors for neuropeptide y (npy), and purinoceptors, sensing neurotransmitters secreted by adjacent sympathetic nerve [25, 26] . recently, studies also provide hints that pvat might be a potential source of micrornas (mirnas) by releasing microvesicles or exosomes, which may either enter circulation or transfer into nearby adipocytes or macrophages for endocrine functions and inflammatory communications [27] [28] [29] . in addition to its well-established roles in providing structural support to vasculature and in thermoregulation of intravascular temperature [30] , pvat plays significant roles in modulating the onset and progression of many cvds, including atherosclerosis and hypertensive heart diseases [5, 31] . various animal models, either after pretreatment protocol (table 1) , with modified pvat content ( table 2) , or of human disease background (table 3) , have been used to emphasize the importance of pvat in cardiovascular events. in this session, we attempt to summarize the major animal models in the context of vascular tone regulation, atherosclerosis, vascular remodeling, angiogenesis, and hypertensive disorders. animals and/or their tissues might undergo acute or chronic procedure prior to experiments on vascular reactivity assay. since the first-time discovery of anti-contractile effect of pvat against norepinephrine on isolated rat aorta by soltis and cassis in 1991 [32] , abundant acute ex vivo experiments investigating the effect of pvat on vascular function have been carried out, using isolated blood vessels from rat, mouse, swine, rabbit, and human [32] [33] [34] [35] [36] [37] . depending on the nature of the study, the interaction between pvat and different regions of vascular bed, like aorta, mesenteric artery, and coronary artery, were investigated [33, 38] . for instance, mouse small mesenteric arteries, together with pvat, were isolated and mounted on a wire myograph to study how adiponectin activates large conductance calcium-activated potassium (bk ca ) channels or other pathways to induce anti-contractile responses ex vivo [39, 40] . pvat surrounding vasculature consists of many types of cells, dominated by perivascular adipocytes (either brown-like or white-like depending on the region of vascular bed). pvat around atherosclerotic aortae recruits immune cells, such as macrophages and t cells, responsible for the production of proinflammatory cytokines during atherogenesis. resident stem cells within pvat show multipotency towards adipocytes, endothelial cells, and smooth muscle cells. pvat also provides an interface for neuron-adipocyte interactions and releases microvesicles and/or exosomes bearing mirnas to either enter circulation or modulate adipose tissue inflammation in addition, chronic diet-induced obese animal model is generally used to provide pathophysiological mimetics to study the vasoregulatory roles of pvat. following the feeding of high-fat diet (hfd), normally lasting for 3 to 4 months, the mouse of diet-induced obesity (dio) is sacrificed to evaluate the impact of pvat on vascular function in the state of obesity [41, 42] . using the c57bl/6j mouse model of dio, li and his group showed the role of endothelial nitric oxide synthase (enos) uncoupling in pvat underlying obesity-related vascular dysfunction [43] . recently, the significance of exercise is increasingly recognized, so research groups started to use chronic exercise animal model in their studies. after male wistar rats were trained on a treadmill designated for small animals with individual lanes for about 8 weeks, the thoracic aortae with pvat were isolated for vascular function assay [44, 45] . however, routine exercise results in broad effects in metabolic profile of trained animals that it may not be easy to draw a direct link between pvat and the improved vascular function. transplantation is a common procedure in exploring the function of pvat in atherogenesis and vascular remodeling. normally, researchers might transplant pvat from an artery to another or replace pvat with viscera and subcutaneous adipose tissues [46, 47] . takaoka et al. demonstrated that transplantation of subcutaneous fat from mice fed on normal chow could attenuate the enhanced neointima formation due to the removal of periadventitial adipose tissue in c57bl/6 mice fed with high-fat high-sucrose (hf/hs) diet [47] . another study transplanted thoracic pvat from donor mice to carotid arteries of recipient apoe −/− mice to indicate the therapeutic potential of pvat in promoting atherosclerotic plaque vulnerability [48] . in addition to the non-genomic experimental models, several transgenic rodent models with reduced or absent pvat have been developed. the a-zip/f mouse is one of the currently • isolated arteries from rat, mouse, swine, rabbit, or human • pvat around aorta, mesenteric artery or coronary artery, etc. • only acute experimental clues [32] [33] [34] [35] [36] [37] [38] [39] [40] diet-induced obesity (dio) • mice subjected to a high-fat diet, normally for 3 to 4 months (≥ 8 weeks) • [46] [47] [48] available models, in the absence of wat, and with dramatically reduced pvat and bat during lifetime, resulting in complex pathological phenotypes including hypertension and diabetes mellitus [49] . in brief, the a-zip/f mouse expresses the dominant negative protein a-zip/f, under the control of an adipose-specific acid-binding protein (ap2) promoter [13, 49] . although a-zip/f mouse exhibits altered vascular contractile activity, ex vivo co-incubation of its aorta with pvat from wild-type (wt) mouse could not restore the impaired vascular function [50] . furthermore, takemori et al. showed that the expression level of angiotensin ii (ang ii) type 1 receptors is higher than that of ang ii type 2 receptors [50] . these observations imply that the absence of pvat may not be the cause of dysfunctional aortae, suggesting that a-zip/f mouse might not be suitable for understanding the interrelationship between pvat and vascular tone regulation. several other mouse models with ablation of adipose tissue might be potentially suitable for pvat study, including those of either inborn or inducible adipose deletion [51] . generated in 1993, the ucp-i-dt transgenic mouse is considered one of the classical rodent models with complete ablation of bat, with physiological phenotypes of hyperphagia, diabetes mellitus, and hypertriglyceridemia [52] . however, it was later reported that ucp-i-dt model still retains a significant number of brown adipocytes, expressing approximately 50% of ucp1 mrna in the brown fat [53] . another potential transgenic model for pvat study is named transgenic skinny, characterized with complete ablation of adipose tissue and increased glucose metabolism [54] . however, this model is also accompanied by overexpression of leptin [54] , meaning that it might complicate the study involving vascular tone and hypertension, in which leptin plays active regulatory roles [55] . in 2005, a relatively new mouse model named fat apoptosis through targeted activation of caspase 8 (fat-attac) was developed, through targeted utilizing of a caspase 8-fkbpv (phe36val mutant fk506 binding protein (fkbp)) fusion protein under the control of adipocytespecific fabp4 promoter [56] . in short, through activation of caspase 8 to induce fat apoptosis, this mouse exhibits phenotypes close to a-zip/f mouse showing reduced wat and bat content, reduced systemic inflammation, and glucose intolerance [56] . despite the lowered fat content in these animals, there are still lacking reports on their pvat status. in 2012, chang and his colleagues developed a murine smooth muscle cell-selective pparγ knockout (smpg ko) model that is deficient in pparγ in vascular smooth muscle cells (vsmcs), showing the presence of bat and wat depots but not pvat [30] . however, smpg ko mice were hypotensive during the resting period of circadian cycle [30, 57] , which might not be suitable for examining the effect of pvat on blood pressure regulation. rodents with susceptibility to human diseases remain the species of choice to address the important roles of pvat in the progression of atherosclerosis, hypertension, and vascular remodeling. both apolipoprotein e-knockout (apoe −/− ) mouse and low-density lipoprotein (ldl) receptor-deficient mouse (ldlr -/-) with significant higher ldl levels, are the standard models used in atherosclerosis research. these mice develop robust lesions to form atherosclerotic plaques on the western or atherogenic diet [58] . hence, many studies examined the role of pvat using apoe −/− mice. for example, transplantation of perivascular visceral adipose tissue from apoe -/donors to the right common carotid artery, a site that typically does not develop spontaneous atherosclerosis, of apoe −/− recipients resulted in a more advanced atherosclerotic lesion with the evidence of atherothrombosis and endothelial dysfunction [59] . another study reported that periaortic adipose tissue (pat) activated the renin-angiotensin system (ras) to accelerate chronic kidney disease-associated atherogenesis [60] . in addition, another study unraveled that pvat contributes to aneurysm formation in an angiotensin ii type 1a (at 1a ) receptor-dependent manner [61] . few studies used ldlr −/− mouse, another well-recognized atherosclerotic model but with much lower total levels of plasma cholesterol than apoe −/− mice under low fat chow diet [62] . however, the studies are not directly related to atherosclerosis that a recent report focused on how pat-derived il-6 contributes to arterial stiffness in ldlr −/− mice [63] . a most recent study shows the protective benefit of pat in endothelial dysfunction and insulin-induced vasodilation of ldlr −/− mice [64] . working on hypertensive rodent models provides useful clues about the contribution of pvat to hypertension-related complications. earlier studies examined the influence of hypertension on the anti-contractile effect of pvat. lu et al. suggested that changes in adipocyte composition of pvat, but not its mass, might exacerbate vascular dysfunction in a spontaneously hypertensive rats (shr) [65] . these studies were paralleled by studies using new zealand obese (nzo) mice that exhibit pvat malfunction on arterial tone regulation despite excess visceral adipose mass. the anti-contractile effects of pvat were diminished in mesenteric arteries of nzo mice with age. voltage-gated potassium channel subfamily q (kcnq) channel openers reduced arterial blood pressure in both models of hypertension independent of ganglionic blockade. thus, our data suggest that kcnq channels play a pivotal role in periadventitial vasoregulation of peripheral skeletal muscle and visceral arteries, and kcnq channel opening may be an effective mechanism to improve impaired pvat vasoregulation and associated hypertension [17, 66] . we also found that bk ca channels and/or k v 1.5 channels in vsmcs are not the downstream mediators involved in the inhibitory effects of 4-pyridinylmethyl-9(10h)-anthracenone (xe991) (a kcnq channel blocker) on pvat-dependent arterial relaxation [67] . kcnq1 channels are apparently not involved in the control of arterial tone by α1-adrenoceptor agonists and pvat [68] . based on preclinical and clinical data, kcnq channels (in particular kcnq2-5 channels (k v 7.2-5)) in vsmcs can now be viewed as potential novel drug targets for treatment of resistant hypertension, particularly in comorbid conditions such as obesity and metabolic syndrome [69] . taken together, the available data allowed us to propose that dysfunctional pvat contributes to cardiovascular risks [11, 17] . accumulating evidence suggests that pvat is a significant autocrine/endocrine/paracrine organ surrounding the vasculature. by secreting a wide spectrum of candidate molecules, including perivascular factors (like pvrfs and pvcfs), adipokines (like adipokines and angiotensins), inflammatory cytokines, and possibly mirnas, pvat plays a pivotal role in regulating vascular tone, vascular inflammation, and vascular remodeling through targeting different cell types in vasculature and even in the pvat microenvironment including perivascular adipocyte itself (fig. 2 ). both pvat and endothelium are now considered important paracrine tissues regulating the contractile state and homeostasis of underlying smcs through the production of various substances [17] . pvat adipocytes release bioactive signaling molecules, majorly categorized into pvrfs and pvcfs, to confer vasoregulatory effect [11] . certain simple molecules (e.g., no, h 2 s), reactive oxygen species (e.g., hydrogen peroxide (h 2 o 2 )), and adipokines (e.g., adiponectin, angiotensin 1-7 (ang1-7)) are notable pvrfs [6, 17] , contributing to the anti-contractile effect of pvat on vascular bed. in 1987, palmer et al. remarkably discovered that endothelial cells produce no, the critical endothelium-derived relaxing factor for proper vascular function [70] . similar to endothelium, pvat was shown to express enos to generate no from l-arginine for the regulation of vascular tone [71] , through no-mediated upregulation of cyclic guanosine monophosphate (cgmp) levels and subsequent activation of protein kinase g [72, 73] . importantly, knockout of protein kinase g accounted for the loss of anti-contractile effect of pvat on the mouse mesenteric arteries [73] . among pvrfs, adipocyte-derived relaxing factor (adrf) is a transferable factor released by pvat, through a ca 2+ -dependent mechanism, to induce vasodilation of surrounded blood vessels [74] ; nevertheless, the nature of adrf has been long debated [17] . the volatile gaseous mediator h 2 s, which mediates the anti-contractile effect by opening kcnq channels in vsmcs, was once suggested to be a primary candidate of adrf [75] . in the cardiovascular system, h 2 s is extensively synthesized by cystathionine gamma lyase (cse) encoded by cth gene in both endothelial cells and perivascular adipocytes [76, 77] . presence of cse inhibitors d lpropargylglycine (ppg) and β-cyano-l-alanine (bca) significantly attenuated the anti-contractile effect of pvat in rat aortae ex vivo [78] . however, gollasch et al. recently showed that cse-derived h 2 s is unlikely to be the universal adrf among different species since cse inhibition by ppg and bca only reduced the anti-contractile effect in rat but not in mouse aortae [79] . lack of cse also did not influence the anti-contractile effect of pvat in mice [80] [71, 72] . earlier than the discovery of endothelium-derived no, the lipid compound prostaglandin (pg) was identified in 1977 to be one of the first critical modulators of vascular function in both physiological and pathophysiological states [84] . synthesized by two cyclooxygenase (cox) enzymes (cox1 and cox2) from arachidonic acid, the five primary pgs bind to specific pg receptors to either induce vasodilation [prostaglandin d2 (pgd 2 ); prostaglandin i2 (pgi 2 )] or vasoconstriction [prostaglandin f2α (pgf 2α ); thromboxane a2 (txa 2 )] or both [prostaglandin e2 (pge 2 )] [85] . similar to that in endothelium, expression of both cox1 and cox2 in pvat bestows the ability to generate pgs, especially the vasodilatory pgi 2 [86] . through binding to the prostacyclin receptor (ip) on vsmc, endothelium-derived pgi 2 stimulates g s subunit to activate adenylate cyclase for higher cyclic adenosine monophosphate (camp) production [87] . subsequently, the adenosine triphosphate (atp)-sensitive potassium channels (k atp ) and delayed-rectifier potassium channels (k dr ) of vsmc sense the increase in intracellular camp levels, resulting in their opening/activation, responsible for prostacyclin-mediated vasodilation [88] . however, although it was shown that pgi 2 from pvat improves hfd-impaired endothelial function of apoe -/mouse carotid artery [30] , whether pvat-derived pgi 2 follows the same mechanistic aspect as endothelium-derived pgi 2 remains is yet to be determined. in contrast, pvat releases another lipophilic and heat-stable factor named methyl palmitate or palmitic acid methyl ester (pame), which is associated with a more welldefined anti-contractile mechanism [89] . pvat-derived pame opens the voltage-gated potassium (k v ) channels on vsmcs to promote vasorelaxation [33] . in shr, both anticontractile effect of pvat and secretion of pame were found to be diminished, indicating a possible role of pame in the pathophysiology of hypertension [33] . more recent studies demonstrate that pame could contribute to pvat-related relaxations by activating kcnq (kv7) channels in rat aortae but not in human mesenteric arteries [90] . the ras of pvat gives rise to the production of the heptapeptide ang1-7, which serves as a vasodilator [91] . particularly in the aortae and mesenteric arteries of humans and rodents, pvat expresses the main ras components, including angiotensinogen, angiotensin-converting enzyme (ace), ace2, renin, (pro)renin receptor, aldosterone, angiotensin ii type 1 (at1), and angiotensin ii type 2 (at2) [10, 92] . the ras components of pvat enable the conversion of ang ii to ang1-7 via ace2 [10] , the relaxing factor that induces no release through activating enos in endothelial cells [93] . therefore, inbalanced release of ang ii and ang1-7 from pvat is closely related to the pathophysiology of vascular dysfunction and hypertension [94] . apart from pvrfs, pvat also produces pvcfs to modulate vasoconstriction. imbalance between pvrfs and pvcfs deteriorates vascular function, which is often observed in pathological conditions such as obesity and hypertension [95] . expressed by pvat, the pvcf chemerin executes the chemerin-axis by docking to the primary chemerin receptor 23 ( c h e m r 2 3 ) o n v s m c s , l e a d i n g t o a u g m e n t e d fig. 2 pvat candidates. perivascular adipocytes secrete a constellation of candidate molecules for autocrine/ paracrine/endocrine regulation of various cellular (e.g., adipocyte formation, browning of wat-like adipocytes), physiological (e.g., vascular tone regulation, lipid metabolism), immune (e.g., monocyte/macrophage infiltration, inflammation) and pathological events (e.g., vascular dysfunction, hypertension, atherosclerosis and coronary heart disease). perivascular adipocytes might be a potential source of cir-mirnas for long-distance endocrine regulation vasocontraction, particularly in obesity [96] . in addition, norepinephrine (ne) released by pvat also accounts for the enhanced vasoconstriction [17] . in 2014, ayala-lopez et al. showed that the non-sympathetic components within pvat microenvironment might supply catecholamines in a tyramine-sensitive manner, causing arterial contraction [97] . later in 2015, the same group provided additional experimental evidence to suggest that pvat adipocyte expresses organic cation transporter 3 (oct3) to serve as the prime candidate transporter, responsible for the uptake of ne into pvat [97] . in other words, the local stores of ne (as catecholamine) in pvat adipocyte contribute to the arterial contraction [97] . owen et al., through comparative proteomic analyses on coronary pvat between lean and obese swine, uncovered that increased contractile effects of obese coronary pvat are associated with the alterations in the pvat proteome, particularly the protein calpastatin encoded by the calpastatin (cast) gene [98] . in the same study, ex vivo verification shows that coronary adipose-derived calpastatin dosedependently increases contractions of swine coronary arteries in the presence of pvat [98] , providing hints that calpastatin might be a novel pvcf. moreover, pvat adipocytes also generate ros, specifically superoxide anion (o 2 − ), by nicotinamide adenine dinucleotide phosphate (nad(p)h) oxidase similar to endothelial cells [99] . through the activation of tyrosine kinase and mitogen-activated protein kinase (mapk)/extracellular signal-regulated kinase (erk) pathway, o 2 − was found to be the augmenting factor of arterial contractile response in rat superior mesenteric artery upon electric field stimulation (efs) [100] . notably, pvat secretes another critical subtype of vasoregulatory candidates, namely adipokines including adiponectin, leptin, tnf-α, and il-6. adiponectin is an abundant adipocyte-derived plasma protein which is reported to elicit vasoprotective benefits influencing the function of nearly all cell types within the vessel wall [101] . basically, adiponectin exerts protective effects on endothelium by activating the amp-activated protein kinase (ampk)-enos pathway to elevate no bioavailability and by improving the redox state in vessel through inhibiting enos uncoupling, a state when enos generates o 2 − instead of no under oxidative stress [37, 102] . these findings are consistent with several longitudinal studies that high plasma adiponectin levels are associated with a lower risk of cardiovascular events, including acute myocardial infarction and acute coronary syndrome [11] . leptin is another crucial adipokine mainly produced by adipocytes proportionally to body fat mass [103] . leptin elucidates vasoregulatory effect through both endothelialdependent and endothelial-independent manners [104] . in short, leptin can directly stimulate the production of endothelial no via the ampk/protein kinase b (akt)/enos signaling cascade to cause peripheral arterial relaxation [105] and can indirectly trigger vasoconstriction via the central activation of sympathetic nervous system [106] . leptin resistance during obesity and hyperleptinemia are correlated with hypertension and coronary artery disease [11, 107] . the pro-inflammatory cytokines, such as tnf-α and il-6, are also bioactive molecules related to vasoregulation. depending on the pathophysiological conditions, the dualacting tnf-α can be either a vasodilator or vasoconstrictor [17] . normally, tnf-α-mediated vasorelaxation involves no and prostaglandin production [108, 109] . however, under conditions like lack of regular exercise, overnutrition, aging, and smoking, tnf-α production is significantly upregulated , which eventually reduces no bioavailability by inhibiting enos activity and aggravating superoxide generation by nad(p)h oxidase [110] . moreover, via the inhibition on cyt o c h r o m e p 4 5 0 ( c y p 4 5 0 ) , t n f -α d i m i n i s h e s epoxyeicosatrienoic acid (eet), one of the endotheliumdependent hyperpolarizing factors (edhf) [111] . as a consequence, the vsmcs fail to hyperpolarize, resulting in diminished relaxation [112] . meanwhile, pvat-derived il-6 binds to corresponding il-6 receptor (il-6r) to cause endothelial dysfunction by reducing bioavailable no [113] . in opposite, il-6 can also upregulate tnf-α expression in endothelial cells, but not vsmcs, to restrain no production [114] . adipocytokines, referred to the constellation of both adipokines and inflammatory cytokines, are important candidates for maintaining the metabolic homeostasis of healthy subjects. deficiencies in these factors, either owing to adipocyte dysfunction or excess adiposity, are intimately correlated with the pathogenesis of multiple diseases related to obesity, especially cvds [115] . pvat secretes an array of a d i p o c y t o k i n e s , i n c l u d i n g a d i p o n e c t i n , l e p t i n , adrenomedullin, vascular endothelial growth factor (vegf), angiotensinogen, plasminogen activator inhibitor-1 (pai-1), resistin, omentin, apelin, adipsin, agouti, acylation stimulation protein (asp), insulin-like growth factor-1 (igf-1), secreted frizzled-related protein 5 (sfrp5), tnf-α, and various interleukins (e.g., interleukin 1 (il-1), il-6) [17, 115, 116] . not only these factors play an autocrine role in the metabolism of pvat adipocyte itself, they also carry out paracrine/endocrine function to regulate other cellular processes. in addition to the beneficial effect on vascular tone, adiponectin exerts many other vasculoprotective and angiogenic properties. in terms of ischemia, adiponectin improves angiogenic repair in adiponectin-knockout (ko) mice by activating ampk signaling in ischemic adductor muscle [117] . in addition, another study using adiponectin-ko mice showed that adiponectin suppresses the neointimal thickening after artery injury [118] . furthermore, adiponectin performs antiinflammatory action by inhibiting the production of cysteine-x-cysteine (cxc) receptor 3 chemokine ligands in macrophages and by reducing the recruitment of t lymphocytes in atheromata using apoe/adiponectin double-deficient (apoe −/− apn −/− ) mice [119] . similarly, leptin also contributes to other aspects of cardiovascular system. structurally similar to the helical cytokine family, leptin acts upon different immune cells, including monocytes/macrophages, t cells, and neutrophils to induce the release of inflammatory cytokines like tnf-α and il-6, promoting vascular inflammation [120, 121] . another study uncovered that via erk1/2 and nuclear factor kappa-light-chain-enhancer of activated b cell (nf-κb) pathway, leptin can expedite vsmc proliferation, which aggravates arterial intimal thickening and vascular remodeling [122] . furthermore, pvat also expresses the angiogenic factor vegf, which promotes the vasculogenesis and angiogenesis of endothelium, hematopoiesis of stem cells, and migration of monocytes/macrophages [123] . vegf was shown to induce bat-like phenotype in wat in vivo [124] , which accounts for the bat-like properties of pvat in an autocrine/ paracrine manner. these experimental evidences shed light on the importance of pvat, which acts as an important source of adiponectin, leptin, and vegf apart from bat and wat. the multiple pvat-derived adipokines are crucial in maintaining the homeostasis of cardiovascular system by endocrinal regulation of different cell types. for instance, resistin promotes smc proliferation and upregulates mrna abundance of fatty acid-binding protein in endothelial cells to affect blood pressure [125] . meanwhile, visfatin is considered one of the important vsmc growth factors via nicotinamide mononucleotide (nmn)-mediated erk1/2 and p38 signaling [126] , whereas omentin suppresses neointimal formation after arterial injury, myocyte apoptosis, and vsmc proliferation via ampk-dependent signaling [127] . apelin is crucial in modulating cardiac development, vasomotor tone, and angiogenesis [128] , and igf-1 exerts cardioprotective effects by inhibiting oxidative stress and apoptosis of cardiomyocytes [129] . pvat-derived angiotensinogen is the critical raw material for ras to synthesize ang ii, which induces gene reprogramming or necrosis in cardiac myocytes, and upregulation of fibrosis-associated genes in cardiac fibroblasts during cardiac dysfunction [130] . during obesity, high circulating level of adipose tissue-derived pai-1 potentially stabilizes the fibrin matrix by suppressing plasminogen-mediated fibrinolysis to contribute to atherosclerotic plaque development and/or to promote the uptake of low-density lipoprotein (ldl) into lesion [131] . in healthy subjects, pvat derives numerous antiinflammatory adipocytokines, such as adiponectin, sfrp5, and adrenomedullin [11, 132] . adiponectin, through binding to the cooh terminal of adiponectin receptor (adipor) of monocytes/macrophages, activates the ampk phosphorylation to suppress i kappa b kinase (ikk)-nf-κb pathway, which hinders pro-inflammatory cytokine production to reduce the risk of acute myocardial infarction and atherosclerosis [133] . sfrp5 exerts an antagonistic effect against wingless-related mmtv integration site 5a (wnt5a) signaling, in which wnt5a exerts pro-inflammatory effects on endothelial cells and pro-atherogenic effect in aortic regions [120, 134] . meanwhile, the bioactive peptide adrenomedullin was shown to combat inflammation by inhibiting the release of pro-inflammatory cytokines (il-6, tnf-α, and interleukin 1β (il-1β)) from lipopolysaccharide (lps)-activated macrophages [132, 135] . in contrast, dysfunctional pvat derives multiple pro-inflammatory adipocytokines, including monocyte chemotactic protein-1 (mcp-1), leptin, tnf-α, il-1β, and il-6 [136] . during obesity, increased fat mass is associated with the alterations in cellular composition, metabolome, and dysregulated adipocytokine secretome, characterized by dysfunctional adipose tissue [137] . in obesity, dysregulated adipocytes including perivascular adipocytes express classical macrophage features, characterized by a secretome of elevated pro-inflammatory and reduced anti-inflammatory adipocytokine contents (e.g., adiponectin, sfrp5) [136, 138] . besides, these pro-inflammatory adipocytokines are responsible for prompting a chronic low-grade inflammation state, endothelial cell proliferation, microvascular remodeling, recruitment of macrophages (cd68+ cells) and t cells to atherosclerotic lesions, and polarization of macrophages towards m2 phenotype during atherosclerotic progression [11, 139] . the small non-coding mirnas are important guide molecules in rna silencing of gene expression [140] . mirnas are crucial in regulating adipogenesis (formation of adipocytes), metabolic homeostasis, and endocrine functions of adipocytes [141] . many mirnas have been identified to be differentially regulated during adipogenesis in human and murine, including let-7c, mir-143, mir-210, mir-221, mir-27, and mir-30a-e [142] . notably, obesity alters the expression profile of mirnas in adipose tissue, influencing the progression of inflammation in human adipose tissue [141, 142] . for instance, the expressions of mir-26b, mir-132, and mir-155 are associated with the number of macrophages infiltrating the fat depot [143] . during obesity, the expression of mir-132 is down-regulated and its expression level is related to the activation of nf-κb signaling and transcription of mcp-1 and interleukin 8 (il-8) [141, 144] . more importantly, in addition to the adipogenesis and modulation of inflammation state of adipocytes themselves, different studies reported that adipose tissue is capable of secreting circulating mirnas (cir-mirnas) to the bloodstream for endocrine functions. the cir-mirnas might be trapped within microvesicles or exosomes [27, 29] . collectively termed as extracellular vesicles, microvesicles (around 100 nm to 1 μm in diameter) and exosomes (around 40-100 nm in diameter) are of different sizes and origins [145] . in 2017, thomou et al. reported that mice with adipose tissue-specific knockout of mirna-processing enzyme dicer (adicerko) display a significant decrease of circulating exosomal mirnas and stated that cir-mirnas should be defined as a previously undescribed form of adipokine [29] . earlier in 2010, ogawa et al. provided in vitro data that 3t3-l1 adipocyte-derived microvesicles contain 143 mirnas in which most of them are adipocyte-specific and that these mirna-containing microvesicles could be transported into cultured macrophages [146] . in the same study, mirnacontaining microvesicles were identified to be present in rat serum in vivo [146] . with close proximity to the circulatory system, it is therefore reasonable to hypothesize that pvat might be one of the closest sources of adipose tissue-derived cir-mirnas, encapsulated in either exosomes or microvesicles, in the endocrine regulation of other tissues. moreover, thomou et al. also reported that dicer-knockout not only modulates fundamental functions of adipocytes like whitening of bat, but also influences other tissues altering circulatory lipids, glucose uptake, and insulin resistance [29, 147] . these studies might shed light on the therapeutic potential of targeting cir-mirnas in cardiovascular complications concerning abnormal energy intake and lipid metabolism. little is known about the effects of pvat on nervous systems controlling blood vessels. a recent review in 2014, by bulloch and daly, highlighted our poor understanding of the link between pvat and the autonomic nervous system, or other nerves, that are involved in controlling the vasculature [25] . in fact, the innervation of pvat was first demonstrated in 1970, when diculescu and stoica produced a set of falk fluorescence plates demonstrating a clear innervation of perivascular fat in the rat mesenteric artery [148] . however, it is only recently that the interest to investigate neurotransmission in pvat has been revived. blood vessels, especially resistant vessels, receive multiple perivascular innervations from autonomic system (sympathetic nerves) and non-adrenergic non-cholinergic (nanc) neurotransmissions, including sensory nerves, peptidergic innervations [calcitonin gene-related peptide (cgrp) and substance p], and nitrergic innervation. in addition, cotransmission concept that includes purinergic signaling and npy also mediates vascular tone. purinergic signaling arises from both sympathetic and sensory nerves while npy is the second main neurotransmitter co-released from sympathetic nerve terminals [149] . due to limited availability of research articles on neuronal expression and activity within pvat, the present article can only review sympathetic and sensory neurotransmissions in pvat. both sympathetic and sensory nerves are well-known as the key players in vascular regulation and interact reciprocally [149] . for instance, activation of sensory nerves reduced adrenergic neurotransmission via pre-junctional inhibition and treatment with cgrp or sp decreased the amplitude of neurogenic-evoked vasoconstriction [150, 151] . conversely, capsaicin-induced transient receptor potential cation channel subfamily v member 1 (trpv1) desensitization and inhibition of cgrp receptors enhanced neurogenic vasoconstriction [152, 153] . the innervation of bat by the sympathetic nervous system (sns) has long been recognized both at the level of blood vessels and also directly on adipocytes [154] . sns innervation plays a critical role in regulating non-shivering thermogenesis in rodents by controlling the release of noradrenaline (na) from sympathetic nerve terminals, which consequently stimulates β 3 -adrenoceptors that turns on a cascade of intracellular events ending in activation of ucp1 and triggering bat thermogenesis [155, 156] . investigation using histofluorescence, electron microscopy, and unilateral denervation model verified the sympathetic innervation of bat [155, 157] . further examination with retrograde viral transneuronal tract tracer, pseudorabies virus, injected into the central sympathetic outflow, revealed that circuits ultimately terminate in bat, indicating that the sns outflow directly originates from the brain [155] . various experimental approaches, including neurochemical (ne turnover), neuroanatomical (viral tract tracing), and functional (sympathetic denervation-induced blockade of lipolysis) studies have revealed sns innervation in wat [158] [159] [160] . in wat, the sns is the principal initiator of lipolysis in mammals [161] . it has been demonstrated that the sympathetic nervous system plays a role in modulating lipolysis in white adipose tissue and the degree of lipolysis appears to be determined by the relative expression of adipose adrenoceptors [158] . adipocytes express receptors for the sympathetic co-transmitters npy and atp (npy receptors and purinoceptors, respectively) and also express muscarinic receptors and receptors for peptides including angiotensin ii and sp [162] . four adrenoceptor subtypes, α 2 -, β 1 -, β 2 -, and β 3 -adrenoceptors, exist in wat [163] [164] [165] , in which all beta (β 1 -, β 2 -, and β 3 -) adrenoceptors stimulate lipolysis while α 2adrenoceptors inhibit the process [166] . the sns innervation in wat also plays a role in the control of lipid proliferation [158, 167] . it has been shown that sympathetic denervation of wat in vivo stimulates fat cell proliferation [160] . in nearly every vascular bed studied across different animal species, the expression of sympathetic nerves has been shown and it accounts for the largest proportion of innervation in the resistance vasculature [149] . sympathetic nerves mainly regulate medium and small arteries, and innervation is sparser in large conduit arteries [168] . it has been reported that vascular responses to sympathetic nerve activity (sna) can vary, depending on the content and composition of vesicles released from specific axon varicosities [169, 170] , the size and location of vessel branches within resistance networks [171, 172] , and the frequency and firing pattern of action potentials [173] . in 2011, dashwood and loesch showed that pvat of human saphenous vein receives direct sympathetic innervation using immunostaining technique [174] . more recently, bulloch and daly also provided evidence of sympathetic innervation in pvat, of mouse mesenteric arteries, using immunofluorescence techniques [25] . adipocytes were observed to be innervated by sympathetic nerves, with mast cells commonly located along the nerves [148] . the same study also found that bat comprises more nerve fibers compared to wat [148] . consistently, another study found that adrenergic nerves innervated only 2-3% of all wat isolated from rats mesentery, epididymis, omentum, and subcutis which were fed, fasted or fasted, and then refed [175] . although it is becoming more apparent that sympathetic nerves are distributed within pvat, however, its influence on vasoregulation is not well-defined yet. in the seminal study of pvat in 1991, the potential involvement of sympathetic nerves in modulating contractile response to electrical field stimulation has been revealed [32] . electric field stimulation (efs) produced a frequencydependent contraction in intact rat aortae but no response observed in cleaned tissues [32] . the authors postulated that this was most likely due to the presence of sympathetic nerves in the pvat [32] . in 2006, gao et al. revisited the neurogenic contractile response in tissues with pvat and their studies were carried out in rat mesenteric arteries [100] . the group produced a similar observation to that of soltis and cassis, in which pvat augmented the arterial contractile response to perivascular nerve stimulation [32, 100] . this was shown to occur through the production of superoxide mediated by nad(p)h oxidase and involved activation of tyrosine kinase and mapk/erk pathways [100] . in 2010, lu et al. further explored a pro-contractile property of pvat on perivascular nerves by providing evidence for the presence of angiotensin ii in mesenteric pvat [176] . this pvat-derived compound was suggested to potentiate efs-evoked neurogenic contractile responses in isolated rat mesenteric arteries, consistent with previous observations that angiotensin ii enhances sympathetic neurotransmission through pre-and post-junctional mechanisms [177, 178] . despite the majority of studies showing that sympathetic activation in pvat promotes vasoconstriction in different vessels [32, 100, 176] , however, there are few recent studies that contradict the role of pvat-derived sympathetic nerves in vascular tone regulation. ayala-lopez et al. demonstrated that removal of pvat did not reduce response of efs stimulus at 20 hz of rat aortae; however, the presence of pvat in rat superior mesenteric artery potentiated a 20-hz-induced contraction [97] . the authors also showed that pvat comprises functional catecholamines and which are independent of sympathetic nerves and tyramine-sensitive [97] . in a more recent publication, the same group also found that pvat of human splanchnic blood vessels contain elements of an adrenergic system with capabilities of synthesis and storage of catecholamines, which may influence vascular tone [179] . however, how pvat catecholamines would be stimulated or released physiologically demands explanation. recently, saxton et al. also supported the role of pvat as a reservoir for ne; however, the group suggested that these catecholamines are released from sympathetic fibers via organic cation transporter 3, thereby preventing ne from reaching the blood vessel and causing contraction [180] . in contrast to previous studies, the group showed that the presence of pvat potentiated vasorelaxation of healthy c57bl/6j mouse mesenteric resistance arteries [180] . the study suggests that electric activation of sympathetic nerves in pvat stimulates the release of a transferable anti-contractile factor (adiponectin) and the release of the adiponectin is dependent on β3-adrenoceptor activation [180] . torok et al. compared the effect of pvat on sympathetic neurotransmission in normotensive and hypertensive rats [181] . the group demonstrated that pvat exhibited inhibitory influence on the contractions to endogenous norepinephrine released from arterial sympathetic nerves during efs in superior mesenteric artery isolated from normotensive wistar-kyoto rats (wky), which is in line with a recent study [180] . however, the anti-contractile effect against sympathetic-induced vasoconstriction was abolished in spontaneous hypertensive rats [181] . in contrast, the abdominal aorta with intact pvat elicited larger contractions to efs and tyramine when compared to the aortae after removing pvat [181] . the authors attributed that the difference in the modulatory effect of pvat on adrenergic contractions between abdominal aortae and superior mesenteric arteries might be due to the endogenous ne of pvat [181] . accumulating data on the effect of sympathetic neurotransmission in pvat marks discrepancy. the diversity of experimental conditions, species, and vessels used may account for this contradictory observations. as for experimental conditions, many studies used different parameters (voltage, frequency, duration, and pulse) for studying direct adrenergic activation. some studies used supramaximal voltage, although majority of studies used nerve blockers such as guanethidine and/or tetradotoxin to verify nerve mediation in efs-evoked responses. it has been proposed that greater voltage potentiates efs-enhanced vasoconstriction in pvat-intact preparations, probably due to generation of superoxide anions [180] . furthermore, certain groups carried out experiments under basal condition while some other groups conducted experiments under raised tone, using different agents. the conflicting data should be addressed and the role of sympathetic innervation in pvat should be elucidated as it may represent a novel target in the treatment of cardiovascular diseases. pvat is well-recognized as a source of numerous vasoactive compounds. it therefore holds promise as a novel therapeutic target. despite this, information on the link between pvatderived mediators and the neurovascular system is sparse. to date, very few in vitro studies that have investigated specifically on the relationship between pvat-derived nerves and pvat mediators [180, 182] , although there are substantial number of studies which have examined the non-pvatspecific interaction between sympathetic activation and adipokines. receptors for adrenergic system and its cotransmission have been detected in adipocytes and stimulation of these receptors modulate adipocytes physiology (e.g., lipolysis and lipogenesis) and contents [162] . hence, it is postulated that sympathetic innervation in pvat modulates pvat mediator release and integral for normal vascular function as well as pathophysiology and vice versa. indeed, our understanding on the complex interaction between pvat-derived nerves and pvat-derived factors is at its infancy, despite it has huge potential in modulating vascular tone. a challenge to scrutinize the crosstalk includes the extremely short duration of neurotransmission and the involvement of non-neuronal effect resulting from the overstimulation of smcs. in addition, technique to elucidate the pvat-specific interaction in vivo proves to be challenging and still have not been developed yet. recently, it has become apparent that the sympathetic system is a key regulator of leptin production in wat [183] . sympathomimetic amines and cold exposure or fasting (which results in sympathetic stimulation in wat) decrease leptin gene expression in the tissue and thus leptin production [183] . furthermore, it has been shown that the β3adrenoceptor is involved in inhibiting insulin-stimulated leptin secretion from isolated rat adipocytes [184] , and it has also been postulated that the sympathetic system might contribute to supporting an inhibitory action on leptin synthesis [183] . conversely, sympathetic blockade often increases circulating leptin and leptin gene expression [183] . in turn, leptin regulates arterial pressure by enhancing sympathetic nerve activity to brown adipose tissue, kidney, and other tissues [185] . acceleration of sympathetic activity is a hallmark of obesity. in obese patients, it has been shown that cytokines derived from adipocytes such as leptin, tnf-α, and il-6 mediate sympathetic activation [186] . some studies indicated that leptin acts both centrally and peripherally to potentiate the sympathetic nervous system, including enhancing catecholamine synthesis in the adrenal medulla [187, 188] , which subsequently activate the renal sympathetic nerves, hence increase blood pressure. resistin, a pro-contractile adipokine, acts directly in the central nervous system to influence the sympathetic nerve activity [189] . there is growing evidence that resistin may have complex interactions with leptin [189, 190] . both of these adipokines synergistically excite sympathetic nerves innervating cardiovascular organs but could antagonize each other's actions on bat, in which resistin inhibits bat activity and vice versa [189] . adiponectin, a protective adipokine, has been shown to be regulated by adrenergic system. imai and co-researchers demonstrated that sympathetic nervous system suppresses serum adiponectin levels and adiponectin mrna expression in mice subcutaneous, epididymal, and mesenteric wat in vivo [191] , and the regulation differs among wat depots of mice. in their study, it was shown that cold exposure suppresses serum adiponectin levels through sympathetic nerve activation [191] . consistently, in healthy male volunteers, exposure to cold for 2 h activated sympathetic nerves, which consequently reduced adiponectin release [192] . nonetheless, subcutaneous adipokine gene expression was unaltered [192] . the same study found that sympathetic stimulation triggered mcp-1 release [192] . on the contrary, a very recent study showed that direct sympathetic stimulation in pvat of mice mesenteric arteries in vitro enhanced the release of adiponectin via β3adrenoceptor activation [180] . il-6 is a pro-inflammatory cytokine shown to be secreted in higher concentrations from pvat compared with other fat depots and potentially has a role in promoting arterial stiffness [193, 194] . an in vivo study by de luigi et al. showed that sympathectomy increased lipopolysaccharide-induced il-1β and il-6 messenger rna in rat adrenals and spleen and concluded that sympathetic nervous system denervation enhances the synthesis and production of peripheral il-1β and il-6 by central lipopolysaccharide [195] . sympathetic neurons have also been shown to stimulate production of il-6 in vitro using cell culture [196] . mcp-1, another pro-inflammatory cytokine, has been reported to be rapidly expressed by sympathetic ganglion neurons following axonal injury [197] . sensory nerves are abundant in wat [158, 160] . sensory innervation of wat was first shown neuroanatomically using an anterograde tract tracer, true blue, which resulted in labeled neurons in the dorsal root ganglia (drg) [198] . further experiments including histology and a transneuronal viral tract tracer, the h129 strain of herpes simplex virus-1 (hsv) [199] , convincingly corroborated the existence of cgrp and sp containing nerves within wat. bat also has significant sensory innervation, as revealed by immunohistochemical staining for cgrp and sp [154, 200] . there is accumulating evidence that bat has marked sensory innervation, as shown by immunohistochemical markers of sensory neuropeptides such as cgrp and sp [155] . denervation of sensory nerves in bat results in general decreases in bat growth and protein content, mitochondrial content such as cytochrome oxidase activity, and thermogenic capacity, such as ucp1 content [201, 202] . collective data obtained from different experimental protocols including anterograde viral tract tracer [198] , co-culture of drg cell and adipocytes, and immunohistochemistry staining strongly indicate that wat is innervated by sensory nerves [203] . recently, murphy et al. showed endogenous leptin secreted from wat activates spinal sensory nerves innervating siberian hamster brain in a paracrine manner [204] . despite the innervation and feedback loops of sensory nerves that have been recognized in wat [205] , the significance of the innervation is still poorly understood and warrants further exploration. evidence has accumulated that nanc nerves play important roles in regulating vascular tone [206] . in nanc neurotransmission, sensory nerves play a vital role in vasoregulation and generally cause vasodilatation [206, 207] . sensory nerves demonstrate physiological antagonism of sympathetic nervedependent vasoconstriction [208] and also possesses both anti-dromic and orthodromic conduction; thus, this allows their participation in local axon reflexes independent of efferent signaling from the cell body [209] . cgrp, a potent vasodilator, has been recognized as the primary neurotransmitter of sensory nerves while sp is a cotransmitter [207, 210] . cgrp is a 37-amino acid neuropeptide that is primarily localized to c and aδ sensory fibers and has two major forms, α and β [211] . αcgrp is predominantly expressed in the nervous system while β-cgrp is primarily expressed in the enteric sensory system [212] . a recent study has provided solid evidence that sensory nerves are intimately linked to the sympathetic and angiotensin signaling systems [211] . in cgrp knockout mice, enhanced hypertension and vascular remodeling/ inflammatory changes in an angiotensin ii-induced hypertension model have been demonstrated [213] . hence, this has created a concept that sensory nerves appear to play a protecting role against the adverse effects associated with the cardiovascular disturbance. in vascular arena, pvat has now has become the subject of intense investigation as it involves in cardiovascular pathophysiological conditions. in spite of the extensive research on pvat, surprisingly, expression and role of sensory nerves in pvat have not been described previously. it was thought that sensory nerves which involved in blood vessel regulation were only confined at the adventitia and relaxation responses to efs in rat isolated mesenteric artery were credited to myogenic tone or dependent on an intact endothelium [214, 215] . nonetheless, it is very intriguing to find out that there is a mismatch of efs experiment outcomes for isolated rat mesenteric arterial bed and rat isolated mesenteric artery. furthermore, evidence of sensory-induced vasorelaxation in other isolated arteries is limited [182] . in rat pvat-intact mesenteric artery beds, efs elicited robust sensory neurogenic relaxation responses but these responses were markedly reduced in pvat-denuded mesenteric artery beds or abolished in clean rat mesenteric artery [182] . the discovery made by abu bakar et al. on the expression of sensory nerves within pvat and functional sensory nerves that are critically dependent on pvat has orientated our understanding on the role of sensory nerves [216] . pvat-denuded vessels that are normally used in conventional vascular experiments have led to a postulation that sensory nerves are not involved in vasodilatation of rat mesenteric artery; however, this concept has been refuted by the recent finding [182] . on contrary to efs-induced sensory stimulation, it is interesting to note that pvat removal does not totally eradicate efs-evoked frequency-dependent vasoconstriction (sympathetic activation) [180, 181] , although some studies demonstrated significant reduction of efsevoked sympathetic vasoconstriction in clean vessels [32, 100, 176] . in addition, while many studies have shown extensive sensory innervation at the adventitia of clean vessels [182, 207, 217] , however, it is unclear why vasodilatation cannot be demonstrated in the absence of pvat. the data however further corroborates that pvat plays a crucial role in sensory-induced vasodilation. in comparison to sympathetic nerves which have been located in pvat in 1970 [148] , it is only recently that sensory nerve innervation has been demonstrated in pvat [182] . a double staining protocol with the presence of anti-cgrp and anti-protein gene product (pgp) 9.5 antibodies was applied to verify the observation [182] . the distribution of nerves was found to be more extensive in small arteries (second-order of mesenteric artery) compared to large arteries (superior mesenteric artery), which is consistent with efs data which showed neurogenic relaxation responses to efs were greater in second-order mesenteric artery compared to superior mesenteric artery [182] . interestingly, cell bodies or neuronal somata were visualized in pvat of rat mesenteric artery [182] . the observation is not in line with the traditional notion, in which cell bodies of cgrp were believed to be located only in drg [218] , and terminate peripherally on blood vessels and other tissues innervated by the sensory nervous system, and also centrally in laminae i/ii of the dorsal horn of the spinal cord [219] . however, a more recent research using dna sequence analysis to show 100% homology with the β-cgrp cdna indicates that mrna encoding β-cgrp is expressed in the vessel, exhibited the presence of adventitial neuronal somata in small rat mesenteric artery [220] , which is consistent with the recent observation [182] . cgrp immunoassay study data further supports the existence of sensory nerves in pvat layer [182] . investigation to elucidate the interaction between pvatderived sensory nerves and pvat-derived mediators has been attempted in the recent study [182] . the attempt has led to a development of a new technique which exclusively study the interaction between pvat-derived nerves and pvat-derived factors; tissues (mesenteric artery beds) in the presence or absence of pvat were perfused under normal or low oxygenation, efs was applied and samples (perfusates) were collected during efs (very short period), and the samples were then subjected to multiplex assay for adipokines (adiponectin, leptin, il-6, mcp-1, tnf-α, interleukin beta (il-β), and total pai-1 analysis [182] . under standard oxygenation, only leptin level was shown to be elevated in pvat-intact preparations [182] , which suggests that there is a link between endogenous leptin and sensory nerves. presumably, cgrp and/or other neurotransmitter(s) released from sensory nerves during efs act at receptors on the adipocytes in pvat to cause a release of leptin. furthermore, exogenous leptin potentiated neurogenicmediated relaxations in mesenteric arteries [182] . this observation is consistent with leptin's effect in the cardiovascular system. under normal conditions, leptin demonstrates vasorelaxing effect [221, 222] . the anti-contractile effect of leptin is achieved via both endothelial-dependent (through involvement of no) and endothelial-independent mechanisms [223, 224] . it has been shown that leptin dilates canine small mesenteric arteries and veins by a mechanism involving endothelial release of nitric oxide [221] . recently, the role of leptin secreted from wat as a paracrine factor to activate spinal sensory nerves has been shown in siberian hamsters [204] . a more recent publication shows that leptin receptors lepr are expressed on sensory neurons, and the deletion of leptin signaling in vagal afferent neurons results in hyperphagia and obesity [225] . hence, the present data adds a new insight on the relationship between leptin and sensory nerves. the release of leptin was virtually abolished by both pvat removal and under conditions of low oxygen. in addition, low oxygen levels were shown to enhance inflammatory mediator, il-6 release [182] . the observation highlights that the level of oxygen plays a role in mediating the release of some pvat mediators. moreover, acute hypoxia model that was employed in the study might be extrapolated for certain disorders, such as hypoxia in obesity, and can be exploited for studying the effect of pvat on neurotransmissions under pathological conditions. the autocrine/paracrine/endocrine functions of pvat have been elucidated by an abundance of experimental and clinical research. however, there still remain some major unanswered questions and emergent future directions about pvat. in the first place, the true identity of adrf still requires confirmation. although certain studies pointed that h 2 s might potentially be the primary adrf candidate of perivascular adipocytes [75] , however, h 2 s might not be the universal adrf since cse inhibition does not diminish the anti-contractile effect in mouse aortae [79] , unlike endothelium-derived no which is the universal vasodilator of different species. furthermore, there is data indicating that adiponectin is not the adrf using an approach of utilizing adiponectin gene-deficient mice [226] . another investigation provided clues that the deficiency of leptin receptors in the zucker fa/fa rat showed no significant modification on the anticontractile effect of pvat, implying that leptin probably is not the adrf [227] . despite the unclear chemical nature of adrf, a promising adrf candidate should at least fulfill the requirement to mimic adrf characteristic effects on k + channels (i.e., it should be a potent opener of kcnq and probably other k v channels), as reviewed in 2012 and 2017 [6, 17] . therefore, future studies need to explore the constellation of adrf-activated kcnq channels and other involved k v channels and to identify the identity of adrf with reference to the stated requirements using gene-silencing or knockout techniques. in addition, more efforts are needed to perform comparative studies to dig out the similarities between the mechanisms of endothelium-induced and pvat-induced relaxation, for the purpose of reductionism. for example, further experiments should be carried out to verify whether pvat-derived pgi 2 follows the same mechanistic aspect as endothelium-derived pgi 2 . multiple rodent models are currently available, characterized by the reduction in the content of bat and/or wat (table 2) . however, the lack of report on the actual reduction of pvat might limit the dependability of certain models such as transgenic skinny and fat-attac mice [54, 56] . hence, further verification on the pvat content of the genetic engineered mice might extend the generality of pvat studies. in 2017, thomou et al. provided experimental clues in hinting the novel endocrine regulatory approach of adipose tissue via cir-mirnas [29] . the close proximity between pvat and vascular tree provides sufficient suspects that pvat might be one of the major sources of cir-mirna factory. however, the field concerning pvat-derived cir-mirna is still largely undefined so we propose the following questions: (1) whether pvat shares similar mirna expression profile as bat or wat, (2) whether exosomes and microvesicles cir-mirnas are the sole packaging approaches of cir-mirnas for long-distance metabolic regulation, (3) what is mirna profile of extracellular vesicles, and (4) how the onset and progression of cvds are mediated by cir-mirnas. according to the genome-wide comparative expressional studies between subcutaneous and coronary perivascular adipose tissue-derived adipocytes, a signature of 307 differentially expressed genes for cardiovascular homeostasis was identified [7] . it is of rationale to hypothesize that pvat should have different mirna expression profile and hence altered cir-mirna expression profile. apart from exosomal and microvesicular cir-mirnas, whether adipose tissue generates other carriers for cir-mirna packaging, such as microparticles, requires further confirmation. previous study showed that adipocytes are able to release microparticles, which act as bfind-me^signals to prompt macrophage migration [228] , and therefore microparticles might be a third option of mirna carrier. past investigation focused on how the generation of fibroblast growth factor 21 is affected by the presence of cir-mirnas [29] ; therefore, future studies can concentrate more on the role of cir-mirnas in cardiovascular complications such as endothelial dysfunction, coronary heart disease, and atherosclerosis. since the identification of anti-contractile effect of pvat in 1991, more and more primary and clinical investigations unravel the roles of pvat in addition to the vasoregulatory effect. accompanied by the increasing rates of obesity worldwide, more scientists and clinicians are now aware of the pivotal endocrine and paracrine roles of adipose tissue in obesity-related cardiovascular events. more importantly, the close proximity between pvat and vascular system facilitates the interaction between pvat and vascular system. pvat, through the production of numerous perivascular contracting/relaxing factors and adipocytokines, acts as an autocrine/paracrine/endocrine regulatory center for the homeostasis of cardiovascular system, sharing the status not less important than that single layer of endothelium. furthermore, pvat provides an interface for active pvat-neuronal communications and may be a potential source of cir-mirnas. therefore, pvat is a master endocrine organ, much more than a mechanistic support to the vasculature. author contributions all the authors researched the data for the article, provided substantial contributions to discussions of its content, wrote the article, and edited/reviewed the manuscript before submission. competing interests the authors declare no competing interests. human perivascular adipose tissue dysfunction as a cause of vascular disease: focus on vascular tone and wall remodeling perivascular adipose tissue: an unique fat compartment relevant for the cardiometabolic syndrome regional differences in perivascular adipose 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emerg microbes infect doi: 10.1038/emi.2013.25 sha: doc_id: 304850 cord_uid: 9xetsc2c nan virologists have been surprised by a recent report that has changed our long-standing conception of the ecology of influenza viruses. potentially, we can no longer rely on waterfowl to be the only source of new flu variants, as bats have now been found to harbor influenza viruses whose internal genes share common ancestry with all known influenza a viruses. 1 other genome portions share even older ancestry, 2 while the main surface protein lies within the known diversity of 'usual' influenza a viruses. the appearance of such a vast mixture of genes suggests that more undiscovered flu strains are lurking in bats. for any virus, the identification of a mammalian reservoir is highly relevant because the 'fitness valley' that viruses need to cross for the conquest of new hosts is shallow if the hosts are genetically related. 3 our knowledge of mammalian viruses is fairly opportunistic, focusing on agents of obvious disease in livestock and pets. the range of viruses carried unnoticed by our phylogenetic next of kin may be huge. for instance, wild small mammals including bats and rodents have now been shown to harbor a tremendous spectrum of relatives of human paramyxoviruses-a family that contains the mumps virus, several different respiratory agents and the measles virus. 4 not all of these have yet been proven to have their cognates in bats, but the sample studied so far is just a tiny fraction of the tremendous bat diversity. some of these agents have already been suggested to cross-infect humans. 5 that is a worrying perspective because the concept of liberating humankind from some of its most notorious viruses by mass vaccination is essentially dependent on the absence of animal sources from which eradicated viruses could be replenished. 6 the implications of recent findings might even reach into the future agenda of virus eradication: the hepatitis c virus, one of the most important human viruses and a prime candidate for eradication pending vaccine availability, has relatives in companion animals including dogs and horses. 7, 8 these and other recent findings remind us of an important issue in viral reservoir ecology: non-persisting viruses are maintained on a social level, requiring large, dense and interconnected host groups for their perpetual transmission. 9 human immunodeficiency virus and its ape reservoir with a rather small group size might have been a decoy rather than a paradigm for this field of research because the virus is able to persist in individuals and depends less on efficient transmission for maintenance. on the contrary, candidates for the next pandemic would be agents that are transmitted efficiently and cause acute disease-such as severe acute respiratory syndrome and flu. the novel human coronavirus emc/2012 with its connection to bats might establish another recent case. 10, 11 within the class of mammals, bats form the largest contiguous social groups. their association with pathogenic viruses has been proposed to be due to specific immune functions, 12 but these remain to be proven. large social group sizes and a migratory lifestyle may suffice to make certain bat species become breeders of viruses. the reliance of bat-borne viruses on transmissibility rather than persistence could explain their high onward transmissibility after host-switching. 13 there are prominent examples of bat-borne viruses that can be passed between humans, including ebola virus, marburg virus, nipah virus and the severe acute respiratory syndrome agent. for comparison, we have few examples of rodent-derived viruses that are routinely passed from human to human. lassa virus may be the only relevant exception, and even there, transmission seems to be possible only under conditions of very close contact. apart from certain bat species, there is only one other mammalian species that forms interconnected social groups of more than one million individuals-humans. we may thus provide a familiar environment for bat-borne viruses that are optimized for transmission in large social groups. in the virus-hunting scene, there is now a rush to study bat-borne viruses, doubtlessly triggered by the finding of severe acute respiratory syndrome-related viruses and the conjecture that bat-borne viruses might spark the next pandemic. however, there remains a large gap between the many studies describing novel reservoir-borne viruses and our capabilities to use this knowledge to predict or prevent future human disease outbreaks. this is not to say there is no progress. there are reports emerging of longitudinal and quantitative studies of reservoir-borne viruses showing potential utility for prevention. for instance, very recent work has identified adolescent bats as pronounced carriers of marburg virus in a crowded bat cave in uganda where at least two well-documented human infections have occurred. 14 interestingly, these adolescents are forced to roost in less preferred places close to the cave's entrance-areas preferentially touched and passed by humans visiting the cave. other studies have convincingly shown that the breeding season is a time when several bat-borne viruses are amplified-a situation that is highly similar to a kindergarten where runny noses are commonplace. 15 however, beyond such practical insight, we still know little about the fundamental ecological mechanisms driving virus emergence. the idea that reservoir-borne viruses should exist peacefully with their hosts is most likely not widely valid. 13 as we dig deeper into viral reservoir ecology, including its man-made modifications, we may find that changes in host populations affect the transmission and maintenance of viruses with possible consequences for their potential to infect humans (figure 1 ). for example, analogously to the dilution effect theory, one could expect that either the reduction or expansion of the host group density would allow more virulent virus variants. obviously, the investigations necessary to probe such effects need to be led by ecologists rather than virus hunters. as for virologists, we will contribute little to the prevention of the next pandemic by piling up virus sequences-we need to generate functional insight to further triage among reservoir-borne viruses with regard to their epidemic risks. for example, we can identify bona-fide interferon antagonists in reservoir-borne viruses using sequence homology and systematically test how potent these proteins are at breaking our innate immunity barrier. 16 beyond innate immunity and receptor-mediated cell entry, there are exciting new results from comparative studies of virushost interactions across the family tree of viruses that identify new cellular pathways that can be hijacked by viruses or that suppress their replication. 17 not only can these additional targets be used as tests for viral cross-host compatibility, but their comparison between mammals may also yield targets for cross-host antiviral drugs. such drugs could confer practical pandemic preparedness. attribution-noncommercial-noderivative works 3.0 unported license. to view a copy of this license, visit http:// creativecommons.org/licenses/by-nc-nd/3.0 habitat fragmentation resource abundance change of social structure risk virus replication / transmission duration of excretion / infectivity figure 1 modification of viral maintenance optimum. the maintenance of non-persisting viruses requires a sufficient rate of transmission (x-axis) within the host population or group. a longer duration of excretion or infectivity (y-axis) allows for a lower virus transmission rate while still successfully maintaining the virus. increased transmission correlates with increased replication and virulence. as virulence kills or incapacitates hosts, it limits the duration of infectivity, leaving a limited space in which maintenance can be optimized (triangle). the optimum is virus-specific (gray curve). changes in host ecology can move the maintenance optimum within the limits of the optimization space (dashed curve). those viruses whose maintenance optimum is moved into the red area of the optimization space may pose increased pandemic risks. emerging microbes and infections a distinct lineage of influenza a virus from bats the neuraminidase of bat influenza viruses is not a neuraminidase host species barriers to influenza virus infections bats host major mammalian paramyxoviruses novel potentially-zoonotic paramyxoviruses from the african straw-colored fruit bat, eidolon helvum measles eradication: past is prologue characterization of a canine homolog of hepatitis c virus serology-enabled discovery of genetically diverse hepaciviruses in a new host infectious diseases in primitive societies isolation of a novel coronavirus from a man with pneumonia in saudi arabia human betacoronavirus 2c emc/2012-related viruses in bats, ghana and europe what links bats to emerging infectious diseases? crossing the line: selection and evolution of virulence traits seasonal pulses of marburg virus circulation in juvenile rousettus aegyptiacus bats coincide with periods of increased risk of human infection amplification of emerging viruses in a bat colony canine hepacivirus ns3 serine protease can cleave the human adaptor proteins mavs and trif viral immune modulators perturb the human molecular network by common and unique strategies the deutsche forschungsgemeinschaft (germany's national research council) has established a priority program on ecology and species barriers in emerging viral diseases (coordinator's grant to christian drosten, dr 772/10-1). key: cord-291965-9r9ll83m authors: pfefferle, susanne; oppong, samuel; drexler, jan felix; gloza-rausch, florian; ipsen, anne; seebens, antje; müller, marcel a.; annan, augustina; vallo, peter; adu-sarkodie, yaw; kruppa, thomas f.; drosten, christian title: distant relatives of severe acute respiratory syndrome coronavirus and close relatives of human coronavirus 229e in bats, ghana date: 2009-09-17 journal: emerg infect dis doi: 10.3201/eid1509.090224 sha: doc_id: 291965 cord_uid: 9r9ll83m we tested 12 bat species in ghana for coronavirus (cov) rna. the virus prevalence in insectivorous bats (n = 123) was 9.76%. cov was not detected in 212 fecal samples from eidolon helvum fruit bats. leaf-nosed bats pertaining to hipposideros ruber by morphology had group 1 and group 2 covs. virus concentrations were <45,000 copies/100 mg of bat feces. the diversified group 1 cov shared a common ancestor with the human common cold virus hcov-229e but not with hcov-nl63, disputing hypotheses of common human descent. the most recent common ancestor of hcov-229e and ghanabt-covgrp1 existed in ≈1686–1800 ad. the ghanabt-covgrp2 shared an old ancestor (≈2,400 years) with the severe acute respiratory syndrome–like group of cov. c oronaviruses (covs) (order nidovirales, family coronaviridae, genus coronavirus) are enveloped viruses with plus-stranded rna genomes of 26-32 kb, the largest contiguous rna genomes in nature (1) . they are classified into 3 groups, which contain viruses pathogenic for mammals (groups 1 and 2) and poultry (group 3) (1). hu-man covs (hcovs)-229e, -nl63, -oc43, and -hku1 are endemic worldwide and cause mainly respiratory infections in children and adults. the severe acute respiratory syndrome (sars) coronavirus (sars-cov) is a novel zoonotic coronavirus that caused an international epidemic in 2002-2003. fortunately, efficient public health management interrupted this epidemic (2) . studies conducted in china in the aftermath of the sars epidemic have identified covs in bats (chiroptera) and implicated this speciose mammalian order as the most likely reservoir of all known coronaviruses (3) (4) (5) (6) (7) . among the most urgent concerns prompted by the sars epidemic is the likelihood of similar future events. thus, it seems highly relevant to study the ecology of bat covs in terms of diversity, host restriction, virus prevalence, risk of exposure, and the circumstances of past host transition events. the genetic diversity of bat-borne covs is currently unclear. preliminary data suggest that covs may be adapted in a stricter sense to a specific host species rather than to specific regions (5, 6, (8) (9) (10) (11) (12) . a variety of pathogenic covs occur in other mammals or poultry. however, the genetic range within these animals is considerably less than that observed in even single bat species or subfamilies (7, 8) . estimates indicate that there are >100 bat species in sub-sahran africa. this finding is in contrast to ≈50 species in the entire western palaearctic region (europe, middle east, north africa) (13, 14) . african bats have been shown to harbor pathogens that are occasionally transmitted to humans. this transmission may result in severe disease outbreaks, e.g., ebola and marburg viruses (15) . because bats are a part of the human diet in wide areas of africa (16) , it appears highly relevant to study covs in african bats. we have demonstrated by serologic studies that african bats have antibodies against covs (10) . antibodies reactive with sars-cov antigen were detected in 47 (6.7%) of 705 bat serum specimens from 26 species (10) . recently, tong et al. detected sequences of covs in bats from kenya (17) . we describe the results of studies on bats in ghana obtained by using noninvasive sampling of frugivorous and insectivorous bats at 2 caves, a lake habitat of diverse insectivorous bats, and a large urban roosting site of frugivorous bats. bayesian inference of diversification dates gave implications on the recency of the introduction of hcov-229e into the human population, irrespective of its original source. in the locations identified in figure 1 , mist netting and sampling were conducted as described (11) . in kumasi zoo, fecal samples were collected with plastic foil under trees occupied by eidolon helvum bats (estimated colony size 300,000). for all capturing and sampling, permission was obtained from the wildlife division of the ministry of lands, forestry, and mines in ghana. research samples were exported under a state agreement between the republic of ghana and the federal republic of germany, represented by the city of hamburg. additional export permission was obtained from the veterinary services of the ghana ministry of food and agriculture. samples (1-4 fecal pellets or swabs suspended in rna stabilization solution [rnalater tissue collection; applied biosystems, foster city, ca, usa]) were tested at the kumasi centre for collaborative research in tropical medicine as described (11, 18) . after initial sequencing, specific primers were designed for each group of cov found. nested reverse transcription-pcr (rt-pcr) primer sets used for sequencing of longer fragments of representative viruses are available upon request. nucleic acid alignments were conducted based on amino acid code by using the clustalw algorithm (www. ebi.ac.uk/clustalw) in the molecular evolutionary genetics analysis version 4.0 software package (www.megasoftware.net) (19) . two gap-free nucleotide alignments (817 bp and 1,221 bp) were generated. tree topologies were determined on both datasets by using mrbayes version 3.1 (20) . the analysis used a general time reversible (gtr) substitution model, with 6 rate categories to approximate a gamma-shaped rate distribution across sites and an invariant site assumption (gtr + γ6 + i). markov chain monte carlo (mcmc) chains of 10 7 iterations were sampled every 500 generations, resulting in 20,000 sampled trees. two metropolis-coupled chains (1 cold and 3 heated chains each) were run in parallel, compared, and pooled. convergence of chains was confirmed by the potential scale reduction factor statistic in mrbayes (21) and by visual inspection of each cold chain using the tracer program (22) . phylogenetic dating was conducted by using bayesian evolutionary analysis sampling trees (beast) (22) . chain lengths in beast were at least 20,000,000 generations with sampling every 500 generations. convergence of the model was checked visually and by the effective sample size statistic with tracer. during february 2008, bats were sampled in the described locations around kumasi, ghana. initially, 7 fecal samples tested positive by pan-cov pcr. products (440 bp, rdrp gene) were sequenced and aligned with prototype cov. neighbor-joining phylogenies indicated 2 distinct groups of sequences that belonged to cov group 1 (n = 4) and group 2 (n = 3), respectively. specific primer pairs for the group 1 and group 2 sequences were designed and applied again to all samples. five additional viruses were found, resulting in a total cov prevalence of 9.76% in insect-eating bats (n = 123). no virus was found in any oral swab. all virus findings in fecal samples are listed by capture site in table 1 . notably, all cov findings were in insect-eating leafnosed bats of the genus hipposideros. within the genus, the species h. abae could be discriminated unambiguously by morphology ( table 1 ). the remaining hipposideros species were assigned to the complex of forms related to currently recognized species h. caffer and h. ruber. because 2 morphotypes were present (figure 2 ), the mitochondrial cytochrome b gene was sequenced as described (23) . both morphotypes belonged to phylogenetic lineages distinct from h. caffer and possibly represented 2 distinct species (p. vallo, personal ongoing investigation). both are collectively referred to as h. caffer (cf.) ruber in this study. a fraction of 15.4 % of h. cf. ruber specimens yielded cov, without a difference between sexes (14%/19%, n = 57/21 [m/f], respectively). only adult males and nonlactating adult females, but no lactating females, juveniles, and subadults of h. cf. ruber were encountered. to estimate the quantity of cov genomes in bat feces, we did end-point dilution experiments with the nested pan-cov rt-pcr (18). the previously determined sensitivity limit of the pcr assay was 5-45 copies/pcr (18) . in the assay, the equivalent of 1 mg feces was tested per pcr tube (100 mg feces collected, 1:10 dilution extracted, 1:10 dilution tested). the highest dilution factor that still yielded an amplification signal in any of the samples was 1:10, which suggested a maximal concentration of 50 to 450 cov rna copies/mg of feces. in h. cf. ruber bats in the kwamang and booyem caves, a diverse group 1 cov was found. further analysis was complicated by the low rna content in samples. based on alignments of prototype group 1 viruses, 5 different nested rt-pcrs were designed and the rdrp fragment could finally be extended by 441 bp to the 5′ end, providing an 817-nt fragment for phylogenetic analysis. all methods of phylogenetic inference placed this virus next to a common ancestor with human coronavirus 229e, which circulates worldwide in humans ( figure 3 ). bootstrap support of the hcov-229e/ghanabt-covgrpi root point in neighbor-joining analysis was 100%. the corresponding bayesian posterior probability was 1.0. the most closely related member of the ghanabt-covgrp1 clade shared 91.90% nucleotide identity with hcov-229e in the analyzed fragment. the most distant member was 86.50% identical. the next phylogenetic neighbor, the human cov hcov-nl63, was only 74.70%-78.60% identical in the analyzed fragment. with the pan-cov screening assay, a group 2 cov was initially found in the kwamang cave. sequences from 3 bats were identical. the secondary group-specific pcr identified 4 additional samples of this virus, 1 of them from booyem cave b and the remaining from kwamang. nucleotide identity among these sequences was 97.2%-100%. phylogenetic analysis with different methods of inference (neighbor-joining nucleotide-based, neighbor-joining amino acid-based, bayesian) yielded variable tree topologies suggesting basal associations with either the 2a, 2d, or 2b subgroups (data not shown) (24) . based on alignments of prototype group ii viruses, 8 additional nested rt-pcr primer sets were designed and 2 of the samples could be amplified. sequences could be extended 520 bp upstream and 383 bp downstream of the initial fragments, yielding 1,221-bp fragments for phylogenetic analysis. bayesian phylogenetic inference with different substitution models and parallel analysis using metropolis coupling now placed the virus reliably next to a common ancestor with the 2b group of cov (sars-like viruses, figure 3 ). the bayesian posterior probability of the cov 2b/ghanabt-covgrp2 clade being monoyphletic was 1.0. a maximum of 72.2% nucleotide identity was shared with sars cov. reliable isolation dates were researched in the literature for each employed virus. because a reliable molecular clock dating existed for the most recent common ancestor (mrca) of the hcov-oc43/bovine cov pair (25) , this date was set as a normal-distributed probabilistic prior within the published ranges (25) for calibration of all analyses. a first analysis was conducted on the 1,225-bp dataset that did not include the novel ghanabt-covgrp1. all virus sequences were assumed to be contemporary. phylogeny was inferred using a gtr + γ4 + i model. the resulting mrca date of the cov2b (sars-like)/ghanabt-covgrp2 clade was 260 ad and that of the hcov-nl63/-229e pair was 981 ad (see table 2 for details). to include the novel ghanabat-covgrp1, we repeated the same analysis by using the 817-bp dataset. the resulting mrca date of the hcov-nl63/229e pair was 816 ad in this analysis, which was in good concordance with results from the 1,221-bp dataset ( table 2 ) and also with previously published data (26) . the diversification estimate for the novel group 1 bat-cov and hcov-229e then was 1803 ad. because it has been suggested that codon-based evolutionary models may be preferred for bayesian phylogenetic inference from protein-coding datasets (27) , analyses on the 817-bp dataset were repeated by using the srd06 substitution model in beast. this analysis did not yield a different substitution rate, but resulted in older resulting mrca dates ( table 2) . a bayes factor test conducted in tracer yielded a strong estimate of superiority of the codon-based model over the gtr + γ4 + i model (log 10 bayes factor 139 [20 is highly significant]). to further optimize the prediction of mrca dates, the constant population size assumption used in all analyses was exchanged against expansion growth or exponential growth assumptions. both assumptions were predicted to fit the data better than the constant size model (bayes factors 13.5 and 13.9). there was no difference between the expansion and exponential models (bayes factor 0.34 in favor of expansion). the mrca date of hcov-229e and the ghanabt-covgrp1 was 1686 (expansion) or 1800 (exponential growth). table 2 summarizes the results. figure 3 shows a dated phylogeny of coronaviruses with mrcas according to the 2 last mentioned analyses. to determine whether cov recombination might play a role in the studied virus population, the structural nucleocapsid gene was amplified using 8 nested rt-pcr primer sets that had been designed on alignments of all available cov group 1 nucleocapsid sequences. using a similar approach, we also tested the same samples for cov group 2 nucleocapsid sequences. only group 1 rt-pcrs yielded fragments. these fragments could be combined into contig*mrca, most recent common ancestor; ci, confidence interval; hpd, high population density; sars, severe acute respiratory syndrome; hcov, human coronavirus; gtr + + i, general time reversible gamma-shaped rate distribution across sites and an invariant site assumption. †estimation of the year (bc) of the most recent common ancestor. ‡estimation of the year of the most recent common ancestor of extant cov. all years ad except as indicated. §cov group 2b without novel bt-cov from this study (figure 2 ). uous 1,030-nt sequences for bt-cov ghanakwam 19 and 1,176 nt for bt-cov ghanaboo 344. as shown in figure 3 , panel b, the resulting phylogenetic placement was exactly matching that of the rdrp fragments, giving no evidence of recombination between the rdrp region located in the middle of the genome and the nucleocapsid gene located at the extreme downstream end. sequencing of the nucleocapsid gene of the ghanabt-covgrp2 was not successful when we used 15 nested rt-pcrs designed on alignments of all available cov 2b nucleocapsid sequences. amplification with above mentioned nested rt-pcrs for cov group 1 was also unsuccessful. in the aftermath of the sars epidemic, bats have been identified as carriers of cov in china (3) (4) (5) (6) (7) . furthermore, in addition to our earlier finding of antibodies against covs in various african bats (10), we have confirmed the presence of cov in bats of ghana. together with recent data from germany, north america, trinidad, and kenya (11, 12, 28) , these findings suggest that the association of cov with bats is a worldwide phenomenon. the prevalence of cov in insect-eating bats (9.76%) matched our previous findings in germany. however, in that study we sampled during the breeding season and showed that covs are most likely amplified in maternity roosts (11) . the composition of the catch in this study (no lactating females, no young bats) suggests sampling outside the breeding season and may not be directly comparable. future studies relating to risks of exposure should address whether virus prevalence may change over time. the risk of exposure was also addressed by investigations of virus concentration. several groups have shown that covs are almost exclusively detected in bat feces and not, as hypothesized earlier, in saliva (3, 4, 28, 29) . surprisingly, little virus was found in all fecal samples tested in our study. we estimated the rna concentration per full sample (100 mg feces = 2-4 fecal pellets) to be only up to 4.5 × 10 4 rna copies. human pathogenic viruses transmitted by the fecal-oral route generate much higher virus concentrations in stool, up to ≈10 12 rna copies/mg, e.g., for different picornaviruses (30) (31) (32) . based on these data it would be difficult to postulate that humans can acquire cov from bat feces. however, studies in other locations and at different times are needed to address virus concentration in bat droppings in more detail. because virus in this study was only observed in insectivorous bats and not in frugivorous bats, future studies should investigate whether insects might constitute a source of cov infection for bats. to achieve a direct prediction of the potential of bat covs to infect human cells, it would be highly relevant to conduct virus isolation studies on bat feces. however, in our study we sampled no more than 100 mg of feces per bat. all samples had to be collected in rnalater solution (0.5 ml) (applied biosystems) for reasons of storage and transportation. although it has been suggested that rnalater solution may preserve virus infectivity (33, 34) , our observations showed that the solution has to be diluted at least 1:20 in cell culture medium to avoid cytotoxicity (data not shown). because of the low virus rna concentrations observed, we did not attempt to isolate the virus. however, the absence of successful virus isolation from bat feces in previous studies (3) (4) (5) (6) 8, 11, 12) may not reflect the incapability of bat cov to infect human cells. recently, a synthetic bat cov complemented with an appropriate spike protein has shown potential to infect human cells (35) . reconstruction of phylogenetic and temporal relationships between bat cov and other mammalian cov is another way to obtain information on their zoonotic potential. unfortunately, for cov long sequence fragments must be analyzed before valid phylogenies can be inferred from the conserved nonstructural genome portion (28, 36) . because of the low concentration of rna in bat samples, generation of long sequences from novel bat cov is tedious and technically demanding, which may be why some published phylogenies of bat cov are based on short datasets, making it difficult to use these data for reference. for molecular clock dating, we have therefore relied on reference viruses mainly from other mammals that covered our 1,221-bp fragment in the conserved rdrp region. we assumed that the rdrp would be under less selective pressure than the structural genes and other nonstructural genes, and therefore could be used to infer nucleotide substitution rates over distantly related covs (7, 25, 26, (36) (37) (38) . we have confirmed all tree topologies using alternative methods of phylogenetic inference, including an mcmc algorithm implemented in mrbayes that eliminates artifacts contributed by fixation of mcmc chains in suboptimal prosterior probability maxima (20) . calibration was conducted on reliable isolation dates of prototype and novel bat cov from the literature, as well as on the mrca of the hcov-oc43/bovine cov clade. for dating of only this specific cov clade, a wide range of dated virus isolates has been available that covered as much as 34% ) of the projected time of virus evolution from root to tip (1890-2004) (25) . a probabilistic calibration prior was used, which is favorable for dating in combination with relaxed molecular clock assumptions (39) . the determined mean substitution rates were in good concordance with earlier studies on non-bat-cov that used maximum likelihood-based methods in addition to bayesian inference (25, 26, 38, 40) . although the exponential growth prior on the virus population seemed equivalent with an expansion growth model by the bayes factor test and produced highly compatible mrcas, the exponential model produced a better match with the previously determined mrca of the hcov-nl63/hcov-229e pair (26) . because pyrc et al. generated these data by 3 alternative approaches (bayesian, serial unweighted pair group method with arithmetic mean, maximum likelihood [26] ), we used their mrca to validate our results, and consequently prefer the mrca dating from the exponential growth population model (as presented in figure 3 in plain type). one earlier study on bat and non-bat cov suggested a much faster evolutionary rate for cov than other studies (7) . as vijaykrishna et al. pointed out, their results were associated with large confidence intervals caused by the lack of available data on bt cov at the time the study was conducted (7) . the increase of available sequence data now enables a better account of cov evolutionary history. all covs in our study were found in members of the genus hipposideros (family hipposideridae). the genus rhinolophus from the sister family rhinolophidae was found to host sars-like viruses in several studies in china. one of our hipposideros covs was in a basal phylogenetic relationship with the sars-like clade (group 2b); their most recent common ancestors date back to ≈400 bc. tong et al. (17) have detected a sequence fragment of a bat cov in kenya that also belongs to the 2b clade but is associated with the genus chaerephon, a free-tailed bat that is rather distantly related to the genus hipposideros. although these authors analyzed only a short sequence fragment, their 2b cov seems to be related more closely to sars cov than the virus found in our study. in the many studies conducted in china, only closely related members of the 2b group were detected, with the most basal members dating back only to the 17th century, according to our analysis. the cooccurrence of basal and closely related viruses in africa, as well as the existence of the same virus clade in bats other than those of the family hipposideridae, may entail speculations about a possible origin of the sars-like group of covs in africa rather than in asia. another result that should be integrated with earlier findings is the surprisingly recent date of the mrca of the novel grp1 bt cov and the human common cold virus hcov-229e. further to the proven recent host switching of sars cov, vijgen et al. have suggested that hcov-oc43 entered the human population ≈120 years ago, causing a pandemic (25) . this virus was most likely acquired by humans from domestic cattle. results of our study show that it is not unlikely that hcov-229e, which today is circulating worldwide in humans, resulted from a host switching event not more than 208-322 years ago. however, as with molecular clock dating of viruses, associated confidence limits should not be overlooked. because h. cf. ruber bats are found only in sub-saharan africa and are not migratory (23) , it would be relevant to know how tightly the associated cov is restricted to its host. despite the statistical limitations of our rather small sample size, the absence of cov in bats of the closely related species h. abae that were tested in our study in 2 different caves speaks in favor of tight host restriction. another supportive argument is the absence of cov in c. afra, a bat species sampled in sufficient numbers at the booyem cave. this cave was coinhabited by cov-positive h. cf. ruber bats. if tight host restriction to nonmigratory h. cf. ruber bats existed, this would indicate an origin of hcov-229e within the geographic range of its host, i.e., the rainforest belt and the wet forested savannahs of sub-saharan africa (23) . unfortunately, it will be difficult to reconstruct whether the projected host transition event might have been associated with human epidemic disease. nidovirales: a new order comprising coronaviridae and arteriviridae identification of a novel coronavirus in patients with severe acute respiratory syndrome severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats bats are natural reservoirs of sars-like coronaviruses identification of a novel coronavirus in bats prevalence and genetic diversity of coronaviruses in bats from china evolutionary insights into the ecology of coronaviruses molecular diversity of coronaviruses in bats bats as a continuing source of emerging infections in humans coronavirus antibodies in african bat species detection and prevalence patterns of group i coronaviruses in bats, northern germany detection of group 1 coronaviruses in bats in north america the world's biogeographical regions: cluster analyses based on bat distributions molecular species identification boosts bat diversity bats: important reservoir hosts of emerging viruses bushmeat trade in techiman detection of novel sars-like and other coronaviruses in bats from kenya generic detection of coronaviruses and differentiation at the prototype strain level by reverse transcription-pcr and nonfluorescent low-density microarray molecular evolutionary genetics analysis (mega) software version 4.0 mrbayes 3: bayesian phylogenetic inference under mixed models markov chain monte carlo methods in biostatistics beast: bayesian evolutionary analysis by sampling trees koubek p variation of mitochondrial dna reveals high cryptic diversity in hipposideros caffer complex comparative analysis of twelve genomes of three novel group 2c and group 2d coronaviruses reveals unique group and subgroup features complete genomic sequence of human coronavirus oc43: molecular clock analysis suggests a relatively recent zoonotic coronavirus transmission event mosaic structure of human coronavirus nl63, one thousand years of evolution choosing appropriate substitution models for the phylogenetic analysis of protein-coding sequences detection and phylogenetic analysis of group 1 coronaviruses in south american bats what links bats to emerging infectious diseases? prevalence, types, and rna concentrations of human parechoviruses, including a sixth parechovirus type, in stool samples from patients with acute enteritis circulation of 3 lineages of a novel saffold cardiovirus in humans detection, genetic characterization, and quantification of norovirus rna from sera of children with gastroenteritis the effect of sample type, temperature, and rnalater on the stability of avian influenza virus rna viral infectivity is maintained by an rna protection buffer synthetic recombinant bat sars-like coronavirus is infectious in cultured cells and in mice unique and conserved features of genome and proteome of sars-coronavirus, an early split-off from the coronavirus group 2 lineage the polymerase gene of corona-and toroviruses: evidence for an evolutionary relationship severe acute respiratory syndrome coronavirus sequence characteristics and evolutionary rate estimate from maximum likelihood analysis relaxed phylogenetics and dating with confidence genetic evolution and tropism of transmissible gastroenteritis coronaviruses we are grateful to the voluntary staff at noctalis for support. dr pfefferle is a physician and molecular biologist at the bernhard nocht institute for tropical medicine, hamburg, germany. her research focuses on coronavirus reverse genetics and molecular aspects of coronaviral host switching. key: cord-277309-kelebqr6 authors: wang, lin-fa; anderson, danielle e title: viruses in bats and potential spillover to animals and humans date: 2019-01-18 journal: curr opin virol doi: 10.1016/j.coviro.2018.12.007 sha: doc_id: 277309 cord_uid: kelebqr6 in the last two decades, several high impact zoonotic disease outbreaks have been linked to bat-borne viruses. these include sars coronavirus, hendra virus and nipah virus. in addition, it has been suspected that ebolaviruses and mers coronavirus are also linked to bats. it is being increasingly accepted that bats are potential reservoirs of a large number of known and unknown viruses, many of which could spillover into animal and human populations. however, our knowledge into basic bat biology and immunology is very limited and we have little understanding of major factors contributing to the risk of bat virus spillover events. here we provide a brief review of the latest findings in bat viruses and their potential risk of cross-species transmission. in the last two decades, several high impact zoonotic disease outbreaks have been linked to bat-borne viruses. these include sars coronavirus, hendra virus and nipah virus. in addition, it has been suspected that ebolaviruses and mers coronavirus are also linked to bats. it is being increasingly accepted that bats are potential reservoirs of a large number of known and unknown viruses, many of which could spillover into animal and human populations. however, our knowledge into basic bat biology and immunology is very limited and we have little understanding of major factors contributing to the risk of bat virus spillover events. here we provide a brief review of the latest findings in bat viruses and their potential risk of crossspecies transmission. although there have been significant advances in diagnostics and medical countermeasures during the past century, the risk of cross-species transmission of known and unknown pathogens has emerged as a threat to human and animal populations due to a various factors, including industrialization, intensive farming, urbanization, rapid transportation and climate change [1, 2 ] . it is generally accepted that approximately 75% of emerging infectious diseases for humans are zoonoses [1, 3, 4] . the rate of emergence of novel viruses appears to be increasing as a result of both increased spillover from their natural reservoirs and our improved ability in detection [3] . among the newly emerged and most deadly zoonotic viruses discovered in the past few decades, bat-borne viruses occupy a greater proportion than viruses from any other mammalian order [5 ,6 ,7,8] . several studies have now concluded that bats are exceptional in their ability to act as natural reservoir of viruses and they are able to harbour more diverse viruses per animal species [6 ,9] . while the underlying biology for this observation is yet to be uncovered, it is certain that we will witness more disease outbreaks from bat-borne viruses in the years to come. at the present time, it is impossible to predict the risk of spillover potential for the vast number of viruses or viral sequences which have been detected in bats around the world. but it will be a good start to focus on the viruses in the 'known unknown' category, that is new or variant strains of bat viruses related to those which have already spilled over into and caused diseases in animals or humans. although bats are known to also carry dna viruses, all of the disease-causing and species-jumping bat-viruses are so far limited to rna viruses. in this brief review, we will focus on the major rna virus families harboured by bats that have demonstrated spillover and severe disease-causing potential. bats, order chiroptera, are the only mammals capable of powered flight and are among the most ancient of mammals and underwent extensive speciation for the last 100 million years. there are currently more than 1000 species of bats, making them the second most diverse mammalian group, after rodents, and representing 20% of extant mammalian species [10] . although the recent surge of interest in bats is mainly driven by their association with many of the most lethal viruses, bats are known for their exceptionally long life span and for being less prone to cancer [8] . bats are not only rich in species diversity, but also have great variation in their geographical locations, dietary preferences, physiological range of body temperatures, social behaviour and navigation and vision systems [11] . it is therefore important to recognise that such immense diversity makes it difficult to generalize-specific associations relating to bats and viruses to all members of the chiropteran order. in addition, due to the large number of bats around the world and the fact that similar bats can live in different geographical locations and multiple bat species can co-exist in similar ecological habitats, any virus surveillance or virome detection study should not be viewed as a holistic examination of any given systems. instead, they are more likely to be a transient snapshot of a specialised system at a given time. while recognising that there are limitations on current investigations of viruses in bats, we are also optimistic that with the increasing interest and research activities in this field, we will gain a more accurate panoramic view of bats and viruses in the not too distant future. as a matter of fact, the findings accumulated in the last few decades have already pointed towards a few virus families as being both more prevalent in bats and with proven potential for spillover into other animal species [5 ,12] . the summary below highlights these virus families, followed by other bat viruses which may possess spillover potential and are considered to be important enough to keep on our watch list. coronaviruses were not known to cause severe diseases in humans before the emergence of severe acute respiratory syndrome (sars) coronavirus (cov). the sars outbreak in 2002-2003 remains as one the most impactful pandemic outbreaks of the 21st century mainly due to the fact that the aetiology was totally unknown during the outbreak, which made accurate diagnosis and effective control impossible [13, 14] . the outbreak lasted more than six months with rapid spread of the virus from southern china to more than 30 countries on all major continents, and resulted in more than 8000 human infections and 774 deaths [14] . multiple international teams spent the next decade hunting for the origin of sars-cov and serendipitously found many sars-cov related viruses in bats, most abundantly from the genus rhinolophus (horseshoe bats) [15] [16] [17] . the most conclusive evidence came from the isolation of a cov from bats in china which was more than 98% identical in genome sequence to sars-cov and capable of using the sars-cov receptor, ace2, on human cells [18 ] . while it is not easy to assess the spillover potential of many sars-cov related bat covs due to unsuccessful attempts to isolate the viruses, it should be noted that a 'consensus' virus constructed via reverse genetics pointed to a high probability of human infection [19] . although a significant amount of attention was focused on sars-cov related viruses, the international community was again caught by surprise with the emergence of the middle east respiratory syndrome (mers)-cov in 2012 [20 ] . mers-cov is genetically quite different from sars-cov (figure 1 ), despite both viruses belonging to the genus betacoronavrius. as of october 1, 2018, mers-cov has infected 2249 people in 27 countries with 35% case fatality [21] . camels have been identified as important reservoir hosts for mers-cov and mers-cov related viruses [22,23,24 ,25,26,27 ] , but there is strong evidence that the evolutionary ancestors of these viruses are bats [28, 29, 30 ] . co-circulation and recombination of covs has been implicated as a mechanism that maintains viral diversity and continuous zoonotic transmission [27 ,31] . severe disease outbreaks caused by covs related to viruses associated with bats are not limited to humans. in 2016-2017, there was a major outbreak of swine acute diarrhoea syndrome (sads) in piglets in multiple southern china farms in a region geographically close to where the sars outbreak began in 2002 [32 ] . the origin of the causative agent, sads-cov, was quickly traced back to a bat colony in the vicinity of the pig farms where bat cov with more than 98% genome sequence identity was detected in rhinolophus spp. bats. sads-cov belongs to the genus alphacoronavirus and is genetically most closely related to hku2, a previously reported bat cov [32 ] . human coronavirus 229e is in the same genus, but is only distantly related to those bat covs. examination of pig farmers with close contact with sick and dying piglets did not yield evidence of human infection, hence humans may not be susceptible to sads-cov. further study is required to determine the true zoonotic potential of sads-cov and closely related bat covs. for unknown reasons, despite of the wide presence of covs in bats of different locations and species with relative high viral genome levels, multiple attempts by different international groups to isolate bat covs have been largely unsuccessful. the only successful isolation was achieved with sars-like viruses by direct isolation using vero e6 cells [18 ] or inoculation into the brain of suckling rats [17] . one of the first bat-borne bsl4 agents identified was hendra virus (hev) in australia in 1994 [33 ] , an emerging pathogen that caused the deaths of 7 people. additionally, 103 equine and 2 canine cases have been reported [34, 35] . in humans the case fatality rate from hev infection is 57%. all four species of flying fox in australia (pteropus poliocephalus, p. alecto, p. scapulatus and p. conspicillatus) have been found to be seropositive for hev antibodies and all have detectable virus in their urine with black flying fox (pteropus alecto) being the major reservoir host [36] . a closely related virus, nipah virus (niv) emerged in 1998 in malaysia, which transmitted from bats to humans via pigs as an intermediate and amplifying host [37] . in total, that outbreak resulted in 283 human cases and 109 deaths (39% case fatality) in malaysia as well as 11 cases and one death in singaporean abattoir workers [38] . a related, but not identical virus, was responsible for multiple niv outbreaks in bangladesh/india [39, 40] , with the latest outbreak that occurred in 2018 in kerala and resulted in 19 human infections and 17 deaths [41] . the reservoir hosts of niv have been identified as the large flying fox (p. vampyrus) and small flying fox (p. hypomenalus) in malaysia [42, 43] and the indian flying fox (p. giganteus) in bangladesh and india [44, 45] . after many years of unsuccessful attempts, niv was isolated from the indian flying fox in bangladesh [46] . the current status of henipavirus transmission during outbreaks is summarised in figure 2 . apart from hev and niv, cedar virus (cedpv) remains the only other isolated henipavirus species and experimental evidence from animal trials suggests that cedpv is non-pathogenic for humans [47] . serological evidence of henipavirus infection has been detected in lyle's flying fox (pteropus lylei) populations in southeast asia [48] , large flying fox in indonesia [49] and may be endemic among bat populations on the african continent [50] [51] [52] [53] . additionally, a ghanaian bat henipavirus, kumasi virus (kumpv), genome has been sequenced [54 ] . cumulatively, these studies indicate a wide global distribution of henipaviruses. menangle virus (menpv) is a zoonotic paramyxovirus, first identified in a disease outbreak of reproductive disease in pigs in 1997 at a piggery in new south wales, australia [55, 56] . the virus was also shown to be zoonotic, with 2 piggery workers with high-level exposure developing a serious influenza-like illness and rash during the outbreak. these individuals also developed neutralizing antibodies to menpv [55] . bats were hypothesized to be the source of the outbreak and menpv-neutralizing antibodies were detected in grey-headed flying foxes (pteropus poliocephalus), black flying foxes (pteropus alecto) and spectacled flying foxes (pteropus conspicillatus) [56] . in 2009, menpv was isolated from a bat roost at cedar grove, australia, where black flying foxes were the predominant species in this colony at the time of sampling [57] . tioman virus (tiopv) was isolated from urine of the small flying fox (pteropus hypomelanus) collected from tioman island, malaysia [58] . due the close relationship of tiopv with the zoonotic menpv, an experimental challenge of pigs was performed [59] and the trial suggested that pigs could act as an intermediate or amplifying host for tiopv and that oral secretion is a possible means of viral transmission. the identification of sequences similar to mumps virus (muv) in bats revealed that a virus that was believed to only infect humans has substantial similarity to a counterpart in bats [54 ] . the genetic and functional viruses in bats and potential spillover to animals and humans wang and anderson 81 aside from menpv and tiopv, other paramyxoviruses from the genus rubulavirus have been isolated from or detected in bats without evidence of zoonotic transmission. porcine rubulavirus (porpv), the causative agent 'blue eye' disease in pigs was first identified in mexico in 1980 [64] and serological surveillance data [65] suggests bats are likely reservoir of this virus. mapuera virus (mprpv), a rubulavirus closely related to porpv [66] that has not been associated with any human disease, was isolated from the salivary glands of a healthy fruit bat (sturnira lilium) captured in brazil in 1979 [67] . tukoko virus (thkpv) 1, 2 and 3 are rubulaviruses that have been detected and sequenced from rousettus leschenaultii in china [68] , but these viruses were unable to be cultured in the laboratory and the potential of these viruses to cause disease in humans and animals is yet to be ascertained. in 2012, metagenomic analysis from rna extracted from blood and serum samples of a patient with severe acute febrile illness revealed a novel paramyxovirus [69 ] most closely related to thkpv-3. the novel paramyxovirus was provisionally named sosuga virus (sospv) in recognition of its probable geographic origin (south sudan, uganda). the patient, a wildlife biologist, developed a severe illness after spending six weeks sampling bats and rodents. symptoms included fever, malaise, headache, generalized myalgia and arthralgia, neck stiffness, a metallic taste, sore throat and a maculopapular rash that was present later in the infection. the biologist was discharged after two weeks of hospitalization, but considerable sequelae (myalgia, arthralgia, headache, malaise, and fatigue) persisted for several months [69 ] . bat tissues collected during the period just before the onset of symptoms were tested for sospv, and several egyptian rousette bats (rousettus aegyptiacus) were found to be positive. four additional sospv-positive samples were found in archived tissues from egyptian rousette bats collected at other locations in uganda, suggesting this species could be a potential natural reservoir for this paramyxovirus [70] . although it has been known for more than four decades that ebola and marburg viruses can cause lethal haemorrhagic diseases in humans, the massive outbreak in west africa during 2014-16 was unprecedented with more than 11 000 human fatalities [71] . the ongoing ebola outbreak in the democratic republic of congo is a further indication that more outbreaks in africa [72] . currently, there are five distinctive species identified in the genus ebolavirus, including bundibugyo (bdbv), reston (restv), sudan (sudv), taï forest (tafv) and zaire (ebov). very recently, a sixth species has been proposed, named bombali virus (bomv), based on genome sequence detected in free-tailed bats in sierra leone (chaerephon pumilus and mops condylurus) [73] . both members of the genus marburgvirus, marburg virus (marv) and ravn virus (ravv), have been shown to cause fatal diseases in humans [74, 75] . the third genus, cuevavirus, contains only one species, lloviu virus (llov), whose disease-causing potential in humans is unknown [76, 77] . our group has recently characterized complete coding genome of a new filovirus named while llov, bomv and mlav sequences were first discovered in bats, and marburg viruses have been isolated directly from bats [79 ,80] , the role of bats as a reservoir for ebolaviruses is still debated mainly due to the lack of direct isolation of ebolaviruses from bats [81] . however, the detection of bomv seems to strengthen the notion that bats are likely natural reservoirs of ebolaviruses [73] . reoviruses (respiratory enteric orphan) were not known to be associated with severe human diseases when they were first discovered in the 1950s and about one third of the human population has been exposed to at least one of the mammalian reoviruses (mrvs) [82] . the outbreak of a severe respiratory and enteric disease among different members of a family in melaka, malaysia, changed our appreciation of both the diversity and the zoonotic potential of this class of viruses in the genus orthoreovirus, family reoviridae [83 ] . since the discovery of melaka virus during the 2006 outbreak investigation, at least 15 different strains have been identified, either from human outbreak investigations or bat virome studies [83 ,84-95] , as summarised in table 1 . serological and molecular detection studies suggest that the prevalence of these viruses could be severely underestimated due to the lack of routine diagnosis in hospitals [96, 97] . a plethora of known and novel viruses were identified in samples collected and metagenomically screened from straw-coloured fruit bat (eidolon helvum) in cameroon [98] and neoromicia species in south africa [99] . these bats were shown to harbor divergent viruses, including members of the families astroviridae, circoviruidae, parvoviridae, partitviridae, coronaviridae, picobirnavirdae, adenoviridae, herpesviridae, papillomaviridae, phenuiviridae, and picornaviridae [98, 99] . these recent studies build upon previous work [100] [101] [102] [103] to further expand the diversity of the bat virome. uniquely, the picobirnaviruses identified utilize an alternative genetic code [98] . similarly, there are many other bat-borne viruses with the potential for zoonotic transmission, but without documented human infection. rotaviruses and noroviruses, members of the families reoviridae and caliciviridae, respectively, are the major etiologic agents of acute gastroenteritis and several reports have identified rotavirus [98, 99, [104] [105] [106] or norovirus [107, 108] viruses in bats and potential spillover to animals and humans wang and anderson 83 different bat species worldwide. the caliciviruses recently discovered in bats were found to be antigenically similar to human noroviruses, again highlighting the potential for cross-species transmission [109] . influenza virus is known for zoonotic transmission and two novel subtypes were discovered in bats. in 2012, a new influenza virus genomic sequence was identified in frugivorous yellow-shouldered bats (sturnira lilium) in guatemala and was designated h17n10 [110] . the following year, a distinct influenza genome, classified as h18n11, was characterized from the flat-faced fruit bat (artibeus planirostris) in peru [111] . although virus isolation was not successful, reverse genetics was used to synthetically generate these viruses [112] and subsequent research highlighted the differences between these viruses and conventional influenza viruses. identification of these viruses in bats not only expanded the host reservoir of influenza and the genetic diversity of the viruses, but immediately raised the question about zoonotic potential. studies using synthetic viruses have allowed the identification and characterization of cellular receptors mediating virus attachment and entry, factors important for understanding the tissue tropism and possible zoonotic transmission [112] [113] [114] . hantaviruses are predominantly rodent-borne pathogens and transmission to humans can lead to severe diseases and death. species of hantaviruses have been identified in bats from africa and asia, expanding the potential reservoirs range and genetic diversity of these viruses [115] [116] [117] [118] [119] [120] . hantaan orthohantavirus (htnv) was isolated from two broadly distributed insectivorous bat species (eptesicus serotinus and rhinolophus ferrumequinum) [117] . evidence of a lethal genotype of andes orthohantavirus (andv), araraquara orthohantavirus (arqv), has been documented among several neotropical bats in brazil [118, 120] . arqv is one of the most virulent and lethal among all hantaviruses in humans and viral rna closely related to arqv was detected in urine of the common vampire bat (desmodus rotundus) [119] . these studies highlight that bats are probably playing an under appreciated part on the maintenance, circulation, and transmission of hantavirus in nature. recently, a group of previous unknown bunyaviruses, including severe fever with thrombocytopenia syndrome (sfts) virus and heartland virus (hrtv), emerged during human disease outbreaks [121, 122] . although their animal origins are not known, the most closely related virus has been found in bats in india. in 2010 a novel phlebovirus was isolated from leschenault's rousette bat (rousettus leschenaultii) in western india. the virus was identified by electron microscopy and phylogenetic analysis of the complete genome showed its close relation to sftsv and hrtv [123] . in contrast to new and emerging viruses where viral pathogenesis and transmission route is unknown, rabies has long been recognized throughout history, due to the characteristic symptoms associated with the disease [124, 125] . infection with lyssaviruses, including rabies virus and australian bat lyssavirus leads to rabies disease [126] . although bites and scratches from infected bats occur, there is an effective vaccine and post exposure prophylaxis available for this deadly disease. we know that there are several groups of bat viruses that can infect and cause severe diseases in humans, as we have briefly covered in this review. it is also well established that there are a large number of related viruses circulating in bats in different parts of the globe, but as of yet we are unable to accurately predict which of these viruses are capable of spillover and whether they will cause diseases in humans. we view these viruses as the 'known unknowns'. among the four families of viruses discussed in this review, each has a different combination of characteristics. in terms of frequency, the reoviruses seem to be the most permissive to spillover, especially in asia. however, to date we have only experienced severe, but not lethal, infections in humans. on the other hand, filoviruses and henipaviruses are far the more deadly but the frequency of spillover is relatively low [7, 12] . rabies virus is highly lethal and responsible for a large number of human deaths. but the direct spillover from bats to humans is limited. in contrast, the known genetic diversity of covs in bats is much greater than any of the other bat zoonotic viruses. covs contain the largest genomes of all known rna viruses, and hence are naturally exposed to a higher chance of genetic mutation per genome. to prevent frequent 'lethal mutations', covs have evolved to contain an exoribonuclease which increases the fidelity of rna genome replication [127, 128] . however, the large positive rna genomes of covs are highly prone to gross genetic changes via recombination, which is elegantly illustrated by two recent studies, one on sars-like viruses [129 ] and another on the discovery of a recombinant cov containing a reovirus p10 gene sequence [130 ,131] . the true rate of cov spillover into humans and livestock animals may be greatly underestimated as these occurrences do not always cause severe or lethal disease, demonstrated by serological surveillance. spillover and zoonotic transmission of covs is not limited to bat covs, rather increasing evidence suggests the emergence of new cov strains and the mutation of existing strains resulting in new disease syndromes in both animals and humans [132, 133] . one of the challenging scientific questions is why many of the bat-borne zoonotic viruses are so lethal when they spill over into human and/or livestock animal populations. up until now, our knowledge was very limited in addressing this question due to lack of research tools to conduct comparative immunology and pathogenesis studies in bats. several recent studies, however, start to reveal that bats may have evolved a more balanced innate defence system. on one hand, bats have elevated level of certain defence genes or pathways from type i interferon [134] to apoptosis [135] , at the same time bats exhibit more immune tolerance in different pathways, from inflammation [136] , sting signalling [137] to nk cell activation [138] . however, it should be cautioned that these are very early and preliminary studies, many of them based on genomics and bioinformatics analysis only, more indepth functional studies are required to get a better understanding of the asymptomatic infection of bats by viruses highly lethal in other mammals. the importance of bats as a source of emerging viruses has been proven from numerous studies in the last two decades. while most of the investigation is triggered by zoonotic spillover of bat viruses, the impact of spillover into livestock animals should not be underestimated as shown by the sads-cov outbreak. from the great genetic diversity and wide geographical locations of the various bat viruses detected so far, it is almost certain that we will see more and more disease outbreaks caused by bat viruses. among the 'known unknowns', bat coronaviruses may be a more likely cause of future spillover into both human and livestock populations due to their greater genetic diversity already known in bats around the world, their large positive strand rna genome size with a high rate of recombination, and proven spillover events in both human and animals. we are still at an infancy stage in terms of understanding bat biology in the context of how some of the most lethal viruses can peacefully co-exist with bats, but most recent research findings suggest the key but not in other mammals. although it has been a decade since its publication, this paper remains to be one of the best reviews on this subject. olival kj, hosseini pr, zambrana-torrelio c, ross n, bogich tl, daszak p: host and viral traits predict zoonotic spillover from mammals. nature 2017, 546:646-650. a milestone paper demonstrating the special status of bats as virus reservoir. viruses in bats and potential spillover to animals and humans wang and anderson 85 www.sciencedirect.com current opinion in virology 2019, 34:79-89 bats as a continuing source of emerging infections in humans mass extinctions, biodiversity and mitochondrial function: are bats' special' as reservoirs for emerging viruses? a comparison of bats and rodents as reservoirs of zoonotic viruses: are bats special? a molecular phylogeny for bats illuminates biogeography and the fossil record bat biology, genomes, and the bat1k project: to generate chromosome-level genomes for all living bat species bats and their virome: an important source of emerging viruses capable of infecting humans the severe acute respiratory syndrome bats, civets and the emergence of sars bats are natural reservoirs of sars-like coronaviruses severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats genomic characterization and infectivity of a novel sars-like coronavirus in chinese bats isolation and characterization of a bat sars-like coronavirus that uses the ace2 receptor after more than ten years of continuous investigation, this paper finally demonstrated bats as the true reservoir of sars related viruses sars-like wiv1-cov poised for human emergence isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study evidence for camel-to-human transmission of mers coronavirus middle east respiratory syndrome coronavirus in dromedary camels: an outbreak investigation an important finding indicating that camels are the reservoir or amplifying host of mers-cov in middle east mers coronaviruses in dromedary camels presence of middle east 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into human populations in africa henipavirus neutralising antibodies in an isolated island population of african fruit bats evidence of henipavirus infection in west african fruit bats henipavirus and tioman virus antibodies in pteropodid bats bats host major mammalian paramyxoviruses a very large study demonstrating the great diversity of bat borne viruses in multiple viral families probable human infection with a newly described virus in the family paramyxoviridae. the nsw expert group infection with menangle virus in flying foxes (pteropus spp.) in australia evidence of bat origin for menangle virus, a zoonotic paramyxovirus first isolated from diseased pigs tioman virus, a novel paramyxovirus isolated from fruit bats in malaysia thong wong k: tioman virus, a paramyxovirus of bat origin, causes mild disease in pigs and has a predilection for lymphoid tissues cross-neutralization between human and african bat mumps viruses for the first time, it is demonstrated that a mumps virus 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3, in fruit bats from china novel paramyxovirus associated with severe acute febrile disease, south sudan and uganda although it was limited to a single patient, it demonstrated that the spill over events of bat viruses may occur more frequently than we expected a recently discovered pathogenic paramyxovirus, sosuga virus, is present in rousettus aegyptiacus fruit bats at multiple locations in uganda reflections on early investigations into the ebola virus dr congo ebola virus outbreak: responding in a conflict zone the discovery of bombali virus adds further support for bats as hosts of ebolaviruses characterization of a new marburg virus isolated from a 1987 fatal case in kenya marburgvirus genomics and association with a large hemorrhagic fever outbreak in angola discovery of an ebolavirus-like filovirus in europe re-emergence of lloviu virus in miniopterus schreibersii bats characterization of a filovirus (m englà virus) from rousettus bats in china this is the first time that a novel filovirus representing a new genus is discovered in asia isolation of genetically diverse marburg viruses from egyptian fruit bats filoviruses and bats testing new hypotheses regarding ebolavirus reservoirs. viruses orthoreoviruses and their replication a previously unknown reovirus of bat origin is associated with an acute respiratory disease in humans nelson bay virus. a novel reovirus pulau virus; a new member of the nelson bay orthoreovirus species isolated from fruit bats in malaysia identification and characterization of a new orthoreovirus from patients with acute respiratory infections a novel reovirus isolated from a patient with acute respiratory disease xi river virus, a new bat reovirus isolated in southern china investigation of a potential zoonotic transmission of orthoreovirus associated with acute influenza-like illness in an adult patient virulence potential of fusogenic orthoreoviruses. emerg infect dis rapid whole genome sequencing of pteropine orthoreovirus by modified rolling circular amplification with adaptor ligation -next generation sequencing a new member of the pteropine orthoreovirus species isolated from fruit bats imported to italy first isolation and characterization of pteropine orthoreoviruses in fruit bats in the philippines isolation of pteropine orthoreovirus from pteropus vampyrus in garut serological evidence of human infection with pteropine orthoreovirus in central vietnam pteropine orthoreovirus infection among out-patients with acute upper respiratory tract infection in malaysia cameroonian fruit bats harbor divergent viruses, including rotavirus h, bastroviruses, and picobirnaviruses using an alternative genetic code a metagenomic viral discovery approach identifies potential zoonotic and novel mammalian viruses in neoromicia bats within south africa discovery and genomic characterization of a novel bat sapovirus with unusual genomic features and phylogenetic position discovery of novel virus sequences in an isolated and threatened bat species, the new zealand lesser short-tailed bat (mystacina tuberculata) molecular survey of rna viruses in hungarian bats: discovering novel astroviruses, coronaviruses, and caliciviruses. vector-borne zoonotic dis molecular detection of viruses in kenyan bats and discovery of novel astroviruses, caliciviruses and rotaviruses novel highly divergent reassortant bat rotaviruses in cameroon, without evidence of zoonosis group a rotaviruses in chinese bats: genetic composition, serology and evidence for bat-tohuman transmission and reassortment characterization of a novel g3p[3] rotavirus isolated from a lesser horseshoe bat: a distant relative of feline/canine rotaviruses detection and characterization of a novel norovirus in bats deciphering the bat virome catalog to better understand the ecological diversity of bat viruses and the bat origin of emerging infectious diseases bat caliciviruses and human noroviruses are antigenically similar and have overlapping histo-blood group antigen binding profiles a distinct lineage of influenza a virus from bats new world bats harbor diverse influenza a viruses synthetically derived bat influenza a-like viruses reveal a cell type-but not speciesspecific tropism characterization of uncultivable bat influenza virus using a replicative synthetic virus novel insights into bat influenza a viruses phylogeny and origins of hantaviruses harbored by bats, insectivores, and rodents hantavirus in bat a new natural reservoir of hantavirus: isolation of hantaviruses from lung tissues of bats evidence of hantavirus infection among bats in brazil detection of hantavirus in bats from remaining rain forest in sao paulo, brazil metagenomic analysis of fever, thrombocytopenia and leukopenia syndrome (ftls) in henan province, china: discovery of a new bunyavirus a new phlebovirus associated with severe febrile illness in missouri malsoor virus, a novel bat phlebovirus, is closely related to severe fever with thrombocytopenia syndrome virus and heartland virus rabies in the vampire bat of trinidad, with special reference to the clinical course and the latency of infection the history of the outbreak of paralytic rabies in trinidad transmitted by bats to human beings and the lower animals from 1925 australian bat lyssavirus: implications for public health coronaviruses lacking exoribonuclease activity are susceptible to lethal mutagenesis: evidence for proofreading and potential therapeutics coronaviruses: an rna proofreading machine regulates replication fidelity and diversity discovery of a rich gene pool of bat sars-related coronaviruses provides new insights into the origin of sars coronavirus a very detailed study to show that all building blocks of sars-cov are present among a diverse group of covs in the same cave a bat-derived putative cross-family recombinant coronavirus with a reovirus gene an important finding demonstrating that covs are highly active in recombination, even with viruses outside the coronavirus family the persistent prevalence and evolution of cross-family recombinant coronavirus gccdc1 among a bat population: a two-year follow-up coronaviruses: emerging and reemerging pathogens in humans and animals animal coronaviruses: what can they teach us about the severe acute respiratory syndrome? contraction of the type i ifn locus and unusual constitutive expression of ifnalpha in bats proteomics informed by transcriptomics reveals hendra virus sensitizes bat cells to trail-mediated apoptosis unique loss of the pyhin gene family in bats amongst mammals: implications for inflammasome sensing dampened sting-dependent interferon activation in bats the egyptian rousette genome reveals unexpected features of bat antiviral immunity we thank xiao fang lim, xinglou yang and anna uehara for help with figure 1 , figure 3 and table 1 key: cord-343206-9tqivs5f authors: pruvot, mathieu; cappelle, julien; furey, neil; hul, vibol; heng, huy sreang; duong, veasna; dussart, philippe; horwood, paul title: extreme temperature event and mass mortality of insectivorous bats date: 2019-04-29 journal: eur doi: 10.1007/s10344-019-1280-8 sha: doc_id: 343206 cord_uid: 9tqivs5f a mass mortality event involving chaerephon plicatus and taphozous theobaldi bats occurred during a heat wave in april 2016 in cambodia. this was investigated to clarify the causes of the die-off and assess the risk to public health. field evidences, clinical signs, and gross pathology findings were consistent with a heat stress hypothesis. however, the detection of a novel bat paramyxovirus raises questions about its role as a contributing factor or a coincidental finding. systematic documentation of bat die-offs related to extreme weather events is necessary to improve understanding of the effect of changing weather patterns on bat populations and the ecosystem services they provide. under a changing global climate, the frequency of extreme weather events is expected to increase, in particular, warm temperature extremes (ipcc 2014) . although the effects of climate change on wildlife populations is increasingly documented (fao 2012) , there is still much to learn about the responses of particular species to extreme temperature events, and the potential consequences for their populations and the ecosystem services they support. in 2015-2016, cambodia experienced the worst drought in 50 years amplified by a strong el niño event in the region (unescap 2015) , with temperatures soaring to an all-time high of 42.6°c. in april 2016, a mass mortality of bats was reported from the phnom bok temple in the angkor wat complex of siem reap province, cambodia. the die-off was suspected to be related to the concurrent extreme temperatures. however, as bats can host diverse pathogens of public health concern (wang 2015) , an investigation was undertaken to clarify the causes of the mass mortality and assess the presence of pathogens. the mortality event was reported by local authorities (ministry of environment) to have begun on 10 april 2016. at the time of the site investigation (26-27 april 2016), they reported that over 500 bats had died, particularly around 1-2 pm. last removal of carcasses had taken place 1 week before the visit, and counts of carcasses during the visit revealed 135 adults and 41 pups. these were mainly females and young of chaerephon plicatus (wrinkle-lipped bats), although the advanced decomposition of most individuals precluded systematic documentation of their sex (fig. 1 ). only two taphozous theobaldi (theobald's tomb bat) individuals were observed in distress but still alive. bat carcasses were only observed at one of the five buildings of the temple, whose roof had collapsed. two other buildings (mostly collapsed) did not have bats. the last two buildings containing live bats were located east of the first three buildings, and had intact roofs covered by large trees (fig. 2) . the temperature profiles of the three buildings containing bats, as measured by a digital thermometer (table 1) , greatly differed due to variations in sun exposure and vegetation cover. clinical signs reported by observers included panting, dropping on the ground, and convulsions. the two distressed t. theobaldi bats were clinging to the lower part of the temple wall in full daylight (abnormal behavior), with wings slightly open and panting moderately. these were caught with a net, examined, anesthetized, euthanized, and necropsied (sr-b01 and sr-b02 in table 2 ). body temperatures were slightly above 38°c (table 2) , compatible with normal body temperature ranges observed in many bat species (o'shea et al. 2014) . necropsy only revealed mild and localized hemorrhage on the liver, spleen, and lungs of one animal (sr-b02). another recently dead c. plicatus bat was found clinging to another wall of the same building (sr-b03) and sampled. nucleic acids were separately extracted from liver, spleen, kidney, lungs, and oral and rectal swabs samples collected from the three individual bats (table 2) using the qiagen rneasy mini kit. extracts were all tested using universal viral family-level rt-pcr assays for filoviruses, lyssaviruses, coronaviruses, paramyxoviruses, influenza viruses, flaviviruses, alphaviruses, and astroviruses (sánchez-seco et al. 2001; vázquez-morón et al. 2006; moureau et al. 2007; zhai et al. 2007; chu et al. 2008; tong et al. 2008; atkins et al. 2009; quan et al. 2010; watanabe et al. 2010; anthony et al. 2012) . only one kidney sample from a t. theobaldi individual (sr-b02) tested positive using the paramyxovirus universal assay. subsequent phylogenetic analysis of a~420 nucleotide region of the rna-dependent rna polymerase (l) gene showed that this was a novel bat paramyxovirus (btpv taphozous-theobaldi sr-b02 mh778310) sharing 79% nucleotide identity with viruses of the proposed genus jeilongviruses across this segment of the gene (fig. 3) . this viral group mostly comprises viruses detected from bats and rodents. the presence of mild hemorrhage in the infected individual was consistent with previous reports of other jeilongvirus infection in rodents (jun et al. 1977) ; however, these lesions have also been found in bats suffering from heat shock (welbergen et al. 2008) , and preserved tissues were not available for further histological examination. previous studies have suggested that nutritional and reproductive stressors may lead to increased excretion of the temperature on the building where mortality occurred exceeded 42°c, often reported as a critical atmospheric temperature threshold for bat thermoregulation (licht and leitner 1967) . the absence of mortality in the cooler buildings, the timing of death in the hottest part of the day, the behavior and clinical signs observed, the sex and age ratios (female and young primarily affected), gamma distribution 4 and 1000 bootstrap. the numbers next to the branches indicate the percentage of 1000 bootstrap replicates that support each phylogenetic branch. the tree is drawn to scale, with branch lengths measured in the number of substitutions per site and the non-specific pathology findings were all consistent with previous reports of heat stress events in bats (welbergen et al. 2008) , although most previous reports involved flying-fox species (pteropus sp.) (welbergen et al. 2008 ). other c. plicatus colonies in the battambang province experienced mass mortalities during the same period (furey, n., unpublished data). the absence of data on the initial population size of both species precluded a conclusive assessment of the relative impact on both species. regardless, c. plicatus is a common bat species in cambodia and southeast asia, and provides important ecosystem services (kunz et al. 2011) . for instance, its services in controlling the rice pest white-backed planthopper (sogatella furcifera) were recently valued at 1.2 million usd/year in thailand alone (wanger et al. 2014 ). in addition, the bat guano produced by insectivorous bats is widely used as fertilizer (thi et al. 2014 ) and represents a profitable market. thus, the inability of c. plicatus to cope with extreme temperature events and the potential consequences on long-term population trends may have major impacts on bat communities, ecosystems, and rural livelihood in the region. our findings imply that maintaining vegetation cover around bat colonies may help them to cope with high temperatures. in the context of rapid land-use change in cambodia (peterson et al. 2015) , it is necessary to understand how vegetation cover influences coping mechanisms. systematically documenting mass mortality events is important to understand how bat populations could be affected by a changing environment and climate (welbergen et al. 2008) . it also provides an important opportunity to conduct surveillance of pathogens circulating in bat populations and understand how heatrelated stress may influence the excretion of pathogens hosted by bats. as such, this report contributes to documenting how changes in land-use and weather patterns influence bat population resilience and health. emergence of fatal avian influenza in new england harbor seals characterization of an outbreak of astroviral diarrhea in a group of cheetahs (acinonyx jubatus) novel astroviruses in 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transcription-pcr assays to detect novel paramyxoviruses niño 2015/2016: impact outlook and policy implications rt-pcr for detection of all seven genotypes of lyssavirus genus bats and viruses: a new frontier of emerging infectious diseases bat coronaviruses and experimental infection of bats, the philippines climate change and the effects of temperature extremes on australian flying-foxes rapid molecular strategy for filovirus detection and characterization publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgments we thank the forestry administration, the ministry of environment, and the angkor center for conservation of biodiversity for facilitating this investigation. all procedures performed in studies involving animals were in accordance with protocol no. 15:04 reviewed and approved by wcs's institutional animal care and use committee. access to site and authorization to conduct the study was granted by the ministry of environment of the royal government of cambodia. the authors declare that they have no conflict of interest. key: cord-316343-u1uup5da authors: luo, yun; li, bei; jiang, ren-di; hu, bing-jie; luo, dong-sheng; zhu, guang-jian; hu, ben; liu, hai-zhou; zhang, yun-zhi; yang, xing-lou; shi, zheng-li title: longitudinal surveillance of betacoronaviruses in fruit bats in yunnan province, china during 2009–2016 date: 2018-02-01 journal: virologica sinica doi: 10.1007/s12250-018-0017-2 sha: doc_id: 316343 cord_uid: u1uup5da previous studies indicated that fruit bats carry two betacoronaviruses, batcov hku9 and batcov gccdc1. to investigate the epidemiology and genetic diversity of these coronaviruses, we conducted a longitudinal surveillance in fruit bats in yunnan province, china during 2009–2016. a total of 59 (10.63%) bat samples were positive for the two betacorona-viruses, 46 (8.29%) for hku9 and 13 (2.34%) for gccdc1, or closely related viruses. we identified a novel hku9 strain, tentatively designated as batcov hku9-2202, by sequencing the full-length genome. the batcov hku9-2202 shared 83% nucleotide identity with other batcov hku9 stains based on whole genome sequences. the most divergent region is in the spike protein, which only shares 68% amino acid identity with batcov hku9. quantitative pcr revealed that the intestine was the primary infection organ of batcov hku9 and gccdc1, but some hku9 was also detected in the heart, kidney, and lung tissues of bats. this study highlights the importance of virus surveillance in natural reservoirs and emphasizes the need for preparedness against the potential spill-over of these viruses to local residents living near bat caves. electronic supplementary material: the online version of this article (10.1007/s12250-018-0017-2) contains supplementary material, which is available to authorized users. coronaviruses are enveloped, single-stranded rna viruses that belong to the subfamily coronavirinae, family coronaviridae, in the order nidovirales. based on the genetic distance and serological characterization, the family consists of four genera: alpha-, beta-, gamma-, and delta-coronaviruses (https://talk.ictvonline.org/ictvreports/ictv_online_report/introduction/). coronaviruses are important human pathogens that cause outbreaks of severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) (de groot et al. 2013; drosten et al. 2003) . six human coronaviruses have been identified: human coronavirus 229e (hcov-229e), hcov-oc43, hcov-hku1, hcov-nl63, sars-cov, and mers-cov (hu et al. 2015) . hcov-229e, hcov-oc43, hcov-hku1, and hcov-nl63 are widespread in human populations and known to cause mild respiratory disease, while sars-cov and mers-cov had led to pandemics (channappanavar and perlman 2017) . stronger evidence showed that the direct ancestor of sars-cov, and likely mers-cov, originated in bats. bats are the only mammals capable of flight and represent approximately 20% species of all mammals (hunter 2007) . according to dietary differences, bats are distinguished as insectivores and frugivores (stuckey et al. 2017) . frugivore bats are ideal bushmeat because of huge body and thick-flesh for local people in some districts in africa and southeast asia (mickleburgh et al. 2009 ). meanwhile, frugivore bats in african or pacific countries harbor diversity of virulent viruses, such as marburgvirus, hendra virus, and nipha virus (shi 2013) . in china, crossreactive antibody or phylogenetically related viruses to henipaviruses, ebolaviruses and rabies virus have been detected in chinese fruit bats (he et al. 2015; jiang et al. 2010; li et al. 2008; yang et al. 2017; yuan et al. 2012 ). in addition, genetically diverse reoviruses, adenoviruses, and coronaviruses have been detected or isolated from fruit bats (du et al. 2010; li et al. 2016; tan et al. 2017) . ro-batcov hku9 and ro-batcov gccdc1 are two closely related but distinct betacoronavirus species found in guangdong and yunnan province, respectively. both were found in the chinese brown fruit bat rousettus leschenaulti (huang et al. 2016; lau et al. 2010; woo et al. 2007 ). hku9 includes more variants and are genetically diverse, while gccdc1 is less diverse. the greatest difference between these two viral species is the presence of p10 gene, which is thought to have been obtained from a reovirus, in the gccdc1 genome (huang et al. 2016; lau et al. 2010) . in yunnan province, there are at least three fruit bat species, eonycteris spelaea, r. leschenaultia, and an unclassified rousettus species (he et al. 2015; yang et al. 2017) . these bats frequently cohabitate in the same cave and can only be distinguished by bat experts or molecular identification. in this study, we conducted a longitudinal surveillance of the two betacoronaviruses in fruit bat samples collected during 2009-2016 in yunnan province and reexamined the prevalence, genetic diversity, and host specificity of these viruses. sampling was conducted as described previously (li et al. 2005) . because of conservation concerns, for most captured bats, we collected fecal or anal samples and released the bats after sampling. several bats were sacrificed for species identification and viral tissue tropism assays. bat species were identified based on morphological characteristics and further confirmed by cytochrome b (cytb) sequencing (agnarsson et al. 2011) . all samples were stored at -80°c until further analysis. all animal sampling processes were performed by veterinarians with approval from the animal ethics committee of the yunnan institute of endemic diseases control and prevention. rna was extracted from bat fecal or anal samples using the high pure viral rna kit (roche, basel, switzerland). partial rdrp was amplified using the superscript iii onestep rt-pcr and platinum taq enzyme kit (invitrogen, carlsbad, ca, usa) by family-specific degenerate seminested pcr (luna et al. 2007 ). expected pcr products were gel-purified and subjected to sequencing using the sanger abi-prism platform (applied biosystems, foster city, ca, usa). to exclude pcr contamination, the nucleotide sequences of the virus and bat cytb of positive samples were evaluated by two independent pcrs by different experimenters. the partial rdrp sequences obtained in this study were submitted to genbank under accession numbers mg762619-mg762664 for batcov hku9 and mg762606-mg762618 for batcov gccdc1. qpcr was used to investigate the tissue tropism of these viruses in various tissues. total rna was extracted from the hearts, livers, spleens, lungs, kidneys, brains, and intestines of six bats infected with bat coronaviruses hku9 or gccdc1 using the high pure viral rna kit. partial rdrp representing hku9 or gccdc1 were cloned into the pgem-t-easy vector (promega, madison, wi, usa) and used as a positive control for quantitative analysis. primers for the two different viruses were designed using idt online software (https://sg.idtdna.com/site) (supplementary table s1 ). the assay was carried out in triplicate on a cfx connect real-time system (bio-rad, hercules, ca, usa) with the one-step rt-pcr sybr green kit (vazyme, nanjing, china). the pcr thermal cycling parameters were 50°c for 5 min, 95°c for 10 min, and 40 cycles of 95°c for 5 min, and 60°c for 30 s. an absolute quantitative method was used to determine the number of copies of the viruses referring to the standard control generated from the positive sets. amplification of full-length s, n, and p10 gene primers targeting the s, n, and p10 gene were designed based on alignment of the reported hku9 or gccdc1 sequences (primer sequences provided upon request). the first round of pcr amplification was performed in a total volume of 25 ll using superscript iii one-step rt-pcr (invitrogen) under the following parameters: 50°c for 30 min, 94°c for 5 min; 35 cycles of 94°c for 30 s, 50°c for 30 s, and 68°c for 3 min; and a final extension at 68°c for 10 min. the second round of pcr amplification was performed in a total volume of 50 ll using the platinum taq enzyme kit (invitrogen) under the following conditions: 94°c for 5 min; 35 cycles of 94°c for 30 s, 50°c for 30 s, and 72°c for 3 min; and a final extension at 72°c for 10 min. expected pcr products were gelpurified and sequenced directly using target primers. weak bands were cloned into the pgem t-easy vector and sequenced using the sanger abi-prism platform. fulllength n and p10 sequences were deposited into genbank under the following accession numbers: mg762665-mg762673, mg762688-mg762692, and mg762675-mg762687. one positive sample (id: 2202) was further sequenced using an illumina platform at novogene (beijing, china). briefly, the supernatant of homogenized intestine was centrifuged at 10,0009g for 10 min at 4°c. the supernatant was filtered through a 0.45-lm polyvinylidene difluoride filter (millipore, billerica, ma, usa) to remove eukaryotic and bacterial-sized particles. the filtered samples were then centrifuged at 100,0009g for 2 h. the pellets were resuspended in 140 ll hanks' solution and rna was extracted with the qiaamp viral rna minikit (qiagen, hilden, germany) according to the manufacturer's protocol. sequence-independent pcr amplification was conducted as previously described (ge et al. 2012) . pcr products greater than 500 base pairs were excised and extracted with a minelute gel extraction kit (qiagen). the pcr products were adaptor-anchored, pooled, and sequenced on an illumina platform. the filtered sequence reads were aligned to sequences in the ncbi nonredundant nucleotide database (nt) and nonredundant protein database (nr) downloaded from the ncbi ftp server using blastn and blastx, respectively. all reads matched to coronavirus were extracted and assembled using megahit and trinity software. based on the partial genome sequences of viruses, the remaining genome sequences were determined by inverse pcr, genome walking, and 5 0 -and 3 0 -rapid amplification of cdna ends (race). next, the nucleotide sequence of the full-genome (accession numbers: mg762674) and deduced amino acid sequences of the open reading frames (orfs) were compared to those of related betacoronaviruses. for coronavirus species demarcation, seven independent replicase domains in the orf1ab of the virus were selected for further analysis. partial rdrp sequences, full-length n gene sequences, and full-length genomic sequences obtained in this study were aligned with those of huk9, gccdc1, and related coronaviruses and representative betacoronaviruses using clustalw. the phylogenetic tree was constructed by the neighbor-joining method with mega7.0 software with 1000 bootstrap replicates. according to the structure of the phylogenetic tree, the identities of all sequences from different lineages were calculated using clustalw in megalign. vero e6 and primary intestine cell lines of e. spelaea and r. leschenaulti were used for virus isolation. cells were cultured and inoculated with viral rna-positive samples after tenfold dilution. the cells were incubated in culture medium containing 5% fetal bovine serum. after three blind passages, the cell culture supernatant was tested for the presence of live virus by nested rt-pcr. a total of 555 fecal or anal samples from fruit bats were collected at four locations in yunnan province, china in 2009-2016 ( fig. 1) . by rt-pcr detection targeting partial rdrp, 46 (8.29%) samples were positive for hku9 and 13 (2.34%) were positive for gccdc1 or closely related viruses (table 1) . different sampling times and sites showed different detection rates for hku9. no positive results were detected in samples collected in mengla, 2011 and mojiang in 2013 (table 1 ). hku9 infection rates in chuxiong, mengla, and jinghong were 18.59% (29/156), 5.32% (10/188), and 6.14% (7/114), respectively. gccdc1 was not detected until 2015, with a positive rate of 5.26% in 2015 and significantly high positive rate in 2016 (18.86%) in mengla. the amplified partial rdrp sequences in this study shared 74.4%-100% identity at the nucleotide (nt) level. a phylogenetic tree was conducted based on the alignment of partial rdrp sequences along with previously reported hku9, gccdc1, and related stains, as well as representative strains of other betacoronaviruses. the results revealed 59 sequences classified as two coronavirus species, hku9 or gccdc1 ( fig. 2a) . all sequences from rousettus bats were hku9-related viruses and those from e. spelaea were gccdc1-related viruses. in contrast to the gccdc1 strains which are highly similar, the hku9-related strains were highly diverse. within the hku9 species, the sequences in this study and previously reported sequences were divided into 5 lineages: lineage 1 comprising 28 sequences and previously reported hku9-10-2, hku9-5-2, and hku9-2 exclusively from r. leschenaulti; lineage 2 comprising 5 sequences and previously reported hku9-1 from r. leschenaulti; lineage 3 comprising 10 sequences and previously reported hku9-4 from unidentified rousettus species r. sp.; lineage 4 comprising the previously detected hku9-3, 9-5, and 9-10 from r. leschenaulti; lineage 5 comprising 3 sequences from rousettus species. the other 13 sequences were exclusively from e. spelaea and grouped with previously reported batcov gccdc1 (huang et al. 2016) . to further characterize the relationships between the newly detected coronaviruses, we amplified the full-length sequences of s, n, and p10 gene from selected positive samples. we amplified n from 9 hku9-related viruses and fig. 2 phylogenetic analysis of the detected coronaviruses in this study. partial rdrp sequences (a), complete nucleoprotein gene sequences (b), and full-length genomic sequence of batcov hku9-2202 (c) were aligned with corresponding sequences of representative viral species in the genus betacoronavirus. phylogenetic trees were constructed using the neighbor-joining method implemented in mega7 and bootstrap values calculated from 1000 replicates. the sequence obtained in this study is labeled in color and named by the sample isolate identifier followed by bat species, location, and collection year. 5 gccdc1-related viruses and p10 from 13 gccdc1related viruses. the amplifications of s failed for all positive samples. p10 amplified from this study shared 99%-100% similarity with previously reported sequences (huang et al. 2016) . the amplified n sequences of hku9 and gccdc1-related viruses showed 74.5%-100% and 95.2%-97.4% nt identity with each other, respectively. the phylogenetic tree constructed based on n showed a topology structure similar to that of rdrp (fig. 2b) . the full-length genome sequence was obtained from one sample (batcov hku9-2202) in lineage 5 by highthroughput sequencing and race. the genome of hku9-2202 is 29,118 nt in length excluding the polya tail, with a g/c content of 42%. the main orfs of hku9-2202 were predicted and deduced in the order: 5 0 -orf1ab-spike (s)-ns3-envelope (e)-membrane (m)-nucleocapsid (n)-ns7a-ns7b-3 0 ( table 2 ). the putative transcription regulatory sequences (trss) and their genomic localization were predicted based on the conserved core sequence (5 0 -acgaac-3 0 ) of the trss of betacoronaviruses. notably, in the putative trs of e, there was a difference of one nucleotide with the consensus core sequences (table 2) . comparative genomic sequence analysis indicated that hku9-2202 shared 83% nt identity with other previously reported batcov hku9 strains. the most divergent regions were located in the s protein, which shared only 68% amino acid (aa) identity with those of other batcov hku9. the aa identities of seven concatenated replicase domains, which were selected to define coronavirus species by the international committee on taxonomy of viruses, shared 93% identity with other batcov hku9, which was higher than the new species demarcation of 90%. thus, the newly identified hku9-2202 likely belongs to the batcov hku9 species. to determine the evolutionary position of hku9-2202, the full genome was subjected to phylogenetic analysis. hku9-2202 formed a separate branch within the clade of batcov hku9 species (fig. 2c ). tissues (heart, liver, spleen, lung, kidney, brain, intestine) from five bats positive for coronavirus were quantified by qpcr (fig. 3) . higher virus genome copies were detected in all intestines and varied from 4.89 9 10 2 to 5.67 9 10 6 copies/g in different tissues. three hku9-positive bats (bt9431, bt9446 and bt9466) showed wider tissue tropism, as demonstrated by the presence of viral rna in the kidney, heart, and lung tissues (fig. 3a) . three gccdc1positive bats (bt9444, bt9463, and bt967) showed exclusive intestine tropism (fig. 3b) . the viral rna was not detected in the brain, spleen, and liver tissues. in this study, we conducted a longitudinal study of batcov hku9 and batcov-gccdc1 as well as related coronaviruses in fruit bats in 2009-2016. highly diverse hku9related covs were found in rousettus bats, while gccdc1-related viruses found in e. spelaea showed high similarity. for hku9-related covs, in addition to four (lau et al. 2010 ), a novel lineage was identified in this study. previous studies reported that all group 2d coronaviruses within the betacoronavirus were from r. leschenaulti. in this study, we identified all bat species positive for coronavirus by sequencing the cytb gene and found that hku9 and gccdc1 were from two different genera, rousettus and eonycteris, respectively. hku9 consists 5 lineages. lineage 1 and 2 are from r. leschenaulti and lineages 3-5 are from an unidentified species rousetta sp. these results suggest that the coronaviruses may undergo host restriction and have a long evolution history with their hosts. we amplified multiple n genes and obtained the fulllength genomic sequence of a novel hku9 of linage 5 (batcov hku9-2202). the most notable sequence difference between this novel hku9 and previously identified batcov hku9s is within the s gene. the s protein of hku9-2202 shares 61%-68% aa identity to those of previously identified hku9. the s protein plays a pivotal role in mediating coronavirus entry into host cells. whether mutations in s are responsible for virulence and tissue tropism of hku9-2202 requires further analysis. coronavirus is known to infect the host through the respiratory system and intestines (masters and perlman 2013) . in this study, we found that intestine tissues are the major target of batcov hku9 and gccdc1. however, some hku9 was also detected in the kidney and lung, suggesting that batcov hku9 has wide tissue tropism and the potential to be transmitted by the oral-fecal route and respiratory routes to infect other animals. there are at least five fruit bat species in china, all which are located in tropical regions. these fruit bats feed on fruits and flowers and have frequent contact with peoples and farms, thus increasing the risk of spillover of bat viruses to domestic animals and humans. in our previous study, we also found that these bats harbor novel genetically diverse filoviruses, some of which were found to coinfect with batcov hku9 or gccdc1 in the same individual (huang et al. 2016; yang et al. 2017) . our results improve the understanding of variable viruses carried by fruit bats in china. further studies are needed to investigate the virome of these bat populations and understand the spillover potential of these bat viruses to other animals and humans. a time-calibrated species-level phylogeny of bats (chiroptera, mammalia) pathogenic human coronavirus infections: causes and consequences of cytokine storm and fig. 3 tissue distribution of batcov hku9 (a) and gccdc1 (b) in positive bat samples. immunopathology middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group identification of a novel coronavirus in patients with severe acute respiratory syndrome xi river virus, a new bat reovirus isolated in southern china metagenomic analysis of viruses from bat fecal samples reveals many novel viruses in insectivorous bats in china filovirus rna in fruit bats bat origin of human coronaviruses a bat-derived putative cross-family recombinant coronavirus with a reovirus gene the nature of flight-the molecules and mechanics of flight in animals seroprevalence of rabies virus antibodies in bats from southern china coexistence of different genotypes in the same bat and serological characterization of rousettus bat coronavirus hku9 belonging to a novel betacoronavirus subgroup bats are natural reservoirs of sars-like coronaviruses antibodies to nipah or nipah-like viruses in bats characterization and pathogenicity of a novel mammalian orthoreovirus from wild short-nosed fruit bats generic detection of coronaviruses and differentiation at the prototype strain level by reverse transcription-pcr and nonfluorescent low-density microarray bats as bushmeat: a global review emerging infectious diseases associated with bat viruses bartonella, bats and bugs: a review novel bat adenoviruses with low g?c content shed new light on the evolution of adenoviruses comparative analysis of twelve genomes of three novel group 2c and group 2d coronaviruses reveals unique group and subgroup features genetically diverse filoviruses in rousettus and eonycteris spp. bats, china serological evidence of ebolavirus infection in bats acknowledgements we thank xing-yi ge, shi-yue li, and hui-min huang for assistance in bat sampling. this work was jointly supported by the china natural science foundation (81290341 and 31621061 to zls), united states agency for international development emerging pandemic threats predict project (aid-oaa-a-14-00102), and national institute of allergy and infectious diseases of the national institutes of health (award number r01ai110964) to zls.author contributions zls and xly designed and coordinated this study. rdj, bjh, dsl, yzz, and gjz collected samples. yl, bl, xly performed most of the experiments. zls, xly, yl, bh, and hzl analyzed the data. yl, xly, and zls drafted the manuscript. all authors read and approved the final manuscript. conflict of interest the authors declare that they have no conflict of interest.animal and human rights statement this study was approved by the animal ethics committee of the wuhan institute of virology. all institutional and national guidelines for the care and use of animals were followed. key: cord-274241-biqbsggu authors: shaw, timothy i.; srivastava, anuj; chou, wen-chi; liu, liang; hawkinson, ann; glenn, travis c.; adams, rick; schountz, tony title: transcriptome sequencing and annotation for the jamaican fruit bat (artibeus jamaicensis) date: 2012-11-15 journal: plos one doi: 10.1371/journal.pone.0048472 sha: doc_id: 274241 cord_uid: biqbsggu the jamaican fruit bat (artibeus jamaicensis) is one of the most common bats in the tropical americas. it is thought to be a potential reservoir host of tacaribe virus, an arenavirus closely related to the south american hemorrhagic fever viruses. we performed transcriptome sequencing and annotation from lung, kidney and spleen tissues using 454 and illumina platforms to develop this species as an animal model. more than 100,000 contigs were assembled, with 25,000 genes that were functionally annotated. of the remaining unannotated contigs, 80% were found within bat genomes or transcriptomes. annotated genes are involved in a broad range of activities ranging from cellular metabolism to genome regulation through ncrnas. reciprocal blast best hits yielded 8,785 sequences that are orthologous to mouse, rat, cattle, horse and human. species tree analysis of sequences from 2,378 loci was used to achieve 95% bootstrap support for the placement of bat as sister to the clade containing horse, dog, and cattle. through substitution rate estimation between bat and human, 32 genes were identified with evidence for positive selection. we also identified 466 immune-related genes, which may be useful for studying tacaribe virus infection of this species. the jamaican fruit bat transcriptome dataset is a resource that should provide additional candidate markers for studying bat evolution and ecology, and tools for analysis of the host response and pathology of disease. bats are an ancient and diverse group [1] and are the second largest taxonomic group of mammals with more than 1,200 identified species among the 5,499 known mammals [2, 3] . bats are the only mammals to have evolved powered flight, which has allowed dispersal across all continents other than antarctica. bats are critical components of ecosystems, serving as major predators of insects, pollinating flowers and dispersing seeds of keystone plant species worldwide. the body sizes of bats range from less than 2 gm with 8 cm wingspans to more than 1 kg with 2 m wingspans. most contemporary species of bats are insect-, nectar-, or fruit-eaters, but about 1% are carnivores, including fish-eating and blood-drinking species. the evolutionary origin of bats remains controversial [4, 5] . in early work, bats were thought to be closely related to rodents and primates [6] . bats are now established within laurasiatheria; however, the placement of bats within laurasiatheria has been difficult to resolve because the major groups diverged from one another within a relatively short period of time [7] . different placements recently hypothesized for bats include: (a) sister to perissodactyla (horse) [8] ; (b) sister to cetartiodacyla (cattle+dolphin) [5] , (c) sister to perissodactyla+cetartiodacyla (horse, cattle, dolphin) [9] , (d) sister to ferungulata (cattle+dolphin, dog+horse) [4, 10] and (e) the pegasoferae hypothesis which places bat with perissodactyla and carnivora (horse+dog) [11] (see [5] for a review). two bat genomes have been sequenced to date [12] , the little brown bat (myotis lucifugus, 76 coverage) and the large flying fox (pteropus vampyrus, 2.66 coverage), but neither has been extensively annotated. these species represent the two major clades within bats: the microbats and megabats. transcriptome sequencing for another megabat species, the australian flying fox (pteropus alecto), has recently been published [13] . thus, a transcriptome for a microbat species is needed. many highly pathogenic viruses are hosted, or suspected to be hosted, by bat reservoirs, including ebolaviruses, marburg virus, hendra virus, nipah virus, rabies virus and coronaviruses [2] . in total, more than 100 viruses have been isolated from, or detected in, bats of dozens of species, yet many of the viruses that cause disease in humans cause little or no disease in the bats. significantly, the great majority of bat species have not been examined for infectious agents and are, thus, likely underappreciated as reservoir hosts. the continued encroachment of humans upon bat habitat and bat migrations caused by climate change may lead to novel infectious diseases among humans and livestock. moreover, some infectious diseases cause significant morbidity and mortality in bats that could have dramatic impacts on population numbers and cascading ecological effects [14] . thus, the study of bats and their infectious agents is an important but neglected aspect of zoonotic and wildlife disease research. jamaican fruit bats (artibeus jamaicensis) are one of the most common bats in the tropical americas, ranging from the caribbean islands, tropical south and central america, mexico and the florida keys [15] . the jamaican fruit bat is a microbat in the family phyllostomidae, which contains 56 genera and 192 species. they are a frugivorous generalist and fig specialist of medium size; about 80 mm in length with a wingspan of 130 mm and mass of about 50 grams. they can readily fly 20 km per night, although they typically maintain a smaller home range as long as food is available, and can live 9 years or more in the wild. females typically produce two offspring per year and provide maternal care for about 50 days, with pups reaching adult body weight by about 80 days. several microbes of interest have been isolated from or detected in jamaican fruit bats, including histoplasma capsulatum, trypanosoma cruzi, eastern equine encephalitis virus, mucambo virus, jurona virus, catu virus, itaporanga virus and tacaiuma virus, suggesting the species may be an important reservoir and vector of infectious diseases [15] [16] [17] [18] . it is unknown what diseases these pathogens may cause in bats. tacaribe virus (tcrv) was isolated from 11 artibeus bats (6 a. lituratus, 5 a. jamaicensis) in the late 1950s in and near port of spain, trinidad [19] . tcrv was the first arenavirus isolated in the americas and during the next decades other arenaviruses with substantial similarity to tcrv were identified that cause the south american hemorrhagic fevers (sahf) [20, 21] . the known reservoir hosts for all other arenaviruses are rodents, making tcrv exceptional for its repeated isolation from artibeus bats. because exhaustive searches for evidence of other potential reservoir hosts of tcrv failed to suggest another reservoir species [19, 22] , it has been suspected that artibeus bats are reservoirs of the virus. however, recent work by us demonstrated that trlv-11573, the only remaining isolate of tcrv, causes a fatal infection resembling the sahf in jamaican fruit bats, one of the species from which tcrv was isolated, or is cleared without disease, suggesting this species is not a suitable reservoir host for tcrv and the virus may be a significant pathogen for bats [23] . because of the equivocal role of artibeus bats as a reservoir host species for tcrv, and because of the similarities with human sahf, the jamaican fruit bat may be a novel model for studying the pathology of the disease. however, as an unusual, non-model organism, very little is known about its physiology, immunology or host response to tcrv. no antibodies are available with known specificity to jamaican fruit bat proteins, which dramatically limits its usefulness. to address some of these deficiencies, we have performed transcriptome sequencing and analysis of spleen, lung, kidney and poly-ic-stimulated primary kidney cells to identify genes of interest for assessing the host response to tcrv infection. more than 240,000 454 reads and 142 million illumina reads were obtained ( table 1 ). the reads were submitted to short read archive (sra) under srr539297 and srr538731. reads from lung, kidney, and poly-ic-stimulated primary kidney cell libraries were pooled for a combined de novo assembly using the 454 gs assembler program, yielding 6,450 contigs. for the illumina spleen sequences, we first corrected reads using the soapdenovo correction tool and further assembled them using soapdenovo, yielding 214,707 contigs. a total of 367,317 snps and 44,679 indels were detected through gigabayes. at least 16 reads covering a site were required to ensure the snp was of high quality. using tgicl, a combined assembly of the 454 and illumina contigs was constructed that contained 102,237 contigs with n10, n50, and n90 of 3,882 bp, 1,004 bp, and 289 bp, respectively. human and mouse genomes were used as references to estimate the distribution of bat contigs within known gene transcripts. human and mouse genomes were chosen for completeness of their annotations. genomic features were divided into 5 groups: 1 kb upstream of 59 utr, 59 utr, cds (coding sequence), 39 utr, 1 kb downstream of 39 utr. we found 58.03% and 23.18% mapped cds region for human and mouse genome respectively ( figure 1a ). because we performed transcriptome sequencing, we expected a majority of the sequences to map to cds and utr regions of the genome. many rna genes were also mapped, including long noncoding rnas and a substantial number of micrornas ( figure 1b ). annotation was concentrated on identifying micrornas because they could be cross validated through their rna secondary structure features. to further obtain a confident set of microrna sequences, a microrna prediction pipeline was used to cross validate the blast mapping of prediction. in the process, 42 confident microrna candidates were found that have been deposited within mirbase [24, 25] . we present the list of predicted micrornas as table s1 . mapping the predicted snps on the genomic features indicates that the vast majority of snps are in the cds region ( figure 1c-1d) . although humans and mice are both outside laurasiatheria the relatively fast rate of molecular evolution of mice is expected to result in more differences between bats and mice than bats and humans [26] [27] [28] [29] . the presence of sequences mapped 1 kb upstream or downstream of the known transcript indicated possible alternative splicing from human and mouse transcripts. blast2go was used to functionally annotate contigs. a total of 20,020 contigs (19.58% overall) had significant matches to known proteins in the ncbi non-redundant protein (nr) database. horse and human were identified as the top two species with best blast hits for bat contigs ( figure 2 ). the blastx annotation process is biased by the completeness of the annotation for each respective genome; therefore, despite the lack of a completely annotated horse genome, a high similarity between bat and horse genomes was apparent. the human genome is well annotated, which explains the high number of blast hits between bat and human. the go annotation divides the functional annotation into three main components: biological process, cellular process, and molecular [30] . a majority of the annotated genes encoding proteins that function within a cell or organelle are involved in metabolic and cellular processes. the primary molecular functions of these genes are catalytic and binding activities ( figures 3a-c) . a total of 466 immune-related genes were annotated by blast2go. these immune genes include toll-like receptors, cytokines, transcription factors, kinases and several chemokine receptors. in addition, categorizer was used to categorize the immune class using the goslim database, resulting in 30 categories representing a broad range of immune activities ( figure 4 ). the immune response and lymphocyte activation genes represented the largest proportion of theses transcripts. there were 82,218 unannotated contigs. a total of 16,869 sequences had open reading frames longer than 300 nt; 5,417 were identified through blastp to the nr database with evalue,1e 23 . for the remaining contigs, 54,892 mapped to the assembled myotis lucifugus genome, and 48,809 mapped to the assembled pteropus vampyrus genome. there were 20,145 contigs that mapped to pteropus alecto, australian flying fruit bat, and 18,359 that overlapped between genomic and transcriptome sequences for all three datasets ( figure 5 ). through this process, we were able to account for 65,828 (80%) unannotated contigs. the completeness of genes mapped to immunological pathways was examined using human and mouse as reference species. based on the ortholog data obtained, all contigs were mapped onto immune system related kegg pathways ( table 2 ) and determined that many genes were missing from these pathways. this could be due to the low expression within bat tissues or due to the overly stringent e-value cutoff of 1e 220 during reciprocal blast annotation that we chose to limit the number of false positives. a kegggraph visual representation of contigs mapped onto the mouse pathway was generated (figures s1, s2, s3, s4, s5, s6, s7, s8, s9, s10, s11, s12, s13, s14, s15, s16, s17, s18). pathways involved in the adaptive immune response, t and b cell signalling pathways, generally had more mapped genes than did those involved in innate response or natural killer (nk) cell-mediated cytotoxicity pathways ( figures 6a and 6b ). the nk cell cytotoxicity pathway appears to have almost half of its genes missing, whereas the b cell receptor pathway appears to have most of its genes present. nucleotide substitution in the coding region can be synonymous or non-synonymous. the ratio between the rate of synonymous (ds) and non-synonymous mutation (dn) can be used to infer the degree of selection operating on the system. we used the human genome as a reference for dn/ds calculations because the human genome is well annotated. reciprocal blast was used to identify human, mouse, and bat orthologs. macse was used to generate codon alignments. the alignments were trimmed for excessive gap codon triplets, and paml was used to calculate dn/ds for each gene. when genes are highly conserved, synonymous mutations (ds) tend to be estimated as 0, resulting in a larger dn/ds ratio, therefore those results were removed from the analysis. after filtering, dn/ds results were obtained for 14,717 genes. the majority of the genes have close to zero dn/ds with clear evidence of purifying selection, a feature common among mammalian genes [31] [32] [33] . for investigation of positive selection, tang et al. [34] have argued that a dn/ds threshold of greater than 1 for positive selection might be overly stringent. because of this, a dn/ds cutoff of 0.7 was chosen to investigate genes that might be experiencing weak purifying selection. a total of 138 genes above the 0.7 threshold were found (table s2) . for genes with evidence of positive selection, 32 exceeded the 1.0 dn/ds threshold (figure 7) . through annotation by david [35, 36] , there were 14 genes involved in transcriptional activation and regulation processes. there were 9 genes associated with cellular signaling. in particular, we found dna-damage-inducible transcript 4 (ddit4) gene with dn/ds 1.4053; this protein is involved in the mtor signaling pathway and it regulates cell growth and promotes neuronal cell death [37, 38] . ectodysplasin a (eda), involved with cytokine:receptor interaction pathways, had a dn/ds value of 1.23. the phylogenetic placement of bats within laurasiatheria is still unresolved. through reciprocal blast, we identified 8,785 putative orthologs across mouse, rat, cattle, horse and human (table s3) . afterward we filtered out alignments with greater than 5% gap, the 2,378 genes remaining were used to construct 500 iteration multilocus bootstrap species tree (see methodology). this resulted in a highly supported species tree placing bat sister to the clade containing cattle, horse, and dog ( figure 8 ). the jamaican fruit bat transcriptome described here is a major new resource for genetic studies of bats. this bat is an important seed dispersing and pollinating species found in most of the tropical americas. it is likely susceptible to infectious diseases, could be a zoonotic reservoir and vector, and may be a suitable model for the pathogenesis of sahf. considering the importance of immunological functions in response to infections, we conducted a transcriptome assessment of genes from spleen, kidney and lungs so that genetic tools and methods can be used to study this species as well as other microbats. genes were identified that mapped to immune response pathways; based on categorize classification of immune classes, we found 40 different immune classes. recently, the transcriptome sequencing for the australian black flying fox was performed [13] . our data contain a greater proportion of lymphocyte related immune classes than does the flying fox's transcriptome dataset. however, our dataset also contained a lesser proportion of cytokine related immune classes than the flying fox's transcriptome dataset. genes involved in adaptive immune response generally had more mapped genes compared to genes involved in innate responses. from figures 6a and 6b , more genes were mapped to the b cell receptor signalling pathway than to the nk cellmediated cytotoxicity pathway. this bias is likely due to the large number of b cells found in the spleen. due to our stringent blast criteria, it is also possible that lowering the e-value threshold could obtain additional genes mapped but at the risk of more false positives. we deposited 42 microrna genes for a. jamaicensis into mirbase, and according to mirbase this gene set is the first deposited bat microrna genes. estimates of substitutions within the orthologous contigs found 32 genes with a dn/ds ratio.1. this ratio provides a guide for indicating potential genes that are under positive selection. many genes were involved in transcriptional activation/regulation processes, suggesting potential differences in the transcriptional regulating architectures of bats and humans. the ddit4 and eda have a dn/ds ratio.1, suggesting these genes are under positive evolutionary selection. ddit4 is involved in regulation of cell death and its positive selection suggests a potential difference in cell death regulation between human and bats; further analysis will need to be performed to verify the functional differences. another potential positively selected gene, eda is associated with ectodermal dysplasia type 1 [39] , a disorder associated with abnormal development of physical structures, including skin, hair, nails, teeth, and sweat glands. we suspect the bat's eda gene could be used as a potential reference for future studies of the disorder. for transcripts that failed to be identified by reciprocal blast searches, we predicted the orf for the unannotated contigs and used blastp against the nr database to identify 5,349 unannotated contigs. for the remaining unannotated contigs, we used genomic data from myotis lucifugus and pteropus vampyrus, as well as transcriptome data from pteropus alecto to identify additional unannotated contigs. existing artibeus contigs that were not present within the nr database, but overlapped among myotis and pteropus genomic and transcriptomic sequences indicated the possibility for bat specific transcripts. we also found contigs that mapped only to the myotis lucifugus genome indicating the possibility for microbat specific contigs. in total, we were able to account for 80% of the unannotated trancripts, and the remaining unannotated transcripts likely include misassembled contigs, contigs not sequenced sufficiently in the other bats to be included in their genome assemblies, as well as a few transcripts specific to artibeus jamaicensis. many additional analyses are warranted to further refine the transcriptome information from artibeus and other bats. phylogenomics is an important tool for resolving the tree of life, and this transcriptome data set provides an opportunity to study the evolutionary history of bats. bats were once thought to be closely related to primates [6] ; however, further work using molecular information placed them within laurasiatheria [40] . our finding of bat as sister to the clade containing horse, dog, and cattle is consistent with the recent study by mccormack et al. [4] and zhou et al [10] . here, we used 2,378 loci from a microbat and species tree analyses to obtain 95% bootstrap support, whereas mccormack et al. use 683 loci to obtain 64%bootstrap support. a recent study by nery et al. [5] obtained a concatenated data from 3,733 loci from megabat with 100% bootstrap support and 1.0 posterior probability placing bat as sister to cattle. our phylogenetic tree is less resolved than nery et al., probably because we did not include the more limited transcriptome data available from dolphin and hedgehog. maximum likelihood analyses are powerful, yet can lead to incorrect conclusions in certain situations, whereas species tree analyses are less powerful but more robust to well-known violations of the models used for maximum likelihood phylogenetic analysis, such as incomplete lineage sorting (see [5] and references therein). additional work is clearly warranted, especially by using additional taxa, testing for convergence and specific violations of gene-tree models, and other sources of conflict among protein-coding genes and other portions of the genome. a principal difficulty for identifying mechanisms of pathogenesis of the sahf, in which the immune response may play a contributory role, is a lack of animal model resources that faithfully recapitulate human disease [41] . although laboratory mice (mus musculus) and rats (rattus norvegicus), which have substantial experimental methodologies and reagents, can be infected with junã­n virus (junv), the etiologic agent of argentine hemorrhagic fever, the pathogenesis is markedly different than human disease. the guinea pig (cavia porcellus) typically exhibits signs of disease that closely resembles human disease; however, there are few immunological or genetic tools for assessing the host response to infection. junv is also a bsl-4 and select agent, thus use of virulent strains is confined to only a few laboratories with highly specialized containment facilities. the pathogenesis of tcrv, a bsl-2 agent, in jamaican fruit bats exhibits many similarities to the sahf in humans, thus the use of transcriptome data could be useful for studying pathogenesis using a variety of new technologies, such as pcr arrays for pathway discovery, and for the development of antibodies to specific artibeus proteins that are important in the pathogenesis of disease. the transcriptome resource provided will facilitate research into artibeus host responses to infectious agents, including mechanisms of pathogenesis of arenavirus disease and will also provide further resource for additional understanding for the bat species evolution and physiological development. all procedures were approved by the unc institutional animal care and use committee (iacuc) and were in compliance with . substitution estimation scatter plot. we calculated the nonsynonymous mutation rate (dn) and synonymous mutation rate (ds) using orthologous genes between bat and human. two lines were drawn representing the two dn/ds cutoffs of 0.7 (green) and 1.0 (blue). doi:10.1371/journal.pone.0048472.g007 figure 8 . unrooted species tree from the orthologous dataset across six boreoeutheria mammals. the species tree was generated from 2378 gene loci. there was 95% bootstrap support for placing bats (chiroptera) sister to perissodactyla, cetartiodactyla, and carnivora. doi:10.1371/journal.pone.0048472.g008 the usa animal welfare act. unc animal care and use committee approval number, 1207c-ra-b-15. five bats from the university of northern colorado jamaican fruit bat colony were used for this work. male and female a. jamaicensis bats were euthanized by respiratory hyperanesthesia followed immediately by thoracotomy. tissues were aseptically removed and flash frozen in liquid nitrogen for subsequent rna extraction. tissues were homogenized in buffer rlt (rneasy kit, qiagen, valencia, ca) containing 2me using a bead beater and silicone beads. the homogenate was passed over a qiashredder column prior to total rna extraction according to manufacturer's instructions. for cell culture, one kidney from one bat was collected in serum-free hbss and minced under aseptic conditions, then trypsinized (trypsin-versene) at room temperature in a sterile 50 ml trypsin flask. cells were washed 36 in 10% fbs-emem, then seeded into a vented t-25 flask. the next day, unattached cells were removed and fresh 10% fbs-emem added. when cells approached confluence they were passaged with trypsin at a split ratio of 1:4. poly-ic was added to 50 mg/ml in two t-75 flasks containing 20 ml each of 10% fbs-emem and incubated for 6 hours, after which rna was extracted according to manufacturer's instructions (qiagen). total rna was extracted using the rneasy minelute cleanup kit (qiagen) and then shipped on dry ice to seqwright (houston, tx) for cdna library construction and sequencing. rna concentrations and quality were assessed by a260/a280 and a260/a230 absorbance values and agarose gel electrophoresis. a260/a280 values were all above 2.0 and a260/a230 were all above 1.9. electrophoresis of the rna samples demonstrated that 28s and 18s rrna were not degraded. libraries for the 454 were prepared from three tissues (kidney, lung, poly-ic-stimulated kidney cells). for 454 library construction, full-length cdna was synthesized with two set of primers for driver and tester cdna [42, 43] . single-stranded cdna was used for hybridization instead of double-stranded cdna. excess amounts of sense-stranded cdna hybridized with antisense-stranded cdna. after hybridization, duplex was removed by hydroxyapatite chromatography. normalized tester cdna was re-amplified with tester specific primer l4n. driver cdna was unable to amplify using l4n. an illumina truseq rna library was made from spleens according to manufacturer's instructions. the libraries were then sequenced according to manufacturer's recommendations: 454 using titanium chemistry and illumina using 26100 nucleotide paired-end sequencing on a hi-seq 2000. the 454 and illumina libraries were assembled individually and also by combining both libraries. bases from the 454 reads were called from the 454 generated sff file using pyrobayes [44] and 454 gs assembler (version 2.5) was used to perform the assembly. soap denovo [45] (version 1.04) was used to assemble reads obtained from spleen (illumina library). only contigs greater than 200 bases were used in the final analysis. prior to performing the combined assembly, duplicates from pre-assembled contigs of lung, kidney and spleen tissues were removed with cd-hit [46] (cd-hit-2009-0427) at default criterion and then combined into longer fragments with tgicl [47] . gigabayes [48] -a short-read snp/indel discovery program was used to detect polymorphisms. snp/indel detection was performed for both libraries separately. to make snp/indel predictions more reliable, we used the criterion that minor allele and major allele (alleles with fewer reads are minor alleles, and alleles with more reads are major alleles) occur at least twice and 8 times for 454 and illumina libraries, respectively. to identify the approximate relative position of conserved mammalian genes, we mapped the bat contigs on to the genome of mouse mm9 and human grch37 (downloaded from the ucsc genome browser) using blat v.34 [49] with a minimum score of 80 used as a filter. coordinates of the protein coding genes were obtained from ensemble (http://uswest.ensembl.org/index.html) xenoref and gtf files. we also normalized the number of blat hits based on the total annotated transcript regions (1000 nt upstream of 59 utr, 59 utr, cds, 39utr, and 1000 nt downstream of 39utr) that were present in the mouse and human. to predict precursor-microrna genes within assembled sequences, we downloaded precursor micrornas for mouse, rat and human from mirbase [24, 25] . we performed a blast search focused on high quality candidates, hits with $95% sequence identity [50] . based upon rnafold [51] secondary structure prediction, we further filtered out sequences that did not possess any hairpin loop structure. previously, it had been demonstrated that micrornas tend to have deterministic folding [52] and, therefore, we used unpaired structural entropy (use) to evaluate the rna secondary structure base pairing distribution (cutoff 0.83 use score). mir-abela, a support vector machine learning program [53] , was used to cross validate the prediction. the final remaining unfiltered sequences are considered as highly confident microrna candidates. orthologous contigs (against human, mouse, dog, cattle and horse) were identified using the reciprocal blast (blastn) approach [54] as it has been found to be superior to sophisticated orthology detection algorithms [55] . a stringent cutoff of 1e 220 was used to separate paralogs from orthologs. cdna sequences from human (homo_sapiens.grch37. 64 the substitution rate is inferred from orthologous genes between bat and mouse. sequences were aligned using macse [56] and an in-house java script was used to trim/remove codon gap triplets from the alignment. substitution rate was estimated using a maximum likelihood method implemented in the codeml program of paml 4.5 [57, 58] . the pairwise maximum likelihood analyses were performed in runmode-2. estimated rates of non-synonymous to synonymous substitutions (dn/ds) were plotted as a scatter plot. blast2go [59] was used to functionally annotate contigs. a combined graph was generated for each go category. to prevent overloading graphs, the sequence filter value was changed to 500 in all 3 categories (biological process, molecular function and cellular component). functional annotation was performed separately for all assembled contigs present in the combined assembly. based on categorizer [60] , we further classified the genes using the go slim database immune classes. the completeness of mapping the bat genes using euarchontoglires as a reference was further examined through kegg. to do this, we first downloaded the xml file of annotated kegg pathways [61, 62] for human and mouse. to identify genes that are functionally important within kegg pathways, kegggraph was used to represent a graph form of the kegg pathway. we further used kegggraph to compute the relative betweenness centrality, which is the algorithmic representation of the involvement of a node within a network. we chose to set a cutoff of grabbing the top 4 nodes within each network, or selecting the top 4 functionally important genes within each pathway [63] . open reading frame was predicted from the assembled contigs through the orfpredictor web server (http://proteomics.ysu.edu/ tools/orfpredictor.html) [64] . a customized java program was used to parse through the prediction to identify sequences longer than 300 nt. to perform additional annotation of the predicted open reading frame we used blastp with an e-value of 1e 23 against the most recent nr database that is available from ncbi during our analysis (august 26 th , 2012). using contigs that were functionally unannotated, we compared the jaimacan fruit bat contigs against three other available bat sequence dataset. myotis lucifugus and pteropus vampyrus genomes were downloaded from the ncbi tracedb ftp server (ftp://ftp.ncbi.nih.gov/pub/tracedb/). the pteropus alecto transcriptome was obtained from dr. a. papenfuss [13] . an evalue threshold of 1e 25 was used to indicate blast hit. we then used an r package venndiagram [65] for displaying the mapped unannotated contigs that overlapped between different bat genome and transcriptomes. to resolve the evolutionary relationship for the artibeus bat species, we filtered the putative bat orthologs between human, mouse, dog, cattle, and horse. insectivores such as hedgehog and dolphin were not used in our analysis due to limited gene annotation in these taxa. to obtain the best multiple sequence alignment for each putative orthologs, we used aqua's pipeline for performing multiple sequence alignment; the pipeline consists of multiple sequence alignment through muscle and mafft which is refined by rascal and assessed by normd [66] [67] [68] [69] . a customized java program was used to filter alignments (obtained through aqua) with greater than 5% gap per sequence. additionally, we filtered for sequences that are at least.1,000 bp long. phyml 3 was used to generate a maximum likelihood gene tree [70, 71] . mraic, a perl script wrapper for phyml, was used to infer the best substitution model for each gene tree based on aic, aicc, bic, and akaike weights [72] . aic was used as the objective function since not much variation was observed across different objective function. njst was used to calculate the unrooted species tree based on our gene trees [73] . a customized rprogram is used for performing a nonparametric bootstrap species tree through resampling nucleotides within loci as well as resampling the loci within the data set as described by seo [74] . fossil evidence and the origin of bats bats: important reservoir hosts of emerging viruses iucn red list version 2011.2: tabel 3a -status category summary by major taxonomic group (animals) ultraconserved elements are novel 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protein-coding sequences the gene ontology categorizer natural selection on protein-coding genes in the human genome a scan for positively selected genes in the genomes of humans and chimpanzees positive selection on the human genome a new method for estimating nonsynonymous substitutions and its applications to detecting positive selection systematic and integrative analysis of large gene lists using david bioinformatics resources bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists rtp801 is elevated in parkinson brain substantia nigral neurons and mediates death in cellular models of parkinson's disease by a mechanism involving mammalian target of rapamycin inactivation rtp801 is a novel retinoic acid-responsive gene associated with myeloid differentiation mutations within a furin consensus sequence block proteolytic release of ectodysplasin-a and cause x-linked hypohidrotic ectodermal dysplasia parallel adaptive radiations in two major clades of placental mammals junin virus. a xxi century update construction of a uniformabundance (normalized) cdna library construction and characterization of a normalized cdna library pyrobayes: an improved base caller for snp discovery in pyrosequences de novo assembly of human genomes with massively parallel short read sequencing cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences tigr gene indices clustering tools (tgicl): a software system for fast clustering of large est datasets a general approach to single-nucleotide polymorphism discovery blat-the blast-like alignment tool a uniform system for microrna annotation fast folding and comparison of rna secondary structures analyzing modular rna structure reveals low global structural entropy in microrna sequence identification of clustered micrornas using an ab initio prediction method gapped blast and psi-blast: a new generation of protein database search programs phylogenetic and functional assessment of orthologs inference projects and methods macse: multiple alignment of coding sequences accounting for frameshifts and stop codons paml 4: phylogenetic analysis by maximum likelihood paml: a program package for phylogenetic analysis by maximum likelihood blast2go: a universal tool for annotation, visualization and analysis in functional genomics research categorizer: a web-based program to batch analyze gene ontology classification categories kegg for integration and interpretation of large-scale molecular data sets kegg: kyoto encyclopedia of genes and genomes kegggraph: a graph approach to kegg pathway in r and bioconductor orfpredictor: predicting proteincoding regions in est-derived sequences venndiagram: a package for the generation of highly-customizable venn and euler diagrams in r muscle: multiple sequence alignment with high accuracy and high throughput mafft: a novel method for rapid multiple sequence alignment based on fast fourier transform aqua: automated quality improvement for multiple sequence alignments rascal: rapid scanning and correction of multiple sequence alignments morephyml: improving the phylogenetic tree space exploration with phyml 3 estimating maximum likelihood phylogenies with phyml model selection and multi-model inference estimating species trees from unrooted gene trees calculating bootstrap probabilities of phylogeny using multilocus sequence data we thank jason shaw for assistance with bat handling and stephanie james for laboratory assistance, and uga's georgia advanced computing resource center and institute of bioinformatics for computational resources and support. we thank anthony papenfuss for providing the assembled transcriptome for p. alecto. contributed reagents/materials/analysis tools: ts tg ll. wrote the paper: tis tg ts. key: cord-284367-cy61pjcb authors: muleya, walter; sasaki, michihito; orba, yasuko; ishii, akihiro; thomas, yuka; nakagawa, emiko; ogawa, hirohito; hang’ombe, bernard; namangala, boniface; mweene, aaron; takada, ayato; kimura, takashi; sawa, hirofumi title: molecular epidemiology of paramyxoviruses in frugivorous eidolon helvum bats in zambia date: 2013-12-31 journal: j vet med sci doi: 10.1292/jvms.13-0518 sha: doc_id: 284367 cord_uid: cy61pjcb in this study, we describe the detection of novel paramyxoviruses from the eidolon helvum species of fruit bats. we extracted rna from 312 spleen samples from bats captured in zambia over a period of 4 years (2008–2011). semi-nested rt-pcr detected a total of 25 (8%) positive samples for paramyxoviruses which were then directly sequenced and analyzed using phylogenetic analysis. among the positive samples, seven novel paramyxoviruses were detected. five viruses were closely related to the genus henipavirus, while two viruses were related to the unclassified bat paramyxoviruses from ghana and congo brazzaville. our study identified novel henipavirus-related and unrelated viruses using rt-pcr in fruit bats from kansaka national park and indicated the presence of similar bat paramyxoviruses originating from wide geographic areas, suggesting the ability of bats to harbor and transmit viruses. the presence of these viruses in fruit bats might pose a public health risk. in the past 10 years, a lot of attention has been given to bats as reservoirs of emerging zoonotic viruses. this has been as a result of the high detection rate of previously unknown viral sequences in bats coupled with the emergence of pathogens, such as hendra, nipah, severe acute respiratory syndrome (sars)-corona, ebola and marburg viruses, all of which are highly virulent and pose a great zoonotic risk [2, 3, 8, 9, 17] . bats, being the only flying mammals with ancient evolutionary origins and long life span, are capable of covering great distances during migrations, rendering them suitable hosts and reservoirs for various viruses. paramyxoviruses from the family paramyxoviridae have been implicated in several human epidemics and mortalities [6, 10, 19] . several studies have indicated bats as potential natural reservoirs of paramyxoviruses, such as henipavirus-, respirovirus-and morbillivirus-related viruses [1, 4] . this undoubtedly presents a threat to the health of the hu-man population in areas where human beings live in close proximity to fruit bat species [7] . in zambia, straw-colored fruit bats (eidolon helvum) annually converge in kasanka national park (knp). in this study, we investigated the presence of paramyxoviruses in the eidolon helvum bats captured over a period of 4 years (2008-2011) from knp (s15:34.688 e28:16.513). during that period, a total of 312 spleen samples were collected from the same number of bats (table 1) . appropriate research permits and hunting licenses were obtained from the zambia wildlife authority (zawa). total rna was isolated from spleen tissues using trizol (life technologies, carlsbad, ca, u.s.a.) according to the manufacturer's instructions. a semi-nested broad spectrum rt-pcr targeting the paramyxovirus polymerase (l) gene was used to screen total rna samples (n=312) for paramyxoviruses using par-f1, par-f2 and par-r primers and pcr conditions described by tong et al. [16] . a total of 25 samples out of 312 bat spleens (8%) were positive for paramyxoviruses on semi-nested pcr. the positive products (584 bp) were then purified using the monofas purification kit (gl sciences, tokyo, japan), according to the manufacturer's instructions. the purified pcr products were then subjected to cycle sequencing reactions using the big dye terminator v3.1 system (life technologies) and the par-f2 and par-r inner primers. ethanol precipitation was used to remove the labeled dntps from cycle sequence products and subjected to electrophoresis in the abi 3130 genetic analyzer (life technologies). phylogenetic analysis was performed using reference sequences and positive samples by aligning all sequences using clustalw1.6 followed by the creation of a mega file format created using mega ver.5.2 [14] . the neighbor joining method was used to generate the phylograms [12] with a 1,000 bootstrap replicate confidence level [5] . to compute the evolutionary distances, the maximum composite likelihood method was used with the number of base substitutions per site as units [15] . samples ab853101, ab853102, ab853104, ab853105 and ab853094 showed a nucleotide homology of 73% with the nipah virus (af212302), while ab853106 and ab853096 had a nucleotide homology of 74% with the unclassified bat paramyxovirus (bat pv) (jn648087) from ghana. the relatively low nucleotide homology might indicate that these sequences originate from novel paramyxoviruses. the samples from zambia formed clusters with the henipavirus-related viruses and with the unclassified bat paramyxoviruses (fig. 1) . within the henipavirus-related virus cluster, two groups (a and b) were observed. group a comprised novel zambian strains closely related to the nipah (nc002728, fn86955 and af212302) and hendra (af017149 and nc001906) viral sequences, while group b comprised a cluster of zambian strains, in close relationship with an unclassified bat pv from ghana (jn648085) and cedar virus (jq001776) (fig. 1) . the remaining zambian strains, including the novel ab853106 sequence, formed a cluster with the unclassified bat pv sequences from ghana (jn648078, jn648081, jn648087 and jn648089) and congo brazzaville (he647835 and he647837) (fig. 1) . the close relatedness of the viral sequence from ghana and congo brazzaville strains with those from zambia might imply the ability of bats to harbor and transmit similar viruses over long and diverse geographical distances. this is facilitated by their ability to migrate, covering thousands of kilometers to their hibernation and feeding sites [11] . along their migratory path, they interact directly or indirectly with several terrestrial mammalian species in different geographical locations, thus enhancing the interspecies transmission of viruses [11] . humans can become exposed to these viruses through environmental contamination with urine and feces from bats. although paramyxovirus infections derived from bats have been reported in humans in bangladesh [13] , none have been reported in africa. the absence of cases might be as a result of under-reporting. as such continued surveillance and assessment of the zoonotic risk posed by these viruses still remains important. in order to isolate the detected viruses, spleens from pcr positive bats were homogenized in minimum essential media (mem) followed by centrifugation at 1,000 × g for 3 min. the supernatant was then applied to vero e6 cell with 70-80% growth confluence. the vero e6 cells were cultured in mem with 2% fetal bovine serum (fbs) and 2% antibiotic-antimycotic (life technologies). the inoculated vero e6 cell cultures were then incubated at 37°c for 21 days, coupled with cell passage and microscopic examination. however, after several passages, cytopathic effects were not observed. we also performed semi-nested rt-pcr to detect paramyxovirus rna in the supernatants of the inoculated cells. however, no positive signals were detected. isolation of paramyxoviruses using vero e6 cells has successfully been reported [18] , implying that vero e6 cell lines might be suitable for isolation of paramyxoviruses. in this study, the small amount of virus in the supernatant or the failure to successfully remove the virus from infected cells using the freeze and thaw technique might be responsible for the absence of cytopathic effects in the cell culture. in a study by wilkinson [18] , out of 8 positive samples, virus isolation was only successfully carried out in two samples. furthermore, serological examination of bat sera may provide important information about their exposure to specific infections. unfortunately, however, our study did not carry out any serological test. in conclusion, we report the identification of novel henipavirus-related (n=5) and unrelated (n=2) viruses in fruit bats from the knp using rt-pcr. the viruses identified in this study were shown to originate from wide geographical areas, and their presence in fruit bat species might pose a public health risk and as such, continued surveillance of these viruses in fruit bats in essential. the results presented are for the semi-nested rt-pcr-based paramyxovirinae detection system. samples shown in this table were collected from the spleens of eidolon helvum fruit bats. co-circulation of diverse paramyxoviruses in an urban african fruit bat population ebola outbreak killed 5000 gorillas nipah virus: a recently emergent deadly paramyxovirus bats host major mammalian paramyxoviruses confidence limits on phylogenies: an approach using the bootstrap henipaviruses: emerging paramyxoviruses associated with fruit bats novel paramyxoviruses in free-ranging european bats fruit bats as reservoirs of ebola virus bats are natural reservoirs of sars-like coronaviruses henipavirus vaccine development food availability and annual migration of the straw-coloured fruit bat eidolon helvum the neighbor-joining method: a new method for reconstructing phylogenetic trees breaking the chain in bangladesh mega5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods prospects for inferring very large phylogenies by using the neighbor-joining method sensitive and broadly reactive reverse transcription-pcr assays to detect novel paramyxoviruses bats and viruses: a brief review identification of novel paramyxoviruses in insectivorous bats of the southwest indian ocean bats as a continuing source of emerging infections in humans acknowledgments. we thank ms. yamanouchi, research center for zoonosis control, hokkaido university, for technical assistance. we also wish to thank the zambia wildlife authority and the kasanka trust for providing permission and the enabling environment for sampling the fruit bats. this study was supported in part by grants from the ministry of education, culture, sports, science and technology (mext); the ministry of health, labour and welfare, japan; the japan initiative for global research network on infectious diseases (j-grid), mext japan. key: cord-265357-3f0xph0y authors: halczok, tanja k.; fischer, kerstin; gierke, robert; zeus, veronika; meier, frauke; treß, christoph; balkema-buschmann, anne; puechmaille, sébastien j.; kerth, gerald title: evidence for genetic variation in natterer’s bats (myotis nattereri) across three regions in germany but no evidence for co-variation with their associated astroviruses date: 2017-01-05 journal: bmc evol biol doi: 10.1186/s12862-016-0856-0 sha: doc_id: 265357 cord_uid: 3f0xph0y background: as bats have recently been described to harbor many different viruses, several studies have investigated the genetic co-variation between viruses and different bat species. however, little is known about the genetic co-variation of viruses and different populations of the same bat species, although such information is needed for an understanding of virus transmission dynamics within a given host species. we hypothesized that if virus transmission between host populations depends on events linked to gene flow in the bats, genetic co-variation should exist between host populations and astroviruses. results: we used 19 nuclear and one mitochondrial microsatellite loci to analyze the genetic population structure of the natterer’s bat (myotis nattereri) within and among populations at different geographical scales in germany. further, we correlated the observed bat population structure to that of partial astrovirus sequences (323–394 nt fragments of the rna-dependent rna polymerase gene) obtained from the same bat populations. our analyses revealed that the studied bat colonies can be grouped into three distinct genetic clusters, corresponding to the three geographic regions sampled. furthermore, we observed an overall isolation-by-distance pattern, while no significant pattern was observed within a geographic region. moreover, we found no correlation between the genetic distances among the bat populations and the astrovirus sequences they harbored. even though high genetic similarity of some of the astrovirus haplotypes found in several different regions was detected, identical astrovirus haplotypes were not shared between different sampled regions. conclusions: the genetic population structure of the bat host suggests that mating sites where several local breeding colonies meet act as stepping-stones for gene flow. identical astrovirus haplotypes were not shared between different sampled regions suggesting that astroviruses are mostly transmitted among host colonies at the local scale. nevertheless, high genetic similarity of some of the astrovirus haplotypes found in several different regions implies that occasional transmission across regions with subsequent mutations of the virus haplotypes does occur. electronic supplementary material: the online version of this article (doi:10.1186/s12862-016-0856-0) contains supplementary material, which is available to authorized users. with their particular social, ecological, physiological and immunological traits, bats provide unique hosts for many viruses to co-evolve with (e.g. [1, 2] ). indeed, bats are increasingly recognized as reservoirs for a wide range of viruses, some of which carry a zoonotic potential, for example rabies and other viruses of the genus lyssavirus, sars-like, mers-like and other coronaviruses [2] [3] [4] [5] . consequently, several studies have investigated the genetic co-variation between different bat species and their associated viruses [6, 7] . however, much less is known about genetic co-variation of viruses and different populations of the same bat species, although such information is required to gain a better understanding of the transmission dynamics within a given host species (e.g. [8, 9] ). the natterer's bat (myotis nattereri kuhl 1817 sensu lato [10] ) is a non-migratory vespertilionid bat that is widespread throughout europe with the exception of the iberian and italian peninsula and the south of france [11] . while this species uses underground sites for hibernation during the winter months, it mostly roosts in trees and buildings during summer forming maternity colonies that consist of female bats and their juveniles as well as occasionally some males [12, 13] . males typically roost either individually or in small groups in the vicinity of the maternity colonies [14] . male and female natterer's bats have been found to exhibit philopatry even though males leave their natal colony but stay in its vicinity [13] . mating takes place during autumn at swarming sites that are typically up to 50 km away from the summer colony [13] . recently, various viruses have been reported to be harbored by m. nattereri [15] , including herpes[16] , lyssa[17] and astroviruses (e.g. [18] ). the astroviridae form a large family of non-enveloped, positive-sense, singlestranded rna viruses [19] . astroviruses are mostly transmitted via the fecal-oral route [20] and may cause diarrhea in many animal species, including humans [21] . however, the route of transmission in bats has not yet been elucidated. even though astroviruses have been detected in a variety of species [22] , bats have been hypothesized to be a potential reservoir host in europe and asia [7, 23] . the high prevalence and diversity of astroviruses harbored by bats is remarkable [18, 21, 23] and their capability to cross species barriers and become adapted to new hosts, including spill-over to other taxa, has been suggested [24] . due to the occurrence of astroviruses in animals in close contact to humans, e.g. livestock and also bats using human habitation as roosting, it has been argued that astroviruses should be considered as potential candidates for zoonotic infections (e.g. [24] ). however, almost nothing is known about the transmission of astrovirus among different populations of their bat hosts. we studied patterns of population genetic structure and dispersal of myotis nattereri within and among three geographic regions of germany using both nuclear and mitochondrial microsatellite markers. the population genetic structure of m. nattereri has previously only been investigated in the united kingdom (uk; [13] ). however, the population genetic structure of bats occurring in the uk may be affected by their insular status, and some important differences between insular and continental populations have been described (e.g. [25] ). thus, our study adds new important insights into the dispersal behavior of m. nattereri in mainland europe. moreover, we investigated for the first time if genetic covariation occurs between populations of a bat host and its harboured astroviruses. fischer et al. [18] reported distinctly higher similarities in astrovirus sequences of samples collected from the same bat species in different geographic localities than between samples from different species sampled at the same locality, whereas different results were obtained in the czech republic for some other european bat species [26] . because our analyses are based on the sequences found by fischer et al. [18] in m. nattereri, we assume that, similarly to coronaviruses in chinese bats [27] , astroviruses are mostly transmitted within natterer's bats rather than within the local bat community as a whole. we hypothesized that if virus transmission between host populations were associated with events linked to gene flow in the bats, e.g. mating [28] , genetic co-variation should be detectable between host populations and astroviruses on a larger scale (e.g. between geographic regions), even though not necessarily within a certain region. many bat astroviruses form distinct phylogenetic clusters [21] , but little is known whether astrovirus population structure matches that of their host species. as astroviruses are hypothesized to be transmitted via the fecal-oral route [20] , both direct and indirect virus transmission within the breeding colonies of bats and at swarming sites during mating seem possible [8] . in comparison, for bat ectoparasites such as bat flies (nycteribiidae) that are also transmitted both by direct body contact and indirectly through the bats' roosts, a more efficient transmission among different bat populations has been detected at swarming sites as compared to breeding colonies [29] . as swarming sites represent the main mating sites for m. nattereri, where gene flow occurs, it is expected that if viruses are also mainly transmitted between conspecifics during that period, the transmission route of the viruses should resemble the pattern of host gene flow. since transmission of astroviruses at swarming sites would cause viruses to be transmitted between members of different colonies visiting a given swarming site, local differentiation should not occur at the colony level but rather on a larger scale. viral genetic patterns should therefore follow an isolation-by-distance pattern using swarming sites as stepping-stones for gene flow, as suggested for the bat host [30] . sampling occurred between 2010 and 2014 from maternity colonies in three regions in germany: bavaria (by, n = 92 adult females, one colony), north rhine westphalia (nrw, n = 100 adult females, five colonies) and mecklenburg western pomerania (mv, n = 172 adult females, ten colonies; fig. 1 ). bats were either taken directly out of the bat boxes provided for the colonies or captured using mist nets or harp traps when leaving the colonies' roosts. three millimeter wing tissue samples were collected and stored in 90% ethanol until dna extraction. in addition, between 2012 and 2014, samples of bat saliva, feces and urine were opportunistically taken within the three regions of interest (by, n = 177; nrw, n = 74; mv, n = 19) and screened for the presence of astrovirus-related rna (additional file 1: table s3 ). genomic dna was extracted using an ammonium acetate precipitation method [31] . individuals were genotyped using 19 nuclear microsatellite markers and one mitochondrial microsatellite marker. the following nuclear markers were used: ef15 [32] ; b22 [33] ; a2-mluc, a13-mluc, e5-mluc, g6-mluc, g30-mluc, g31-mluc [34] , d15, h19, h23, h29 [35] ; mnatt-1, mnatt-2 [36] ; mschreib3 [37] ; mnatt-8, mnatt-11 [38] ; fv5ap [39] and gzbyr (5′-tccttgtcactataagctcagtgg-3′ (forward); 5′-ccaggcaatagtctcctagcac-3′ (reverse)). the 5′ end of the reverse primers fv5ap and g30-mluc were pig-tailed [40] with the sequence gttt and the 5′ end of the reverse primer g31-mluc with the sequence gtttt to facilitate adenylation. these 19 autosomal microsatellite markers were amplified in two multiplex polymerase chain reactions (pcrs, table 1 ). pcrs were carried out in 9 μl reaction volumes using the qiagen multiplex kit (qiagen, hilden, germany). each multiplex reaction contained 1 x qiagen multiplex master mix and between 0.11 μm and 1.06 μm of each primer. after drying 1 μl of dna (approximately 10 ng) for 15 min at 52°c in a 96-well pcr plate (vwr), multiplex reactions were performed. the pcr amplification was carried out in a 2720 thermal cycler (applied biosystems), with an initial 5 min denaturation at 95°c, followed by 30 cycles with denaturation at 95°c for 30 s, annealing at 60°c for 90 s and extension at 72°c for 1 min. final incubation occurred at 60°c for 30 min. the mitochondrial dna marker at-2 [41] was amplified in a separate pcr. after drying 1 μl of dna (approximately 10 ng) for 15 min at 52°c in a 96-well pcr plate (vwr), this pcr was carried out in 10 μl reaction volume which contained 0.2 μm of primer at-2, 1.0 mm dntps, 0.8 mm of mgcl 2 , 1.0 μl 10 x taq buffer b1 (solis, biodyne, tartu, estonia) and 1 unit of taq hot firepol® dna polymerase (solis, biodyne, tartu, estonia). this pcr amplification was carried out in a 2720 thermal cycler (applied biosystems), with an as in by only one colony was sampled, no close-up is provided initial 15 min denaturation at 95°c, followed by 40 cycles with denaturation at 95°c for 30 s, annealing at 53°c for 60 s and extension at 72°c for 1 min. final incubation occurred at 72°c for 7 min. pcr products were separated using an abi 3130 genetic analyzer (applied biosystems) together with the internal size standard genescan 500 liz (applied biosystems) and analyzed using genemapper v 5.0 (applied biosystems). since it has been reported that the presence of closely related individuals within populations can bias bayesian multi-locus clustering methods [42] , we removed closely related individuals from the dataset before conducting population genetic structure analyses using the program structure [43] . for this purpose, we first determined the degree of relatedness between all pairs of individuals within a population using trioml [44] , as implemented in the coancestry 1.0.1.5 software package [45] . relatedness densities were further plotted using r (r core [46] ) in order to determine the relatedness threshold for excluding individuals from the analyses. this threshold was selected manually by best separating the first peak of the plotted distribution (i.e. closely related individuals) from the rest (unrelated individuals). from every pair of individuals with a relatedness value exceeding the determined threshold (0.3), one individual was randomly removed, respectively. all other analyses, except for structure, were performed on the whole data set. as uneven sampling can bias inferences on the number of clusters in the program structure [47] , efforts were made to have comparable number of individuals from the three regions investigated after the removal of closely related individuals (71 from by, 73 from nrw and 104 from mv; cf. results). as preliminary runs using the original structure model showed limited population structure (additional file 1: figure s2 ), structure [43] was run on the nuclear dna dataset assuming admixture and correlated allele frequencies using the locprior model that allows for the use of sample group information (here the colony) in the clustering process [48] . thus, twenty independent runs of k = 1-10 were conducted for the whole dataset after removing closely related individuals as mentioned above. additionally, twenty independent runs of k = 1-10 were run for each dataset within a sampling region (nrw, mv and by), respectively. all runs used 10 6 iterations after a burn-in period of 10 5 . for each of the genetically distinct populations inferred by structure the significance of deviations from hardy-weinberg equilibrium (hwg, [49] ) and linkage disequilibrium between loci was tested in genepop 4.0.7 [50] . the false discovery rate (fdr) correction method was used to deal with multiple testing [51] . to assess the level of genetic diversity, the observed (h o ) and expected heterozygosity (h ex ) for each locus as well as for each population inferred by structure for the complete data set were calculated using genetix 4.05.2 [52] . the mean number of alleles (a) and the allelic richness (a r ) were calculated for each locus and each subpopulation using fstat v.2.9.3 [53] (table 1) . differences in the number of alleles per locus, allelic richness and expected and observed heterozygosities between the populations inferred by structure were tested for significance using the wilcoxon signed-rank test in r. micro-checker 2.2.3 [54] , set for 10 000 iterations and a 95% confidence interval, was used to test for null alleles. population pairwise f st values [55] on the whole dataset including closely related individuals, were used to measure the level of genetic differentiation between the populations inferred by structure. for both the nuclear and the mitochondrial dna data set, a hierarchical population structure was assumed where colonies were clustered within regions. f st values were thus calculated using hierarchical analyses within the r-package hierfstat [56, 57] . genetic structure was tested among colonies within sampling regions with more than 1 colony (mv and nrw) and among sampling regions. the significance of the f-statistics was tested by 10,000 permutations. isolation-by-distance for the entire set of 17 colonies was tested via mantel's test [58] from the comparison of all pairwise f st /(1-f st ) values with pairwise geographic distances using the r package ecodist [59] with 10,000 permutations. the test was performed for the whole dataset as well as within regions consisting of several colonies (nrw and mv). a total of 270 samples obtained from saliva, feces and urine of myotis nattereri were screened for the presence of astroviruses by using a published hemi-nested pcr protocol [21] for the amplification of a highly conserved region of the rna dependent rna polymerase gene (rdrp). this pcr assay enables the detection of a variety of bat-associated astroviruses by using degenerated primers. further details of the virus-related sampling and laboratory are presented in fischer et al. [18] . a total 57 sequences representing 19 different astrovirus haplotypes (=sequences having 100% identity) (n1-19, fig. 4) were identified from the screening of 270 samples. three haplotypes were excluded from the analyses (n17-19, fig. 4 ) as they were phylogenetically too distant from the remaining 16 and too rarely encountered (only once per haplotype) to make biologically meaningful inferences about their correlation to the bat host's population genetic structure. out of the 270 samples, fischer et al. [18] were able to assign 73 individual sequences to a specific haplotype (n1-19, fig. 4) , whereas the remaining astrovirus positive samples were shorter than 279 nt and could therefore not be assigned to a haplotype. as in this study we only analyzed individually marked adult female bats that were clearly identifiable via their rfid-tag, in total 57 individual sequences, representing these 16 different haplotypes, were used (n1-16, fig. 4) . the genetic distances between the different astrovirus haplotypes were calculated using the tamura-nei model implemented in mega 6.0 [60] . further, the sequences were translated into amino acids and amino acid genetic distances were computed using the p-dist method implemented in mega 6.0 [60] . this latter measure was tentatively used to differentiate viral species following the recommendation of the astroviridae working group of the international committee for taxonomy of viruses (ictv astroviridae working [61] ), proposing that mean amino acid genetic distances (p-dist) of the full length orf2 larger than 0.284 distinguish different species. as we found no shared identical sequences between regions (by, mv and nrw), a permutation test was conducted using r (r core [46] ) to test how likely such a pattern would be expected by chance (additional file 1: database s1). moreover, the program poptree2 [62] was used to generate genetic distance matrices for the nuclear and mitochondrial dna datasets based on the d a distances [63] of population microsatellite allele frequencies within a priori populations. for the astrovirus dataset, we first used the program jmodeltest 2.1.7 [64] to calculate likelihood scores for substitution model selection. genetic and amino acid distances were then calculated using mega 6.0 [60] based on the maximum composite likelihood substitution model with gamma correction for among-site rate heterogeneity and an estimated proportion of invariable sites. associations between astrovirus and bat host genetic distances (both mitochondrial and nuclear) were first analyzed via a mantel test [58] using the software genalex 6.501 [65] . to control for the possible effect of geographic distance, we also carried out partial mantel tests in passage v 2.0.11.6 (9999 permutations, [66] ). the geographic distance matrix used was calculated from the gps coordinates of the different sampling sites using the create option in passage. for the genetic correlations between host and astrovirus sequences the nrw dataset was modified. due to significant population genetic structuring on the basis of mtdna and the fact that the sampled colonies in nrw are up to 46 km apart from each other compared to maximally 6.5 km in mv (see fig. 1 ) we split the nrw data set in four separate sampling units (nrw2, nrw3, nrw5 and nrw6; fig. 1 ). here, we only used genetic data from the colonies within nrw where virus sequences were detected. together with mv and by, the total dataset for comparing host and virus population structures now consisted of six populations between which pairwise genetic distances were computed as mentioned above. after removing closely related individuals at the threshold of 0.3, the nuclear dna dataset consisted of samples from 248 bats in total, including 71 from by, 73 from nrw and 104 from mv. using the 19 autosomal microsatellite loci, structure inferred the presence of three distinct genetic clusters ( fig. 2 ; additional file 1: figure s1 ), splitting our data set into the three sampled regions nrw, mv and by. no additional sub-structuring was detected by structure within any of these three sampling regions (data not shown). the 19 autosomal microsatellite loci had between 4 and 24 alleles and an average of 10.2-11.7 alleles per each of the three genetically distinct populations inferred by the program structure (table 1) . no significant deviations from hardy-weinberg equilibrium were detected in these three populations. deviations from linkage disequilibrium at the α = 0.05 level (after fdr correction) did occur consistently in all three regions between two loci (mnatt-2 and mnatt-11, table 1 ) but were not detected in all respective colonies (linkage occurred in by, in three out of ten colonies in mv and in three out of five colonies in nrw). because of the inconsistencies at the colony level, we decided to nevertheless keep both loci for further analyses. no marker with consistently appearing null alleles was found within nrw and mv, whereas in by the two loci ef15 and fv5ap showed the presence of null alleles. however, since the estimated frequency of null alleles per locus was low (<0.1), we kept those loci. mean expected and observed heterozygosity were globally similar across colonies and regions (tables 1 and 2 ). the number of alleles found in mv was significantly higher than in by (p = 0.0020) and nrw (p = 0.0284), but by and nrw did not differ significantly from each other. moreover, allelic richness was significantly higher in mv as compared to by (p = 0.0124) but no significant differences were found between by and nrw and between nrw and mv. the overall level of differentiation among the three regions was weak but significant (f st = 0.0088, p = 0.0001) based on nuclear dna estimated using hierarchical f-statistics. significant genetic differentiation was further identified among colonies within regions (f st = 0.0194, p = 0.0001). when analyzing the data obtained by the mitochondrial dna marker at-2, pairwise genetic differentiation was found to be much higher at both geographic scales (among regions: f st = 0.4979, p = 0.0001; among colonies within regions: f st = 0.3657, p = 0.0001). when correlating genetic differentiation f st /(1-f st ) with ln of geographic distances between all colonies within our study area, a significant pattern of isolationby-distance was detected (mantel r = 0.2989, p = 0.0160; fig. 3 ). in contrast, the isolation-by-distance patterns within the nrw (mantel r = -0.2762, p = 0.7869) and the mv population (mantel r = 0.2275, p = 0.0535) were not significant, even though in mv there was a similar trend visible as in the entire data set. we found no overlap in the detected haplotypes between the three regions (fig. 4) . the permutation test revealed that the probability of having no such overlap by chance was very low (p = 0.007; 100,000 permutations). however, a closer look at the genetic relationship of the different astrovirus haplotypes (fig. 4) revealed that sequences of high similarity occur in different geographic regions. genetic distances in astrovirus haplotypes that cluster together (n1-3, n4-6, n7-9, n10-11, n12-15; fig. 4 ) ranged between 0.0033 and 0.0313, whereas genetic distances among clusters were distinctly higher (0.2093-0.6895). as for the amino acid genetic distances (p-dist), distances ranged between 0.0221-0.0588 and 0.5732-0.6786 within and between clusters, respectively, the latter being typical of species differences. rough estimations of divergence times using an astrovirus mutation rate of 3 × 10 −3 [67] indicate that differences within clusters have occurred within 1-10 years, versus 70-230 years for between-cluster divergence time. furthermore, the astrovirus genetic distances neither correlated with those of the host's nuclear dna nor with those of the host's mitochondrial dna. this was true both for the mantel tests and for the partial mantel test correcting for geographic distance (table 3 ). in this study, we analyzed the population genetic structure of m. nattereri within and among summer colonies at different geographical scales in germany and correlated it with that of the astrovirus sequences found in the respective host colonies. our aim was to assess whether the population structure and dispersal patterns of host populations can explain the genetic structure of astrovirus sequences and thus ultimately shed light on the virus transmission dynamics within a given bat species [8, 9] . our findings show significant population structure in m. nattereri with the detection of three genetic clusters which correspond to the three regions sampled (nrw, mv and by). both the results obtained using the bayesian clustering approach and the f st values between the three genetic clusters show the existence of significant population genetic structure. the detection of an isolation-by-distance pattern over the whole study area combined with the continuous presence of the species across germany suggests that the levels of gene flow are not high enough to prevent some population differentiation [68] . the observed strong mitochondrial substructure and weak but significant structure at the nuclear level, likely results from differences in effective population size and female philopatry combined with male-mediated gene flow. differences in patterns of genetic structure in mitochondrial and nuclear dna have been reported in many european bat species and were associated with male-biased dispersal [41, 69] . according to previous studies in m. nattereri in the uk [13, 30] , both sexes fig. 3 isolation-by-distance analysis for data from 19 nuclear microsatellite loci from myotis nattereri within germany. the graph displays the significant correlation between genetic differentiation and ln of geographical distance (in km) for all pairwise comparisons of colonies. genetic distance was measured as rousset's f st /(1-f st ) and the relationship with geographic distance was tested using a mantel test with 1000 permutations. the line represents a linear regression of this relationship and only serves an illustrative purpose appear to be highly philopatric to their natal area but visit central swarming sites during autumn for mating. according to rivers et al. [30] , this pattern results in the same genetic pattern as permanent male dispersal [13] . in connection with the existing overall population isolation-by-distance pattern detected and the absence of a significant pattern on a local scale, i.e. within a region, we suggest that individuals from different summer colonies meet and mate at swarming sites within each region (e.g. [13, 70] ). this would result in gene flow following a stepping-stone model and would both lead to a significant isolation-by-distance over larger scales [71] and to the absence or a weak signal on a local scale [30, 33] . within the uk, isolation-by-distance was not detected between summer colonies of m. nattereri unless distances exceeded 100 km [30] . our results agree with those obtained by rivers et al. [30] as we also did not detect significant genetic isolation-by-distance between summer colonies within a given region in germany (even though there is a trend in mv), but over the study area as a whole. therefore, we suggest that the isolation-by-distance pattern observed here is generated by swarming sites acting as stepping-stones for gene flow. in our study area, identical astrovirus haplotypes harbored by m. nattereri do not overlap between geographic regions (by, mv and nrw, fig. 4 ). based on the partial sequence of the conserved rdrp gene analysed in this study [21] , where the mean amino acid distances ranged from 0.022 to 0.059 within and 0.573-0.679 between clusters, the analyzed haplotypes form six different groups which might represent six different viral species [61] (c1-6, fig. 4 ). the detection of the same putative viral species in the different regions combined with the estimated divergence times (<10 years for within species) suggests occasional transmission between host populations. both the observed genetic population structure of m. nattereri and existing data from ringing studies in germany [72] show that natterer's bats rarely if ever move over long distances (more than 100 km). thus, it is unlikely that individual bats directly transmit a certain virus haplotype between the three geographic regions analyzed (nrw, by and mv). since viruses have a considerably higher mutation rate [73] compared to the bat host, mutations in the virus sequences do occur within much shorter time scales than mutation in the bats' genome [74, 75] . as bats need to transmit the astroviruses directly, the movement of viruses across the landscape should mirror the movement of the bats and hence occur successively over large distances following the stepping stone model of the host. as a consequence, virus transmission over large distances is likely to take multiple years. in the course of these successive transmissions events, mutations will occur in the virus which will lead to viral population differentiation as we observe within the putative viral species. no association was found when correlating the genetic distances of the different astrovirus sequences with their bat host genetic distances. we had originally expected that if the transmission route of astrovirus sequences resembles the pattern of host gene flow, genetic covariation between astroviruses and host populations should be detected, especially across regions. the reason why no such correlation was found for m. nattereri and its associated astroviruses could be due to strong differences in population size (hence genetic drift) and mutation rate between bats and viruses. in contrast to higher eukaryotes such as the bat host, rna viruses are subject to higher selective pressures and combined with a high mutation rate allow continuous and rapid adaptation to changing environmental conditions [73, 76, 77] . coupled with large population sizes, virus evolution can thus already be observed within very short time scales of weeks to months [74, 75] . the frequent fluctuations in the prevalence of viral populations (e.g. bottlenecks) and hence the higher genetic drift they face might prevent these populations from showing patterns of isolation-bydistance [78] . moreover, our virus sampling could only be performed during the summer period since only some of the autumn swarming sites are known so far. additionally, due to logistic reasons exact sampling dates differed between the sampling localities, which could blur the signal if some haplotypes or putative viral species are more abundant in different periods (see additional file 1: table s1 ). finally, although large from a virology perspective, the number of samples with viral material was relatively limited to perform population genetics analyses. this combined with the variations in sampling times could confound our analysis if viruses show quick temporal variation in prevalence and/or turnover. a further possible explanation for the lack of a genetic correlation between hosts and viruses is that at swarming sites bats may not only interact in ways that lead to gene flow. multiple mating and contact between individuals that does not result in successful mating might also represent transmission opportunities for viruses that are not reflected within the host genetic pattern. in summary, our findings suggest that for m. nattereri within germany, swarming sites act as steppingstones for gene flow, as indicated by an overall isolation-by-distance pattern and the absence of such a significant pattern on a local scale. the observed population genetic structure indicates that no apparent strong barriers to gene flow exist within our study area for the bat host. while putative viral species were mostly shared between geographic regions, no haplotypes were shared for any putative viral species. despite the observed genetic differentiation between the three geographic regions in the bat host and to a certain extent also in the harbored astroviruses, we did not detect a correlation between host and virus genetic distances. this could potentially be due to differences in genetic drift, selective pressure, population size and mutation rate between bats and viruses. further studies with a higher astrovirus sample size and with specific simultaneous sampling during autumn mating at swarming sites are required to shed further light on the host-virus relationship between bats and their astroviruses. additional file 1: supplementary material. figure s1 . summary of the log-likelihood values from the 20 independent runs conducted with structure for the number of genetic clusters (k) set to a minimum of 1 and a maximum of 10. the left graph shows the log-likelihood results of the runs for each k, whereas the right graph shows delta k plotted against k. the most likely number of genetic clusters is three using both methods. figure s2 . comparisons of the structure runs for k = 3 with (top) or without (bottom) the locprior option. table s1 . life history, ecology and longevity in bats bats as reservoirs of severe emerging infectious diseases lagos bat virus transmission in an eidolon helvum bat colony bats: important reservoir hosts of emerging viruses bat origin of human coronaviruses host phylogeny constrains cross-species emergence and establishment of rabies virus in bats detection of diverse astroviruses from bats in china population genetic structure of the daubenton's bat (myotis daubentonii) in western europe and the associated occurrence of rabies spatial epidemiology: an emerging (or reemerging) discipline genetic analyses reveal further cryptic lineages within the myotis nattereri species complex die fledermäuse europas: kennen, bestimmen, schützen: kosmos uk: harpercollins autumn swarming behaviour of natterer's bats in the uk: population size, catchment area and dispersal habitat preference, range use and roosting ecology of natterer's bats (myotis nattereri) in a grassland-woodland landscape european bats as carriers of viruses with zoonotic potential discovery of herpesviruses in bats novel papillomaviruses in free-ranging iberian bats: no virus-host co-evolution, no strict host specificity, and hints for recombination insectivorous bats carry host specific astroviruses and coronaviruses across different regions in germany astrovirus research: essential ideas, everyday impacts, future directions ecological drivers of virus evolution: astrovirus as a case study novel astroviruses in insectivorous bats pathogenesis of astrovirus infection amplification of emerging viruses in a bat colony astrovirus infections in humans and animals -molecular biology, genetic diversity, and interspecies transmissions evidence of recent population bottlenecks and inbreeding in british populations of bechstein's bat, myotis bechsteinii detection of diverse novel bat astrovirus sequences in the czech republic. vector borne zoonotic dis prevalence and genetic diversity of coronaviruses in bats from china parasite co-structure: broad and local scale approaches host and parasite life history interplay to yield divergent population genetic structures in two ectoparasites living on the same bat species genetic population structure of natterer's bats explained by mating at swarming sites and philopatry the evolution of cooperative and pair breeding in thornbills acanthiza (pardalotidae) characterization of dinucleotide microsatellite loci in big brown bats (eptesicus fuscus), and their use in other north american vespertilionid bats mean colony relatedness is a poor predictor of colony structure and female philopatry in the communally breeding bechstein's bat (myotis bechsteinii) development of conserved microsatellite markers of high cross-species utility in bat species (vespertilionidae, chiroptera, mammalia) characterization of highly variable microsatellite loci in the bat myotis myotis (chiroptera : vespertilionidae) empirical assessment of non-invasive population genetics in bats: comparison of dna quality from faecal and tissue samples isolation and characterization of highly polymorphic microsatellite loci in schreibers' long-fingered bat, miniopterus schreibersii (chiroptera : vespertilionidae) isolation and characterization of eight polymorphic microsatellite loci for natterer's bat, myotis nattereri (vespertilionidae, chiroptera) genetic diversity is maintained in the endangered new zealand long-tailed bat (chalinolobus tuberculatus) despite a closed social structure and regular population crashes modulation of non-templated nucleotide addition by taq dna polymerase: primer modifications that facilitate genotyping extreme sex-biased dispersal in the communally breeding, nonmigratory bechstein's bat (myotis bechsteinii) the effect of close relatives on unsupervised bayesian clustering algorithms in population genetic structure analysis inference of population structure using multilocus genotype data triadic ibd coefficients and applications to estimating pairwise relatedness coancestry: a program for simulating, estimating and analysing relatedness and inbreeding coefficients team rc: r: a language and environment for statistical computing. r foundation for statistical computing the program structure does not reliably recover the correct population structure when sampling is uneven: subsampling and new estimators alleviate the problem inferring weak population structure with the assistance of sample group information estimation of average heterozygosity and genetic distance from a small number of individuals genepop (version-1.2) -population-genetics software for exact tests and ecumenicism controlling the false discovery rate -a practical and powerful approach to multiple testing genetix 4.05, logiciel sous windows pour la génétique des populations. france: laboratoire génome populations interactions fstat (version 1.2): a computer program to calculate f-statistics micro-checker: software for identifying and correcting genotyping errors in microsatellite data the genetical structure of populations hierfstat, a package for r to compute and test hierarchical f-statistics a step-by-step tutorial to use hierfstat to analyse populations hierarchically structured at multiple levels the detection of disease clustering and a generalized regression approach the ecodist package for dissimilarity-based analysis of ecological data mega6: molecular evolutionary genetics analysis version 6.0 group iaw. ictv official taxonomy: updates since the 8th report poptree2: software for constructing population trees from allele frequency data and computing other population statistics with windows interface accuracy of extimated phylogenetic trees from molecular data, ii. gene-frequency data phylogenetic model averaging genalex 6.5: genetic analysis in excel. population genetic software for teaching and research-an update passage: pattern analysis, spatial statistics and geographic exegesis. version 2 high evolutionary rate of human astrovirus using spatial bayesian methods to determine the genetic structure of a continuously distributed population: clusters or isolation by distance? contrasted patterns of mitochondrial and nuclear structure among nursery colonies of the bat myotis myotis high gene diversity at swarming sites suggest hot spots for gene flow in the endangered bechstein's bat the stepping stone model of population structure and the decrease of genetic correlation with distance 40 jahre fledermausmarkierungszentrale dresden: methodische hinweise und ergebnisübersicht: saxoprint different rates of (non-)synonymous mutations in astrovirus genes; correlation with gene function rna virus mutations and fitness for survival models of rna virus evolution and their roles in vaccine design future issues in rna virus evolution rates of spontaneous mutation correlation of pairwise genetic and geographic distance measures: inferring the relative influences of gene flow and drift on the distribution of genetic variability we thank the local forestry and conservation departments as well as the "fledermausforschungsprojekt wooster teerofen e.v." for support and the following people for help in the field: daniela fleischmann, lena grosche, ralf koch, markus melber, and jaap van schaik. for general support during the lab work we thank ina römer, and katja böhm and franziska neitzel who helped to design the primers fv5ap and gzbyr. we are grateful to stefan braendel, kathleen drescher, nicola fischer, alain frantz, darren obbard, jaap van schaik, caroline schöner, eric witt and one anonymous referee for providing comments on the manuscript. this work was funded by the deutsche forschungsgemeinschaft (ke 746/6-1 and ke 746/7-1) within the priority programm "ecology and species barriers in emerging viral diseases (spp 1596)". the datasets supporting the conclusions of this article are included within the article (and its additional files 1 and 2). the genbank accession numbers for the astrovirus sequences detected to be harboured by m. nattereri are kt894898 -kt894916. authors' contributions th and gk designed the study with input from ab-b, sjp and kf. gk, vz, fm and ct provided samples. th and rg performed the laboratory work. th performed data analyses on bats and viral sequences and drafted the manuscript with input from ab-b, kf, sjp and gk. sjp contributed to analysis. all authors have commented on the manuscript, read and approved the final version of the manuscript. the authors declare that they have no competing interests. submit your next manuscript to biomed central and we will help you at every step: key: cord-017785-zwnkrs23 authors: baker, michelle l.; schountz, tony title: mammalia: chiroptera: immunology of bats date: 2018-03-10 journal: advances in comparative immunology doi: 10.1007/978-3-319-76768-0_23 sha: doc_id: 17785 cord_uid: zwnkrs23 bats are a large and diverse group comprising approximately 20% of all living mammalian species. they are the only mammals capable of powered flight and have many unique characteristics, including long lifespans, echolocation, and hibernation, and play key roles in insect control, pollination, and seed dispersal. the role of bats as natural reservoirs of a variety of high-profile viruses that are highly pathogenic in other susceptible species yet cause no clinical disease in bats has led to a resurgence of interest in their immune systems. equally compelling is the urgency to understand the immune mechanisms responsible for the susceptibility of bats to the fungus responsible for white syndrome, which threatens to wipe out a number of species of north american bats. in this chapter we review the current knowledge in the field of bat immunology, focusing on recent highlights and the need for further investigations in this area. bats (order chiroptera) are a diverse group of nocturnal mammals comprising approximately 20% of all mammalian taxa and consisting of more than 1300 species across 21 families (simmons 2005) . phylogenetic analysis places bats within the superorder laurasiatheria, sister to carnivores (e.g., cats, dogs), ungulates (e.g., horses, cows), and cetaceans (e.g., dolphins) ( fig. 1) (tsagkogeorga et al. 2013) . bats are believed to have diverged from other eutherian mammals approximately 88 million years ago (mya) (lei and dong 2016) . the traditional classification system divided bats into two suborders: microchiroptera (microbats) and megachiroptera (megabats). microbats are defined by their smaller size (4-16 cm), the use of echolocation, and the use of hibernation during the winter for many species. megabats consist of the flying foxes (also called fruit bats) and are larger nonecholocating bats (up to 1.6 kg with wingspans of 1.7 m) belonging to the pteropodidae family. however, more recent phylogenetic analyses based on molecular data have led to a reclassification of bats into the suborders yinpterochiroptera and yangochiroptera. the yinpterochiroptera suborder includes the nonecholocating pteropodidae family (flying foxes) and the echolocating rhinolophoidea family, while the yangochiroptera suborder consists of the remaining echolocating microbats (teeling et al. 2005 (teeling et al. , 2016 . the two suborders of bats are estimated to have diverged approximately 63 mya (lei and dong 2016) . although the new classification has strong statistical support, it remains controversial as it suggests that laryngeal echolocation evolved twice in chiroptera, once in yangochiroptera and once in the rhinolophoids (teeling et al. 2000) . of all the mammals, bats are the most ecologically diverse. they are the only mammals that have evolved powered flight and have adapted to a variety of environments across all continents with the exception of the polar regions. their diets are equally diverse, including fruits, pollen, insects, small vertebrates, and even blood, and they play important roles in the ecosystem through seed dispersal, pollination, and insect control. bats have longer lifespans relative to other mammals, typically living 3.5 times longer than mammals of similar size (austad 2010) . maternal investment is generally high, with most species giving birth to a single pup per year (from tsagkogeorga et al. 2013 with permission) and pups averaging approximately 23% of maternal body weight at birth (barclay and harder 2003) . curiously, despite their longer lifespans, there is anecdotal evidence that bats are resistant to tumors . the characteristic that has drawn the most attention in recent decades is their role as natural reservoirs for a variety of viruses that are highly pathogenic in other species yet rarely cause clinical disease in bats. this characteristic in particular has led to renewed interest in the immune systems of bats. bats are highly gregarious mammals, with most species living in high-density colonies, providing ideal environments for transmission and maintenance of pathogens within populations. combined with their frequent movement between roosts, transmission of viruses, bacteria, parasites, and fungi could potentially occur readily between individuals and populations, resulting in a situation of constant pathogen exposure. approximately 200 viruses have been detected across different bat species, and many of the viruses identified in bats are highly pathogenic in other species, including humans (moratelli and calisher 2015) ; however, they likely host many more (anthony et al. 2013) . examples include high-profile viruses such as the severe acute respiratory syndrome coronavirus (sars-cov), marburg virus, and hendra and nipah paramyxoviruses. these viruses occasionally spill over to other susceptible hosts, causing severe disease and mortality yet causing no disease in bats. the long coevolutionary history of bats and viruses has likely resulted in the establishment of a state of equilibrium, allowing both the viruses and their host to coexist in a disease-free state typical of natural reservoirs. bats also host a variety of other pathogens, including parasites, bacteria, and fungi. unlike viral infections, there are examples of these pathogens causing disease among bats. the fungus that causes white nose syndrome (wns), pseudogymnoascus destructans, has resulted in mass mortalities among a number of north american microbat populations, with some species now threatened with extinction (blehert et al. 2009 ). evidence for lower fungal loads consistent with the development of resistance to the fungus have been observed in some bat populations, providing hope that selection on immune genes may lead to the development of resistance or tolerance mechanisms (langwig et al. 2017) . however, it is unlikely that this will occur rapidly enough for many affected populations. several bacterial infections, including tick-borne spirochaete bacteria, borrelia spp., and some enteric bacteria, have also been associated with pathology in bats (reviewed in brook and dobson, 2015) . brooks and dobson (2015) presented evidence that bats may have evolved mechanisms to eliminate intracellular pathogens such as viruses at the expense of their ability to eliminate extracellular pathogens (bacteria, parasites, and fungi) and hypothesize that mitochondrial adaptations may play a role. in light of the increasing emergence of infectious diseases and the impacts of pathogens such as wns, deciphering the immune systems of bats has never been more critical and offers potential for identifying novel antiviral therapies and approaches to the conservation of bats threatened by diseases such as wns. fortunately, progress in the area of bat immunology is rapidly advancing as new groups enter the field and advances in technology provide opportunities for more rapid discovery. several reviews that have appeared over the last 5 years have described the various aspects of the immune systems of bats butler et al. 2014; schountz 2014; baker and zhou 2015; schountz et al. 2017) . in this chapter we provide a broad overview, with a focus on recent highlights in bat immunology and areas for future research. although few studies have examined the histology of bat lymphoid tissues, from an anatomical perspective, bats appear to have the majority of primary and secondary lymphoid organs present in other mammals, including thymus, bone marrow, spleen, and lymph nodes (papenfuss et al. 2012; zhou et al. 2016b) . bone marrow has been isolated from long bones, including humerus and radius, and from the ribs but appears to be absent in the distal wing bones (papadimitriou et al. 1996; zhou et al. 2016b) . notably absent, at least in the species that have been examined to date, are peyer's patches, which are generally located in the submucosa and lamina propria of the small intestine. no peyer's patches were present in the horseshoe bat, rhinolophus hildebrandtii, or the common pipistrelle bat, pipistrellus pipistrellus (strobel et al. 2015; makanya and john 1994) . the submucosa of the intestine of the horseshoe bat was devoid of lymphoid tissue, with the exception of a few aggregations of lymphoid nodules in the rectal submucosa (makanya and john 1994) . a range of immune cell types also appear to be present in bats. morphological characteristics have been used to identify lymphocytes, neutrophils, eosinophils, basophils, and macrophages in the brazilian free-tailed bat, tadarida brasilensis (turmelle et al. 2010a) . macrophages and t-and b-cell populations have also been identified in the indian flying fox, pteropus giganteus, based on cellular adherence and scanning electron microscopy (sarkar and chakravarty 1991) . more recently, the phenotype, morphology, and function of dendritic cells and macrophages have been characterized from bone marrow from the black flying fox, pteropus alecto (zhou et al. 2016b) . cells resembling follicular dendritic cells (fdcs) have also been described in the indian flying fox (sarkar and chakravarty 1991) . unlike dendritic cells that originate in the bone marrow, fdcs are of mesenchymal origin and are found in primary and secondary lymphoid follicles in b-cell areas of lymphoid tissue. fdcs are essential for high-affinity antibody production and for the development of b-cell memory. they also have the ability to maintain intact antigen for extended periods (van nierop and de groot 2002; heesters et al. 2014) . whether they play the same role in bats remains to be determined but presents an interesting possibility for the maintenance of persistent viral infections. the lack of species-specific reagents has often been a hindrance to comparative immunologists. however, bat immunology made a resurgence in an age of rapid advances in species-independent approaches such as next-generation sequencing, proteomics, and gene editing technologies such as crispr/cas9. rnaseq studies on tissues and cells from a variety of different species of bats have provided evidence that bats have nearly all of the major components of the immune system that are present in other mammals, including receptors and molecules associated with innate and adaptive immunity and micrornas (papenfuss et al. 2012; shaw et al. 2012; cowled et al. 2014) . rnaseq data from virus-infected bat cells and wnsinfected bat tissues have also offered insights into the genes associated with hostpathogen responses (wynne et al. 2014 (wynne et al. , 2017 field et al. 2015) . to date, partial genome sequences of 14 bat species are available in the ncbi database, providing valuable insights into the evolution of immune genes and essential sequence information for the design of primers and the development of reagents essential for studies of the immune responses of bats. the bat1k project, which aims to sequence the genomes of the approximately 1300 species of bats, will no doubt provide a valuable resource for comparing the immune repertoire of different species of bats (teeling et al. 2018) . the genomes of bats are condensed compared to other mammals, ranging from 1.6-3.54 gb. smaller genome sizes in both bats and birds have been hypothesized to be associated with the metabolic requirements of flight (kapusta et al. 2017 ). a number of genomic regions associated with immunity have been analyzed in detail, in particular in the black flying fox (p. alecto), using a combination of wholegenome data and additional sequencing. these include regions associated with innate, for example, type i interferon (ifn), and adaptive immunity, for example, major histocompatibility class i (mhc-i) and mhc-ii. consistent with the smaller size of the genomes of bats, these regions are also condensed and contain fewer genes compared with the corresponding region from other mammals (ng et al. , 2017 zhou et al. 2016a) . for example, the type i ifn locus of the black flying fox is highly condensed and contains fewer ifn genes than any other species sequenced to date (fig. 2) . the description of the genomes of two divergent bat species, the australian black flying fox (p. alecto) and david's myotis (mytois davidii), provided the first glimpse into unique genetic signatures within immune pathways of bats, lending support to the idea of inadvertent changes in the immune system associated with the evolution of flight (zhang et al. 2013 ). these include changes in the genes associated with dna response/dna repair pathways that are tightly linked with innate immune pathways (fig. 3) . the dna damage sensor, dna-dependent protein kinase catalytic subunit (dna-pkcs), which is also part of the cytoplasmic microbial nucleic acid sensing complex, was among the genes that have undergone selection in bats (ferguson et al. 2012) . accelerated evolution of innate immune genes including nuclear factor-kb (nf-kb) family member rel, ifnar1, toll-like receptor 7 (tlr7), ifn stimulated gene 15 (isg15), interleukin-18 (il-18), and nucleotidebinding oligomerization domain-like receptor (nlr) family, pyrin domain containing 3 (nlrp3) were also observed in the genomes of the two bats, an observation that may be a consequence of the coevolution of bats with viruses (zhang et al. 2013) . notably absent from the black flying fox and david's myotis genomes is the pyrin and hin domain (pyhin) gene family, which are involved in the recognition of foreign dna (zhang et al. 2013 ). this finding was recently confirmed in eight additional bat species across both suborders (ahn et al. 2016 ). the family member, absent in melanoma 2 (aim2), is a cytosolic dna sensor and also part of the inflammasome complex that results in the activation of inflammatory cytokines, including il1β and il18. a second component of the inflammasome, nlrp3, has undergone positive selection in the black flying fox and david's myotis, consistent with the possibility that the formation of inflammasomes is impaired in bats, which may in turn dampen the inflammatory response against pathogens (zhang et al. 2013) . the absence of a number of natural killer (nk) cell receptors from bat rnaseq and genome data sets is also striking (papenfuss et al. 2012; shaw et al. 2012; zhang et al. 2013) . genes that encode mammalian nk cell receptors are located within the leukocyte receptor complex (lrc) and the natural killer complex (nkc) of the genome. the two families have undergone convergent evolution to bind mhc-i molecules for the control of nk cell function. genes within the lrc encode immunoglobulin (ig)-like genes, including the killer cell ig-like receptors (kir), leukocyte ig-like receptors (lilrs), and leukocyte-associated ig-like receptors (lairs). those within the nkc encode lectin-like receptors, including the ly49 c-type lectin family. the composition of the lrc and nkc varies considerably among species. while most species have expanded either their lrc or nkc gene families, there are exceptions to this rule. in humans and nonhuman primates, the main nk cell receptors are encoded in the lrc and belong to the ig superfamily. rodents and horses have only expanded their ly49 c-type lectin family of nk receptors (kelley et al. 2005) . in contrast, cattle appear to have diversified nk genes within both the nkc and lrc regions, whereas domestic dogs and four species of marine carnivores contain single copies of kir and ly49 genes (hammond et al. 2009; schwartz et al. 2017) . in bats, kirs and ly49-like receptors appear to be absent from transcriptome and genome data sets from the black flying fox, and only a single pseudogene of ly49 was identified in the genome of david's myotis bat (papenfuss et al. 2012; zhang et al. 2013) . two kirs have been identified in the genome of the big brown bat, eptesicus fuscus, but whether they are functional remains to be determined (guethlein et al. 2015) . overall, evidence to date is consistent with the contraction of both kir and ly49 families of receptors in bats. other nk cell coreceptors have been identified in bat genome and rnaseq data sets, hinting at some level of nk cell function in bats. these include the presence of cd94 and nkg2c, which form heterodimers to generate inhibitory signals. the more divergent nkg2d, which binds mhc-i chain-related genes, mica/b, and the ul16 binding proteins (ulbps) in humans (kelley et al. 2005) , was also detected. coreceptors, including cd16, cd56, and cd244, were also transcribed in the black flying fox (papenfuss et al. 2012) . the failure to identify a number of nk cell receptors in several bat species supports the hypothesis that bats may have atypical nk cell responses or use different subsets of receptors. the availability of rnaseq and genomic data has also accelerated the characterization of a variety of immune genes and provided opportunities to examine transcription in various tissues and cells. molecular information exists for a variety of mammalian cytokines that have been described in bats including interleukins (il2, il4, il6, il10, il12), cytokines (tnfα, tgfβ), and ifns (types i, ii, and iii) (iha et al. 2009; he et al. 2010 he et al. , 2014 kepler et al. 2010; zhou et al. 2011a zhou et al. , 2016a janardhana et al. 2012; loria-cervera et al. 2014) . detailed descriptions of pattern recognition receptors, tlrs, and rig-i like helicases have also been reported (iha et al. 2010; cowled et al. 2011 cowled et al. , 2012 although only a few studies have examined the nature of ig genes in bats, a few unusual characteristics have already emerged that have been extensively reviewed elsewhere (butler et al. 2014) . the constant regions of bat igs appear to correspond to the canonical structure and repertoire found in other eutherian mammals. bats transcribe igm, igd, iga, ige, and multiple subclasses of igg (baker et al. 2010; butler et al. 2011; wynne et al. 2013) , although some species do not have ighδ genes and others have only a single ighγ gene gerrard et al. 2017) . studies of the heavy chain variable (vh) region repertoires of black flying foxes and little brown bats (myotis lucifugus) suggest bats may have the greatest number of vh gene segments among mammals (baker et al. 2010; bratsch et al. 2011) . furthermore, evidence from little brown bats indicates that bats may depend more on combinatorial diversity and less on somatic hypermutation . the antigen-binding region of black flying fox vh genes contains amino acids typically associated with lower antigen avidity but greater specificity (baker et al. 2010) . this, combined with the lack of evidence for somatic hypermutation, is consistent with the possibility that highly specific vh segments are encoded in the genomes of bats because of the long coevolutionary history of bats and viruses. the availability of cell lines from a range of different bat species has provided opportunities to study several aspects of the immune response of bat cells in vitro. this has been particularly useful for studying host-virus interactions. ifn responses of bat cells and cell lines following stimulation with viruses and synthetic tlr ligands, including polyinosinic:polycytidylic acid (polyi:c) and bacterial lipopolysaccharide (lps), have demonstrated that ifn production pathways are functional in bat cells and supernatant from stimulated cells has antiviral activity (stewart et al. 1969; omatsu et al. 2008; crameri et al. 2009; kepler et al. 2010; zhou et al. 2011b ). significantly, ifnα and ifn signaling molecules, such as ifn regulatory factor 7 (irf7), are constitutively expressed in unstimulated pteropid bat tissues and cells, consistent with the possibility that the innate immune systems of bats are at higher states of activation than other mammals, presumably allowing bats to rapidly respond to microbial infection (zhou et al. , 2016a . the constitutive expression of ifnα has been described in two species of pteropid bats (p. alecto and cynopterus brachyotis) and is a first for any species. curiously, fetal and kidney cell lines from a third pteropid bat species, the egyptian rousette bat (rousettus aegyptiacus), have low constitutive expression of ifnα, indicating that high baseline levels of ifnα may not be a feature of all bat species (kuzmin et al. 2017) . the downstream signaling events triggered by ifn result in the induction of hundreds of ifn-stimulated genes (isgs), which are responsible for the antiviral state induced by ifns. the profile of isgs in unstimulated bat cells and the kinetics of isg induction following stimulation with ifn also differs from other species. unstimulated cells from the black flying fox have higher levels of isgs compared to human cells. the isg profile of bat cells consists predominantly of a subset not associated with the acute inflammatory responses that often accompany elevated ifn activity (cheon et al. 2013; zhou et al. 2016a ). stimulation of cells from the black flying fox with ifnα also leads to the induction of novel subsets of isgs, including ribonuclease l (rnasel), that are not known to be induced by ifn to other species and the isg response is elevated for a shorter period of time in bat compared to human cell lines (de la cruz-rivera et al. 2017; zhang et al. 2017) ; rnasel is also elevated in bats that die from experimental tacaribe virus infection (gerrard et al. 2017) . overall, these studies point to differences in the regulation and profile of bat isgs as being central to the ability of bats to tolerate constitutive ifnα expression without pathology. consistent with the nature of the isg response, additional evidence is also accumulating for differences in the activation of other components of the inflammatory immune response in bats. comparison of the inflammatory cytokine production of polyi:c-stimulated cell lines from big brown bats (e. fuscus) and humans have demonstrated that the induction of high levels of proinflammatory cytokines, tnfα and il8, occurs in human but not in bat cells (banerjee et al. 2017 ). this result again demonstrates that bats may regulate their immune response more tightly compared to other species. experimental infections of bat cells and cell lines have also provided insight into the antiviral response of bats, revealing differences in the responses to different viruses and between cell types. infection of black flying fox splenocytes with the bat paramyxovirus, tioman virus, resulted in the downregulation of type i ifns and the upregulation of type iii ifns, indicating that type iii ifns may play an important role in the ability of bats to coexist with viruses (zhou et al. 2011a ). in contrast, henipavirus infection antagonizes type i and type iii ifn production and signaling in black flying fox cells but only ifn production in human cells (virtue et al. 2011a, b) . the difference in the behavior of bat ifns upon tioman and henipavirus infection may reflect different ifn production mechanisms in splenocytes, which are professional immune cells, and cloned bat cells, which are predominantly fibroblastlike (crameri et al. 2009 ). infection of cells from the black flying fox with henipavirus and the egyptian rousette bat with ebola or marburg results in the induction of ifnβ, but curiously no increase in ifnα has been observed, at least at the time points examined in these studies (zhou et al. 2016a; kuzmin et al. 2017) . as described earlier, p. alecto has high constitutive ifnα, which may account for its low induction, but this does not appear to be the case for the rousette bat. both marburg and ebola viruses, but particularly marburg, induced a potent innate immune response in rousette cells, which was generally stronger than that in human cells. the timing of induction of ifns and isgs in ebola-virus-infected cells was also delayed compared to cells infected with marburg virus (kuzmin et al. 2017) . the natural reservoir for marburg virus is known to be the rousette bat, but the reservoir for ebola is unknown and believed to be another bat species. the differences in host response of rousette bat cells to the two filoviruses may therefore reflect adaptations associated with the role of this species as a natural reservoir for marburg but not ebola. although isg responses have also been examined following viral infections in vitro, their ability to restrict viral replication has only been examined for a few isgs (de la cruz et al. 2017; zhou et al. 2013 ). the best-characterized isgs include myxovirus resistance (mx) genes and 20-50-oligoadenylate synthetase 1 (oas1). mx proteins are large gtpases that were initially described as inhibitors of influenza viruses and act by detecting viral replication and then trapping viral components. the oas1 proteins are activated by dsrna leading to the activation of rnase l, which then degrades both cellular and viral rna (sadler and williams 2008) . mx1 and oas1 from the black flying fox have been demonstrated to be highly upregulated by pteropine orthoreovirus nb (prv1nb) virus infection, an orthoreovirus carried by pteropid bats . furthermore, bat mx1 proteins from pteropidae, phyllostomidae, and vespertilionidae demonstrate antiviral activity against ebola and bat influenza-like viruses. however, thogoto virus, a tick-transmitted orthomyxovirus that is not known to infect bats, was not inhibited by bat mx1 despite the ability of human mx1 to inhibit thogoto virus replication. evidence for positive selection in two variable and surface-exposed regions of bat mx1 genes were hypothesized to explain some of the species-specific antiviral activities of these proteins (fuchs et al. 2017 ). however, antiviral activity of black flying fox rnasel has been demonstrated against the yellow fever flavivirus, which is carried by mosquitoes, consistent with differences in specificity among different bat isgs (de la cruz et al. 2017 ). cell-mediated immune (cmi) responses are controlled by cd8 + cytotoxic and cd4 + helper t-lymphocyte populations and result in the killing of virus-infected cells or activation of the antibody and cytokine response. fewer studies have examined cmi in bats. the single type ii ifn, ifnγ, is produced by black flying fox bat cells stimulated with mitogens such as phytohaemagglutinin (pha) and cona, and recombinant bat ifnγ has antiviral activity against semliki forest virus and hev in vitro . at least in vitro, ifnγ from the black flying fox appears to have activity similar to that of ifnγ from other mammals, consistent with its role in the cmi response. curiously, in rousette bat cell lines, ifnγ is induced following infection with marburg virus but not following infection with ebola virus, indicating there may be differences in the cmi response induced by these two closely related viruses (kuzmin et al. 2017) . a number of earlier studies have described the in vitro responses of pteropid bats and microbats to t-cell mitogens and mixed lymphocyte responses in pteropid bats (mcmurray and thomas 1979; chakraborty and chakravarty 1983; chakravarty and paul 1987; paul and chakravarty 1987) . although these studies have been relatively crude due to the absence of specific reagents, they have all reported delayed responses compared with those of conventional laboratory animals. the presence of regulatory t cells was implicated in the delay in mitogenic responses of b cells in bats . whether these cells are involved in the delay in t-cell-mediated immune responses observed in bats remains to be determined. more recent studies have used proteomics to functionally characterize black flying fox mhc-i molecules and identify endogenous and viral peptide ligands. peptides derived from bat mhc-i molecules display a relatively broad length distribution, consistent with earlier observations based on sequence information demonstrating relatively large peptide binding grooves in the bat class i molecules wynne et al. 2016) . furthermore, an unusual preference for a c-terminal proline residue was identified in endogenous and hendra virus (hev)-derived peptides presented by bat mhc-i molecules, consistent with the possibility that differences in antigen presentation or processing may exist in bats ). bats are capable of mounting antibody responses to viruses and model antigens, and the appearance of antibodies appears to follow the same succession as that of other mammals with the early appearance of igm followed by igg (hatten et al. 1968 (hatten et al. , 1970 chakraborty and chakravarty 1983; wellehan jr et al. 2009 ). although all of the ig isotypes have been detected at the mrna level in a variety of bat tissues, iga protein appears to be present at surprisingly low levels in tissues and secretions from the black flying fox, which may have implications for its role in mucosal immunity in bats ). there are also differences in the time course, quantity, and duration of antibody responses, and questions exist over the protective nature of antibodies in bats (hatten et al. 1968; mcmurray et al. 1982; chakraborty and chakravarty 1984; davis et al. 2007; wellehan jr et al. 2009; turmelle et al. 2010b) . responses to antigens such as ϕx174 bacteriophage and sheep red blood cells have demonstrated that the generation of neutralizing antibodies is delayed in the big brown bat, the pteropid bat, and the indian flying fox (pteropus giganteus) (hatten et al. 1968; chakraborty and chakravarty 1984) . isotype switching from igm to igg also appears to be delayed in the big brown bat (hatten et al. 1968 ). despite genetic evidence for limited somatic hypermutation in the little brown bat, an increase in antibody affinity as measured by the ability of antibodies to dissociate from ϕx174 increased following secondary immunization in the big brown bat (hatten et al. 1970) . measures of cmi in bats have been crude relative to studies in other species and are limited to studies demonstrating t-cell-mediated inflammation to protein antigens such as purified protein derivative (ppd), pha, and bovine serum albumin (bsa). such skin sensitivity tests in two bat species, the common vampire bat (desmodus rotundus) and seba's short-tailed bat (carollia perspicillata), immunized with ppd or bsa revealed delayed responses in both species compared to similar reactions in mice (mcmurray and thomas 1979) . lack of inflammatory responses have also been reported in most indian flying foxes subjected to skin sensitivity tests using the contact allergen 2-4 dinitrofluorobenzene (chakraborty and chakravarty 1983) . unlike conventional laboratory animals, few "clean" captive colonies of bats exist, and experimental infections often rely on the use of wild caught individuals, which represent a mixed population of unknown age, susceptibility, and prior viral exposure. experimental infections have been performed on a number of species of bats using rabies virus, australian bat lyssavirus (ablv), marburg, hev, nipah virus (niv), japanese b encephalitis (je) virus, and tacaribe virus (tcrv) (williamson et al. 1998 (williamson et al. , 1999 almeida et al. 2005; davis et al. 2007; middleton et al. 2007; turmelle et al. 2010b; halpin et al. 2011; cogswell-hawkinson et al. 2012; paweska et al. 2012) . although the only immune parameter measured during these studies has been antibody responses, these experiments have provided valuable information on the kinetics of viral infection, the timing and duration of antibody responses and the nature of protective immunity following reinfection. with the exception of rabies virus, ablv and tcrv, bats generally show no clinical signs of disease following infection. neutralizing antibodies to a variety of viruses have been detected in wild caught bats, demonstrating they are capable of mounting an antibody response to viruses (halpin et al. 2000; lau et al. 2005; leroy et al. 2005) . the transfer of maternal antibody to pups occurs in bats, and the decline of maternal antibodies has been examined in captive black flying, variable flying foxes (pteropus hypomelanus), and straw-colored fruit bats (eidolon helvum) baker et al. 2014 ). however, whether bats transfer maternal antibody both pre-and postpartum and the isotypes involved is unknown. the interpretation of antibody responses in bats is extremely challenging, and, as described earlier, the nature of antibody responses in bats often differs both qualitatively and quantitatively compared to other species. rabies and ablv are among the only viruses known to result in clinical disease in naturally infected and experimentally infected bats. however, not all bats develop disease, and the mechanisms responsible for differences in disease outcome between individuals are not understood. evidence from experimental infections has demonstrated that even the development of neutralizing antibodies does not always provide protection from reexposure. for example, a group of wild caught bats (12 big brown bats, e. fuscus, and 12 mexican free tailed bats, tadarida brasiliensis) challenged by oral-nasal inoculation with rabies virus all developed antirabies neutralizing antibodies within 3 months. rechallenge by intramuscular inoculation 6 months later resulted in an amnestic response in 21 animals, including 9 that developed clinical rabies (davis et al. 2007 ). low seroconversion rates have also been reported in big brown bats inoculated with rabies by intramuscular challenge with only 15 of 43 inoculated animals developing antibodies. this study also reported clinical disease following secondary or tertiary infections in bats that had seroconverted following primary inoculation (turmelle et al. 2010b) . similarly, almeida et al. (2005) described the intramuscular challenge of 40 vampire bats (d. rotundus) with rabies virus, of which 30 bats survived. once again, there was no correlation between the level of neutralizing antibody and survival. many bats that developed low or undetectable antibodies, as well as those with high antibody titers, survived infection. infection of gray-headed flying foxes, pteropus poliocephalus, with rabies or ablv results in similar rates of mortality and seroconversion. mccoll et al. (2002) reported clinical signs of disease in three of ten ablv-infected and two of four rabies-infected gray-headed flying foxes, none of which seroconverted prior to euthanasia. five of the ablv-infected survivors seroconverted by 23 dpi, with titers waning by 50 dpi. one of the rabies-infected survivors also seroconverted, but not until 70 dpi (mccoll et al. 2002) . these studies indicate that antibodies may not provide protection and support a role for other components of the immune response in those animals that survive infection. unlike rabies and ablv infections, clinical disease has not been reported in any bat species either naturally or experimentally infected with a variety of other bat-borne viruses, including hev, niv, marburg, ebola, and je viruses. however, similar to rabies infection, the role of the antibody response in providing protection remains unclear, and many animals survive infection but fail to seroconvert. the henipaviruses hev and niv are carried by pteropid bats. in australia, hev antibodies have been identified in all four species of australian flying foxes (p. alecto, p. poliocephalus, p. scapulatus, and p. conspicillatus) . niv antibodies have been identified in bats from southeast asia and africa. in malaysia, two pteropid species, small flying foxes (p. hypomelanus) and malayan flying foxes (p. vampyrus), are considered to be the reservoir hosts . a number of experimental infections of pteropid bat species have been performed to understand the nature of viral infection in the natural reservoir of these viruses. niv infection of 11 gray-headed flying foxes by subcutaneous injection resulted in the production of neutralizing antibody in all individuals inoculated, but in a separate study, only 4 of 8 malayan flying foxes that were infected by the oral-nasal route produced a neutralizing antibody response (middleton et al. 2007; halpin et al. 2011) . both subcutaneous and oral-nasal routes of infection have also been used for hev inoculation of pteropid bats. neutralizing antibody responses were detected in 10 of 20 black flying foxes inoculated oral-nasally with hev (halpin et al. 2011) . similarly, in gray flying foxes challenged with hev, neutralizing antibodies were detected in two of four bats inoculated by subcutaneous injection and three of the four bats inoculated by the oral-nasal route, with none of the bats displaying clinical signs of disease (williamson et al. 1998) . a study of four gray-headed flying foxes in late gestation infected subcutaneously with hev also described the presence of neutralizing antibodies in all four bats, and no abnormalities were observed in the fetuses or adults at necropsy (williamson et al. 1999) . in other mammals, pregnancy results in a bias in the immune response toward humoral immunity and away from cmi, which could be harmful to a fetus (szekeres-bartho 2002) . whether the nature of the maternal immune response facilitates greater production of antibody in infected bats during pregnancy remains to be investigated. a natural reservoir of marburg virus are the egyptian rousette bats (r. aegyptiacus) (towner et al. 2009) , and a number of experiments have been performed to study the nature of viral transmission and infection in this species (paweska et al. 2012; schuh et al. 2017a, b) . marburg virus is capable of horizontal transmission between inoculated and naïve r. aegyptiacus. all inoculated bats seroconverted, with igg antibodies peaking between 14-28 dpi. marburg virus antibody titers in both inoculated and in contact bats declined within 1 month following attainment of peak levels and were undetectable after 2 months (schuh et al. 2017a) . a subsequent study revealed that bats rechallenged with marburg virus 17-24 months following primary experimental infection developed virus-specific secondary antibody, indicative of the development of long-term protective immunity (schuh et al. 2017b) . clearly, additional work is needed to understand the antibody responses of bats and the nature of antibody-mediated protection against various viruses. given what we have learned about innate immunity, particularly in pteropid bats, it is possible that innate immune mechanisms, such as ifn, reduce viral replication to low levels, delaying the generation and magnitude of an antibody response. evidence for a highly diverse germline repertoire of antibodies and the absence of somatic hypermutation could indicate that bats have evolved a repertoire of antibodies that are highly pathogen specific. such antibodies may provide some level of early protection without reaching the higher titers observed in other species. although no studies have examined the cmi responses of bats to viral infections, the generation of an ifnγ reagent for pteropid bats has been described and will assist in future studies to examine cmi in bats ). wns is caused by a cold-loving (pyrophilic) and keratinophilic fungus (p. destructans) first identified in north american bats in 2006 that infects the epidermis and dermis of the muzzle, ears, and wings. since its discovery, it has been detected in six species of north american bats, and infected populations have undergone a decline of up to 90%, with several species threatened with regional extinction within the next decade. p. destructans infects bats during hibernation, causing them to arouse early, leading to depletion of energy reserves and ultimately leading to a severe inflammatory response and resulting histopathology. the fungus is widely distributed in north america and europe and has recently been found in asia (hoyt et al. 2016) . although naturally infected european bats also develop histopathological lesions in response to p. destructans, no mass mortality is observed in european or asian bats (zukal et al. 2016) . similar to the situation with viruses, the long coevolutionary relationship of european and asian bats with p. destructans has presumably led to an equilibrium between the host and pathogen. in the longer term, this may also evolve in north american bats, and evidence of some level of resistance has been reported in some populations (langwig et al. 2017) . however, the rate of mortality among some bat species is too high to ignore. understanding the hostpathogen relationship and the genes and pathways associated with disease tolerance and resistance will be important for identifying viable treatments and assessing the immune responses of bats to drugs or vaccines. earlier reports describing the immune response of bats during hibernation indicate that, like other hibernating mammals, their immune responses are suppressed during torpor when they are initially infected with p. destructans. for example, hibernating e. fuscus bats maintained at 8 °c fail to generate antibodies in response to infection with je virus (sulkin et al. 1966 ). in addition, activation plasma complement against bacteria (escherichia coli, staphylococcus aureus) and fungi (candida albicans) is lower in hibernating little brown bats compared to nonhibernating bats (moore et al. 2011) . several studies have now begun to examine the host response of bats to p. destructans to determine the level of immune activation that occurs during torpor and after arousal. p. destructans begins to colonize bat skin during hibernation, yet visible signs of inflammation are characteristically absent in torpid animals, and neutrophils and macrophages are absent from sites of pathogen invasion in hibernating bats with wns. in little brown bats, overt skin damage does not occur until 2-3 weeks after bats have emerged from hibernation with intense neutrophilic inflammation associated with invasive p. destructans infection (meteyer et al. 2012) . studies of bats from wns-affected and unaffected sites have also demonstrated significantly higher circulating leukocyte counts in wns-affected bats with elevated body temperatures (above 20 °c). the latter is consistent with the mobilization of cells associated with arousal from torpor and euthermia (moore et al. 2013) . the absence of neutrophil and t-cell infiltration has been confirmed through rnaseq analysis of wns-infected little brown bat wing tissues during hibernation (field et al. 2015) . despite the absence of neutrophil invasion, increases in gene expression for inflammatory cytokines have been detected in wing tissues from hibernating wns infected bats compared to hibernating bats not affected by wns. these include il1β, il6, il17c, il20, il23a, il24, and g-csf and chemokines, such as ccl2 and ccl20. hibernating little brown bats exhibiting visible fungal infections elevated levels of transcripts for proinflammatory cytokines, il23 and tnfα, the anti-inflammatory cytokine il10, and the antimicrobial peptide cathelicidin in lung tissue compared to hibernating uninfected bats (rapin et al. 2014) . overall, these studies are consistent with the induction of an innate antifungal response in wns-infected bats prior to emergence from hibernation followed by infiltration of immune cells and, presumably, activation of adaptive immune responses following arousal. overactivation of the immune response following arousal from torpor, combined with a depletion of energy reserves, appears to be the main cause of mortality. in contrast to p. destructans, bats are known to carry other fungal pathogens, such as histoplasma capsulatum, without disease. h. capsulatum is a pathogenic fungus that causes pulmonary and systemic infections in humans. bats are considered to be the main reservoir of this fungus, and it is commonly found in bat guano (taylor et al. 2005) . although bats are susceptible to infection, mortality is rare in bats that are inoculated intranasally, which is the most likely route of natural infection. higher mortality rates are observed in bats inoculated intraperitoneally (mcmurray and greer 1979; greer and mcmurray 1981) . great fruit eating bats (artibeus lituratus) respond to infection with the generation of complement fixing antibodies by 3 weeks and precipitating antibodies by 5 weeks post infection (mcmurray and greer 1979) . natural infection rarely results in disease, indicating that, similarly to the situation with most viruses, bats have likely evolved mechanisms to control infection, at least under conditions where they are infected under nontorpid conditions. the field of bat immunology is very much in its infancy, and significant opportunities exist for future research. thanks to advances in technology, such as wholegenome sequencing and rnaseq, considerable progress has been made, in particular with regard to our understanding of the immune system of the black flying fox, p. alecto. however, as bats are a highly diverse group of mammals that have evolved independently for a long period of time, it is possible that different immune mechanisms exist between the two suborders and across species. there is likely much more to be learned from comparative studies across different bat species. comparative genomics of bats have provided important clues to the adaptations that may allow bats to coexist with viruses in the absence of disease. these include evidence for positive selection on a variety of immune genes and differences in the repertoires of nk cell receptors. additional genomic data, including long read assemblies, will be required to resolve highly repetitive regions such as the lrc and nkc to confirm the absence of important receptor families and to resolve other repetitive regions of the bat immunome. a number of genomic regions also remain largely unexplored, partly owing to their repetitive nature. these include b-and t-cell receptor (bcr and tcr) regions. examining the repertoire and diversity of these regions will provide opportunities to examine their functional activities and importance. for example, no information exists on the repertoire of tcrs in bats and the relative importance and roles of αβ and γδ t cells. observations from genomic data sets pave the way to further addressing the role of different components of the immune system in the responses of bats to infection. the mechanisms involved in tcr and bcr diversification also remain unknown. the roles of terminal deoxynucleotidyl transferase (tdt), recombination activating gene (rag), and activation-induced cytidine deaminase (aid) on recombination, somatic hypermutation, gene conversion, and class switching remain to be explored. a number of important differences in the innate immune system have also been identified in bats that are at odds with the responses in humans and other species. in particular, the constitutive activation of ifnα in the black flying fox is striking. in other mammals, constitutive ifn expression can have implications for inflammation and autoimmunity. identifying the mechanisms responsible for the ability of bats to tolerate high levels of ifn in the absence of inflammation has significant potential for identifying novel therapeutics to treat viral diseases in humans and other species. to this end, functional characterization of the different subsets of isgs already identified in both unstimulated and stimulated cells would provide valuable insights into the mechanisms responsible for the control of viral infection in the absence of inflammation. additionally, dissection of the signaling pathways responsible for the control of ifn response will contribute to our understanding of differences in the regulation of ifn in bats compared to other species. as described earlier, a number of functional differences have been identified in the immune system of bats compared to other species. these include the nature of cell-mediated and antibody responses of bats. to advance our understanding of the nature of these responses, appropriate bat-specific reagents will be required. some commercially available human and mouse antibodies generated against highly conserved intracellular proteins are cross reactive with bat proteins and have already proven useful (zhou et al. 2016b) . a handful of bat-specific antibodies have also been generated wynne et al. 2013) . additional reagents will be necessary to advance the field, including monoclonal antibodies for use in flow cytometry, immunohistochemistry, and elisas, to dissect the roles of different cell types, including b and t cells, dendritic cells, and macrophages. reagents will also be required to examine the responses of various cytokines to examine proinflammatory and anti-inflammatory pathways for comparison to other species and to answer specific questions, including the confirmation of cytokine expression at the protein level (e.g., ifnα to confirm its constitutive expression). recombinant cytokines and growth factors will also be important for examining the responses of cells to cytokine stimulation and the expansion of specific subsets of antigenspecific lymphocytes. lastly, the development of closed breeding colonies of bats will be essential in progressing research into immunity in bats, overcoming the issues associated with wild caught individuals of unknown age and history of infection. renewed interest in bat immunology emerged following the identification of bats as reservoirs for a number of viruses, including sars-cov and ebola, that are highly pathogenic in other species. prior to the emergence of these viruses, few studies had examined any aspect of bat immunology. a number of important observations have already been made through studies of the immune systems of bats, with evidence for adaptations not observed in any other species. significant progress has now been made in the identification of genes and pathways associated with immunity, and one of the recurring themes that is emerging with regard to viral infections is the ability of bats to control inflammatory responses. regulation of the immune system is likely an important mechanism for preventing pathology associated with infection. however, bats are an extraordinarily diverse group of mammals, and the adaptions identified to date may not apply across all bat species. in contrast to the apparent regulation of the immune response during viral infections, uncontrolled inflammatory responses due to infection with pathogens such as wns clearly demonstrate that bats are capable of overactivating their immune system, causing immunopathology. as described earlier, there are still gaps in our understanding of the immune systems of bats, and significant opportunities exist. studies of bat immunology provide opportunities to identify novel mechanisms that could be applied to redirecting the immune system of other species to prevent disease and to the conservation of bats affected by pathogens such as wns. unique loss of the pyhin gene family in bats amongst mammals: implications for inflammasome sensing experimental rabies infection in haematophagous bats desmodus rotundus a strategy to estimate unknown viral diversity in mammals 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characterisation of immunoglobulins from the australian black flying fox (pteropus alecto) using anti-fab affinity chromatography reveals the low abundance of iga proteomics informed by transcriptomics reveals hendra virus sensitizes bat cells to trail mediated apoptosis characterization of the antigen processing machinery and endogenous peptide presentation of a bat mhc class i molecule comparative transcriptomics highlights the role of the ap1 transcription factor in the host response to ebolavirus nipah virus infection in bats (order chiroptera) in peninsular malaysia comparative analysis of bat genomes provides insight into the evolution of flight and immunity ifnar2-dependent gene expression profile induced by ifn-α in pteropus alecto bat cells and impact of ifnar2 knockout on virus infection type iii ifns in pteropid bats: differential expression patterns provide evidence for distinct roles in antiviral immunity type iii ifn receptor expression and functional characterisation in the pteropid bat, pteropus alecto bat mx1 and oas1, but not pkr are highly induced by bat interferon and viral infection irf7 in the australian black flying fox, pteropus alecto: evidence for a unique expression pattern and functional conservation contraction of the type i ifn locus and unusual constitutive expression of ifn-α in bats unlocking bat immunology: establishment of pteropus alecto bone marrow-derived dendritic cells and macrophages white-nose syndrome without borders: pseudogymnoascus destructans infection tolerated in europe and palearctic asia but not in key: cord-310061-nro623aa authors: valitutto, marc t.; aung, ohnmar; tun, kyaw yan naing; vodzak, megan e.; zimmerman, dawn; yu, jennifer h.; win, ye tun; maw, min thein; thein, wai zin; win, htay htay; dhanota, jasjeet; ontiveros, victoria; smith, brett; tremeau-brevard, alexandre; goldstein, tracey; johnson, christine k.; murray, suzan; mazet, jonna title: detection of novel coronaviruses in bats in myanmar date: 2020-04-09 journal: plos one doi: 10.1371/journal.pone.0230802 sha: doc_id: 310061 cord_uid: nro623aa the recent emergence of bat-borne zoonotic viruses warrants vigilant surveillance in their natural hosts. of particular concern is the family of coronaviruses, which includes the causative agents of severe acute respiratory syndrome (sars), middle east respiratory syndrome (mers), and most recently, coronavirus disease 2019 (covid-19), an epidemic of acute respiratory illness originating from wuhan, china in december 2019. viral detection, discovery, and surveillance activities were undertaken in myanmar to identify viruses in animals at high risk contact interfaces with people. free-ranging bats were captured, and rectal and oral swabs and guano samples collected for coronaviral screening using broadly reactive consensus conventional polymerase chain reaction. sequences from positives were compared to known coronaviruses. three novel alphacoronaviruses, three novel betacoronaviruses, and one known alphacoronavirus previously identified in other southeast asian countries were detected for the first time in bats in myanmar. ongoing land use change remains a prominent driver of zoonotic disease emergence in myanmar, bringing humans into ever closer contact with wildlife, and justifying continued surveillance and vigilance at broad scales. infectious diseases are considered to be "emerging" if they appear in a new population or geographic region or are occurring with greater frequency than the expected background rate [1] [2] [3] . emerging infectious diseases (eids) are capable of causing debilitating health effects and financial instability, especially in less developed countries with insufficient capacity to mount health interventions, and thus pose a significant global public health challenge in the 21 st [4] . an estimated 60-75% of eids are comprised of zoonotic diseases; of these, more than 70% have purportedly originated in wildlife species [3] [4] [5] . spillover has been largely attributed to changes in anthropogenic activity subsequent to exponential human population growth since the latter half of the 20 th century. large-scale land use change, such as deforestation and land conversion for agriculture, can alter host-pathogen relationships and increase human encounter rates with wildlife and their pathogens, making cross-species transmission events more likely [6, 7] . for established pathogens, human-mediated biodiversity loss often leads to reduced populations of suboptimal host species and increased numbers of competent or amplifying hosts, potentially precipitating higher infection rates in people [8] . in addition, intensification of livestock and poultry production systems results in artificially dense populations of domestic animals, which can lead to pathogen amplification and spillover to humans [7] . approximately two-thirds of human pathogens occupy complex, multi-host systems, and pathogens with multiple animal hosts, including some wildlife species, are more likely to become emergent [9] . bats are increasingly recognized as the natural reservoirs of viruses of public health concern [10] [11] [12] [13] . the capacity of bats to carry and transmit zoonotic pathogens has been hypothesized to be due to their unique life history traits, including their ability for sustained flight, potential for long-distance dispersal, aggregation into densely populous colonies, and adaptation to peri-urban habitats [11, 12] . historically, bats have been linked to highly pathogenic viruses that pose a serious threat to human health, including the coronaviruses responsible for severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers), the hemorrhagic ebola and marburg filoviruses, and paramyxoviruses such as nipah virus [10, 11, [13] [14] [15] [16] [17] [18] . more recently, a pandemic of an acute respiratory syndrome originating in wuhan, china in december 2019 was linked to a coronavirus (designated "sars-cov-2") that shared 96% identity with a bat-borne coronavirus at the whole-genome level [19] . in some cases, these viruses can subsequently spread through person-to-person contact following spillover from animals, increasing their epidemic potential [10, 11, 19] . the 2002-2003 sars epidemic, the emergence of mers in people in 2012, and the ongoing covid-19 pandemic have prompted substantial interest in detecting coronaviruses of bat origin due to public health concern and their pandemic potential [10, [13] [14] [15] [16] [17] [18] . coronaviruses (cov) are a family of enveloped, single-stranded rna viruses that commonly infect the respiratory and gastrointestinal tracts of their mammalian and avian hosts [10] . the alphacoronaviruses and betacoronaviruses are of particular importance to human health, with sars-cov, sars-cov-2, and mers-cov-which have caused the most severe disease in humans to datebelonging to the latter group [10, 20, 21] . mounting evidence indicates that bats are the evolutionary hosts and origin for these cov lineages [10, [19] [20] [21] [22] . in addition to human-associated covs, bats are also hosts of coronaviruses that infect production animals, and have been implicated in the emergence and origin of swine acute diarrhea syndrome (sads), transmissible gastroenteritis virus (tgev) in pigs, and porcine epidemic diarrhea (ped), which can cause considerable losses [23] [24] [25] [26] . thus, bat-borne covs can pose a significant threat to human health and food production. in spite of these infectious disease threats, bats are an indisputably essential component of ecosystems. they provide critical services such as seed dispersal, pollination, control of insect populations (including crop pests and disease vectors), and fertilization via guano, making them invaluable assets to agricultural industries and small-holder farming [27] . the importance of bats to ecosystems and human communities while being the natural reservoirs of many zoonotic pathogens presents a challenge for disease control. the potential threats posed by bat-borne coronaviruses to human and livestock health necessitate the identification and characterization of these viruses at high-risk interfaces among humans, domestic animals, and wildlife. particular attention is needed in developing regions of high biodiversity, where eids are most likely to arise, and where substantial losses in agricultural production may be a source of financial insecurity [28] [29] [30] [31] [32] . myanmar is a particularly vulnerable country due to the interplay of ecological and human factors, which increase opportunities for viral spillover. the nation is situated in the heart of the southeast asia region, a hotspot for eids, including some neglected tropical diseases and some of pandemic potential like sars and h5n1 influenza [31, 32] . a combination of biological, ecological, socioeconomic, and anthropogenic factors renders the region particularly susceptible to emerging zoonoses that could impart a considerable public health and economic burden [31, 32] . our study aimed to detect coronaviruses in free-ranging bats living in close proximity to human communities. (fig 1) . these sites were targeted as potential high-risk human-animal interfaces due to land use change increasing human proximity to wildlife and potential human exposures through livelihood, recreational, commercial, and religious or cultural activities. two of these sites also featured popular cave systems where people were routinely exposed to bats through guano harvesting, religious practices, and ecotourism. sites 1 and 2 consisted of several smaller sub-sites where bat capture and sampling events occurred. all surveillance activities were conducted in collaboration with three of myanmar's government ministries: (1) the ministry of livestock, agriculture, and irrigation; (2) the ministry of health and sports; and (3) the ministry of natural resources and environmental conservation. all work conducted was approved through a letter of agreement, ethical review committee, and memorandum of understanding, respectively. bat sampling was performed by trained field personnel in collaboration with myanmar's ministry of agriculture, livestock and irrigation (moali) and ministry of natural resources and environmental conservation (monrec). all bats were captured using mist nets, with each individual manually restrained for species identification, morphometric evaluation, and sample collection. no anesthetic or immobilization agents were used during capture or handling. oral and rectal swabs were collected when possible using sterile polyester-tipped applicators (animal size often precluded rectal swab collection). naturally voided guano samples consisting of combined urine and feces were also collected from the environment using plastic tarps. at site 2, the tarps were placed on the floor of the caves and left overnight, with sample collection occurring the following morning. at sites 1 and 3, the tarps were placed at cave entrances and under roosting areas in the evening as the bats emerged to forage, and samples were collected immediately. guano pellets were collected randomly from the tarps and pooled. tarps were disinfected between each use and gloves were changed in between each sampling event. pooled guano samples were attributed to a presumptive host species based on field identification of species in caves when possible, otherwise were designated as "unidentified chiropterans" when multiple species were present. all sample types were collected into 500 μl viral transport medium (thermoscientific microtest tubes, fisher scientific, pittsburgh, pa, usa) or 500 μl trizol reagent (invitrogen trizol reagent, fisher scientific, pittsburgh, pa, usa), transported from the field in liquid nitrogen, and transferred to a -80˚c freezer within five days and stored until time of testing. bats were humanely trapped, handled, and sampled from according to protocols approved by the institutional animal care and use committee of the university of california at davis (protocol 19300) and smithsonian institution (protocol 16-05) and with approvals from moali and monrec. bats were released within 1 km of the sample testing was performed at the uc davis one health institute laboratory and the veterinary diagnostic laboratory, livestock, breeding, and veterinary department (lbvd) in myanmar. 250 μl was used from each sample for rna extraction per kit instructions, and to ensure availability of an additional aliquot should a second extraction or other downstream analyses be needed. rna was extracted using direct-zol rna columns (zymo research corp), and 8 μl rna was used for cdna transcription using superscript iii (invitrogen). samples were screened for coronaviruses using two broadly reactive consensus conventional polymerase chain reaction (pcr) assays targeting two non-overlapping fragments (434 bp and 332 bp) of the rna-dependent rna polymerase (rdrp) of orf1ab of covs [33, 34] . bands of the expected size were cloned (pcr4-topo vector; invitrogen corp.) and sanger sequenced (abi 3730 capillary electrophoresis genetic analyzer; applied biosystems, inc., foster city, ca). sequences were analyzed and edited using geneious prime (version 2019.1.3), uploaded to genbank (s1 table) , and compared with known sequences in the database. coronavirus sequences were classified as belonging to viral taxa according to established cut-offs and methods [28] . virus sequences that shared less than 90% identity to a known sequence were labelled sequentially as predict_cov-1, -2, -3 etc; while groups sharing �90% identity to a sequence already in genbank were given the same name as the matching sequence. based on these criteria, the cov sequences detected were assigned to discrete viral taxa. viral culture and isolation were not attempted for any positive samples. bat samples positive for a cov-including positive pooled guano samples-were barcoded to confirm the host species using pcr assays targeting fragments of the cytochrome b gene (cytb) and the cytochrome oxidase subunit 1 genes (co1) [35] . one pcr amplicon was selected for sequencing and compared to reference sequences in genbank using blast tools. a threshold of 97% sequence identity was used to confirm the species. sequences with <95% sequence identity were classified to the genus. dna barcoding was also performed on a subset of the cov-negative pooled guano samples. pooled guano samples were assigned a presumptive origin species based on host barcoding. a total of 464 bats representing at least 11 species across eight genera from six families were captured and sampled (table 1 ). both insectivorous microbats and fruit bats were represented in our study population. a total of 759 samples were collected and tested (464 oral swabs, 140 rectal swabs, 155 guano samples). a total of 461 samples were collected in the dry-season sampling (244 oral swabs, 117 rectal swabs, and 100 guano samples) and 298 samples (220 oral swabs, 23 rectal swabs, and 55 guano samples) in the wet season. covs were detected in 48 samples: one oral swab and seven rectal swabs from seven individual bats and 40 pooled guano samples (table 1 ). viral fragments were detected from one unidentified tomb bat (taphozous sp.), three horsfield's leaf-nosed bats (hipposideros larvatus), and three greater asiatic yellow house bats (scotophilus heathii). thirty-six of the 40 positives detected in guano were attributed to h. larvatus, while the host species for the remaining four positive pooled guano samples was identified as wrinkle-lipped free-tailed bats (chaerephon plicatus). overall viral prevalence across all bat taxa and all coronaviral genotypes was approximately 1.5%. the vast majority of positive detections (83.3%) were made from pooled guano samples, while oral swabs had the lowest yields. positive detections were made from 40 samples collected during the dry season (83.3%), while wet-season sampling resulted in positive detections from eight samples (16.7%). both sites 1 and 2 accounted for positive detections, while no coronaviral sequences were detected at site 3. fifty-four total sequences were recovered, clustering within seven distinct coronaviral genotypes. using established cut-offs and methods [28] , we detected four alpha coronaviruses (predict_cov-35, 47, 82, and 90) and three betacoronavirues (predict cov-92, 93, and 96). of these, the alphacoronavirus predict_cov-35 was previously known, having been found in scotophilus kuhlii, unidentified myotis, and other unspeciated host bats in the neighboring countries of cambodia and vietnam from 2013 to 2017 [36] . the remaining six coronaviruses were novel (three alphacoronaviruses and three betacoronaviruses). predict_cov-92 was the most commonly detected coronavirus, found in 36 pooled guano samples attributed to h. larvatus (table 1) . interestingly, three coronaviruses were only found as co-infections: predict_cov-90 was detected with predict_cov-35, predict_cov-93 with -96, and predict_cov-96 also with -92. 3 indicates at least one instance of co-infection. 4 did not meet the 95% nt identity threshold for identification to the taxonomic level of species. https://doi.org/10.1371/journal.pone.0230802.t001 three new alphacoronaviruses, three new betacoronaviruses, and one previously described alphacoronavirus were detected in bats in myanmar. none of the viruses appeared to be closely related to sars-cov, mers-cov, or sars-cov-2. guano samples accounted for the majority of positives, suggestive of an important transmission route for cov shedding from bats [29, 28, 29] and a possible risk to people during the act of guano harvesting [37, 38] . viral detection in guano also has implications for future surveillance, as our study demonstrates the value of non-invasive collection of guano for viral surveillance, potentially obviating the need for handling individual bats for coronaviral detection. our findings supplement those of he et al., who profiled the virome of insectivorous bats from northern myanmar but did not detect coronaviruses in that study [40] . a difference was found in positives for cov by species, as samples from h. larvatus represented 83% of positives. a wide diversity of covs has been found in hipposiderid bats [28, 34, 39, 41] , and our study is consistent with those findings. four covs detected in our surveillance study were found in a single host species each: predict_cov-90 was found only in s. heathii; and predict_cov-92, -93, and -96 were found only in h. larvatus (table 1) . these findings may possibly suggest limited host-switching and viral sharing for certain viruses within our study populations, a pattern consistent with prior observations that viral groups are likely significantly associated with host taxa at the family level [28] . however, further evidence is needed to elucidate host-viral relationships and ecology in the region. our findings also likely reflect a bias in our sampling effort. although h. larvatus samples accounted for the most positives, these were largely detected in guano samples collected from the environment, as individuals were not frequently caught by mist net. overall in our study, the numbers of individual bats handled and sampled per species were relatively low, ranging from one to 218 (table 1 ). viral prevalence may vary widely with the species of host and pathogen. anthony et al. suggested a sample size of at least 154 individuals per species in order to maximize our ability to detect covs. targeting more host species, specific taxa (hipposideridae), and larger sample sizes might have improved our detection rate in the species where no covs were found [28, 29] . currently, active pathogen surveillance at human-wildlife interfaces in myanmar is limited. despite relatively small sample sizes, our study detected several coronaviruses in insectivorous bats, suggesting that more may remain to be uncovered. given the potential consequences for public health in light of expanding human activity, continued surveillance for coronaviruses is warranted, especially in other species and human-wildlife interfaces. anthony et al. estimated that over 3,200 covs occur in bats, most of which remain undiscovered [28] . enhancing our sampling effort to incorporate more diverse bat families and larger sample sizes may enable us to identify more covs in bats in myanmar. additionally, because only short fragments of the conserved rdrp gene (328 bp and 434 bp) were amplified in this study, protein sequence and phylogenetic analyses were not pursued, and identification of recombination events was not possible. while this is an inherent shortcoming of our methodology, the purpose of this study was not to fully characterize specific viruses, but to broadly screen for viruses in bats living in proximity to human communities to better understand potential sources of zoonotic transmission in the context of these human-wildlife interfaces. further studies may consider complete genomic sequencing for more comprehensive profiling of the bat viromes in this ecosystem. in particular, evaluation of the spike gene sequences may provide insights into host range, including potential viral host-sharing or host-switching events [42] . land use change will likely continue bringing people into closer proximity with bats, raising encounter rates and opportunities for spillover, facilitating the emergence of zoonotic viruses, and supporting the need for surveillance [12, 43] . historically, human activities have arguably played a significant role in interspecies transmission events. following the sars outbreak, coronaviruses have since been detected in numerous bat species globally, including in asia, africa, europe, the americas, and the australasian region [28, [44] [45] [46] [47] [48] [49] . mounting evidence supports the role of bats in the transmission of viruses of public health concern-including sars--cov and mers-cov-and the zoonotic potential of unknown bat-borne coronaviruses warrants vigilant, continued surveillance [10] . understanding their ecology and prevalence in their natural hosts can improve our ability to detect, prevent, and respond to potential public health threats. finally, given the essential ecosystem services provided by bats, public health efforts should advocate for preventative measures to protect people against disease transmission while enabling human communities and bats to coexist on a shared landscape. supporting information s1 fauci as the challenge of emerging and reemerging infectious diseases factors in the emergence of infectious diseases host range and emerging and reemerging pathogens global trends in emerging infectious diseases risk factors for human disease emergence human ecology in pathogenic landscapes: two hypotheses on how land use change drives viral emergence ecology of zoonoses; natural and unnatural histories impacts of biodiversity on the emergence and transmission of infectious diseases diseases of humans and their domestic mammals: pathogen characteristics, host range and the risk of 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deltacoronavirus fatal swine acute diarrhea syndrome caused by an hku2-related coronavirus of bat origin a new bat-hku2-like coronavirus in swine prevalence and genetic diversity of coronaviruses in bats from china bat coronavirus in brazil related to appalachian ridge and porcine epidemic diarrhea viruses ecosystem services provided by bats global patterns in coronavirus diversity optimizing viral discovery in bats global hotspots and correlates of emerging zoonotic diseases emerging infectious diseases in southeast asia: regional challenges to control zoonoses in south-east asia: a regional burden, a global threat bat coronaviruses and experimental infection of bats, the philippines identification of a severe acute respiratory syndrome coronavirus-like virus in a leaf-nosed bat in nigeria identification of mosquito bloodmeals using mitochondrial cytochrome oxidase subunit i and cytochrome b gene sequences predict 1 & 2 test data healthmap database group c betacoronavirus in bat guano fertilizer coronaviruses in guano from pteropus medius bats in peradeniya diversity of coronavirus in bats from eastern thailand virome profiling of bats from myanmar by metagenomic analysis of tissue samples reveals more novel mammalian viruses circulation of alphacoronavirus, betacoronavirus and paramyxovirus in hipposideros bat species in zimbabwe recombination, reservoirs, and the modular spike: mechanisms of coronavirus cross-species transmission bats, coronaviruses, and deforestation: toward the emergence of novel infectious diseases? front microbiol detection of group 1 coronaviruses in bats in north america detection of novel sars-like and other coronaviruses in bats from kenya detection and prevalence patterns of group i coronaviruses in bats coronavirus infection and diversity in bats in the australasian region coronaviruses in bats from mexico detection of coronaviruses in bats of various species in italy we also thank the livestock breeding and veterinary department (lbvd) within the ministry of agriculture, livestock, and irrigation (moali); ministry of natural resources and environmental conservation (monrec); and the department of medical research (dmr) within the ministry of health and sports (mohs), myanmar, with whom we collaborated closely on surveillance activities. thanks also to the invaluable field and laboratory staff who provided technical skill and expertise and were critical in the research process. key: cord-284015-vvtv492b authors: nikaido, masato; kondo, shinji; zhang, zicong; wu, jiaqi; nishihara, hidenori; niimura, yoshihito; suzuki, shunta; touhara, kazushige; suzuki, yutaka; noguchi, hideki; minakuchi, yohei; toyoda, atsushi; fujiyama, asao; sugano, sumio; yoneda, misako; kai, chieko title: comparative genomic analyses illuminate the distinct evolution of megabats within chiroptera date: 2020-09-23 journal: dna res doi: 10.1093/dnares/dsaa021 sha: doc_id: 284015 cord_uid: vvtv492b the revision of the sub-order microchiroptera is one of the most intriguing outcomes in recent mammalian molecular phylogeny. the unexpected sister–taxon relationship between rhinolophoid microbats and megabats, with the exclusion of other microbats, suggests that megabats arose in a relatively short period of time from a microbat-like ancestor. in order to understand the genetic mechanism underlying adaptive evolution in megabats, we determined the whole-genome sequences of two rousette megabats, leschenault’s rousette (rousettus leschenaultia) and the egyptian fruit bat (r. aegyptiacus). the sequences were compared with those of 22 other mammals, including nine bats, available in the database. we identified that megabat genomes are distinct in that they have extremely low activity of sine retrotranspositions, expansion of two chemosensory gene families, including the trace amine receptor (taar) and olfactory receptor (or), and elevation of the dn/ds ratio in genes for immunity and protein catabolism. the adaptive signatures discovered in the genomes of megabats may provide crucial insight into their distinct evolution, including key processes such as virus resistance, loss of echolocation, and frugivorous feeding. bats belong to the order chiroptera and have the ability of powered flight. accounting for one-fifth of all mammals in terms of the number of species, bats are one of the most successful groups of mammals. 1 it is of primary interest for biologists to identify the processes and mechanisms of dynamic adaptation in bats. traditionally, morphological and paleontological analyses placed the order chiroptera within the superorder archonta (primates, dermoptera, chiroptera, and scandentia). 2 however, dna sequencing data has challenged the validity of the archonta, and alternatively proposed the inclusion of bats into laurasiatheria (cetartiodactyla, perissodactyla, carnivora, pholidota, chiroptera and eulipotyphla). [3] [4] [5] [6] although laurasiatheria is now considered to be a natural assemblage, the phylogenetic position of bats within laurasiatheria remains to be resolved. 7, 8 the paraphyly of microbats is also under debate. traditionally, morphological studies proposed the sub-division of the order chiroptera into two suborders: microchiroptera (microbats) and megachiroptera (megabats or old-world fruit bats). 9 microbats use ultrasonic echolocation for flight and for foraging in the night, whereas megabats do not echolocate, and primarily use vision to fly and feed on fruits and/or nectars. megabats are also neuroanatomically distinct from microbats, as megabats have a developed visual system. 10 molecular data suggests that five lineages of microbats, including rhinopomatidae, rhinolophidae, hipposideridae, craseonycteridae, and megadermatidae, are more closely related to megabats than to other microbats. therefore, the five lineages of rhinolophoid microbats and megabats were re-classified as 'yinpterochiroptera' and the remaining microbats as 'yangochiroptera'. 5, 11, 12 thus, recent molecular studies suggest that several adaptive characteristics specific to megabats have emerged within a short period of time from a microbat-like ancestor. genome-wide analyses have been used to identify the unique evolution of bats in several studies. seim et al.'s 13 study determined the genome sequence of one microbat (brandt's bat) and found the signatures for adaptive evolution in genes related to physiology and longevity. zhang et al. 14 determined the genome sequences of one microbat (david's myotis) and one megabat (black flying fox) and found that genes for flight and immunity evolved due to positive selection. parker et al. 15 identified the genomes of three microbats, including the greater horseshoe bat, the greater false vampire bat, and parnell's mustached bat, and one megabat, the straw-coloured fruit bat. in comparing the genomes of these bats with those of other mammals, this study identified that genes related to hearing/deafness showed convergent evolution among echolocating mammals. pavlovich et al. 16 recently determined the whole genome of the egyptian fruit bat (r. aegyptiacus), which is a natural reservoir for the marburg virus, and revealed that the genes for immunity were expanded and diversified, suggesting an antiviral mechanism that is used to control viral infection. especially, as bats are natural hosts for zoonotic virus including henipaviruses, filoviruses, and coronaviruses, which are emerging viruses with high rates of fatality, the comparative genomic study in bats may provide an effective solution against the current global pandemics of coronavirus disease-2019 . 17 in this study, we determined the genome sequences of two rousette megabats, leschenault's rousette (rousettus leschenaultia) and the egyptian fruit bat (r. aegyptiacus). we assessed the genomic signatures for the process of natural selection that facilitates the dynamic and adaptive evolution of megabats. in particular, the main aim to determine the whole-genome sequence of egyptian fruit bat in addition to the previous study 16 is to obtain higher quality genome data, which facilitates more accurate and comprehensive gene annotations, especially for multi-gene families. in addition, the genome sequences of leschenault's rousette belonging to the same genus as the egyptian fruit bat is of our interest to identify genomic differences in closely related bat species. these genome sequences were compared with those of 22 mammals, including six microbats and three megabats, available in the database. we used genome-wide phylogenetic analyses, followed by candidate gene analyses focussed on retroposons and chemosensory multi-gene families for taste, olfaction, and pheromone detection. in addition, we also performed global positive selection analyses. as a result, the inter-relationships among laurasiatheria were consistently reconstructed, with the order eulipotyphla diverging first, followed by the divergence of chiroptera and the remaining groups, including cetartiodactyla, perissodactyla, pholidota, and carnivora. the reciprocal monophyly of yinpterochiroptera and yangochiroptera was also shown with reliable statistical support. we revealed several notable distinct features in megabat genomes, including extremely low activity of sine retrotranspositions and the expansion of the genes for the trace amine receptor (taar) and olfactory receptor (or). additionally, the signatures for positive or relaxed selection were observed in genes for immunity and protein catabolism. thus, our comparative genomic analyses may illuminate the genetic mechanisms underlying the dynamic adaptation of megabats during diversification in the order chiroptera. egyptian fruit bats (r. aegyptiacus) and leschenault's rousettes (r. leschenaulti), both of which were provided by ueno zoo, were maintained under controlled conditions using an air conditioner and moisture chamber. the animals were kept in steel cages and fed fruit and water at the same time every day. all experiments were performed in accordance with the animal experimentation guidelines of the university of tokyo and were approved by the institutional animal care and use committee of the university of tokyo. as for egyptian fruit bats, we prepared kidney-derived primary cultured cells. a pregnant egyptian fruit bat was deeply anesthetized with isoflurane, the uteri were surgically removed, and the animal was euthanized by bleeding. the kidney from the fetus was fragmented using scissors and treatment with tryple (gibco). the fragmented kidney was then cultured in dmed containing 5% fetal calf serum to obtain primary cultured cells. genomic dna was extracted from the frozen spleen tissue or cultured kidney cells of two individuals of egyptian fruit bat, and frozen kidney tissue from one individual of leschenault's rousette, using a blood & cell culture dna kit (qiagen, hilden, germany), according to the manufacturer's protocol with minor laboratory customizations, the information can be available upon request. the dna samples (>20 kb) were subjected to the sequencing as described below after quality and quantity check. to construct paired-end sequencing libraries, the genomic dna was fragmented using a covaris s2 focussed-ultrasonicator (covaris, woburn, ma, usa). the paired-end libraries were constructed using the truseq dna pcr-free library prep kit (illumina, san diego, ca, usa). mate pair libraries were prepared from genomic dna using the nextera mate pair sample preparation kit (illumina, san diego, ca, usa). all libraries were sequenced on an illumina-hiseq 2500 system using rapid-mode chemistry with paired-end sequencing. prior to assembly, data pre-processing was performed. first, the adapter sequences were trimmed using the fastq-clipper ea-utils v1.1.2, 18 setting the parameters to '-p 10 -m 1 -l 0'. second, we filtered the reads mapped to the mitochondrial genome using bwa-aln v0.6.2 19 with default parameters. finally, we performed base error correction using soapec v2.01 20 with the parameters '-k27 -l 150'. we then assembled the reads using platanus v1.2.1 21 with default parameters. contamination candidates were removed by mapping to escherichia coli and phix genomes using blastn v2.2.9, 22 setting the parameters to '-e 1e-30'. the statistics of the genome assemblies and the information of sequence libraries are summarized in supplementary tables s1-1 and s1-2. in order to test the quality of the reference assembly in the egyptian fruit bat, we additionally constructed a fosmid library, which was end-sequenced using abi 3730xl sequencers. the protein-coding genes in the genomes of egyptian fruit bat and leschenault's rousette were identified based on the alignment with annotated gene sequences of 14 mammals (cat, dog, horse, cow, hedgehog, human, macaque, mouse, rat, black flying fox, little brown bat, brandt's bat, david's myotis, and large flying fox; supplementary table s2 ) that are available in the database. the sequences for each gene of the 14 mammals were aligned to the two bat genomes by using blat 23 to identify approximate gene loci. the blat alignments of the gene sequences to the genomes were refined by the exonerate software to estimate the exon-intron boundaries. 24 in addition to the homology-based identification, rna-seq-based transcript reconstruction and ab initio gene prediction were performed to identify the protein coding genes. rna of primary culture cells from the kidney of the egyptian fruit bat was extracted by using trizol reagent (thermo fisher). a total of 122,017,734 paired-end reads of mrna (illumina-hiseq, 101 bp) were aligned to the genomes using tophat. 25 in total, 97,696,475 and 88,528,280 paired-end reads could be mapped to the genome sequences of r. aegyptiacus and r. leschenaultii, respectively. transcript structures were reconstructed using augustus 26 based on the tophat alignment of the illumina reads to the bat genomes. the expression levels of the reconstructed genes were computed using cufflinks 27 based on the tophat alignment of the illumina reads to the genomes. a total of 8,079 genes were expressed with fragments per kilobase of transcript per million of reads mapped (fpkm) ! 1 in the kidney-derived primary cultured cells. examples are shown in supplementary fig. s1 . ab initio genes were obtained by using genscan 28 and snap. 29 the genomic sequences were cut to seven megabase-long fragments, and genscan was run on each fragment. the genes identified were assigned to gene loci based on the overlap of exons on the same strand, and the redundancies of the transcripts were removed. only transcripts annotated with the start codon (atg) and introns flanked by canonical splice dinucleotide pairs (gt-ag, gc-ag, and at-ac) were retained. a total of 46,249 and 47,073 transcripts were annotated over 20,005 and 20,913 gene loci, respectively, on the genomes of the egyptian fruit bat and leschenault's rousette. the completeness of the gene determination was evaluated by using busco. 30 similarly, we assessed protein-coding genes on the genomes of four other bat species, the straw-coloured fruit bat, the greater false vampire bat, the greater horseshoe bat, and the strawcoloured fruit bat. 15 due to fragmental nature of these genome assemblies (n50: 15-27 kb), however, we did not use the thresholds of initial codon and splice sites as used in the annotation of the genomes of the egyptian fruit bat and leschenault's rousette. we identified the longest orf in each transcript mapped by exonerate by using transdecoder (https://github.com/transdecoder/transdecoder/ blob/master/transdecoder.longorfs) and used it as the gene annotation. we identified 28,367-31,441 transcripts in 19,296-20,272 gene loci on these genomes (supplementary table s3 ). the ratio of complete genes of the annotated genes evaluated by busco was 53.8-76.0%. additional to this annotation, the tandemly duplicated receptor genes, including ors, taste receptors (t1rs and t2rs), vomeronasal receptors (v1rs and v2rs), formyl peptide receptors (fprs), and taars, were annotated separately. olfactory receptors were identified by the method described previously. 31, 32 the other receptor genes were identified using another protocol. 33 in short, we obtained protein sequences of mammalian t1rs, t2rs, fprs, v1rs, v2rs, and taars from the ncbi refseq database (https://www.ncbi.nlm. nih.gov/refseq/). the redundant sequences, which contain more than 80% identity as identified by cd-hit 34 , were removed to establish representative query sequences. for t1rs, we used only the transmembrane regions as query sequences. we used the ncbi conserved domains database to annotate the 7-transmembrane domains of t1rs. using the query sequences, we performed a tblastn search against the whole-genome sequence assemblies available in genbank (https://www.ncbi.nlm.nih.gov/genbank/). the taxonomic classification and the accession numbers of the whole-genome sequences are summarized in supplementary table s2 . the exon-intron structure of each sequence, which was obtained by tblastn, was predicted with the exonerate program 24 using translated query sequences as protein models. the resulting hit sequences were classified into 'intact', 'truncated', and 'pseudo-genes'. due to an assembly issue, the 'truncated' genes included poly 'n' sequences. in order to estimate the gene copy numbers, in these analyses, we treated the 'truncated' genes as 'putatively intact'. the pseudo-genes include inactivating mutations in the coding region. the resulting genes were assessed to determine whether they encode the chemosensory receptors of interest using blastx searches and ghostz 35 against the uniref50 database (https://www.uniprot.org/help/uniref). we used the framework for annotating translatable exons (fate), which is available in github (https://github.com/hikoyu/fate), for the automation of the procedures described above. we constructed a phylogenetic tree based on the single-copy orthologous gene sets of mammals, as previously reported by wu et al., 36 to elucidate the phylogenetic relationships of megabats with other mammals. briefly, the nucleotide sequences of the 6,365 proteincoding genes of the two megabat species and 22 other mammalian species (supplementary table s2 ) were aligned using the prank software v.170427 37 in codon level. sites that are shared by <70% of the species were removed from the alignment. among the 6,365 genes, 2,093 genes were listed for all species, and were used for the analyses. the gene tree was constructed using raxml software, v8.1.12 38 using the gtrþcþi model with 1,000 bootstrap replicates for each 2,093 gene. we collected the best tree for all 2,093 genes, which were used to infer the coalescent species tree with branch length by astral-iii. 39 the node support of the species tree was obtained by 1,000 replicates of bootstrapping. branch length shown in the tree indicates the branch length in coalescent units. 40 we used the genome of leschenault's rousette for the identification of tes, based on two approaches, including de novo characterization of tes and identification of homologous copies of known tes in another megabat, the large flying fox (supplementary table s2 ). in the first approach, repeatmodeler ver. 1.0.8 (http://www.repeatmasker. org/repeatmodeler.html) was used to obtain a collection of repetitive sequences. for each of the preliminary consensus sequences, we conducted a local nucleotide blast search (r ¼ 2, g ¼ 5, e ¼ 2, with an e-value cutoff of 10 à10 ) and collected 80-100 copies along with their 10-kp flanking sequences. the copy sequences were aligned using mafft ver. 7. 41 the alignment was manually modified using mega 5.0 42 and a consensus sequence was re-constructed. the consensus sequence was used for the next round of the blast search, as described above, to obtain additional copies. this procedure was repeated until a full-length consensus sequence was completed. the full-length tes were characterized and classified based on the sequence structure, including terminal inverted repeats and long terminal repeats (ltrs), coding proteins such as transposase and reverse transcriptase, and by comparison with known elements using repeatmasker ver. 4.0.6 (http://repeatmasker.org), censor, 43 and rtclass1. 44 for the second approach, a te library of another megabat, the large flying fox, including 65 te families which were obtained from repbase, 45 was used as a query for a homology search against leschenault's rousette. the local nucleotide blast search, alignment of the copy sequences, and reconstruction of the consensus sequence were conducted as described above. a similar blast search was also conducted using 102 te families of a microbat (the little brown bat, supplementary table s2) library; however, no additional novel tes were found except in the results from the two approaches listed above. all of the newly characterized 118 te (sub) families were designated in conformity with the repbase classification. the repeat contents of the two rousettus genomes were estimated using repeatmasker with the sensitive option (-s) of cross-match search using the rousettus repeat library which we developed here. the te contents, such as the number of copies and length, were summarized based on their divergence (%) from the consensus sequence at the family/subfamily levels by using in-house perl scripts. the te contents of other species were summarized based on the repeatmasker output (http://www.repeatmasker.org/genomic datasets/rmgenomicdatasets.html). orthologous genes under relaxed selection on megabat lineages were identified from the aligned 6,365 single-copy genes. on every alignment, we used the codeml branch model in paml 4.8 46 to detect the elevation of the dn/ds ratio (the non-synonymous substitution rate to the synonymous substitution rate) on stem and crown megabat branches. the species tree shown in fig. 1 was used as a guide tree in the analysis. likelihood ratio tests and inspections of the p-value were used to compare likelihoods between two models: (i) that assumed the megabat lineages as foreground branches; and (ii) that assumed the dn/ds ratio was not altered in all branches (null hypothesis), to evaluate the significance of the elevation of the dn/ds ratio for megabat branches. we performed further analyses for the genes of interest using the codeml branch-site models for analysing the positive selection on each site. in the branch-site test, we tested stem and crown megabats as the foreground branches and used microbats and outgroup species, including human, macaque, mouse, rat, cat, dog, chinese pangolin, sunda pangolin, bottlenose dolphin, cow, horse, hedgehog, asian musk shrew, and common shrew, as background branches. for the branch-site test, we used two models for the analysis, including one model of a null hypothesis that assumes that the gene was under two types of selective pressures (purifying selection and neutral selection), and one model that used an alternative hypothesis to assume the gene was under three categories of selective pressures, including positive selection on the megabat branches. the likelihood ratio test comparing the likelihoods of these two models was used to evaluate the significance of the alternative model. to assess the functionality of positively selected sites, protein structure deposited in protein data bank (pdb) was used. the protein structures were depicted using the open-source version of pymol. 47 we constructed draft genomes of the egyptian fruit bat and leschenault's rousette by assembling short read data into contigs and scaffolding them using platanus v1.2.1. 21 the genome of the egyptian fruit bat is composed of 1.90 gbp with 4,974 scaffolds (n 50 ¼ 37.2 mbp) and the genome of leschenault's rousette is composed of 1.90 gbp with 8,141 scaffolds (n 50 ¼ 32.7 mbp) (supplementary tables s1-1 and s1-2). the high qualities of the two genomes are demonstrated by the ratios of complete genes, which are 98.1 and 97.9%, respectively, as evaluated by busco 30 (supplementary table s3 ). the quality of both genomes in terms of the continuity of the scaffolds and the rate of n is high enough to facilitate genome-wide evolutionary analyses and characterization of multi-gene families. in addition, independent genome assemblies and gene annotations of the two individuals of egyptian fruit bat determined in the previous study 16 and this study may be utilized as an initial step towards the identification of the genotypic, transcriptomic, and phenotypic variation of this species in the future research. figure 1 shows the maximum likelihood phylogenetic tree with the time scale for 24 mammals, including 11 bats (five megabats and six microbats) based on 2,093 single-copy orthologous gene sets. four species of euarchontoglires, including humans, macaca, mouse, and rat, were used as outgroups. the tree successfully highlights the evolutionary history of laurasiatherian mammals in that eulipotyphla diverged first among them. in this phylogenetic tree, chiroptera diverged after eulipotyphla; however, the bootstrap probability (bp) supporting this node was not so high (63.5) . in addition, the grouping of pegasoferae (chiroptera, perissodactyla, and carnivora), which was originally proposed by the insertion of retroposons 7 and supported by several genome-wide analyses, 13, 14 was not supported. given that the bps for the inter-relationships of cetartiodactyla, perissodactyla (carnivora þ pholidota), and chiroptera were relatively low (63.7, 63.5) and the branch lengths were markedly short, it is highly likely that the initial divergence of laurasiatherian mammals occurred rapidly during evolution. such rapid speciation events may hamper reconstruction of the consistent tree topology for these groups. 8, 48 importantly, as it was shown in the previous studies, 5,49,50 the reciprocal monophyly of yangochiroptera and yinpterochiroptera was successfully supported in this analysis, suggesting that the megabats are nested in microbat lineages. although it is difficult to estimate the ancestral state in the megabat ancestors due to the rarity of the fossil record, the phylogenetic tree suggests that several distinct characteristics in megabats, including the welldeveloped visual system, frugivorous diet, and the absence of echolocation, evolved in a short period of time during evolution from a 'microbat-like' ancestor. we next focussed on assessing the signatures for such adaptive evolution in these groups based on the genome-wide comparative analyses. in both the leschenault's rousette and egyptian fruit bat genomes, tes account for $35% of the genome, including sines (3.9%), lines (21%), ltr retrotransposons (6.2%), and dna transposons (4.0%) (supplementary table s4 and fig. s2 ). it is notable that the proportions of tes in megabats, including the two rousettus species and the large flying fox, are considerably lower as compared to the levels in other mammals, such as humans, where nearly half of the genome is covered by tes ( fig. 2a and supplementary fig. s2) . consistent with the previous observations, it is also interesting that the proportion of tes is generally correlated with the genome size in mammals 51, 52 (supplementary fig. s2 ). co-variation between an accumulation of tes and dna loss by large segmental deletions is considered a major contributing factor to determine the genome size. 50 therefore, the smaller genome sizes in the megabats may be due to a lower activity of tes, at least in part. indeed, our analysis revealed that the number of young (recently retrotransposed) te copies in the megabat genomes is very small (fig. 2b and supplementary fig. s3 ). as exemplified by the microbat myotis lucifugus, where the number of tes representing <5% divergence from the consensus sequence is 180,000 (6.5% among all te copies; supplementary fig. s3 ) consistent with the previous studies, 53 in general, young tes constitute a few percent among all tes in mammalian genomes. however, the copy numbers of young tes is only 2,900 (0.15%) and 7,300 (0.38%) for the rousettus species and large flying fox, respectively ( fig. 2b supplementary fig. s3 ). the small proportion of young tes is partly accounted for the low frequency of retrotransposition events in megabat-specific sines (fig. 2) . in general, different types of sine families are distributed for each mammalian clade, such as order, sub-order, or family. 52 in megabats, the only known active sines are the 5s rrna-derived meg sines. 54 it should be noted that rousettus genomes contain no more than 23,000 copies of the meg-related sines, which cover 0.21% of the genome. however, clade-specific sines are, in general, retrotranspositionally highly active, with 10 5 -10 6 copies present in each mammalian genome (fig. 2a) . the large flying fox (pteropus vampyrus) also has only 22,000 copies of meg-related sines. based on the wide distribution of meg sines in megabats, including rousettus, macroglossus, eonycteris, and cynopterus, 54 the origin of meg can be traced back to the common ancestor of megabats, which existed at least 24 million years ago. 49 it is possible that such a low retrotranspositional activity of the sines found in rousettus and pteropus is observed widely among megabats. it has been demonstrated that flying vertebrates, including bats, have substantially lost tes and have smaller genome sizes in association with cellular metabolic constraints. 55, 56 the small proportion of meg sines in the megabats may also be a result of the constraint related to their powered flight. another notable te family is line-1 (l1), as it has been reported that the retrotranspositional activity of l1 has been lost in megabats. 57 it is unlikely that the extinction of l1 resulted from the quiescence of l1 itself, because a synthesized sequence of the reconstructed megabat l1 is capable of retrotransposition in human hela cells. 58 in addition, we identified that in addition to l1, all types of tes have the least activity in megabats among the mammals investigated (fig. 2b) . this low activity of young tes may be due to an unknown megabat-specific mechanism for te repression or a result of extensive dna loss during the past tens of millions of years. one of the possible mechanisms by which te activity may be tightly repressed is an antiviral immune system in megabats. suggesting that the egyptian fruit bat may possess a novel mode of antiviral defense, 16 several antiviral-related genes are known to have expanded in this bat. for example, ribonuclease l, an interferoninducible endoribonuclease that cleaves viral rnas, 59 evolved under relaxed selective constraint in bats. 16 ribonuclease l is also known to restrict retrotransposition of human l1 and mouse iap elements in human cells. 60 in addition, several other factors that restrict retrotransposition in humans and mice are known to be involved in an antiviral immune system. 61 thus, it is possible that a unique antiviral mechanism against exogenous parasites (i.e. viruses) is secondarily used for the restriction of the endogenous retroelements. as general mobilization of sines in mammals relies on the l1 machinery, the restriction of megabat l1 could limit the meg sine activity. 62 the low activity of tes may partly contribute to the small genome size ( supplementary fig. s2 ), which could also be advantageous with respect to cell size and metabolic constraints in megabats as well as other flying vertebrates 55, 56 . therefore, the unusual characteristics of the tes, likely shared among megabats, are an important example to study the molecular mechanisms underlying restriction of retrotransposition. such future studies may shed light on the reason why bats have such compact genomes. it also remains unknown why ves sines in microbats are active, whereas the genome size is relatively small among mammals (fig. 2) . the difference in the sine activity between megabats and microbats may be affected by a possibly distinct antiviral immune system between the two groups, given that expansion of some antiviral-related genes occurred specifically in megabats. 16 most of the chemosensory receptors are encoded by multi-gene families, allowing animals to detect highly diversified chemicals in the environment. the previously published studies have shown that the collections of the chemosensory receptor genes are flexible and highly variable among mammals, including the ors, taste receptors (t1rs and t2rs), vomeronasal receptors (v1rs and v2rs), fprs, and taars. 63 the number of certain chemosensory receptor gene families has been shown to have a strong correlation with the degree of dependence on these ligand chemicals for survival. 32, 64, 65 several studies have revealed that bats lost several chemosensory receptor genes, such as t1r1 for umami, 66 and v1rs for pheromone(s) 67 that may be due to the specific sensory adaptation in the ancestor of these groups. it is possible that megabats re-allocated the diversity in chemosensory receptor genes as a sensory trade-off, given that megabats have experienced the secondary loss of echolocation ability, which is one of the most specialized senses in bats. 68 to examine this possibility, we comprehensively characterized the chemosensory receptor genes and compared their diversity by focussing on whether or not the repertoires in megabats show notable differences from those in microbats. our comparative genomic analyses of chemosensory receptor genes in the genomes of 25 mammals revealed that the copy number of the intact genes and pseudo-genes show a certain variation among bat species. in t1rs, the absence of t1r1, the umami receptor, in all of the bats that we analysed is consistent with the findings of the previous studies. 66 all megabats possess two t1rs (t1r2 and t1r3), whereas microbats are somewhat variable, in that they can possess no (greater false vampire bat), one (little brown bat), or two (brandt's bat, greater horseshoe bat) t1rs (fig. 3a and supplementary table s5 ). it is noteworthy that all megabats possess t1r2, which is the sweet receptor, suggesting the importance of sweet taste for their frugivorous lifestyle. no intact t1rs in the greater false vampire bat could be explained by their specific adaptation for a carnivorous diet, which resembles the blood-feeding activity of the vampire bat (desmodus rotundus), which also lost t1rs. 66, 69 as for t2rs, which are bitter taste receptors, the copy numbers are relatively smaller in megabats than those in microbats ( fig. 3a and supplementary table s5 ). the smaller number of t2rs in megabats can also be explained by their frugivorous diet, as compared with that of microbats, which are mostly insectivores. indeed, the repertoires of t2rs in primates have a strong correlation with their diet, 70 suggesting the importance of t2rs for feeding adaptation in mammals. we identified little variation between megabats and microbats in fprs, which are expressed in the sensory neurons of the vomeronasal organ and mediate innate avoidance behaviours (fig. 3a supplementary table s5 ). 71 suggesting that fpr-mediated chemodetection is not directly linked with the difference in their habitats, mega-and microbats both possess two to eight fprs. however, a previous study, by comparing the orthologous sequences among a broad range of mammals, found the signatures for the operation of positive selection in fprs. 72 therefore, to examine the possible contribution of fprs to the adaptive evolution of megabats, more detailed investigation is necessary by focussing on the dn/ds values among orthologous fpr sequences of many bat species, which are lacking at present. there was an extensive reduction in v1rs, which are known to be expressed in vno neurons of mammals and detect various pheromones, [73] [74] [75] in both megabats and microbats ( fig. 3a and supplementary table s5) . especially, only one v1r was found in the genomes of megabats. the reduction of v1rs revealed in this study is consistent with the findings of the previously published studies. 67 the inactivation of trpc2s 76,77 and ancv1rs, 78,79 which is responsible for vno function, suggested the degeneration of vnos in most bat lineages including megabats. although most bats do not possess intact v1rs, parnell's mustached bat possesses four intact v1rs (fig. 3a and supplementary table s5) , which is consistent with the presence of the vno in this species. 80 in addition, recent study has suggested that there are a substantial number of v1rs in distantly related groups of phyllostomids and miniopterids, which possess an intact vno, suggesting that they retained v1r-mediated chemical communication. 77, 81 therefore, the ancestor of all extant bats is expected to possess an intact vno, as well as a certain number of v1rs, that were independently degenerated after the divergence of each family, including megabats (pteropodidae). namely, the loss of echolocation and the degeneration of the vno occurred spontaneously in the ancestor of megabats. v2rs are expressed in the basal region of the vno neurons 74, 82, 83 and peptide pheromones were detected in mice. 84, 85 however, intact v2rs have been identified only in a limited number of mammals, such as rodents, 63 mouse lemurs, 86 and opossum. 87 our comprehensive analysis failed to find intact putative v2rs in the genomes of all bats and most of other mammals. this result suggests that, before the acquisition of the echolocation ability, the v2r-mediated pheromone detection system has already been lost in the common ancestor of all extant bat lineages. it is noteworthy that the hedgehog and the horse possess seven and one intact v2rs, respectively ( fig. 3a and supplementary table s5 ). this provides the first description of intact v2rs in the genomes of laurasiatherian mammals. more detailed analyses may provide insight into the v2r-mediated pheromone detection system in these species. one of the most intriguing results in the chemosensory receptor genes was obtained from taars. trace amine receptors have been believed to function as receptors for trace amines, for example, tyramine and octopamine in the brain. 88 however, a recent study revealed that taars may be expressed primarily or exclusively in the moe, 89 and are responsible for detecting volatile amines, including ethological odors that evoke innate animal behavioural responses. 90 in this study, we revealed that the number of taars was increased in the common ancestor of megabats. in particular, the number of taars, which were identified to be from five to seven copies in microbats, increased to more than 15 copies in megabats. in particular, leschenault's rousette possess 38 putatively intact (29 intact and 9 truncated) taars, which is the largest number the number of intact, truncated, and pseudo-genes is indicated in blue, yellow, and red, respectively. we treated the truncated genes as 'putatively intact'. the dotted lines show the variation in the number of intact þ 'putatively intact' genes among mammals. it should be noted that the number of taars is obviously higher in megabats than in microbats. (b) phylogenetic tree of intact taars in 24 mammals. only the intact genes were included in the tree. the taars of the egyptian fruit bat and leschenault's rousette are indicated by the square (green) and triangle (blue). it is obvious that the taars of subfamilies seven and eight were expanded in two rousettus bats. zebrafish taar13c in the ncbi database was used as an outgroup. mouse taar1-9 in the ncbi database was used as an indicator for each taar subfamily. accession codes for these database-derived genes are available in supplementary fig. s4 . identified among mammals ( fig. 3a ; supplementary tables s5 and s6 ). the phylogenetic analyses of intact taars for the 24 mammals clearly demonstrated that the expansion of the genes in the two rousettus bats, including the egyptian fruit bat and leschenault's rousette, occurred in subfamilies seven and eight in a species-specific manner ( fig. 3b; supplementary fig. s4 and table s6 ). eyun et al. 91 also reported a high copy number of taars in one megabat, the large flying fox; however, the repertoire was quite different from that of these two rousettus bats ( fig. 3a ; supplementary tables s5 and s6 ). although taars were expanded in subfamilies seven and eight in the two rousettus species, they were expanded only in subfamily seven in the java fruit bat. the number of intact genes, as well as the pseudo-genes, was highly variable among the megabats, suggesting that birth and death of taars were quite active. phylogenetic, as well as copy number, analyses suggest that taars have provided a large contribution to some process of adaptive evolution and diversification of megabats. interestingly, pavlovich et al. 16 revealed the gene expansion of mhc genes in the genomes of the egyptian fruit bat, suggesting novel modes of antiviral defense. thus, the mhc genes and taars were both expanded in megabats. santos et al. 92 reported that taars may be a key mediator in mhc-dependent mating choices in the sac-winged bat (saccopteryx bilineata). based on these findings, it is possible that the megabats use diversified taars for mate choice, by taking advantage of mhc-related molecules that are also diversified. functional experiments investigating taars and mating in megabats may provide insight into the possible link between taars and mhc genes. ors, which are expressed in the moe, have undergone extensive expansion and contraction that may be associated with environmental adaptations. in ors, we also revealed the notable increase of the genes in megabats, which is more evident in two rousettus bats, including the egyptian fruit bat and leschenault's rousette ( fig. 3a and supplementary table s5 ). although the copy numbers of putatively intact (intact and truncated) ors span from 249 to 543 in microbats, those of megabats ranges from 401 to 740. the increase in the number of ors in megabats may be the signature for the reallocation in response, leading to the loss of the echolocation ability in the megabat ancestor. hayden et al. 65 identified convergent or patterns linked to frugivorous diet in megabats and new world fruit-eating microbats (phyllostomids). given that the increase in the ors is more extensive, these patterns of ors are not only linked to the frugivorous diet, but also to some other roles, such as predator avoidance and social communication. by extensively analysing the copy-number variations of chemosensory receptor genes between megabats and microbats, we revealed obvious differences in taars and ors, both of which are expressed in the moe. it is possible that the contraction of vnomediated chemo-detection and echolocation in megabats may lead to the expansion of chemo-detection genes expressed in the moe. in addition, it is noteworthy that the repertoires of taars and ors function was deduced by enrichment analysis in webgestalt. 93 were obviously differentiated even between closely related two species belonging to the rousettus, suggesting that birth and death of these genes are quite active in this genus ( fig. 3a and b ; supplementary tables s5 and s6 ). the results propose the possibility that two rousettus bats are particularly dependent on olfaction through taars and ors. in addition to the candidate approach, which focussed on retroposons and chemosensory receptor genes, we also performed global analyses on the protein-coding genes of megabats. the elevation of dn/ds ratios were examined for the 6,365 single-copy orthologous genes 35 using the branch model of codeml implemented in paml4.8. 46 the likelihood ratio tests and the inspection of p-value identified that the elevation of dn/ds ratios (p < 0.05) was significant in 246 genes (supplementary table s7 ). as shown by the enrichment analyses for the resultant 246 genes using webgestalt, 93 the elevation of the dn/ds ratios in megabats was remarkable in genes related to the immune system and protein catabolism (table 1 and supplementary table s8 ). the elevation of the dn/ds ratios in immune system genes has been reported in several comparative genomic analyses on mammals, including the pangolin, 94 microbat, 14 and megabat. 16 notably, microbats and pangolins have recently begun to attract attention as possible host reservoirs of sars-related coronaviruses responsible for the current outbreak of coronavirus disease-2019 (covid-19). 95, 96 pavlovich et al. 16 revealed the episodic evolution of immune response genes in egyptian rousette, a natural reservoir of marburg virus, by showing an unusual expansion of ngk2, cd94, mhc, and ifn gene families. we revealed the episodic evolution by showing the elevation of dn/ds ratios in many immune response genes in megabat lineages ( table 1 ). the tolerance for zoonotic viruses without overt pathology in bats are consistent with the episodic evolution in immune response genes. namely, co-evolution of viruses and immune system in these species may be facilitated by the adaptive evolution. further molecular biological and physiological investigations of these candidate genes are of primary importance in elucidating how bats tolerate infections by various zoonotic viruses. interestingly, the elevation of the dn/ds ratio of protein catabolism was also reported in the tyrosine aminotransferase gene (tat) in megabats. 97 to further investigate the evolution of the protein catabolism pathway in megabats, we focussed on another representative gene, 3-hydroxyacyl-coa dehydrogenase (hadh), in which the elevation of the dn/ds ratio was significant in the branch model (table 1; supplementary tables s9 and s10 ). hadh is involved in the degradation of ile, val, lys, and tyr to convert them into energy via the citric acid (tca) cycle (fig. 4a) . the branch-site test for hadh ( fig. 4 and supplementary table s10) revealed that seven sites were positively selected with a posterior probability (p) of >95%, including three sites with a p of >99% (fig. 4b) . the likelihood for the operation of positive selection was not significant, as only a few sites were detected as positively selected (11%, figure 4 . positively selected sites in hadh on megabat lineages. (a) in protein metabolism, hadh is involved in the degradation of ile, val, lys, tyr and transforms these factors into acetyl-coa or succinyl-coa for the tca cycle (https://www.genome.jp/dbget-bin/www_bget?hsa: 3033). (b) the sequence alignment between the positively selected sites in hadh in the megabat lineages and microbats and human hadh. the codon alignment of all hadh sequences used in this study is available in supplementary alignment file s3. the sites were identified by the branch-site model on paml. positively selected sites are highlighted in yellow (p, >95%) and red (p-value,p >99%). (c) positively selected residues on megabat lineages are mapped on the human hadh dimer (pdb: 1f0y). the a chain is presented as a spherical model (yellow and red). the hadh dimer a chain is shown as a cartoon model (white) and the b chain is shown as a surface model (gray). the ligands of hadh, nad, and acetoacetyl-coa are shown as a stick model (blue and orange, respectively). supplementary table s10). we then mapped the positively selected sites on the human hadh dimer structure (pdb: 1f0y, fig. 4c ). although the positively selected sites were not located on the ligand (nad and caa) binding sites, it was of interest that four sites (r221, e229, a247, and l286) were located on the dimer interface (fig. 4c) . the mutations on these four residues change electric charges or polarities, such as r221y, e229n, a247s, and l286s, suggesting that dimer formation is likely to be interrupted and enzyme catalysis is degraded. shen et al. 97 identified the significantly low activity of tat in megabats and discussed that the elevation of the dn/ds ratio in tat may be the relaxation of purifying selection in response to their frugivorous diet. megabats may utilize the ingested proteins for the synthesis of new proteins, rather than for energy production through catabolism, as their diets, which include fruits and nectar, are rich in carbohydrates but poor in protein. accordingly, it is possible that the megabats are less dependent on the protein catabolism pathway. in this study, we provide additional and inclusive evidence which suggests that the evolutionary constraints on genes for protein catabolism were relaxed due to the adaptation for frugivorous diets. in summary, our comparative genomic analyses revealed several distinct signatures for adaptive evolution in megabats. (i) the activity of tes is considerably lower compared to other mammals, which is possibly related to a defense mechanism against viruses. the small size of the genomes, which may be due to the low activity of tes, could be advantageous in association with cellular metabolic constrains of flying organisms. (ii) taars and ors, which function in the neurons of moe, show specific expansions, implying the important contribution of olfaction in their adaptation processes. (iii) positive selection in genes for immunity may suggests the coevolution of immune system and viruses, providing crucial insights into the mechanism of asymptomatic infection of bats for zoonotic viruses as a host reservoir. 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trace-amine-associated receptor gene in a mammal webgestalt, a more comprehensive, powerful, flexible and interactive gene set enrichment analysis toolkit pangolin genomes and the evolution of mammalian scales and immunity isolation of sars-cov-2-related coronavirus from malayan pangolins identifying sars-cov-2-related coronaviruses in malayan pangolins adaptive evolution in the glucose transporter 4 gene slc2a4 in old world fruit bats (family: pteropodidae) the authors thank mr. yujiro kawabe for the illustration of the animals in fig. 1 . computations were partially performed on the nig supercomputer at rois national institute of genetics. all nucleotide sequence reads and the genome assembly have been deposited in the ddbj sequence read archive (sra) for egyptian fruit bat (dra001680) and for leschenault's rousette (dra010589). the sequences of fosmid library of egyptian fruit bat were also deposited in the database (ddbj accession nos. ga901612-ga919400). the raw data for the rna-seq analyses of the kidney-derived primary cultured cells of the egyptian fruit bat has been deposited in ddbj sra (dra010068). none declared. the supplementary data are available at dnares online. key: cord-347532-n51qv9pp authors: wacharapluesadee, supaporn; sintunawa, chirapol; kaewpom, thongchai; khongnomnan, kritsada; olival, kevin j.; epstein, jonathan h.; rodpan, apaporn; sangsri, paiboon; intarut, nirun; chindamporn, ariya; suksawa, kanyarat; hemachudha, thiravat title: group c betacoronavirus in bat guano fertilizer, thailand date: 2013-08-17 journal: emerg infect dis doi: 10.3201/eid1908.130119 sha: doc_id: 347532 cord_uid: n51qv9pp nan to the editor: bats play a critical role in the transmission and origin of zoonotic diseases, primarily viral zoonoses associated with high casefatality rates, including those caused by nipah virus (niv) and severe acute respiratory syndrome (sars)-like coronavirus (cov) infections (1) . recently, the world health organization (who) reported 44 confirmed cases of human infection with middle east respiratory syndrome cov, resulting in 22 deaths. full-genome and phylogenetic analyses of these middle east respiratory syndrome covs have been published elsewhere (2) . the identified viruses from 2 patients (previously referred to as england/ qatar/2012 and emc/2012) are genetically related and belong to group c betacoronavirus, which is most related to covs from nycteris bats in ghana and pipistrellus bats in europe (2, 3) . in addition, bat covs hku4 and hku5 originated from tylonycteris pachypus and pipistrellus abramus bats, respectively, in the people's republic of china (4) . bats are also known to harbor and transmit nonviral zoonotic pathogens, including the fungal pathogen histoplasma capsulatum, which causes histoplasmosis in humans (5) . bat guano is sold for use as a fertilizer in several countries, including thailand, indonesia, mexico, cuba, and jamaica. the practice of collecting and harvesting bat guano may pose a considerable health risk because guano miners have a high level of contact and potential exposure to bat-borne pathogens. to assess pathogens in bat guano, we examined bat guano from a cave in the khao chong phran non-hunting area (kcp-nha) in ratchaburi province, thailand, where bat guano was sold as agricultural fertilizer, for the presence of niv, cov, and h. capsulatum fungi. bats from 14 species in 7 families have been found roosting within this area. tadarida plicata bats are the most abundant species (2,500,000 bats), and 3 other species of bats found at the site each had thousands of members: taphozous melanopogon, taphozous theobaldi, and hipposideros larvatus. a random sample of dry bat guano, ≈100 g, was collected in a sterile plastic bag weekly from the main cave at kcp-nha from september 2006 through august 2007. the specimens were sent for analysis by express mail (at room temperature within 2-3 days) to the who collaborating centre for research and training in the viral zoonoses laboratory at chulalongkorn university. samples were frozen immediately at -80°c until nucleic acids were extracted and pcr assays were run. a total of 52 collected bat guano specimens were examined in this study. two aliquots of feces from each weekly specimen (104 samples total) were screened for cov, niv, and h. capsulatum by pcr. rna was extracted from 10 mg of fecal pellet by using the qiaamp viral rna mini kit (qia-gen, hilden, germany). cov rna was detected by using nested reverse transcription pcr with the degenerated primers to amplify the rna-dependent rna polymerase (rdrp) gene (6) . niv rna was detected by duplex nested reverse transcription pcr (7). to detect h. capsulatum and other fungi, we extracted genomic dna directly from bat guano by using the silica-guanidine thiocyanate protocol, nuclisense isolation reagent (biomérieux, boxtel, the netherlands), according to the manufacturer's protocol. we tested for fungal ribosomal dna (rdna) in extracted total nucleic acid specimens by using the pcr protocol designed to amplify all rdna from 4 major fungus phyla at the internal transcribed spacer 1 and 2 regions (8) . four ( and phylogenetically clustered with the group c betacoronavirus (figure) , with 76%, 80%, and 77% nt identity to bat cov hku4, bat cov hku5, and human cov emc and england1_cov, respectively. the other cov sequence (kcp31: showed 82% nt identity with bat cov hku8, an alphacoronavirus. although we recognize that longer sequences or full genomes may alter the topology of the phylogeny slightly and give stronger branch support, we expect that the overall topology and placement of these covs would remain consistent. samples from particular bat species could not be identified because bats of different species roost in this cave, and samples were pooled during collection for bat guano fertilizer. the detection of covs in bat guano from the kcp-nha cave in ratchaburi was consistent with the previous finding of alphacoronavirus from hipposideros armiger bats from the same province in 2007, but those researchers tested fresh bat feces (9) . all bat guano samples screened by pcr were negative for niv and histoplasma spp. but were positive for group c betacoronavirus. the natural reservoir and complete geographic distribution of this cov are currently unknown. although we did not isolate live virus from these samples, the detection of nucleic acid and previous isolation of viruses from bat feces and urine (10) warrants some concern that guano miners might be exposed to bat pathogens in fresh excreta as well as in soil substances. we suggest that guano miners use preventive measures of personal hygiene and improved barrier protection to reduce the possibility of exposure to zoonotic pathogens. fields virology proposal for a unified norovirus nomenclature and genotyping evolutionary dynamics of gii.4 noroviruses over a 34-year period epochal evolution of ggii.4 norovirus capsid proteins from 1996 to indications for worldwide increased norovirus activity associated with emergence of a new variant of genotype ii.4, late 2012 molecular detection and epidemiology of astrovirus, bocavirus, and sapovirus in italian children admitted to hospital with acute gastroenteritis recombinant norovirus gii.g/gii.12 gastroenteritis in children nationwide surveillance study of human astrovirus infections in an italian paediatric population an automated genotyping tool for enteroviruses and noroviruses rapid emergence and antigenic diversification of the norovirus 2012 sydney variant in denmark new directions in conservation medicine: applied cases of ecological health full-genome deep sequencing and phylogenetic analysis of novel human betacoronavirus human betacoronavirus 2c emc/2012-related viruses in bats, ghana and europe molecular diversity of coronaviruses in bats. virology histoplasmosis associated with exploring a batinhabited cave in costa rica detection of novel sars-like and other coronaviruses in bats from kenya duplex nested rt-pcr for detection of nipah virus rna from urine specimens of bats amplification and direct sequencing of fungal ribosomal rna genes for phylogenetics sars-coronavirus ancestor's foot-prints in south-east asian bat colonies and the refuge theory isolation of nipah virus from malaysian island flying-foxes address for correspondence: supaporn wacharapluesadee, who collaborating centre for research and training on viral zoonoses kcp12, and kcp15); 19 additional human and animal covs from the national center for biotechnology information database are included. construction of the tree was based on 152 nt of the rna-dependent rna polymerase gene region by maximum-likelihood method and gtr+i model with the 1,000 bootstrap resampling method mhv, murine hepatitis virus (nc001846) rousettus bat cov hku9 tylonycteris bat cov hku4 (nc009019) pipistrellus bat cov hku5 sars_batcov hku3, severe acute respiratory syndrome (sars)-related rhinolophus bat cov hku3 (dq022305) sars-related chinese ferret badger cov sars-related palm civet cov an expanded version is online at wwwnc key: cord-315972-5g2hnk1x authors: tong, suxiang; conrardy, christina; ruone, susan; kuzmin, ivan v.; guo, xiling; tao, ying; niezgoda, michael; haynes, lia; agwanda, bernard; breiman, robert f.; anderson, larry j.; rupprecht, charles e. title: detection of novel sars-like and other coronaviruses in bats from kenya date: 2009-03-17 journal: emerg infect dis doi: 10.3201/eid1503.081013 sha: doc_id: 315972 cord_uid: 5g2hnk1x diverse coronaviruses have been identified in bats from several continents but not from africa. we identified group 1 and 2 coronaviruses in bats in kenya, including sars-related coronaviruses. the sequence diversity suggests that bats are well-established reservoirs for and likely sources of coronaviruses for many species, including humans. t he 2003 outbreak of severe acute respiratory syndrome (sars) generated renewed interest in coronaviruses (cov) and the source for the sars cov that caused the outbreak in humans (1) . serologic studies demonstrated that the virus had not previously circulated in human populations to any large extent and suggested a source of zoonotic origin (2) (3) (4) . a likely natural viral reservoir for the virus was not identifi ed until horseshoe bats (rhinolophus spp.) in several regions in the people's republic of china were demonstrated to harbor sars-like covs (5) . subsequently, a number of other sars-like covs, as well as covs from antigenic groups i and ii, were identifi ed from bats in asia, europe, and north america, and coronavirus antibodies were detected in african bat species (6) (7) (8) (9) (10) (11) . it is not surprising that a growing number of covs have been detected in bats. to date, >60 viral species have been detected in bats because their biodiversity (second only to rodents), high population densities, wide distribution, and ability to fl y over long distances allow them to harbor and easily spread multiple infectious agents. bats have long been known as natural hosts for lyssaviruses and more recently have been recognized as potential reservoirs for emerging human pathogens, including ebola, marburg, nipah, and hendra viruses in addition to sars-cov (12, 13) . given the association of bats with emerging infectious diseases, fi eld surveys were performed during july-august 2006 in the southern portion of kenya ( figure 1 ). the selection of sites was based on preliminary data regarding bat roost locations and observations of bats in the fi eld during the survey. attempts were made to collect specimens from 10-20 animals of each species present in each location. bats were captured manually and by using mist nets and hand nets; adults and subadults of both sexes were captured. each bat was measured, sexed, and identifi ed to the genus or species level when possible. blood samples and oral and fecal swabs were collected; the animals were then euthanized in compliance with fi eld protocol. blood, fecal swabs, and selected tissue samples were transported on dry ice from the fi eld and stored at -80°c. fecal swabs (n = 221; table) were screened for the presence of cov rna using 2 semi-nested reverse transcription-pcr (rt-pcr) assays. for the pan-coronavirus rt-pcr, conserved primers were designed from highly conserved regions of the rna-dependent rna polymerase (rdrp) gene 1b based on available cov sequences (1st and 2nd round forward 5′-atgggitgggay tatccwaartgtg-3′; 1st round reverse 5′-aattat arcaiacaacisyrtcrtca-3′; 2nd round reverse 5′-ctagticcacciggyttwanrta-3′). for the pan-bat coronavirus rt-pcr, conserved primers were designed from the same highly conserved regions based on available bat cov sequences and presumed to be more specifi c to bat coronaviruses (1st and 2nd round forward 5′-atgggitgggaytatccwaartgtg-3′; 1st round reverse 5′-tattatarcaiaciacrccatcrtc-3′; 2nd round reverse 5′-ctggticcacci ggyttnacrta-3′). total nucleic acids were extracted from 200 μl of a phosphate buffered saline suspension of each swab by using the qiaamp mini viral elute kit (qiagen, santa clarita, ca, usa), according to the manufacturer's instructions. the seminested rt-pcr was performed by using the superscript iii one-step rt-pcr kit and platinum tag kit (invitrogen, san diego, ca, usa). the positive pcr products were purifi ed by gel extraction by using the qiaquick gel extraction kit (qiagen) according to the manufacturer's instructions; they were then sequenced on an abi prism 3130 automated sequencer (applied biosystems, foster city, ca, usa), according to the manufacturer's instructions. of 221 bat fecal swabs examined, 41 (19%) were positive by at least 1 of the 2 seminested rt-pcr assays (table) . one specimen had 2 distinct cov sequences, each amplifi ed by 1 of the 2 pcr assays, giving a total of 42 distinct cov sequences. to characterize the overall diversity of cov sequences, in this study a phylogenetic tree (figure 2 ) of the 121-bp fragment of rdrp was generated from 39 coronaviruses from bats in kenya and 47 selected human and animal coronaviruses from the national center for biotechnology information database based on the bayesian monte carlo markov chain method (14) . three of the 42 sequences were not of suffi ciently high quality to include in this tree. some nodes had low bayesian posterior probabilities ( figure 2 ). longer sequences from these viruses are needed to refi ne their phylogenetic relationships. among the 39 sequences in the tree, 23 belonged to previously defi ned group 1 and were mapped into 5 different sequence clusters. the 121-bp sequences in these 5 clusters had an average nucleic acid (na) sequence identity of 88%, 85%, 81%, 77%, and 80% when compared with the next closest previously characterized covs (i.e., btcov1a, bthku8, bthku7, hcov229e, and btcova970, respectively). the remaining 16 sequences would likely be placed into group 2. two sequences from chaerophon spp. bats (location 17) were closely related to a sars-like cov cluster, including 1 sequence shown in figure 2 (btky15) and another (btky16) that was 1 of the 3 low-quality sequences excluded from the tree. these 2 na sequences show ≈89% identity with the nearest previously characterized bat: sars-like cov, btcovrf1, shows ≈80% na sequence identity to sars cov (urbani strain) and ≈63% na sequence identity to the human group 2 cov hcovoc43. the 15 remaining na sequences were grouped into 2 clusters. one cluster contains the recently described bthku9 with >95% na sequence identity, and the other cluster (btky18-like cluster) contains no other previously known covs, with <75% na sequence identity to bthku9. the pattern of cov detections by bat species and location demonstrates several features concerning coronaviruses in bats. a given bat species in the same location can harbor several distinct covs as noted for chaerophon spp. (location 17), miniopterus infl atus (location 5), and rousettus aegyptiacus (location 2 and 16); similar covs can also been seen in the same type of bat in different locations, as noted for btcov1a-like cluster covs being detected in miniopterus spp. bats of 4 species from different locations. one m. infl atus bat from location 5 harbored 2 different, but closely related, covs, 1 (btcov 36) from the btco-v1a-like cluster and 1 (btcov 35) from the bthku8-like cluster ( figure 2 ). covs of these 2 closely related clusters were detected in miniopterus spp. bats, but not detected in other bat genera, including those that shared roosts with miniopterus spp. bats. this fi nding is consistent with studies from china in which btcov1a-like and bthku8-like covs were frequently identifi ed but only in miniopterus spp. bats (15) . this may suggest that viruses of the btco-v1a-like cluster and the bthku8-like cluster are specifically adapted to miniopterus spp. bats and not easily transmitted to other bat species. in contrast, other genetically similar covs were detected in several different bat species. for example, covs from th bthku7-like cluster were detected in both chaerophon spp. and otomops martinsseni bats; covs from the btco-va970-like cluster were detected in cardioderma cor and rousettus aegyptiacus bats; covs from the btky18-like cluster were detected in chaerophon spp., eidolon helvum, and r. aegyptiacus bats; and covs from the bthku9-like cluster were detected in hipposidereos commersoni and r. aegyptiacus bats. these data demonstrate that the cov diversity in bats previously detected in asia, europe, and north america is also present, possibly to a greater extent, in africa. the extent of this diversity among covs may be shown more clearly through additional studies in bats, and increased demonstration of cov diversity in bats may require a reconsideration of how they should be grouped. the frequency and diversity of cov detections in bats, now in multiple continents, demonstrate that bats are likely an important source for introduction into other species globally. understanding the extent and diversity of cov infection in bats provides a foundation for detecting new disease introductions that may, like sars, present a public health threat. a novel coronavirus associated with severe acute respiratory syndrome antibody response of patients with severe acute respiratory syndrome (sars) to nucleocapsid antigen of sars-associated coronavirus infectious diseases emerging from chinese wet-markets: zoonotic origins of severe respiratory viral infections isolation and characterization of viruses related to the sars coronavirus from animals in southern china severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats identifi cation of a novel coronavirus in bats coronavirus antibodies in african bat species detection and prevalence patterns of group i coronaviruses in bats, northern germany detection of group 1 coronaviruses in bats in north america prevalence and genetic diversity of coronaviruses in bats from china molecular diversity of coronaviruses in bats bats as a continuing source of emerging infections in humans fruit bats as reservoirs of ebola virus beast: bayesian evolutionary analysis by sampling trees coronaviruses in bent-winged bats (miniopterus spp we thank janet c. beagley, evelyne mulama, heather burke, dorine bonyo, edwin danga, leonard nderitu, solomon gikundi, lydia kigo, m. kariuki njenga, and olga urazova for excellent technical and logistical assistance. we thank yu li, jane iber, and cara burns for their discussion and support in the phylogenetic analysis.the study was supported in part by the global disease detection program (centers for disease control and prevention, atlanta, georgia).dr tong is a virologist in the gastroenteritis and respiratory virus laboratory branch of the division of viral diseases, centers for disease control and prevention. her interests are in novel emerging covs. key: cord-313472-skcaw9ls authors: suwannarong, kanokwan; chanabun, sutin; kanthawee, phitsanuruk; khiewkhern, santisith; boonyakawee, paisit; suwannarong, kangsadal; saengkul, chutarat; bubpa, nisachon; amonsin, alongkorn title: risk factors for bat contact and consumption behaviors in thailand; a quantitative study date: 2020-06-03 journal: bmc public health doi: 10.1186/s12889-020-08968-z sha: doc_id: 313472 cord_uid: skcaw9ls background: bats serve as an important reservoir for emerging infectious diseases. bat contact and consumption, which persists in asia, poses risks for the transmission of bat-borne infections. methods: an analytical cross-sectional survey for risk factors associated with bat contact and consumption behaviors was conducted in ten provinces of thailand from may 2016 to december 2017. a standardized questionnaire administered through face-to-face interviews was used to collect information from 626 villagers who lived in or nearby areas of high bat density. the questionnaire contained 23 independent variables related to sociodemographic, knowledge, attitudes, practices, and perceptions. results: the respondents (n = 626) were 285 females and 341 males, mean age of respondents was 47.58 years-old and lived in rural setting. our results showed that 36.42% of respondents (n(1) = 228) in 10 provinces reported bat contact during the past 6 months. furthermore, 15.34% of respondents (n(2) = 96) in 9 out of 10 provinces reported of having consumed bat meat in the past 6 months. risk factors for bat contact included sex (male) (or = 1.56, 95% ci 1.09–2.28), educational attainment (lower than secondary school) (or = 1.45, 95% ci 1.02–2.18), and the consideration of bats as being economically beneficial to the community (or = 3.18, 95% ci 2.03–4.97), while agriculture-related occupation (or = 0.54, 95% ci 0.37–0.79), knowledge that it is safe to eat bats (or = 0.58, 95% ci 0.37–0.93), practice of allowing children to play with bats (or = 0.65, 95% ci 0.44–0.96), and attitude of feeling safe in areas where bats live (or = 0.56, 95% ci 0.38–0.86) were statistically significant protective factors against bat contact. risk factors for bat consumption included sex (male) (or = 2.48, 95% ci 1.49–4.11) and educational attainment (lower than secondary school) (or = 2.21, 95% ci 1.27–3.85), while knowledge of whether bats are safe to eat (or = 0.04, 95% ci 0.01–0.25), knowledge of whether there are laws pertaining to hunting bats for consumption (or = 0.35, 95% ci 0.18–0.71), and the practice of allowing children to play with bats (or = 0.51, 95% ci 0.31–0.81) were statistically significant protective factors against bat consumption. conclusions: this study provides a better understanding of the sociodemographic factors, knowledge, attitudes, perceptions and practices that might influence bat contact and bat consumption behaviors. information on risk factors can be used for the development of appropriate education and communication interventions to promote proper knowledge, attitudes and practices regarding bats and bat-borne zoonotic diseases in thailand and other areas in the southeast asia region with similar environmental and cultural characteristics. the human and wildlife interface has been a global concern in the past decade due to several zoonotic disease outbreaks related to wildlife contact. pathogen transmission may occur through several routes, e.g., inhalation [1] ; bites [2] [3] [4] ; scratches [5] ; hunting [6] [7] [8] [9] ; guano use as fertilizer [10] ; food consumption [6, 11, 12] ; handling, slaughtering and butchering; drinking water or food contaminated with saliva or feces [13] ; and possible human-to-human transmission. bats are important reservoirs of several zoonotic pathogens. bat-borne diseases in humans have been reported worldwide and have been considered global concerns; these include the nipah virus [13, 14] , the hendra virus [15] , ebola [16] , lyssaviruses [17] , and severe acute respiratory syndrome coronavirus (sars-cov) [2, 18, 19] . it has been documented that several factors influence human contact with bats. for example, previous studies in asia, and north america reported that sex [6] , occupation [4] , and locations of bat habitats [6] influence human-bat interactions. bats play a cultural role in thailand including medicinal, religious, and culinary [10] . a better understanding of human-bat interactions is critical to understanding zoonotic disease spillover and has been insufficiently studied [20] . especially in thailand, the understanding of human-bat interactions and the epidemiological links involved in bat-borne disease and bat consumption is still insufficient. the objective of this study was to determine the factors associated with bat contact and bat consumption behaviors in ten provinces of thailand from may 2016 to december 2017. our results will support the development of appropriate education and communication interventions to promote proper knowledge, attitudes and practices regarding bats and bat-borne zoonotic diseases in thailand and southeast asia. an analytical cross-sectional study was conducted to identify the factors associated with bat contact and bat consumption behaviors among persons who lived in or near areas of high bat density within at least 6 months before the study. the questionnaire interviews were conducted from may 2016 to july 2017. the chulalongkorn university institution review boards (irbs) and chiang rai provincial health office approved the human study (ref no . 034/59 and 26/ 2559). this study obtained agreements from local administrative offices and the chiefs of villages to conduct data collection at the study sites. written informed consent forms were signed by the participants after they received information about the objectives of the study and prior to the interviews. different sampling methods were applied to select the study sites and study populations. the whole country was first stratified into four regions; central, northern, northeastern and southern thailand. provinces, districts and villages in each region were then purposively selected based on; 1) a high density of bats in the villages/ study areas, 2) a potential bat-human interface was observed by researchers during scoping visits, discussed with some villagers and/or local authorities, and 3) information obtained from the relevant local and national authorities such as the ministry of natural resources and environment (mnre), and the ministry of public health (moph). lastly, a simple random sampling method (srs) was used to select respondents from official household registry records that were obtained from the local health promotional hospitals in the villages. in this study, 10 provinces were selected as representatives of regions in thailand that have different living characteristics and practices. the provinces were ang thong, ayutthaya, lopburi and saraburi (central provinces); chiang mai and chiang rai (northern provinces); khonkaen and ubonratchathani (northeastern provinces); and krabi and surat thani (southern provinces) ( fig. 1 and table 1 ). bats from each study site were collected for species identification using physical characteristics and dna sequence variations in mitochondrial cytochrome-b (cytb) [21] . the inclusion criteria for respondents were males or females between 20 and 75 years of age who had lived in the selected areas for at least 6 months before data collection and were willing to participate in this study. the sample size calculation for this study was as follows: here, p = proportion of participants with bat contact. based on a previous study, 23.00% of respondents reported bat consumption at some time in their lives [22] . the variable p is 0.23 for this calculation, z = 1.96 (95% confidence interval), and d (margin of error) = 5%. therefore, the calculated sample size was 340. to increase the power, the sample size was elevated to 626. simple random sampling (srs) was used on the lists of the respondents retrieved from health promotional hospitals in the study areas. after srs of respondents was conducted, the participants were contacted by trained researchers for data collection. using a standardized questionnaire, a face-to-face interview was conducted with the respondents in a place that was not too secluded but still free from disturbances. a questionnaire was designed to collect information on bat contact and bat consumption behaviors. this questionnaire was modified from previous reports [10, 23, 24] . the questionnaire interview was administered to collect information on sociodemographic factors, knowledge, attitudes, practices and perceptions for bat contact and bat consumption behaviors. the questionnaire was pretested with 30 respondents in a district with the same environmental and population characteristics as the actual selected sites. the questionnaire was refined per the pretest results. field researchers were trained in its administration and in conducting questionnaire interviews with written informed consent forms obtained prior to the interviews. after the interviews, the information was checked for validity and precision before it was entered into spss software version 22 (chicago, il). independent variables (n = 23) were included in the analysis. the variables were based on previous relevant studies [10, [25] [26] [27] [28] [29] . the independent variables, including the sociodemographic information, knowledge, attitudes, practices, and perceptions were as follows: -humans can get diseases from bats (true vs false) -there are no concerns about getting diseases from bats (true vs false) -bats can transmit diseases to humans (true vs false) -bats are economically beneficial to the community (agree vs disagree) -one can contract diseases if exposed to bats (true vs false) -one can contract diseases by drinking water from the same places as bats (true vs false) -one can contract diseases by eating fruits left by bats (true vs false) -bat guano is safe to use (true vs false) -it is safe to eat bats (true vs false) -there are laws related to hunting bats for consumption (true vs false) -children are allowed to play with bats (true vs false) -dead bats that are found are brought home for food (true vs false) -one feels safe in areas where bats live (true vs false) the dependent variable was a report of either one of bat contact experiences by the study respondents. list of bat contact experiences included the following: -found dead bat(s) in house -found live bat(s) in house, the community or tourist location -cleaned bat guano from house or the community -cleaned bat carcasses from house or the community -bat guano mining/collecting -use of bat guano -bitten by a bat -consumed bats for food -other activities, e.g., hunted bats, exposed to urine of bats the interviewed data were entered into and analyzed by spss software version 22 (chicago, il). after data cleaning, the dependent and independent variables were assessed. data were analyzed in 2 steps. first, bivariate analysis was performed, in which the degree of association of each variable was computed and each of the independent variables was analyzed separately. second, a stepwise logistic regression model was constructed including independent variables that had p values ≤0.15 results in bivariate analysis. this second step used p ≤ 0.05 as the cutoff point for identifying statistically significant variables. in this study, 626 respondents participated in the questionnaire interview. these respondents from 37 villages of 16 districts in 10 provinces in thailand ( fig. 1 and table 1 ). bats species from each study site were identified: chiang mai (scotophilus heathii and megaderma spasma), chiangxs rai (taphozous melanopogon), lopburi (taphozous melanopogon and chaerephon plicatus), khon kaen (chaerephon plicatus), and ayutthaya, ang thong, saraburi (pteropus lylei). of the 626 respondents who participated in the questionnaire interview, 228 (36.42%) respondents from 10 provinces reported having experienced bat contact during the past 6 months before data collection. moreover, 96 (15.34%) respondents in 9 out of 10 provinces reported having consuming bat meat in the past 6 months. the 626 respondents were 285 females and 341 males. the mean age of the respondents was 47.58 years. approximately 21.09% of respondents were aged > 36 years. most of them lived in rural settings (93.29%) and were married or cohabiting (84.03%). respondents worked as farmers (rice, grains or vegetables) (35.78%), followed by temporary employees (22.68%), government officers (10.54%), housewives (7.03%), and shop vendors/owners (5.59%). most of the respondents (65.65%) had attained educational levels lower than secondary school, and 80.35% had families composed of more than 2 persons. most respondents had a monthly family income of ≤15, 000 baht (500 usd) (69.97%). most respondents owned a motorcycle (91.69%), while fewer owned a car (50.16%). in this study, 228 respondents in 10 provinces reported a bat contact experience during the past 6 months before data collection. among those 228 respondents, 56.14% reported encountering live bats in a house, the community or a tourist location, while 42.10% reported eating bats for food, 34.21% found bat guano in a house or the community, 28.51% found dead bats in the house, 20.18% participated in bat guano mining/collecting, 19.74% cleaned bat carcasses from a house or the community, 10.96% used bat guano as fertilizer, 17.98% were involved in other contact activities (e.g., took bats from nets), and 7.02% been bitten by a bat in the past 6 months (table 2) . in this study, the questionnaire interview contained 10 sociodemographic questions and 13 questions related to the knowledge, attitudes, practices and perceptions regarding bat contact experiences. bivariate analysis was performed to determine the associations between bat contact experiences and twenty-three independent variables. of the 23 independent variables, 11 variables had significantly associations in the bivariate analysis, using p ≤ 0.15 as a cutoff point: sex (male), age group (> 36 years), occupation (agriculture-related occupation), educational attainment (< secondary school), family monthly income (< 15,000 baht), no concerns about getting diseases from bats, considered bats to be economically beneficial to the community, believed it is safe to eat bats, allowed children to play with bats, and felt safe in areas where bats live (table 3 ). all 11 variables were included in the stepwise logistic regression analysis for bat contact experiences. the results showed that sex (male) (or = 1.56, 95% ci 1.09-2.28, p = 0.014), educational attainment (< secondary school) (or = 1.45, 95% ci 1.02-2.18, p = 0.041), and considered bats to be economically beneficial to the community (or = 3.18, 95% ci 2.03-4.97, p < 0.001) were statistically significant associated with bat contact experiences, while occupation (agriculture-related occupation) (or = 0.54, 95% ci 0.37-0.79, p = 0.002), believed it is safe to eat bats (or = 0.58, 95% ci 0.37-0.93, p = 0.023), allowed children to play with bats (or = 0.65, 95% ci 0.44-0.96, p = 0.031), and felt safe in areas where bats live (or = 0.56, 95% ci 0.38-0.86, p = 0.007) were statistically significant protective factors against bat contact experiences (table 4) . among the 626 respondents, 96 respondents (15.34%; 28 females and 68 males) reported eating bats in the past 6 months. of the respondents who reported contacts with bats, 42.10% (96/228) reported eating bats in the past 6 months. they also reported that bats were hunted, butchered, slaughtered, and cooked by several categories of persons (e.g., husbands, wives, neighbors, children, or hunters) in their communities. in this study, 170 out of 626 respondents (27.16%) reported having eaten bats in their lifetime, of whom 62.35% (106/170) reported consuming bats more than 10 years ago, 11.18% (19/170) who reported eating bats during the last 1-10 years, 20.00% (34/170) who reported eating bats within the past 12 months, 2.94% (5/ 170) who reported eating bats in the past month, 2.35% (4/170) who reported eating bats within week of data collection, and 1.18% (2/170) who reported eating bats within the preceding week. of the 96 respondents who reported eating bats, 70.83% were male and 83.33% were < 36 years old (mean age of respondents 47.21 years old). most respondents were married or cohabiting (85.42%) and had more than one child (69.79%). the occupations of respondents were farmers (rice, grains or vegetables) (39.58%), followed by temporary employees (29.17%). most respondents had attained an educational level < secondary school (79.17%) and had families composed of more than 2 persons (76.04%). most of the respondents (66.67%) had monthly family income < 15,000 baht (500 usd). some respondents owned a car (42.71%), majority owned a motorcycle (96.88%). of the 96 respondents, those in the northern region, chiang mai (28.13%) and chiang rai (13.54%), reported more bat consumption than those in other provinces, including those in the northeastern region, ubon ratchathani (18.75%), southern region, surat thani (16.67%) and krabi (14.58%). on the other hand, respondents in the central region reported less bat consumption (table 1) . with regard to the details of bat consumption behaviors, we found that 26.04% (25/96) of respondents reported killing bats themselves, followed by vendors (15.63%), hunters (13.54%), and neighbors (11.56%). the participants reported that no children aged 0-10 years old killed bats. with respect to preparing and cooking bats for food, 34.38% (33/96) of respondents prepared bat meat by themselves, followed by spouses (14.58%), neighbors (11.56%), and children (1.04%). persons reported that the bats were eaten by spouses (48.96%), neighbors (39.58%), and children (13.54%). most respondents reported eating cooked bat meat (98.96%, 95/96), while only one respondent, a 63-year-old female from chiang rai province, reported eating raw bat meat. bats were obtained by hunting in caves (30.21%), purchasing from local markets (19.79%), hunting by themselves (13.54%), and hunting by neighbors (8.33%). dishes containing bat were "kang om" (spicy vegetable soup) (18.75%), followed by spicy stir fry (13.54%), "kua kling" (dry spicy fry with herbs), and deep-fried bat meat (8.33%). the preferred recipes varied among provinces or regions; for example, people in the northern region, chiang mai, preferred to cook "kang om" while those in southern provinces, krabi, preferred spicy stir fry to other dishes. the field investigation showed that a typical bat preparation process included butchering the bats, selecting the parts for consumption, adding additional herbs for flavor, and boiling the ingredients in a large pot (fig. 2) . in this study, 23 variables (sociodemographic factors, knowledge, attitudes, practices and perceptions) were involved in the bivariate analysis, and eleven independent variables were eligible for the stepwise logistic regression analysis. these included sociodemographic variables (sex, educational attainment, owning a car, and owning a (table 6 ). this is the first quantitative study regarding the risk factors for bat contact and bat consumption behaviors in thailand. our study showed that 36.42% of 626 respondents reported bat contact experiences during the past 6 months. the respondents reported finding live bats in houses, the community or tourist locations; eating bats for food; cleaning bat guano from the house or community; finding dead bats in the house; participating in bat guano mining/collecting; cleaning bat carcasses from the house or community; using bat guano as fertilizer; participating in other contact activities; and having been bitten by a bat. this current study showed an incidence of bat contact experiences (36.42%) that was higher than that reported in a study in canada [30] , which showed that 16% of participants had direct contact with bats, and 4% found bats in their houses. however, the frequency of reports of being bitten by bats in the current study was lower (7.02%) than that in the canadian study (39.00%). interestingly, bat consumption behavior was shown in the second rank of frequent exposure behavior (42.10%). from the stepwise logistic regression analysis on bat contact behavior, male, low education attainment and considering bats to be economically beneficial to the community were risk factors for contact with bats. in contrast, farming or agriculture-related occupations were a protective factor against contacting bats, which was different from a study in guatemala [31] . this might be because other occupations, e.g., temporary workers, had more chances and/or free time to hunt or purchase bats. our results also showed inappropriate knowledge and attitudes with regard to feeling that it is safe to eat bats, allowing children to play with bats, and feeling safe in areas where bats live, which were influenced by the villagers' contact with bats. our observations agreed with the findings from a study in australia in which respondents had inappropriate perceptions that could lead to more bat contact/exposures [12] . the consumption of wild animals, including bats, a product often called bushmeat, poses challenges for both wildlife conservation and human health [24] . this study showed that 96 (15.34%) of the 626 respondents reported bat consumption during the past 6 months. however, 170 respondents (27.16%) in this study reported eating bats in their lifetime, which was higher than the studies in the republic of ghana (23.00%) [22] and madagascar (25.8%) [23] . thus, bat consumption incidences in thailand should be considered a matter for concern. this study showed that males reported more bat consumption behavior. our findings were comparable to previous studies in which males were more likely to consume wildlife in thailand and the lao pdr [10, 12] . low education attainment was one of the risk factors that lead to bat contact and consumption. in addition, inappropriate knowledge and attitudes regarding whether it was safe to eat bats, legal to hunt bats, and safe to allow their children to play with bats could also be factors affecting bat consumption. among the provinces, the respondents from the northern region (chiang mai, and chiang rai) reported eating bats more than those in other regions. while, the central provinces reported less bat consumption due to their beliefs and social norms per the qualitative study results. regular law enforcement activities in the central provinces might be one of the contributing factors. in conclusion, this study has provided information related to sociodemographic factors, knowledge, attitudes, perceptions, and practices that may influence bat contact and bat consumption behaviors among thai villagers. the information from this study can be used in the development of communication interventions for zoonotic diseases related to bat contact and bat consumption behaviors in areas with similar environmental and cultural characteristics. supplementary information accompanies this paper at https://doi.org/10. 1186/s12889-020-08968-z. additional file 1. rodent-borne diseases and their risks for public health human-bat interactions in rural west africa cross sectional survey of human-bat interaction in australia: public health implications rabies-related knowledge and practices among persons at risk of bat exposures in thailand bushmeat hunting, deforestation, and prediction of zoonoses emergence characteristics and risk perceptions of ghanaians potentially exposed to bat-borne zoonoses through bushmeat bushmeat hunting and zoonotic transmission of simian tlymphotropic virus 1 in tropical west and central africa factors determining the choice of hunting and trading bushmeat in the kilombero valley hunting, food preparation, and consumption of rodents in lao pdr bat consumption in thailand primates on display: potential disease consequences beyond bushmeat rodent consumption in khon kaen province transmission of human infection with nipah virus nipah virus infection outbreak with nosocomial and corpse-to-human transmission hendra virus: a one health tale of flying foxes, horses and humans fruit bats as reservoirs of ebola virus australian bat lyssavirus infection: a second human case, with a long incubation period a sars-like cluster of circulating bat coronaviruses shows potential for human emergence diversity of coronavirus in bats from eastern thailand a framework for the study of zoonotic disease emergence and its drivers: spillover of bat pathogens as a case study a test of the genetic species concept: cytochrome-b sequences and mammals knowledge, perceptions and attitude of a community living around a colony of straw-coloured fruit bats (eidolon helvum) in ghana after ebola virus disease outbreak in west africa analysis of patterns of bushmeat consumption reveals extensive exploitation of protected species in eastern madagascar uncovering the fruit bat bushmeat commodity chain and the true extent of fruit bat hunting in ghana importance of rodents as a human food source in benin conservation strategies for understanding and combating the primate bushmeat trade on bioko island, equatorial guinea wildlife trade and human health in lao pdr: an assessment of the zoonotic disease risk in markets the socio-economic drivers of bushmeat consumption during the west african ebola crisis children's traditional ecological knowledge of wild food resources: a case study in a rural village in northeast thailand bat rabies in the united states and canada from 1950 through 2007: human cases with and without bat contact knowledge, attitudes and practices regarding rabies and exposure to bats in two rural communities in guatemala publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we would like to thank dr. kallaya harnpicharnchai, mr. vichien patchamit, mr. buncha muankla, and mr. suphat hlikthuk for their support in the fields. we also thank the chief medical officers of the provincial health offices and their staff as well as the local authorities for their cooperation and assistance during research. we would also like to thank the department of natural parks, wildlife and plant conservation for their support on site and during the field bat sample collection. this research was supported by the chulalongkorn university, national research university fund, health research (nru59-028-hr). chulalongkorn university provided financial support to the center of excellence for emerging and re-emerging infectious diseases in animals and the one health research cluster. the thailand research fund provided financial support to the trf senior scholar to the corresponding author (rta6080012). we would like to thank the rachadapisek sompote fund and the second thailand. 6 authors' contributions ks1, pk, sk, pb, ks2, cs, and nb conducted and coordinated the field study and questionnaire interviews. ks1 and sc conducted the data analysis. ks1 and aa drafted and revised the manuscript. aa supervised the study and served as principle investigator of the project and corresponding author of the manuscript. all authors read and approved the final manuscript. the first author (kanokwan suwannarong) is a postdoc associate at the center of excellence for emerging and re-emerging infectious diseases in animals, faculty of veterinary sciences, chulalongkorn university. she received the postdoctoral fellowship from the second century fund (c2f), chulalongkorn university. her research interest is social epidemiology and community studies related to emerging infectious diseases and health managements. century fund (c2f), chulalongkorn university for the first author's postdoctoral fellowship as well as for study designs, data collection and analysis of this project. all data generated or analyzed during this study are included in this published article and supplement tables.ethics approval and consent to participate the chulalongkorn university institution review boards (irbs) and chiang rai provincial health office approved this human study (ref no. 034/59 and 26/ 2559, respectively). this study obtained agreements from local administrative offices and the chiefs of villages to conduct data collection at the study sites. written informed consent forms were obtained after describing the objectives of the study to the participants and prior to the interviews. the faculty of veterinary science, chulalongkorn university's animal care and use committee, approved the animal study (iacuc no. 1531035 and 1631006) . the department of national park, wildlife and plant conservation, ministry of natural resources and environment approved the bat sample collection (no. ts0907.4/25574). not applicable. the authors declare that they have no competing interests. key: cord-352361-jh31omg2 authors: nobach, daniel; herden, christiane title: no evidence for european bats serving as reservoir for borna disease virus 1 or other known mammalian orthobornaviruses date: 2020-01-30 journal: virol j doi: 10.1186/s12985-020-1289-3 sha: doc_id: 352361 cord_uid: jh31omg2 background: the majority of emerging infectious diseases are zoonotic in nature and originate from wildlife reservoirs. borna disease, caused by borna disease virus 1 (bodv-1), is an infectious disease affecting mammals, but recently it has also been shown to cause fatal encephalitis in humans. the endemic character of borna disease points towards a nature-bound reservoir, with only one shrew species identified as reservoir host to date. bats have been identified as reservoirs of a variety of zoonotic infectious agents. endogenous borna-like elements in the genome of certain bat species additionally point towards co-evolution of bats with bornaviruses and therefore raise the question whether bats could serve as a potential reservoir of orthobornaviruses. methods: frozen brain samples (n = 257) of bats of seven different genera from germany were investigated by orthobornaviral rt-pcr. additionally, tissue slides of formalin-fixed paraffin-embedded material of a subset of these bats (n = 140) were investigated for orthobornaviral phosphoprotein by immunohistochemistry. results: the brain samples were tested by rt-pcr without any evidence of orthobornavirus specific amplicons. immunohistochemistry revealed a faint immunoreaction in 3/140 bats but with an untypical staining pattern for viral antigen. conclusions: rt-pcr-screening showed no evidence for orthobornaviral rna in the investigated bats. however, immunohistochemistry results should be investigated further to elucidate whether the reaction might be associated with expressed endogenous bornaviral elements or other so far unknown bornaviruses. the increasing incidence of emerging infectious diseases (eid) represents a threat to public health. interestingly, the majority of these eids are zoonotic in nature and originate from wildlife reservoirs. due to their biological characteristics, particularly bats have been identified as reservoirs for many emerging viruses [1] . many of these emerging viruses are rna-viruses of the order mononegavirales. in this order, the virus family bornaviridae has been growing remarkably during recent years due to the discovery of several new species and genera. as of 2019, the taxonomy comprises three genera: carbovirus, cultervirus and orthobornavirus [2] . of the genus orthobornavirus, two species, mammalian 1 orthobornavirus and mammalian 2 orthobornavirus, are known to affect mammals. belonging to mammalian 1 orthobornavirus, borna disease virus 1 (bodv-1) is still the most prevalent bornavirus in mammals. bodv-1 is well known to cause severe and fatal neurological borna disease in a variety of mammals, mainly horses and sheep. recently bodv-1 was identified as causal agent in fatal human encephalitis cases [3, 4] . this underlines the need to unravel potential sources of infection in order to prevent further animal and human cases, especially since no curative therapy or vaccination exists [3, 4] . endemic areas for borna disease are located in central europe, such as bavaria, saxony-anhalt and saxony in germany, st. gallen and canton of grisons in switzerland and vorarlberg and upper austria in austria [5, 6] . phylogeny of bodv-1 isolates reflects their geographical origin and respective endemic regions regardless of the host species they have been isolated from [5] . the endemic character, strong conservation of the viral genome and seasonal occurrence of the disease already pointed to a potential wildlife reservoir [7] . as the sequences of bd cases from neighbouring locations are particularly stable even over years, this wildlife reservoir was assumed to be territorially bound [5] . in several of the endemic areas, the bicolored white-toothed shrew (crocidura leucodon), belonging to the order of eulipotyphla, has been identified as natural reservoir of bodv-1 [6, 8, 9] . however, beside the bicolored white-toothed shrew, no other reservoir, neither eulipotyphla nor rodent, has been found yet. bank voles have been experimentally proven to be susceptible to bodv-1 [10] and serum antibodies against bornaviruses have been detected in free ranging bank voles [11] . nevertheless, there is no evidence for naturally infected bank voles in endemic areas to date. serological data have shown that several other free ranging small mammals, mostly belonging to the order of rodentia, can also exhibit serum antibodies against bornaviridae, but without any other evidence of bodv-1-infection [9] . only bicolored white-toothed shrews display a disseminated virus distribution and harbour bodv-1 in excretory and secretory organs [9, 12] and shed infectious virus which suggests that they can transmit bodv-1 [13] . although several rodents and other small mammals are known as important reservoirs for many viruses, bats (order: chiroptera) represent the vast majority of identified natural reservoirs of several virus families/species to date [1, 14] . for example, bats are known reservoirs for a growing list of rna viruses, including rabies virus and other lyssaviruses, henipaviruses, coronaviruses and ebola virus [15] . virus infections in bats follow the typical pattern of reservoirs with a persistent course lacking clinical disease [1, 16] . several biological characteristics including gregariousness with large colonies, seasonal migrating and long life span make bats suitable to carry and spread viruses [16] . seasonal migrating and wide hunting territories of bats can lead to wide distribution of harboured viruses [1] , however, shedding in pulses and additional local factors can lead to local transmission events [16] . in europe, 35 bat species from the order chiroptera can be found [17] . these european bats share the same biological characteristics, but transmission events of zoonotic viruses are rare due to smaller dimensions of populations and colonies [18] . in germany, 25 bat species can be encountered from the family rhinolophidae and vespertilionidae [19] . they include common native species, such as members of the genera pipistrellus, myotis and vespertilio, and some seriously endangered species like rhinolophus hipposideros, myotis emarginatus and barbastella barbastellus [19] . several bat species, for example myotis natteri, use roosts in barns and stables [20] , which facilitates the possibility of virus transmission to livestock [18] . in addition, endogenous bornavirus-like elements (ebl) have been detected in the genome of several bat species [21] [22] [23] [24] . these ebl are dna-sequences in bat genomes displaying considerable sequence identities to present-day bornaviral genes on amino acid level of about 30-50% [25] . in some bat species, transcription of ebl similar to the bornaviral rna-dependent rnapolymerase has been reported [22] . further analysis of these ebl strongly hint at ancestral and repeated contact between bats and bornaviruses during their evolution at least 11,8 million years ago [23, 26] . the function of these ebl is still discussed and an immunological benefit in the interaction with bornaviruses has been suggested in some mammalian species [22, 27] . as the retaining of the expressed ebl in the bat genome despite evolutionary selection requires resources, a benefit of the ebl and regular encounters between bornaviruses and bats during evolution seem plausible [22] . in conclusion, due to the continuous detection of new viruses in bats, the unclear situation regarding additional potential bodv-1-reservoirs and molecular evidence for co-evolution of bats and bornaviruses, this study was conducted to investigate the potential presence of the most common orthobornaviruses in bats from endemic and non-endemic areas in germany. two hundred fifty-seven brain samples of bats from germany (97 from endemic regions in bavaria) were provided by the leibniz institute for zoo and wildlife research (leibniz-izw), berlin (79 pipistrellus sp., 67 nyctalus sp., 57 myotis sp., 22 eptesicus sp., 17 vespertilio sp., 12 plecotus sp., 2 barbastella sp., 1 bat without species identification), and stored frozen at − 80°c. additionally, bat organs from diagnostic necropsies archived as formalin-fixed paraffin-embedded (ffpe) material of 101 bats from the german federal state of bavaria (31 pipistrellus sp., 14 vespertilio sp., 12 eptesicus sp., 12 nyctalus sp., 10 plecotus sp., 8 myotes sp.) and 39 bats from the federal state of hesse (5 myotis sp., 2 pipistrellus sp., 2 plecotus sp., 1 nyctalus sp., 29 bats without species identification) were provided by the leibniz-izw, the state veterinary institute of giessen (landesbetrieb hessisches landeslabor) and the department of animal ecology and systematics, giessen. all organ tissues were retrieved from diagnostic necropsy material from carcasses submitted by bat rehabilitation centres and bat researchers in germany to the respective institution. samples from the leibniz-izw were archived materials from a previous larger study on disease and causes of death in european bats from germany [28] . for screening for orthobornaviral rna (200 bp of x/ p-orf), brain samples were analysed by a two-step rt-pcr detecting a broad spectrum of orthobornaviruses (see below). rna isolation and rt-reaction was performed with rneasy mini kit (qiagen) and quantitect reverse transcription kit (qiagen) according to manufacturer's instructions, respectively. pcr was performed with mytaq hsmix (bioline) under manufacturer's standard condition with degenerated primers (additional file 2: table s1 ) [29] . these primers were designed to detect viruses of seven species of the genus orthobornavirus (mammalian 1 orthobornavirus, mammalian 2 orthobornavirus, passeriform 1 orthobornavirus, passeriform 2 orthobornavirus, psittaciform 1 orthobornavirus, psittaciform 2 orthobornavirus and waterbird 1 orthobornavirus), but not viruses of the species elapid 1 orthobornavirus of the genus orthobornavirus or viruses of the genera carbovirus or cultervirus. the applied rt-pcr assay has been proven to detect several known pathogenic members of the genus orthobornavirus (bodv-1, variegated squirrel bornavirus 1 (vsbv-1), parrot bornavirus 2 (pabv-2), parrot bornavirus 4 (pabv-4)) [29] . as internal control, glyceraldehyde-3phosphate-dehydrogenase-(gapdh)-amplification (402 bp) was included. as positive control, isolated rna from a bodv-1-positive mouse was used, and a formerly negatively tested bat served as negative control. lengths of amplicons were visualized with gel electrophoresis (2% agarose gel with 3% midori green (biozym)) according to manufacturer's instructions and commercial sanger sequencing of orthobornaviral amplicons was performed for positive controls (gatc, eurofins genomics). bodv-1 negatively-tested bat-rna was spiked with serial dilutions of either bodv-1-rna, vsbv-1-rna, pabv-2-rna or pabv-4-rna to assess specificity and sensitivity. to screen for bornaviral antigen, immunohistochemistry was performed using a polyclonal antibody for the detection of bornaviral phosphoprotein (antibody p24). this antibody is known for its cross-reactivity also with the phosphoprotein of pabv-2 and pabv-4 of the species psittaciform 1 orthobornaviruses [30] and vsbv-1 [31] . all reactions were compared to a negative control slide incubated with a rabbit serum (rabbit immunoglobulin fraction, dako). organs with positive immunostaining were further examined with a panel of antibodies to examine specificity of this reaction. the panel included two antibodies directed against the viral nucleoprotein of bodv-1 (monoclonal antibody bo18 [32] and polyclonal antibody anti-bodv-n [4] ) and a mix of polyclonal antibodies detecting vsbv-1nucleoprotein and phosphoprotein [provided by dennis tappe, bernhard nocht institute hamburg]. to exclude unspecific reaction of the polyclonal rabbit-antibodies, a polyclonal antibody detecting rabies virus as well as a second control rabbit serum (thermofisher) were used as additional negative controls (details on immunohistochemistry protocols in additional file 4: table s2 ). by rt-pcr-screening, in 239/257 samples gapdhamplicons could be obtained, the other 19 samples were excluded due to insufficient quality. these 239 samples were tested for orthobornaviral rna and no specific amplicons regardless of origin from endemic or nonendemic areas were observed. the control consisting of rna of a bodv-1 infected mouse was correctly amplified as verified by correct size on the gel and respective sequences (additional file 1: figure s1 ). spiking of bat rna with serial dilutions of various orthobornavirus-rna demonstrated the detection limit of 5000 orthobornavirus copies in 660 ng rna. by immunohistochemistry applying the polyclonal antibody p24 specific for the phosphoprotein, a faint reaction was found in 3/140 animals, in particular located in the cytoplasm of smooth muscle cells of the intestine. all respective negative control slides were without any immunoreaction regardless which control antibody was used. no immunoreactivity was found using the monoclonal antibody bo18 specific for the bodv-1nucleoprotein in these samples. immunoreactivity was found using the polyclonal antibody detecting bodv-1nucleoprotein in one sample and using the polyclonal antibodies detecting vsbv-nucleoprotein and phosphoprotein in two samples. however, in 17/140 other animals a comparable immunoreaction was observed in negative control slides using control rabbit serum or polyclonal anti-rabies antibodies (details in additional file 5: table s3 and additional file 3: figure s2 ). recent cases of fatal encephalitis in humans due to bodv-1 infection strengthen the need to survey potential wildlife reservoirs and identify potential risk factors for infection. although the bicolored white-toothed shrew has been identified as indigenous reservoir of bodv-1, other potential reservoirs or animal carriers are still unknown so that further investigations of small mammals including bat species are urgently needed. bats have already been discovered as reservoir of emerging and highly pathogenic viruses. many factors, such as their gregarious way of life, can facilitate pathogen transmission to other bats and virus persistence in the population. in european bats, only few zoonotic viruses have been discovered [18, 33, 34] and the overall hazard for humans is comparably low [18] . as some bats take roosts in barns and stables [20] and bat carcasses are found in close proximity to agriculture [35] , a risk of sporadic transmission events to livestock animals can be assumed if viruses can be detected. animal movement across borders of endemic regions during hunting and migration of bats seems to contradict stable geographical clustering of bodv-1 isolates and the hypothesis of a territorially bound reservoir [5] . nonetheless, consistent usage of the same roosts as summer or winter quarter may support observed clustering and could facilitate rare endemic transmission. additionally, the molecular evidence for co-evolution of bats and bornaviruses [22, 27] could suggest the possibility of infections of other potentially so far unknown bornaviruses beside the tested orthobornaviruses. therefore, this study aimed to examine the possible role of bats as carrier and reservoir of orthobornaviruses such as bodv-1 as one of the most common virus. all samples originated from bats which died because of injuries or disease. they were part of a previous study on diseases and causes of death in european bats, where traumatic injuries and inflammatory lesions, partly due to bacterial infections, were the major cause of deaths in these animals [28] . since borna disease in animals is known to be endemic in specific areas of germany [5] , samples were sorted by regional origin corresponding to known endemic regions (bavaria) and other nonendemic regions in germany. however, as some bat species tend to have wide hunting territories or migrate during the year and can cross the borders of endemic regions, this sorting might bear a risk of bias. the study includes samples from available bat species and is not limited to bat species suspected to interact with bornaviruses [23, 24] as interspecies virus transmission has already been observed [1, 14] . interestingly, the screening by rt-pcr for orthobornaviral rna provided no evidence of orthobornavirus infection in the investigated bats. the detection limit of the applied orthobornavirus rt-pcr was 0.01 ng/μl orthobornaviral rna in 700 ng/μl mammalian rna according to literature [29] and 5000 copies in 660 ng rna in our own testing. therefore, already low amounts of viral rna should have been detected as verified by the correct amplification of all control orthobornavirus species. spillover host [36] and reservoir species [12] regularly yield high amount of viral rna, much higher than the detection level of the applied rt-pcr assay. however, the presence of previously undiscovered bornavirus species, such as the ones recently described in reptiles and classified as carboviruses [37] cannot be excluded and could be further investigated by broad and undirected approaches, such as metagenomics. in contrast to the rt-pcr results, the faint immunohistochemical reaction in smooth muscles of three animals raises the question whether an antigen with cross-reactivity or a bornaviral phosphoprotein is present. however, in several other animals similar reactions were detected applying unrelated polyclonal antibodies produced in rabbits and unspecific rabbit serum. moreover, the staining pattern is rather untypical for bodv-1 but occurs regularly in avian bornavirus infections [30] . the immunohistochemical reaction was not observed using a monoclonal antibody against the nucleoprotein of bodv-1. it was observed in one animal using a polyclonal antibody against the nucleoprotein of bodv-1 and in two animals using polyclonal antibodies against proteins of variegated squirrel bornavirus-1. thus, an infection with another, so far unknown bornavirus could not completely be excluded and has to be investigated in further studies. scarcity and limited quality of material impeded further immunohistochemical and molecular investigations. as endogenous bornaviral elements similar to bornaviral rna-dependent rnapolymerase have been found in bats [22] , a translated endogenous element could also have been detected by immunohistochemistry due to cross-reactivity. however, an endogenous bornaviral-phosphoprotein-like protein has not been found yet and some authors have discussed a deleterious effect of endogenization of bornaviral phosphoprotein [38] . on the contrary, an endogenous bornaviral-p-like protein might also help prevent against bornavirus infection as the bornaviral polymerase is inhibited by a disturbed nucleoprotein-phosphoproteinration [39] . to summarize, rt-pcr-screening of tissues from european bats revealed no evidence for orthobornaviral rna. further studies could unravel whether the immunohistochemical reactions might be due to expression of endogenous sequences gained during evolution of the bat species or even new bornaviruses. supplementary information accompanies this paper at https://doi.org/10. 1186/s12985-020-1289-3. additional file 1: figure s1 . gel electrophoresis of pcr products. 1-10: borna-negative samples of sufficient quality with gapdh-band at 402 bpamplicon length; 11: sample of insufficient quality without gapdh-band; ntrt: no template reverse transcription-reaction control; pc: positive control (bodv-1-positive mouse); nc: negative control (bodv-1 negative bat); ntc: no template control of pcr; bp: base pairs. additional file 2: table s1 . primer sequences. information about primers. additional file 3: figure s2 . immunohistochemistry reactions in the intestine of eptesicus nilssonii. 2a polyclonal antibody p24 specific for orthobornavirus phosphoprotein, staining of smooth muscle cells; 2b polyclonal antibody anti-bodv-n specific for bodv-1 nucleoprotein, no immunoreaction; 2c polyclonal antibodies vsbv-n and vsbv-p specific for vsbv-1 nucleoprotein and phosphoprotein, faint staining of smooth muscle cells; 2d negative control: rabbit immunoglobulin fraction, no immunoreaction; 2e negative control: control rabbit serum, no immunoreaction; 2f negative control: polyclonal antibody specific for rabies virus, no immunoreaction. additional file 4: table s2 . immunhistochemistry details. information about antibodies used in the immunohistochemistry. additional file 5: table s3 . bats with positive immunoreactivity. information about all bats with immunoreaction with antibody p24. bats: important reservoir hosts of emerging viruses taxonomy of the order mononegavirales: update 2019 fatal encephalitis associated with borna disease virus 1 fatal encephalitic borna disease virus 1 in solid-organ transplant recipients genetic clustering of borna disease virus natural animal isolates, laboratory and vaccine strains strongly reflects their regional geographical origin infections of horses and shrews with bornaviruses in upper austria: a novel endemic area of borna disease epidemiological pattern of classical borna disease and regional genetic clustering of borna disease viruses point towards the existence of to-date unknown endemic reservoir host populations shrews as reservoir hosts of borna disease virus bicolored white-toothed shrews as reservoir for borna disease virus intracerebral borna disease virus infection of bank voles leading to peripheral spread and reverse transcription of viral rna serological evidence for borna disease virus infection in humans, wild rodents and other vertebrates in finland distribution of borna disease virus antigen and rna in tissues of naturally infected bicolored white-toothed shrews, crocidura leucodon, supporting their role as reservoir host species shedding of infectious borna disease virus-1 in living bicolored white-toothed shrews biological characters of bats in relation to natural reservoir of emerging viruses bats as reservoirs of severe emerging infectious diseases ecological dynamics of emerging bat virus spillover international union for conservation of nature and nature resources (iucn): the iucn red list of threatened species european bats as carriers of viruses with zoonotic potential the itinerant natterer: physical and therman characteristics of summer roosts myotis nattereri (mammalia: chiroptera) genomic mining reveals deep evolutionary relationships between bornaviruses and bats an rna-dependent rna polymerase gene in bat genomes derived from an ancient negative-strand rna virus systematic estimation of insertion dates of endogenous bornavirus-like elements in vesper bats a preliminary study of viral metagenomics of french bat species in contact with humans: identification of new mammalian viruses unexpected inheritance: multiple integrations of ancient bornavirus and ebolavirus/marburgvirus sequences in vertebrate genomes endogenous non-retroviral rna virus elements in mammalian genomes the biological significance of bornavirus-derived genes in mammals diseases in free-ranging bats from germany screening red foxes (vulpes vulpes) for possible viral causes of encephalitis pathogenesis of avian bornavirus in experimentally infected cockatiels a variegated squirrel bornavirus associated with fatal human encephalitis restricted expression of borna disease virus glycoprotein in brains of experimentally infected lewis rats no virological evidence for an influenza a -like virus in european bats failure to detect viral rna in bat samples collected in the balkan region novel paramyxoviruses in free-ranging european bats reverse transcription real-time pcr assays for detection and quantification of borna disease virus in diseased hosts divergent bornaviruses from australian carpet pythons with neurological disease date the origin of extant bornaviridae prior to the end-cretaceous extinction paleovirology of bornaviruses: what can be learned from molecular fossils of bornaviruses active borna disease virus polymerase complex requires a distinct nucleoprotein-tophosphoprotein ratio but no viral x protein publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors want to thank silke engel for help with the rt-pcr, silke gantz and diana waldschmidt for help with the immunohistochemistry and jana müller for fruitful discussions during writing and proofreading. the authors are grateful to marion biering, kristin mühldorfer and gudrun wibbelt, leibniz institute for zoo and wildlife research, berlin, jorge a. encarnação, department of animal ecology and systematics, and anne nesseler, state veterinary institute of giessen, for sharing their archival samples. authors' contributions dn contributed to the conception of the work, performed the laboratory work (rt-pcr, ihc) and analysed the data and wrote the manuscript. ch contributed to the conception of the work, the acquiring of samples and the interpretation of data and manuscript writing. all authors read and approved the final manuscript. the authors declare that they have not received any funding. all data generated or analysed during this study are included in this published article and its supplementary information files.ethics approval and consent to participate all work was done according to the respective laws of the federal republic of germany. no additional ethic approval was required. not applicable. the authors declare that they have no competing interests. key: cord-318080-cmx3q2sc authors: amoroso, maria grazia; russo, danilo; lanave, gianvito; cistrone, luca; pratelli, annamaria; martella, vito; galiero, giorgio; decaro, nicola; fusco, giovanna title: detection and phylogenetic characterization of astroviruses in insectivorous bats from central‐southern italy date: 2018-06-12 journal: zoonoses public health doi: 10.1111/zph.12484 sha: doc_id: 318080 cord_uid: cmx3q2sc in recent years, bats have been found to harbour many viruses, raising several questions about their role as reservoirs and potential disseminators of zoonotic viruses. we investigated the presence of six virus families in bats in three regions of central‐southern italy. astroviruses were identified in seven of 13 bat species. sequence analysis revealed marked genetic heterogeneity among the astroviruses identified, with nucleotide identity ranging between 60.26% and 87.62%. astrovirus diversity was not associated with the bat species, the geographic areas or the bat colony, suggesting the circulation of several astrovirus strains in italian ecosystems. genetic diversification and interspecies transmission appear common in bat astroviruses and could provide, potentially, the bases for transmission to humans and other mammals. yet overemphasizing this risk might have detrimental consequences for bat conservation and preservation of the important ecosystem services bats provide. risk is crucial in western countries. several bat species avoid human settlements and have little chance to enter into contact with humans (russo & ancillotto, 2015) , and other species are at risk of extinction (e.g. conenna, rocha, russo, & cabeza, 2016; o'shea, cryan, hayman, plowright, & streicker, 2016) , making the risks negligible. however, increasing anthropization of the environment is altering the ecosystems, disrupting usual habitats and/or creating novel ecological niches that may overlap with human activities. it should be considered, however, that bats provide important ecosystem services (aizpurua et al., 2018; ancillotto et al., 2017; boyles, cryan, mccracken, & kunz, 2011; kunz, braun de torrez, bauer, lobova, & fleming, 2011) . overemphasizing the potential risks posed by bats to humans may generate unjustified alarmism, a fact that has raised considerable concern in conservationists (lópez-baucells, rocha, & fernández-llamazares, 2017) , as also highlighted by the eurobats agreement on the conservation of bat populations in europe (working group on "communication, bat conservation and public health"). under this perspective, surveillance may still be important to avoid or mitigate potential conflicts and eventually improve bat conservation policies. in italy, a few studies have been carried out to assess the presence of viruses in bats lelli, papetti, et al., 2013) but only in the northern regions. these studies detected orthoreoviruses and coronaviruses in various bat species. however, there are no data for the central-southern italian regions, which are characterized by a warmer, drier climate and a different biogeography. in this study, we screened 13 bat species living in these geographic areas. the bat species were selected ad hoc to represent a range of environmental and behavioural differences. our data set covered species that form large colonies in caves and show high fidelity to their roosting sites (e.g. miniopterus schreibersii); species that roost in trees in small groups and switch roosts frequently (e.g. barbastella barbastellus); and synanthropic species that are more likely to enter into contact with humans (e.g. pipistrellus kuhlii). the bats were screened for a large panel of viruses, including rabies viruses, coronaviruses, reoviruses, caliciviruses, astroviruses and enteroviruses. we overall sampled 147 individuals from three regions of central-southern italy. most bats we screened were caught on emergence from their roosts with harp traps or at drinking sites with mist nets. in such cases, bats were promptly removed from the trap or the net and their forearm length and body mass were measured, respectively, with a caliper to the nearest 1 mm and a digital scale to the nearest 0.1 g. sex was assessed by inspecting genitalia (racey, 1988) , and wings were trans-illuminated to distinguish juveniles from adults (anthony, 1988) . in females, we ascertained pregnancy by palpation (racey, 1988) , and lactation by the presence of enlarged nipples surrounded by a hairless skin area and by extruding milk with a gentle finger pressure on the nipple base. bats were immediately released after processing. a few samples came from animals hosted at rehabilitation centres. saliva was sampled from each bat with a dry sterile swab, which was placed in viral transport medium (chu, poon, guan, & peiris, 2008) , and transported in a cool box to the laboratory for the analysis. bat droppings expelled during manipulation of specimens were collected, stored in sterile vials and preserved in cool boxes during transportation. nucleic acids were extracted with the magmax automated system the presence of rabies virus and mers-coronavirus (mers-cov) was investigated by real-time rt-pcr using two commercial kits, that is rabies virus real time rt-pcr kit (shanghai zj bio-tech co., ltd) and mers-cov real time rt-pcr kit (shanghai zj bio-tech co., • identification of astroviruses in four bat species never found positive before. • identification of astrovirus in bats living in italy. • strains of astrovirus identified revealed a high degree of genetic diversity, not related to bat species. • mechanisms other than host specificity may drive virus genetic diversification. ltd). the reaction was carried out following the manufacturer's instructions. all the tests were performed with a 7500 fast real time pcr system (applied biosystems, monza, italy). positive and negative controls were included in the kits. the primers employed for detection of the different viruses are indicated in table 1 . all the assays were carried out with the flexid mastercycler nexsusx2 (eppendorf) using the superscript™onestep rt-pcr kit (life technologies italia). coronaviruses (covs) were searched as described by drosten et al. (2003) (table 1) . for reovirus detection, the nucleic acids were preventively denatured (2 μl viral extract with 1.4 μl of dmso at 97°c for 5 min) and then reversetranscribed and amplified using a nested protocol, as described previously (leary et al., 2002; all the pcr products were analysed by tape station 2200, an automated platform for electrophoresis, (agilent technologies), using the d1000 screentape system. amplicons of the pcr-positive samples were sequenced as previously described (amoroso et al., 2013) . the nucleotide sequence similarity searches were performed using the blast server (http:// www.ncbi.nlm.nih.gov/genbank/index.html). nucleotide sequences of astvs were aligned using the program clustal w (larkin et al., 2007) with reference sequences of mamastrovirus (mastv), using an avian astrovirus (avastv) jf414802 as outgroup (supporting information table s1 ). phylogenetic analyses were carried out by mr bayes (huelsenbeck & ronquist, 2001; ronquist & huelsenbeck, 2003) program implemented in the software package geneious v. 9.1.8 (biomatters, new zealand). bayesian inference was performed using four "chains" run over one million generations (with the first 2000 trees discarded as "burn-in") and supplying statistical support with subsampling over 200 replicates. jmodeltest (posada, 2008) all the screened bats were negative to reoviruses, caliciviruses, enteroviruses, rabies viruses and mers-cov. when analysed for the bat species b. barbastellus (7.14%) and in an additional four bat species (table 2) . geographic location of sampling sites from which positive samples were taken is indicated in figure 1 . upon interrogation (november 2017) of genbank sequence database ( (table 3) . ta b l e 3 interrogation by blast search of ncbi nucleotide database (december 2017) based on the orf1b (rdrp) sequences generated in this study. the strain with the highest % nucleotide identity (% pi) and e value (e-v) found in the database (bm, best match) is shown for each bat sequence generated in this study (table 3) . upon sequence comparison, a marked genetic heterogeneity was information table s1 ) and supported by bootstrap values ≥95. in this study, we investigated the presence of various human viral pathogens in 14 different species of bats captured in central and southern italy. none of the samples was positive to rabies virus that is the most serious concern for the potential human transmission. from an epidemiologic point of view, bat-associated rabies cases are rare, with the incidence rates in canada and the unites states being as low as 2.2-6.7 human cases per billion persons/year over a 57-year period (velasco-villa et al., 2017) . however, there is still a potential risk that needs attention, especially for bat specialists and rehabilitators, who handle bats and are often exposed to bites. our study, in spite of the relatively small number of sampled animals, confirms that this risk is negligible in italian territories. consensus diagnostic molecular assays are useful to detect novel viral species or genetically different viral strains, but they are usually not highly sensitive. using broadly reactive consensus primers, cov rna was detected in 10/147 (6.8%) samples. these results could not be confirmed with sequence analysis. however, the samples also tested negative by a quantitative assay specific for mers-cov, thus ruling out the presence of this virus in the sampled population. using consensus primers universal for the astroviridae family, in europe and asia (drexler et al., 2011; fischer et al., 2016 fischer et al., , 2017 kemenesi et al., 2014; zhu et al., 2009) . the rt-pcr prevalence (22.45%) observed in our study was higher than that observed in hungary (6.94%) (kemenesi et al., 2014) and similar to that found in germany (25.8%; fischer et al., 2016) . astv in 46% of the tested bats (chu et al., 2008; xiao et al., 2011) . the prevalence was also found to greatly vary by bat species (fischer et al., 2017) . as an example, a study carried out in china revealed the presence of astrovirus in 83.3% of the samples of myotis pylosus analysed, whilst the prevalence was found much lower for other species (chu et al., 2008) . to detect and characterize the astv strains, we amplified and sequenced a fragment of 422 bp of the rdrp gene, which represents the most conserved region of the astv genome. upon sequence analysis, we observed a remarkable genetic diversity among the various bat astv strains detected in italy. such a high degree of variation was observed also within species and in the same geographic area or colony. these findings do not confirm the bat species specificity of astv, proposed by other authors (fischer et al., 2016 astrovirus infection is associated with gastro-enteritis in most animal species, and humans astvs are regarded as a common cause of viral diarrhoea in children (mendez, aguirre-crespo, zavala, & arias, 2007; xiao et al., 2017) . avian astvs have also been associated with extra-intestinal diseases, such as nephritis in chicken (imada et al., 2000) and hepatitis in ducks (todd et al., 2009) . recently, astvs have been also detected in the nervous tissues of minks with shaking disease (blomstrom, widen, hammer, belak, & berg, 2010) and of bovines with neurological disorders (bouzalas et al., 2014; li et al., 2013) . neurological disease in immunocompromised human patients has been associated with astv infection (brown et al., 2015; fremond et al., 2015; lum et al., 2016; quan et al., 2010) . in our research, all the animals positive to astv appeared healthy, as also reported in previous studies in bats (fischer et al., 2017) . astroviruses could therefore simply be nonpathogenic members of the bat virome. however, more information is needed on bat-borne immune response to state whether these viruses are really nonpathogenic for bats. bats could play an important role in transmitting such viruses to humans, as astv transmission usually follows an oral-faecal route. contamination of food or drinking water could for example occur by bat droppings. on this regard, it is however important to underline that the probability that human ingest food and water contaminated by astv coming from human faeces looks much higher-see, for instance, the high percentage (28.70%) of human astvs recently found in mussels harvested in the gulf of naples, italy (fusco et al., 2017) . interspecies transmission of astv has been documented on more occasions (de battisti et al., 2012; de benedictis, schultz-cherry, burnham, & cattoli, 2011; mihalov-kovacs et al., 2017; nagai et al., 2015) . also, novel human astvs (mlb1, mlb2, va1, hmo-c, hmo-b, hmo-a, va-2) have been identified that are genetically unrelated to "classical" human astvs (banyai, meleg, moschidou, & martella, 2010; finkbeiner, le, holtz, storch, & wang, 2009; finkbeiner, li, et al., 2009; kapoor et al., 2009 ) and closer to animal astvs. the origin of animal-like human astvs has not been deciphered yet. the potential zoonotic risks associated with bats have attracted the attention of researchers, mostly after the discovery of sars-like and mers-like covs (two coronaviruses highly pathogenic for humans) in european bat species, although the zoonotic risks posed by bat viruses, likely very limited, should be assessed more properly (kohl & kurth, 2014) , in large structured studies. thanks go to luciano bosso for helping with the preparation of figure 1 . maria grazia amoroso http://orcid.org/0000-0002-8663-7445 agriculture shapes the trophic niche of a bat preying on multiple pest arthropods across europe: evidence from dna metabarcoding bubaline herpesvirus 1 associated with abortion in a mediterranean water buffalo effects of free-ranging cattle and landscape complexity on bat foraging: implications for bat conservation and livestock management age determination in bats detection of newly described astrovirus mlb1 in stool samples from children detection of a novel astrovirus in brain tissue of mink suffering from shaking mink syndrome by use of viral metagenomics neurotropic astrovirus in cattle with nonsuppurative encephalitis in europe conservation. economic importance of bats in agriculture bats as 'special' reservoirs for emerging zoonotic pathogens astrovirus va1/hmo-c: an increasingly recognized neurotropic pathogen in immunocompromised patients bats: important reservoir hosts of emerging viruses dbatvir: the database of bat-associated viruses novel astroviruses in insectivorous bats investigation of a potential zoonotic transmission of orthoreovirus associated with acute influenza-like illness in an adult patient insular bats and research effort: a review of global patterns and priorities genetic characterization of astroviruses detected in guinea fowl (numida meleagris) reveals a distinct genotype and suggests cross-species transmission between turkey and guinea fowl astrovirus infections in humans and animals -molecular biology, 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caliciviruses among children hospitalized for acute gastroenteritis in the united states additional supporting information may be found online in the supporting information section at the end of the article. how to cite this article key: cord-286708-igu984oc authors: chua, kaw bing; voon, kenny; crameri, gary; tan, hui siu; rosli, juliana; mceachern, jennifer a.; suluraju, sivagami; yu, meng; wang, lin-fa title: identification and characterization of a new orthoreovirus from patients with acute respiratory infections date: 2008-11-25 journal: plos one doi: 10.1371/journal.pone.0003803 sha: doc_id: 286708 cord_uid: igu984oc first discovered in the early 1950s, reoviruses (respiratory enteric orphan viruses) were not associated with any known disease, and hence named orphan viruses. recently, our group reported the isolation of the melaka virus from a patient with acute respiratory disease and provided data suggesting that this new orthoreovirus is capable of human-to-human transmission and is probably of bat origin. here we report yet another melaka-like reovirus (named kampar virus) isolated from the throat swab of a 54 year old male patient in kampar, perak, malaysia who was suffering from high fever, acute respiratory disease and vomiting at the time of virus isolation. serological studies indicated that kampar virus was transmitted from the index case to at least one other individual and caused respiratory disease in the contact case. sequence analysis of the four small class genome segments indicated that kampar and melaka viruses are closely related. this was confirmed by virus neutralization assay, showing an effective two-way cross neutralization, i.e., the serum against one virus was able to neutralize the other. although the exact origin of kampar virus is unknown, epidemiological tracing revealed that the house of the index case is surrounded by fruit trees frequently visited by fruit bats. there is a high probability that kampar virus originated from bats and was transmitted to humans via bat droppings or contaminated fruits. the discovery of kampar virus highlights the increasing trend of emergence of bat zoonotic viruses and the need to expand our understanding of bats as a source of many unknown viruses. reoviruses (respiratory enteric orphan viruses), members of the family reoviridae, are a large and diverse group of non-enveloped viruses with segmented dsrna genomes, which are taxonomically classified into ten genera [1, 2] . members of the genus orthoreovirus contain 10 genome segments and have been isolated from a broad range of mammalian, avian and reptilian hosts. although orthoreoviruses have been identified as the causative agents of diseases in animals, infections in humans are generally benign with very rare cases of mild upper respiratory tract illness or enteritis in infants or children [3] . orthoreoviruses are divided into two subgroups, fusogenic and nonfusogenic, based on the ability of the virus to induce cell-cell fusion and syncytium formation [4] . a fusogenic orthoreovirus, the melaka virus (melv), was isolated from a human patient suffering acute upper respiratory disease [5] . melv was shown to be capable of human-to-human transmission and has close sequence relatedness to two bat-borne orthoreoviruses, the nelson bay virus (nbv) isolated from fruit bats in australia and the pulau virus (pulv) isolated from fruit bats in malaysia [6, 7] . epidemiological tracing suggested that melv originated from bats and was transmitted directly to the index case, followed by subsequent transmission to other members of the same family [5] . bats have been shown to be the reservoir hosts of many recently emergent zoonotic viruses, including hendra virus, nipah virus, menangle virus, and potentially sars and ebola viruses [8] [9] [10] [11] [12] [13] . nbv was the first reovirus of bat origin, which was isolated in 1968 from the heart blood of a flying fox (pteropus poliocephalus) in new south wales, australia [6, 14] . nbv was also the first mammalian reovirus to display fusogenic properties [6] , a characteristic previously only known for avian reoviruses (arvs). in 1999, during a search for nipah virus in pteropid bats on tioman island, pulv was isolated from pteropus hypomelanus [7, 15] . here, we report the discovery and characterization of kampar virus (kamv), the fourth member in the nbv species group and its isolation from a human patient with fever and acute respiratory illness. although there is no direct evidence to suggest that kamv originated from bats, the close relationship of kamv with other members of the nbv group and preliminary epidemiological data suggest that kamv is most likely a batborne orthoreovirus. to mitigate the potential health as well as socio-economic impact of emerging diseases, in particular, the potential emergence of pandemic influenza, the ministry of health malaysia undertook nation-wide influenza-like illness surveillance for early detection, identification and control of these emerging diseases. in august 2006, as part of the surveillance process, a patient with acute influenza-like illness was investigated in kampar, a town situated in the north-western part of peninsular malaysia, about 36 kilometres south of ipoh, the capital city of the state perak. case 1 (index case). subject 1 (s1), a 54-year old chinese man, was well until 19 august 2006 when he developed sudden onset of high fever with chills and rigor. this was associated with cough, sore-throat and headache. there was no associated dyspnoea, tachypnoea, haemoptysis or chest pain on coughing. besides the severe frontal throbbing headache, he had generalized body aches, myalgia and severe malaise. on the following day, he developed nausea, vomiting and diarrhoea. the vomitus consisted of food taken and was not bile-stained. the stool was described as watery without excessive mucous and it was non-malenic. the gastrointestinal symptom was associated with abdominal pain and loss of appetite. his illness was not relieved with self-medication of antipyretics. on review, there was no associated giddiness, blurring of vision, photophobia, skin bleeding or arthritis. he sought medical treatment at the government health clinic in kampar on 21 august 2006. at the outpatient clinic, he was noted to be febrile (an axillary temperature of 40.1 degree celsius), illlooking with a generalized body erythema that blanched on pressure and was more prominent over the face and upper trunk. he had mild conjunctivitis but there was no jaundice. his tonsils were enlarged and injected but there was no white exudate noted over the tonsils. he was not in any respiratory distress and his lungs were clear with good air entry on auscultation. other systemic examination was essentially normal and there was no significant lymphadenopathy noted. a provisional diagnosis of influenza-like illness was made at which he was given a higher dose of anti-pyretic. his illness was noted to resolve on 23 august 2006 although he still appeared weak and lethargic. venous blood samples were taken from the patient for full blood count analysis, and the results are shown in table 1 . these results indicated that his white blood cell and platelet counts were within normal limits although there was a relative lymphopenia in the blood sample taken at first examination. his throat swab was taken on august 21 for virus isolation as described below. case 2 (contact case). subject 2 (s2) is a 28 year old female chinese medical officer who attended to the index patient in the government health clinic in kampar on 21 august 2006. on 25 august, she developed nasal obstruction with mild runny nose. this was associated with a mild sore-throat and hoarseness of voice. there was no associated cough or breathing difficulty. apart from the subjective feeling of mild lethargy and general unwellness, she did not experience fever, headache or myalgia. her upper respiratory symptoms resolved within three days. case 3 (suspected contact case). subject 3 (s3), a 46 year old female, is the wife of the index patient. she regularly stays and takes care of the family home. due to the nature of their work in other parts of the country, her husband and their 17-year old son only come back intermittently to stay in the same house. she was unable to re-call having a similar illness as her husband previously, although she did remember experiencing incidents of mild headache and fever in late august to early september, which resolved with self-medication. since she did not seek medical help, there was no record of medical examination at the time of suspected infection. on august 21, during the examination by the local doctor, a throat swab was taken from the index case patient (s1) and sent in viral transport medium (vtm) to the national public health laboratory for virus isolation. after 3 days of culturing, a syncytial cytopathic effect (cpe) was noted in mdck and vero cells, but not in hep-2 cells. after 2 passages in mdck cells, the virus was able to replicate and cause syncytial cpe in all types of mammalian cell-lines available in the laboratory inclusive of c6/36 (atcc crl-1660) cell-line which is of mosquito cell in origin (data not shown). the virus was named kampar virus (kamv) after the location of the index case. due to the similar cpe morphology ( figure 1 ) and cell line susceptibility patterns between kamv and the recently discovered melaka virus (melv), which also causes acute respiratory diseases in humans [5] , immunofluorescent antibody testing was conducted to examine cross reactivity. as shown in figure 2 , the strong cross reactivity between kamv antigen and human anti-melv sera, and vice versa, suggested these viruses are closely related. this was later confirmed by further serological characterization and molecular analyses (see below). the small township of kampar is bordered by forested limestone hills on the eastern side and a scattering of abandoned old tin mining ponds on the other side. s1 is a part-time carpenter and lives with his wife and their youngest son (17 years old) in an old wooden house with metal zinc roof and plain cement floor. his house is situated on reclaimed old tin mining land which is about 80 metres from the central market of kampar town. many tall trees, comprising of mango trees, coconut palms and natural wild fruiting trees (in particular, the ''sea-almond'' or locally named ketapang trees), are found surrounding his house and between neighbouring houses. a tall fruiting mango tree is planted close to the house which is of less than three feet from the side wall of the house and directly adjacent to the window of the living room. at night, fruit bats were noted to use this mango tree to feed on the ''sea-almond'' fruits which they obtained from the nearby ketapang trees. partially eaten fruits and leftover stones of the fruits abandoned by the fruit bats were noted on the ground just outside the window of the living room or on the roof of the house. only occasionally, the fruit bats were noted to feed on the ripened mangoes. during night time, s1 and his wife often rested in the living room while watching television. there was no recollection of fruit bats flying into his house or roosting inside the house. there was also no record of dead fruit bats being found in the house compound. it is not clear at the present time whether kamv is carried by a specific fruit bat species or by multiple bat species circulating in the region. a preliminary survey of bat sera collected in peninsular malaysia from our previous studies indicated a low prevalence of kamv-specific antibodies in at least two different bat species, pteropus vampyrus (1/55) and pteropus hypomelaunus (2/77). considering that the closely related pulv virus was isolated from pteropus hypomelaunus [7] and nbv from pteropus poliocephalus [6] , it seems likely that these orthoreoviruses may have a broad host range among different bats. it should be noted that smaller fruit bats, such as eonycteris spelaean and cynopterus brachyotis, are more commonly found in the areas where kamv and melv were discovered. further field study is required to elucidate the bat species mainly responsible for the spill over of these viruses into human population in malaysia. the family keeps a couple of chickens in the backyard and a few pet birds in the patio. a week prior to the onset of s1's illness, his wife bought 6 chickens from a neighbour and one of them died on day 2 of illness. on follow-up investigation, venous blood samples were taken from s1, his wife (s3) and the doctor (s2) who examined him on august 21. all sera were tested for the presence of antibodies against kamv by immunofluorescent antibody testing using kamv-infected mdck cells. as indicated by the results in table 2 , anti-kamv antibodies were present in all three subjects, with the index case patient showing the highest level of igg antibodies. to exclude the possibility that kamv was just a passenger virus which might be commonly present in the human population in this region and was not the cause of the observed sickness in s1 and s2, we have since conducted serological surveillance of 31 human serum samples collected in the same region for an unrelated study in early 2008. none of these sera had any specific igm antibodies against kamv and only one serum sample contained low level (1:80) of kamv-reactive igg antibodies. to further establish the antigenic relationship between kamv and three other known orthoreoviruses in the nbv species group, a four way cross-neutralization was conducted as described in the materials and methods with the results presented in table 3 . it is clear that all of the four viruses share significant antigenic relatedness as evident from the cross-neutralization activities of each serum. the failure of any of the four sera to neutralize the control virus, mammalian reovirus 3 (mrv3), and of the mrv3 serum to neutralize any of the four viruses confirmed the specificity of the assay. while the kamv human serum neutralized the homologous virus better than heterologous viruses, such a difference was not observed for the melv human serum, which seemed to be equally effective in neutralizing all four viruses. the close genetic relationship between kamv and melv was confirmed by the comparison of genome segment mobility or electropherotypes using sds-page. the two viruses have similar but not identical rna genome segment profiles (figure 3) . the genetic relatedness of kamv and three other viruses in the nbv species group was further corroborated by the following molecular characteristics: (i) the deduced protein products encoded by the four small (s) genome segments of these viruses are very similar in size and share significant levels of sequence identity (table 4 ); (ii) one s-class genome segment of orthoreoviruses may be polycistronic, and its coding arrangement is used as a useful molecular marker for differentiation of different species groups (duncan et al., 2004) . the s1 segment of kamv has an identical coding arrangement to that of the three other viruses (data not shown); (iii) orthoreoviruses have highly conserved genome terminal sequences at the 59 end of the positive sense rna which can be used as a genetic marker for virus classification [3] . for kamv, the consensus sequence is 59 gcuuww (w = u or a), which is identical to that of melv, pulv and nbv (figure 4 ), but different from other orthoreoviruses. the evolutionary relationship between kamv and other known orthoreoviruses was investigated by conducting phylogenetic analyses for all the proteins encoded by the s-class genome segments. representative phylogenetic trees based on deduced amino acid sequences of the major outer capsid protein and the major inner capsid protein are shown in figure 5a and 5b, respectively. it is evident that kamv is a close member of the nbv species. similar trees were obtained based on other proteins (data not shown). respiratory tract infections remain the main infectious disease of humans and account for a large proportion of public health spending worldwide. despite recent success in discovery of novel respiratory pathogens during the last decade [16] [17] [18] , there is still a substantial proportion of rtis remaining undiagnosed. isolation of melv from a patient suffering acute respiratory disease marked a new beginning of linking orthoreoviruses to acute human rtis [5] . this was especially significant in that epidemiology studies indicated that melv originated from bats. the last decade has experienced a surge in the discovery of emerging viruses of bat origin, several of which have had significant impact on human and animal heath, tourism and trade [8, [19] [20] [21] . although the exact reason is not fully known, it seems that bats are an ideal reservoir for a number of zoonotic viruses covering many different virus families. in this context, the isolation of kamv confirms the observed trend of zoonotic virus emergence out of bat populations in the pacific region. although there is no direct evidence to prove the origin of kamv, the close genetic relationship of kamv with melv, pulv and nbv and the location of the house of the index case in close proximity to fruit trees frequently visited by fruit bats indicated a high probability that kamv is also a bat-borne virus. subsequent serological surveillance confirmed low levels of antibodies to kamv circulating in two bat species commonly found in peninsula malaysia. retrospective serological studies indicated that the index has transmitted the virus to at least one other person, his doctor, presumably via contact droplet nuclei or aerosol. it is more difficult to assess how his wife was infected by kamv although serological results suggested she could have been infected earlier in the same manner as her husband. the risk of virus spillover from bats as a result of increasing encroachment by animals and humans into bat habitats is enhanced by the wide distribution of a large number of bat species and the seemingly great genetic diversity of newly emergent bat-borne viruses. this is true for henipaviruses [22] [23] [24] [25] , coronaviruses [26, 27] , and now orthoreoviruses. henipaviruses were initially discovered in pteropid bats, but a subsequent seroprevalence survey indicated that henipaviruses or henipa-like viruses are also circulating among non-pteropid bats [28] [29] [30] . since the discovery of sars-like coronaviruses in horseshoe bats, a large number of new coronaviruses have been detected in many different bat species [26, 27, [31] [32] [33] [34] [35] . it is important to conduct similar serological and virological surveillance studies for bat orthoreoviruses to better understand their geographic distribution, their host range and their genetic diversity. such information will be essential for an effective risk assessment of future spill over events and potential large scale outbreaks. during the preparation of this manuscript, the partial s segment sequences of a new orthoreovirus were made available in the genbank by a group in hong kong (genbank accession numbers eu165526 and eu170365-7). this virus, tentatively named reovirus strain hk23629/07, was isolated from a patient suffering an acute respiratory infection. phylogenetic analysis of the available sequences confirmed that the hk strain is very closely related to other members of the nbv group. a closer look at the phylogenetic tree based on the most variable protein, cell attachment protein sigma c (figure 6 ), indicated that the malaysian bat isolate (pulv) is more closely related to the three human isolates (melv, kamv and hk) than to the australian bat isolate (nbv). this suggests that geographic location, rather than the host of isolation, is a more important determinant for genetic relatedness. although the exact reservoir species of kamv is yet to be determined, preliminary serological studies suggest that kamv is likely able to infect multiple fruit bat species in malaysia. the isolation of three related viruses from human patients within such a short period and the close relation between bat and human isolates would suggest that spill over by this group of orthoreoviruses is not an uncommon event and systematic surveillance among rti patients is needed to provide a more accurate assessment of this newly discovered viral infection among human population in different countries in this region and beyond. in conclusion, the discovery and characterization of kamv corroborate our previous work on melv and demonstrate the increasing risk posed by unknown bat viruses which are capable of infecting and causing disease in humans. this further highlights the urgent need to systematically survey bat-borne viruses in the international community so as to enable us to conduct more effective risk assessment, to provide forecast for potential future outbreaks and to devise better prevention and control strategies. a throat swab was taken from the patient at the time of his first clinical examination and transported in viral transport medium (vtm) to the national public health laboratory for virus isolation. the sample was treated with antibiotics (c. penicillin 100,000 i.u./ml and streptomycin 100 mg/ml) for an hour before being inoculated in duplicate (100 ml and 200 ml, respectively) into freshly confluent monolayers of mdck (atcc, ccl-34), vero (atcc, ccl-81) and hep-2 (atcc, ccl-23) cells cultured in a 24-well tissue culture plate. the plate was incubated at 37uc in 5% co 2 and examined daily for the presence of cpe in cultured cells. supernatant from cultures with visible syncytial cytopathic effect (cpe) after 3 days was taken for further analysis by serial passage in different cell lines available in the laboratory. the investigation conducted in this study was approved by the ethics committee of the malaysian national public health laboratory. all patients (subjects) in this manuscript have given written informed consent (as outlined in the plos consent form) to publication of their case details. no identification of the subjects is to be revealed in any publication. immunofluorescence antibody testing (ifat) and virus neutralization assay were conducted as previously described [5] . briefly, for ifat a freshly confluent monolayer of mdck was infected with kamv and at full cpe, the infected cells were harvested, washed four times and suspended in sterile pbs at a cell concentration of approximately 3000 cells per millilitre. an aliquot of the infected cell suspension was carefully spotted onto each well of teflon coated slides, followed by air-drying over a warm plate and subsequent fixation in cold acetone for 10 min. serial 2-fold dilutions of serum samples were then added to detect specific reactivity. for detection of igm, igg was removed by absorption with protein a prior to serum dilution. bound antibodies were detected using fluorescein conjugated rabbit anti-human igm or igg (dako, usa). specific reactivity/labelling were read under a uv fluorescence microscope (olympus bx50, japan). for vnt, serial 2-fold dilutions of control and test sera were prepared in duplicate starting at 1:10. an equal volume of virus working stock containing 150 tcid 50 was added to the diluted sera and incubated for 30 min. the pre-incubated virus/ serum mix was added to confluent cell monolayers and incubated for 1 h. the inoculum was removed, monolayers washed three times with pbs and cell media replaced. ability of sera to neutralize virus was determined by scoring the extent of cpe observed in duplicate wells three days later. virion from culture supernatant was harvested and concentrated by centrifugation and viral rna was extracted and purified using the qiamp viral rna kit (qiagen, germany). for each virus, an aliquot of 15 mg rna was run on a 10% sdspolyacrylamide/bis gel under denaturing and reducing conditions at 150 v for 4 hrs at room temperature. the gel was washed with distilled water, stained with ethidium bromide before the photo was taken. extraction and purification of dsrna and synthesis of randomly primed cdna were carried out as previously described [5, 7, 36] . the majority of sequence information was obtained by pcr with primers designed from conserved regions of melv, pulv and nbv (primer sequences will be supplied upon request). to obtain the genome segment terminal sequences, a two-step pcr strategy was used. first, the single primer amplification technique or spat [36] was used to generate primary pcr product followed by a semi-nested pcr using the combination of kamv genome segment-specific primers and the adaptor-specific primer used in spat. pcr products were sequenced directly without cloning. all regions of the genome segments were sequenced at least three times. phylogenetic analysis was conducted using the mega4 software package [37] . phylogenetic trees were constructed using the neighbour-joining algorithm with bootstrap values determined by 1,000 replicates. complete genome sequences of the four s segments were deposited in genbank under accession numbers eu448334 to eu4488337 for s1, s2, s3 and s4 segments, respectively. family reoviridae orthoreoviruses and their replication genus orthoreovirus extensive sequence divergence and phylogenetic relationships between the fusogenic and nonfusogenic orthoreoviruses: a species proposal a previously unknown reovirus of bat origin is associated with an acute respiratory disease in humans structure and cytopathic effects of nelson bay virus pulau virus; a new member of the nelson bay orthoreovirus species isolated from fruit bats in malaysia bats: important reservoir hosts of emerging viruses a novel morbillivirus pneumonia of horses and its transmission to humans nipah virus: a recently emergent deadly paramyxovirus an apparently new virus (family paramyxoviridae) infectious for pigs, humans, and fruit bats bats are natural reservoirs of sars-like coronaviruses fruit bats as reservoirs of ebola virus nelson bay virus. a novel reovirus a novel approach for collecting samples from fruit bats for isolation of infectious agents severe acute respiratory syndrome characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia identification of a new human coronavirus anthropogenic deforestation, el nino and the emergence of nipah virus in malaysia zoonotic viruses of wildlife: hither from yon host range and emerging and reemerging pathogens molecular biology of hendra and nipah viruses bat nipah virus genetic characterization of nipah virus nipah virus in lyle's flying foxes prevalence and genetic diversity of coronaviruses in bats from china molecular diversity of coronaviruses in bats antibodies to nipah-like virus in bats (pteropus lylei) the natural history of hendra and nipah viruses nipah virus infection in bats (order chiroptera) in peninsular malaysia covdb: a comprehensive database for comparative analysis of coronavirus genes and genomes evolutionary relationships between bat coronaviruses and their hosts detection and prevalence patterns of group i coronaviruses in bats coronavirus antibodies in african bat species detection of group 1 coronaviruses in bats in north america strategies for the sequence determination of viral dsrna genomes mega4: molecular evolutionary genetics analysis (mega) software version 4.0 we thank g. marsh and m. tachedjian for critical reading of the manuscript, k. selleck for technical assistance, and t. pye and e. hansson for help with dna sequencing. key: cord-294698-mtfrbn87 authors: kim, h. k.; yoon, s.‐w.; kim, d.‐j.; koo, b.‐s.; noh, j. y.; kim, j. h.; choi, y. g.; na, w.; chang, k.‐t.; song, d.; jeong, d. g. title: detection of severe acute respiratory syndrome‐like, middle east respiratory syndrome‐like bat coronaviruses and group h rotavirus in faeces of korean bats date: 2016-05-23 journal: transbound emerg dis doi: 10.1111/tbed.12515 sha: doc_id: 294698 cord_uid: mtfrbn87 bat species around the world have recently been recognized as major reservoirs of several zoonotic viruses, such as severe acute respiratory syndrome coronavirus (sars‐cov), middle east respiratory syndrome coronavirus (mers‐cov), nipah virus and hendra virus. in this study, consensus primer‐based reverse transcriptase polymerase chain reactions (rt‐pcrs) and high‐throughput sequencing were performed to investigate viruses in bat faecal samples collected at 11 natural bat habitat sites from july to december 2015 in korea. diverse coronaviruses were first detected in korean bat faeces, including alphacoronaviruses, sars‐cov‐like and mers‐cov‐like betacoronaviruses. in addition, we identified a novel bat rotavirus belonging to group h rotavirus which has only been described in human and pigs until now. therefore, our results suggest the need for continuing surveillance and additional virological studies in domestic bat. bats are considered reservoirs of several emerging infectious disease. recently emerging human viruses, such as severe acute respiratory syndrome coronavirus (sars-cov), middle east respiratory syndrome coronavirus (mers-cov), nipah virus, hendra virus and ebola virus, are thought be bat-borne viruses (han et al., 2015) . in addition, new species of influenza a viruses, lyssaviruses, paramyxoviruses and coronaviruses continue to be discovered in bats around the world (tong et al., 2013; banyard et al., 2014; yang et al., 2014; mortlock et al., 2015) . reassortant group a rotavirus was also detected in the faeces of a straw-coloured fruit bat (esona et al., 2010) . therefore, viruses that originate in bats may be a source of additional spill over from wildlife into domestic animals and humans (plowright et al., 2015) . since the outbreak of mers-cov in korea in 2015, the prediction and control of newly emerging viruses has gained urgency. however, in korea, there have been a few studies on viruses in domestic bat species. serological evidence of japanese encephalitis virus (jev) was reported in the early 1990s (lee and lee, 1992) . in 1995, there was a report on the isolation and genetic characterization on a hantavirus from bats (jung and kim, 1995) . more information of viruses in bats is needed to clarify the role of these animals in infectious diseases epidemiology. therefore, in this study, we investigated viruses in bat species in korea, using 49 faecal samples collected from july to december 2015 in 11 sites in natural bat habitats. from july to december in 2015, 49 faecal samples were collected at 11 sites in natural bat habitats, such as caves, an abandoned mine and under a bridge (table 1) . fresh bat faeces were placed into transport medium in a 10% suspension and were transported to the laboratory for further laboratory analysis. the major bat species at the collection sites were determined based on morphology and on previous data from bats' roosting sites (han et al., 2012) . in this study, the major bat species in the collecting sites were rhinolophus ferrumequinum, myotis macrodactylus, myotis aurascens kuzyakin, myotis petax and miniopterus schreibersii. consensus primer-based reverse transcriptase polymerase chain reactions (rt-pcrs) were performed to detect influenza a viruses, coronaviruses, lyssaviruses and paramyxoviruses (fouchier et al., 2000; poon et al., 2005; v azquez-mor on et al., 2006; tong et al., 2008) . the detailed information on the rt-pcrs is in table s1 . a total of 49 bat faecal samples were tested. the amplicons from positive rt-pcrs were sequenced using target-specific forward and reverse primers synthesized by cosmogenetech co. ltd (daejeon, korea). the nucleotide data obtained from sanger sequencing of the pcr fragments were further analysed with related sequences in genbank using bioedit (hall, 1999) and mega version 6 (tamura et al., 2013) . in total, 16 bat faecal samples that were collected in four sites were used for high-throughput sequencing ( table 1) . the supernatants from suspensions of one, four, five and six faecal samples collected in danyang, wonju, taebaek and uljin, respectively, were pooled by sampling site. each of the four pooled samples was filtered through a 0.2-lm filter and ultracentrifuged at 252 000 g for 1 h. each pellet was resuspended in 500 ll of 19 digestion buffer (turbo dna free kit; ambion, darmstadt, germany), and a total of 10 ll of turbo dnase was added. after incubating for 30 min at 37°c, suspensions were transferred to a 1.5-ml reaction tube and centrifuged at 1500 g for 3 min. the supernatants were used for rna extraction. rna was extracted using trizol ls (invitrogen corp., carlsbad, ca, usa), following the manufacturer's manual. the extracted rna was submitted to macrogen (seoul, korea) for high-throughput sequencing in a hiseq 2000 sequencing system based on the transcriptome de novo sequencing platform. the obtained viral contigs were analysed and annotated by the mg-rast server (meyer et al., 2008) . the cut-off for the annotation was 10 à5 , 60% and 15 for maximum e-value, minimum percentage identity and minimum alignment length, respectively. the contigs annotated as mammalian viruses were validated through blastn (http:// www.ncbi.nlm.nih.gov). the genbank accession numbers for the nucleotide sequences in this study are ku528584-ku528594. there were no samples positive for influenza a viruses, lyssaviruses or paramyxoviruses. among 14 cases of the submitted samples, three cases were positive for coronavirus by rt-pcr, showing the expected 440 nt target. these included the following: (case no. 1) one of four samples from ho cave in wonju where rhinolophus ferrumequinum and myotis macrodactylus were the major species present, (case no. 6) one of six samples bm abandoned mine in inje where rhinolophus ferrumequinum was the major species found and (case no. 12) two of four samples bgs (case no. 12) cave in munkyung where miniopterus schreibersii was found to be the major species (table 1) . in the ho cave, one of four faecal samples (b15-8) was positive by coronavirus-specific rt-pcr. when the pcrpositive fragment was sequenced, it was found to be a bat coronavirus (bat cov) high-throughput sequencing from the pooled bat faecal samples as shown in table 1 , cases 1, 2, 3 and 4 were further analysed by high-throughput sequencing. by hiseq 2000 sequencing, 4341, 162 559, 7690 and 16 657 contigs were obtained from cases 1, 2, 3 and 4, respectively. based on the annotation by the mg-rast system, several viral sequences that are related to mammalian viruses, insect viruses, plant viruses, fungal viruses and bacteriophages were identified. the mammalian viral sequences annotated by the mg-rast system were further validated by blastn (http:// www.ncbi.nlm.nih.gov). the blastn result is presented in table 2 . fifteen viral sequences from case no. 1 were similar to porcine rotavirus ska-1 (69-73% nucleotide identity), bat cov sc2013 (70-91% nucleotide identity), bat cov hku5-5 (82% nucleotide identity), alphacoronavirus eptesicus fuscus-related strains (95-96% nucleotide identity) and rhinolophus pearsoni bunyavirus shaanxi 2011 (72% nucleotide identity). three viral sequences from case no. 2 were similar to banna virus 02vn078b (90% nucleotide identity), bat cov cdphe15 (80% nucleotide identity) and bat cov neixiang-31 (82% nucleotide identity). there was no mammalian viral sequences in cases 3 and 4. partial rna-dependent rna polymerase (rdrp) sequences obtained from b15-8 (sample in case no. 2), b15-21 (sample in case no. 6), b15-40 and b15-41 (sample in case no. 12) were phylogenetically compared with other reference sequences (fig. 1a) . three of these sequences clustered with the alphacoronaviruses (b15-8, 40 and 41 because an rdrp sequence from betacoronavirusrelated bat cov b15-1-6 (detected from the pooled sample in case no. 1) was not obtained, a partial spike gene sequence (c10151) was compared with the spike genes of reference betacoronaviruses (fig. 1b) . in addition, the full spike gene sequence obtained from bat cov b15-21 was also analysed. bat cov b15-1-6 grouped with mers-covlike bat covs (bat cov strains hku5, a434, sc2013 and hku4), showing 80.9-82.2% nucleotide identities, and was found to be closely related to mers-cov emc, showing 76.6% of nucleotide identity. the amino acids sequence encoded by the full spike gene of b15-21 was aligned and compared with human sars-cov and sars-cov-like bat cov sequences ( figure s1 ). the s2 domain is more conserved than the s1 domain in the spike protein. the receptor binding domain (rbd) of the sars-cov-like bat covs, except for rashc014, had two major deletion sites, tgnyn (446-450) and pfspdgkpctppa (472-484), compared to human sars coronaviruses sequences. the receptor binding motif (rbm) and human ace2 (hace2)-interacting residue were highly variable between human sars-covs and sars-cov-like bat covs but rashc014 strain. porcine rotavirus-related sequences from pooled bat faeces in case no. 1 were analysed with reference sequences of rotaviruses in human and animals (fig. 2) . based on the partial sequences of the vp1, vp3 and vp4 regions, the rotavirus-related sequences were found to belong to rotavirus group h. the group h bat rotavirus in this study according to a recent report, there are 23 species of bats in korea (han et al., 2012) . most korean bats are thought to be insectivores, as no fruit bats have been found in korea to date, except for some fruit bats as pets or indoor exhibitions (han et al., 2010 (chu et al., 2008) . as bat covs b15-40 and 41 were also found in faeces in a cave where miniopterus schreibersii was dominant, bat cov strains in the same lineage with bat cov 1a and 1b may be adapted to the miniopterus spp. it was interesting that bat cov b15-21 found in this study was closely related to sars-cov-like bat covs, such as bat cov rf1, based on the partial rdrp and s genes, respectively. the newly identified bat cov b15-21 clustered with the sars-cov-like bat cov group in the phylogenetic analysis. the major bat species in the site where bat cov b15-21 was collected was rhinolophus ferrumequinum, which is consistent with a previous report that sars-covlike bat covs were mostly found in rhinolophus spp. (yuan et al., 2010) . the amino acids of its full spike gene showed that the b15-21 strain is closely related to sars-cov-like bat covs rp3, rf1, hku3-2 and 273 rather than human sars-covs and bat cov rashc014, which was reported to have high potential for human emergence (menachery et al., 2015) and showed two specific deletion sites in the rbd of the spike protein. one additional betacoronavirus (b15-1-6), which was not detected by the consensus primer-based rdrp-specific rt-pcr (poon et al., 2005) , was detected by high-throughput sequencing as shown in table 2 . three contigs were similar to bat cov sc2013, which is a mers-related betacoronavirus that was found in bats in china (yang et al., 2014) . one contig was also similar to bat cov hku5-5, which clustered with mers-cov (woo et al., 2007; annan et al., 2013) . phylogenetic analysis based on partial spike gene sequences of bat cov b15-1-6 showed that it belonged to a mers-cov-like clade with previously known mers-cov and mers-cov-like bat covs. although information of the associated bat species was not clearly available, this study first report that sars-cov and mers-cov-like bat covs exist in korea. this may imply that other strains or types of those coronaviruses may circulate in bat species in korea. therefore, we cannot ignore appearance of novel or variant coronavirus through local emergence in korea, like sars and mers in china and middle east, respectively. in addition to coronaviruses, this study is the first to determine the existence of group h rotaviruses in bat faeces collected in korea. group h rotavirus is a recently proposed group of rotaviruses that include strains adrv-n and b219, which infect human adults (alam et al., 2007; matthijnssens et al., 2012) . so far, group h rotaviruses have only been reported in human and pigs (molinari et al., 2015) , but this study provides evidence that bat species may be a host of group h rvs. to confirm that, there should be follow-up studies including virus isolation and characterization, genomic analysis, continuous surveillance and vp6-based classification (matthijnssens et al., 2012) to find its prevalence, epidemiology and zoonotic potential. localized emergences of zoonotic diseases are usually due to expansion of the wildlife-human interface (morse et al., 2012) . the human-bat interface in korea has not been fully described. people usually recognize that bats are flying and roosting in the deep forest or caves. however, the sites where the bat faecal samples were collected showed some evidence of human-bat contact ( figure s2 ). some caves or abandoned mine where bats are roosting are located near human habitats. people living there frequently visit into the caves or the abandoned mine to take a rest or to ferment foods. some caves were also used for some religious ceremony by korean traditional shamans. in this study, sars-cov-like and mers-cov-like bat covs and group h rotavirus were detected for this first time in korea, which may be of interest because of their zoonosis potential. several kinds of evidence for humanbat contact were also found. therefore, the potential for novel or variant human viruses from bats in korea should not be ignored. continuous surveillance and additional virological research should be a priority to prevent future emergences of zoonotic viral diseases. intraspecies diversity of sars-like coronaviruses in rhinolophus sinicus and its implications for the origin of sars coronaviruses in humans. j. gen. virol. 91, 1058-1062. additional supporting information may be found in the online version of this article: table s1 . information on targets and primers for viral detection. figure s1 . amino acid-based multiple alignment of spike proteins from b15-21 and other reference strains. figure s2 . possible evidence of human-bat interaction in the bat habitats in this study. genetic analysis of an adrv-n-like novel rotavirus strain b219 detected in a sporadic case of adult diarrhea in bangladesh ska-1(porcine)-ab576629 j19(human)-dq113897 b219(human)-ef453355 03v0567(chicken)-nc_021590 cal-1(human)-eu490414 bang373(human)-nc_021541 03v0568(chicken)-nc_021625 05v0049(chicken)-nc_014511 osu(porcine)-gu199514 j19(human)-dq113900 b219(human)-ef453357 ska-1(porcine)-ab576631 03v0567(chicken)-nc_021589 cal-1(human)-eu490417 bang-373(human)-nc_021551 osu(porcine)-ay277921 human betacoronavirus 2c emc/2012-related viruses in bats lyssaviruses and bats: emergence and zoonotic threat genomic characterizations of bat coronaviruses (1a, 1b and hku8) and evidence for co-infections in miniopterus bats reassortant group a rotavirus from straw-colored fruit bat detection of influenza a viruses from different species by pcr amplification of conserved sequences in the matrix gene bioedit: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/ nt nucl isolation of a zoonotic pathogen kluyvera ascorbata from egyptian fruit-bat rousettus aegyptiacus sounds of the bats in korea. national institute of biological resources press bats as reservoirs of severe emerging infectious diseases genomic characterization of m and s rna segments of hantaviruses isolated from bats detection of antibodies in korean bats to japanese encephalitis virus vp6-sequence-based cutoff values as a criterion for rotavirus species demarcation 2015: a sars-like cluster of circulating bat coronaviruses shows potential for human emergence the metagenomics rast server -a public resource for the automatic phylogenetic and functional analysis of metagenomes genetic variability of vp6, vp7, vp4, and nsp4 genes of porcine rotavirus group h detected in brazil 2012: prediction and prevention of the next pandemic zoonosis novel paramyxoviruses in bats from sub-saharan africa ecological dynamics of emerging bat virus spillover identification of a novel coronavirus in bats mega6: molecular evolutionary genetics analysis version 6.0 sensitive and broadly reactive reverse transcription-pcr assays to detect novel paramyxoviruses rt-pcr for detection of all seven genotypes of lyssavirus genus comparative analysis of twelve genomes of three novel group 2c and group 2d coronaviruses reveals unique group and subgroup features mers-related betacoronavirus in vespertilio superans bats key: cord-304481-yqc8r3ll authors: luis, angela d.; o'shea, thomas j.; hayman, david t. s.; wood, james l. n.; cunningham, andrew a.; gilbert, amy t.; mills, james n.; webb, colleen t. title: network analysis of host–virus communities in bats and rodents reveals determinants of cross‐species transmission date: 2015-08-24 journal: ecol lett doi: 10.1111/ele.12491 sha: doc_id: 304481 cord_uid: yqc8r3ll bats are natural reservoirs of several important emerging viruses. cross‐species transmission appears to be quite common among bats, which may contribute to their unique reservoir potential. therefore, understanding the importance of bats as reservoirs requires examining them in a community context rather than concentrating on individual species. here, we use a network approach to identify ecological and biological correlates of cross‐species virus transmission in bats and rodents, another important host group. we show that given our current knowledge the bat viral sharing network is more connected than the rodent network, suggesting viruses may pass more easily between bat species. we identify host traits associated with important reservoir species: gregarious bats are more likely to share more viruses and bats which migrate regionally are important for spreading viruses through the network. we identify multiple communities of viral sharing within bats and rodents and highlight potential species traits that can help guide studies of novel pathogen emergence. most emerging infectious diseases are zoonotic (passed from animals to humans), with > 70% originating in wildlife (jones et al. 2008) . cross-species transmission is one of the most challenging and least studied aspects of disease ecology, yet it is the defining process in zoonotic disease emergence (lloyd-smith et al. 2009 ). bats are the reservoirs of a number of emerging viruses, including ebolaviruses, sars coronavirus and nipah and hendra paramyxoviruses (calisher et al. 2006 ). viral and malarial parasite diversity in bats is high (drexler et al. 2012; luis et al. 2013; quan et al. 2013; schaer et al. 2013; drexler et al. 2014) , and bats appear to be major and ancient natural reservoirs of several viral families, including hepaciviruses, pegiviruses, paramyxoviruses, coronaviruses and influenza a viruses ( drexler, et al. 2012 ( drexler, et al. ,, 2014 quan et al. 2013; tong et al. 2013) . since cross-species transmission can be common among bats (streicker et al. 2010; cui et al. 2012) , understanding their role as reservoir hosts requires examining them in a community context rather than concentrating on individual species. several traits have been hypothesised to make bats particularly suited to hosting and transmitting viruses, including life history traits such as relatively long lifespans for their body size (munshi-south & wilkinson 2010) , which may facilitate viral persistence for chronic infections; their reliance on torpor, which can decrease viral replication and immune function (dempster et al. 1966; prendergast et al. 2002) ; and flight, which can facilitate transmission of viruses to new areas. in addition, many bat species are gregarious, living in dense aggregations, and some roosting sites can house diverse assemblages of bat species (kunz 1982; kuzmin et al. 2010) , which could facilitate transmission of pathogens and sustain acute-immunising infections. in evolutionary terms, bats are ancient mammals, and it has been hypothesised that viruses that evolved in bats may use conserved cellular receptors enhancing their ability to transmit viruses to other mammals (calisher et al. 2006) . the diet of some species has been hypothesised to facilitate transmission of viruses; after feeding, frugivorous bats often leave partially eaten fruit behind which can be contaminated with viruses (chua et al. 2002; dobson 2005) . bats are the second most diverse mammalian order with over 1100 species, with many overlapping species distributions; multiple regions across the globe are home to more than 40 bat species, allowing for species to interact and potentially spread viruses between them ( fig. 1 ; calisher et al. 2006; luis et al. 2013) . here, we examine the structure of host-virus communities in bats and rodents and how host traits, such as those above, correlate with propensity to host and transmit viruses. rodents are a suitable group for comparison because they also host many important zoonotic viruses and share many of the characteristics hypothesised to make bats suitable as viral reservoirs. for example, rodents are also evolutionarily ancient; they are older than bats and more closely related to humans (dos reis et al. 2012) . rodents are the most diverse mammalian order with approximately twice the number of rodent species as bat species, many of which express torpor, and display a wide range of life history traits, including some long-lived species. networks have been used extensively in disease ecology to model the finite and heterogeneous number of social contacts between individuals. here, we use networks to describe sharing of viruses between different species, rather than individuals. this is akin to other ecological networks, such as food webs, mutualistic, or host-parasite networks (montoya et al. 2006) . however, we collapse the bipartite network (separate nodes for viruses and host species) to a unipartite projection, weighting contacts between host species by the number of viruses they share. in the same way social networks can highlight the disproportionate importance of specific individuals, such as 'super-spreaders', in population-level disease dynamics, our networks can highlight the importance of certain species in host-virus communities and disease emergence. in addition, we can use other network methods to look at substructures, or 'communities' in the network, highlighting sets of highly interconnected species (newman 2004) . here, we use databases of bat and rodent viruses spanning 78 years of publications (luis et al. 2013) to create networks that connect host species via their shared viruses and examine cross-species transmission at several scales. a database of bat and rodent viruses compiled from the literature (luis et al. 2013 ) was used to create separate networks of viral sharing for bats and rodents, with each node representing a host species and edges between two nodes representing the presence of virus(es) in both species. edges were weighted by the number of viruses shared between the two species. see supplementary methods in supporting information for more details on network formulation. to account for sampling bias in the analyses, we used the number of citations on web of science (http://thomsonreuters.com/products_services/science /science_products/a-z/ web_of_science/) with the host species' latin species name for the search term (e.g. altizer et al. 2011) . the distribution was highly skewed, therefore we used the log of this number to normalise the distribution. we also performed the analyses using the species name and 'virus' as the search terms. all results were qualitatively similar. we calculated various node and network statistics ( table 1 ). the node statistics that we calculated were degree (the number of links a node has), weighted degree (incorporating the number of viruses shared) and betweenness (the number of shortest paths that go through a node (weighted)). the network statistics that we calculated included the degree distribution, transitivity (if two nodes are connected, the probability that their adjacent nodes are also connected), degree assortativity (likelihood of high-degree nodes connecting to other high-degree degree nodes and low-degree nodes to other low-degree nodes) and connectance (links per species 2 , or the proportion of links that are present out of all possible links). we also calculated quantitative connectance, which is the quantitative linkage density (which takes into account the weights of the edges; bersier et al. 2002) divided by the number of species in the network. to account for sampling bias, we additionally calculated the quantitative linkage densities and connectance for sampling effort-corrected networks. see supporting information for more details. as another test to account for bias, we calculated the quantitative connectance of the jaccard matrices (the intersection divided by the union of the viruses for each pair of host species). this gives the proportion of the viruses shared rather than the absolute number. the multiple regression on matrices and gls analyses described below use the original networks (absolute number of viruses not weighted by sampling effort) because for those, we take sampling effort into account as a covariate. to calculate p-values for the network statistics in table 1 , we performed 10 000 permutations of the networks, where we shuffled the edge weights. see supporting information for more details. species traits were compiled from online databases and the literature as described in luis et al. (2013) . we examined body mass, number of litters per year, litter size, maximum longevity, torpor use, international union for conservation of nature (iucn) conservation status, geographical distribution area, latitude of the midpoint of the species distribution, number of other species in the same taxonomic order that are sympatric, and for bats only, migratory classification, diet, gregariousness and propensity to roost in caves. see supplemental figures s4-s7 in luis et al. (2013) for plots of the raw data. bat species traits that were new to these analyses were gregariousness, propensity to roost in caves and diet (see supporting information for more details). virus and host trait data and r code are provided online as supporting information. multiple regression on distance matrices (mrm) (lichstein 2007 ) was used to detect significant correlations between viral sharing, phylogeny, sympatry and similarity in host traits using 'mrm' in the r package 'ecodist' (goslee & urban 2007 ; r core team 2012). the response variable was the viral sharing matrix (adjacency matrix of the network), with each entry giving the number of viruses shared between each pair of host species. the 'ape' package in r (paradis et al. 2004 ) was used to calculate a phylogenetic correlation matrix based on species' shared branch lengths of the most complete mammalian phylogenetic supertree available containing bats and rodents (bininda-emonds et al. 2007 ). see figs s4 and s5 in supporting information. using the species distribution shape files from the iucn website (http://www.iucnredlist.org/technical-documents/spatial-data) and the r packages 'sp' and 'rgeos' (pebesma & bivand 2005 ), a sympatry matrix was created. each entry of the matrix was either a 1 if the two species' distributions overlap or a 0 if they do not. matrices of sampling effort were created by multiplying the logged number of citations for each species together. matrices showing similarity in host traits were also calculated. for example for each pair of species, the difference in the number of litters per year was calculated. this matrix was standardised so values ranged between 0 and 1, and the matrix used in the multiple regression models was 1 minus this matrix (so it would be a correlation matrix similar to the phylogenetic and sympatry matrices). host species were grouped into 'communities', partitions of highly interconnected nodes, by using the community detection algorithm described in blondel et al. (2008) , which maximises the modularity between groups of the weighted networks. see supporting information for more details. as an alternative to using modularity to assign nodes to communities, we also used clique percolation theory (palla et al. 2005) . in this method, nodes can belong to multiple communities. see supporting information for more details. ‡quantitative connectance, which takes into account edge weights. §quantitative connectance, with weights adjusted according to sampling intensity. ¶quantitative connectance, where the weights are the proportion of viruses shared rather than the absolute number (see methods). generalised least squares (gls) were used to examine correlates of viral richness per host species, degree and betweenness, while allowing for phylogenetic correlation to be incorporated into the error structure as described below. because many life history traits are correlated, we performed principal components analyses (pca) on the life history traits: logged body mass, maximum longevity, number of litters per year and litter size (separately for bats and rodents). the variables were rescaled to have unit variance before analysis in r using the 'prcomp' function (r core team 2012), and these principal components were then used in subsequent analyses. see fig. s6 for pca plots and table s1 for loading values and variance explained by each principal component. because closely related species share traits, we tested for phylogenetic dependence by setting the error term for the gls to the phylogenetic correlation matrix multiplied by an additional parameter, pagel's k, that was estimated to determine the strength of phylogenetic dependence (pagel 1999; freckleton et al. 2002) . a k estimate of 1 indicates that the error structure of the model was directly proportional to the species shared branch lengths. a k of 0 indicates the error structure of the model was not related to the species shared branch lengths (i.e., phylogeny does not explain any additional variation). note that phylogeny is not a covariate in this analysis, and may not be significant if the model covariates themselves have phylogenetic dependence. models were ranked by their aicc (akaike information criterion adjusting for finite sample sizes) values. correlation coefficients (r) were pearson's product moment correlation comparing observed values to model predictions. we also ran these models using mcmc generalised linear mixed effects models (hadfield, 2010) . see the supporting information for more details. maps were made using the r packages 'maptools ', 'maps', 'sp' and 'pbsmapping' (pebesma & bivand 2005; becker et al., 2012; schnute et al. 2012; bivand & lewin-koh 2013) . viruses were mapped by the union of their hosts' distributions using the command 'gunioncascaded' from the 'rgeos' package (pebesma & bivand 2005) . the assumption was made that the virus exists in the entire distribution of its host(s), analogous to the fundamental niche concept (hutchinson 1957; harris & dunn 2010) . to visually assess whether sampling effort had a strong effect on the virus distributions, we also plotted these distributions discounting by sampling effort of the host species (figs s7 and s8). the bat network had 143 host species connected by 110 viruses, and the rodent network had 196 host species connected by 185 viruses (figs 3a, 4a, s1 and s2). a species' degree is the number of other host species it is connected to by shared viruses. degree in bats ranged from 0 to 53 with a mean of 15.0 and in rodents ranged from 0 to 78 with a mean of 12.5 (table 1 ; see fig. s3 for degree distributions). the mean degree and mean weighted degree were significantly higher in bats than in rodents. there was significant transitivity (the probability that the adjacent nodes of a node are also connected) in both bats and rodents. assortativity in bats was significant, suggesting that bat species with high degree tend to interact with other bat species of high degree (and low degree with low degree), but there is no evidence of this in rodents (table 1) . the bat network was 1.64 times more connected (connectance = links/species 2 ) than the rodent network (table 1) . this was statistically significantnot once in 10 000 permutations was the difference between connectance for the bat and rodent networks at least the observed difference. the quantitative connectance adjusted for sampling effort and the quantitative connectance on the jaccard matrices were also higher in bats (table 1) . next, we examined the connections between species, using multiple regression on matrices (lichstein 2007) to determine the correlates of the number of viruses that two host species share. both phylogeny (relatedness) and sympatry (geographic range overlap) were important predictors of viral sharing (table 2) . species with overlapping distributions and closer phylogenetic relationships shared more viruses. sympatry was more important than phylogeny for viral sharing in both orders, but both explained more variation in bats. sampling effort (by number of citations on web of science) was also important. well-studied pairs of species were found to have more viruses shared between them. the amount of variation explained by sympatry, phylogeny and citations together was 21% in bats and 14.3% in rodents. similarity in life history traits explained a small amount of additional variation (table s2) . although bats generally have received attention as disease reservoirs, some species are more important for disease emergence. we determined which species have the most viruses, the highest degree (the most connections) and the highest betweenness in the network (a measure of network centrality the number of shortest paths between any two nodes that go through the node of interest) and host traits associated with these network metrics. for all analyses of the bat and rodent networks, sampling effort was importantthe more a species was studied, the more viruses, higher degree and higher betweenness. consequently, we adjusted for sampling effort by number of citations on web of science. the most important variable associated with hosting more viruses in bats was diet (fig. 2a, table s3 and s4). host traits that correlated with the highest degree within the bat network (the most connections or viruses shared), in order of importance, were gregariousness and sympatry; diet was marginally important (fig. 2b, table s6 and s7). migration was the most important host trait associated with betweenness in bats (fig. 2c, table s9 and s10). in these analyses, phylogeny is not a covariate, but incorporated into the error structure, and an additional phylogenetic correlation parameter (k; pagel 1999) is estimated, to determine if phylogeny can explain variation unexplained by the model covariates. for bats, k, was 0 for the best models for number of viruses and degree (table s3 and s6), indicating that phylogeny did not explain any additional variation. a likely explanation is that the best models included diet, which is strongly related to phylogeny. however, k for the best model for betweenness was 0.79 (table s9 ), indicating that phylogeny explained additional variation. for rodents, sympatry was the most important host trait; species whose distributions overlapped with a greater number of other rodent species had more viruses and higher degree and betweenness (fig. 2d-f and table s11-s19). in addition, rodent species that are larger, longer lived and with higher reproductive rates (e.g. sigmodon hispidus, rattus norvegicus) had more viruses (pc3; table s1, s11-s13). species at higher latitudes had marginally higher degree (after controlling for sympatry: table s14-s16). the results from the mcmc generalised linear mixed effects models were largely similar to these results (see tables s5, s8 , s13, s16 and s19). the only significant difference was the best model for the number of viruses in bats included only citations and phylogeny (table s5 ). see the supporting information for more details. although 88 and 69% of all possible pair-wise combinations of species are connected by < 8 degrees of separation in the bat and rodent networks, respectively, the influence of a single species may be more localised. we detected distinct 'communities' of viral sharing within orders (figs 3a and 4a) , by maximising the modularity (blondel et al. 2008) . modularity can attain values in the range from à0.5 to 1. if positive, there are more within-group connections than would be expected at random, and in practice, values > 0.3 indicate significant community structure (newman 2004) . modularity scores were 0.54 and 0.55 for the bat and rodent networks respectively. in the bat network, there were 10 viral sharing communities. each species had distributions that on average overlapped with 85% of the species within its community vs. 18% of species outside (t = 8.82, d.f. = 12.54, p = 9.8 9 10 à7 ; fig. 3b-f ). the largest and most densely connected community (fig. 3b) consisted of 34 bat species in the americas, largely connected by vector-borne viruses. the remaining communities were connected by a mixture of vector-borne and non-vector-borne viruses. the green community (fig. 3c ) was broadly distributed from europe to australia, and approximately half of the species in this community also shared viruses with species in other communities that overlapped spatially. there were two globally distributed communities of rodents with 61 and 44 species (fig. 4b,c) and two communities that were more spatially restricted. each rodent species on average overlapped with 68% of the species within its community vs. 7% outside of its community (t = 4.66, d.f. = 6.12, p = 0.003; fig. 4b-e) . the house mouse, mus musculus, has spread across the globe along with its viruses (fig. 4b) . removing this species results in largely the same community structure, indicating this species has already facilitated the transmission of viruses to multiple sympatric species across the globe. the black rat, rattus rattus, and the brown rat, r. norvegicus, are also widely distributed and are the main species holding the green community together (fig. 4c) . when either rattus species is removed from this community, it splits into several smaller communities, suggesting less secondary viral spread to sympatric rodent species. overall properties of the bat and rodent networks provide a global view of viral sharing within each order, to our current knowledge. the number of viruses per host species, the number of hosts per virus, the mean degree and connectance were higher in the bat network than the rodent network. this suggests that viruses may pass more easily between bat species than between rodent species. our analyses indicate that characteristics unique to bats, such as gregariousness and migration, may facilitate this increased transmission. previous studies hypothesised frugivory as an important viral transmission mechanism among bats and from bats to other species, because virus has been isolated from partially eaten fruit, which is often shared and dropped to the ground (chua et al. 2002; dobson 2005) . our study is consistent with that hypothesis, with frugivores hosting more viruses than nectivores, insectivores and sanguivores. however, diet is strongly correlated with phylogeny, and the best model using mcmc glmm only included phylogeny. therefore, these results could indicate the importance of diet or, alternatively, some other factor correlated with phylogeny, such as immunological functioning, for example. the most important host characteristics associated with degree in bats were gregariousness and sympatry. these characteristics have been hypothesised as important mechanisms for viral maintenance and spread (calisher et al. 2006; luis et al. 2013 )bat species with distributions that overlap with a greater number of other bat species, and particularly those which are gregarious, will have greater interspecific contacts and chance for cross-species transmission. at some roosting sites, bat densities can be as high as 3000 bats per square metre, and some roosts house diverse assemblages of species (constantine 1967; kunz 1982) . high densities can lead to high contact rates, facilitating pathogen transmission and persistence. these dense roosting sites are often caves, therefore we had hypothesised that propensity to roost in caves would be an important predictor of viral sharing in bats. however, colony size (gregariousness) was more important than where the colony roosts. large colony sizes do not occur on the same scale in rodents and may have led to the higher mean degree and connectance in bats compared to rodents. species with high betweenness might not have or share the most viruses, but could play a key role in disseminating viruses because they can connect disparate regions of the network. migration was the most important host trait associated with betweenness in bats. long-distance animal movement has been hypothesised to enhance geographic spread of infectious disease, however, evidence in most cases is lacking (altizer et al. 2011 ). in the bat network, many of the shortest network paths went through regional migrants (moving 100-500 km annually), which were more important than long-distance migrants (> 500 km). this is consistent with the idea of 'migratory culling' (bradley & altizer 2005) in which the physiological stress of long-distance migration can increase mortality of infected individuals. for rodents, sympatry was the most important covariate, again emphasising the importance of spatial overlap in cross-species transmission. we found that species that had higher reproductive rates and body mass had more viruses (pc3 of the pca), which is in accordance with the findings of han et al. for all rodent pathogens and parasites (2015). this is the first analysis we are aware of that attempts to identify communities of pathogen sharing at a global scale. these communities reveal which species are most highly connected, providing insight into virus maintenance and spread. they may also be useful in guiding public health strategies. for example if a new zoonotic virus is identified in hipposideros speoris, it may be useful to look for it in other species of the green community (fig. 3c ) as well. the links in our networks represent real connections, the sharing of viruses, and may be evidence of cross-species transmission, coevolution of host and virus lineages, or indirect links of cross-species transmission through an intermediate host or vector. the importance of phylogeny for viral sharing could indicate the virus originated in a common ancestor and was maintained or coevolved in separate lineages, or the greater ability of viruses to transmit between closely related species. however, the greater importance of sympatry in viral sharing indicates that cross-species transmission is at least as important if not more important than coevolution in establishing the network. for cross-species transmission to take place, there must be contact, direct or indirect, between two host species, and therefore, it is not surprising that sympatry is important in the sharing of viruses. the importance of sympatry is reflected in similarities between viral and host species richness maps (fig. 1 ). locations where host species diversity is highest generally have more viruses. however, bats in europe and india have greater viral richness than predicted by the host species richness map (fig. 1a,c) , and this does not appear to be due to sampling bias (fig. s7) . the community context may provide additional insight. there are two host-virus communities connecting europe to east asia where species richness is high (fig. 3c,f) , and these communities may provide a bridge. higher viral richness than expected from host species richness in india may also reflect connections with areas of high species richness in s.e. asia (fig. 3c) . for the viral richness maps, we assumed that the virus exists throughout the distribution of its host(s) analogous to the fundamental niche concept (hutchinson 1957; harris & dunn 2010) and representing the broadest possible distribution. however, there may be barriers that restrict the realised niche of a virus. for example rhinolophus ferrumequinum hosts sars-like coronavirus and japanese encephalitis virus, and its range extends from e. asia to w. europe, but the populations in e. asia and europe do not appear to mix (flanders et al. 2009 ). thirteen viruses were present in both bats and rodents. ten of these were vector transmitted. therefore, direct transmission between bats and rodents appears rare. of the three directly transmitted viruses, only ebola could have been transmitted from bats via fruit. the other viruses shared between bats and rodents in this study, hantaan and puumala, were found in insectivorous bats, and transmission may have occurred from rodents, as they are currently thought to be the reservoirs of these hantaviruses. however, a recent study indicates bats may be important and ancient reservoirs of hantaviruses more generally (guo et al. 2013) . a recent study examining the sharing of parasites in primates using similar network methods used an overall metric for network centrality which included weighted degree, betweenness and several other network measures of the importance of a node (g omez et al. 2013) . in this study, the authors found that hosts with denser populations living in larger groups and having broad distributions had higher centrality. although overall, degree and betweenness were correlated in our study, the metrics were different enough to be best predicted by different covariates in batsgregariousness for degree, and migration for betweenness. however, in rodents, the most important factor was the same for the different metricssympatry best predicted the number of viruses, degree and betweenness. for our analysis we used a database covering 78 years of publications . subsequent analyses, particularly focusing on bats as reservoirs of viruses, have been published (e.g. drexler et al. 2012; quan et al. 2013 ). studies such as drexler et al.'s (2012) report short genome fragments that are yet to be categorised by the ictv, which makes it difficult to discern what sequences should be considered the same virus for classification of viral sharing, and thus we have not included them in our analyses. however, molecular studies increase our ability to distinguish viruses and understand virus-host relationships. as interest in reservoirs of emerging infectious diseases increases, especially in bats, we predict that results such as these will further strengthen our findings and allow for more refined analyses at the community level. indeed, several recent studies support our conclusions, showing a preponderance of host switching between bats and from bats to other mammals (drexler et al. 2012; guo et al. 2013; drexler et al. 2014 ). this study is based on a large literature search, and therefore there are necessarily constraints on inference, given different motivations for, and methods used during studies of both rodent and bat viruses through time. we were concerned that the recent attention on bats as reservoirs may have biased the sampling and artificially increased the apparent connectance of the bat network; therefore as a secondary analysis, we also created networks using only viral accounts before the year 2000. we found that although both the bat and rodent networks were smaller (the bat network was reduced to 120 species and 744 links; and the rodent network to 145 species and 738 links), the connectance remained relatively steady (5.2% in bats and 3.5% in rodents), suggesting that the apparent greater viral sharing in bats compared to rodents is not merely a function of recent attention on bats. however, the importance of citations in our analyses for both orders highlights the importance of sampling effort overall. although we were able to account for sampling effort in the analyses examining species traits associated with viral sharing, it is difficult to account for sampling effort in creating the networks themselves. one approach which we implemented was creating networks using weighted edges based on sampling effort; this gave us confidence that among the species in our database, the connectance is higher in bats than in rodents. although our database only has 9% of extant rodent species and 13% of extant bat species, the rodents in our database have been more studied overall and more studied for viruses than bats even when removing the most highly studied, lab rodents -mus musculus and rattus norvegicus (significant by t-test on log number of citations for the species name, means = 4.90 & 4.44, p = 0.002; and the species name + virus, means = 1.99 & 1.43, p < 0.001). we would expect more of the total viral community to have been identified in the more highly studied group of species (rodents), thus the sampling bias should increase the apparent connectance in rodents compared to bats. however, this does not account for species not in our database. if the rodent species that have not been sampled share more viruses than the bat species that have not been sampled, then these results may not hold. similarly, the species trait data was only available for roughly 37% of the bat and rodent species in our networks, which corresponds to 5 and 3% of known extant bat and rodent species respectively; therefore, the species traits associated with important hosts may also change as our knowledge increases. new viruses are being discovered frequently, suggesting that there are many more viruses yet undetected in both bats and rodents. therefore, our conclusions and the maps of viral diversity for both orders (fig. 1 ) may change significantly as our knowledge increases. we hope that this first, broad examination of viral sharing will help motivate future efforts including more directed data collection. another potential source of bias may be reports of spillover or incidental hosts that are not important reservoirs but are treated with equal weight in these analyses. we took care to distinguish the hantaviruses and arenaviruses, for which there is relatively good knowledge of host-virus relationships. however, we found that this did not impact the qualitative conclusionswhen using ictv classifications the rodent network still had lower mean degree (13.8), mean weighted degree (19.01), connectance (3.51%) and transitivity (0.57) than the bat network. however, for most of the reported viruses in bats and rodents, there is little knowledge of the ecology and epidemiology in these hosts. this again highlights the importance of further study of both bats and rodents as reservoirs of viruses. our networks connect hosts by their shared viruses. for a given pair of connected species, we do not know in which direction the cross-species transmission may have taken place, or if transmission even occurred between these species. there may have been a third species that transmitted the virus to both species. as more viral sequences become available, molecular methods, such as those used by streicker et al. (2010) , may be used to infer directionality in cross-species transmission, and help resolve these issues. our study highlights the interconnectedness of species with respect to viruses and shows the benefits of examining pathogens in a community ecology context at several scales. our analyses suggest that unique bat characteristics figure importantly in sharing and spreading viruses, lending quantitative support for bats' special status in zoonotic virus emergence and demonstrating which characteristics affect reservoir potential. practically, our analyses may help guide future surveillance for optimal prevention of emerging zoonoses. this could help campaigns aimed at preventing spillover between bats and humans to benefit human health while conserving bats' important roles in ecosystem services such as pest management, plant pollination and seed dispersal (boyles et al. 2011) . animal migration and infectious disease risk & r version by ray brownrigg quantitative descriptors of 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virus in bats new world bats harbor diverse influenza a viruses adl, ctw, jlnw and aac designed study. adl, tjo, dtsh, atg and jnm compiled data. adl performed analyses and wrote the manuscript, and all other authors contributed substantially to editing. key: cord-333914-c150ki1n authors: koba, ryota; suzuki, satori; sato, go; sato, shingo; suzuki, kazuo; maruyama, soichi; tohya, yukinobu title: identification and characterization of a novel bat polyomavirus in japan date: 2020-08-20 journal: virus genes doi: 10.1007/s11262-020-01789-7 sha: doc_id: 333914 cord_uid: c150ki1n a novel polyomavirus (pyv) was identified in the intestinal contents of japanese eastern bent-wing bats (miniopterus fuliginosus) via metagenomic analysis. we subsequently sequenced the full genome of the virus, which has been tentatively named miniopterus fuliginosus polyomavirus (mfpyv). the nucleotide sequence identity of the genome with those of other bat pyvs was less than 80%. phylogenetic analysis revealed that mfpyv belonged to the same cluster as pyvs detected in miniopterus schreibersii. this study has identified the presence of a novel pyv in japanese bats and provided genetic information about the virus. electronic supplementary material: the online version of this article (10.1007/s11262-020-01789-7) contains supplementary material, which is available to authorized users. bats are considered the natural reservoirs of a variety of zoonotic rna viruses, such as ebola viruses, marburg viruses, and severe acute respiratory syndrome coronavirus [1] [2] [3] . several dna viruses, including adenoviruses, herpesviruses, and polyomaviruses (pyvs), have also been detected [4] [5] [6] . however, the pathogenic and zoonotic roles of these dna viruses have been not clarified. pyvs are small double-stranded dna viruses with a circular genome of approximately 5 kbp. the viral genome consists of three regions: regulatory, early, and late regions. the regulatory region is responsible for transcription from both the early and late promoters and the initiation of viral dna synthesis. the early region contains genes encoding the large t antigen (tag) and small t antigen (tag). the late region contains the structural proteins vp1, vp2, and vp3 [7] . although pyv diversity in bat populations in north, central, and south america, africa, indonesia, and new zealand were investigated in previous studies [6, [8] [9] [10] [11] [12] , the prevalence and genetic diversity of pyvs in japanese bats remain unclear. the aims of this study were to (i) determine the presence of pyvs in japanese bats, (ii) characterize the genomic structure of bat pyvs, and (iii) analyze the evolutionary relationships between the bat pyv detected in this study and other known bat pyvs. eighteen bats (miniopterus fuliginosus) were collected in wakayama prefecture, japan. the pooled intestinal contents obtained from each bat (approximately 1 g/body) were prepared as a 10% suspension in sterilized phosphate-buffered saline (pbs a total of 10,136,210 reads and 5362 contigs were obtained in a pool of sample from 18 bats. to identify homologous sequences, the obtained genomic data were analyzed via a blastn search using the dna data bank of japan database in accordance with a previously reported method [13, 14] . virus-related sequences were identified in 123 contigs. of these, 14 contigs contained pyv-like sequences with high identities. other contigs contained the sequences of eel river basin pequenovirus, montastraea cavernosa colony-associated virus, and grapevine-associated totivirus-1. to determine the complete viral genome of these pyv-like sequences, pcr was performed using la taq dna polymerase (takara bio, otsu, japan) in accordance with the manufacturer's instructions. specific pcr primers were designed on the basis of the sequences obtained from the contigs. the primer sequences were as follows: pyv-f1 (sense, 5′-aag ttt gca gta gtc ttt gaa gat gtg aag ggt c-3′), pyv-r1 (antisense, 5′-cac tcc tgg gct ttc ctg ctc ata ttt atg ca-3′), pyv-f2 (sense, 5′-cat aaa cag ggt caa acc ac-3′), and pyv-r2 (antisense, 5′-aag cac tcc acc aaa gga aa-3′). dna extracted from the pooled sample of bat intestinal contents was used as the template. the pcr products were visualized via electrophoresis on a 1% agarose gel stained with sybr safe (life technologies, carlsbad, ca, usa). the full genomic dna could be amplified by two independent pcr using the aforementioned primers. the amplified dna was cloned by inserting the pcr product into the pcr2.1 topo vector (life technologies) in accordance with the manufacturer's instructions. the obtained sequences were analyzed using the bigdye terminator v3.1 cycle sequencing kit (life technologies), and nucleotide sequences were assembled using atgc computer software (genetyx corporation). a homology search was performed using ncbi blast. the genome of miniopterus fuliginosus polyomavirus (mfpyv) has a length of 4956 bp (accession number: lc529726). the genome organization includes an early region coding for tag and tag on one strand and a late region encoding the capsid proteins vp1, vp2, and vp3 on the opposite strand. a noncoding regulatory region (nccr) was located between the start of the early region and that of the late region, in line with previous findings for bat pyvs (fig. 1a and supplementary table 1) [9] [10] [11] [12] . interestingly, open-reading frames encoding vp2 and vp3 of mfpyv did not overlap with that of vp1. the stop codons of vp2 and vp3 are located at base positions 1184-1186, whereas the start codon of vp1 is located at base positions 1188-1190. a single nucleotide (guanine) at 1187 separates the vp2/3 and vp1 regions ( fig. 1a and supplementary fig. 1 ). therefore, genomic composition of mfpyv is genetically different from those of typical pyvs in terms of non-overlapping vp regions. figure 1b and c present the phylogenetic trees of vp1 and tag of mfpyv and 28 other bat pyvs constructed using neighbor-joining analysis. based on phylogenetic analyses of the vp1 and tag amino acid sequences, both regions of mfpyv are closely related to those of other miniopterus pyvs and group b bat pyvs (fig. 1b and c) . vp1 is a major pyv structural protein that is indispensable for entry of the virus into host cells [7] . mfpyv vp1 displayed less than 72% nucleotide sequence identity with other bat pyvs (supplementary table 2 ). tag is a multifunctional protein that plays important roles in viral dna replication and the regulation of viral and cellular gene expression [15] [16] [17] . the predicted mfpyv tag exhibited low similarity (< 73%) with those of other pyvs (supplementary table 2 ). mfpyv tag sequences contained features known to be conserved in tags of other bat pyvs, including the highly conserved dnaj domain (hpdkgg), a retinoblastoma (rb)-binding motif (lycne), and several functional motifs (supplementary fig. 2 ). according to a previous report, these elements work together to bind rb and interrupt its interaction with the e2f transcription factor to promote viral replication and cell cycle progression [18] . tag is generated via alternative splicing of the early mrna transcript [11, 19] . in the early region of the mfpyv genome, conserved predicted splice donor sites are located at base positions 4729-4734 (cct gag /gta agg ) and 4346-4351 (ttt cag /gtc ttc ) (fig. 1a) . in the deduced nccr region of the mfpyv genome, several conserved elements were identified, including several copies of the consensus tag binding site gaggc and its reverse complement gcctc supplementary fig. 3) . these elements are likely to comprise the core of the replication origin [20] . comparison of the full-length genome sequence of mfpyv with those of other bat pyvs revealed that mfpyv is most closely related to the ky156 strain with 70% nucleotide sequence identity (supplementary table 2 ). according to the polyomaviridae study group of the international committee on taxonomy of viruses, a novel pyv species should have < 81% nucleotide sequence identity to other known pyvs [7] . mfpyv exhibited less than 81% nucleotide sequence homology to the known reference pyvs including previously reported bat pyvs. in line with the nomenclature of the other bat pyvs, we propose the name mfpyv for the newly discovered virus. for virus isolation, we attempted to propagate the mfpyv strain using the tb1-lu cell line derived from the lungs of the free-tailed bat tadaria brasiliensis (atcc #ccl-88). however, a cytopathic effect was not observed in the cells following serial passage of the cultures. viral dna replication was also not detected in the cells and supernatant collected at each passage. there is a need for additional research to identify efficient cell culture systems for bat pyvs to elucidate the viral infection/replication mechanisms and their pathogenicity. in conclusion, we detected a novel pyv genome sequence in japanese bats. further epidemiological investigations are needed to determine the extent of pyv genetic variation in various bat species in japan. bats: important reservoir hosts of emerging viruses from sars to mers: 10 years of research on highly pathogenic human coronaviruses large serological survey showing cocirculation of ebola and marburg viruses in gabonese bat populations, and a high seroprevalence of both viruses in rousettus aegyptiacus first detection of adenovirus in the vampire bat (desmodus rotundus) in brazil a novel bat herpesvirus encodes homologues of major histocompatibility complex classes i and ii, c-type lectin, and a unique family of immunerelated genes discovery of diverse polyomaviruses in bats and the evolutionary history of the polyomaviridae taxonomical developments in the family polyomaviridae detection of polyoma and corona viruses in bats of canada novel polyomaviruses in south american bats and their relationship to other members of the family polyomaviridae detection of novel polyomaviruses in fruit bats in indonesia genomic characterization of two novel polyomaviruses in brazilian insectivorous bats discovery of novel virus sequences in an isolated and threatened bat species, the new zealand lesser short-tailed bat (mystacina tuberculata) evaluation of rapid and simple techniques for the enrichment of viruses prior to metagenomic virus discovery the fecal virome of pigs on high-density farm sitespecific binding of wild-type p53 to cellular dna is inhibited by sv40 t antigen and mutant p53 sv40 large t antigen targets multiple cellular pathways to elicit cellular transformation cellular transformation by sv40 large t antigen: interaction with host proteins the molecular chaperone activity of simian virus 40 large t antigen is required to disrupt rb-e2f family complexes by an atp-dependent mechanism rna processing in the polyoma virus life cycle sequences flanking the pentanucleotide t-antigen binding sites in the polyomavirus core origin help determine selectivity of dna replication publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-308932-pp8etmwq authors: baker, m. l.; schountz, t.; wang, l.‐f. title: antiviral immune responses of bats: a review date: 2012-08-01 journal: zoonoses public health doi: 10.1111/j.1863-2378.2012.01528.x sha: doc_id: 308932 cord_uid: pp8etmwq despite being the second most species‐rich and abundant group of mammals, bats are also among the least studied, with a particular paucity of information in the area of bat immunology. although bats have a long history of association with rabies, the emergence and re‐emergence of a number of viruses from bats that impact human and animal health has resulted in a resurgence of interest in bat immunology. understanding how bats coexist with viruses in the absence of disease is essential if we are to begin to develop therapeutics to target viruses in humans and susceptible livestock and companion animals. here, we review the current status of knowledge in the field of bat antiviral immunology including both adaptive and innate mechanisms of immune defence and highlight the need for further investigations in this area. because data in this field are so limited, our discussion is based on both scientific discoveries and theoretical predictions. it is hoped that by provoking original, speculative or even controversial ideas or theories, this review may stimulate further research in this important field. efforts to understand the immune systems of bats have been greatly facilitated in recent years by the availability of partial genome sequences from two species of bats, a megabat, pteropus vampyrus, and a microbat, myotis lucifugus, allowing the rapid identification of immune genes. although bats appear to share most features of the immune system with other mammals, several studies have reported qualitative and quantitative differences in the immune responses of bats. these observations warrant further investigation to determine whether such differences are associated with the asymptomatic nature of viral infections in bats. despite being the second most species-rich and abundant group of mammals, bats are also among the least studied, with a particular paucity of information in the area of bat immunology. although bats have a long history of association with rabies, the emergence and re-emergence of a number of viruses from bats that impact human and animal health has resulted in a resurgence of interest in bat immunology. understanding how bats coexist with viruses in the absence of disease is essential if we are to begin to develop therapeutics to target viruses in humans and susceptible livestock and companion animals. here, we review the current status of knowledge in the field of bat antiviral immunology including both adaptive and innate mechanisms of immune defence and highlight the need for further investigations in this area. because data in this field are so limited, our discussion is based on both scientific discoveries and theoretical predictions. it is hoped that by provoking original, speculative or even controversial ideas or theories, this review may stimulate further research in this important field. efforts to understand the immune systems of bats have been greatly facilitated in recent years by the availability of partial genome sequences from two species of bats, a megabat, pteropus vampyrus, and a microbat, myotis lucifugus, allowing the rapid identification of immune genes. although bats appear to share most features of the immune system with other mammals, several studies have reported qualitative and quantitative differences in the immune responses of bats. these observations warrant further investigation to determine whether such differences are associated with the asymptomatic nature of viral infections in bats. little importance as vectors or reservoirs. much of our knowledge of bats and viruses is from studies of rabies virus and other lyssaviruses (calisher et al., 2006) . however, in recent years, many novel viruses of human and veterinary importance have been discovered that are hosted, or suspected to be hosted, by bats. in the 1990s, novel paramyxoviruses, hendra and nipah viruses, caused outbreaks of fatal disease in australia and malaysia (murray et al., 1995; chua et al., 2000) . both viruses are hosted by species of pteropid bats. severe acute respiratory syndrome, caused by a coronavirus, was identified during an outbreak in china and hong kong in the early 2000s. subsequent research has indicated its ancestor is a batborne virus (lau et al., 2005; li et al., 2005) . also in the 2000s, marburg virus was demonstrated to be hosted by fruit bats, and there is compelling evidence that ebolaviruses are also hosted by fruit bats (leroy et al., 2005; towner et al., 2009 ). in addition, melaka and kampar viruses and related bat reoviruses from malaysia are associated with respiratory disease in humans (chua et al., 2007 (chua et al., , 2008 (chua et al., , 2011 . currently, more than 100 viruses have been isolated from or detected in bats. the diversity of viruses found in bats is matched only by rodents that are the most abundant and diverse group of mammals and are reservoir hosts to a large number of viruses that also cause disease in humans and other species (meerburg et al., 2009) . it is now clear that bats have been substantially underappreciated as reservoirs of viruses important to human and veterinary health (calisher et al., 2006; wang et al., 2011) . the peridomestic nature of some bat species and the encroachment of humans upon bat habitats make it likely that new human pathogens from bats will be discovered after spillover events. bats have often been vilified by much of the public and have been frequently targeted for extermination, despite their critical roles in many ecosystems. because of this, many bat biologists, whose help laboratory scientists will need to answer many questions about bats, are often reluctant to engage or assist in biomedical research. in addition, obtaining bats for biomedical research is extremely challenging. most captive colonies are maintained by zoos, which are unwilling or unable to donate their excess animals for such research because of long-standing policies. the establishment of colonies from wild bats is also problematic because they require specialized housing and diets. moreover, the animals must be evaluated for possible pathogens, including rabies virus. therefore, most research is conducted on wild bats in their natural environments, or captured animals in laboratory settings, which introduces additional problems with housing, diet and occupational health (e.g. animal stress and rabies immunization for personnel). this practice severely limits experimental examination of viruses hosted by bats. although bats may be persistently infected with many viruses, evidence from experimental and naturally infected bats has demonstrated that they rarely display clinical symptoms (sulkin et al., 1966; swanepoel et al., 1996; williamson et al., 1998 williamson et al., , 2000 leroy et al., 2005 leroy et al., , 2009 middleton et al., 2007; towner et al., 2009) . experimental infection of bats has included viruses such as hendra and nipah viruses that are known to result in high disease mortality in other mammals including humans. these studies have confirmed the virulence of the viruses used for experimental infections using conventional laboratory mammals such as guinea pigs that succumb to the same dose of virus infection that bats respond to in the absence of disease (williamson et al., 2000; middleton et al., 2007) . the only viruses that have been demonstrated to cause clinical signs of disease in bats are rabies virus and the closely related australian bat lyssavirus (field et al., 1999; mccoll et al., 2002) . however, results of experimental infections are inconsistent, with only a small proportion of bats succumbing to infection (mccoll et al., 2002) . in addition, very few viruses have been shown to have negative impacts on natural bat populations. one exception is tacaribe virus, an arenavirus closely related to the south american haemorrhagic fever viruses, which caused the deaths of many artibeus bats in trinidad in the 1950s (downs et al., 1963) and in experimental infections (cogswell-hawkinson et al., 2012) . it is unknown whether this virus is still circulating or what impact it may have on artibeus bats today. overall, these results demonstrate that for the most part, bats are able to coexist with viruses and may have evolved mechanisms to control viral replication more effectively than most other mammals. a similar situation exists in rodents that also show limited or no signs of disease in response to the viruses they harbour (fulhorst et al., 1999; botten et al., 2000) . all viruses must evade the immune response for a sufficient period of time to allow transmission to other susceptible hosts, and many viruses possess immunemodulating genes that provide a competitive advantage over the immune response. much of what has been learned about immune responses has been based upon pathology models; however, many zoonotic viruses have coadapted with their vertebrate hosts to cause persistent, apathogenic infections. because bats have not been examined in great detail, and because there are so many species of bats, virtually nothing is known about the role of their immune responses in control of viral infections, nor how viruses manipulate the immune responses of bats. fortunately, much of what is known about other natural zoonotic virus-reservoir relationships, particularly rodent hosts (fulhorst et al., 1999; easterbrook et al., 2007; schountz et al., 2007) , and the availability of novel and increasingly less-expensive deep sequencing methods (glenn, 2011) makes the study of specific bat reservoirs and viruses highly tractable. furthermore, the recent availability of partial genome sequences has provided important resources to study various aspects of bat biology, including genes associated with the immune system. two bat genomes have been sequenced as part of the us national institutes of health funded mammalian genome project, one from the megabat pteropus vampyrus and a second from the microbat myotis lucifugus. although both bat genomes are low coverage (2.6· for p. vampyrus and 1.7· for m. lucifugus), these projects have an important role to play in revealing the mechanisms that have evolved to allow bats to remain asymptomatic when infected by so many viruses. although few bat-specific reagents exist to identify specific cell types in bats, a variety of cells have been described based on morphological and physiochemical characteristics, demonstrating the presence of similar populations of cells in bats to other mammals. macrophages, b cells and t cells have been identified in the spleen and lymph nodes from the indian fruit bat (pteropus giganteus) using scanning electron microscopy and cellular adherence properties. these cells displayed similar characteristics to those from other mammals including humans and mice. in p. giganteus, the ratio of macrophages/b cells/ t cells was 1 : 2 : 9, similar to that of mice in which the ratio was approximately 1 : 1 : 8 (sarkar and chakravarty, 1991) . a variety of immune cells including lymphocytes, neutrophils, eosinophils, basophils and macrophages have also been identified by morphology in histological sections from the brazilain free tailed bat (tadarida brasiliensis) following injection of the t-cell mitogen, phytohaemagglutinin (pha) (turmelle et al., 2010a) . cells resembling follicular dendritic cells (fdcs) have also been described in p. giganteus (sarkar and chakravarty, 1991) . follicular dendritic cells are capable of capturing and retaining antigen in the form of immune complexes that can persist for months or even years and are important for the induction and maintenance of memory immune responses (mandels et al., 1980; . evidence for the ability of some viruses to retain infectivity when complexed within fdcs has been demonstrated (keele et al., 2008) . however, whether fdcs play a role in the persistence of viral infections in bats awaits further investigation. one hypothesis for the ability of bats to remain asymptomatic to viral infection is that they are able to control viral replication very early in the immune response through innate antiviral mechanisms. the recent description of a variety of innate immune genes in bats provides the first step in understanding the role of the innate immune system in antiviral immunity in bats. the recognition of pathogens by pattern recognition receptors (prrs) including toll-like receptors (tlrs) and retinoic acid inducible gene-like helicases (rlhs) provides the first line of defence against infection (xiao, 2009 ). toll-like receptors have been described in two species of fruit bats, pteropus alecto and rousettus leschenaultia (iha et al., 2009; cowled et al., 2011) . not surprisingly given the role of tlrs in the recognition of conserved molecular patterns, the bat tlrs were highly conserved between bats and other mammals cowled et al., 2011) . evidence from p. alecto for the presence of transcripts corresponding to tlrs 1-10 and 13 provides evidence that bats are capable of recognizing a range of pathogens including viruses, bacteria and fungi. however, the p. alecto tlr13 transcript contained stop codons within its open reading frame and may represent a transcribed pseudogene (cowled et al., 2011) . to date, the only other mammals in which tlr13 has been identified are rodents. although the ligand for tlr13 is unknown, knockdown of tlr13 in mouse cells results in greater susceptibility to vesicular stomatitis virus (vsv), indicating it likely has a role in viral recognition (shi et al., 2011) . the transcription of a tlr13 pseudogene in pteropid bats may indicate that this gene has only recently undergone inactivation and at one time may have encoded a functional protein involved in viral sensing and may still be intact and functional in other bat species. the cytoplasmic rlhs, retinoic acid inducible gene i (rig-i), melanoma differentiation-associated protein 5 (mda5) and laboratory of genetics and physiology 2 (lgp2) have also been described in p. alecto, providing evidence for a similar repertoire of rlhs in megabats to other mammals (cowled et al., 2012) . overall, these reports provide evidence for the presence of the two major families of virus sensing prrs in bats consistent with recognition of a similar range of pathogens to other species of mammals. the interferon (ifn) response represents a potent first line of defence against viral infection conferring cells with an 'antiviral state' and preventing the spread of viral infection (randall and goodbourn, 2008) . the ifn signalling and production pathway is therefore a logical starting point in understanding the asymptomatic nature of viral infection in bats. three classes of ifn have been identified, designated types i, ii and iii, which differ in their amino acid sequences and the receptor complex they signal through (pestka et al., 2004; schroder et al., 2004) . type i (including a and b) and iii (k) ifns are induced directly in response to viral infection and thus play an important role in innate immunity. although they differ in the receptor complex they signal through, type i and iii ifns result in the induction of an overlapping set of ifn stimulated genes (isgs) that are responsible for the antiviral activity of ifns (sadler and williams, 2008) . type i ifns have been described in three species of fruit bats, rousettus aegyptiacus, the malaysian flying fox, p. vampyrus, and the greenish naked-backed fruit bat, dobsonia viridis, and from the microbat, m. lucifugus (omatsu et al., 2008; he et al., 2010; kepler et al., 2010) . in humans and mice, there are 13 and 14 ifna genes, respectively, but in bats, only seven ifna genes have been identified in the p. vampyrus genome and only ifna pseudogenes have been identified in the m. lucifugus genome sequence (van pesch et al., 2004; kepler et al., 2010) . however, as both currently available bat genomes are low coverage genome sequences, it is possible that some members of the type i ifn family are absent, despite the sequences being inferred from the unassembled trace archives that should contain a broader representation of the genome than the assembly. he et al. (2010) described the cloning of seven ifna subtypes and one pseudogene from d. viridis with evidence for positive selection among this gene family. both m. lucifugus and p. vampyrus also appear to have expanded the ifnw family of genes, with up to a dozen ifnw members in each species of bat. humans have only a single functional ifnw family member and at least two pseudogenes, and mice have a single ifnw pseudogene (hardy et al., 2004; kepler et al., 2010) however, this family has expanded in cats that have 13 ifnw subtypes and in cattle that have 24 potentially functional ifnw genes (yang et al., 2007; walker and roberts, 2009 ). furthermore, cat (felis catus) ifnw has been implicated in protection against parvovirus infection (paltrinieri et al., 2007) . thus, the expansion of the ifnw family in bats may also have implications for antiviral immunity. type iii ifns have also been identified in the m. lucifugus genome with the identification of a single full-length ifnl locus (fox et al., 2009 ). in the pteropid bat p. alecto, two ifnl genes (il28a and il29) and the two chains of the type iii ifn receptor complex (il10r2 and ifnkr1) have been characterized, and ifnkr1 has been demonstrated to act as a functional receptor (zhou et al., 2011a,b) . furthermore, unlike the type iii ifn receptor of mammals such as mice and humans, in p. alecto, the type iii ifn receptor displays a wide tissue distribution consistent with a more significant role for the type iii ifns in antiviral immunity in bats (sommereyns et al., 2008; witte et al., 2010; zhou et al., 2011a) . pteropid bat cells and cell lines readily secrete ifn in response to stimulation with synthetic tlr ligands including polyinosine-polycytidylic acid (polyic) and lipopolysaccharide (lps), demonstrating that ifn production pathways are functional in bat cells (stewart et al., 1969a; crameri et al., 2009; kepler et al., 2010; zhou et al., 2011b) . bats also demonstrate an ifn response following viral infection in vivo and in vitro. the earliest work on ifn production in bats described the detection of ifn in spleen and brain tissues from microbats (t. brasiliensis) infected with japanese b encephalitis (je) virus. although ifn was detected in both tissues during the first week of infection, it was detected only in brain tissue during the second week of infection despite the presence of virus in both tissues (stewart et al., 1969a,b) . the persistence of the virus in certain populations of cells even in the presence of ifn has been speculated to reflect the presence of populations of cells that may be insensitive to the action of ifn (sulkin and allen, 1974) . in vitro studies have demonstrated the induction of ifnb in vsv-infected peripheral blood mononuclear cells (pbmcs) from p. vampyrus, demonstrating a delay in the ifn response in comparison with stimulation with the tlr ligands, polyic or lps, a result that is consistent with the mechanisms of ifn signalling of other mammals (kepler et al., 2010) . recently, evidence for differences in the ifn responses of bats and the ability of viruses to evade the ifn response of bat cells have also been described. in p. alecto splenocytes, type i and iii ifns appear to be differentially induced following infection with the bat-borne paramyxovirus tioman virus with type i ifns downregulated and type iii ifns upregulated following viral infection (zhou et al., 2011b) . in contrast, henipavirus infection antagonized both type i and iii ifn production in human cell lines and ifn production and signalling in pteropid bat cell lines (virtue et al., 2011a,b; zhou et al., 2011b) . the ability of viruses to antagonize both the ifn signalling and production pathways in bat cells is intriguing and may indicate that factors other than ifn play a key role in antiviral immunity in bats. thus, these results demonstrate not only differences in the ifn response following infection with different viruses but also differences between bats and humans which may be significant in terms of the ability of bats to control viral replication. few studies have examined the ifn signalling pathway following ifn production to determine the ability of ifns to induce an 'antiviral state' in bat cells. ifns exert their antiviral actions through binding to cell surface receptors, which in turn activate the jak-stat pathway. activation of the receptor-associated janus family of tyrosine kinase enzymes results in the phosphorylation of latent cytoplasmic signal transduction and activator of transcription (stat) family of transcription factors. the phosphorylated stat1 and stat2 dimerize to interact with ifn regulatory factor 9 and translocate to the nucleus resulting in isg production and the induction of an antiviral state (samuel, 2001) . the stat1 protein is the only component of this signalling pathway that has been characterized in bats. stat1 is present in the egyptian fruit bat, r. aegyptiacus, and is phosphorylated and translocated to the nucleus following stimulation with human ifna consistent with its activation in a similar manner to other mammals. rabies virus infection of human and bat cells antagonizes stat1 function, resulting in failure of stat1 to be translocated to the nucleus (brzó zka et al., 2006; fujii et al., 2010) . overall, this study demonstrated that the stat1 signalling pathway in r. aegyptiacus cells is similar to that of other mammals. further characterization of other signalling molecules involved in the ifn response will play an important role in understanding the nature of innate antiviral immunity in bats. the ability of ifn to induce an antiviral state through the induction of isgs is the hallmark of the ifn response (sadler and williams, 2008) . stewart et al. (1969a) used ifn containing supernatant prepared from polyic stimulated t. brasiliensis embryo cells to compare the antiviral activity of bat ifn with ifns prepared from cells from other species. this study demonstrated each species of ifn has a characteristic spectrum of antiviral activity. although bat ifn displayed antiviral activity within a similar range to other species, this study did not examine the effect of bat ifn on any bat-borne viruses. more recently, recombinant p. alecto type iii ifn demonstrated antiviral activity against the bat-borne orthoreovirus, pulau virus (zhou et al., 2011b) . the induction of isgs has also been demonstrated in bats with bat type iii ifn, resulting in the induction of isg56 and rig-i production in bat cell lines (zhou et al., 2011a) . pteropid bat cell lines also produce isg54 and isg56 following stimulation with universal type i ifn that is an ifna hybrid constructed from recombinant human ifna a/d (virtue et al., 2011a) . the induction of 2¢,5¢-oligoadenylate-synthetase 2 (oas2) has also been detected in p. vampyrus pbmcs following infection with vsv or stimulation with polyic or lps. the results of this study demonstrated a higher induction of oas2 by vsv compared with either polyic or lps (kepler et al., 2010) . these results provide evidence that the signalling molecules downstream of the ifn response are likely similar in bats to other mammals. the complement cascade kills foreign microbes by disrupting the microbial plasma membrane following activation through the binding of complement to antibodies that have attached to microbial surfaces (prodinger et al., 2003) . a variety of assays have been used to measure complement activity in bats to assess immunity under various environmental conditions. a comparison of complement activity in three microbats (eptesicus fuscus, m. lucifugus and t. brasiliensis) and one megabat (p. vampyrus) demonstrated higher levels of complement activity in the microbats by immune haemolysis but not by immune adherence. furthermore, complement activity in eptesicus microbats was relatively insensitive to changes in temperature above or below 37°c, whereas activities of guinea pig and pteropid bat complements decreased at temperatures above or below 37°c (hatten et al., 1973) . the ability to maintain complement activity may be a biological necessity in hibernating animals such as microbats where body temperatures can vary extensively and over prolonged periods of time. allen et al. (2009) used complement activity as a measure of bactericidal activity in t. brasiliensis bats, demonstrating variation in activity with roosting ecology, providing evidence for the influence of environmental factors on immune function. studies of bat adaptive immunity have provided evidence for the presence of both antibody and cell-mediated immunity in bats. however, several reports have demonstrated qualitative and quantitative differences in adaptive immune responses and in the generation and maintenance of immunological memory. these findings warrant further investigation to determine the relevance of these findings to the maintenance of viruses in bats. butler et al. (2011) demonstrated that four species of bats, including one megabat (cynopterus sphinx) and three microbats (carollia perspicillata, m. lucifugus and e. fuscus) transcribe igm, ige, iga and multiple igg classes, the latter of which appears to have diversified after speciation as in other mammals. serum fractionation using normal serum from the neotropical species, artibeus lituratus and from p. giganteus has confirmed that bat igm, igg and iga are homologous to corresponding human immunoglobulins (mcmurray et al., 1982; chakravarty and sarkar, 1994) . however, evidence so far indicates that igd may be unique to microbats, with igd present at the genomic and transcriptional level in m. lucifugus but not in the megabats. igd is an apparently ancient isotype and has a spotty distribution among vertebrates (ohta and flajnik, 2006) . among mammals, igd was once believed to be present only in humans and rodents until its more recent identification in various classes of animals including artiodactyls and monotremes (zhao et al., 2002 (zhao et al., , 2009 . although a broader survey of megabats will be required to rule out the presence of igd in this group, given that igd is not present in all mammals, it may not be surprising to find that megabats have lost this immunoglobulin isotype. immunoglobulin genes are assembled by recombination of germline-encoded gene segments: variable (v), diversity (d) and joining (j) for heavy (h) chains and v and j for light (l) chains. variation in the amino acid residues at the n terminal ends that are encoded by the v regions of both h and l chains contribute to antibody diversity and establishes antibody specificity (max, 2003) . to obtain insight into the antigen-binding capability and specificity of bat antibodies, several studies have examined the diversity of the vh regions of bat immunoglobulin genes. evidence from both megabats and microbats has demonstrated a highly diverse antibody repertoire, exceeding that of most of species and on par only with humans and mice (baker et al., 2010; bratsch et al., 2011) . in the pteropid bat, p. alecto, the amino acid sequence composition of the antigen-binding site of the expressed vh region is enriched in arginine and alanine residues and has a lower proportion of tyrosines compared to other mammals (baker et al., 2010) . tyrosines are directly involved in antigen binding and confer structural diversity, while arginines have been reported to be detrimental to antigen binding and may contribute to self-reactivity (radic et al., 1993; birtalan et al., 2008) . whether these characteristics are associated with differences in antigen-antibody interactions in bats awaits further functional characterization. however, differences in antigen binding may help to explain previous observations of the simultaneous presence of virus and antibody in bats (sulkin et al., 1966) . comparison of the germline and expressed vh repertoire of m. lucifugus has revealed a very low mutation rate consistent with the possibility that this species relies on combinatorial and junctional diversity rather than somatic hypermutation . all other mammals studied thus far use post-combinatorial mechanisms to fine tune their antibody repertoire resulting in antibodies that recognize fewer epitopes per antigen but do so with greater specificity and affinity. this result may provide evidence that bats rely solely on combinatorial mechanisms. however, as this study focused only on vh sequences expressed with igg in a single m. lucifugus, further work is required to confirm this result across multiple individuals and species using all of the immunoglobulin subclasses. the effector functions mediated by antibodies include neutralization, precipitation, agglutination, opsonization, antibody-dependent cellular cytotoxicity and the activation of the classical complement pathway. neutralizing antibodies to viruses including hendra virus, ebolaviruses and sars-like cov have been detected in wild-caught bats, demonstrating that bats are capable of mounting an antibody response (halpin et al., 2000; lau et al., 2005; leroy et al., 2005) . some of the earliest experiments performed on bat immune systems were measuring antibody responses. early studies of antibody responses in bats were consistent with differences in both the kinetics and magnitude of antibody responses compared with other mammals. several studies have used model antigens such as sheep red blood cells (srbcs), /x174 bacteriphage and 2,4-dinitrophenylated bovine serum albumin (dnp-bsa) to compare the nature of the antibody response of bats with that of conventional laboratory animals (hatten et al., 1968 (hatten et al., , 1970 chakraborty and chakravarty, 1984; wellehan et al., 2009) . hatten et al. (1968) reported that the magnitude and duration of the neutralizing antibody response of big brown bats (eptesicus fuscus fuscus) maintained at 24 and 37°c to immunization with /x174 bacteriophage was lower than that of guinea pigs and rabbits. a delay in attaining a peak in the primary antibody response was also reported in pteropid bats immunized with srbcs (chakraborty and chakravarty, 1984) . secondary responses also appeared to be slower or non-existent. a more pronounced igm response was observed in e. fuscus, and the appearance of igg appeared to be slower supporting poor isotype switching (hatten et al., 1968) . secondary immunization with / x174 bacteriophage has demonstrated an anamnestic response only in bats housed at 24°c but not at 37°c (hatten et al., 1968 (hatten et al., , 1970 . however, evidence for an increase in the affinity of antibodies for /x174 has been reported in e. fuscus (hatten et al., 1970) . clearly, further work is needed to understand the nature of antibody responses in bats. however, overall these studies demonstrate differences in both primary and secondary antibody responses in bats compared to conventional laboratory mammals. experimental infections and vaccinations have also been performed in bats to provide information on the kinetics and nature of antibody responses to viruses. consistent with the results obtained from bats immunized with /x174 or srbc antigens, vaccination and experimental viral infections have provided evidence for quantitative and qualitative differences in antibody responses in bats compared with other mammals. in addition, results from experimental infections appear to vary between species and viral infections. big brown bats (e. fuscus) experimentally infected with je virus generally demonstrate a neutralizing antibody response within 20 days of infection. however, these studies have failed to detect evidence of complement fixation (cf) or haemagglutination (hi) by je virus antigen (sulkin et al., 1966; leonard et al., 1968) . as cf and hi responses were demonstrated in guinea pigs and rabbits during these experiments, the failure to detect a response in bats was considered to reflect a difference in the host antibody response rather than the assay. experimental infection of neotropical bats with venezuelan equine encephalitis virus resulted in a high hi and neutralizing antibody response in artibeus fruit bats but low or undetectable response in phyllostomus discolour (seymour et al., 1978) . furthermore, bats exposed to prolonged periods of cold (8°c) likely to be encountered during hibernation failed to develop an antibody response to je virus despite the persistence of the virus in various tissues but developed detectible antibody within 1 week following transfer from 8 to 24°c (sulkin et al., 1966) . these results are consistent with the ability of bats to maintain viruses, which are likely biochemically inert for long periods of time under states of immunosuppression. the ability of antibody to provide long-lasting protection is one of the hallmarks of the adaptive immune response. vaccination of bats against rabies virus appears to confer resistance to challenge compared to unvaccinated bats that succumb to disease. however, several studies have demonstrated that vaccinated bats are capable of clearing viral infection even in the absence of detectible neutralizing antibody (seymour et al., 1978; sétien et al., 1998; aguilar-setien et al., 2002; turmelle et al., 2010b) . although these studies provide evidence that bats develop protective immunity following vaccination, the failure to detect an antibody response in some bats is striking and may indicate that the nature of protective immunity in bats differs from other mammals. evidence for viral recrudescence has also been reported in a captive p. vampyrus, which displayed changes in neutralizing antibody to nipah virus, providing evidence of the maintenance of virus in bats in a manner that does not sustain an antibody response. one individual was initially seropositive, became seronegative within 1-2 months and remained seronegative for 11 months before displaying a gradual increase in neutralizing antibody and viral excretion (sohayati et al., 2011) . these results demonstrate that failure to detect specific antibodies may be insufficient evidence for excluding prior exposure. however, as this event was observed in only one individual, the significance of viral recrudescence in bat populations remains to be investigated and will require long-term studies of captive individuals of known history of viral exposure. differences in the numbers of cells expressing surface immunoglobulin (sig) have also been observed in p. giganteus with a higher number of sig-positive cells in peripheral blood (82%) compared to humans and mice (15-30%) (chakravarty and sarkar, 1994) . as no batspecific reagents existed to further characterize the nature of this population of cells, the significance of this result remains unknown. further studies to characterize the nature of b cells in bats may assist in resolving whether differences in the numbers of b cells are a general characteristic of bats and whether this plays a role in the observed differences in antibody responses of bats. cell-mediated responses are controlled by t lymphocytes, which include cytotoxic and helper functions. the different populations of t cells in bats have not been characterized to date, and only one t-cell coreceptor, cd4, has been characterized (omatsu et al., 2006) . however, a number of reports have described in vitro responses of lymphocytes to t-cell mitogens in pteropid bats and microbats (mcmurray and thomas, 1979; chakravarty and paul, 1987; paul and chakravarty, 1987) . these studies have indicated that bats display evidence for delayed responses to t-cell mitogens, pha and concanavalin a (cona) with a peak at 120 h compared to 48 h in mice (mcmurray and thomas, 1979; chakravarty, 1986, 1987) . a delay in mixed lymphocyte responses (mlr) have also been observed with a peak at 7 days for p. giganteus in comparison with 5 days in mice, thus providing further evidence that cell-mediated immunity in bats is slower than that of other mammals (chakraborty and chakravarty, 1983) . the presence of suppresser t cells has also been implicated in the delay in mitogenic responses of b cells in bats . whether these cells are involved in the delay in t-cell-mediated immune responses observed in bats remains to be determined. more recently, an ifnc response was demonstrated following stimulation of pteropid bat lymphocytes with the t-cell mitogens pha and cona, demonstrating that bats are capable of a similar ifnc response to other mammals (janardhana et al., 2012) . in vivo cell-mediated responses in bats have been measured using delayed-type hypersensitivity (dth) tests using the pha skin test or skin sensitivity to 2-4 dinitrofluorobenzene (dnfb) (christe et al., 2000; allen et al., 2009; turmelle et al., 2010a) . delayed-type hypersensitivity of p. giganteus to dnfb resulted in a characteristic dth response within 48 h, similar to other mammals. however, only three of twelve bats tested in this study responded to treatment with dnfb, suggesting that bats may not be sensitive to dnfb to the same extent as other mammals (chakraborty and chakravarty, 1983) . a time series of histological skin sections taken from skin biopsies of t. brasiliensis following pha or saline injection has also demonstrated substantial individual variation but overall has provided evidence for a strong cell-mediated immune response (turmelle et al., 2010a) . cell-mediated immunity has also provided evidence for changes in immunocompetence because of environmental and physiological factors. differences in immunocompetence because of roost ecology were observed in t. brasiliensis using subcutaneous pha injection as a measure of in vivo t-cell-mediated immunity, providing evidence for the effect of environment on immune responsiveness (allen et al., 2009) . christe et al. (2000) used pha skin tests to demonstrate that greater mouse-eared bats, myotis myotis, mount weaker cell-mediated responses during pregnancy compared with that of non-reproductive and lactating females. immunocompetence was also lower in early pregnancy than at later stages of gestation (christe et al., 2000) . this result is consistent with changes in immunocompetence reported in other mammals that undergo a shift in the immune response towards a humoral immune response and away from cell-mediated immunity during pregnancy (szekeres-bartho, 2002) . changes in immune function during pregnancy have been speculated to favour replication of viruses including zaire ebolavirus in bats. high titres of virus present in birthing fluids, blood and placental tissues may then be a source of infection to terrestrial mammals including apes (leroy et al., 2005) . further studies into the antiviral immune response during pregnancy may provide insights into whether changes in immune function influence viral infections in bats and/or correlate with spillover events from bats to other susceptible species. although cell-mediated responses both in vivo and in vitro have provided important information on the t-cellmediated responses of bats, no reagents currently exist to identify different populations of t cells in bats. the ability to identify and sort different populations of t cells would provide valuable insight into the role of t cells in antiviral immunity in bats. the mhc plays an important role in resistance to infectious diseases, autoimmunity, transplantation and reproductive success (kumánovics et al., 2003) . despite the importance of the mhc, no work has been reported on the mhc class i genes of bats and only a few studies have provided information on mhc class ii polymorphism in bats (mayer and brunner, 2007; richman et al., 2010; schad et al., 2011) . the earliest evidence for the degree of mhc polymorphism in bats came from mlr assays. mixed lymphocyte responses test the recognition and proliferation of t cells from different individuals, and this response is highly dependent on mhc class ii polymorphism (derks and burlingham, 2005) . pteropus giganteus lymphocytes undergo delayed and lower levels of proliferation in mlr tests compared to their responses to mitogens such as cona (chakraborty and chakravarty, 1983; chakravarty and paul, 1987) . as the proliferation of cells in mlrs correlates with the degree of genetic difference in mhc loci between individuals, delayed and weaker mlr responses in bats may be evidence for low mhc polymorphism. only recently has genetic evidence for the degree of mhc class ii polymorphism in bats been reported. the class ii dr beta (drb) locus is the most extensively studied of the mhc loci in mammals because of its high diversity and has been the focus of all of the mhc class ii analyses performed on bats to date (mayer and brunner, 2007; richman et al., 2010; schad et al., 2011) . richman et al. (2010) demonstrated extreme differences in polymorphism between bat species with extensive polymorphism at the mhc class ii drb locus in myotis velifer compared to the extremely limited polymorphism in myotis vivesi. m. velifer is a geographically widespread continental species compared to m. vivesi that is a narrowly distributed and endangered island endemic species. the lower population size of m. vivesi may have relaxed selection for the maintenance of many alternative alleles in the population, thus lowering mhc polymorphism. a single drb locus has been described in the bulldog bat, noctilio albiventris displaying moderate allelic variability within the range of other mammals. in addition, males displayed a significantly higher heterozygosity rate and genetic variability compared to female bats (schad et al., 2011) . the single drb locus of the sac-winged bat, saccopteryx bilineata, displayed low heterozygosity and evidence for diversifying selection. substantial nucleotide sequence variation between the drb alleles of s. bilineata was consistent with a history of balancing selection, but there was no evidence for ongoing balancing selection acting to maintain alternative alleles at intermediate frequency. in addition, unexpected homozygosity for a common allele was observed in this population of s. bilineata, consistent with pathogen-driven positive selection playing a role in the evolution of mhc genes in this species (mayer and brunner, 2007) . drb intron sequences from three species of bats (r. aegyptiacus, c. perspicillata and phyllostomus discolour) have also been used to infer phylogenetic relationships and demonstrate the monophyly of chiroptera (kupfermann et al., 1999) . overall, studies of drb polymorphism in bats provide evidence for the influence of factors such as population size and pathogen pressure on the diversification of class ii genes. the degree of variation in drb polymorphism described above may be consistent with wide variation in the mhc variability in bats that may in turn influence the ability of different populations of bats to respond to infections. a number of bat cytokine genes have now been characterized including cdnas corresponding to interleukin (il)-2, il-4, il-6, il-10, il-12p40 and tumour necrosis factor (tnf) from rousettus leschenaultii . partial cdnas for il-10, il-23a, tnf and granulocyte macrophage colony-stimulating factor have been cloned from seba's fruit bat (c. perspicillata) (cogswell-hawkinson et al., 2011) . these cytokines appear to be highly conserved with those from other mammals. kepler et al. (2010) described the in silico identification of ifnc from p. vampyrus and m. lucifugus, confirming that both bats appear to have a single ifnc locus similar to other mammals. more recently, p. alecto ifnc has been described, including the characterization of its antiviral activity against semliki forest virus and hendra virus. this study included the generation of important bat-specific reagents for the detection of ifnc, an important step for future studies of the role of ifnc and t-cell-mediated immunity during viral infections in bats (janardhana et al., 2012) . although only limited work has been performed on bat cytokines, these studies pave the way for examining the role of cytokines in antiviral immunity in bats. as discussed above, functional and genome sequence analyses of bats have revealed some surprises, but overall, it appears that bats share many of the immunological features of other mammals. it is evident they have similar antibody and t-cell receptor genes, cytokines and chemokines, transcription factors, cluster of differentiation (cd) markers and activation pathways found in the immune responses of other mammalian species. bats have molecules involved in cell self-defence against viruses, innate response mechanisms and adaptive responses. defining these molecules will be relatively easy; understanding how the viruses and bat reservoirs have shaped one another, and the tempo and mode of immune responses will be more challenging. such functional studies will likely result in significant insights into host-virus relationships, the implications of which will have impacts on the development of novel therapeutics for other species and the ability to predict viral spillover events. the variability in the results obtained from studies involving wild-caught bats emphasizes the need for captive colonies of bats of known age and history of infections for focused studies of bat immunology. although challenging, information from such colonies would greatly assist in the interpretation of data obtained from wild-caught individuals. the development of cell lines also plays an important role in this regard. although cell lines have now been generated from a number of tissues from the pteropid bat, p. alecto (crameri et al., 2009) , the development of additional cell lines from immune relevant cells will also assist in developing assays for studying various aspects of bat immune function. future studies using expression tools, such as real-time pcr arrays, can be rapidly and inexpensively deployed to study particular species of bats and their viruses, and they can be highly informative regarding the genetic responses of bats during infection. however, transcription data are limited because many genes are post-transcriptionally regulated. furthermore, post-translational modification events such as phosphorylation cannot be assessed by transcriptional analysis. thus, it will be necessary to determine which antibodies currently available are crossreactive with bat cells and to generate antibodies for those proteins that appear important based upon transcriptional analysis. because most intracellular signalling proteins are often highly conserved between mammalian species, it is likely that some antibodies specific for those proteins in humans or mice will be useful for bat studies. however, most cytokines and cd molecules are often quite divergent between species and will likely require development of new reagents. vaccination of vampire bats using recombinant vaccinia-rabies virus roosting ecology and variation in adaptive and innate immune system function in the brazilian free-tailed bat (tadarida brasiliensis) immunoglobulin heavy chain diversity in pteropid bats: evidence for a diverse and highly specific antigen binding repertoire the intrinsic contributions of tyrosine, antiviral immune responses of bats m serine, glycine and arginine to the affinity and specificity of antibodies experimental infection model for sin 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bats (pteropus poliocephalus) il-28a, il-28b, and il-29: promising cytokines with type i interferon-like properties innate immune recognition of nucleic acids cloning and characterization of a novel feline ifn-omega artiodactyl igd: the missing link ornithorhynchus anatinus (platypus) links the evolution of immunoglobulin genes in eutherian mammals and nonmammalian tetrapods type iii ifn receptor expression and functional characterisation in the pteropid bat, pteropus alecto type iii ifns in pteropid bats: differential expression patterns provide evidence for distinct roles in antiviral immunity key: cord-293946-4bquxdqa authors: huong, nguyen quynh; nga, nguyen thi thanh; long, nguyen van; luu, bach duc; latinne, alice; pruvot, mathieu; phuong, nguyen thanh; quang, le tin vinh; hung, vo van; lan, nguyen thi; hoa, nguyen thi; minh, phan quang; diep, nguyen thi; tung, nguyen; ky, van dang; roberton, scott i.; thuy, hoang bich; long, nguyen van; gilbert, martin; wicker, leanne; mazet, jonna a. k.; johnson, christine kreuder; goldstein, tracey; tremeau-bravard, alex; ontiveros, victoria; joly, damien o.; walzer, chris; fine, amanda e.; olson, sarah h. title: coronavirus testing indicates transmission risk increases along wildlife supply chains for human consumption in viet nam, 2013-2014 date: 2020-08-10 journal: plos one doi: 10.1371/journal.pone.0237129 sha: doc_id: 293946 cord_uid: 4bquxdqa outbreaks of emerging coronaviruses in the past two decades and the current pandemic of a novel coronavirus (sars-cov-2) that emerged in china highlight the importance of this viral family as a zoonotic public health threat. to gain a better understanding of coronavirus presence and diversity in wildlife at wildlife-human interfaces in three southern provinces in viet nam 2013–2014, we used consensus polymerase chain reactions to detect coronavirus sequences. in comparison to previous studies, we observed high proportions of positive samples among field rats (34.0%, 239/702) destined for human consumption and insectivorous bats in guano farms (74.8%, 234/313) adjacent to human dwellings. most notably among field rats, the odds of coronavirus rna detection significantly increased along the supply chain from field rats sold by traders (reference group; 20.7% positivity, 39/188) by a factor of 2.2 for field rats sold in large markets (32.0%, 116/363) and 10.0 for field rats sold and served in restaurants (55.6%, 84/151). coronaviruses were also detected in rodents on the majority of wildlife farms sampled (60.7%, 17/28). these coronaviruses were found in the malayan porcupines (6.0%, 20/331) and bamboo rats (6.3%, 6/96) that are raised on wildlife farms for human consumption as food. we identified six known coronaviruses in bats and rodents, clustered in three coronaviridae genera, including the alpha-, beta-, and gammacoronaviruses. our analysis also suggested either mixing of animal excreta in the environment or interspecies transmission of coronaviruses, as both bat and avian coronaviruses were detected in rodent feces on wildlife farms. the mixing of multiple coronaviruses, and their apparent amplification along the wildlife supply chain into restaurants, suggests maximal risk for end consumers and likely underpins the mechanisms of zoonotic spillover to people. human-wildlife contact with a bat or an intermediate host species in china likely triggered a coronavirus spillover event that may have involved wildlife markets and led to the pandemic spread of sars-cov-2 [1, 2] . the pandemic risk of commercial trade in live wildlife was first recognized during the 2002-2003 severe acute respiratory syndrome (sars) outbreak due to sars-cov [3] . this virus spread to countries in asia, europe, and the americas with 8,096 people infected and 774 deaths, costing the global economy about $us 40 billion in response and control measures [4, 5] . unfortunately, the impact of covid-19, the disease caused by sars-cov-2, has reached nearly every country and greatly surpassed those numbers by many orders of magnitude [6] . while bats are thought to be the ancestral hosts for all groups of coronaviruses [7] , for both sars-cov and sars-cov-2 wildlife trade supply chains are suspected to have contributed the additional conditions necessary for the emergence, spillover, and amplification of these viruses in humans [8, 9] . in viet nam, between 2013 to 2014, we conducted coronavirus surveillance to understand the presence and diversity of coronaviruses in wildlife at sites identified as high-risk interfaces for viral spillover from wildlife to humans [10] . we sampled at three sub-interfaces along the live field rat trade (rattus sp. and bandicota sp.) including field rats sold by rat traders, by vendors in large markets, and rats butchered and sold in restaurants as prepared dishes. we also sampled rodents raised on wildlife farms to assess risk from different wildlife supply chains destined for human consumption. we sampled bat guano, primarily on bat guano farms to assess the potential occupational risk of this practice given that bat guano farm artificial roost structures are often erected near human dwellings. in the early 2000s, the vietnamese field rat trade was estimated to process 3,300-3,600 tons of live rats annually for consumption, a market valued at us$2 million [11] . although rats are still commonly traded in wet markets and sold live for food consumption along the mekong delta in southern viet nam, no recent published data on the scale and scope of the trade is available [12] . this human-wildlife interface involves the capture of wild free-ranging field rats, subsequent trade, and consumption along a supply chain involving the entire mekong delta region, particularly cambodia and viet nam [13] . driving this trade are consumers in viet nam and cambodia, some of whom report eating rats at least once per week because of their good flavor, low cost, and perception of rats as 'healthy, nutritious, natural, or disease free' [13] . rat parts (heads, tails, and internal organs discarded at slaughter) are also often fed to domestic livestock or herptiles raised in captivity including frogs, snakes, and crocodiles [12] . over the past three decades, commercial wildlife farming has developed in many countries in southeast asia, including viet nam. although there are historic references to the occurrence of wildlife farms in viet nam dating back to the late 1800s, the rapid expansion in terms of farm numbers, species diversity, and scale of operations has occurred in recent decades in response to growing domestic and international demand for wildlife [14] . a 2014 survey across 12 provinces in southern viet nam identified 6,006 registered wildlife farms of which 4,099 had active operations. the surveyed farms were stocked with approximately one million wild animals including, rodents, primates, civets, wild boar, oriental rat-snakes, deer, crocodiles, and softshell turtles. ninety-five percent of the farms held 1-2 species of wildlife, and 70% of the farms also raised domestic animals on the same premises [15] . a key component of the wildlife farm industry in viet nam is the raising of wild species for meat for human consumption [15] . these farms sell to urban wild meat restaurants serving increasingly affluent populations throughout the country and also supply international markets with wild meat [16] . commercial wildlife farming in viet nam is part of the expanded international trade of wildlife that has been hypothesized to contribute to the cause of global epidemics, such as sars [17] and now covid-19. emerging evidence suggests zoonotic virus spillover risk is a concern at bat-human interfaces in asia. guano harvested from a cave in thailand were positive for a group c betacoronavirus, which includes mers-cov, and 2.7% of 218 people living in close proximity to bats known to carry viruses related to sars-cov tested positive for sars-related antibodies in china [18, 19] . the traditional practice of guano farming in parts of cambodia and viet nam involves the construction of artificial bat roosts in gardens or backyard farms, under which domestic animals and crops are raised, and children often play [20, 21] . cambodian development programs promoted the practice in 2004 to enhance soil fertility, reduce reliance on chemical fertilizers, generate income ($us 0.50/kg), control insect pests, and protect the lesser asiatic yellow bats (scotophilus kuhlii) that were being hunted [20] [21] [22] . no personal protection measures are taken when harvesting the guano, which is reported to improve the growth rate in five economically important plant species [23] . in this study we investigated the presence and diversity of coronavirus sequences in the field rat trade distribution chain, wildlife farms specializing in raising rodents for human consumption, and bat guano "farms" and roosts near human dwellings to better understand the natural hosts of coronaviruses and the risk for these interfaces to facilitate spillover into humans. sampling was performed at multiple sites representing several high-risk interfaces for contacts among people, rodents, and bats. rodent sampling focused on the live field rat trade supply chain and wildlife farms specializing in raising rodents [malayan porcupines (hystrix brachyura) and bamboo rats (rhizomys sp.)] for meat. along the field rat supply chain, we targeted eight sites involved in the private sale and butchering of rats for consumption, defined as 'traders' for the purpose of this study in dong thap and soc trang provinces, 14 large market sites where rats were butchered and sold in dong thap and soc trang provinces (>20 vendors), and two restaurant sites in soc trang province where live rats were kept on the premises and butchered and served as food (fig 1) . the 28 rodent farm sites targeted in dong nai province produced malayan porcupines and bamboo rats for human consumption (fig 2) . other species observed or raised at the wildlife farm sites included dogs, cattle, pigs, chickens, ducks, pigeons, geese, common pheasant, monitor lizards, wild boar, fish, python, crocodiles, deer, civets, non-human primates as pets or part of private collections, free-flying wild birds, and free-ranging peri-domestic rats. bat sampling occurred at bat guano "farms" and a natural bat roost located at a religious site. bat guano farms consisted of artificial roosts constructed with a concrete base and pillars topped with fronds of coconut palm or asian palmyra palm (borassus flabellifer) (fig 3) . seventeen bat guano farms were sampled in the two provinces of dong thap and soc trang. the natural bat roost was located at a religious site in soc trang province known as the "bat pagoda", where pteropus sp. have historically roosted in trees protected from hunting, and light and noise pollution [24] . all study sampling occurred from january 2013 to march 2014 at 41 sites in the wet (south viet nam: may 1 st -november 30 th ) and 30 in the dry (south viet nam: december 1 st -april 30 th ) seasons. given the distances between sites, 69 sites were sampled once except the bat pagoda natural roost in soc trang province, which was visited three times and sampled in both seasons. samples from animals were humanely collected using standard and previously published protocols and no animals were purchased for this study (s1 table) [25]. samples from rats at all three sub-interfaces in the field rat trade were collected from individual carcasses after the rats were slaughtered by a trader, market vendor, or restaurant kitchen staff as part of the rat meat preparation process during normal sales to customers. oral swabs were collected from the severed heads of all the rats, with at least one additional tissue sample collected from the discarded internal organs of each individual. the small intestine was the additional tissue most frequently collected with brain, kidney, lung, rectal swab, and urine also collected from some individuals. rats were usually butchered at a common site for each observed time period that was only cleaned intermittently following the trader's, vendor's, or restaurant's regular practices. feces, urine, and swabs of the pen floors (environmental samples), were collected non-invasively (without handling the animals) from rodents on wildlife farms. samples were classified as 'fecal sample' or 'urine sample' when voided feces or urine was collected from an animal housed individually in its own cage, and as 'environmental sample' when collected as a swab from cages housing multiple individuals. fecal samples and a small number of urine samples excreted by bats in guano farms and the natural roost site were collected on clean plastic cover sheets within 1-2 hours after placement under bat roosts, and thus each sample may represent one or multiple bats. oral and rectal swabs were also collected from live-captured bats during one sampling visit at the natural pagoda roost site. all animals were identified in the field to the lowest taxonomic level possible based on morphological characteristics, and species was identified in a subset of animals through genetic barcoding [15] . due to difficulty of morphologic identification in the field, unless barcoded, rodents (rattus argentiventer, r. tanezumi, r. norvegicus, r. exulans, r. losea, and bandicota indica; [12, 26] ) were categorized as "field rats". bats were classified as "microchiroptera" following the traditional taxonomic classification (new classification of two new suborders yangochiroptera and yinpterochiroptera, was only published near the end of the study, so for consistency we used the historical classification [27] ). all samples were collected in cryotubes containing rnalater (rna stabilization reagent, qiagen), and stored in liquid nitrogen in the field before being transported to the laboratory for storage at -80˚c. samples were tested by the regional animal health office no. 6 (raho6) laboratory in ho chi minh city. the study and sampling activities for specified dates and locations were approved by the department of animal health of the ministry of agriculture and rural development and animal sampling protocols were reviewed by the institutional animal care and use committee at the university of california at davis (protocol number 16048). rna was extracted (rna miniprep kit, sigma-aldrich) and cdna transcribed (superscript iii first strand cdna synthesis system, invitrogen). coronavirus rna was detected using two broadly reactive consensus nested-pcr assays targeting the rna dependent rna polymerase (rdrp) gene [28, 29] . the positive control was a synthetic plasmid containing the primerbinding sites for both assays. distilled water was used as a negative control and included in each test batch. pcr products were visualized using 1.5% agarose gels, and bands of the correct size were excised, cloned, and sequenced by sanger dideoxy sequencing using the same primers as for amplification. for sequence analysis and classification operating taxonomic units were defined with a cut off of 90% identity, i.e. virus sequences that shared less than 90% identity to a known sequence were labelled sequentially as predict_cov-1, -2, -3, etc. and groups sharing � 90% identity to a sequence already in genbank were given the same name as the matching sequence [7] . a phylogenetic tree was constructed for sequences amplified using the watanabe protocol, as this pcr protocol yielded longer sequences and more positive results than the quan protocol. several representative sequences for each viral species found in our study were included for analysis and are available in genbank (s3 table) . alignments were performed using muscle, and trees were constructed using maximum likelihood and the tamura 3-parameter model in mega7 [30] . the best-fit model of dna substitution was selected in mega7 using bic scores (bayesian information criterion) and maximum likelihood values (lnl). bootstrap values were calculated after 1,000 replicates. in addition, a median-joining network was constructed using network 5.0.0.3 [31] to explore phylogenetic relationships among bat coronavirus 512/2005 sequences at the intraspecies level, as haplotype networks may better represent the relationships among viral sequences with low sequence diversity compared with phylogenetic trees [32] . visualization of sampling locations in provinces in viet nam, along with the distribution by species and interface was constructed with the ggplot2 and sf packages [33] . the source of the viet nam provincial map is geoboundaries v. 3.0.0 (https://www.geoboundaries.org; [34] ) and open development mekong (https://vietnam.opendevelopmentmekong.net). all analyses were done using r version 3.5.0 or higher (r development core team, vienna, austria). data (s1 data) and code (s1 r code) are available in the supplementary materials. the effect of risk factors (season, sub-interface type) was examined and limited to interfaces for which the distribution of samples across factors could support the analysis. these included season for pteropus bat samples collected in the bat pagoda natural roost and the effect of season and sub-interface for samples collected in the rodent trade in southern viet nam. given the low sample size, the effect of season for pteropus bats samples positive for coronaviruses was assessed using a fisher exact test. the effect of season (dry, wet, with dry season as reference category) and sub-interface type (trader, large markets, restaurants, with trader as reference category) in traded rodent samples positive for coronaviruses was assessed with a mixed effect multivariable logistic regression, with sites as random effect (i.e. grouping variable) using the lme4 r package [35] . a p-value of less than 0.05 was considered statistically significant. the 95% binomial confidence intervals for proportions were calculated using binom.test in r. the comparison of the proportion of coronavirus positives in different sample types was performed on positive individuals sampled in the live field rat trade with multiple sample types collected per individual. we then calculated the proportion of individuals positive for each sample type, as a proxy for the probability of detection by each sample type. a total of 2,164 samples collected between january 2013 and march 2014 from rodents and bats were tested for coronaviruses (table 1, s1 table) . assuming that non-invasive samples from bats and farmed rodents represented unique distinct individuals, these samples came from 1,506 individuals, including 1,131 rodents (702 field rats and 429 wildlife farm rodents) and 375 bats from 70 sites sampled in dong thap, soc trang, and dong nai provinces in the southern region near the mekong river delta (fig 4) . out of 70 sites, coronavirus positives were detected at 58 including 100% (24/24) of live rat trade sites, 60.7% (17/28) of rodent wildlife farm sites, 94.1% (16/17) of bat guano farm sites, and at the one natural pteropid bat roost. wildlife farms were only sampled in dong nai province and the live rat trade and bat interfaces were sampled in dong thap and soc trang provinces (fig 4) . coronaviruses were detected in the field rat trade (a mix of rattus and bandicota genera) at all sites in dong thap (n = 16) and soc trang (n = 8) provinces, with 34.6% (95% ci 29. table) . it should be noted, however, that since sites were only visited during one season, both independent variables were defined at the site level and confounding effects with other site-level characteristics cannot be excluded. among the positive field rats with more than one sample tested (n = 220), the proportion positive by sample type was 79.9% (95% ci 73.9-84.9%, 175/219) in oral swabs, 52.9% (95% ci a field rat here refers to a mix of rattus sp. and bandicota sp. b this environmental sample collected from a porcupine cage on a porcupine farm was barcoded as rattus sp., suggesting this species was free-ranging at the site (fig 2) . 50 .0% in feces (1/2), 100% in spleen (1/1), and 0% in urine/urogenital swabs (0/1). at the rodent wildlife farm interface, 6.0% (95% ci 3.8-9.3%, 20/331) of hystrix brachyura and 6.3% (95% ci 2.6-13.6%, 6/96) of rhizomys sp. were positive. the overall proportion of positives was 6.3% (95% ci 4.3-9.1%, 27/429) ( table 1 and fig 4) . there was no difference among species or season and proportion positive in rodent farms, and low sample size and unequal sampling limited analysis. the proportion of coronavirus positives at the two bat interfaces differed by an order of magnitude as 74.8% (95% ci 69.5-79.4%) of the non-invasive samples collected from microchiroptera bats at bat guano farms were positive, and 6.7% (95% ci 2.2-17.0%) of the pteropus predict_cov-17 and predict_cov-35 were first reported by anthony et al. [17] . we found predict_cov-17 in pteropus bats and in microchiroptera ( table 1 ). the predict_ cov-17 sequences from pteropus detected in this study clustered closely with predict_ cov-17 sequences from pteropus giganteus bats in nepal and pteropus lylei bats in thailand [36] (fig 6, s3 table) . predict_cov-35 was found in microchiroptera in bat guano farms the analysis included 17 sequences from this study (red from bat hosts, blue from rodent hosts), six sequences (in gray) from a previous study in viet nam [26] , and 26 reference sequences (in black) available in the genbank database (s3 table) . the tree was rooted by a strain of night-heron coronavirus hku19 (genbank accession no. nc_016994). https://doi.org/10.1371/journal.pone.0237129.g006 and in a pteropid bat (table 1) . predict_cov-35 sequences from viet nam clustered with other predict_cov-35 sequences found previously in samples from hunted scotophilus kuhlii bats in cambodia (s3 table; dr. lucy keatts personal communication), and with sequences found in bats from an earlier study in the mekong delta region in viet nam (fig 6) . bat coronavirus 512/2005 was detected in microchiroptera bat guano; and in h. brachyura (feces and environmental samples), r. pruinosus (feces barcoded), and r. argentiventer (barcoded environmental sample) in wildlife farms (table 1 and s1 table) . in microchiroptera, bat coronavirus 512/2005 was frequently found in co-infection with predict_cov-35 (table 1 , s1 table) . network analysis showed the relationships among the bat coronavirus 512/2005 sequences from the three provinces in south viet nam (fig 7) . we observed two main clusters and a shallow geographic structure of genetic diversity, perhaps illustrative of sampling effort but also of localized transmission and circulation of bat coronavirus 512/2005 strains in these provinces. one cluster was exclusively detected in microchiroptera and mostly restricted to dong thap province and another cluster included sequences shared among all hosts and distributed in the three provinces (fig 7) . parts of the network showed a star-like topology (fig 7) , typical of populations in expansion that have recently increased size. there were two sequence types that were shared among microchiroptera and farmed rodents. the remaining 11 sequence types isolated from rodents on wildlife farms were not identical to those isolated from bats and were characterized by several nucleotide differences (fig 7) . murine coronavirus and longquan aa coronavirus were detected in 209 and 56 field rat samples, respectively, and 26 were coinfected with both (table 1) . two sequences of ibv were detected in rodent feces collected on two wildlife farms, one in a bamboo rat and another in a malayan porcupine. the rodent farm interface where bat and avian coronaviruses were detected in feces were not full containment facilities and possibly had bats and birds flying and roosting overhead (fig 2) . the ibv positives were detected in fecal samples from wildlife farms that had chickens, pigs, and dogs on site. significant findings of this study are the high proportion of coronavirus positive wildlife (bats and rodents) and the increasing proportion of positives found along the rat trade supply chain from sub-interfaces close to the capture site (rat traders) to restaurants. the transit of multiple rat species through the supply chain, and admixing with other species and taxa at sub-interfaces along the supply chain, offers opportunities for inter-and intra-species viral exchange and recombination. capture and transport of wildlife combined with overcrowding and close confinement of live animals in cages results in increased animal contact, likely leading to stress. while methodologically similar to rodent surveys in zhejiang province, china (2%), dong thap province, viet nam (4.4%), and globally (0.32%), our overall proportion of coronavirus positives was much higher among field rats (34.5%) and somewhat higher among farmed rodents (6.3%) [7, 26, 37] . stress, dehydration, and poor nutrition reduce animal condition and alter immune function and likely contribute to both increased shedding of viruses by infected animals, and increased susceptibility to infection of animals in the wildlife trade chain for human consumption [38] . the amplification of coronavirus along the supply chain may be seasonal as field rats were significantly more positive in the wet season. rattus argentiventer generally reproduce yearround in viet nam, but are particularly abundant in the wet season (may through october) following the rice harvest when an abundance of food supports the population increase [39] . if these seasonal population increases affect density dependent contact, there could be increased coronavirus prevalence and shedding in wild field rats during certain times of the year, which could then be further amplified along the trade. our survey was not a comprehensive multi-year evaluation of the field rat supply chain and was restricted to two provinces with this human-wildlife interface. these limitations mean we are not able to make inferences about larger spatial patterns or the inter-annual variability of coronavirus prevalence in wildlife populations found in this interface, which spans into neighboring cambodia. field rat carcasses were sampled immediately after they were slaughtered by traders, market vendors, or restaurant kitchen staff to optimize viral detection. some viral cross contamination of carcasses during the butchering process may have increased the proportion of coronaviruses detected in individual animals. the degree to which cross-contamination may have elevated the proportion of coronaviruses detected in individual animals is unknown, however, this proportion accurately reflects the risk of human exposure from handling and consumption of field rats at sub-interfaces along this wild meat food chain. from a mechanistic perspective as animals progress along the wildlife supply chain, opportunity for human contact increases, including close direct contact with traders, butchers, cooks, and consumers [40] . the combination of increased coronavirus prevalence in traded wildlife and greater opportunity for human-wildlife contact as well as intra-and inter-species contact in trade systems is likely to increase the risk of zoonotic transmission of coronaviruses in wildlife markets, restaurants, and other trade interfaces. we detected avian and bat coronaviruses in rodents raised on wildlife farms, including malayan porcupines and bamboo rats, but we did not detect rodent-associated coronaviruses. the only previously published coronavirus testing of malayan porcupine samples carried out in china were negative [41] . it is unclear if the malayan porcupine samples from animals screened in this study were infected with the avian or bat viruses or if environmental contamination or mixing occurred with avian and bat guano. chickens were present at the two sites where the ibv-positive rodents were detected, and bats fly and potentially roost overhead at most farms. 'artificial market' studies of influenza a viruses have found cage-stacking of species on top of other species and shared water sources facilitate viral transmission [42, 43] . nevertheless, viral sharing between species and environmental contamination or mixing (i.e. bat/ bird guano landing on rat feces) are two equally likely explanations for the presence of bat and avian coronaviruses detected in rodent fecal and environmental samples. the field rats were co-infected with the longquan aa coronavirus and the murine coronaviruses, both of which are from the lineage a (embecovirus) betacoronavirus genus. co-infections with multiple coronaviruses deserve particular attention as this co-occurrence may facilitate viral recombination leading to the emergence of new viruses [44, 45] . at the very least, we conclude that rodents in the field rat and farmed rodent supply chains are being exposed to coronaviruses from rodents, bats, and birds and perhaps creating opportunities for coronavirus recombination events, which may lead to viruses that could spill over into humans [46, 47] . our findings indicate a high risk of consumer exposure even if crosscontamination due to shared butchering materials influenced the proportion of positive individuals. repeated and more direct individual sampling of these species at these interfaces would be advantageous to determine if viral sharing was occurring versus environmental contamination of samples. the high proportion of positive bat feces at bat guano farms indicates the potential risk of bat guano farmers, their families, and their animals being exposed to bat coronaviruses. the overall proportion of positives (74.8%) was higher than previous studies using similar testing methods targeting bats in viet nam (22%), thailand (7.6%), lao pdr (6.5%), and cambodia (4.85%) [26, 48, 49] . in this region of viet nam, artificial roosts are typically erected in backyard family owned plots that incorporate a mosaic of duck, goat, or pig production and crops such as guava tress or other fruit trees and large scale kitchen gardens. bats have been shown to be an important evolutionary hosts of coronaviruses, including those infecting humans [7, [50] [51] [52] [53] . both predict_cov-17 and predict_cov-35 have been detected previously in the pteropus and microchiroptera bats in viet nam, cambodia, and nepal, which confirms that coronaviruses are capable of infecting distantly related hosts [7] . the finding of the same virus in different bat species raises the question of whether they coroost and/or share viruses through contact during other activities. utilizing shared resources such as water or feeding on and around crops and fruit could lead to contact and facilitate a host jump. the presence of the same virus in bat species in multiple neighboring countries supports the suggestion by others that virus distribution coincides with their bat host distribution [7, 54, 55] . while there has been no testing of the pathogenicity of these bat coronaviruses in humans or animals, they are found at close contact bat-human interfaces and further characterization is needed to understand their host range and potential for spillover. any general persecution of bats because of zoonotic viruses they may carry can actually increase the number of susceptible bats and increase transmission risk to people [56] , and would interfere with the important ecosystem services that bats provide, such as controlling insect pests of rice fields [57] , plant pollination, and seed dispersal. beyond the viral findings, this work represented an important opportunity for capacity development in field, laboratory, and scientific disciplines, as well as opportunities for social engagement and education of high-risk communities on zoonotic disease threats. the consensus pcr approach for viral detection provides a cost-effective tool to detect emerging viruses in low-resource settings. our work adds to the growing body of research demonstrating the utility of this approach to detect both known and novel viruses and co-infections in a variety of taxa, sample types, and interfaces. in viet nam, the direct result is an enhanced one health surveillance capacity to detect important emerging or unknown viruses in humans, wildlife, and livestock. in the communities with which we partnered, strong engagement enabled teams to sample a wide diversity of wild animals at high-risk interfaces. importantly, we have returned to these same communities to share the viral findings and to educate participants with an outreach program on how to live safely with bats [58] . large percentages of coronaviruses were detected in bats and rodents at sites where people have close contact and interact with wildlife including sub-interfaces along wildlife trade chains, wildlife farms, and artificial bat roosts where bat guano is collected for use as fertilizer. the high proportion of coronavirus positive samples at these human-wildlife interfaces highlights the potential for human exposure to wildlife origin coronaviruses. the observed viral amplification along the wildlife trade supply chain for human consumption, illustrated by the field rat trade in this study, likely resulted from the admixing of different species or sub-populations, and the close confinement of stressed live animals. this highlights the potential for coronavirus (and other virus) shedding and amplification along other wildlife supply chains (e.g., civets, pangolins) where similarly large numbers of animals are collected from a wide range of locations, transported, and confined. the detections of rodent, bat, and avian coronaviruses confirm concerns about productions systems and supply chains that increase contact between wildlife and domestic species. livestock and people living in close contact with rodents, bats, and birds shedding coronaviruses provides opportunities for intra-and interspecies transmission and potential recombination of coronaviruses. human behavior is facilitating the spillover of viruses, such as coronavirus, from animals to people. the wildlife trade supply chain from the field to restaurant and end consumer provides multiple opportunities for such spillover events to occur [1] . since the sars outbreak, broad scientific consensus exists that long term, structural changes, and wildlife trade and market closures will be required to prevent future epidemics. to minimize the public health risks of viral disease emergence from the consumption of wildlife and to safeguard livestock-based production systems, we recommend precautionary measures that restrict the killing, commercial breeding, transport, buying, selling, storage, processing and consuming of wild animals. the time has come for the global community to collectively assume responsibility through targeted wildlife trade reform. the world must also increase vigilance through building and improving detection capacity; actively conducting surveillance to detect and characterize coronaviruses in humans, wildlife, and livestock; and to inform human behaviors in order to reduce zoonotic viral transmission to humans. the more opportunities we provide for humans to come into direct contact with a multitude of wildlife species, the higher the likelihood of another spillover event. the costs of inaction are astronomically high and we must ensure that future food production and security is sustainable, just, and supports global health. supporting information s1 table. summary of all testing results by genus, interface, sub-interface, sample types, sites, percentage of samples testing positive, and viral species. (pdf) s2 table. multivariate mixed effect logistic regression showing the association between season and sub-interface with coronavirus positives in field rats. (pdf) s3 table. genbank accession numbers for coronavirus sequences detected in this study and for reference sequences. (pdf) s1 data. (txt) s1 r code. code used to conduct the analysis described. 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resurgence in kitaka mine bat population after extermination attempts the wrinkle-lipped free-tailed bat (chaerephon plicatus buchannan, 1800) feeds mainly on brown planthoppers in rice fields of central thailand living safely with bats. usaid we are thankful to the government of viet nam, the wildlife conservation society health team for conducting field sampling, partnering laboratories for running diagnostic tests, and many other agencies for collaborations on this project. specifically, we would like to acknowl key: cord-305857-2409me0p authors: lópez-roig, marc; bourhy, hervé; lavenir, rachel; serra-cobo, jordi title: seroprevalence dynamics of european bat lyssavirus type 1 in a multispecies bat colony date: 2014-09-04 journal: viruses doi: 10.3390/v6093386 sha: doc_id: 305857 cord_uid: 2409me0p we report an active surveillance study of the occurrence of specific antibodies to european bat lyssavirus type 1 (eblv-1) in bat species, scarcely studied hitherto, that share the same refuge. from 2004 to 2012, 406 sera were obtained from nine bat species. blood samples were subjected to a modified fluorescent antibody virus neutralization test to determine the antibody titer. eblv-1-neutralizing antibodies were detected in six of the nine species analyzed (pipistrellus pipistrellus, p. kuhlii, hypsugo savii, plecotus austriacus, eptesicus serotinus and tadarida teniotis). among all bats sampled, female seroprevalence (20.21%, 95% ci: 14.78%–26.57%) was not significantly higher than the seroprevalence in males (15.02%, 95% ci: 10.51%–20.54%). the results showed that the inter-annual variation in the number of seropositive bats in t. teniotis and p. austriacus showed a peak in 2007 (>70% of eblv-1 prevalence). however, significant differences were observed in the temporal patterns of the seroprevalence modeling of t. teniotis and p. austriacus. the behavioral ecology of these species involved could explain the different annual fluctuations in eblv-1 seroprevalence. wildlife plays a key role in emerging infectious diseases by providing a -zoonotic pool‖ from which pathogens may emerge [1] . zoonotic pathogens represent approximately 60% of all pathogens able to infect humans [2] . in recent years, bats have been implicated in numerous emerging infectious disease events and have been recognized as important reservoir hosts for viruses that can cross the species barrier to infect humans and other domestic and wild mammals [3] . the role of bats in viral diseases is well established, particularly their role as hosts for lyssaviruses, coronaviruses, flaviviruses, astroviruses and adenoviruses [3] [4] [5] . bats have several unique features that may maximize their effectiveness as reservoir hosts for viruses. bats are the second largest order of mammals. currently, there are about 1200 recognized bat species worldwide, accounting for approximately 21% of all mammalian species. bats have the potential to rapidly and widely spread viruses (having a high mobility, they are the only mammals capable of flight). they have a long lifespan and a high survival rate, and many bat species have a gregarious behavior. bats can fly long distances between their summer and overwintering sites, permitting the exchange of viruses between conspecifics or bats of other species, i.e., in france, rabies virus infections have been associated with the migratory routes of nathusius' pipistrelle (pipistrellus nathusii) bats [6] . persistent viral infections occurring among long-lived bats, coupled with their often gregarious roosting behavior, could greatly increase the potential for intra-and inter-species transmission of viruses [7] , especially in summer and winter periods. seasonality in temperate zone bats includes birthing periods, migration, gregarious behavior and torpor. each of these strategies may affect population density, contact rates and immune response, thus leading to spatiotemporal variation in infection dynamics [8, 9] . numerous bat species have been found to be infected by lyssaviruses [10] . bats serve as reservoirs of 13 of the 15 lyssavirus species described (the only lyssavirus species that have not been isolated from bats, to date, are mokola virus and ikoma virus). furthermore, recently described lyssavirus species enlarged the genetic diversity of lyssaviruses found in bats [11] [12] [13] , suggesting that the lyssaviruses originated in these mammals and progressively diverged from a common ancestor [14, 15] . in europe, four of the lyssavirus species recognized, european bat lyssavirus types 1 and 2 (eblv-1 and eblv-2, respectively), bokeloh bat lyssavirus (bblv), the west caucasian bat virus (wcbv) and one tentative species, lleida bat lyssavirus, circulate among several bat species [12, 16, 17] . eblv-1 is widely distributed throughout europe, and two variants have distinct distributions and evolution histories: one is eblv-1a, which has an east-west distribution from russia to france, with very little genetic variation; and the other is eblv-1b, which exhibits a south-north distribution and far more genetic diversity [18] . different studies showed that lyssavirus dynamics exhibits a strong seasonal pattern [8] and that the breeding period could favor the infection of bats [19] [20] [21] . many bat species roost in very large and dense maternity colonies. this dense clustering of individuals can provide large opportunities for viral exchange in bat colonies [10] . previous studies have observed a higher seroprevalence in multispecies colonies compared to monospecific colonies, suggesting that interspecific virus transmission plays an important role in eblv-1 dynamics [22] . however, in some cases, infection cycles may be maintained among specific host species and transmission may be minimal among sympatric bats [9] . furthermore, differences in the ecological behavior of species (e.g., migration, torpor) can drive different bat infection dynamics. in this sense, a higher number of species might not only increase the rates of contact between bat groups, but could also facilitate virus entry or spread through the higher mobility of individuals among colonies, especially if there are migratory species involved [22] . few studies have addressed the inter-annual dynamics of lyssavirus among bat multispecies that are roosting in the same refuge, despite these studies giving a better understanding of the dynamics of bat lyssaviruses. our previous investigations have analyzed the temporal dynamics of lyssavirus in one bat species (myotis myotis) roosting in two colonies [23, 24] . the present report is based on a long-term (nine years) longitudinal study of the prevalence of eblv-1 neutralizing antibodies and provides the first report on the inter-annual dynamics of eblv-1 in p. austriacus and t. teniotis, both being bat species scarcely studied hitherto. we chose this locality, because we found three species (p. pipistrellus, p. austriacus and t. teniotis) that were eblv-1 rna-positive by nested reverse transcriptase-polymerase chain reaction in the first year of study [22] . our specific goals were: (i) to provide information about eblv-1 seroprevalence in the wild bat community where several european bat species share the same refuge; and (ii) to compare the temporal patterns of seroprevalence mainly in two less-studied bat species that, moreover, exhibit different ecological strategies. this study was carried out at the san pedro de los griegos pothole (41°1' n, 0°38' e; elevation: 550 m), situated 5 km from oliete village (teruel province). the cavity is an enormous hole with an entrance of 65 × 75 m and a 108-m maximum depth. crevices in the walls are optimal roost sites for many birds and bat species. however, the pothole is totally illuminated and shows a large lagoon inside ( figure 1 ). around the cavity, the vegetation is dominated by a mix of low growing stipa sp., brachypodium retusum, rosmarinus officinalis and thymus vulgaris. local weather is characterized by continental climate with a mean annual temperature of 14.60 °c and a mean annual precipitation of 278 mm (mainly in spring). however, mean daily temperature is over 20 °c between june and august (with 15.70 °c and 32.13 °c as the mean minimum and maximum temperatures, respectively). the permanent availability of water and nutrients, the dampening of hard external climatic conditions and the suitability of the habitat for the reproduction of various vertebrate species make the san pedro pothole a site of unprecedented high biodiversity in europe [25] . bats were captured in summer (from june to july) over a 9-year period (2004-2012). mist nets were employed to capture bats at sunset when emerging from the pothole to forage. all bats were identified to species based on published identification keys of the bats of europe [26] . individuals were sexed, and the reproductive status of adult females was classified as pregnant or lactating, based on palpation of the abdomen and nipple condition [27] . blood samples were obtained by a small puncture made in the median artery. the amount of blood sampled varied from 0.2 ml to 0.5 ml, according to the size of the animal. pressure with a sterilized absorbent hemostatic sponge impregnated with gelatin was applied to prevent bleeding and facilitate healing. the bats were given 10% glucose water to drink to prevent dehydration and to provide rapidly assimilated compounds for energy. once bleeding ceased, the bat was released. vials containing blood were stored at 4 °c for a few hours. samples were centrifuged for 20 minutes at 9660× g, and the serum was extracted with a micropipette. serum samples and blood pellets were stored at -20 °c before analysis. all animals were handled in strict accordance with good animal practices, as defined by current european legislation. bat capture and blood sampling were authorized by permit from the spanish regional committee for scientific capture. the technique used to detect eblv-1 neutralizing antibodies is an adaptation of the rapid fluorescent focus inhibition test (rffit) [23, 28] . a constant dose of a previously titrated (calibrated to give 80% fluorescent foci-infected cells), cell culture-adapted eblv-1 challenge virus (8918 fra) was incubated with 3-fold dilutions of the sera to be labelled. after incubation of the serum-virus mixtures, a suspension of bsr cells (a clone of bhk 21 cells) was added. after 24 hours incubation, the cell monolayer was acetone-fixed and labelled with a fluoresceinated anti-nucleocapsid antibody (bio-rad, marnes-la-coquette, france). the optimal challenge dose (the dilution giving 80% infected cells for each virus production) was calculated. titers are presented as an arithmetic mean of two independent repetitions. serum samples with antibody titers <27 are considered negative for eblv-1neutralizing antibodies. this cut-off value is similar to that applied in other studies [23, 24, 28, 29] . to study the variation in eblv-1-antibody prevalence, we conducted two analyses: first, three explanatory variables (sex, species and year) were first screened using a univariate analysis and a chi-square test to check for statistically significant associations with serological status (0: negative; 1: positive). in the second analysis, we used a generalized additive model (gam) to study the temporal patterns of eblv-1-antibody prevalence in only two species (p. austriacus and t. teniotis). more specifically, we used a generalized additive model with the binomial error distribution, where the seroprevalence was the response variable and sex, species and year (2004-2012) were the explanatory variables. the -year‖ variable was modeled as a covariate fitted with penalized cubic regression splines and sex and species as a fixed categorical factor. to avoid over-fitting and to retain more easily interpretable relationships in the gam smoothing function, an upper limit of 4 degrees of freedom was set for the year variable when fitting the models. we used an information-theoretic procedure and the akaike information criterion corrected for small sample sizes (aicc) to compare models [30] . modeling was performed using the -lme4‖ and ‗‗mgcv'' packages in the r program v. 2.14 [31] . we report the results of the prevalence of specific eblv-1 neutralizing antibody analysis from the 2004-2012 period in nine bat species roosting in the same refuge. five of these species (eptesicus serotinus, p. kuhlii, p. pygmaeus, myotis myotis and m. daubentonii) were captured sporadically (sample size <10 individuals during the whole study period), while the rest of the species sampled (p. pipistrellus, hypsugo savii, plecotus austriacus and tadarida teniotis) were captured often. the larger samples (>100 individuals) were obtained in p. austriacus and t. teniotis, because they form large colonies in this cavity. t. teniotis form a colony of several hundred individuals. the colony of p. austriacus is smaller and consists of 150 individuals, approximately [32] . we observed pregnant females in all bat species, except in e. serotinus, p. pygmaeus and m. myotis, where females were never captured, indicating that this cavity is a breeding roost for the rest of the species found. males were also captured during the breeding period, indicating that males, either as solitary individuals or forming part of the maternity colonies (e.g., p. austriacus), are present during the breeding period in the cave. among the 406 sera obtained, 71 (17.49%) were positive for eblv-1-neutralizing antibodies. eblv-1 antibodies were detected in 6 (66.67%) of the nine species analyzed (p. pipistrellus, p. kuhlii, h. savii, p. austriacus, e. serotinus and t. teniotis) ( table 1) . no significant differences in eblv-1 seroprevalence were detected among seropositive bat species (χ 2 = 1.67, df = 5, p = 0.89). the highest seroprevalence was observed in h. savii. we did not find any difference in eblv-1 seroprevalence between females (20.21%, 95% ci: 14.78%-26.57%) and males (15.02%, 95% ci: 10.51%-20.54%) (χ 2 = 1.88, df = 1, p = 0.17) when all species were analyzed together and when only bat species with a large sample size-p. austriacus and t. teniotis-were considered (table 1) . capture-mark-recapture of some bats during the study period allowed the tracking of temporal changes in eblv-1 seroneutralization titers. seven p. austriacus were captured and analyzed almost two times at intervals of one or several years. four of these seven bats showed positive antibody titers, becoming negative in the following recapture sessions after some years, indicating that these bats survive at least several years after their seroconversion (table 2) . the models that incorporate sex and species variables were not significantly different from the model without these variables (δaicc < 2) ( table 6 ). the best model showed a significant different nonlinear pattern in the eblv-1 seroprevalence along p. austriacus and t. teniotis. the effect of year fitted with the spline was highly significant for two species (p. austriacus: df = 2.92, p < 0.001 and t. teniotis: df = 3.87, p = 0.026), suggesting a different inter-annual pattern among these species (figure 3 , table 6 ). although no positive sera were detected in three bat species (m. myotis, m. daubentonii and p. pygmaeus), this result is probably due to the very low sample size. the high percentage (67%) of seropositive species found and the lack of significant differences in eblv-1 seroprevalence among seropositive species suggest that most of the bat species can be exposed to eblv-1 in this pothole although most of these species are not considered as lyssavirus reservoirs by previous studies [12, 13, 16, 33] . previous studies have shown higher prevalence in females than in males [33, 34] . this difference may be due to the gregarious behavior of female bats in summer (nursing colonies are composed almost exclusively of adult females). in these colonies, virus transmission may be favored by high contact rates during social grooming, nursing or olfactory or lingual contact with body fluids. reproductive activity may also play a role in virus transmission [19] , because an increased susceptibility to infectious disease during pregnancy and lactation has been demonstrated in bats [34] and other mammals [35] . however, we report in this study no sex differences of eblv-1 seroprevalence. the presence of males in this cavity during summer could indicate that males also are present in maternity colonies, as observed in p. austriacus colonies, or roost near these colonies. significant fluctuations in the percentage of seropositive bats are indicative of several different episodes of eblv-1 infection occurring in p. austriacus and t. teniotis colonies during the period of study. a quick increase and a high seropositive percentage after a lyssavirus episode are not unusual in a gregarious behavior species and could explain the sudden increase in the percentage of seropositive bats in t. teniotis and p. austriacus colonies. a similar quick increase with seropositive peaks of 60%-70% was observed in different colonies of m. myotis in mallorca [23, 24] . however, in m. myotis colonies, the evolution of seroprevalence after infection peaks follows a more gradual decline over subsequent years, until a new episode takes place, very different from what is observed here. the delay between the waves is then dependent on the rate of inflow of susceptible bats into the colonies as a consequence of new births, bat immigration from neighboring colonies and the expiration of eblv-1specific immunity in previously infected animals [23] . when a sufficient fraction of susceptible bats in the colony is reached, the virus spreads again if infected individuals join the colony. in the t. teniotis and p. austriacus colonies, the increase of seroprevalence is followed by a rapid decline until seropositive bats are not detected. the difference in the seropositive percentage evolution can be due to a higher rate of inflow of individuals in colonies of t. teniotis and p. austriacus. no data of inflow are available on t. teniotis, but very few recaptures were obtained during the study, indicating probably a high inflow rate in this colony. however, recapture rates in the p. austriacus colony were higher, suggesting a lower inflow in this species. another hypothesis could be a different lifespan of immunity in these species. recent studies estimated the lifespan of the m. myotis immunity from eblv-1 to be around two years [36] . in this respect, it is possible that the immunity lifespan would be shorter in p. austriacus and t. teniotis than in m. myotis. the best model obtained by gam analysis indicated that inter-annual patterns of seroprevalence evolution were significantly different for t. teniotis and p. austriacus. annual fluctuations could result from the behavioral ecology of the species involved [9] . t. teniotis and p. austriacus are two species with a different social organization and behavior. while t. teniotis forms large maternity colonies and can make long seasonal movements, p. austriacus forms smaller maternity colonies constituted by both sexes and makes shorter seasonal movements [37] . different host ecology, behavior and movement could explain the different temporal variations in seroprevalence in these two species. changes in density during migration or colony formation may affect contact rates and, thus, disease dynamics [9, 38] . differences in eblv-1 exposure dynamics could also be related to host community composition and inter-species interaction. higher eblv-1 seroprevalence was observed in large and multispecies colonies compared to smaller and monospecific colonies, suggesting that interspecific virus transmission plays an important role in dynamics. a higher number of species might not only increase the rates of contact between bat groups, but could also facilitate virus entry or spread through the higher mobility of individuals among colonies, especially if there are migratory species [22] . in this sense, m. schreibersii (a species that often shares roost with m. myotis) has been considered as a regional reservoir and an essential species for eblv-1 persistence in the balearic islands [36] . other bat species present in the san pedro pothole, such as p. pipistrellus and p. kuhlii, showed lower eblv-1 seroprevalence than p. austriacus and t. teniotis. however, previous studies of bat rabies surveillance in europe did not find eblv-1-neutralizing antibodies in both species of pipistrellus (for review see [39, 40] ). these results could be indicative of a low public health risk associated with these synanthropic species. furthermore, the lack of a standardized serological test procedure, including arbitrary cut-off values, makes the comparison between previous european studies difficult. however, the higher values of eblv-1 seroprevalence in our study could be due to differences in virus circulation and dynamics resulting from regional differences or selection of different types of colony (large multispecies maternity colonies in this case) [39, 40] . research programs that focus mainly on multi-host systems will help advance our understanding of the ecology of bat diseases. this research addresses the role of multiple hosts in the infection dynamics of lyssavirus. to advance our understanding of the ecology of bat lyssavirus, we report the results of specific eblv-1 neutralizing antibody analysis in nine bat species roosting in the san pedro de los griegos pothole. these results suggest that most bats species roosted in this cave were exposed to the eblv-1 lyssavirus. the evolution of seroprevalence in t. teniotis and p. austriacus colonies after infection peaks is different from that observed in m. myotis colonies. differences in behavior ecology and population dynamics among bat species could explain 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host switching in lyssavirus history from the chiroptera to the carnivora orders genomic diversity and evolution of the lyssaviruses phylogenetic relationships of irkut and west caucasian bat viruses within the lyssavirus genus and suggested quantitative criteria based on the n gene sequence for lyssavirus genotype definition phylogeography, population dynamics, and molecular evolution of european bat lyssaviruses ecology of rabies virus exposure in colonies of brazilian free-tailed bats (tadarida brasiliensis) at natural and man-made roosts in texas. vector borne zoonotic dis amplification of emerging viruses in a bat colony urban habituation, ecological connectivity and epidemic dampening: the emergence of hendra virus from flying foxes (pteropus spp.) ecological factors associated with european bat lyssavirus seroprevalence in spanish bats european bat lyssavirus infection in spanish bat populations temporal dynamics of european bat lyssavirus type 1 and survival of myotis myotis bats in natural colonies espagne): un refuge de biodiversité sans é quivalent en europe (in french) illustrated identification key to the bats of europe ageing and assessment of reproductive status of pipistrelle bats, pipistrellus pipistrellus in commission des laboratoires de référence et d'expertise, editors. méthodes de laboratoire pour le diagnostique de la rage model selection and multimodal inference: a practical information-theoretic approach r: a language and environment for statistical computing. r foundation for statistical computing aná lisis demográ ficos y sanitarios en las colonias de plecotus austriacus y tadarida teniotis de la sima de san pedro (oliete, parque cultural del rí o martí n) (in spanish) active surveillance of bat rabies in france: a 5-year study reproduction and nutritional stress are risk factors for hendra virus infection in little red flying foxes (pteropus scapulatus) immunosuppression during pregnancy and lactation insights into persistence mechanisms of a zoonotic virus in bat colonies using a multispecies metapopulation model bat migrations in europe. a review of banding data and literature; federal agency for nature conservation animal migration and infectious disease risk bat rabies surveillance in europe twenty years of active bat rabies surveillance in germany: a detailed analysis and future perspectives the authors wish to thank sergi vives, departament d'estadística de la facultat de biologia, university of barcelona, for his mathematical support. we thank pepe royo of the centro de arte rupestre -antonio beltrán‖ del parque cultural del río martín of ariño (teruel, spain) for providing access to installations and for support during sample collection. we thank xavier bayer and cisco guasch for sharing his team's fieldwork.the research leading to these results has received funding from ministerio de sanidad y servicios sociales e igualdad, dirección general de salud pública y sanidad exterior. the authors declare no conflict of interest. key: cord-295727-s63lffi8 authors: lima, luciana; espinosa-álvarez, oneida; hamilton, patrick b; neves, luis; takata, carmen sa; campaner, marta; attias, márcia; de souza, wanderley; camargo, erney p; teixeira, marta mg title: trypanosoma livingstonei: a new species from african bats supports the bat seeding hypothesis for the trypanosoma cruzi clade date: 2013-08-03 journal: parasit vectors doi: 10.1186/1756-3305-6-221 sha: doc_id: 295727 cord_uid: s63lffi8 background: bat trypanosomes have been implicated in the evolutionary history of the t. cruzi clade, which comprises species from a wide geographic and host range in south america, africa and europe, including bat-restricted species and the generalist agents of human american trypanosomosis t. cruzi and t. rangeli. methods: trypanosomes from bats (rhinolophus landeri and hipposideros caffer) captured in mozambique, southeast africa, were isolated by hemoculture. barcoding was carried out through the v7v8 region of small subunit (ssu) rrna and fluorescent fragment length barcoding (fflb). phylogenetic inferences were based on ssu rrna, glyceraldehyde phosphate dehydrogenase (ggapdh) and spliced leader (sl) genes. morphological characterization included light, scanning and transmission electron microscopy. results: new trypanosomes from bats clustered together forming a clade basal to a larger assemblage called the t. cruzi clade. barcoding, phylogenetic analyses and genetic distances based on ssu rrna and ggapdh supported these trypanosomes as a new species, which we named trypanosoma livingstonei n. sp. the large and highly polymorphic sl gene repeats of this species showed a copy of the 5s ribosomal rna into the intergenic region. unique morphological (large and broad blood trypomastigotes compatible to species of the subgenus megatrypanum and cultures showing highly pleomorphic epimastigotes and long and slender trypomastigotes) and ultrastructural (cytostome and reservosomes) features and growth behaviour (when co-cultivated with hela cells at 37°c differentiated into trypomastigotes resembling the blood forms and do not invaded the cells) complemented the description of this species. conclusion: phylogenetic inferences supported the hypothesis that trypanosoma livingstonei n. sp. diverged from a common ancestral bat trypanosome that evolved exclusively in chiroptera or switched at independent opportunities to mammals of several orders forming the clade t. cruzi, hence, providing further support for the bat seeding hypothesis to explain the origin of t. cruzi and t. rangeli. trypanosomes (euglenozoa: kinetoplastea: trypanosomatidae) are blood parasites widespread in all continents, adapted to all classes of vertebrates and transmitted by leeches and a variety of bloodsucking arthropods. although chiroptera harbour numerous trypanosome species with a high prevalence and worldwide distribution, species diversity, vectors, life cycles, distribution and trypanosome evolution remain poorly understood [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] . the majority of trypanosomes reported in bats have not been cultivated, and their classification has been based exclusively on the morphology of blood trypomastigotes. large blood trypanosomes of the subgenus megatrypanum, followed by small blood forms of the subgenus schizotrypanum, comprise the majority of the trypanosomes reported in bats throughout south america, asia, europe and, especially, africa [1, 3, 5, 7, 8, [10] [11] [12] [13] [14] . the subgenus megatrypanum, originally comprising large blood trypanosomes from artiodactyls [15] , was amended exclusively on a morphological basis to include any large trypanosome found in bats, monkeys and rodents [1, 2, 4, 6] . molecular phylogenetic analysis has demonstrated the polyphyly of the traditional subgenus megatrypanum, which was revised as a clade comprising trypanosomes from ruminants headed by the type species t. theileri, a cosmopolitan parasite of cattle [16] [17] [18] [19] . however, in the reappraisal of this subgenus, other species from nonruminant hosts that putatively belong to this subgenus need to be phylogenetically positioned, especially those from bats, which together with trypanosomes from artiodactyls, account for most of the species assigned to this subgenus [1, 4, 7] . most bat trypanosome species that have been characterised by molecular approaches belong to the subgenus schizotrypanum [8] [9] [10] [11] 13, 14, [20] [21] [22] [23] [24] [25] . with the exception of t. cruzi, there are no schizotrypanum species in hosts other than bats. t. rangeli was found in brazilian bats [26] , this species is infective to several mammals and comprises distinct genotypes [26, 27] , which clustered into a clade containing t. conorhini from rats, t. vespertilionis from a european bat and two african trypanosomes from monkey and civet. although t. rangeli, t. conorhini and t. vespertilionis were morphologically classified into the subgenera herpetosoma, megatrypanum and schizotrypanum, respectively, molecular phylogenies demonstrated that they clustered together forming the strongly supported sister clade of the schizotrypanum clade. trypanosoma sp. (t. sp. bat) from an african megabat (suborder megachiroptera) originally assigned to the subgenus megatrypanum was positioned at the edge of this clade [8] [9] [10] 18, 20, 23, 24] . the major assemblage formed by the subgenus schizotrypanum and the clade t. rangeli/t. conorhini was designated as the t. cruzi clade. the positioning of a kangaroo trypanosome at its edge in association with vicariance has supported the southern supercontinent hypothesis for the origin of t. cruzi. accordingly, this species could have originated in marsupials at a time when south america, australia and antarctica formed a single continent. however, in conflict with this hypothesis, some australian trypanosomes from marsupials are more related to trypanosomes from non-australian hosts [9, 18, 20, 21, 23, 24] . the discovery of african terrestrial mammals infected with trypanosomes placed in the t. cruzi clade [18] has complicated the southern supercontinent hypothesis. taken together, the findings that t. c. marinkellei from south american bats is the closest living relative of t. cruzi [8, 10, 22, 25, 28] followed by t. erneyi from african bats [10] , the close phylogenetic relationship between t. dionisii from europe and south america [8, 9, 24] , the presence of t. rangeli in brazilian bats [26] and the relationships of this species with african (t. sp. bat) and european (t. vespertilionis) bat trypanosomes, and the discovery of tcbat, a bat-associated t. cruzi genotype found in south and central america [14, 22] all support the bat seeding hypothesis for the origin of the t. cruzi clade [24] . in this scenario, which is the most parsimonious for explaining the relationships observed within the t. cruzi clade, an ancestral trypanosome parasite in bats diverged to lineages that evolved exclusively in bats, giving rise to the bat-restricted species, or evolved through multiple switches at independent times in hosts of other mammalian orders, including the generalists t. cruzi and t. rangeli, which also infect bats. multiple trypanosome jumps between hosts were most likely facilitated by the sharing of niches by bats, haematophagous insects (vectors) and terrestrial mammals. oral infection through the predation of infected bats by other mammals and by the consumption of insect vectors by bats probably played important roles in the colonisation of new hosts by bat trypanosomes. transmission of trypanosomes among bats is likely to occur by an oral route when the vector insects are eaten by insectivorous bats. the grooming habits of the bats probably facilitates the infection by the bat trypanosomes transmitted by ectoparasite cimicids [3, 6, 7] . both vectorial and oral transmission routes are important in the natural transmission cycles of t. cruzi and other trypanosomes nested in the t. cruzi clade [1, 3, [6] [7] [8] . with the discovery of t. erneyi [10] and a new genotype of t. dionisii in the uk [9] , bat trypanosomes from the old world revealed to be more closely related to south american bat trypanosomes than showed by previous studies [8, 22, 26] . these findings suggested movement of bat trypanosomes between the new and old worlds occurred in a relatively more recent time than bat fossil records suggested [9, 24] . trypanosomes from the t. cruzi clade are likely to have started to diversify sometime after the great diversification of bats in the eocene (70-58 mya) [29] [30] [31] . however, the extant species of bat trypanosomes appear to have emerged during a short period and much more recently than expected based on the fragmented paleontological history of bats [9, 24] . in this study, we isolated and characterised 14 new trypanosomes from african bats captured in mozambique, southeast africa, by inferring phylogenetic relationships using ribosomal ssu rrna, ggapdh and sl genes. sequences from the new bat isolates were compared to those from other bat trypanosomes determined in this and in previous studies (including other isolates morphologically assignable to the subgenus megatrypanum) to address taxonomic questions about bat trypanosomes. comparison of bat trypanosomes by combining molecular, morphological and behavioural information provides new information on the evolutionary history of bat trypanosomes and the origin of the t. cruzi clade. bats were captured in mozambique, southeastern africa, in the district of chupanga (s18°02′ e35°34′), zambezi valley, and the gorongosa national park (s18°58′ e34°21′), both of which are located in the province of sofala in central mozambique (table 1 ; figure 1 ). captures were carried out with mist nets; bats were anaesthetised and blood samples were collected by cardiac puncture as previously described [8, 22] . for the molecular identification of bats, liver tissue samples were fixed in 100% ethanol, processed for genomic dna and used to sequence the cytochrome b gene (cyt b) as previously described [32] . sequences were analysed by blast search in genbank. bat blood samples were examined for the presence of trypanosomes by using the microhaematocrit (mh), giemsa-stained blood smear examination and haemoculture methods as employed before for trypanosomes from different vertebrate hosts [8, 10, 22, 33, 34] . for the haemoculture, bat blood samples were transferred to tubes containing a medium consisting of solid phase blood agar base (bab) with an overlay of lit (liver infusion tryptose) medium containing 10% foetal bovine serum (fbs); the tubes were maintained at 25-28°c for 10-15 days. positive cultures were transferred to culture flasks containing a monolayer of insect cells (hi-5 from trichoplusia ni) and epimastigotes from log-phase cultures were then transferred to tc100 medium (= grace's medium) containing 10% fbs, with incubation at 25°c. the utilization of insect feeder cells for the isolation in culture of trypanosomes largely improves the differentiation of blood trypomastigotes to epimastigotes and the multiplication of epimastigotes in primary cultures [10, 33, 34] . the isolates were grown in tc100 with 5.0% fbs for dna preparation and cryopreservation at the trypanosomatid culture collection (tcc) of the department of parasitology, university of são paulo, brazil. amplification, sequencing and data analysis of ssu rdna and ggapdh dna was extracted from cultured bat trypanosomes by classical phenol-chloroform method and used as templates for the pcr amplification of dna sequences. pcr amplification, cloning and sequencing of the variable v7-v8 region of ssu rrna (employed as barcodes), whole ssu rrna and ggapdh genes were determined as before [35] [36] [37] . sequences were aligned using clustal x [38] and the resulting alignments were manually refined. we created the following alignments for phylogenetic inferences: a) the v7v8 region of ssu rrna sequences (~880 bp) from the new bat trypanosomes aligned with their closest australian and bat trypanosomes, yielding a high similarity index for the new bat trypanosomes by blast search; b) ggapdh sequences (~830 bp) of trypanosomes representing all major clades in the phylogenetic tree of trypanosoma, using nontrypanosome trypanosomatids as an outgroup; c) concatenated ggapdh and ssu rrna sequences (~3.3 kb) from 6 new isolates and several species of t. cruzi clade using t. lewisi as an outgroup. the species included in the phylogenetic trees and their respective host, geographical origin and genbank accession numbers are shown in table 1 . phylogenies were inferred by using maximum likelihood (ml), bayesian inferences (bi) and parsimony (p) analyses. parsimony and bootstrap analyses were carried out using paup version 4.0b10 [39] with 500 replicates of a random addition sequence followed by branch swapping (ras-tbr) as previously described [36, 37] . the ml analyses were performed using raxml v.2.2.3 [40] . tree searches were performed with gtrgamma, with 500 maximum parsimony starting trees. model parameters were estimated in raxml for the duration of the tree search. nodal support was estimated with 500 bootstrap replicates in raxml using gtrgamma and maximum parsimony starting trees. mrbayes v3.1.2 [41] was used for bi inferences as described previously [36, 37] . dna from cultured trypanosomes and from bat blood samples were tested by fflb carried out using four primer sets and pcr conditions described previously [10, 42, 43] . amplification, sequencing and data analysis of spliced leader (sl) sequences the amplification and sequencing of whole sl gene repeats from bat trypanosomes were performed using primers and reaction conditions as previously described [17, 27] . pcr-amplified whole sl repeats were purified from agarose gels and cloned and at least 3 clones from each isolate were sequenced. the resulting sequences were aligned with clustalx and the resulting alignment was manually refined. the phylogenetic analysis of sl sequences was performed using the nj method as previously described [17, 19, 26] . two new isolates from bats (tcc1270 and 1271) were compared for their growth behaviour in tc100 and lit media during the logarithmic and stationary phases. cultures containing a large number of trypomastigotes at stationary phase were transferred to monolayers of hela cells to verify their ability to invade and develop within cells [10, 22] . epimastigotes of logarithmic cultures were transferred to monolayers of mammalian cells (llc-mk2) and incubated at 37°c to assess the differentiation of epimastigotes into large and wide trypomastigotes resembling blood forms. to analyse mouse infectivity, balb/c mice were inoculated (i.p.) with t. livingstonei cultures containing trypomastigote forms (~10 6 /mouse) from tc100 cultures. mouse blood samples were examined weekly from 3 to 20 days p.i. by mh, and at the 20 th day p.i. by haemoculture method (he). to evaluate the behaviour of t. livingstonei in triatomines, 15 4 th -5 th instar nymphs of each rhodnius neglectus and triatoma infestans were inoculated with stationary phase cultures containing epi-and metacyclic trypomastigotes, dissected at 10 and 30 days p.i., and the contents of their digestive tubes were examined for trypanosomes. for light microscopical analysis, blood smears from naturally infected bats and logarithmic and stationary phase cultures in tc100 medium were fixed with methanol and giemsa-stained. for the analyses of the ultrastructural organization by tem and sem, cultures from two isolates were processed as previously described [10, 36, 37] . tem was performed with a jeol 100cx electron microscope. for the scanning electron microscopy (sem), flagellates fixed with glutaraldehyde were adhered to poly-l-lysine-coated coverslips and processed for observation on a zeiss dsm 940 microscope as previously detailed [10, 34] . the capture and handling of bats was performed in accordance with the research project approved by the scientific boards of the veterinary faculty of the universidade eduardo mondlane, maputo, mozambique and the ethic committee in animal experimentation from the institute of biomedical center, university of são paulo, são paulo, brazil. in this study, we evaluated trypanosome infection in 79 bats from mozambique: 48 rhinolophus landeri from chupanga, and 31 hipposideros caffer from gorongosa (table 1; figure 1 ). we determined the cyt b gene sequences from bat liver dna to confirm the morphological identification and ascertain the bat species by blast analyses from genbank ( table 1) . the examination of blood samples from 37 r. landeri by microhaematocrit revealed the presence of trypanosomes in 15 bats, yielding a prevalence of~40%. however, the parasitemia was low and few trypomastigotes could be found in blood smears. other blood samples from this species and from h. caffer could not be examined by this method because of fieldwork complications. the blood samples from all bats were examined by haemoculture, and cultures of 11 r. landeri isolates and three from h. caffer were established. these new trypanosomes from bats were first cultivated with a monolayer of hi-5 cells in tc100 medium, and then gradually adapted to tc100 dispersing feeder cells. table 1 ). species from the t. lewisi clade were used as outgroups. numbers are bootstrap values derived from 500 replicates in the p/ml/bi analyses. the analysis of the v7v8 variable region of the ssu rrna gene for barcoding trypanosomes has demonstrated that this sequence is sufficiently polymorphic to distinguish all species from the several vertebrate classes examined to date [8, 10, 19, 33, 34] . in this study, barcoding using v7v8 ssu rrna revealed that all new isolates from african bats shared high sequence similarity; 2-3 cloned sequences were determined for each isolate, and they tightly clustered together and were virtually identical (~0.2% of divergence) and different from any previously reported trypanosome species. regarding their closest relatives, the new trypanosomes diverged~9.5% from australian trypanosomes from kangaroo, possums (marsupials) and rodents, and~12% from t. sp. bat (gabon, africa) and t. vespertilionis (uk bat). dna from bat blood samples with negative haemoculture results were also used as a template for barcoding, and revealed trypanosome sequences identical to those of cultivated trypanosomes (figure 2a) . the fflb techniques relies on the amplification and fluorescence detection of four small regions of rrna genes of variable length according to the species/isolates, and have been valuable to distinguish a wide range of trypanosomes from cultures, blood and insect samples [10, 42, 43] . here, we barcoded the new bat trypanosomes from culture and directly from blood samples. a comparison was made of their fflb profiles with those from several previously barcoded trypanosomes, including the following species found in bats: t. cruzi, t. c. marinkellei, t. dionisii, t. rangeli and t. erneyi [10, 43] , resulting in unique profiles for each species. highly similar but nonidentical fflb profiles were found for all the new cultivated bat trypanosomes and the isolates from bat blood samples. the fflb patterns permitted to distinguish the new trypanosomes from all trypanosomes from bats and other hosts investigated in this (figure 2b ) and in previous studies [10, 42, 43] . phylogenies based on ssu rrna and ggapdh have been used for evolutionary and taxonomic studies of trypanosomatids and it has been recommended that all new trypanosome species are phylogenetically validated using at least these two genes [10, 34, 36, 37] . here, the new bat isolates were initially positioned using independent ggapdh ( figure 3 ) and ssu rrna (data not shown) sequences in phylogenetic trees comprising representative species of all major trypanosome clades. concordant tree topologies from ml, p and bi analyses were obtained by using these two genes. in all phylogenetic trees, the new bat isolates formed a well-supported clade close to australian trypanosomes (10% divergence) and basal to the t. cruzi clade (figure 3) . we selected 6 new isolates (tcc1270, 1271, 1295, 1298, 1304 and 1953) to be positioned in the phylogeny of trypanosoma using concatenated data set from whole ssu rrna and ggapdh sequences (figure 1 ). the use of these combined genes corroborated all the clades and their phylogenetic relationships as demonstrated in broader phylogenies [10, 23] . the new bat isolates are highly homogeneous, diverging by only 0.3% in their ggapdh sequences. the clade formed by the new bat trypanosomes was basal to the t. cruzi clade (100% bootstrap); their closest relatives were the australian trypanosomes, whereas t. vespertilionis and t. sp. bat were more closely related, despite being separated from the new bat trypanosomes by~13% ggapdh sequence divergence (figures 1 and 3) . taken together, barcoding and phylogenetic analyses demonstrated that the new african bat isolates belong to only one species, which are exclusive to african bats so far and display intra-specific variability (genotypes) insufficient to represent more than one species. the results supported the classification of this trypanosome as a new species designated as trypanosoma livingstonei n. sp., which did not belong to any known subgenus. we determined 3-4 sequences of full-length sl unit repeats from each of three selected isolates from t. livingstonei. the results showed large repeats, varying among and within the isolates as follows: 1315 bp for tcc1304, 1322 and 1326 bp for tcc1933, and 1323, 1347 and 1363 bp for tcc1948. the sl sequence alignment revealed that the 39 bp exon, which is conserved in all trypanosome species, can display different nucleotides as observed in one sequence of the isolate tcc1933 (figure 4a ), whereas the intron sequences (110 bp) were identical for all three isolates (figure 4a ). the intergenic regions were quite variable in length and sequence inter-and intra-isolates; tcc1948 showed the most highly divergent sequences (figure 4b) . alignments restricted to the exon and intron sequences (sl transcript) enabled an evaluation of the genetic relatedness of t. livingstonei with all available trypanosomes permitting reliable alignments, namely t. sp. bat, t. vespertilionis, t. rangeli and t. conorhini. t. dionisii and t. erneyi sequences could be partially aligned whereas all other species, including t. cruzi, resulted in inconsistent alignments. t. livingstonei largely diverged in their intergenic sequences from all these species (data not shown). all sl transcript sequences from t. livingstonei isolates clustered together and their relationships with the other species (figure 4c ) corroborated ssu rrna and ggapdh data (figures 1 and 3) . additionally, we inferred the putative sl secondary structure (sl transcripts) from t. livingstonei and compared it with that from t. sp. bat. the results showed a similar general secondary structure as a consequence of their similar sl transcript sequences (figure 4d ). the sl secondary structure of t. livingstonei slightly differed from those inferred for t. vespertilionis and t. rangeli, whereas t. dionisii, t. cruzi and t. erneyi exhibited clearly different sl secondary structures [44; data not show]. the sequence of whole sl repeats from t. livingstonei revealed a copy of the 5s ribosomal rna (5s rrna) gene inserted into the intergenic region in the same orientation as the sl gene. the same arrangement was demonstrated in this study on the sl repeats from t. sp. bat and t. vespertilionis. the 5s rrna sequences from t. livingstonei were almost identical to those from most trypanosomes [19] . however, one nucleotide substitution (a/g at position 18) was found in the sequences from two (tcc1304 and 1948) of the three isolates from this species; this polymorphism was not detected in any other trypanosome 5s rrna. light microscopy of t. livingstonei blood and culture forms and behaviour in cultures trypanosomes found in bat blood from which the t. livingstonei isolates were derived were large trypomastigotes with a broad body and a pointed posterior end, a markedly frilled undulating membrane and a short free flagellum. the small kinetoplast occupied a marginal position adjacent to the rounded and nearly central nucleus and several surface striations (figure 5a,b) . dividing forms were not observed in blood smears. the first forms observed in haemoculture were epimastigotes arranged in rosettes and attached by their flagella (figure 5c ). free epimastigotes in the supernatant predominated during the log-and mid-phase cultures, and these forms are pleomorphic, with bodies varying in length from 16.0 to 29.0 μm (with an average of 22.3 μm) and from 1.1 to 2.0 μm in width (average of 1.56 μm), with most forms displaying long free flagellum (average 15.5 μm length) (figure 5d ). both rounded forms and large epimastigotes were observed as dividing forms at log-phase (figure 5d) . flagellates from the mid-log cultures are mostly large epimastigotes with a prominent undulant membrane and long flagella, and they were long and slender trypomastigotes; both are dividing forms (figure 5e ). at stationary cultures, small and slim flagellates with a large and nearly terminal kinetoplast resembling metacyclic trypomastigote forms were predominant (figure 5f ). when co-cultivated with a monolayer of hela mammalian cells at 37°c, t. livingstonei epimastigotes developed into wide free-swimming trypomastigotes in the supernatant (figure 5g) , with a resemblance but a smaller size than the bat blood forms (figure 5a,b) . these trypomastigotes were dividing in early cultures (figure 5g ), but attempts to successively culture them were unsuccessful. the development of epimastigotes into broad trypomastigotes under similar culture conditions is a feature of megatrypanum spp. [1, 45] . t. livingstonei was unable to invade and develop within mammalian cells, a feature of all schizotrypanum species [10] . scanning electron microscopy (sem) analyses of t. livingstonei cultures showed a diversity of forms including the following: a) large rosettes of epimastigotes united by flagella with one to three body torsions ( figure 6a) ; b) a ruffled area near the cytostome, an invagination of the membrane close to the flagellar pocket shown by sem as a small opening near the emergence of the flagellum (figure 6b,c) ; c) long and slender epimastigotes with noticeable body torsions and a dilated anterior extremity constituted by the joining of the flagellum and cell membranes ("undulant membrane") before the emergence of the flagellum (figure 6d,e) . the ultrastructural organisation of t. livingstonei epimastigotes (tem analysis) revealed all common organelles of trypanosomatids. however, some features should be mentioned as follows: a) the cytostome (figure 7a,b,c) , which forms together with the flagellar pocket the main structure involved in the endocytic process; b) a large number of reservosomes, which are compartments that accumulate endocytosed macromolecules found at the posterior region of epimastigotes (figure 7a) ; c) the compacted diskshaped kinetoplast structure (figure 7a,b,c) . the small trypomastigotes from end-phase t. livingstonei cultures (tc100 medium), which most likely correspond to metacyclic forms, were incapable of infecting balb/c mice. this absence of infection was confirmed by blood examination using the microhaematocrit from two to 15 days post-inoculation and, after that, by haemoculture. epi-and trypomastigotes were unable to infect triatomines (rhodnius neglectus and triatoma infestans). 1295, 1298, 1304, 1902, 1933, 1935, 1947, 1948, 1953, 1954 , 2339 and 2348, whose bat hosts and locality of collection in mozambique, africa, are in table 1 . type host: chiroptera, rhinolophidae, rhinolophus landeri. additional host: chiroptera, hipposideridae, hipposideros caffer. locality: mozambique, province of sofala, district of chupanga (s18°02′ e35°34′), zambezi valley and the gorongosa national park (s18°58′ e34°21′). morphology: t. livingstonei exhibits large and wide blood trypomastigotes (average 32.4 μm length and 7.8 μm wide) with several striations, small kinetoplast, frilled undulating membrane and a free flagellum averaging 11.0 μm in length. epimastigotes from log-phase cultures are mostly slender and pointed at posterior ends, ranging from 16 etymology: the name was given because trypanosoma livingstonei n. sp. was first discovered in bats captured in chupanga, mozambique, a small village in the margin of the zambezi river, where mary livingstone, the wife of david livingstone, died of "fevers" in 1862; her grave remains in an small cemetery from a portuguese mission practically destroyed by the mozambique wars. for a better appraisal of the genetic diversity and evolutionary history of trypanosomes, and for their reliable classification and phylogenetic inferences, studies must include trypanosomes from all vertebrate classes, representative of orders, genera and species, by using molecular phylogenetic approaches. bats are among the most common hosts of a large variety of trypanosomes in africa, asia, south america and europe. however, our knowledge of their genetic diversity, hosts, vectors, life cycles, pathology, distribution and phylogenetic relationships is restricted to a few species. almost all available data are about the species of the subgenus schizotrypanum because bat trypanosomes in this subgenus are the closest relatives of the human pathogen t. cruzi [8] [9] [10] [11] 13, 14, 22, 25, 28, 46] . however, several bats around the world harbour a plethora of trypanosome species, most of which are morphologically assigned to the subgenus megatrypanum [1, 2, 4, 6, 7, 47] . in this study, we surveyed trypanosomes in blood samples from bats of old world-restricted families rhinolophidae and hipposideridae captured in mozambique. we obtained 11 haemocultures from r. landeri and 3 from h. caffer. morphologically, the large trypomastigotes found in bat blood smears would be assigned to the subgenus megatrypanum. however, multilocus phylogeny validated in this subgenus only the trypanosomes from ruminants allied to t. theileri [16, 17, 19, 45] . with the exception of artiodactyls, bats were the main hosts of trypanosomes morphologically classified in the subgenus megatrypanum [1, 7] , so a thorough phylogenetic analysis of bat trypanosomes was required to warrant their exclusion from this subgenus. the phylogenetic positioning of t. livingstonei and t. sp. bat, both of which are morphologically compatible with the subgenus megatrypanum, support the exclusion of bats as hosts of species in this subgenus. the morphology of t. livingstonei blood and culture forms largely differs from those of the megatrypanum species. however, epimastigotes of this species developed into large trypomastigotes resembling blood forms under a monolayer of mammalian cells at 37°c, a process also observed for the megatrypanum trypanosomes [1, 45] . in fact, t. livingstonei blood and culture forms exhibited unique morphological features as shown by light and sem microscopy. this new species exhibited a cytostome, reservosomes and a disk-shaped kinetoplast, all of which are absent in species of the megatrypanum and common to those of schizotrypanum. this is the first study to use tem and sem to analyse a bat trypanosome not classified into schizotrypanum. there are no unambiguous differences in the overall ultrastructural organisation that would be useful for distinguishing t. livingstonei from schizotrypanum trypanosomes, even though the new species strongly diverged with all molecular markers and in several biological features such as the inability to develop inside mammalian cells and lack of infection in mice and triatomine bugs. the vectors of t. livingstonei are so far unknown; in this work we demonstrated its inability to infect t. infestans and rhodnius neglectus in accordance with the fact that triatomines cannot be the vectors of this species because they do not occur in africa. in africa and europe, bat bugs (cimicids) are the vectors of t. dionisii and t. (megatrypanum) incertum [6, 7] . the bat restricted stricticimex brevispinosus was found to be infected by a megatrypanum trypanosome in africa [48] . in addition, sand flies were incriminated as vectors of t. (megatrypanum) leonidasdeanei in south america [49] . the barcoding of new african bat trypanosomes morphologically assignable to the subgenus megatrypanum through both v7v8 ssu rrna and fflb has shown similar sequences and profiles for all the new isolates, which were shown to be highly different from the barcodes of other trypanosomes from bats and other hosts [10, 42, 43] . in all the inferred phylogenetic analyses, the new bat trypanosomes always tightly clustered together, forming a homogeneous clade separated by sufficient genetic distances from all other trypanosomes to allow their description as a new species, that is, t. livingstonei n. sp., which does not nest within any known subgenera. for insect and plant trypanosomatids, sl genes have proven to be valuable for identifying the genera and species of cultivated flagellates, as well as for the barcoding of trypanosomatids directly from their hosts [50] [51] [52] [53] . sl rna genes have also been used for species identification and genotyping of t. cruzi and t. rangeli [22, 26, 27, 54] , t. vivax [55] and t. theileri [17, 19] . the characterisation of whole sl gene repeats in t. livingstonei showed a larger length and more polymorphic sequences among isolates of the same species and repeats of the same isolate, when compared to other trypanosome species. in addition, this species enclosed a copy of 5s rrna within its intergenic region, as reported before for t. vivax, t. conorhini, t. rangeli, t. desterrensis, t. theileri and t. melophagium, but absent in t. cruzi, t. cruzi marinkellei, t. brucei and t. lewisi [17, 19, 21, 44, 45, 55, 56] . here, the comparison of primary and secondary structures from the sl rrna of t. livingstonei and other trypanosomes corroborated its close relationships with the trypanosomes that nested into a strongly supported (despite being highly heterogeneous) major clade containing african, europe and south american species from bats (t. sp. bat and t. vespertilionis), t. rangeli, t. conorhini and trypanosomes from monkeys and civets. the trypomastigotes we found in blood smears from bats infected with t. livingstonei resembled those denominated as t. heybergi-type and described for the african megatrypanum trypanosomes t. leleupi, t. mpapuense, t. morinorum and t. thomasi. these species, which could all be synomies, were reported in bats from congo, zambia, kenya and tanzania [1, 3, 7, 47] . our findings corroborated that african species of rhinolophus and hipposideros bats harbour trypanosomes morphologically similar to t. heybergi. however, t. (megatrypanum) leonidasdeanei and t. (megatrypanum) pessoai were reported in south american bats and also described as resembling t. heybergi [49, 57] . nevertheless, no cultures, dna sequences or blood smears were available from any t. heybergi-type trypanosomes, which prevented the molecular comparison between previously reported species and our new isolates. bat species harbouring t. livingstonei are endemic to sub-saharan african bats, although their genera, rhinolophus and hipposideros, are widespread throughout asia, oceania, europe and africa, but both are absent from the new world. bats may have originated in laurasia (~65 mya), and bat trypanosomes should have diverged since the great diversification/expansion of bats in the eocene [29] [30] [31] . a long past and extensive bat radiation, recent movement of bats across large geographic distances (even large oceanic barriers but not across the atlantic ocean), and incomplete bat palaeontology have complicated the studies about the origin and dispersion of bat trypanosomes. associations between bats and their trypanosomes, and an evaluation of possible paleontological and eco-biogeographical scenarios could account for the origin, genetic diversity, relationships and current distribution of these parasites and are crucial for understanding their evolutionary history. there is an urgent need for an extensive taxonomic revision of the genera trypanosoma on a strongly supported phylogenetic basis that firstly requires the molecular analyses of a large sampling representative of host species and geographic ranges. this may allow for the description of several new species and the creation of new subgenera to accommodate new species that formed clades without correspondence to any subgenera previously proposed by hoare [1] . to meet these objectives, new trypanosome cultures should be obtained and deposited in reference collections. the naming of any new trypanosomatid species should be considered valid only when supported by sound and broad phylogenies (using at least ssu rrna and ggapdh genes). however, the description of new trypanosome species based on small dna sequences, accompanied or not by the morphology of blood flagellates (mostly because cultivation have failed), have been accepted [58] [59] [60] . we are designating the new african bat isolates as t. livingstonei on the basis of its position in the trypanosoma phylogenetic trees inferred using ssu rrna and ggapdh genes, its genetic distances from other species and also taking into account its peculiar sl rna gene repeats. morphological features and information regarding host species, and its behaviour in culture and in mice complement the species description. these data can be valuable for comparative studies of the cellular biology, host-parasite interactions, ecology and evolution of trypanosomes. the phylogenetic evidence produced by this study underscores the great genetic diversity of trypanosomes in bats around the world. t. livingstonei fell at the edge of the t. cruzi clade, which comprises all bat trypanosomes sampled to date regardless of whether they are from africa, europe or south america. the position of t. livingstonei at the base of the t. cruzi clade further supports the hypothesis that the clade was ancestrally a group of bat-restricted parasites that evolved exclusively in these hosts and later jumped at independent times to mammals of other orders. in the most likely scenario, the trypanosomes from several mammalian orders nested into this clade, including those from african and australian terrestrial mammals, evolved from a bat trypanosome. other explanations require multiple jumps into bats, which seem less probably. apparently, this ancestral bat trypanosome gave rise morphologically, biologically (different life cycles and vectors), ecologically and genetically distinct species. the positioning of t. livingstonei in all inferred phylogenies provides evidence that the t. cruzi clade derived from a bat trypanosome, 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(euglenozoa: trypanosomatidae) and the extension of the host range of trypanosoma copemani to include the koala (phascolarctos cinereus) trypanosoma irwini n. sp (sarcomastigophora: trypanosomatidae) from the koala (phascolarctos cinereus) morphological polymorphism of trypanosoma copemani and description of the genetically diverse t. vegrandis sp. nov. from the critically endangered australian potoroid, the brush-tailed bettong (bettongia penicillata) (gray,1837) trypanosoma livingstonei: a new species from african bats supports the bat seeding hypothesis for the trypanosoma cruzi clade we would like to thank many people who kindly helped us with the fieldwork in mozambique. we are particularly grateful to our friends carmen e josé martins for their delightful company and hospitality in their house in chupanga. we are deeply indebted to carlos pereira and the staff of the gorongosa national park. we are also grateful to laerte b. viola, arlei marcili, bruno r. fermino and many other students for their efforts in bat capture and sample collection. this work was supported by grants from the conselho nacional de desenvolvimento científico and tecnológico (cnpq) within the proafrica, protax, and universal programs to mmgt and epc. ll is a postdoctoral fellow sponsored by são paulo state research support foundation (fapesp) process nº 2012/14985-6, espinosa-álvarez o is a phd student sponsored by cnpq (protax). the authors declare that they have no competing interests. key: cord-329050-vzsy6xw1 authors: nabi, ghulam; wang, yang; lv, liang; jiang, chuan; ahmad, shahid; wu, yufeng; li, dongming title: bats and birds as viral reservoirs: a physiological and ecological perspective date: 2020-09-22 journal: sci total environ doi: 10.1016/j.scitotenv.2020.142372 sha: doc_id: 329050 cord_uid: vzsy6xw1 the birds (class aves) and bats (order chiroptera, class mammalia) are well known natural reservoirs of a diverse range of viruses, including some zoonoses. the only extant volant vertebrates, bats and birds have undergone dramatic adaptive radiations that have allowed them to occupy diverse ecological niches and colonize most of the planet. however, few studies have compared the physiology and ecology of these ecologically, and medically, important taxa. here, we review convergent traits in the physiology, immunology, flight-related ecology of birds and bats that might enable these taxa to act as viral reservoirs and asymptomatic carriers. many species of birds and bats are well adapted to urban environments and may host more zoonotic pathogens than species that do not colonize anthropogenic habitats. these convergent traits in birds and bats and their ecological interactions with domestic animals and humans increase the potential risk of viral spillover transmission and facilitate the emergence of novel viruses that most likely sources of zoonoses with the potential to cause global pandemics. the coronavirus disease 2019 caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) was characterized as a global pandemic on 11th march 2020 (world health organization, 2020) . the covid-19 pandemic has had a dramatic socio-economic impact due to its exceptionally rapid spread and higher number of deaths (cash and patel, 2020; weiss and murdoch, 2020) , particularly among older age groups (mahase, 2020) . epidemiological research has revealed that free-living bats are likely the native host of the sars-cov-2 (zhou et al., 2020) . other recent viral epidemics are also believed to have originated from either bats or birds (calisher et al., 2006; chan et al., 2015; olival et al., 2017; nabi et al., 2020) . indeed, bats or birds are thought to host many pathogens (morse et al., 2012; chan et al., 2013; hayman, 2016; olival et al., 2017; woolhouse and brierley, 2018) . the relatively high number of zoonoses carried by birds and mammals is highly correlated with their diversity at the order level (mollentze and streicker, 2020) . birds (class aves) and bats (order chiroptera, class mammalia) have both undergone dramatic adaptive radiations; there are over 10,000 bird species (avibase; https://avibase.bsc-eoc.org/) and 1,400 bat species, and both taxa have a global distribution (wilson and mittermeier, 2019) . the high diversity of birds and bats provides an abundance of potential reservoirs for a diverse range of viruses (calisher et al., 2006; hayman, 2016) , especially recently emerging, high-profile zoonoses j o u r n a l p r e -p r o o f (olival et al., 2017; woolhouse and brierley, 2018) . why are birds and bats reservoirs of so many zoonotic viruses chen et al., 2014; hayman, 2016; olival et al., 2017; miłek and blicharz-domańska, 2018; venkatesh et al., 2018; wong et al., 2019) ? one explanation lies in their many shared, convergent features, such as small body size, high population densities, close social interaction, spatial mobility, and the ability to colonize anthropogenic environments chan et al., 2015) . these features predispose birds and bats to act as viral reservoirs and to transmit viruses to other vertebrates, including humans. however, few studies have compared the physiology and immunology of these ecologically, and medically, important taxa (caviedes-vidal et al., 2007; mollentze and streicker, 2020; song et al., 2020) . this paper reviews convergent traits in the physiology, immunology and flight-related ecology of birds and bats with the aim of a better understanding of why these species are such important reservoirs of viral zoonoses, and the potential risk of bat and bird viruses infecting humans. wild birds are reservoirs of many emerging zoonotic viruses (reed et al., 2003; abulreesh et al., 2007) . for example, a large variety of influenza a viruses are hosted by wild aquatic birds in the orders of anseriformes and charadriiformes (olsen et al., 2006) . approximately 300 avian species have been confirmed to carry the west nile virus (cdc, 2019) and also others gamma-and delta-covs have been detected in multiple avian orders on all continents hughes et al., 2009; chu et al., 2011; chamings et al., 2018) . the majority of viral infections in birds are either typically of low pathogenicity or asymptomatic (olsen et al., 2006; kuiken, 2013; lycett et al., j o u r n a l p r e -p r o o f journal pre-proof 2019). however, in recent years, several birds-borne viruses, such as the highly pathogenic avian influenza virus (hpaiv) a (e.g., h5n1 and h7n9) and infectious bronchitis viruses (ibv), have caused major epidemics and mortality among humans or domestic animals (alexander, 2007; bui et al., 2016; wang et al., 2020) . bats, primates, and rodents not only have the greatest viral richness among mammals but also harbor a higher proportion of zoonotic viruses than other mammalian taxa (olival et al., 2017; mollentze and streicker, 2020) . bats host a greater diversity of viruses than non-flying mammals, including the paramyxoviruses (drexler et al., 2012) , the rhabdoviruses (rupprecht et al., 2017) , the hepaciviruses, the pegiviruses (quan et al., 2013) , and the influenza a viruses (tong et al., 2013) . the updated bat-virus database indicates that 301 bat species host viruses with all known viral genomic structures and replication strategies according to the baltimore classification system (http://www.mgc. ac.cn/dbatvir/; (chen et al., 2014; hayman, 2016) . bats are believed to host the ancestors of all major mammalian paramyxoviruses (drexler et al., 2012; hayman, 2016) ; those hosted by non-flying mammals and birds originated from bats (drexler et al., 2012) . in the past two decades, domestic mammals and humans have contracted several viruses from bats including sars-cov-2 (zhou et al., 2020) , the virus that is causing the current global pandemic, but also sars-cov, the middle east respiratory syndrome coronavirus (mers-cov), the ebola virus, the marburg virus, and the rabies virus (cui et al., alpha-and beta-covs (woo et al., 2012) . birds (particularly aquatic birds) are natural hosts of the influenza a virus (olsen et al., 2006; webster et al., 1992) , but bats also host influenza a-like viruses (tong et al., 2013; zhu et al., 2013) and the conventional influenza a virus can infect bat cells (zhou et al., 2014) . the fact that they are both capable of flight means that birds and bats have coexisted within a broad range of spatial niches for over 50 myr (veselka et al., 2010) . it is therefore unclear whether coronaviruses and influenza a virus were transmitted from birds to bats or vice versa (brunotte et al., 2016) . it is worth noting that the human influenza a virus is thought to have come from an avian ancestor, with pigs as an immediate host, approximately 100 years ago (gammelin et al., 1990; scholtissek, 1996) . because they are both endothermic vertebrates, birds and bats should be subject to similar selective pressures on flight-related morphological and physiological traits (mcguire and guglielmo, 2009 ). the convergent traits of miniaturized body size, enhanced metabolic rate and antioxidant capacity, prolonged lifespan, a short but efficient digestive tract, and possessing some specific immunological features relative to non-flying mammals are thought to be the result of functional constraints on evolution imposed by the demands of powered flight (thomas and suthers, 1972; norberg, 1990; caviedes-vidal et al., 2007; costantini, 2008; munshi-south and wilkinson, 2010; song et al., 2020; ) . unlike non-flying mammals which tend to increase in size over evolutionary time (cope's rule; laurin, 2004) , the evolutionary trend in birds and bats has been towards miniaturization; the mass of flying birds ranges from 1.5 g to 15 kg, and for bats from 1.5 g to 1.5 kg ( fig. 1 ; norberg, 1990) . in the volant groups, although the j o u r n a l p r e -p r o o f energetic costs (per unit body weight) can vary with the type of flight (e.g., sustained flapping flight, short flight, and gliding flight; guigueno et al., 2019) , in general, the energetic cost (per unit body weight) of flight is approximately comparable in birds and bats (thomas and suthers, 1972; munshi-south and wilkinson, 2010) . compared with non-flying mammals, both birds and bats have significantly higher metabolic demands for volant flight (norberg, 1990; guigueno et al., 2019) . the enhanced metabolic demands for powered flight in birds and bats are thought to have driven the evolution of reduced cell and genome sizes (gregory, 2001; 2002; organ et al., 2007) . for example, bats have the smallest genome size (∼1.6 to 3.54 gb) of all mammals, and their dna loss/gain ratio is ∼4.3-fold greater than that of other mammals (kapusta et al., 2017; teeling et al., 2018) . similarly, birds have the smallest genome size of all vertebrates (tiersch and wachtel, 1991; gregory et al., 2009 ). furthermore, the reduction in dna in both birds and bats has proceeded mostly through the deletion of large segments (>10 kb) events and gene loss (zhang et al., 2014; kapusta et al., 2017) . the acquisition of powered flight and the evolution of smaller genome size in birds and bats is believed to have been achieved by streamlining genomic structure and reducing genomic redundancy (teeling et al., 2018) . in endotherms, body size is positively correlated with life span (speakman, 2005) . however, bats and birds have a substantially longer life span than non-flying mammals of similar body size ( fig. 1 ; austad and fischer, 1991; prinzinger, 1993 , healy et al., 2014 . on average, birds live up to four times longer than similar-sized mammals (lindstedt and calder, 1981; holmes and ottinger, 2003) , and bats live 3.5 times longer than similar-sized, non-flying, placental mammals (austad and fischer, 1991; wilkinson and south, 2002) . the mechanisms underlying the relatively long j o u r n a l p r e -p r o o f life spans of birds and bats are associated with their enhanced capability for preventing oxidative damage to mitochondria and nuclear dna (costantini, 2008; munshi-south and wilkinson, 2010) . because of their longer life spans, widespread distributions and population connectivity, both bats and birds are exposed to a broader range of environments which may increase the probability of these taxa accumulating zoonotic pathogens over time (figuerola and green, 2000; munshi-south and wilkinson, 2010; lucas, 2016) . in both birds and bats, the size and mass of the digestive tract have been minimized to reduce weight during the flight (caviedes-vidal et al., 2007) . however, both birds and bats have more efficient digestive systems than non-flying mammals (caviedes-vidal et al., 2007) . there has also been a striking convergence in the gut microbiomes of birds and bats that tends to be independent of diet or phylogeny (song et al., 2020) . despite their smaller body size, the capacity of bats and most birds to fly allows them to easily escape from unfavorable conditions and predation (healy et al., 2014) . given that the immune system is highly conserved in amniotes, the basic structure and function of responses to viruses are broadly similar in mammals and birds (schat and kaiser, 2014; wigley, 2017) . however, birds and bats as volant groups, some specific immunological features absent in non-flying mammals have enabled them to coexist with viral pathogens (table 1 ; zhang et al., 2013; schat and kaiser, 2014) and act as natural reservoirs for emerging viruses (brook and dobson, 2015; chan et al., 2013) . birds lack lymph nodes but have a specific primary lymphoid organ, the bursa of fabricius. birds have heterophil in their white blood cells that is the functional equivalent of mammalian neutrophil (schat and kaiser, 2014; wigley, 2017) . compared to mammals, birds have different repertoires of j o u r n a l p r e -p r o o f journal pre-proof toll-like receptors (tlrs), inflammatory cytokines and other immune molecules . genes related to innate immunity in birds are initiated immediately during antiviral responses. for example, the duck major histocompatibility complex type i (mhc-i) and interferon-induced protein with 5 tricopeptide repeats (ifit5) are initiated in response to h 5 n 1 virus inoculation (vanderven et al., 2012) . avian immunoglobulin y (igy) is a functional counterpart of mammalian igg and ige, providing defense against infections. however, igy can not activate the complement system and promote hemagglutination inhibition (warr et al., 1995; zhang et al., 2017) . these specific features of the avian immunological system enable birds to be tolerant of many viruses . such notion has been validated by among mammalia groups, bats possess very similar virus-sensing pattern recognition receptors (prrs) and conserved immune systems. however, bats appear to control viral replication by initiating an innate immune response earlier than non-flying mammals brook and dobson, 2015) . there are other critical differences in the adaptive immune response between bats and non-flying mammals. first, bats have a diverse antibody repertoire with relatively lower avidity and a weaker association with antigens . second, bats exhibit a delayed, or differential, peak of primary antibody response and a slow secondary antibody response relative to rodents, primates, and ungulates chan et al., 2013; pavlovich et al., 2018) . in bat genomes, genes in the type i interferon family, the mhc-i, and natural killer-cell receptors, are known to be highly expanded . it has recently been suggested that the low expression j o u r n a l p r e -p r o o f of c-1-tetrahydrofolate synthase in the cells and tissues of bats compared to humans is due to antiviral replication (anderson et al., 2020) . notably, several genes, such as c-rel (a vital gene for maintaining lymphoid cell function) and the ataxia-telangiectasia mutated gene (atm) in the dna damage checkpoint-dna repair pathway, were positively selected in bat ancestors . compared with non-flying mammals, these special immunological features allow bats to mount efficient immune responses against a diverse range of viruses (banerjee et al., 2020) . the ability to fly not only significantly reduces the risk of predation but also significantly increases the ability to colonize new niches and habitats (norberg, 1990) . this enhanced mobility also means that birds and bats transport viruses over hundreds, even thousands of kilometers during migration (hill et al., 2012; prosser et al., 2013) . approximately 20% of birds are migratory (kirby et al., 2008; newton, 2008) . the migrations of billions of birds worldwide also transport viruses to stopover sites, overwintering and breeding habitats (hill et al., 2012) , although migrations can also lower infection risks by escaping from habitats where pathogen stages have accumulated and eliminating infected individuals during strenuous journey (satterfield et al., 2018) . wild birds are associated with the dispersal of covs (georgopoulou and tsiouris, 2008; chamings et al., 2018) , the influenza a virus (hill et al., 2012) , the arboviruses (west nile virus), the usutu virus, the newcastle disease virus, the avian pox virus, and the duck plague virus (georgopoulou and tsiouris, 2008; verhagen et al., 2015; satu et al., 2017) . fewer bats migrate (krauel and mccracken, 2013) but some undertake migrations of over 1,000 km (plowright j o u r n a l p r e -p r o o f allocati et al., 2016) . such migrations allow bat viruses, such as covs, the rabies virus, the hendra, and nipah viruses, to spread over long distances (calisher et al., 2006) . although migratory bird and bat species have different migration patterns, their movements, particularly during intercontinental migration, contribute to viral dissemination and also facilitate viral recombination, mutation and evolution (bahl et al., 2013; hill et al., 2016; plowright et al., 2017; lycett et al., 2019) . given that bats and birds have evolved to compete for spatial niches such as roosting sites they may interact with each other, either directly or indirectly. the bird-bat interactions include co-occurrence in the same nest (e.g., between starling sturnus vulgaris and noctule bats nyctalus noctule (myczko et al., 2016) , the predation of eggs, nestlings, or adult birds by bats (medellín, 1988; ibáñez et al., 2001; perrella et al., 2020) and the predation of bats by birds (fenton and fleming, 1976; camargo and laps, 2016; mikula et al., 2016) . perrella et al. (2020) found about 8% of bird nests were preyed by bats and 2% by reptiles. both bats and birds species richness increases in proximity to the equator due to higher ecological productivity (brown, 2014; de oliveira et al., 2018) . similarly, pathogen diversity is also greater in tropical areas compared to temperate regions, and therefore, pathogen richness in birds and bats could be higher near the equator compared to temperate regions (guernier et al., 2004) . this coexistence could allow the mixing of bats and birds viruses for the generation of recombinant, novel mutant, or reassortment of rna viruses perrella et al., 2020) . furthermore, a large number of birds and bats are gregarious with high population densities. for example, colonies of the mexican free-tailed bat (tadarida brasiliensis mexicana) can contain up to one j o u r n a l p r e -p r o o f million individuals per roost at an average density of about 4,000 bats/m 2 (mccracken and gustin, 1991) . indirect bird-bat interactions include competition for food, and for temporal, and spatial, niches (fenton and fleming, 1976; goldingay, 2009 ). therefore, these convergent features of small body size, high population densities, and spatial mobility, and the bird-bat social interactions provide the opportunity for exchanging viruses, thereby facilitating the emergence of highly pathogenic, new viruses. human activity, such as agriculture and urban development, is causing significant degradation, loss and fragmentation of bird and bat habitat (voigt and kingston, 2016; walsh et al., 2017) . although the majority of the birds and bats are susceptible to anthropogenic change, some flourish in anthropogenic environments, including cities (duchamp and swihart, 2008; johnson and munshi-south, 2017) and are well adapted to urban environments by exhibiting a suite of phenotypic traits in morphology, physiology, and behavior (macgregor-fors et al., 2012; magle et al., 2012; jung and threlfall, 2016; afelt et al., 2018; isaksson, 2018 ) . anthropogenic environments provide some human commensal species with an abundance of food and reduced numbers of parasites and predators, thereby increasing reproductive output and winter survival (minias, 2016) . the often high densities of birds and bats in anthropogenic environments facilitate viral transfer to humans (plowright et al., 2015; afelt et al., 2018) . domestic fowl and livestock are often also at high density in anthropogenic environments, in some countries close to captive wild game. this juxtaposition of domestic and wild animals provides an abundance of immediate hosts for bird and bat-derived viruses, many of which can become pathogenic once transmitted to humans . furthermore, many birds and bats are j o u r n a l p r e -p r o o f caught and transported to markets to be sold for food, traditional medicine, ornamentation, as pets or for sport hunting (mildenstein et al., 2016) . for example, at least 167 species of bats (92 species of large-bodied fruit bats and 75 insectivorous species) are reported to be hunted in africa, asia, central and south america, and across the islands of oceania (mildenstein et al., 2016) . similarly, 4,561 bird species (45.7% of all species) are caught by humans for different purposes (butchart, 2008) . generally, these captured bats and birds are sold and kept in close contact with humans and other taxa in overcrowded and unhygienic wet markets that have become epicenters for the mixing and transmission of viral pathogens aguirre et al., 2020) . the high human population density in cities increases the risk of humans becoming infected with recombinant viruses from intermediate hosts, some of having already caused global pandemics (sehgal, 2010) . the risk of cross-species transmission depends on the spatio-temporal network connecting viral reservoirs to intermediate, and final, hosts (hassell et al., 2017; plowright et al., 2017) . anthropogenic environments can, therefore, be regarded as a viral nexus where bird and bat-derived viruses, a diverse range of potential intermediate hosts, and humans, all exist in close proximity. because their primary and secondary hosts are either mildly symptomatic or asymptomatic, many bird and bat-derived viruses may spread and diversify unnoticed in anthropogenic environments (afelt et al., 2018) . the occasional transmission of viral pathogens from asymptomatic host species to new hosts can lead to either asymptomatic infection, severe disease, or death. birds and bats share a variety of flight-related physiological and ecological traits that predispose them to harbor, disperse, and transmit viruses. special features of their j o u r n a l p r e -p r o o f immune systems enable them to function as asymptomatic carriers of a diverse range of viruses. close interactions between birds and bats in the course of competition for spatial niches, further increases the probability of viral transmission, recombination, and mutation, while the migrations undertaken by many birds and bats disperse viruses over long distances. the ability of some birds and bats to flourish in anthropogenic environments increases the probability of the viral transmission to domestic animals or captive wild game, which facilitates the emergence of novel viruses pathogenic to humans. by bringing together birds, bats, domestic animals, wild game and humans, urban environments provide the ideal conditions for acquiring new viral genes, and harboring high viral burden with strains of higher transmission efficiency, thus facilitating transmission of the viruses to humans. considering that many ongoing bird and bat-derived zoonotic viruses are probably circulating, diversifying, and spreading unnoticed, the risk of such pandemics is ongoing. more transdisciplinary and interdisciplinary investigations are warranted to unravel the complex interactions connecting bat and bird-derived viruses to immediate hosts and to humans and shed light on the origin of the current covid-19 pandemic and reduce the risk of future pandemics. the relationships between body mass and life span in birds (n=1660), bats (n=260), and non-flying mammals (n=2293). birds (p < 0.001) and bats (p < 0.001) have significantly longer lifespans than non-flying mammals when controlled for body mass in a general linear model. black lines around the fitted line are the 95% confidence intervals for each taxon. body mass and lifespan data for each group were taken from myhrvold et al. 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biodiversity investigation, observation, and assessment key: cord-283756-ycjzitlk authors: simons, robin r. l.; gale, paul; horigan, verity; snary, emma l.; breed, andrew c. title: potential for introduction of bat-borne zoonotic viruses into the eu: a review date: 2014-05-16 journal: viruses doi: 10.3390/v6052084 sha: doc_id: 283756 cord_uid: ycjzitlk bat-borne viruses can pose a serious threat to human health, with examples including nipah virus (niv) in bangladesh and malaysia, and marburg virus (marv) in africa. to date, significant human outbreaks of such viruses have not been reported in the european union (eu). however, eu countries have strong historical links with many of the countries where niv and marv are present and a corresponding high volume of commercial trade and human travel, which poses a potential risk of introduction of these viruses into the eu. in assessing the risks of introduction of these bat-borne zoonotic viruses to the eu, it is important to consider the location and range of bat species known to be susceptible to infection, together with the virus prevalence, seasonality of viral pulses, duration of infection and titre of virus in different bat tissues. in this paper, we review the current scientific knowledge of all these factors, in relation to the introduction of niv and marv into the eu. bat-borne viruses with pandemic potential have been identified as the origin of a number of recent human disease outbreaks. examples include the paramyxoviruses nipah virus (niv) in malaysia and [2] and the filoviruses ebola (ebov) and marburg viruses (marv) in africa [3] . bats have also been linked with the more recent middle east respiratory syndrome coronavirus (mers-cov) [4, 5] . nipah virus, in particular, has been suggested to have pandemic potential as it is capable of limited human-human transmission and rna viruses in general have particularly high mutation rates. a human-adapted strain of niv, were it to emerge in asia, could spread rapidly due to high population densities and global interconnectedness [1] . a large, and still increasing, number of different viruses have been isolated from bats, many of which are asymptomatic in the host and are closely related to human pathogens. these viruses have the potential for cross-species transmission (i.e., -spillover‖) to other mammalian species, for example, marv in monkeys [6] and niv in pigs [7] , and to directly or indirectly infect humans [2] . a recent paper describes the infection of a wildlife biologist with a novel paramyxovirus during a field trip to south sudan and uganda [8] . a recent study found that bats have, on average, significantly more zoonotic viruses per species than rodents, which are also known to host a large range of viruses [9] . additionally, the authors estimated that viruses had a broader host range in bats, averaging 4.51 host species per virus. bat-borne paramyxoviruses have been identified in various bat species across africa, australia, south america and asia and recently the detection and characterization of paramyxoviruses in free-ranging european bats has also been reported [10] . further to this, recent evidence places bats as tentative hosts at ancestral nodes to paramyxoviridae and pneumoviridae [11] . bat species can have very broad geographic ranges [9, 12] and multiple species can share the same habitats and even roost sites [13] . studies of pteropus bats in australia and asia found they could travel hundreds of kilometers between roosting sites with their home ranges extending across national boundaries and over sea [14, 15] . factors that affect the degree and rate of contact between animals and humans are important for spillover of any zoonotic emerging infectious disease. most human outbreaks of bat-borne zoonotic diseases have been suggested to be as a consequence of human activities. for example, outbreaks of marv in africa have been linked to human contact with bat caves, for reasons such as mining operations [16] or tourism [17, 18] . deforestation has also led to bat colonies moving closer to areas inhabited by humans in search of food and roosting sites [19] . bats are known to have varying degrees of contact with domestic animals and commercial food crops [20, 21] , in particular contact of pteropus giganteus bats with date palm sap producing trees in bangladesh is considered a risk factor for human niv infection [22] . livestock can act as an intermediate host between bats and humans. the outbreak of niv in malaysia in 1998 was linked to infection of pigs via fruit bats and resulted in the culling of over one million pigs and the deaths of over 100 people [7] . similarly, in australia hendra virus is transmitted to humans via horses; to date 82 horses and seven people have been infected (four people have died). bats themselves are a known food source for humans in some areas of africa [23] and asia [24, 25] . while bats in the european union (eu) are known to harbor zoonotic viruses that can be transmitted via close contact, such as the european bat lyssaviruses 1 and 2 (eblv1 and 2), there is currently no confirmation of the presence of viruses with pandemic potential in bats in the eu (i.e., at least capable of sustained human-human transmission). however, it is important to note that this should not be taken as proof of absence of such viruses, but rather that they have not been detected during surveillance of bat populations in the eu to date. historically, the first reports of human marv cases were in laboratory workers in germany and yugoslavia in 1967, through direct contact with blood from african green monkeys (cercopithecus aethiops) imported from uganda [6, 26] . however, more recent cases of bat-borne viruses affecting humans in the eu have been isolated incidents, notably the case of a dutch tourist returning from uganda after visiting a bat cave in which marv-infected bats roost [17, 18] . similarly, as of november 2013, 10 cases of mers-cov have been detected in europe [27] , several clusters of which represent chains of transmission in which the primary case had been infected in the middle east. previous studies have demonstrated the presence of similar viruses in eu bat populations, suggesting there is a risk of spillover of related viruses in the future. the first filovirus discovered in europe that was not directly imported from an endemic area of africa was lloviu virus (llov), detected in dead insectivorous bats in massive bat die-offs in caves in spain in 2002 [28] . simultaneous bat die-offs were observed in portugal and france, although a causal relationship between llov and mortality was not shown. countries in the eu have strong historical links with many of the countries where zoonotic bat-borne viruses such as niv and marv are present and, consequently, commercial trade and human travel pose a potential route of introduction of such viruses into the eu. many patients in the 2007 ebov outbreak in uganda presented with mild clinical symptoms raising concerns that travel is possible whilst infected, [29] . other factors, such as the importation of bushmeat, including bats and body parts of primates [29] , could be potential routes of virus introduction. a qualitative risk assessment for the introduction of henipaviruses to the uk concluded that there was a low level of risk from southern asia, south-east asia and australia, through import of fruit and bushmeat and a very low level of risk through import of bat meat, horses and companion animals and through human travel [30] . however, the assessment highlighted the high levels of uncertainty, reflecting the limited data and specific details of the routes involved. a risk assessment for niv establishing in australia also identified a lack of relevant data in various areas, reflected in the high levels of uncertainty in the risk estimates [31] . in this paper we review the scientific literature relating to the risk of introduction of niv and marv into the eu, but do not formally assess the risk. we begin by reviewing the current evidence for the geographical location of the viruses and thus where a potential risk of introduction to eu member states (mss) may exist. next, we review the evidence for factors which may affect the probability of an eu introduction via the various routes, such as prevalence and transmission dynamics in bat species and survival and transmission potential of the viruses. finally we review the evidence directly relating to potential routes for introduction into the eu. the main routes we consider in detail are human travel, trade of fruit and pig products and illegal importation of bushmeat. other routes such as bat migration, the unintentional introduction of living or dead bats by aircraft and the effect of climate change are also given consideration. identification of countries which have had human cases of niv or marv is important for assessing the risk of introduction to the eu, as it identifies the areas from which human travel may be a particular risk. knowledge of the risk factors regarding human infection in these countries is also of relevance as they may suggest other potential routes of introduction, highlight similar factors which are present in the eu and may facilitate spread of the viruses, or indicate potential control measures. niv: studies suggest that human outbreaks are linked to one of two distinct nipah virus strains; niv-malaysia or niv-bangladesh [32] [33] [34] . the malaysian strain emerged in 1998 with an outbreak in commercially farmed pigs, resulting in >250 human cases reported in malaysia and singapore with a case-fatality rate approaching 40% [33] . pteropus vampyrus and pteropus hypomelanus were subsequently identified as likely natural reservoir hosts for the virus [35, 36] . in this instance, pigs were implicated as amplifier hosts with viral isolates from both sick pigs and humans showing identical nucleotide sequences [7] . the presence of large commercial pig farms with fruit trees in the vicinity meant that foraging bats could drop partially eaten fruit contaminated with niv into pig farms. pigs could then have access to this fruit and become infected with niv [37] . transmission was attributed to direct contact with infective excretions and secretions and viral spread among pig farms was due to movement of pigs [38] . there were no reported incidences of human-human transmission and there have been no further acute human cases attributed to niv-malaysia since 1999, although relapsed and late onset cases have been reported [39] . laws in malaysia preventing fruit farming in pig farming areas may have prevented further niv outbreaks. in contrast, there have been regular seasonal outbreaks of niv-bangladesh since its apparent emergence in 2001, predominantly in bangladesh, although two outbreaks have also been reported in west bengal, india, close to the border of bangladesh [40] . up to january 2014, there have been 304 reported human cases linked to niv-bangladesh with 232 deaths, giving a reported case fatality rate of 76% [40, 41 ].this appears much higher than the case-fatality rate of niv-malaysia, although direct comparison may be complicated by various biases such as method of surveillance and reporting. while human-human transmission is considered a major pathway for human infection with this strain [42] , studies in india and bangladesh suggest the main risk factor is consumption of raw date palm sap [22, 43, 44] . date palm sap is harvested from december through to march by cutting into the tree trunk and allowing the sap to flow overnight into an open clay pot [37] . infrared camera studies have demonstrated that p. giganteus bats frequently visit date palm sap trees and lick the sap during collection [45] , potentially contaminating it with niv from saliva and/or urine. other reported risk factors for human infection include consumption of alcoholic beverages made from date palm sap [46, 47] , climbing trees and contact with other niv infected patients [48] or animals [49] . a recent study investigating the role of landscape factors in niv spillover risk in bangladesh [50] found a significant correlation between niv spillover and villages with higher human population density, more fragmented forest cover and p. giganteus roost sites containing the tree species polyalithia longifolia or bombax ceiba. the geographical distribution of cases within bangladesh is shown in figure 1 . cases generally occur in areas near inland water, predominantly the ganges, which provides a suitable habitat for date palm trees. in 2013, there were cases reported from 14 districts, more than in any other year, but generally only a few cases per district; the largest number was five cases reported from manikganj [41] . the pattern of cases suggests multiple small outbreaks in different regions, rather than large outbreaks caused by one source. [40, 41] . initial laboratory investigations at the time of the niv outbreak in siliguri (india) in 2001 did not identify an infectious agent. retrospective analysis, however, identified the presence of niv antibodies in serum samples by enzyme-linked immunosorbent assay (elisa) and rna by real-time pcr (rt-pcr) in (stored) urine samples [46, 51] . sequence analysis confirmed that the pcr products were more closely related to the bangladesh strain (97.5% identity) than the malaysia strain. to date, there is no reported evidence of niv outbreaks in humans emerging in other parts of india or in any other countries. however, given the delay in identification of the siliguri outbreak and large distribution of bats that can carry niv, it is possible that more human cases have occurred where niv has not been detected or reported. additionally, surveillance for niv in bangladesh may be more sensitive due to the previous cases reported to the authorities each year. as such, wider geographical distribution of human cases of niv should not be ruled out. marv: since the 1967 outbreak of marv in laboratory workers in europe [6] , outbreaks have been confined to sub-saharan africa, although there have been reported cases of individuals acquiring infection in uganda and then travelling to the netherlands [18] and the usa [52] . human cases of filovirus infection in africa have been associated with hunting fruit bats for meat and with entry to mines or caves where large populations of fruit bat species, such as rousettus aegyptiacus, are present [18, 53, 54] . it has been suggested that human infection could be through exposure to the excretions from bats roosting in the caves [52] , although an experimental study on r. aegyptiacus bats inoculated with the hogan marv strain (originally from the south africa outbreak [55] ) did not detect virus in the faeces of infected bats [56] . one study reported -working as a miner‖ as a significant risk factor for a positive antibody result to marv, with 87% of the population who tested positive for marburg antibodies working in the local gold mines [16] . there has not been a direct food consumption transmission route reported for marv, although index cases of filovirus infection have often had suspected contact with dead primates found in the forest, with exposure thought to occur during the butchering process [29, 57] and the hunting process, which may involve the use of shotguns, potentially causing spray of body tissue and fluids [53] . identification of countries where niv and marv have been detected in bats is important to understand the potential for infected bats from these areas to directly enter the eu, contaminated trade products destined for the eu, or infected humans who may travel to the eu. knowledge of the species of bats that are susceptible to niv and marv is also a relevant factor for assessing the susceptibility of bat species present in the eu. there is a growing body of literature on the geographical distribution and range of niv and marv in animal species with particular reference to bats. a complicating factor in defining the range is that studies in bats typically report serological or rna detection results, rather than detection of infectious virus. while a seropositive result is strong evidence of historical exposure to a virus, there may be cross reactivity with related viruses, precluding exact identification of the virus to which exposure has occurred, as seen for niv and hev in australasia [58] . detection of rna demonstrates the presence of genetic material, but does not prove current presence of infectious virus. additionally, there can be difficulties in using serological assays outside their original, validated scope, for example when an existing assay is used with samples from an alternative species [59] . here, the absence of positive and negative control samples and -gold standard‖ diagnostic assays makes it hard to determine an appropriate cutoff point to distinguish between seropositive and seronegative individuals [60] . as such, positive identifications do not confirm active virus infection at the current time and, in some cases, could only be an indication of historical exposure to a related virus. in the absence of virus isolation or full genomic characterization, it cannot, therefore, be definitely confirmed whether the virus is currently present. niv: table 1 shows a summary of reported testing for niv in bat species. niv is predominately associated with asian fruit bats of the genus pteropus, which have been suggested as the natural reservoir for henipaviruses [61] . only a few studies have successfully isolated niv virus from bats. isolation has been reported from the urine of p. vampyrus [62] and p. hypomelanus [35] in malaysia and p. lylei in cambodia [63] , but at very low prevalence, with only 1/769 samples yielding a virus isolate in the cambodia study. such low prevalence could be a factor for the inability to isolate niv in test samples of bats in other studies. rna positive pcr results have been obtained for p. vampyrus in indonesia [64] and p. lylei in thailand [65] , which identified both niv-malaysia and niv-bangladesh rna sequences. niv rna has also been detected in p. giganteus in india [66] and p. vampyrus and rousettus amplexicaudatus in east timor [58] . of particular interest to the eu is the identification of henipavirus antibodies in myotis daubentonii in china [67] , as this species is also found across much of europe, although it should be noted that niv specific rna was not detected in this study and virus isolation was not attempted. while niv is predominantly associated with asia there is increasing evidence for the presence of related viruses in africa. paramyxovirus rna related to hev and niv has been detected in eidolon helvum bushmeat in the republic of congo [68] and in the faeces and urine from roosting e. helvum bats in ghana [61, 69] . viral concentrations were estimated to be low using rt-pcr assays [61] . other studies have identified henipavirus antibodies in eidolon dupreanum and pteropus rufus in madagascar [70] . a recent study combined genetic and serological analyses to determine the extent of connectivity among e. helvum populations across central africa. antibodies to henipaviruses were present in bats from all locations with henipavirus seroprevalences reported to be between 25%-85.7%, with an overall average of 41.6% [71] . however, the presence of infection on isolated islands suggested that large population size and connectivity may not be responsible for viral persistence. these studies do not confirm the specific presence of infectious niv virus in bats in continental africa and madagascar, but they now constitute a reasonably substantial body of work, from a number of independent sources, which suggest increasingly strong evidence for the presence of henipaviruses in bats that have a geographical range outside of asia and oceania. marv: table 2 shows a summary of reported testing for marv in bat species. there are several studies reporting the prevalence of marv in bats in caves in africa including the countries of gabon, uganda and the democratic republic of congo (drc) [13, [72] [73] [74] [75] [76] . marv is now considered endemic in r. aegyptiacus bats in gabon [76] and, in general, those bat species which serve as potential reservoirs for marv are endemic to regions of central africa. there is little evidence for the potential of marv to occur outside africa at this point, although there are few published reports of testing for this virus on other continents; a study in india showed that none of 140 bats tested, including p. giganteus (n = 31), cynopterus sphinx (n = 30) and megaderma lyra (n = 79), were positive by pcr for marv rna [66] . within africa, there are also reports of antibody or rna evidence of marv infection in bat species other than r. aegyptiacus, such as rhinolophus eloquens, miniopterus inflatus [13] and hypsignatus monstrosus [75] , but reports are less frequent. this highlights the importance of knowledge on the exact species of bats for the purpose of risk assessment, suggesting the main zoonotic risk is likely from r. aegyptiacus. in an experimental study, marv was found to be present in the blood and saliva of viraemic r. aegyptiacus bats but not in their faeces or urine [56] , suggesting that close contact between adjacent bats of the same species within the roost may be important for marv transmission. marv rna has also been reported in a pooled liver, spleen, lung extract from a female r. aegyptiacus fruit bat in kenya in 2007, although tissues from other bats including 75 r. aegyptiacus from two locations were negative [77] . from an eu perspective, r. aegyptiacus are known to be present in cyprus [78] and turkey [79] and populations were found in the wild in tenerife in the early 2000s, as a result of escaped captive animals [80] , before being effectively eradicated by 2009. there is no reported evidence to suggest presence (or absence) of marv in these populations. bat host heterogeneity of virus prevalence is important both in terms of further spread of infection within the roost and spill-over to humans, e.g., through being hunted for bushmeat. some fruit bat colonies in trees in ghana have up to 1 million bats, so the prevalence may vary spatially within the colony [81] . fruit bat colonies in caves with more than 40,000 r. aegyptiacus bats are structured with juveniles more likely to be exposed to bat droppings due to their peripheral positions within the colony [74] . a study on active infection of marv in a bat cave in uganda found a higher prevalence in older juvenile bats (12.4%) than younger juveniles (2.65%) or adults (2.4%), the older juveniles were six months old at the time of capture and younger juveniles three months old [74] . thus, an important consideration is whether juveniles and adult bats have different behaviors that would affect the onward transmission of marv. for example, are older juvenile bats and non-breeding adult bats more likely to range further in migration (and hence spread disease to other hosts) than younger juveniles or the breeding adults, or to be caught by bushmeat hunters (as they are less experienced in survival)? based on data gathered in tables 1-2, those countries of the world where there is evidence of recent niv or marv infection in humans or bats are highlighted in figure 2 . we define that a country is positive for human infection only if it has had a reported human case in the last 10 years (i.e., since 2003). such a period of time without a reported case suggests that while there may still be potential for a human case in the country itself, the risk of import to the eu is extremely low. thus, malaysia and singapore are not considered positive for niv and south africa and kenya are not considered positive for marv. given the issues regarding use of serological positive results as an indicator of current virus presence, we do not consider serological positive results alone to be an indication of current viral presence in bats for this analysis. information from the iucn red list website is used to determine the geographical range of those bat species known to have been naturally infected [12] , as there is a potential for undetected viral presence in these countries. it can be seen that while recent human infections of both niv and marv appear to be limited in geographical range (the red areas in figure 2 ), there are a number of countries where bats have been identified as having the virus, but no human infection has been reported. it is also noted that the full geographical range of these bat species is extensive and in the case of r. aegyptiacus encroaches on the south-east boundary of europe, although the range of pteropus bats is much further east. however, if species serologically positive for henipaviruses are considered then m. daubentonii would be included and the geographical range would be much wider, encompassing europe and australia. viral load is a measure of the number of viral particles present in an organism or bodily fluid, e.g., the mass/volume of bat faeces, urine, saliva or bushmeat. the virus may be quantified in a number of ways including plaque-forming units (pfu), tissue culture infectious dose 50% units (tcid 50 ) or number of genome copies. currently there are no published dose-response curves that convert pfu or tcid 50 units in to risk of infection in humans or livestock animals. furthermore, the genomic copies may not all be equally infectious (due to the mutant spectrum) and some may be defective. it is not clear whether dispersion of the virions lowers the risk of transmission. however, the viral load is an important factor in a release assessment for any virus because it directly affects the risk of transmission. niv: while the studies mentioned previously demonstrate the likelihood of a continual reservoir of niv in many countries, the actual prevalence of bats currently shedding virus may be very low. as such, data on viral load is limited. however, with the application of real-time pcr, henipavirus-related sequences ranging from 110 to 34,000 genome copies per 0.3 cm 3 and 1.8 × 10 6 per ml of bat urine have been reported [68] . experimental studies have also been conducted in other animals. titres of up to 10 5 pfu/ml from brain and basal turbinates and 10 4 pfu/ml from trachea swabs were obtained from niv infected piglets [96] , with lower levels found in lung and spleen and shedding peaks during the first week post inoculation. titre data are also available for niv strains from bangladesh and malaysia in experimentally infected rodents [33, 34] . marv: one study reported that no viraemia or presence of marv rna could be detected in various tissues collected from r. aegyptiacus bats experimentally inoculated through oral or nasal routes [56] , but subcutaneous and intraperitoneal inoculation resulted in high levels detected in plasma (10 3 to 10 4 tcid 50 /ml) for five to nine days post inoculation, with titres up to 10 6.5 and 10 6.0 tcid 50 /g in the liver and spleen respectively. virus was also occasionally detected in lung, heart, kidney and salivary glands with loads up to 10 5.6 tcid 50 /g. ranges for tcid 50 /ml of marv in tissues of naturally-infected r. aegyptiacus in uganda have also been derived from a standard curve of diluted stock virus using q-rt-pcr [74] . high values of 50,000-1,500,000 tcid 50 /ml were obtained from liver, spleen and lung whilst values of 5-100 tcid 50 /ml were obtained from multiple tissues including blood and intestines. a potential factor affecting the prevalence of viruses, regarding the risk of zoonotic transmission, is seasonal pulsing, or oscillations of prevalence, with peaks in prevalence at specific times of the year. periods of higher risk are relevant to eu incursion as they will affect factors such as the probability of eu tourists contacting an infected bat and thus impact on routes such as human travel to and from niv and marv areas. indeed, seasonal pulses of marv circulation in juvenile r. aegyptiacus bats coincide with periods of increased risk of human infection [74] . the influx of susceptible young is a crucial driver of infection dynamics and bat reproduction and survival are thought to be major drivers of bat disease dynamics [97] . many bat species exhibit highly synchronised parturition which can dramatically alter population contact rates and susceptibilities. sex differences in behaviour and distribution of bats during times of the year when the potential for disease transmission is greatest may also have important implications for disease dynamics [97, 98] . the role of bat torpor in infection dynamics is largely unstudied [97] . torpor typically reduces pathogen replication rates and hence lengthens the incubation periods. a study found a clear indication for torpor being a key factor in allowing perpetuation of rabies virus through the hibernation period, through prolonged incubation period and reduced mortality [99] . this enabled the virus to persist in the population until susceptible individuals from the annual birth pulse could become infected and continue the cycle. migration and coloniality may also be important drivers of disease dynamics [97] , altered migration behaviour may result in declining immunity within specific colonies which could lead to more explosive hev epidemics [100] . niv: there is evidence of a seasonal pattern for spillover of niv to humans; a review of all human outbreaks of niv between 1998 and 2009 found that, except for the initial event in malaysia, they all occurred in the first five months of the year [65] . a longitudinal study in thailand found the bangladesh strain of niv was dominant in the urine of p. lylei bats, with highest recovery of rna in may [65] . in two sites, the bangladesh strain was almost exclusively detected between april and june while the malaysian strain was found dispersed during december to june. breeding of the bats (including mating and birthing) occurs in december to april, and may not be the only factor involved in bat transmission. there is some evidence that pregnant and lactating pteropus scapulatus and p. conspicillatus females had a significantly higher risk of hev infection [98, 100] resulting in a seasonal pattern due to seasonality of reproduction of these bats. a study on an orchard in new south wales investigated the legal shooting of pteropus poliocephalus [20] , found that the majority of bats shot were female (ratio 1:1.75) and that 65% of these females were lactating. this suggests that pregnant and/or lactating females are more likely to be foraging for food and coming into contact with crops/orchards, which could not only be eaten by horses, but also could contribute to seasonality of human spillover for viruses such as niv bangladesh, for which oral transmission to humans through date palm sap is a route. the wild date palm produces sap seasonally from mid-october to mid-march and winter (december to early february) is the traditional date palm sap gathering season in bangladesh. outbreaks of niv generally coincide with this season, appearing between december and may. marv: a study of marv in r. aegyptiacus in the python cave in uganda predicted an oscillating biannual pattern of bat prevalence in the cave, with peaks in february and march. these peaks in prevalence coincided with the birthing seasons of the bats in the cave and the temporal clustering of previous reported spillover events of marv into humans [74] . pcr data showed distinct oscillating pulses of marv infection in older juvenile bats (~six months of age) peaking in february and august that temporarily coincided with the peak twice-yearly birthing seasons. the authors speculate that the marv pulses reflect the pulses of newly weaned bats which populate the -low-lying‖ roosting areas where they are infected and may pass infection amongst themselves [74] . as they age, and are recruited into the adult population, their colony positions are taken by the next generation of juvenile bats. it is not clear whether the oscillation peaks in juvenile bats coincide with other environmental/ecological factors affecting the bats such as local shortage of fruit or migration. knowledge of survival of virus in different media and under different environmental conditions is important for assessing the concentrations of virus on contaminated fruit and infected bushmeat over time and ultimately the risk to humans. this can be used to predict the concentrations of virus on the surface of fruit after export by taking into account the duration of transport to the eu. duration of infection in both humans and bats is also important when considering the probability of shedding infectious virus on arrival in the eu. niv: the incubation period for niv in humans has been reported to be as much as 10 days [46] . surveillance in bangladesh in 2013 found that among secondary cases, who had a single exposure to niv, the delay between exposure to onset of illness ranged from 6-16 days, with a median incubation period of nine days [41] . the incubation period following a single intake of raw date palm sap to onset of illness varied between 2-12 days, with a median of seven days. a laboratory study on persistence of henipaviruses under various environmental conditions found that they were sensitive to ph, temperature and desiccation [101] . the study showed a 3-4 log 10 inactivation of henipaviruses in fruit juice (lychee, pawpaw and mango) over three to four days, although titres were still detectable after three days. there were also large variations in the half-life of the virus at different temperatures and ph values; e.g., in mango flesh, the half-life of niv was 0.2 hours for ph 3.5 at 37 °c but 30.3 hours for ph 5 at 22 °c. for the purpose of risk assessment it is the rate of inactivation which is important, rather than the limit of survival, which depends on the starting titre. marv: investigation of the 1975 outbreak of marv in germany suggested that the incubation period could be as much as nine days [26] . an experimental study on the effects of marv on the common marmoset (callithrix jacchus) found that animals became febrile after 5-6 days [102] . an experimental study looking at marv, zaire ebolavirus (zebov) and reston ebolavirus (rebov), demonstrated survival for long periods in liquid media at both room temperature and 4 °c, with virus recoverable from glass and plastic surfaces over three weeks after the start of the experiment [103] . similar decay rates were found for marv and zebov, while rebov had significantly better survival within an aerosol. although data for survival of filoviruses on fruit are not available, a study looking at survival of poliovirus, simian rotavirus and feline calicivirus in the uk found prolonged periods of survival on fresh fruit and vegetable produce at refrigeration temperatures (4-8 °c), extending beyond the shelf life of the product [104] . survival at 22 °c was poorer, but some viruses remained viable for over a week. removal of viruses using conventional chlorine washing could give more than 3 log 10 reduction, but was only <1 log 10 for poliovirus. however, it should be noted that these are non-enveloped viruses, and may, therefore, have different survival properties to the enveloped filoviruses. human-human transmission has been identified for both niv and marv. this, combined with incubation periods that could be over a week [26, 46] , suggest that human travel could be an important route for transmission of bat-borne zoonotic viruses into the eu. the recent mers-cov cases in the eu highlight the risk of virus introduction from human travel [27] . there has been one high profile case of tourism leading to an introduction of marv into the eu [17, 18] . a similar, but non-fatal, incident from a person who visited the same bat cave in uganda also occurred in the usa [52] . neither incident resulted in identified infection in other individuals. data from eurostat show that there are large numbers of people travelling between the eu and areas where niv and marv have been reported, both by air and by sea [105] . the number of immigrants from these areas settling in eu countries is generally increasing and they will naturally have strong ties to their homeland. for example, the uk censuses of 2001 and 2011 show an increase in both number and percentage of the population of england reporting to be of indian, pakistani, bangladeshi and african ethnicity [106] . combined, the indian, pakistani and bangladeshi ethnicity groups make up 5.5% of the england population and 12% of the population of london in 2011 and similar data show that there has been an increase in the number of people reporting to be born in these countries [106] . one could generally expect the individuals and their friends and families to have frequent trips to and from their native countries. data from the uk in 2006 suggested that out of 58,100 trips to the uk made by individuals using a bangladeshi passport, 20,600 were made by people returning after a temporary leave of absence [107] . figure 3 shows the migration into the eu from the niv and marv countries identified in figure 2 . it is apparent that, in terms of migration from niv countries, the uk has the highest influx of migrants of all eu countries, while from marv countries it is france. further analysis showed that the majority of bangladeshi migrants go to the uk. this might suggest that when considering the risk from humans entering the eu from bangladesh, the uk is more likely to be at risk (before considering the impact of border control measures). previous research suggests that historically the bangladeshis that travelled to the eu were predominantly from sylhet [107] , an area in the north east with very few reports of human niv cases, although more recently this may no longer be the case. it should be noted that some airports, such as heathrow, london, act as hubs for passengers going on to other destinations, which may lead to an overestimate for individual mss. however, an infected individual may be a risk even if only passing through the airport, as they will still likely have contact with airport staff and other passengers. a study looking at the risk of human-human transmission of viral haemorrhagic fevers (vhf), including marv, on airplanes found only a few events of vhf cases in the literature and no documented infection in follow up contacts [108] . the study suggested that contact trace back should be undertaken for passengers and crew with direct contact with an infected individual, passengers seated within one seat of the case and cleaning staff responsible for cleaning the section occupied by the case. however, trace back of passengers seated more than one seat away from the infected individual was not considered necessary. this suggests that close contact is thought necessary for human-human transmission and so not everyone on an aircraft with an infected individual is likely to be at risk. as such, this would mean that spread of the virus to multiple locations in an eu ms, due to the dispersion of multiple individuals infected during the flight, is unlikely. however, the lack of data regarding vhf on flights and subsequent reliance on expert opinion in this study suggests that there is fairly high uncertainty surrounding the conclusions and further evidence should be sought, particular with regard to other viruses; factors such as stronger capability for airborne transmission could lead to different conclusions. tourism may have specific risks independent of other human travel. people who travel to foreign countries on holiday are likely to be there for only short periods of time, e.g., 1-2 weeks and some, particularly ecotourists, may visit bat caves or colonies, returning home soon afterwards. entering such areas carries the potential risk of direct contact with infected bats and contamination of shoes and clothing with potentially contaminated bat guano/faeces. there is a documented incident, in the python cave in uganda, of this leading to marv infection [17, 18] , but there are numerous unofficial reports of similar such events. tourists are perhaps also more likely to be unaware of the risks of virus transmission, and therefore unaware of the appropriate precautionary measures. an author of this paper recently returned from west africa where they witnessed tourists entering an occupied bat cave and having contact with fresh bat guano. the tourists were not aware of the potential risks of virus transmission. however, the recent case of infection of a wildlife biologist with a novel paramyxovirus highlights that there is still a risk for people who are aware of and carrying out appropriate safety precautions [8] . the short duration that tourists generally spend away means that, if infected, it is likely that they will return to the eu before clinical symptoms have developed, and there is little time for decay of pathogens in guano or indeed loss of guano from the clothing or shoes. it is well established that foodborne zoonoses can pose a threat to human health. pathogens may be present in products destined for human consumption either through infection of the source product in the natural environment (e.g., contamination of growing crops by infected animals or infection of animal tissues to be consumed while the animal was alive) or cross contamination of the product during processing, typically with urine or faeces. for viruses such as niv and marv, while cross-contamination during transportation could result in the presence of virus in other products, the most likely products to be contaminated are those that are associated with outbreaks, i.e., fruit and pig meat. while pig meat has not been directly associated with human infection, live pigs were identified as the source of human infection in the niv-malaysia outbreak, although pigs in malaysia are now considered free of niv [7] . marv has not been associated with infection in any livestock animals to our knowledge (marv is known to infect primates which have been found in bushmeat seizures [109] , but in this section we only consider animal products that would be traded legally for food). drinking raw data palm sap, or alcoholic beverages made from it, have been identified as risk factors for human niv infection in bangladesh [22, 46, 47] , primarily due to the risk of direct contamination of the sap by the local pteropus bats. we have found no evidence of official trade of either of these products to the eu, although it is possible that individuals may bring alcoholic beverages with them in their personal belongings (the raw date palm sap ferments very quickly so is less likely to be brought over to the eu). while there are a number of products that involve its use, such as palm sugar, there are no reports of human infection as a result of consumption of such products. this suggests that the processing that takes place during the preparation of such products, in the case of palm sugar the sap is generally boiled, mitigates the risk. fruit bats are known to feed on a wide range of crops and they are often considered pests due to feeding in commercial orchards, although their importance in pollination is recognized [110, 111] . a study on a vineyard in india found the old world fruit bat c. sphinx was responsible for >90% damage to crops at the periphery of the vineyard [21] . as such, it is common practice to protect commercial crops through the use of measures such as netting or shooting; one study on a stone fruit orchard in sydney, australia, consisting of four hectares of nectarine trees, where shooting was known to occur in order to protect the orchard, found a total of 164 dead or injured flying foxes over 14 days at the time when the nectarine crop was ripening and being harvested [20] . niv-malaysia was isolated from fruit on tioman island, and contamination of fruit by bats is thought to be a potential route for the infection of pigs during the malaysian niv outbreak [35] . a number of outbreaks in bangladesh have been linked with consumption of date palm sap [22] , with the sap likely being contaminated with bat urine or saliva. while the date palm sap is the only identified foodborne source of human niv infection in bangladesh, bats could potentially have contact with, and contaminate with saliva or urine, any unprotected fruit grown in the region. while unlikely, if these crops are exported, there could be a risk of virus introduction into the eu. transport times can be less than 24 hours by air travel, not long enough to allow significant decay of the virus. this route is less likely for marv as, to date, it has not been detected in the faeces or urine of either experimentally or naturally infected r. aegyptiacus bats [56, 74] . faostat databases contain details of volumes of trade between eu mss and extra eu countries [112] . the eu has strong trade links with the niv and marv identified areas in figure 2 . while these databases show that there is little trade of pig products from niv and marv regions to the eu there is trade of fruit products (e.g., dates, apples, fruit juice). figure 4 shows the relative volume of trade of fruit products from these areas and eu mss. the biggest importers from these areas are the netherlands and the uk, with germany having a relatively high volume from niv areas and france from marv areas. as with the human travel, it should be noted that some countries may act as hubs for trade products, with subsequent further distribution to other destinations. figure 2 . trade data from faostat [112] . trade of infected animals for non-food purposes could also pose a risk of viral introduction. marv has been identified in the african green monkey (cercopithecus aethiops), which have historically been traded for research purposes. there is considerable movement of horses around the world, primarily for sporting events. horses are known to be susceptible to hev with infections in australia [2] , but to date there have been no reported cases of niv in horses. pet travel could also be a risk as the pets could potentially be infected with bat-borne zoonotic viruses in endemic countries. recently a kitten, infected with rabies virus, entered france from morocco demonstrating that such events can occur, even though the accompanying certificate of good health did not meet the regulatory provisions for the import of domestic carnivores from morocco [113] . data from traces suggests that movement of live animals such as pets between niv or marv countries (as defined in figure 2 ) and the eu are primarily animals not considered a risk of carrying the viruses (e.g., tropical fish) [114] . however, there are a number of movements of cats and dogs. an experimental infection study of two cats with the malaysian strain of niv found that they started to develop clinical symptoms after five days [96] . one cat developed acute clinical disease while the other recovered. virus was recovered from the tonsils and urine up to eight days post inoculation. while a very small sample size, and being aware that the experimental challenge dose is likely much higher than would be received in nature, this demonstrates that there is a potential risk of pets bringing the virus back into the eu. there is currently no quarantine regulation for third country pets, although the risk of bringing in pets from niv and hev areas is recognized by at least some mss [115] . bat guano is also a potential trade product; it is sold for use as a fertilizer in several countries including thailand, indonesia, mexico, cuba and jamaica [116] , and in theory could be imported into the eu. one study reported four of 104 bat guano samples from a bat cave in ratchaburi province, thailand, were positive for group c betacoronavirus rna, although none contained niv rna [116] . the legal importation of bats could also be a risk. the emergence of wild r. aegyptiacus bats in tenerife was believed to be a result of the escape of captive bats [80] . there are no instances of live bat imports into the eu from niv countries on traces, but there are many instances of bats used in scientific research and zoos. r. aegyptiacus bats, known to be susceptible to marv, have been kept in zoo's in the eu; in 1997 two such bats died of rabies after being imported from a dutch zoo to a danish zoo [117] . additionally there are reports of r. aegyptiacus being kept as pets. however, given the low numbers and the likely increased testing/surveillance of animals destined for these purposes the risk of importation from this route is likely very low. bushmeat is a term used to capture a variety of raw, smoked or partially processed meat that originates from the hunting of a variety of wild animals, including bats. it is well documented that bushmeat is illegally imported into both europe and the usa [109, 118, 119] and, as such, it could act as a conduit for pathogen spread. in a recent study, illegal bushmeat imported into the united states was found to contain retroviruses and/or herpesviruses [109] and henipavirus-like rna has been detected in internal organs of bat bushmeat sampled in the republic of congo [68] . the perception of bushmeat as having zoonotic potential is not well recognized among bushmeat hunters, traders and consumers; one study reported that only 24% of bushmeat hunters in sierra leone are aware of the zoonotic disease risk [120] and in a survey on bushmeat in the usa, participants in a focus group considered bushmeat to be a wholesome healthy and safe alternative to commercially produced meat from a shop [121] . in an experimental study of r. aegyptiacus bats, marv was not detected in muscle, brain or skin tissues collected after cardiac exsanguination [56] . this suggests that these tissues (including muscle) are not heavily infected, and that positive results in liver, spleen and kidney were not due to the presence of blood. if confirmed to be the case in naturally occurring infections, it could mitigate the risk of marv infection from the consumption of bushmeat if internal organs are not eaten. hunting of wildlife for food is a widely distributed practice in many parts of the world and constitutes an important source of animal protein for some rural communities. one paper reported that 83.3% of households in brazzaville, congo consumed bushmeat [122] and a survey of municipal markets identified 35 different animals species, nine of which it is prohibited to hunt [123] . economic recession over the past 20 years has driven the commercialisation of bushmeat as a trade item; bushmeat now reaches the international markets as part of the $159 billion annual global wildlife trade. the commercial trade in bushmeat occurs across almost all of tropical africa, asia and the neotropics, notably in the densely forested regions of west africa [124] . estimates of bush meat harvests in ghana are around 385,000 tons annually [125] . the bushmeat markets across west africa are nowadays dominated by small bodied, fast reproducing species such as rodents like the grasscutter (thryonomys swinderianus) [126] . there is little officially reported information on the use of bats as bushmeat, a review of 31 survey papers on bushmeat did not report anything on bats [23] , but unofficial reports and eye witness accounts suggest it is not uncommon to see bats for sale in african markets. it is possible that bats do not follow a typical bushmeat commodity chain and amounts are therefore underestimated in standard bushmeat surveys [23] . bats are often hunted for pre-arranged orders and regular customers rather than sale through wholesalers who may prefer to concentrate on larger animals with a higher value-to-weight ratio. one study estimates that 128,000 e. helvum are sold each year in southern ghana [23] . this involves a commodity chain stretching up to 400 km and involving multiple vendors. no official data regarding the size of the bushmeat trade exist as much of the trade is informal or illegal. while much trade is intra-country, trans-border trade does occur through known trade routes throughout the region and there is a limited amount of inter-continental trade from africa to europe [127] . recently a quantity of bushmeat thought to be from the central african republic was seized by french police, and was reported to include bats, although the species were not named [119] . imports of bushmeat into the uk do occur and mostly take place from those parts of africa with which the uk has close historical connections, in particular west africa [124] . residents of the uk who have their ethnic and cultural origins in central and west africa and who are returning from a visit there often bring bushmeat into the uk for their own consumption. in comparison with the domestic market in bushmeat in central and west africa the amount of bushmeat coming into the uk represents only a very tiny fraction of the total turnover [128] . a wildlife policy briefing report, which sets out bushmeat preferences in urban liberia provides a good indication of the sort of bushmeat likely to be imported into the uk, since returnees and visitors to the uk are most likely to buy their bushmeat in urban markets and are likely to reflect current local preferences [129] . the list comprises ungulates, rodents, primates and pangolins. bats do not feature in the 15 most preferred animals for taste from urban communities in west africa or in the more generic list of animal involved in the bushmeat trade in west africa. chaber et al. sampled 134 passengers from 29 flights from central and west africa to france over 17 days in june 2008 [118] . fifty-five passengers were found to be carrying fish or domestic meat and nine were carrying bushmeat. average individual consignments of bushmeat were over 20 kg, compared with 4 and 9 kg for livestock and fish. most illegal imports detected by uk border agency are small amounts and continue to be typically gifts by travellers visiting family (or returning from visiting family abroad), or seizures from tourists, business people and students travelling to the uk for the first time. most do not involve deliberately smuggled goods but are made from passengers who are not aware of the current rules and prohibitions in place for products of animal origin (poao) imports [130] . as well as personal carriage, bushmeat may be imported either by postal carriage or commercial freight to the eu. hm revenue and customs found bushmeat to constitute 3% of poao customs seizures for the period 2003-2004. some bushmeat samples entering eu states from africa do so from european transit flights, as under the single market goods can travel freely from one member state to another without checks. thus the situation in any specific member state depends on the effectiveness of border controls in other member states. the bushmeat from animals hunted in tropical forests destined to be carried to the eu is likely to be preserved in some form for the duration of the journey. the bushmeat consumed in the uk imported from west africa is most often either smoked, dried or salted [128] . because of this processing the initial load of viable organisms on the bushmeat would be expected to be reduced significantly. the average duration of smoking of bushmeat was found to be about 6 hours 30 minutes per day at a maximum temperature of 126.3 °c [131] . to preserve the bushmeat it may be frozen on arrival in the uk. freezing in general promotes virus survival and a laboratory study suggested long survival times of marv at 4 °c [103] . throughout africa and asia, bats have been used in zootherapy, which is the treatment of human ailments with remedies made from animals and their products. around 80% of the population in africa uses traditional medicine and there is also a growing interest in many developed nations [132] . there is evidence of bats being used for specific ailments in zootherapy and it is possible that they may still be used by migrants in european countries. treatment of ailments with bats include disorientation in patients with mental illness [133] , fertility medicines and post birthing remedies, [134] , the use of bat droppings of p. giganteus to treat patients with alcohol and drug addiction, [135] , and night blindness [136] . in asia, asthma is the most frequently cited disease for which bats are used as a remedy [137] [138] [139] . these therapies are frequently practiced in countries where there is evidence of niv infection in bats. kanda tribal healers in bangladesh use p. giganteus in formulations for the treatment of fever [140] , one pharmaceutical company in vietnam reportedly imported 50 tonnes of faeces of rhinolophus bats [141] . in a survey of 802 asthma patients in singapore primary care clinics on the use of complimentary therapies, 222 patients (27.7%) had used complimentary medicine out of which 99 (12.3%) used animal products, 33 (4.1%) of which had used fruit bats [142, 143] . whilst there is evidence that bats, as bushmeat, are eaten extensively in africa and asia there is little evidence of them being internationally traded or brought to the eu in personal possessions; a number of studies have investigated illegal imports of bushmeat, but rarely have bats been among the samples seized. however, these are relatively small studies and do not confirm the extent to which bats are exported as bushmeat. additionally, other animals, such as monkeys, were identified in the seized samples and are known to be susceptible to viruses such as marv. a review of possible microbiological hazards associated with the illegal importation of bushmeat concluded that although there was a lack of quantitative data relating to the microbiological risks, the risk of foodborne illness from consumption of bushmeat appeared to be very low and the risk of foodborne illness from cross contamination was also minimal [128] . normal cooking would probably destroy any viruses and bacteria present although there were no data presented to verify this. the risk from use of bats in zootherapy is not as well understood. however, while the risk of contaminated bushmeat may be low, the consequence could be very high. migration is a seasonal, usually two-way movement from one place or habitat to another, to avoid unfavourable climatic conditions and/or to seek more favourable energetic conditions [144] . some bat species are known to migrate large distances and cross national borders [15] . such behaviour will connect seemingly distant bat populations, and an infected individual could therefore act as a vector to introduce a new virus into a naï ve population. bat flights are generally short distances for the purpose of foraging, hunting, changing roost sites or social behaviour. indeed, the majority of bat species in the world are sedentary. some bats, however, particularly those in the temperate regions of the world, perform annual long distance flights [145] . bat migration typically occurs along rivers, as shown for bats in poland and central slovakia [57, 146] and tends to avoid mountainous areas [146] . with regards to bat species and geographical areas relevant to niv and marv, in congo a massive annual fruit bat migration takes place up the lulua river with hunting of the bats by villagers. direct exposure to the fruit bats may have led to an outbreak of ebov in 2007 [53] . regular mass long-distance migrations have not been reported for r. aegyptiacus [147] and a sedentary life history for r. aegyptiacus is also supported by the morphological record [148] . in contrast, some e. helvum individuals migrate more than 1000 km [145] , in some cases following the seasonal fluctuation in fruit abundance [149] . thus, one study reported that that 16 out of 85 (22%) e. helvum (22%) were migratory, although 78% (69 of 85) were non-migratory [150] . the median travel distance of the non-migratory bats was 15 km (compared to 860 km for the migratory bats) and similar to the observed daily commuting distances of r. aegyptiacus [147] . based on available data and their own capture information, it was assumed that e. helvum has a core distribution in equatorial africa, with migrations in the northern direction, e.g., mauritania and niger from may to september and towards the south e.g., tanzania, zimbabwe and zambia during the months of october and december [150] . thus, e. helvum from regions of africa north of the equator will generally migrate south in the autumn, away from europe. there is no evidence to suggest that the return migration routes in the spring would take the bats north of the sahara desert or that bats that might accidentally fly north (instead of south) in the autumn and reach europe. a review of data collected over 73 years from banding of some one million bats within europe, provides information on which bats cross national borders [145] . these data suggest there are a number of european bat species which migrate seasonally in the range of a few hundred kilometers and four species that are considered long distance migrants (regularly 3000 to 4000 km in one return flight). the migration routes are generally limited to europe, with the general trend from north-east to south-west europe. however, there are data showing movements of nyctalus noctula from russia into bulgaria [145] and it is reported that pipistrellus nathusii killed in summer and autumn at german wind turbines originated from estonia or russia [151] . an occurrence of vespertilio murinus on a north sea drilling rig confirmed that bats can fly across large bodies of sea [152] . this raises the question of whether migration of bats from africa to europe can occur, for example, across the strait of gibraltar. there have been studies in relation to the genetic diversity in ibero-moroccan bats, but this does not address the frequency of vagrant african bats flying from morocco into southern europe. colonies of r. aegyptiacus, known hosts of marv, occur in cyprus and southern turkey. no banding studies have been done and existing knowledge is based on field observations in europe [145] . in cyprus, no long distance flights are known, but seasonal altitudinal shifts have been observed [145] , which could alter contact rates with other bat species. thus, despite the growing evidence on migration of bat species within europe, there are no data to suggest whether migration of bats into europe from niv or marv endemic areas (as outlined in figure 2 ) could occur. a longer term risk factor is the gradual spatial creep of viruses due to transmission to previously uninfected species whose habitat spatially overlaps that of known infected species. for example p. vampyrus are known hosts of niv. they are not found outside of asia, according to iucn red list (see figure 2 ), but have been reported in the shaanxi region of china, close to where m. daubentonii have also been recorded [12] . m. daubentonii are also known to be present across europe and there is a report of henipavirus antibodies in three of four myotis bat species at a location in yunnan province, southern china in 2006 and 2007. this included nine of 79 m. daubentonii bats [67] . although pteropid bats are not widespread in china, henipaviruses could be introduced to china by other susceptible bat species whose habitats and ranges overlap those of pteropid bats in neighbouring countries. this raises the question of whether henipaviruses could eventually emerge in european bats. however, there are a number of additional factors that may delay and/or prevent this from occurring, such as mountainous areas providing geographical barriers to interaction of neighbouring bat populations. indeed according to the iucn redlist the populations of m. daubentonii in china and europe are not contiguous. it would be interesting to know if bats in south-east asia migrate in a north-westerly direction to the same regions as those migrant european bat species to give a -virus cross-roads‖. the risk of eu bat infection with marv due to overlapping species populations is potentially higher than niv, due to shorter geographical distances, r. aegyptiacus are already present in some european countries where their range may overlap with some migratory european bat species, and the fact that some african fruit bat species (e.g., e. helvum) migrate large distances, although generally within the sub-saharan african continent [150] . however, marv has not been isolated from any bats in cyprus or indeed northern africa, although there have been few published reports of attempts to find marv outside its normal range. additionally, marv has not been isolated from as many different bat species as henipaviruses, so the risk of virus transfer between species may be more limited. this may reflect the ubiquity of molecular receptors for henipaviruses among mammal species. there are a number of less obvious routes by which bat carcasses or products could enter europe. for example a bat strike on a long haul aircraft may result in the carcass of the bat being carried long distances across international boundaries. the remains of a bat were found in the wing flap of a boeing 767 that had flown from heathrow (uk) to ben-gurion airport in israel [153] . the plane had previously flown from ghana to london and pcr was used to identify the bat as having highest similarity with e. helvum. flying foxes and other bats were the animal species most often involved in aircraft strikes in australia between 2002 and 2011 with the majority of air strikes occurring at locations on the east coast of australia [154] . for the 16 year period (1990-2005) 66 ,392 strikes were reported to the united states federal aviation authority of which bats were involved in 0.2% [155] . this raises the question of what happens to the bat carcass remains and in particular how it is disposed of. in theory it could drop off the plane on coming into land at the destination airport as the carcass thaws or the wing flaps change position. this raises the possibility of the carcass being eaten by scavenging animals or even pet dogs or cats. accidental translocations of bats between land masses by ships or aircraft have also been known to occur, almost certainly with a far greater frequency than is actually reported [156] . as some viruses such as coronaviruses can survive for long periods in water [157] , bat guano or even dead bats transported in bilge waters of ships could, in theory, serve as route of transport of bat viruses around the world. another route, again involving aircraft, is where the bat is a stowaway either in the aircraft hold, or even the cabin itself. for example, in 2011, a bat flew through the cabin of a commercial airliner minutes after takeoff during an early morning flight from wisconsin to georgia [158] . the emergence of new viruses typically reflect change and combinations of events [159, 160] . in this respect, anthropogenic changes, and in particularly globalization, are drivers. other changes including farming practice, environmental and climate change not only affect land use but also influence zoological and ecological factors including habitat and food supply. thus, over time, there may be changes in both the range and distribution of species and intensity and nature of species' interactions. climate change is associated with extreme weather events such as drought and flood. it is most likely to be linked to the geographical distribution of fruit bats through availability of food sources; the species p. nathusii has been observed to be adapting its range in response to recent climate changes [161, 162] . this raises the question of how the range and population of fruit bats will change; ultimately, warming could convert forests to grassland savannas which are unsuitable habitats. a shift in the range of pteropid bats due to climate change could have an impact on the circulation of henipaviruses, by putting bats under stress [163] . pteropus spp. may excrete viruses more often than usual in stressful situations such as when their food is destroyed by climatic events and extreme stress can result in immune suppression which can facilitate increased shedding of the virus [100] . bats may also spread the virus between regions if they search for food in areas unaffected by flooding. additionally one study found a significant association with the dry season for spillover events [153] . in this paper we have discussed factors that should be considered when assessing the risk of introduction of two bat-borne viruses, nipah virus and marburg virus, into the eu. the routes considered to pose a significant risk of introduction into europe include human travel, legal trade and illegal importation of bushmeat. a number of other potential routes should also be considered, including, bat strikes on aircraft and bat migration, although migration may not be significant as currently there is little evidence of significant migration pathways into europe. however, it is unclear whether the absence of knowledge of migration routes into the eu from the countries identified as having infection in bats from figure 2 is because they do not exist or because their existence has not been comprehensively investigated. additionally, if niv or marv were to spread to areas on a european migration route, such as russia, then bat migration could become a greater risk. another, more long term risk for introduction to the eu could be transmission between bat species with overlapping distributions; r. aegyptiacus are hosts of marv and present in cyprus (although marv is not known to be present in bats in cyprus), where the range of this species may overlap with some migratory european bat species. it should also be noted that migration could pose a risk for other bat viruses which may be present on these migration routes. the two viruses discussed in this paper were chosen as they are not known to be present in eu bat populations, but published literature indicates their potential for causing large scale human outbreaks. there are many other bat-borne viruses of similar potential that we do not cover in detail here, but also require in depth consideration, such as ebola virus, hendra virus and mers-cov. at the time of writing there was limited and not conclusive evidence that mers-cov was a bat-borne virus [4, 5] . while the risks of introduction of other bat borne zoonotic viruses should be considered on a case by case basis, there will likely be a degree of commonality with the factors and routes discussed in this paper, especially for viruses within the same family as marv or niv, namely filoviruses such as ebov and paramyxoviruses such as hev. while there is serological evidence of henipaviruses and filoviruses on multiple continents, the isolation of infectious virus in either bats or humans is currently limited to more confined geographical areas; niv in asia and marv in central africa. human infection of niv in particular is currently limited to bangladesh and west bengal in india. given the more widespread identification of niv amongst bat species and countries in asia, it is not clear why human outbreaks appear to be confined to this region. this could reflect the route of transmission, sensitivity of surveillance and also perhaps the greater titre of niv-bangladesh in bat saliva or urine compared to niv-malaysia [33] . further knowledge of why these viruses do not currently seem to be spreading further, could help in assessing the risk of further spread, including the risk of reaching the eu. while a number of studies report high serological prevalence, actual virus infection in bats is rarely detected. this could explain why human spillover events of niv-bangladesh are fairly localised. p. giganteus roosts have been identified within 5 km of villages in bangladesh and can consist of around 650 individual bats [50] , so even a low prevalence of infection within the roost can mean that there are still sufficient numbers of infected individuals able to contaminate local food sources such as date palm sap. in this paper we have discussed risks posed by bats, regarding entry of zoonotic viruses to eu, but the ecological importance of bats should also be recognized. insectivorous bats are responsible for controlling populations of other species considered to be pests such as mosquitoes and other insects, while fruit bats feed on nectar and pollen and so provide an important function as pollinators and/or seed dispersers [164] . while the mass culling of pigs in malaysia undoubtedly helped to control the niv outbreak there, culling, or relocation, of wild bats could potentially increase levels of infection [165] . for example, research in peru found that culling campaigns failed to reduce the seroprevalence of rabies among the studied vampire bat colonies [166] . additionally culling of bats is considered by many to be unethical and methods are unavailable that comply with current standards of animal welfare. there are many alternative methods to help control virus disease, such as the use of bamboo skirts to prevent niv contamination of data palm sap in bangladesh [45, 167] , limiting potential for indirect contact between livestock and bats at a local level, use of personal protective equipment by investigators dealing with suspect cases and a vaccine against hev in horses in australia [165, 168] . this review identifies those routes which could provide a potential for introduction of niv and marv into the eu, but does not formally assess the risk associated with each route. for niv we have shown that, of the eu mss, the uk has the highest volume of relevant human travel (figure 3 ), but the netherlands has the highest volume of relevant trade (figure 4) , suggesting that the most probable route for introduction may vary between eu mss. however, to formally assess this it will be important to also take into account virus specific factors such as prevalence, titre and survival and ms specific factors such as border inspections or controls. therefore, it would be preferential to develop a quantitative risk assessment (qra), which would require large amounts of data. this review suggests that while data may be lacking to fully assess the risk for routes such as bushmeat, or indeed any other illegal activity, there are sufficient data available to assess legal routes such as volume of trade and human travel. in general, we found no evidence to suggest that the risk of niv release to the uk has changed from that reported in a previous qualitative risk assessment [30] . reported human cases of niv continue to be limited to bangladesh and an increase in the number of those cases may be due to enhanced awareness and surveillance. a number of human cases of marv have been reported in uganda recently, but again this could be attributed to better surveillance. while there is evidence to suggest henipavirus infection of m. daubentonii in china and the presence of r. aegyptiacus in the eu country of cyprus, these are not sufficient factors on their own to warrant undue concern. however, it should be noted that there is a lack of research and surveillance in this area and the evidence for absence of niv or marv in bats present in the eu is limited. human migration patterns continue to change across some areas of the eu, suggesting the frequency of human travel to niv or marv areas and corresponding illegal imports of products such as bushmeat may change. this could increase the probability of a -rare event‖ occurring, such as importation of a bushmeat sample contaminated with virus and, as has been observed in the past, a single introduction event can be enough to cause an outbreak of disease in humans. a better understanding of surveillance sensitivity and biases in reporting, and further investigations of the presence and prevalence of these viruses in both bats and humans should be carried out, as high uncertainty remains about the risks associated with these diseases and how best to prevent or limit the risk of an introduction event. this work was funded by the european union fp7 project antigone (anticipating global onset of novel epidemics 278976) and the uk department for environment, food and rural affairs (defra) project se4213. the authors would also like to thank trevor drew and tony fooks (ahvla) for their valuable contributions to this paper. the content of the manuscript was researched, written and prepared by robin simons, paul gale and verity horigan, with ideas and contributions by emma snary and andrew 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investigation of the climatic and environmental context of hendra virus spillover events bat strikes in the australian aviation industry wildlife strikes to civil aircraft in the united geographic translocation of bats: known and potential problems survival of surrogate coronaviruses in water rabies risk assessment of exposures to a bat on a commercial airliner-united states predicting the impact of climate change on livestock disease in great britain horizon scanning for emergence of new viruses: from constructing complex scenarios to online games hibernation record of pipistrellus nathusii in southern moravia climate change-linked range expansion of nathusius' pipistrelle bat, pipistrellus nathusii (keyserling & blasius, 1839) interdisciplinary approaches to understanding disease emergence: the past, present, and future drivers of nipah virus emergence the effect of bat (rousettus aegyptiacus) dispersal on seed-germination in eastern mediterranean habitats flying foxes carrying hendra virus in queensland pose a potential problem for other states ecological and anthropogenic drivers of rabies exposure in vampire bats: implications for transmission and control piloting the use of indigenous methods to prevent nipah virus infection by interrupting bats' access to date palm sap in bangladesh guidelines for veterinarians handling potential hendra virus infection in horses the authors declare no conflict of interest. key: cord-332088-5c77h0of authors: beena, v.; saikumar, g. title: emerging horizon for bat borne viral zoonoses date: 2019-10-26 journal: virusdisease doi: 10.1007/s13337-019-00548-z sha: doc_id: 332088 cord_uid: 5c77h0of bats are the only flying placental mammals that constitute the second largest order of mammals and present all around the world except in arctic, antarctica and a few oceanic islands. sixty percent of emerging infectious diseases originating from animals are zoonotic and more than two-thirds of them originate in wildlife. bats were evolved as a super-mammal for harboring many of the newly identified deadly diseases without any signs and lesions. their unique ability to fly, particular diet, roosting behavior, long life span, ability to echolocate and critical susceptibility to pathogens make them suitable host to harbor numerous zoonotic pathogens like virus, bacteria and parasite. many factors are responsible for the emergence of bat borne zoonoses but the most precipitating factor is human intrusions. deforestation declined the natural habitat and forced the bats and other wild life to move out of their niche. these stressed bats, having lost foraging and behavioral pattern invade in proximity of human habitation. either directly or indirectly they transmit the viruses to humans and animals. development of fast detection modern techniques for viruses from the diseased and environmental samples and the lessons learned in the past helped in preventing the severity during the latest outbreaks. bats are the only flying placental mammals that present all around the world except in arctic, antarctica and a few oceanic islands. they are the second largest order of mammals that evolved from one of the oldest fossil, icaronycteris, during eocene period (50 million years ago) and diverged into 925 known species which constitute 20% of [ 4800 mammalian species [29] . although the bats attribute advantages in the diverse ecosystem as pest controller (insectivorous bats) and pollinators (frugivorous bats); the worrisome fact is they act as natural reservoirs for a large number of emerging as well as re-emerging pathogens that other animals and humans can contract. moreover they gained a bad reputation in classical literatures, in which bats are associated with evils-lucifer, darkness, dracula-blood fed vampires and as omens and in modern scientific society they were obligatorily dangerous, as evolved as a super-mammal for harboring many of the newly identified deadly diseases without any signs and lesions. recent database on bat viruses from 69 countries worldwide comprises more than 4100 bat-associated animal viruses belonging to 23 viridae detected in 196 bat species [6] . recently reported 43% of the emerging and reemerging pathogens included in bioterrorism list as category a, b, c were recognized in different bat species. the emergence of bat borne zoonotic viruses significantly arise a global public health impact. many of the emerging and reemerging viruses are formidable foes for the physicians putting them into confusion due to their mutagenic nature. best example is the recent report of zika virus in india, which doesn't cause any developmental mutagenicity in children compared to the outbreak in brazil in 2015. in asia and pacific regions, bats were demonstrated as natural reservoirs for a large number of this types of emerging as well as re-emerging pathogens such as sars, ebola, marburg, nipha, hendra, tioman, menangle, australian bat lyssa virus, rabies and many encephalitis causing viruses in humans and animals [2] . sub saharan africa, where people hunt bats as bush meat is the biggest hot spot for viral spill over from bats to humans and other mammals. southeast asia is also been considered as another danger zone. a change in agent, host and environment is responsible for the emergence and re emergence of various diseases. from bats the pathogen get transmitted to humans via intermediate hosts like horses(hendra) and pigs(nipah) and different species of animals get infected by consumption of partially eaten fruits of bats and the chewed out materials of bats after extracting the juice. studies suggest bats can travel a long distance (2000-3000 km) which also develops issues of introducing new disease to the place unknown earlier. phylogenetic analysis suggests a co evolutionary relationship between viruses and the existing bats [18] . all these facts arose international scientific attention for the study on bats and bat associated viruses and it suggests that a series of events happened to precipitate the emergence of the viruses which were ancient and circulating in the bats for a long time. recent applications of conventional pcr/rt pcr, metagenomics and next-generation sequencing (ngs) technologies revealed the complexity of the bat virome, which may impact upon its reservoir capacity and consequently affect vector-reservoir host interactions. several studies showed bats as an important reservoirs for a number of rna viruses (including, lyssa, corona, paramyxo, filo and astro viruses) and dna viruses (including, parvo, circo, herpes and adeno) [3] . variation in the incidence and diversity of viruses in bats suggests that some species of bats are reservoir host and some others are incidental hosts [36] . the bat virome in frugivorous bats are less compared to the insectivorous bats [57] . more than 200 viruses were reported in bats wherein most are rna viruses. out of 60 viruses found to be associated with bats, 59 were rna viruses due to high degree of mutations and recombination [28, 56] . the first report of a transmission of a viral disease from bats to humans was a rabies virus (rabv) belonging to the lyssa virus genus [5] . rio bravo virus was the first non rabies virus to be recognized as originating from bats in 1960s [35] . majority of viruses identified in bats were belonging to flavi virus group including west nile virus and kyasanur forest disease virus [39] and the application of metagenomics helped to identify picorna viruses in bats. since a large number of different types of virus were identified in bats it is better to understand the spectrum and characteristics of viruses that bats carry. it may help to prevent and control potential emerging bat-borne diseases. further as bats are acknowledged for emerging zoonoses, identification and characterization of novel viruses from bats is needed. unlike other animals the detailed information regarding bat anatomy, ecology, importance in ecosystem and their ability to act as reservoirs for a large number of viruses which are potentially harmful for humans and animals have to be studied. moreover, knowledge regarding the antibody and cytokine synthesis in bats, pathogenicity and the pathology associated with infections are lagging. some of important pathogenic rna viruses identified in bats so far with emphasis on nipha virus transmission and few more bat borne viruses are discussed below. the oldest and most eloborately studied viruses in bat was the bat lyssa virus of family rabdoviridae. rabies virus was detected in both haematophagus and non haematophagus bats [24] . in australia, in 1998, a new lyssa virus from pteropid bats was characterized by sequencing, electron microscopy and by mouse model study [17] . australian bat lyssa virus produces non suppurative meningo-encephalomyelitis and negri bodies were detected in brain of some bats with lesions similar as in rabies [22] . in western europe, when rabies incidence was found declining among terrestrial mammals, new cases in cats were reported. later it was discovered as european bat lyssa virus 1 (eblv1) circulating among european bats got transmitted to cats [10] . the virus is also being reported in sheep, stone marten and in humans presuming cross transmission from bats to other terrestrial mammals [11] . in straw-colored fruit bats rabies virus was injected intracerebrally and it was found to be suffered with diffuse, mild to moderate meningoencephalitis with two to three cell layer thick perivascular cuffing [45] . earlier rabies bat virus (rabv) and eblv was restricted to america and europe respectively. but now many reports are there about the presence of lyssa viruses in bats of other countries including india. in asia, only few reports of rabies virus isolates; one from india and in thailand but were not confirmed by further studies. recently in india, bats sampled from nagaland hills were tested positive for rabies antibodies, but the epidemiology, prevalence distribution and pathogenicity is still incomplete. all these evidences of rabies virus antibodies in different places give a room for speculation for the presence of lyssa virus in free ranging bats in india. india reported the first bat parainfluenza virus of paramyxoviridae from a rousettus leschenaulti bat in 1966 [21] . hendra, henipa and nipha viruses of paramyxovirus group attain recognition as the twenty-first century emerging diseases [66] . hendra and nipah viruses have regular spill over from pteropus bats to humans and domestic animals. the first outbreak of hendra virus was reported in 1994, in brisbane, australia killing 21 horses and 2 humans. in addition to the affected target species, the seropositivity was reported from flying foxes which serve as the major reservoir host of the virus [16] . nipha virus disease is a heart breaking disaster in which more than one million pigs were culled. it's a newly emerged deadly infection characterized by fever, respiratory distress and encephalitis and was reported in 1999 from pigs and humans in the south east and south asian region. later a series of outbreaks were reported in various countries like bangladesh, malaysia, singapore and in india at different time points [32] . direct contacts with infected pigs were main source and a case fatality rate of 40-100% was noticed during the outbreak. the case fatality rate in 2001 outbreak in siliguri, india was found to be 68% [52] . during initial outbreak in 2001, neutralizing antibodies for nipha virus were detected in bats present throughout peninsular malaysia residing around the infected pig farms [63] . nipha virus was first isolated from urine of pteropus hypomelanus and pteropus vampyrus [51] . two strains of the virus were isolated from the pteropus lylei in cambodia and from different species of bats in indonesia, thailand and philippines [50] . all these reports suggest that the henipa virus is distributed throughout the pteropid bats in the world. an encephalitis outbreak that occurred in people of southern phillipines in 2014 is considered mostly due to horse meat consumption and to horses the virus get transmitted by eating the fruits partly eaten by bat [7] . therefore the bats are proved to be the global reservoir hosts for the paramyxo viruses. diagrammatic representation of bat transmitted nipha viral zoonoses is depicted in fig. 1 . in india a survey on 140 bats of 3 different species for the pathogenic nipha, ebola and marburg using enzyme linked immunosorbant assay showed nipha virus specific igg from serum sample of a bat [59] . the amino acid and nucleotide sequences derived from pteropus showed 100% homology to the nipha virus reported in 2001 and 2007 outbreak in india and bangladesh. seropositivity for nipha virus in pteropus giganteus in the northern region of india was tested using indirect elisa method and recorded 95% sensitivity and 75% specificity when compared with cnt [14] . bats were again proved to be the source for the recent nipha outbreak in kerala, india [26] . during the survey on bats for nipha virus in malaysia in 2001, researchers isolated a novel virus and named as tioman virus. later tioman virus was isolated from the bat tissue specimen (pteropus giganteus) in the north east region of india. yaiw et al. [60] experimentally proved that the tioman virus capable of infecting and replicating in lymphocytes of lymphnodes, payers patches, spleen, thymic epithelioreticular cells and tonsillar tissue of pigs. philbey et al. [40] and wong et al. [56] separately infected pigs oronasally with tioman virus and postulated as this could be the possible source of paramyxovirus transmission from bats to pigs. from three species of fruit bats, three new henipa virus and two new rubula viruses were detected from indonesia [44] . identification and complete genome analysis of three tuhoko viruses (the university of hong kong) thkpm 1, thkpm2 and thkpm3 in roseuttues leschnaulti was studied in china [29] . these three viruses have a common origin and in phylogenetic analysis they were found closely related to menangle and tioman viruses. hendra, nipha and menangle virus are the only three virus of the paramyxoviridae affect humans recovered from bats so far. in china, a novel toti virus like virus was identified and isolated from the bat guano and its cytopathic effects were studied in sf9, hz and c6/36 cell lines [62] . since these are bsl4 agents, their incidence prevalence study and pathology reports are limited in most of the asian countries. filo viruses are the most lethal haemorrhagic fever causing pathogen in human and non human primates. the filoviridae family includes ebola virus (zaire, sudan, reston, tai forest and bundybugai), marburgh and recent cueva virus. in philippian, during an outbreak in 2008 reston ebola is found circulating in pigs and among nonsymptomatic human cases [34] . these suggest that there are multiple hosts involved in filovirus. in bangladesh, 5 rousettus leschenaultii bats were found seropositive against ebola zaire and reston viruses [38] . in china, r. leschenaultii was found seropositive for reston and zaire ebola viruses along with other insectivorous bat species (yuan et al. [64] ). detection of filoviruses in pigs in philippines, fruit bats in china and bangladesh and orangutans from indonesia reveal the extent of spread of the filoviruses among different species in asia [37] . a panviral microarray study reported the presence of reston ebolavirus co-infected with porcine reproductive and respiratory syndrome in pigs in philippines. towner et al. [48] using immunoperoxidase method demonstrated the peri mebranous localization of marburg virus antigen in the liver of infected roseuttues aegyptius bats and presence of small collections of mononuclear inflammatory cells and hepatocyte necrosis. the epidemiological role of bats in transmission of flavi viruses is not yet clear. in bangladesh while screening for nipha virus a novel gb like virus was identified from sera of a single colony of pteropus giganteus [14] . novel gb virus d, belongs to a new group in flaviviridae, pegi group. serosurveillance of around 1650 bats from 7 different countries using unbiased high throughput sequencing method revealed the presence of sequences showing 25-100% similarity to viruses in hepaci and pegi group. this study concluded that bats are the natural reservoir for the entire hepaci and pegi groups [41] . in southern china, seroprevalance study against je, detected the presence of antibodies in 12% (43/336) of bats by micro-seroneutralization test and 11 bats out of this were have neutralizing antibodies [9] . during the period from 1986 to 2009, four isolates of je virus such as b58 from roseuttues leschnaulti and gb30 from murina aurata were reported in china and these isolates were similar to human liyujie and mosquito bn19 isolates [53] . as a part of investigation on bat borne je in japan, a virus different to je was accessed in serological study and named as yokosu virus [27] . international scientific community gained interest to do research on bats after the discovery of sars in horseshoe bats. in addition to sars-cov, five human coronaviruses (hcovs), including two alpha named as -229e, -nl63, and beta corona virus like oc43, -hku1 and a mers cov were identified [65] . bats are considered as the ancestor for sars cov, mers cov, human cov 229e and nl63 [13, 23] . sars cov like virus antibody prevalence was found to be 84% in chinese horse shoe bats and roseuttues sp. but pathology associated with these viruses in bats was not noticed. large diversity of corona virus was noticed in south east asian countries like china, philippines, japan and thailand. majority of corona viruses were discovered from tissues, blood samples and faeces, indicate an enteric tropism of this virus in bats. detection of corona viral genome fragments in spleen and brain of indonesian bats suggests that the virus is not restricted to the respiratory and enteric epithelium [1] . search for the herpes viruses in bats dated back in 1996 when a cytomegala virus particle was demonstrated in the acinar cells of submandibular gland of two myotis lucifugus bats [47] . a novel beta herpes virus was detected from the lung, kidney, spleen, liver and blood of a number of insectivorous bats by blast search of the complete gb sequence and phylogeny base studies [55] . by targeting glycoprotein b (gb) and dna-directed dna polymerase (dpol) genes of hvs, two gamma and two beta herpes virus were identified in bats in china [57] . out of 520 faecal sample from 8 different species of bats in southern china, 73 samples were positive for herpes virus (69 gamma herpes virus and 4 were beta herpes virus). detection of herpes virus in the digestive tract and anal swabs of bats suggests oral faecal route as main mode of transmission for herpes virus in bats [67] . in philippines during 2012, 70 bats belonging to megabat species were examined and a novel gamma herpes virus was identified from 20% of the intestine (14/70) and 10% of the lung (7/ 69) and serum samples (5/52) [43] . bat adenovirus was first isolated in japan from a fruit bat (pteropus dasymallus yayeyamae) and named as ryukyu virus 1 -rv1 [33] . kohl et al. [25] carried out genome pyrosequencing of bat adenovirus 2 and found there was an interspecies transmission during evolution of canine adenovirus 1 and 2 from vespertilionid bat adenovirus. also, the adenovirus infected bats showed virus tropism in intestine, liver and kidneys but the lesions were noticed in spleen and lungs like follicular hyperplasia and non suppurative interstitial pneumonia respectively. later the adenovirus was isolated during the surveillance programme for nipha, ebola and sars in 2007-2008 periods in china from myotis bats and grouped the novel bat adeno virus tjm (bt-adv-tjm) in mast adenovirus genus [31, 30] . this report also demonstrates the epidemiology of bats adenovirus is prevalent in myotis species and scotophilus kuhlii out of the 19 bat species surveyed in china. the fourth mastadenovirus was isolated from r. leschenaultii bat in india and it showed approximately 20% divergent at the nucleotide level from its closest known relative, japanese batadv [42] . the average prevalence of adeno associated virus was found to be 22.4% from 5 provinces in china [31, 30] . tan et al. [46] performed next generation sequencing for three adenovirus isolates from rhinolophus sinicus bats in china and reported an unusually large e3 genome present in these isolates. the bat hepadna can infect human hepatocyte and are antigenically related with the hepatitis b virus. even though an effective hepatitis b vaccine is available, the virus is not globally eradicated. recent studies suggest hepatitis b virus is present in a number of free ranging bats [19] . in asia, america and africa during the period from 2002 to 2011, 3080 individual bats (199 liver and 2881 sera) from 54 different bat species were collected and screened using nested pcr for hepadna virus. the result shows that there was a low prevalence rate (0.3%). also the histological studies reveals like in all other host, only few inflammatory cells mainly lymphocytes were seen in the portal triad of the infected bats without any symptom and have high viremic form [12] . in myanmar, out of 853 individual bats of 6 species, bat hepatitis virus (bthv) was detected in the pooled liver tissue of long fingered bats (miniopterus fuliginosus) by electron microscopy and also the full genome of bthv [19] . in china, a novel bat hepatitis virus was identified by full genome sequencing of the identified virus in pomona roundleaf bats [20] . recently in china out of 78 bat liver screened by rt-pcr with degenerate primers followed by sequencing and phylogentic analysis, one hepaci virus and 4 novel hepadna viruses were identified [54] . some of the less significant viruses identified in different bat species are shown in table 1 . viral diseases are rising up day by day and majority are zoonotic and about 30-40% are where the bats as host. sometimes the viruses disappear and remain passive for long time and re emerge with increased virulence. researchers trying to develop vaccines against many viruses, but in a developing country with huge population like india, it is not advisable. for the last few decades bats are found as plethora for many numbers of viruses, but the exact role in maintenance and transmission of these viruses to terrestrial animals has not been studied yet clearly. besides, there doesn't exist a proper pathogen database for indian bats. by making the public aware about the major yang et al. [61] emerging horizon for bat borne viral zoonoses 325 factors associated with bat borne zoonotic disease transmission such as magnitude and frequency of bat human interaction and public reminders like not to touch bats, it is easy to control and prevent the bat borne diseases to a large extent. after ebola outbreak in west africa, peoples in different communities were advised about the safety measures to be taken while the bush meat consumption, bat hunting and during frequent cave visits. handling bat guano also arise health risk because in brazil while screening of 533 fruit eating bats it was found 2 bats have high concentration of influenza a subtype (hl18nl11) in their intestines and faeces [4] . in addition to viral zoonoses, non viral zoonotic pathogens like histoplasma capsulatum causing histoplasmosis (fungal infection) and countless number of zoonotic bacteria were reported in bat faeces. high prevalence and diversity of these viruses and its low pathogenicity in bats raises questions like whether virus was coevolved along with bats. along with detection and characterization of bat born viruses, the pathology associated with these viruses in the host and the environmental factors which trigger these virus spills over will be found helpful in preventing epidemic. so as to counteract the future outbreak we have to conduct active pre emergence research such as surveillance, identification of the potent pathogens, susceptible hosts, pathogenicity on the natural hosts and the various factors triggering cross species transmission. deforestation and urbanization are the major factors for all these deadly outbreaks. the human encouragement to forest areas and close contact with wild animals, increased breeding of mosquitoes in congested areas and change in climate and ecology become precipitating factors for the bat borne zoonotic viral outbreaks. all the bats borne disgusting outbreaks became a lesson for future and after the deadly viral outbreaks in different parts of the world, our country become alert to begin a fight against the viruses and it found useful in preventing a huge outbreak in recent years, nipha and zika outbreaks in india. modern molecular diagnostics, the potential and biology of the agent, host immune response studies and vaccine developments helped to prevent further outbreaks to great extend. control and management measures such as campaining the public regarding the issues, and practice personal hygiene, washing hands properly before food esp in rainy seasons and avoid eating fallen fruits and vegetables without proper washing should be followed. detection of coronavirus genomes in moluccan naked-backed fruit bats in indonesia viral zoonoses that fly with bats: a review bats: important reservoir hosts of emerging viruses bat influenza a (hl18nl11) virus in fruit bats sur une grande ã©pizootie de rage dbatvir: the database of batassociated viruses outbreak of henipavirus infection a previously unknown reovirus of bat origin is associated with an acute respiratory disease in humans endogenous hepadnaviruses in the genome of the budgerigar (melopsittacus undulatus) and the evolution of avian hepadnaviruses european bat lyssavirus transmission among cats phylogeography, population dynamics, and molecular evolution of european bat lyssaviruses bats carry pathogenic hepadnaviruses antigenically related to hepatitis b virus and capable of infecting human hepatocytes genomic characterization of severe acute respiratory syndrome-related coronavirus in european bats and classification of coronaviruses based on partial rna-dependent rna polymerase gene sequences henipavirus infection in fruit bats (pteropus giganteus) discovery of an endogenous deltaretrovirus in the genome of long-fingered bats (chiroptera: miniopteridae) natural hendra virus infection in flying-foxes-tissue tropism and risk factors characterisation of a novel lyssavirus isolated from pteropid bats in australia phylogeny and origins of hantaviruses harbored by bats, insectivores, and rodents hepatitis virus in long-fingered bats identification of a novel orthohepadnavirus in pomona roundleaf bats in china bat parainfluenza virus. immunological, chemical and physical properties histopathology and immunohistochemistry of bats infected by australian bat lyssavirus close relative of human middle east respiratory syndrome coronavirus in bat vampire bat rabies: ecology, epidemiology and control genome analysis of bat adenovirus 2: indications of interspecies transmission deadly nipah outbreak in kerala: lessons learned for the future phylogeny of the genus flavivirus severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats identification and complete genome analysis of three novel paramyxoviruses, tuhoko virus 1, 2 and 3, in fruit bats from china prevalence and genetic diversity of adeno-associated viruses in bats from china bat guano virome: predominance of dietary viruses from insects and plants plus novel mammalian viruses origin and evolution of nipah virus isolation of novel adenovirus from fruit bat (pteropus dasymallus yayeyamae) ebola reston virus infection of pigs: clinical significance and transmission potential bats and zoonotic viruses: can we confidently link bats with emerging deadly viruses? novel paramyxoviruses in bats from sub-saharan africa filoviruses in bats: current knowledge and future directions ebola virus antibodies in fruit bats kysanuar forest disease virus infection in the frugiviorous bats, cynopterus sphinx an apparently new virus (family paramyxoviridae) infectious for pigs, humans, and fruit bats bats are a major natural reservoir for hepaciviruses and pegiviruses isolation of a novel adenovirus from rousettus leschenaultii bats from india detection of a novel herpesvirus from bats in the philippines molecular detection of a novel paramyxovirus in fruit bats from indonesia lagos bat virus infection dynamics in free ranging straw coloured fruit bats (eidolon helvum) novel bat adenoviruses with an extremely large e3 gene cytomegalovirus in the principal submandibular gland of the little brown bat, myotis lucifugus isolation of genetically diverse marburg viruses from egyptian fruit bats identification of a novel bat papillomavirus by metagenomics duplex nested rt-pcr for detection of nipah virus rna from urine specimens of bats molecular characterization of nipah virus from pteropus hypomelanus in southern thailand nipah virus: an emergent deadly paramyxovirus infection in bangladesh japanese encephalitis viruses from bats in yunnan detection and genome characterization of four novel bat hepadnaviruses and a hepevirus in china bat coronaviruses and experimental infection of bats, the philippines nipah virus infection: pathology and pathogenesis of an emerging paramyxoviral zoonosis virome analysis for identification of novel mammalian viruses in bat species from chinese provinces the complete genome sequence of a g3p[10] chinese bat rotavirus suggests multiple bat rotavirus inter-host species transmission events emerging horizon for bat borne viral zoonoses 327 detection of nipah virus rna in fruit bat (pteropus giganteus) from india tioman virus, a paramyxovirus of bat origin, causes mild disease in pigs and has a predilection for lymphoid tissues isolation and characterisation of novel bat corona virus closely related to the direct progenitor of severe acute respiratory syndromecorona virus a novel totivirus-like virus isolated from bat guano nipah virus infection in bats (order chiroptera) in peninsular malaysia serological evidence of ebolavirus infection in bats, china isolation of a novel coronavirus from a man with pneumonia in saudi arabia emerging paramyxoviruses: receptor tropism and zoonotic potential high prevalence and diversity of viruses of the subfamily gammaherpesvirinae, family herpesviridae, in fecal specimens from bats of different species in southern china publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord-297790-tpjxt0w5 authors: mandl, judith n.; schneider, caitlin; schneider, david s.; baker, michelle l. title: going to bat(s) for studies of disease tolerance date: 2018-09-20 journal: front immunol doi: 10.3389/fimmu.2018.02112 sha: doc_id: 297790 cord_uid: tpjxt0w5 a majority of viruses that have caused recent epidemics with high lethality rates in people, are zoonoses originating from wildlife. among them are filoviruses (e.g., marburg, ebola), coronaviruses (e.g., sars, mers), henipaviruses (e.g., hendra, nipah) which share the common features that they are all rna viruses, and that a dysregulated immune response is an important contributor to the tissue damage and hence pathogenicity that results from infection in humans. intriguingly, these viruses also all originate from bat reservoirs. bats have been shown to have a greater mean viral richness than predicted by their phylogenetic distance from humans, their geographic range, or their presence in urban areas, suggesting other traits must explain why bats harbor a greater number of zoonotic viruses than other mammals. bats are highly unusual among mammals in other ways as well. not only are they the only mammals capable of powered flight, they have extraordinarily long life spans, with little detectable increases in mortality or senescence until high ages. their physiology likely impacted their history of pathogen exposure and necessitated adaptations that may have also affected immune signaling pathways. do our life history traits make us susceptible to generating damaging immune responses to rna viruses or does the physiology of bats make them particularly tolerant or resistant? understanding what immune mechanisms enable bats to coexist with rna viruses may provide critical fundamental insights into how to achieve greater resilience in humans. an estimated ∼60% of emerging infectious diseases are caused by pathogens which originate from a non-human animal source, referred to as zoonoses (1) (2) (3) . moreover, the frequency of outbreaks caused by zoonotic pathogens has been increasing over time in the human population, with viruses being the most successful at crossing the species barrier (2) (3) (4) . given the impact of viral zoonoses on global public health, considerable resources have been invested into better understanding patterns in their emergence to improve predictions of where they might arise. one key variable in such predictions is to determine the animal reservoir populations within which these novel viruses can be maintained indefinitely (with or without disease) and which therefore act as sources for transmission to humans (5) . in some instances, epidemiological associations may provide clues to identifying a reservoir host species, and the detection of natural infection through seroconversion or the virus itself provides further evidence. recently, phylogenetic analyses have also been used to investigate viral origins-with a presence of greater diversity and of strains ancestral to those in humans being indicative of a virus circulating within a particular natural host population (6) . once identified, viral reservoirs have historically been critical levers through which to reduce human cases (5) . however, reservoir hosts may also provide us with fundamental insights into host-pathogen interactions and are a rich opportunity to examine the immunological processes that contribute to patterns governing which pathogens cross into humans, cause disease and why (7, 8) . this can be particularly informative as in many instances, the zoonotic viruses that are so pathogenic in humans do not cause disease in the reservoirs with which they coexist. bats have been confirmed as reservoir hosts for many viruses, several of which are associated with fatality rates as high as 90% among diagnosed human cases. it has long been appreciated that rabies and other lyssaviruses causing lethal encephalitis can be transmitted from numerous bat species (9, 10) . live marburg virus (marv) has been isolated from rousettus aegyptiacus fruit bats which, jointly with epidemiologic evidence and detection of viral rna, strongly suggests that r. aegyptiacus is a reservoir host of this filovirus (11) . the related ebolavirus (ebov) likely also circulates in african fruit bats, with a few species having been implicated so far-the mobility of which accounts for the sudden appearance of ebola in west africa during the 2014 outbreak, a region where ebolavirus had not previously been detected (12, 13) . the highly pathogenic henipaviruses, of which hendra virus emerged in australia and nipah virus in south-east asia via horse and pig intermediate hosts respectively, have been shown to be transmitted from pteropus bats (14, 15) . in china, horseshoe rhinolophus bats have been identified as the reservoirs for sars coronavirus via palm civet intermediate hosts, the cause of a large outbreak of atypical pneumonia across several countries that began in 2002 in china (16) (17) (18) . more recently, mers coronavirus that has caused lethal respiratory infections mostly in saudi arabia, likely transmitted via dromedary camels, was shown to be closely related to several bat coronaviruses, including those sequenced from neoromicia capensis, pipistrellus abramus, and vespertilio superans bats (19, 20) . moreover, additional viruses may continue to emerge from bats, as in the single case of sosuga virus infection in a wildlife biologist collecting bats in south sudan (21) . in addition to these emerging zoonotic viruses, bats may be the source of a number of viruses with which humans have older evolutionary associations. for instance, bats harbor viruses closely related to both mumps (rubula virus) and measles (morbilli virus) and have likely been donors of these viruses to other mammalian groups, possibly including humans (6, 22) . furthermore, both old and new world bats carry diverse hepadnaviruses, some of which are related to hepatitis b virus and can infect human hepatocytes (23) . hepaciviruses that are related to hepatitis c virus and pegiviruses that are related to human gb viruses were detected in the sera of many different bat species, and given the basal position of these bat viruses in phylogenetic trees, may also represent strains ancestral to those found in humans (24, 25) . the preponderance of links between bat and human pathogens has led to a debate about whether bats disproportionately contribute to emerging viral infections crossing the species barrier into humans (26) (27) (28) (29) (30) . given the diversity of the chiroptera order (figure 1) , we may simply see more bat viruses because there are so many (>1,300) species of bats (31) . however, even when accounting for the fact that they make up ∼20% of extant terrestrial mammals, bats are overrepresented as reservoir hosts of pathogens with a high potential for spilling into human populations (32, 33) . in fact, no known predictors that have been described to impact the likelihood of crossing the species barrier, including reservoir host ecology, phylogenetic relatedness to humans or frequency of reservoir-human contact, explain this pattern (32) . thus, why bats are such a frequent source of pathogenic human viruses remains a tantalizing mystery. among viruses, those that have genomes encoded by rna generally jump across species boundaries more frequently, presumably due to their inherently greater mutation rates that facilitate the rapid adaptation to replicating within new hosts (34) . interestingly, all pathogenic viruses that have made the jump to humans for which bat species may be reservoirs share the common feature that they have single-stranded rna genomes (with the exception of hepadnaviruses which have a dna genome but replicate via an rna intermediate). so far, available evidence suggests that bats remain disease-free when infected with the rna viruses they carry-even those highly pathogenic to humans-and are able to coexist with them without detectable fitness costs using measures such as changes in temperature, loss of body weight, or overt signs of inflammation (35) . indeed, so far only one rna virus studied which circulates in a bat population has been shown to consistently cause significant morbidity and mortality: tacaribe virus in the jamaican fruit bat (artibeus jamaicensis), which recent evidence suggests is not a reservoir host for this virus (36) . data from experimental rabies and lyssavirus infections suggests that rhabdoviruses may also cause disease in bats, although experimental infection outcome is very dependent on the infection route. intracerebral infection with different strains and in different bat species invariably led to death (37, 38) . in contrast, intramuscular infection led to muscle weakness, paralysis and visible histological cns lesions in 30% of experimentally infected flying foxes (pteropus poliocephalus) (39) . similarly, a subset of vampire bats (desmodus rotundus) experimentally infected intramuscularly with a high dose of rabies virus remained healthy despite viral shedding in the saliva and survived (40) . naturally infected bats are thought to either die or remain healthy and seroconvert, but transmission in freeranging populations remains incompletely understood (41) . while bats seem to be frequent hosts for rna viruses, current available data indicates that primates and humans disproportionately harbor dna viruses such as herpesviruses (32) . interestingly, it is these dna viruses that can persist in an individual which can also be found in isolated, small indigenous groups-perhaps suggestive of humans having a more ancient relationship with such dna viruses (42) . it may even be the case that persistent dna viruses in humans impact immune responses specifically to rna viruses, but this has not yet been examined. it is likely that differences in evolutionary history of pathogen exposure between bats and humans have led to distinct adaptations in anti-viral immune responses and the ability to tolerate certain infections without disease while being susceptible to others. importantly, bats differ in many aspects of their physiology and behavior from humans that may have direct or indirect effects on immune function. bats are a monophyletic mammalian group traditionally divided by morphological data into two suborders, the megabats and microbats, which more recent molecular data has revised into the yinpterochiroptera and yangochiroptera suborders (figure 1 ). bats possess a suite of traits that make them distinct from other mammals in a number of ways. these unique life history traits may play a role in understanding which pathogens bats have evolved to coexist with and why. in particular, such traits may explain the ability of bat populations to maintain particular viral pathogens indefinitely, and may have effects on immune function through specific energetic or evolutionary trade-offs we have yet to better define. despite the diversity of viruses carried by bats, they are not typically known to cause mass bat die-offs or reduce bats' remarkable longevity. in this respect, bats represent a potential opportunity for long-term persistence of viruses within a population and across generations. bats live significantly longer than similarly-sized terrestrial mammals and, despite their small size, are characterized as "slow" mammals in the slow-fast continuum (43, 44) . although their weights range from 2 grams to 2 kilograms, with respect to longevity bats group with large mammals such as humans and non-human primates (45) . aerial living has an obvious advantage in avoiding predation, but bats outlive even birds. for example, the brandt's bat (myotis brandtii) lives up to 41 years, compared to selasphorus platycercus, a bird species of similar size that lives for ∼14 years (45, 46) . thus, flight can only partially account for their extraordinarily long lives. initially, the longevity of some bats was attributed to seasonal hibernation, as temperate-zone species enter continuous torpor of up to 75 days, with a dramatic drop in metabolic rate such that small fat reserves can sustain them throughout the entire hibernating season (43) . however, even non-hibernating bat species live three times longer, on average, than predicted by their size, and heterothermy is not an accurate predictor of lifespan in other mammalian orders, suggesting that the driving force behind their surprising longevity is intrinsic to bats as a group (47) (48) (49) . like other "slow" mammals, bat females typically only have one offspring per year, perhaps because the volant lifestyles of bats make it difficult to rear more than one offspring, as pregnant females and those with recent births must navigate and forage with added weight; on average, neonatal bat pups are ¼ of their mother's weight (50) . the physical and energetic constraints of rearing multiple offspring may necessitate small litters, which would in turn require prolonged reproductive capability and enhanced longevity to ensure maintenance of the population over generations. thus, in bats, the dependence of colony survival as a whole may depend upon enhanced individual survival and delayed senescence (51) . genetic analyses of several bat species have shown differences in the growth hormone (gh)/insulin-like growth factor 1 (igf1) axis which in humans is associated with aging, resistance to diabetes and cancer (52) . the determinants of adult survival in bats have been historically difficult to identify, as this requires tracking individuals over many years, and until recently longitudinal studies of bat mortality were conducted using tagged bats, of which only a fraction were recovered (53) . recently, a 19year study of a colony of bechstein's bats demonstrated that unlike terrestrial mammals, survival could not be predicted by common indicators such as season, age, and body size. instead, the only accurate predictor of mortality was a single cataclysmic weather event that affected multiple countries in north-central europe. additionally, even the oldest female bats were reproductively capable, indicating that bat survival is primarily affected by catastrophic natural events rather than factors that normally dictate an individual's fitness (45) . molecular phylogenetic studies of bats suggest that there are massive gaps in bat fossil records. as bats are the second most diverse order of mammals, outnumbered only by rodents, the number of species unrepresented in the fossil records is staggering. over half of microbat and nearly all of megabat fossil histories are missing (31, 54) . the enormous incompleteness of the fossil records has made it difficult to identify when specific morphological traits of bats arose. as molecular phylogeny groups two echolocation-reliant microbat species with megabats (also called old world bats or pteropodids), which do not rely on echolocation, there is some debate as to whether echolocation first arose in the common ancestor of bats and was subsequently lost in megabats, or whether it arose twice, independently (31) . pteropodids have adaptations that enhance visual acuity at night (55), and they do not require echolocation for foraging (56) . there are multiple types of echolocation that can be partially delineated by species, but are more clearly categorized by the type of environment. divergent species that inhabit the same type of environment, such as those that hunt in large, open spaces, often use the same form of echolocation, suggesting that habitat has a greater influence on echolocation than phylogeny (31) . importantly, echolocation can result in the production of droplets or small-particle aerosols of oropharyngeal fluids, mucus, or saliva, thus facilitating transmission of viruses between individuals in close proximity (57, 58) . the unique navigation tactic of many bat species may inadvertently facilitate virus transmission among bats in the same habitat. bats are the only mammal capable of powered flight, which likely evolved ∼65 million years ago alongside birds following radical ecological changes that resulted in the extinction of the dinosaurs (54, 59) . during flight, bats consume approximately four times as much oxygen, and they have a markedly higher concentration of red blood cells compared to small terrestrial mammals (60). bat flight is markedly different from that of birds and insects, whose wing surfaces are typically composed of inflexible material, such as feathers or chitin. bat wings are constructed from live skin stretched across elongated arm and finger bones, making them extraordinarily malleable and sensitive to environmental cues (59) . the plasticity of bats' wings allows them to navigate and inhabit diverse ecospheres, contributing to their extensive speciation. moreover, the capability of powered flight can allow the efficient spread of viruses and thus the introduction of pathogens to which colonies may otherwise have remained naïve. as flight is extremely metabolically demanding, in addition to evolving the physical mechanisms required for flight, bats have also evolved necessary underlying molecular mechanisms. the mitochondrial respiratory chain accounts for nearly all atp required for mobility in eukaryotes, and genetic analysis of both micro-and megabat species revealed an enrichment of genes specific to the oxidative phosphorylation (oxphos) pathway. specifically, 4.9% of nuclear-encoded and 23% of mitochondrial oxphos genes have evidence of positive selection in bats, which is markedly higher than the expected 2% of orthologous genes in previous genome-wide studies that show evidence of positive selection (61) . genomic analysis of pteropus alecto and m. davidii suggests positive selection for the dna damage checkpoint pathway and changes in overlapping aspects of this pathway with the innate immune system, indicating that evolutionary adaptations important for flight may have secondarily affected bat immunity (62). as a group, bats exhibit the greatest diversity of social systems in mammals. tropical species are primarily responsible for this diversity, as temperate species are more restricted in their social behavior. generally, however, bats are extremely social creatures that tend to form dense roosting colonies (63) , and almost all temperate-zone species live in closed societies with very little infiltration of foreign bats into established roosts (63, 64) . in particular, female bats form maternity colonies in which males do not take part. as bats are capable of longdistance flight, dispersal barriers cannot explain the philopatry of females. instead, benefits such as knowledge of foraging areas and social thermoregulation likely selected for these colony types. additionally, there is evidence that forming closed societies limits the potential invasion of new pathogens, thereby protecting colony members that would otherwise be vulnerable to infection. for example, pseudogymnaoscus destructans has decimated north american bat populations that do not live in the type of closed societies observed elsewhere (64) . dna analysis of a closed society of bechstein's bats revealed extraordinarily high conservation of mitochondrial dna and relatively low conservation of nuclear dna, suggesting stable maternal populations within colonies and gene flow between colonies via promiscuous mating with males. it is possible that the mating patterns of temperate-zone species may allow transmission of pathogens between colonies via traveling males while the frontiers in immunology | www.frontiersin.org more insular females may allow viruses to persist throughout generations within a colony. an important commonality among pathogenic rna viruses in humans presenting with disease is that the host response is an important contributor to the disease process, with dysregulated and excessive innate immune responses being particularly important drivers of tissue damage during infection (8) . given the general absence of clinical signs of disease in bats infected with the same viruses that are so lethal in humans or other non-natural hosts infected experimentally, a critical question has been to understand whether bats might establish effective disease tolerance, thus maintaining fitness despite pathogen replication, or whether bats are more resistant to infection through more successful control of pathogen replication and what the contribution of the immune response is (65, 66) . the lack of many fundamental immunological tools enabling the probing of bat immune responses has meant that truly mechanistic studies of bat immunity have been very limited, although recently there has been some progress in establishing approaches such as flow cytometry to identify distinct bat immune cell populations (67, 68) . so far, studies of bat immunity have primarily taken one of three approaches, whereby each comes with important strengths and weaknesses that have to be kept in mind: (i) comparative genome studies, (ii) in vitro cell culture assays, and (iii) experimental infections. comparative genome studies have confirmed that the critical components of the innate and adaptive immune system are conserved in bats at the gene level and that bats have the machinery for innate responses to pathogen-associated molecular patterns (pamps), the production of anti-viral effector molecules such as type i interferons (ifn), t cell responses (variable t cell receptors, mhci and mhcii), and b cell responses [reviewed in (35) ]. interestingly, based on the 10 bat genomes sequenced so far, the only family of genes lost entirely in all of them are pyhin genes (69) . members of the pyhin family are dna sensors capable of recognizing foreign dna, including dna viruses and damaged self dna which can be generated by rna viral infection. recognition of dna results in production of ifn through interaction with stimulator of interferon genes (sting). the pyhin family also encode the only identified class of dna sensors capable of activating the inflammasome. it has been hypothesized that the absence of the pyhin family may allow bats to limit activation of the innate immune response to damaged self-dna generated by rna viral infection, thus avoiding excessive inflammation (69, 70) . genome comparisons highlighting contractions or expansions of specific gene families, specific genes under positive selection, or nonconserved sequence differences in critical protein domains can thus provide the basis for hypotheses worth testing further. however, it is important to note that much can be missed in absence of data on gene regulation, especially during infection when gene expression kinetics can make a critical difference to the infection outcome. moreover, the absence of a gene or gene family does not rule out that other proteins have evolved to compensate for their loss of function. thus, while whole genome analyses can provide a context for specific questions or be hypothesis-generating, on their own they cannot distinguish tolerance from resistance mechanisms. the repeated identification of signatures of positive selection in innate immune genes in particular, does however lend credence to the idea that bats have specific adaptations as a result of a long co-evolutionary history with viruses. cell culture assays with bat cell lines, or, in some instances, primary bat cells, have been used to assess whether bats are permissive for viral replication and to determine whether particular immune receptor signaling pathways are intact. as discussed below, such studies have probed the type i ifn pathway in particular, revealing some possible species-specific differences among bats (71) (72) (73) (74) (75) (76) (77) (78) (79) (80) (81) (82) (83) . however, it is important to note that in some instances immortalized cells can behave differently from primary cells and that such cultures may miss additional differences imposed by changes in cell localization, cell recruitment or cell-cell interactions in a whole animal. careful experiments measuring the quality, magnitude, and kinetics of immune responses in bats during infection and upon administration with defined stimuli for which we have comparative information from humans remain to be done to provide additional evidence that specific innate immune pathways are wired differently. experimental infections come with the enormous challenge of having to house and/or breed colonies of bats and to have biosafety-level 4 facilities in place to perform infections with viruses lethal to humans. moreover, some trial and error is involved in determining which route and dose leads to viral replication, establishing a source of the virus (humanadapted strains tend to replicate less well in bats than strains obtained from naturally infected bats), and amplifying this viral stock without extensive tissue culture passaging. studies to date have examined the kinetics of viral replication by quantifying the extent of viremia and dissemination to other tissues, and assessing changes in white blood cell counts, body mass, and temperature. given the generally low levels of viral shedding and short infectious periods observed so far it remains poorly understood how transmission occurs in the wild to sufficient levels that cross-species jumps occur. some infection experiments have also provided evidence that a particular bat species is unlikely to be a reservoir despite epidemiological evidence, for example for r. aegyptiacus and ebolavirus. certainly, once good experimental infection models are established, such studies have the potential to be hugely informative with regard to anti-viral immune responses elicited using, for instance, comparative transcriptome analyses. one drawback may be that experimental infections do not mimic the impact of chronic stress arising from the disruption of wildlife populations, which bats are particularly sensitive to jones et al. (84) . comparison of either cave-roosting or foliage-roosting species in areas of malaysian borneo designated as actively logged forest, recovering forest, or fragmented forest revealed varying impacts of habitat disturbance on stress and circulating white blood cells (85) . overall, the limited studies of bat immunity that have been done have focused largely on 2 species: p. alecto and r. aegyptiacus. we summarize this work below, but comparisons of observations made across species suggest that although a number of species appear to be capable of avoiding the pathological effects of rna virus infection, each bat species may have achieved this through distinct pathways, possibly involving changes to both increase pathogen replication control and to mitigate any immunopathology through decreased inflammatory responses and hence increased disease tolerance. the most well studied bat species with regard to antiviral immune responses is the australian black flying fox (p. alecto). this interest has stemmed from the fact that pteropid bats have been identified as the natural reservoirs for the deadly hendra and nipah viruses (86) , which continue to cause outbreaks [such as most recently in india in may 2018 (87)]. to date, several studies have examined the kinetics of viral infection in pteropus bats and the nature of transmission and replication in other susceptible species (88) (89) (90) (91) . in australia, all four species of pteropid bats (p. alecto, p. poliocephalus, p. scapulatus, and p. conspicillatus) have antibodies to hendra virus but only p. alecto and p. conspicillatus are considered to be the primary reservoir hosts (14, 92, 93) . in south east asia, both pteropus spp. occurring in malaysia have been found to be seropositive for nipah virus neutralizing antibodies, and the virus has been isolated from p. hypomelanus and p. vampyrus (15, 94) . experimental infections of pteroid bats with hendra or nipah virus result in sub-clinical infection with short periods of virus replication and shedding, and low antibody titres (88) (89) (90) (91) . upon subcutaneous infection of p. poliocephalus with hendra virus, viral antigen was detected by immunohistochemistry at 10 dpi in blood vessels of spleen, kidney and placenta (89) . similarly, oronasal hendra virus infection of p. alecto led to the presence of viral genome in lung, spleen, liver and kidney 3 weeks later, but virus isolation was unsuccessful at this timepoint (89, 91) . the malaysian flying fox, p. vampyrus and the australian species, p. poliocephalus demonstrate similarly short periods of viremia upon infection with nipah virus. in subcutaneously infected p. poliocephalus, virus was isolated from the kidney and uterus of bats euthanized at 7dpi, but no virus was isolated at any of the other timepoints examined (3, 5, 10, 12, or 14 dpi) and there was no evidence of antigen in any tissue by immunohistochemistry, including tissues collected at 7 dpi. in this study, low neutralizing antibodies were detected in all bats with the exception of one individual that developed a significant neutralizing antibody titre -possibly reflecting the fact that p. poliocephalus is not the natural host for nipah virus (90) . in p. vampyrus challenged by oronasal nipah inoculation, viral genome was detected in a throat swab at 4 dpi and a rectal swab of the same individual at 8 dpi but virus was undetectable in tissues collected at postmortem from all individuals (49, 50, or 51dpi), consistent with a short period of viremia. similar to previous studies, antibody titres were low in all p. vampyrus bats (91) . overall, these results are consistent with bats controlling replication rapidly, at least following experimental infections which involve higher doses of virus compared to what bats would likely be naturally exposed to in the wild. the absence of a robust antibody response also appears to be typical of all experimental hendra and nipah virus infections performed to date. since antibody responses are the only immune parameter that has been measured during experimental infections of bats so far, it is difficult to speculate on the mechanisms responsible for control of viral infections in vivo. pteropus alecto was among the first bat species to have its genome described in detail. genomic studies provided initial clues for possible differences in the innate immune system of bats, with evidence for selection of key innate immune genes and the expansion or contraction of specific immune gene families (62, 68, 95) . the mhci region is contracted (96) , as is the type i ifn locus, which in p. alecto contains fewer ifn genes than any other mammalian species sequenced, with only three functional ifn-α loci (68) . in contrast, pteropid bats have the largest and most diverse family of apobec (apolipoprotein b mrna editing enzyme, catalytic polypeptide-like) proteins identified in any mammal (95) . apobecs interfere with the replication of retroviruses by deaminating cytosine residues in nascent retroviral dna. this is notable, as bats are an important source of mammalian retroviruses, many of which have been transmitted to other mammals (97, 98) . apobec diversification may therefore have occurred to counteract the effect of retroviruses and possibly other viruses, as apobecs have been shown to restrict the replication of other virus families including hepadnaviruses, and parvoviruses (99, 100) . members of the apobeca3 protein family exhibit direct antiviral activity through dna cytosine deamination which results in hypermutation of the nascent retroviral dna which is then degraded or rendered non-functional (101) . the mechanism of antiviral activity against non-retroviruses remains largely unknown. for parvovirus adeno-associated virus, apobec meditated inhibition has been speculated to involve direct interaction with the viral dna or the replication machinery (102) . whether the expanded family of abobecs in bats have evolved other mechanisms to control dna and rna viruses remains to be determined. as apobecs can be induced by even low levels of type i ifn (103) , one hypothesis to be tested is that bats, through their multiple apobecs, are able to restrict viral replication without causing inflammation. pteropus alecto is the only bat species to date in which apobec genes have been mapped, and whether the expansion of this gene family extends to other bat species remains to be determined. in addition to the identification of putative immune pathways distinct in p. alecto through genome studies, differences have been identified in the activation of innate immune effectors in p. alecto from studies performed in vitro, primarily using cell lines derived from tissues including the kidney and lung. ifns are the first line of defense following viral infection and unsurprisingly, because of this, they have been the most extensively studied group of genes in bats. both type i (ifna and ifnb) and iii (ifnl) ifns are detectable in bat cells. curiously, a unique characteristic of pteropid bats is the constitutive expression of mrna for ifna and the signaling molecule, ifn regulatory factor 7 (irf7) in unstimulated tissues and cells [75, 68a] . constitutively expressed ifna and irf7 may allow bats to respond more rapidly to infection, thus avoiding the lag time between pathogen detection and response. furthermore, viral infection or stimulation with synthetic ligands result in little ifna induction in pteropid bat cells (68) . the constitutive expression of ifna has been described in two species of pteropid bats (p. alecto and cynopterus brachyotis) and is a first for any species. ifnb and ifnl are activated following stimulation of cells from p. alecto and p. vampyrus with synthetic ligands such as polyic (71) (72) (73) (74) . moreover, bat ifns demonstrate antiviral activity (68, (71) (72) (73) (74) 104) . however, viral infection of p. alecto splenocytes results in induction of ifnl but not ifnb, hinting at differences in the function of type i and iii ifns (74) . in humans and mice, ifnl has recently been demonstrated to have a role not only in controlling virus replication, but also in dampening damage-inducing neutrophil functions and in modulating tissue-damaging, transcriptionindependent responses such as production of ros (77, 80) . a hypothesis yet to be tested is whether upregulation of ifnl rather than ifnb has a similar function in bats. the endoplasmic reticulum (er) membrane protein, sting, is involved in induction of type i ifn by cytosolic dna (105) . stimulation of bat splenocytes with gmp-amp, which is produced following sensing of cytosolic dna by cgas, results in little induction of ifn compared to responses observed in mouse splenocytes (83) . bat sting contains an amino acid substitution of the highly conserved and functionally important serine residue s358 which may be responsible for dampening sting-dependent ifn activation in bat cells in response to dna. however, comparable levels of ifn induction in mouse and bat cells in response to the rna viral mimic polyic indicate that sting-associated inhibition of the ifn response does not extend to rna viruses (83) , thus the relevance to rna viruses in bats remains unknown. downstream of the induction of ifns, novel subsets of ifn stimulated genes (isgs) have been detected in unstimulated and stimulated pteropid bat cells indicative of a response that is less damaging to the host. furthermore, the isg response is elevated for a shorter period of time in p. alecto compared to human cell lines which again may be a strategy to avoid tissue damage (78, 81) . the less inflammatory profile of isgs may be the key to the ability of bats to tolerate higher ifn expression without adverse consequences. the balance between resistance and tolerance may therefore be achieved through careful selection of the pathways that are activated and shorter periods of activation or limited activation to prevent inflammation. in this regard, studies of the regulation of ifn signaling in bats is likely to provide important additional insights. a second bat species whose host responses to viral infections has been studied more recently is the egyptian fruit bat (r. aegyptiacus). marburg virus (marv) has been repeatedly isolated from this species with demonstrated seasonal pulses of active marv replication in juvenile bats living in caves in uganda (11, 106) . moreover, r. aegyptiacus were a suspected reservoir for ebolavirus (ebov) based on epidemiological evidence and detected seroreactivity to ebov, but no infectious virus has been isolated thus far from wild rousettus bats (107) . indeed, while cell lines from r. aegyptiacus are equally susceptible to marv and ebov (79, 108) , experimental infections of r. aegyptiacus seem to confirm that it is a reservoir for marv, but is unlikely to be the source of ebov spillover to humans. subcutaneous ebov infection results in very low viral replication, no viremia, little dissemination to other tissues, and no viral shedding, although some animals seroconvert, suggesting that r. aegyptiacus are unlikely to perpetuate ebov in the wild (109, 110) . in contrast, experimental marv infection of r. aegyptiacus resulted in acute viremia that peaked on days 5-6 post-infection (although generally at lower levels than in humans), oral shedding that peaked on days 7-8 postinfection, and dissemination to other tissues including spleen, liver, kidney and salivary glands (109, (111) (112) (113) . interestingly, viral replication was not associated with increases in white blood cell counts, any clinical signs of infection such as changes in body temperature or body weight, and infected tissues showed little evidence of inflammatory infiltrates (109) . in all experiments, viremia was cleared by day 13 and oral shedding ceased by day 19. intriguingly, a cohousing experiment resulted in marv transmissions to uninfected bats 4-7 months after experimental infection, raising the question of whether persistent infection with intermittent shedding is possible or whether very long latent periods without detectable viral replication could follow exposure (114) . upon secondary challenge of previously marv-infected bats, none showed any detectable viral replication or shedding, providing evidence that protective immunity is established (115) . unlike for pteropus bats, no constitutive expression of type i ifns has been detected in r. aegyptiacus (79) , but type i ifns are induced in r. aegyptiacus cell lines upon stimulation with sendai virus as seen in other mammals (82) . furthermore, in r. aegyptiacus the type i ifn genes are expanded, again in contrast to p. alecto (82), but like for p. alecto a number of genes in the type i ifn pathway or involved in innate immune recognition of pamps show signs of having been under positive selection (82) . whether positive selection of genes in either bat species is associated with tolerance remains to be determined, especially given that innate immune genes in humans have also been under positive selection (116) . a transcriptome study which generated 20 rna sequencing libraries from 11 tissues taken from 1 female and 1 male r. aegyptiacus found a reduced coverage of nk cell related genes compared to other mammals, but confirmed that in these bats the predominant t cells had an αβ t cell receptor, and showed that ige, igg, igm, and iga, as well as a number of pro-and anti-inflammatory cytokines, were all detectable (117) . the recently sequenced r. aegyptiacus genome revealed substantial differences in the repertoire of nk cell receptors, with this bat species entirely lacking functional killer cell immunoglobulin receptors (kirs) and with all killer lectinlike receptors (klrs) encoding either activating and inhibitory interaction motifs, or inhibitory interaction motifs only (82) . nk cells are important immune cell players in an antiviral response but without assessment of the consequences of these genomic differences it is difficult to draw any specific conclusions with regard to viral control or the magnitude of inflammation elicited upon infection with viruses like marv. nonetheless, these genomic data provide some interesting hypotheses to be tested in the future. some additional studies probing the induction of cytokines upon stimulation of bat cells with defined innate immune stimuli provides some evidence that innate immune recognition of viruses may be altered, leading to a reduction in proinflammatory responses. stimulation of kidney and myeloid cells from the big brown bat (eptesicus fuscus) with polyinosinicpolycytidylic acid (polyi:c) resulted in only limited activation of the inflammatory cytokine, tumor necrosis factor alpha (tnfα) compared to human cells which display a robust tnfα response. induction of tnfα is controlled by transcription factors, including the nf-kappa b (nf-κb) family which consists of five members, [rela (p65), relb, c-rel, nfκb-1 (p50), and nfκb-2 (p52)] which form homo-or hetero-dimers that are bound by molecules of the inhibitor of nfκb (iκb) family and retained in the cytoplasm of the cell in an inactivated state (118) . in e. fuscus, a potential repressor (c-rel) binding motif was identified in the tnfα promoter region which may explain the difference in induction of tnfα in e. fuscus cells. consistent with this hypothesis, partial knockdown of c-rel transcripts significantly increased basal levels of tnfα transcripts in e. fuscus cells (104) . the transcription factor, c-rel has also undergone positive selection in the bat ancestor which may indicate that this mechanism is common to other species of bats (62) . of note, low levels of tnfα induction have also been associated with tolerance in european bank voles which are a natural reservoir for puumala hantavirus (puuv) (119) . stimulation of macrophages from the greater mouse eared bat (myotis myotis) suggested that this species may have also evolved mechanisms to avoid excessive inflammation caused by cytokines. while high levels of tnfα, il1β, and ifnβ were produced in response to in vitro challenge with lipopolysaccharides (lps) and polyi:c, there was also a sustained, high-level transcription of the anti-inflammatory cytokine il-10, which was not observed in mouse macrophages (120) . furthermore, unlike in the mouse, m. myotis macrophages did not produce the proinflammatory and cytotoxic mediator, nitric oxide, in response to lps. the same study also showed evidence of bat specific adaptations in genes involved in antiviral and proinflammatory signaling pathways through comparison with other mammalian taxa, including rig-i, il1b, il-18, nlrp3, sting, and casp1, further supporting the evolution of adaptations associated with reducing inflammatory responses in bats (120). even less is known about immune responses of bats to nonviral pathogens than to viral pathogens, but it is clear that while anti-inflammatory responses may be characteristic of antiviral responses in bats, they are susceptible to disease upon infection with particular pathogens-in some instances due to dysregulated and damaging immune responses. one particular example of this is the emerging infectious disease, white nose syndrome (wns), that has decimated north american bat populations beginning in 2006, in what will likely rank as one of the most devastating wildlife diseases in history (121) (122) (123) . for reasons that remain poorly understood, the psychrophilic fungus pseudogymnoascus destructans (formerly geomyces destructans) causes no mass mortality in european bats despite being abundantly detected (124, 125) . indeed, evidence suggests that a single p. destructans genotype was introduced to north american bat species from europe (125) . in north america, p. destructans infection is not specific to a particular bat genus, replicating in many different bat species during hibernation and targeting the furless skin of the wings, ears, and muzzle (126) . distinct hypotheses have been proposed for why p. destructans is so deadly in north american bats, ascribing the impaired tolerance to infection compared to european bat counterparts to either physiological or immunological factors. on the one hand, more frequent arousal, electrolyte depletion, and dehydration are thought to contribute to mortality following infection (127, 128) . the destruction of wing tissue in wns results in a marked electrolyte imbalance, as the wings play a critical role in maintaining water levels, especially during hibernation, during which bats are particularly vulnerable to dehydration (129, 130) . dehydration catalyzes arousal in hibernating bats, which is extraordinarily metabolically costly and rapidly depletes the fat reserves necessary to survive until spring (127) . an alternative hypothesis posits that the restoration of the immune system following emergence from hibernation induces the fatal pathology of wns. during hibernation, destruction of cutaneous tissue is limited and infiltrating immune cells are entirely absent, yet in the weeks following arousal, infected bats exhibit overt wing damage and corresponding neutrophilic and lymphocytic infiltration (131) . hibernation does not preclude a localized immune response to p. destructans at the site of infection and transcriptomic analysis of infected tissue showed upregulation of some acute inflammatory genes in infected tissue (132, 133) . however, the observed immune responses likely occur during arousal periods, which are more common in infected bats. ultimately, immunosuppression during torpor allows p. destructans to colonize infected bats relatively unchecked (124) , and upon emergence from hibernation, the exuberant immune response may result in deadly immunopathology during wns (131) . in addition to general studies of immune cell recruitment and transcriptional responses during wns, body mass and white blood cell counts were examined following lps administration in four bat species (134) (135) (136) (137) . subcutaneous lps challenge in of pallas's mastiff bats (molossus molossus) led to a loss of body mass of ∼7% within the first day, but did not result in changes in circulating white blood cell counts or body temperature (135) . seba's short-tailed fruit bat (carollia perspicillata) also showed a decrease in body mass following lps challenge, but this was associated with increases in white blood cell counts as well as increases in derivatives of reactive oxidative metabolites (drom) (134) . subdermal lps challenge of fish-eating myotis (myotis vivesi) led to body mass decreases, increased resting metabolic rate and skin temperature (136) , while intraperitoneal lps challenge of wrinkle-lipped bats (chaerephon plicatus) caused an increase in circulating leukocytes, but did not result in a reduction in body mass compared to controls (137) . the differential responses to lps challenge suggest that the immune response to bacterial infection varies across species. of note, postmortem examinations of ∼500 dead bats comprising 19 species from germany revealed inflammatory lesions, many of which had evidence of underlying bacterial or parasitic infections, particularly in the lung (138) . bats have an array of unique life history characteristics that not only allow them to be particularly good reservoirs for viruses that are highly pathogenic in other species, but also appear to have shaped their immune systems. although research on bat antiviral immunity has focused on only a few species to date, at the genomic level, selection on genes is concentrated on the innate immune system across both suborders of bats. however, while these studies have provided a rich source of hypotheses, the majority remain to be tested at the functional level and many questions remain that cannot be answered from comparative genome studies. experimental studies to date have demonstrated some functional differences between bat species, with the common emerging theme that the overall antiviral response appears to converge on a lower inflammatory profile, with tight regulation of the cytokine and inflammatory response key to clearing viral infection without the pathological outcomes typically associated with infection. however, whether this is due to specific tolerance mechanisms that are at play or increased resistance to rna virus replication still remains unclear. fewer studies have examined the adaptive immune system than those probing innate immune pathways, but experimental infections with bat borne viruses have demonstrated that bats generate low or absent antibody responses which often wane rapidly. this is reminiscent of the response of another reservoir host, the sooty mangabey which is the natural reservoir for simian immunodeficiency virus (siv) and for yellow fever virus. sooty mangabeys given an attenuated yellow fever virus vaccine strain generate much lower, transient antibody responses as compared to humans or rhesus macaques. changes to innate immune responses are also evident in sooty mangabeys (139) . thus, intriguingly, different reservoir hosts may have arrived at similar solutions to avoid the pathological consequences that follow viral infection in non-natural hosts. despite the ability of bats to avoid disease associated with viral infection, this trait does not extend to all pathogens, as evidenced by the severe consequences associated with infection of north american bats with the fungus that causes wns. thus, the pathways associated with the control of other pathogens have not been under the same selection pressures as those responsible for controlling infections with rna viruses-or there are immunological trade offs involved which lead to greater susceptibilities to some pathogens than others. overall, it is clear that studying host-pathogen interactions in reservoir hosts has considerable potential to provide novel insights into host 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2018 mandl, schneider, schneider and baker. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-334628-axon4jdc authors: lee, saemi; jo, seong-deok; son, kidong; an, injung; jeong, jipseol; wang, seung-jun; kim, yongkwan; jheong, weonhwa; oem, jae-ku title: genetic characteristics of coronaviruses from korean bats in 2016 date: 2017-07-19 journal: microb ecol doi: 10.1007/s00248-017-1033-8 sha: doc_id: 334628 cord_uid: axon4jdc bats have increasingly been recognized as the natural reservoir of severe acute respiratory syndrome (sars), coronavirus, and other coronaviruses found in mammals. however, little research has been conducted on bat coronaviruses in south korea. in this study, bat samples (332 oral swabs, 245 fecal samples, 38 urine samples, and 57 bat carcasses) were collected at 33 natural bat habitat sites in south korea. rt-pcr and sequencing were performed for specific coronavirus genes to identify the bat coronaviruses in different bat samples. coronaviruses were detected in 2.7% (18/672) of the samples: 13 oral swabs from one species of the family rhinolophidae, and four fecal samples and one carcass (intestine) from three species of the family vespertiliodae. to determine the genetic relationships of the 18 sequences obtained in this study and previously known coronaviruses, the nucleotide sequences of a 392-nt region of the rna-dependent rna polymerase (rdrp) gene were analyzed phylogenetically. thirteen sequences belonging to sars-like betacoronaviruses showed the highest nucleotide identity (97.1–99.7%) with bat-cov-jtmc15 reported in china. the other five sequences were most similar to mers-like betacoronaviruses. four nucleotide sequences displayed the highest identity (94.1–95.1%) with bat-cov-hku5 from hong kong. the one sequence from a carcass showed the highest nucleotide identity (99%) with bat-cov-sc2013 from china. these results suggest that careful surveillance of coronaviruses from bats should be continued, because animal and human infections may result from the genetic variants present in bat coronavirus reservoirs. coronaviruses are large, pleomorphic, enveloped viruses containing a single linear, positive-sense single-stranded rna molecule. coronavirus genomes are approximately 27-32 kb in length, the largest continuous rna genomes among mammalian viruses [1] . coronaviruses are classified into four genera: alpha, beta, gamma, and delta [2] . coronaviruses are the second most prevalent cause of the common cold in humans [3] . in livestock and poultry, coronaviruses are recognized causes of enteric and respiratory infections that are often fatal in young animals [4] . an outbreak of severe acute respiratory syndrome (sars) in 2002 and 2003 resulted in infection of 8096 people worldwide, with 774 (9.5%) of them dying from this novel human coronavirus [5] . sars coronavirus is believed to have been acquired from an animal species, and the chinese horseshoe bat may have been the original source of sars infection [6] [7] [8] [9] . the emergent middle east respiratory syndrome coronavirus (mers-cov) may also have originated in bats, with fragments of mers-cov genes being identified in bats from both saudi arabia [10] and africa [11] . more than 200 viruses have been isolated from and detected in bats [12] . the ability of bats to fly and migrate, as well as the large sizes of their social groups, predisposes bats to acquire and maintain viruses [13] . given the import of mers into south korea [14] and the presence of sars in the relatively close geographic location of china [9] (fig. 3) , together with the fact that bats are a reservoir for coronaviruses, the prevalence of coronavirus infection in korean bat species should provide valuable information. although sars-like and mers-like bat coronaviruses have already been reported in the feces of korean bats [14] , the prevalence and presence of coronaviruses in other bat samples have not been described. therefore, we investigated the prevalence of coronaviruses in korean bat species using various kinds of samples, including oral swabs, fecal and urine samples, and carcasses. the coronaviruses found were characterized for their relationships to each other and to coronaviruses isolated in south korea and in other countries. a total of 672 samples (332 oral swabs, 245 fecal samples, 38 urine samples, and 57 bat carcasses) were collected at 33 sites of natural bat habitat from january 2016 to september 2016 (table 1 ). bats were captured by net for collection of oral swabs, fecal samples, and urine samples, and released immediately after sampling. fecal samples were also collected from guano. the bat carcasses collected were naturally dead bats found at the site where the other samples were taken. swab samples were kept in viral transport medium at 4°c, and organs collected from carcasses and other samples were stored at −80°c before processing. all fecal and tissue samples were resuspended in 1% antibiotic-antimycotic solution (corning, usa) diluted in phosphate-buffered saline (pbs), and clarified by centrifugation at 3500×g for 10 min. rna from 200 μl sample was extracted with the qiaamp® viral rna mini kit (qiagen, germany) and eluted in 60 μl rnase-free water. cdna was synthesized by primescript first strand cdna synthesis kit (takara, japan) following the manufacturer's instructions. bat-cov screening was performed by a pancoronavirus pcr method based on primers used by poon et al. (corona 1 forward, 5′-ggttgggactatcc taagtgtga-3′, and corona 2 reverse, 5′-ccat catcagatagaatcatcata-3′) [15] , followed by sequencing of the amplified product to confirm bat-cov identification. the pan-coronavirus primers were used to amplify and sequence a 440-bp segment of the highly conserved rna-dependent rna polymerase (rdrp) gene. cycle sequencing reactions were performed using the bigdye® terminator cycle sequencing kit version 1.1 (applied biosystems, foster city, ca, usa). reactions were purified using sigmaspin™ post-reaction clean-up columns (sigma, st. louis, mo, usa) and sequenced on an abi prism 3130 automated capillary dna sequencer (applied biosystems) according to the manufacturer's instructions. primer sequences were excluded from the raw sequence data, and the sequences we analyzed were 392bp long. all sequences obtained in this study were submitted to genbank (accession numbers ky432454-432471). the nucleotide sequences were aligned and compared to 52 selected human and animal cov sequences available from the genbank database using clustalw software implemented in bioedit version 7.0.9.0. the phylogenetic trees were drawn using the neighbor joining method using the maximum composite likelihood model with mega 7 software. coronaviruses were detected in 13 oral swabs, one carcass (intestine), and four fecal samples from a total of 672 samples, giving an overall detection rate of 2.7% (18/672). four of the 14 bat species tested were found to harbor coronavirus. thirteen positive oral swabs were detected from one species of the family rhinolophidae, which accounted for 24.3% (163/672) of the samples, and the other positive samples were from vespertilionidae bats that comprised 55.5% (373/672) of the samples. the 13 coronavirus-positive oral swabs were collected from bats at an abandoned mine in jeonbuk province ( fig. 1 ). oral swabs and other samples (n = 60) were obtained from three species of bats, rhinolophus ferrumequinum, miniopterus schreibersii, and myotis macrodactylus, but coronaviruses were only detected in samples from r. ferrumequinum (table 2 ). one carcass was collected from a cave in chungbuk province (fig. 1 ). in total, 94 samples were obtained from six bat species (m. schreibersii, myotis petax, vespertilio sinensis, murina leucogaster, r. ferrumequinum, and m. macrodactylus), and coronavirus was detected from a v. sinensis sample. four coronaviruspositive fecal samples were collected in four different regions, in gyeongbuk province (andong, yeongdeok, and gyeongju) and in the metropolitan city gwangju (fig. 1 ). in the forest of andong, 43 samples in total were collected from three species of bats (eptesicus serotinus, m. petax, and pipistrelus abramus), and coronavirus was detected from one p. abramus bat. in yeongdeok, a total of five samples was collected from p. abramus living in the forest, and one sample was positive for coronavirus. in the forest of gyeongju, five samples in total were collected only from p. abramus bats, and one of the samples was positive for coronavirus. in the forest of gwangju, 18 samples in total were collected from two species of bats (e. serotinus and myotis aurascens) and coronavirus was detected from one e. serotinus bat ( table 2) . to determine the genetic relationships between the 18 bat coronaviruses obtained in this study and previously known coronaviruses, the 392-nt rdrp sequences were analyzed phylogenetically. all the sequences obtained in this study belonged to the betacoronavirus genus (fig. 2) . the sequences (fig. 2) . the 13 sequences from oral swabs were 97.1-100.0% identical with each other, and the closest known cov strain, with 97.1-99.7% nucleotide identity, was bat-cov-jtmc15 that was recently isolated from r. ferrumequinum in china. the nucleotide identity with sars-cov was only 86.7-89.0%. one sequence from a carcass showed the highest identity (99%) with bat-cov-sc2013 that was isolated from vespertilio superans (synonym of v. sinensis), also from china [16] . the nucleotide identity with mers-cov was 83.9%. four sequences from fecal samples shared 96.9-100.0% identity with each other and showed 94.1-95.1% identity with bat-cov hku5 [17] . the nucleotide identity with mers-cov was 84.1-84.4%. due to shorter reported sequences from kim et al. [14] , another phylogenetic tree was constructed from the 355-bp rdrp sequences of korean bat coronaviruses obtained by kim et al. [14] and in this study to analyze the genetic relation between bat-covs in south korea (fig. 3) . thirteen sequences from oral swabs were clustered with bat-cov b15-21, which was detected in fecal bat samples collected from an abandoned mine in gangwon province. the major bat species present there was r. ferrumequinum, but the coronaviruspositive bat species were not specified [14] . nucleotide identity between bat-cov b15-21 and the 13 clustered sequences was 97.7-100.0%. the other three korean bat coronaviruses (bat-cov b15-8, b15-40, and b15-41) belonged to the alphacoronavirus genus, but we did not detect coronaviruses belonging to this group in our study. there are approximately 24 species of bats in south korea, and most are insectivorous and relatively small [18] . bats in south korea usually do not migrate over distances greater than 1000 km [19], but apart from two species found only in south korea, they are generally distributed in neighboring countries such as china, east siberia, japan, and taiwan. although it is not clear if bats go between china and other countries, some species that migrate over 500 km can carry pathogens to south korea. the fact that the 18 bat coronaviruses (bat-covs) detected in this study were all in this study, the prevalence of coronavirus in korean bat samples was 2.7% overall. the prevalence by sample type was 5.4% (13/332) for oral swabs, 1.8% (1/57) for carcasses, 1.6% (4/245) for fecal samples, and 0.0% (0/38) for urine samples. reported rates from other countries for coronavirus prevalence in bats were 6.5% (64/985) and 5.3% (50/951) for china, 8.2% (16/195) for italy, and 5.3% (32/606) for mexico [20] [21] [22] [23] , not greatly different from our figure of 2.7% for prevalence in south korea. fourteen out of 24 korean bat species were screened for covs in this study. the species positive for bat-covs were r. ferrumequinum, v. sinensis, p. abramus, and e. serotinus. the bat-covs that showed the highest nucleotide identity with our isolates were obtained from the same species that we identified as positive, except for e. serotinus. to our knowledge, this is the first time a bat-cov has been detected from e. serotinus in asia. in europe (italy), a lineage c betacoronavirus was detected from e. serotinus in 2014 [24] . bat-covs were usually detected at higher rates in alimentary samples (fecal samples, rectal swabs, and intestine) than in respiratory samples (oral and throat swabs) [7, 15, 17, 22, 25, 26] . we collected alimentary and respiratory samples and urine samples. however, bat-covs were not detected in urine samples. animal coronaviruses usually produce either enteric or respiratory infections. animal models show similar clinical features to sars and mers patients; the majority of whom presented with respiratory symptoms but in some cases also suffered from enteric complications [27] . unlike other human coronavirus infections, a number of mers cases were associated with acute renal failure. mers-cov replication in the human kidney suggests the potential of shedding viruses in urine [28] . bat-cov isolate bat sl-cov-wiv1 (kf367457) showed infectivity to rhinolophus sinicus kidney cell lines, which means it may be possible to detect bat-cov in urine [6] . the current study revealed that korean bats have sarsand mers-related bat-covs, consistent with earlier work [ 1 4 ] . t h e p r e v i o u s s t u d y d e t e c t e d a l p h a -a n d betacoronaviruses only from fecal samples in four regions, two in gangwon, one in chungbuk, and one in gyeongbuk [14] . the only betacoronavirus rdrp sequence bat-cov b15-21 (ku528590.1) was clustered with 13 bat-covs β-cov lineage b β-cov lineage c α-cov fig. 3 phylogenetic tree of korean bat coronaviruses detected by kim et al. [13] and in this study, based on 355-nt sequences. phylogenetic trees were constructed by using the neighbor joining method and bootstrap values were determined by 3000 replicates. scale bar the estimated genetic distance of these viruses. black circle sequences from oral swabs. black up-pointing triangle sequences from fecal samples. white uppointing triangle sequences from carcass (16bo122, 129, 132, 133, 134, 135, 139, 141, 143, 145, 150 , 154, and 155) detected from danyang, chungbuk province. the identity between the 14 sequences was very high at 97.7-100%, even though the sample types and collection sites were different. the species of bat infected with bat-cov b15-21 was not specified in the paper, but the major bat species of the sample collection site was r. ferrumequinum; the same species in which we detected 13 bat-covs. the same bat-covs were detected from fecal samples in the prior study [14] and from oral swabs in our study. spike proteins, which are translated from spike genes, define viral tropism by receptor specificity and by membrane fusion activity during entry into cells [29] . although the spike genes were not identified in this study, full spike gene analysis of bat-cov b15-21 suggested a low potential for human emergence. one mers-like betacoronavirus, bat-cov b15-1-6 (ku528586.1, ku528585.1), was identified by kim et al. [14] , but the similarity of this isolate to our isolates could not be analyzed because the rdrp sequence was not obtained. high nucleotide identity between bat-cov b15-1-6 and bat-cov-sc2013 (kj473821) suggests that bat-cov b15-1-6 and 16bt3 might be very similar. bat-covs have been identified in many countries including the australasian region [10, 11, 14-16, 20-26, 30, 31] . but bat-covs identified in korea were genetically close to bat-covs from china, a close neighbor of korea. according to the results of bat-cov research in china, the degree of host restriction for coronaviruses in bat populations was high. for example, similar viruses were detected in the same species of bat in different regions, approximately 1600 km apart, while two different bat species in the same habitat had different coronaviruses [22] . in our results, three bat-covs (16bf109, 211, and 244) from p. abramus were clustered with each other, even though samples were collected from different habitats in different regions (andong, yeongdeok, and gyeongju). the bat-cov detected from e. serotinus bat in the metropolitan city gwangju (16bf104) was also clustered with these three bat-covs mentioned above. however, there was no matching virus for 16bf104 found in the same species of bat in other locations, nor in other species in the region where samples were collected. our data could support the possibility of relationships between genetic variation, geographic locations where bat-covs were isolated, and bat host species, but more data on bat-covs in korea would be needed to address this. in summary, the prevalence of coronavirus in korean bats was determined and found to be comparable to or lower than the rates in other countries. we detected sars-related and mers-like bat-covs that were genetically very similar to bat-covs identified in china. for the study of bat-cov seasonal occurrence and transmission, continuous monitoring during all seasons is required, and ideally alimentary, respiratory, and other samples need to be collected from each bat. future work to determine the complete genome sequences of bat-covs from south korea would give a more complete picture of their pathogenicity and the possibility of infectious spread to other animals and humans. bat cov-jtmc15(ku182964) bat cov-273(dq648856) sars bat cov-lingbao(kf294456) sars bat cov-rf1(dq412042) bat cov-rp(jx993987) bat cov hku3-1(dq022035) bat cov-rp3(dq071615) bat cov rsshc014(kc881005) sars cov-ma15(jf292915) sars cov-tor2(jx163928) sars cov-urbani(ay278741) sars-cov(nc004718) bat cov bm48-31/bgr/2008(gu190251) bat cov slo1a0082(gq404797) phev(nc007732) mhv(nc001846) hcov-hku-1(nc006577) bat cov-hku9(nc009021) tcv(nc010800) ibv(nc001451) bat cov-hku4(nc009019) kbat cov-16bf104 kbat cov-16bf109 kbat cov-16bf221 kbat cov-16bf244 bat cov hku5-1(dq249217) bat cov-sc2013(kj473821) kbat cov-16bt3 bat cov(kc243390) bat cov btcov/ukr-g17(kc243392) mers-cov(nc019843) human beta-cov(jx869059) human 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mechanism for acute renal failure during middle east respiratory syndrome (mers) coronavirus infection coronavirus spike proteins in viral entry and pathogenesis coronavirus infection and diversity in bats in the australasian region ecohealth detection of group 1 coronaviruses in bats in north america emerg acknowledgements we thank dr. c.w. jeong and his colleagues for their efforts in the collection of wild bat carcasses and samples. this research was supported by grant no. 2016-01-01-033 from the nier of the republic of korea. the funders had no roles in the study design, data collection and analysis, decision to publish, or the preparation of the manuscript. key: cord-338400-30vl2hks authors: epstein, jonathan h.; quan, phenix-lan; briese, thomas; street, craig; jabado, omar; conlan, sean; ali khan, shahneaz; verdugo, dawn; hossain, m. jahangir; hutchison, stephen k.; egholm, michael; luby, stephen p.; daszak, peter; lipkin, w. ian title: identification of gbv-d, a novel gb-like flavivirus from old world frugivorous bats (pteropus giganteus) in bangladesh date: 2010-07-01 journal: plos pathog doi: 10.1371/journal.ppat.1000972 sha: doc_id: 338400 cord_uid: 30vl2hks bats are reservoirs for a wide range of zoonotic agents including lyssa-, henipah-, sars-like corona-, marburg-, ebola-, and astroviruses. in an effort to survey for the presence of other infectious agents, known and unknown, we screened sera from 16 pteropus giganteus bats from faridpur, bangladesh, using high-throughput pyrosequencing. sequence analyses indicated the presence of a previously undescribed virus that has approximately 50% identity at the amino acid level to gb virus a and c (gbv-a and -c). viral nucleic acid was present in 5 of 98 sera (5%) from a single colony of free-ranging bats. infection was not associated with evidence of hepatitis or hepatic dysfunction. phylogenetic analysis indicates that this first gbv-like flavivirus reported in bats constitutes a distinct species within the flaviviridae family and is ancestral to the gbv-a and -c virus clades. bats (order chiroptera), after rodents, comprise the most diverse group of mammals with more than 1,100 species. they are present on six continents, often have substantial habitat overlap with humans [1] and harbor several zoonotic viruses causing significant human morbidity and mortality, including ebola-and marburgvirus, nipah virus (niv), and sars-like coronaviruses [2] [3] [4] [5] . proximity of bats to human populations may facilitate the zoonotic transmission of viruses either through direct contact, via amplifying domestic animal hosts, or through food-borne routes [6] [7] [8] . the current study was set up as part of a viral discovery effort to target key wildlife reservoirs in emerging disease hotspots. bangladesh is a 'hotspot' for emerging zoonotic diseases [9] , with a relatively high diversity of wildlife that likely harbors new zoonotic pathogens, one of the densest human populations on the planet, and a high level of connectivity between people, domestic animals and wildlife. in bangladesh and india, frugivorous pteropus giganteus bats have been identified as a reservoir for niv [10, 11] , which has been recognized as the cause of several outbreaks of encephalitis [12] [13] [14] . pteropus giganteus bats are common throughout the indian subcontinent, living in close association with humans and feeding on cultivated fruit [14] . niv transmission from bats to humans has been linked with the harvest and consumption of raw date palm sap, which becomes contaminated with bat feces, urine or saliva overnight when bats such as p. giganteus come to feed from the collecting pots [14, 15] . date palm sap or other foods eaten by both bats and people, may also serve as a vehicle for transmission of other bat-borne agents. several zoonotic flaviviruses, including japanese encephalitis virus, west nile virus, and kyasanur forest virus have been identified in bats; however, to date, gb viruses have not [1] . gb viruses a and c (gbv-a and -c) represent two recently identified species that are currently unassigned members of the family flaviviridae [16] . gbv-a viruses have been described in new world primates and are not known to infect humans [17] [18] [19] , while gbv-c (also known as hepatitis g virus (hgv)) have frequently been isolated from humans in many regions of the world, including india and bangladesh [19] [20] [21] [22] [23] , and from wild chimpanzees (pan troglodytes) in africa [24, 25] . here we describe discovery of a virus in the serum of healthy bats in bangladesh, tentatively named gb virus d (gbv-d), that is distantly related to gbv-a and -c and represents a new member of the family flaviviridae. every effort was made to minimize bat stress and avoid injury during capture, restraint, and sampling procedures. this study was conducted following wildlife trust institutional guidelines under iacuc approval g2907 issued by tufts new england medical center, boston, massachusetts. as part of a longitudinal surveillance study of nipah virus in bats, 98 free-ranging p. giganteus bats were caught from a colony of approximately 1800 individuals in the faridpur district of bangladesh in december 2007 ( figure 1 ). each bat was anesthetized using isoflurane gas; morphometric measurements (weight, forearm length, head length, and body condition) were taken and bats were aged [10] . each bat was marked for future identification using an rfid microchip (avid corp, www.avidid. com) implanted subcutaneously between the scapulae. three ml of blood were collected and placed into serum separator tubes (vacutainer; becton dickinson, franklin lakes, nj, usa). serum was allowed to separate overnight at 4uc then drawn off without centrifugation and immediately frozen using a liquid nitrogen dry shipper. to inactivate potentially infectious agents, serum samples were heat-treated at 56uc for 30 min and then stored at 270uc. for rna extraction, 250 ml of serum was added to 750 ml tri-reagent ls (molecular research center, cincinnati, oh, usa). saliva was collected from the bat's throat using a sterile cotton swab. urine was collected either by catching urine in a 1.0 ml sterile cryovial while the bat was urinating, or by urethral swab. urine and saliva swabs were immediately placed into 1 ml tri-reagent ls and frozen in liquid nitrogen. total rna from serum was extracted for uhts analysis to screen for the presence of microorganisms. five microliters of total rna from each bat were combined into 4 pools: 4 pregnant bats; 4 non-pregnant female bats, and 2 pools of 4 adult male bats, respectively. reverse transcription (rt) was performed on dnase i-treated (dna-free, ambion inc., austin, tx, usa) rna pools to generate cdna using superscript ii rt (invitrogen, carlsbad, ca, usa) and random octamers linked to a defined arbitrary, 17-mer primer sequence tail (mwg, huntsville, al, usa) [26] . after rnase h treatment cdna was amplified by the polymerase chain reaction (pcr), applying a 9:1 mixture of the defined 17-mer primer sequence and the random octamer-linked 17-mer primer sequence, respectively [27] . products of .70 base pairs (bp) were selected by column purification (minelute, qiagen, hilden, germany) and ligated to specific linkers for sequencing on the 454 genome sequencer flx (454 life sciences, branford, ct, usa) without dna fragmentation [28, 29] . sequences were analyzed using software applications implemented at the greeneportal website (http:// tako.cpmc.columbia.edu/tools/). multiple forward and reverse primers for rt-pcr (available upon request) were designed using the sequences obtained by uhts in order to fill gaps between fragments. amplifications were performed with bio-x-act (bioline, london, uk) according to manufacturer's protocols. products were size fractionated by electrophoresis and directly sequenced in both directions with abi prism big dye terminator 1.1 cycle sequencing kits (perkin-elmer applied biosystems, foster city, ca, usa) at a commercial facility (genewiz, south plainfield, nj, usa). additional methods applied to obtain the genome sequence included touch-down pcr [30] , 2-step walking pcr [31] , and 39-and 59-race (invitrogen). a real time taqman pcr assay was developed to screen bat samples for gbv-d. reactions were performed in a 25 ml volume by using commercial taqman universal master mix (applied biosystems, foster city, ca, usa). primers and probe were designed to target a 60 nt region in the ns4a gene region: fadiforward, 59-gcagctgcgtgtgcca; fadi-reverse, 59-acacc-catgatgttaccacgac; fadi-probe, 59-fam-aggacccgg-tcgctccagca-t-bqx (tib molbiol, adelphia, nj, usa). cycling conditions were: 50uc for 2 min, and 95uc for 10 min, followed by 45 cycles at 95uc for 15 sec and 60uc for 1 min. thermal cycling was performed in an abi 7300 real-time pcr system (applied biosystems). a liver function panel was conducted at the international center for diarrheal disease research (dhaka, bangaldesh) using non heat-treated bat sera (automated chemistry analyzer au 640, olympus corporation, tokyo, japan). the following parameters were analyzed: total protein, albumin, globulin, albumin:globulin ratio, total cholesterol, total bilirubin , alkaline phosphatase, alanine transferase, aspartate aminotransferase, gamma glutamyltransferase , and lactate dehydrogenase. sequence alignments were generated with clustalw software [32] and phylogenetic relationships deduced using geneious software [33] . statistical significance was assessed by bootstrap re-sampling of 1000 pseudoreplicate data sets. sequence relations were determined from p-distance matrices calculated with pairwise deletion for missing data and homogeneous patterns among lineages based on clustalw alignments as implemented in mega software [34] . sliding window similarity analysis was performed using simplot [35] . potential signalase cleavage sites, glycosylation sites, and phosphorylation sites were analyzed using the respective prediction servers available at the center for biological sequence analysis (http://www.cbs.dtu.dk/services/). bats are important reservoirs for emerging zoonotic viruses with significant impact on human health including lyssaviruses, filoviruses, henipaviruses and coronaviruses. opportunities for transmission to humans are particularly prominent in countries like bangladesh, where people live in close association with bats. whereas previous studies of bats have employed assays that test for known pathogens, we present the first application of an unbiased molecular approach to pathogen discovery in this reservoir for emerging zoonotic disease. unbiased pyrosequencing of serum from pteropus giganteus bats enabled identification of a novel flavivirus related to hepatitis c and gb viruses. viral nucleic acid was present in 5 of 98 (5%) sera, and in the saliva of one animal. sequence identification of two strains of the virus, tentatively named gbv-d, suggests p. giganteus as a natural reservoir. detection of viral nucleic acid in saliva provides a plausible route for zoonotic transmission. phylogenetic analysis indicates that gbv-d is ancestral to gbv-a and -c, and separate from the recently classified genus hepacivirus. our findings provide new insight into the range of known hosts for gb-like viruses and demonstrate the power of unbiased sequencing to characterize the diversity of potentially zoonotic pathogens carried by bats and other reservoirs. total rna from the serum of healthy bats captured at a roost in the faridpur district of bangladesh was extracted for uhts analysis. extracts of 16 individual bats were combined into 4 pools consisting of 4 pregnant adult bats, 4 non-pregnant adult female bats, or 264 adult male bats. each pool yielded between 1,400 and 2,000 assembled contigs or singlton reads (representing 50,000-75,000 reads ranging in size from 31-328 nt). two reads of 238 and 215 nucleotides (nt) derived from the pregnant bat pool had distant homology to gbv-a sequences at the deduced amino acid (aa) level in the e2 and ns4a gene regions respectively (blastx); no homology was detected by searches at the nt level (blastn; local copy of the executables with standard settings except that the reward for a nucleotide match was set to 2 instead of 1). no viral sequences were detected in other pools at the nt or aa levels. screening of the individual rna preparations from the pregnant bat pool using primers derived from the uhts reads confirmed the presence of the gbv-like sequence in the serum of bat 93. a quantitative real time pcr assay indicated a load of approximately 30 000 rna copies in bat-93 serum extract, and identified an additional 4 positive bat sera from the original 98 samples (5/98; 5%), indicating serum loads ranging from 350 to 70,000 rna copies per assay. these positive samples came from male bats that were not included in the initial uhts pools. extracts of saliva from the five positive bats indicated a load of approximately 200 rna copies in bat 93; no signal was obtained with urine extracts from the five positive bats. near full-length genome sequence was generated from bat-93 and a second positive serum (bat 68), applying primers crossing mature structural proteins in gb viruses, as well as other flaviviruses, are the product of cleavage by host signal peptidase [36] . in gbv-d the first potential signal sequence cleavage site is present after a stretch of 57, largely basic aa (6 kda, pi = 12), followed by sequence homologous to e1 (pfam 01539, http:// pfam.sanger.ac.uk/) ( figure 3 ). the single glycosylation site n 177 it present in that sequence is located in a position comparable to gbv-c, -a, -b and hcv glycosylation sites. identification of the downstream e2 termini is less apparent as the next 580 aa contain multiple potential signal sequences and 10 potential glycosylation sites that indicate no homology to hepaciviral e2/ns1 (pfam 01560), until the sequence aligns with n-terminal ns2 motifs (pfam 01538) (figure 2, figure 3 ). however, despite similarity to pfam 01538 no signal sequence compatible with cleavage at a 759 /a was found; cleavage may occur at g 826 /r, which combined with potential signalase cleavage at a 584 /f may indicate the existence of a heavily glycosylated potential 26 kda product instead of the p7 transmembrane protein identified in hcv [37] [38] [39] or the 13 kda variant described in gbv-b [40, 41] . conserved c-terminal motifs of the autocatalytic ns2/ns3 endoprotease domain are compatible with ns2/ns3 cleavage at s 1067 /a and comparable to other gbv and hcv [42] . figure 3 indicates potential cleavage sites for ns3 (peptidase s29, pfam 02907; dead box helicase, pfam 07652; helicase c, pfam 00271), ns4a (pfam 01006), ns4b (pfam 01001), ns5a (domain-1a zinc finger, pfam 08300; domain-1b, pfam 08301), and ns5b (pfam 00998). conserved aa motifs were recognized in ns proteins. rnadependent rna polymerase (rdrp) motifs in rdrp block iii that are conserved with respect to other gbv and hepaciviruses were identified in ns5b (figure 3 ) [43] [44] [45] [46] . potential phosphorylation sites are present at multiple serine (9), threonine (14) and tyrosine (4) residues in ns5a, compatible with its possible function as a phosphorylation-regulated mediator of viral replication [47] . however, significant conservation of primary sequence is not obvious for phosphorylation sites, proline-rich, or interferon-sensitivity determining region motifs [48] [49] [50] . the c-terminal portion of ns3 has homology to conserved ntpase/helicase motifs [51] ; the nterminal portion includes conserved active triad residues h 1123 , d 1147 , s 1204 of serine protease [52] , the viral protease responsible for cleavage of mature non-structural proteins [53] . likewise, the active triad h 991 , e 1011 , c 1032 of the cis-acting protease activity in the cterminal portion of ns2 is conserved with respect to other gbv and hcv [42] . the only other discernable motif identified was a wellconserved n 75 c/d c motif at the n-terminus of e1 (figure 3 ) [54] . phylogenetic analysis of gbv-d was performed in comparison to selected representatives of gbv-a, gbv-b, gbv-c and hcv. analysis of ns5b aa sequence ( figure 4a ) confirmed a closer relationship of gbv-d to gbv-a and -c than to gbv-b or hcv as also indicated by pairwise sequence comparisons ( table 1) . the same relationships were also apparent when ns3, or the complete polyprotein sequence were analyzed ( figure 4b and c, respectively). all three trees show gbv-d consistently at the root of the gbv-a/-c viruses, indicating an independent phylogenetic clade compatible with a separate species distinct from the recently created genus hepacivirus [16] . a liver serum chemistry panel was conducted on sera from 15 bats, the five gbv-d infected and 10 non-infected animals. standard assays to detect hepatitis and/or impaired liver function were performed [55] . levels of total protein, alanine transferase, aspartate aminotransferase and total cholesterol were within published ranges reported for p. giganteus, except for bat 33 (infected) and bat 73 (uninfected), which had modest elevation in aspartate aminotransferase. reference values for albumin, globulin, albumin:globulin ratio, total bilirubin, alkaline phosphatase, gamma glutamyltransferase and lactate dehydrogenase are not available for p. giganteus, however, values were comparable to those reported for other pteropus species [56] . mean values did not significantly differ between infected and uninfected bats ( table 2 ). molecular analyses of sera from pteropus giganteus bats from faridpur, bangladesh led to the identification of a 9,633 nt sequence consistent in genomic organization with known gbv and other species within the family flaviviridae [16] . whereas previous studies of bats have employed assays that test for known pathogens, ours is the the respective sequences are indicated. entebbe bat virus was used as an outgroup; distance in substitutions per site is indicated by scale bars; percent bootstrap support for values greater than 85% is indicated at respective nodes. doi:10.1371/journal.ppat.1000972.g004 table 2 . liver function values from pteropus giganteus bats. no indication of hepatitis or impaired liver function was observed; no significant differences between mean values for infected (bold) or non-infected bats were apparent. the first report of an unbiased molecular approach to pathogen discovery in this important reservoir of emerging infectious diseases. the modest yield of novel microbial sequences may reflect the choice of sample (e.g., serum vs feces, tissue or another specimen), competition between host and microbial template during unbiased amplification, or both. efforts to address template competition are under way that include subtraction of host nucleic acids or the use of semi-random primers that do not amplify host sequences. such efforts will likely enhance the sensitivity and throughput of unbiased sequencing technologies for pathogen discovery. the discovery of this chiropteran flavivirus broadens both the taxonomical and geographical distribution of gb-like viruses. three types of gb viruses have been described: gbv-a, -b and -c [18, 19, 24, 25, 54, 57] . gbv-b, which has never been found in humans and was only reported in captive tamarins after serial passage of the original human gb serum [58] , is most closely related to hcv and was recently classified together with hcv into a new genus, hepacivirus, within the family flaviviridae [16] . gbv-a and -c remain unclassified members of the family. gbv-a have been isolated from several new world monkeys. different genotypes appear to be associated with specific monkey species of the genera saguinus, callithrix (callitrichidae family) and aotus (aotidae family), without any clinical signs associated with infection [24, 54, 57] . gbv-c have been isolated from humans with non-a-e hepatitis; however, its pathogenicity is unknown and the virus is widespread in the human population [21, [59] [60] [61] . population studies showed that gb viruses are enzoonotic and species-specific within both old and new world nonhuman primates as well as humans, and have likely co-evolved with their hosts over long periods of time [62] . previously, the only gbv found in the old world was gbv-c from chimpanzees (in africa) and humans. although gbv-c were found in humans, gb viruses have not been previously reported in primates or other animals on the indian subcontinent. gbv-c and -a are remarkable for a truncated or missing capsid (c) protein [18, 19] . due to exhaustion of our samples we were unable to complete assessment of the 59-terminal sequence; nonetheless, race experiments suggest that gbv-d likely codes for a short basic peptide, instead of a full-length c protein. the first methionine (m 1 ) predicts a peptide of 57 aa (pi = 12); however, the more favorable kozak context [63] of m 3 indicates a 55 aa peptide. after signalase cleavage from the polyprotein precursor, this peptide may be functional, possibly influencing maturation of, or directly binding to, the e1 and/or e2 glycoproteins. phylogenetic analyses of ns5b, ns3 and complete polyprotein sequence place gbv-d at the root of the gbv-a and -c clades and are consistent with a model wherein gbv-d is ancestral to gbv-a and -c clades. mixed relationships indicative of recombination events [64] were not evident (figure 2, figure 4 ). both pteropid bats and chimpanzees are restricted to the old world. while the range of chimpanzees (africa) and p. giganteus (the indian subcontinent) do not overlap, it is possible that other primate species in bangladesh or india, such as macaques, or other fruit bats in africa such as eidelon spp., whose range overlaps that of chimpanzees, may carry related viruses. while gbv-a is only known from primates of the new world, an african origin has been suggested for gbv-c based on a 12-aa indel sequence in ns5a [65] . although the ns5a sequence of gbv-d, similar to that of gbv-a, appears elongated in the indel region, compatible with their respective earlier phylogenetic branching compared to gbv-c, little sequence conservation is observed in that region. the bats in this study, like primates infected with their associated gbv [66] , all appeared to be healthy. the lack of chemical evidence of hepatic inflammation or dysfunction suggests that this virus may not target hepatic cells in bats. this is consistent with the behavior of gbv-a in its natural primate hosts [54] . in contrast, elevated alanine transferase levels and mild hepatitis are observed in experimental infections of macaques with gbv-c isolates from humans [67] . five percent of the bats we studied were infected with one of at least two different strains of gbv-d, which suggests widespread viral circulation within this species. the observation that bats are asymptomatically infected with diverse strains that constitute a distinct phylogenetic clade is compatible with a co-evolutionary relationship between gbv and their hosts [57, 62] , and supports the hypothesis that p. giganteus bats may be a natural reservoir for gbv-d. in one case we were able to detect gbv-d nucleic acid in saliva. this suggests a potential route for viral transmission via fighting or grooming behavior, or via food shared by bats. pteropus giganteus is a frugivorous bat species that carries niv, a zoonotic paramyxovirus [10, 11] . this species lives in close association with humans in bangladesh and bats have been observed drinking from (and urinating into) date palm sap collecting pots [14] . human consumption of contaminated palm juice is proposed to be a major route of niv transmission [68] . although it is unclear whether infectious virus was present in bat saliva, the observation that saliva can contain gbv-d nucleic acids provides a biologically plausible 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constitute a separate genus of the flaviviridae studies on transmission of human viral hepatitis to marmoset monkeys. 1. transmission of disease serial passages and description of liver lesions acute non-a-e hepatitis in the united states and the role of hepatitis g virus infection gb virus type c/hepatitis g virus clinical impact of gb virus c viremia on patients with hiv type 1 infection in the era of highly active antiretroviral therapy phylogenetic analysis of gb viruses a and c: evidence for cospeciation between virus isolates and their primate hosts initiation of translation in prokaryotes and eukaryotes homologous recombination in gb virus c/ hepatitis g virus african origin of gb virus c hepatitis g virus species-specific variants of gb virus a in captive monkeys serological and histological findings in infection and transmission of gbv-c/hgv to macaques transmission of human infection with nipah virus we thank the forestry department of bangladesh for permission to conduct this research; md. sheikh gofur and md. pitu biswas for help in sampling bats; a. bennett, a. tashmukhamedova, and r. tokarz for technical support, and k. olival for critical comments on the manuscript. conceived and designed the experiments: jhe tb pd wil. performed the experiments: jhe plq dv sh. analyzed the data: jhe plq tb cs oj dv skh me pd. contributed reagents/materials/analysis tools: jhe tb cs sc sak mjh skh me spl wil. wrote the paper: jhe plq tb wil. coordinated field and lab activities and logistics for work in bangladesh; contributed to paper-writing: mjh. coordinated permissions, field activities and logistics for work in bangladesh; contributed to paperwriting: spl. key: cord-338055-2d6n4cve authors: hassan, sk. sarif; ghosh, shinjini; attrish, diksha; choudhury, pabitra pal; seyran, murat; pizzol, damiano; adadi, parise; el-aziz, tarek mohamed abd; soares, antonio; kandimalla, ramesh; lundstrom, kenneth; tambuwala, murtaza; aljabali, alaa a. a.; lal, amos; azad, gajendra kumar; uversky, vladimir n.; sherchan, samendra p.; baetas-da-cruz, wagner; uhal, bruce d.; rezaei, nima; brufsky, adam m. title: a unique view of sars-cov-2 through the lens of orf8 protein date: 2020-08-26 journal: biorxiv doi: 10.1101/2020.08.25.267328 sha: doc_id: 338055 cord_uid: 2d6n4cve immune evasion is one of the unique characteristics of covid-19 attributed to the orf8 protein of severe acute respiratory syndrome coronavirus 2 (sars-cov-2). this protein is involved in modulating the host adaptive immunity through downregulating mhc (major histocompatibility complex) molecules and innate immune responses by surpassing the interferon mediated antiviral response of the host. to understand the immune perspective of the host with respect to the orf8 protein, a comprehensive study of the orf8 protein as well as mutations possessed by it, is performed. chemical and structural properties of orf8 proteins from different hosts, that is human, bat and pangolin, suggests that the orf8 of sars-cov-2 and bat ratg13-cov are very much closer related than that of pangolin-cov. eighty-seven mutations across unique variants of orf8 (sars-cov-2) are grouped into four classes based on their predicted effects. based on geolocations and timescale of collection, a possible flow of mutations was built. furthermore, conclusive flows of amalgamation of mutations were endorsed upon sequence similarity and amino acid conservation phylogenies. therefore, this study seeks to highlight the uniqueness of rapid evolving sars-cov-2 through the orf8. severe acute respiratory syndrome-coronavirus-2 (sars-cov-2) is a novel coronavirus whose first outbreak was reported in december 2019 in wuhan, china, where a cluster of pneumonia cases was detected, and on 11th march, 2020, who declared this outbreak a pandemic [1, 2, 3] . as of 22nd august 2020, a total of 22.9 million confirmed covid-19 cases have been reported worldwide with 7,99,350 deaths [4] . sars-cov-2 belongs to the family coronaviridae and has 55% 5 nucleotide similarity and 30% protein sequence similarity with sars-cov, which caused the previous outbreak of sars in 2002 [5, 6] . sasr-cov2 is a single-stranded rna virus of positive polarity whose genome is approximately 30 kb in length and encodes for 16 non-structural proteins, four structural and six accessory proteins [7, 8] . the sars-cov-2 has a total of six accessory proteins including 3a, 6, 7a, 7b, 8 and 10 [9, 10] . among these accessory proteins, orf8 is a significantly exclusive protein as it is different from another known sars-cov and thus associated with high efficiency 10 in pathogenicity transmission [11, 12] . the sars-cov-2 orf8 displays arrays of functions; inhibition of interferon 1, promoting viral replication, inducing apoptosis and modulating er stress [13, 14, 15] . the sars-cov-2 orf8 is a 121 amino acid long protein, which has an n-terminal hydrophobic signal peptide (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) aa) and an orf8 chain (16-121 aa) [16, 17] . the functional motif (vlvvl) of sars-cov-orf8b, which is responsible for induction of cell stress pathways and activation of macrophages is absent from the sars-cov-2 orf8 protein [18] . in 15 the later stages of the sars-cov epidemic it was found that a 29 nucleotide deletion in the orf8 protein caused it to split into orf8a (39 aa) and orf8b (84aa) rendering it functionless while the sars-cov-2 orf8 is intact [19] . also, the sars-cov orf8 had a function in interspecies transmission and viral replication efficiency as a reported 382 nucleotide deletion, which included orf8ab resulted in a reduced ability of viral replication in human cells [20] . however, the sars-cov-2 orf8 mainly acts as an immune-modulator by down-regulating mhc class i molecules, therefore protecting the 20 infected cells against cytotoxic t cell killing of target cells. simultaneously it was proposed forth that it is a potential inhibitor of type 1 interferon signalling pathway which is a key component of antiviral host immune response [21, 22] . the orf8 also regulates unfolded protein response (upr) induced due to er stress by triggering atf-6 activation, thus promoting infected cell survivability for its own benefit [23] . since this protein impacts various host pathogen processes and developed various strategies that allow it to escape through host immune responses, it becomes important to study the 25 mutations in the orf8 to develop a better understanding of the viral infectivity and for developing efficient therapeutic drugs against it [24] . in this present study, we identified the distinct mutations present across unique variants of the sars-cov-2 orf8 and classified them according to their predicted effect on the host, i.e disease or neutral and the consequences on protein structural stability. furthermore, we compared the orf8 protein of sars-cov-2 with that of bat-ratg13-cov and 30 pangolin-cov orf8 and tried to determine the evolutionary relationships with respect to sequence similarity and discussed the rising concern on its originality. in addition we made a study on polarity and charge of the sars-cov-2 orf8 mutations in two of its distinct domains and explored the possible effect on functionality changes. following this, we present the possible flow of mutations considering different geographical locations and chronological time scale simultaneously, validating with sequence-based and amino acid conservation-based phylogeny and thereby predicted the possible route 35 taken through the course of assimilation of mutations. as of 14th august, 2020, there were 10,314 complete genomes of sars-cov-2 available in the ncbi database and accordingly each genome contains one of the accessory proteins orf8 and among them only 127 sequences were found to 40 be unique. the amino acid sequences of the orf8 were exported in fasta format using file operations through matlab [25] . among these 127 unique orf8 protein sequences, only 96 sequences possess various mutations and the remaining sequences do not either possess any mutations or possess ambiguous mutations. in this present study, we only concentrate on 96 orf8 proteins, which are listed in table 1 . in order to find mutations, we hereby consider the reference orf8 protein as the orf8 sequence (yp 009724396.1) of the sars-cov-2 genome (nc 045512) from china: wuhan [26] . 45 only the mutations l3f, t14a, k44r, f104y and v114i are embedded in the orf8 proteins of bat-cov. mutations in proteins are responsible for several genetic orders/disorders. identifying these mutations requires novel detection methods, which have been reported in the literature [27] . in this study, each unique orf8 sequence was aligned using ncbi protein p-blast and sometimes using omega blast suites to determine the mismatches and thereby the missense mutations (amino acid changes) are identified [28, 29] . for the effect of identified mutations, a web-server 'meta-snp' was used and also for the structural effects of mutations, another web-server 'i-mutant' was used [30, 31] . the web-server 60 'quark' was used for prediction of the secondary structure of orf8 proteins [32, 33] . a protein sequence of orf8 is composed of twenty different amino acids with various frequencies. per-residue disorder distribution in sequences of query proteins was evaluated by pondr-vsl2 [40] , which is one of the more accurate standalone disorder predictors [41, 42, 43] . the per-residue disorder predisposition scores are on a scale from 0 to 1, where values of 0 indicate fully ordered residues, and values of 1 indicate fully disordered residues. values above the threshold of 0.5 are considered disordered residues, whereas residues with disorder scores between 0.25 and 0.5 are considered highly flexible, and residues with disorder scores between 0.1 and 0.25 are taken as moderately flexible. the sars-cov-2 orf8 protein (yp 009724396) is a 121 amino acid long protein which has an n-terminal hydrophobic signal peptide (1-15 aa) and an orf8 chain (16-121 aa). a schematic representation of orf8 (sars-cov-2) is presented in fig.2 . it was observed that the total number (63) of hydrophilic residues is more than that (58) of the hydrophobic residues. however, from the predicted secondary structure (fig.3) , it was found that the highest solubility score is four, indicating that although hydrophilic residues are higher in number, however, they are not highly exposed to the external environment since they are folded inside of the protein therefore this protein is poorly soluble. we further predicted the secondary structure as well as solvent accessibility of orf8 proteins of sars-cov-2, batratg13-cov and pangolin-cov using the ab-initio webserver quark (fig.3 ) and tried to perceive the differences. comparing the orf8 secondary structures of sars-cov-2, bat-ratg13-cov and pangolin-cov, the following changes are found at four different locations as presented in table 2 . table 2 , it is inferred that the secondary structures of orf8 (sars-cov-2) and that of bat-ratg13 are much closer related than the orf8 of pangolin-cov. 90 it was obtained that the largest conserved region in the orf8 protein of sars-cov-2 is 'pftincqe' (in d2), eight amino acids long (table 3 ) as seen in fig.4 . it turned out that the 6.6% region is 100% evolutionary conserved across all 96 distinct variants of the 121 amino acid long orf8 protein. most of the conserved regions in sars-cov-2 orf8 (table 3 and fig.4 ) lie around helix-coil and strand-coil junctions signifying the functional importance of these regions. helix regions also have conserved amino acids. it can be hypothesised 95 that these junctions are involved in protein-protein interactions. therefore, these regions are conserved in nature. the orf8 protein of sars-cov-2 has only 55.4% nucleotide similarity and 30% protein identity with that of sars-cov as shown in fig.5 . although the sars-cov-2 orf8 has differentiating genome characteristics but exhibit high functional similarity with that of sars-cov orf8ab: • the sars-cov orf8ab original protein was found to have an n-terminal hydrophobic signal sequence that directs its transport to the endoplasmic reticulum (er). however, after deletion of 29 nucleotides, which split the orf8ab protein into orf8a and orf8b, it was found that only orf8a was able to translocate to the er while orf8b remained distributed throughout the cell. likewise, the sars-cov-2 orf8 protein also contains an n-terminal hydrophobic signal peptide (1-15 aa), which is involved in the same function. • the er has an internal oxidative environment akin to other organelles, which is necessary for protein folding and oxidation processes. due to this oxidative environment, formation of intra or inter-molecular cysteine bonds between unpaired cysteine residues takes place as the sars-cov orf8ab protein is an er resident protein and there are ten cysteine residues present, which exhibit disulphide linkages and form homomultimeric complexes within the er. similarly, the orf8 of sars-cov-2 has also seven cysteines, which may be expected to form these types of disulphide 110 linkages. • the sars-cov orf8ab is characterized by the presence of an asparagine residue at position 81 with the asn-val-thr motif responsible for n-linked glycosylation, whereas sars-cov-2 orf8 has an n-linked glycosylation site at asn (78) and the motif is asn-tyr-thr. • the sars-cov-2 orf8 is found to have both protein-protein and protein-dna interactions while sars-cov orf8ab shows only protein-protein interactions. further, it was found that the orf8 protein of sars-cov-2 is very much similar (95%) to that of bat-cov ratg13 on the basis of sequence similarity as well as on the basis of phylogenetic relationships as shown in fig.6 . as we can see from the sequence alignment, there are only six amino acid differences between the sars-cov-2 orf8 and bat-cov ratg13. all of these mutations in the orf8 protein with respect to the reference orf8 sequence of 120 bat-ratg13 were found to be neutral type as predicted through webserver meta-snp and all of them had a decreasing effect on stability of the protein as determined using the server (i-mutant). we have also aligned the pangolin-cov orf8 sequence with that of sars-cov-2 and found that there is a sequence similarity of 88%, as depicted in fig.7 . we observed a difference of 15 amino acid residues between the pangolin-cov and the sars-cov-2 orf8. it was 125 analysed that in both the bat-cov ratg13 and the pangolin-cov orf8 protein the mutations l10i, v65a and s84l are occurred. so, it can be hypothesised that sars-cov-2 may have originated from pangolin-cov or bat-cov ratg13 orf8. also, the comparison of frequencies of the hydrophobic, hydrophilic and charged amino acids was performed, which are present among the four different orf8 proteins of sars-cov, sars-cov-2, bat ratg13-cov and pangolin-cov was 130 made. as seen in table 4 , sars-cov-2 orf8, sars-cov orf8ab, bat-cov ratg13 orf8 and pangolin-cov orf8, are all similar in terms of hydrophobicity and hydrophilicity and it is known that hydrophobicity and hydrophilicity play an important role in protein folding which determines the tertiary structure of the protein and thereby affect the function. the orf8 sequences of sars-cov-2, bat-cov ratg13 and pangolin-cov have almost the same positive and negative charged amino acids, therefore we can say that probably they have similar kind of electrostatic and hydrophobic interactions, 135 which also contribute to the functionality of the proteins. again, for the sars-cov orf8ab it was found that the number of positive and negative charged amino acids are closely similar to the sars-cov-2 orf8. although, the sars-cov sequence bears less similarity with the sars-cov-2 but they are probably similar in terms of electrostatic and hydrophobic interactions. furthermore, we analysed the three orf8 sequences and checked for their molecular weight, isoelectric point (pi), 140 hydropathy, net charge and extinction coefficient using a peptide property calculator (https : //pepcalc.com/) and found that all the properties are almost identical, which shows that the orf8 protein of bat-cov ratg13, pangolin-cov and sars-cov-2 are very closely related from the chemical aspects of amino acid residues (fig.8 ). indicating that orf8 of the pangolin-cov is more negatively charged as compared to the sars-cov-2 orf8. also, since these three proteins have a similar molecular weight, pi, extinction coefficient and nature of hydropathy plot, it would be difficult to differentiate these three proteins by biophysical techniques on the basis of these properties. the orf8 of sars-cov-2 is significantly different from the orf8 of pangolin-cov and it seems that the orf8 150 protein (sars-cov-2) is imitating the properties as well the structure of orf8 of bat ratg13-cov as a blueprint. the differences between the orf8 of sars-cov-2 and bat-cov ratg13 and pangolin-cov can be further demonstrated by the analysis of the per-residue intrinsic disorder predispositions of these proteins. results of this analysis are shown in fig.9a , which illustrates that the intrinsic disorder propensity of the orf8 from sars-cov-2 is closer to that of the orf8 from bat-cov ratg13 than to the disorder potential of orf8 from pangolin-cov. this is in agreement 155 with the results of other analyses conducted in this study. analysis is conducted using pondr-vsl2 algorithm [40] , which is one of the more accurate standalone disorder predictors [41, 42, 43] . a disorder threshold is indicated as a thin line (at score = 0.5). residues/regions with the disorder scores > 0.5 are considered as disordered. each of the orf8 amino acid sequences (fasta formatted) are aligned with respect to the orf8 protein (yp 009724396.1) from china-wuhan using multiple sequence alignment tools (ncbi blastp suite) and found the mutations and their associated positions were detected accordingly [29] . it is noted that a mutation from an amino acid a 1 to a 2 at a position p 160 is denoted by based on observed mutations, it is noticed that amino acids threonine (t) and tryptophan (w) are found to be most vulnerable to mutate to various amino acids. it is noteworthy that the sars-cov-2 orf8 is rapidly undergoing different type of mutations, indicating that it is a highly evolving protein, whereas the bat-cov orf8 is highly conserved (fig.6) and the pangolin-cov orf8 is 100% conserved (fig.7) . a pie chart presenting the frequency distribution of various mutations is shown in fig.11 . the n-terminal signal peptide of orf8 (d1) of sars-cov-2 is hydrophobic in nature. we further analysed (table 5) the mutations and observed that hydrophobic to hydrophobic mutations are dominating, indicating that hydrophobicity of the domain is maintained and thus we can postulate that there is probably no functional change in the hydrophobic n-terminal signal peptide. further, it was found that there was a change from hydrophilicity to hydrophobicity in two positions, thus enhancing distinct non-synonymous mutations and the associated frequency of mutations, predicted effect (using meta-snp) as well as the predicted change of structural stability (using i-mutant) due to mutation(s) are presented in table 6 . the most frequent mutation in the orf8 proteins turned out to be l84s (hydrophobic (l) to non-charged hydrophilic (s)) which is a clade (s) determining mutation with frequency 23 [44] . it is observed from based on predicted effects and change of structural stability, mutations are grouped into four classes as shown in table 190 7. in table 8 , the list of unique orf8 protein ids and their associated mutations with domain(s) and the predicted effects and changes of structural stability are presented. further, based on the three different types of mutations viz. neutral, disease and mix of neutral & disease, all the orf8 proteins are classified into three groups which are adumbrated in table 9 . also the corresponding pie chart is given in fig.12 . note that, other than these two strains, there are other sixty-four other orf8 sequences, which do not possess any of these two strain-determining mutations. this clarifies that the orf8 protein is certainly one of the fundamental proteins which directs the pathogenicity of a variety of strains of sars-cov-2. to study the evolution of mutations and to observe the relationships among three orf8 proteins, we compared sarscov-2 orf8 with that of bat-cov ratg13 and pangolin-cov orf8. the detailed analysis of all mutations is presented in table 12 . based on table 12 , it can be suggested that reverse mutations will lead to the same sequence for the sars-cov-2 orf8 protein will have the same sequence as that of bat-cov ratg13 and pangolin-cov orf8 proteins in the near future. we can also conclude that sars-cov-2 is showing genetically reverse engineering when compared with bat-cov 220 ratg13 and pangolin-cov orf8. the reversal of mutation also happened here and it was found that the frequency of leucine to serine mutation at 84th position is quite high. predisposition in the vicinity of residue 50, whereas the lowest disorder propensity in the vicinity of residue 70 is found in variants qkv06506.1 and qkq29929.1. finally, although variant qmu91370.1 is almost indistinguishable from variant qjs56890.1 within the first 40 residues, its intrinsic disorder predisposition in the vicinity of residue 70 is one of the lowest among all the proteins analysed in this study. interestingly, comparison of the fig.9 (a) and fig.9 (b) show that the variability in the disorder predisposition between many variants of the protein orf8 from sars-cov-2 isolates is 235 noticeably greater than that between the reference orf8 from sars-cov-2 and orf8 proteins from bat-cov ratg13 and pangolin-cov. here we present five different possible mutation flows according to the date of collection of the virus sample from patients [45] . sequence homology-and amino acid compositions-based phylogenies have been drawn for the orf8 proteins 240 associated in each flow. in this flow of mutations (fig.13 in order to support these mutation flows, we analysed the protein sequence similarity based on phylogeny and amino acid 255 composition. the reference orf8 sequence yp 009724396 is found to be much more similar to the variants qmt48896.1 and qmi92505.1, which are more similar to each other as depicted in the sequence based phylogeny (fig.14 (left) ). this sequence based similarity of the orf8 proteins qmt48896.1 and qmi92505.1 is illustrated in the chronology of mutations as shown in fig.13 . similarly, the mutation flow of the sequences qmt96239.1 and qmu92030.1 is supported by the respective sequence-based similarity. the network of five orf8 protein variants from the us is justified based on similar amino acid compositions/conservations across the five sequences as shown in fig.14 (right) . in this flow of mutations (fig.15 ), we observed one sequence with first-order mutations, i.e where only one mutation accumulated in the sequence. additionally, four sequences (all are from the us) were identified with second order mutations 265 stating that four sequences were found to have two mutations. to the hydrophobic phenylalanine (f), so it may account for disrupting the ionic interactions. as it is a neutral mutation, the sequence accumulated two neutral mutations. the protein sequence qlh58953.1 acquired a second mutation, p38s, which was found to be of disease-increasing type and the polarity also changed from hydrophobic to hydrophilic, thus 270 indicating these mutations may have some significant importance. the protein sequence qlh58821.1 possesses a second mutation, v62l, which was found to be of neutral type with no change in polarity. here, this sequence accumulated two neutral mutations, which may account for some functional changes. by comparing both the sequence-based phylogeny ( fig.16 (left) ) and amino acid conservation-based phylogeny (fig.16 (right)), we found that according to sequence-based phylogeny the australian sequence is closely related to the orf8 275 wuhan sequence. however, according to the pathway, it should be closely related to both the wuhan sequence and the sequences having second order mutation. this can be attributed to the presence of 119 amino acid residues instead of 121 aa residues. in this case, the sequence has a two amino acid deletion, therefore, it is present at first node. here we analysed the us sequences considering the wuhan sequence (yp 009724396.1) as the reference and found one sequence, qkc05159.1, with a single mutation and seven sequences with two mutations each (fig.17 ). the first sequence, qkc05159.1, contained the l84s mutation (strain determining mutation), which was of neutral type. however, the polarity changed from hydrophobic to hydrophilic, which may account for some significant change of function. the sequences that accumulated 2nd mutation along with l84s are as follows: • qms53022.1: this protein sequence acquired a second mutation at position 11, which changed the hydrophilic amino acid threonine (t) to hydrophobic amino acid isoleucine (i) therefore affecting the ionic interactions. this 290 mutation was found to be a diseased-increasing type, so it may affect the structure of the protein. • qmt50804.1: this sequence gained a second mutation, e19d, which was predicted to be of disease-increasing type with no change in polarity. the sequence first accumulated a neutral mutation then a disease-increasing mutation, signifying that these mutations may have some functional importance. • qjd48694.1: h112q occurred as second mutation in this sequence, which was found to be of disease-increasing 295 type with no change in polarity. consequently, these mutations may contribute to immune evasion property of the virus. • qkv06506.1: this sequence possesses the s67f mutation, which was predicted to be of neutral type, which changed the hydrophilic serine (s) to the hydrophobic phenylalanine (f), thus interfering with the ionic interactions that may increase or decrease the affinity of the viral protein for a particular host cell protein. • qkv40062.1: this sequence acquired a second mutation at q72h, which was found to be a neutral mutation and no change in polarity was observed. as this sequence accumulated two neutral mutations, it can be assumed that neutral mutations also have a significant importance. • qkv07730.1: the t11a mutation occurred as the second mutation in this sequence, which was predicted to be of disease-increasing type and the polarity was changed from hydrophilic to hydrophobic, hence the structure and 305 function of the protein are expected to differ. from the sequence based phylogeny (fig.18 (left) ) it was observed that the wuhan sequence was the first to originate. although, qkc05159.1 is the first sequence in our flow considering the time, it was found that in the phylogenetic tree it is present at fourth node instead of second node, which is probably due to the presence of ambiguous mutations in this sequence. it was also determined that qkv07730.1 is very similar to qmt50804.1 and again qmt28672.1 is more similar 310 to both of them. all the other sequences having second order mutations are closely related to each other and follow the chronology. from the amino acid based analysis (fig.18(right) ) it was found that the wuhan sequence has a high conservation similarity with that of qkv06506.1, thus proving that this sequence was identified chronologically after the wuhan sequence followed by qkc05159.1 and qmt28672.1, which again are very similar to each other. in the possible flow of mutations (fig.19) , we have found one sequence with a single mutation, six sequences with two mutations, and another two sequences with three mutations. the us sequence qkc05159.1 was identified to have the l84s mutation, which is a strain determining mutation and was predicted to be a neutral mutation where polarity was changed from hydrophobic to hydrophilic. the sequences that accumulated second mutations along with l84s are as following and 320 it should be noted that the mutational accumulation occurred in a single strain: • qlh01196.1: the a65s mutation occurred as a second mutation in this us sequence, which was found to be 325 of neutral type. however, the polarity changed from hydrophobic to hydrophilic, thus potentially influencing the function of the protein. • qmu91334.1: this us sequence possesses the d63g mutation, which was predicted to be of neutral type. however, the polarity changed from hydrophilic to hydrophobic so, this sequence accumulated two neutral mutations, which may allow the virus to evolve in terms of virulence. serine (s). the mutation was neutral, thus accumulating two neutral and one disease-increasing mutations, being of 335 significant importance for the evolution of the virus. we identified one more sequence qlh57924.1, which possesses another third mutation, f16l, which was predicted to be a neutral mutation and no polarity change was observed. this sequence acquired three neutral mutations that may promote virus survival. • qku37052.1: this sequence with the w45l mutation was reported in saudi arabia, which was found to be of disease-increasing type with no polarity change. therefore, this sequence also accumulated one neutral and one 340 disease-increasing mutation, which may affect both the structure and function of the protein. • qmt96539.1: the f104s mutation was reported in the us sequence, which was found to be of disease-increasing type and the polarity changed from hydrophobic to hydrophilic. altogether, the sequence possesses one neutral and one disease-increasing mutation that may allow the virus to acquire new properties for better survival strategies. sequence-based phylogeny (fig.20 (left) ) suggested that the wuhan sequence originated first. due to the presence 345 of an ambiguous amino acid sequence, qkc05159.1 did not show close similarity to the wuhan sequence. based on sequence based phylogeny (fig.20 (left) ) it was observed that wuhan sequence originated first. due to presence of ambiguous amino acids sequence qkc05159.1 was not observed in close proximity to wuhan sequence. qkg86865.1 and qlh57924.1 were found to have third order mutations and they are assumed to be closely related by the flow and the same has been supported by amino acid conservation-based phylogeny (fig.20 (right) ). flow-v qjr88780.1 (australia) possesses the mutation l84s with reference to the wuhan orf8 sequence yp 009724396.1 (fig.21) . another sequence qjr88936.1 was reported, which possesses a second mutation, v62l. this mutation was predicted to be neutral with no change in polarity. however, the hydrophobicity increased. this sequence belongs to a particular strain and acquired two neutral mutations, indicating that these mutations may play some important role in the function of orf8a. as can be seen from both the sequence-based phylogeny and amino acid conservation-based phylogeny (fig.22) , the wuhan sequence has originated earlier and the sequences qjr88780.1 and qjr88936.1 are more closely related to each other than to the wuhan sequence as both sequences have one common mutation not present in the wuhan sequence. among sars-cov-2 proteins, the orf8 accessory protein is crucial because it plays a vital role in bypassing the host immune surveillance mechanism. this protein is found to have a wide variety of mutations and among them l84s (23) and s24l (7) have highest frequency of occurrence, which bears distinct functional significance as well. it has been reported that l84s and s24l show antagonistic effects on the protein folding stability of sars-cov-2 [44]. l84s destabilizes protein folding, therefore up-regulating the host-immune activity and s24l favours folding stability positively, thus enhancing the 365 functionality of orf8 protein. l84s is already established as a strain determining mutation and since according to our studies both l84s and s24l do not occur together in a single sequence of sars-cov-2 orf8 protein, it is proposed that virus with the s24l mutation is a new strain altogether. we also observed that hydrophobic to hydrophobic mutations are dominant in the d1 domain. therefore, hydrophobicity is an important property for the n-terminal signal peptide. however, in the d2 domain, hydrophobic to hydrophilic mutations are observed more frequently, consequently making the 370 ionic interactions more favourable and allowing the protein to evolve in terms of better efficacy in pathogenicity. the orf8 sequence of sars-cov-2 shows 93% similarity with the bat-cov ratg13 and 88% similarity with that of the pangolin-cov orf8. thus, the orf8 protein of sars-cov-2 can be considered as a valuable candidate for evolutionary deterministic studies and for the identification of the origin of sars-cov-2 as a whole. we also analysed a wide variety of mutations in the sars-cov-2 orf8, where we compared it with the orf8 of bat-cov ratg13 and 375 pangolin-cov in relation to charge and hydrophobicity perspectives and we found that the bat-cov ratg13 orf8 protein exhibits exactly the same properties as that of the sars-cov-2 orf8 protein, whereas the properties of the pangolin-cov orf8 are relatively less similar to the sars-cov-2 orf8. furthermore, to study the evolutionary nature of mutations in the orf8, we aligned three bat sequences and found that two of them were exactly the same and there were only six amino acid differences in the third with respect to the other two sequences. so, only two variants were identified for bat-cov. therefore, it shows that the rate of occurrence of mutations is slow in the bat-cov ratg13 orf8. however, for pangolins no differences were observed among four pangolin-cov orf8 sequences and therefore only a single variant of orf8 is present. based on sequence alignment, biochemical characteristics and secondary structural analysis, the bat-cov, the pangolin-cov and the sars-cov-2 orf8 displayed a high similarity index. additionally, in the orf8 of sars-cov-2, certain mutations were found to exhibit exact reversal with respect to bats and pangolins and therefore pointing towards the genomic origin of sars-cov-2. however, unlike bat-cov and pangolin-cov, the mutational distribution of the orf8 (sars-cov-2) is widespread ranging from the position 3 to 121, having no defined conserved region. this surprises the scientific community enormously. further this property differentiates the sars-cov-2 orf8 from that of bat-cov and pangolin-cov, thus raising the question over the natural trail of evolution of mutations in sars-cov-2. we further predicted the types and effects of mutations of 95 sequences and grouped them into four domains and found that diseased type mutations with decreasing effect on stability are more prominent. consequently, it is hypothesized that these mutations are promoting the viral survival rate. furthermore, we tracked the possible flow of mutations in accordance to time and geographic locations and validated our proposal with respect to sequence-based and amino acid conservation-based phylogeny and therefore putting forward the order of accumulation of mutations. the authors do not have any conflicts of interest to declare. the epidemiology and pathogenesis of coronavirus disease (covid-19) outbreak the explosive epidemic outbreak of novel coronavirus disease 2019 (covid-19) and the persistent threat of respiratory tract infectious diseases to global health security the effect of human mobility and control measures on the covid-19 epidemic in china coronavirus disease 2019 (covid-19): situation report editorial-differences and similarities between severe acute respiratory syndrome (sars)-coronavirus 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methods for prediction of intrinsic disorder and its 490 molecular functions comprehensive comparative assessment of in-silico predictors of disordered regions accurate prediction of disorder in protein chains with a comprehensive and empirically designed consensus implications of sars-cov-2 mutations for genomic rna structure and host microrna targeting pathogenetic perspective of missense mutations of orf3a protein of sars-cov2 authors thank prof. bidyut roy of human genetics unit, indian statistical institute, kolkata, indis for his kind support for structure predictions. key: cord-323307-nu9ib62h authors: dong, dong; lei, ming; hua, panyu; pan, yi-hsuan; mu, shuo; zheng, guantao; pang, erli; lin, kui; zhang, shuyi title: the genomes of two bat species with long constant frequency echolocation calls date: 2016-10-26 journal: mol biol evol doi: 10.1093/molbev/msw231 sha: doc_id: 323307 cord_uid: nu9ib62h bats can perceive the world by using a wide range of sensory systems, and some of the systems have become highly specialized, such as auditory sensory perception. among bat species, the old world leaf-nosed bats and horseshoe bats (rhinolophoid bats) possess the most sophisticated echolocation systems. here, we reported the whole-genome sequencing and de novo assembles of two rhinolophoid bats – the great leaf-nosed bat (hipposideros armiger) and the chinese rufous horseshoe bat (rhinolophus sinicus). comparative genomic analyses revealed the adaptation of auditory sensory perception in the rhinolophoid bat lineages, probably resulting from the extreme selectivity used in the auditory processing by these bats. pseudogenization of some vision-related genes in rhinolophoid bats was observed, suggesting that these genes have undergone relaxed natural selection. an extensive contraction of olfactory receptor gene repertoires was observed in the lineage leading to the common ancestor of bats. further extensive gene contractions can be observed in the branch leading to the rhinolophoid bats. such concordance suggested that molecular changes at one sensory gene might have direct consequences for genes controlling for other sensory modalities. to characterize the population genetic structure and patterns of evolution, we re-sequenced the genome of 20 great leaf-nosed bats from four different geographical locations of china. the result showed similar sequence diversity values and little differentiation among populations. moreover, evidence of genetic adaptations to high altitudes in the great leaf-nosed bats was observed. taken together, our work provided a useful resource for future research on the evolution of bats. bats (order chiroptera) are one of the largest monophyletic clades in mammals (order chiroptera), and constitute nearly 20% of living mammalian species. they can perceive their surroundings using a wide range of sensory systems, and have long been regarded as the most unusual and specialized species of all mammals. most bats are sophisticated echolocators and rely on their echolocation systems for navigation. however, old world fruit bats have no laryngeal echolocating ability, and navigate largely by vision. based on overwhelming molecular genetic evidence, it has been proposed that echolocating bats are paraphyletic (teeling, et al. 2005) . old world fruit bats and some laryngeal echolocators (including rhinolophidae, hipposideridae, craseonycteridae, megadermatidae, and rhinopomatidae families) are a natural group -the suborder yinpterochiroptera, and the remaining laryngeal echolocating bats are grouped to another suborder yangochiroptera . two distinct navigation approaches can be employed by echolocating bats: low duty cycle (ldc) echolocation and high duty cycle (hdc) echolocation (teeling 2009 ). ldc echolocators can separate pulse and echo in time to avoid forward masking, whereas some species of hdc echolocators separate pulse and echo in frequency. it has been documented that rhinolophoid bats might possess the most sophisticated echolocation systems (jones and teeling 2006) . recently, results from some hearing-related genes suggested sequence convergence in laryngeal echolocating bats (li, et al. 2008; davies, et al. 2012) . we attempted to investigate whether similar patterns can be detectable in other hearing-related genes. furthermore, a sensory trade-off between investment in vision and echolocation has been identified (dechmann and safi 2009) . loss-of-function in short-wave sensitive opsin (sws1 gene) occurred in rhinolophoid bats, which use hdc echolocation and can emit long constant frequency calls (zhao, et al. 2009 ). although several bat genomes have been sequenced (zhang, et al. 2013) , the evolutionary mechanisms of the rhinolophoid bats remains unclear. comparative genomics will provide us opportunities to investigate whether similar patterns can be detectable in other sensory genes. the great leaf-nosed bat (hipposideros armiger) and the chinese rufous horseshoe bat (rhinolophus sinicus) are two important species of rhinolophoid bats. first, these are model organisms with remarkable hdc echolocation ability and can emit continuous ultrasonic calls of long constant frequency with remarkable acoustic features (doppler-shift compensation) (schnitzler, et al. 2003) . we can comprehensively explore how rhinolophoid bats evolved a specialized form of echolocation. second, they are important reservoir hosts of emerging viruses, and the chinese rufous horseshoe bat has been suggested to carry the direct ancestor of severe acute respiratory syndrome (sars) coronavirus (ge, et al. 2013) . in this work, we presented the genomes of the great leaf-nosed bat and the chinese rufous horseshoe bat using the next generation sequencing platform (illumina hiseq 2500). the result revealed the adaptation of auditory sensory perception in hdc echolocators, and showed an extensive contraction of olfactory receptor gene repertoires as well as pseudogenization of some vision-related genes. furthermore, we performed genome re-sequencing to analyze the population genetic structure of the great leaf-nosed bats. the genomic data provide genetic evidence of adaptive evolution in rhinolophoid bats. a female great leaf-nosed bat (hipposideros armiger) and a female chinese rufous horseshoe bat (rhinolophus sinicus) were captured from a cave (n 30°20.497′ genomic dna was extracted from bat muscle using the qiagen dneasy blood and tissue kit. six paired-end libraries with insert size of 170 bp, 500 bp, 800 bp, 3k bp, 8k bp and 20k bp were constructed and sequenced for the great leaf-nosed bat and the chinese rufous horseshoe bat, respectively. the libraries were sequenced using illumina hiseq2500 platform, which has a read length of 101 bp. low quality sequencing reads were filtered out and potential sequencing errors were removed. the following filtering criteria were carried out: 1) filter reads with >5% unidentified nucleotides; 2) filter reads with >10 nucleotides aligned to the adapter sequence, allowing <3 mismatches; 3) remove putative pcr duplicates generated by pcr amplification in the library construction process. finally, we generated 476.5 gb and 288.5 gb of sequences for the great leaf-nosed bat and the chinese rufous horseshoe bat, respectively. the genome sequences were assembled using allpaths software (butler, et al. 2008 ). briefly, contigs were generated by constructing a de bruijin graph with the sequencing reads from short-insert library data. the graph was simplified to generate the contigs by removing tips, merging bubbles and solving repeats. the sequencing reads were mapped to the assembled contigs, and the scaffolds were constructed by weighting the rates of consistent and conflicting paired-end relationships. at last, we retrieved the read pairs with one end that uniquely mapped to the contigs and the other end located in the gap region, a local assembly for these collected reads was performed to fill the gaps. a more detailed genome assembly method is provided in supplementary methods. total rnas of the two bats were extracted from brain, cerebellum, heart, liver, stomach, kidney, lung and muscle tissues for the generation of transcriptome data. paired-end libraries for rna sequencing were constructed using the illumina mrna-seq prep kit. the quality and integrity of the rna samples were determined using the agilent2100 bioanalyzer. poly(a) mrnas were isolated using oligo(dt) beads, fragmented, and converted to cdnas followed by end repair, adaptor ligation, and pcr amplification. the libraries thus generated were sequenced using the illumina hiseq2500 platform as described above. we searched for tandem repeats across the genomes using tandem repeats finder. transposable elements were predicted in the genomes by homology search against the known transposable elements (te) in repbase (jurka, et al. 2005 ) (version 20110920) using repeatmasker version 3.3.0 (tarailo-graovac and chen 2009). the protein-coding genes of the bat genomes were annotated by combining homology-based, ab initio and rna-seq gene prediction methods. at first, rna-seq data were assembled using the trinity package (trapnell, et al. 2013) . pasa (version r2012-06-25) (haas, et al. 2003) was then used to map the assembled transcripts. based on the set of gene models, a training set was constructed for de novo predictors by selecting the genes with complete structures and at least 100% mapping rate for uniprot vertebrate proteins. for the ab initio prediction, augustus (stanke and waack genes with the training set generated by pasa. for homology-based gene prediction, the protein sequences of human, mouse, dog, cow, little brown bat and large flying fox were downloaded from ensembl release 72 and mapped onto the repeat-masked genome using genblasta (she, et al. 2009 ). rna-seq data were mapped to the genome using tophat (trapnell, et al. 2009 ), and the transcription-based gene structure were generated by cufflinks (trapnell, et al. 2013) . the final gene set was generated by merging all genes predicted using glean software (http://sourceforge.net/projects/glean-gene/). to infer gene function, it was based on the best match of the alignment to the swissprot and translated embl nucleotide sequence data library databases using blastp. interproscan (mulder and apweiler 2007 ) was used to determine motifs and domains in the final gene set. to evaluate completeness of the genomes and annotations, cegma method (parra, et al. 2007) was employed. we used the treefam methodology (li, et al. 2006) to define gene families in 14 mammalian genomes (human, macaque, mouse, rat, dog, cat, horse, rhinoceros, cow, pig, little brown bat, large flying fox, great leaf-nosed bat and chinese rufous horseshoe bat). the protein sequences of other 12 mammalian species were obtained from ensembl database (release 72). gene family expansion and contraction analysis was performed by café software (de bie, et al. 2006) . a random birth and death model was proposed to study gene gain and loss in the gene families across a user-specified phylogenetic tree. a global parameter λ (lambda), which described both gene birth (λ) and death (μ = -λ) rates across all branches of all gene families was estimated using the maximum likelihood method. a conditional p-value was calculated for each gene family, and families with conditional p-values less than 0.05 were considered to have a significantly accelerated rate of expansion and contraction. protein sequences of the aforementioned 14 mammals were aligned using muscle software (edgar 2004) . all orthologous genes were concatenated to one super gene for each species. raxml (stamatakis 2014 ) was applied to build phylogenetic trees. we partitioned the data by coding genes, and evaluated the model parameter independently for each partition. in all partitioned analyses, the empirical base frequencies and the evolutionary rates were estimated independently for every partition. bootstrap support was obtained by repeating the original partitioned ml raxml analysis on 100 bootstrap replicates for each dataset using different random number seeds in each repetition. next, we inferred the species tree using coalescent method: maximum pseudo-likelihood estimation of species tree (mp-est) (liu, yu, et al. 2010) . individual gene tree for each gene was estimated using the maximum-likelihood method and rooted by an outgroup (human). species trees were estimated from the rooted gene trees in the program mp-est with 100 bootstrap replicates. the results supported that bats are member of scrotifera (chiroptera + carnivores + perissodactyla + cetartiodactyla) with bat lineage diverging from fereuungulata (carnivores + perissodactyla + cetartiodactyla). the values of ka, ks, the ka/ks ratio were estimated for each gene using the codeml programs nested in the paml package (yang 2007) . in order to detect positively selected genes, optimized branch-site likelihood model (zhang, et al. 2005) was used. we separately explored the positively selected genes in the great leaf-nosed bat and the chinese rufous horseshoe bat. for each analysis, only one bat species was selected as foreground branches, and all other species were regarded as the background branches. the revised branch-site model a was employed, which attempts to identify positive selection acting on some sites on the "foreground branches". using an likelihood ratio test (lrt), the alternative hypothesis that positive selection occurs on the foreground branches (ka/ks > 1) is compared with the null hypothesis (ka/ks=1). bayesian empirical bayes values were used to identify sites under positive selection. then, branch two-ratio model was applied to detect accelerated evolved genes in specific lineage. the one-ratio model assumed an equal ka/ks ratio for all lineages in the phylogeny, and the two-ratio model assumed two ka/ks ratios: one branch for the background, one for the foreground branch leading to the specific species. then, clade model c was employed to test for positive selection along the rhinolophoid bats. the two clades were assumed to share sites under purifying selection and neutral evolution, but to differ at a third site partitions under divergent selection. the null model used for the clade model c was m2a_rel (weadick and chang 2012) , whose lrt has a relatively lower false-positive rate. go annotations were downloaded from ensembl databases and were assigned to these orthologous genes. the binomial test was used to identify go categories with more than 20 gene that had an excess of non-synonymous changes in bat lineages. next, we used the program mapp (multivariate analysis of protein polymorphism) (stone and sidow 2005) to evaluate the physicochemical impact of these convergent amino acid substitutions on bats. physicochemical variations can be used to predict how these particular convergent amino acid substitutions might affect protein function. in this work, we performed a probabilistic analyses of the sequence convergence in echolocating bats. a maximum likelihood approach, implemented in the software package codeml ancestral, was used. we compared the pair-wise branches of two echolocating bat in the phylogeny, and posterior probabilities of all possible amino acid substitutions were calculated. the probabilities of divergent and convergent substitutions were calculated as the sum of joint probabilities of substitutions between the two branches of echolocating bats. convergence and divergence estimates were based on posterior distributions of ancestral states and substitutions. the same state (same amino acid) represents convergent substitutions, and the different state represents divergent substitutions. finally, to further validate that the convergence between two branch pairs of echolocating bats was significant, we performed the simulation analysis to compare the observed probabilities against that of the null hypothesis. simulated sequences were generated using evolver, another package from paml package (yang 2007) . the branch-wise convergence probabilities were calculated with 1,000 replicates. we used the similar in silico method as previously reported in dong et al. (dong, et al. 2009 ). at first, we used previously published or genes in vertebrates as query sequences (niimura and nei 2007) and conducted a tblastn search against the genome sequences with a cutoff e value of 1e-10 to identify the or gene repertoires. here, we totally identified or gene repertories from eight mammalian genomes (the site (http://genome.ucsc.edu). then, the non-redundant blast-hits were extended to the 5' and 3' directions along the genome sequences, and the potential coding regions were extracted from these sequences. the chemosensory receptor genes in mammals have high sequence similarity. here, we re-performed a tblastn against the genome sequences using or coding genes identified from each species, and the non-redundant blast-hits were used to identify the or pseudogenes containing interrupting stop codons or frameshifts. to identify partial or genes from these sequences, we extracted the sequences that did not have any nonsense or frameshift mutations. we then constructed a multiple alignment of these sequences together with functional or genes by the program e-ins-i in mafft version 5.8 (katoh, et al. 2002) . from those alignments, we extracted partial or sequences that meet the following criteria. when the c-terminal region of an or gene is missing from the genome sequence, the n-terminal region should contain an initiation codon at a proper position and should not contain any nonsense mutations, frameshifts, or long gaps. when the n-terminal region is missing, the c-terminal portion should have a stop codon at a proper position and should not contain any nonsense mutations, frameshifts, or long gaps. we also identified 6 and 10 sequences with nonsense stop codon in the great leaf-nosed bat and chinese rufous horseshoe bats, which miss both a start and stop codons. however, these sequences were removed because they have relatively short sequence length (~400 bp) and have strong sequence similarity with bitter taste receptor genes. to assign identified or genes into distinct or gene families, a collection of protein sequences from horde database version 42 (safran, et al. 2003 ) was used. to detect the extensive gain and lose of or gene repertories, we employed the reconciled tree method (nam and nei 2005) , in which the topology of a gene tree is reconciled with that of a species tree. an in-house program was applied. briefly, based on the phylogenetic tree of or genes, we compared the condensed gene tree and the species tree under the parsimony principle. the number of ancestral genes can be estimated, and the information of the past occurrence of gene expansion and contraction. here, we used a 70% condensed tree of or genes for analyses. a list of vision-related genes were obtained from go category of visual perception (go:0007601). we subjected human vision-related proteins to tblastn against the genomes with cutoff threshold of e-value 1e-5. we found that best-hits for each human protein by using the criteria that more than 30% of the aligned sequences showed an identity above 30%. genewise algorithm was employed to identify potential pseudogenes with parameters -genesf -for -quiet. those genes with frame shifts or pre-mature stop codons were considered as candidates. we then filtered them as follows: 1) we aligned all human proteins to their corresponding genomic loci, and those genes with frameshifts or premature stop codons in human-to-human alignments were removed; 2) as for the human-to-human alignments, those genes with obvious splicing errors near their frameshifts or premature stop codons were removed; 3) candidate pseudogenes with a low number of sequencing reads covering their frameshift or premature stop codon sites were regarded assembly error. those genes with a number of reads containing genotype variations at these sites were considered as heterozygous and were also removed. we used a method based on ka/ks to identify go categories that significantly above average in the great leaf-nosed bat genome and chinese horseshoe bat genome. at first, the ka and ks rates are calculated by paml package from all aligned bases with quality score larger than 20 in orthologs, using the f3x4 codon frequency model and the rev substitution matrix. in order to examine the evolution function catalog, we downloaded the go annotation of human gene from the ensembl biomart database (release-71). we estimated the average ka and ks values for those genes which have annotated go as following equations (s1, s2). where t is the number of annotated genes within go categories, i a and i a are the numbers of non-synonymous substitutions and sites, i s and i s are the numbers of synonymous substitutions and sites in gene i, as estimated by paml, respectively. the expected proportion of non-synonymous substitutions a p in a go category was then calculated (s3). for a given go category c, the probability c p of observing an equal or higher number of non-synonymous substitutions and synonymous substitutions was calculated assuming a binominal distribution (s4). where c a and c s are the total number of non-synonymous and synonymous substitutions in go category c, respectively. we applied an approach to the binomial test described above to identify go categories that have an excess of non-synonymous changes on one lineage. for lineages x and y, the average proportion of non-synonymous substitutions were calculated by the following formula (s5). x is the total number of non-synonymous substitutions in the x lineage, y is the total number of non-synonymous substitutions in the y lineage, and the divergence of the proportion of non-synonymous substitution numbers in different lineages between the observed and expected obeys binomial distribution, the formula is as in the following equation (s6). as described for the absolute rate tests, we then computed this statistic for every go category, as well as for every category in 10,000 randomly permuted data sets. we sampled a total of 20 great leaf-nosed bats distributed in four different locations. genomic dna was extracted from wing membranes of each individual. paired-end sequencing library with an insert size of 500 bp was constructed for each sample, and sequenced on the illumina hiseq 2500 platform with 2×101 bp mode. duplicate sequencing reads were filtered out according to the following criteria: 1) any reads with >10% unidentified nucleotides; 2) reads with >10 nt aligned to the adapter sequence, allowing <10% mismatches; 3) reads with 50% bases having phred quality <5. the filtered reads were mapped to the genome using bwa, and samtools were used to call snps. then, we filtered snps using vcftools and gatk under the following criteria: 1) coverage depth >4 and <10000; 2) root mean square mapping quality >10; 3) the distance of adjacent snps >10 bp; 4) the distance to a gap > 10; 5) read quality value >30. to estimate phylogenetic relationships, the genetic distances were calculated among all samples to generate a neighbor-joining (nj) tree using phylip. we performed a principal component analysis using the package gcta. the population structure was inferred using frappe (v1.1) with a maximum likelihood method (tang, et al. 2005) . sliding-window approach (10 kb window sliding in 10 kb step) was employed to quantify polymorphism levels (θ π , pairwise nucleotide variation as a measure of diversity) and genetic differentiation (fst) between the high altitude region (dq) and low altitude regions (tw, jx and gz). to detect significant signatures of selective sweep, z-transformed fst values was calculated. next-generation genome sequencing was carried out, generating 476.5 gb and 288.5 gb of sequences for the great leaf-nosed bat and the chinese rufous horseshoe bat (supplementary table s1 ), respectively. the genome size was estimated to be 2.18 gb and 2.07 gb for the great leaf-nosed bat and the chinese rufous horseshoe bat ( supplementary fig.1 supplementary fig.3 ). known transposon-derived repeats account for 25.8% and 28.5% of the genomes in the great leaf-nosed bat and the chinese rufous horseshoe bat, respectively, which are lower than other non-bat mammalian species (supplementary table s5 ). to facilitate the genome annotation, we generated a high-depth transcriptome data from these two rhinolophoid bats. with repeats masked, the genome was annotated by integrating the homologous prediction, ab initio prediction and transcription-based prediction methods. as a result, a non-redundant reference gene set of 22,009 and 23,152 protein-coding genes were generated for the great leaf-nosed bat and the chinese rufous horseshoe bat ( supplementary fig.4) , respectively. we employed cegma method to evaluate the completeness of genome annotation. the result showed that the vast majority of the core genes were present in our predicted gene sets (97.08% for the great leaf-nosed bat and 96.14% for the chinese rufous horseshoe bat), indicating the completeness of gene sets identification. next, we aligned the transcriptome sequencing reads to the predicted genes, and the result showed that approximately 96% of exons are accurately covered (96.8% for the great leaf-nosed bat and 97.1% for the chinese rufous horseshoe bat). comparative analysis showed a high gene sequence similarity between them (91%, supplementary fig.5 ). we next examined the level of homology between our predicted genes and sequences in the uniprot database. the result showed that >92% of the genes were functionally annotated (94% for the great leaf-nosed bat and 92.2% for the chinese rufous horseshoe bat). compared with the gene families in other three mammalian species -the little brown bat, large flying fox and human, we identified 8,792 homologous gene families shared by five species. a total of 975 gene families were specific to the rhinolophoid bats ( fig. 1) . further functional annotation indicated that the rhinolophoid bats specific gene families were significantly over-represented in two major functional categories: atp binding (43 genes, f.d.r.= 0.0002) and immunity and host defense (25 genes, f.d.r.= 0.0051; supplementary table s6) . until now, the relationship of bats to other members of superorder laurasiatheria has proven difficult to resolve. some studies insisted that bats belong to the clade of pegasoferae which comprises chiroptera, carnivores and odd-toed ungulates (lindblad-toh, et al. 2011; meredith, et al. 2011; mccormack, et al. 2012) , whereas others proposed that bats are a sister group to the clade comprising carnivores and euungulata (pumo, et al. 1998; murphy, et al. 2001; murphy, et al. 2007; song, et al. 2012; zhang, et al. 2013) . to determine the phylogenetic position of bats within the superorder laurasiatheria, a total of 4,569 single-copy 1:1 orthologous genes were fig.6 ). the result based on nucleotide data was in line with previous analysis that bats are a sister group to odd-toed ungulates, whereas the result based on amino acid data supported that bat bats are sister group to the fereuungulata (carnivores + perissodactyla + cetartiodactyla). to account for the tree discordance among loci, coalescent method was applied. coalescent trees were highly consistent with the result inferred from amino acid data using partitioned method ( supplementary fig.7) . to dissect the phylogenetic signal, previously published eight different phylogenetic hypotheses ( supplementary fig.8) were proposed (waddell, et al. 1999; murphy, et al. 2001; nishihara, et al. 2006; prasad, et al. 2008; lindblad-toh, et al. 2011; meredith, et al. 2011; mccormack, et al. 2012 table s7 , supplementary fig.9 ). the result is consistent after incorporating the data from eulipotyphyla group ( supplementary fig.10) . we subsequently estimated the divergence time among these 14 mammalian species. the bat lineage seems to be diverged from fereuungulata around 81 million years ago, and the rhinolophoid bats seem to be diverged from the old world fruit bats around 68 million years ago. comparative genome analyses were carried out to assess the evolution and innovation within the rhinolophoid bats. we next determined the expansion and contraction of gene orthologous clusters during evolution. the result identified 48 significantly expanded and 65 significantly contracted gene families in the great leaf-nosed bat, 46 significantly expanded and 54 significantly contracted gene families in the chinese rufous horseshoe bat (fig. 2) . functional annotation showed that gene family contraction mainly included many olfactory receptor gene families in both rhinolophoid bat lineages (supplementary table s8 ), which is consistent with the result that the olfactory system is aberrant in some echolocating bats. many of the expanded gene families in both rhinolophoid bats are significantly enriched in immune-related functional categories (supplementary table s9 ). moreover, we identified 577, 453 and 182 positively selected genes in the great leaf-nosed bat, the chinese rufous horseshoe bat and the large flying fox, (supplementary tables s10, 11, 12), respectively. olfaction is of great importance in the lives of bats. many bats can use olfaction for mother-pup recognition, find food and avoid danger. in old world fruit bats, olfaction appears to be of particular importance, and fruit bats can find food from scent cues. animals that rely heavily on the sense of smell tend to have large numbers of or genes, while species that always use other senses have fewer functional or genes (niimura and nei 2007) . it has been suggested that bats displayed a diverse olfaction abilities. in order to describe the diversity of bat or gene repertoires, we identified the entire set of or genes of four bat species (supplementary methods, supplementary table s13 ). in line with previous work (hayden, et al. 2014) , we observed that echolocating bats have less fraction of or pseudogenes (18% for the great leaf-nosed bat, 16% for the chinese rufous horseshoe bat and 14% for the little brown bat) than non-echolocating bats (26% for the large flying fox). however, further analysis showed that the large flying fox and little brown bat have more than 400 intact or genes while these two rhinolophoid bats only have <300 intact or genes. this finding is consistent with the result that rhinolophoid bats have a relatively small olfactory epithelium than the frugivorous pteropodidae (neuweiler 2000) . next, we reconstructed a protein neighbor-joining tree of all newly identified intact or genes in bats (fig. 3a) . it is obvious that or genes can be classified into two distinct classes based on sequence similarity: class i, postulated to bind to water-borne molecules, and class ii, hypothesized to bind to airborne molecules. the exact number of or genes in each class/or family are shown (supplementary table s14 , table s15 ). it seems that four bat species contain similar number of or genes in class i, while or gene contraction occurred in two rhinolophoid bats in class ii . previous works have documented that the number of or genes varies extensively among mammalian species, and extensive gains and losses of or genes have been observed (niimura and nei 2007) . to further understand the evolutionary changes of or gene repertoires, we estimated the gains and losses of the or genes in a diverse range of mammals (supplementary methods). evolutionary changes in the number of or genes in mammals have been shown in fig. 3b . we can clearly identify an extensive or gene contraction events occurred to the branch leading to the common ancestor of bats. further extensive gene contractions can be observed in the branch leading to the rhinolophoid bats. this finding also suggests massive "birth-and-death" of or genes in the bat species. table s18 ). since high omega may be due to stochastic effect caused by extremely small sample size, we removed these genes with omega value of 999. the result is also stable that more positively selected genes were detected in the branches leading to echolocating bats (12 genes, great leaf-nosed bat, p = 9.1e-5; 10 genes, chinese rufous horseshoe bat, p = 8.8e-3; 10 genes, little brown bat, p = 0.011). next, branch model (two-ratio model) was carried out with the attempt to detect genes with accelerated evolution in the bat species. the result further indicated that more hearing-related genes have higher ɷ values on the branches leading to echolocating bats than all other lineages (supplementary table s19) . clade model c implemented in paml was employed (weadick and chang 2012) , and the result also persisted that more positively selected genes were detected in the branches leading to echolocating bats (supplementary table s20 ). moreover, a significant association between the average number of non-synonymous substitutions for all the hearing-related genes leading to each mammalian species and the estimated frequency of best hearing sensitivity for that species (r = 0.84, p = 0.00032, fig. 4 ) was observed. no significant correlation between such hearing frequencies and number of synonymous changes was observed (p = 0.132). a significant association between the number of non-synonymous changes between sister taxa was observed (r = 0.67, p = 0.006). it is obvious that echolocating bats have typically undergone many more non-synonymous changes in the hearing-related genes than non-echolocating mammals. these results indicated the evolution of ultrasonic hearing in the rhinolophoid bats has involved in adaptive amino acid replacements in the hearing-related genes, which provided evidence conferring greater auditory sensitivity to ultrasonic frequency. previous works have documented that seven hearing-related genes underwent convergent evolution in echolocators (li, et al. 2008; liu, cotton, et al. 2010; davies, et al. 2012; shen, et al. 2012 ). here, genome-wide signatures of convergent evolution were examined in laryngeal echolocating bats. except for the previously reported seven hearing-related genes, we totally identified 10 genes examined in the sound of perception category containing potential sequence convergent loci (site-wise convergence posterior probabilities > 0.5). to confirm our result, we amplified and sequenced these 10 hearing-related genes from another two echolocating bats (eptesicus fuscus and miniopterus natalensis). the result also showed that these 10 genes have higher convergence probabilities occurred in echolocating bats from a wider range of taxa, and the convergence probabilities between branches were significant based on simulations (supplementary table s21 ). however, maximum likelihood trees recovered the topology that all laryngeal echolocating bats formed a monophyletic clade for only four genes (col1a1, icam1, bsnd and strc, supplementary fig.11 ). further analyses showed that echolocating bats are paraphyletic based on synonymous substitutions, whereas the non-synonymous trees revealed monophyly of laryngeal echolocators for only one hearing-related genes (strc gene, supplementary fig.12 ). next, multivariate analyses of protein polymorphism (mapp) was employed to detect the physicochemical impact of convergent substitutions in echolocating bats. mapp scores were estimated for the amino acid variants nested in the strc gene, and the result showed that these replacements had important functional effects (mapp score = 18.61, p = 1.44e-4 for h28q; mapp score = 10.33, p = 3.98e-3 for a39t; mapp score = 7.37, p = 2.27e-2 for v169i). we further measured the number of sites with convergent amino acid substitutions along the branches as a direct measurement of sequence convergence, and found that the number of convergent sites in the branch pairs is proportional to the number of divergent sites ( supplementary fig.13 ). the number of convergent sites in the laryngeal echolocating bats does not significantly exceed that between the branch pair of the little brown bat and large flying fox, given their numbers of divergent sites (supplementary table s22 ). no significant differences was observed in the total number of sites that have experienced convergent substitutions from hearing-related genes. this result indicated that there is no exceptional genomic signature indicative of adaptive convergence between laryngeal echolocating bats, and genes with adaptive convergent substitutions might confine to few specific genes. bats are nocturnal mammals. the eyes of most echolocating bats are relatively small and poorly developed, whereas old world fruit bats often have excellent eyesight . rhinolophoid bats have the most sophisticated echolocation ability, and have been proposed that some genes involved in visual perception may have undergone relaxed selection (zhao, et al. 2009 ). we next examined the molecular basis for the poor visual perception in the echolocating bats. of bats have long been regarded as important reservoir hosts of emerging viruses (calisher, et al. 2006) . to examine population dynamics and understand evolutionary processes, we sampled 20 great leaf-nosed bats from 4 major distributed locations in china, including one group from high-altitude region (fig. 6a, table s28) are located at the intergenic regions. in order to resolve their phylogenetic relationships, we constructed a neighbor-joining (nj) tree based on pairwise genetic distances (fig. 6b) . this result showed that the great leaf-nosed bats formed separate groups according to the different locations. principal component analysis clearly divided these samples into four groups (dq, gz, jx and tw, fig. 6c) . these results suggested that there were significant population structures among the great leaf-nosed bat populations. furthermore, we performed population structure analysis. when k=4, all these four populations were clearly separated (fig. 6d) . next, we measured the genetic diversity values (θ π ) of four populations, and found similar sequence diversity values (dq: 0.0012, gz:0.0009, jx:0.0009 and tw:0.0011, supplementary fig. s14 ). we further observed that the population differentiation statistic (fst) between populations, and the result showed little differentiation among populations (fst ranging from 0.013 between jx and tw to 0.057 between tw and dq, supplementary table s29) , which suggests universal inter-region gene flows. since the method of population differentiation has been widely used to detect selective sweeps (akey, et al. 2010; axelsson, et al. 2013; gou, et al. 2014 table s31 ). the result showed that genes related to catabolic process are likely to have been targets of recent positive selection. interestingly, we found that five genes (epas1, plxnd1, gja1, sell and chdh) belong to hypoxia response related go categories (pugh and ratcliffe 2003; storz and moriyama 2008) , including 'angiogenesis', 'blood coagulation', 'blood vessel morphogenesis' and 'oxidoreductase activity'. epas1 can respond to the changes in available oxygen in the cellular environment under the high-altitude conditions. our work suggested that epas1 is involved in a selective sweep during the move of bats from low to high altitude. although hypoxia go categories are not over-represented, these highlighted hypoxia-related genes gave us a clue that genetic adaptations might be associated with high altitude. using deep sequencing and de novo assembly, we generated two genomes of rhinolophoid bats. rhinolophoid bats can perceive the world by using a wide range of sensory mechanisms, some of which have become highly specialized. these genome data provided useful resources to decipher the molecular adaptations of phenotypic traits. rhinolophoid bats arguably possess the most sophisticated echolocation systems, and can emit relatively long calls adapted to detect and classify the wing beats of insects. they are heavily reliant on hearing for a variety of ecologically important roles. previous works have documented that hearing-related genes are predominantly evolutionarily conserved in mammals (kirwan, et al. 2013) . here, we found evidence that some hearing-related genes have undergone darwinian selection associated with the evolution of specialized constant frequency echolocation. positive selection acting on hearing-related genes in rhinolophoid bats might result from the extreme selectivity used in auditory processing by these bats. many previous works have reported the sequence convergence of some hearing-related genes reuniting echolocating bats (li, et al. 2008; liu, et al. 2011; davies, et al. 2012; . we found no genome-wide sequence convergence for echolocation, indicating erroneous phylogenetic grouping are still rare it has been suggested that the enlargement of one area of brain might be associated with the reduction in size of other brain area (harvey and krebs 1990) . the auditory cortex and the inferior colliculus are extremely enlarged in the volume in laryngeal echolocating bats (especially in rhinolophoid bats), while visual brain areas are relatively enlarged in old world fruit bats (dechmann and safi 2009 ). the trade-off has been proposed in investment in brain tissues because of the extreme energetic demands imposed by neural processing. our result showed more visual perception genes have become pseudogenes in rhinolophoid bats, and it is reasonable to speculate that some visual perception gene may have undergone relaxed natural selection in echolocating bats. meanwhile, positive selection acting on some hearing-related genes was identified. such concordance suggests that some genes are impacted by natural selection, which raised the possibility that changes at the sensory genes will have direct consequences for those genes controlling for other sensory modalities, perhaps via trade-offs. this finding supports the longstanding but weakly supported assumption that bats are experiencing trade-off between vision and audition . olfaction is of great importance in the lives of bat species. previous works have identified olfactory receptor (or) gene repertoire in the little brown bat and the large flying fox using the profile hidden markov model (hayden, et al. 2010; hayden, et al. 2014 in specific gene family. a possible explanation is that the little brown bat has no well-developed olfaction ability, but tends to recognize specific odorants after recent or gene duplication. these comparative analyses have provided great insights into adaptation to their specialized sensory mechanisms. in this work, we re-sequenced the genome of 20 great leaf-nosed bats from four distributed locations. the genome re-sequencing analysis has been performed based generally on the following considerations: 1) to characterize the genetic diversity and patterns of evolution; 2) to understand the genetic bases of adaptation to high altitude in the great leaf-nosed bats. efforts for the conservation measures will benefit from the knowledge of population genetic structure of the great leaf-nosed bats. here, we found very little differentiation among populations, which suggests universal inter-region gene flows or incomplete lineage sorting. a broader geographical scale analysis is needed in the future. furthermore, we provided evidence of genetic adaptation in the great leaf-nosed bat that are associated with high altitude. selective sweep mapping was conducted for populations from different altitudes, and identified several hypoxia-related genes with a high extent of differentiation on the genome scale. epas1 is transcription factor that respond to the changes in the available oxygen in the cellular environment under high-altitude conditions, and mutations at epas1 are tightly associated with hematologic phenotypes (van patot and gassmann 2011). previous works have documented that epas1 polymorphisms are associated with tibetan people with lower hemoglobin concentrations (beall, et al. 2010) . a loss-of-function role of epas1 might exist in high-altitude adaptation. so, our result indicated potential high-altitude hypoxia adaptation mechanisms of the great leaf-nosed bat. our work is based on a limited genome re-sequencing resource, and data from more samples are necessary for future work. however, false positives notwithstanding, the results provided valuable staring points for experimental follow-up, and suggested an initial evolutionary scenario of bats in adaptation to high-altitude hypoxia. to the best of our knowledge, it is the first time to report the de novo assembled genome and genome re-sequencing of bats with long constant frequency echolocation calls. these data are essential for us to understand the evolution of bats. tracking footprints of artificial selection in the dog genome the genomic signature of dog domestication reveals adaptation to a starch-rich diet natural selection on epas1 (hif2alpha) associated with low hemoglobin concentration in tibetan highlanders prediction of 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of a neotropical fruit bat, artibeus jamaicensis, and a new hypothesis of the relationships of bats to other eutherian mammals human gene-centric databases at the weizmann institute of science: genecards, udb, crow 21 and horde from spatial orientation to food acquisition in echolocating bats genblasta: enabling blast to identify homologous gene sequences parallel evolution of auditory genes for echolocation in bats and toothed whales parallel and convergent evolution of the dim-light vision gene rh1 in bats (order: chiroptera) consel: for assessing the confidence of phylogenetic tree selection resolving conflict in eutherian mammal phylogeny using phylogenomics and the multispecies coalescent model raxml version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies gene prediction with a hidden markov model and a new intron submodel physicochemical constraint violation by missense substitutions mediates impairment of protein function and disease severity mechanisms of hemoglobin adaptation to high altitude hypoxia estimation of individual admixture: analytical and study design considerations using repeatmasker to identify repetitive elements in genomic sequences hear, hear: the convergent evolution of echolocation in bats? a molecular phylogeny for bats illuminates biogeography and the fossil record differential analysis of gene regulation at transcript resolution with rna-seq tophat: discovering splice junctions with rna-seq hypoxia: adapting to high altitude by mutating epas-1, the gene encoding hif-2alpha towards resolving the interordinal relationships of placental mammals an improved likelihood ratio test for detecting site-specific functional divergence among clades of protein-coding genes paml 4: phylogenetic analysis by maximum likelihood comparative analysis of bat genomes provides insight into the evolution of flight and immunity evaluation of an improved branch-site likelihood method for detecting positive selection at the molecular level the evolution of color vision in nocturnal mammals this project is supported by key construction program of the national '985' project of east china normal university to dong dong (79633006), and the national natural science foundation of china (no. 31570382) to shuyi zhang. we thanks shanghai majorbio bio-pharm biotechnology co., ltd. for genome sequencing and dr.chao-hung lee for providing valuable advices.. dd designed the study, and dd, ml, ph, yp, sm, gz, ep, kl and sz carried out the data analysis. dd wrote the manuscript. all authors read and approved the final manuscript. the authors declare no competing financial interests. key: cord-338586-rkrkofri authors: shi, zhengli title: bat and virus date: 2010-02-01 journal: protein & cell doi: 10.1007/s13238-010-0029-7 sha: doc_id: 338586 cord_uid: rkrkofri bat, the only flying mammal and count more than 20% of the extant mammals on earth, were recently identified as a natural reservoir of emerging and reemerging infectious pathogens. astonishing amount (more than 70) and genetic diversity of viruses isolated from the bat have been identified in different populations throughout the world. many studies focus on bat viruses that caused severe domestic and human diseases. however, many viruses were found in apparently healthy bats, suggesting that bats may have a specific immune system or antiviral activity against virus infections. therefore, basic researches for bat immunology and virus-host interactions are important for understanding bat-derived infectious diseases. ✉ correspondence: zlshi@wh.iov.cn bat, the only flying mammal and count more than 20% of the extant mammals on earth, were recently identified as a natural reservoir of emerging and reemerging infectious pathogens. astonishing amount (more than 70) and genetic diversity of viruses isolated from the bat have been identified in different populations throughout the world. many studies focus on bat viruses that caused severe domestic and human diseases. however, many viruses were found in apparently healthy bats, suggesting that bats may have a specific immune system or antiviral activity against virus infections. therefore, basic researches for bat immunology and virus-host interactions are important for understanding bat-derived infectious diseases. bats, originated about 50 million years ago, are currently widely distributed and make up more than 20% of extant mammals on earth (simmons, 2005; teeling et al., 2005) . bats are traditionally grouped into two suborders: megachiroptera (megabat), which contains a single family pteropodidae (42 genera, including 166 species), and microchiroptera (microbat), which contains 16 bat families (135 genera, including 759 species) within the order chiroptera (simmons, 2005) . megabat mainly feed on fruit, and some bats, such as pteropus, have long distance migration capability. most microbats feed on insects, while some feed on birds, lizards, frogs, fish or blood (vampire bat) (aguirre et al., 2003; patterson et al., 2003) . bats have various habitats, including caves, trees, roof and discarded tunnels, with a large group from hundreds to thousands of individuals in the same habitat. because of many unique biological properties such as echolocation, hibernation, flying ability and living environment, bats are considered as enigmatic mammals. bats as reservoirs of human viruses were recognized as early as 1920s when rabies was found in bat in south and central america (sulkin and allen, 1974 ), but bats as carriers of emerging zoonotic viruses were not well acknowledged until the mid-1990s. the number of identified bat viruses rapidly increased after the discovery of henipavirus and severe acute respiratory syndrome coronavirus (sars-cov). up to today, more than 80 viruses, including members from families of rhabdoviridae, orthomyxoviridae, paramyxoviridae, coronaviridae, togaviridae, flaviviridae, bunyaviridae, reoviridae, arenaviridae, herpesviridae, picornaviridae, hepesviridae and adenoviridae, have been isolated from different bat species (calisher et al., 2006) . since the discovery of bat borne rabies virus in 1920s, different genotypes of rabies or rabies-related viruses within the genus lyssavirus of the family rhabdoviridae have been documented in bats. there are seven recognized genotypes of lyssavirus (bourhy et al., 1993) : rabies virus (genotype 1), lagos bat virus (genotype 2), mokola virus (genotype 3), duvenhage virus (genotype 4), european bat lyssavirus type 1 (eblv-1; genotype 5), european bat lyssavirus type 2 (eblv-2; genotype 6), and australian bat lyssavirus (genotype 7). all the genotypes except mokola virus have bat reservoirs (badrane and tordo, 2001) . human rabies caused by bat lyssavirus was incessantly reported in south and north america, europe, australia and africa (hanna et al., 2000; favi et al., 2002; badilla et al., 2003; fooks et al., 2003; nathwani et al., 2003; paweska et al., 2006; blanton et al., 2008; van thiel et al., 2008) . henipavirus the henipavirus is a new genus with the family paramyxoviridae and contains only two closely related but distinct members, hendra virus (hev) and nipah virus (niv) . restricted only to australia, hev was initially described after an outbreak of severe respiratory disease in horses during september 1994, which ultimately results in deaths of 13 horses and 1 horse trainer in hendra, australia, a suburb of brisbane (murray et al., 1995) . since then, several sporadic outbreaks have been reported in australia in 1999 , 2004 -2008 (field, 2008 . niv was discovered in malaysia during a major outbreak of acute respiratory syndrome in pigs occurring from september 1998 to june 1999, resulting in 265 cases of encephalitis in humans and 105 deaths (chua et al., 2000) . the similar disease outbreak has also been documented in singaspore, india in 2001 , bangladesh in 2001 -2008 (paton et al., 1999 chadha et al., 2006; eaton et al., 2006) . serologic and virologic studies suggest that bats are the principle reservoir hosts of henipaviruses. numerous frugivorous bat species of the genus pteropus (family pteropodidae) exhibit a high seroprevalence for henipaviruses. in addition, hev has been isolated from p. poliocephalus and p. alecto in australia (halpin et al., 1999) , niv has been isolated from p. hypomelanu in malaysia (chua et al., 2002) and p. lylei in cambodia (reynes et al., 2005) . antibodies to henipavirus have also been detected among both frugivorous and insectivorous bats in thailand (wacharapluesadee et al., 2005) , indonesia (sendow et al., 2006) , madagascar (lehle et al., 2007) and china ; however, no virus has been isolated from those tested bat samples. bats are recently identified as natural reservoir of sars-cov that caused outbreaks of human respiratory disease during 2002-2003 in china and resulted in 8422 cases with 916 deaths in 29 countries and regions (chan-yeung and xu, 2003) . antibody and genomic sequences similar to sars-cov were discovered in rhinolophus bat, including r. sinicus, r. pearsoni, r. macrotis and r. ferrumequinum and r. pusillus poon et al., 2005; ren et al., 2006) . the nucleotide sequences of bat sars-like covs (sl-cov) have 78%-92% identities to sars-cov and among themselves, and display great genetic diversity. the phylogenetic analysis indicated that rhinolophus bat may carry the direct progenitor of human sars-cov (hon et al., 2008) . filoviruses marburg virus and ebola virus, belonging to the family filoviridae, are two emerging viruses that cause human hemorrhagic fever disease (hf) (sanchez et al., 2007) . the marburg virus was recognized during 2-center outbreaks in marburg, germany, and belgrade, former yugoslavia, in 1967 (malherbe and strickland-cholmley, 1968 ). the disease was associated with laboratory work using african green monkeys (cercopithecus aethiops) imported from uganda. subsequently, outbreaks and sporadic cases have been reported in angola, democratic republic of the congo, kenya, south africa (in a person traveled to zimbabwe) and uganda (http:// www.who.int/csr/disease/marburg/en/). the ebola virus was first identified in a western equatorial province of sudan and in a nearby region of zaïre (now the democratic republic of the congo) in 1976 after significant epidemics in yambuku in northern democratic republic of the congo, and in nzara in southern sudan (emond et al., 1977) . since then, numerous outbreaks have been documented in democratic republic of the congo, uganda and sudan (http://www.who.int/csr/ disease/ebola/en/). four different ebola virus strains were identified: zaire, sudan, reston and côte d'ivoire (ivory coast) ebolaviruses, and the former two are associated with hf outbreaks in africa with high case fatality (53%-90%) (sanchez et al., 2007) . both zaire and sudan strains have been detected in bats. marburg virus was detected in fruit bat rousettus aegyptiacus in gabon, uganda and democratic republic of the congo and in insectivorous bat rhinolophus eloquens towner et al., 2007 towner et al., , 2009 ). zaire ebola virus was detected in 3 bat species (epomops franqueti, hypsignathus monstrosus, and myonycteris torquata) in gabon and the republic of the congo (leroy et al., 2005; biek et al., 2006; gonzalez et al., 2007; pourrut et al., 2007 pourrut et al., , 2009 . the available sequence data demonstrated that both ebola and marburg virus in bats display genetic diversity. the melaka virus, a novel reovirus, was isolated from a 39-year-old male patient in melaka, malaysia, who was suffering from high fever and acute respiratory disease upon virus isolation. two of his family members developed similar symptoms approximately 1 week later and had serological evidence of infection with the same virus. epidemiological tracing revealed that the family was exposed to a bat in the house approximately 1 week before the onset of the father's clinical symptoms. genome sequence analysis indicated a close genetic relationship between melaka virus and pulau virus, which is a also reovirus isolated in 1999 from fruit bats in tioman island, malaysia (chua et al., 2007) . in addition to bat borne virus that caused severe human diseases, many other bat viruses that has no evidence to cause animal or human diseases have been discovered in bat populations throughout the world. these viruses were regularly detected in apparently healthy bats and display genetic diversity. other bat covs during the search for the origin of sars-cov, numerous novel covs were detected in large numbers of bat species in china, south and north america, germany and africa (poon et al., 2005; chu et al., 2006; ren et al., 2006; tang et al., 2006; dominguez et al., 2007; lau et al., 2007; muller et al., 2007; woo et al., 2007; carrington et al., 2008; gloza-rausch et al., 2008; misra et al., 2009; pfefferle et al., 2009) . the phylogenetic analysis based on the fully sequenced bat-cov indicated high genetic diversity that of bat covs, which were grouped into cov group 1, group 2a, 2c and 2d (ren et al., 2006; tang et al., 2006; woo et al., 2007; lau et al., 2007) . bat astroviruses a group of novel astroviruses was found in apparently healthy insectivorous bats, particularly in the genera miniopterus and myotis (36%-100% and 50%-70%, respectively), in hong kong (chu et al., 2008) . similar viruses were detected in a large numbers of bat samples in mainland china (zhu et al., 2009) . the phylogentic analysis revealed a remarkably high genetic diversity of bat astroviruses that form five monophyletic groups clustered in the genus mamastrovirus within the family astroviridae. some bat astroviruses may be phylogenetically related to human astroviruses, implying potential risk of inter-species transmission of mamastrovirus. herpesvirus herpervirus was initially discovered in little brown bats (myotis lucifugus) by virus morphology (tandler, 1996) and later detected in different bat species in the philippines, africa and europe with molecular detection methods (wibbelt et al., 2007; molnar et al., 2008; razafindratsimandresy et al., 2009; watanabe et al., 2009) . the phylogenetic analysis based on obtained partial gene sequences indicated that bat herpesviruses display genetic diversity and form distinct clade within the subfamily alhaherpesvirinae, betaherpesvirinae and gammaherpesvirinae. adenovirus a novel adenovirus was isolated by a japanese scientist during tissue cultures derived from bat spleen from fruit bat p. dasymallus yayeyamae. the partial polymerase sequence of this bat adenovirus showed homolog to tree shrew adenovirus 1 (70% amino acid sequence identity). a germany group later reported another novel adenovirus in german bats (sonntag et al., 2009) . recently, our group has also detected genetic diversity adenoviruses in 5 bat species in china (unpublished results), indicating a wide distribution of adenovirus in bat populations. bats viruses did not attract human attention until recent years when several emerging and reemerging human viruses were associated with bats. however, information about the natural history of most viruses in bats is limited. it is evident that most of the bat viruses have existed in bats for long time. why did some of them cause severe disease in domestic animals and human in recent years? the invasion of bat habitats and expanding agricultural industry increase the close contact opportunity between bats and human, thus increase the opportunity of virus transmission from bat to domestic animals and human. the molecular detection technology significantly contributes to the discovery of more bat viruses. considering the diversity of bat species in the world, the currently identified bat viruses may represent only a small part of bat reservoir. therefore, a wide-ranged screening of bat viruses is required to prepare specific diagnostic methods for preventing future emerging viral diseases in animals and human. viruses usually have narrow host ranges, which restrict them transmitted from one animal to others. however, zoonotic viral diseases consist of more than 70% among the emerging and remerging viral diseases (jones et al., 2008) . the first step for successful interspecies transmission is that the virus can use the same receptor as found in their original host of other animals for their entering the invading host cells. some bat borne viruses, such as henipaviruses, have wide host ranges and can easily transmit from bats to animal and human as their cellular receptor ephrin b2 and b3 are highly conserved in different animals. this may explain why hendrs virus and nipah virus continually revisiting human and caused disease outbreaks in recent years. unlike henipaviruses, sars-cov, which use the angiotensin converting enzyme 2 (ace2) as cellular receptor, has a relatively narrow host ranges. the ace2 protein of different origins are variable at the n-terminal ends that contact directly the receptor binding domain (rbd) of the sars-cov spike protein (s). a minor amino acid change in the rbd or ace2 n-terminal end can abolish the entry of sars-cov ren et al., 2008) . this may partially explain why sars-cov completely disappeared when the transmission chain (civet and other small mammals as intermediate hosts) was interrupted. thus, further studies should focus on the interspecies transmission of bat viruses and evaluate the potential risk of bat viruses to domestic animals and human. most bat viruses known today were discovered in apparently healthy bats. experimental infection to bat by henipavirus virus and rabies virus also demonstrated that bat shed the virus but did not produce any clinical syndrome as did in other animals and human (almeida et al., 2005; hughes et al., 2006; tjornehoj et al., 2006; middleton et al., 2007) , suggesting that bats may have specific immune system or antiviral activity against virus infections. 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of two sars-like coronaviruses in horseshoe bats and genetic variation analysis difference in receptor usage between severe acute respiratory syndrome (sars) coronavirus and sars-like coronavirus of bat origin nipah virus in lyle's flying foxes filoviridae: marburg and ebola viruses henipavirus in pteropus vampyrus bats order chiroptera. in mammal species of the world: a taxonomic and geographic reference, 3 edn new adenovirus in bats virus infction in bats studies of reservoir hosts for marburg virus cytomegalovirus in the principal submandibular gland of the little brown bat, myotis lucifugus prevalence and genetic diversity of coronaviruses in bats from china a molecular phylogeny for bats illuminates biogeography and the fossil record natural and experimental infection of sheep with european bat lyssavirus type-1 of danish bat origin isolation of genetically diverse marburg viruses from egyptian fruit bats marburg virus infection detected in a common african bat fatal case of human rabies (duvenhage virus) from a bat in kenya: the netherlands bat nipah virus detection of a new bat gammaherpesvirus in the philippines discovery of herpesviruses in bats comparative analysis of twelve genomes of three novel group 2c and group 2d coronaviruses reveals unique group and subgroup features detection of diverse astroviruses from bats in china key: cord-354738-4rxradwz authors: kohl, claudia; kurth, andreas title: european bats as carriers of viruses with zoonotic potential date: 2014-08-13 journal: viruses doi: 10.3390/v6083110 sha: doc_id: 354738 cord_uid: 4rxradwz bats are being increasingly recognized as reservoir hosts of highly pathogenic and zoonotic emerging viruses (marburg virus, nipah virus, hendra virus, rabies virus, and coronaviruses). while numerous studies have focused on the mentioned highly human-pathogenic bat viruses in tropical regions, little is known on similar human-pathogenic viruses that may be present in european bats. although novel viruses are being detected, their zoonotic potential remains unclear unless further studies are conducted. at present, it is assumed that the risk posed by bats to the general public is rather low. in this review, selected viruses detected and isolated in europe are discussed from our point of view in regard to their human-pathogenic potential. all european bat species and their roosts are legally protected and some european species are even endangered. nevertheless, the increasing public fear of bats and their viruses is an obstacle to their protection. educating the public regarding bat lyssaviruses might result in reduced threats to both the public and the bats. the european continent is inhabited by 52 hibernation. many bat species migrate over vast distances while others are rather territorial. all bats in europe utilize echolocation to navigate. contrary to the worldwide efforts in protecting bats, they have been increasingly gaining attention as potential reservoir hosts of some of the most virulent viruses we know. various publications reviewed bats globally as carriers and potential reservoir hosts of human-pathogenic and zoonotic viruses [3] [4] [5] [6] [7] [8] [9] [10] , while hardly anything is known about human-pathogenicity of european bat viruses apart from lyssaviruses. in this review, we discuss a selection of viruses as possible threats posed by european bats to the public from our point of view. a summary of viruses that have been detected in european bats is given in table 1 at the end of the manuscript. a more comprehensive and up-to-date list of bat-associated viruses can be found online at the database of bat-associated viruses (dbatvir) [11] . european bat lyssaviruses (family rhabdoviridae) are the most important zoonotic bat-borne viruses in europe and have been comprehensively reviewed by banyard et al. in this special issue on bat viruses (title: lyssavirus infections of bats: emergence and zoonotic threat) [12] . therefore, we will provide a short overview. [13, 14] nyctalus noctula rhinolophus ferrumequinum hungary pcr [15] myotis myotis germany pcr [16] astroviridae myotis myotis germany pcr [16] mamastrovirus italy pcr [22, 28] the postulates drafted by jacob henle and robert koch in the late 19th century constitute a framework regarding the principles of cause-and-effect in microbiology [55] . back then, it was comparatively straightforward to limit cause-and-effect to four postulates, although viruses had not yet been discovered nor was molecular biology developed ( table 1 ). all of the postulates are hard to fulfill for viruses, as they do not grow on nutrient media, but require living cells for replication. when looking for viruses on a molecular level, it is necessary to consider that only the first postulate can be accomplished. studies identifying a host-pathogen relationship solely at the molecular level do not take into consideration that detection does not equal etiology. even though polymerase chain reaction (pcr) screening and metagenomic studies are indispensable and valuable tools, virologists should stay close to the henle-koch postulates when assuming a possible virulence of viruses detected in bat hosts. a plethora of coronaviruses has been detected in bats, mostly belonging to the alphaand betacoronaviruses [11, 56] . the genus alphacoronavirus hosts human-pathogenic strains (i.e., human cov 229e and nl63); however, in this review, we will focus on selected highly human-pathogenic betacoronaviruses and their european bat virus relatives [56] . from november 2002 until july 2003 the world was confronted with the first pandemic of the new millennium, caused by a novel coronavirus (cov) inducing the severe acute respiratory syndrome in humans (sars) [57] [58] [59] . the pandemic spread from its origin, a wet-market in the guangdong province in china, through 33 countries on five continents resulted in more than 8000 infected humans of whom more than 700 eventually died [60, 61] . the search for the animal reservoir began, identifying masked palm civets and bats as possible sources. subsequently, a plethora of diverse coronaviruses of distinct groups have been detected in various bat species around the world via molecular-biological techniques. in 2012, another human-pathogenic coronavirus, called middle east respiratory syndrome coronavirus (mers-cov), began spreading from the arabian peninsula, so far resulting in globally 707 laboratory-confirmed cases of infection with mers-cov, including at least 252 deaths [62] . dromedaries and bats are suspected as reservoirs for mers-cov [63] . recent findings support the plausibility of dromedaries as reservoir species [64] . although numerous studies in european bats report the presence of sars-like-cov and mers-like-cov sequences [21, [24] [25] [26] 65] , no final conclusion can be drawn regarding their zoonotic potential. a related virus detected in bats cannot necessarily be considered as zoonotic. a few alterations in the sars-cov spike protein enabled its binding to the host receptor ace-2, thus sars-cov became capable of infecting humans [66] . so far, the sars-like cov detected in european bats lack these alterations and thus are not predicted to be capable of infecting humans. although virus strains might be similar or related on a nucleic acid level, the distinct function of proteins is crucial when determining the host range. therefore, mere similarity is not sufficient to examine the potential of viruses to infect humans or even predict their virulence. it took ten years from the emergence of sars-cov for the first bat cov to be isolated from rhinolophus bats in china, that displayed the human ace-2 receptor, which enabled the virus to infect human cells [67] . these findings provide evidence for the reservoir theory. from the european perspective, nevertheless, no sars-like cov or mers-like cov has been isolated from any european bat, nor has any transmission of sars-like cov or mers-like cov to humans been reported. the case of mers-cov is slightly different, as a sequence of 190 base pairs with 100% identity to mers-cov was detected in a bat (taphozous perforates-the species identification performed was not beyond doubt, as it was based on exclusion criteria (no cytochrome b sequence of taphozous perforates is available in genbank [68] )) in saudi arabia [8] . this finding initiated a controversy among leading cov experts, as the journal nature recently reported [69] . they discussed that the complete genome sequence of mers-cov obtained from the bat should confirm that the virus was indeed identical and not coincidentally just a short conserved region of the virus genome. furthermore, a prevalence study might provide insights into the distribution of mers-cov in bat populations. although taphozous perforates are not abundant in europe, climate change and environmental factors may have an effect on the future distribution of this bat species (figure 1 ) [70] . the case of mers-cov emergence impressively demonstrates the necessity of virus discovery and prevalence studies. with the first sequence of mers-cov that became available, bats were suspected as reservoir hosts, not only because mers-cov is a sars-cov relative, but also because previous bat virus discovery studies had provided eligible sequences of bat cov to genbank, allowing for correlations with the novel mers-cov. recently, a quasi-species of mers covs was recovered from nasal swabs of dromedaries of the kingdom of saudi arabia [64] . the mers cov consensus genome variants from dromedaries and humans are indistinguishable, supporting the plausibility of dromedaries in the role of transmission [64] . in 2002, the first reported outbreak of filovirus, named lloviu virus (llov), in a european bat population occurred in france, spain, and portugal [29] . several colonies of schreiber's bats (miniopterus schreibersii) suddenly declined due to an unknown disease. llov was found in animals that showed signs of viral infection, but not in healthy bats co-roosting in the caves (myotis myotis). llov is distinctly related to filoviruses found in african bats and was classified in 2013 as type species of the novel genus cuevavirus [56] . unfortunately, the lack of successful isolation of llov prohibits the experimental infection of schreiber's bats to clarify whether llov is the first filovirus capable of inducing disease in bats. this would challenge the hypothesis of bats as potential reservoir hosts for other filoviruses like ebola and marburg virus. schreiber's bats are distributed in distinct lineages throughout oceania, africa, southern europe, and south-east asia (figure 2 ) [72] . they are thought to transmit and maintain llov across different lineages throughout their habitats, although no studies are available to prove this hypothesis. consequently, the sole demonstration of a novel filovirus sequence does not provide evidence of a possible public health threat. following the henle-koch postulates, the virus should be isolated and further characterized to draw conclusions on the evolution of filoviruses in their respective bat host. as most filoviruses are described as highly pathogenic for humans, the occurrence of llov should be carefully monitored by prevalence studies in the highly abundant miniopterus schreibersii (figure 2 ). in 2012, three distinct paramyxoviruses were detected in german bats, two of which were related to the proposed genus jeilongvirus (myotis mystacinus, pipistrellus pipistrellus) and one was related to the genus rubulavirus (nyctalus noctula) [34] . another study published in the same year described another 12 different paramyxoviruses in bats from germany (myotis bechsteinii, m. daubentonii, m. myotis, and m. mystacinus) and bulgaria (myotis alcathoe and m. capaccinii), all of which belong to the genus morbillivirus [35] . none of the novel bat paramyxoviruses are closely related to viruses of the highly pathogenic genus henipavirus or other human-pathogenic paramyxoviruses [34, 35] . there is no evidence to suggest that any of these novel paramyxoviruses are capable of infecting humans. similar to the case of the llov filovirus, virus isolates and prevalence studies in both humans and bats could improve knowledge and clarify their zoonotic potential. few studies have documented the negative results from pcr testing of european bats for other human-pathogenic viruses. for instance following generic pcr screening for flavi-, hanta-and influenza-a viruses in 210 european bats in 2011 [73] , testing of another 1369 central european bats for influenza-a viruses [74] and testing 42 european bats for hepadnaviruses in 2013 did not lead to the detection of any viral nucleic acids [75] . pcr screening of 468 european bats for orthopoxviruses has not revealed any known or novel virus sequences [76] . so far, the only virus isolates (beside lyssaviruses) obtained from european bats are one bunyavirus, one adenovirus and 22 orthoreoviruses [13, 19, 36, 37] . these represent the only isolates that would allow for further characterization and potential clarification of their zoonotic potential. nevertheless, recombinant viruses, constructed on sequence information, are also valuable tools to study prevalence and pathogenicity in vitro. toscana virus (tosv) was isolated from a bat's brain in 1988, while simultaneously tosv was isolated from sandflies in the laboratory [19] . as tosv has never been reported in bats afterwards and no hemagglutination-inhibiting antibodies has been initially found in the bat, there is a reasonable chance that this tosv isolation may have been a cross-contamination [77] . bat adenovirus 2 (bat adv-2) was isolated from a bat's intestine in 2009 [13] , and the whole genome was obtained and circumstantially analyzed [14, 78] . bat adv-2 displays a monophyletic relationship to the adenoviruses of canids (cadv). moreover, open trading frames (orf) in the bat adv-2 genome and the cadv are identical and not present in other members of the mastadenoviruses. the closely related canine adv contribute to the severe kennel cough syndrome in canids and show an unusually broad host range [79] . this provides evidence suggesting an ancestral inter-species transmission of mastadenoviruses between bats and canids. like in the case of rabies virus, which is prevalent in both bats and terrestrial mammals (e.g., dogs, raccoons, skunks, and foxes) of the americas, a continuing exchange and transmission between bats and canids or other terrestrial animals might be possible [80] . there is no evidence of a zoonotic potential of bat adv-2. in 2012, three novel orthoreoviruses were isolated from plecotus auritus and myotis mystacinus in germany [36] . a subsequent pcr screening obtained identical viral sequences also in other bat species: pipistrellus pipistrellus, pipistrellus nathusii, pipistrellus kuhlii, and nyctalus noctula. at the same time, a group in italy detected further 19 orthoreoviruses in myotis kuhlii, rhinolophus hyposideros, tadarida teniotis, and vespertilio murinus [37] . summing up the data for the reovirus isolates from germany and italy, a close relationship was revealed to the genus mammalian orthoreovirus (mrv), in particular to an orthoreovirus obtained from a dog (strain t3/d04) with hemorrhagic enteritis in italy [36, 37, 81] . no ancestral relationship was assumed here, but rather an opportunistic -behavior‖ of the novel closely related mrvs, as they were detected in various different bat species. moreover, the newly isolated mrvs are phylogenetically related to viruses capable of inducing severe meningitis in humans [82] . recently, a study published by steyer et al. described the detection of an mrv from a child hospitalized with acute gastroenteritis in slovenia [83] . the causative agent was determined to be an mrv with the highest similarity of 98.4%-99.0% in the respective segments to a bat mrv (t3/bat/germany/342/08) [83] . this might indicate a human-pathogenic potential of strain t3/bat/germany/342/08. as the case of sars-cov has shown that even small changes in the genome are important for determining the host range, this has to be determined for the bat mrvs in further studies. interestingly, no contact was reported between the infected child and bats, but contact to a domestic dog was assumed [83] . the isolated viruses will allow for a seroprevalence study (cross-reactivity and cross-neutralization with other strains) in humans, which shall be initiated to examine the prevalence of specific antibodies to bat mrvs in germany and italy (where these viruses have been found) to clarify their zoonotic potential. this is especially interesting as asian bat orthoreoviruses of the genus pteropine orthoreovirus have already been linked to potentially zoonotic respiratory diseases in humans [84, 85] . rhabdoviruses of the genus lyssavirus that have been detected in europe are considerably harmful and truly zoonotic agents, inevitably causing the death of unvaccinated humans if not treated in time before onset of the rabies disease [86] . even though bat-transmitted lyssaviruses have a fatality rate of virtually 100% and are suspected to be transmissible by bat biting and scratching, the reported total number of human fatalities in europe is low (n = 2-5 since 1963) [86] [87] [88] . all described hosts of european bat lyssaviruses (eblv-1 and eblv-2) are synanthropic, hence sharing their habitats with humans [87] . eblv-1 has been predominantly detected in eptesicus serotinus and e. isabellinus in europe, both living in buildings, roofs, and attics usually in the southern regions of europe (e. serotinus until 55° north, e. isabellinus in southern portugal-e. isabellinus is a north african population of e. serotinus that is controversially but not concludingly discussed as a novel species [1]), and male bats are reported to co-roost with multiple bat species [90] . eblv-1 was also detected in v. murinus, m. schreibersii, m. myotis, m. nattereri, r. ferrumequinum, and t. teniotis. whether these bat species constitute accidental hosts infected by spillover from co-roosting e. serotinus species, or whether they are additional reservoirs, has not yet been determined [38] [39] [40] [41] 91] . two human cases described by johnson et al. were confirmed as infected with eblv-2, which is prevalent in european m. daubentonii and m. dasycneme [40, 86] . m. daubentonii is prevalent in north-eastern europe and is frequently found co-roosting with p. pipistrellus and m. nattereri, whereas m. dasycneme is found throughout europe and in the mediterranean, co-roosting with m. capaccinii. so far, none of the co-roosting bats have been reported to carry eblv-2 [90] . however, spillover transmission to other animals (stone-marten, sheep, and cat) was described for eblv-1 [92] [93] [94] . overall, lyssaviruses prevalent in european bats pose a risk to public health, and preventive measures have already been implemented by many european countries for decades (e.g., surveillance, vaccination plans, and post exposure prophylaxis) [87] . especially the high-risk occupational groups (i.e., bat workers, bat carers in bat bat hospitals) are at increased risk. however, lyssavirus prevalence in european bats is very difficult to determine and results are very heterogenic [40] . the lyssavirus prevalences are considerably low, but changes of behavior as a result of a lyssavirus infection may be more likely to bring bats into contact with humans. however, it is necessary to balance the risk with the total number of fatal human cases during the last 35 years (five cases in 590 million people living in greater europe) [87] . accordingly, the risk is relatively low and would probably fall to zero if people were educated appropriately. direct contact (bites and scratches) with certain bat species might be risky and require post exposure prophylaxis. only few of the european bat species are known to be reservoirs of eblv-1 and eblv-2, but all of the european species are endangered or close to extinction. relocation or culling of bat colonies, in spite of being an obvious solution from the viewpoint of the general public, increases the risk of lyssavirus exposure and transmission and should not be considered [95] . only education can channel public fear to avoid further threats to the bats and the general public. alexander von humboldt discovered the latitudinal gradient in species diversity as early as 1799 [96] : the richness of species is subject to a global diversity gradient, abating from the species-rich tropics toward the higher latitudes [97] . bats influence this gradient significantly. more than 1100 bat species have been described worldwide. although they are abundant worldwide except for the polar regions, a steep diversity gradient is present from the tropics towards the poles [97] [98] [99] [100] . are fewer viruses prevalent in european bats because of the lower abundance of species in the more temperate europe? and is the zoonotic risk posed by bats decreased accordingly? only few studies on the biogeography of microorganisms are available. these studies indicate that the latitudinal diversity gradient has either no or a top-down effect on microbial diversity [101] [102] [103] [104] [105] . two studies hypothesized that the local diversity and dispersal of viruses is very high, though overall, the viral diversity is limited on the global scale [106, 107] . therefore, no assumptions can be made regarding the viral diversity in species abundant in temperate climates. as the total number of abundant species might not be essential, the change in biodiversity might play a role. the effect of decline in biodiversity on the emergence of diseases is subject of numerous publications [108] [109] [110] [111] [112] [113] [114] . basically, there are arguments in favor of two controversial theories; reduced biodiversity could either increase (dilution effect) or decrease the risk of disease transmission. for almost half of the zoonotic diseases that have newly emerged by spillover since 1940, a preceding change in land-use, agriculture, and wildlife hunting was reported [108] . all of the above-mentioned effects contribute to changes in biodiversity and increased contact situations between human and animal hosts, also in europe. once spillover in novel hosts has occurred, a high density of the novel host population eventually facilitates the establishment in the novel niche. thus, human overpopulation and a decreased biodiversity might be mutual factors promoting the establishment of emerging infectious diseases. in conclusion, the baas becking hypothesis from 1932 might still be appropriate: -everything is everywhere, but the environment selects‖ [115] . until now, lyssaviruses have been the only proven zoonotic viruses in european bats and may cause rabies in humans. however, only few bat species are known to transmit lyssaviruses in europe, and the number of human cases is rather low. nevertheless, education of the general public should be intensified to avoid easily preventable infections. although viruses with zoonotic potential have been detected in european bats, no clear assumption can be made without further studies. sero-prevalence studies should be conducted on the orthoreoviruses isolated from european bats, especially as a closely related virus was detected in a diseased child in slovenia [83] . other bat viruses detected by using molecular techniques should be isolated (e.g., mers-like cov or bat bunyavirus) to allow for characterization and follow-up sero-prevalence studies. in general, bats are special reservoir hosts because of their biological features, long-time co-evolution and high diversity of viruses that can be found. furthermore, there is neither a clearly decreased risk in the emergence of zoonotic viruses in temperate climates compared to 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biogeography of soil bacterial communities body size determines the strength of the latitudinal diversity gradient microbial biogeography: putting microorganisms on the map biogeography of diseases: a framework for analysis here a virus, there a virus, everywhere the same virus? microbial biogeography? impacts of biodiversity on the emergence and transmission of infectious diseases biodiversity loss and emerging infectious disease: an example from the rodent-borne hemorrhagic fevers biodiversity loss and the rise of zoonotic pathogens pangloss revisited: a critique of the dilution effect and the biodiversity-buffers-disease paradigm a meta-analysis suggesting that the relationship between biodiversity and risk of zoonotic pathogen transmission is idiosyncratic host range and emerging and reemerging pathogens population biology of multihost pathogens geobiologie of inleiding tot de milieukunde the authors are grateful to ursula erikli for copy-editing and the two anonymous reviewers for their valuable comments. both authors reviewed the literature and wrote the manuscript. the authors declare no conflict of interest. key: cord-342124-jdv17u86 authors: nieto‐rabiela, fabiola; wiratsudakul, anuwat; suzán, gerardo; rico‐chávez, oscar title: viral networks and detection of potential zoonotic viruses in bats and rodents: a worldwide analysis date: 2019-06-20 journal: zoonoses public health doi: 10.1111/zph.12618 sha: doc_id: 342124 cord_uid: jdv17u86 bats and rodents are recognized to host a great diversity of viruses and several important viral zoonoses, but how this viral diversity is structured and how viruses are connected, shared and distributed among host networks is not well understood. to address this gap in knowledge, we compared the associative capacity of the host–virus networks in rodents and bats with the identification of those viruses with zoonotic potential. a virus database, detected by molecular methods, was constructed in the two taxonomic groups. we compiled 5,484 records: 825 in rodents and 4,659 in bats. we identified a total of 173 and 166 viruses, of which 53 and 40 are zoonotic viruses, in rodents and bats, respectively. based on a network theory, a non‐directed bipartite host–virus network was built for each group. subsequently, the networks were collapsed to represent the connections among hosts and viruses. we identified both discrete and connected communities. we observed a greater degree of connectivity in bat viruses and more discrete communities in rodents. the coronaviridae recorded in bats have the highest values of degree, betweenness and closeness centralities. in rodents, higher degree positions were distributed homogeneously between viruses and hosts. at least in our database, a higher proportion of rodent viruses were zoonotic. rodents should thus not be underestimated as important reservoirs of zoonotic disease. we found that viruses were more frequently shared among bats than in rodents. network theory can reveal some macroecological patterns and identify risks that were previously unrecognized. for example, we found that parvovirus in megabats and gbagroube virus in rodents may represent a zoonotic risk due to the proximity to humans and other zoonotic viruses. we propose that epidemiological surveillance programmes should consider the connectivity of network actors as a measure of the risks of dispersion and transmission. bats and rodents are hosts of a significant proportion of zoonoses, higher than any other mammalian order. over 200 viruses belonging to 27 viral families have been isolated or detected in bats; however, bat-human transmission has only been observed for 11 viruses, belonging to four different viral families: rhabdoviridae, filoviridae, coronaviridae and paramyxoviridae (allocati et al., 2016) . some examples of those viruses are as follows: sars-related coronavirus, sosuga rubulavirus, ebola virus and marburg virus, rabies lyssavirus, nipah henipavirus and hendra henipavirus (allocati et al., 2016; calisher, childs, field, holmes, & schountz, 2006; hayman, 2016; o'shea et al., 2014; plowright et al., 2015) . rodents have similar zoonotic potential to bats and are associated with a large number of zoonotic viruses, such as sin nombre virus, puumala virus, crimean-congo hemorrhagic fever virus, kyasanur forest virus, tick-borne encephalitis virus, lassa fever virus and venezuelan equine encephalitis virus, among others. all of the aforementioned bat-and rodent-associated viruses have a large impact on public health. however, it is important to take into count that not all of these viruses are obligate pathogens; some are generally commensal. previous studies have explored viral associations on relatively restricted spatial or phylogenetic scales. for example, hayman (2016) propose maps of viral distributions according to the distribution of hosts' families, streicker et al. (2010) explored rabies viruses using a phylogenetic approach, and cui, tachedjian, and wang (2015) compared retrovirus associations between bats and rodents. anthony et al. (2017) explored coronavirus networks at the level of host family, and bordes, caron, blasdell, garine-wichatitsky, and morand (2017) analyse the relationships among zoonotic diseases in southeast asia. luis et al. (2015) analyse viral networks between rodents and bats at global scale identifying several ecology factors to explain virus-host associations. recently, works explored the specificity and frequency of sharing dna and rna viruses among carnivores and bats (wells, morand, wardeh, & baylis, 2018) and the importance of the phylogeny to explain the viral richness associated with bats and rodents (guy, thiagavel, mideo, & ratcliffe, 2019) . however, there are currently no studies at a global level that incorporate the human influence in the viral networks. while some authors consider bats and rodents to belong to a similar category of high zoonotic risk potential (han, schmidt, bowden, & drake, 2015) , other work examines the differences between bats and rodents (luis et al., 2015) . several different distinctive features of bats have been hypothesized to explain their particularly high viral richness, such as their ability to fly, long migrations, high trophic diversity and social structure (brook & dobson, 2015; moratelli & calisher, 2015) . however, currently the viral diversity and connectivity among different species of bats are not well understood (moratelli & calisher, 2015; o'shea et al., 2014) (luis et al., 2013) . rodents, like bats, have been recognized as reservoirs for several zoonotic viruses (han et al., 2015) , such as virus of hantaviridae (schmaljohn & hjelle, 1997) and arenaviridae families (charrel & de lamballerie, 2010) . however, there are differences in rodent-virus associations that impact their zoonotic potential compared with bats. in disease ecology, analytical tools have been used to holistically explain the dynamics of infections and provide novel hypothesis to explain macroecological patterns (johnson, roode, & fenton, 2016) . one of the theories that helps to predict dynamic changes in host-pathogen systems is graph theory, also known as network theory (bordes et al., 2017; johnson et al., 2016) . this approach can be used to gain better understanding of how interactions take place within pathogen communities, how hosts are connected with pathogens, their preferred association and patterns of pathogen transmission (godfrey, 2013; white, forester, & craft, 2017 ). the graphs, better known as networks, focus on the interactions between entities (newman, 2014) , and they have the potential to infer relationships within a larger framework (hossain & feng, 2016; luke & stamatakis, 2012) . a network is capable of emphasizing the preferred union as a process (hartonen & annila, 2011) and capturing both the individual elements in a system as well as their relevant interconnections (kolaczyk & csardi, 2014) . in disease ecology, this kind of analysis could be applied to describe viral diversity associated with different hosts and detect hosts and viruses that share associations, and therefore identify groups that share similar characteristics (white et al., 2017) . in network theory, centrality and dispersion metrics quantify the importance of each component member (martínez-lópez, perez, & sánchez-vizcaíno, 2009; newman, 2014; opsahl, agneessens, & skvoretz, 2010) . the parameter "betweenness" can be used to impacts • the analysis of virus and host networks (rodents and bats) allows us to measure the potential risk of zoonotic diseases. • measuring network connectivity can be a useful tool for identifying hosts and viruses of potential importance in the transmission dynamic of zoonotic diseases. • bats presented twice as many connections between virus and host as rodents, indicating a higher zoonotic potential transmission. recognize dispersing hosts and key viruses in the evolution or viral transmission (opsahl et al., 2010; white et al., 2017) , while "closeness" can indicate hosts and viruses that may have little direct connectivity but are surrounded by important highly connected nodes (opsahl et al., 2010; white et al., 2017) . in terms of disease ecology, we can employ these and other parameters to explore the host-host, virus-host and virus-virus interactions by collapsing the networks and identifying communities. network analysis thus offers the opportunity to recognize highly diverse viruses and hosts based on a high degree of connectedness. bats and rodents are excellent taxa in which to implement this tool because they harbour a large number of highly adaptable viruses and hosts with high resistance. therefore, in this study we aimed to compare and recognize the differences in the associative capacity of the host-virus networks in rodents and bats worldwide, as well as to identify the viruses that may shift across species, including humans, suggesting zoonotic potential. does not exist in the ictv classification, we assigned all reports from astroviridae family as astrovirus. to increase the certainty of identification of the viruses, only studies that used molecular methods to detect viruses were included in the database we compiled. subsequently, each of the viruses identified in rodents or bats was classified as direct zoonotic or non-zoonotic pathogens (allocati et al., 2016; calisher et al., 2006; han et al., 2015) . an independent undirected bipartite network was built for each of the orders of rodentia and chiroptera. that is to say, each network included two types of nodes-viruses and hosts. viral nodes were connected to host nodes when the virus indicated by the node has been detected in the species indicated by the host node. we included a human host node, which was connected to viruses that have been classified as zoonotic. this helped us to group and identify zoonotic viruses and viruses close to them (which could have zoonotic potential). then, host-to-host and virus-to-virus networks were constructed in order to explore these networks in different dimensions. the "bipartite.projection" function in the igraph package implemented in r software version 3.4.2 (r core team, 2017) was used to collapse the bipartite network. basically, in the collapsed networks a host was connected to another host when they shared a common virus and a virus to a virus when they shared a common host ( figure 1 ). in each host-host collapsed network, the host nodes were conserved and the virus nodes were transformed using the weight of corresponding links in order to illustrate the relationships among different hosts (figure 1a ,b). in each virus-virus network (one for bats and one for rodents), the virus-virus relationship was highlighted by collapsing the host nodes into the weighted links ( figure 1c ). we measured the networks on two levels: individual node and the entire network. at the node level, we measured different centrality values including: degree (number of links that a node has), betweenness (number of times a node is an intermediary to connect each possible pair of nodes) and closeness (the degrees of average separation in relation to other nodes) (martínez-lópez et al., 2009; newman, 2014) . at the network level, we measured density and diameter. network density is the proportion of links that are actually observed in the network divided by those that could possibly occur and network diameter is the length of the longest geodesic distance (newman, 2014) . network level measurements were useful for summarizing the "big picture" of the network and identify the key nodes that are closely related to humans. network level measurements were generated using the algorithms provided in the packages "igraph" (csárdi table 1 ), and plots were produced using the packages "igraph" and "ggplot2" (gómez-rubio, 2017) in r (r development core team, 2011). communities were detected using the maximization of modularity method (newman, 2016) , which recognize nodes with dense and weak connections between groups. we used the function "cluster_ edge_betweenness" (girvan, girvan, newman, & newman, 2002; newman & girvan, 2003) in the "igraph" package (csárdi & nepusz, 2006) to identify the nodes with dense connections with humans, which is based on the following equation: where m denotes the total number of links in the network, a ij refers to the actual number of links between nodes i and node j , γ is a parameter calculated by the algorithm; k, degree of i; δ gigj is a randomized number of links between a pair of nodes. community detection facilitates the recognition of groups of hosts that share viruses and viruses that share hosts, and which therefore may continue to enter in contact with each other because they share similar characteristics. a subnetwork was built by choosing communities with more than four host-virus pairs, which is above the minimum number accepted in statistical normality samples (n = 3) (hammer, harper, & ryan, 2001; royston, 1982) . these subnetworks were illustrated to gain better community visualization and recognize the most relevant communities for the detection of potentially zoonotic viruses. from the subnetwork, the most important communities were chosen using to the measures of the members (top five nodes) and the number of zoonotic viruses (80%) as selection criteria. then, a sociogram representing the preferential unions and the complex interaction on the largest communities was constructed using the package "visnetwork" (almende, thieurmel, & robert, 2016) in r. this choice of subnetworks helps us to focus and observe in more detail the interactions within these important communities. the rodent database contained 825 records including 172 rodent species and 123 viruses, of which 53 are zoonotic viruses. the bat database contained 4,659 records, consisting of 220 bats species associated with 166 viruses, of which 40 viruses were classified as zoonotic. both databases are detailed in appendix s1. table 2 , and centrality values are in appendix s2. 78.06% of the nodes had a degree value of 1 or 2, making them uninformative in terms of epidemiological information, though they may be involved in co-evolutionary processes. thirty-nine different communities were detected. sixteen communities included only two members, while the largest group consisted of 32 members. this particular group included humans, as shown in appendix s2. ten communities with at least eight members were selected in the subnetwork (figure 2b ). we excluded communities 1 and 9 despite fulfilling the inclusion criteria because they are linear, with a single virus that influences the whole. (1) ta b l e 1 formulas applied to calculate networks parameters degree centrality (d) freeman (1978) closeness centrality (c) this network contained 147 nodes of rodent species with 502 links. the diameter of the network was 5, and the density was 0.0627 ( figure 3a) . the top five nodes in terms of degree, betweenness and closeness are shown in table 2 , and the remaining values are in appendix s3. thirty-five different communities were detected, and the largest of which contained 43 members, followed by a group of 21 members (appendix s3). a total of 387 nodes (220 bat species, 166 viruses, human node) and 736 links were contained in the bipartite bat network. the network diameter was 10, and the density was 0.0049 ( figure 4a ). three zoonotic viruses had the highest degree and betweenness values; these were bat coronavirus, rabies lyssavirus and bat paramyxovirus (table 4 ). 65.71% of the nodes had degrees of 1 or 2, so they do not provide much information to the network but they may be involved in co-evolutionary processes. all centrality values are given in appendix s2. twenty-nine different communities were detected; four of those had only two members, and the largest community contained 38 members (appendix s2). eleven communities contained eight or more members (figure 4b ). communities 1, 3 and 4 were linear and possessed simple edges, with a virus that influenced the whole community. community 7 was a homogeneous community with rich ecological interactions, but which was not highly related to zoonotic viruses. the last community was associated with humans had the highest number of zoonotic viruses involved (figure 4c ,d, details below). a low number of zoonotic viruses were found in homogeneous bat network communities. we focused only on the community that included the human node. in figure 4a , three red lines representing the three highly connected viruses in the network followed by a homogeneous community and later in green the human node connections. the community that included humans (figure 4c ,d) has 13 host nodes (including human) and 23 virus nodes with 45 links. fruit bat parvovirus was the only non-zoonotic virus in that community. miniopterus, mormopterus and saccolaimus were the only bat genera that directly shared viruses with humans. the host-host chiroptera network contained 221 nodes and 6,949 links. the diameter and density of the network were 5 and 0.2911, respectively ( figure 5a ). the five nodes with the highest centrality values are shown in table 5 , and detailed results are in appendix s4. sixty-seven different communities were detected; the largest had 69 members. the second largest community consisted of 56 members including humans (appendix s4). the virus-virus network contained 164 nodes and 2,132 links. the diameter of the network was 4, and the density was 0.1655 bats are well-known as excellent reservoirs for zoonotic viruses that usually result in high public health impact (gay et al., 2014; luis et al., 2013 luis et al., , 2015 plowright et al., 2015) . nevertheless, in our database, (tables 3 and 5 ). however, the clusters in the bat network are four times denser compared with rodents; this makes it easier to continue sharing the viruses. in addition, they can act as virus mixers, allowing the viruses to acquire characteristics that allow them to infect other host species, including humans. in addition, the human node is more closely connected to bats. it shows that viruses are shared to a greater degree among bats as discussed earlier (figures 3a and 5a) . one plausible explanation is that many species of bats live in high-density populations, with many individuals in close proximity to each other, such as in caves and roosts sites. indeed, there are always a larger number of bat species than rodent species in a given area (kerth, perony, & schweitzer, 2011) . the difference in connectivity between the bat and rodent hostvirus networks has implications for the zoonotic potential of each taxon. high connectivity facilitates viral transmission within and between species, and so, bats are expected to have higher zoonotic pofor that reason, deeper comprehension is required to unravel this entanglement. one of the objectives covered in this work was to recognize non-zoonotic viruses that may be strongly connected with humans and therefore have zoonotic potential. the bat-virus community that contained humans was composed of bats distributed in africa and australia may be explained by high rates of human-bat contact (allocati et al., 2016; rupprecht, 2009 ). one non-zoonotic virus that was included in the bat-virus community that included humans is fruit bat parvovirus. the parvoviridae family were transmitted from bats to other mammals by a viral ancestor suggesting their zoonotic capacity, and groups of genes in their genome denote this potential (canuti et al., 2011) . even though the virus currently infects only bats (canuti et al., 2011) , it is firmly connected with the human node in our network by pteropus poliocephalus, a species endemic to eastern australia (lunney, richards, & dickman, 2008) . future studies are recommended to elucidate its potential for zoonosis. andes viruses (rodents), cowpox (rodents) and rabies lyssavirus (bats) were defined as main actors (high values of degree, betweenness and closeness) in the bipartite networks. however, their importance disappeared when the network was collapsed to virus-virus interactions, likely because their geographical restriction may limit their viral connectivity. andes virus is only distributed in south america (martinez et al., 2005) , cowpox in europe (vorou, papavassiliou, & pierroutsakos, 2008) and rabies lyssavirus in america (moratelli & calisher, 2015) . therefore, these viruses in the rodent community selected, community 3 has members with predominantly european distribution. further, two non-zoonotic viruses are included among the nine zoonotic viruses, but we do not think they are likely to have zoonotic potential because they do not have direct contact with the human node. therefore, in this community we do not find viruses with zoonotic potential. in the rodent community 5, m. musculus has high values of connectivity but in the sociogram (figure 2d) , it is evident that the connectivity is with non-zoonotic viruses. this host species thus is likely less important in public health terms, but highly relevant for disease ecology. in addition, two zoonotic viruses are included in the community 5, and while they can transfer their zoonotic potential to other non-zoonotic viruses using m. musculus as a virus mixer, we consider this unlikely because the proportion of zoonotic viruses is low, adding the specificity of rodents' viruses and the associative characteristics founding in the rodents. thus, the one-to-one virushost species relationship suggested by the node-to-edge ratio suggests that spillover is unlikely, though not impossible. in the human-rodent community, gbagroube virus is noteworthy because it is the only non-zoonotic virus found in the community. gbagroube virus should be closely monitored along with its host, mus setulosus, found in central africa (granjon, 2016) . in our study, the human is the most relevant and largest node connected in both groups. the relevance of the human in the network is explained by several factors. first, humans' enormous population and globalization push human populations to nearly everywhere on earth and greatly increases the probability of contact with innumerable organisms, resulting in the emergence of zoonotic diseases (kock, 2014) . secondly, because zoonotic diseases have clear social implications, once detected in one species, they are much more likely to be tested for, and thus detected, in others (oliver-morales & abarca garcía, 2016). it is therefore possible that the high apparent importance of humans in the networks is more due to the over-representation of zoonotic viruses in the literature than to humans actually being particularly highly. in the database, we do not have ebola virus reports because in the database the dbatvir database did not identify the host species from which the virus was isolated. similarly, we found that 78.06% of rodent nodes and 65.71% of bats nodes were poorly connected (1-2 degree). surely, bats have associations that we do not recognize. in addition, there are associations that could occur but do not, but these cannot be identified because cases in which viruses were tested for and not detected are not often reported. we therefore think it is important to report negative samples and the number of animals tested in meticulous reports. a future study may complement and compare our study with models where the influence of humans is omitted. we must take into account that the human node influences the network structure, and the ecological relationships must be analysed without this influence. surely, laxer networks will be observed when the connective force of humans is removed. however, our study does need to include both to identify potentially zoonotic viruses. it is pertinent that, in future investigations, different characteristics of the viruses must be considered simultaneously and not only by their connectivity in the network such as gene sequence, type of transmission and virulence. in the present study, we focus only on viral host capability, not on the symbionts and their associative nature. spatial analysis may help to further explain how our findings apply among different regions of the world. graph theory, beyond allowing the visualization of complex interactions, allows the quantification of many aspects of connectivity and structure. rodents should be taken into account as important reservoirs for zoonotic viruses, since in our database, a greater proportion of the total viruses reported were zoonotic viruses in rodents than in bats. fruit bat parvovirus in bats and gbagroube virus in rodents should be monitored to elucidate their zoonotic potential. in the present study, we only assessed their network proximity to humans and other zoonotic viruses, and molecular genetic approaches may help to confirm our results. counting the number of zoonotic symbionts associated with each order is not a conclusive estimate of their zoonotic potential. our findings reveal that viruses were more frequently shared among bats than rodents. for that reason, bats have more zoonotic potential that the rodents. however, potential emerging zoonotic diseases may arise from both taxonomic groups. we are very grateful to papiit (project ia206416), programa castillo for their contribution in the construction of the databases. we declare that we have no conflict of interest. gerardo suzán https://orcid.org/0000-0002-9840-2360 oscar rico-chávez https://orcid.org/0000-0002-9833-1101 bat-man disease transmission: zoonotic pathogens from wildlife reservoirs to human populations visnetwork: 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wibbelt, gudrun title: diseases and causes of death in european bats: dynamics in disease susceptibility and infection rates date: 2011-12-28 journal: plos one doi: 10.1371/journal.pone.0029773 sha: doc_id: 289555 cord_uid: 1z4vbldd background: bats receive increasing attention in infectious disease studies, because of their well recognized status as reservoir species for various infectious agents. this is even more important, as bats with their capability of long distance dispersal and complex social structures are unique in the way microbes could be spread by these mammalian species. nevertheless, infection studies in bats are predominantly limited to the identification of specific pathogens presenting a potential health threat to humans. but the impact of infectious agents on the individual host and their importance on bat mortality is largely unknown and has been neglected in most studies published to date. methodology/principal findings: between 2002 and 2009, 486 deceased bats of 19 european species (family vespertilionidae) were collected in different geographic regions in germany. most animals represented individual cases that have been incidentally found close to roosting sites or near human habitation in urban and urban-like environments. the bat carcasses were subjected to a post-mortem examination and investigated histo-pathologically, bacteriologically and virologically. trauma and disease represented the most important causes of death in these bats. comparative analysis of pathological findings and microbiological results show that microbial agents indeed have an impact on bats succumbing to infectious diseases, with fatal bacterial, viral and parasitic infections found in at least 12% of the bats investigated. conclusions/significance: our data demonstrate the importance of diseases and infectious agents as cause of death in european bat species. the clear seasonal and individual variations in disease prevalence and infection rates indicate that maternity colonies are more susceptible to infectious agents, underlining the possible important role of host physiology, immunity and roosting behavior as risk factors for infection of bats. bats are among the most successful and diverse mammals on earth. approximately 1230 chiropteran species are found on every continent except antarctica and inhabit a multitude of diverse ecological niches [1] . bats play essential roles in maintaining healthy ecosystems, as they act as plant pollinators, seed dispersers, and predators of populations of insects including harmful forest and agricultural pests [2] . most bat species are listed in the iucn red list of endangered species and almost half of these are considered threatened or near-threatened [3] . to estimate and prevent further population declines, research has been primarily focused on bat biology, ecology and behavior, while disease aspects were largely neglected [4] . in the last two decades, the importance of chiropteran species as potential vectors of significant viral diseases especially in regard to zoonoses has received growing attention. besides bat rabies that has been studied for more than half a century, extensive research efforts identified a large number of microbial agents [5] including important emerging zoonotic viruses detected in bats across the world [6] [7] [8] [9] [10] [11] [12] . however, most studies are limited to the identification of microorganisms detected and investigations regarding infectious diseases and causes of death in bats are sparse [13] [14] [15] [16] . in europe, research is predominantly focused on european bat lyssaviruses [17, 18] and coronaviruses [19, 20] , but first indications of bat-pathogenic bacteria [13, 14, [21] [22] [23] and novel viruses [24, 25] isolated from deceased bats in germany and great britain were found. in this study, we provide new data on infectious diseases in european bat species, considering factors likely to affect the susceptibility of bats to infectious agents including effects of seasonality, individual and species-specific heterogeneities, and possible intra-and inter-species transmission dynamics. all bat species in europe are strictly protected under the flora-fauna-habitat guidelines of the european union (http://ec.europa. eu/environment/nature/legislation/habitatsdirective/index_en.htm) (92/43/eec) and the agreement on the conservation of populations of european bats (www.eurobats.org) that prohibit invasive sampling of bats for research purposes. for the animals investigated in this study, carcasses of deceased bats found in germany were kindly provided by bat researchers and bat rehabilitation centers of different federal states. between 2002 and 2009, a total of 486 deceased bats of 19 european vespertilionid species (i.e., family vespertilionidae) were investigated (fig. 1a , [26] ). the bat carcasses originated from 6 different geographic regions in germany, i.e. berlin greater metropolitan area (n = 223), bavaria (n = 165), brandenburg (n = 38), lower saxony (n = 36), thuringia (n = 21), and baden-wuerttemberg (n = 3), and were collected by bat researchers and bat rehabilitation centers. most animals represented individual cases that were found dead, injured or moribund near human habitation. thus, the species composition in this study predominately reflected the urban and suburban bat fauna, which is characterized by a disproportionate abundance of a few bat species (fig. 1a , [27, 28] ). two groups of 2 and 21 adult noctules (nyctalus noctula), respectively, were collected from tree hibernacula destroyed during wood logging. a further group of 25 deceased adult n. noctula originated from a colony that was trapped in a rain pipe in december. nine dead juvenile pipistrellus pipistrellus were collected from a nursery roost. if bats died in care or had to be euthanized for animal welfare reasons, the carcasses were immediately stored at 220uc and were shipped to the leibniz institute for zoo and wildlife research, berlin, germany, for diagnostic investigations. of all carcasses examined histo-pathologically, about 90% were suitable for bacteriological investigation. a lesser extend (43%) was also examined for selected viral agents at the robert koch institute, berlin, germany. in addition, a brain sample of each animal was submitted to the friedrich-loeffler-institute, wusterhausen, germany, for rabies diagnosis. a full necropsy was performed on each bat and all macroscopic findings including ectoparasite infestation were recorded. for histo-pathological examination, small slices of multiple organ tissues (i.e., lung, liver, heart, kidney, adrenal gland, spleen, intestine, pancreas, brain, tongue, larynx, salivary gland and pectoral muscle) and tissues conspicuous for pathological changes were fixed in buffered 4% formalin, processed using standard methods and embedded in liquid paraffin. sections were cut at 2-5 mm and routinely stained with hematoxylin-eosin (he). in addition, special histological staining methods were used depending on microscopic findings, i.e. for the detection of bacteria (gram or giemsa staining), fungi (periodic acid schiff or grocott's gomori methenamine silver nitrate staining), iron (prussian blue stain), mineralization (von kossa staining), connective and collagen tissue (trichrome staining). details on pathological results are published elsewhere [26] . the causes of mortality were rigorously standardized with the primary cause of death identified for each bat as the most serious injury, disease or event subsequently fatal to the animal. to ensure independence of primary and contributing causes of death, the categorization was based on the severity of pathological findings. samples of lung, liver, heart and kidney, and tissues conspicuous for pathological changes (e.g. enlarged spleen) of 430 bats were plated onto columbia (5% sheep blood), chocolate, gassner, and macconkey agar (oxoid, germany) and were incubated at 37uc (chocolate agar 5% co 2 ) for 24-48 h. specific culture media and conditions for the isolation of yersinia, salmonella and anaerobic bacteria were used if appropriate. primary identification of bacterial strains was based on colony morphology, hemolysis, gram-staining, indol production, catalase and oxidase reaction. bacterial species identification was carried out using the relevant commercial api test system (biomérieux, germany). additional conventional biochemical tests [29, 30] were applied to confirm api test results where necessary. in case of ambiguous biochemical test results, 16s rdna gene analysis was performed for final identification [23] . salmonella isolates were characterized at the national reference laboratory for the analysis and testing of zoonoses (salmonella) at the federal institute for risk assessment, berlin, germany. identification and characterization of yersinia and pasteurella species have been reported earlier [22, 23] . homogenized organ tissue of lung, liver, heart, kidney, spleen, brain and salivary gland of 210 bats were pooled for each individual and used for rna/dna extraction and further molecular analysis by generic pcr assays detecting flavi[31] , hanta[32] , corona[33] , and influenza a-viruses [34] . also, pcr assays specific for 8 previously described herpesviruses [24] from european vespertilionid bats were used. for this purpose, rna/ dna was isolated using the nucleospinh rna ii kit (macherey-nagel, germany) and the nucleospinh tissue kit (macherey-nagel), respectively, according to the manufacturer's instructions. because of limitations in sample volume, for 180 out of the 210 bats pcr assays could only be applied for 4 different bat herpesviruses. internal controls were used for all pcr assays to test for inhibition. for confirmation, all retrieved fragments of bat herpesvirus-specific pcr assays were checked for sequence identity to previously published isolates [24] . for detection of lyssavirus antigen in brain tissue the fluorescent antibody test (fat) using a polyclonal antirabies conjugate (sifin, germany) was used [35] . fat-positive brain tissues were subject of virus isolation in murine neuroblastoma cell culture (na 42/13) using the rabies tissue culture infection test (rtcit) as described elsewhere [36] . lyssaviruses isolated in cell culture were characterized using both a panel of 10 anti-nucleocapsid monoclonal antibodies (mab) [37] and partial sequencing of a fragment of the nucleoprotein gene after rna extraction using trizol (invitrogen, germany) essentially as described [18] . genomic dna was extracted from organ homogenates using the nucleospinh tissue kit (macherey-nagel) according to manufacturer's recommendations. genetic identification of the bat species was performed by amplification and sequencing of a 241 bp fragment of the cytochrome b (cytb) gene [38] using primers fm up (59-ccc chc chc aya tya arc cmg art gat a -39) and fm down (59-tcr acd ggn tgy cct ccd att cat gtt a -39). in addition, for differentiation of the 2 distinct pipistrellus species, p. pipistrellus and p. pygmaeus, a rapid multiplex pcr assay was performed as described by kaňuch et al. the bat data were categorized in regard to different explanatory numeric and factor variables, e.g. bat species, sex and age class. the variable 'age class' ranked between 1 and 4 with increasing age (i.e. neonates, juveniles, subadults, and adults) and was used as numeric variable. for endoparasitic analysis, we defined a 3 level variable 'bat size' according to the body size of a certain bat species to reduce the degrees of freedom of the full model, i.e. large species (n. noctula, eptesicus serotinus, and vespertilio murinus), medium-sized species (e. nilssonii, plecotus auritus, myotis daubentonii, m. nattereri, and p. nathusii) and small species (p. pipistrellus, and m. mystacinus). to detect effects of seasonality, 4 different activity periods were specified according to the date of sampling, i.e. hibernation period (november to march), post-hibernation period (april/may), maternity period (june to august), and swarming period (september/october). as dependent binary variable for the respective models we either classified the mortality cause being disease or not (i.e. trauma), or the presence-absence of bacterial, ecto-and endoparasitic infections. we formulated 4 different hypotheses to test for individual and species-specific differences in disease susceptibility and infection rates: (a) disease-related mortality in bats is influenced by sex, age and species-specific differences, and degree of endoparasitic infection. (b) bacterial infection in bats is influenced by sex, age and species-specific differences, occurrence of traumatic injuries and cat predation. (c) ecto-or (d) endoparasitic infection in bats is affected by age, sex and species-specific differences. seasonal effects were not analyzed because of too many missing data points. because the long-term dataset was highly biased towards sampling procedure, preservation of bat carcasses and following diagnostic investigations, we split and filtered the full data into several subsets reflecting the different analyses (table 1) . all statistical analyses were performed using the r software v. 2.13.1 (r development core team 2011, vienna, austria). we used the chi-square test for given probabilities to evaluate significant differences in the sex ratio among bats of different species. for hypotheses a and b, we used a generalized linear mixed modeling approach (binomial glmm using function lmer in library lme4) with bat species included as random effect. this variable had not been significant as fixed effect (results not shown), but from other studies we can assume that there are speciesspecific differences in susceptibility of bats to certain infectious agents and therefore included it as random effect. we further used generalized linear models (glm with logit link and binomial error structure; for datasets with bat species .10 individuals) to test for individual and species-specific differences in parasite infection rates (hypotheses c and d). we created a full model for each hypothesis (a-d) to examine multiple and interaction effects of the specified variables. to select the final model variables, we used a stepwise backward algorithm (function stepaic in library mass) based on akaike's information criterion (aic) [40] . the daic of the final model was calculated relative to a random intercept model to demonstrate the effect size of the selected variables. results of the diagnostic analyses follow the full data splitting into several subsets (see section 'statistical analysis' in material and methods; table 1 ). all sampled bats belonged to 7 different genera (i.e. pipistrellus, nyctalus, myotis, eptesicus, plecotus, vespertilio, and barbastella) and 19 european vespertilionid species (fig. 1a) . three bat species, the common pipistrelle (p. pipistrellus, n = 138), the noctule bat (n. noctula, n = 92), and the serotine bat (e. serotinus, n = 53) constituted about 60% of all bat carcasses investigated in this study, whereas p. pygmaeus, nyctalus leisleri, myotis brandtii, m. bechsteinii, m. dasycneme, plecotus austriacus and barbastella barbastellus were represented in small numbers of 1 to 4 animals. the overall sex ratio was 1.5 males to 1 female with significant species-specific differences (fig. 1b) . animals in their first year of life (neonates, juveniles, and subadults) represented one third (32.5%, n = 158) of bat samples (fig. 1c) . overall, we were able to assign a cause of death to 70% (n = 304) of bats investigated in this study. two thirds of mortality were due to trauma (n = 145) or disease (n = 144), while almost 4% of bats had died of other non-infectious causes like pulmonary edema, dehydration and hypoglycemia (table 2 ). in 30% (n = 129) no significant pathological findings could be found. among the 145 traumatized bats, additional mild (n = 42), moderate (n = 28) and severe (n = 4) inflammatory organ changes were noted in one half (50.9%) of individuals, and 23% of the bats revealed bacterial (n = 19) and/or parasitic infections (n = 15) ( table 3) . of the 144 bats considered as dying of disease, fatal bacterial (n = 54), viral (n = 5) and parasitic infections (n = 2) were observed in 42%. besides, amniotic fluid aspiration was noted in a neonate noctule bat (n. noctula), and a juvenile common pipistrelle (p. pipistrellus) was euthanized because of severe forearm bone deformation. the remaining 81 bats (56.3%) revealed moderate to severe pathological changes of unknown etiology or unconfirmed bacterial or viral cause ( table 2 ). based on the glmm analysis, significant age-and sexdependent differences (daic = 23.13) were detected between the general causes of mortality, disease and trauma ( table 4 ). the disease presence in bat samples decreased continuously with increasing age. neonates and juveniles of both sexes were significantly more affected by disease than older age classes (table 4 ; fig. 2a) . we also found a significant trend in diseaseassociated mortality between the sexes, with adult females displaying higher disease prevalence (52.5%) than males (36.4%) ( table 4 ). no significant association was observed between a certain cause of mortality (i.e. disease or trauma) and severity of endoparasitic infection (daic = 0.75, result not shown). the seasonal distribution of disease-related mortality cases (fig. 2b ) described a trimodal pattern, with peaks in spring (april), summer (june to august) and winter (december). the proportion of traumatized individuals also increased obviously during the summer months up to and including the swarming period, but was low during the rest of the year. about 90% (n = 430) of bat samples were examined bacteriologically. among these, 42 different bacterial genera with more than 53 bacterial species were identified (table s1 ). predominant bacteria isolated were enterococcus faecalis (14.7%, n = 63), hafnia alvei (11.2%, n = 48), serratia liquefaciens (10%, n = 43), and pasteurella multocida (7.7%, n = 29). in 37% (n = 157) of bats no bacterial growth was observed at all. comparative bacteriologic and histo-pathologic analysis identified 22 different bacterial species that were clearly associated with pathological lesions and/or systemic infection, found in 17% (n = 73) of bats investigated bacteriologically ( table 5) . members of the families pasteurellaceae (above all p. multocida) (41.1%, n = 30), enterobacteriaceae (various bacterial species) (28.8%, n = 21), and streptococcaceae (above all enterococcus spp.) (21.9%, n = 16) were predominant bacteria associated with disease. more than half (54.8%, n = 40) of bacterial infections were observed in bats with traumatic injuries. the glmm analysis revealed low sex-and age-dependent differences in bacterial infection (daic = 1.97, result not shown). female bats (21.9%) and adults (21.6%) showed marginally higher prevalence of bacterial disease compared to males (18.3%) and to other age classes (15.6%), respectively. however, we found a strong influence of cat predation (daic = 16) associated with bacterial infection in bats (table 4 ). testing for human-pathogenic zoonotic viruses, no examined bat sample (0/210) was positive for influenza a virus, corona-, hanta-and flaviviruses, respectively. no inhibition of the pcr assays was notified. out of 486 bats tested for rabies virus infection, 2 serotine bats (e. serotinus) were positive for lyssavirus by fat and rtcit. the viruses were identified as european bat lyssavirus type 1 (eblv-1) sublineage a, both using mabs and sequencing. applying bat herpesvirus-specific pcr assays, 63 out of 210 bats proved to be infected with 7 of the previously described 8 bat herpesviruses ( table 6 ). the highest prevalence of 65% (24/37) was observed for bat gammaherpesvirus 6 (batghv6) in common pipistrelle bats (p. pipistrellus), followed by bat gammaherpesvirus 5 (batghv5, 42.1%) in nathusius' pipistrelle bats (p. nathusii) and bat gammaherpesvirus 4 (batghv4, 33.8%) in noctule bats (n. noctula). co-infection with different bat herpesviruses were recognized in 4 n. noctula (7.4%) infected with batghv3 and batghv4, and in one n. noctula (1.5%) infected with batghv4 and batghv5. batghv5 was not only detected in its initially specific host p. nathusii, but also in 3 other bat species, i.e. n. noctula, myotis myotis and m. mystacinus. although the prevalence of batghv3 (13.0%) and batghv4 (33.8%) differed significantly within its migrating host n. noctula, no difference was observed between the sexes. two juvenile n. noctula were found to be infected with batghv4. interestingly, for the sedentary bat species p. pipistrellus being infected with batghv6, a considerably higher prevalence was observed in 22 juvenile bats (72.7%) resulting in an overall prevalence of 65% also without difference between adult male and female bats. ectoparasites (mites, fleas, and ticks) were noted in 14% (n = 62) of bats, but a potential bias in ectoparasite numbers collected from dead animals in comparison to ectoparasite abundance on live animals has to be taken in account. female bats (17.1%) were slightly more infested by ectoparasites than males (14.7%), whereas in different age classes ectoparasite prevalence was almost balanced. the glm analysis revealed significant species-specific differences in ectoparasite infestation (daic = 14.58, table 4 ). most bat species revealed low ectoparasite prevalence (range 5.3-11.8%), while almost 43% (n = 20) of n. noctula were infested with mites and/or fleas (fig. 3a) . microscopic examination of organ tissues revealed endoparasitic infection in 29% (n = 124) of investigated bats, involving different protozoan (families eimeriidae and sarcocystidae) and helminth parasites (trematodes, cestodes, and nematodes). helminthes were predominantly found in the gastro-intestinal tract of the bats, while in some animals, granulomatous organ lesions were associated with larval migration of nematode species. based on the glm analysis, clear age-and species-specific differences (daic = 24.95) were observed between infected and non-infected bats ( table 4 ). the prevalence of endoparasitic infection in bat samples increased significantly with increasing age, whereas the increase in prevalence was more rapid between juveniles and subadults (8.5%) compared to the older age classes (4.5%). marginal differences were also observed between the sexes, with female bats showing slightly higher (30.4%) endoparasite prevalence than males (24.4%). regarding species-specific differences, large bats like n. noctula, e. serotinus and v. murinus revealed higher endoparasite prevalence compared to individuals of medium-sized or small vespertilionid species (table 4 ; fig. 3b ). this study was based on a passive surveillance sampling strategy that inherently influences the composition of bats sampled for diagnostic investigations [27] and might also effect the data presented on causes of death by ecological and anthropogenic impacts of urban landscapes [41] . trauma and disease represented the most important causes of mortality in deceased bats from germany, which differ from results of previous studies [13] [14] [15] where disease-related mortality often played a subordinate role. young bats and adult females were significantly more affected by disease, indicating that sex-and age-related disease prevalence in table 3 . pathological findings and bacterial, viral and parasitic infections specified for the general causes of mortality, trauma and disease. bats are strongly correlated with the maternal season. this assumption is further supported by the distinct increase of diseaserelated mortality from june to august, which corresponds to the maternity period of central european bat species. similar seasonal prevalence patterns in bats have also been described for parasitic (e.g. [42] [43] [44] [45] ) and viral infections (e.g. [19, 46, 47] ). in contrast, the increase of trauma-associated mortality cases from july to october resembles 4 major behavioral activity patterns of european bat species (i.e. weaning, mating, pre-hibernal fat storage, and migration) [48] and could therefore predispose bats to trauma. however, both seasonal peaks also coincide with time and locations where sick, injured or dead bats are more likely to be discovered as well as with the seasonal roosting behavior of bats adapted on urban habitats [27] . the additional seasonal peaks of disease-associated mortality corresponded to the post-hibernal and the early hibernal period of temperate zone bats. currently, there is a lack of knowledge of bat immunology. it is known for other mammalian species that hibernation reduces the innate and adaptive immune response; likewise an increasing risk of infection could be assumed for hibernating bats [49] . with the start of the hibernation season, large aggregations of bats originating from various colonies might enhance the risk of spreading infectious agents similar to maternity colonies. equally, the post-hibernal increase of disease-related mortality is suggestive for reduced immunity in association with prolonged fasting during hibernation. bacterial diseases have rarely been documented in bats. pasteurella spp., here identified in 7% of bats, were the predominant bacterial pathogens reported in european bats and infection appears to be strongly correlated with cat predation [13, 14, 23, 26] . in our study, bacterial infections were confirmed in 17% of bats investigated bacteriologically. most of these bacterial isolates represented opportunistic pathogens that usually do not harm the host unless the immune system is weakened [50] or preceding injury to natural host barriers (e.g. skin abrasion). primary bacterial pathogens like samonella enterica serovar typhimurium, s. enteritidis and yersinia pseudotuberculosis [22] were identified in almost 12% of affected bats. some of the bacterial species (e.g. burkholderia sp., cedecea davisae and clostridium sordellii) are newly described in bats. nevertheless, bacteriologic analyses can markedly be influenced by post-mortem bacterial invaders, freezing and storage of bat carcasses and the inability to detect certain bacteria by routine culture methods, resulting in some bacterial species that might have escaped detection. we found a strong association between cat predation and bacterial infection in bats as almost one half of bats (44%) caught by cats were affected by bacterial disease. various bacteria can be transmitted via cat bites [51] ; hence bats attacked by cats are likely to succumb to bacterial infection even if non-fatal injuries were present. this relation has been proven for p. multocida infections in european bat species [13, 14, 23, 26] . on the other hand, bats already debilitated by disease might easier fall prey to predators like cats. consequently, bats may also act as vectors for zoonotic pathogens, as domestic cats could pass these infectious agents on to humans. such cross-species transmission events from bats to domestic animals are well documented [9, 52] . for all tested human-pathogenic zoonotic viruses no infected bat could be detected in this study except lyssaviruses. bat rabies is the only bat transmitted zoonosis in europe that is known to have resulted in human cases [53] . unlike in other mammals table 4 . result of the final model variables corresponding to 4 different analyses: (a) disease-vs. trauma-related mortality, and presence-absence of (b) bacterial, (c) ecto-and (d) endoparasitic infection. where lyssaviruses ultimately cause lethal rabies, in bats nonlethal lyssavirus infections may also lead to the development of immunity [47] . with the detection of eblv-1 we confirm that this lyssavirus circulates among e. serotinus as previous studies showed [18] . in germany, bat rabies is also caused by eblv-2 and bokeloh bat lyssavirus (bblv) [54, 55] , but while the latter was recently isolated from m. nattereri, eblv-2 is associated with m. daubentonii and m. dasycneme [56] . the apparent absence of eblv-2 and bblv in the sampled bats is likely due to the fact that lyssavirus infections have a very low incidence in bat populations [18] and that the sample size was too limited, especially concerning the relevant species. there is a high prevalence for herpesviruses in different insectivorous bat species in germany (this study, [24] ). most of the previously described bat herpesviruses have been detected in low numbers in more than one bat species [24] . here, we observed a high species-specific prevalence among herpesvirusinfected bats, indicating that a certain type of european bat herpesvirus is primarily associated with a single bat species. this is supported by batghv6 and batghv7 that were again only identified in their initial hosts p. pipistrellus and p. auritus (both sedentary), respectively, underlining the typical strong speciesspecificity of mammalian herpesviruses. however, species' range overlap and close inter-species contacts in bat roosts may result in cross-species transmission and could explain the observed overcoming of the species barrier (this study batghv5, [24] ). interspecies transmission have also been discussed for other mammalian herpesviruses, i.e. bovine and equine herpesviruses (e.g. [57, 58] ). furthermore, for rna viruses (i.e. rabies virus) phylogenetic distance between different host species and overlap in geographic range have recently been demonstrated as strong predictors of host shifts and cross-species transmission in bats [59] . some of the bat species (i.e. n. noctula, p. pipistrellus, and p. nathusii) in this study appear to be more susceptible to herpesvirus infection. in n. noctula, 3 different gammaherpesviruses (batghv3, 4, 5) with significant prevalence differences were recognized. such type-specific differences in prevalence between the phylogenetically distant viruses batghv3 (13.0%) and batghv4 (33.8%) within one bat species indicates co-evolutionary virus-regulated mechanisms. parasite infestation in wildlife often occurs without clinical effects, but severe infection can reduce host fitness either in terms of survival or reproductive success [60] . most data on infection dynamics in bats came from parasite studies focusing on individual and seasonal variations in ectoparasite prevalence (e.g. [43] [44] [45] 61] ). host density, roost preference and movement pattern seem to be important factors explaining individual and speciesspecific parasite infestation rates in bats [43] [44] [45] . in european vespertilionid species, female-biased parasite loads are most likely associated with host physiology and differences in roosting behavior [42, 44] . we also found species-specific seasonal variations in ectoparasitic infestation, with n. noctula and m. daubentonii showing higher ectoparasite prevalence in spring and autumn compared to the breeding season (data not shown), which is in accordance with zahn and rupp [43] . additional findings of our parasite analyses are distinct variations in ecto-and endoparasite prevalence in relation to bat species. bats primarily roosting in trees or nest boxes were more frequently infested with ectoparasites like n. noctula (43%) and m. daubentonii (25%) compared to other species (range 5-12%) investigated in this study. high ectoparasite loads have generally been described in bats preferring enclosed roosts like burrows and cavities [61, 62] , suggesting that structural characteristics and the microclimate of roosting habitats influence ectoparasite survival and re-infection of bat hosts. this assumption is in accordance with results of pearce and o'shea [63] who found differences in ectoparasite prevalence and intensity in eptesicus fuscus in relation to environmental factors (i.e. temperature and humidity) of different roost sites. in contrast to these results, the endoparasite prevalence in european vespertilionid bats seems to be correlated with the body size of the bat species [26] . species-specific variations in diet and prey selection could possibly effect endoparasite prevalence in insectivorous bats [64] , as larger bats feed on insects of a wider size range including hard-bodied prey [65, 66] . this assumption is supported by the clear prevalence increase in subadult and adult bats compared to low endoparasite infection rates in juveniles primarily feeding on milk. a multitude of publications is restricted to pathogen presence or absence in different chiropteran species; here we demonstrate the impact of diseases and infectious agents on bats themselves. alongside to trauma-associated mortality and undefined mortality cases, disease aspects represented one third of mortality causes in 486 investigated bats of 19 european vespertilionid species. by comparing pathology and bacteriology results, we were able to detect 22 different bacterial species that were clearly associated with disease in bats. at least 12% of all bats had died due to bacterial, viral and parasitic infections. finally, we found clear seasonal and individual variations in disease prevalence and infection rates, indicating an increased susceptibility to infectious agents in female bats and juveniles during the maternity season. our data emphasize and provide the basis for disease related studies in bat species on population level to elucidate the complexity of the ecology of infectious agents and host species likewise. table s1 bacteria isolated from bats found in germany. 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analysis of infected dog and cat bites european bat lyssavirus transmission among cats human rabies due to lyssavirus infection of bat origin first isolation of eblv-2 in germany novel lyssavirus in natterer's bat bat rabies serological survey of herpesvirus infections in wild ruminants of france and belgium new hosts for equine herpesvirus 9 host phylogeny constrains cross-species emergence of rabies virus in bats behavioral adaptations to parasites: an ethological approach relationships between roost preferences, ectoparasite density, and grooming behaviour of neotropical bats roosting habits of bats affect their parasitism by bat flies (diptera: streblidae) ectoparasites in an urban population of big brown bats (eptesicus fuscus) in colorado when parasites become prey : ecological and epidemiological significance of eating parasites the implications of food hardness for diet in bats prey consumed by eight species of insectivorous bats from southern illinois the authors would like to thank berliner artenschutz team-bat-e.v., f. key: cord-324295-9c1zxjng authors: bonilla-aldana, d. katterine; jimenez-diaz, s. daniela; arango-duque, j. sebastian; aguirre-florez, mateo; balbin-ramon, graciela j.; paniz-mondolfi, alberto; suárez, jose antonio; pachar, monica r.; perez-garcia, luis a.; delgado-noguera, lourdes a.; sierra, manuel antonio; muñoz-lara, fausto; zambrano, lysien i.; rodriguez-morales, alfonso j. title: bats in ecosystems and their wide spectrum of viral infectious threats: sars-cov-2 and other emerging viruses date: 2020-08-20 journal: int j infect dis doi: 10.1016/j.ijid.2020.08.050 sha: doc_id: 324295 cord_uid: 9c1zxjng bats have populated earth for approximately 52 million years, serving as natural reservoirs for a variety of viruses through the course of evolution. transmission of highly pathogenic viruses from bats has been suspected and linked to a spectrum of emerging infectious diseases in humans and animals worldwide. examples of such viruses include marburg, ebola, nipah, hendra, influenza a, dengue, equine encephalitis viruses, lyssaviruses, madariaga and coronaviruses, involving the now pandemic severe acute respiratory syndrome coronavirus 2 (sars-cov-2). herein, we provide a comprehensive review on the diversity, reservoirs, and geographical distribution of the main bat viruses and their potential for cross-species transmission. j o u r n a l p r e -p r o o f bats have populated earth for approximately 52 million years, serving as natural reservoirs for multiple viruses through the course of their existence 7, 8 . the evolution of their physical, physiological and behavioral characteristics has allowed them to expand to all continents except antarctica, with ecological niches located in urban or rural areas, and especially in caves, mines and some types of foliage 9 . evolutional changes have also determined their current eating patterns and their role within the ecosystem 10 . bats are the only mammals capable of flight; they have nocturnal habits during which they either feed or mate and a layer of short fur that protects them from humidity and cold temperatures 11 . bats belong to the order chiroptera (wings on the upper extremities), with approximately 1,100 species subclassified into two suborders: the megachiroptera and microchiroptera. this classification allows a better understanding of some of their behavioral patterns 8, 12, 13 . bats from the megachiroptera suborder, commonly known as megabats, account for approximately 170 species mainly located in asia, africa and oceania or pacific region ( figure 1 ); their size can vary between 40 and 150 cm with their wings spread, and they weigh 1 kg on average. megabats feed exclusively on fruit, seeds and pollen and their main habitats include caves, mines, trees and some buildings. megachiroptera bats cannot echo localize 8 . the microchiroptera or microbats include over 930 species distributed throughout the entire planet with the exception of some islands and the poles. their size ranges from 4 to 16 cm and feed mostly on flowers and fruit. they possess an echolocation system that allows hematophagous bats to search and capture small prey such as lizards, small mammals, and arthropods. their primary habitats include forests and tropical areas although they are also capable of coexisting with humans in some urban settings 8, 12, 13 . j o u r n a l p r e -p r o o f another notable feature is that microbats can travel long distances of up to 2000 km during migratory season to fulfill their nutritional needs, which is relevant to understand local and intercontinental spread of colonies and coexistence with other animals of the same species. microbats also play a role as pollinators as their feces fertilize and distribute seeds among the areas they inhabit, in addition to serving as plague controllers by feeding on insects, frogs, and rats 8, 14 . bats that coexist within the same geographical area often host common microorganisms 11, 12 . contagion rates depend on contact speed and susceptibility to infections of a specific population 12 . evolutionary processes granted bats with hollow bones to facilitate air maneuvering. this hollowness results in the absence of bone marrow and thus the inability to produce b cells necessary for an efficient immune response, making bats asymptomatic carriers for a long list of viruses 8 . additionally, several species are facultative heterotherms capable of entering profound lethargy during periods of physiological stress to compensate for energy and water deficits, favoring viral persistence 12 . continuous physical contact within bats of the same colony facilitates viral circulation, especially during breeding and migration seasons 15 . the proposed mechanisms of viral transmission between microbats is aerosol release produced by larynx vibrations that occur during echolocation in addition to close contact with other types of secretions such as fecal matter and urine 16 . then, bats carry multiple emerging and reemergin pathogens, especially viral threats ( table 1) . studies from the ecohealth alliance suggest that bats are one of the leading carriers of emerging infectious agents that can potentially affect other mammals, including humans [17] [18] [19] . although the precise reservoir of sars-cov-2 has not been established, a sensible hypothesis is that bats of the genus rhinolophus ferrumequinum could be at fault: chinese studies have reported that sars-cov-2 is very similar to coronaviruses naturally found in bats; however, these viruses are constantly evolving and mutating, making it difficult to pinpoint an exact reservoir ( figure 1 ). since there is no effective treatment or vaccine for covid-19 to date, strong regulations---including isolation, quarantine and social distancing---have been established by many countries in an effort to reduce expansion of the disease given the high person-to-person transmissibility of sars-cov-2, either directly by respiratory droplets with infective particles or indirectly by fluid-contaminated objects. j o u r n a l p r e -p r o o f a study in indonesia identified cov genes in bareback fruit bats, where partial rna-dependent rna polymerase (rdrp) sequences and regions between helicase and rdrp genes were detected and amplified in faeces and tissue samples 22 . another study conducted in zhoushan city, zhejiang province, found that out of 334 bats sampled, approximately 26% were naturally infected with coronaviruses 23 . marburg virus is an rna virus belonging to the filoviridae family (genus marburgvirus) ( further molecular testing conducted on liver, spleen and lung samples of egyptian fruit bat rousettus aegyptiacus reported the presence of marv rna ( figure 1 ). possible routes of transmission include fruit contamination and its consumption by humans or direct contact with bat's infected organs. marv can also spread from human to human through contact with bodily fluids or fomites from sick patients (table 3 ). in humans, the incubation period ranges from 3 to 9 days, and clinical presentation usually involves flulike symptoms, fever between 39 and 40 degrees celsius, conjunctivitis, cramps, cervical lymphadenopathy, and hemorrhagic manifestations. death occurs as a consequence of cardiocirculatory collapse and multiple hemorrhages in the digestive tract and lungs. a study detected a high seroprevalence of antibodies against marburg virus in fruit bats in south africa, with a 19.1% seroconversion rate in recaptured bats 24 ; another study detected marv genome in bats captured in zambia 25 ; and a posterior serosurvey identified filovirus-specific immunoglobulin g antibodies in 71 out of 748 serum samples collected from migratory fruit bats 26 . currently, there is no effective treatment for marv infection other than symptomatic support and rehydration, but some hematologic, immunologic, and pharmacologic actions are under development to improve survival rates. ebola virus also belongs to the filoviridae family and displays a negative-stranded single rna genome. first described in 1976 in the democratic republic of the congo, this notorious virus has decimated populations of gorillas, chimpanzees and humans in africa with mortality rates ranging from 25% to 90% (table 2) . evidence of infection has been reported in three different species of frugivorous bats associated with large outbreaks of ebola hemorrhagic fever in 2014-2015, which later escalated to pandemic proportions resulting in the death of 11,301 people 8, 27 . multiple studies point at bats of the genus myotis as the main reservoir for ebola virus given that these bats carry a copy of viral gene vp35 (table 3) studies in africa analyzed 4,022 blood samples from bats, detecting antibodies against ebola virus in one genus of insectivorous bats and six species of fruit bats 29 . another study conducted in sierra leone identified the complete genome of a new ebola virus, the bombali virus, in free-tailed bats resting inside human dwellings, suggesting potential human transmission 30 . in kenya, researchers also identified the bombali virus in the organs and excreta of free-tailed bats (mops condylurus) 31 . in malaysia, an outbreak in pigs and humans took place between september 1998 and april 1999, affecting 283 people and causing 109 human deaths and the slaughter of more than one million pigs (table 2) . initially, the outbreak was attributed to the japanese encephalitis virus; but later, researchers demonstrated that the causal agent was a virus that belonged to the henipavirus genus, family paramyxoviridae, closely related to the hendra virus. fruit bats (genus pteropus) are the main natural reservoir for nipah virus (niv), while pigs serve as intermediate hosts ( table 3 ). the infection is transmitted from bats to pigs and subsequently from pigs to humans ( figure 1 ). on the island of malaysia, researchers found niv in the urine and saliva of flying foxes (pteropus hypomelanus and pteropus vampyrus). initial circulation of niv likely occurred in late 1997 through contaminated food debris from migrating flying foxes on which pigs fed. fruit bat migration to cultivated orchards and pig farms was a consequence of the lack of fruit during droughts related to el niño phenomenon and wild fires in indonesia. a study conducted in 324 bats (predominantly pteropus, p. vampyrus and p. hypomelanus) from malaysia found that 6.48% had neutralizing antibodies against niv and the virus was subsequently isolated from the urine and fruits consumed by p. hypomelanus. previous human studies showed that most cases had a history of direct contact with live pigs. in humans, the disease can be fatal and is characterized by respiratory and particularly severe neurological manifestations, such as encephalitis and coma. clinical signs in animals may vary, including agitation, spasms, seizures, rapid breathing, and harsh cough. evidence of infection (virus isolation, immunohistochemistry, serology) and neurological involvement has been reported in dogs and horses. transmission studies in australia established that niv could rapidly spread through pigs via oral and parenteral inoculation. neutralizing antibodies were detectable 10-14 days after infection [32] [33] [34] [35] [36] . influenza a viruses (iav) are one of the leading causes of disease in humans, with important animal reservoirs including birds, pigs, and horses that can potentially produce new zoonotic variants (table 2) . (table 3) . notably, small yellow-shouldered bats in central america have been proposed as potential mammalian reservoirs of influenza (figure 1 ). research has shown that bats are susceptible to iav infection. a seroprevalence study identified iav h9 in 30% of fruit bats sampled in africa. bats are believed to have the ability to harbor more genetic diversity of the influenza virus than any other mammal and bird species. j o u r n a l p r e -p r o o f hendra virus, formerly known as equine morbillivirus, is a henipavirus of the paramyxoviridae family for which bats of the genus pteropus serve as main vectors (table 2) . this virus is endemic in australian flying foxes which are currently in danger of extinction and it can be detected in blood, urine, faeces, and fetal and uterine tissue. different studies affirm that hendra virus is horizontally transmitted from bat to bat, but in rare instances vertical transmission has been reported ( figure 1 ). although horses are usually accidental hosts that can contract the virus from these megabats, it has been suggested that the virus could be found in the environment, entering equines through the upper airways and oropharynx. transmission to humans occurs with close contact with infected horses, either through bodily fluids or aerosols; studies have ruled out person-to-person transmission. hendra virus was discovered after an outbreak that killed 20 equines and their trainer in queensland, australia, in 1994 37 . the virus remains viable for approximately four days in bat urine and fruit juice, but fails to survive in temperatures above 37 degrees celsius. the disease has an incubation period of 5-12 days, and its main symptoms are similar to influenza but with a mild, progressive encephalitis. since this infection is uncommon in humans, an effective antiviral has not been developed (table 3) . lyssaviruses are a wide range of pathogens that cause rabies ( table 2 ). the first case of human rabies was reported in ukraine in 1977. hematophagous bat desmodus rotundus, mainly located in latin america ( figure 2) , is considered the primary vector of this disease, which historically has resulted in large economic losses by causing the death of cattle and horses that get infected through bat bites. occasionally, those bites are also seen in human beings (figures 3-4) . lyssaviruses have also been identified in other hematophagous bats such as diphylla ecaudata and diaemus youngi and frugivorous species such as artibeus, planirostris trinitalis, diclidurus albus, hemiderma sp. and phyllostoma superciliatum (table 3) . a colombian study performed on 286 brains from bats of six families and 23 species showed that two species, artibeus lituratus and artibeus planirostris, were positive for the rabies virus 38 . another study conducted in chile reported that 9.5% of 15,000 bats captured were naturally infected 39 as a consequence of the destruction of natural habitats, closer interaction between bats and humans has grown significantly, especially in mining areas 44 . rabies transmission primarily occurs via direct bites or scratches from infected bats 38 ; secondary transmissions in humans takes place through contact with infected pets. these viruses cause acute progressive encephalitis that is inevitably fatal from the onset of clinical signs. initial symptoms are similar to influenza and evolve in a few days to severe neurological involvement that ultimately leads to death 45, 46 . currently, only preventive vaccines are available [47] [48] [49] [50] [51] . dengue virus (denv) belongs to the flaviviridae family, which is transmitted by mosquitoes, most commonly aedes aegypti ( table 2) 52 . there are four denv serotypes (denv-1, denv-2, denv-3 and denv-4) that can cause febrile syndromes in humans. dengue fever is recognized as an epidemic reemerging disease that has affected many countries in recent decades 53-57 . there is evidence that bats could naturally become infected with denv 58 . in mexico, a study evaluated 162 bats and identified denv nucleic acid and anti-denv antibody 59 . another study in costa rica evaluated 12 species of bats, reporting a cumulative seroprevalence of 21.2% (51/241) by prnt and a j o u r n a l p r e -p r o o f prevalence of 8.8% (28/318) in organs tested by rt-pcr 60 . in french guiana, denv nucleic acid was detected in the liver and sera of wild-caught bats 61 . recently in colombia, viral rna was obtained from bat tissues, and a nested-rt-pcr detected amplicons of 143 fragment of the ns5 gene that were then sequenced by the sanger method. in non-hematophagous bats such as carollia perspicillata and phyllostomus discolor captured in ayapel and san carlos (córdoba) respectively, an amplicon corresponding to ns5 was detected; these amplicons showed high similarity with denv-2 57 . yet, the clinical relevance of denv isolation from bats is unclear, as well as the implications in transmission to humans. similarly occurs with other arboviruses, such as madariaga and the equine encephalitis viruses. so far, the have been confirmed as infected hosts, probably reservoirs, but not a direct source for human infections (figure 1 ). the equine encephalitis group involves rna viruses belonging to the togaviridae family, of the alphavirus (table 3 ). these findings suggest that fruit bats from the caribbean region in colombia could be involved in the enzootic cycle of eev 62 (figure 1 ). the madariaga virus is a strain of the eastern equine encephalitis virus. rats and bats presumably serve as the main vectors given the reported seropositivy of brown short-tailed bats (carollia castanea), lanza bats (phyllostomus discolor) and seba's short-tailed bats (carollia perspicillata) 65 . several factors increase the interaction between humans and bats. in most cases, this is due to the intrusion of humans into virgin territories inhabited by bats, fueled by the search of economical resources. this j o u r n a l p r e -p r o o f phenomenon forces bats to adapt to new settings occupied by men, such as buildings, tombs, mines and bridges. additional illegal trading of bats for human consumption and traditional medicine in asia, coupled with poor sanitation and hygiene practices, has facilitated the emergence of zoonotic diseases, some with pandemic potential. financial openness and globalization involve close connections between countries and across continents and can serve as a mean of transportation that accelerates the spread of these emerging diseases. emerging diseases are a global matter of concern, especially during the last decades, and now even more with the occurrence of the 2020 pandemic of covid-19. sars-cov-2 and other emerging pathogens are significantly present in bats. recognizing bats as potential reservoirs or transmission sources for several pathogens that can be a threat to human beings is of upmost importance for global health since many of these conditions may cause severe damage and even led to death, in some cases in a significant proportion of individuals. however, we should not neglect the responsibility of the active role of humans in the invasion of natural habitats and illegal trafficking of bats. the one health approach on this [67] [68] [69] , became critical, the balance of human, animal and environmental health is of utmost importance in the assessment of emerging diseases. we consider that conducting new investigations centered around the role of bats and their ecosystems in the transmission of emerging diseases should be a priority to global health. close contact with infected animals, body fluids such as blood, organs, urine, faeces and aerosols generated during defecation, consuming food contaminated with these viruses, direct bite or scratches from these mammals, or even eating it with poor cooking. work in forests, agriculture and fishing covid-19: zoonotic aspects the next big threat to global health? coronavirus infections reported by promed villamil 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from sars, mers and recent advances to combat this pandemic virus none. key: cord-329617-gzivtsho authors: lee, albert k.; kulcsar, kirsten a.; elliott, oliver; khiabanian, hossein; nagle, elyse r.; jones, megan e.b.; amman, brian r.; sanchez-lockhart, mariano; towner, jonathan s.; palacios, gustavo; rabadan, raul title: de novo transcriptome reconstruction and annotation of the egyptian rousette bat date: 2015-12-07 journal: bmc genomics doi: 10.1186/s12864-015-2124-x sha: doc_id: 329617 cord_uid: gzivtsho background: the egyptian rousette bat (rousettus aegyptiacus), a common fruit bat species found throughout africa and the middle east, was recently identified as a natural reservoir host of marburg virus. with ebola virus, marburg virus is a member of the family filoviridae that causes severe hemorrhagic fever disease in humans and nonhuman primates, but results in little to no pathological consequences in bats. understanding host-pathogen interactions within reservoir host species and how it differs from hosts that experience severe disease is an important aspect of evaluating viral pathogenesis and developing novel therapeutics and methods of prevention. results: progress in studying bat reservoir host responses to virus infection is hampered by the lack of host-specific reagents required for immunological studies. in order to establish a basis for the design of reagents, we sequenced, assembled, and annotated the r. aegyptiacus transcriptome. we performed de novo transcriptome assembly using deep rna sequencing data from 11 distinct tissues from one male and one female bat. we observed high similarity between this transcriptome and those available from other bat species. gene expression analysis demonstrated clustering of expression profiles by tissue, where we also identified enrichment of tissue-specific gene ontology terms. in addition, we identified and experimentally validated the expression of novel coding transcripts that may be specific to this species. conclusion: we comprehensively characterized the r. aegyptiacus transcriptome de novo. this transcriptome will be an important resource for understanding bat immunology, physiology, disease pathogenesis, and virus transmission. electronic supplementary material: the online version of this article (doi:10.1186/s12864-015-2124-x) contains supplementary material, which is available to authorized users. and pteropus whereas yangochiroptera includes the family myotidae and genus myotis [3] . unlike most mammals, bats can fly and this ability enabled their wide geographical range and increased metabolism [2] . interestingly, bats have recently come to the forefront of zoonotic disease research with vast number of pathogens identified in a wide variety of bat species [2] . upwards of 85 different viruses, primarily rna viruses, have been detected and/or isolated from bats [2, 4] . amongst these are emerging viruses that cause lethal disease in humans and nonhuman primates including nipah virus [5, 6] , hendra virus [7] , severe acute respiratory syndrome (sars)-like coronavirus [8] , middle east respiratory syndrome coronavirus (mers-cov) [9] , marburg virus (marv) [10] [11] [12] [13] , and ebola virus (ebov) [14] [15] [16] . despite the severe virulence of these viruses in humans, infected bats are often asymptomatic [13, [17] [18] [19] [20] [21] [22] . nipah virus and hendra virus interactions with their natural reservoir hosts, pteropus vampyrus and pteropus alecto, respectively, are well characterized. experimental infections of bats with high doses of henipaviruses have shown virus replication and shedding with little to no disease [20] [21] [22] . remarkably, the only viruses known to have induced any observable pathology in bats are rabies virus and australian bat lyssavirus [2, 23] . understanding mechanisms of disease and differential responses to infection in asymptomatic reservoir host species compared to species that exhibit severe pathology will help inform the development of novel therapeutics and disease prevention approaches. rousettus aegyptiacus, commonly known as the egyptian rousette bat, has been identified as a natural reservoir host for marv through ecological, epidemiological, and experimental studies [10, 12, 13, 18, 19, 24] . furthermore, it has been speculated this bat could host ebola virus [12, [25] [26] [27] , although recent experimental infection studies have shown ebola virus does not replicate well in r. aegeyptiacus [28] . the majority of human outbreaks due to marv have been associated with caves inhabited by r. aegyptiacus. furthermore, epidemiological surveillance of the r. aegyptiacus colony located in the python cave in uganda revealed a biannual spike in marburg virus prevalence. this pattern correlated strongly with spillover transmission events in humans [24] . initial studies in captive bats evaluated clinical signs, virus dissemination, and virus shedding patterns during experimental infection with a marv isolate derived from wild bats [13] . consistent with a natural reservoir host, the bats showed little to no evidence of disease even though the virus disseminated throughout their body and was actively shed [13] . these results were confirmed when bats were infected with marv angola, a strain isolated from a lethal human case [18] . in the absence of genetic and transcriptomic information for r. aegyptiacus and with limited available reagents, studying this reservoir host animal model has been challenging. the rapid expansion in genomic knowledge for different bat species has facilitated comparative studies that rely on the identification of genes and gene families, and has established a framework for developing necessary reagents. full genome annotations for pteropus vampyrus (2.63x, [29] ), myotis lucifugus (6.6x, [29] ) pteropus alecto (110x, [30] ), myotis davidii (110x, [30] ), and myotis brandtii (77.8x, [31] ) are now available. additionally, transcriptomic annotations for pteropus alecto [32] and artibeus jamaicensis [33] have been published. in particular, the complementary genome and transcriptome annotations for p. alecto has aided studies on henipavirus infections in its reservoir host [30, 32] . the host transcriptional response to different viruses was also recently assessed in a kidney cell line derived from p. vampyrus utilizing the previously annotated genome [34] . in this manuscript, we report the transcriptomic annotation of r. aegyptiacus from a de novo assembly of rna sequencing data from 11 tissues isolated from a male and a female bat. we identified 24,118 canonical coding transcripts whose expression profiles were consistent with the corresponding tissues of origin. in addition, we identified and validated novel coding transcripts that do not have any homology with the known sequences. furthermore, we evaluated the annotation for immune-related genes and assessed the presence and expression of genes associated with a variety of immune functions. we employed a de novo assembly approach to generate a comprehensive transcriptome without relying on a genome reference. first, we generated 20 rna-seq libraries consisting of 11 tissue types (table 1 , fig. 1a ) each collected from one male and one female r. aegyptiacus bat, which yielded approximately 2.1 billion reads. we then assembled the high quality reads using trinity [35] (fig. 1b) . this process generated 14,796,219 contigs. the assembly had high continuity and coverage with a median number of 718,807 contigs and median n50 of 1,540 [36] , which leverages the homology of known sequences of related species. we assigned gene symbols to contigs when this information was available. this process clustered the contigs into isoform groups (fig. 1c) . we compared our assembly to the transcriptomes of three related bat species --m. davidii, p. alecto, and m. brandtii. using blast, we recovered 90. tissue-specific transcriptome assemblies contained different numbers of contigs, due to their different levels of expression and sequencing depth. without a common ground for comparison, it was difficult to perform downstream comparative analyses such as differential gene expression analysis; therefore, we combined contigs from all tissues into one unified, nonredundant reference transcriptome (fig. 1d) . to this end, we iteratively merged the assemblies two at a time, similar to the approach employed in [37] (fig. 1d) . we obtained 4,746,293 nonredundant contigs. among the nonredundant contigs, 974,765 (20.54 %) of the sequences were annotated by bat sequences, 860,578 (18.13 %) by primate sequences, and 104,796 (2.2 %) by sequences in nt database (fig. 2a) . the nonredundant contig set had slightly lower sensitivity, though it still remained high; 86.60 % of m. davidii, 85.95 % of m. brandtii, and 95.30 % of p. alecto transcripts were recovered. the resulting annotated contigs were assigned gene names and combined using the longest annotated orf as the transcript. this resulted in an annotation for r. aegyptiacus that contained a total of 24,118 genes. to determine the efficiency of using the msa pipeline, we determined that 84 % (20,207 genes) of the contigs were annotated using the bat database and 16 % (3,911 genes) were subsequently annotated using the primate database. these data show that the msa pipeline, which utilizes known transcripts from related species only, is a sensitive and efficient method for de novo transcriptome annotation. we evaluated biological validity of the reconstructed transcriptome by analyzing global expression patterns across the different tissues. if the transcriptome assembly and annotations were accurate, the expression profiles of a given tissue should cluster with those of the same tissue origin and segregate from those of different origins [36, 38] . a gene can result in more than one transcript isoform; therefore, to capture the highest amount of information, for each gene, we focused on the transcript with the longest open reading frame (orf) (fig. 2a) . after normalizing the expression values, we performed multidimensional scaling (mds) to determine the relationships between the gene expression patterns in different tissues. as expected, mds showed a clear separation of the samples according to the tissue of origin (fig. 3a) and explains 74 % of the variance in the data. to examine the evolutionary relationship among tissues, we performed hierarchical clustering of the gene expression profiles (fig. 3b ). the brain, which has a different developmental pathway compared to the other organs, was classified as an outgroup. the spleen, lymph node, and bone marrow are all organs of the immune system and, as expected, clustered near each other. the peripheral blood contains some of the same cell types as the immune organs, thus, clustered near these tissues. lastly, the gonads and kidney, which develop from the intermediate mesoderm, were grouped as neighbors in the tree. these results suggest that our transcriptome captured sufficient heterogeneity of gene expression to distinguish individual tissues while preserving their developmental relationships. we further assessed biological validity of our transcriptome assembly through gene ontology (go) analysis of tissue-specific expression profiles. we compared expression profile of each tissue with the average expression in the whole dataset, and identified the top 200 most differentially expressed genes based on a generalized linear modeling framework. using this list, we examined the enriched go biological process (bp) terms. figure 4 shows the top 10 go bp terms from the bone marrow, spleen, lymph nodes, and peripheral blood mononuclear cells (pbmcs). (for other tissues, see additional file 1). terms enriched for each tissue are consistent with their expected physiological functions. r. aegyptiacus is a natural reservoir host for marv, allowing for virus replication and dissemination with little to no pathological consequences [13, [17] [18] [19] [20] [21] [22] . one important aspect of reservoir host biology is how their immune response compares to that of animal species that experiences severe disease, such as humans. therefore, we examined the transcriptome for the presence of immune-related genes. we associated the r. aegyptiacus gene set with go terms based on the human-specific gene ontology annotation. this resulted in 14,781 genes that mapped to 14,817 go terms. we used categorizer [39] and applied the immune class goslim terms to identify immune-related genes from this set. similar to previous studies in p. alecto and a. jamaicensis, we found that out of 14,817 go terms, approximately 2.75 % were associated with immune response [32, 33] . amongst the most represented go terms were cytokine production, lymphocyte activation, t cell activation, regulation of apoptosis, and regulation of lymphocyte activation (fig. 5) . we next searched for specific genes related to various aspects of the immune response in other mammals, primarily mice and humans. we first evaluated the annotation of the transcriptome for the presence of anti-viral genes. a multitude of pattern recognition receptors were identified including toll-like receptors (tlrs) 1-9, rig-i, mda5, and lgp2 along with the important scaffold and signaling molecules myd88 and mavs. a variety of antiviral molecules were also found, including mx1 and mx2, pkr, sting, irf3, irf5, irf7, members of the ifit and ifitm families, and isg15. we also looked for the presence of type i, ii, and iii interferons (ifn). we were able to identify ifngamma, ifngamma2, and ifnalpha. transcripts corresponding to the ifn receptor subunits ifnar1 and ifnar2 were also identified. ifnalpha and ifnbeta have been previously characterized by cloning from stimulated cells [40] . we, however, did not find any contigs corresponding to ifnb. to eliminate the possibility of an impaired assembly, we aligned the processed rna-seq reads to the ifnb sequence from p. alecto [41] (additional file 2 and additional file 3). we detected only 2 reads from r. aegyptiacus,which did not provide sufficient coverage to construct the transcript. these data suggest that ifnb expression in healthy tissues of r. aegyptiacus is low, consistent with other mammals in which ifnb is primarily expressed after exposure to a stimulus. we also searched the transcriptome for genes associated with innate immune cells. we found the transcripts for the cd14 and cd11c genes, which are commonly used for phenotyping macrophages and dendritic cells, as well as transcripts for the cd80 and cd86 genes, which are useful for evaluating the activation status of these cells. genes associated with natural killer (nk) cells, however, were less evident. we were able to identify transcripts of co-receptor gene cd56, but not cd16. transcripts of genes encoding for molecules in the killer cell lectin-like receptor (klr) family, including nkg2a and nkg2d, were also not found. in other bat transcriptomes, such as p. alecto and a. jamaicensis, coverage of nk cellrelated genes was more sparse than that of other mammals [32, 33] . a similar observation was made in the genome of m. davidii [30] . the absence of nk cell-related genes in the r. aegyptiacus transcriptome further strengthens the theory that bats might contain a different nk cell receptor repertoire than other species. next, we examined the repertoire of genes associated with adaptive the immune response. we identified a variety of transcripts associated with t cell identification, activation, inhibition, and differentiation including cd3 , cd4, cd8a, cd25, cd69, ccr7, pd-1, ctla4, gata3, foxp3, and tbet. interestingly, we were able to identify transcripts for the tcrα and tcrβ chains, but were unable to find transcripts for the tcrδ and tcrγ chains. the transcriptome annotation for p. alecto included these genes, but they were present at low levels [32] . this supports the notion that αβ t cells are the predominant t cell subset in bats. we also looked at genes associated with b cells and were able to find transcripts for cd19, cd20, cd27, as well as transcripts that were similar to the frequency shown is the percent of immune class go slim terms associated with that particular pathway out of all the go terms that were identified the immunoglobulin heavy chains a, e, g, and m and the immunoglobulin light chains κ and λ. future analysis of the r. aegyptiacus genome is required to fully evaluate the immunoglobulin gene repertoire. finally, we studied the cytokine and chemokine repertoire, important for shaping both innate and adaptive immune responses. we found a variety of transcripts corresponding to a wide array of both pro-inflammatory and anti-inflammatory cytokines. these included il-2, il-4, il-5, il-6, il-12a, il-12b, il-17a, il-23, il-10, tgfβ, tnf, ifnγ , il-1β, ccl2, ccl5, and cxcl10. altogether, the reference transcriptome generated for r. aegyptiacus provides an excellent foundation for investigating reservoir host immunology in bats. there were 2,806,154 unannotated contigs from the nonredundant contig set (fig. 2b) . of those, 71.6 % (2,008,503 contigs) did not have an orf suggesting the majority of these contigs may be noncoding transcripts. to determine if the unannotated contigs were real or artifacts from the assembly, we used blast to align this set of contigs to the p. alecto genome and found that 96 % (2,706,432 contigs) were aligned. to evaluate the possibility of an incomplete or impaired assembly, we grouped the aligned contigs into a total of 1,012,664 clusters based on the presence of overlapping sequences. this reduction suggests that multiple isoform expression patterns between different tissues may have affected our assembly or that our short read assembly may have been incomplete. nonetheless, the number of unannotated contigs that aligned to the p. alecto genome suggests that these contigs, either coding or noncoding, may be novel transcripts shared within the order pteropodinae. future studies evaluating the conservation and possible functions of these sequences are essential to determine the importance of these genetic elements. to validate novel contigs in r. aegyptiacus that appeared to be coding we utilized pcr. primers were designed to produce amplicons for eight highly expressed, unannotated contigs that contained orfs longer than 400 bp. using rna isolated from the spleen, we were able to produce amplicons of the expected size from at least one bat ( fig. 6 and additional file 4). the sequences of these amplicons were found to fig. 6 unannotated, novel transcripts from r. aegyptiacus were validated of by rt-pcr. rna from the spleen of both bats was reverse transcribed to make cdna. the cdna was amplified using primers specific for one of 8 novel transcripts that were unannotated in the assembly, but contained a complete orf larger than 400 nucleotides. the expected product sizes were: transcript 1, 457 bp; transcript 2, 450 bp; transcript 3, 419 bp; transcript 4, 548 bp; transcript 5, 469 bp, transcript 6, 277 bp; transcript 7, 507 bp; and transcript 8, 301 bp match the expected sequence from the assembled orf of the unannotated contig. these contigs also showed high sequence similarity to the p. alecto genome. in particular, six of the 8 validated transcripts showed sequence similarity higher than 75 % at a query coverage greater than 64 %. the other two validated transcripts had a query coverage of 23 with 78.36 % identity (transcript 1 in fig. 6 ) and a query coverage of 7 with 91.27 % identity (transcript 2 in fig. 6 ) (additional file 5); therefore, we hypothesize that these transcripts might be specific to r. aegyptiacus. further investigation is needed to fully understand the characteristics and biological functions associated with the proteins these contigs encode. in this paper, we presented the comprehensively annotated of transcriptome of r. aegyptiacus and assessed its quality and biological validity. this transcriptome will be an important resource to study bat immunology. in particular, it will facilitate the process of investigating differences in host responses between asymptomatic reservoir host species and species that exhibit severe pathology. it will also pave the way for the development of novel therapeutics and prevention approaches against emerging zoonotic virus outbreaks. tissues and blood were collected from one male and one female adult r. aegyptiacus bats that were bred and housed at the colony established at the center for disease control and prevention, atlanta, ga, usa (amman et al. 2015 [13] ). approximately 100 mg of the following tissues were collected and homogenized in 1 ml of trizol ls (invitrogen, carlsbad, ca): liver (bat id:bat7, bat17), lung (bat05, bat15), heart (bat03, bat13), kidney (bat04, bat14), brain (bat02, bat12), axillary lymph nodes (bilateral, pooled) (bat06, bat16), spleen (bat10, bat19), bone marrow (bat01, bat11), and gonad (bat08, bat20). pbmcs (bat08, bat18) were isolated from the blood and stored in trizol ls as well. rna was extracted using the purelink rna mini kit (invitrogen, carlsbad, ca). cdna was synthesized using the truseq stranded total rna sample prep kit (illumina, san deigo, ca) according to the manufacturer's protocol. the libraries were evaluated for quality using the agilent 2100 bioanalyzer (agilent, santa clara, ca). after quantification by real-time pcr with the kapa qpcr kit (kapa biosystems, woburn, ma), libraries were diluted to 10 nm. cluster amplification was performed on the illumina cbot and libraries were sequenced on the illumina hiseq 2500. eight of the female bat libraries were single-end, while the remaining tissues from the female bat and all tissues from the male bat were paired-end. all of the libraries sequenced were 125 bp in length. the average library depth was 66 m reads (minimum 16 m and maximum 98 m). all experimental procedures were conducted with approval from the centers for disease control and prevention (cdc, atlanta, ga, usa) institutional animal care and use committee, and in strict accordance with the guide for the care and use of laboratory animals (committee for the update of the guide for the care and use of laboratory animals 2011). the cdc is an association for assessment and accreditation of laboratory animal care international fully accredited research facility. no human patient-derived clinical materials were used in these studies. we first examined the quality of the reads using fastqc v0.11.3 [42] . we also preprocessed the reads to remove the adapter sequence using cutadapt v1.5 [43] . we removed "agatcggaagagcacacgtctgaactcc agtcac" from the forward strand and "agatcggaa-gagcgtcgtgtagggaaagagtgt-agatctcgg-tggtcgccgtatcatt" from the reverse strand. we performed strand-specific de novo transcriptome assembly using trinity r20140413p1 [35] with the parameters: "-normalize_reads" and "-ss_lib_type fr", along with its default parameters for all of our samples. for annotation of contigs and clustering them into a gene model, we used multiple species annotation pipeline, an nucleotide-based annotation approach that is more efficient and faster than blastx [36] . to make a blast [44] database for bats, we started with the complete "nucleotide collection" (nt) database. we exported all accession numbers of the bat sequences at ncbi and made a subset database from nt using "blastdb_aliastool -db nt -dbtype nucl -gilistbats.sequence.gi.txt -title bats -out bats". using the same type of query, we also created a database for primates including humans due to their extraordinarily well-annotated transcriptomes, which will maximize the power of our annotation pipeline. we then used blast to iteratively align the contigs to the bat db, the primate db, and finally nt using a subtractive approach: what did not align to the bat db was aligned to the primate db, and what did not align to the primate db was aligned to nt. to assess the coverage of our transcriptome, we downloaded the m. davidii, p. alecto, and m. brandtii transcriptomes from ncbi eukaryotic genomes annotations [41] . we generated a blast index out of union of all contigs from our samples, and aligned the three bat contigs to our blast databases. we chose the alignment with 70 % of sequence identity with maximum evalue of 1e-4. to generate a nonredundant set of contigs, we iteratively merged individual assemblies using the the methods similar to the [37] employed to merge the kmers. using cd-hit-est v4.6 [45] with sequence identity threshold of 0.99, we merged the first two pairs of contig sets (of sample i and sample i + 1) upto the final sample n. after each iteration, we merged the resulting merged contig sets using a similar approach until only one contig set remained. for the expression profiling, we generated a reference transcriptome consisting of transcripts each representing a gene model according to the following method: we first used transdecoder (r20140413p1) [46] to find the orf of all transcripts. then, based on the msa pipeline, we chose a transcript with gene symbols and the longest orf in each gene cluster to capture the most information for downstream expression analysis. we did not consider the contigs mapped to nt database in this manuscript because obtaining feature files for all sequences as required by the msa pipeline was computationally impractical, and a majority of the gene symbols (24, 118) are captured in the bat and primate databases. after a canonical transcript set was obtained, we used this as a transcriptome reference for expression analysis. we mapped the preprocessed reads to this reference using rsem v1.2.19 [47] and obtained a gene-to-count matrix. we removed the transcripts with expression variance equal to zero or with low expression (count <= 10). for mds plot, we used the spearman correlaton as a distance measure and "cmdscale" from the "stats" package in r [48] . to explore the biological processes in each gene expression profile, we employed a oneto-all sample comparison using the edger generalized linear model framework [49, 50] . for each tissue, we compared individual gene expression within the tissue versus the average expression of all other tissues. with each tissue having differently ranked gene lists, we then selected top 200 genes and ran gene ontology analysis using topgo [51] with human-specific gene ontology annotation [52] . we used blast [44] to align unannotated contigs to the genome of p. alecto with the evalue of 1e-4 and query coverage of 40 %. to cluster the aligned contigs into groups, we used bedtools [53] setting the distance threshold parameter at 0. for transcripts that did not align with any similarity to bat, primate, or nt blast databases, we applied a series of filters to select for the coding transcripts to be validated. we used the following criteria: an orf that was complete with both a start and stop codon, an orf that was at least 400 bp in size, and a transcript that was expressed (a read count > 0). we further selected for the novel transcripts with usuable primers using primer-blast [54] . using these criteria, the number of novel transcripts was narrowed down to a total of 8. the primers and expected amplicon size are listed in additional file 4. for validation, rna was extracted from the spleen tissue of both the male and female bats using trizol ls (invitrogen, carlsbad, ca). cdna was synthesized from 2.5 μg of rna using the superscript iii first-strand synthesis supermix (invitrogen, carlsbad, ca). amplicons for each of the primer sets were generated using phusion hotstart flex dna polymerase (new england biolabs, ipswitch, ma) and run on a 1.5 % agarose gel for visualization. the correct size amplicon was gel extracted, quantified, and sanger sequenced on the applied biosystems 3730×1 dna analyzer. bats and zoonotic viruses: can we confidently link bats with emerging deadly viruses? memórias do instituto oswaldo cruz microbat paraphyly and the convergent evolution of a key innovation in old world rhinolophoid microbats bats: important reservoir hosts of emerging viruses serologic evidence for the presence in pteropus bats of a paramyxovirus related to equine morbillivirus identifying hendra virus diversity in pteropid bats nipah virus: a recently emergent deadly paramyxovirus bats are natural reservoirs of sars-like coronaviruses middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group studies of reservoir hosts for marburg virus marburg virus infection detected in a common african bat isolation of genetically diverse 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captive juvenile black flying fox seasonal pulses of marburg virus circulation in juvenile rousettus aegyptiacus bats coincide with periods of increased risk of human infection ebola haemorrhagic fever. the lancet large serological survey showing cocirculation of ebola and marburg viruses in gabonese bat populations, and a high seroprevalence of both viruses in rousettus aegyptiacus ebola virus antibodies in fruit bats experimental inoculation of egyptian rousette bats (rousettus aegyptiacus) with viruses of the ebolavirus and marburgvirus genera comparative analysis of bat genomes provides insight into the evolution of flight and immunity genome analysis reveals insights into physiology and longevity of the brandt's bat myotis brandtii the immune gene repertoire of an important viral reservoir, the australian black flying fox transcriptome sequencing and annotation for the jamaican fruit bat (artibeus jamaicensis) transcriptome profiling of the virus-induced innate immune response in pteropus vampyrus and its attenuation by nipah virus interferon antagonist functions full-length transcriptome assembly from rna-seq data without a reference genome transcriptome reconstruction and annotation of cynomolgus and african green monkey de novo assembly and analysis of rna-seq data the evolution of gene expression levels in mammalian organs categorizer: a web-based program to batch analyze gene ontology classification categories induction and sequencing of rousette bat interferon α and β genes cutadapt removes adapter sequences from high-throughput sequencing reads gapped blast and psi-blast: a new generation of protein database search programs cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences rsem: accurate transcript quantification from rna-seq data with or without a reference genome r: a language and environment for statistical computing edger: a bioconductor package for differential expression analysis of digital gene expression data differential expression analysis of multifactor rna-seq experiments with respect to biological variation topgo: topgo: enrichment analysis for gene ontology gene ontology: tool for the unification of biology bedtools: a flexible suite of utilities for comparing genomic features submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution we thank thomas kepler, stephanie d'souza, adam hume, elke muhlberger, jenna kelly for comments and discussion on the project. we also thank ahhyun kim for the illustration of a bat in fig 1a. this work was funded by the defense threat reduction agency (dtra) grant hdtra1-14-1-0016 and the training program in computational biology 5t32gm082797-07. the findings and conclusions in this report are those of the authors and do not necessarily represent the views of the centers for disease control and prevention or the u.s. army. the authors declare that they have no competing interests. rr and gp designed the study. akl assembled the sequence data and constructed the assembly. akl and oe annotated the assembly. akl and hk examined the assembled data and assessed the quality. kak and ern performed and interpreted the molecular studies. akl and kak analyzed and interpreted the expression studies. mebj, bra and jst provided biological material for analysis. akl and kak wrote the manuscript. all authors read, edited, and approved the final manuscript. key: cord-334027-xhfmio7k authors: fagre, anna c.; kading, rebekah c. title: can bats serve as reservoirs for arboviruses? date: 2019-03-03 journal: viruses doi: 10.3390/v11030215 sha: doc_id: 334027 cord_uid: xhfmio7k bats are known to harbor and transmit many emerging and re-emerging viruses, many of which are extremely pathogenic in humans but do not cause overt pathology in their bat reservoir hosts: henipaviruses (nipah and hendra), filoviruses (ebola and marburg), and coronaviruses (sars-cov and mers-cov). direct transmission cycles are often implicated in these outbreaks, with virus shed in bat feces, urine, and saliva. an additional mode of virus transmission between bats and humans requiring further exploration is the spread of disease via arthropod vectors. despite the shared ecological niches that bats fill with many hematophagous arthropods (e.g., mosquitoes, ticks, biting midges, etc.) known to play a role in the transmission of medically important arboviruses, knowledge surrounding the potential for bats to act as reservoirs for arboviruses is limited. to this end, a comprehensive literature review was undertaken examining the current understanding and potential for bats to act as reservoirs for viruses transmitted by blood-feeding arthropods. serosurveillance and viral isolation from either free-ranging or captive bats are described in relation to four arboviral groups (bunyavirales, flaviviridae, reoviridae, togaviridae). further, ecological associations between bats and hematophagous viral vectors are characterized (e.g., bat bloodmeals in mosquitoes, ingestion of mosquitoes by bats, etc). lastly, knowledge gaps related to hematophagous ectoparasites (bat bugs and bed bugs (cimicidae) and bat flies (nycteribiidae and streblidae)), in addition to future directions for characterization of bat-vector-virus relationships are described. bats and the viruses they harbor have been of interest to the scientific community due to the unique association with some high consequence human pathogens in the absence of overt pathology. virologic and serologic reports in the literature demonstrate the exposure of bats worldwide to arboviruses (arthropod-borne viruses) of medical and veterinary importance [1] . however, the epidemiological significance of these observations is unclear as to whether or not bats are contributing to the circulation of arboviruses. historically, a zoonotic virus reservoir has been considered a vertebrate species which develops a persistent infection in the absence of pathology or loss of function, while maintaining the ability to shed the virus (e.g., urine, feces, saliva) [2] [3] [4] . haydon et al. extended this definition of a reservoir to include epidemiologically-connected populations or environments in which the pathogen can be permanently maintained and from which infection is transmitted to the defined target population. the significance of the relative pathogenicity of the infectious agent to the purported reservoir host has been debated [5] . in the case of bats as a reservoir species, rigorous field and experimental evidence now exist to solidify the role of the egyptian rousette bat (rousettus aegyptiacus) as the reservoir for marburg virus [6] [7] [8] . considering arboviruses, additional criteria must be met in order to consider a particular vertebrate species a reservoir. reviewed by kuno et al., these criteria include the periodic isolation of the infectious agent from the vertebrate species in the absence of seasonal vector activity, and the coincidence of transmission with vector activity [9] . further, the vertebrate reservoir must also develop viremia sufficient to allow the hematophagous arthropod to acquire an infectious bloodmeal [10] in order for vector-borne transmission to occur. bats have long been suspected as reservoirs for arboviruses [11] , but experimental data that would support a role of bats as reservoir hosts for certain arboviruses remain difficult to collect. here we synthesize what information is currently known regarding the exposure history and permissiveness of bats to arbovirus infections, and identify knowledge gaps regarding their designation as arbovirus reservoirs. the order bunyavirales is divided into eight families, four of which pose threats to public health and veterinary medicine-families nairoviridae, peribunyaviridae, phenuiviridae, and hantaviridae [12] . while bats have been demonstrated to host hantaviruses, these viruses do not rely on an arthropod in their transmission cycle and thus will not be discussed [13] . viruses in order bunyavirales that have been experimentally examined in bats or described in field studies are descried in table 1 . members of the genus orthonairovirus of medical and veterinary significance include crimean congo hemorrhagic fever virus (cchfv) and nairobi sheep disease virus (nsdv) [12] . cchfv is transmitted by ticks in genera rhipicephalus and hyalomma [14] . while neither live virus nor nucleic acid of cchfv has been detected from bats, serologic evidence suggests past infection of populations of bats across a diverse geographic range [15] [16] [17] . further, bats are often parasitized by both soft and hard ticks, which occupy a diverse range of ecological niches in endemic countries [18] [19] [20] . a 2016 seroprevalance study by müller and colleagues examining 16 african bat species (n = 1, 135) found that the prevalence of antibodies against cchfv was much higher in cave-dwelling bats (3.6%-42.9%, depending on species) than foliage-living bats (0.6%-7.1%) [15] . they also screened 1,067 serum samples by rt-pcr, but all were negative for cchfv nucleic acid [15] . experimental studies to assess the ability of bats to support replication of cchfv have not been published. members of the genus orthobunyavirus include many viruses of importance to human and veterinary medicine, including bunyamwera virus, california encephalitis virus, jamestown canyon virus, kaeng khoi virus, and la crosse encephalitis virus [12] , but limited evidence exists regarding the exposure or potential involvement of bats in the circulation of viruses in this family. kaeng khoi virus (kkv) has been isolated from cimicid bugs (order: hemiptera, family: cimicidae) (stricticimex parvus and cimex insuetus) and from suckling wrinkle-lipped bats (tadarida plicata ) in caves in thailand, but was not isolated from soft ticks tested in the same area (ornithodorus hermsi) [21] . additionally, kkv has been implicated in the case of several mine workers who reported illness and were discovered to have seroconverted [22] , demonstrating spillover of this virus to humans in association with the cave environment, and suggesting that cimicids may play a role in vectoring virus between bat and human hosts. to date, no experimental data have been generated to address this hypothesis. spence and colleagues attempted to experimentally infect jamaican fruit bats (artibeus jamaicensis) via intramuscular injection with nepuyo virus (group c serogroup), yet no infectious virus was subsequently recovered from the bats [23] . this is interesting considering two strains of nepuyo virus were isolated from jamaican fruit bats (artibeus jamaicensis) and great fruit-eating bats (artibeus literatus) in honduras, and protective sera were found in jamaican fruit bats in trinidad. [24, 25] . bats of undetermined species were involved in a large serosurvey in brazil that examined antibodies in wildlife against the gamboa serogroup orthobunyaviruses, though none were found to be positive [26] . seven and twelve species of trinidadian bats were examined for antibodies by hi against caraparu (group c serogroup) and maguari (bunyamwera serogroup) viruses, respectively, and were all found to be negative [25] . viruses in the genus phlebovirus (family phenuiviridae) of importance to human and animal health include rift valley fever virus (rvfv) and severe fever with thrombocytopenia syndrome virus (sftsv) [12] . bats of the species miniopterus schreibersii (n = 1) and eptesicus capensis (n = 2) were experimentally infected with rvfv and the m. schreibersii bat's urine and liver tested positive for antigen [27] . a recent study by balkema-buschmann and colleagues experimentally infected egyptian rousette bats (rousettus aegyptiacus) with vaccine strain mp-12 and recovered infectious virus from spleen and liver of some animals [28] . oelofsen & van der ryst (1999) examined 350 samples from 150 field-caught bats in africa, yet none were positive for antigen by use of elisa [27] . kading et al (2018) detected neutralizing antibodies against rvfv in egyptian rousette bats and little epauletted fruit bats (epomophorus labiatus) in uganda, a country that has recently experienced human cases of rvfv [29, 30] . whether or not bats serve as a reservoir of rvfv during interepidemic periods remains to be determined. bangui virus (bgiv) is an unclassified bunyavirus and was isolated from an unidentified bat in the central african republic (car) [31] . mojuí dos campos virus (mdcv) is another ungrouped bunyavirus isolated from an unidentified bat species [32, 33] . the family flaviviridae includes many high-consequence emerging arboviruses, including zika virus (zikav), yellow fever virus (yfv), and dengue virus (denv). flaviviruses associated with bats that do not appear to utilize an arthropod vector ("no-known vector flaviviruses") have been reviewed elsewhere [56] . viruses in family flaviviridae that have been experimentally examined in bats or described in field studies are descried in table 2 . interestingly, despite denv isolations from artibeus spp. bats in the wild, experimental infections of great fruit-eating bats (a. intermedius) with denv-2 and jamaican fruit bats with denv serotypes 1 and 4 resulted in low levels of viremia, low rates of seroconversion, and lack of detection of viral rna in the organs via rt-pcr, indicating that bats may not act as a suitable reservoir host [57] [58] [59] . experimental infection of the indian flying fox (pteropus giganteus) resulted in no viremia or clinical signs, but intracerebral inoculation of little brown bats (myotis lucifugus) resulted in irritability, paralysis, and death [60, 61] . denv nucleic acid and anti-denv antibodies have been detected in mexican bats on the gulf and pacific coast, and nucleic acid has been detected in the liver and/or sera of wild-caught bats in french guiana [62, 63] . anti-denv antibodies have been detected in multiple bat species in uganda [29] . however, a survey in central and southern mexico analyzing 240 individuals representing 19 bat species by rt-pcr resulted in no detection of viral nucleic acid [64] . a 2017 study by vicente-santos and colleagues examined 12 bat species from costa rica and found a cumulative seroprevalence of 21.2% (51/241) by prnt and a prevalence of 8.8% (28/318) in organs tested by rt-pcr [65] . no infectious virus was isolated and viral loads were considered too low for the bats to function as amplifying hosts. rather, vicente-santos and colleagues surmised a spillover event from humans to bats, with bats functioning as a dead-end host [65] . the serum of jamaican fruit bats (artibeus jamaicensis) and great fruit-eating bats (a. literatus) from grenada (n = 50) were also tested for antibodies against denv 1, 2, 3, and 4, and none were seropositive [66] . while field evidence supports the exposure of bats to denv in multiple geographic areas, experimental infections conducted to date are consistent in that bats are not likely to support denv replication and circulation to levels high enough to infect blood-feeding mosquitoes. multiple studies conducted experimental infections of insectivorous bats with japanese encephalitis virus (jbev) and found that bats were susceptible to infection with this virus. three species of bats (big brown bats (eptesicus fuscus), little brown bats (myotis lucifigus) and eastern pipistrelles (pipistrellus subflavus)) were inoculated with jbev in the laboratory and maintained infection while held under simulated hibernation conditions. bats infected prior to hibernation were viremic upon arousing from hibernation, with circulating virus detectable as long as 112 days after the initial infection [67] . big brown bats also demonstrated recurrent viremia in the absence of clinical signs in a subsequent study [68] . importantly, researchers demonstrated a mosquito-bat-mosquito transmission cycle and postulated this may be an overwintering mechanism for jbev since mosquitoes did successfully transmit jbev to bats at low temperatures [67] . eastern pipistrelles also became infected with jbev after consumption of infected mosquitoes, demonstrating that bats could be infected orally as well as through a mosquito bite [67] . no demonstrable pathologic effects noted during infection of three bat species [big brown bats (eptesicus fuscus), little brown bats (myotis lucifigus) and mexican free-tailed bats (tadarida brasiliensie mexicana) with various strains of jbev or st. louis encephalitis virus (slev) [69] . no pathology nor viremia was appreciated when pipistrelles (pipistrellus abramus) were infected with jbev [70] . while experimental data demonstrated that some bat species can sustain jbev infections and support mosquito-borne transmission of this virus, the epidemiological significance of these observations in the field remains unclear. jbev has been isolated from wild-caught bats in taiwan (miniopterus fuliginosus and hipposideros armiger terasensis [32, 71] , china (rousettus leschenaultia and murina aurata [72, 73] , japan (miniopterus schreibersi fuliginosus and rhinolophus cornutus cornutus [74] . antibodies against jbev have been detected in wild-caught bats in indonesia (unspecified species) [75] , china (rousettus leschenaultia, cynopterus sphinx, taphozous melanopogon, miniopterus schreibersii, pipistrellus abramus, rhinolophus macrotis and miniopterus fuliginosus [76, 77] , australia (pteropus scapulatus and pteropus gouldi) [78] , taiwan (unspecified species) [79] , india (pteropus giganteus, hipposideros pomona, hipposideros speoris, hipposideros bicolor, hipposideros cineraceus, megaderma lyra, cynopterus sphynx, and rhinolophus rouxi) [80] [81] [82] , and japan (miniopterus schreibersi fuliginosus, rhinolophus ferrum equinum nippon, vespertilio superans, myotis macrodactylus, rhinolophus cornutus cornutus, pipistrellus abramus, myotis mystacinus, plecotus auritus sacrimontis, and murina leucogaster hilgendorfi) [83] . multiple isolations of jbev from locations where the virus is endemic, in addition to the fact that genetic characterization of isolates has supported their similarity to strains identified from human and mosquito isolates, support the role of bats in ongoing circulation of jbev [84] . another medically-important flavivirus with both field-obtained information and in vivo experimental inoculation is slev. a 1983 study by herbold and colleagues demonstrated that 9% of wild-caught eptesicus fuscus and myotis lucifugus (n = 390) in ohio possessed neutralizing antibodies to slev [85] . other serosurveillance efforts in north america and grenada focused on detection of slev in free-ranging bat populations have resulted in largely negative findings [66, 86] . following experimental infection, viremia and transplacental transmission (albeit infrequent) was appreciated in mexican free-tailed bats (tadarida brasiliensis) [69, 87] . the viremia in these bats reached 4 log units, likely too low a titer to facilitate transmission to a blood-feeding mosquito [10] . upon inoculation, little brown bats (myotis lucifugus) appear to be resistant or only slightly susceptible to slev [69] . herbold and colleagues (1983) demonstrated that inoculation of eptesicus fuscus with slev results in infection and virus was maintained throughout hibernation (70 days), with viremia developing four days following arousal (105 days post-infection) [85] . low levels of viremia upon experimental inoculation in conjunction with low seroprevalence data indicate this virus likely does not utilize bats as a reservoir host in nature. to date, biosurveillance testing of bats in central america for wnv have turned up negative results. grenadian artibeus jamaicensis and artibeus literatus (n = 50) bats were negative for wnv neutralizing antibodies by prnt [66] , 14 trinidadian bat species (n = 384) were negative by elisa for wnv antibodies [88] , and 16 mexican bat species (n = 146) tested for wnv rna by rt-pcr were negative [89] . in north america, results have been negative or indicative of low levels of circulation in bat populations tested. tissues from 312 field-collected bats representing seven species in illinois tested by rt-pcr were all negative for wnv, and the same study reported one big brown bat (eptesicus fuscus) with neutralizing antibodies (n = 97) [90] . a field survey taking place in new jersey and new york reported one big brown bat and one northern long-eared bat (myotis septentrionalis) with neutralizing antibodies to wnv (n = 83) [86] . in another field study, only two of 149 free-tailed bats (tadarida brasiliensis) possessed neutralizing antibodies against wnv [91] . in uganda, kading et al. (2018) detected neutralizing antibodies to wnv in 2/8 african straw-colored flying foxes (eidolon helvum), and 3/44 little epauletted fruit bats (epomophorus labiatus) [29] . simpson and o'sullivan (1968) demonstrated experimental inoculation of african straw-colored flying foxes did not result in viremia though two of three bats developed neutralizing antibody. in the same study, two of three egyptian rousette bats were infected but only trace viremia was detected and seroconversion was not appreciated [43] . experimental inoculation of free-tailed bats (tadarida brasiliensis) did not result in viremia, and infection of big brown bats resulted in low titers (10-180 pfu/ml) [91] , not capable of supporting transmission to feeding mosquitoes [10] . attempts to experimentally infect vampire bats (desmodus rotundus) and black mastiff bats (molossus rufus) by mosquito bite (aedes aegypti) were unsuccessful [11] . experimental inoculation of multiple bat species (eumops perotis, carollia perspicillata, phyllostomus hastatus and bats in the genus mollosus) were similarly unsuccessful [92] . still, kading et al. detected a significant neutralizing antibody titer against yfv in one egyptian rousette bat in uganda in 2012, indicating bats are exposed to this virus in nature [29] . uganda has experienced outbreaks of yfv in recent years [93] . while multiple african bat species (eidolon helvum, rousettus aegyptiacus, and rousettus angolensis) demonstrated viremia following inoculation with zikav, mops condylurus did not become viremic, although did contain low virus titers in the kidney [43, 44] . experimentally-infected little brown bats were susceptible to the zikav by the intraperitoneal, intradermal, intracerebral and intrarectal routes of exposure, but not susceptible intranasally [94] . however, it is unclear how zikav could circulate in bat populations. kading et al. (2018) did not detect neutralizing antibodies to zikav among 292 ugandan bats screened. flavivirus infections of bats with an emphasis on the potential role in zika virus ecology has been reviewed elsewhere [95] . flavivirus serology has been historically challenging due to the cross-reactivity of viral epitopes to circulating antibodies [96] . therefore, the results of serologic surveillance studies must be interpreted cautiously [29, 97] . further, multiple methods exist for antibody detection (e.g., hi, prnt, elisa), and the biological significance of neutralizing vs. non-neutralizing antibodies must be taken into account. in 2010, the serum of 140 mexican bats from three species (glossophaga soricina, artibeus jamaicensis, and artibeus literatus) was assayed by prnt using wnv, slev, and denv 1-4, and 26 were positive for flavivirus-specific antibodies (19%). none of the titers exceeded 80, and all samples were also negative when tested for flavivirus nucleic acid by rt-pcr [97] . in a 2015 serosurvey, eight bats (2.6%) displayed non-specific hemagglutination-inhibition (hi) results indicating cross-reactivity or antibodies against an undetermined flavivirus [88] . kading and colleagues performed a serosurveillance study in ugandan bats and identified 13.6% (85/626) had non-specific flavivirus antibodies by plaque reduction neutralization assay (chaerephon pumilus, hipposideros ruber, mops condylurus, nycteris macrotus, eidolon helvum, epomophorus minor, and rousettus aegyptiacus) [29] . still, results generally supported the widespread exposure of bats in uganda to flaviviruses [29] . in 2018, sotomayor-bonilla and colleagues reported that liver and spleen samples from 12 mexican bat species tested negative using pan-flavivirus ns5 primers [98] . a recent study in brazil suggested a lack of arboviral circulation in bat populations, as 103 individuals from 9 species were tested for molecular and serologic evidence of alphavirus and flavivirus infection and all were negative [99] . results of experimental infection of egyptian rousette bats with wnv and of angolan free-tailed bats (mops condylurus) with ntaya virus resulted in very low levels of viremia, while infection of african straw-colored fruit bats with ntaya virus resulted in neither pathology nor detectable viremia [43] . few studies have examined the presence of viruses in genus coltivirus in bat populations, and to date, a single isolation has been made (table 3 ) [127] . a 1984 study by chastel and colleagues failed to detect antibodies to eyach virus (reoviridae, colorado tick fever group) in the serum of two field-caught bats [128] . to date, five orbiviruses have been isolated from wild-caught bats and serologic evidence exists for exposure of australian and south american bats to orbiviruses (table 3) . while no evidence of human exposure exists for these bat-associated orbiviruses, bukakata (bukv) and fomede (fomv) appear to be strains of the chobar gorge species [129] . cgv was isolated from ornithodoros species ticks in nepal, and serum from nearby humans and ruminants possessed anti-cgv antibodies, indicating past exposure [130] . further investigation is warranted to determine the true vector-host association of these viruses and their zoonotic potential. viruses in family reoviridae that have been experimentally examined in bats or described in field studies are descried in table 3 . viruses in genus alphavirus (family togaviridae) that have been experimentally examined in bats or described in field studies are descried in table 4 . enzootic circulation of chikv is understood to occur among non-human primates and forest-dwelling mosquitoes [142] , but other vertebrates including rodents, bats, reptiles and amphibians have been shown to support chikv replication [143, 144] . the range of peak viremia developed by big brown bats was relatively low, but within the range of infectivity to blood feeding mosquitoes [10, 143] . when indian flying foxes (pteropus giganteus) and big brown bats were experimentally infected with chikv, bats developed viremia but no clinical signs of disease, indicating they could play a role in the natural transmission of this virus [60, 143] . experimental infection of african straw-colored flying foxes did not result in viremia or seroconversion to chikv, supporting a separate study which reported lack of viremia in experimentally infected egyptian rousette bats and african straw-colored flying foxes [43, 44] . in 2015, the serum of 42 wild-caught grenadian bats (genus artibeus) were subjected to prnt and 15 (36%) were found to possess neutralizing antibody to chikv [66] . chikv has been circulating in central and south america since 2013 [145] . whether or not bats are contributing to the natural circulation of chikv in endemic areas or areas of introduction remains to be determined. serological evidence exists supporting exposure of bats to encephalitic alphaviruses in the field, and experimental data demonstrate the susceptibility of bats to infection with alphaviruses including veev. four mexican bat species were examined for molecular evidence of infection with venezuelan equine encephalitis virus (veev), western equine encephalitis virus (weev), and eastern equine encephalitis virus (eeev). no individual bats were positive for weev or eeev, but 3% (5/150) representing all four species were positive for veev [89] . field-caught jamaican fruit bats (artibeus jamaicensis) and great fruit-eating bats (artibeus literatus) were negative by prnt for eeev and weev antibodies, but 2.6% (1/38) had neutralizing antibodies to veev [66] . similarly, the serum of 384 bats representing 14 species was subjected to elisa, and 2.9% (11/384) contained veev-specific antibodies. elisa and hi assays for eeev and weev antibodies, respectively, were all negative [88] . four species of wild-caught bats from the northeastern united states were tested for neutralizing antibody against eeev and weev. samples were negative for antibodies against weev, but 1.3% of the 128 bats tested did possess eeev-neutralizing antibody [47] . bats of the genera myotis and eptesicus were experimentally infected with eeev, and developed viremia but failed to develop neutralizing antibodies. infection of big brown bats by bite of culiseta melanura and aedes aegypti mosquitoes was successful. more non-hibernating than hibernating bats were seropositive for eeev [146] . in a recent serosurveillance study, 2/432 bats were seropositive by plaque reduction neutralization assay to babanki virus (bbkv) and 9/626 egyptian rousette bats had non-specific alphavirus antibodies (table 4 ) [29] . multiple isolates of bbkv were obtained from cx. perfuscus mosquitoes collected from multiple locations in uganda during this same sampling period as when bats were sampled [147] . mosquito blood meals from bats comprised 7.5% of the total blood meals identified from the species cx. perfuscus [148] . it is unclear whether bats contribute to the transmission cycle of bbkv or are merely incidentally exposed through mosquito bites ten pteropus poliocephalus bats were experimentally infected with ross river virus, and five developed low (log 10 2.2 tcid 50 /100 µl) detectable and short-lived (2 days) viremia. still, 2% of the colonized mosquitoes (aedes vigilax) that fed on the bats between days 1-4 post-infection became infected [148] . kading et al. (2014) modeled that for viremias <log 10 2.0/ml, the probability of a mosquito becoming infected was around 0.1 or less given the low circulating titer and the volumetric constraints of a small mosquito blood meal; therefore, at least 10 mosquitoes would need to feed on an animal with a low viremia in order for one mosquito to ingest virus [10] . in the case of rrv, published data demonstrate that infection of mosquitoes fed on rrv-viremic bats is still possible despite a viremia and low titer [149] . therefore, if bat and mosquito populations are in high numbers, 50% of bats develop a detectable viremia, and 2% of mosquitoes become infected, mosquito-borne transmission could take place even though experimentally-determined efficiencies are low [149] . antibodies against mayaro virus were detected by hi in 37 trinidadian bat species tested [25] . a number of hematophagous arthropods feed on bats, including bat flies (genera nycteribiidae and streblidae), bat bugs and bed bugs (family cimicidae), and ticks (families argasidae and ixodidae) [18, 20, 21, [167] [168] [169] [170] [171] [172] . viruses of medical and veterinary significance have also been isolated from these arthropods [21, [173] [174] [175] . however, the contribution that these ectoparasites play in the circulation of medically important viruses among bats and other hosts is unclear and necessitates further investigation. kading and schountz (2016) reviewed instances in the literature where mosquito blood meals have been identified as originating from bats [95] . information on primary mosquito vectors feeding on bats is very limited. tiawsirisup et al. 2012 collected mosquitoes from five genera inside a bat cave in thailand to investigate sylvatic circulation of jbev. while these collections included arbovirus vectors cx. quinquefasciatus and cx. tritaenhiorhynchus, the only blood-fed mosquitoes collected from the cave were cx. quinquefasciatus, at least 20 of which had fed on leschnault's rousette (rousettus leschenaulti) bats [176] . culiseta morsitans theobald mosquitoes (vector of eastern equine encephalitis virus) were found to have fed on eastern pipistrelle bats (pipistrellus subflavus), but these blood meals comprised only 1% of the total blood meals identified from this mosquito species [177] . no information was found on any blood meals from bats being detected in aedes (stegomyia) species, but it is unclear how much investigation has been done in this area. sixteen of 20 field-collected ae. funereus mosquitoes (vector of rrv) had fed on pteropus alecto bats [149] . in africa, mosquito species in which bat blood meals have been identified and are known to be associated with a number of medically-important arboviruses include: coquillettidia (cq.) fuscopennata (theobald) (yfv, sindbis, chikungunya viruses), culex (cx.) perfuscus edwards (wnv, oropouche, sindbis, wesselsbron, usutu, babanki viruses), cx. (cx.) neavei theobald (wnv, babanki, spondweni, sindbis, koutango viruses), and cx. (cx.) decens group (wnv, chikungunya, babanki viruses [148, 178] . while mosquitoes in the subgenus cx. (cx.) are recognized as primary vectors of wnv, sindbis, babanki, and usutu viruses, only for babanki virus has additional field data been collected so far that support a potential role for bats in virus circulation (discussed above) [29] . bat flies (order: diptera; families: nycteribiidae, streblidae) are highly host-specific obligate hematophagus ectoparasites of bats [179, 180] . a novel fusogenic orthoreovirus, tentatively named mahlapitsi virus (mahlv), was discovered in bat flies (eucampsipoda africana) associated with egyptian rousette bats [181] . a novel orthobunyavirus, tentatively named wolkberg virus (wbv), was isolated from the same species of bat flies in a similar geographic region [182] . dengue virus rna was detected by rt-pcr in bat flies (strebla wiedemanni, trichobius parasiticus) associated with common vampire bats (desmodus rotundus) [100] . bartonella spp. bacteria have also been found infecting both fruit bats and the bat flies parasitizing them in madagascar, providing a documented example of a pathogen-vector-bat association involving bat flies [183] . more research in this area is needed to elucidate the role of bat ectoparasites in spreading pathogens among individual bats in a roost. in addition to parasitizing humans by infesting their dwellings, bed bugs and other arthropods in family cimicidae are found in close association with bat populations [170] . some cimicids are known to play a role in alphavirus transmission. cliff swallow bugs (oeciacus vicarious) transmit fort morgan virus and buggy creek virus [173, 174, 184, 185] among passerine birds. tonate virus, which is closely related to veev, and also causes fatal encephalitis in man, has also been isolated from swallow bugs in the 1970's [186] . as previously described, bat bugs (cimex insuetus and strcticimex parvus) host the orthobunyavirus kkv which likely causes disease in humans [21, 22] , but this also remains a largely understudied research area. ticks and mites are known to parasitize bats and they both fill similar ecological niches. many studies have identified viruses in bats that cluster phylogenetically with other arboviruses transmitted by ticks. other isolations are indicative of bats playing a role in viruses isolated from either hard-bodied or soft-bodied ticks. for example, estero real virus was first isolated from ticks (ornithodoros tadaridae) obtained from a palm tree colonized by cuban bats [187] . due to the close ecological association between egyptian rousette bats (r. aegyptiacus) and soft-bodied ticks in caves, the argasid tick ornithodoros faini was screened for marburgvirus rna but all pools were found to be negative [19] . to date, evidence does not support the role of arthropods in the transmission of filoviruses in nature. bats are also known to host mite species in families macronyssidae, dermanyssidae, and spinturnicidae, though the significance of these arthropod's role in virus transmission between bats and other vertebrates is largely unknown [188] [189] [190] . species in macronyssidae and dermanyssidae have also been demonstrated to efficiently transmit arboviruses (further reviewed in [191] ). a recent study explored viruses of potential arthropod-origin in the fruit bat hypsignathus monstrosus and identified five viruses: one dicistrovirus (family dicistroviridae, order picornavirales), one nodavirus (family nodaviridae), and two tombus-like viruses. they also detected a related tombus-like virus isolated from fig wasps and primitive crane flies co-habitating with bats, suggesting the ability of arthropods to host "bat-associated" viruses [192] . tombus viruses typically infect plants, but tombus-like viruses infect a wide range of hosts, including arthropods and marine invertebrates [193] [194] [195] . ingestion of arthropods by fruit bats has been documented [196, 197] , and could be a potential mechanism explaining the association between viromes of arthropods and frugivorous bat species. with regard to experimental infections, one must take into account the method of inoculation (e.g., intracerebral, subcutaneous, intraperitoneal, intramuscular), the inoculating titer, as well as whether or not vector bites were associated with the infection. in analyzing serologic results, one must take into account whether the assay is detecting neutralizing or non-neutralizing antibodies, and consider the dynamics of antibody decay which may impact detection sensitivity [198] . arthropod saliva has been shown to potentiate arbovirus infection, affecting not only the magnitude of peak viremia but also the viral loads in certain organs [199] . the inoculating viral strain used is also important, as it should align geographically with where the species under study resides to truly characterize their potential as a reservoir species. komar et al. (2003) presented detailed methods for estimating the reservoir competence of vertebrate species for mosquito-borne viruses [200] , including calculating a reservoir competence index based on the susceptibility to infection, mean daily infectiousness, and the duration of infectiousness. this reservoir competence index is interpreted as the relative number of infectious vectors that derive from a particular vertebrate species [200] , and would be easily transferrable to evaluating the reservoir competence of bats. further, bats are very long-lived (up to 25 years) compared to other small mammals, potentially increasing the chances of exposure and subsequent transmission to other vertebrates or invertebrate vectors [1] . cell culture also offers a viable starting point for initial assessments of viral replication in a particular host species, particularly when in vivo studies are not feasible. to date, many cell lines derived from the organs of bats have been established [201] . importantly, the intent of this review is not to vilify bats, but to analyze the existing literature and its support for bats as arboviral reservoirs, in addition to identifying areas for future study. bats provide vital ecosystem services, such as arthropod suppression, pollination, and seed dispersal [202] . past depopulation efforts in response to viral outbreaks resulted in increased viral spillover, and are not a viable means of disease control and have even resulted in higher virus infection rates in the bat species when colonies repopulated from neighboring roosts [203] . further work should encompass directed field surveillance complemented by both in vitro and in vivo approaches, with field surveillance efforts focused on optimization of non-lethal and non-invasive sampling [204] [205] [206] [207] . when possible, longitudinal sampling of identifiable animals through use of marking or tagging allows for monitoring of seroconversion and viremia persistence, providing a chance to better characterize transmission periodicity and seasonal changes in viremia profiles. to truly elucidate the role of bats as reservoirs for arboviruses, field surveillance studies documenting natural infection and transmission dynamics among vector and vertebrate species must be supplemented with experimental infections to characterize viremia profiles and infectiousness to vectors, virus-induced pathology, and immune kinetics following infection. with bats, these tasks are not trivial, and carry significant challenges in both the field and the lab. these challenges are evidenced by the few arboviruses for which there are substantial field and laboratory data involving the infection of bats. while many studies have presented serologic data indicative of past exposure of free-ranging bats to arboviruses of medical and veterinary importance, these studies should be followed up with laboratory assessments of reservoir host competence to shed light on the true epidemiological significance of the field data. further, the detection of viral nucleic acid in free-ranging bats does not necessarily implicate the species as an arboviral reservoir. rather, recovery and isolation of live virus at biologically relevant titers, and demonstrating the persistence of the pathogen in nature among connected populations of a potential reservoir species is more definitive. unfortunately, few established bat colonies exist for use in vivo viral pathogenesis studies, limiting the achievability of these studies. to fulfill the vertebrate reservoir host paradigm, in vivo infections must support findings in the field. the isolation of marburg virus from egyptian rousette bats in uganda in addition to experimental infections demonstrating viremia and shedding in the absence of overt pathology support the role of this bat species as the reservoir for marburg virus [6, 7, 208] . for arboviruses, the combination of field work and in vivo pathogenesis studies are lacking. still, several examples have emerged from this review that point to bats as potentially competent amplifying hosts for arboviruses. kaeng khoi virus was isolated from both bats and cimicid bugs in thailand, and was also shown to be the causative agent behind sick mine workers [21] . some bat species did develop a viremia at a level that would be infectious to mosquitoes when inoculated experimentally with chikv, and bats have been found exposed to chikv during field investigations [66, 143] . experimental evidence supporting transmission of rrv from pteropus poliocephalus to recipient mosquitoes in addition to identification of rrv-seropositive bats trapped in australia and indonesia highlights the need for further investigation into the role of bats in the ecology of this disease [75, 149, 157] . a mosquito-bat-mosquito transmission cycle was established in the lab for jbev [67] . serological evidence from the field documenting bat exposure to jbev, the sustained viremia in bats during hibernation, and demonstration of mosquito transmission from and to bats in the laboratory collectively demonstrate the capability of bats to function as reservoir hosts for this virus. circumstantial evidence from field-sampled mosquitoes and bats supports the cycling of babanki virus among bats and mosquitoes in uganda [29] , but experimental data are still lacking. for rvfv, bats evaluated in the lab have supported virus replication, and multiple populations of bats in the field have been found with neutralizing antibodies or natural infection with rvfv [1, 28, 29] . additional studies are warranted on these and other viruses for which additional field or experimental data are needed to support the role of bats as a possible reservoir. author contributions: a.c.f. and r.c.k. designed the study. a.c.f. and r.c.k. analyzed the data. a.c.f. and r.c.k. prepared the manuscript. funding: this research received no external funding. the authors declare no conflict of interest. bats: 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fever virus from bats in the republic of guinea the mammals of guinea as reservoirs and carriers of arboviruses the role of wild mammals in the maintenance of rift valley fever virus ecological and epidemiological studies of toscana virus istituto super. sanit ecology of viruses isolated from sand flies in italy and characterized of a new phlebovirus (arabia virus) a review of flaviviruses that have no known arthropod vector experimental infection of artibeus intermedius bats with serotype-2 dengue virus experimental inoculation of artibeus jamaicensis bats with dengue virus serotypes 1 or 4 showed no evidence of sustained replication dengue virus in bats from southeastern mexico attempts at experimental infection of the indian fruit-bat pteropus giganteus with chikungunya and dengue 2 viruses and antibody survey of bat sera for some viruses studies of dengue fever virus in the cave bat (myotus lucifugus) dengue virus in mexican bats dengue infection in neotropical forest mammals. vector-borne zoonotic dis no evidence of dengue virus infections in several species of bats captured in central and southern mexico neotropical bats that co-habit with humans function as dead-end hosts for dengue virus serological evidence of widespread exposure of grenada fruit bats to chikungunya virus japanese b encephalitis in bats during simulated hibernation the bat as a reservoir of viruses in nature studies of arthropod-borne virus infections in chiroptera. i. susceptibility of insectivorous species to experimental infection with japanese b and st. louis encephalitis viruses susceptibility of bats to japanese b encephalitis virus japanese encephalitis virus surveillance in taiwan. ii. isolations from mosquitoes and bats in taipei area 1969-1970 japanese encephalitis viruses from bats in yunnan isolation of japanese encephalitis virus from brain tissues of bat in yunnan province studies of arthropod-borne virus infections in chiroptera. vi. isolation of japanese b encephalitis virus from naturally infected bats a survey for arboviral antibodies in sera of humans and animals in lombok, republic of indonesia detection of japanese encephalitis virus antibodies in bats in southern china detection of serum antibodies against japanese encephalitis virus in bats in hainan and guangdong provinces of relationship between some coastal fauna and arthropod-borne fevers of north queensland encephalitis on taiwan. v. animal and bird serology serological evidence of arbovirus activity in birds and small mammals in japanese encephalitis affected areas of bankura district japanese encephalitis studies in vellore, south india. part iv. search for virological and serological evidence of infection in animals other than man antibodies against japanese encephalitis virus in insectivorous bats from karnataka studies of arthropod-borne virus infections in chiroptera. vii. serologic evidence of natural japanese b encephalitis virus infection in bats comparison of genomic and amino acid sequences of eight japanese encephalitis virus isolates from bats louis encephalitis virus in ohio bats nile virus antibodies in bats from new jersey and new york studies of arthropod-borne virus infections in chiroptera. ii. experiments with japanese b and st. louis encephalitis viruses in the gravid bat. evidence of transplacental tranmission seroepidemiology of selected alphaviruses and flaviviruses in bats in trinidad potential sympatric vectors and mammalian hosts of venezuelan equine encephalitis virus in southern mexico a survey for west nile virus in bats from illinois experimental and natural infection of north american bats with west nile virus the reaction of certain species of bats to yellow fever virus1 outbreak of yellow fever in central and southwestern uganda effect of zika virus and bwamba virus in the cave bat (myotus lucifugus) flavivirus infections of bats: potential role in zika virus ecology antigenic relationships between flaviviruses as determined by cross-neutralization tests with polyclonal antisera serologic evidence of flavivirus infection in bats in the yucatan peninsula of mexico survey of mosquito-borne flaviviruses in the cuitzmala river basin, mexico: do they circulate in rodents and bats? trop lack of serological and molecular evidence of arbovirus infections in bats from brazil detection of dengue virus in bat flies (diptera: streblidae) of common vampire bats, desmodus rotundus detection of dengue virus neutralizing antibodies in bats from costa rica and ecuador relationships between the flying fox (genus pteropus) and arthropod-borne fevers of north queensland studies of arthropod-borne virus infections in chiroptera. iv. the immune response of the big brown bat (eptesicus f. fuscus) maintained at various environmental temperatures to experimental japanese b encephalitis virus infection studies of arthropod-borne virus infections in chiroptera. iii. influence of environmental temperature on experimental infection with japanese b and st. louis encephalitis viruses transmission of japanese encephalitis virus from the black flying fox, pteropus alecto, to culex annulirostris mosquitoes, despite the absence of detectable viremia susceptibility of cynopterus sphinx (frugivorus bat) & suncus murinus (house shrew) to japanese encephalitis virus isolation of kyasanur forest disease virus from the insectivorous bat, rhinolophus rouxi and from ornithodoros ticks kyasanur forest disease virus infection in the frugivorous bat, cynopterus sphinx demonstration of antibodies against the virus of kyasanur forest disease (kfd) in the frugivorous bat rousettus leschenaulti, near poona studies of arboviruses in western australia studies of the epidemiology of arthropod-borne virus infections at mitchell river mission serologic survey of neotropical bats in guatemala for virus antibodies isolation of st. louis encephalitis virus from bats (tadarida b. mexicana) in texas studies of potential avian, arthropod and mammalian hosts of mosquito-borne arboviruses in the minnesota area lipotropism in pathogenesis of encephalitis viruses in insectivorous bats studies of arthropod-borne virus infections in chiroptera persistence of tick-borne encephalitis virus in hibernating bats the demonstration of antibodies against the virus of the tick-borne encephalitis in certain bats experimental infection of bats species myotis myotis borkhausen with the czechoslovak tick-borne encephalitis virus prevalence of antibodies to arboviruses in various animals in israel studies on yellow fever in ethiopia. 3. serological and virological studies of the woodland fauna isolation of the west nile virus from the frugivorous bat, rousettus leschenaulti twelve isolations of zika virus from aedes (stegomyia) africanus (theobald) taken in and above a uganda forest part ii-investigations of vertebrate hosts and arthropod vectors study by electron microscopy of erythrocytes from cave bats (myotus lucifugus) infected intraperitoneally with yellow fever virus (17d strain). texas rep a novel coltivirus-related virus isolated from free-tailed bats from côte d'ivoire is able to infect human cells in vitro isolation of eyach virus (reoviridae, colorado tick fever group) from ixodes ricinus and i. ventalloi ticks in france discovery and characterization of bukakata orbivirus (reoviridae:orbivirus), a novel virus from a ugandan bat arbovirus catalog: chobar gorge peruvian horse sickness virus and yunnan orbivirus, isolated from vertebrates and mosquitoes in peru and australia isolation of the fomédé virus from chiroptera, nycteris nana, in the republic of guinea arbovirus circulation in the republic of guinea viruses isolated from bats in japan identification of novel viruses using virushunter-an automated data analysis pipeline ife: a new african orbivirus isolated from eidolon helvum bats captured in nigeria, cameroon and the central african republic electron microscopy of japanaut and tilligerry viruses: two proposed members of the orbivirus group matucare virus, a new agent recovered from ornithodoros (alectorobius) boliviensis. am chikungunya virus and its mosquito vectors. vector borne zoonotic dis viremia in north american mammals and birds after experimental infection with chikungunya viruses reptiles and amphibians as potential reservoir hosts of chikungunya virus chikungunya virus infections among travelers-united states eastern equine encephalomyelitis virus in experimentally infected bats arboviruses isolated from mosquitoes collected in uganda identification of host blood from engorged mosquitoes collected in western uganda using cytochrome oxidase i gene sequences investigation of gray-headed flying foxes (pteropus poliocephalus) (megachiroptera: pteropodidae) and mosquitoes in the ecology of ross river virus in australia isolation of chikungunya virus from bat in yunnan province and serological investigations les arbovirus au senegal. ii. isolement de plusieurs souches technique pour l'étude de i'infestation naturelle des chauves-souris par les arbovirus experimental viraemia with japanese encephalitis virus in certain domestic and peridomestic vertebrates infections in wildlife with the viruses of vesicular stomatitis and eastern equine encephalomyelitis a search for encephalitis and rabies in bats of eastern massachusetts virus research in amazonia annually recurrent epidemic polyarthritis and ross river virus activity in a coastal area of new south wales. ii. mosquitoes, viruses, and wildlife studies of the sindbis virus. part i. susceptibility of the cave bat by various routes of exposure isolation of sindbis virus from bat organs eco-epidemiology of the venezuelan equine encephalitis virus in bats of cordoba and sucre, colombia aislamiento del virus de la encefalitis venezolana de un murciélago frugivoro (artibeus turpis) en méxico epidemiologic studies of venezuelan equine encephalitis virus in almirante, panama susceptibility of bats to certain encephalitis viruses bats in relation to the health, welfare, and economy of man epidemic strain of venezuelan equine encephalomyelitis virus from a vampire bat captured in oaxaca prevalence of selected zoonotic diseases in vertebrates from haiti biology and ecology of bat flies (diptera: streblidae) on bats in the genus carollia roosting habits of bats affect their parasitism by bat flies (diptera: streblidae) bat flies (diptera: streblidae, nycteribiidae) parasitic on bats (mammalia: chiroptera) at parque estadual da cantareira, são paulo, brazil: parasitism rates and host-parasite associations mitochondrial dna and morphology show independent evolutionary histories of bedbug cimex lectularius (heteroptera: cimicidae) on bats and humans heteroptera: cimicidae) are morphologically, but not genetically differentiated among bat hosts seasonal dynamics in the numbers of parasitic bugs (heteroptera, cimicidae): a possible cause of roost switching in bats (chiroptera, vespertilionidae) overwintering of infectious buggy creek virus (togaviridae: alphavirus) in oeciacus vicarius (hemiptera: cimicidae) in north dakota transmission of an arbovirus by a member of the family cimicidae correlation of severity of human tick-borne encephalitis virus disease and pathogenicity in mice mosquito distribution and japanese encephalitis virus infection in a bat cave and its surrounding area in lopburi province molecular identification of blood-meal sources in culiseta melanura and culiseta morsitans from an endemic focus of eastern equine encephalitis virus in new york mosquitoes of western uganda bats and their bat flies: community composition and host specificity on a pacific island archipelago bat flies: obligate ectoparasites of bats isolation of a novel fusogenic orthoreovirus from eucampsipoda africana bat flies in south africa isolation of a novel orthobunyavirus from bat flies (eucampsipoda africana) bartonella spp. in fruit bats and blood-feeding ectoparasites in madagascar role of the cliff swallow bug (oeciacus vicarius) in the natural cycle of a western equine encephalitis-related alphavirus relationship of cliff swallows, ectoparasites, and an alphavirus in west-central oklahoma bijou bridge" strain), a member of the venezuelan equine encephalomyelitis virus complex, from cliff swallow nest bugs (oeciacus vicarius) and nestling birds in north america estero real virus: a new virus isolated from argasid ticks ornithodoros tadaridae in cuba richness, infestation and specificity of spinturnicid mites (acari: spinturnicidae) on bats in southern oaxaca bat flies (diptera: streblidae, nycteribiidae) and mites (acari) associated with bats (mammalia: chiroptera) in a high-altitude region in southern minas gerais ectoparasites of arizona bats vectorial role of some dermanyssoid mites (acari, mesostigmata, dermanyssoidea). parasite diverse rna viruses of arthropod origin in the blood of fruit bats suggest a link between bat and arthropod viromes a whole genome perspective on the phylogeny of the plant virus family tombusviridae metagenomics reshapes the concepts of rna virus evolution by revealing extensive horizontal virus transfer redefining the invertebrate rna virosphere deliberate insectivory by the fruit bat rousettus aegyptiacus deliberate insectivory by the fruit bat pteropus poliocephalus by aerial hunting viral antibody dynamics in a chiropteran host the enhancement of arbovirus transmission and disease by mosquito saliva is associated with modulation of the host immune response experimental infection of north american birds with the new york 1999 strain of west nile virus tools to study pathogen-host interactions in bats ecosystem services provided by bats marburgvirus resurgence in kitaka mine bat population after extermination attempts empirical assessment of non-invasive population genetics in bats: comparison of dna quality from faecal and tissue samples empirical evaluation of non-invasive capture-mark-recapture estimation of population size based on a single sampling session optimizing viral discovery in bats optimizing non-invasive sampling of an infectious bat virus experimental inoculation of egyptian rousette bats (rousettus aegyptiacus) with viruses of the ebolavirus and marburgvirus genera key: cord-352563-yb446bap authors: li, ya; li, jialu; liu, yunbo; shi, zhengli; liu, haizhou; wei, yuquan; yang, lin title: bat adeno-associated viruses as gene therapy vectors with the potential to evade human neutralizing antibodies date: 2019-05-20 journal: gene ther doi: 10.1038/s41434-019-0081-8 sha: doc_id: 352563 cord_uid: yb446bap the prevalence of adeno-associated virus (aav) has been investigated in bat populations, but little is known about the biological properties of this virus. in this study, four full-length bat aav capsid genes were isolated in china, with their amino acid sequences sharing 61% identity with those of aav2 on average. these capsid genes could package aav particles in combination with aav2 rep and itrs, albeit at a lower efficiency. bat aavs could only slightly infect mouse liver but could transduce mouse muscle to some extent after systemic administration with a higher muscle/liver ratio than that of primate aavs. bat aav 10hb showed moderate muscle transduction, similar to that of aav2, during direct intramuscular injection and, compared with other aav serotypes, was also relatively efficient in resisting human antibody neutralization after intramuscular injection. evolutionary analysis revealed a number of codons in bat aav capsid genes subject to positive selection, with sites corresponding to v259 and n691 in 10hb capsids being localized on the surface of the aav2 capsid. mutagenesis studies indicated that the positive selection in bat aav capsids is driven by their tropism evolution in host species. taken together, the results of this study indicate that bat aav 10hb vector has the possible applications for muscular gene therapy, especially in the presence of human aav neutralizing antibodies. adeno-associated virus (aav) is a dependovirus genus parvovirus that was originally identified as a contaminant in a simian adenovirus preparation [1] . aav requires the assistance of other viruses, such as adenovirus and herpes simplex virus, for competent replication. in the absence of a helper virus, wild-type aav2 can integrate its genome into the human chromosome 19. the aav genome encodes at least three open-reading frames, with the rep gene encoding the replication proteins, the cap gene encoding the capsid proteins, and the recently identified assembly-activating protein (aap) being related to virion assembly [2] . aavs have been universally employed as gene therapy vectors because of their stable transduction of numerous tissues and relative scarcity of the immune response. thirteen aav serotypes and over 100 aav variants have been discovered from non-human primate and human tissue samples [3, 4] , with variable tissue tropism and immune profiles. some representative aav serotypes have been utilized in clinical trials for gene therapy of genetic diseases, such as aav1 for lipoprotein lipase deficiency [5] , aav2 for leber's congenital amaurosis [6] , aav8 for hemophilia b [7] , and aav9 for spinal muscular atrophy [8] . despite the universal application of aavs in gene therapy, an important concern for the efficacy of aav is the high prevalence of neutralizing antibodies within human populations. a prevalence of up to 59% was observed for neutralizing antibodies against aav2 in an investigated french population, and even the aav8 originally isolated from primates had a prevalence of 19% [9] . furthermore, different aav serotypes showed some common footprints for antibody binding, suggesting cross-reactivity among primate aav serotypes and their neutralizing antibodies [10] . the discovery of novel aav serotypes and directed evolution remain two important genetic approaches to enhance aav in its capacity to evade preexisting human neutralizing antibodies. for instance, numerous aav serotypes have been successively isolated from cattle, goats, and pigs [11] [12] [13] . the bovine and caprine aavs showed potential to evade neutralization of human antibodies, while the porcine aav demonstrated distinct tissue tropism from primate aavs. these results suggest the potential application of nonprimate mammalian aavs as gene therapy vectors to overcome the human immunological barrier. at the same time, some notable studies have been performed to evolve aav for optimized immunological properties [14, 15] , which further expanded the application of natural aav serotypes. bats are a large reservoir for different viruses, including the notorious sars coronavirus [16] . in our previous work, we sequenced the complete rep and cap sequences of a bat aav yunnan strain and investigated primarily the epidemiology of bat aavs in several chinese provinces [17] . however, the potential of bat aav to be used as a gene therapy vector has not been thoroughly elucidated to date. in this study, a number of bat aav capsid genes will be cloned and characterized with a focus on their tissue tropism and immunological properties, which will establish a foundation for their further application in gene therapy, especially in the presence of human aav antibodies. for this study, 293 (human embryo kidney cells), a549 (human lung carcinoma cells), huh7 (human hepatoma cells), ht29 (human colon cancer cells), and u87 (human glioma cells) cells were maintained in dulbecco's modified eagle's medium (dmem) supplemented with 10% fetal bovine serum, 100 u of penicillin/ml, and 0.1 mg of streptomycin/ml. cells were maintained at 37°c in a 5% co 2 humidified atmosphere. the bat total fecal samples were collected in 2007 and 2009 from yunnan province and in 2010 from hubei province in china. the bat intestinal tissues were collected from yunnan in 2007. viral nucleic acid was extracted from the fecal samples with a qiaamp rna mini kit (qiagen) following the manufacturer's instructions. these rna samples have been used for epidemiological studies on rna viruses. however, since bat genomic dna was detected from them, they were also used for amplification of aav capsid genes in this study. viral dna was also directly isolated from the intestinal samples using the dneasy blood and tissue kit (qiagen). a set of primers was selected from the bat aav yunnan strain genomic sequence (genbank no. gu226971) published previously: bt-cap5′ (5′-cccaagcttcgggggaatctgactccgtga acttcgccgaga-3′) and bt-cap3′ (5′-ggaagatctc gcagagaccaaagttcaactgaaacga-3′). these primers were used to amplify the complete capsid gene from different bat aav fecal samples. a 2.5-kb pcr product was obtained, treated successively with bglii, klenow fragment, and hindiii and inserted into the uf1-aav8 vector treated successively with noti, klenow fragment, and hindiii. the uf1-aav8 vector contains the aav2 rep gene and itr sequences and was used for cloning shuffled aav capsid genes [18] . this bat aav capsid gene was designated 07yn considering its sampling from yunan in 2007. to adapt the pcr to more bat dna samples, a degenerate primer dcap3′ was designed based on alignment of the 3′ untranslated region of mammalian aav genomes (5′-ggaagatctactga-macgaat(h/-)ammcggtttattg-3′, modified from bello et al. [19] . by the use of the bt-cap5′/dcap3′ primers, two additional bat aav capsid gene sequences were amplified from bat fecal samples and cloned into the uf1-aav8 vector. the sequences were designated 09yn and 10hb according to their sampling time and regions. furthermore, a bt-cap3′-n1 primer was selected from the downstream sequence of the 07yn capsid gene obtained by 3′ rapid amplification of genomic dna ends (rage; data not shown): (5′-ggaa-gatctacgcgagagaccggacttgactgaaacgaa-3′). a fourth bat aav capsid gene was amplified from the myotis ricketti intestinal dna sample using bt-cap5′/bt-cap3′-n1 primers and designated as 1285 according to the bat code. the 09yn, 10hb, and 1285 capsid genes were cloned into the uf1-07yn plasmid using hindiii/bglii digestion to generate uf1-09yn, uf1-10hb, and uf1-1285, respectively. all bat aav capsid genes were sequenced using the abi automated sequencer. a series of cloning experiments were performed to prepare for the production of recombinant bat aavs. the 07yn bat aav capsid gene was released from the uf1-07yn plasmid by treatment by successive treatment with bglii/klenow/hindiii and ligated into the aav2/9 vector treated successively with noti/klenow/hindiii to obtain aav2-07yn. the 09yn, 10hb, and 1285 capsid genes were similarly cloned into the aav2-07yn vector via hindiii/ bglii digestion to obtain aav2-09yn, aav2-10hb, and aav2-1285, respectively. with respect to aav2-10hb, due to its lower packaging efficiency, we attempted to mutate the start codon of its aav2 rep gene from atg to acg by transferring the rep gene from uf1-10hb to this plasmid via spei/hindiii digestion. the uf1-serial plasmids contained the aav2 rep gene, bat aav cap genes and aav2 itr. these plasmids were used together with the human adenovirus 5 helper plasmid phelper for the generation of wild-type bat aav vectors. moreover, the aav2-serial plasmids contained the aav2 rep gene and bat aav cap genes. these plasmids were used together with phelper and a plasmid encompassing a reporter gene expression cassette and aav2 itr, aav-cmv-luc1, or aav2.1-cmv-lacz (kind gifts from dr. xiao xiao) for the generation of recombinant bat aav vectors. thus, two or three plasmids were used for ca 3 po 4 transfection of 293 cells [20] . in addition, recombinant aavs, including aav2, aav8, and aav1, were generated for use as controls in bat aav transduction experiments. aav vectors were purified by fractionation with cscl gradient centrifugation. dnase-resistant genome copy titers of the vector preparations were determined by dot blots with probes specific to the aav2 rep gene or the cmv promoter. titration of recombinant aavs was performed in huh7, u87, a549, 293, and ht29 cells. cells were maintained in dmem complete medium and plated into a 24-well plate at a 1:3 ratio. twenty-four hours later, aav2-, aav8-, 1285-, 09yn-and 10hb-cmv-lacz were used for infection in duplicate at a multiplicity of infection (moi) of 5 × 10 3 , 5 × 10 4 , and 5 × 10 5 , respectively. forty-eight hours after infection, the cells were resuspended in 250 µl of lysis buffer (100 mm k 3 po 4 , ph 7.8; 0.2% triton x-100) and centrifuged at 10,000 × g for 3 min. the supernatants were used for detection of β-galactosidase activity using the tropix galacto-light kit (applied biosystems). the protein quantitation was accomplished using the bca protein assay kit (beyotime). animal use in this study was approved and monitored by the sichuan university institutional animal care and use committee. to investigate the transduction efficiency of aav vectors in systemic administration, 5 × 10 11 vector genomes (v. g.) of aav2-, aav8-, 1285-, 09yn-, or 10hb-cmv-luc vectors were injected into male 7-week-old c57b6 mice by tail vein with four mice per group. two weeks later, mouse tissues, including the heart, liver, brain, lung, kidney, spleen, muscle and testis, were collected for the luciferase assay. one hundred milligrams of the tissue samples was homogenized using a tissue-tearor device (biospec) in 0.8 ml of lysis buffer on ice, and the lysates were centrifuged at 10,000 × g for 3 min. the supernatants were used for detection of luciferase activity using the luciferase assay system (promega). the protein concentration was determined using the bradfold assay (beyotime). genomic dna was extracted from mouse tissues for quantitative detection of vector genome copies by taqman probes (applied biosystems) with a single-copy endogenous gene (glucagon gene) as the diploid cell number reference. to study the direct muscle transduction of aav vectors, 10 10 v.g. of aav2-, aav1-, 07yn-, 1285-, 09yn-, or 10hb-cmv-luc vectors were directly injected into the gastrocenemius muscles of male 7-week-old c57b6 mice with bilateral muscles of three mice treated as a group. two weeks later, the mouse muscles were collected for luciferase assays and real-time pcr analysis as described above. male 7-week-old cb17 scid mice (vital river) were injected with 294 µl of 50 mg/ml intravenous human immunoglobulin (ivig; shuanglin biopharmaceutics, s20003024) by tail vein injection. twenty-four hours later the amount of ivig circulating in the mouse blood was expected to decrease by half to an approximate antibody concentration of 5 mg/ml, which is close to the physiological level of the normal human body [21] . next, 5 × 10 11 v.g. of aav2-, 09yn-, or 10hb-cmv-luc vectors were injected into the scid mice by tail vein with four mice per group. furthermore, for each aav vector, an identical number of mice were injected with the same vector dose but without ivig pretreatment. two weeks later, gastrocenemius muscles were collected from the mouse groups for detection of luciferase activity. to investigate the effects of human antibody on direct intramuscular injection, 602 µl of 50 mg/ml ivig was injected into male 7-week-old cb17 scid mice to yield an approximate antibody concentration of 10 mg/ml 24 h after treatment. 10 10 v.g. of aav2-, 09yn-and 10hb-cmv-luc vectors were directly injected into the gastrocenemius muscles with four mice per group. furthermore, for each aav vector, an identical number of mice were injected with the same vector dose but without antibody treatment. two weeks later, gastrocenemius muscles were collected from each mouse group for the luciferase assay as described above. twenty-eight complete coding sequences of dependovirus capsids [22] were downloaded from genbank in april 2014 (http://www.ncbi.nlm.nih.gov/; table s1 ). in addition to the four bat aav capsid genes listed in this study, a total of 32 dependovirus sequences were aligned using clustalx2.1 [23] . the resulting fasta file was further aligned using the codon algorithm implemented in pranster to improve the quality of alignment [24] . geneconv [25] , maxchi [26] , and gard [27] were used to detect recombination breakpoints among dependovirus capsid gene sequences. the sequence alignment was therefore partitioned into 10 larger segments that contained no recombination breakpoints to exclude the disturbance of recombination on detection of positive selection [28] . tree topologies were inferred from these sequence segments by the maximumlikelihood method implemented in dnaml of phy-lip3.69 [29] . the codeml program of paml4.6 [30] was used for the detection of positive selection across dependovirus gene lineages. the nested evolution models m1a/m2a and m7/ m8 were compared using likelihood ratio tests to assess the significance of positive selection, and empirical bayes methods enabled the identification of individual positively selected codon sites a posteriori. furthermore, positive selection could occur episodically in the specific dependovirus capsid gene lineage. the branch-site test implemented in codeml and mixed effects model of evolution implemented in meme [31] allow selection pressure to vary both among protein sites and across tree branches. these methods were used for detection of episodic diversifying selection across bat aav lineages. conditions existed in which an identical serine residue was observed through all of the gene lineages and was detected as positively selected by the programs. however, this finding was attributed to toggling selection or doublet mutation [32] but not strict positive selection, because eventually, no replacement could be observed. t250 and e694 were inferred to be subject to positive selection in bat aav capsid gene lineages and to be exposed on the exterior capsid surface. the bat aav 10hb was selected as a prototype for evolutionary studies, and the corresponding v259 and n691 in its capsid were mutated by pcr methods. the mutagenesis primers for the v259 site were as follows: v259t-1: 5′-ccaaagt acgtcgcgtccccggttccgctgccgtt-3′; v259h-1: 5′-ccaaagtagtgcgcgtccccggttccgctgcc gtt-3′; v259-2: 5′-ttacagcacgccttggggata ctt-3′. the mutagenesis primers for the n691 site were as follows: n691-1: 5′-ccgtgtactggatctcggggtt cc-3′; n691e-2: 5′-ccaacttcgagaacagcgccaa cgcgcagttct-3′; n691a-2: 5′-ccaacttcgccaa-cagcgccaacgcgcagttct-3′; n691k-2: 5′-cca acttcaaaaacagcgccaacgcgcagttct-3′; n691s-2: 5′-ccaacttcagtaacagcgccaacgcgc agttct-3′. these six mutated 10hb capsid genes were used for the production of recombinant bat aav encompassing the luciferase reporter gene. these modified vectors were compared to the parental 10hb and control aav2 vectors on their capacity to transduce mouse skeletal muscle after intramuscular injection using the methods described above. after a series of pcr experiments, four full-length bat aav capsid genes were amplified and sequenced (fig. 1 ). the bat aav capsid genes shared an amino acid sequence identity of 60.7-61.4% with that of the aav 2 capsid gene (table 1) . moreover, different sequence homology was shown within the bat aav group. the 07yn and 1285 capsids shared 99.6% amino acid sequence identity, with replacement of only three residues. the 09yn capsid had a weaker relationship with 07yn with 99.0% of sequence identity and seven replacements. the close relationships of 07yn, 1285, and 09yn capsids inferred them as a single serotype. by contrast, the 10hb capsid had a lower (82%) sequence identity with the other three bat aav variants, inferring it as a distinct serotype. generally, the~60% sequence identity between bat aavs and aav2 capsid vp1 proteins suggested their markedly different transduction and immunological properties. to produce bat aav vectors for additional studies, we employed the pseudotyping strategy. the bat aav capsid genes were cloned into the wild-type aav plasmid uf1-aav8 with the aav2 rep gene and itrs, or the recombinant aav plasmid aav2/9 with the aav2 rep gene. the uf1-series plasmids were used in combination with the adenovirus helper plasmid to generate of the wild-type bat aavs, and the aav2-series plasmids were used in combination with the phelper and the plasmid encompassing the aav2itrs and a reporter gene, i.e., luciferase or βgalactosidase. the generated recombinant aav particles were then detected by dot blotting ( table 2 ). all bat aav capsid genes were competent in packaging wild-type and recombinant aav vectors. however, these genes displayed different efficiencies for vector production. specifically, the 07yn, 1285, and 09yn had similar capsid coding sequences, but the 07yn vector showed a lower production titer; thus, only 1285 and 09yn were selected representatively for comparison of their tissue tropism and the underlined numbers represent the highest and lowest similarity of aav capsids a panel of the following human cell lines was transduced with serial dilutions of aav2, aav8, or bat aavs: hepatoma (huh7), glioma (u87), lung carcinoma (a549), embryo kidney (293), and colon cancer (ht29) cells. generally, aav2 exhibited the highest transduction for most of the cells (data not shown). by contrast, aav8 could only efficiently transduce huh7 cells with similar βgalactosidase activity as that of aav2. furthermore, none of the three bat aav variants could efficiently transduce any of the five human cell lines tested. interestingly, the bat aavs were not capable of infecting the bat kidney cell line bk from myotis davidii (data not shown). thus, the nondetection of bat aav infection on human cell lines might infer a potential host species barrier for bat aavs to infect human cells. however, the different properties of human cells in vitro and in vivo could also be the reason for their nonpermissiveness for bat aav infection. we next investigated bat aavs to determine their systemic gene delivery efficiency and tissue tropism. the aav luciferase reporter gene cassette was packaged in the viral particles of aav2, aav8, and bat aavs for a side-by-side comparison. at 2 weeks after tail vein injection of aav vectors in 7week-old c57b/6 mice, luciferase activities and vector dna copy numbers in various tissues were analyzed (fig. 2) . consistent with a previous report [33] , the aav8 vector transduced mouse liver as well as heart and muscle efficiently (fig. 2a) . aav2 predominantly infected the liver with considerably lower transduction in other organs. in contrast, the bat aavs only slightly infected mouse liver but could transduce the heart and muscle to some extent. in keeping with the transgene expression, bat aav vector copy numbers in the liver were considerably lower than those of aav2 and aav8, and considerably lower than those of aav8 in the heart and muscle (fig. 2b) . however, in the kidney and spleen, the bat aav 1285 vector showed a significantly higher vector copy number than aav2 and aav8, although no obvious transgene expression was observed in these organs. furthermore, the muscle versus liver transduction ratio was calculated for the five aav vectors examined (fig. 2c) . interestingly, in contrast to the muscle/liver ratio of 0.4 observed for aav2 and aav8, the ratios of 11.0, 30.2, and 8.8 were observed for bat aav 1285, 09yn, and 10hb, respectively. these data showed the distinct in vivo tissue distribution of bat aavs from that of the primate aavs given their weak transduction in mouse liver and lower transduction in other tissues. since bat aavs showed muscle transduction to some extent in systemic administration, their performance in direct intramuscular injection was further investigated. the aav2, aav1, and four bat aav vectors expressing luciferase were used for direct intramuscular injection of mouse gastrocnemius muscle (fig. 2d, e) . at 2 weeks post injection, the highest transgene expression was observed in aav1-treated mouse muscles, moderate luciferase expression was observed in aav2-transduced and 10hbtransduced muscles, while transgene expression was only slightly detected in 07yn-, 1285-, and 09yn-injected mouse tissues (fig. 2d) . intriguingly, no association was observed between the vector genome copies and transgene expression levels. most vector genome copies were detected in mouse muscles injected with the 1285 vector, while the other five vectors showed the muscle gene copy numbers at the same level (fig. 2e) . these data reiterated the results from systemic administration of aav vectors in that bat aav 1285 might be persistent in mouse tissues, such as kidney, spleen, or muscle for a long time, but the ratelimiting factors after cellular entry might hinder their efficient transduction. notably, the vector genome copy number and transgene expression in skeletal muscle of the 10hb vector were similar to those of aav2, which inferred its potential application as a vector for muscular gene therapy. we considered utilizing the in vitro neutralization assay to study the immunological properties of the novel bat aavs. however, none of the available cell lines, including those from humans or bats, was permissive for bat aavs. in contrast, bat aavs could transduce mouse muscle in vivo both by systemic administration and by direct intramuscular injection. furthermore, the scid mice infused with ivig, the pooled human antibodies, have been demonstrated as an excellent model to assess the immune evasion of antibody neutralization by aavs [12, 21] . we thus sought to inject the bat aavs into the scid mice to evaluate their capacity to transduce muscle in the presence of ivig. to evaluate the neutralization properties of the aav vector in systemic administration, 294 µl of 50 mg/ml ivig was injected into the male scid mice, which should result in an~5 mg/ml antibody concentration after 24 h mimicking the lower antibody level in human plasma. scid mice were then injected intravascularly with 5 × 10 11 v.g. of aav2, bat aav 09yn or 10hb-cmv-luciferase vectors. without ivig treatment, the aav2 transduction of the mouse muscle was 4.2-fold of that of the 09yn and 1.7-fold of that of 10hb (fig. 3a) . however, after ivig treatment, the aav2 transduction in the mouse muscle decreased to approximately one-sixth of the untreated control, and the transduction of 09yn and 10hb, respectively, decreased to approximately one second and one-third of the untreated controls. the 10hb subsequently had the muscle transduction efficiency similar to that of the aav2. thus, the capacity to resist human antibody neutralization for the three vectors in the case of intravenous injection appeared to be 09yn > 10hb > aav2. to assess the three aav vectors on their neutralization properties in intramuscular injection, 602 µl of 50 mg/ml of ivig was injected into the scid mice, corresponding to añ 10 mg/ml antibody concentration 24 h later, mimicking the higher antibody level in human blood. in the absence of ivig pretreatment, the bat aav09yn and 10hb vectors produced luciferase activity that was 98.6-fold and 3.7-fold lower than that produced by the aav2 vector, respectively. in the presence of ivig, the luciferase activity produced by the aav2 and 09yn vectors was completely inhibited close to the baseline (fig. 3b) . by contrast, the transgene expression from the 10hb vector was reduced to 14.4% of the level produced in the absence of ivig, with absolute luciferase activity over 2.5 × 10 5 rlu/mg protein. therefore, the muscle transduction by bat aav 10hb was considerably more resistant to ivig neutralization than the 09yn and aav2. the significant transgene expression in the presence of human antibodies suggested the application potential of the 10hb vector for muscular gene therapy in the case of preexisting aav immunity. the evolution of bat aavs has been primarily investigated in our previous report [17] . we were interested in the evolutionary mechanism of bat aavs with respect to their unique capsid sequences, tissue tropism, and immunological properties. using the maximum-likelihood method, we constructed a phylogenetic tree for bat aavs and other representative dependovirus taxa based on their complete capsid coding sequences (fig. 4) . the four bat aav variants formed a monophyletic group distinct from other dependovirus taxa, which inferred their independent evolutionary origin. a branch composed of rat aav/poaav1/poaav5/ caprine aav/aav5 was directly clustered with the bat aavs in the molecular phylogeny which demonstrated their closer phylogenetic relationship with bat aavs. in accordance with the condition in primate aavs [3] , a number of recombination events were detected among ) . however, no recombination event was detected during the evolution of the four bat aav capsid genes. since recombination could interfere with the detection of positive selection [28] , the dependovirus capsid sequence alignment was partitioned into 10 segments according to the detected recombination breakpoints. these sequence segments were then subjected to selection analysis using the branch-site test in the paml program [30] or the meme method in the hyphy program [31] . with respect to the branch site test, two sites corresponding to s238 and m420 in the 09yn capsid were subject to positive selection in the whole bat aav lineage; one site corresponding to n344 in the 09yn capsid was selected within the 07yn/ 09yn/1285 lineage; and four sites corresponding to q213, r297, a422, and m664 sites in the 10hb capsid were selected in the 10hb branch (table 3) . interestingly, the results from the meme analysis differed partially from those obtained from the branch-site test. the site corresponding to s238 in the 09yn capsid was positively selected for the whole bat aav lineage, and the three sites corresponding to t259, n344, and e694 in the 09yn capsid were selected for the 07yn/09yn/1285 lineage. the site corresponding to s214 in the 10hb capsid was selected for the 10hb branch. these results suggest that positive selection but not genetic recombination play the crucial role in the speciation of bat aav variants. for a better comprehension of their functional roles, the positively selected sites of bat aavs were projected onto the aav2 structure [34] . all three bat aav sites from the 10hb branch were projected onto the interior surface of the aav2 capsids (data not shown). the sites corresponded to q213, s214, and r297 in the 10hb capsid and n223, s224 and k309 in the aav2 capsid. by contrast, both bat aav sites from 07yn/09yn/1285 lineage were projected onto the exterior surface of the aav2 capsids (fig. 5) . the sites corresponded to t259 and e694 in the 09yn capsid, v259 and n691 in the 10hb capsid, and h271 and n705 in the aav2 capsid. unfortunately, no functional information is currently available for the three bat aav sites localized on the interior surface of aav2 capsids. however, mutations of h271 and n705, two projected sites on the exterior surface of aav2 capsids, were associated with the altered transduction or neutralization properties of aav2 [35] . fig. 4 maximum-likelihood phylogeny based on coding sequences of vertebrate dependovirus capsid genes. the tree topology was estimated using the dnaml program in the phylip package [29] thus, it was plausible that positive selection within the corresponding sites in bat aavs was related to their adaptation to uncharacterized bat species. to test this hypothesis, it would be interesting to perform mutagenesis experiments on the bat aav 10hb capsid gene to examine their effects in muscle transduction in mice. the 10hb variant was selected because of its moderate muscle transduction and effective evasion of antibody neutralization (figs. 2and 3) . the evolutionary pathway of the v259 site in the 10hb capsid (corresponding to t259 in the 09yn capsid and h271 in the aav2 capsid) is demonstrated in fig. 6a . positive selection was detected in two dependovirus lineages with this codon being mutated from t to h in the aav1/aav6 lineage and from v to t in the bat aav 07yn/1285/09yn lineage. moreover, the evolutionary pathway of the n691 site in the 10hb capsid (corresponding to the e694 site in the 09yn capsid and the n705 site in the aav2 capsid) is demonstrated in fig. 6b . this codon was replaced from the ancestral n to e in bat aav 07yn/1285/09yn lineage and aav7, to k in aav4, to a in bovine aav and aav1/aav6 lineage, and to s in rat aav. based on these data, the v259 site in the 10hb capsid gene was mutated to t or h, and the n691 site was mutated to e, k, a, or s to evaluate the effects on aav transduction (fig. 7) . moreover, when the six bat aav 10hb mutants were compared side-by-side with the wild-type 10hb virus, they showed variable transduction properties in mouse muscle. the transduction of 10hb v259t and v259h fell to approximately one-seventieth and one-fortieth of that of wild-type 10hb, respectively (fig. 7a) . this result was consistent with previous observations of aav2 vector with mutations at the orthologous h271 site [35] . however, mutations at the n691 site within the 10hb capsid only slightly reduced the muscle transduction of the 10hb vector, which was also in keeping with the results in the aav2 vector with mutations at the orthologous n705 site. intriguingly, the 10hb v259h and v259t mutants showed only slightly reduced aav genome copy numbers in mouse muscle (fig. 7b) . furthermore, the 10hb mutants n691a, n691k, and n691s exhibited highly similar vector genome copies to those of the wild-type 10hb vector. the 10hb n691e mutant was distinct in its significantly improved genome copy number in mouse muscle. the v259h and v259t mutations dramatically altered the intracellular although the cellular entry of the 10hb mutants n691a, n691k, and n691s was not affected, their intracellular trafficking was slightly reduced. with respect to the 10hb n691e mutant, it was unique in that the increased vector genome copies balanced its reduced intracellular trafficking such that no alteration in transgene expression was observed in muscle for this vector. in this study, four aav variants were isolated from bat fecal or intestinal samples from two regions of china. the 07yn, 1285, and 09yn were sampled from the same location in yunnan province at different times. consequently, the variants showed only a limited number of sequence variations among one another. specifically, 1285 capsid differed from the 07yn capsid by three amino acid residues, while 09yn capsid differed from 07yn by seven amino acid replacements (fig. 1 ). by contrast, the 10hb capsid from hubei province differed from 07yn by 132 residues. these results inferred that the genetic diversity of the bat aav capsid gene was dictated by the geographic distribution of the original bat populations. furthermore, the bats might be an important source for the isolation of novel aav serotypes because the four aav variants were isolated from a limited number of bat samples and because aav sequences were highly prevalent in bat samples from different regions of china [17] . the bat aav capsids showed~60% sequence identity with that of aav2 (table 1) , which demonstrated the distinct biological properties of bat and primate aavs. in another aspect, the bat aav capsid genes could be used in combination with the aav2 rep gene and itr sequences to generate recombinant aav particles, albeit with the lower titers, which suggested a structural and functional conservation of aav capsid genes across a large number of dependovirus species. the pseudotyping strategy was successfully applied to the production of bat aav vectors to investigate their transduction and immunological properties. intriguingly, the bat aavs could not efficiently infect the human cell lines (data not shown), which might be attributable to their high sequence variation from human aavs and a potential interspecies barrier for transmission of bat aav to human populations. furthermore, when used for systemic administration, the bat aavs could not transduce most of the organs including the liver in mice, but the 1285 vector could persist in the kidney and spleen for at least 14 days without significant transduction (fig. 2a, b) . this unique property of 1285 inferred that its capsids retained high stability in mouse tissues without efficient capsid uncoating and transgene expression. furthermore, the bat aav 10 hb vector could moderately transduce the mouse muscle after intramuscular injection, exhibiting the efficiency comparable to that of aav2 (fig. 2d, e) . notably, the muscle versus liver ratio of transgene expression for bat aavs was over 24 times higher than that of aav2 and aav8. this characteristic of bat aavs might favor their further genetic modification for gene vehicles specifically targeting to mouse skeletal muscle. in accordance with their distinct capsid sequences from primate aavs, the bat aavs also appeared more resistant to neutralization by human aav antibodies after both systemic and local administration (fig. 3) . significantly, the bat aav10hb vector could still efficiently transduce the skeletal muscle in a high dose of ivig after intramuscular injection in mice, which was in contrast to the complete loss of transgene expression from the aav2 vector at the same antibody titer. considering both its moderate transduction of mouse muscle and efficient evasion of human antibody neutralization, the bat aav 10hb vector exhibited the potential to be used for muscular gene therapy in humans. numerous genome sequences have been reported for dependoviruses, a parvovirus genus with the unique life cycle requiring helper viruses [22] . however, little is known about their evolutionary mechanism. bioinformatics analysis revealed a limited number of recombination events among dependovirus species (data not shown), distinct from the frequent occurrence of homologous recombination in primate aavs [3] . furthermore, a number of positively selected sites were detected during the speciation of bat aavs (table 3 ; fig. 5 ). mutagenesis experiments of two of these selected sites in the 10hb capsid gene supported their functional roles in mouse muscle transduction (fig. 7) . these mutations appeared to affect muscle transduction of the 10hb vector primarily by lowering their intracellular trafficking or virion uncoating with no significant alteration of the tissue distribution of the vector genome. interestingly, mutation of the n705 site in the aav2 capsid, which is orthologous to n691 in the 10hb capsid altered its neutralization property [35] . thus, positive selection in bat aav capsid genes might be driven spontaneously by selection pressure from infection and immunity similar to that occurring in canine parvovirus [36] . the bats remain an important reservoir for virus transmission and spreading. some of these viruses could be pathogens representing potential threatening to human health [16] . however, from another perspective, the bats could also be a potential source for virus isolation and vector development. novel aav vectors have also been isolated from other nonprimate mammals including cattle, goats, and pigs [11] [12] [13] . a common attribute of these aav vectors was their efficient evasion of preexisting immunity from human aav antibodies. unfortunately, these vectors might not be superior to primate aavs in their transduction properties, and to the best of our knowledge, no reports have described their further genetic engineering. ideally, these vectors could be further tailored to optimize their transduction and immunological properties [37] , which could ultimately benefit their application to human gene therapy. adenovirusassociated defective virus particles a viral assembly factor promotes aav2 capsid formation in the nucleolus adeno-associated viruses undergo substantial evolution in primates during natural infections clades of adeno-associated viruses are widely disseminated in human tissues immune responses to intramuscular administration of alipogene tiparvovec (aav1-lpl(s447x)) in a phase ii clinical trial of lipoprotein lipase deficiency gene therapy safety and efficacy of gene transfer for leber's congenital amaurosis long-term safety and efficacy of factor ix gene therapy in hemophilia b single-dose gene replacement therapy for spinal muscular atrophy prevalence of serum igg and neutralizing factors against adeno-associated virus (aav) types 1, 2, 5, 6, 8, and 9 in the healthy population: implications for gene therapy using aav vectors capsid antibodies to different adeno-associated virus serotypes bind common regions cloning and characterization of a bovine adeno-associated virus novel caprine adeno-associated virus (aav) capsid (aav-go.1) is closely related to the primate aav-5 and has unique tropism and neutralization properties novel adeno-associated viruses derived from pig tissues transduce most major organs in mice directed evolution of adeno-associated virus yields enhanced gene delivery vectors structure-guided evolution of antigenically distinct adeno-associated virus variants for immune evasion isolation and characterization of a bat sars-like coronavirus that uses the ace2 receptor prevalence and genetic diversity of adeno-associated viruses in bats from china a myocardium tropic adeno-associated virus (aav) evolved by dna shuffling and in vivo selection isolation and evaluation of novel adeno-associated virus sequences from porcine tissues production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus human immunoglobulin inhibits liver transduction by aav vectors at low aav2 neutralizing titers in scid mice the family parvoviridae clustal w and clustal x version 2.0 an algorithm for progressive multiple alignment of sequences with insertions statistical tests for detecting gene conversion analyzing the mosaic structure of genes gard: a genetic algorithm for recombination detection robust inference of positive selection from recombining coding sequences phylogeny inference package) version 3.6. distributed by the author paml 4: phylogenetic analysis by maximum likelihood detecting individual sites subject to episodic diversifying selection frequent toggling between alternative amino acids is driven by selection in hiv-1 adenoassociated virus serotype 8 efficiently delivers genes to muscle and heart the atomic structure of adeno-associated virus (aav-2), a vector for human gene therapy mutations on the external surfaces of adenoassociated virus type 2 capsids that affect transduction and neutralization high rate of viral evolution associated with the emergence of carnivore parvovirus engineering adeno-associated viruses for clinical gene therapy acknowledgements this work was funded by grants from the national natural science foundation of china (31170157 and 81471776) and the national science and technology major project of the ministry of science and technology of china (2018zx09201018-013) to ly and from the national natural science foundation of china (81290341) to zs. we are grateful to dr. xiao xiao for his constructive advice in the construction and production of bat aav vectors. we extend our appreciation to dr. juan li for her technical support in intramuscular injection in mice. we are grateful for the localization of selected sites of bat aav capsid genes on the aav2 structure by drs. lin-ya huang and mavis agbandje-mckenna, and for the helpful comments on our manuscript provided by dr. mario mietzsch. conflict of interest the authors declare that they have no conflict of interest.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-345695-5vi9wibk authors: hicks, lorin l.; schwab, nathan a.; homyack, jessica a.; jones, jay e.; maxell, bryce a.; burkholder, braden o. title: a statistical approach to white-nose syndrome surveillance monitoring using acoustic data date: 2020-10-22 journal: plos one doi: 10.1371/journal.pone.0241052 sha: doc_id: 345695 cord_uid: 5vi9wibk traditional pathogen surveillance methods for white-nose syndrome (wns), the most serious threat to hibernating north american bats, focus on fungal presence where large congregations of hibernating bats occur. however, in the western usa, wns-susceptible bat species rarely assemble in large numbers and known winter roosts are uncommon features. wns increases arousal frequency and activity of infected bats during hibernation. our objective was to explore the effectiveness of acoustic monitoring as a surveillance tool for wns. we propose a non-invasive approach to model pre-wns baseline activity rates for comparison with future acoustic data after wns is suspected to occur. we investigated relationships among bat activity, ambient temperatures, and season prior to presence of wns across forested sites of montana, usa where wns was not known to occur. we used acoustic monitors to collect bat activity and ambient temperature data year-round on 41 sites, 2011–2019. we detected a diverse bat community across managed (n = 4) and unmanaged (n = 37) forest sites and recorded over 5.37 million passes from bats, including 13 identified species. bats were active year-round, but positive associations between average of the nightly temperatures by month and bat activity were strongest in spring and fall. from these data, we developed site-specific prediction models for bat activity to account for seasonal and annual temperature variation prior to known occurrence of wns. these prediction models can be used to monitor changes in bat activity that may signal potential presence of wns, such as greater than expected activity in winter, or less than expected activity during summer. we propose this model-based method for future monitoring efforts that could be used to trigger targeted sampling of individual bats or hibernacula for wns, in areas where traditional disease surveillance approaches are logistically difficult to implement or because of human-wildlife transmission concerns from covid-19. introduction north american bat species face several contemporary conservation challenges throughout their range, including threats to habitat from loss and alteration of forested environments, and direct mortality from development of wind energy infrastructure [1, 2] . however, transmission of pseudogymnoascus destructans (pd), the pathogen that causes white-nose syndrome (wns), has emerged as the most serious threat to over a dozen species of north american bats that use caves and mines for hibernacula [3] . the pd fungus invades skin tissues (e.g., wing membrane) which disrupts homeostasis and causes dehydration that corresponds to an abnormally high frequency of arousals from torpor [4] . bats with wns increase arousal [5] and acoustic activity rates [6] above baseline levels during the normal hibernation period, which depletes critical energy reserves and can ultimately cause mortality. wns has caused precipitous declines in populations of hibernating bats and has contributed to the federal listing of the northern longeared bat (myotis septentrionalis) as threatened under the u.s. endangered species act [7] and endangered listings for three bat species in canada under the species at risk act [northern long-eared bat, little brown bat (myotis lucifugus), and tri-colored bat (perimyotis subflavus)] [8] . monitoring for wns is a critical management action and primarily has focused on detecting the pd pathogen either directly from bats captured in hibernacula or with environmental samples from occupied caves and mines [9] . since its emergence in eastern north america in 2006, wns has spread westward with some models predicting wns to arrive to montana by 2026 [10] . in june 2020, sampling conducted at bridges in 3 eastern montana counties confirmed the presence of pd, but not wns [11] . further, wns was detected in a little brown bat in washington state in 2016 and pd detection continues to increase among sites and across different species within washington [12] . thus, there is a second wns epicenter, which could affect known and previously unrecognized susceptible bat species and increase the spread of wns through western north america from both the west and the east [13] . the presence of wns in the pacific northwest opens another front in critical efforts to combat bat declines and shortens the timeline for implementing conservation strategies to protect vulnerable bat species. this effort is complicated by the fact that winter hibernation behavior for most western species is poorly understood [14] , yet this is when wns has the greatest negative impacts on bat populations [2, 15, 16] . further, little is known about winter roost sites and activity patterns of many bats in western north america. a recent review indicates that wns-susceptible species rarely congregate in large numbers in the western u.s. and that known winter roosts may be relatively uncommon or difficult to identify [17] . thus, typical surveillance approaches of estimating hibernating bat numbers and sampling for pd implemented in eastern north america where large hibernacula are more common does not translate well to all ecoregions [18] . therefore, alternate approaches to facilitate pathogen surveillance, monitor disease impacts, and conduct mitigation efforts for wns are urgently needed [13] . in addition to wns, bats can be influenced by habitat alteration from anthropogenic activities, such as forest harvesting [19] . forested areas provide essential habitat features that support bat diversity, including roost sites (e.g., trees) and foraging areas (e.g., riparian zones, wetlands) [20] [21] [22] . sustainable management of these forest resources may minimize the environmental stress on bats outside of the winter season (when wns most severely impacts bats). despite a robust network of public lands managed for preservation in western north america [23] , bat species richness is low in some protected areas, such as glacier national park [24] and lower bat activity is commonly reported in undisturbed habitats compared to disturbed [19] . managed forests support many species of bats not found or poorly represented in gov/reqapp/usermain.asp. these data were produced as part of a regional bat acoustic monitoring network and we had no special privileges to access these data and obtained them via request from the montana natural heritage program. funding: funding was provided by national council for air and stream improvement, inc., plum creek timber, weyerhaeuser, stimson lumber, f. h. stoltze land and timber. weyerhaeuser provided access and logistical support to collect empirical data and provided support in the form of salary for authors lorin hicks and jessica homyack. weyerhaeuser also contracted with tetratech for data collection and analysis, completed by author nathan schwab. the funder provided support in the form of salaries for authors [lh, jh], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. plum creek timber, stimson lumber, f. h. stoltze land and timber also provided funding and support in the form of salary for lorin hicks. weyerhaeuser also contracted with tetratech for data collection and analysis and nathan schwab received support in the form of salary. the specific roles of these authors are articulated in the 'author contributions' section. the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. weyerhaeuser provided access and logistical support to collect empirical data and provided support in the form of salary for lorin hicks and jessica homyack. plum creek timber, stimson lumber, f. h. stoltze land and timber also provided funding and support in the form of salary for lorin hicks. weyerhaeuser also contracted with tetratech for data collection and analysis and nathan schwab received support in the form of salary. this does not alter our adherence to plos one policies on sharing data and materials. protected areas on public lands [1] and some forest management practices that reduce understory vegetation such as commercial thinning and prescribed burning can increase bat activity and occupancy [19, 25, 26] . nationally, public and private forestlands are managed under state forest practices rules, water quality best management practices, and forest sustainability programs (i.e., sustainable forestry initiative, forest stewardship council, american tree farm) to protect water quality and quantity, which includes riparian buffers that often provide habitat for bats. we quantified presence of bats across forested sites in montana, usa to document baseline levels of species diversity and activity rates prior to presence of wns. our objective was to evaluate long-term acoustic monitoring as a surveillance approach to monitor for potential wns presence in overwintering bat populations on both managed and unmanaged forest. specifically, we developed a statistical method to evaluate whether activity data occur outside the range of normal conditions, thus warranting more intensive sampling for the presence of pd on bats or in the environment. we predicted that bat populations in the western u.s. would have low levels of activity during winter months, high levels of activity during the summer, and that acoustic monitoring could be used as an effective approach for disease surveillance in remote areas of the intermountain west. our study area included forested areas across montana, usa (fig 1) . the forested areas in the western portion of the state were douglas fir (pseudotsuga menziesii)/snowberry (symphoricarpos albus) forest type, which generally characterized the region [27] and included locations of our acoustic detector stations. these forests had a mixed overstory, including douglas-fir, ponderosa pine (pinus ponderosa), grand fir (abies grandis), western larch (larix occidentalis), lodgepole pine (pinus contorta), and engelmann spruce (picea engelmannii), with understory vegetation comprised of snowberry, huckleberry (vaccinium spp.) and beargrass (xerophylum tenax). westside elevations ranged from 800 to 1150 meters. the climate in these ecoregions was continental-maritime with relatively low annual precipitation, with most precipitation occurring as snow [28] . the forested locations in eastern montana were characterized by limber pine (pinus flexilus) and ponderosa pine (pinus ponderosa) forest with understory of idaho fescue (festuca idahoensis) and bluebunch wheatgrass (agropyron spicatum). eastside sample site elevations ranged from 850-1520 meters. climate in the eastern ecoregions was continental with dramatic fluctuations in winter temperatures and longer, hotter, more arid summers than western ecoregions [29] . we selected 41 sampling sites that were near lentic or lotic systems and had �25% forest cover within a 1-kilometer buffer, estimated from the montana land cover data set in a geographic information system [30] (fig 1) . we did not randomly select sites for acoustic detectors but installed them in areas with potential for high bat activity that included proximity to standing live and dead trees, talus and rock cliffs, and waterbodies. we located four sites on intensively managed forest owned and managed by plum creek timber company (after 2016, weyerhaeuser) for production of sawtimber and other forest products. the remaining 37 sampling sites were selected from a regional bat acoustic monitoring network organized by the montana natural heritage program [31, 32] . these locations are primarily represented by federal and state landownership, but also include some tribal and private land. we we installed a single, full-spectrum acoustic recording unit (aru) at each sampling site. not all arus recorded data simultaneously or across the entire 2011-2019 study period, but we installed units for year-round recording of bat echolocation. each aru station consisted of a sm3bat or sm2bat+ acoustic detector (wildlife acoustics, maynard, massachusetts) and a microphone (u1 or smx-us) mounted on a pole at a height of approximately 3 meters. the arus operated on a 6-or 12-volt charging system (20-watt solar panel, 36-amp hour battery, and a charge controller) and housed in a weather-proof container on the ground below the microphone. we set the arus to record from sunset to sunrise to maximize battery life and balance with other logistical restraints of year-round sampling in remote locations. the units employed a sampling rate (samples per second) of 196,000, 256,000 or 384,000 khz. all arus used a minimum frequency filter of 8 khz, a trigger window of 2 seconds and a maximum file length of 5-8 seconds. most arus recorded in the wac0 compression format while four arus recorded directly to.wav format. the individual aru equipment and settings varied between the hardware platforms (sm3bat vs. sm2bat+), with detection distances of approximately 20-30 meters [33] , depending on the frequency of the source and other environmental variables (i.e., temperature and humidity) [34] . the equipment and settings were consistent for a given site throughout the study. the sensitivities of all microphones were within the manufacturer's specifications at the beginning of the deployment at each sampling site and replaced approximately annually. we examined equipment for functionality, checked microphone sensitivity, and exchanged data storage cards approximately once per month during summer and up to 6 months between visits during winter due to logistical constraints of access. each aru recorded a site-specific temperature approximately once every 1 or 5 minutes. at the end of our study, we parameterized acoustic files with sonobat 4.1.0 (sonobat, arcata, california usa) using a high-performance computing cluster at montana technological university. the parameters were then input into the sonobat montana [20160912] classifier to automatically assign a species classification to each bat pass. the montana classifier uses two regional species suites, each tuned to the species potentially present in that portion of the state. in cases where a bat pass was unable to be classified to species, a generic "bat" label was assigned. similar to the north american bat monitoring program [35] , we defined a bat pass as a sequence of echolocation pulses separated by more than 2 seconds of silence. we limited the maximum file duration to 5-8 seconds to reduce potential for recording more than one bat per sequence [36] . to quantify activity rates of bats, we analyzed the number of bat passes per detector-night (the number of operational detectors multiplied by the number of nights). for a night to be included in our estimates of detector-nights, the detector needed to register an internal temperature in the status file. we excluded data that did not meet this criterion from analysis. we manually verified a subset of diagnostic bat passes (approximately monthly for each species) with sonobat to ensure that species diversity of bats was accurate at all sampling stations [37] . we calculated the average number of passes per night over each month, by species, site and year using the ratio of total monthly qualified passes and the number of nights in the month. similarly, we calculated the average number of passes per night across all species, including passes without a species classification (total bat activity). a mean monthly temperature (˚c) was estimated from the mean of temperatures recorded at each site between sunset and sunrise across all nights in a month. we used a statistical model to describe variation in monthly-average nightly bat activity associated with several sources. to address our research questions, we aimed to characterize the extent to which activity varied among sampling sites, across years, by season, and in association with temperature. we fit a linear mixed-effects model with fixed effects for monthlyaverage mean temperature, season, and their interaction. we included random intercepts by site and year and random slopes for the temperature effect by site and year. thus, this model allowed for different overall levels of bat activity by site and year, and different temperature trends by site and year, while estimating population-average effects and quantifying the variation among sites and years. bat activity was log-transformed prior to analysis, and a constant of 0.01 was added prior to transformation to accommodate values of zero. we defined season by assigning each month to one of four calendar-based seasons (winter = dec, jan, feb; spring = mar, apr, may; summer = jun, jul, aug; fall = sep, oct, nov). we fit all models in r [38] using package lme4 [39] . we excluded sites with < ten months with at least one bat detection across all species from data used to fit the model. additionally, only data from 2012-2016 were used to fit the model, as limited data were available outside of this range. we note that both data restriction choices were performed to ensure sufficient data were available to estimate site-specific effects in the mixed effects model, but that no such restrictions were used in our raw data summaries. in addition to using the fitted model to describe variation in bat activity, we also used the model to generate prediction intervals [40] for new or future observations of bat activity in our region. we generated prediction intervals based on the model output using the 'predictinterval' function in package mertools [41] . we collected acoustic data used for our analysis between 28 sept 2011 and 4 august 2019, totaling 24,850 detector-nights at 41 sites. stations operated for an average of 606 (sd = 307) nights through the study. there were gaps in recordings because of equipment malfunction and wildlife encounters, but more than 5.37 million bat passes were recorded. after removing months with erroneous data due to equipment malfunction, 868 months of bat pass and temperature data were analyzed (per site mean: 21.2 months, sd = 10.7 months). we manually vetted acoustic files to confirm 13 species across the study, 11 of which were observed on managed forest sites (table 1) . three species [big brown bat (eptesicus fuscus), silver-haired bat (lasionycteris noctivagans), and western small-footed myotis (myotis ciliolabrum)] had confirmed activity in all calendar months, indicating that some bat species maintained some level of flight activity year-round. three presumed migratory species-spotted bat (euderma [2] . due to the high percentage of missing species information, our analyses include both estimates of total bat activity, i.e., total activity over all species, including bat passes not identified to species, and estimates for individual species where possible. across 24,850 detector-nights of sampling ambient temperatures, mean monthly temperatures ranged from -16.5˚c to 23.3˚c. mean monthly temperatures (± sd˚c) in montana varied across seasons [winter -1.7˚c (± 3.9˚c); spring 5.8˚c (± 4.3˚c); summer 16.2˚c (± 3.2˚c); and fall 6.7˚c (± 5.7˚c]). bat activity tended to be highest during summer months with most sites averaging >100 passes/night during july and august (fig 2) . additionally, most sites showed some activity during december and january, with one site averaging >10 passes/night. species-specific results show similar trends. we observed a strong positive association between average total bat activity and mean monthly temperature (fig 3) . the relationship was approximately loglinear for all 41 study sites, although the level of activity varied by site. results from our fitted model indicated a strong positive association between mean monthly temperature and total bat activity that varied by season (figs 4 and 5) . the estimated trends during the fall and spring indicated approximately 12.0-and 8.5-times greater numbers of bat passes per day, respectively, for every 5˚c increase in mean monthly temperature, on average, after adjusting for site and inter-annual differences. the association between bat activity and temperature was less pronounced during the winter and summer, where in both seasons we estimated approximately 2.8-fold greater activity, respectively, for 5˚c increases in temperature. overall levels of activity at a similar temperature also varied by season, although the difference was strongly dependent on temperature (fig 6) . we used the model random effects to estimate among-site and among-year variation in the mean response and temperature trends. we estimated considerable among-site variation in total bat activity after adjusting for temperature and seasonal effects, with site-specific means varying approximately 3.3-fold (1 sd) and among-year variation within a site of approximately 1.8-fold (fig 4) . estimated temperature trends among sites and among years were generally consistent, with variation in slopes of approximately 12% and 1% respectively. we fit this same model form to three individual species-eptesicus fuscus, myotis lucifugus, and m. californicus-to explore the possibility of seasonal differences in temperature associations among a species less vulnerable to wns [e. fuscus; [43] ], one highly vulnerable (m. lucifugus; [44] and one of unknown vulnerability (m. californicus). we note that since the majority of the bat passes were unidentified, the species-specific estimates may contain substantial unknown biases, as an unknown proportion of the unidentified bat passes consist of these species. our results suggest that winter activity for m. californicus is less strongly associated with mean monthly temperature than e. fuscus, while the reverse is suggested for the fall (fig 5) . we used the model fit to e. fuscus activity data (i.e., detections classified to the species e. fuscus) to illustrate a proposed monitoring approach with a statistical model for a single wnssusceptible species. the model was fit to data from all 41 sites to estimate among-site and among-year variation in activity, but we use the results to generate prediction intervals for four forest sites to depict how a landowner might implement this approach for wns surveillance monitoring. most observed activity data points fell within the 95% prediction interval, as expected (fig 7) . the prediction intervals were constructed to incorporate the estimated among-year variation in activity within a site. future observations of activity for these sites, along with model predictions, could be plotted to compare with prediction intervals. rapidly evolving technologies to passively observe and monitor wildlife concurrent with computational improvements in processing and analyzing large quantities of data are shifting management and research techniques from an animal-in-hand approach to indirect measurements of presence, abundance, diversity, and health [45, 46] . although acoustic monitoring has been implemented to investigate habitat relationships and community composition of bats [36] , uses of this technology are expanding into other objectives. here, we describe how yearround activity rates of bat species derived from acoustic monitoring can be used for surveillance monitoring for wns. we developed a simple and repeatable statistical modeling approach for wns surveillance monitoring with bat activity data collected from a broad geographic and temporal scale prior to known occurrence of wns in the intermountain west. this statistical approach can be applied elsewhere and may be particularly relevant for geographic locations where prominent hibernacula are inaccessible, rare, or unidentified [18] . further, our long-term and year-round collection of acoustic data for bats expands on limited knowledge of activity rates of species through time, seasons, across locations, and prior to wns. very little is known about the winter ecology of many species, specifically in western north america because of the limited sampling that has occurred during that season. technological advances in passive monitoring devices and identification software and an interest in wns has contributed to increased monitoring for bats in winter, when previously it was assumed they were rarely active [6, 47] . across our broad selection of 41 forested sites in montana, we documented a diverse community of bats (13 confirmed species), including at sites with a history of forest management. several species maintained some level of activity through winter, despite cold temperatures that averaged -1.0˚c in this season, which was below temperatures where at least some insect prey could sustain flight [48] . as expected, bat activity was positively related to warmer temperatures, although the strength of the relationship varied by season, with the strongest relationships with temperatures in spring and fall. in more temperate climates, other species of bats also displayed year-round activity that varied by season, sites, and species, including both migratory and non-migratory bats [2, 6] . the implications of winter activity of bats is not well-understood, particularly as it relates to body condition and energy stores or susceptibility to wns [6] . the ecology of bats infected with pd may lead individuals to change behaviors, such as higher activity rates during winter when bats typically use torpor to conserve energy [6] . aberrant behaviors by bats, including daytime activity out of roosts or hibernacula and during subfreezing temperatures increased after detection of wns in the southeastern u.s. [6] . however, the effects of winter bat activity on individual fitness have not been well-quantified. in north carolina, it was posited that year-round activity of typically migratory bats, including l. borealis, whose non-migratory behaviors could increase survival by reducing mortality from threats during migration, such as strikes from wind turbines [2] . alternatively, year-round activity by bats could be energetically costly if food resources do not meet metabolic demands and reduce reproductive output by individuals [2] . where underground caves or human structures are uncommon as hibernacula (e.g., the intermountain west), bats may congregate in smaller numbers in rock crevices during winter ( [49] ; unpublished data from this study). these wintering behaviors may provide protection against bat to bat transmission of pd. however, talus slopes can provide suitable temperature and humidity profiles for growth of pd, albeit below optimal levels [50] . the variability of relationships among prey availability, yearround activity, individual fitness, and susceptibility to wns remain a knowledge gap for north american bats [51, 52] . in addition to describing presence and seasonal activity rates of bats across forested sites in montana, we developed a process for using acoustic monitoring for disease surveillance that can be applied elsewhere. whereas approaches to incorporate uncertainty in estimates of population trends from bat count data in hibernacula are established, less information is available for managers to identify early-warnings from acoustic monitoring data [18] . here, we suggest the same basic framework that we used for the descriptive model of bat activity could be applied to an acoustic monitoring program aimed at identifying changes in bat activity consistent with wns. the approach could be used for single or multiple species and on individual sites or a collection of sites. additionally, the modeling approach is useful for detecting aberrant behaviors that could be associated with wns, including higher than normal activity rates during seasons where bats should be in torpor, or low activity rates due to population-level declines in bats. the general approach we consider is as follows: 1. collect baseline (pre-wns) data within a season or seasons of interest, and across several years and optionally across several sites. 2. fit a mixed-effects model to the data, incorporating variation with temperature, among years and among sites. optionally include other covariate information predictive of bat activity that can be easily gathered. 3. generate a population or site-level prediction interval that includes among-year variation. 4. monitor the collection of new data for excessive departures from the model prediction interval. in particular, look for bat activity in excess of the predicted range during winter months followed by bat activity below the predicted range during the following spring and summer [53] . we emphasize that our modeling approach was descriptive in nature and that we only recorded bat activity during nighttime. however, the approximately log-linear relationship with mean monthly temperature was strong in our dataset and may be useful in other investigations of bat activity. bat activity also showed approximately log-linear relationships with mean daily-max and daily-min temperatures. the non-random selection of sites included in our analysis limits the interpretation of the estimated model random effects or their application to other populations of sites. another limitation of species-specific results in this study relates to the large proportion of bat detections where no species label was assigned, where unknown biases could impact model estimates. we suggest that future site-specific surveillance models may be improved by including acoustic monitoring in daytime and by obtaining more high-quality species-specific call data with updated microphone technology [6] . we used prediction intervals to characterize expected ranges of among-year activity at the site level. one advantage of this approach in a monitoring program is that one set of intervals can be applied across multiple years without the need for annual refitting, provided suitable baseline data were used for model fitting. it is important to note that prediction intervals for mixed-effects models can be defined for any level of the random-effects structure [41] , with the appropriate level dependent on the end-use application. alternatives to prediction intervals could be employed in a monitoring program, including refitting a model each year to directly compare new data with a reference time period. currently, most conservation and management actions related to wns in north america are related to known hibernacula. for regions where large hibernacula are uncommon or rare, but where bats may still be susceptible to this threat, alternative approaches are necessary to ensure early detection of wns outbreaks and prevent further losses with targeted management actions. we propose a proactive and simple monitoring approach that could assist in managing risk of further loss of bat populations, which is especially relevant given current restrictions on field research due to concerns about transmission of covid-19 from humans to north american bats and among humans [54] . deploying passive arus to quantify bat activity, developing a predictive model of activity rates for one or more sites (including managed and unmanaged forest), and examining future data for deviations from these prediction intervals can all assist with a rapid response, including wns confirmation that could assist in recovery efforts. strong geographic and temporal patterns in conservation status of north american bats winter activity of coastal plain populations of bat species affected by white-nose syndrome and wind energy facilities white-nose syndrome: the devastating disease of hibernating bats in north america wing pathology of white-nose syndrome in bats suggests life-threatening disruption of physiology frequent arousal from hibernation linked to severity of infection and mortality in bats with white-nose syndrome winter behavior of bats and the progression of white-nose syndrome in the southeastern united states endangered and threatened wildlife and plants; 4(d) rule for the northern long-eared bat bats and white-nose syndrome 2020 determinants of pseudogymnoascus destructans within bat hibernacula: implications for surveillance and management of whitenose syndrome spread of white-nose syndrome on a network regulated by geography and climate fungus that causes white-nose syndrome found in eastern montana bat-killing disease white-nose syndrome confirmed east of the cascade range in washington first detection of bat white-nose syndrome in western north america. msphere activity following arousal in winter in north american vespertilionid bats western crevice and cavity roosting bats winter foraging of silver-haired and california myotis bats in western washington a review of bat hibernacula across the western united states: implications for white-nose syndrome surveillance and management improved analysis of long-term monitoring data demonstrates marked regional declines of bat populations in the eastern united states bently wigley t. site occupancy of foraging bats on landscapes of managed pine forest use of modified water sources by bats in a managed pine landscape bats in forests: what we know and what we need to learn bat activity in harvested and intact forest stands in the allegheny mountains us protected lands mismatch biodiversity priorities a macroecological perspective on strategic bat conservation in the u bat response to prescribed fire and overstory thinning in hardwood forest on the cumberland plateau effects of fire and its severity on occupancy of bats in mixed pineoak forests department of agriculture, forest service, intermountain forest and range experiment station ecoregions of the conterminous united states forest regions of montana land cover/land use theme montana natural heritage program montana bat and white-nose syndrome surveillance plan and protocols 2012 -2016 a directory of reports on long-term acoustic monitoring for bats at sites across the northern rocky mountains and great plains do you hear what i hear? implications of detector selection for acoustic monitoring of bats weather conditions determine attenuation and speed of sound: environmental limitations for monitoring and analyzing bat echolocation a plan for the north american bat monitoring program (nabat) on the importance of articulating assumptions when conducting acoustic studies of habitat use by bats montana bat and white-nose sydrome surveillance plan and protocols r: a language and environment for statistical computing. r foundation for statistical computing statistial intervals: a guide for practitioners mertools: tools for analyzing mixed effect regression models white-nose syndrome response team. bats affected by wns 2020 the resistance of a north american bat species (eptesicus fuscus) to white-nose syndrome (wns) an emerging disease causes regional population collapse of a common north american bat species computational sustainability: computing for a better world anda sustainable future how to learn to stop worrying and love edna monitoring winter acoustic activity of bats in montana effects of the environmental temperature on aedes aegypti and aedes albopictus mosquitoes: a review. insects winter bat activity in the canadian prairies unsuspected retreats: autumn transitional roosts and presumed winter hibernacula of little brown myotis in colorado frequent arousals from winter torpor in rafinesque's big-eared bat (corynorhinus rafinesquii) identifying research needs to inform white-nose syndrome management decisions. conservation science and practice going, going, gone: the impact of white-nose syndrome on the summer activity of the little brown bat (myotis lucifugus) fish & wildlife health committee and wildlife resource policy committee, bat working group, guidance for bat-related activities in response to covid-19. association of fish and wildlife agencies key: cord-317813-sisfxdso authors: banskar, sunil; bhute, shrikant s.; suryavanshi, mangesh v.; punekar, sachin; shouche, yogesh s. title: microbiome analysis reveals the abundance of bacterial pathogens in rousettus leschenaultii guano date: 2016-11-15 journal: sci rep doi: 10.1038/srep36948 sha: doc_id: 317813 cord_uid: sisfxdso bats are crucial for proper functioning of an ecosystem. they provide various important services to ecosystem and environment. while, bats are well-known carrier of pathogenic viruses, their possible role as a potential carrier of pathogenic bacteria is under-explored. here, using culture-based approach, employing multiple bacteriological media, over thousand bacteria were cultivated and identified from rousettus leschenaultii (a frugivorous bat species), the majority of which were from the family enterobacteriaceae and putative pathogens. next, pathogenic potential of most frequently cultivated component of microbiome i.e. escherichia coli was assessed to identify its known pathotypes which revealed the presence of virulent factors in many cultivated e. coli isolates. applying in-depth bacterial community analysis using high-throughput 16 s rrna gene sequencing, a high inter-individual variation was observed among the studied guano samples. interestingly, a higher diversity of bacterial communities was observed in decaying guano representative. the search against human pathogenic bacteria database at 97% identity, a small proportion of sequences were found associated to well-known human pathogens. the present study thus indicates that this bat species may carry potential bacterial pathogens and advice to study the effect of these pathogens on bats itself and the probable mode of transmission to humans and other animals. of previous reports of bacterial identification from bat guano or the bats intestinal content 17, 18 there remains an immense scope of identifying additional bacterial species from bats. hence, a serious need of detailed and thorough study of bat guano associated bacteria was experienced. such studies are important to catalogue the various bacteria from bats, so that their potential role in zoonosis can be recognized, interpreted and can be comprehended during the zoonosis. metagenomic studies using 16 s rrna gene sequencing have been used to characterize gut microbial communities of different bats species 19, 20 . a report from philips et al. 19 mentioned that, the herbivory dietary strategy and the reproductively active bats carried more diverse microbiota than carnivore and reproductively inactive individuals 19 . similarly, the carrillo-araujo et al. reported the diet to be a primary factor to define the gut microbiome of bats 20 . further, our previous study reported the considerable similarity of microbial communities of frugivorous and insectivorous bats indicating that there was dietary overlap among the bats of different feeding habits 21 . in addition to the diet, the decaying guano communities were also explored revealing a higher abundance of bacterial communities involved in nitrogen cycling 22 . interestingly, using the same approach, veikkolainen et al. 12 reported and confirmed the presence of important human bacterial pathogen bartonella mayotimonensis 12 . there are more than 1230 bats species worldwide 21 which should be explored for their microbial communities to understand a comprehensive 'bats' microbiome' from every aspect including host-microbe relationship as well as their possibility of carrying putative bacterial pathogens. this study deals with the isolation and identification of general bacterial communities and potential bacterial pathogens present in rousettus leschenaultii guano. further, to evaluate the pathogenic potential, virulence genes were positively identified from the most commonly isolated bacteria from the gut microbiome i.e. escherichia coli. additionally, 16 s rrna gene sequencing was performed using ion torrent pgm to identify the bacterial communities and screened for the presence of putative human pathogens. therefore, this study discourages the human interference in the wildlife which otherwise may cause irreparable loss to the ecosystem and severe human health hazards. bat guano collection. fresh bat guano pellets were collected at the robber's cave, mahabaleshwar, maharashtra, india. collection plates were left inside the cave for two hours to capture the guano dropping. this way, a total of 31 fresh guano pellets were collected from which 17 guano of sufficient quantity and free from contaminations were used for the further study. bat identification. sequence analysis of the cytochrome b (cytb) gene amplified from total extracted dna of the guano pellets revealed that the collected fecal pellets were from a single bat species i.e. rousettus leschenaultii, a predominant bat species reported from the robber's cave 23 . this confirmed that the collected guano pellets were from single bat species. total viable counts and identification of bacteria. the total viable count of bat guano was performed, it ranged from 1.71 × 10 7 to 3.34 × 10 10 cfus/gram of the guano among four different bacterial media used. all bacterial isolates were identified by 16 s rrna gene sequencing followed by the blast analysis at ncbi. a total of 922 bacterial isolates from bat guano and 143 bacterial isolates from cave environment samples (total 1065) were identified (the entire list of 1065 bacteria identified is provided in supplementary table s1 ). all the isolates showed 99% or above similarity except five isolates. all 16s rrna gene sequences of isolates were further analyzed for media-wise genus level distribution. maximum numbers of bacterial genera (i.e. 26) were obtained using luria hiveg agar (la) media followed by zobell's marine agar (zma) (21 genera), arret and kirshbum agar (ak) (19 genera) then streptomycetes isolation agar (str) (12 genera). similarly, la was found to be the most efficient in capturing unique bacterial genus (7) followed by zma (3) then ak (2) and str (1) (fig. 1) . thus, total 105 species (supplementary figure s2 ) belonging to 37 different genera (fig. 2 ) and 4 bacterial phyla i.e. proteobacteria, firmicutes, actinobacteria and bacteroidetes were isolated from bat guano. the most dominating bacterial genera obtained was enterobacter (151 isolates) followed by enterococcus (134 isolates) and escherichia (133 isolates). three unique bacterial genera i.e. aquitalea, cedecea and pontoea were obtained from cw (cave stream water) sample but not obtained in any other samples whereas only one bacterial genera i.e. staphylococcus was obtained from bcs (bats' cave ceiling soil) sample (supplementary figure s3 ). literature survey for bacterial pathogens. as taxonomic affiliation of most of the isolates (~60%) was proteobacteria and majority of pathogens belong to phylum proteobacteria, we speculated that guano could contain a higher abundance of pathogenic bacteria. hence, a detailed literature survey was performed for all 105 bacterial species obtained from r. leschenaultii guano, to identify their pathogenic potential. only bacteria belonging to risk group-2 or above were considered a pathogen. this survey revealed that only two bacteria belongs to rg (risk group) 2 (table 1) , although other 54 bacterial species were also reported to have association with different opportunistic infections. to cause an infection successfully, bacteria must possess some virulence factors. in this study, e. coli was represented by more than 10% of total isolates. although, it is a common component of a healthy gut microbiome, many virulent strains of e. coli (pathotype) also exist. hence, 96 e. coli isolates were assessed for their pathogenic potential by the identification of eight virulence genes (supplementary table s4 ). this assessment revealed 40.6% of isolates to be ibea and hlya gene positive (indicative of extraintestinal pathogenic e. coli (expec)) and 31.3% were east1 gene positive (indicative of enteroaggregative e. coli (eaec)). additionally, one isolate possessed the enterotoxigenic e. coli (etec) heat-stable enterotoxin-b (estii) gene. interestingly, one of the e. coli isolates was entero-hemorrhagic (ehec) scientific reports | 6:36948 | doi: 10.1038/srep36948 bearing shiga toxin-ii (stx 2 ) gene which additionally co-harbored east1 gene. seven other cultivated isolates were found positive for ibea and east1 genes ( fig. 3) while none of the e. coli isolates belonged to enteroinvasive (eiec) pathotype (ipah negative). on comparison with the previous studies on avian pathogenic strains of e. coli 24 , a higher prevalence of the ibea gene was revealed in this study (37.5% vs previous ~26%). nonetheless, e. coli strains isolated from human vagina and neonatal meningitis appears to have similar prevalence of ibea gene 25, 26 . in present study the prevalence of east1 gene in e. coli strains was quite similar to the previous study (31.3%) in weaned pigs with the diarrheal infections 27 . thus, a comparable frequency of virulent genes was observed. ion torrent sequence analysis. further, to get an in-depth overview of total bacterial communities of r. leschenaultii guano, 16s rrna gene sequencing was performed using ion torrent pgm. the generated sequences were analyzed using qiime (quantitative insight into microbial ecology) package 28 . the results indicated the presence of 31 different bacterial phyla in bat guano which revealed the huge uncultivated bacterial communities present in the bat guano (fig. 4) . samples g15 and g28 showed the abundance of proteobacteria (~98%) while g9 had the abundance of actinobacteria (~78%) and g26 and g8 had the abundance of firmicutes (92% and 55% respectively). the cave ground surface soil (cgs) sample showed the abundance of proteobacteria (~54%) followed by bacteroidetes (~28%) and actinobacteria (~7.4%). further, a huge inter-individual variation in bacterial communities was also observed. the various indices of alpha diversity indicated the cgs to be the most diverse sample, whereas, among fresh guano samples g9 had maximum bacterial diversity (supplementary figures s5 and s6 ). further, computation of core microbiome from ion torrent sequences of fresh guano revealed the presence of five bacterial phyla i.e. proteobacteria, tenericutes, candidate division tm7, firmicutes and actinobacteria but in different proportions (fig. 5, supplementary figure s7 ). bat guano microbiome comparison. two previous bat microbiome studies were compared with the present bat guano microbiome study. to accomplish this, fastq files were retrieved from sra (sequence read archive) ncbi, quality filtered and analyzed using qiime pipeline. the analysis revealed that these three bat microbiome studies are quite different in terms of composition and the relative proportion of bacterial communities. only three phyla i.e. chlamydiae (~63.6%), proteobacteria (~27.3%) and bacteroidetes (~10%) were represented in veikkolainen et al. 12 study (location: finland; bat species: myotis daubentonii). on contrary, the study by de mandal et al. 22 (location: india; bat species: not available) and present study (location: india; bat species: r. leschenaultii) comprised of 21 and 27 bacterial phyla predominated by actinobacteria (~38.7%) and firmicutes (~42.1%) respectively (fig. 6 ). in an attempt to identify the core bacterial microbiome, at a broader taxonomic level, two bacterial phyla were identified in all three studies (supplementary figure s8) . as sample cgs of the present study and composite guano samples used in de mandal et al. study 22 are representatives of decaying guano hence a comparison between them was performed. the beta diversity analysis using the jaccard distance revealed that the cgs and the composite guano contained different bacterial communities which in turn were different from the rest of fresh guano samples (fig. 7 ). pathogen identification from ion torrent sequences. the presence of potential bacterial pathogens in culture-based study prompted us to assess ion torrent data for their presence. hence, ion torrent sequences were searched against the available human bacteria pathogen database 29 . upon analysis, at 97% sequence identity cutoff and ≥ 99% query coverage 2692 (about 0.6504%) sequences showed identity to the well-known pathogens belonging bats are crucial and the integral part of healthy ecosystem, serving it in many ways such as chiropterophily, a mode of pollination exclusively carried by the bats. apart from their ecological services, the bio-fertilizers made from the bat guano serve as the rich source of nutrients for better crop production, farming and gardening. bats also has promising prospects for human health. 'draculin' (desmoteplase), a glycoprotein from the vampire bat's saliva, a natural anti-coagulant, is currently in trial to be used as the medication to treat patient of ischaemic stroke. it can reopen the clogged blood vessels so that the damage can be prevented 30 hence, it can prove to be a life saver. despite grandness of bats their every aspect including their gut microbial communities need to be addressed. the present study has attempted to identify the bacterial communities present in rousettus leschenaultii's (a frugivorous bat species) guano using culture-based approach and has led to the identification of about a thousand bacteria belonging to more than a hundred different bacterial species. a majority of them have been isolated for the first time from bats. since all cultivated bacteria were identified using nearly full length 16 s rrna gene sequencing, therefore, identity was ensured. additionally, ion torrent sequencing of 16 s rrna gene provided information about the general bacterial communities with specific emphasis on pathogens among the total communities present in bat guano. our observation of bacterial count for the r. leschenaultii guano is in the range of 1.71 × 10 7 -3.34 × 10 10 cfus/gram, which is higher than previously reported count i.e. 2 × 10 5 for the guano of myotis sp. of bats; possibly the use of fresh guano here as against the old and dried pellet used previously could be the reason 31 . the reported bacterial count for the stomach and intestinal contents of different species of bats was in the range of 10 5 -10 15 cfus 10, 15, 16 indicating that the main differences, apart from the sample itself, could be because of inter-individual variation due to host species, diet, body size, specificity and geographical location 32 . so far, most studies have used one or two bacteriological media to identify the bacterial communities associated with bats 15,31 , hence were confined. few of such studies have reported about 17, 26 and 25 bacterial species from 4, 6 and 10 species of bats respectively 17,32,33 . in contrast, using four different bacteriological media, we were able to acquire four different bacterial phyla belonging to 37 genera and 105 different species, thus, signifying the importance of traditional approach in microbial communities' exploration. this fact is further strengthened by the presence of unique 66 bacterial species not reported earlier from bats. interestingly, only a single genus of bacteria i.e. staphylococcus was found in bcs. therefore, the possibility of bcs contaminating the guano was excluded and assumed that all bacterial species isolated from bat guano are autochthonous community. such rock dwelling staphylococcus has been associated with manganese mobilization in basalt rocks 34 , hence, its high abundance in bcs is explained. while guano shared some bacterial genera with those observed in cw and cgs; the possibilities of these contaminating the guano was taken care during the sampling. the next generation sequencing platforms are rapidly changing the ways of characterizing microbial community from various sources. metagenomic studies using these sequencing technologies have contributed enormously to our understanding of structure and functioning of a given system. accordingly, using ion torrent sequencing, it was observed that two guano samples has more than 98% sequences representing the proteobacteria, one showed the higher abundance of actinobacteria and another one has the firmicutes in abundance. previous studies have demonstrated the huge variation in gut bacterial communities 22 even in genetically similar human individuals 30 . therefore, the major reason appears to be the host specificity, individual's diet, health and physiological state 27 . recently, bat guano microbiome was reported from the composite guano collected from cave floor 22 , hence, no information was available about the host bats. conversely, in addition to multiple guano microbiomes, we identified the bat to be rousettus leschenaultii, a most dominating frugivorous bat in robber's cave 23 . additionally, alpha diversity and rarefaction analysis indicated the significant microbial enrichment in cgs sample indicating that the decaying process may have led to the increased bacterial diversity 35 . the decaying or the disintegration process is affected by the various physical and environmental factors. additionally, the biogeochemical processes crucial for the recycling of the organic and inorganic matters are primarily carried out by the microorganism which causes the change in ph (due to the release of ammonia) and nutritional contents which further allow the growth of diverse bacteria 35 . hence, the enrichment of bacterial communities in the decaying guano is explained. further, the underlying soil may also cause an increase in the microbial load. the comparison between composite guano and samples from this study revealed that both cgs and composite guano are quite dissimilar in microbial composition from each other as well as rest of the fresh guano samples which further indicates the bacterial communities of decaying guano is rather different from the fresh guano. we speculate that the differences in two different decaying guano could be because of their origin, host bats' species, duration of the decaying process and other physicochemical properties leading to the different trajectories of development of microbial communities. at the broader taxonomic assignments, we were able to detect 31 bacterial phyla as against only 18 phyla in composite guano samples 22 . while this holds true, the core microbiome contained only five different phyla: actinobacteria, candidate division tm7, firmicutes, proteobacteria and tenericutes, which indicates probably all fresh guano of r. leschenaultii are characterized by the presence of these five phyla. we believe that the other bacterial phyla that we were able to recover could be derived from the consumed food material, as bats have a huge diet which remains only transiently in the gastrointestinal tract 10 . further, on a comparison between ion torrent sequencing results and culture based finding, it was observed that three major bacterial phyla i.e. actinobacteria, firmicutes and proteobacteria were shared confirming the stable nature of these bacterial phyla in bat gut environment. a higher abundance of actinobacteria was observed in ion torrent sequence data, whereas, the higher relative proportion of firmicutes and proteobacteria was obtained in culture, suggesting fewer actinobacteria were cultivable on the employed media. similarly, due to the stringent growth requirements, tenericutes could not be obtained in culture 36 even though it was one of the members of core communities. furthermore, an absence of bacteroidetes in the core microbiome and its presence in culture indicated the individual variations of guano samples. comparison of the present study with the two previously published microbiome studies revealed that this study reported a higher number of bacterial phyla, probably the fresh guano samples collected for the study appears to be the prime reason. further, in an attempt to find the core microbiome from different microbiome studies indicated that only two phyla i.e. proteobacteria and bacteroidetes are present commonly in three compared studies. the other two recent microbiome studies showed the strong influence of host phylogeny, dietary pattern, physiology and geography on the gut microbial communities of bats 19, 20 . earlier bats have been suspected as the reservoir of the human bacterial pathogens 12 and their guano was considered important in pathogen dissemination 37 . here, two bacterial species i.e. escherichia coli (risk group-i/ii) and staphylococcus aureus (risk group-ii) were cultivated. additionally, 54 bacterial species obtained from guano have been reported to cause various human infections. except acinetobacter johnsonii (cause fish infection) and staphylococcus lentus (cause mastitis in goats) others were mainly found involved in the sepsis, urinary tract infections (utis) and other infections especially in immune compromised individuals. in the present study, a majority of the cultivated bacteria belong to the family enterobacteriaceae and most of these isolates were identified as escherichia coli (116, ~12.6%), a well-known commensal gut inhabitant and opportunistic rg-i/ii pathogen 38 . hence, a high percent of expec and eaggec pathotype in r. leschenaultii guano could be a serious health concern. additionally, shiga toxin producing strain has been linked with the severe outbreak in the germany, leading to more than 4000 cases and more than 50 deaths 39 . these results are quite staggering, indicating that the e. coli recovered from r. leschenaultii guano may prove to be pathogenic. few studies have indicated the presence of virulence genes in wild animals 40, 41 . cabal et al. 42 compared the virulence gene profile of e. coli isolated from cattle, swine and broiler. the results indicated the presence of stx1 and stx2 gene (from both o157:h7 and non-o157:h7 e. coli serotypes) in cattle and swine 42 . similarly, post weaning diarrhea in piglets is a common problem in piggery farms and the major reason appears to the epec/etec carrying different virulence genes especially the heat labile/stable toxins (sta and stb) 43, 44 . recently, even the healthy dairy cows have been reported to carry virulent genes (stx2, st and lt) in e. coli isolated from their dung 45 . additionally, the humans are reported to be the prime reservoirs for eaec, epec and eiec 46,47 , though they remain healthy. hence, commensals like e. coli may carry virulence genes but often do not cause the infections as the appropriate combination of virulent genes, required to cause the infection, is not available 38 . likewise, merely the presence of rg-2 bacteria from the host does not indicate it to be the pathogen as all strains of a species may not be the pathogen. nevertheless, a possibility of gradually acquiring the additional virulence genes, required to cause a successful infection cannot be ignored. in the light of presence of various pathotypes of e. coli, it is essential to discuss other potentially pathogenic bacteria as well. in the discussion that follows numbers in the parentheses (in bold) indicate an abundance of the said bacterial species. the other isolated member of genus escherichia is escherichia fergusonii (16) , a close relative of e. coli which has been isolated from the feces of many warm blooded animals. it has also been recovered from the wound infections, utis, diarrhea etc. 48 and reported to cause the bacteremia in diabetic patient 49 . the other dominating genera cultivated was enterobacter e.g. includes enterobacter asburiae (35) , enterobacter ludwigii (21) , enterobacter cloacae (102) and members of enterococci i.e. enterococcus faecalis (37) and enterococcus hirae (81) which causes various infections especially in immunocompromised individuals [50] [51] [52] [53] . notably, e. faecalis is among the most common species isolated from human clinical samples. both citrobacter koseri (41) and citrobacter freundii (29) are the opportunistic pathogens causing neonatal infections but the neonatal meningitis caused due to c. koseri have a high mortality rate 54 . similarly, serratia marcescens (24) is a common cause of nosocomial, urinary tract and wound infections 55, 56 . relatively fewer gram-positive bacteria were obtained from guano. the main genus among these is staphylococcus. it included staphylococcus aureus (a rg-2 organism), staphylococcus nepalensis (13) and staphylococcus lentus (49) , which are well-known to cause various infections in humans as well as in other animals 57, 58 . therefore, it may pose a threat to the herds of sheep and goats grazing nearby bats' roosting sites. similarly, potential bacterial pathogens were also searched in ion torrent data using the human bacteria pathogen database. this database was created from the 'human pathogenic bacteria virulence factor database' and only the bacterial species whose proteins were experimentally validated were considered for its creation 29 . although, the ion torrent generates the sequences of shorter read length (here ~200 bp), the use of v3 region for the identification ensured maximum coverage of diversity (~96-99%) 59 . in addition, the only sequence which had high query coverage (≥ 99%) and high similarity value (> 97%) during the blast search against the database was considered for the analysis 60 . hence, the possibility of the presence of these pathogens may not be ignored. pathogens identified from ion torrent sequencing includes pseudomonas aeruginosa, and clostridium perfringens etc. p. aeruginosa (240) is a gram-negative pathogen responsible for several nosocomial infections and opportunistic infections 61, 62 . the bacterial species salmonella enterica (347) and shigella flexneri (174) causes gastroenteritis (food poisoning) and shigellosis 63 , respectively. these can be transmitted by the direct contact, fecal or oral route. hence, the consumption of food contaminated with bat guano should be avoided. the other species found scientific reports | 6:36948 | doi: 10.1038/srep36948 i.e. streptococcus pneumoniae (375), enterococcus faecalis (240), staphylococcus aureus (647) which may cause streptococcal pneumonia, meningitis, bacteremia, infectious lesions, neonatal infections and septicemia [64] [65] [66] . the important observation was the presence of sequences affiliated to mycobacterium tuberculosis (38) and corynebacterium diphtheriae (32) . mycobacterium tuberculosis is an airborne disease and spread through the droplet nuclei. similarly, corynebacterium diphtheriae, a causative agent of diphtheria, may cause skin infections and septicemia by the droplets, secretions or direct contact 67 . hence the proper caution should be taken when entering a cave having bats' roosting site. additionally, bartonella henselae (12) , one of the causative agents of bartonellosis (cat scratch disease), has been suspected earlier from bats 11 which can be transmitted to humans by accidental bite or scratches. additionally, it can also be transmitted by the ectoparasites harbored by the infected animals 12 . some sequences were also found affiliated to brucella melitensis (202) which is the most pathogenic brucella species to humans 68 . it principally affects the goats and sheep, hence, the grazing goats and sheep which comes in contact with bats' guano may get infected. hence, these may cause zoonotic infections in humans by direct contact, aerosol inhalation 69 or indirectly by consumption of improperly cooked food products from diseased animals. in conclusion, the present study revealed that each gram of r. leschenaultii's guano contains millions of bacteria, belonging to hundreds of different bacterial species, some of which can be potentially pathogenic to humans. additionally, computed core microbiome revealed five bacterial phyla, three of them are found in the culture-based study. further, a fairly large inter-individual variation in microbiome composition of guano was observed indicating the individual specificity. the virulence gene profiling of cultivated e. coli strains from guano revealed the presence of many virulent genes, though it may not be declared pathogens. this study reports the largest culture-based study from r. leschenaultii bat guano to identify the cultivable bacterial diversity and potential human pathogenic bacteria. thus it is strongly advised that the humans should not interfere with the wild life otherwise it may cause huge loss to the environment and health related issues to the humans. sampling site and sample collection. guano samples were collected from the 'robbers cave' (17°52′ 57"n and 73°40′ 35"e) situated in the basalt rock structure of western ghats, of maharashtra state, india. collection plates were placed on cave ground surface under the roosting bats' colony and were left for about 2 hours. after 2 hours individual guano pellets were observed and collected separately. thus, a total of 31 guano pallets were collected and kept at 4 °c for transportation to the laboratory for further processing. only 17 guano samples with sufficient quantity were used for the bacterial isolation. other potentially interfering and guano contaminating factors were also included in the study e.g. cave water (cw, from a stream which flows from a side of the cave, cave ceiling soil (bcs, which continuously fall on the ground and hence in guano pellets) and cave ground surface soil (cgs). sample processing. about the half quantity of all selected samples was suspended in 1 ml of water and mixed thoroughly. the supernatant was serially diluted up to 10 −5 followed by plating of dilution 10 −4 in different media plates (dilutions 10 −4 and 10 −5 were used for la media) and incubation for 16-18 hours at 37 °c incubators. post incubation plates were observed and total colony forming units (cfus) were counted. then total viable counts were calculated by considering the number of cfus observed, dilution factor, the volume of supernatant (inoculum) and the weight of guano used. subsequently, about 21 random colonies of guano sample from each media were sub-cultured in their respective medium. post-incubation a loop-full of each colony was suspended in te (tris-ethylenediamine tetra acetate) buffer (ph 8.0) to use further for dna isolation. the remaining guano sample was used for the total bacterial community dna extraction for ion torrent sequencing. bacterial dna isolation and species identification. for bacterial identification, dna of pure cultures was extracted using purelink ® pro 96 genomic dna purification kit (invitrogen, inc. usa). pcr amplification of 16 s rrna gene was performed in triplicates using 10x pcr buffer, 0.2 mmol/l of dntps, 0.5 units of taq polymerase (bangalore genei) and 10 picomols of each primers 8 f (5′-aga gtttgatcctggctcag -3′) and 1492 r (5′-cggttaccttgttacgactt-3′). pcr parameters include initial denaturation at 95 °c for 5 min followed by 1 min at 95 °c denaturation, 55 °c annealing and extension at 72 °c for 35 cycles followed by final extension at 72 °c for 10 minutes then incubation at 4 °c. pcr amplified products were gel checked for positive products. the positive products were purified using 20% peg-nacl (polyethylene glycol-nacl). the purified products were processed and sequenced using abi 3730xl dna analyzer (applied biosystems, usa). to obtain nearly full-length sequence (1.5 kb) of bacterial 16 s rrna gene, internal primers, 704 f (5′-gtagcggtgaaatgcgtaga-3′) and 907 r (5′-ccgtcaattcmtttgagttt-3′) were also used. the sequences were concatenated using chromaspro v1.4 (http://www.technelysium.com.au/chromaspro.html), followed by blast analysis at ncbi. a blast hit, with sequence similarity ≥ 97% and query coverage ≥ 99 is considered as the species of the isolate. identification of bacterial pathogens. only bacterial species belonging to risk group-2 or above were considered pathogen in present study (nih guidelines; http://osp.od.nih.gov/sites/default/files/nih_guidelines. html#_to c446948381). table s4 ) were used to identify the five different pathotype of the e. coli 33 . the dna extracted from the 96 e. coli isolates was used to amplify and check the pathotype of e. coli. the pcr was performed using 10x pcr buffer, 0.2 mmol/l of dntps, 0.5 units of taq polymerase (thermo scientific inc. usa) and 10-15 picomols of respective primers. pcr parameters include initial denaturation at 95 °c for 5 min followed by 1 min at 95 °c denaturation, different annealing temperatures 33 for 45 seconds and extension at 72 °c for 1 minutes for 35 cycles followed by a final extension at 72 °c for 10 minutes then incubation at 4 °c. bat guano community dna extraction and pcr amplification of 16s rrna gene. total community dna extracted from bat guano using stool dna isolation kit (qiagen, the netherlands), was used for the ion torrent analysis. the extracted dna was quantified using nanodrop (nanodrop, thermo scientific, usa), followed by pcr amplification of v3 region of 16 s rrna using hi-fidelity amplitaq gold (invitrogen inc., usa) and eubacterial universal primers 341 f (5′-cctacgggaggcagcag-3′) and 518 r (5′-attaccgcggctgctgg-3′). resulting pcr products were purified using agencourt ampure xp dna purification beads (beckman coulter, usa) which were then subjected to end repair. the blunt ended products were used as a substrate for sample-specific barcode and adapter ligation reaction as per the manufacturer's instructions. prior to the sequencing, all amplicons were assessed for size distribution and molar concentrations using bioanalyzer 2100 (agilent technologies, usa). the concentration of all the amplicons was adjusted to lowest dna concentration and subsequently, amplicons were pooled in an equimolar ratio and diluted so as to obtain the pooled amplicons of around 26 pm. the pooled amplicons were attached to ion sphere particles (isps) and used for emulsion pcr using ionxpress template-200 kit using iononetouch system. next, template-positive isps were enriched using iononetouch es system. the enriched isps were then loaded onto 316 chip and sequencing was performed on ion torrent personal genome machine (life technologies, usa) for 130 cycles. ion torrent data analysis. barcode specific fastq files were processed using mothur pipeline 57 to obtain fasta and quality file. these two types of the files were quality filtered using following conditions: size 150-200 bp, homo-polymer max. 5, ambiguity max 0, and average quality score 20. this way we were able to obtain 874999 good quality sequences. all these reads were pooled into a single fasta file and analyzed using qiime (quantitative insight into microbial ecology) package. briefly, otus picking was done using open reference approach at 97% sequence similarity cutoff. a representative sequence of each otu was picked up and lowest possible taxonomic rank was assigned to each of them by using rdp classifier (v2.7) 71 and silva database (silva_111) 72 as reference. in order to assess the relationship between sequencing depth and discovery of new otus, rarefaction analysis was performed. further, diversity measurements were made using alpha diversity indices such as shannon, simpson and chao1. for computing core otus, cgs sample was excluded and a core otu was defined as an otu present in all (100% of) the samples. microbiome data was retrieved from sra (sequence read archives) ncbi in fastq format to compare present bat guano microbiome studies with the previously published microbiome studies. thus including ours, a total three studies i.e. de mandal et al. 22 , veikkolainen et al. 12 were compared. all the files were quality filtered using mothur pipeline and only good quality sequence having a length more than 100 bases and homopolymers allowed no more than 5 bases and having 0 (zero) ambiguity were used in the comparison. later all the filtered, good quality sequences were compiled into a single fasta file and processed using qiime pipeline. a closed reference approach using silva_111_database 72 as reference was used to pick otus so that different studies which has utilized the different region of 16 s rrna gene can be compared. pathogen identification from ion torrent sequencing data. bacterial pathogen database 29 was used for the search of the bacterial pathogens from ion torrent sequencing reads. all the 16 s rrna gene sequences of bat guano (excluding cgs) were compiled into a single fasta file (containing 441560 sequences) and searched against the above mentioned database for the sequence similarity using ncbi-blast-2.2.30+ algorithm 73 . thus, every sequence was assigned the closest hit. only blast hit showing query coverage ≥ 99% and maximum identity with maximum score value and lowest e-value was considered. the number of sequences having blast hit with ≥ 97% identity were short-listed and calculated the proportion of sequences affiliated to the bacterial pathogens. statistical analysis. statistical analysis was performed in graphpad prism (v6.0) and the venn diagram was prepared in the venny 2.0 (http://bioinfogp.cnb.csic.es/tools/venny/). the heat maps were constructed using mev software. fruit bats (chiroptera: pteropodidae) as seed dispersers and pollinators in a lowland malaysian rain forest seed dispersal by bats and birds in forest and disturbed habitats of chiapas economic importance of bats in agriculture bats: important reservoir hosts of emerging viruses update: outbreak of severe acute respiratory syndrome-worldwide link to mers virus underscores bats' puzzling threat virus disease in west africa -the first 9 months of the epidemic and forward projection 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characterization of surface-located pullulanase of streptococcus pneumoniae endocarditis and biofilm-associated pili of enterococcus faecalis brucellosis: an overview establishment of systemic brucella melitensis infection through the digestive tract requires urease, the type iv secretion system, and lipopolysaccharide o antigen cryptic diversity in european bats naive bayesian classifier for rapid assignment of rrna sequences into the new bacterial taxonomy the silva ribosomal rna gene database project: improved data processing and web-based tools basic local alignment search tool escherichia coli endocarditis: seven new cases in adults and review of the literature virulence factors in escherichia coli urinary tract infection staphylococcus aureus infections s.b. and y.s.s. designed the study. s.b. did the sampling from the field, bacteria isolation and identification, all pre-sequencing work for the ion torrent sequencing and virulence gene profiling. s.s.b. and m.v.s. assisted in ion torrent sequencing. m.v.s. assisted in initial ion torrent data analysis. s.b. and s.s.b. performed the data analysis and manuscript writing. s.p. assisted in the conceptualization of the study. all authors commented on the final draft. key: cord-327534-f2wvh6la authors: zhou, peng; cowled, chris; mansell, ashley; monaghan, paul; green, diane; wu, lijun; shi, zhengli; wang, lin-fa; baker, michelle l. title: irf7 in the australian black flying fox, pteropus alecto: evidence for a unique expression pattern and functional conservation date: 2014-08-06 journal: plos one doi: 10.1371/journal.pone.0103875 sha: doc_id: 327534 cord_uid: f2wvh6la as the only flying mammal, bats harbor a number of emerging and re-emerging viruses, many of which cause severe diseases in humans and other mammals yet result in no clinical symptoms in bats. as the master regulator of the interferon (ifn)-dependent immune response, ifn regulatory factor 7 (irf7) plays a central role in innate antiviral immunity. to explore the role of bat irf7 in the regulation of the ifn response, we performed sequence and functional analysis of irf7 from the pteropid bat, pteropus alecto. our results demonstrate that bat irf7 retains the ability to bind to myd88 and activate the ifn response despite unique changes in the myd88 binding domain. we also demonstrate that bat irf7 has a unique expression pattern across both immune and non-immune related tissues and is inducible by double-strand rna. the broad tissue distribution of irf7 may provide bats with an enhanced ability to rapidly activate the ifn response in a wider range of tissues compared to other mammals. the importance of irf7 in antiviral activity against the bat reovirus, pulau virus was confirmed by sirna knockdown of irf7 in bat cells resulting in enhanced viral replication. our results highlight the importance of irf7 in innate antiviral immunity in bats. bats have been implicated in the spillover of many deadly viruses including rabies, henipaviruses (hendra and nipah), ebola virus, and the coronaviruses (cov): severe acute respiratory syndrome (sars-cov) and the recently emerged middle eastern respiratory syndrome virus (mers-cov), all of which impose a significant threat to human health [1, 2, 3, 4, 5, 6] . as natural hosts, bats rarely show clinical signs of disease during infection [7] . how bats co-exist with viruses and the role of the bat innate immune system in controlling viral replication remain poorly understood [8] . identifying the mechanisms responsible for controlling viral replication in bats has profound implications for the development of therapeutic strategies targeting viral infections in humans and other species. one of the most important early anti-viral defenses in mammals is the ifn system, which not only provides pivotal protection immediately following infection but also shapes the adaptive immune response [9] . of the three ifn families discovered, type i (including a and b) and type iii (l) ifns respond directly to viral infection. due to the importance of ifns in controlling viral replication, the regulation of the ifn response has been extensively studied in humans and other mammals. key to the regulation of ifn production and signaling is the ifn regulatory factor (irf) transcription factor family. the irf family consists of nine members which share functional and structural characteristics. however, only irf1, irf3, irf5 and irf7 have been implicated as positive regulators of type i ifn transcription, and only irf3 and irf7 are designated as antiviral irfs [10, 11] . since their first discovery within the biological context of epstein-barr virus latency, irf7 was identified as the master regulator of the type i ifn-dependent immune response, and perhaps that of type iii ifn as well [12, 13, 14] . irf7 is expressed only at low levels in most cells but is constitutively expressed in certain immune cells such as plasmacytoid dendritic cells (pdc) which specialize in ifn production. correspondingly, the tissue distribution of human irf7 is restricted to immune tissues which contain large numbers of specialized immune cells including spleen, thymus and peripheral blood lymphocytes whereas non-immune tissues including the intestine and colon express almost undetectable levels of irf7 [12] . although irf7 is expressed at low levels in most cell types, it is induced strongly by type i ifn mediated signaling in all cells [15] . interestingly, multiple fish species (japanese flounder, crucian carp, mandarin fish, snakehead fish and atlantic salmon) have been demonstrated to express irf7 constitutively in all tissues including both immune and non-immune tissues [16, 17, 18, 19, 20] . viral sensing either by toll like receptors (tlrs) or retinoic acid-inducible gene 1 (rig-i)-like receptors can result in the activation of irf7 and subsequent induction of ifns [21, 22] . all tlrs with the exception of tlr3 activate irf7 through the adaptor protein, myd88 (myeloid differentiation primary response gene 88) through the myd88-dependent pathway. myd88 forms a complex with the kinases irak-4 (interleukin 1 receptor associated kinase 4), irak-1 and traf-6 (tnf receptor associated factor). this complex binds directly to irf7 leading to ubiquitination by traf-6 and phosphorylation by irak1 or ikk-1 (ikb kinase-1) and translocation from the cytosol to the nucleus where irf7 binds to promoter elements inducing ifn production [21, 23] . tlr3 and tlr4 activate irf7 through the myd88-independent pathway through the adaptor molecule trif (tir-domain-containing adapter-inducing ifn-b) which forms a complex with tbk1 (tank binding kinase 1), ikk-e (inhibitor of nuclear factor-kb kinase e) and irf7. in this case the phosphorylated irf7 forms a homodimer or heterodimer with irf3 and translocates to the nucleus where it binds to the ifn promoter via its dna-binding domain to induce type i or type iii ifn [24] . in irf7 knockout mice, viral induced ifn production through the tlr3 (myd88-independent) pathway is greatly impaired. as a result, mice become more susceptible to viral infection [13] . although irf3 has been reported to preferentially activate ifn-b over ifn-a genes, irf7 is believed to efficiently activate both ifn-a and ifn-b [25] . the human ifn-b promoter region contains four positive regulatory domains (prds 1 to iv) that serve as binding sites for irfs. in the human ifn-a promoter region there may be two or three prd modules depending on the ifn-a subtype [26] . due to the importance of irf7 in the innate immune response, it is an active target for viruses to evade the host immune response [27] . a role for irf7 in immunosurveillance has also been identified in breast cancer [28] . using the australian black flying fox (p. alecto) as a model species we have begun to explore the role of the ifn system in the control of viral replication in bats. we have demonstrated that tlrs, rig-ilike receptors, and some ifn stimulated genes (pkr, mx1 and oas1) appear to be conserved in sequence compared to other mammals [29, 30, 31] . however, bats appear to have relatively higher expression of type iii ifn and wider distribution of type iii ifn receptors consistent with a role for type iii ifns in antiviral immunity [32, 33] . bat genome analysis has also provided evidence for positive selection of genes within the ifn pathway, including tlr7, c-rel, tbk-1, ifn-c, isg15 and rig-i [34] . these changes may have occurred in response to the co-evolution of bats with viruses and may have consequences for the clearance of viral infections and the ability of bats to coexist with viruses. due to the central role of irf7 in the regulation of the ifn response, we performed sequence and functional analysis of p. alecto irf7. our results provide the first description of irf7 in any species of bat and evidence for conserved irf7 functional activity despite variation at the sequence level in the bat irf7 gene. all animal experiments were approved by the australian animal health laboratory (aahl) animal ethics committee (protocol number 1389). immortalized and cloned p. alecto kidney (pakit03) and lung (palut02) cells established previously [35] were cultured in dmem/f12-hams (sigma), supplemented with 10% foetal calf serum (fcs, hyclone), 100 units/ml penicillin, 100 mg/ml streptomycin and 50 mg/ml gentamycin (sigma). human embryonic kidney hek293t cells were cultured in dmem supplemented with 10% fcs (hyclone), 15 mm lglutamine, 100 mg/ml penicillin, neaa/na-py/fungizone. all cells were maintained in a humidified atmosphere of 5% co 2 at 37uc. sendai virus (sev, cantell strain) was prepared in chicken embryos as described previously [29] . pulau virus (pulv) was prepared and titered as described previously [33] . for infection of cells, virus was incubated with cells for one hour at 37uc, then replaced with normal cell culture medium for the indicated time. full-length irf7, irf3 and myd88 open reading frames (orfs) were identified in the p. alecto genome (ncbi id p. alecto asm32557v1) [34] using blastx. for comparative purposes, sequences were obtained from the current genome assemblies from the ensembl database for the following species: ensp00000380697, homo sapiens (human); ensmusp00000095565, mus musculus (mouse); ensecap00000007698, equus caballus (horse); enssscp00000013664, sus scrofa (pig); ensbtap00000056564, bos taurus (cow). the microbat myotis davidii irf7 amino acid sequence was deduced from irf7 orf annotated from the published m. davidii genome (ncbi id m. davidii asm32734v1) [34] . the p. alecto irf7 sequence has been submitted to genbank under accession number kj534586. sequence alignment was performed using clustalx and visualized using genedoc (http://www.nrbsc.org/gfx/genedoc/ index.html). alignment files were visualized using emboss plotcon to determine the conservation of irf7 proteins among different species. genomic intron-exon maps of the genes were drawn using fancy gene v1.4 by comparing individual irf7 orfs of p. alecto, horse and human (http://host13.bioinfo3.ifomieocampus.it/fancygene/). phylogenetic trees were constructed using the neighbour joining method and mega4.1 program with 1000 bootstrap replicates [36] . primers listed in table 1 were designed based on the p. alecto genomic sequences and used in rt-pcr to amplify irf7, irf3 and myd88 from rna extracted from freshly isolated bat splenocytes. to construct expression plasmids, pcr products corresponding to full-length irf3 and irf7 were ligated directly to vivid colors pcdna 6.2/emgfp topo vector (life technologies) with an n-terminal gfp tag. the myd88 orf was ligated to the pflag-cmv2 expression vector (sigma) using restriction enzymes noti and sali with an n-terminal flag tag for detection. to generate a truncated bat irf7 (tirf7) that lacked the myd88 binding region at amino acids (aa) 234-298, overlapping pcr was performed [37] . the resulting tirf7 pcr product was ligated to the pcdna 6.2/emgfp topo vector. the human irf7 (hu-irf7) and hu-myd88 plasmids have been described previously [38] . hu-irf7 is in the pegfp-n1 vector and hu-myd88 is in the pef-bos vector with an n-terminal flag tag. mouse ifn-a4, ifn-a6 and human ifn-b promoter plasmids, abbreviated as mu_ifn-a4p, -a6p and hu_ifn-bp respectively, have been described previously [21] . the bat ifn-b promoter plasmid was constructed using sequence 1000 bp upstream from the start codon of ifn-b orf from the p. alecto genome. promoter prediction was performed using the online transcriptional start site prediction tool, matinspector in the genomatix software suite (http://www.genomatix.de/cgi-bin//matinspector_prof). regions containing putative irf3 or irf7 binding sites were identified from 2221 to 270 bp from the atg of the bat ifn-b gene by comparison with human ifn promoters and cloned into the pgl4.1 expression vector (promega). a transfection control prl-tk plasmid containing renilla luciferase was obtained from promega. details of primers used during plasmid construction can be found in table 1 . hek293t cells were transfected using fugene 6 (promega) according to the manufacturer's instructions. approximately 2610 5 cells per well in a 24-well plate were co-transfected with 100 ng of the relative ifn promoter plasmids, 50 ng of prl-tk (promega) served as an internal control. where indicated, expression plasmids for bat irf7, human irf7 or bat myd88 were included in the transfection mix. cells were harvested 30 h post-transfection and lysed using passive lysis buffer provided in the following kit. luciferase activities were determined using the dual-luciferase assay system (promega) using a thermo fluoroskan ascent fl machine. for promoter experiments in pakit03 cells, similar transfections were performed using lipofectamine 2000 (life technologies). a smartpool consisting of four small interfering rnas (sirnas) targeting bat irf7 (siirf7) was designed based on the full-length irf7 orf sequence using dharmacon custom services (thermo). information of the four siirf7 can be found in table 1 . transfection of siirf7 was performed in pakit03 cells using the neon transfection system (life technologies) according to the manufacturer's instructions. briefly, 20 nm of each of the four siirf7 was used for every 10 5 cells. cells were harvested into rlt lysis buffer (rneasy kit, qiagen) 48 h post-transfection and stored at 280uc prior to rna extraction. rna was extracted from cell lysate from the sirna knockdown experiment using a qiagen rneasy kit and converted to cdna using the quantitect reverse transcription kit for real-time pcr (qiagen). all experiments were performed according to manufacturer's protocols. preparation of cdna from 12 p. alecto tissues including brain, kidney, liver, lung, lymph nodes, spleen, heart, small intestine, wing, salivary gland, thymus and testis from three individual bats and from polyi:c stimulated palut02 cells has been described previously [33] . for each sample, 1 mg rna was applied to reverse transcription using the quantitech reverse transcription kit (qiagen). irf7 qrt-pcr primers were designed using primer express 3.0 (applied biosystems) with default parameter settings and are listed in table 1 . primers for ifn-b and 18s rrna have been described previously [33] . reactions were carried out using express sybr greener qpcr supermix universal (life technologies) in an applied biosystems 7500 fast real-time qrt-pcr instrument. for each reaction from cdna, 2 ml of 1:5 diluted cdna were used with a final concentration of 200 nmol of each primer. the cycling profile for cdna samples consisted of an initial denaturation at 94uc for 2 minutes followed by 40 cycles of 94uc for 15 seconds, 60uc for 1 minute, followed by melt curve analysis. expression levels of target genes were calculated using the standard curve method after normalisation to the housekeeping gene 18s rrna. pakit03 cells were seeded onto glass coverslips in 24-well plates at 10 5 cells per well one day before transfection. they were transfected with 200 ng each of gfp-irf7 and flag-tagged myd88 (human or bat) using lipofectamine 2000 (life technologies). at 16 h post-transfection, cells were fixed in 4% paraformaldehyde in pbsa at room temperature for 40 minutes. after removal of fixative, cells were washed three times with pbsa, followed by treatment with 0.1% triton x-100 for 10 minutes and blocked with 0.5% bsa in pbsa for 30 minutes. mouse anti-human myd88 antibody (santa cruz, cat. sc11356) was diluted 1:1000 in 0.5% bsa and applied to cells and incubated at 37uc for 1 h. cells were then incubated with alexa 488 conjugated goat anti-mouse secondary antibody at 37uc for 1 h and washed three times in pbsa. nuclei were labelled with dapi and coverslips were mounted on glass slides for analysis. all slides were examined under a leica confocal microscope (leica, germany). the immunoprecipitation method for analysis of irf7 and myd88 has been described previously [38] . in brief, 2610 6 hek293t cells were seeded 24 h prior to co-transfection with 1.25 mg of both flag-myd88 and gfp-irf7 (human or bat) using fugene 6 (promega). cells were then lysed 24 h later with lysis buffer (50 mm tris (ph7.4), 1.0% triton x-100, 150 mm nacl, 1 mm edta, 2 mm na 3 vo 4 , 10 mm naf, 1 mm pmsf and protease cocktail inhibitor mixture; promega). cell lysates were cleared by centrifugation (9000 g, 10 min, 4uc), and then pre-cleared with protein g-sepharose beads for 30 min at 4uc and flag-myd88 immune complexes were immune-precipitated from supernatant using anti-flag m2-agarose (sigma) conjugated beads for 2 h at 4uc. beads were washed with 3 x lysis buffer and eluted by boiling beads in 5 volumes of sds page sample buffer. lastly, sds page and western blot analysis were performed using the samples obtained. for the blotting, antihuman myd88 rabbit antibody and anti-human irf7 goat antibody (both from santa cruz) were used at 1:1000 dilution during primary antibody incubation and ap-conjugated goat antirabbit or rabbit anti-goat secondary antibody (both from life technologies) were used at 1:2000 dilution. to explore possible differences in the bat antiviral immune system which may influence the association between bats and viruses, irf family members were chosen as primary targets due to their importance in ifn induction and signaling [10] . all irf members from irf1 to irf9 were identified in the bat genome, indicating their relative conservation at the family level. we next performed sequence analysis on the four known positive regulators of type i ifn transcription in humans; irf1, irf3, irf5 and irf7. comparison of the sequence similarity of the deduced protein sequence of human and bat irfs demonstrated significant conservation of irf1 (96%), irf3 (88%) and irf5 (87%). in contrast, bat irf7 shares only 64% and 55% amino acid similarity to human and mouse respectively. to determine whether the relatively low sequence conservation of the bat irf7 gene compared to human and mouse affects the functional activity of bat irf7, this gene was chosen for further functional analysis. the recently published p. alecto whole genome sequence and transcriptome data were used to identify the irf7 gene [39, 40] . primers based on the genomic irf7 sequence were used to amplify full-length irf7 by pcr from bat spleen cdna resulting in the identification of a single full length irf7 transcript. by aligning the bat irf7 cdna sequence with the corresponding region in the p. alecto genome, we were able to determine the intron and exon structure of this gene. as shown in figure 1 , bat and horse irf7 have nine exons compared to the ten exon structure of human irf7. horse was included in this comparison due to the close phylogenetic relationship between bats and horses (figure s1 and [34] ). nine exons were also identified in irf7 genes from other laurasiatheria species, including pig, cow and dog. of the sequences available in the ensembl database, the ten exon structure appears to be typical only among primate irf7 genes (data not shown). analysis of the putative bat irf7 promoter region around 1000 bp upstream of the start site of the orf resulted in the identification of two ifn stimulated response elements (isres) and one nuclear factor kappa b (nf-kb) binding site. to determine whether the presence of two isre sites was unique to bats, the irf7 promoters of other species (human, mouse, horse, cow, dog, cat and rat) were also examined using the publicly available databases. all species examined contain two isre sites (one isre and one irf binding site) with the exception of human which has a single isre (data not shown). based on the deduced protein sequence, p. alecto irf7 was aligned with six other species: human, mouse, pig, cow, horse and the microbat, david's myotis (m. davidii) available from the recently completed genome sequence [40] . these species were chosen because human and mouse have been well studied, while all other species are phylogenetically close to p. alecto [34] . fulllength irf7 contained multiple functional domains including an n-terminal dna-binding domain (dbd), followed by a constitutive activation domain (cad), virus-activated domain (vad), inhibitory domain (id), and c-terminal serine-rich region ( figure 1 ; [22, 37] . the vad is responsible for binding to the upstream activators of irf7: myd88, traf6 or tbk-1, while the serine-rich region is the target for virus-inducible phosphorylation [21, 41] . from the alignment, bat irf7 appears to have a conserved dbd ( figure s2 ), c-terminal serine-rich domain which is the target of phosphorylation, and auto-inhibitory domain [37] . however, the region between amino acids 200-300, which corresponds to the vad and contains the myd88 binding site is not conserved in sequence among all species ( figure 1b) . furthermore, in the putative myd88 binding region, both bats are less conserved not only to human but also to other laurasiatheria species shown here (pig, cow and horse). as the myd88-irf7 pathway is critical to ssrna-induced human ifn production, alignment was performed between bat myd88 and myd88 from human, mouse, pig, cow and horse. no significant change that would potentially alter the functionality of bat myd88 was identified based on sequence analysis (figure s3). to determine the tissue distribution of bat irf7, its transcription was examined in a range of immune and non-immune associated bat tissues. the tissues were from three apparently healthy bats caught from the wild in which the irf7 level should represent the normal expression pattern in wild bats. as shown in figure 2a , bat irf7 is widely expressed among all bat organs at the mrna level, with spleen, small intestine and lung having the highest irf7 expression and wing and salivary gland the lowest. with the exception of the wing, all other tissues showed similar expression levels of irf7 with around 10-fold difference between spleen which had the highest expression and salivary gland with the lowest. this pattern differs from the transcription pattern of bat tlr7, 8 and 9, which appear to be predominantly expressed in immune tissues [30] . next, the inducibility of irf7 by a known virus mimic was explored by testing irf7 transcription using our cloned and immortalized bat lung cell line (palut02 cells) following stimulation with the double stranded rna (dsrna) ligand, alecto irf7 with irf7 from human, mouse, cow, pig, horse and m. davidii using a 20 amino acid sliding window and created using emboss plotcon (http://emboss.bioinformatics.nl/cgi-bin/emboss/plotcon). the similarity across the whole orf between these species is shown by scores. the lower the score the lower the sequence conservation. the least conserved region is boxed. doi:10.1371/journal.pone.0103875.g001 polyi:c. the palu02 cell line has previously been demonstrated to produce ifn in response to polyi:c in a dose dependent manner [42] . as shown in figure 2b , stimulation by either treatment or transfection with polyi:c, which mimics ifn production through tlr3 or rlh pathways respectively resulted in strong induction of irf7. notably, irf7 mrna was induced at the earliest time point of 3 h following stimulation, to a peak level of around 1000 times higher than mock treated cells, highlighting the importance of irf7 in early antiviral defense in bats. these data are consistent with bat irf7 being inducible by both tlr and rlh pathways. in summary, bat irf7 is constitutively transcribed in all tissues and cell lines tested (palut02 cells and pakit03 cells, below) and can be strongly induced by dsrna. due to the limitations of the human irf7 antibody used in this study, the expression of irf7 at the protein level awaits the development of a suitable bat specific reagent. to test the ability of irf7 to induce ifn-b production a bat ifn-b promoter assay was used. the putative promoter region of the bat ifn-b gene was examined and predicted to contain irf7 and irf3 binding modules ( figure s4 ). this region was ligated to the pgl4.1 luciferase reporter vector. interestingly, the bat ifn-b promoter contains one residue difference in its prdi which may abolish its ability to bind to irf3 or irf7 [43] . in contrast, the second prd (prdiii) is almost identical to the corresponding domain of the human ifn-b promoter region and was therefore predicted to be functional [44] . comparison of the prdi domain of other closely related species available in the ensembl database and the microbat, m. davidii, revealed the disruption of prdi is unique to p. alecto ( figure s4 ). in the bat immortalized kidney cell line (pakit03 cells), a plasmid encoding bat irf7 was cotransfected with the bat ifn-b promoter plasmid for 24 h and cells were then infected with sev for another 6 h before performing the luciferase test. the pakit03 cells were chosen for these experiments due to their ability to be successfully transfected and sev was chosen due to its potent ability to induce ifns and other cytokines in the absence of viral products that block the ifn response [45] . results clearly indicate that irf7 alone can induce ifn-b activation and sev induces enhanced activation of irf7 in a dose dependent manner ( figure 3a) . to examine whether the poorly conserved myd88-binding region in bat irf7 influenced its transactivation potential, bat irf7 was compared with that of human irf7 using ifn promoter assays. this experiment was designed to test the hypothesis that the sequence differences identified in the bat irf7 region do not affect its ability to activate ifn transcription. this response was tested using mouse ifn-a4, mouse ifn-a6 or human ifn-b promoter plasmids co-transfected with increasing doses of bat or human irf7 expression plasmids in hek293t cells. as shown in figures 3b-d , bat irf7 activates all three promoters in a dosedependent manner. together with the results described above using the bat ifn promoter, these results demonstrate that bat irf7 is capable of activating ifn in bat cells following stimulation with sev or in human cells co-transfected with human or mouse ifn promoters. cells transfected with mock or empty vector failed to activate the ifn promoters significantly. although identical doses of human and bat irf7 plasmid were used in these experiments, the results are not statistically comparable due to the difference in irf7 protein expression from the two plasmids. of note, we used human hek293t cells and mouse ifn-a and human ifn-b promoters in this assay because they are a well established system for testing the activation of ifn [21] . given the high conservation in the dna-binding domain of bat irf7, it was predicted to be capable of binding to the human and mouse ifn promoters followed by activation by downstream factors. to explore the importance of bat irf7 in the ifn production pathway, a knockdown approach was used in our p. alecto pakit03 cells. transfection of sirna targeting bat irf7 (siirf7) for 24 h resulted in a reduction of native irf7 mrna expression to approximately 20% compared to mock transfected cells. the statistical analysis of the knock down effects was calculated by comparing mrna expression in the knock down samples to mock transfected cells. to exclude the possibility of off-target effects of sirna transfection, the expression of a closely related gene, irf3 was examined in transfected cells. as shown in figure 4a , there was a decrease in irf3 transcription in siirf7 transfected cells due to possible toxic effects and/or off target effects of the sirna smartpool but this change was not statistically significant. having confirmed the knockdown effect of siirf7, we then explored the downstream effect of reduced irf7 on ifn-b production and viral replication. two experiments were performed; firstly, 24 h after siirf7 transfection, cells were stimulated with sev for 6 h and ifn-b mrna was detected by qpcr. knockdown of irf7 impaired the induction of ifn-b by sev by 2.5 fold relative to untransfected cells ( figure 4b) . notably, bat cells maintained some ifn-b induction in siirf7 cells, a result which likely reflects insufficient knockdown of irf7 or ifn-b induction through alternative (irf3 or nf-kb) pathways. to examine the effect of ifn knockdown on the replication of a bat-borne virus, pulv, a dsrna reovirus originating from pteropid bats, was used to infect siirf7-transfected bat cells [46] . a dose of 10 moi was used to infect pakit03 cells one day after siirf7 transfection (or mock transfection). cell supernatant containing virus was collected 24 h after infection and applied to a tcid 50 test. figure 4c shows that when bat irf7 was knocked down, pulv replicated to a titer more than four-fold higher than in mock-transfected cells. these data demonstrate that bat irf7 is functionally important in sev induced ifn-b production and antiviral defense against pulv infection of bat cells. we next wanted to determine whether bat irf7 is involved in the production of ifn-a and ifn-b by the myd88 despite the divergent nature of its myd88 binding domain. the transactivation activity of bat irf7 was compared to that of human irf7 using expression plasmids containing bat or human myd88 and irf7 co-transfected with mouse ifn-a4 or ifn-a6 promoter plasmids. in mice, ifn-a4 is the earliest ifn-a induced by viral infection, while ifn-a6 is induced later in the response. a dose of 10 ng or 100 ng of irf7 was co-transfected with myd88 for the ifn-a4p and ifn-a6p promoter assay respectively in hek293t cells. these doses of irf7 were chosen as they do not result in huge induction of the native ifn promoter. as shown in figure 5a , the activation of ifn-a4p by both human and bat irf7 was increased by co-transfection with myd88. co-transfection of cells with bat myd88 and irf7 resulted in a higher response compared to co-transfection with the corresponding human plasmids. our results demonstrate that even with a significant difference in its myd88 binding region, bat irf7 is still capable of inducing ifn-a transcription via myd88 ( figure 5 ). some differences were observed between ifn-a4 and ifn-a6 inducibility which may be due to differences in their irf or nf-kb binding motifs. no ifn-a4 activation occurred following cotransfection of bat myd88 with bat irf3 confirming that irf3 is myd88 independent ( figure s5a) . a similar experiment was performed to confirm that bat irf3 is capable of activating the human ifn-b promoter confirming the activity of bat irf3 ( figure s5b ) [21] . thus, only ifna production by irf7 is dependent on myd88. to confirm that bat irf7 interacts with bat myd88, experiments were performed to examine the interaction between the two proteins. firstly, hek293t cells were co-transfected with plasmids encoding flag-tagged myd88 (either human or bat, as indicated) and gfp-tagged irf7 (human or bat). cells were lysed and protein immunoprecipitated with anti-flag antibody conjugated beads followed by immunoblotting with anti-flag or anti-human irf7 antibody. human irf7 protein was successfully captured by human myd88 which was detected in ip samples demonstrating protein interaction between human myd88 and human irf7 ( figure 6a, panel 1) . the detection of only a faint band may be due to the relatively low expression of the input human irf7 and myd88 proteins from these expression plasmids. clear signals were detected for irf7 and myd88 following co-ip of bat myd88 with bat or human irf7 ( figure 6a ). there is a slight difference in the molecular weights of human and bat myd88 and irf7 which are reflected on the blot (human and bat myd88 are 33.3 and 33.6 kd respectively and human and bat irf7 have a molecular weight of 54.2 and 55.6 kd respectively). these results clearly demonstrate that bat myd88 protein is capable of binding both bat and human irf7 proteins. confocal microscopy was used to determine the colocalisation of the two proteins, to further confirm protein interaction. bat kidney pakit03 cells or human kidney hek293t cells were used to examine colocalisation of irf7 with myd88. a dose of 200 ng/ well of either human or bat myd88 and irf7 plasmids were used to transfect cells grown overnight on coverslips in 24-well plates. sixteen hours later, cells were fixed and stained with anti-human myd88 antibody and examined under the confocal microscope. myd88 transfection alone resulted in the formation of very large condensed aggregates in the cytoplasm of both human and bat cells. human myd88 and human irf7 colocalised in a manner similar to previous studies ( figure 6b ) [21, 41] . similarly, bat myd88 and irf7 proteins also demonstrated clear co-localisation. as shown in figure 6b , bat irf7 appeared to be surrounded by myd88 in an aggregated form, which is the typical myd88 structure [47, 48] . in addition, bat myd88 also colocalised with human irf7, which is consistent with our ip results ( figure 6a ). as expected, no such aggregated structure was observed following co-expression of bat myd88 with bat irf3, ruling out the possibility of interaction between these two proteins. having confirmed the functional reliance and binding capability of bat irf7 to bat myd88, it appeared that the poorly conserved myd88-binding region (between aa 200-300) retains a similar function to the corresponding human domain. to further confirm this was the case, an internal deletion mutant of bat irf7, tirf7 was constructed ( figure 7a ). this form of bat tirf7 contained a deletion of aa 225-331 which has been shown to abolish the ability of human irf7 to transactivate the ifn-a1 promoter [22] . the mouse ifn-a4 promoter transactivation activity by tirf7 was compared to the full-length irf7 protein using a luciferase assay. having confirmed successful expression, figure 7b clearly demonstrates that tirf7, was unable to activate the mouse ifn promoter, even in the presence of myd88 ( figure 7b ). these data are consistent with functional conservation of the region corresponding to aa 233-298 in bats with that of human irf7 despite significant sequence variation. irf7 is a master regulator of ifn expression in mammals and is therefore central to the innate antiviral immune response. in humans, irf7 acts predominately in pdcs via activation of tlr7/9 and the myd88 dependent signaling pathway [49] . regulation of the ifn response may play an important role in the ability of bats to coexist with viruses in the absence of clinical signs of disease. this report describes the analysis of irf7 from our model bat species, the australian black flying fox, p. alecto, an important reservoir for viruses including hendra virus, which has resulted in the deaths of numerous horses and humans since its discovery in 1994 [4, 46] . our results support the conservation of functional activity of irf7 in p. alecto but provide evidence of a wider tissue distribution which has implications for broader activation of the ifn response in bats. our results provide the first functional characterization of irf7 in any species of bat and contribute to our understanding of the function and evolution of irf7 in mammals. bat irf7 was identified from the bat genome and bat transcriptome data [39, 40] , together with rt-pcr results from spleen cdna resulting in the identification of a single full length variant of irf7. in humans and mice, irf7 expression is very low in most tissues and cells with the exception of pdcs and cells that have been activated by ifn [12, 15] . in contrast, the transcription of p. alecto irf7 was detected not only in immune-related tissues but comparable expression was observed in many other organs as well. although there is a lack of data on the tissue distribution of irf7 in mammals besides human and mouse, there have been several studies on fish irf7. interestingly, at least five species of fish including crucian carp, mandarin fish, snakehead fish, atlantic salmon and japanese flounder express irf7 constitutively in a wide variety of tissue types although different irf7 transcripts were expressed in each species. these tissues were neither primarily immune-related nor serve as portals for microbial infection, where the immune response is easily initiated. since these fish irf7s can also be induced by dsrna, they were hypothesised to play an important role in fish immunity [16, 17, 18, 19, 20] . although further analysis of the cell types responsible for constitutive irf7 expression in bats is required, a constitutively expressed irf7 in a broad range of cells and organs may result in faster and stronger ifn production upon viral infection [49] . this observation is similar to the pattern of type iii ifn receptor expression which has a wide distribution in bats but only limited distribution in other mammals [50] . thus, bats may maintain the potential to rapidly activate the innate immune response in a broader subset of tissues and cells than other mammals. induction of irf7 by treatment or transfection of our bat kidney cell line with the dsrna ligand, polyi:c resulted in a peak in the induction of irf7 at 9 h post-treatment, which is 3 h later than the peak in bat type i and type iii ifns but similar to that of isgs mx1, oas1 and pkr described previously in bat cells [29, 33] . this result is consistent with the induction of irf7 through type i ifn feedback similar to other species. in humans, irf7 is generated through multiple pathways following ifn induction. following the production of ifn and binding to the . bat irf7 interacts with bat myd88. (a) binding of bat irf7 to bat myd88. hek293t cells were transfected with gfp-tagged irf7 and flag-tagged myd88 (human or bat, as indicated). at 24 h post-transfection, whole cell lysate was prepared and immunoprecipitated with anti-flag antibody m2. immunoprecipitated complexes (ip) were analysed by immunoblot for irf7 and myd88 expression using an anti-human irf7 antibody (top panel), and an anti-flag antibody m2 for detection of flag tagged myd88 (middle panel). whole cell lysate (10 ul) was also run on an sds-page gel and subsequently analysed for irf7 expression using anti-human irf7 antibody (bottom panel). (b) bat irf7 co-localizes with bat myd88. pakit03 cells were transfected with bat gfp-irf7/irf3 and bat flag-myd88 and hek293t cells were transfected with human gfp-irf7 and human or bat flag-myd88. 16 hours post-transfection (or 24 h), cells were fixed for indirect immunofluorescence assay using an anti-human myd88 antibody and red fluorescence-conjugated secondary antibody. pictures show co-localisation of bat irf7 or bat irf3 with bat myd88 in pakit03 cells and human irf7 or human irf3 with human myd88 in hek293t cells. doi:10.1371/journal.pone.0103875.g006 ifn-ar, a complex consisting of activated stat1, stat2 and irf9, called the ifn stimulated gene factor 3 (isgf3) is formed, which in turn binds to the isre on the irf7 promoter and induces irf7 transcription. the human irf7 promoter region contains an nf-kb binding site and a single functional isre approximately 1.3-kb upstream from the atg start site, both of which are important in the induction of irf7 [49, 51] . analysis of the putative bat irf7 promoter region resulted in the identification of two isres and one nf-kb binding site indicating that multiple mechanisms for irf7 activation may also exist in bats. however, two isre sites were also identified in the irf7 promoters of other species examined (mouse, horse, cow, dog, cat and rat). thus, whether the broad distribution of constitutively expressed irf7 is the result of the presence of a more efficient irf7 promoter region driven by transcription factors other than irfs, or simply due to enriched immune-related cells in all tissues will require further study. sequence differences in the myd88 binding domain of bat and human irf7 led to the hypothesis that there may be functional differences in the activation of bat irf7 and the regulation of the ifn response that may contribute to the ability of bats to resist the clinical outcomes of viral infection. our results demonstrate that these sequences differences do not appear to affect irf7 function either in ifn transactivation activity or activation by myd88. bat irf7 was capable of activating both ifn-a and ifn-b promoters and the levels of transactivation were equivalent to or higher than that of human irf7. similarly, bat myd88 and bat irf7 maintained binding capability similar to their human counterparts. deletion of the myd88-binding region of bat irf7 impaired its ability to activate ifn, demonstrating functional conservation of the myd88 binding domain with that of human irf7. collectively, these data demonstrate that bat irf7 is capable of inducing ifn and myd88 binding in a similar manner to human irf7. although the myd88 binding domain of bat irf7 has low sequence conservation with the equivalent region in human irf7, experimental data demonstrate a fully functionally irf7 exists in pteropid bats. our results describing the experimental knockdown of irf7 using sirna is to our knowledge the first description of the use of sirnas in bat cells. the successful knockdown of irf7 is consistent with the presence of an rna-silencing mechanism in bats similar to that in other mammals. irf7 knockdown resulted in impaired ifn-b induction in sev infected cells and enhanced pulv replication. although further work will be required to determine whether irf7 is the master regulator of the bat ifn response, these results confirm that irf7 plays an important role in anti-viral defense and the early innate immune response in bats [13] . analysis of the bat ifn-b promoter also revealed one residue difference in the prdi module, known to be associated with activation by irf3 or irf7 [43] . a similar change in the human ifn-ap impairs its inducibility by irfs [26] . although bat ifn-b is strongly inducible in bat cells following either stimulation or viral infection, further work will be necessary to determine whether this mutation affects the induction of ifn-b under conditions other than those described here [29] . the presence of this mutation may also indicate that transcription factors other than irf3 and irf7 are involved in the regulation of ifns in bats. therefore, future work focusing on ifn promoters (including ifn-a and ifn-l) will be necessary to explore whether the bat irf-ifn induction pathway is as critical to ifn induction as it is in other species. in humans, there are around 1400 transcription factors that have been recognized [52] . whether these factors play similar roles in bats or whether they perform different functions resulting in differences in the expression of downstream genes remains to be determined. understanding the mechanisms responsible for the regulation of the ifn response will assist in discovering how bats successfully coexist with viruses. in conclusion, our data clearly demonstrate that bat irf7 exhibits a constitutive expression pattern across a broad range of immune and non-immune related organs, intact ifn transactivation function, and binds to and is activated by bat myd88. these findings not only provide the first information on the myd88-irf7 dependent ifn production pathway in bats, but also explore the functionality of bat irf3 and identify critical point mutations in the bat ifn-b promoter. ultimately, we hope these advances may help uncover the mechanisms underlying the ability of bats to co-exist with deadly viruses, and that this will in turn lead to the development of potential therapeutic strategies targeting viral infections in other mammals. severe respiratory illness caused by a novel coronavirus nipah virus: a recently emergent deadly paramyxovirus isolation and characterization of a bat sars-like coronavirus that uses the ace2 receptor isolation of hendra virus from pteropid bats: a natural reservoir of hendra virus fruit bats as reservoirs of ebola virus public health awareness of emerging zoonotic viruses of bats: a european perspective bats: important reservoir hosts of emerging viruses antiviral immune responses of bats: a review interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures the irf family, revisited type i interferon [corrected] gene induction by the interferon regulatory factor family of transcription factors irf-7, a new interferon regulatory factor associated with epstein-barr virus latency irf-7 is the master regulator of type-i interferon-dependent immune responses ifn regulatory factor family members differentially regulate the expression of type iii ifn (ifn-lambda) genes positive feedback regulation of type i ifn genes by the ifn-inducible transcription factor irf-7 molecular cloning and characterization of interferon regulatory factor molecular cloning and characterization of crucian carp (carassius auratus l.) interferon regulatory factor 7 gene structure and transcription of irf-1 and irf-7 in the mandarin fish siniperca chuatsi gene structures and promoter characteristics of interferon regulatory factor 1 (irf-1), irf-2 and irf-7 from snakehead channa argus regulation and function of interferon regulatory factors of atlantic salmon interferon-alpha induction through toll-like receptors involves a direct interaction of irf7 with myd88 and traf6 multiple regulatory domains control irf-7 activity in response to virus infection ikappab kinase-alpha is critical for interferon-alpha production induced by tolllike receptors 7 and 9 involvement of the ubiquitin-like domain of tbk1/ikk-i kinases in regulation of ifn-inducible genes differential viral induction of distinct interferon-alpha genes by positive feedback through interferon regulatory factor-7 the role of differential expression of human interferon-a genes in antiviral immunity interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures silencing of irf7 pathways in breast cancer cells promotes bone metastasis through immune escape bat mx1 and oas1, but not pkr are highly induced by bat interferon and viral infection molecular characterisation of toll-like receptors in the black flying fox pteropus alecto molecular characterisation of rig-i-like helicases in the black flying fox, pteropus alecto type iii ifn receptor expression and functional characterisation in the pteropid bat, pteropus alecto type iii ifns in pteropid bats: differential expression patterns provide evidence for distinct roles in antiviral immunity comparative analysis of bat genomes provides insight into the evolution of flight and immunity establishment, immortalisation and characterisation of pteropid bat cell lines mega4: molecular evolutionary genetics analysis (mega) software version 4.0 phosphorylation-induced dimerization of interferon regulatory factor 7 unmasks dna binding and a bipartite transactivation domain suppressor of cytokine signaling 1 negatively regulates toll-like receptor signaling by mediating mal degradation the immune gene repertoire of an important viral reservoir, the australian black flying fox comparative analysis of bat genomes provides insight into the evolution of flight and immunity role of a transductional-transcriptional processor complex involving myd88 and irf-7 in toll-like receptor signaling type iii ifns in pteropid bats: differential expression patterns provide evidence for distinct roles in antiviral immunity regulation of virusinduced interferon-a genes evidence for a nuclear factor(s), irf-1, mediating induction and silencing properties to human ifn-beta gene regulatory elements sendai virus defective-interfering genomes and the activation of interferon-b pulau virus; a new member of the nelson bay orthoreovirus species isolated from fruit bats in malaysia regulation of myd88 aggregation and the myd88-dependent signaling pathway by sequestosome 1 and histone deacetylase 6 distinct roles of tir and non-tir regions in the subcellular localization and signaling properties of myd88 irf7: activation, regulation, modification and function type iii ifn receptor expression and functional characterisation in the pteropid bat, pteropus alecto regulation of the promoter activity of interferon regulatory factor-7 gene. activation by interferon snd silencing by hypermethylation a census of human transcription factors: function, expression and evolution we thank drs prasad paradkar and glenn marsh for critical review of the manuscript. key: cord-310734-6v7oru2l authors: bolatti, elisa m.; zorec, tomaž m.; montani, maría e.; hošnjak, lea; chouhy, diego; viarengo, gastón; casal, pablo e.; barquez, rubén m.; poljak, mario; giri, adriana a. title: a preliminary study of the virome of the south american free-tailed bats (tadarida brasiliensis) and identification of two novel mammalian viruses date: 2020-04-09 journal: viruses doi: 10.3390/v12040422 sha: doc_id: 310734 cord_uid: 6v7oru2l bats provide important ecosystem services as pollinators, seed dispersers, and/or insect controllers, but they have also been found harboring different viruses with zoonotic potential. virome studies in bats distributed in asia, africa, europe, and north america have increased dramatically over the past decade, whereas information on viruses infecting south american species is scarce. we explored the virome of tadarida brasiliensis, an insectivorous new world bat species inhabiting a maternity colony in rosario (argentina), by a metagenomic approach. the analysis of five pooled oral/anal swab samples indicated the presence of 43 different taxonomic viral families infecting a wide range of hosts. by conventional nucleic acid detection techniques and/or bioinformatics approaches, the genomes of two novel viruses were completely covered clustering into the papillomaviridae (tadarida brasiliensis papillomavirus type 1, tbrapv1) and genomoviridae (tadarida brasiliensis gemykibivirus 1, tbgkyv1) families. tbrapv1 is the first papillomavirus type identified in this host and the prototype of a novel genus. tbgkyv1 is the first genomovirus reported in new world bats and constitutes a new species within the genus gemykibivirus. our findings extend the knowledge about oral/anal viromes of a south american bat species and contribute to understand the evolution and genetic diversity of the novel characterized viruses. bats belong to the order chiroptera, which is the second-largest mammalian group, comprising 21 families and 1411 species distributed globally, with the exception of polar areas [1, 2] . approximately 25% of the world's bat species are endangered, causing concerns about the negative conservation impact and its influence on the ecosystem services these bats provide, such as arthropod regulation, seed dispersal, and pollination [1, 3] . on the other hand, certain specific aspects of bats-including their relatively long lifespan in relation to their body size [4] , the reliance of some species on prolonged torpor [5] , and flight-may make them suitable for hosting a wide variety of viruses [6] , including zoonotic viruses highly pathogenic to humans [6] , such as severe acute respiratory syndrome (sars)-related coronavirus, ebola virus, nipah virus, and hendra virus [7] [8] [9] [10] . nevertheless, little is known about their own pathogens [3] . in addition, the gregarious behavior of many bat species, such as free-tailed bats tadarida brasiliensis (i. geoffroy saint-hilaire, 1824), may facilitate rapid transmission of pathogens between bats and other species [6] . t. brasiliensis is the most abundant migratory and cosmopolitan species of the new world bats, widespread throughout the americas [11] [12] [13] and protected by international agreements [14] . using next-generation sequencing (ngs) technologies, an enormous variety of viral species and genotypes [15, 16] have been identified in the tissues and feces of bats mainly inhabiting asia [17, 18] , africa [19, 20] , europe [21, 22] , and north america [23, 24] . on the other hand, the viromes of bats from south america remain understudied [25, 26] . for example, the identification of viruses infecting t. brasiliensis is principally limited to detection of specific viral families, such as rabies lyssavirus [27] , alphacoronaviruses [28] , polyomaviruses [29] , circoviruses [30] , and anelloviruses [31] . in order to contribute to the preservation of t. brasiliensis and to evaluate its possible role as a pathogen reservoir, greater efforts directed at identifying the viruses present in this species are needed. in this study we report a detailed description of two novel complete genome sequences, one describing a new papillomavirus genus and the other representing a novel variant of an existing gemykibivirus species. in addition, we report a preliminary overview of the t. brasiliensis virome composition. altogether, our findings add to the knowledge of viral diversity in a south american bat species, providing insights for understanding their role as reservoirs, as well as their own pathogens, which may have consequences for the animals' health. the bat colony investigated occupies the attic of the law school building at the universidad nacional de rosario in downtown rosario, argentina (32 • 56 36.76 s 60 • 39 02.09 w) [32] . in this place, t. brasiliensis (molossidae) establishes a maternity colony every year that can reach about 30,000 individuals during the maternity season (november to march), after which they migrate [32, 33] . a total of 98 swab samples (49 oral and 49 anal) were collected from 49 adult female specimens inhabiting this colony from december 2015 to february 2016. briefly, bats were manually captured from the walls and held in individual cotton bags for determination of their species based on anatomical and morphological characters, reproductive condition, and age [33] . the oral cavity and anal regions of each individual were sampled using individual sterile cottontipped swabs (deltalab, barcelona, spain), rolled back and forth (10 times), suspended in 200 µl of saline solution (nacl 0.9%), and stored at 4 • c until further processing. the bats were rehydrated and released. during this study, every effort was made to minimize interference with and suffering of the animals; no breeding or pregnant females were captured, and no animals were sacrificed. sample collection was conducted by trained professionals as approved by the ministry of environment of the argentinian santa fe province (file 021010016257-1) and facultad de ciencias bioquímicas y farmacéuticas (universidad nacional de rosario) animal ethics committee (file 6060/243, 20 march 2015). samples were processed according to previously published protocols that have been successfully applied for identification of papillomavirus (pv) in human skin swab samples [34] [35] [36] . briefly, the cells were centrifuged at 13,000× g for 5 min and the pellets were resuspended in 100 µl te buffer (qiagen, hilden, germany) containing 100 µg of proteinase k (qiagen), and incubated overnight at 55 • c. following proteinase k inactivation (95 • c for 10 min), the lysates were stored at −20 • c. subsequently, the obtained samples were tested for the presence of pv dna using improved versions of fap [37, 38] and cut pcrs [35] , as described previously [36, 39] . circular dna molecules in lysates of five selected pv-positive samples (four anal and one oral swab) were enriched using rolling-circle amplification (rca) with the illustra templiphi 100 amplification kit (ge healthcare, chicago, il, usa) [40] [41] [42] . the pool of rca-enriched samples was sequenced on an illumina hiseq 4000 instrument at the sequencing facility of gatc biotech (ebersberg, germany). sequencing libraries were prepared using the gatc automatic library preparation approach, and the sequencing reads were sequestered in the format of 2 × 150 bp. reads were subjected to quality trimming and filtering using the bbduk program (bbtools v38.42). end trimming was performed on the first and last 15 bases of each read, clipping bases with phred scores below 15. trimmed reads shorter than 120 bp and with an average phred score below 20 were discarded. the read pairs contained in the metagenomic sample, which shared k-mers, sliding-window subsequences of 27 nt, with the sequencing datasets of samples (six in total) that were processed and analyzed in the same sequencing batch, were discarded using the bbduk program (referred to as laboratory-batch background screen in figure 1 ). the primary purpose of this step was to conservatively limit the possibility of falsely identifying viral taxa that did not originate from the bat metagenomics sample and that could have been introduced by aerosol during sample processing or index hopping during sequencing. in order to limit the content of bacterial reads, the metagenomic dataset was mapped to the bacterial reference-index files (obtained 6 november 2017, from ftp://ftp.ccb.jhu.edu/pub/infphilo/ centrifuge/data/p_compressed.tar.gz) using the centrifuge sequence classification system (centrifuge version 1.0.3-beta) [43] . reads not mapping to any bacterial taxon were used in further metagenomic analyses (unless stated otherwise). two types of metagenomic characterization workflows were used: (1) taxonomic classification of ngs read pairs and (2) taxonomic classification of contigs assembled de novo from ngs read pairs. in both cases, the centrifuge metagenomic classification system with the reference nucleic sequence index files, obtained from ftp://ftp.ccb.jhu.edu/pub/infphilo/centrifuge/data/p_compressed+h+v.tar.gz (version 12 june 2016, downloaded 6 november 2017), was used to obtain the final taxonomic calls (default parameter settings). taxonomic classification of sequences was further summarized to the taxonomic level of family using pavian [44] . de novo assembly was performed with two different de brujin graph assembly tools: spades (v3.11) [45] and unicycler (obtained from github: 27 october 2017; github commit: 220d5daebc8267d3 7378f191e14acb5c5a1ff757), adapting various parameter settings. altogether, six different metagenomic de novo assemblies were constructed, using settings specified in table s1 . all contigs assembled de novo by any of the six approaches exhibiting a minimum length of 500 nt were collected and subjected to taxonomic classification (workflow 2). the circularity of the complete genome assemblies was determined by matching the sequence stretches (minimum match length 50 nt) at their 5 and 3 ends. coverage statistics of the novel complete viral genome sequences were obtained by remapping the trimmed read dataset to the constructed genome assemblies using bowtie2 (v2.2.6) [46] . (1) and contigs assembled de novo (2) . pair reads quality filtering and trimming were performed with the bbduk program (bbtools v38.42). the centrifuge metagenomics classification system was used for the taxonomic classification of pair reads and contigs (centrifuge version 1.0.3-beta) [43] . de novo assembly was performed using spades v3.11 [45] and unicycler. (1) and contigs assembled de novo (2) . pair reads quality filtering and trimming were performed with the bbduk program (bbtools v38.42). the centrifuge metagenomics classification system was used for the taxonomic classification of pair reads and contigs (centrifuge version 1.0.3-beta) [43] . de novo assembly was performed using spades v3.11 [45] and unicycler. sequences of the e1, e2, l2, and l1 genes of 376 reference pv genomes, downloaded from pave (http://pave.niaid.nih.gov/ on 13 march 2019), and the corresponding genes from the novel pv, tadarida brasiliensis papillomavirus type 1 (tbrapv1), were used in the phylogenetic analysis. additional information on nucleotide sequences (genbank accession number and virus name abbreviations) used in these analyses is summarized in table s2 . the e1e2l2l1 concatenation was constructed by first obtaining the amino acid-guided multiple sequence alignments of each gene. multiple sequence alignments were obtained using muscle (v3.8.31) [47] . as suggested by bernard et al. (2010) [48] , the multiple sequence alignments used for phylogenetic analysis of tbrapv1 were guided by the amino acid alignments and the pv identity calculation was based on patristic distance measurements, as determined by seaview [49] . phylogenetic clustering was conducted using iq-tree [50] . the most appropriate substitution models were determined using modelfinder [51] , according to the bayesian information criterion. branch support values were calculated using uf bootstrap (1000 replicates) [52] , sh-alrt (1000 replicates), and abayes tests [53] . phylogenetic analysis of genomoviridae was conducted using a reference dataset of 166 complete genome nucleotide sequences and 166 rep protein sequences downloaded from ncbi genbank (19 march 2019). the complete genome sequences were rotated to all begin in the start codon of the rep gene using circulator (version: github commit a4befb8c9dbbcd4b3ad1899a95aa3e689d58b638) [54] , and the two subunits of the rep gene were concatenated into a single protein sequence in which the genbank record indicated them as parts of different genes/coding sequences. pairwise sequence identity values used for taxonomic classification of tadarida brasiliensis gemykibivirus 1 (tbgkyv1) were obtained using sequence demarcation toolkit (sdt v1.0) [55] . in this scope, the pairwise sequence alignments were produced using muscle (v3.8.1) [47] . phylogenetic trees were rendered using figtree (v1.4.4) (http://tree.bio.ed.ac.uk/software/figtree/), and sequence identity histograms were visualized using gnuplot (v1.5). open reading frames (orfs) of novel viral genome sequences were marked using orffinder (ncbi); the manual annotation process of the orfs was guided by the use of ncbi blastp, and the identification of viral-family specific sequence motifs was performed using regular expressions with the linux grep utility (v2.16). members of papillomaviridae and genomoviridae identified in bat species so far are summarized in table s3 . the complete genome sequence of the novel pv type (tbrapv1) was obtained by generating four overlapping amplicons in different pcr reactions, using 1 x pcr buffer, 3.5 mm of mgcl 2 , 200 µm of each dntp (thermo fisher, walthem, ma, usa), 1.25 u of gotaq hotstart polymerase (promega, madison, wi, usa), and 0.8 µm of each of the primers (tbrapv1-1f 5 -cagggtattcagggtgtttctcc-3 and tbrapv1-1r 5 -aatgtttctaatctgcaacc-3 ; tbrapv1-2f 5 -gtgcgcggcgacttctcatactta-3 and tbrapv1-2r 5tcagcctcattgtcctcatcattg-3 ; tbrapv1-3f 5 -tgggcttgaaacctggacactaca-3 and tbrapv1-3r 5 -atgcccgggaa tatggatgga-3 ; tbrapv1-4f 5 -ggcctgcaagaccacctac-3 and tbrapv1-4r 5 -gggggcatctgacctgtta-3 ). cycling conditions for the four reactions were the same and were performed as follows: initial denaturation at 95 • c for 2 min, followed by 45 cycles of 40 s at 94 • c, 40 s at 50 • c, and 2 min at 72 • c, with a final extension at 72 • c for 5 min. the amplicons were resolved in a 1% agarose gel electrophoresis and the~2 kb fragments were gel purified, ligated into the pgem-t easy vector (promega), and transformed into e. coli cells. sanger sequencing was performed using sequencing facilities at macrogen inc. (seoul, korea). in august 2019, dna clones and the corresponding nucleotide sequences were subsequently submitted to the animal papillomavirus reference center (http://www.animalpv.org/) for its confirmation and official designation. the genbank/embl/ddbj accession numbers for the novel viruses reported in this paper are tbrapv1 (mn329804) and tbgkyv1 (mn329805). the relevant raw high throughput sequencing data obtained in this study was deposited at the ncbi sequence read archives (sra) with the following accession number: prjna615356. the contigs, obtained by de novo assembly as part of the metagenomic workflow (2), have also been made available for download (supplementary data s1). ngs data analysis workflows and centrifuge-based taxonomic assignments of reads and contigs are depicted in figure 1 and table 1 , respectively. briefly, a total of 10,409,798 read pairs were sequestered from the rca-enriched samples, and 10,220,118 of them passed the quality filtering and trimming procedures. out of these, 6,738,566 read pairs were removed during the laboratory-batch background screen and an additional 878,852 read pairs were identified as originating from bacteria. the final metagenomic characterization was carried out using the remaining 2,602,700 read pairs ( figure 1 ). metagenomic analysis revealed that only a small proportion of read pairs (13,897 read pairs; 0.534%) and de novo assembled contigs longer than 500 nt (153 out of total 42,891; 0.357%) mapped to viral taxa. overall, a large number of phage-related sequences were detected (77.3% of viral read pairs and 39.9% of viral contigs), likely representing the most abundant entities infecting bacteria present in the bat digestive system, which exhibited similarity mostly to the families inoviridae, siphoviridae, and myoviridae ( table 1 ). the eukaryotic viral sequences (insect, invertebrate, plant, protist, and vertebrate viruses) could be summarized into a total of 35 viral families, 22 corresponding to viral families with dna genomes, and 13 to families with rna genomes. sequences of 10 different viral families infecting insects and crustaceans, mostly found related to the families baculoviridae, ascoviridae, iridoviridae, and nimaviridae, were detected (0.900% of viral read pairs and 12.4% of assembled viral contigs). on the other hand, sequences related to viruses infecting plants (five viral families, 0.446% of viral read pairs, and 4.57% of viral contigs) were mostly associated with phycodnaviridae or potyviridae, whereas those related to viruses infecting protists (five viral families, 13.6% of viral read pairs, 0.654% of viral contigs) clustered predominantly in the family mimiviridae. sequences classified as originating from vertebrates, predominantly mammalian viruses, were represented by 7.07% of viral read pairs and 35.9% of viral contigs. the principal viral families identified included retroviridae, genomoviridae, herpesviridae, papillomaviridae, and poxviridae. the analysis also identified (although in low counts) viral sequences related to the family alloherpesviridae, which infects fish and amphibians. of note, the metagenomic analysis indicated that 205 out of 461 read pairs were assigned to the family retroviridae (1.48% of viral read pairs), and 22 assembled contigs (14.4% of viral contigs; table 1 ) exhibited resemblance to the nucleotide sequence of desmodus rotundus endogenous retrovirus isolate 824 (genbank accession number: nc_027117) [56] . however, a more detailed analysis of this sequence revealed the presence of two flanking regions at the 5 and 3 ends of approximately 1000 nt, which probably derived from the host genome (desmodus rotundus). in fact, contigs and reads previously classified as retroviridae in our study aligned with these flanking regions. this finding explained the initial misclassification and indicated that great care should be taken when using genbank sequence data as reference material because a large portion of sequences and their respective annotations may not have been curated adequately. finally, a total of 96 read pairs and 10 assembled contigs were classified as similar to viruses unassigned to taxonomical families (table s4 ). the metagenomic analysis suggested that a total of 90 read pairs (workflow 1) and 14 contigs (workflow 2) could be attributed to pvs (table 1 ). the longest contig obtained by assembly de novo covered the complete genome of tbrapv1, which was subsequently confirmed by conventional molecular methods (pcr, cloning, and sanger sequencing; data not shown). remapping read pairs (quality-, background-, and bacteria-filtered read pairs) to the confirmed tbrapv1 genome sequence indicated a complete sequence length of 8151 nt, with a gc content of 46%. the complete novel genome sequence was covered on average 252.6× by a total of 6935 read pairs (13,870 reads) . detailed analysis of the tbrapv1 viral genome (table 2 and figure 2a) , showed a typical genomic organization of bat pvs, potentially encoding four early genes (e6, e7, e1, and e2) and two late genes (l2 and l1) [57, 58] . a putative e4 gene was found overlapping the e2 gene ( figure 2a) , with its own start and stop codons, and the presence of e4-characteristic proline-rich stretches (12.7%) with an important role in cell cycle arrest was found [59] . typical domains were additionally identified in the putative viral proteins encoded by tbrapv1. the e6 protein contained two characteristic zinc-binding domains, separated by 36 amino acids [61] , and four internal and likely not functional pdz-binding motifs (rtnv, isdl, ssil, lssl) [62] . the e7 protein contained a prb-binding motif (lwcde) [63] and a single zinc-binding domain ( table 2) . analysis of the e1 protein, the largest protein encoded by tbrapv1 (table 2) , showed the typical atpbinding site of the atp-dependent helicase (gpsnsgks) [64] , and several cdk-phosphorylation and the upstream regulatory region (urr) of tbrapv1 contained two typical tata boxes, three putative e2 protein binding sites, an e1 protein binding site [60] , and three putative polyadenilation sites for late gene transcripts (table 2) . multiple potential binding sites for transcriptional regulatory factors, such as ap-1, nf-1, and sp-1, were also present within the urr (data not shown). typical domains were additionally identified in the putative viral proteins encoded by tbrapv1. the e6 protein contained two characteristic zinc-binding domains, separated by 36 amino acids [61] , and four internal and likely not functional pdz-binding motifs (rtnv, isdl, ssil, lssl) [62] . the e7 protein contained a prb-binding motif (lwcde) [63] and a single zinc-binding domain ( table 2) . analysis of the e1 protein, the largest protein encoded by tbrapv1 (table 2) , showed the typical atp-binding site of the atp-dependent helicase (gpsnsgks) [64] , and several cdk-phosphorylation and cyclin-binding sites. a highly conserved bipartite-like nuclear localization signal (nls) and a leucine-rich crm1-dependant nuclear export signal (nes) (lspvlekvti), which together allow shuttling of the e1 protein between the cell nucleus and the cytoplasm in most human pvs [65] [66] [67] , were identified at the n-termini of the e1 protein. no conserved leucine zipper domain was present at the c-termini of the putative tbrapv1 e2 protein, in agreement with other bat pvs (espv2 and rfpv1) [58] . at the n-termini of the l2 protein, a highly conserved furine cleavage motif (rrkr), as well as a transmembrane-like domain (gtggggrgvpigprvatgrpggpinsvg) [68] , were identified. in addition, a canonical polyadenylation site, necessary for regulation of early viral transcripts [69] , was also found in the tbrapv1 l2 gene (table 2) . phylogenetic analysis, based on 377 pv l1 gene nucleotide sequences, indicated peak sequence identities of tbrapv1 to hpv41 and rfpv1, which amounted to 61.5 and 60.6%, respectively (figure 3 , charts a2 and a4). maximum likelihood phylogenetic clustering of l1 sequences (figure 4 ) suggested common ancestry of tbrapv1, edpv1, and hpv41, and that tbrapv1 branched away prior to the delineation of edpv1 and hpv41, with high sh-alrt, abayes, and uf bootstrap support values. further phylogenetic analyses were conducted based on the concatenated alignments of 377 e1, e2, l2, and l1 gene nucleotide sequences, and they indicated a peak sequence identity of tbrapv1 to rfpv1 (54.1%, figure 3 , charts a1 and a3), whereas the maximum likelihood phylogenetic clustering indicated analogous common ancestry to the concatenated pv genes ( figure 5) , with high sh-alrt, abayes, and uf bootstrap support values. the metagenomic analysis indicated the presence of several genomovirus-related read pairs and sequence contigs (table 1) , and the complete genome sequence (2196 nt) of tbgkyv1 was recovered using de novo assembly. remapping to the tbgkyv1 genome sequence indicated a mean coverage of 59.4× by a total of 430 read pairs (860 paired reads + 7 unpaired reads) and did not reveal any abnormalities that would indicate misassembly. completeness of the circular genome sequence was determined by matching 5 and 3 ends, and the sequence was rotated to begin with the characteristic genomoviridae nonanucleotide motif [70] . three non-overlapping orfs, with a minimum protein length of 120 aa, were found, exhibiting peak amino acid similarities to the genomoviridae rep and cp/cap proteins. the rep protein of tbgkyv1 was encoded across two different rep-encoding orfs separated by an intron, representing a catalytic and a central protein domain ( figure 2b , table 3 ), which is characteristic for replication-associated proteins encoded by single-stranded (cress) dna viruses [71] . phylogenetic analysis and pairwise sequence comparison demonstrated that tbgkyv1 shares its highest identity in the rep protein (77.5%) and the complete genome level (nucleotide sequence, 77.2%) with the "mongoose associated gemykibivirus 1" (genbank accession number kp263545) [70] ( figures 3b and 6) . nucleic sequence identity histograms of the 377 pv nucleotide sequences (a1 and a2) and tbrapv1 (a3 and a4) based on the concatenated e1, e2, l2, and l1 gene sequences (a1 and a3) and on the l1 gene sequence (a2 and a4). multiple sequence alignments were constructed using muscle (v3.8.31) [47] , and the distance matrices were estimated using seaview v4.7 [49] , as suggested in bernard et al. (2010) . red arrows indicate the maximum sequence similarities of tbrapv1 for each of the sequence contexts (e1, e2, l2, and l1 concatenation and the l1 gene sequence). the blue arrows (a1 and a2) indicate the overall maximum sequence identity in the depicted context (e1, e2, l2, and l1 concatenation and the l1 gene sequence). the histograms were visualized using gnuplot (v5). (b) pairwise sequence identity histograms based on 167 complete genome nucleotide (purple) and rep gene (green) amino acid sequences from the genomoviridae family, based on the entire pairwise identity distance matrices (b1) and on the matrix slices representing pairwise identities of tbgkyv1 to all other 166 genomoviridae sequences (b2). the pairwise similarity matrices were obtained through pairwise sequence alignments (muscle v3.8.31) [47] , using sequence demarcation toolkit (sdt v1) [55] . for the arrows in the histogram: red = the species demarcation threshold/criteria (sdc) for genomoviridae [70] ; green and blue = maximum pairwise identity values of tbgkyv1 for the complete genome (green) and the rep protein sequence contexts (blue). histograms were visualized using gnuplot (v5). **nt = nucleotide, aa = amino acid, scd = sequence demarcation threshold/criteria [70] . cg: complete genome. nucleic sequence identity histograms of the 377 pv nucleotide sequences (a1 and a2) and tbrapv1 (a3 and a4) based on the concatenated e1, e2, l2, and l1 gene sequences (a1 and a3) and on the l1 gene sequence (a2 and a4). multiple sequence alignments were constructed using muscle (v3.8.31) [47] , and the distance matrices were estimated using seaview v4.7 [49] , as suggested in bernard et al. (2010) . red arrows indicate the maximum sequence similarities of tbrapv1 for each of the sequence contexts (e1, e2, l2, and l1 concatenation and the l1 gene sequence). the blue arrows (a1 and a2) indicate the overall maximum sequence identity in the depicted context (e1, e2, l2, and l1 concatenation and the l1 gene sequence). the histograms were visualized using gnuplot (v5). (b) pairwise sequence identity histograms based on 167 complete genome nucleotide (purple) and rep gene (green) amino acid sequences from the genomoviridae family, based on the entire pairwise identity distance matrices (b1) and on the matrix slices representing pairwise identities of tbgkyv1 to all other 166 genomoviridae sequences (b2). the pairwise similarity matrices were obtained through pairwise sequence alignments (muscle v3.8.31) [47] , using sequence demarcation toolkit (sdt v1) [55] . for the arrows in the histogram: red = the species demarcation threshold/criteria (sdc) for genomoviridae [70] ; green and blue = maximum pairwise identity values of tbgkyv1 for the complete genome (green) and the rep protein sequence contexts (blue). histograms were visualized using gnuplot (v5). nt = nucleotide, aa = amino acid, scd = sequence demarcation threshold/criteria [70] . cg: complete genome. figure 6 . phylogenetic tree of the genomovirus rep amino acid sequence. the tree was constructed using the lg+i+g4 substitution model, and branches are annotated with sh-alrt (1000 replicates), abayes, and uf bootstrap support (1000 replicates) values, respectively. maximum support values are shown with asterisks (*). novel tbgkyv1 is depicted in bold. genomoviridae genera were classified according to varsani and krupovic (2017) . unclassified sequences were not depicted. table 3 and figure 2b . the large intergenic region of the novel virus contains the characteristic nonanucleotide (tataaatag) motif, which is likely to be important for rolling-circle replication initiation [72] [73] [74] . the rep protein's catalytic domain of tbgkyv1 contained rolling circle replication motif i (lftysq), possibly involved in the recognition of iterative dna sequences associated with the origin of replication [70] , motif ii (thlhv), which may regulate the nicking/joining endonuclease activity at the origin of dna replication [73, 75] , motif iii (yatk), involved in the double-strand dna cleavage [73, 76] , and a geminivirus rep protein sequence (grs) (rlfdvenfhpnivpsr), which allows appropriate spatial arrangements of motifs ii and iii [77] . furthermore, rep helicase motifs walker-a (gpsrtgkt), walker-b (vfddi), and walker-c (wlmn) [78] [79] [80] [81] were identified in the central rep protein domain. walker motifs contribute to atp binding, which is used as an energy source to unwind the dsdna intermediate in the 3 -5 direction by the rep helicase [80, 81] . more than 200 viruses from 27 taxonomic families have been isolated from or detected in bats so far [16] , with a few of them implicated in the etiology of several severe diseases in humans. except for rabies, no direct evidence of zoonotic diseases transmitted by new world bats has been found [27] . new world, especially south american, and old world bat species had different evolutionary histories, leading to distinct immunological features [3, 16] . viruses infecting south american bat species have been poorly studied [24, 26] , and further research focused on evaluating their viromes is required. here, a first attempt to assess to the virome composition in pooled oral and anal swabs of t. brasiliensis was presented. during our study a total of 6,738,566 read pairs were removed during the laboratory batchbackground screening, due to traces of sequences from co-processed samples. the power of ngs stems from non-specific sampling of nucleic acids and automated repetition, yielding vast numbers of sequencing reads, providing the opportunity to characterize populations of nucleic acids with unprecedented sensitivity, accuracy, and non-specificity. due to its super-sensitivity, even the slightest addition of environmental nucleic acids to a sample may be detected using ngs and can potentially further complicate the interpretation of the results. laboratory background and/or dna isolation kit-derived contamination has been addressed previously as a major factor that can severely impede the interpretation of high throughput sequencing data, and the use of negative controls has been proposed [82] . moreover, positive/negative control samples have been recommended in metagenomic experiments aimed at detecting pathogens in clinical samples [83] . in this study, the metagenomic sample was processed alongside six samples of molluscum contagiosum skin lesions, and laboratory background filtering was initially considered due to the detection of approximately 90 molluscum contagiosum virus read pairs in the trimmed read dataset. in order to prevent the identification of human skin microflora in the pooled bat swab sample, a strict k-mer based negative filtering was used, effectively removing any read pair that contained at least one 27 bp subsequence that could be identified in any of the read pairs from the six background datasets. because the background samples also originated from mammalian (human) skin, it could be that a large portion of mammalian reads were removed during this step, explaining the somewhat extreme number of read pairs classified as laboratory-batch background. moreover, it is also likely that the viral composition of human and bat skin could be shared to some degree, but due to the filtering scheme reads originating from bats' anal and oral microflora sharing nucleotide sequence similarity to that of human skin may have also been removed prior to metagenomic classification. as a consequence, taken strictly, only the subset of viruses that are present in t. brasiliensis, but not in human skin, was explored here. although dna spillovers could be suspected and controlled for to some degree, in this case it may be a greater challenge in studies where multiple samples from different species or anatomical sites of bats are processed. in light of the present results, it would likely be beneficial to process the samples in such studies as independently as possible and to include negative controls that would characterize the laboratory background, such as sequencing libraries of buffer solutions that underwent the same treatment as the samples, as suggested previously [84, 85] . specifically, a total of 13,897 virus-related read pairs (0.53% out of 2,602,700) and 153 virus-related contigs (0.36% out of 42,891) were assembled de novo, mapped to viral taxa, and identified by ngs. although the proportion (and number) of virus-related sequences detected in this study (<1%) is comparable to reports of previous studies of bat viromes based on illumina sequencing [19, 21, 86] , it may be that additional physical viral dna/rna enrichment steps, such as centrifugation, filtration, and/or nuclease-treatment, could further augment the viral read yields, as suggested previously [87] . initially, this study was focused on the identification of pvs in t. brasiliensis, in order to explore their diversity in different hosts. accordingly, swab samples included in this work were first processed using experimental protocols, designed previously to suit our aforementioned initial aim [34] [35] [36] . viral dna was enriched using rca, as suggested previously by others [40] [41] [42] . however, it should be noted that rca may have favorably facilitated the amplification of circular genomes and, as a consequence, hindered the detection of linear genomes. thus, more than 80% of the classified viral sequences (11,162/13,801) identified in our analysis corresponded to circular viral genomes. in this study about 4.5% of viral reads and 60% of contigs corresponded to sequences from 35 eukaryotic viral families, mostly with dna genomes. interestingly, virus-associated sequences from rna viruses belonging to 14 families were also detected, most likely reflecting the presence of traces of viral rna. limited but highly accurate reverse transcriptase activity has indeed previously been reported for the phi29 dna polymerase, used in rca [88] . most of the insect-infecting viral sequences detected belonged to viral families infecting lepidopteran adults or larvae, which may represent the diet of t. brasiliensis, as detected previously in feces and anal swabs from various insectivorous bat species (myotis sp., rhinolophus sp., molossus sp., neoromicia sp.), including t. brasiliensis [19, 21, 23, 26] . in addition, the detection of various plant viral families in this study could reflect the plant diet of the insects ingested by the bats. a total of 15 different mammalian viral families were identified in t. brasiliensis samples, representing approximately 43% (15/35) of the eukaryotic viral families interrogated herein. several mammalian viral families, supported by the contigs and sequencing reads, have been identified previously in new world [23, 24, 26] and old world [17, 18, 89] bat species. the mammalian viral families identified in t. brasiliensis included typical zoonotic viruses identified previously in bats, such as polyomaviridae [29] , rhabdoviridae [90] , coronaviridae [23, 28] , poxviridae, flaviviridae, and adenoviridae [23] . the identification of circoviridae and astroviridae in t. brasiliensis was also in line with the results of previous studies [23, 31] . on the other hand, this study indicated the presence of genomoviridae, alloherpesviridae, papillomaviridae, herpesviridae, paramyxoviridae, and reoviridae in t. brasiliensis for the first time. notably, the presence of incorrect annotations in public databases, such as the sequences assigned to the retroviridae family in this study, highlight the need for the curation of data (whenever possible) to avoid the under-and/or overestimation of the classified sequences derived from metagenomics studies. pvs are a highly diverse family of non-enveloped and double-stranded circular dna viruses that are known to infect a wide variety of mammals, as well as birds, reptiles, and fish [91, 92] . various human and non-human pvs, including bat pvs, have frequently been identified in healthy epithelia and may represent part of the native epithelial microflora [34, 57, 58] . several studies have suggested the presence of pvs in old world bat species using conventional [57, 58, 93] or ngs aproches [20, 41, 94, 95] . the only pv type (mmopv1) identified in a new world bat species (molossus molossus) has recently been described [26] , suggesting a crude sampling imbalance and a severe lack of information to elucidate the evolutionary mechanisms driving pv diversification on the global scale. in this study, tbrapv1 has been identified in pooled oral and anal swabs of t. brasiliensis by ngs, and its sequence has been completely characterized by conventional molecular techniques. tbrapv1 is the first reported pv type found in t. brasiliensis and the second pv type identified in new world bat species. according to the current ictv papillomaviridae classification guidelines (published in june 2018), based on the nucleotide sequence of the l1 gene [96] , tbrapv1 is the founding member of a novel pv genus in the firstpapillomavirinae taxonomical subfamily, sharing more than 45% sequence identity to other pv types included in this subfamily. although nucleotide sequences analysis in the l1 gene indicated that tbrapv1 shares a 61.5% identity with hpv41 (nu-pv) and should be included within the same genus (more than 60% of nucleotide identity in l1 gene) [91] , the mentioned demarcation criteria suggests a visual inspection of phylogenetic trees derived from concatenated e1, e2, l1, and l2 nucleotide sequences to delineate pv genera [96] . in the present study, such analysis clustered tbrapv1 basal to the delineation of hpv41 (nu-pv) and edpv1 (sigma-pv), identified in a north american porcupine (erethizon dorsatum) and, therefore, may represent a novel genus within the papillomaviridae family. this phylogenetic clustering also indicated that tbrapv1 shares common ancestry with other bat pvs such as espv1, espv3, rfpv1, ehpv1, and mscpv2. on the other hand, tbrapv1 is only distantly related to rapv1, ehpv2, ehpv3, espv2, mscpv1, and mrpv1, which have been identified from different tissues and bats species. the idea that different bat pvs evolved during a process of strict host coevolution is further refuted by the observation that different bat pvs appear scattered around the papillomaviridae phylogenetic tree in a highly polyphyletic manner [57, 58] . in addition, under strict host coevolution it would be expected for tbrapv1 and mmopv1, both molossid pvs, to be closely related; nevertheless, mmopv1 has a basal taxonomic position with respect to tbrapv1. these observations support the idea of multiple evolutionary forces as drivers of pv evolution, including coevolution, adaptive radiation, broad host range, host switch, and recombination [58] . genomoviruses are single-stranded circular dna viruses that belong to the recently proposed genomoviridae family [97] . members of this family encode two genes-the cap/cp and the rolling-circle replication-associated protein (rep)-and an intergenic region. it has been proposed that a novel viral complete genome sequence of the same species exhibits more than 78% similarity to any other complete genomovirus genome [70] . in addition, in previous studies, the authors aimed to establish nine genera within the family genomoviridae based on pairwise comparisons of complete genome sequences [70] . cress dna viruses, including genomoviruses, have been found in association with a great variety of animal species, such as camels [98] , bats [89] , mongooses, badgers [99] , wolves [100] , pigs [101] , and humans [102] , as well as in environmental-associated [103] and plant-associated [104] samples. however, no direct implication with a disease has been demonstrated so far. in particular, bat-associated genomoviruses have been identified from feces [105, 106] or pharyngeal and anal swab samples [89] of asiatic [89, 105] or european [106] bat species and have been attributed to various taxonomic genera of the genomoviridae family [70] . tbgkyv1 is a novel species within the gemykibivirus genus according to the classification criteria [70] . it should be noted that the rep and cap proteins of tbgkyv1 exhibited different percentages of similarity, the cap being considerably more divergent than the rep, indicating differences in their evolutionary histories due to their respective molecular functions [70, 106] . to the best of our knowledge, this is the first report demonstrating the presence of genomoviral sequences in mucosal swab samples of a new world bat species. finally, it is worth noting that the results of our metagenomic screening of the pooled t. brasiliensis oral and anal swab samples is effectively provided at three different levels of specificity/sensitivity. the two complete genome sequences (tbrapv1 and tbgkyv1), that have been described at the highest level of detail, also confer the highest level of confidence. in other words, we have complete confidence that these two viruses were present in the pooled nucleic acid samples. assigning sequences that did not assemble at the level of complete viral genomes to taxonomical families could therefore be a valid approach offering a higher level of sensitivity than only the complete genome assemblies, but at the cost of diminished specificity. in addition, identifying a sequence fragment that resembles a known viral genome in a given genomic region, may not always be sufficient to infer that that specific virus was present in the sample. viruses are highly promiscuous entities, which can easily exchange parts of their genomes with their hosts, be integrated or even naturalized into the host genomes [107] . taxonomical viral families identified among the assembled contigs could be interpreted as viral families that were probably represented in our samples. lastly, and due to the low number of assembled contigs, that were found related to known viral sequences, we attempted to increase the sensitivity even further by obtaining taxonomical family mappings also for the source read pairs. these results, however, should be interpreted with utmost caution, because they likely confer a very low level of specificity due to the limited sequence length (2 × 150 bp). taxonomical viral families identified by read-pair taxonomy mapping only, merely suggest the possibility that these families were present and should be replicated in the future by taxonomical mappings conferring greater specificity, for example with longer sequences, such as those assembled from illumina or nanopore reads. this study presents an initial description of the oral/anal virome composition of t. brasiliensis, a widely-distributed new world bat species living in close contact with the human population, for the first time. although their biological significance is not clear, this work contributes to a better understanding of the evolution and genetic diversity of these viruses. using conventional nucleic acid detection techniques and/or bioinformatics approaches, the whole genomes of two novel viruses were completely covered, tbrapv1 and tbgkyv1, clustering into the papillomaviridae and genomoviridae families. tbrapv1 is the first pv type identified in this host and the prototype of a novel genus in the firstpapillomavirinae taxonomic subfamily. tbgkyv1 is the first genomovirus reported in new world bats and constitutes a new species within the genus gemykibivirus. future studies are required to investigate the possible health impact of the viruses described on bat colonies and to identify the factors that contribute to their dispersal. supplementary materials: the following are available online at http://www.mdpi.com/1999-4915/12/4/422/s1, supplementary data 1: details of the assembled nucleotide sequences obtained by de novo assembly and used in the taxonomic classification as a part of the metagenomic workflow (2) (figure 1 ). table s1 : de novo assembly settings in metagenomic workflow: taxonomic 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monitoring of viruses: insights from an endangered wolf population small circular single stranded dna viral genomes in unexplained cases of human encephalitis, diarrhea, and in untreated sewage detection and molecular characterization of gemycircularvirus from environmental samples in brazil identification and molecular characterization of a single-stranded circular dna virus with similarities to sclerotinia sclerotiorum hypovirulence-associated dna virus 1 genome sequences of poaceae-associated gemycircularviruses from the pacific ocean island of tonga diverse replication-associated protein encoding circular dna viruses in guano samples of central-eastern european bats global organization and proposed megataxonomy of the an integrated tool for comprehensive microbial variant detection and genome assembly improvement this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors thank irene villa, german saigo, mauricio taborda, and violeta di domenica for collecting bat samples. the authors declare no conflict of interest.viruses 2020, 12, 422 key: cord-355737-o0y4rn0z authors: ng, melinda; ndungo, esther; kaczmarek, maria e; herbert, andrew s; binger, tabea; kuehne, ana i; jangra, rohit k; hawkins, john a; gifford, robert j; biswas, rohan; demogines, ann; james, rebekah m; yu, meng; brummelkamp, thijn r; drosten, christian; wang, lin-fa; kuhn, jens h; müller, marcel a; dye, john m; sawyer, sara l; chandran, kartik title: filovirus receptor npc1 contributes to species-specific patterns of ebolavirus susceptibility in bats date: 2015-12-23 journal: elife doi: 10.7554/elife.11785 sha: doc_id: 355737 cord_uid: o0y4rn0z biological factors that influence the host range and spillover of ebola virus (ebov) and other filoviruses remain enigmatic. while filoviruses infect diverse mammalian cell lines, we report that cells from african straw-colored fruit bats (eidolon helvum) are refractory to ebov infection. this could be explained by a single amino acid change in the filovirus receptor, npc1, which greatly reduces the affinity of ebov-npc1 interaction. we found signatures of positive selection in bat npc1 concentrated at the virus-receptor interface, with the strongest signal at the same residue that controls ebov infection in eidolon helvum cells. our work identifies npc1 as a genetic determinant of filovirus susceptibility in bats, and suggests that some npc1 variations reflect host adaptations to reduce filovirus replication and virulence. a single viral mutation afforded escape from receptor control, revealing a pathway for compensatory viral evolution and a potential avenue for expansion of filovirus host range in nature. doi: http://dx.doi.org/10.7554/elife.11785.001 ebola virus (ebov) and some of its relatives in the family filoviridae (filoviruses) cause sporadic outbreaks of a highly lethal disease. these outbreaks are thought to be initiated by viral spillover from an animal reservoir to a highly susceptible accidental host, such as a human or nonhuman primate (feldmann and geisbert, 2011; leroy et al., 2005; towner et al., 2009) . recent work suggests that some filoviruses infect bats in nature, and that these viruses may be distributed more widely than previously recognized. very short rna fragments corresponding to portions of ebolavirus genomes were detected in several frugivorous bats of the family pteropodidae ('old world fruit bats') in both africa and asia (leroy et al., 2005; jayme et al., 2015) , and longer filovirus rna fragments and near-complete rna genomes were isolated from insectivorous schreibers's long-fingered bats in asia and europe, respectively (negredo et al., 2011; he et al., 2015) . however, despite considerable efforts, infectious ebolaviruses have never been recovered from bats. by contrast, marburg (marv) and ravn (ravv) viruses were found to circulate in egyptian rousettes (rousettus aegyptiacus), indicating that these bats are susceptible to marv/ravv and encounter them frequently in nature. egyptian rousettes have been proposed as natural hosts for these viruses (amman et al., 2012; towner et al., 2009) . this progress notwithstanding, many key questions remain. for example, the biological factors that influence filovirus host range and interspecies transmission are still poorly understood, as are the virus-host relationships that determine which species of bats are susceptible to infection by ebov and other filoviruses. viral entry receptors are key determinants of tissue tropism and host range (radoshitzky et al., 2008; sheahan et al., 2008; hueffer et al., 2003; demogines et al., 2013) . niemann-pick c1 (npc1), a highly conserved endo/lysosomal protein involved in cellular cholesterol trafficking, was recently identified to be an essential entry receptor for all known filoviruses (cô té et al., 2011; carette et al., 2011; ng et al., 2014) . in this study, we uncover a pattern of virus and host species specificity in the filovirus susceptibility of bat cells, which can be explained by elife digest ebola virus and other filoviruses can cause devastating diseases in humans and other apes. numerous small outbreaks of ebola virus disease have occurred in africa over the past 40 years. however, in 2013-2015, the largest outbreak on record took place in three western african nations with no previous history of the disease. human outbreaks of ebola virus disease likely begin when a person encounters an infected wild animal. though it remains unclear precisely which animals harbor ebola virus between outbreaks, and how they transmit the virus to humans or other primates, recent work showed that some filoviruses do infect specific types of bats in nature. ng, ndungo, kaczmarek et al. sought to identify the genes that influence whether or not a type of bat is susceptible to infection by ebola virus and other filoviruses. several filoviruses, including ebola virus, were tested to see if they could infect cells that had been collected from four types of african fruit bats. these bats are all found in areas where outbreaks have occurred in the past. the tests revealed that a small change in the sequence of the npc1 gene in some bat cells greatly reduced their susceptibility to ebola virus. npc1 encodes a protein that mammals need in order to move cholesterol within their cells. in humans, the loss of the protein encoded by npc1 causes a rare but very severe disease called niemann-pick type c disease. this protein also turns out to be a receptor that the filoviruses must bind to before they can infect the cells. further analysis then revealed that npc1 has evolved rapidly in bats, with changes concentrated in the parts of the receptor that interact with ebola virus. ng, ndungo, kaczmarek et al. went on to discover some changes in the genome sequence of ebola virus that could compensate for the changes in the bat's npc1 gene. these findings hint at one way that a filovirus could evolve to better infect a host with receptors that were less than optimal. following on from this work, the next challenges will be to expand the investigation to include additional types of bats, other types of mammals, and other host genes that could influence filovirus infection and disease. further studies could also examine the other side of the arms race -that is, the evolution of viral genes in bats. however, such studies would be complicated by the lack of viral sequences that have been collected from bats, because to date most have been isolated from humans and other primates instead. changes in the affinity of the essential interaction between npc1 and the filovirus entry glycoprotein, gp. crucially, genetic analyses reveal that npc1 is under positive selection in bats, with a strong signature of selection at precisely the same residue that influences the filovirus-receptor interaction. our findings suggest that amino acid sequence changes in npc1 at these positively-selected sites represent host adaptations to resist filovirus infection, and reveal one pathway by which a filovirus could escape from receptor control. in sum, our results support the hypothesis that bats and filoviruses have been engaged in a long-term co-evolutionary relationship, one facet of which is a molecular arms race between the viral glycoprotein and its entry receptor, npc1. means ± sd (n = 3-4) from two biological replicates are shown. in panels c and d, the infectivity of each virus was normalized to that obtained in vero grivet monkey cells. means for infection of the different cell lines by each virus were compared by one-way anova (p-value indicated above each group of bars). tukey's post hoc test was used to compare infection means on hypsignathus monstrosus vs eidolon helvum cells (*p < 0.05; ****p < 0.0001; ns, no statistical significance). doi: 10.7554/elife.11785.003 the following figure supplements are available for figure 1 : we first explored the possibility that there exist virus-and/or bat species-dependent differences in the cellular host range of filoviruses. kidney fibroblast cell lines derived from three african pteropodids whose ranges overlap the locations of known african filovirus disease outbreaks ( figure 1a ,b) were exposed to authentic ebov and marv ( figure 1c ). we observed a large ebov infection defect in african straw-colored fruit bat (eidolon helvum) cells but not in cells from bü ttikofer's epauletted fruit bats (epomops buettikoferi) and egyptian rousettes. by contrast, cells from bats of all three species were similarly susceptible to infection by marv ( figure 1c) . thus, cells from african straw-colored fruit bats appear to be selectively refractory to ebov infection. an npc1-dependent block to cell entry accounts for the ebov infection deficit in african straw-colored fruit bat cells the viral spike glycoprotein, gp 1,2 (herein termed gp) mediates all steps of filovirus entry into the cytoplasm of host cells . vesicular stomatitis viruses bearing filovirus gp proteins (vsv pseudotypes) provide a highly validated surrogate system to recapitulate filovirus entry under biosafety level 2 containment (takada et al., 1997; jangra et al., 2015) . to assess whether the ebov infection defect in the african straw-colored fruit bat cells occurs at the viral entry step, we exposed an expanded panel of kidney fibroblast cell lines from four african pteropodids to vsv pseudotypes bearing gp spikes (vsv-gp) from seven filoviruses, including two non-african viruses, reston virus (restv) and lloviu virus (llov) ( figure 1d ). as observed with authentic ebov, vsv-ebov gp infection was substantially reduced in the african straw-colored fruit bat cells; however, this virus could efficiently infect cells derived from the other pteropodids, including those of a proposed ebov host, the hammer-headed fruit bat (hypsignathus monstrosus) (leroy et al., 2005) . strikingly, only vsvs bearing ebov gp, and to a lesser degree, those bearing bdbv and tafv gp, were deficient at infecting african straw-colored fruit bat fibroblasts. similar strong but ebov-specific reductions in infection were measured in two kidney and lung cell lines derived from additional african straw-colored fruit bats (figure 1-figure supplement 1) . therefore, reduced infection of these bat cells by ebov reflects a virus-and host species-specific restriction at the cell entry step. we surmised that the filovirus receptor, npc1, might explain the selective resistance of the african straw-colored fruit bat cells to ebov entry and infection. accordingly, we engineered these cells to stably express human npc1 (hsnpc1) (figure 2-figure supplements 1,2), and then exposed them to ebov ( figure 2a ). provision of hsnpc1 substantially enhanced authentic ebov infection in the african straw-colored fruit bat cells. by contrast, we found no evidence that either marv infection in these cells, or ebov/marv infection in permissive bü ttikofer's epauletted fruit bat cells was limited by receptor availability (figure 2a ). finally, similar results were obtained with vsvs bearing filovirus glycoproteins ( figure 2b ). taken together, these findings indicate that ebov infection is reduced in african straw-colored fruit bat cells because of a specific molecular incompatibility between the ebov glycoprotein and the filovirus entry receptor. npc1-dependent cell entry is reduced, but not completely eliminated, in african straw-colored fruit bat cells although ebov entry and infection in african straw-colored fruit bat cells was consistently reduced to 0.1-1% relative to that in cells from the other pteropodids, we noted that infection was not completely blocked. to determine if ebov could inefficiently infect these bat cells via an npc1-independent mechanism, we used crispr/cas9 genome engineering to derive an african straw-colored fruit bat cell line fully deficient in npc1. we identified a single cell clone (eidolon helvum npc1-#1 [ehnpc1-#1]) in which all npc1 alleles bore insertions or deletions (indels) at the expected site ( figure 3a ). these indels were predicted to frameshift the npc1 open reading frame at amino acid position 81 (homo sapiens hsnpc1 numbering), generating truncated polypeptides of 82, 83, and 109 residues that lacked the majority of the 1278-amino acid npc1 sequence. ehnpc1-#1 cells were deficient in clearance of lysosomal cholesterol, a well-established cellular function of npc1 (carstea et al., 1997) , but could be rescued by ectopic hsnpc1 expression, confirming that npc1 had indeed been disrupted in these cells ( figure 3b ). we next exposed wild-type (wt) and ehnpc1-#1 fibroblasts to vsvs bearing ebov or marv gp. no detectable infection was obtained with either virus in npc1-deficient cells, indicating that filovirus entry into these cells is absolutely dependent on the e. helvum npc1 ortholog ( figure 3c ). moreover, ebov gp-dependent infection in ehnpc1-#1 cells reconstituted with hsnpc1 was dramatically enhanced over that observed in wt cells, whereas marv gp-dependent infection was rescued by hsnpc1 expression to a level resembling that in wt cells ( figure 3c ). therefore, the low levels of ebov infection in african straw-colored fruit bat cells likely arise from the weak, but nonzero, activity of ehnpc1 as an ebov entry receptor. filovirus gps must directly engage the second luminal domain of npc1, domain c, during cell entry . accordingly, we postulated that the african straw-colored fruit bat npc1 ortholog is poorly recognized by ebov gp. to test that hypothesis, we generated and sequenced npc1 cdnas from all four pteropodid cell lines. alignment of their domain c amino acid sequences with that of hsnpc1 revealed a high degree of conservation (>90%), with identical arrangements of cysteine residues and similar predicted secondary structures suggestive of a similar overall fold (figure 3-figure supplement 1) . to examine gp-npc1 binding, we engineered and expressed soluble forms of the four pteropodid npc1 domain cs, as described for hsnpc1 (figure 4 -figure supplement 1) . a cleaved form of ebov gp could capture hsnpc1 domain c in an elisa, as shown previously . ebov gp bound with similar avidity to npc1 domain cs derived from egyptian rousettes (ranpc1), hammer-headed fruit bats (hmnpc1) and bü ttikofer's epauletted fruit bats (ebnpc1), but poorly or not at all to that of african straw-colored fruit bats (ehnpc1) ( figure 4a ). like the infection defect in african straw-colored fruit bat cells, this receptor binding defect was selective for ebov gp, since gps derived from marv and the european filovirus, llov (ng et al., 2014) , bound equivalently to all four pteropodid domain cs ( figure 4a ). these findings the restriction in ehnpc1-ebov gp binding can be mapped to a single amino acid change in ehnpc1 to define the molecular basis of the defect in interaction between ebov and ehnpc1, we generated a panel of npc1 domain c chimeras comprising sequences from permissive ranpc1 and nonpermissive ehnpc1, and tested them in the gp-binding elisa. a single chimera, ehnpc1 domain c containing four ehnpc1firanpc1 amino acid residue changes, regained the capacity to efficiently recognize ebov gp ( figure 4b ). further dissection revealed that only a single amino acid change, f502d, in a central region of npc1 domain c was needed to effect this complete restoration in gpfigure 3 . the incompatibility between ebov gp and eidolon helvum npc1 reduces, but does not eliminate, ebov entry into african straw-colored fruit bat cells. (a) crispr/cas9 genome engineering was used to knock out the npc1 gene in african straw-colored fruit bat kidney fibroblasts. wt npc1 gene sequence aligned with the sequences of all three alleles in the knockout (npc1-#1) cell clone. the grna target sequence is marked in red, and the protospacer adjacent motif (pam) sequence of the grna target site is underlined. (b) the capacity of wt and npc1-#1 cells, and npc1-#1 cells stably expressing hsnpc1, to clear lysosomal cholesterol was determined by staining with filipin iii complex from streptomyces filipensis, as described (carette et al., 2011) . . we conclude that a species-specific defect in virus-receptor interaction, caused by a single amino acid residue change in ehnpc1 relative to other, permissive african pteropodid npc1 orthologs, reduces ebov infection in african straw-colored fruit bat cells. moreover, because residues in the npc1-binding site are conserved among all available ebov gp sequences (supplementary file 1), this restriction is almost certain to be encountered by all known ebov variants and their isolates, including those detected in ebov disease patients during the recent epidemics in western and middle africa gire et al., 2014; tong et al., 2015; carroll et al., 2015; kugelman et al., 2015) . previous work has led to the hypothesis that bats in equatorial africa and elsewhere harbor filoviruses (reviewed in [wahl-jensen et al., 2013] ). these results, together with our findings for virusand host species-specific differences in cellular susceptibility to filovirus infection, hinted at the possibility of a deeper co-evolutionary relationship between filoviruses and bats. one hallmark of such a relationship between a virus and its host is the evolution, under selective pressure to resist infection, of host genes encoding proviral and antiviral factors. to evaluate whether the npc1 gene has evolved under positive selection in bats, we combined the npc1 sequences obtained in this study with those of bats from six other species (two non-african pteropodids, two phyllostomids, and two vespertilionids) compiled through assembly of publicly available rnaseq data ( a mutation in ehnpc1 reduces receptor binding to ebov gp and viral infection, a phenotype that could reasonably produce a selective advantage (figure 4) . other codons identified in only some of the tests for dn/ds>1, or at slightly lower significance levels, may still have functional significance. for example, additional codons were identified in two regions of domain c that may form a part of the recognition surface for ebov gp ( figure 5c ). our finding that signatures of accelerated sequence evolution localize to structural features in npc1 that are important for virus binding (domain c and position 502) leads us to postulate that mutations at these sites can protect bats from infection or severe disease caused by filoviruses and/or other intracellular microbes. a single mutation at residue 141 in ebov gp enhances viral entry by strengthening its interaction with ehnpc1 co-evolutionary arms races between hosts and pathogens are thought to be driven by cycles of genetic adaptation and counter-adaptation (meyerson and sawyer, 2011; daugherty and malik, 2012; demogines et al., 2013) . in this context, we postulated that mutation of residue 502 in ehnpc1 could be countered by viral mutation. to identify such putative compensatory viral changes, figure 6 . a sequence polymorphism in the npc1-binding site of filovirus gp influences gp-ehnpc1 binding and ehnpc1-dependent filovirus entry. (a) binding of ebov gp (wt and mutant v141a) to soluble npc1 domain c proteins derived from african pteropodids measured by an elisa. ranpc1, egyptian rousette; ebnpc1, bü ttikofer's epauletted fruit bat; hmnpc1, hammer-headed fruit bat; ehnpc1, african straw-colored fruit bat. (b) infection of african straw-colored fruit bat cells with vsv pseudotypes bearing ebov gp (wt or v141a). means ± sd (n = 3-4) from a representative experiment are shown in each panel. means for vsv-ebov gp wt vs v141a infection were compared by unpaired two-tailed student's t-test with welch's correction (**p < 0.01). (c) surface-shaded representation of a single gp1-gp2 monomer (pdb id: 3csy highlighting key residues in the npc1-binding site (yellow) and residue 141 (red). gp1, blue. gp2, grey. (d) alignments of gp1 sequences from a panel of filoviruses. v141, orange; a141, white text on blue shading; other residues divergent from consensus sequence, black text on green shading. (e) infection of african pteropodid cells with vsv pseudotypes bearing sudv gp (wt or a141v). means ± sd (n = 4) from two biological replicates are shown. means for vsv-sudv gp wt vs a141v infection on each cell line were compared by unpaired two-tailed student's t-test with welch's correction (*p < 0.05, **p < 0.01, ****p < 0.0001). doi: 10.7554/elife.11785.015 we screened a panel of point mutants in the npc1-binding site of ebov gp by elisa for enhanced binders to ehnpc1 domain c. while no single point mutant bound to ehnpc1 as well as it did to the other pteropodid npc1s or to hsnpc1, gp(v141a) partially restored ehnpc1 binding ( figure 6a ). infection by vsv particles bearing ebov gp(v141a) was substantially enhanced in african straw-colored fruit bat cells, commensurate with this mutant gp's increased binding affinity for ehnpc1 ( figure 6b) . examination of the x-ray crystal structure of ebov gp revealed that v141 is located at the edge of the putative npc1-binding site, where it forms part of a raised rim ( figure 6c ). the v141a mutation likely creates a more sterically favorable (open) npc1binding site that can overcome the structural mismatch at the gp-npc1 binding interface ( figure 6c ). naturally-occurring sequence variation at residue 141 in gp contributes to virus-and bat species-specific patterns of cellular susceptibility to filoviruses although no known ebov isolate contains the v141a mutation, we observed that llov and sudan virus (sudv) gp naturally possess a141 ( figure 6d ). because both gp proteins could mediate efficient viral entry into african straw-colored fruit bat cells ( figure 1d ) and bind to ehnpc1 ( figure 4a data not shown for sudv), we postulated that amino acid changes at position 141 in the gp receptor-binding site broadly influence the capacity of filovirus glycoproteins to utilize ehnpc1 for viral entry. accordingly, we exposed pteropodid kidney fibroblasts to vsv pseudotypes bearing sudv gp(wt) or sudv gp(a141v) ( figure 6e ). consistent with our hypothesis, the a141v mutation substantially reduced sudv gp-dependent infection in african straw-colored fruit bat cells. unexpectedly, this mutant virus also infected egyptian rousette cells significantly less well than wt, pointing to the existence of sequence context-dependent effects that selectively affect sudv gp(a141v) binding to ranpc1 ( figure 6e ). these findings provide evidence that gp residue 141 can influence cellular susceptibility to infection by modulating npc1 recognition in a manner that depends on the sequences of both proteins. we speculate that sequence variation at residue 141 and potentially other positions in the receptor-binding site of filovirus glycoproteins has been shaped by selective pressure to utilize restrictive npc1 receptors, with potential consequences for viral host range and virulence. the ongoing, unprecedented ebola virus disease epidemic in western africa highlights the urgent need to uncover the biological and ecological factors that underlie the distribution, evolution, and emergence of filoviruses. while a full answer to this question will require the integration of knowledge across multiple levels of biological organization, from genes to populations to ecosystems, previous work has shown that studies of molecular interactions between viruses and their host cells can contribute important pieces to this puzzle. the essential interactions between viruses and their entry receptors provide particularly cogent examples. a switch in receptor binding from the feline to the canine ortholog of the transferrin receptor drove the emergence of a new virus, canine parvovirus, and fueled a global disease pandemic in dogs (allison et al., 2014) . analyses of interactions of sars-like coronaviruses with their receptor ace2 have helped to trace the emergence of sars coronavirus from bats to humans, and its use of civets as intermediate amplifying hosts ge et al., 2013; ren et al., 2008) . in this study, we show that interactions between filoviruses and their entry receptor npc1 can influence the cellular susceptibility of bats to infection. this observation is especially striking in light of previous findings that filoviruses could efficiently infect a broad range of mammalian cells, including some derived from bats (kuhn, 2008; kuhl et al., 2011) . indeed, this prior work and the results of experimental infection studies in rodents and bats have led to the hypothesis that interactions between viral components and those of the host innate and adaptive immune systems constitute the primary molecular variables influencing filovirus host range in nature (ebihara et al., 2006; volchkov et al., 2000) . here, we propose that npc1 is also a genetic determinant of filovirus susceptibility in bats. the essential nature of npc1 for infection in cells derived from mammals of multiple species, including bats (figure 3) , and for infection and in vivo pathogenesis in lethal ebov infection mouse models argues against the existence of alternative filovirus entry receptors (carette et al., 2011; herbert et al., 2015) . therefore, strong reductions in the affinity of virus-npc1 recognition are predicted to reduce or eliminate infection in whole bat hosts, as observed in npc1deficient mice (carette et al., 2011; herbert et al., 2015) , barring viral mutation to enhance this affinity. it is conceivable that even modest defects or delays in viral multiplication through such a mechanism could help determine host range by accelerating viral immune clearance, as recently observed in npc1-heterozygous mice (herbert et al., 2015) , or by synergizing with other host-virus barriers. the highly virus-and host species-specific nature of the virus-receptor mismatch uncovered in this study warrants the determination of more bat npc1 sequences for inclusion in genetic analyses (see below), and a more comprehensive phenotypic examination of virus-bat pairs. such studies maydiscover additional interesting bat-filovirus dynamics, including incompatibilities between filoviruses and npc1 or other proviral/antiviral host factors. such discoveries have potential implications for our understanding of the molecular basis of filovirus infection, virulence, and host range. we found that a single amino acid change, at residue 502, in the african straw-colored fruit bat ortholog of npc1 (ehnpc1) greatly diminished the susceptibility of cells from multiple tissues and individuals to ebov. these migratory pteropodids are widely distributed across sub-saharan africa ( figure 1a) , roost in large colonies near human settlements, and host other rna viruses with zoonotic potential peel et al., 2013) . moreover, they are extensively hunted for bushmeat in western africa (kamins et al., 2011) , making them ideal candidates to transmit viruses directly to humans. unfortunately, there is little information currently available on the susceptibility of african straw-colored fruit bats to ebov or their potential role as filovirus hosts. serologic surveys have found some evidence for exposure to one or more filovirus; however, neither infectious virus nor coding-complete or full viral genomes-the gold standards-have been successfully obtained from these bats, indicating they may only have been exposed to filoviruses, rather than being productively infected (reviewed in [wahl-jensen et al., 2013; olival and hayman, 2014] ). while more extensive wildlife sampling and, if feasible, experimental infections of african straw-colored fruit bats will be required to clarify this picture, we can extrapolate to several possible scenarios. first, these bats are fully resistant to ebov, and therefore cannot be the source of this virus in the 2013-present ebov disease outbreak in western africa or the 2014 outbreak in middle africa. second, because african straw-colored fruit bat cells do remain weakly susceptible to ebov ( figure 3c ), it is conceivable that they support ebov replication at low levels. indeed, this is one hallmark of a sustaining viral reservoir. third, the filoviruses circulating in these bats, whether ebov or otherwise, bear one or more gp mutations (e.g., v141a) that circumvent the infection barrier imposed by ehnpc1. assessing this last hypothesis and understanding the nature of the selection pressures that drive gp evolution in vivo will require the isolation of ebolavirus gp sequences from bats-there are none currently available. although these results suggest that african straw-colored fruit bats are selectively refractory to ebov, our genetic findings indicate that this is not merely a special relationship between one host and one virus. rather, we used a diverse set of bat npc1 sequences, only one of which is from african straw-colored fruit bats, to show that a number of codons, including residue 502, have evolved under recurrent positive selection. this is a process in which resistant npc1 variants are 'serially replaced' in response to compensating viral mutations that restore susceptibility. we provide evidence that the filovirus gp interaction surface in the second luminal domain of npc1, domain c, is a hotspot for such positive selection ( figure 5 ). by contrast, the vast majority of codons in mammalian npc1 have evolved under purifying selection. we propose that this pattern of selection is the signature of a long-term genetic conflict between filoviruses and npc1 in bats, superimposed over the normal evolutionary signature of a housekeeping gene with a critical role in cellular cholesterol trafficking. similar signatures of recurrent positive selection have been identified in other housekeeping genes that encode viral receptors, including the transferrin receptor (kaelber, et al., 2012; demogines et al., 2013 ) (tfr; receptor for new world arenaviruses [radoshitzky et al., 2007] , the betaretrovirus murine mammary tumor virus [ross et al., 2002] , and parvoviruses [parker et al., 2001] ), bat angiotensin-converting enzyme-2 (demogines et al., 2012) (ace2; receptor for sars-like coronaviruses [li et al., 2003] ), and mammalian dipeptidyl peptidase-4 (cui et al., 2013) (dpp4; receptor for mers-like coronaviruses [raj et al., 2013] ). in these cases as well, the preponderance of positively-selected residues localize to virus-receptor interfaces. interestingly, the sequence polymorphism at npc1 residue 502 did not impair cholesterol clearance from lysosomes ( figure 3) , and none of the residues under positive selection were found to be mutated in niemann-pick type c disease patients (runz et al., 2008; vanier and millat, 2003) . thus, despite being constrained by its housekeeping function, npc1 appears to retains a sizeable sequence space accessible to adaptive mutation. it is tempting to speculate that sequence variation at residue 141 ( figure 6 ) and potentially other positions in the receptor-binding site of filovirus glycoproteins represents the other half of the genetic arms race, shaped by selective pressure to utilize restrictive npc1 receptors. although more data, especially filovirus sequences from bats, are needed, our findings raise the tantalizing possibility that filoviruses, including those yet undiscovered, are each adapted to specific bat hosts, with co-evolved virus-receptor interactions constituting one potential biological barrier to interspecies viral transmission. alternatively, it is conceivable that repeated contacts between unknown (non-bat) reservoir hosts carrying specific filoviruses, and bats of particular species, have driven positive selection in bat npc1 to limit infection (and selection of filoviruses with compensating sequence changes in gp). in this scenario, detection of anti-filovirus antibodies or filovirus genome-derived oligonucleotides may reflect a type of spillover event from the actual filovirus reservoir hosts into bats. our hypothesis that npc1 in bats has been genetically sculpted by filoviruses (and vice versa) presupposes not only a long-term coevolutionary relationship, but also one in which these viruses have imposed selective pressure on bats to limit or eliminate infection. the discovery of filovirus np-and vp35-related endogenous viral elements (eves) in bat genomes is consistent with such a long-term relationship (taylor et al., 2010; katzourakis and gifford, 2010) . to further investigate the deeper origins of filoviruses in bats, we screened all available bat genomes for filovirus-related eves. we obtained evidence for synteny between a filovirus nucleoprotein (np)-like eve in the genome of the big brown bat (eptesicus fuscus) and those previously identified in three, more distantly-related, myotis bats (figure 7 and supplementary file 5) (taylor et al., 2011) . this new discovery strongly suggests that all four eves resulted from a single insertion event prior to the divergence of the myotis and eptesicus genera, »25 million years ago (miller-butterworth et al., 2007) . therefore, bats may have been exposed to filovirus-like agents for far longer than previously recognized (»13 million years ago [taylor et al., 2011]) . available experimental exposure studies, although limited in number and scope, suggest that some filoviruses isolated from humans can replicate in bats without causing substantial host pathology (e.g., marv and ravv in egyptian rousettes jones et al., 2015; paweska et al., 2012] ). these observations therefore prompt a key question: what is the origin and nature of the selective pressure that has driven accelerated npc1 evolution in bats? our scant understanding admits a number of possibilities. first, it is conceivable that some filoviruses do indeed replicate in a manner that is deleterious to their specific bat hosts-we may simply not have identified the viruses and hosts in question. indeed, the filovirus llov, discovered in schreibers's long-fingered bat carcasses in spain and portugal, may exemplify this possibility (negredo et al., 2011) . alternatively, in some cases (e.g., ebolaviruses and egyptian rousettes), the human viral isolates used in challenge studies may differ from these bat isolates in important respects due to human adaptation (human ebov, bdbv, tafv, restv, and sudv isolates do not infect egyptian rousettes ). second, filoviruses may have been more virulent in bats in the past. thus, the positive selection signatures observed in bat npc1, which cannot be accurately dated, may represent fixed alleles that are the consequence of a selective process driven by ancient filoviruses with properties distinct from their modern counterparts. indeed, the lack of virulence observed in some bats may reflect a dé tente that was shaped by precisely these historic genetic conflicts between filoviruses and bats. third, we cannot rule out the (unlikely) possibility that the evolution of npc1 in bats was driven by an entirely different infectious agent that also utilizes (or utilized) npc1 to multiply in its hosts. regardless of the mechanisms that genetically shaped npc1, we propose that polymorphisms in this gene nevertheless impose host barriers that impede the colonization and spread of present-day filoviruses in bats in africa and elsewhere. our findings set the stage for broader explorations of species-specificity in filovirus interactions with proviral and antiviral host factors, with an eye to uncovering new molecular arms races between filoviruses and bats and new genetic determinants of filovirus host range and host switching. the following immortalized pteropodid fibroblast cell lines were used: roni/7.1 (kidney; rousettus aegyptiacus), hypni/1. (biesold et al., 2011) . the species origin of each cell line was confirmed in the publication in which it was first described (kuhl et al., 2011) . bat cell populations stably expressing human npc1 (hsnpc1) were generated as described previously (carette et al., 2011) . briefly, subconfluent monolayers of cells were transduced with a retroviral vector expressing hsnpc1 modified at the c-terminus with a triple flag epitope tag. transduced cells were selected by puromycin treatment (10 mg/ml). licenses for capturing and export of bats, as well as ethical review and clearances of animal handling procedures were obtained from the ghana forestry commission of the ministry of food and agriculture. bat organ samples were obtained as described (drexler et al., 2012) . bats were caught, anesthetised with ketamine/xylazine and exsanguinated by heart puncture. carcasses were transported on ice to a nearby laboratory facility, and organs were dissected and immediately snap-frozen for long-term storage. animals were typed morphologically and genetically as described previously (kuhl et al., 2011) . vero african grivet kidney cells and 293t human embryonic kidney fibroblast cells were obtained from atcc. cell lines were maintained in dulbecco's modified eagle medium (dmem) (life technologies, grand island, ny) and supplemented with 10% fetal bovine serum (atlanta biologicals, flowery branch, ga), and 1% penicillin-streptomycin (life technologies). all cell lines were maintained in a humidified 37˚c, 5% co 2 incubator. we knocked out the npc1 gene in the eidni/41.3 cell line by crispr-cas9-mediated genome editing as described previously (mali et al., 2013) . a crispr guide rna (grna) sequence to target 5'-gttgtgatgttcagcagcttcgg-3' in the e. helvum npc1 mrna was cloned into the grna cloning vector (addgene plasmid #41824). eidni/41.3 cells were co-transfected with plasmid encoding human codon-optimized endonuclease cas9 (hcas9, addgene plasmid #41815), grna cloning vector encoding the e. helvum npc1-specific grna, a monomeric red fluorescent protein (mrfp1) expression plasmid (to monitor transfection efficiency), and pmx-ires-blasti (confers blasticidin resistance to transfected cells) using lipofectamine 2000 (life technologies). at 24 hr post-transfection, transfected cells were selected with 50 mg/ml of blasticidin for 24 hr and then allowed to recover in the absence of the selection agent. total rna was isolated from surviving cells with the rnaeasy mini kit (qiagen, valencia, ca) as per the manufacturer's directions. the e helvum npc1 mrna sequence flanking the grna target site was amplified with the one-step rt-pcr kit (qiagen) and the following primers: forward: 5'-at-tctggactaccaaaatctttgcc-3', and reverse: 5'-acatggcatccaagcccaag-3'. thermocycling conditions used for the rt-pcr were: 50˚c for 30 min (reverse transcription), followed by 95˚c for 15 min (initial pcr activation), then 30 cycles of 94˚c for 30 sec, 60˚c for 30 sec, 72˚c for 1 min, then a final extension of 72˚c for 10 min. amplified pcr products were tested for indels at the target site with the surveyor mutation detection kit for standard gel electrophoresis (transgenomic, omaha, ne), as per the manufacturer's instructions. once indels were confirmed, amplified pcr products from single cell clones were cloned into a topo-ta vector (life technologies). multiple clones for each single cell population were sequenced to confirm disruption of npc1 alleles. recombinant vesicular stomatitis indiana viruses (vsvs) expressing egfp, and ebov, marv, or llov gp in place of vsv g have been described previously wong et al., 2010; ng et al., 2014) . vsv pseudotypes bearing glycoproteins derived from vsv, ebov, bdbv, tafv, sudv, and marv were generated essentially as described previously (takada et al., 1997) . vsv particles containing gp cl were generated by incubating rvsv-gp-ebov with thermolysin (200 mg/ml) (sigma-aldrich, st. louis, mo) for 1 hr at 37˚c. the protease was inactivated by addition of phosphoramidon (1 mm) (sigma-aldrich), and reaction mixtures were used immediately. infectivities of vsv pseudotypes were measured by manual counting of egfp-positive cells using fluorescence microscopy at 16-24 hr post-infection, as described (chandran et al., 2005) . infectivities of rvsvs were measured in a similar manner, except that nh 4 cl (20 mm) was added to infected cell cultures at 1-2 hr post-infection to block viral spread, and individual egfp-positive cells were manually counted at 12-14 hr post-infection. the wild-type filoviruses ebola virus/h.sapiens-tc/cod/1995/kikwit-9510621 (ebov/kik-9510621; "ebov-zaire 1995") and marburg virus/h.sapiens-tc/deu/1967/hesse-ci67 (marv/ci67) used in this study were described previously swenson et al., 2008) . cells were exposed to virus at an moi of 1 pfu/cell ( figure 1c ) or 3 pfu/cell ( figure 2a ) for 1 hr. viral inoculum was then removed, and fresh culture media was added. at 48 hr (figure 2a ) or 72 hr ( figure 1c ) post-infection, cells were fixed with formalin and blocked with 1% bovine serum albumin (bsa). ebov-infected cells and uninfected controls were incubated with ebov gp-specific monoclonal antibody kz52 (maruyama et al., 1999) . marv-infected cells and uninfected controls were incubated with marv gp-specific monoclonal antibody 9g4 (swenson et al., 2004) . cells were washed with pbs prior to incubation with either goat anti-mouse igg or goat anti-human igg conjugated to alexa 488. cells were counterstained with hoechst stain (invitrogen, carlsbad, ca), washed with phosphate-buffered saline (pbs), and stored at 4˚c. infected cells were quantitated by fluorescence microscopy and automated image analysis. images were acquired at 20 fields/well with a 20â objective lens on an operetta high content device (perkinelmer, waltham, ny). operetta images were analyzed with a customized scheme built from image analysis functions available in harmony software. from bats of four species (hypsignathus monstrosus, eidolon helvum, epomops buettikoferi, and, rousettus aegyptiacus), mrna was collected from cell lines (or spleen samples for additional eidolon helvum npc1 domain c sequences; figure 4 -figure supplement 2), cdna libraries were constructed, and the npc1 transcript was sequenced (see supplementary file 3 for primers). using available rnaseq read data (supplementary file 2), we assembled bat transcriptomes and identified npc1 sequences in bats of six additional species (myotis brandtii, artibeus jamaicensis, cynopterus sphinx, myotis lucifugus, pteropus alecto, and desmodus rotundus) . transcriptome data were cleaned with trimmomatic (bolger et al., 2014) and assembled using trinity (grabherr et al., 2011) and trans-abyss (robertson et al., 2010) . the 10-species npc1 alignment (supplementary file 4) was analyzed for positive selection using the m8 codon model in the codeml package in paml (yang et al., 2000) , rel, and fel (pond and frost, 2005) , and meme (murrell et al., 2012) available at http://datamonkey.org/ (delport et al., 2010) . all evolutionary analyses were done using both the npc1 gene tree and a species tree ( figure 5-figure supplement 1) . the species tree represents the accepted relationships amongst the bats analyzed (agnarsson et al., 2011; almeida et al., 2011) . to identify orthologous filovirus-related eve insertions, we screened bat genomes in silico for eves. a representative set of filovirus protein sequences was obtained from genbank, supplemented by the putative protein sequences of previously identified filovirus eves (taylor et al., 2014; taylor et al., 2011; taylor et al., 2010; katzourakis and gifford, 2010) . these sequences were used as 'probes' in tblastn screens of whole genome shotgun (wgs) sequence data derived from bats of ten species (eidolon helvum, eptesicus fuscus, myotis brandtii, myotis davidii, myotis lucifugus, pteropus alecto, pteropus vampyrus, megaderma lyra, pteronotus parnellii, and rhinolophus ferrumequinum) . statistically significant matches to filovirus probes were extracted, conceptually translated, and aligned with homologous filovirus proteins. orthologous flanking sequences were identified by blast comparison of eve-containing contigs. an alignment of the identified eves, along with the flanking information in the relevant bat genomes, is shown in supplementary file 5. a construct engineered to encode hsnpc1 domain c (residues 372-622) flanked by sequences that form a stable, antiparallel coiled coil, and fused to a preprotrypsin signal sequence with flag and hexahistidine tags at its n-terminus has been described (deffieu and pfeffer, 2011; . similar constructs bearing bat npc1 domain cs were generated by replacing the human domain c sequence with a sequence encoding domain c from each bat npc1 ortholog. soluble domain c was expressed in human 293-freestyle cells (invitrogen) and purified from supernatants by nickel affinity chromatography, as described previously . alternatively, cell supernatants containing soluble domain c were used directly for gp-npc1 binding elisas following calibration for domain c concentration (see below). npc1 domain c concentrations used in the elisas were normalized as follows. proteins were resolved by sds-page followed by immunoblotting with an anti-flag antibody followed by an antimouse alexa-680 secondary antibody (invitrogen). blots were visualized using the li-cor odyssey imager, and the domain c band was quantified using the li-cor image studio package (li-cor biosciences, lincoln, ne). thermolysin-cleaved vsv-ebov gp particles were captured onto high-binding 96-well elisa plates (corning, corning, ny) using kz52, a conformation-specific anti-ebov gp monoclonal antibody. plates were blocked with pbs containing 3% bsa, and serial dilutions of npc1 domain c protein were then added. bound domain c was detected with an anti-flag antibody conjugated to horseradish peroxidase (sigma-aldrich) and ultra-tmb substrate (thermofisher, grand island, ny). all binding steps were carried out at 37˚c for 1 hr or at 4˚c overnight. elisas with vsvs bearing llov and marv gp were performed as above, but with the following modifications. vsv-llov gp particles were cleaved by incubation with a reduced concentration of thermolysin (12.5 mg/ml, 37˚c, 1 hr) due to its enhanced protease sensitivity relative to ebolavirus gps, as described (ng et al., 2014) . the viral envelope was then labeled with biotin using a function-spacer-lipid construct (fslbiotin) (sigma-aldrich), as described previously (ng et al., 2014) . biotinylated viral particles were captured onto streptavidin-coated elisa plates (0.65 mg/ml). the remainder of the steps in the elisa were performed as described above for vsv-ebov gp. vsv-marv gp particles were cleaved by incubation with trypsin (150 mg/ml, 37˚c, 5 min; sigma-aldrich), modified as above using fslbiotin, and captured onto streptavidin-coated magnetic beads (spherotech, lake forest, il). beads were then aliquotted into a 96-well round-bottomed plate for the remaining elisa steps. pbs containing 5% nonfat dry milk was used for blocking and washing steps. binding curves were generated by nonlinear regression analysis using prism (4-parameter logistic equation; graphpad software, la jolla, ca). to detect npc1 in primate or bat kidney fibroblasts, whole cell lysates were prepared as previously described . briefly, cells were washed with pbs and lysed in nte-chaps buffer (10mm tris [ph 7.5], 140mm nacl, 1mm edta, 0.5% vol/vol 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) (sigma-aldrich) containing a protease inhibitor cocktail (roche, basel, switzerland), and placed on ice for 30 min. to assist in cell lysis, cell suspensions were vortexed every 10 min, and then placed on ice for 30 min. samples were spun at 14,000 âg for 10 min, and supernatants harvested for western blot. in some experiments, proteins were deglycosylated with protein n-glycosidase f (new england biolabs, ipswich, ma) according to the manufacturer's instructions. proteins were resolved in 8% sodium dodecyl sulfate (sds)-polyacrylamide gels and transferred to nitrocellulose membranes. endogenous npc1 was detected using an anti-niemann pick c1 polyclonal antibody (1:1,000 dilution; ab36983, abcam, cambridge, ma), followed by incubation with a donkey anti-rabbit antibody conjugated to horseradish peroxidase (1:5,000 dilution, santa cruz biotechnology, dallas, tx). endogenous cyclin-dependent kinase 4 (cdk4; loading control) was detected with a rabbit polyclonal antibody (1:1,000 dilution; sc-260, santa cruz biotechnology). ectopic expression of hsnpc1-flag was detected with an anti-flag antibody conjugated to horseradish peroxidase (sigma-aldrich). bands were visualized by incubation with an enhanced chemiluminescence reagent (thermofisher) followed by exposure to x-ray film. in figure 3 , cells were visualized using an inverted fluorescence microscope under illumination with a 63x high-numerical aperture oil objective ( figure 3b ) or a 10x air objective ( figure 3c ). images were captured with an axiocam mrm ccd camera using axiovision software (zeiss usa, thornwood, ny), and imported into photoshop (adobe systems, san jose, ca) for processing. images were cropped, inverted ( figure 3b) , and subjected to linear adjustment for overall brightness and contrast using the levels tool. developed x-ray films were digitized with a flatbed scanner and processed in photoshop as described above. statistical comparison of means among multiple independent groups was carried out by one-way analysis of variance (anova) with tukey's post hoc test for multiple comparisons. in some figures (see figure legends) , an unpaired two-tailed student's t-test with welch's correction for unequal variances (ruxton, 2006) was used for pairwise comparison of independent groups. all statistical analyses were performed in graphpad prism. the albert einstein college of medicine. opinions, conclusions, interpretations, and recommendations are those of the authors and are not necessarily endorsed by the us department of the army, the us department of defense, or the us department of health and human services. the funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. author contributions mn, en, mek, jmd, sls, kc, conception and design, acquisition of data, analysis and interpretation of data, drafting or revising the article; ash, rjg, acquisition of data, analysis and interpretation of data, drafting or revising the article; tb, aik, rmj, acquisition of data, analysis and interpretation of data; rkj, acquisition of data, analysis and interpretation of data, drafting or revising the article, contributed unpublished essential data or reagents; jah, my, acquisition of data, analysis and interpretation of data, contributed unpublished essential data or reagents; rb, acquisition of data, contributed unpublished essential data or reagents; ad, analysis and interpretation of data, drafting or revising the article; trb, conception and design, analysis and interpretation of data, drafting or revising the article; cd, mam, drafting or revising the article, contributed unpublished essential data or reagents; lfw, jhk, analysis and interpretation of data, drafting or revising the article, contributed unpublished essential data or reagents a time-calibrated species-level phylogeny of bats (chiroptera, mammalia) host-specific parvovirus evolution in nature is recapitulated by in vitro adaptation to different carnivore species evolutionary relationships of the old world fruit bats (chiroptera, pteropodidae): another star phylogeny? seasonal pulses of marburg virus circulation in juvenile rousettus aegyptiacus bats coincide with periods of increased risk of human infection oral shedding of marburg virus in experimentally infected egyptian fruit bats 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forward genetic strategy reveals destabilizing mutations in the ebolavirus glycoprotein that alter its protease dependence during cell entry codon-substitution models for heterogeneous selection pressure at amino acid sites we thank tyler krause and cecelia harold for technical support, and gary crameri, shawn todd, and mary tachedjian for help with sourcing bat cdna for npc1 gene amplification. we also thank margaret kielian, jack lenz, max nibert, vinayaka prasad, deeann reeder, nancy simmons, and susan tsang for useful discussions. we thank laura bollinger, integrated research facility at fort detrick, for critically editing this manuscript.supported by grants from the us national institutes of health (ai101436 to kc, gm093086 to sls), the us defense threat reduction agency (hdtra1-11-c-0061 to sls, cb3948 to jmd), eu fp-7 antigone (grant 278976) and the ebokon project (to cd and mam). lfw is supported in part by an nrf-crp grant (nrf2012nrf-crp001-056) in singapore. jhk performed this work as an employee of tunnell government services, inc., a subcontractor to battelle memorial institute, under battelle's prime contract with niaid (no. hhs27220070016i). sls is a burroughs wellcome fund investigator in the pathogenesis of infectious disease. kc is additionally supported by a harold and muriel block faculty scholarship and an irma t. hirschl/monique weill-caulier research award at key: cord-355075-ieb35upi authors: papenfuss, anthony t; baker, michelle l; feng, zhi-ping; tachedjian, mary; crameri, gary; cowled, chris; ng, justin; janardhana, vijaya; field, hume e; wang, lin-fa title: the immune gene repertoire of an important viral reservoir, the australian black flying fox date: 2012-06-20 journal: bmc genomics doi: 10.1186/1471-2164-13-261 sha: doc_id: 355075 cord_uid: ieb35upi background: bats are the natural reservoir host for a range of emerging and re-emerging viruses, including sars-like coronaviruses, ebola viruses, henipaviruses and rabies viruses. however, the mechanisms responsible for the control of viral replication in bats are not understood and there is little information available on any aspect of antiviral immunity in bats. massively parallel sequencing of the bat transcriptome provides the opportunity for rapid gene discovery. although the genomes of one megabat and one microbat have now been sequenced to low coverage, no transcriptomic datasets have been reported from any bat species. in this study, we describe the immune transcriptome of the australian flying fox, pteropus alecto, providing an important resource for identification of genes involved in a range of activities including antiviral immunity. results: towards understanding the adaptations that have allowed bats to coexist with viruses, we have de novo assembled transcriptome sequence from immune tissues and stimulated cells from p. alecto. we identified about 18,600 genes involved in a broad range of activities with the most highly expressed genes involved in cell growth and maintenance, enzyme activity, cellular components and metabolism and energy pathways. 3.5% of the bat transcribed genes corresponded to immune genes and a total of about 500 immune genes were identified, providing an overview of both innate and adaptive immunity. a small proportion of transcripts found no match with annotated sequences in any of the public databases and may represent bat-specific transcripts. conclusions: this study represents the first reported bat transcriptome dataset and provides a survey of expressed bat genes that complement existing bat genomic data. in addition, these data provide insight into genes relevant to the antiviral responses of bats, and form a basis for examining the roles of these molecules in immune response to viral infection. bats make up approximately 20% of the extant mammalian diversity and are the second most species rich mammalian lineage after rodents [1] . the order chiroptera is divided into two suborders: the megachiroptera and microchiroptera. these two lineages are estimated to have diverged approximately 58 million years ago [2] . megachiroptera consists of a single family, the old world fruit bats, while microchiroptera includes 17 families of echolocating bats. bats have a wide geographic distribution and exploit a variety of environmental niches, being absent only from the polar regions. bats are also hosts to numerous viruses, many of which are highly pathogenic to humans and other mammals yet appear to cause no clinical consequences in bats [3] [4] [5] [6] [7] [8] . this group of mammals also shares a variety of unique characteristics that likely facilitate the persistence and spread of the viruses they carry. highly social species, bats live at much higher densities than other mammals. they are the only mammals capable of powered flight and have long lifespans relative to their body size [9] . despite their diversity, unique characteristics and role as natural reservoirs for viruses, bats are also the least studied of all mammalian taxa and there is little information available on antiviral immunity in any bat species. bats are the natural reservoir hosts of more than 80 viruses, with new viruses or viral sequences of bat origin being discovered each year [9, 10] . rna viruses account for the overwhelming majority of known bat viruses, many of which are among the most deadly known to man, including ebola, hendra, nipah and sars-like coronaviruses [9] . many of these viruses, which cause severe morbidity and mortality in humans and other mammals, appear to cause no clinical diseases in bats under natural or experimental infection. the most studied example is the henipaviruses (hendra and nipah viruses) which are members of the family paramyxoviridae. nipah virus has a mortality rate of 40-90% in humans and close to 100% in experimental animal models (cats and hamsters). yet, infection of pteropus vampyrus (the natural reservoir host of nipah virus in malaysia) and p. poliocephalus (a related bat species native in australia) by a high dose of nipah virus, failed to result in clinical signs of disease [7, 8, 11] . other examples of experimental infections of bats including ebola zaire, japanese encephalitis and st. louis encephalitis viruses have not resulted in any symptoms of disease despite the presence of viral rna in tissues [3] [4] [5] [6] . experimental infections of p. poliocephalus with nipah virus have demonstrated the presence of serum antibody and viral shedding in the absence of clinical symptoms of disease [11] . the only viruses that have been demonstrated to cause clinical symptoms of disease in bats are rabies virus and the closely related australian bat lyssavirus [12, 13] . however, results of experimental infections are inconsistent with only a small proportion of bats succumbing to infection, and rates of sero-conversion and virus recovery from tissues were reported to be very low [13] . the long co-evolutionary history of bats with viruses has likely resulted in the adaptation of the bats immune system to cope with viral infection. one hypothesis is that the innate immune system rapidly controls viral replication to very low levels that cause no clinical consequences to bats, but still results in viral shedding and subsequent spillover to other species. however, as little information currently exists on any aspect of bat immunology and few bat-specific reagents exist, this hypothesis remains untested. recent years have seen a surge in the availability of whole genome sequence data. bats were among the organisms sequenced as part of the us national institutes of health (nih)-funded mammalian genome project. these genomic resources are an important step forward in identifying the genes that are involved in antiviral immunity in bats and in providing insights into other unique life history characteristics. there are currently two publicly available bat genome sequences: one from the megabat p. vampyrus and a second from the microbat myotis lucifugus. both bat genomes were initially sequenced to low coverage (2.6x for p. vampyrus and 1.7x for m. lucifugus, though a draft quality assembly of the m. lucifugus genome based on 7x coverage sequencing is now available). additionally, the annotations were predominantly based upon comparative data. despite these shortcomings, these projects have an important role to play in revealing the mechanisms that have evolved to allow bats to remain asymptomatic to so many viral diseases. in order to understand bat-virus interactions, we are developing the australian black flying fox, p. alecto, as a model bat species. p. alecto belongs to the family pteropodidae and is closely related to p. vampyrus [14] . these two species are reservoirs for a variety of closely related viruses, the most important of which include the henipaviruses, hendra virus in p. alecto and nipah virus in p. vampyrus [10] . a number of important resources have now been developed for p. alecto, including cell lines from a variety of tissues [15] . we have also begun to identify some of the genes involved in immune responses in this species and carry out functional studies in bat cells [16] [17] [18] [19] [20] [21] . to begin to characterise the immune gene repertoire of p. alecto, we sequenced the transcriptome of bat immune tissues and mitogen-stimulated cells using the illumina platform. to our knowledge, this study represents the first analysis of the transcriptome of any species of bat. our analysis of the p. alecto transcriptome provides information on a variety of immune genes not previously identified in any bat species and represents an important starting point for examining the antiviral activity of these molecules. overview of the bat transcriptome two separate transcriptomic datasets were generated and raw sequences from each database were submitted to the sequence read archive [sra: srr350710.3 and srr351237.2]. the first was obtained using total rna extracted from a juvenile male flying fox thymus. due to its role in central tolerance, the thymus expresses a large proportion of the proteome and therefore allows for the identification of a broad range of genes, including those involved in the immune response. to enrich for sequences corresponding to cytokines and innate immune genes, the second dataset was derived from pooled total rna obtained from mitogen-stimulated spleen, white blood cells and lymph node and unstimulated thymus and bone marrow obtained from one pregnant female and one adult male flying fox. cells were stimulated with lipopolysaccharide (lps) and ionomycin, which stimulate the production of pro-inflammatory cytokines; polyic, a tlr3 ligand; pha, which triggers t cell activation and pma, which activates t and b cells. about 12.5 million 65 bp long reads were obtained from the thymus dataset, while 23.9 million 76 bp long reads were generated from the stimulated pooled sample. prior to assembly, the raw reads were trimmed of low quality sequence and polya/t tails, uninformative strings of 'n' and primer/adapter contaminants were cleaned. the filtered dataset consisted of 12,399,095 reads from the thymus (between 20-63 bp) and 22,577,294 reads from the stimulated pooled dataset (between 20-73 bp). the filtered reads were de novo assembled using the software packages velvet and oases. the resulting oases assemblies consisted of 247,909 contigs (n50 1244 bp) from the thymus and 313,641 contigs (n50 733 bp) from the pooled samples. the largest contigs in the thymus and pooled samples were 11.8 kb and 8.9 kb respectively, both of which correspond to the dna-dependent protein kinase catalytic subunit (dna-pkcs) which is represented by a 12.4 kb transcript in other species, including horse. for comparative purposes, an assembly using mira was also generated. summary statistics from the velvet, oases and mira assemblies are listed in additional file 1: table s1 . all subsequent analyses were performed using the oases assemblies. to identify orthologues of known mammalian protein coding genes, the bat contigs were used to search the kegg and ncbi non-redundant (nr) protein databases with blastx (e-value < 0.001). of the 247,801 contigs longer than 50 bp in the thymus sequence assembly, about 46% matched annotated proteins in the nr database. for the pooled dataset, about 51% of the 313,528 loci matched proteins in nr. similar results were obtained for both assembled libraries against the kegg database. of the assembled thymus transcripts annotated using kegg, 36% of all transcripts were more similar to horse sequences than to any other species, followed by dog (16%) and cow (12%) (figure 1 ). similar results were obtained for the pooled tissue dataset (not shown). this result is consistent with the now generally accepted view that bats belong within laurasiatheria, which includes carnivora, cetartiodactyla (whales and even toed ungulates), eulipotyphla (moles and shrews), pholidota (scaly anteater) and perissodactyla (odd toed ungulates) [22] [23] [24] [25] [26] [27] . however, until recently, the phylogenetic relationships within laurasiatheria have been controversial. conflicting results have been reported using complete mitochondrial genome sequences to infer phylogenetic relationships with support for a sister relationship between chiroptera and fereungulata (carnivora, pholidota, perissodactyla and cetartiodactyla) or a relationship between chiroptera and eulipotyphla [28] [29] [30] . analysis of the nuclear gene, protamine p1, as well as large genomic datasets, has provided evidence that bats are sister to a clade containing perissodactyla, carnivora, and cetartiodactyla [31, 32] . the volume of sequence data generated by transcriptome sequencing provides the opportunity for larger scale sequence comparisons than previously possible using the few full length bat genes available or by comparison with the limited whole genome sequence data. our results support the comparative analysis of retroposon loci which has also demonstrated that bats share a sister relationship with horses, forming a clade with carnivora [27] . alignment of contigs from the thymus and pooled datasets to the kegg database identified 178,554 and 285,268 contigs respectively with homology to 16,863 and 16,927 unique human proteins. to explore gene function, gene ontology (go) terms were used. of contigs that matched proteins in the kegg database, 86% were assigned go terms and 78% could be mapped to go slim terms using go term mapper (additional file 2: figure s1 ). genes with go slim terms were further classified into twelve selected classes ( figure 2 ). the most abundant go terms found in the thymus dataset were involved in cell growth and maintenance (16.8%), enzyme activity (14.8%), cellular components (14.3%) and metabolism and energy pathways (14.5%). similar results were obtained for the pooled tissue dataset (data not shown). the go classification demonstrates that a diverse range of genes were identified in each of our two datasets providing a broad survey of bat genes. a goal of the present study was to identify immune transcripts, particularly those that may play a role in antiviral immunity. only 3.5% of the bat transcribed genes from each of the datasets showed homology to genes associated with immune function. this represents about 500 different immune-related genes ( figure 2 ). the bat immune transcripts were further categorised using go terms to annotate the transcripts into 40 immune categories. represented in the datasets were genes involved in a broad range of immune activities with lymphocyte activation, cytokine production and t cell activation making up the largest proportions of immune transcripts ( figure 3 ). using kegg codes to identify immune genes, our data revealed 70 genes involved in toll-like receptor (tlr) cascades, 50 genes involved in b cell activation, 79 involved in t cell activation, 72 involved in natural killer cell cytotoxicity and 41 involved in antigen presentation. additional immune genes not identified in the kegg database were obtained by searching sequences from the nr database. the sequences of all genes described in the text are provided in the additional file 3. one hypothesis for the ability of bats to resist the pathological effects of viral infection is that they are able to rapidly control viral replication early in the immune response through innate antiviral mechanisms. the bat transcriptome contained representatives of a variety of immune genes including pattern recognition receptors, interferons, interferon stimulated genes and natural killer cell receptors. pattern recognition receptors (prr) including tlrs, rig-i like helicases (rlhs) and nucleotide oligomerisation domain (nod) like receptors (nlrs) recognise conserved molecular patterns associated with a broad range of pathogens. both tlrs and rlhs initiate signalling pathways that result in the induction of similar immune and inflammatory responses but are expressed in different locations within the cell and differ in the pathogens they recognise. tlrs are transmembrane proteins expressed by the plasma membrane or endosome and recognise a broad range of pathogens including viruses, bacteria and fungi. of eleven previously identified p. alecto tlr genes [18] , only tlr5 was absent from the oases assemblies, however it was present in the mira assembly, which used a lower coverage cut-off and is useful for identifying genes with low expression levels. rlhs are expressed in the cytoplasm where they recognise viral rna and dna [33, 34] . three bat rlh genes, retinoic-acid-inducible protein i (rig-i), melanoma-differentiation-associated gene 5 (mda5) and laboratory of genetics and physiology 2 (lgp2) were identified in our transcriptome datasets and have recently been described in p. alecto [17] . these results provide further evidence that bats are able to recognise a broad range of pathogens, similar to other species. nlrs are a diverse family of cytoplasmic prrs involved in the activation of a variety of signalling pathways. nlrs are primarily involved in bacterial recognition, although more recently, evidence for recognition of viral rna and dna by some members of the nlr family has been reported [35] [36] [37] . the only nlrs identified in the bat transcriptome datasets were nod-like receptor family card domain containing 5 (nlrc5) and nlr family, pyrin domain containing 3 (nlrp3). nlrc5 is a recently identified nlr proposed to function as a positive and negative regulator of antiviral immune responses [36] . nlrp3 (also known as nalp3) is activated by a variety of danger signals including viral and bacterial infections and environmental irritants. activation of nlrp3 in turn activates caspase-1 in the inflammasome which proteolytically cleaves the cytokines il-1î² and il-18 into active mature peptides [37] . the identification two nlrs with associations with antiviral immunity in the bat transcriptome is remarkable and provides a starting point for understanding the role of nlrs in antiviral immunity in bats. the interferon (ifn) response is a key component of the innate immune system and the first cytokines induced against viral infection. since the ifn response is important in the control of viral replication in other mammals, we searched the bat transcriptome for ifns and ifn stimulated genes (isgs) that may be critical to the ability of bats to remain asymptomatic to viral infections. type i (including ifnî± and î²) and iii (ifnî») ifns are induced directly in response to viral infection and play a role in the earliest stages of the innate immune response. type i (î±) ifn and its receptor (ifnar1 and ifnar2) were identified in the bat transcriptome datasets (additional file 3). although type iii ifns, ifnî»1 and ifnî»2 are upregulated in stimulated bat cells [21] , neither of these genes were identified in our datasets, likely reflecting a low expression level in our samples. the il-10r2 chain of the type iii ifn receptor was present in the bat transcriptome, but its partner chain ifnlr1 was not found. both il-10r2 and ifnlr1 were recently described in p. alecto and ifnlr1 was demonstrated to act as a functional receptor for ifnî» [20] . the induction of type i and type iii ifns results in the transcription of hundreds of isgs including prrs that detect viral rna, transcription factors that result in the amplification of the ifn response and a small number of proteins that are directly responsible for inducing an antiviral state. the isgs, myxovirus resistance (mx) gtpases, protein kinase r (pkr), 2'-5' oligoadenylate synthetases (oas), ribonuclease l (rnasel) and isg15 are among the proteins with confirmed antiviral activity in other mammals [38] . the bat transcriptome datasets contained genes orthologous to mammalian mx1, mx2, oas1, oas2, oas3, oas-like (oasl), pkr, rnasel and isg15 consistent with the presence of an isg repertoire in bats that is similar to that of other species. these results provide the first evidence that the pathways activated by the ifn response are likely similar in bats to those described in other mammals. the mx gene family is among the best characterised isgs, first identified as antiviral proteins following the observation that the sensitivity of many inbred mouse strains to orthomyxovirus was solely due to mutations within the mx locus [39] . the mx family of gtpases trap essential viral components, and in so doing prevent viral replication at early time points. although the full spectrum of mx antiviral activity is unknown, representatives of both rna and dna viruses have been shown to be sensitive to the effects of mx [40] . a full length transcript, encoding a 667 amino acid protein was identified in our bat transcriptome datasets and found to be orthologous to mx1 based on comparison with known mammalian mx1 and mx2 family members (figure 4a and data not shown). bat mx1 contained the highly conserved tripartite gtp-binding domain found in all mammalian mx proteins. in addition, a dynamin family signature and putative leucine zipper motif were found near the c terminal end, represented by a stretch of evenly spaced leucine residues. the bat protein was also conserved in the region identified as the stalk of human mxa including loop 2 which is associated with antiviral activity. consistent with other species, loop 4 of the mxa stalk is the least conserved region of the bat mx protein [41] . loop 4 has been reported to be proteinase k sensitive and may play a role in lipid binding [42, 43] (figure 4b ). bat mx1 does not contain the stretch of basic amino acids (k/r) near the c terminal end associated with nuclear localisation of mouse mx1, consistent with the bat protein remaining localised within the cytoplasm [44] . the conservation of key residues important in antiviral activity is consistent with the bat mx1 playing a role in antiviral immunity similar to other species. the identification of the sequences of important isgs will now allow us to determine whether functional differences in the initiation and regulation of these proteins account for the differences in susceptibility of bats to viral infections compared to other mammals. natural killer (nk) cells are an important component of the innate immune response, providing a first line of defence against viruses and tumours. to our knowledge, no investigations of nk cell receptors from any species of bat have been reported previously. nk cells express cell surface receptors that recognise major histocompatibility complex (mhc) class i or class i like molecules on the surface of cells and lyse infected or abnormal cells by cytotoxicity. two families of nk receptors that bind classical mhc class i ligands have been identified: the killer immunoglobulin like receptors (kirs), which are encoded by genes in the leukocyte receptor complex (lrc), and the killer cell lectin like receptors (klrs), which are encoded by genes in the natural killer complex (nkc). different lineages of mammals make use of genes from the two different superfamilies to carry out analogous functions. kirs are used preferentially by primates, cattle, domestic cats, dogs and pigs [45, 46] . similarly, the kir-like receptors, marsupial immunoglobulin-like receptors (mairs) and chicken immunoglobulin-like receptors (chirs), have expanded in marsupials and chickens respectively [47, 48] . although chir-ab binds igy, the ligand for the majority of chirs is unknown and the presence of a charged transmembrane residue and a cytoplasmic immunoreceptor tyrosine-based inhibition motif (itim), are consistent with the possibility that they play a role in nk activity [49] . rodents, horses and platypus are the only species so far described that have expanded the klrs, represented by the ly49 family [50] [51] [52] . in the bat transcriptome dataset, no transcripts with homology to kirs or ly49 receptors were identified. in bony fish, novel immune type receptors (nitrs) which contain an n terminal variable domain and a c terminal ig domain have been identified as the primary activating and inhibitory receptors expressed by nk cells [53] . nitrs were also used to search the bat transcriptome but failed to identify any orthologous transcripts. the failure to find kir or ly49 like receptors in the bat transcriptome may reflect low expression levels of these genes resulting in their absence from our datasets. however, blast searches of the publicly available whole genome sequence of the closely related pteropid bat, p. vampyrus revealed no evidence of kirs or ly49 receptors. as this is a low coverage genome (2.63x), further work is required to determine whether pteropid bats have kir and/or ly49 receptors. overall, the absence of these important nk receptors from our datasets warrants further investigation into the nature of nk cells in bats. nk cells in a wide range of mammalian species additionally express cd94/nkg2 (also called klrd1/ klrc) lectin-like receptor heterodimers. unlike the kir and ly49 receptors, which bind (classical) mhc class ia ligands, the cd94/nkg2 heterodimer binds the (non-classical) mhc class ib ligands hla-e and qa-1 in humans and mice respectively [54] . the cd94/ nkg2a heterodimer generates inhibitory signals whereas the cd94/nkg2c heterodimer generates activating signals within nk cells. both cd94 and nkg2a were identified in the bat transcriptome, however nkg2c transcripts were not identified, possibly reflecting the low abundance of transcripts of this gene in our datasets. two and 37 nkg2a transcripts were identified in the thymus and pooled datasets respectively and six transcripts corresponding to cd94 were identified in the pooled dataset. two of the longest nkg2a sequences were aligned with nkg2a and nkg2c sequences from human and mouse. as shown in figure 5a , the bat genes display highest conservation with other nkg2a genes including the presence of conserved itim motifs in their cytoplasmic domains, designated by i/v/l/sxyxxl/v indicating that they are likely functional inhibitory receptors [55] . the more divergent nkg2d, which binds mhc class i chain-related genes, mica/b, and the ul16 binding proteins (ulbps) in human [46] , was also detected. two distinct bat cd94 contigs were identified, one of which is missing two conserved cysteines in the stalk region, the first of which forms an interchain disulfide bond with nkg2 and the second which forms an intrachain disulphide bond. the second bat cd94 sequence is missing a conserved cysteine in the extracellular domain that forms an intrachain disulphide bond (figure 5b ). the absence of key cysteines in both of the bat cd94 sequences may have implications for the formation of heterodimers with nkg2 and for the unique folding of the cd94 chain. combined with our failure to detect kirs or ly49 receptors, our data may provide the first evidence for the presence of atypical nk cell responses in bats. however, confirmation of the nature of the nk response and the composition of receptors used by bat nk cells awaits further investigation. other nk receptors were also identified in our datasets including cd244 which acts as an activating or inhibitory receptor on human and mouse nk cells respectively [56] and the natural cytotoxicity receptors expressed by nk cells. co-receptors including cd16 and cd56 expressed by subsets of nk cells in other species were also identified in the bat transcriptome. identification of nk cell receptors and co-receptors provides information for the development of reagents to identify bat nk cells and paves the way for further studies of nk cell function during viral infection in bats. genes involved in the adaptive immune system, including mhc class i and ii genes and t and b cell receptors and co-receptors were highly represented in both the thymus and pooled datasets providing evidence that bats have all of the components necessary to mount an adaptive immune response. mhc class i molecules play an important role in the initiation of the adaptive immune response through recognition of endogenously-derived peptides from viruses and other pathogens. in the thymus dataset, 46 contigs had homology to mammalian mhc class i proteins, while 24 were homologous in the pooled data. other transcripts in the mhc class i antigen-loading and presentation pathway were also identified, including beta-2-microglobulin, transporter associated with antigen processing 1 (tap1), calnexin and tapasin. class irelated genes were also present in the bat transcriptome dataset including cd1a, cd1b, cd1d, mr1, hfe, fcrn and ulbps, which have a variety of immune and nonimmune functions. the presence of ulbps is consistent with the expression of nkg2d, but orthologues of mica/b or mill were not observed. the presence of nkg2d suggests bats should have a mic homologue, but these may not be detected possibly due to low or tissue-specific expression. to our knowledge, these sequences provide the first class i and class i-associated transcripts from any species of bat. of the 46 contigs with homology to mhc class i genes in the thymus dataset, 29 contained in-frame stops. these may be expressed pseudogenes, represent assembly or sequencing errors or result from reading frame shifts due to the presence of unprocessed transcript. as the sequences were obtained from multiple individuals, it is not possible to confidently distinguish between alternative isoforms, alleles and in some cases, loci. however, clustering the remaining contigs with open reading frames (orfs), there are clearly at least 9 distinct mhc class i genes expressed. the majority of class i contigs contained the î±1 or î±2 domain or partial sequence corresponding to both domains and were used for further sequence analysis. the deduced amino acid sequence of contigs with the most complete î±1 or î±2 domains were aligned with human hla-a ( figure 6 ). all of the bat class i sequences contained a unique three amino acid insertion in the î±1 domain that appears to be bat specific. as shown in figure 6 , the bat transcripts display amino acid variation in their î±1 and î±2 domains, corresponding to the peptide binding region. however, they appear to be remarkably conserved from residues 131 to 175 of the î±2 domain. these results may indicate that bats contain a very closely related class i gene repertoire that have coevolved with the specific viruses they carry. some of the class i transcripts represented in the thymus and pooled datasets contained an 84 bp insertion at the end of the î±1 domain. the longest of these transcripts corresponded to the leader peptide through to 71 amino acids of the î±2 domain and is shown in figure 6 . the insertion at the end of the î±1 domain is not present in class i sequences from other mammals and includes two in frame stop codons that would prevent translation beyond the î±1 domain ( figure 6 ). this sequence was figure 5 a. alignment of deduced amino acid sequences of bat nkg2 with human and mouse nkg2a and nkg2c. sequences are divided into cytoplasmic, transmembrane, stalk, and lectin domains. the predicted itim motifs in the cytoplasmic domain are shaded. the conserved cysteine residue in the stalk predicted to be involved in interchain disulphide bond formation with cd94 is shaded and indicated with an asterisk. dashes indicate similarity and dots indicate gaps. b. alignment of the deduced amino acid sequences of bat cd94 with the human and mouse orthologues. sequences are divided into cytoplasmic, transmembrane, stalk, and lectin domains. conserved cysteines predicted to be involved in disulphide bond formation are shaded. cysteine pairs are indicated by identical numbers below the cysteine. the cysteine predicted to form a disulphide bond with nkg2 is indicated with an asterisk. confirmed by race pcr and transcripts were detected in a variety of tissues including lymph node, spleen, liver, lung, heart, kidney, small intestine, brain and salivary glands, thus providing evidence that they are not an artefact of the transcriptome assembly (data not shown). comparison with the closely related p. vampyrus whole genome sequence available in ensembl revealed that the 84 bp insertion is identical to the beginning of intron 2 of a p. vampyrus class i gene. mhc class i splice variants that retain intron sequence and result in the translation of a truncated protein have been identified in other mammals, including soluble splice variants of human hla-g that plays a role in immunoregulation at the feotal-maternal interface [57] . further investigation will be required to determine whether the bat gene encodes a soluble protein corresponding only to the î±1 domain or whether it represents a transcribed pseudogene. however, given the abundance of this transcript in our datasets it is possible that it plays a role in immune regulation in p. alecto. unlike class i molecules, which are ubiquitously expressed, class ii molecules are expressed only by antigen presenting and b cells and present exogenously derived peptides to t cells. the mhc class ii molecules are composed of an î± and a î² chain encoded by a and b genes respectively [58, 59] . eutherians have three main classical class ii gene clusters: dp, dr, and dq, as well as the nonclassical dm and dn/do gene clusters [60, 61] . sequences corresponding to exon 2 of mhc class ii drb genes have been described in four species of microbats [62] [63] [64] . however, prior to the present study no class ii genes have been reported from any species of megabat. sequences corresponding to genes involved in the class ii antigen processing and presentation pathway were also identified in our datasets including the class ii invariant (cd74) chain and cathepsin s (additional file 3). in the p. alecto thymus and pooled datasets we identified 78 and 238 contigs respectively that were homologous to class ii sequences. phylogenetic analysis revealed that the alpha chain sequences were homologous to dma, doa, dqa and dra from other mammals (figure 7a ) and the beta chain sequences were homologous to dmb, dob, dqb and drb (figure 7b ). these results are consistent with orthologous relationships between the bat class ii genes and those from other mammals. t cell receptor (tcr) genes corresponding to all four chains of the t cell receptor were present in our datasets, consistent with bats having both î±î² and î³î´ t cells. sequences corresponding to the constant and variable domains of the tcr were identified including many tcrî± related contigs, tcrî² related contigs, a few tcrî³ and tcrî´ chain related contigs. in humans and mice approximately 95% of circulating t cells express the î±î² t cell receptor. in contrast, î³î´ t cells account for up to 70% of circulating t cells in young ruminants, rabbits and chickens [65, 66] . the low abundance of tcrî³ and tcrî´ related transcripts in our datasets is consistent with the possibility that î±î² t cells may be the predominant tcr present in bats. in addition, a variety of t cell co-receptors, including the accessory tcrî¶ chain, cd3, cd4, cd8 and cd28 were identified in our datasets. we previously described the immunoglobulin heavy chain diversity of p. alecto, revealing the presence of a highly diverse variable region gene repertoire [16] . sequences encoding the variable and constant domains of immunoglobulin heavy and light chains were represented in our datasets. these included heavy chain genes encoding iga, igg, igm and ige, which have previously been described in the megabat, cynopterus sphinx. no evidence for the transcription of igd was observed in the p. alecto transcriptome, a result which is consistent with c. sphinx [67] . the two light chain subtypes, kappa and lambda and a variety of b cell co-receptors including cd19, cd22, cd72, cd79a and cd79b were also identified in our datasets (additional file 3). many of the bat immune transcripts showed high levels of sequence similarity compared to homologues from other mammals. among the most conserved bat innate immune genes were the prrs; the tlrs, rig-i helicases and nlrs, which displayed >80% amino acid identity with homologues. this likely reflects their roles in the recognition of conserved pathogen motifs. members of the oas family were also highly conserved, in particular oas1 which shared 87% amino acid identity with the dog oas1 sequence. in addition, the nk co-receptor, cd56 shared 93% amino acid identity with mouse, hamster, guinea pig and human sequences. among the adaptive immune genes, mhc associated proteins, calnexin, tap1 and cathepsin s shared 89-95% identity with corresponding sequences from other mammals reflecting their conserved roles in the antigen processing and presentation pathway. several members of the mhc class i and ii families were also highly conserved, including cd1b and cd1d which shared 88 and 89% amino acid identity with horse and chimp sequences respectively. the bat mhc class ii doa and dra shared 91 and 89% amino acid identity with orthologous sequences in other mammals. the t cell co-receptor, cd28 shared 90% identity with the rhinoceros cd28 sequence and the constant domain of igm shared 92% identity with camel igm. there were >77,000 unannotated contigs in the thymus and pooled datasets. only about 3% of these contigs matched predicted cdnas from the p. vampyrus genome sequence, which are annotated using orthologous sequences from other species [68] . the unannotated contigs contained a total of 3266 open reading frames (orfs) longer than 300 bp. of these, 92.6% (e-value < 10 -3 ) aligned to the closely related p. vampyrus whole genome sequence and represent highly divergent homologues or bat specific genes. the remaining loci represent either misassembled contigs or bat-specific transcripts that are located in sequencing gaps in the low coverage p. vampyrus genome sequence. the 3266 long (>300nt) orfs were searched for conserved domains using profile hidden markov models with hmmscan (hmmer v3; http://hmmer.org/) obtained from the pfam database [69] . this identified 345 orfs containing 214 unique domains, including several defensins, antimicrobial peptides and dna-binding domains. searches using domain models from the pfam-b database, identified a further 437 unique, predicted-conserved domains in 733 orfs. a further 2188 orfs remained unannotated. a high proportion of these were rich in cysteine, tryptophan and proline, and prolines frequently appeared in low complexity regions (additional file 4: figure s2a and b) . further characterisation of these unannotated transcripts will provide insight into whether they are functionally significant and in particular whether any unique bat specific transcripts are involved in the antiviral immune response. bats are a highly diverse, species rich group of mammals that have evolved a variety of distinctive characteristics since their divergence from other mammals [70] . despite the central importance of bats in harbouring a variety of viruses with the potential to spillover to other species, very little is known about antiviral immunity in bats. next generation sequencing provides the opportunity to survey genes that are conserved between distantly related species as well as to provide insights into novel adaptations through the identification of previously unidentified transcripts. to identify genes involved in the immune response, we carried out a transcriptome analysis of thymus and immune cells and tissues of the australian black flying fox, p. alecto. this study represents the first survey of expressed bat immune genes and complements existing low coverage bat genome sequences. our analysis provides a broad overview of the bat transcriptome and contains representatives of all of the major classes of immune genes. the results are consistent with bats having all of the components of the immune system present in other mammals. the majority of these correspond to genes that have not previously been described in any species of bat and thus represent an important resource for future investigations into antiviral immunity in bats. animals p. alecto bats used in this study were wild caught from east brisbane, queensland, australia. bats were handled and euthanised as previously described [15] . all experiments were approved by the australian animal health laboratories animal ethics committee (protocol aec1281). the thymus was removed from a juvenile male bat and immediately stored in rnalater (ambion) for rna extraction. the spleen, lymph nodes (ln), thymus, bone marrow and peripheral blood were collected from one adult male and one pregnant female bat. single cells were extracted from the spleen, thymus and ln by tissue extrusion through a 70 î¼m sterile sieve (bd biosciences) in the presence of dmem supplemented with 15 mm l-glutamine, 100 units/ml penicillin and 100 units/ml streptomycin (invitrogen). splenocytes and peripheral blood lymphocytes (pbmls) were isolated by density centrifugation over lymphoprep (nycomed, oslo) as described previously [21] . cells were resuspended in dmem with 10% fcs, 15 mm l-glutamine, 100 units/ml penicillin and 100 units/ml streptomycin and cell numbers were determined using a haemocytometer with trypan blue exclusion. the thymus and bone marrow cells were stored in rnalater (ambion) for rna extraction and the spleen, thymus and ln were cultured with a variety of stimulants. the isolated splenocytes, ln and pbmls from each bat were pooled and were then seeded at 1 x 10 7 cells per well in 24 well tissue culture plates (nunc) with pha (10ug/ml; sigma) and lps (10 î¼g/ml; sigma); pma (50 î¼g/ml; sigma) and ionomycin (2nm/ml; sigma); or polyic (30 î¼g/ml; invivogen) and incubated in a humidified atmosphere of 5% co 2 in air at 37â°c. cells were harvested in rlt buffer (qiagen) at 0, 1, 4 and 18 hours and homogenised using a qiashredder (qiagen) following the manufacturer's instructions. the lysate was then stored at â��80â°c and total rna extracted the next day (0, 1, and 4 hours) or processed immediately (18 hours). rna extraction was carried out as previously described using the rneasy mini kit (qiagen) with removal of genomic dna with dnase i digestion [16] . total rna from the thymus of a juvenile male bat was used for illumina sequencing separately from all other samples. total rna obtained from the stimulated and unstimulated cells from the two adult bats was pooled as follows: 22% thymus total rna (11% from each bat) and 78% pooled total rna from the rest of the mitogen stimulated and unstimulated cells/tissues (~3.45% for each sample; total of 22 samples). sequencing mrna isolation from total rna, library preparation and single-end read sequencing was performed by geneworks pty ltd, thebarton south australia using the illumina genome analyser iix sequencing platform. library preparation was performed as per illumina's mrna sequencing sample preparation guide (part # 1004898 rev. d) except 5 î¼g of total rna was used for mrna selection using poly-t oligo-attached magnetic beads. the thymus library was run on a single lane of a flow cell resulting in more than 12.5 million 65-base sequences for a total of about 0.82 gigabases (gb) of sequence. the pooled library consisted of 4 lanes resulting in 24 million 76 bp sequences for a total of about 1.8gb of sequence. sequence pre-processing and de novo assembly the quality of the sequences were evaluated using fastqc [71] . sequences were pre-processed in two stages. first, all bases at the 3' end of the reads with quality scores of 3 or lower were removed. second, poly a/t tails, uninformative sequences (ns) and primer/adaptor contaminants were trimmed using snowhite (version 1.1.3) [72], a cleaning pipeline for next-generation cdna sequences, which includes seqclean [73] and tagdust [74] . we ran snowhite with two runs of seqclean and one run of tag-dust and a final minimal length cutoff of 20 bp was used. the pre-processed sequences were de novo assembled using two different approaches. (1) the reads were assembled with velvet (version 1.0.12) [75] using individual kmers from 19 bp to 31 bp. next, the contigs produced by velvet were processed using oases (version 0.1.15) [76] . oases loci were then merged using cd-hit-est (version 4.0) [77] with a global sequence identity threshold of 1.0. finally, a length cutoff was set to 50 bp and the default coverage cutoff of 3 was used. we term the final result of this process a contig (2) . the reads were also assembled using mira 3 (v3.2.0rc3) [78] with default setting for est and illumina reads assembly, i.e. maximum front and end gap clip is 2 bp, maximum length of the possible vector leftover allowed is 18 bp, minimum quality score, window length and read length were all set to 20, allowed to clip poly a/t at ends, and minimum read coverage per contig was 2. the bat contigs were firstly annotated by using the best hits of blastx [79] search against nr protein database and kegg pathway database with an e-value cutoff of 0.001 for annotating the protein coding contigs that were conserved with other species. then the unannotated contigs were further annotated by using blastn search against refseq_rna database with an e-value cutoff of 10 -5 for the contigs containing conserved utrs and without significant protein coding regions. the contigs not annotated by the above two steps were further analysed by using blastn against the cdnas from megabat (p. vampyrus) and microbat (m. lucifugus). we translated the un-annotated transcripts into protein sequences from 6 frames, extracted the orfs longer than 300 bp. this was performed separately for the 2 datasets. these orfs were searched against pfam-a and pfam-b databases to identify conserved domains. the two sets of long orfs were pooled and clustered based on cd-hit with sequence identity of 50% [77] . the amino acid compositions were further analysed for the nonredundant longer orfs with composition profiler [80] . all the kegg ids of the human proteins identified by the blastx searches were extracted from the annotation process and were mapped to uniprot ids. then the go analysis for the uniprot proteins (uniprotkb-goa: gene_association.goa_human) was used to assign the go terms for the transcripts. the number of genes in categories of the go slim database was counted using the go term classification counter, categoriser [81, 82] and the immune category of the bat genes was annotated using the generic gene ontology term mapper [83] . the go classifications were further grouped into twelve broad categories as follows: cell death and apoptosis go:0005783; extracellular matrix go:0005739; cell, go:0005623 and nucleoplasm, go:0005654). binding (binding, go:0005488; protein binding, go:0005515 go:0003677; nucleotide binding go:0004872; actin binding, go:000377; calcium ion binding, go:0005509; chromatin binding, go:0003682; carbohydrate binding, go:0030246 and rna binding, go:0003723). reproduction and development (development enzyme activity (catalytic activity go:0016787 ca), based on the protein alignment to retain codon positions. based on the nucleotide and protein alignments, phylogenetic trees were constructed by the neighbour joining method [85], maximum parsimony and minimum evolution using the mega4 program the genbank accession numbers for sequences used in the sequence and phylogenetic analysis are as follows: mhc class i: (cap58485) hla-a; mhc class iia: human, homo sapiens (hosa) dma (nm_006120) dqa (m21931), dra (u13648) dra (nm_001113706), dma (nm_001004039) gallus gallus (gaga) b-la (ay357253) mhc class iib: hosa dqb (m20432), drb (nm_021983), dob (l29472), dpb (m57466), dmb (u15085) rattus norvegicus (rano) dqb (x56596) equus caballus (eqca) dqb (l33910) dmb (dq431246), drb (ay191776) ovis aries (ovar) dqb (l08792) mumu (nm_001136068) hosa (af135187_1) avian mx: gaga (np_989940) mammal species of the world: a taxonomic and geographic reference in the timetree of life human ebola outbreak resulting from direct exposure to fruit bats in luebo, democratic republic of congo swanepoel r: fruit bats as reservoirs of ebola virus studies of arthropod-borne virus infections in chiroptera. iv. the immune response of the big brown bat (eptesicus f. fuscus) maintained at various environmental temperatures to experimental japanese b encephalitis virus infection experimental inoculation of plants and animals with ebola virus transmission studies of hendra virus (equine morbillivirus) in fruit bats, horses and cats experimental hendra virus infection in pregnant guinea-pigs and fruit bats (pteropus poliocephalus) bats: important reservoir hosts of emerging viruses bats as a continuing source of emerging infections in humans experimental nipah virus infection in pteropid bats (pteropus poliocephalus) australian bat lyssavirus infection in a captive juvenile black flying fox pathogenesis studies with australian bat lyssavirus in grey-headed flying foxes (pteropus poliocephalus) a phylogenetic supertree of the bats (mammalia: chiroptera) establishment, immortalisation and characterisation of pteropid bat cell lines immunoglobulin heavy chain diversity in pteropid bats: evidence for a diverse and highly specific antigen binding repertoire molecular characterisation of rigi-like helicases in the black flying fox, pteropus alecto molecular characterisation of toll-like receptors in the black flying fox pteropus alecto interferon production and signaling pathways are antagonized during henipavirus infection of fruit bat cell lines type iii ifn receptor expression and functional characterisation in the pteropid bat, pteropus alecto type iii ifns in pteropid bats: differential expression patterns provide evidence for distinct roles in antiviral immunity complete mitochondrial genome of a neotropical fruit bat, artibeus jamaicensis; and a new hypothesis of the relationships of bats to other eutherian mammals parallel adaptive radiations in two major clades of placental mammals molecular phylogenetics and the origins of placental mammals resolution of the early placental mammal radiation using bayesian phylogenetics molecules consolidate the placental mammal tree pegasoferae, an unexpected mammalian clade revealed by tracking ancient retroposon insertions the phylogenetic position of the talpidae within eutheria based on analysis of complete mitochondrial sequences monophyletic origin of the order chiroptera and its phylogenetic position among mammalia, as inferred from the complete sequence of the mitochondrial dna of a japanese megabat, the ryukyu flying fox (pteropus dasymallus) maximum likelihood analysis of the complete mitochondrial genomes of eutherians and a reevaluation of the phylogeny of bats and insectivores confirming the phylogeny of mammals by use of large comparative sequence data sets characterization and phylogenetic utility of the mammalian protamine p1 gene differential roles of mda5 and rig-i helicases in the recognition of rna viruses shared and unique functions of the dexd/h-box helicases rig-i, mda5, and lgp2 in antiviral innate immunity function of nod-like receptors in microbial recognition and host defense regulation of immune pathways by the nod-like receptor nlrc5 nlrp3 inflammasome activation: the convergence of multiple signalling pathways on ros production? interferon-inducible antiviral effectors transgenic mice with intracellular immunity to influenza virus the mx gtpase family of interferon-induced antiviral proteins. microbes and infection the interferon-induced mx protein of chickens lacks antiviral activity stalk domain of the dynamin-like mxa gtpase protein mediates membrane binding and liposome tubulation via the unstructured l4 loop structural basis of oligomerization in the stalk region of dynamin-like mxa transport of the murine mx protein into the nucleus is dependent on a basic carboxy-terminal sequence natural killer cell receptors in cattle: a bovine killer cell immunoglobulin-like receptor multigene family contains members with divergent signaling motifs comparative genomics of natural killer cell receptor gene clusters characterization of the opossum immune genome provides insights into the evolution of the mammalian immune system the leukocyte receptor complex in chicken is characterized by massive expansion and diversification of immunoglobulin-like loci the chicken leukocyte receptor complex encodes a family of different affinity fcy receptors the ever-expanding ly49 gene family: repertoire and signaling natural killer cell receptors in the horse: evidence for the existence of multiple transcribed ly49 genes identification of natural killer cell receptor clusters in the platypus genome reveals an expansion of c-type lectin genes the phylogenetic origins of natural killer receptors and recognition: relationships, possibilities, and realities nk gene complex dynamics and selection for nk cell receptors signaling pathways engaged by nk cell receptors: double concerto for activating receptors, inhibitory receptors and nk cells of mice and men: different functions of the murine and human 2b4 (cd244) receptor on nk cells biology and functions of human leukocyte antigen-g in health and sickness* nomenclature for factors of the hla system three-dimensional structure of the human class ii histocompatibility antigen hla-dr1 sequence organisation of the class ii region of the human mhc evolutionary relationships of class ii majorhistocompatibility-complex genes in mammals class ii drb polymorphism and sequence diversity in two vesper bats in the genus myotis non-neutral evolution of the major histocompatibility complex class ii gene drb1 in the sac-winged bat saccopteryx bilineata mhc class ii drb diversity, selection pattern and population structure in a neotropical bat species, noctilio albiventris prominence of gamma delta t cells in the ruminant immune system characterization of avian t-cell receptor î³ genes the two suborders of chiropterans have the canonical heavy-chain immunoglobulin (ig) gene repertoire of eutherian mammals the pfam protein families database a molecular phylogeny for bats illuminates biogeography and the fossil record tagdust-a program to eliminate artifacts from next generation sequencing data velvet: algorithms for de novo short read assembly using de bruijn graphs oases: de novo transcriptome assembler for very short reads cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences using the miraest assembler for reliable and automated mrna transcript assembly and snp detection in sequenced ests gapped blast and psi-blast: a new generation of protein database search programs composition profiler: a tool for discovery and visualization of amino acid composition differences categorizer categorizer: a web-based program to batch analyze gene ontology classification categories generic gene ontology (go) term mapper multiple sequence alignment using clustalw and clustalx the neighbor-joining method: a new method for reconstructing phylogenetic trees mega4: molecular evolutionary genetics analysis (mega) software version 4.0 statistics of local complexity in amino acid sequences and sequence databases accuracy of protein flexibility predictions the universal protein resource (uniprot) submit your next manuscript to biomed central and take full advantage of: â�¢ convenient online submission â�¢ thorough peer review â�¢ no space constraints or color figure charges â�¢ immediate publication on acceptance â�¢ inclusion in pubmed, cas, scopus and google scholar â�¢ research which is freely available for redistribution we thank craig smith, carol de jong, deborah middleton low complexity regions in protein sequences were detected with the seg program with default parameters [87] . the transcription of a bat mhc class i gene was examined using quantitative pcr (qpcr) as described previously [18] . briefly, total rna was prepared from lymph node, spleen, liver, lung, heart, kidney, small intestine, brain and salivary glands using the rneasy mini kit (qiagen) as described above. cdna was generated using a quantitect reverse transcription kit for rt-pcr (qiagen). qpcr primers were designed using primer express 3.0 (applied biosystems) with default parameter settings (5'-acgactcctattccccaggatag-f and 5'-gaaagc cactggtacctgtgaga-r). reactions were carried out using express sybr w greener tm qpcr supermix universal (invitrogen) and an applied biosystems 7500 fast real-time qpcr instrument. additional file 1: table s1 . summary of additive multiple-kmer velvet/ oases/mira3 assembly.additional file 2: figure s1 . overview of the bat transcriptome. the distribution of 178,554 and 285,268 transcriptome sequences that have mapped to human orthologues from p. alecto thymus and pooled tissue datasets based on go slim terms. sequences within the three areas of gene ontology: molecular function, biological process and cellular component are further divided into subgroups at the go slim level.additional file 3: sequences of all genes described in the manuscript.additional file 4: figure 2 . amino acid composition of large unannotated orfs. the horizontal axis shows amino acids sorted by flexibility index [88] .a. amino acid composition of 1656 large unannotated non-redundant orfs relative to proteins in the swissprot database [89] . the amino acids trp, cys and pro have twice the abundance in unannotated orfs compared to swissprot proteins.b. amino acid composition of 1195 low complexity regions in unannotated orfs relative to 1656 unannotated non-redundant orfs. prolines are abundant in low complexity regions, but trp and cys are not. the authors declare that they have no competing interests. key: cord-350286-n7ylgqfu authors: giri, rajanish; bhardwaj, taniya; shegane, meenakshi; gehi, bhuvaneshwari r.; kumar, prateek; gadhave, kundlik; oldfield, christopher j.; uversky, vladimir n. title: when darkness becomes a ray of light in the dark times: understanding the covid-19 via the comparative analysis of the dark proteomes of sars-cov-2, human sars and bat sars-like coronaviruses date: 2020-04-03 journal: biorxiv doi: 10.1101/2020.03.13.990598 sha: doc_id: 350286 cord_uid: n7ylgqfu recently emerged coronavirus designated as sars-cov-2 (also known as 2019 novel coronavirus (2019-ncov) or wuhan coronavirus) is a causative agent of coronavirus disease 2019 (covid-19), which is rapidly spreading throughout the world now. more than 9,00,000 cases of sars-cov-2 infection and more than 47,000 covid-19-associated mortalities have been reported worldwide till the writing of this article, and these numbers are increasing every passing hour. world health organization (who) has declared the sars-cov-2 spread as a global public health emergency and admitted that the covid-19 is a pandemic now. the multiple sequence alignment data correlated with the already published reports on the sars-cov-2 evolution and indicated that this virus is closely related to the bat severe acute respiratory syndrome-like coronavirus (bat sars-like cov) and the well-studied human sars coronavirus (sars cov). the disordered regions in viral proteins are associated with the viral infectivity and pathogenicity. therefore, in this study, we have exploited a set of complementary computational approaches to examine the dark proteomes of sars-cov-2, bat sars-like, and human sars covs by analysing the prevalence of intrinsic disorder in their proteins. according to our findings, sars-cov-2 proteome contains very significant levels of structural order. in fact, except for nucleocapsid, nsp8, and orf6, the vast majority of sars-cov-2 proteins are mostly ordered proteins containing less intrinsically disordered protein regions (idprs). however, idprs found in sars-cov-2 proteins are functionally important. for example, cleavage sites in its replicase 1ab polyprotein are found to be highly disordered, and almost all sars-cov-2 proteins were shown to contain molecular recognition features (morfs), which are intrinsic disorder-based protein-protein interaction sites that are commonly utilized by proteins for interaction with specific partners. the results of our extensive investigation of the dark side of the sars-cov-2 proteome will have important implications for the structural and non-structural biology of sars or sars-like coronaviruses. significance the infection caused by a novel coronavirus (sars-cov-2) that causes severe respiratory disease with pneumonia-like symptoms in humans is responsible for the current covid-19 pandemic. no in-depth information on structures and functions of sars-cov-2 proteins is currently available in the public domain, and no effective anti-viral drugs and/or vaccines are designed for the treatment of this infection. our study provides the first comparative analysis of the orderand disorder-based features of the sars-cov-2 proteome relative to human sars and bat cov that may be useful for structure-based drug discovery. intrinsically disordered proteins (idps) and intrinsically disordered protein regions (idprs)), in order to better understand an interplay between the ordered and disordered components of the proteome. in classical structure-function-paradigm, it is believed that a unique, stable, and well-defined 3-dimensional structure is a prerequisite for a protein to accomplish its unique biological function. although this notion dominated scientific minds for over the hundred years, eventually an idea of the presence of functional intrinsic disorder in proteins came to the attention of the structural biologists. according to this "heretic" viewpoint, a noticeable amount of biologically active proteins (of protein regions) fail to fold into the well-defined structures and instead remain disordered, existing as highly dynamic ensembles of rapidly interconverting conformations under the physiological conditions. these proteins and protein regions are known now as intrinsically disordered proteins (idps) and intrinsically disordered protein regions (idprs), respectively. the propensity of being functional intrinsically disordered proteins (similar to the propensity of forming unique biologically active structures of ordered proteins) is determined by the amino acid sequences [11] [12] [13] . idps exhibit their biological functions in numerous biological processes commonly associated with cellular signalling, gene regulation, and control by interacting with their physiological partners [14] [15] [16] [17] [18] . these functions of idps and idps are regulated by their protein-protein, protein-rna, protein-dna interactions [19, 20] . molecular recognition features (morfs) are the regions in idps implicated in regulation of idps function by protein-protein interactions and serve as the primary stage in molecular recognition. zhang and colleagues have reported the genomic sequence of sars-cov-2 with genbank accession number nc_045512 having 29,903 nucleotides. the virus was isolated from the bronchoalveolar lavage fluid of a patient, went through a circle or renaming, from novel wuhan seafood market pneumonia virus to deadly wuhan coronavirus, to the 2019 novel coronavirus (2019-ncov) or the wuhan-2019 novel coronavirus (wuhan-2019-ncov, and was eventually named sars-cov-2 by the who [21] . it is known that the idps/idprs are present in all three kingdoms of life, and viral proteins often contain unstructured regions that have been strongly correlated with their virulence [22] [23] [24] [25] . in this report, we investigated the disordered side of the sars-cov-2 proteome using a complementary set of computational approaches to check the prevalence of idprs in various sars-cov-2 proteins and to shed some light on their disorder-related functions. we also have comprehensively analyzed idprs among the closely related viruses, such as human sars cov and bat sars-like cov. furthermore, we have also identified protein functions related to protein-protein interactions, rna binding, and dna binding from all three viruses. since these three viruses are closely related, our study provides important means for a better understanding of the sequence and structural peculiarities of their evolution. we believe that this study will help the structural and non-structural biologists to design and perform experiments for a more in-depth understanding of this virus and its pathogenicity. this also will have long-term implications for developing new drugs or vaccines against this currently unpreventable infection. sequence retrieval and multiple sequence alignment. the protein sequences of bat cov (sars-like) and human sars cov were retrieved from uniprot (uniprot ids for individual proteins are listed in table 1 ). the translated sequences of sars-cov-2 proteins [genbank database [26] (accession id: nc_045512.2)] were obtained from genbank. we used these sequences for performing multiple sequence alignment (msa) and predicting the idprs. we have used clustal omega [27] for protein sequence alignment and esprit 3.0 [28] for constructing the aligned images. for the prediction of the intrinsic disorder predisposition of cov proteomes, we have used multiple predictors, such as members of the pondr ® (predictor of natural disordered regions) family including pondr ® vls2 [29] , pondr ® vl3 [30] , pondr ® fit [31] , and pondr ® vlxt [32] , as well as the iupred platform for predicting long (≥30 residues) and short idprs (<30 residues) [33] . these computational tools predict residues/regions, which do not have the tendency to form an ordered structure. residues with disorder scores exceeding the threshold value of 0.5 are considered as intrinsically disordered residues, whereas residues with the predicted disorder scores between 0.2 and 0.5 are considered flexible. complete predicted percent of intrinsic disorder (ppid) in a query protein was calculated for every protein of all the three viruses from outputs of six predictors. the detailed methodology has been given in our previous reports [34, 35] . and disopred3 [42] . the protein residues with anchor, morfpred, and disopred3 score above the threshold value of 0.5 and morfchibi_web score above the threshold value of 0.725 are considered morf regions. idprs facilitate interactions with rnas and dnas and regulates many cellular functions [43] . thus, for predicting the dna binding residues in cov proteins, we have used two online servers: drnapred [19] and disordpbind [43] . for rna binding residues, we used pprint (prediction of protein rna-interaction) [44] and disordpbind [43] . the mean values of the predicted percentage of intrinsic disorder scores (mean ppids), that were obtained by averaging the predicted disorder scores from six disorder predictors (supplementary table 1 -3) for each protein of sars-cov-2 as well as human sars, and bat cov are represented in table 1 . * these sequences are based on genome annotations conducted by wu et al. [45] . proteins and their ppids are coloured to reflect their disorder status (ordered -blue, moderately disorderedpink, highly disordered -red) figures 2a, 2b , and 2c are 2d-disorder plots generated for sars-cov-2, human sars and bat cov proteins, respectively, and represent the ppid pondr-fit vs. ppid mean plots. based on their predicted levels of intrinsic disorder, proteins can be classified as highly ordered (ppid < 10%), moderately disordered (10% ≤ ppid < 30%) and highly disordered (ppid ≥ 30%) [46] . from the data in table 1 , figures 2a, 2b , and 2c, as well as the ppid based classification, we conclude that the nucleocapsid protein from all three strains of coronavirus possesses the highest percentage of the disorder and is classified as highly disordered protein. orf3b protein in bat cov, orf6 protein in sars-cov-2, human sars, and bat cov, and orf9b protein in human sars and sars cov belong to the class of moderately disordered proteins. while the structured proteins, namely, spike glycoprotein (s), an envelope protein (e) and membrane protein (m) as well as accessory proteins orf3a, orf7a, orf8 (orf8a and orf8b in case of human sars) of all three strains of coronavirus are ordered proteins. orf14 and orf10 proteins also belong to the class of ordered proteins. in ch-cdf plot of the proteins of (d) sars-cov-2 (e) human sars and (f) bat cov, the y coordinate of each protein spot signifies distance of corresponding protein from the boundary in ch plot and the x coordinate value corresponds to the average distance of the cdf curve for the respective protein from the cdf boundary. in order to further investigate the nature of the disorder in proteins of sars-cov-2, human sars, and bat cov, we utilized the combined ch-cdf tool that uses the outputs of two binary classifiers of disorder, charge hydropathy (ch) plot and cumulative distribution function (cdf) plot. this helped in retrieving more detailed characterization of the global disorder predisposition of the query proteins and their classification according to the disorder "favours". the ch plot is a linear classifier that differentiates between proteins that are predisposed to possess extended disordered conformations that include random coils and premolten globules from proteins that have compact conformations (ordered proteins and molten globule-like proteins). the other binary predictor, cdf is a nonlinear classifier that uses the pondr ® vlxt scores to discriminate ordered globular proteins from all disordered conformations, which include native molten globules, pre-molten globules, and random coils. the ch-cdf plot can be divided into four quadrants: q1 (bottom right quadrant) is an area of ch-cdf phase space that is expected to include ordered proteins; q2 (bottom left quadrant) includes proteins predicted to be disordered by cdf and compact by ch (i.e., native molten globules and hybrid proteins containing high levels of both ordered and disordered regions); q3 (top left quadrant) contains proteins that are predicted to be disordered by both ch and cdf analysis (i.e., highly disordered proteins with the extended disorder); and q4 (top right quadrant) possesses proteins disordered according to ch but ordered according to cdf analysis [34] . figures 2d, 2e and 2f represent the ch-cdf analysis of proteins of sars-cov-2, human sars, and bat cov and show that all the proteins are located within the two quadrants q1 and q2. the ch-cdf analysis leads to the conclusion that all proteins of sars-cov-2, human sars, and bat cov are ordered except nucleocapsid protein, which is predicted to be disordered by cdf but ordered by ch and hence lies in q2. molecular recognition features (morfs) are short interaction-prone disordered regions found within idps/idprs that commence a disorder-to-order transition upon binding to their partners [47, 48] . these regions are important for protein-protein interactions and may initiate an early step in molecular recognition [48] . in this study, we have analyzed and compared morfs (protein-binding regions) in sars-cov-2 with human sars and bat cov. the results of this analysis are summarized in table 2 , which clearly shows that most of the sars-cov-2 proteins contain at least one morf, indicating that disorder does play an important role in the functionality of these viral proteins. in addition to protein-protein interactions/protein-binding functions, idps and idrs also mediates functions by facilitating their interactions with nucleotides such as dna and rna [20, 49] . therefore, we have used a combination of two different online servers for locating protein residues that are showing the propensity to bind with dna as well as rna. nucleotide-binding residues in proteins of three studied coronaviruses are listed in supplementary coronaviruses encode four structural proteins, namely, spike (s), envelope (e) glycoprotein, membrane (m), and nucleocapsid (n) proteins, which are translated from the last ~10kb nucleotides and form the outer cover of the covs, encapsulating their single-stranded genomic rna. s protein is a large multifunctional protein forming the exterior of the cov particles [50, 51] . it forms surface homotrimers and contains two distinct ectodomain regions known as s1 and s2. in some covs, the s protein is actually cleaved into these subunits, which are joined non-covalently, whereas an additional proteolytic cleavage within the n-terminal part of the s2 subunit that takes place upon virus endocytosis generates spike proteins s2'. subunit s1 initiates viral infection by binding to the host cell receptors, s2 acts as a class i viral fusion protein that mediates fusion of the virion and cellular membranes and thereby promotes the viral entry into the host cells, whereas s2' serves as a viral fusion peptide [52, 53] . spike binds to the virion m protein through its c-terminal transmembrane region [54] . belonging to a class i viral fusion protein, s protein binds to specific surface receptor angiotensin-converting enzyme 2 (ace2) on host cell plasma membrane through its n-terminal receptor-binding domain (rbd) and mediates viral entry into host cells [55] . the s protein consists of an n-terminal signal peptide, a long extracellular domain, a singlepass transmembrane domain, and a short intracellular domain [56] . a 3.60 å resolution structure (pdb id: 6acc) of s protein from human sars complexed with its host binding partner ace2 has been obtained by cryo-electron microscopy (cryo-em the biophysical analysis reported in previous study has also revealed that the s protein from sars-cov-2 has a higher binding affinity to ace2 than s protein from human sars [58] . which has been calculated by averaging the disorder scores from all six predictors is represented by a short-dot line (sky-blue line) in the graph. the light sky-blue shadow region signifies the mean error distribution. the residues missing in the pdb structure or the residues for which pdb structure is unavailable are represented by the grey-coloured area in the corresponding graphs. (e) aligned disorder profiles generated for spike glycoprotein from sars-cov-2 (black line), human sars (red line), and bat cov (green line) based on the outputs of the pondr ® vsl2. msa analysis among all three coronaviruses demonstrates that s protein of sars-cov-2 has a 77.71% sequence identity with bat cov and 77.14% identity with human sars (supplementary figure s1a) . all three s proteins are found to have a conserved c-terminal region. however, the n-terminal regions of s proteins display noticeable differences. given that there is significant sequence variation rbd located at the n-terminal region of s protein, this might be the reason behind variation in its virulence and its receptor-mediated binding and entry into the host cell. according to our intrinsic disorder propensity analysis, s protein from all three covs analysed in this study are highly structured, as their predicted disorder propensity lies below 10% ( table 1 ). in fact, the mean ppid scores of sars-cov-2, human sars cov, and bat cov are calculated to be 1.41%, 1.12%, and 1.85%, respectively. figures 3b, 3c , and 3d represent the intrinsic disorder profiles of s proteins from sars-cov-2, human sars and bat cov obtained from six disorder predictors. finally, figure 3e shows aligned disorder profiles of s proteins from these covs and illustrates remarkable similarity in their disorder propensity, especially in the c-terminal region. it is of interest to map known functional regions of s proteins to their corresponding disorder profiles. the maturation of s protein requires specific posttranslational modification (ptm), proteolytic cleavage that happens at two stages. first, host cell furin or another cellular protease nicks the s precursor to generate s1 and s2 proteins, whereas the second cleavage that takes place after the viral attachment to host cell receptors leads to the release of a fusion peptide generating the s2' subunit. in human sars cov, the first and second cleavage site is located at residues r 667 and r 797 , respectively, whereas in bat cov, the corresponding cleavage sites are residues r 654 and r 784 . as it follows from figure 3 , these cleavage sites are located within the idprs. in human sars cov s protein, fusion peptide (residues 770-788) is located within a flexible region, is characterized by the mean disorder score of 0.232±0.053. similarly, in bat cov s protein, fusion peptide (residues 757-775) has a mean disorder score of 0.320±0.046. s protein contains two heptad repeat regions that form coiledcoil structure during viral and target cell membrane fusion, assuming a trimer-of-hairpins structure needed for the functional positioning of the fusion peptide. in human sars cov s protein, heptad repeat regions are formed by residues 902-952 and 1145-1184, which have mean disorder scores of 0.458±0.067 and 0.353±0.062, respectively. the analogous situation is observed for the s protein from bat cov, where these heptad repeat regions are positioned at residues 889-939 (0.44±0.11) and 1132-1171 (0.353±0.062). another functional region found in s proteins is the receptor-binding domain (residues 306-527 and 310-514 in human sars cov and bat cov, respectively) containing a receptor-binding motif responsible for interaction with human ace2. in human s protein of human sars cov this motif (residues 424-494) is not only characterized by structural flexibility, possessing a mean disorder score of 0.30±0.16, but also contains a disordered region (residues 461-466). since s protein is known as spike glycoprotein, it contains numerous glycosylation sites. due to rather close similarity of disorder profiles of s proteins analysed here, we can assume that all the aforementioned indications of the functional importance of disorder and flexible regions in s proteins from sars cov and bat cov are also applicable to sars-cov-2 s protein. finally, table 2 shows that s protein from sars-cov-2 contain one morf region at its cterminal (residues 1265-1272) by morfchibi_web, two morf regions ((residues 2-6) & (residues 819-823)) by morfpred, and one morf region at n-terminal (residues 1-10) by disopred3. these results indicating that intrinsic disorder is important for its interaction with binding partners. interestingly, the n-terminal region of s protein (residues 1-10) from all three viruses are observed to be a disorder-based protein binding region by two predictors (morfpred and disopred3). n-terminal morf displays its role in viral interaction with host receptor and c-terminal morf displays its role in m protein interaction and viral assembly. moreover, morf region mainly lies in the n-and c-terminal regions suggesting a possible role during cleavage as well. in addition to protein-binding regions, s protein also shows many nucleotide-binding residues. tables 9, 10, and 11 shows that numerous rna binding residues predicted by pprint in all three viruses and a single rna binding residue were predicted by disordpbind in human sars. further, drnapred and disordpbind predicted the presence of many dna binding residues in s protein of all three viruses. these results signify the role of s protein functions related to molecular recognition (protein-protein interaction, rna binding, and dna binding) such as interaction with host cell membrane and further viral infection. therefore, identified idps/idprs and residues/regions from s protein crucial for molecular recognition can be targeted for disorder-based drug discovery. envelope (e) protein is a small, multifunctional inner membrane protein that plays an important role in the assembly and morphogenesis of virions in the cell [59] [60] [61] . e protein consists of two ectodomains associated with n-and c-terminal regions, and a transmembrane domain. it homo-oligomerize to form pentameric membrane destabilizing transmembrane (tm) hairpins to form a pore necessary for its ion channel activity [62] . figure 4a shows the nmr-structure (pdb id: 2mm4) of human sars envelope glycoprotein of 8-65 residues [63] . msa results illustrate ( figure 4b ) that this protein is highly conserved, with only three amino acid substitutions in e protein of sars-cov-2 conferring its 96% sequence similarity with human sars and bat cov. bat cov shares 100% sequence identity with human sars. mean ppid calculated for sars-cov-2, human sars, and bat cov e proteins are 5.33%, 6.58%, and 6.58% respectively ( table 1) . the e protein is found to have a reasonably well-predicted structure. our predictions suggest that the residues of n-and c-terminals are displaying a higher tendency for the disorder. the last 18 hydrophilic residues (residues 59-76) have been reported to adopt a random-coil conformation with and without the addition of lipid membranes [64] . literature suggests that the last four amino acids of the c-terminal region of e protein containing a pzd-binding motif are involved in protein-protein interactions with a tight junction protein pals1. our results support literature as we identified long n-terminal region of approximately 30 residues long as disorder-based protein binding region in all three viruses (see table 2, supplementary table 7 and 8). pals1 is involved in maintaining the polarity of epithelial cells in mammals [65] . respective graphs in figures 4c, 4d , and 4e show the predicted intrinsic disorder profiles for e proteins of sars-cov-2, human sars, and bat cov. we speculate that the disordered region content may be facilitating the interactions with other proteins as well. in agreement with this hypothesis, table 2 shows that in e protein from sars-cov-2, the c-terminal domain serves as protein-binding region. we found that the residues from 45-75 is a long morf in e proteins of all three viruses as predicted by morfchibi_web ( table 2, supplementary table 7 and 8). as aforementioned, these randomly-coiled binding-residues at c-terminus may gain structure while assisting the protein-protein interaction mediated by e protein. one more morf region (residues [26] [27] [28] [29] [30] in the transmembrane domain was observed by disopred3 in the e protein of all three viruses. since these residues are the part of ion channel, we speculate that these residues do specific interactions and may be guiding the specifi functions of ion channel activity. few rna binding residues by pprint and disordpbind and several dna binding residues by drnapred are predicted for e protein in all three viruses. assembly by interacting with the nucleocapsid (n) and e proteins [66] [67] [68] . protein m interacts specifically with a short viral packaging signal containing coronavirus rna in the absence of n protein, thereby highlighting an important nucleocapsid-independent viral rna packaging mechanism inside the host cells [69] . it gains high-mannose n-glycans in er, which are subsequently modified into complex n-glycans in the golgi complex. glycosylation of m protein is observed to be not essential for virion fusion in cell culture [70, 71] . cryo-em and tomography data indicate that m forms two distinct conformations, a compact m protein having high flexibility and low spike density, and an elongated m protein having a rigid structure and narrow range of membrane curvature [72] . some regions of m glycoproteins might serve as important dominant immunogens. although no structural information is available for the full-length m protein as of yet, a short peptide of the membrane glycoprotein (residues 88-96) from human sars cov was co-crystallized with a complex between a-2 alpha chain of the hla class i histocompatibility antigen and β2microglobulin (pdb id: 3i6g) [73] . figure 5a shows that within this complex, the cocrystallized m protein region exists in an extended conformation. m protein of sars-cov-2 has a sequence similarity of 90.1% with bat cov and 89.6% with human sars m proteins ( figure 5b ). our analysis revealed that the intrinsic disorder levels in m proteins of sars-cov-2, human sars cov, and bat cov are relatively low since these proteins show the ppid values of 2.70%, 1.36%, and 1.36% respectively. this is in line with the previous publication by goh et al. on human sars hku4 where they found the mean ppid of 4% using additional predictors such as topidp and foldindex along with the predictors used in our study [74] . figures 5c, 5d , and 5e represent per-residue disorder profiles generated for m proteins of sars-cov-2, human sars cov, and bat cov and show that with the exception to their n-and c-terminal regions, these proteins are mostly ordered. the last 20 residues of mers-cov m protein are important for intracellular trafficking and contains a determinant that localizes it into the golgi network [75] . our results in table 2 illustrates that the disordered c-tail of the m protein is predicted to have disorder based protein-binding region and therefore can serve as a binding site for its specific partner required for its localization inside the host cell. a long morf region (residues 186-220) at the c-terminal of m protein in all three viruses were observed by morfchibi_web. two morf regions (one at n-terminus (residues 1-16) and one at c-terminus (residues 205-221)) was observed by disopred3 in human sars and bat cov. however, single morf (residues 117-132) observed in sars-cov-2 by disopred3. morfpred also predicts a short morf at c-terminus of sars-cov-2 (residues 214-222), human sars (residues 214-221), and bat cov (residues 211-221) ( table 2, supplementary tables 7 and 8) . furthermore, the m protein from all three viruses displays strong tendency to bind with rna (as predicted by pprint and disordpbind) and dna (as predicted by drnapred and disordpbind) (see supplementary tables 9, 10 , and 11). our understanding on m protein of covs (idps and morf at c-terminus and molecular recognition) elucidates its crucial role in interaction with the n and e proteins for viral assembly. nucleocapsid (n) protein: nucleocapsid (n) protein is one of the major viral proteins playing several significant roles in transcription, and virion assembly of coronaviruses [76] . it binds to viral genomic rna forming a ribonucleoprotein core required for the rna encapsidation during viral particle assembly [77] . sars-cov virus-like particles (vlps) formation has been reported to depend upon either m and e proteins or m and n proteins. for the effective production and release of vlps, co-expression of e or n proteins with m protein is necessary [78] . n protein of human sars consists of two structural domains, the n-terminal rna-binding domain (ntd: 45-181 residues) and the c-terminal dimerization domain (ctd: 248-365 residues) with a disordered patch in between these domains. n protein has been demonstrated to bind viral rna using both ntd and ctd [79] . figure 6a1 displays the nmr solution structure of the ntd of human sars cov nucleocapsid protein (45-181 residues) (pdb id: 1ssk) [80] . figure 6a2 shows an x-ray crystal structure of the ctd of human sars cov nucleocapsid protein (270-366 residues) (pdb id: 2gib) [81] . a model of the domain organization of the n-protein from sars-cov-2 is shown in figure 6b . the 419 amino acid-long n protein of sars-cov-2 shows a percentage identity of 88.76% with n protein of bat cov n protein and 89.74% with human sars n protein (supplementary figure s1b) . our analysis revealed that the n proteins of coronaviruses contain the highest levels of intrinsic disorder (see figure 6 and table 1 ). in fact, n proteins from sars-cov-2 human sars cov, and bat cov are characterized by the mean ppid of 64.91%, 71.09%, and 65.80%, respectively. in accordance with the previously evaluated intrinsic disorder predisposition [74] , n protein is highly disordered in all three sars viruses analysed in this study ( table 1) . graphs in figures 6c, 6d , and 6e depict the disorder profiles of sars-cov-2, human sars cov, and bat cov nucleocapsid proteins and show that their n-and c-terminal regions are completely disordered, and all three proteins also contain the central unstructured segment. as expected, the intrinsic disorder predisposition of the n protein of sars-cov-2 is remarkably similar to that for the n protein of human sars cov as reported in a previous study [74] . this is further supported by figure 6f , where pondr ® vsl2-generated disorder profiles of these three proteins are overlapped to show almost complete coincidence of their major disorder-related features. it is clear that in n-proteins, the n-and c-termini and a log central segment are completely disordered. figure 6c shows that in the n protein from sars-cov-2, residues 1-57, 64-102, 145-162, 166-289, and 362-422 are found to be disordered. many of these residues are lying within the ntd and ctd regions, and which, due to their structural plasticity, were not crystallized in human sars cov n protein. sars-cov-2 has a disordered segment from 168-289 residues while human sars has predicted to have an unstructured segment from 145-289 residues. overall, all three n proteins are found to be highly disordered. the n protein from human sars cov has one phosphorylation site (residue s 177 ) and several regions with compositional biases, such as ser-rich (residues 181-213), poly-leu, poly-gln, and ploy-lys (residues 220-225, 240-245, and 370-376), all predicted to be disordered. similarly, in n protein from bat cov, s 176 is phosphorylated, and this protein has ser-rich, poly-leu, and ploy-lys regions (residues 176-206, 219-224, and 369-375, respectively), all of which are disordered. it has been reported to interact using the central disordered region with m protein, hnrnp a1, and self n-n interaction [82] [83] [84] . the middle flexible region is also responsible for its rna-binding activity [85] . deletion of 184-196 residues, 169-308 residues, 161-210 residues of n abolishes its multimerization, rnabinding capacity, and hnrnp a1 interactions respectively. supplementary table 7 and 8, and table 2 shows that n protein is heavily decorated with numerous morfs, suggesting that this protein is a promiscuous binder. long disorder-based protein bonding regions at nand c-terminus of n protein of all three viruses were observed by all four predictors (morfchibi_web, anchor, morfpred, and disopred3). indeed, this is the single protein where we found many morfs as compared with the other structural, non-structural and accessory proteins of covs. the morfs present in these regions may mediate the abovementioned interactions of n proteins. figure 6a2 represents another important disorderrelated functional feature of the n protein. in fact, the ctd homodimer shown there is characterized by highly intertwined morphology, which is typically a result of bindinginduced folding [86] [87] [88] , indicating that a very significant part of ctd gains structure during dimerization. we identified numerous rna binding residues in all three viruses using pprint server. this finding supports the function of n protein as it interacts with genomic rna for a ribonucleoprotein core formation which is crucial step for rna encapsidation during viral particle assembly. in addition, drnapred and disordpbind predicts multiple dna binding residues for n protein in sars-cov-2, human sars, and bat cov. the long flexible (idprs) regions at n and c-terminus of sars-cov-2 have long protein-binding as well as nucleotide-binding regions that may have important role in its interaction with viral rna. these flexible regions can be targeted to inhibit interaction of n protein with viral genomic rna. literature suggests that some viral proteins are translated from the genes interspersed in between the genes of structural proteins. these proteins are known as accessory proteins, and many of them are proposed to be involved in viral pathogenesis [89] . proteins orf3a and orf3b. orf3a is a multifunctional protein with the molecular weight of ~31 kda that has been found to localize in different organelles inside the host cells. also referred to as u274, x1, and orf3, the gene for this protein is present between the s and e genes of the sars-cov genome [90] [91] [92] . the homo-tetrameric complex of orf3a has been demonstrated to form a potassium-ion channel on the host cell plasma membrane [93] . it performs a major function during virion assembly by co-localizing with e, m, and s viral proteins [94, 95] . orf3b protein can be found in the cytoplasm, nucleolus, and outer membrane of mitochondria of the host cells [96, 97] . in huh 7 cells, its over-expression has been linked with the activation of ap-1 via erk and jnk pathways [98] . transfection of orf3b-egfp leads to cell growth arrest at the g0/g1 phase of vero, 293, and cos-7 cells [99] . orf3a induces apoptosis via caspase 8/9 directed mitochondrial-mediated pathways, while orf3b is reported to affect only the caspase 3-related pathways [100, 101] . on performing msa, results of which are shown in figure 7d , we found that orf3a protein from sars-cov-2 is slightly evolutionary closer to the orf3a of bat cov (73.36%) than to the orf3a of human sars cov (72.99%). graphs in figures 7a, 7b , and 7c depict the propensity for disorder in orf3a proteins of novel sars-cov-2, human sars cov, and bat cov (sars-like), respectively. mean ppids in these orf3a proteins are 9.1% (sars-cov-2), 8.8% (human sars), and 6.2% (bat cov (sars-like)). orf3a of sars cov-2 shows protein-binding regions at its n-terminus (by morfchibi_web (residues 1-6), morfpred (residues 7-12), and disopred3 (residues [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] ) and at c-terminus (by morfchibi_web (residues 261-268) and morfpred (residues 259-263)) ( table 2) . similarly, orf3a of human sars and bat cov also shows morfs at n-and c-terminus with the help of morfchibi_web and morfpred (supplementary tables 7 and 8 ). these protein-binding regions in orf3a may have role in its co-localization with e, m, and s viral proteins. apart from morfs, it also displays several nucleotide-binding residues in all three viruses (see supplementary tables 9, 10 , and 11). in fact, this represents maximum number of rna and dna binding residues as compared with all other accessory proteins. these results indicate that the idps/idprs of this protein could be utilized in molecular recognition (protein-protein, protein-rna, and protein-dna interaction). according to the intrinsic disorder predisposition analysis of orf3b proteins, their mean ppid values in sars-cov-2, human sars cov, and bat cov are 0%, 7.1%, and 23.1% respectively, as represented in figures 8a, 8b , and 8c. msa results ( figure 8d ) demonstrate that orf3b of sars-cov-2 is not closer to orf3b protein of human sars and orf3b protein of bat-cov, having a sequence similarity of only 54.6% and 59.1%, respectively. as we can see in table 2 , there is not a single morf found in orf3b of sars-cov-2. however, for human sars we identified three morfs (residues 32-37, 41-70, and 125-153) and for bat cov one morf at n-terminus (residues 1-38) by morfchibi_web server. protein orf6. orf6 is a short coronavirus protein with just 63 residues. also known as p6, this membrane-associated protein serves as an interferon (ifn) antagonist [102] . it downregulates the ifn pathway by blocking a nuclear import protein, karyopherin α2. using its c-terminal residues, orf6 disrupts karyopherin import complex in the cytosol and, therefore, hampers the movement of transcription factors like stat1 into the nucleus [102, 103] . it contains a ysel motif near its c-terminal region, which functions in protein internalization from the plasma membrane into the endosomal vesicles [104] . another study has also demonstrated the presence of orf6 in endosomal/lysosomal compartments [104, 105] . msa results demonstrate that (figure 9d) , sars-cov-2 orf6 is closer to orf6 protein of human sars cov, having a sequence similarity of 68.85% than to the orf6 of bat cov (sars-like) (67.21%). novel sars-cov-2 orf6 is predicted to be the second most disordered structural protein, with ppid of 22.95%, and with especially disordered cterminal region. our analysis of the intrinsic disorder predisposition using six predictors revealed the mean ppid in orf6 proteins of sars-cov-2, human sars, and bat cov to be 22.95%, 20.63%, and 20.63%, respectively ( table 1) . graphs in figures 9a, 9b and 9c illustrate that orf6 proteins from all three studied coronaviruses are expected to be moderately disordered proteins with the high disorder content in their c-terminal regions. these disordered regions are important for the biological activities of orf6. as aforementioned, this hydrophilic region contains lysosomal targeting motif (ysel) and diacidic motif (ddee) responsible for binding and recognition during translocation [104] . however, the n-terminal region does not contain a noticeable disorder. the 1-38 residues of the n-terminal region of human sars cov orf6 was shown to be α-helical and embedded in the membrane, although orf6 is not a transmembrane protein [106] . a long morf region ((residues 26-61 in sars-cov-2), (residues 31-63 in human sars), and (residues 30-60 in bat cov)) is also present at cterminus of orf6 proteins which are tabulated in table 2 , and supplementary tables 7 and 8. no predictor other than morfchibi_web has located morfs in this protein. supplementary table 9 , 10, and 11 shows nucleotide-binding residues in orf6 of all three viruses. it represents very few rna binding residues by pprint and few dna binding residues by drnapred. orf7a and orf7b proteins. alternatively called u122, orf7a is a type i transmembrane protein [107, 108] . it has been proven to localize in er, golgi, and peri-nuclear space. the presence of a krkte motif near the c-terminal region is needed for importing this protein from the er to the golgi apparatus [107, 108] . orf7a contributes to viral pathogenesis by activating the release of pro-inflammatory cytokines and chemokines, such as il-8 and rantes [109, 110] . in another study, overexpression of bcl-xl in 293t cells blocked the orf7a mediated apoptosis [111] . on the other hand, orf7b is an integral membrane protein that has been shown to localize in the golgi complex [112, 113] . the same reports also confirm the role of orf7b as an accessory as well as a structural protein in sars-cov virion [112, 113] . figure 10d represents the 1.8 å x-ray crystal structure of the 14-96 fragment of the orf7a from human sars cov (pdb id: 1xak) and demonstrates the compact seven-stranded topology of this protein, which is similar to that of the ig-superfamily members [114] . importantly, in this crystal structure, residues 82-96 constituted the region with missing electron density, indicating high structural flexibility of this segment. in line with this hypothesis, the nmr solution structure of the 16-99 fragment of the orf7a from human sars cov (pdb id: 1yo4) showed that residues 81-99 are highly disordered [115] . at the domain level, the structure of the orf7a protein includes a signal peptide, a luminal domain, a transmembrane domain, and a short cytoplasmic tail at the c-terminus [95, 114] . we found that 121-residue-long orf7a protein of sars-cov-2 shares 89.26% and 85.95% sequence identity with orf7a proteins of bat cov and human sars cov, respectively ( figure 10e) . on the other hand, the orf7b of sars-cov-2 is found to be closer to orf7b of human sars than to orf7b of bat cov, showing sequence identities of 81.40% and 79.07%, respectively (see figure 11d ). as can be observed from table 1 , our disorder predisposition analyses resulted in the overall ppid for orf7a proteins of 1.65% for sars-cov-2, 0.82% for bat cov and 0.82% for human sars cov. mean ppids estimated for orf7b proteins are 9.30% for sars-cov-2, 4.55% for bat cov and 4.55% human sars cov. figures 10a, 10b , and 10c represent the residues predisposed for disorder in orf7a proteins of sars-cov-2, human sars cov, and bat cov, respectively. table 2 shows that orf7a protein is expected to have several morfs indicating the potential involvement of this protein in disorder-dependent proteinprotein interactions. at the n-terminus, we observed one morf region (residues 1-10) with the help of disopred3 in all three viruses. in addition to protein binding regions, orf7a also contains several rna and dna binding residues. analysis also represents, orf7b proteins from all three viruses have low disorder content, likewise, they are not predicted to contain any morf by any of the predictors used in this study ( table 2, supplementary table 7 and 8). although the orf7b does not contain protein-binding regions, it was found to contain nucleotide (rns and dna) binding regions in the protein figures 11a, 11b , and 11c depict the residues predisposed for disorder in orf7b proteins of sars-cov-2, human sars cov, and bat cov, respectively. according to our analysis, both proteins in all three studied coronaviruses have a mostly ordered structure. proteins orf8a and orf8b. in animals and isolates from early human infections, the orf8 gene codes for a single orf8 protein. however, in late infections, more specifically, at middle and late stages, a 29 nucleotide deletion in the orf8 gene led to the formation of two distinct proteins, orf8a and orf8b containing 39 and 84 residues respectively [116, 117] . both proteins have conformations different from that of the longer orf8 protein. it has been reported that overexpression of orf8b resulted in the downregulation of e protein while the proteins orf8a and orf8/orf8ab have no effect on the expression of protein e. also, orf8/orf8ab was found to interact very strongly with proteins s, orf3a, and orf7a. orf8a interacts with s and e proteins, whereas orf8b protein interacts with e, m, orf3a and orf7a proteins [118] . the disorder-based protein binding regions of this protein identified in this study may have important role in interaction with other proteins. orf8 protein found in early sars-cov-2 isolates having 121 residues and according to our analysis, it shares a 90.05% sequence identity with orf8 protein of bat cov ( figure 12c) . furthermore, figures 12a and 12b show that there is no intrinsic disorder in both orf8 proteins from sars-cov-2 and bat cov. therefore, these two proteins predicted to be completely structured having a mean ppid of 0.00%. in orf8a and orf8b proteins of the human sars, the predicted disorder is estimated to be 2.56% and 2.38%, respectively (table 1) . graphs in figures 13a and 13b illustrate the presence of some disorder near the n-and c-terminals of orf8a and orf8b proteins. table 2 shows the identified morf regions in orf8 of sars-cov-2. it shows three morf regions (residues 1-5, 26-52, and 69-91) by morfchibi_web and one morf region (residues 1-10) by disopred3. in human sars, the n-terminus of both orf8a (residues 1-39) and orf8b (residues 1-83) was found to be morf by morfchibi_web server (supplementary table 7) . further, four proteinbinding regions (residues 26-53, 70-91, 98-104, and 113-130) were identified by morfchibi_web server in bat cov (supplementary table 8 ). apart from protein-binding, orf8 of sars-cov-2, orf8a and orf8b of human sars, and orf8 of bat cov also comprise several nucleotide-binding residues (see supplementary table 9 , 10, and 11). this protein is expressed from an alternative orf within the n gene through a leaky ribosome binding process [119] . inside the host cells, orf9b enters the nucleus, which is a cell cycle-independent process and represents a passive entry. this protein was shown to interact with a nuclear export protein receptor exportin 1 (crm1), using which it translocate out of the nucleus [120] . our morfs analysis shows the presence of disorderbased protein binding regions in orf9b protein which may have role in its interaction with crm1 and further translocation outside the nucleus. a 2.8 å resolution crystal structure of orf9b protein from human sars cov (pdb id: 2cme) shows the presence of a dimeric tent-like -structure along with the central hydrophobic amino acids ( figure 14d) . the published structure has the highly polarized distribution of charges, with positively charged residues on the one side of the tent and negatively charged on the other [121] . based on the sequence availability of accession id nc_045512.2, the translated protein sequence of orf9b is not reported for the sars-cov-2 as of yet. however, based on the report by wu and colleagues [45] , the sequences of the sars-cov-2 are already annotated. therefore, we took the corresponding amino acid sequences from that study and conducted the intrinsic disorder analysis. according to the msa, results shown in figure 14e , orf9b protein from sars-cov-2 shares 73.2% identity with human sars and 74.23% identity with bat cov. our idp analysis ( table 1) shows that orf9b from human sars is a moderately unstructured protein with a mean ppid estimated to 26.53%. as depicted in figure 14a , 14b, and 14c, disorder mainly lies near the n-terminal end 1-10 residues and 28-40 residues near the central region with a well-ordered inner core of human sars orf9b protein. the x-ray crystal structure of orf9b has a missing electron density of the first 8 residues and 26-37 residues near the central region. this indicates that the corresponding regions are disordered, which are difficult to crystallize due to their highly dynamic structural organization. sars-cov-2 orf9b protein with a mean ppid of 10.31% has an n-terminal (1-10 residues) predicted disordered segment. orf9b of bat cov is shown to have an intrinsic disorder content of 9.28%, comparatively lower than that of the human sars orf9b protein. morfs lies in the n-terminal region of orf9b proteins ( table 2, supplementary table 7 and 8). in the absence of other viral proteins, its first 41 residues have been demonstrated to induce membranous structures similar to dmvs [106] . the available crystal structure also has the missing electron density in the n-terminal region suggests that these flexible amino acids are likely to interact with host lipids. the first 3-29 residues of sars-cov2 are identified as disorder-based protein binding region that may have role in its interaction with host lipids and formation of dmvs. supplementary tables 9, 10, and 11 represents nucleotide-binding residues for orf9b of sars-cov-2, human sars, and bat cov. the newly emerged sars-cov-2 has an orf10 protein of 38 amino acids. orf10 of sars-cov-2 has a 100% sequence similarity with orf10 of bat cov strain bat-sl-covzc45 [21] . however, we did not conduct the disorder analysis for orf10 from the bat-sl-covzc45 strain, since all our studies reported here are related to a different strain of bat cov (reviewed strain hku3-1). therefore, we report here only the results of disorder analysis for the orf10 protein from sars-cov-2, according to which this protein has a mean ppid of 0.00% (see also figure 15 for disorder profile of orf10). this protein contains a morf from 3-7 residues at its n-terminus as predicted by morfchibi_web. further, we found its tendency to nucleotides and found the presence of few rna binding sites, however, it does not contain dna binding residues. protein orf14. this is a 70-amino-acid-long uncharacterized protein of unknown function, which is present in human sars and bat cov. in sars-cov-2, orf14 is a 73-amino-acidlong protein. according to the msa, orf14 of sars-cov-2 has 77.1% identity with human-sars and 72.9% identity with bat cov as represented in figure 16d . we have performed the intrinsic disorder analysis to see the peculiarities of the distribution of disorder predisposition in this protein. figures 16a, 16b, and 16c show the resulting disorder profiles of orf14 of sars-cov-2, human sars cov, and bat cov. although these proteins have calculated mean ppid values of 0.00%, 2.86%, and 0.00% respectively, figure 16 shows that they have flexible n-and c-terminal regions. this protein can use intrinsic disorder or structural flexibility for protein-protein interactions since it possesses morfs. it mainly contains morfs at n-and c-terminal regions as tabulated in (table 2, supplementary table 7 and 8). it was also found to contain several rna and dna binding residues (supplementary table 9, 10, and 11) . these results indicating its vital role in protein function related to molecular recognition such as protein-protein, protein-rna, and protein-dna interaction. in coronaviruses, due to ribosomal leakage during translation, two-third of the rna genome is processed into two polyproteins: (i) replicase polyprotein 1a and (ii) replicase polyprotein 1ab. both contain non-structural proteins (nsp1-10) in addition to different proteins required for viral replication and pathogenesis. replicase polyprotein 1a contains an additional nsp11 protein of 13 amino acids, the function of which is not investigated yet. the longer replicase polyprotein 1ab of 7073 amino acids accommodates five other non-structural proteins (nsp12-16) [122] . these proteins assist in er membrane-induced vesicle formation, which acts as sites for replication and transcription. in addition to this, non-structural proteins work as proteases, helicases, and mrna capping and methylation enzymes, crucial for virus survival and replication inside host cells [122, 123] . global analysis of intrinsic disorder in the replicase polyprotein 1ab table 3 represents the ppid mean scores of 15 non-structural proteins (nsps) derived from the replicase polyprotein 1ab in sars-cov-2, human sars cov, and bat cov. these values were obtained by combining the results from six disorder predictors (see supplementary table s4-s6) . figures 17a, 17b , and 17c represent the 2d-disorder plots of the nsps coded by orf1ab in sars-cov-2, human sars cov, and bat cov, respectively. based on the mean ppid scores in table 3 , figures 17a, 17b, 17c , and taking into ppid based classification [46] , we conclude that none of the nsps in sars-cov-2, human sars cov, and bat cov are highly disordered. the highest disorder was observed for nsp8 proteins in all three coronaviruses. both nsp1 and nsp8 are moderately disordered proteins (10% ≤ ppid ≤ 30%). we also observed that nsp2, nsp3, nsp5, nsp6, nsp7, nsp9, nsp10, nsp15, and nsp16 have less than 10% disordered residues and hence, belong to the category of mostly ordered proteins. other non-structural proteins, namely, nsp4, nsp12, nsp13, and nsp14 have negligible levels of disorder (ppid < 1%), which tells us that these are highly structured proteins. the ch-cdf analysis of the nsps from sars-cov-2, human sars and bat cov have been represented in figures 17d, 17e , and 17f respectively. it was observed that all the nsps of the three coronaviruses are located within the quadrant q1 of the ch-cdf phase space, indicating that all the nsps are predicted to be mostly ordered. replicase polyprotein 1ab. the longer replicase polyprotein 1ab is a 7,073 amino acid-long polypeptide, which contains 15 non-structural proteins listed in table 3 . nsp1, nsp2, and nsp3 are cleaved using a viral papain-like proteinase (nsp3/pl-pro), while the rest of nsps are cleaved by another viral 3c-like proteinase, nsp5/3cl-pro. we mapped the cleavage sites of the replicase 1ab polyprotein from human sars cov to the disorder profile of this polyprotein. figure 18 represents the results of this analysis by showing zoomed-in regions surrounding all the cleavage sites with few residues spanning at both terminals. interestingly, we observed that all the cleavage sites are largely disordered, suggesting that intrinsic disorder may have a crucial role in the maturation of individual non-structural proteins. as the nsps of human sars cov are evolutionary close to the nsps of sars-cov-2, we hypothesize that the cleavage sites in the sars-cov-2 replicase 1ab polyprotein are also intrinsically disordered or flexible. to shed more light on other implications of idprs, the structural and functional properties of nsps and their predicted idprs are thoroughly described below. this protein acts as a host translation inhibitor as it binds to the 40s subunit of the ribosome and blocks the translation of cap-dependent mrnas as well as mrnas that uses the internal ribosome entry site (ires) [124] . figure 19d shows the nmr solution structure (pdb id: 2gdt) of human sars nsp1 protein (13-128 residues), whereas residues 117-180 were not included in this structural analysis [125] . sars-cov-2 nsp1 shares 84.44% and 83.80% sequence identity with nsp1s of human sars cov and bat cov, respectively. its n-terminal region is found to be more conserved than the rest of the protein sequence ( figure 19e ). mean ppids of nsp1s from sars-cov-2, human sars cov, and bat cov are 12.78%, 14.44%, and 12.85%, respectively. figure 19a , 19b, and 19c represent the graphs of predicted per-residue intrinsic disorder propensity of these nsp1s. according to the analysis, the following regions are predicted to be disordered: sars-cov-2 (residues 1-7 and 165-180), human sars cov (residues 1-5 and 165-180), and bat cov (residues 1-5 and 165-179). nmr solution structure of nsp1 from human sars revealed the presence of two unstructured segments near the n-terminal (1-12 residues) and c-terminal (129-179 residues) regions [125] . the disordered region residues) at c-terminus is important for nsp1 expression [126] . based on sequence homology with human sars cov nsp1, the predicted disordered c-terminal region of sars-cov-2 nsp1 may play a critical role in its expression. alanine mutants at k164 and h165 in the cterminal region of nsp1 protein is reported to abolish its binding with the 40s subunit of the host ribosome [127] . in conjunction with this data, several morfs are present in the unstructured segments of nsp1 proteins. these regions are tabulated in table 2, and supplementary tables 7 and 8 . this protein functions by disrupting the host survival pathway via interaction with the host proteins prohibitin-1 and prohibitin-2 [128] . reverse genetic deletion in the coding sequence of nsp2 of the sars virus attenuated little viral growth and replication and allowed the recovery of mutant virulent viruses. this indicates the dispensable nature of the nsp2 protein for sars viruses [129] . the sequence identity of the nsp2 protein from sars-cov-2 with nsp2s of human sars cov and bat cov amounts to 68.34% and 68.97%, respectively (supplementary figure s2a) . we have estimated the mean ppids of nsp2s of sars-cov-2, human sars cov, and bat cov to be 5.17%, 2.04%, and 2.03% respectively (see table 3 ). the per-residues predisposition for the intrinsic disorder of nsp2s from sars-cov-2, human sars cov, and bat cov are depicted in figures 20a, 20b , and 20c. according to this analysis, the following regions in nsp2 proteins are predicted to be disordered, residues 570-595 (sars-cov-2), residues 110-115 (human sars), and residues 112-116 (bat cov). as listed in table 2 , and supplementary tables 7 and 8, human sars cov does not contain morf while sars-cov-2 and bat cov have an n-terminally located morf region predicted by morfchibi_web. nsp3 is an almost 2,000-residue-long viral papain-like protease (plp) that affects the phosphorylation and activation of irf3 and therefore antagonizes the ifn pathway [130] . it was also demonstrated that nsp3 works by stabilizing nf-inhibitor further blocking the nf-pathway [130] . figure 21d represents the 1.85 å resolution x-ray crystal structure of the catalytic core of nsp3 protein from human sars cov (pdb id: 2fe8), which was obtained by andrew and colleagues [131] . this structure consists of the residues 723-1036 of nsp3. the structure revealed folds similar to a deubiquitinating enzyme in-vitro deubiquitinating activity of which was found to be efficiently high [131] . nsp3 protein of sars-cov-2 contains several substituted residues throughout the protein. it is equally close with both nsp3 proteins of human sars and bat cov sharing respective 76.69% and 76.31% identity (supplementary figure s2b) . according to our results, the mean ppids of nsp3 proteins of sars-cov-2, human sars, and bat cov are 7.40%, 7.91%, and 7.78% respectively ( table 3) . graphs in figures 21a, 21b , and 21c portray the tendency of nsp3 proteins of sars-cov-2, human sars, and bat cov for the intrinsic disorder. nsp3 proteins of all three studied sars viruses were found to be highly structured and characterized by rather similar disorder profiles. this is further supported by figure 21e , where pondr ® vsl2-generated disorder profiles of these three proteins are overlapped to show almost complete coincidence of their major disorder-related features. according to the mean disorder analysis (see figures 21a, 21b, and 21c) , nsp3 proteins are predicted to have the following idprs, sars-cov-2 (1-5, 105-199, 1221-1238), human sars (102-189, 355-384, 1195-1223) and bat cov (107-182, 352-376, 1191-1217) . the first 112 residues in nsp3 represent a ubiquitin-like globular fold while 113-183 residues form the flexible acidic domain rich in glutamic acid. it is thought to bind and ubiquitinate viral e protein using the n-terminal acidic domain [132, 133] . this unstructured segment has many morfs predicted by anchor and morfpred servers which may facilitate the protein-protein interaction ( table 2) . interestingly, nsp3 of all three viruses was found with highest number of rnabinding residues (supplementary tables 9, 10, and 11) . nsp4 has been reported to induce the formation of the double-membrane vesicles (dmvs) with the co-expression of full-length nsp3 and nsp6 proteins for optimal replication inside host cells [134] [135] [136] . it localizes itself in ermembrane, when expressed alone but is demonstrated to be present in replication units in infected cells. it was observed that nsp4 protein contains a tetraspanning transmembrane region having its n-and c-terminals in the cytosol [137] . no crystal or nmr solution structure is reported for this protein as of yet. nsp4 protein of sars-cov-2 has multiple substitutions near the n-terminal region and has a quite conserved c-terminus (supplementary figure s2c) . it is found to be closer to nsp4 of bat cov (81.40% identity) than to human sars nsp4 (80%). mean ppids of nsp4s from sars-cov-2, human sars, and bat cov are estimated to be 0.80%, 0.60%, and 0.60% respectively. the low level of intrinsic disorder is further illustrated by figures 22a, 22b , and 22c. with ppids around zero, nsp4 were classified as highly structured proteins, which, however, contain some flexible regions. likewise, table 2 shows the presence of only nand c-terminal morfs which possibly assist in cleavage of nsp4 protein from long polyproteins 1a and 1ab. also referred to as 3cl-pro, nsp5 works as a protease that cleaves the replicase polyproteins (1a and 1ab) at 11 major sites [138, 139] . x-ray crystal structure with 1.5 å resolution (pdb id: 5c5o) obtained for human sars cov nsp5 is shown in figure 23d . here, 3cl-protease is bound to a phenyl-beta-alanyl (s, r)-n-declin type inhibitor. another crystal structure resolved to 1.96 å revealed a chymotrypsin-like fold and a conserved substrate-binding site connected to a novel α-helical fold [140] . recently, the x-ray crystal structure (resolution 2.16 å) was solved for the sars-cov-2 nsp5 in complex with an inhibitor n3 (pdb id: 6lu7) ( figure 23e ). nsp5 protein is found to be highly conserved in all three studied cov viruses. sars-cov-2 nsp5 shares a 96.08% sequence identity with human sars nsp5 and 95.42% with nsp5 of bat cov (supplementary figure s2d) . therefore, it not surprising that our analysis demonstrated the identical mean ppid values of 1.96% for nsp5s from sars-cov-2, human sars, and bat cov ( table 3) . the predicted per-residue intrinsic disorder propensity of sars-cov-2, human sars, and bat cov nsp5s are presented in figures 23a, 23b , and 23c, respectively. as the graphs depict, nsp5s have several flexible regions and n-terminally idpr of six residues. due to the low flexibility of this protein, a single morf predicted by morfchibi_web is present in the n-terminal region (residues 3-8) in nsp5s of all three viruses (table 2, supplementary tables 7 and 8) . further, the identified nucleotide-binding residues in nsp5 of all three viruses are tabulated in supplementary tables 9, 10, and 11 . non-structural protein 6 (nsp6). nsp6 protein is involved in blocking er-induced autophagosome/autolysosome vesicle formation that functions in restricting viral production inside host cells. it induces autophagy by activating the omegasome pathway, which is normally utilized by cells in response to starvation. sars nsp6 leads to the generation of small autophagosome vesicles thereby limiting their expansion [141] . nsp6 of sars-cov-2 is equally close to nsp6s from both human sars and bat cov, having a sequence identity of 87.24% ( figure 24d ). according to our analysis, mean ppids for nsp6s are calculated to be 1.03%, 1.03%, and 1.03% for sars-cov-2, human sars cov, and bat cov, respectively. figures 24a, 24b, and 24c show the corresponding graphs of intrinsic disorder tendency of nsp6s from sars-cov-2, human sars cov, and bat cov and demonstrate that these proteins are highly ordered and show low flexibility. as it is a membrane protein, nsp6 proteins are predicted to have only a single morf near the nterminal region (residues 1-19 in sars-cov-2, residues 1-22 in human sars, and residues 1-21 in bat cov) by the disopred3 server (table 2, supplementary tables 7 and 8) . the role of these protein-binding regions for the induction of autophagy is need to be elucidated. nsp7 and 8) . the ~10 kda nsp7 helps in primaseindependent de novo initiation of viral rna replication by forming a hexadecameric ring-like structure with nsp8 protein [142, 143] . both non-structural proteins 7 and 8 contribute 8 molecules to the ring-structured multimeric viral rna polymerase. site-directed mutagenesis in nsp8 revealed a d/exd/e motif essential for the in vitro catalysis [142] . figure 25d depicts the 3.1 å resolution electron microscopy-based structure (pdb id: 6nur) of the rdrp-nsp8-nsp7 complex bound to the nsp12. the structure identified conserved neutral nsp7 and nsp8 binding sites overlapping with finger and thumb domains on nsp12 of the virus [144] . we found that nsp7 of sars-cov-2 share 100% sequence identity with nsp7 of bat cov and 98.80% with nsp7 from human sars (figure 25e) , while sars-cov-2 nsp8 is closer to nsp8 of human sars (97.47%) than to nsp8 of bat cov (96.46%) ( figure 26d ). due to the high levels of sequence identity, mean ppids of all nsp7s were found to be identical and equal to 9.64%. both sars-cov-2 and human sars nsp8 proteins were calculated to have a mean ppid of 23.74% and, for nsp8 of bat cov mean disorder is predicted to be 22.22%. figures 25a, 25b , and 25c display the intrinsic disorder profiles for nsp7s, whereas figures 26a, 26b , and 26c represent the predicted intrinsic disorder propensity of nsp8s. as our analysis suggests, nsp7s might have a well-predicted structure, while nsp8s are moderately disordered. nsp8s are predicted to have a long idpr (residues 44-84) in both sars-cov-2 and human sars, and a bit shorter idpr in bat cov (residues . furthermore, sars-cov nsp7 using its n-terminus residues (v11, c13, v17, and v21) forms a hydrophobic core with nsp8 residues (m92, m95, l96, m99, and l103). additionally, h-bonding takes place between nsp7 q24 and nsp8 t89 residues [143] . these amino acids are the part of morfs predicted in nsp7 and nsp8 proteins. the results are tabulated in both supplementary tables 9, 10 , and 11). nsp9 protein is a single-stranded rna-binding protein [145] . it might protect rna from nucleases by binding and stabilizing viral nucleic acids during replication or transcription [145] . our results on nucleotide-binding tendency of nsp9 shows the presence of several rna binding and few dna binding residues in nsp9 of sars-cov-2, human sars, and bat cov (supplementary tables 9, 10, and 11) . presumed to evolve from a protease, nsp9 forms a dimer using its gxxxg motif [146, 147] . figure 27d shows a 2.7 å crystal structure of the homodimer of human sars nsp9 (pdb id: 1qz8) that identified a unique and previously unreported for other proteins, oligosaccharide/oligonucleotide fold-like fold [145] . here, each monomer contains a coneshaped β-barrel and a c-terminal α-helix arranged into a compact domain [145] . nsp9 of sars-cov-2 is equally similar to nsp9s from both human sars and bat cov, having a percentage identity of 97.35%. the difference in three amino acids at 34, 35 and 48 positions accounts for these similarity scores ( figure 27e ). as calculated, the mean ppids of nsp9s of sars-cov-2, human sars cov, and bat cov are 7.08%, 7.96%, and 7.08% respectively. figures 27a, 27b , and 27c depict the predicted intrinsic disorder propensity in the nsp9 protein from sars-cov-2, human sars, and bat cov. according to our analysis, all three nsp9s are rather structured but contain flexible regions. nsp9 contains conserved residues (r10, k52, y53, r55, r74, f75, k86, y87, f90, k92, r99, and r111) of positively charged side chains suitable for binding with the negatively charged phosphate backbone of rna and aromatic side-chain amino acids providing stacking interactions [145] . these residues are a part of multiple disorder-based binding sites predicted by morfchibi_web server ( table 2, supplementary table 7 and 8) . nsp10 performs several functions for sars-cov. it forms a complex with nsp14 for dsrna hydrolysis in 3′ to 5′ direction and activates its exonuclease activity [148] . it also stimulates the methyltransferase (mtase) activity of nsp14 required during rna-cap formation after replication [149] . figure 28d represents the x-ray crystal structure of the nsp10/nsp14 complex (pdb id: 5c8t) [150] . in agreement with the results of previous biochemical experimental studies, the structure identified important interactions with the exon (exonuclease domain) of nsp14 without affecting its n7-mtase activity [148, 149] . sars-cov-2 nsp10 protein is quite conserved having a 97.12% sequence identity with nsp10 of human sars and 97.84% with nsp10 of bat cov (figure 28e) . mean ppids of all three studied nsp10 proteins are found to be 5.04%. figures 28a, 28b , and 28c represent disorder profiles of nsp10s and signify the lack of long idprs but presence flexible regions in these proteins. furthermore, [11] [12] [13] [14] [15] [16] [17] [18] was predicted by morfpred server. interestingly, the sars-cov nsp10 residues f16, f19, and v21 form van der waals interactions with many of the nsp14 amino acids [150] and one residue (f16) is located in morf region which we have identified. furthermore, many nucleotide-binding residues which are found in all three viruses (supplementary table 9 , 10, and 11) and above-mentioned residues are not found to interact with dna/rna. in coronaviruses, nsp12 is an rna-dependent rna polymerase (rdrp). it carries out both primer-independent and primer-dependent synthesis of viral rna with mn 2+ as its metallic co-factor and viral nsp7 and 8 as protein co-factors [151] . as aforementioned, a 3.1å resolution structure of human sars nsp12 in association with nsp7 and nsp8 proteins (pdb id: 6nur) has been reported using electron microscopy ( figure 25d ). nsp12 has a polymerase domain similar to "right hand", finger domain (398-581, 628-687 residues), palm domain (582-627, 688-815 residues) and a thumb domain (816-919) [144] . sars-cov-2 nsp12 protein has a highly conserved c-terminal region (supplementary figure s2e ). it is found to share a 96.35% sequence identity with human sars nsp12 and 95.60% with bat cov nsp12. mean ppid values for all three nsp12s are estimated to be 0.43% (table 3) . figures 29a, 29b , and 29c show that although these proteins are mostly ordered, they have multiple flexible regions. as rdrp protein is observed to be mostly structured, significant morfs in disordered regions are not found ( table 2, supplementary table 7 and 8). nsp13 functions as a viral helicase and unwinds dsdna/dsrna in 5' to 3' direction [152] . recombinant viral helicase expressed in e.coli rosetta 2 strain was reported to unwind ~280 bp per second [152] . figure 30d represents a 2.8 å x-ray crystal structure of human sars nsp13 (pdb id: 6jyt) [153] . this helicase contains a 19-20 loop on 1a domain, which is primarily responsible for its unwinding activity. furthermore, the study revealed an important interaction of nsp12 with nsp13 that further enhances its helicase activity [153] . the 601-amino-acid-long nsp13 of sars-cov-2 is almost completely conserved, as it shares 99.83% with nsp13 of humans sars and 98.84% with nsp13 of bat cov (supplementary figure s2f) . in accordance with our results, the mean ppids of all three nsp13 proteins are estimated to be 0.67%. figures 30a, 30b , and 30c show that nsp13s contain multiple flexible regions but do not possess significant disorder. as expected, being a low disorder protein nsp13 does not contain any morf region and not a single bindingregion is located by any server used in all three viruses ( table 2, supplementary table 7 and 8). it has many nucleotide-binding residues (rna and dna) which are tabulated in supplementary tables 9, 10, and 11. nsp14 is a multifunctional viral protein that acts as an exoribonuclease (exon) and methyltransferase (n7-mtase) in sars coronaviruses. it's 3' to 5' exonuclease activity lies in the conserved dedd residues related to exonuclease superfamily [154] . its guanine-n7 methyltransferase activity depends upon the s-adenosyl-lmethionine (adomet) as a cofactor [149] . as aforementioned, nsp14 requires nsp10 for activating its exon and n7-mtase activity inside the host cells. figure 28d depicts the 3.2 å crystal structure of human sars nsp10/nsp14 complex (pdb id: 5c8t), where amino acids 1-287 form the exon domain and 288-527 residues form the n7-mtase domain of nsp14. a loop (residues 288-301) is essential for its n7-mtase activity [150] . figure s2g) . mean ppid values for nsp14s from sars-cov-2 and human sars is calculated to be 0.38%, while the nsp14 from bat cov has a mean ppid 0.57%. predicted per-residue intrinsic disorder propensity of nsp14s from sars-cov-2, human sars, and bat cov is represented in figures 31a, 31b , and 31c, respectively. as can be observed from these plots and corresponding ppid values, all nsp14s are found to be highly structured. likewise, table 2 shows nsp14 contains two protein binding regions (residues 8-13 and 441-445) predicted by the morfpred server in all three viruses. as shown in supplementary tables 9, 10, and 11, the orf14 represents multiple nucleotide-binding residues. nsp15 is a uridylate-specific rna endonuclease (nendou) that creates a 2′-3′ cyclic phosphates after cleavage. its endonuclease activity depends upon mn 2+ ions as co-factors. conserved in nidovirus, it acts as an important genetic marker due to its absence in other rna viruses [155] . figure 32d represents a 2.6 å crystal structure of uridylate-specific nsp15 (pdb id: 2h85) that was deduced by bruno and colleagues using x-ray diffraction [156] . the monomeric nsp15 has three domains: nterminal domain (1-62 residues) formed by a three anti-parallel -strands and two α-helices packed together; a middle domain residues) that contains an α-helix connected via a 39-amino-acid-long coil to an ordered region containing two α-helices and five -strands; and a c-terminal domain (192-345 residues) consisting of two anti-parallel three -strand sheets on each side of a central α-helical core [156] . the nsp15 is found to be quite conserved across human sars and bat covs. sars-cov-2 nsp15 shares an 88.73% sequence identity with nsp15 of human sars and 88.15% with nsp15 of bat cov (supplementary figure s2h) . calculated mean ppids of nsp15s from sars-cov-2, human sars, and bat cov are 1.73%, 2.60%, and 2.60%, respectively. similar to many other non-structural proteins of coronaviruses, nsp15s from sars-cov-2, human sars, and bat cov are predicted to possess multiple flexible regions but contain virtually no idprs (see figures 32a, 32b, and 32c) . similarly, no significant disorderbinding regions are predicted in nsp15 proteins ( table 2) . sars-cov-2 contain one morf (residues 9-13) predicted by morfpred server. human sars do not have a single morf while bat cov possesses two very short binding regions (supplementary table 7 and 8). supplementary table 9 , 10, and 11 depicts the presence of many rna binding residues and few dna binding residues in nsp15 of all three viruses. nsp16 protein is another mtase domain-containing protein. as methylation of coronavirus mrnas occurs in steps, three proteins nsp10, nsp14, and nsp16 acts one after another. the first event requires the initiation trigger from nsp10 protein, after which nsp14 methylates capped mrnas forming cap-0 (7me) gpppa-rnas. nsp16 protein, along with its co-activator protein nsp10, acts on cap-0 (7me) gpppa-rnas to give rise to final cap-1 (7me)gpppa(2'ome)-rnas [149, 157] . a 2 å x-ray crystal structure of the human sars nsp10-nsp16 complex is depicted in figure 33d (pdb id: 3r24) [158] . the structure consists of a characteristic fold present in class i mtase family comprising of α-helices and loops surrounding a seven-stranded β-sheet [158] . nsp16 protein of sars-cov-2 is found to be equally similar to nsp16s from human sars and bat cov (93.29%) (supplementary figure s2i) . mean ppids for nsp16s from sars-cov-2, human sars, and bat cov are 5.37%, 3.02%, and 3.02%, respectively. in line with these ppids values, figures 33a, 33b, and 33c show that nsp16s are mostly ordered proteins containing several flexible regions. correspondingly, no significant morfs are present in this protein ( table 2, supplementary table 7 and 8). a single morf (residues [151] [152] [153] [154] [155] [156] were found with the help of morfpred in all three viruses. further, several rnabinding and few dna-binding residues are also identified (supplementary table 9 , 10, and 11). replicase polyprotein 1a. since replicase polyprotein 1a contains non-structural proteins 1-10 identical to those found in replicase polyprotein 1ab, we did not perform their disorder analysis separately. however, replicase polyprotein 1a has one additional non-structural protein designated as nsp11. nsp11 is a small uncharacterized protein cleaved from the replicase polyprotein 1a. this small protein with unknown function requires experimental insights to further characterize this protein. the intrinsic disorder predicting software used in this study requires amino acid sequences, which are at least 30-residue long. therefore, because of their short sequences (just 13 residues) nsp11s from all three studied coronaviruses were not checked for the intrinsic disorder, disorder-based protein binding regions, and nucleotide-binding residues. based on the msa outputs, nsp11 from sars-cov-2 was found to have a sequence identity of 84.62% with nsp11s from human sars and bat cov (figure 34 ). the emergence of new viruses and associated deaths around the globe represent one of the major concerns of modern times. despite its pandemic nature, there is very little information available in the public domain regarding the structures and functions of sars-cov-2 proteins. based on its similarity with human sars cov and bat cov, the published reports have suggested the functions of sars-cov-2 proteins. in this study, we utilized information available on sars-cov-2 genome and translated proteome from genbank, and carried out a comprehensive computational analysis of the prevalence of the intrinsic disorder in sars-cov-2 proteins. additionally, a comparison was also made with proteins from close relatives of sars-cov-2 from the same group of beta coronaviruses, human sars cov and bat cov. our analysis revealed that in these three covs, the n proteins are highly disordered, possessing the ppid values of more than 60%. these viruses also have several moderately disordered proteins, such as nsp8, orf6, and orf9b. although other proteins have shown lower disorder content, almost all of them contain at least some idprs, and all cov proteins analysed in this study definitely have multiple flexible regions. importantly, our study provides novel information on presence of intrinsic disorder at the cleavage sites of the replicase 1ab polyprotein of covs. this observation confirms the crucial role of idprs in maturation of individual proteins. we also established that many of these proteins contain disorder-based binding motifs. since idps/idprs might undergo structural transition upon association with their physiological partners, our study generates important grounds for better understanding of the functionality of these proteins, their interactions with other viral proteins, as well as interaction with host proteins in different physiological conditions. this will also guide structural biologists to carry out a structure-based analysis of sars-cov-2 proteome to explore the path for the development of new drugs and vaccines. the periodical outbreaks of pathogens worldwide always remind the lack of suitable drugs or vaccines for proper cure or treatment. in 2003, nearly 750 deaths were reported due to the sars outbreak in more than 24 countries. but this time, the outbreak of wuhan's novel coronavirus (sars-cov-2) has quickly surpassed this number, indicating more causalities soon. the lack of accurate information and ignorance of primary symptoms are major reasons, which cause many infection cases. although efficient transmission from human to human is confirmed, the actual reasons for fast sars-cov-2 spread are still unknown, but some assumptions were made by researchers and chinese authorities. the fast spread of sars-cov-2, covid-19 pandemic, and associated introduction of quarantine also have made major impacts on economy and education worldwide due to several restrictions, such as limited transportation, restrained or frozen traveling, halted attendance of mass events, the introduction of distant teaching and learning, etc. due to advancements in sequencing techniques, the full genome sequence of sars-cov-2 was made available in a few days of the first infection report from wuhan, china. however, massive subsequent research needs to be done to identify the actual cause of sars-cov-2 infectivity and to design suitable treatment in the coming future. certain possibilities can be explored with the available information. the mutational pressure study on this virus will be very interesting to see if this virus transforms from bat sars to human sars to sars-cov-2. more in-depth experimental studies using molecular and cell biology techniques to establish structurefunction relationships are required for a better understanding of the functioning of sars-cov-2 proteins. additionally, based on the sequence homology and information on proteinprotein interactions, the associated viral and host proteins should be explored, for finding means suitable for limiting replication, maturation, and ultimately pathogenesis of this virus. although structural biology techniques (so-called rational drug design) can be used in drug development utilizing high throughput screening of compounds virtually or experimentally, the applicability of these techniques is limited by the presence of intrinsic disorder in target proteins. therefore, the thorough disorder analysis of three coronaviruses conducted in this study will help structural biologists to rationally design experiments keeping this information in mind. authors contribution: rg: conception and design, interpretation of data, writing, and review of the manuscript, and study supervision. vnu: conception and design, acquisition and interpretation of data, writing, and review of the manuscript. ms, tb, pk, brg, kg: acquisition and interpretation of data, writing of the manuscript. table 6 . evaluation of intrinsic disorder in non-structural proteins of bat cov. table 7 : predicted morf residues in human sars proteins. supplementary table 8 : predicted morf residues in bat cov proteins. supplementary table 9 : predicted nucleotide-binding residues in sars-cov-2 proteins. supplementary table 10 : predicted nucleotide-binding residues in human sars proteins. supplementary table 11 : predicted nucleotide-binding residues in bat cov proteins. supplementary figures s1. multiple sequence alignment of structural proteins of all three studied coronaviruses are generated using clustal omega. the aligned images are created using esprit 3.0. figure s1a . msa of sars-cov-2, human sars, and bat cov spike glycoproteins. figure s1b . msa of sars-cov-2, human sars, and bat cov nucleoproteins. supplementary figure s2 . multiple sequence alignment of non-structural proteins of all three studied coronaviruses are generated using clustal omega. the aligned images are created using esprit 3.0. figure s2a . msa of sars-cov-2, human sars, and bat cov nsp2 proteins. figure s2b . msa of sars-cov-2, human sars, and bat cov nsp3 proteins. figure s2c . msa of sars-cov-2, human sars, and bat cov nsp4 proteins. figure s2d . msa of sars-cov-2, human sars, and bat cov nsp5 proteins. figure s2e . msa of sars-cov-2, human sars, and bat cov nsp12 proteins. figure s2f . msa of sars-cov-2, human sars, and bat cov nsp13 proteins. figure s2g . msa of sars-cov-2, human sars, and bat cov nsp14 proteins. figure s2h . msa of sars-cov-2, human sars, and bat cov nsp15 proteins. figure s2i . msa of sars-cov-2, human sars, and bat 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spike protein the role of severe acute respiratory syndrome (sars)-coronavirus accessory proteins in virus pathogenesis mitochondrial location of severe acute respiratory syndrome coronavirus 3b protein nucleolar localization of nonstructural protein 3b, a protein specifically encoded by the severe acute respiratory syndrome coronavirus sars-cov accessory protein 3b induces ap-1 transcriptional activity through activation of jnk and erk pathways g0/g1 arrest and apoptosis induced by sars-cov 3b protein in transfected cells over-expression of severe acute respiratory syndrome coronavirus 3b protein induces both apoptosis and necrosis in vero e6 cells the 3a protein of severe acute respiratory syndrome-associated coronavirus induces apoptosis in vero e6 cells severe acute respiratory syndrome coronavirus open reading frame (orf) 3b, orf 6, and nucleocapsid proteins function as interferon antagonists severe acute respiratory syndrome coronavirus orf6 antagonizes stat1 function by sequestering nuclear import factors on the rough endoplasmic reticulum/golgi membrane enhancement of murine coronavirus replication by severe acute respiratory syndrome coronavirus protein 6 requires the n-terminal hydrophobic region but not c-terminal sorting motifs a putative diacidic motif in the sars-cov orf6 protein influences its subcellular localization and suppression of expression of cotransfected expression constructs the n-terminal region of severe acute respiratory syndrome coronavirus protein 6 induces membrane rearrangement and enhances virus replication characterization of a unique group-specific protein (u122) of the severe acute respiratory syndrome coronavirus severe acute respiratory syndrome coronavirus 7a accessory protein is a viral structural protein augmentation of chemokine production by severe acute respiratory syndrome coronavirus 3a/x1 and 7a/x4 proteins through nf-kappab activation chemokine upregulation in sars-coronavirus-infected, monocyte-derived human dendritic cells induction of apoptosis by the severe acute respiratory syndrome coronavirus 7a protein is dependent on its interaction with the bcl-xl protein the orf7b protein of severe acute respiratory syndrome coronavirus (sars-cov) is expressed in virus-infected cells and incorporated into sars-cov particles 7a protein of severe acute respiratory syndrome coronavirus inhibits cellular protein synthesis and activates p38 mitogen-activated protein kinase structure and intracellular targeting of the sars-coronavirus orf7a accessory protein solution structure of the x4 protein coded by the sars related coronavirus reveals an immunoglobulin like fold and suggests a binding activity to integrin i domains the 29-nucleotide deletion present in human but not in animal severe acute respiratory syndrome coronaviruses disrupts the functional expression of open reading frame 8 molecular evolution of the sars coronavirus during the course of the sars epidemic in china the human severe acute 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coronavirus the envelope protein of severe acute respiratory syndrome coronavirus interacts with the non-structural protein 3 and is ubiquitinated severe acute respiratory syndrome coronavirus nonstructural proteins 3, 4, and 6 induce double-membrane vesicles mobility and interactions of coronavirus nonstructural protein 4 two-amino acids change in the nsp4 of sars coronavirus abolishes viral replication localization and membrane topology of coronavirus nonstructural protein 4: involvement of the early secretory pathway in replication ligand-induced dimerization of middle east respiratory syndrome (mers) coronavirus nsp5 protease (3clpro): implications for nsp5 regulation and the development of antivirals a novel mutation in murine hepatitis virus nsp5, the viral 3c-like proteinase, causes temperature-sensitive defects in viral growth and protein processing structure of coronavirus main proteinase reveals combination of a chymotrypsin fold with an extra alpha-helical domain coronavirus nsp6 restricts autophagosome expansion the sars-coronavirus nsp7+nsp8 complex is a unique multimeric rna polymerase capable of both de novo initiation and primer extension insights into sars-cov transcription and replication from the structure of the nsp7-nsp8 hexadecamer structure of the sars-cov nsp12 polymerase bound to nsp7 and nsp8 co-factors the severe acute respiratory syndrome-coronavirus replicative protein nsp9 is a singlestranded rna-binding subunit unique in the rna virus world variable oligomerization modes in coronavirus non-structural protein 9 severe acute respiratory syndrome coronavirus nsp9 dimerization is essential for efficient viral growth rna 3'-end mismatch excision by the severe acute respiratory syndrome coronavirus nonstructural protein nsp10/nsp14 exoribonuclease complex in vitro reconstitution of sars-coronavirus mrna cap methylation structural basis and functional analysis of the sars coronavirus nsp14-nsp10 complex biochemical characterization of a recombinant sars coronavirus nsp12 rna-dependent rna polymerase capable of copying viral rna templates mechanism of nucleic acid unwinding by sars-cov helicase delicate structural coordination of the severe acute respiratory syndrome coronavirus nsp13 upon atp hydrolysis discovery of an rna virus 3'->5' exoribonuclease that is critically involved in coronavirus rna synthesis major genetic marker of nidoviruses encodes a replicative endoribonuclease crystal structure and mechanistic determinants of sars coronavirus nonstructural protein 15 define an endoribonuclease family coronavirus nonstructural protein 16 is a cap-0 binding enzyme possessing (nucleoside-2'o)-methyltransferase activity biochemical and structural insights into the mechanisms of sars coronavirus rna ribose 2'-o-methylation by nsp16/nsp10 protein complex key: cord-324324-8ybfiz8f authors: decaro, nicola; lorusso, alessio title: novel human coronavirus (sars-cov-2): a lesson from animal coronaviruses date: 2020-04-14 journal: vet microbiol doi: 10.1016/j.vetmic.2020.108693 sha: doc_id: 324324 cord_uid: 8ybfiz8f the recent pandemic caused by the novel human coronavirus, referrred to as severe acute respiratory syndrome coronavirus 2 (sars-cov-2), not only is having a great impact on the health care systems and economies in all continents but it is also causing radical changes of common habits and life styles. the novel coronavirus (cov) recognises, with high probability, a zoonotic origin but the role of animals in the sars-cov-2 epidemiology is still largely unknown. however, covs have been known in animals since several decades, so that veterinary coronavirologists have a great expertise on how to face cov infections in animals, which could represent a model for sars-cov-2 infection in humans. in the present paper, we provide an up-to-date review of the literature currently available on animal covs, focusing on the molecular mechanisms that are responsible for the emergence of novel cov strains with different antigenic, biologic and/or pathogenetic features. a full comprehension of the mechanisms driving the evolution of animal covs will help better understand the emergence, spreading, and evolution of sars-cov-2. eighteen years after the emergence of severe acute respiratory syndrome (sars) in china and 8 years after the emergence of middle east respiratory syndrome (mers) in saudi arabia, a novel coronavirus (cov) epidemic, recently classified as pandemic by the who, is threatening the human population worldwide (zhou et al., 2020) . the disease, now referred to as coronavirus disease 2019 , is caused by a novel human cov, which was initially denominated 2019 novel coronavirus (2019-ncov) and later renamed as sars coronavirus 2 (sars-cov-2) the by coronavirus study group of the international committee on taxonomy of viruses (gorbalenya et al., 2020) . covid-19 emerged in december 2019 in wuhan city, hubei province, china, in humans exposed to wildlife at the huanan seafood wholesale market, which is the largest seafood market in central china, and where different species of farm and wild animals are commonly sold (lorusso et al., 2020) . the epidemic has then expanded not only to neighbouring asian countries, but also to other continents (https://www.who.int/ docs/default-source/coronaviruse/situation-reports/20200415-sitrep-86-covid-19.pdf?sfvrsn=c615ea20_2). . a list of human covs is showed in table 1 . historically, only two human covs (hcovs) had been known before the sars emergence, namely hcov-229e, an alphacoronavirus originated in bats and transmitted to humans through alpacas, and hcov-oc43, a betacoronavirus which had passed from rodents to humans through cattle (corman et al., 2015 (corman et al., , 2018 . after 2002-2003 sars epidemic, the renovated interest in hcovs allowed the discovery of two additional viruses, the alphacoronavirus hcov-nl63 and the betacoronavirus hcov-hku1, derived from bats and rodents, respectively (tao et al., 2017) . all these four viruses are usually responsible for mild respiratory symptoms in immunocompetent patients. sars-cov and mers-cov are two unrelated betacoronaviruses originated in bats and transmitted to humans by wild carnivores and dromedary camels, respectively. in contrast to other hcovs, these two viruses displayed an increased virulence, causing severe pneumonia and even the death of affected people, with mortality rates of about 10 % and 30 %, respectively (guarner, 2020) . the occurrence of three highly pathogenic covs with a zoonotic origin in less than two decades, highlights the role of animals in generating covs with increased virulence that can adapt to humans, causing epidemics (and eventually pandemics) with high impact on human health. indeed, cov infections of veterinary interest have been known since almost a century (cavanagh, 2007; pedersen, 2014; decaro et al., 2020) , so that animal covs are paradigmatic of how this large family of viruses evolves, generating strains with different biological properties. in addition, the efforts done in veterinary medicine https://doi.org/10.1016/j.vetmic.2020.108693 received 10 march 2020; received in revised form 10 april 2020; accepted 10 april 2020 evolve rapidly, changing their antigenic profile, tissue tropism or host range by means of two distinct mechanisms. the viral replicase (an rna dependent-rna polymerase) does not possess a good proof reading activity, therefore the incorporation of wrong nucleotides at each replication cycle and the consequent accumulation of mutations in the viral genome lead to a progressive differentiation of the viral progeny from the parental strain. this mechanism, which is well known for influenza viruses being responsible for the so called antigenic drift, may cause the progressive adaptation of the viral surface proteins to the cell receptors of new animal species, increasing the viral fitness. in addition, the particular replicating machinery of covs facilitates recombination events due to the presence of consensus sequences upstream each gene. therefore, in the case of coinfection by more than one cov strain, the rna polymerase can jump from the rna of a strain to that of the other one, synthetizing a hybrid rna containing sequences from both viruses. recombination can occur not only with genomic sequences of other covs (homologous recombination), but also with rnas of different viruses and other organisms (heterologous recombination) (luytjes et al., 1988; banner and lai, 1991; lai, 1996; zeng et al., 2008; huang et al., 2016) . recombination is an alternative mechanism that let covs acquire novel biological properties in terms of virulence, host range and tissue tropism, so that cov strains, which are non-pathogenic or lowpathogenic in the original host, may increase their pathogenicity in the same species or adapt to different species spreading in the new host with exceptional rapidity (banner and lai, 1991) . the occurrence of three human cov epidemics in less than 20 years, along with the emergence of less pathogenic human covs, arises some questions on how these viruses that have their reservoirs in bats and rodents may overcome the species barriers jumping to humans. the animal-to-human transmission of viruses has been already occurred in the past, but it seems that its frequency has been increased in the last decades, involving in a short time span not only covs, but also a plethora of genetically and biologically different viruses with zoonotic potential, such as ebola virus, influenza viruses, flaviviruses, hendra and nipah viruses (mcmahon et al., 2018) . climate changes that are intensifying in this first quarter of the 21st century are favouring the spread of vector-borne diseases through increasing the proliferation of vectors and predisposing to their occupation of new ecological niches. the emergence in temperate climate areas such as europe of vectorborne diseases caused by viruses considered exotic until few years ago (west nile virus, usutu virus, chikungunya virus) accounts for a progressive geographic expansion of tropical diseases thanks to the ongoing phenomenon of tropicalisation (mcmahon et al., 2018) . deforestation and urbanization are other major factors that facilitate the spill-over of zoonotic agents to humans by reducing the habitat of wildlife and increasing the chances of contacts between wild animals (like bats, rodents and birds) and human beings (beena and saikumar, 2019; lorusso et al., 2020) . this could be the case of ebola virus, hendra and nipah viruses, hantavirus and coronavirus infections. in addition, the close contact between human beings and different animal species sold at the wet markets of east asia represents the optimal situation for the host species jump and adaptation to humans of potentially zoonotic agents like covs. it is not a coincidence that two of the most severe zoonoses of the last two decades (highly pathogenic h5n1 avian influenza and sars) have emerged in the same chinese province of guangdong where the contact between humans and animals is closer (lorusso et al., 2020) . table 2 reports the most important avian cov species recognised so far and their associated diseases. the number of avian species in which covs have been detected in the last years is humongous. since the emergence of sars-cov in 2002, there has been increased interest in table 2 main coronaviruses in domestic and domesticated avian species. schalk and hawn (1931); beach and schalm (1936) ; beaudette and hudson (1937) turkey ( respiratory and kidney disease spackman et al. (1983) n. decaro and a. lorusso veterinary microbiology 244 (2020) 108693 covs in other species, including birds. prior to that time, our knowledge of covs in avian species was limited largely to three birds of the order galliformes, i.e., domestic fowl (gallus gallus), turkeys (genus meleagris) and pheasants (phasianidae), with their infectious bronchitis virus (ibv), turkey coronavirus (tcov), and pheasant coronavirus (phcov), respectively. these three viruses were considered for a long-time different species for several reasons such as the diverse pathotype (enterotropic or respirotropic), host range and genetic relatedness of the s protein (cavanagh, 2007) . this scenario radically changed after the discovery of several novel covs with high genetic diversity from different avian species and the novel rules for species designation of the coronavirus study group (csg https://talk.ictvonline.org/ictv-reports/ ictv_9th_report/positive-sense-rna-viruses-2011/w/posrna_viruses/ 222/coronaviridae). all these viruses as well as analogous ibv-like covs detected in other birds including penguins, pigeons, peafowl, parrots, waterfowl, teal, quail, duck and whooper swan (cavanagh et al., 2002; circella et al., 2007; domanska-blicharz et al., 2014; torres et al., 2013; hughes et al., 2009; liu et al., 2005; wille et al., 2016; jordan et al., 2015; bande et al., 2016; suryaman et al., 2019) have been assigned to the same viral species known as avian coronavirus (acov) within the subgenus igacovirus of genus gammacoronavirus. ibv and ibv-like strains are commonly detected in both gallinaceous and non-gallinaceous birds, also asymptomatically (cavanagh, 2005) . this might suggest that these species would act as wild reservoirs, spreading ibv strains over the world (de wit et al., 2011) . as for the huge economic impact of the disease it causes, ibv is one of the most studied covs over the last decades. ibv causes the infectious bronchitis (ib), a term adopted in 1931 for describing the main clinical characteristics of a transmissible respiratory disease of poultry detected for the first time in north dakota (usa). ib has been now diagnosed worldwide and is one of the most important viral diseases of poultry characterised by respiratory signs, but it can also affect the kidneys and reproductive tract following viremia with a severity that differs depending on the involved viral strain (cavanagh and gelb, 2008) . the disease also affects wild and ornamental birds chen et al., 2013) . ib control has been hampered by the intricate ibv evolution, which has been entailed, over the years, by the emergence of many different antigenic or genotypic types, commonly referred to as variants, with divergent molecular, biological, and antigenic properties. being a cov, ibv has, indeed, a considerable ability to change both by mutation and by homologous recombination events, which may cause, along with replicase stuttering or slippage, also insertions and deletions in the genome (cavanagh and gelb, 2008) . if these mechanisms involve the hypervariable region s1, they frequently result in the emergence of new ibv variants. although many new variants are not successful, a few may emerge, spread, causing devastating disease either worldwide or in limited geographic areas. currently 32 lineages have been recognized, categorized into six genotypes (gi to gvi) (valastro et al., 2016) . through their s protein, ibv and ibv-like viruses recognise as cellular host receptor the α2,3-linked sialic acid glycan, widely distributed in the respiratory tract and in several other host tissues, factor which may explain the tropism also for several organs of the infected host (winter et al., 2006 (winter et al., , 2008 shahwan et al., 2013; ambepitiya wickramasinghe et al., 2011) . extensive use of vaccines has greatly contributed to the high variability of ibv strains thorough recombination between vaccine and field viruses and viral selection pressure resulting from vaccination and presence of partially immune birds (gandon and day, 2008; gandon et al., 2001; bande et al., 2017) . important ibv-like strains are tcov, responsible for enteritis in turkey (also known as bluecomb disease), guinea fowl coronavirus (gfcov) and quail coronavirus (qcov) responsible for fulminating enteric disease in guinea fowl and quail, respectively cavanagh, 2005; liais et al., 2014) . tcov, gfcov and qcov are evolutionarily distant from acov based on the s protein. while ibv is a primarily respiratory pathogen, tcov causes gastrointestinal disease (liais et al., 2014; guy, 2008) . enterotropism has also been observed for some ibv serotypes; however, all ibv strains infect primarily the respiratory tract, resulting in mild to severe inflammation of the nasal and tracheal epithelia (cavanagh, 2005 (cavanagh, , 2007 . the s1 domain of the s protein is highly variable, with the amino acid sequences of ibv and tcov from the usa sharing < 25 % sequence identity. phylogenetic analysis of the s1 gene shows, indeed, grouping of ibv and ibv-like viruses on the one hand and tcov-us, gfcov and qcov on the other hand (ambepitiya wickramasinghe et al., 2015a) . accordingly, the emergence of covs in turkeys in the usa was proposed to have resulted from recombination events involving ibvs and an as-yet-unidentified cov donating a novel s gene. this switch contributed largely to determine the in vivo tissue tropism of tcov and related viruses. intriguingly, the s protein of these covs requires nonsialylated type 2 poly-lacnac structures on n-glycan cores for binding. this is in marked contrast to the α2,3-linked sialic acid glycan binding of ibv and ibv-like viruses (ambepitiya wickramasinghe et al., 2015b) . the s1 subdomain of a tcov isolate from france in 2008 (tcov-fr) had only 42 % sequence identity to that of the tcov-us strain (maurel et al., 2011) . this diversity was biologically evident by the prominent tropism for the epithelium of the bursa of fabricius and only mild tropism for the small intestine of turkey. tcov-fr s1 protein did not show, indeed, affinity for nonsialylated type 2 poly-lacnac (ambepitiya wickramasinghe et al., 2015a) . this genetic diversity between tcovs is in accordance with several recombination events involving ibvs on different continents with several unknown covs. on the one hand, the s genes of gfcov/fr/2011 (isolated in france in 2011) and tcov-us share significant genetic relationships, and thus these viruses must have acquired their s gene from a common ancestor. on the other hand, gfcov/fr/2011 and fr tcov have a very similar genetic background in other genes. two recombination events may be responsible for the genesis of tcov-us and fr tcov. a first event occurred between an ibv eu recipient strain and an unknown acov donor, resulting in a virus with a new s gene, whose evolution would have resulted in fr tcov and gfcov/fr/2011. a second recombination event involving a us ibv recipient and gfcov/fr/2011 would have generated us tcov viruses, which share a stronger s gene similarity with gfcov/fr/2011 than with fr tcov . additional covs distinct from acovs and mainly circulating in ducks (duck coronavirus, dcov), pigeons (pigeon coronavirus, pcov), or geese (goose coronavirus, gcov) have been identified (cheng et al., 2013; jonassen et al., 2005; muradrasoli et al., 2010; kim and oem, 2014; zhuang et al., 2015; papineau et al., 2019) . although their genome seems to fulfill the official ictv criteria required to distinguish a new species within the gammacoronavirus genus, ictv approval is still pending. historically, covs of birds were all included in the gammacoronavirus genus and, in turn, all covs belonging to this genus were identified only in birds. however, this suggestion was rebutted by the evidence of a cov belonging to the gammacoronavirus genus in a beluga whale first discovered in 2008 (viral species beluga whale coronavirus sw1 species, subgenus cegacovirus, genus gammacoronavirus) (mihindukulasuriya et al., 2008) , and of three novel covs, bucov hku1, thcov hku12, and mucov hku13 in birds of the order passeriformes, namely bulbuls (pycnonotus jocosus), thrushes (turdidae) and munias (lonchura punctulate), respectively, which did not cluster phylogenetically with extant covs identified in birds. these latter three viruses were distinct from known covs forming a unique cluster in the phylogenetic tree, which was the basis for generation of the deltacoronavirus genus (woo et al., 2009) . importantly, additional novel viruses belonging to this novel genus were detected in wild birds chu et al., 2011; durães-carvalho et al., 2015; torres et al., 2016) . these viruses cluster with previously unclassified covs detected in various asian carnivores, i.e., the asian leopard cat (prionailurus bengalensis) and chines ferret badger (nyctereutes procyonoides) (dong n. decaro and a. lorusso veterinary microbiology 244 (2020) 108693 et al., 2007) . covs belonging to the betacoronavirus genus, which are strictly related to mouse hepatitis virus (mhv), were also described in wild birds, including parrots, in brazil (durães-carvalho et al., 2015) . interestingly, this was not the first detection of viruses belonging to the betacoronavirus genus in birds. often overlooked is the discovery over 38 years ago of a cov from the manx shearwater (puffinus puffinus), a bird that visits the shores of britain in summer (nuttall and harrap, 1982; cavanagh et al., 2007) . this virus was also related to mhv. however, at that time, considering the unusual finding and that the virus was isolated by passage of shearwater material in the brains of mice, it was speculated that the detected virus was an mhv strain already present in the mice before inoculation (cavanagh, 2007) . bats are an ancient and heterogeneous group of ecologically important mammals, representing nearly a quarter of all mammalian diversity on earth. they belong to the order chiroptera and further classified in two suborders yinpterochiroptera and yangochiroptera. the first includes the non-echolocating pteropodidae family (megabats) and five echolocating rhinolophoidea microbat superfamilies. yangochiroptera contain thirteen echolocating microbat families (tsagkogeorga et al., 2013) . bats are thought to host a large plethora of viruses. these include, amongst the others, lyssaviruses, filoviruses, henipaviruses, and reoviruses (calisher et al., 2006) . before sars-cov epidemic, bats were not known to host covs. indeed, the first evidence of a bat cov was published in 2005 . after the sars epidemic, there was a boost in interest regarding searching for novel covs in various animals, including bats. to date, over 200 novel covs have been identified in bats and approximately 35 % of the bat virome sequenced to date is composed of covs (chen et al., 2014) . this data has been made available following the massive surveillance, coupled with the advent of next-generation sequencing (ngs) technology, which has been performed in wild animals banerjee et al., 2019) . just a small portion of these covs have been officially recognised by the ictv; many others are still pending for official designation. cov species detected in bats and officially recognised by the ictv are listed in table 3 and the following chapter reasonably discusses only officially recognized bat cov species. bats can carry and transmit covs into local bat populations via migration even though little is known about the migratory patterns of these animals. closely related covs can be detected in the same bat species living at locations separated by thousands of miles (drexler et al., 2010) and different cov species or genera can be found in different bat species living at the same roosting sites. however, some covs have been shown to be species-specific. accordingly, regional patterns of bat cov outbreaks at species level can be deduced from the population distribution of their respective bat hosts. although bats seem to develop clinical diseases induced by several viruses and bacteria (mühldorfer et al., 2011) , generally covs do not cause apparently overt disease in these mammals, also experimentally. this phenomenon seems to be related with peculiar characteristics of their immune system (ahn et al., 2019; brook et al., 2020) . based upon genomic data available so far, it is widely accepted that while birds represent the reservoir for covs belonging to genera gammacoronavirus and deltacoronavirus, bats are the natural reservoir for alpha-and betacoronaviruses. however, only betacoronaviruses of subgenera sarbecovirus, merbecovirus, nobecovirus and hibecovirus have been detected in bats so far. given that several betacoronaviruses from the subgenus embecovirus have been discovered in rodents, it was speculated that rodent covs may be the ancestors of currently circulating viruses belonging to this subgenus . covs have been detected at high frequency in bats in all continents, with alphacoronaviruses being more widespread than betacoronaviruses . subgenus colacovirus (genus alphacoronavirus) officially comprises the viral species bat coronavirus cdphe15, so far composed by two bat covs strains named cdphe15/usa/2006 and myotis lucifugus cov (myl-cov), which share a 98.2 % nucleotide identity across the whole genome. both strains have been detected in myotis lucifugus bats (vespertilionidae) also known as the northern american little brown bats. the former was detected in 2006 in colorado (genbank acc. no. kf430219), while the latter was reported in 2010 in canada. this virus was identified in the intestines and lungs and associated with minimal pathology or inflammation (subudhi et al., 2017) . subgenus decacovirus (genus alphacoronavirus) comprises the species rhinolophus ferrumequinum alphacoronavirus hub-2013 composed so far by btms-al-phacov/gs2013 and btrf-alphacov/hub2013 strains discovered in china in myotis spp. and rhinolophus ferrumequinum bats, respectively. these two viruses share very high sequence identities (higher than 98 %), which dramatically decrease in the s genes (only 85% nucleotide identity) (wu et al., 2016) . woo et al., 2006) shares an 86 % sequence identity with severe acute diarrhoea syndrome-coronavirus (sads-cov) of pigs . these two viruses are now included in the same viral species rhinolophus bat coronavirus hku2 (subgenus rhinacovirus, genus alphacoronavirus). viral strains btkynl63-9a, btkynl63-9b, btkynl63-15 and btkynl63-9a, identified in 2010 in triaenops afer bats from kenya, form the viral species nl63-related bat coronavirus strain btkynl63-9b that is part of the subgenus setracovirus (genus alphacoronavirus) along with human coronavirus nl63 (tao et al., 2017) . in this regard, a bat origin has been strongly suggested for two of the less-pathogenic hcovs causing mild respiratory symptoms in immunocompetent people, namely hcov-229e and hcov-nl63, both belonging to the alphacoronavirus genus. whereas hcov-229e (subgenus duvinacovirus) recognises as direct ancestor an alphacoronavirus from alpacas, which in turn derives from 229e-related covs identified in hipposiderid bats (corman et al., 2015) , hcov-nl63 is likely a recombinant virus originating from the distantly related 229e-related covs associated with hipposiderid bats and covs associated with triaenops afer bats (tao et al., 2017) (table 1 ). the s protein of hcov-nl63 is more closely related to that of 229e-related covs, whereas the rest of the genome with covs included in the nl63-related bat coronavirus strain btkynl63-9b species (tao et al., 2017) . different from the bovine coronavirus (bcov)-like viruses that cause enteric disease, in 2007 a novel alpaca cov was associated to respiratory disease in california, usa. full-length genome analysis showed that this respiratory alpaca cov was closely related to the alphacoronavirus hcov-229e (subgenus duvinacovirus) (crossley et al., 2012) . more recently, close relatives of hcov-229e were detected in african hipposiderid bats. interestingly, both bat and alpaca viruses displayed an intact accessory gene orf8 located at the genomic 3' end, while hcov-229e retained only a conserved trs preceding remnants of this orf, suggesting its loss after acquisition of a 229e-related cov by humans. therefore, hcov-229 is likely a descendant of the alpaca alphacoronavirus (corman et al., 2015) . strains forming the viral species bat hp-betacoronavirus zhejiang2013 (subgenus hibecovirus, genus betacoronavirus) were discovered in hipposideros pratti bats from china in 2013 (wu et al., 2016) . strain ro-batcov gccdc1 356 was identified from stools of rousettus leschenaultii, a species of fruit bats (pteropodidae) of southern asia, which were collected in yunnan province, china, in 2014 (huang et al., 2016) . ro-batcov gccdc1 356 shows a small intact orf of 276 nucleotides embedded between the n and ns7a genes. this orf has no homology to any known coronavirus, and the encoded protein exhibited 54.9 % amino acid identity with the p10 protein encoded by the first orf of segment s1 of bat fusogenic orthoreoviruses (genus orthoreovirus, species nelson bay orthoreovirus, also known as pteropine orthoreovirus). these viruses are double-stranded segmented rna viruses, belonging to the family reoviridae, and are able to cause severe pneumonia in humans (chua et al., 2007; lorusso et al., 2015) . ro-batcov gccdc1 356 is included in the viral species rousettus bat coronavirus gccdc1 within the subgenus nobecovirus, genus betacoronavirus. rousettus bat coronavirus hku9, belonging to subgenus nobecovirus, was also identified in rousettus leschenaultii and in other bat species (mendenhall et al., 2017) . this virus was first detected in 2007 in guangdong province in china (woo et al., 2007) . subsequent studies suggested that the virus was widely distributed and is circulating in different bat species (ge et al., 2012) . covs from the bthku9-like cluster were also detected in hipposidereos commersoni and rousettus aegyptiacus bats in kenya (tong et al., 2009) . being a fruit bat, rousettus leschenaultii has a wider flying range than most of the insectivorous bats in china, thus it may carry viruses over long distances. a comparison of the reported hku9-cov sequences showed a high genetic diversity within this viral species (luo et al., 2018a, b; lau et al., 2010; ge et al., 2012) . when mers-cov was first isolated in the middle east in 2012 and its genome sequenced, it was found that it was most closely related to ty-batcov hku4 discovered in tylonycteris pachypus and pi-batcov hku5 discovered in pipistrellus abramus, which were the only known members of subgenus merbecovirus at that time. these two viruses are now the prototype strains of tylonycteris bat coronavirus hku4 and pipistrellus bat coronavirus hku5 viral species, respectively, within subgenus merbecovirus, genus betacoronavirus. although mers related covs (mers-rcovs) were lately discovered, mers-cov was much closer in the s1 region to hku4-cov than to mers-rcov or hku5-cov. indeed, dipeptidyl peptidase 4 (dpp4), the receptor for mers-cov, is also the receptor for hku4, but neither for hku5 nor for early discovered mers-rcovs. however, hku4 prefers bat dpp4 over human dpp4, whereas mers-cov shows the opposite trend . so far, hku4-covs are only carried by tylonycteris spp. bats (t. pachypus and t. robustula) and are relatively conserved; hku5-covs are found in different pipistrellus spp. bats, including p. abramus, p. pipistrellus and p. minus . due to the current sars-cov-2 pandemic, attention should be given to the viral species severe acute respiratory syndrome-related coronavirus (sars-rcov,subgenus sarbecovirus, genus betacoronavirus) and middle east respiratory syndrome-related coronavirus (mers-rcov, subgenus merbecovirus, genus betacoronavirus), which enclose sars-cov and mers-cov, the first two highly pathogenic covs that were discovered in humans. in 2002, at the beginning of the sars epidemic, almost all early human index patients had animal exposure in a market place, in guangdong province, before developing disease. after sars-cov was identified, its rna and/or specific antibodies were found in masked palm civets (paguma larvata) and animal handlers in a market place. however, later investigations of farmed and wild-caught civets revealed that sars-cov strains found in market civets were transmitted to them by other wild animals (tu et al., 2004; kan et al., 2005) . subsequently, novel covs related to human sars-cov (sars-rcovs) were discovered in horseshoe bats (genus rhinolophus) in china and hong kong lau et al., 2005) . these sars-rcovs showed genome sequence identity of 88-90 % among themselves and 87-92 % identity to human or civet sars-cov isolates. sars-rcovs were detected in rhinolophus spp. bats of other regions of china (tang et al., 2006; woo et al., 2006; yuan et al., 2010; ge et al., 2013) . sars-rcovs with higher genetic diversity with respect to chinese strains were also detected in rhinolophid bats from slovenia, bulgaria and italy in europe (drexler et al., 2010; rihtaric et al., 2010; balboni et al., 2011) . covs related to sars-rcov were also detected in hipposideros spp. and chaerophon spp. bats from ghana, kenya and nigeria (hu et al., 2015) . these evidences suggested that bats may be the natural hosts for sars-cov and that wild carnivores were only intermediate hosts. although these sars-rcovs showed high sequence identity to sars-cov, they were demonstrated to be unable to bind to the human cell angiotensin converting enzyme ii (ace2) receptor, the receptor of sars-cov, as a consequence of deletions in their s protein (ren et al., 2008) . besides, the theory of bat origin of sars-cov lacked a powerful support due to the failure of direct isolation of this virus from bats. thus, considering that no direct progenitor of sars-cov was found in bats and that rna recombination is the fuel for cov evolution, it has been proposed that sars-cov emerged through recombination of bat sars-rcovs. this hypothesis was made after the evidence of a single bat cave in yunnan, china, with very high covs diversity and considering that, within the identified covs, all genetic elements needed to form sars-cov have been identified in that single cave (ge et al., 2013) . recombination analysis also strongly supported the hypothesis that the civet sars-cov strain sz3 originated following a recombination event of two existing bat strains, wiv16 and rf4092 (hu et al., 2017) . moreover, wiv1, the closest relative to sars-cov that has been found in bats so far (more than 95 % nucleotide identity, higher than that of any other bat sars-rcovs (76-92 %)), likely arose through recombination of two other prevalent bat sars-rcov strains. the most frequent recombination breakpoints were within the s gene and upstream of orf8, which encodes an accessory protein. these genes were also involved in the crucial adaptation pathways of sars-cov from bats to wild carnivores, from wild carnivores to humans, and from human to human (cui et al., 2019) . wiv1 has been shown to have the capacity to bind to the human, civet and bat cell ace2 receptor (ge et al., 2013) . the isolation in cellculture of a highly related sars-cov strain, coupled with the evidence of a functional s protein capable of using the same ace2 receptor, provided robust and conclusive evidence for the bat origin of sars-cov. an additional sars-rcov strain has been shown, by reverse genetics studies, to have the capacity to bind to the human ace2 receptor (menachery et al., 2015) . quite the opposite, a direct bat cov highly related to mers-cov of humans was never detected. indeed, the genome sequences of mers-cov in human and dromedaries possess only around 65-80 % nucleotide identities to those of the other members of subgenus merbecovirus from different bats. human mers-covs were instead almost identical to mers-covs identified in dromedary camels (camelus dromedaries). lately, genomic sequence analyses indicated that covs now belonging to the mers-rcov species were found in several bat species from two bat families, vespertilionidae and nycteridae (lelli et al., 2013; de benedictis et al., 2014; corman et al., 2014a, b; anthony et al., 2017; moreno et al., 2017; wong et al., 2019) . however, none of these mers-rcovs is a direct progenitor of mers-cov, as their s proteins differ substantially from that of the human virus. the closest relative to mers-cov of humans and dromedary camels is mers-rcov strain neoromicia/5038 isolated from neoromicia capensis bats in south africa (geldenhuys et al., 2018, table 1 ). a short sequence (around 200 nucleotides) of viral rna identical to that of mers-cov was also detected in a taphozous perforates bat in saudi arabia (memish et al., 2013) . overall, although it is widely accepted that mers-cov ancestor is in bats, further studies are warranted in order to discover the precise mechanisms of its emergence in dromedary camels and humans. it was suggested that mers-cov ancestors had been circulating in bats for very long time. mers-cov has evolved to adapt to use human receptor and the dpp4-recognising bat coronaviruses like hku4 may follow up, thereby posing a serious risk to human health. recent mers-rcovs were shown to have the capacity to bind to the dpp4 as entry cell receptor as they acquired the s1 through recombination with hku4-like viruses (luo et al., 2018a, b) . as for the recent and threatening covid-19 outbreak in humans, we certainly know that sars-cov-2 belongs to the species sars-rcov together with sars-cov from humans and sars-rcovs from wild carnivores and horseshoe bats (genus rhinolophus) (gorbalenya et al., 2020; zhou et al., 2020; wu et al., 2020) . epidemiological investigations revealed that many initial patients were exposed to wildlife at the huanan seafood wholesale market (south china seafood market), which is the largest seafood market in central china (lorusso et al., 2020) . sars-cov-2 has been assigned to an existing species of hundreds of known viruses largely isolated from bats. these viruses have names derived from sars-cov, but only the viral isolates originating from the 2002-2003 outbreak have been confirmed to cause sars in humans (gorbalenya et al., 2020) . importantly, it has also been confirmed that sars-cov-2 uses the ace2 receptor through the receptor binding domain (rbd) of the s protein (hoffmann et al., 2020; zhou et al., 2020) . likely, also sars-cov-2 has a bat origin. according to genome sequences available so far, the most closely related virus (96.2 % of nucleotide sequence identity) to sars-cov-2 is strain batcovratg13 identified from a bat, rhinolophus affinis, from yunnan province, china, followed by sars-rcovs identified from pangolins (tang et al., 2020) . the receptor-binding spike protein of sars-cov-2 is highly divergent from other covs with less than 75 % nucleotide sequence identity to all previously described sars-rcovs, except for a 93.1 % nucleotide identity to batcovratg13 (zhou et al., 2020) . although sars-cov-2 uses the ace2 receptor, five out six critical amino acid residues in rbd were different between sars-cov-2 and sars-cov; the same residues were instead identical to those of pangolin sars-rcovs and, in turn, only one of these residues was identical to those of batcovratg13 (tang et al., 2020) , although this latter shows the highest nucleotide sequence identity with sars-cov-2 along the whole genome. thus, it was tempting to speculate that sars-cov-2 rbd region might have originated from recent recombination event in pangolins or that sars-cov-2 and sars-rcovs of pangolins represent the result of coincidental evolution (lam et al., 2020; tang et al., 2020) . overall, it remains to be solved whether also sars-cov-2 needed an intermediate (and amplification) host before being able to infect humans as it was the case for sars-cov and other hcovs. since a mammal reservoir has not yet been identified, a prudent use of specific antigens is strongly recommended for serological diagnosis of sars-cov-2 in animals as cross-reactions with viruses of the alphacoronavirus genus, widespread in animals, might occur (sun and meng, 2004) . analogously to bats, but with a lesser extent, also rodents have been recently demonstrated to play a significant role in the evolution of cov, in particular of those belonging to subgenus embecovirus of genus betacoronavirus. rodentia (rodents) is the largest order of mammals with more than 2000 species worldwide, representing a major source of zoonotic infectious diseases (han et al., 2015) . for decades, only one species of coronavirus, murine coronavirus (subgenus embecovirus, genus betacoronavirus), has been associated with rodents. the prototype virus, which was named mouse hepatitis virus (mhv), was first isolated in mice in 1949 (cheever et al., 1949) . a mhv variant was lately identified in rats in 1970 (parker et al., 1970) . rat coronavirus (rcov) causes epidemics of respiratory disease in laboratory rat colonies. the two prototype strains of rcov are sialodacryoadenitis virus (sdav) and parker's rcov (rcov-p) (bhatt et al., 1972; parker et al., 1970) . both strains infect the respiratory tract, and sdav can also infect the eye, salivary and lacrimal glands. young rats are especially susceptible to rcov with the infection occurring in the lower respiratory tract and developing into interstitial pneumonia (parker et al., 1970) . together with feline infectious peritonitis virus (fipv) and ibv, mhv has been one of the most strictly animal cov studied ever. mhv is a natural pathogen of mice, normally infecting the liver, gastrointestinal tract, and central nervous system, causing a wide range of disease, including hepatitis, gastroenteritis, and acute and chronic encephalomyelitis. importantly, it served as model for cov replication and pathogenesis, with emphasis for neuro-invasion and neurovirulence (weiss and navas-martin, 2005) . as for the additional structural protein he, some strains (such as jhm) of mhv contain the he protein, while others (such as a59) do not yokomori et al., 1989) . the role of rodents in the evolution of covs belonging to embecoviruses has been recently highlighted by means of the discovery of a novel betacoronavirus in norway rats (rattus norvegicus) in china. this virus forms a separate species named china rattus coronavirus hku24 (chrcov hku24) within the embecovirus subgenus. although designated as a novel species, this virus possessed genome characteristics that resemble to those of both betacoronavirus-1 and murine coronavirus, suggesting that chrcov hku24 represents the murine origin of betacoronavirus-1, with interspecies transmission from rodents to other mammals having occurred centuries ago (lau et al., 2015) . genus betacoronavirus consists of five subgenera, with bat covs being including in all but one of subgenus embecovirus, where rodent, human and bovine covs are included (https://talk.ictvonline.org/ taxonomy/). this supports the hypothesis that rodent covs were the ancestors of embecoviruses of other animals, while bats are the natural reservoirs for all other betacoronaviruses. importantly, rodent covs are not restricted to genus betacoronavirus. a deep virological screening was performed in 1465 rodents sampled in zhejiang province, china, during 2011-2013, with nearly 2% of rodents testing positive for cov . in particular, covs were detected in 10 striped field mice (apodemus agrarius), 4 norway rats, 14 lesser ricefield rats (rattus losea), 1 asian house rat (rattus tanezumi) and 1 chinese white-bellied rat (niviventer confucianus). amplicons of the replicase gene sequences were recovered from 21 (70 %) of the cov rna positive rodent samples described above and whole genome or nearly whole genome sequences (> 98 %) were recovered from 1 and 4 cov positive samples, respectively. by means of whole genome sequence analysis, authors were able to identify a divergent alphacoronavirus, which was lately officially designated as species lucheng rn rat coronavirus (lrnv) within the subgenus luchacovirus, and two novel betacoronaviruses termed longquan aa mouse coronavirus (lamv) and longquan rl rat coronavirus (lrlv) and assigned to the two established species betacoronavirus-1 and murine coronavirus, respectively . moreover, lrnv seems to be a recombinant virus as its n protein gene is more closely related to those of the genus betacoronavirus. overall, the discovery of rodent-associated covs belonging to subgenera that are distinct from those including bat covs warrants further investigations upon the role played by rodents in the evolution and emergence of these viruses. sars-cov replication has been studied in mice, syrian golden and chinese hamsters. the most severe symptoms of sars were observed in aged animals. indeed, aged mouse model of sars-cov has been generated (gretebeck and subbarao, 2015) . transgenic mice expressing human ace2 were also developed to closely mimic sars-cov infection in humans. some animal models have been tested and analysed on the genomic and proteomic level to study the pathogenesis of sars-cov. therefore, we have reason to believe that such models would work also for sars-cov-2. quite the opposite, studies have demonstrated that mice, guinea pigs and hamsters are not susceptible to experimental mers-cov infection, mainly because their homologous dpp4 molecules table 4 coronaviruses in domestic swine and associated diseases. do not function as receptors for mers-cov entry (cockrell et al., 2014) . the first mouse model of mers infection reported in 2014 involved transducing animals with recombinant adenovirus 5 encoding human dpp4 (hdpp4) molecules intranasally, and this resulted in replication of mers-cov in the lungs. this mouse model also showed clinical symptoms of interstitial pneumonia, including inflammatory cell infiltration, and thickened alveolar and mild oedema (song et al., 2019) . currently, six covs are circulating in swine (table 4 ). these include four alphacoronaviruses, transmissible gastroenteritis virus of swine (tgev) and its derivative porcine respiratory coronavirus (prcov) (subgenus tegacovirus), porcine epidemic diarrhoea virus (pedv) (subgenus pedacovirus) and sads-cov (subgenus rhinacovirus), one betacoronavirus, porcine haemagglutinating encephalomyelitis virus (phev) (subgenus embecovirus), and one deltacoronavirus, porcine deltacoronavirus (pdcov) (subgenus buldecovirus). tgev, pedv, sads-cov and pdcov are responsible for acute gastroenteritis in swine, with fatal infections in piglets born to seronegative sows, prcov causes a mild respiratory disease and phev is the causative agent of neurological and/or digestive disease in pigs (mora-díaz et al., 2019; wang et al., 2019). tgev was first described in uk in 1950s, representing the oldest known swine cov. tgev and prcov are closely related to canine coronavirus (ccov) and feline coronavirus (fcov) forming with these carnivore covs a unique species, referred to as alphacoronavirus-1. based on the analysis of the accessory protein gene orf3, it has been postulated that tgev has originated from ccov type ii (ccov-ii), since while ccov type i (ccov-i) exhibits an intact gene, both ccov-ii and tgev, which are strictly related in the s gene, have only remnants of orf3 (lorusso et al., 2008) . prcov, in turn, has derived from tgev through the deletion of ≈600 nucleotides at the 5' end of the s gene (corresponding to ≈200 amino acids at the n-terminus of the spike protein) and consequent change of the major tissue tropism from the enteric to the respiratory epithelium. this large deletion caused the loss of sialic acid binding activity that allows the attachment to mucins and mucin-type glycoproteins, so that tgev but not prcov is able overcome the intestinal mucus barrier, having access to the gut mucosa . prcov shares some epitopes for neutralising antibodies with tgev, so that its extensive circulation in swine herds has resulted in a drastic reduction of tge outbreaks worldwide. pedv was introduced in the pig population in the 1970s, likely as a consequence of a spillover event from bats. the virus was first described in europe and had been primarily maintained as an endemic pathogen in european and asian swine populations until its introduction into north america in 2013. pedv is more strictly related to a scotophilus bat coronavirus 512 than to other known alphacoronaviruses, including tgev and human alphacoronaviruses hcov-229e and hcov-nl63. therefore, pedv and btcov/512/2005 likely have a common evolutionary precursor and a cov cross-species transmission may have occurred between bats and pigs (banerjee et al., 2019) . accordingly, pedv contains signature motifs at the 5′-untranslated region that are shared by bat covs, thus providing further support of the evolutionary origin of pedv from bats and potential cross-species transmission (huang et al., 2013) . currently, different pedv genotypes are described based on the s gene: i) g1a pedv, including classical european and asian strains with moderate virulence; ii) g2 pedv, also called "original us pedv", comprising highly virulent strains that originated in asia and are now widespread in the usa; iii) g1b pedv, which is represented by the so-called s-indel strains, i.e., strains presenting insertions and deletions in the s gene that are associated with mild clinical outbreaks. these strains are natural recombinant pedvs with a g2-like genomic backbone carrying an s1 region of g1a strains; iv) s1 n-terminal domain-deletion (ntd-del) strains that are g2-like strains containing a 194 to 216-aa deletion within the n-terminal domain of the s1 subunit, also associated to mild clinical forms (hou and wang, 2019) . recombinant strains between pedv and tgev have been also reported in europe (akimkin et al., 2016; belsham et al., 2016; boniotti et al., 2016) . sadv-cov, now referred to as swine enteric alphacoronavirus (seacov), is another virulent swine enteric alphacoronavirus that originated from bats, sharing an 86 % sequence identity with a bat alphacoronavirus hku2-cov. since viruses displaying a 96-98 % sequence identity to sads-cov were detected in rhinolophus spp. bats, sads-cov and hku2-cov likely descend from a common ancestor . accordingly, both viruses now belong to the unique species rhinolophus bat coronavirus hku2. in contrast, phev, which was first described in 1957 in nursery pigs with encephalomyelitis in ontario, canada, has not derived from bat covs, but its evolutionary history is tightly intermingled with other two closely related betacoronavirus, hcov-oc43 and the oldest known bcov, with which phev may have common ancestors (vijgen et al., 2006) and is included in the same viral species, betacoronavirus-1 (corman et al., 2018) . most probably, hcov-oc43 and phev descend from a rodent betacoronavirus through preliminary adaptation to bcov, from which they may have emerged in the context of a pandemic recorded historically at the end of the 19th century (corman et al., 2018) . pdcov was recently detected in 2012 in hong kong during cov molecular surveillance in avian and mammalian species. this swine deltacoronavirus seems to recognise another different ancestor, likely emerging from a host-switching event between avian and mammal covs. the most closely related pdcov relative has been identified in quail deltacoronavrus uae-hku30 and the virus has been proposed to be a recombinant between other two avian deltacoronaviruses, sparrow cov hku15 and bulbul cov hku11. all these deltacoronavirus are now members of the same species coronavirus hku15 (lau et al., 2018) . pigs were found to be susceptible to experimental infection with the betacoronavirus mers-cov (vergara-alert et al., 2017), while sars-cov rna was detected in pigs and wild boars wang et al., 2005) . in contrast, a recent experimental infection demonstrated that pigs are not susceptible to sars-cov-2 . few studies have been carried out to assess the circulation of covs in farmed or free-ranging wild boars (sus scrofa). antibodies against tgev/prcov were detected in some animals in slovenia (vengust et al., 2006) and croatia (roic et al., 2012) and pedv rna was demonstrated in south korea (lee et al., 2016) . a wild boar sold at a live animal market of guangzhou, china, was positive for sars-cov rna . the main covs infecting ruminants are reported in table 5 . the oldest known ruminant cov is bcov, which is also the prototype of the species betacoronavirus-1 (subgenus embecovirus, genus betacoronavirus). this virus is able to cause a variety of clinical forms, including enteric disease with high mortality rates in neonate calves, winter disease (a severe enteric form) in lactating cows (decaro et al., 2008b) , and a respiratory disease, also known as shipping fever, in cattle of all ages, with a higher prevalence in 2-3 month-old calves (decaro et al., 2008a) . it was postulated that the presence of genetic signatures differentiates enteric and respiratory bcovs (hasoksuz et al., 1999) , but it was ultimately evident that the same virus strain could be responsible for simultaneous appearance of enteric and respiratory disease in the same animals (chouljenko et al., 2001) . it has been postulated that bcov originated from a rodent cov (corman et al., 2018) . very recently, a novel cov, representing a new viral species, referred to as china rattus coronavirus hku24 (chrcov-hku24), was detected in norway rats in china. this virus was phylogenetically distinct from mhv and hcov-hku1 and displayed genome features that were intermediate between bcov and mhv. therefore, chrcov hku24 may represent the murine origin of bcov and rodents are likely an important reservoir for ancestors of subgenus embecovirus (lau et al., 2015) . bcov is paradigmatic of how covs are able to cross the interspecies barriers, establishing its derivatives as separate viral lineages affecting the respiratory and/or enteric tract of humans (hcov-oc43), swine (phev), horses (equine coronavirus, ecov), and dogs (canine respiratory coronavirus, crcov). a number of bcov-related viruses, all currently included in the unique species betacoronavirus-1, have been detected in the enteric and/or respiratory tract of domestic and wild ruminants. these bcov-like covs include viruses of domestic and domesticated ruminants that were reported in sheep and goats (reinhardt et al., 1995; yang et al., 2008) , water buffalo (bubalus bubalis) (decaro et al., 2008c) , llamas (lama lama) and alpacas (vicugna pacos) (cebra et al., 2003; jin et al., 2007) . in the wild, bcov-like covs were demonstrated in six species of the cervidae family, which are caribou/ reindeer (rangifer tarandus caribou), elk/wapiti (cervus elephus), samber deer (cervus unicolor), white-tailed deer (odocoileus virginianus), sika deer (cervus nippon yesoensis) and water deer (hydropotes inermis) (amer, 2018) . similar viruses were also found to circulate in the giraffe (giraffa camelopardalis) (hasoksuz et al., 2007) , several species of antelopes (alekseev et al., 2008; chung et al., 2011) , wisent (bison bonasus), himalayan tahr (hemitragus jemlahicus) (chung et al., 2011) , and dromedary camels (camelus dromedarius) (woo et al., 2014) . the last strain, detected in the united arab emirates and consequently named dromedary camel coronavirus uae-hku-23 (dccov uae-hku23), was slightly divergent from other bcov-like viruses (woo et al., 2014) . dromedary camels are susceptible to mers-cov infection, developing asymptomatic infections or mild upper respiratory disease, so that they are considered the natural host of mers-cov, with adult animals in many countries in the middle east as well as in north and east africa showing > 90 % seroprevalence to the virus (hemida et al., 2017b) . although human-to-human transmission has occurred outside middle east due to travel-associated patients with mers and has caused large clusters of human cases within healthcare facilities in saudi arabia, jordan and united arab emirates, it remains inefficient and sustained community transmission has not being documented so far, thus suggesting multiple virus introduction into the human population by infected dromedaries (hemida et al., 2017b) . more recently, a phylogenetic study of 173 mers-cov full-genome sequences revealed recombination signatures that defined five major phylogenetically stable lineages, all of which contained human and camel mers-cov sequences (sabir et al., 2016) . in the same study, an alphacoronavirus strictly related to hcov-229e was found in the respiratory tract of dromedary camels of saudi arabia (sabir et al., 2016) . although some studies ruled out the susceptibility of other domestic ruminants to mers-cov (reusken et al., 2013; adney et al., 2016) , a recent study detected specific antibodies and rna in sera and nasal secretions, respectively, of domestic ruminants raised in africa, including sheep, goats and cattle (kandeil et al., 2019) . llamas were found to be susceptible to experimental infections with mers-cov (vergara-alert et al., 2017). the only cov that has been so far known in horses is ecov, which is a bcov-descendant betacoronavirus (subgenus embecovirus). ecov was first isolated from the faeces of a diarrhoeic foal in 1999 (ecov-nc99) in north carolina, usa (guy et al., 2000) , and was initially believed to only affect foals. since 2010, the virus has been recognised in japan, europe and the usa as a new, clinically important, enteric virus of adult horses (pusterla et al., 2018) . despite mers-cov was successfully adapted to the in-vitro growth in equine cell lines (meyer et al., 2015) , serological and molecular table 5 coronaviruses in domestic and domesticated ruminants and associated diseases. sabir et al. (2016) n. decaro and a. lorusso veterinary microbiology 244 (2020) 108693 investigations have demonstrated that horses are not naturally infected by mers-cov (meyer et al., 2015; hemida et al., 2017a) , nor they are susceptible to experimental infection (adney et al., 2016; vergara-alert et al., 2017) . however, surprisingly, mers-cov rna was detected in respiratory specimens of three donkeys of 42 from egypt (kandeil et al., 2019) , a finding that requires further confirmation. a molecular survey aimed to assess cov circulation in horses in saudi arabia and oman has detected two dccov uae-hku23 strains in enteric samples of horses (hemida et al., 2017a) . scarce data are available about cov circulation in donkeys. these equids are susceptible to ecov infection since positive rt-pcr results were obtained from a donkey in ireland (nemoto et al., 2019) . in addition, three donkeys (7.1 %) of 42 from egypt tested positive for mers-cov rna in their nasal secretions (kandeil et al., 2019) . covs of carnivores are listed in table 6 . three covs are known in dogs, i.e., two alphacoronaviruses of the subgenus tegacovirus, namely ccov-i and ccov-ii, and one betacoronavirus of the subgenus embecovirus, namely crcov. ccovs (species alphacoronav-irus-1) are commonly responsible for mild, self-limiting enteritis in pups . although they are neglected viruses and vaccination is not recommended due to the absence of an effective challenge model, two independent studies have demonstrated their significant involvement in the onset of acute canine enteritis (duijvestijn et al., 2016; dowgier et al., 2017) . the evolutionary history of ccovs is tightly intermingled with that of tgev and fcovs. ccov-i possesses a divergent spike protein and the intact form of an additional gene, orf3, whose remnants are present in ccov-ii and, at a lesser extent, in tgev. therefore, ccov-ii has likely emerged as a consequence of recombination between the original ccov-i and an unknown cov in the s gene and of progressive loss of orf3 (lorusso et al., 2008) . a further recombination occurred in the very 5' end of the s gene between ccov-ii and tgev, giving rise to back recombinant ccov-ii strains, also known as tgevlike ccovs, having a spike protein n-terminus of tgev in a ccov-ii backbone (decaro et al., , 2010 . consequently, the ccov taxonomy was revised, with classical and tgev-like strains being referred to as ccov-iia and ccov-iib, respectively. while ccovs are usually involved in mild forms of diarrhoea, there are some hypervirulent strains that are associated to severe, haemorrhagic, sometimes fatal gastroenteritis. in addition, ccov-iia strains, designated pantropic ccov, that are able to spread systemically and cause severe disease and the death of infected dogs have been reported in italy (buonavoglia et al., 2006; alfano et al., 2020) , other european countries (decaro et al., 2013) and south america (pinto et al., 2014) . genomic sequences from pantropic ccovs were analysed, but no obvious genetic signatures that may have caused the switch in pathogenicity were found (decaro and buonavoglia, 2011; decaro et al., 2013) . different from ccov-i and ccov-ii, the betacoronavirus crcov is associated with mild respiratory signs and has been proposed as an etiological agent of canine infectious respiratory disease (cird) together with other viral and bacterial agents . the virus was first detected firstly in uk in 2003 (erles et al., 2003) and subsequently in other european and extra-european countries (decaro et al., 2007 (decaro et al., , 2016 mitchell et al., 2017; maboni et al., 2019; piewbang et al., 2019; more et al., 2020) . being a bcov derivative, crcov possesses the same genomic organisation, with some differences in accessory orfs located between the s and e protein genes. in particular, while some crcovs possess a unique 8.8 kda protein gene directly downstream of the s protein gene, other canine bcov-like covs display the canonical set of bcov accessory genes but with truncated forms of the 4.8 kda protein gene . in cats, two alphacornavirus-1 genotypes are known, namely fcov type i (fcov-i) and fcov type ii (fcov-ii), the latter being generated as table 6 coronaviruses in domestic and domesticated carnivores and associated diseases. jakob, 1914jacob (1914 , pedersen et al. (1984) cat ( erles et al. (2003) n. decaro and a. lorusso veterinary microbiology 244 (2020) 108693 a consequence of recombination events between ccov-ii and fcov-i that generated viruses with a ccov-ii genomic region, encompassing orf1b, orf2 (s gene), orf3abc, orf4 (e gene), and partial orf5 (m gene), in the context of an fcov-i backbone (pedersen, 2014) . both genotypes are involved in the development of feline infectious peritonitis (fip), a perivascular pyogranulomatosis of cats that may occur in two clinical forms, effusive and non-effusive fip, which are characterised by prevalence of effusions in the body cavities and of pyogranulomatous lesion in organs, respectively. fip occurs as a consequence of a change in tissue tropism of an enteric fcov strain (feline enteric coronavirus, fecv), infecting enterocytes of the intestinal villi, that acquires the ability to infect monocytes/macrophages switching to the more virulent fipv, which is responsible for systemic infections and dysregulation of the proinflammatory cytokines (addie et al., 2009) . the changes responsible for the pathogenetic shift have been investigated for many decades, being suggested to be variably represented by point mutations located in the s gene (rottier et al., 2005) , deletion/insertion in the group-specific genes 3c (vennema et al., 1998; chang et al., 2010) , 7b (vennema et al., 1998) or 7a (kennedy et al., 2001a) . however, none of these differences appeared to consistently correlate with disease phenotype. more recent studies have identified specific genetic signatures in the s gene of fcov-i that are implicated in monocyte/macrophage tropism. two amino acid substitutions, m1058 l and/or s1060a, corresponding to nucleotide mutations a23531 t/c and t23537 g, respectively, in the viral genome, together distinguished fcovs found in the tissues of fip cats from those found in the faeces of healthy cats without fip in > 95 % of cases (chang et al., 2012) . however, subsequent studies concluded that these mutations are likely to be markers of systemic fcov infection rather than fip per se (porter et al., 2014; barker et al., 2017) . two alphacoronaviruses, both belonging to subgenus minacovirus, are currently known in mustelids, namely mink coronavirus 1 (mcov-1) and ferret coronavirus (frcov). mcov-1 has been recently identified as the etiological agent of mink epizootic catarrhal gastroenteritis (ecg), an infectious disease of farmed american (neovison vison) and european (mustela lutreola) mink first described in 1975 (larsen and gorham, 1975) and later affecting several million mink in different countries (vlasova et al., 2011) . the disease is observed at greater frequency in mink of ≥4 months and is characterised by seasonality, high morbidity (approaching 100 %) and low mortality (< 5 %). recent full-genome analysis demonstrated that mcov-1 is phylogenetically distant from ccovs and fcovs, being closely related to frcov (vlasova et al., 2011) . presently, the two viruses are considered separate species within subgenus minacovirus (https://talk.ictvonline.org/taxonomy/). frcov has been recognised as the causative agent of epizootic catarrhal enteritis (ece), first described in 1993 in domestic ferrets (mustela putorius furo) in the eastern part of the usa (williams et al., 2000) and subsequently reported in domestic and laboratory ferrets throughout the world (murray et al., 2010) . analogous to fcov, frcov exists in two different pathotypes: i) ferret enteric coronavirus (frecv) is associated to ece, a highly contagious diarrhoeal disease also known as green slime disease, which affects mainly young ferrets with morbidity and mortality rates similar to those of ecg; ii) ferret systemic coronavirus (frscv) is responsible for a systemic diseases of ferrets, which is characterised by pyogranulomatous perivasculitis and peritonitis resembling to those of fip (murray et al., 2010) . similar to fip, wise et al. (2010) have shown that frecv and frscv differ significantly in spike protein and that deletions in frcov 3c may also correlate with the severe pathotype of frscv. recombination in the s, 3c and e genes between different frcov has been also reported (lamers et al., 2016) . different covs were found to circulate in wild carnivores. ccovs were detected in wolves (canis lupus), red foxes (vulpes vulpes), eurasian otters (lutra lutra), common genets (genetta genetta) (alfano et al., 2019; rosa et al., 2020) . ccov-like viruses were also found in african wild carnivores, including spotted hyenas (crocuta crocuta) and silver-backed jackals (canis mesomelas) (goller et al., 2013) . fcovs have a wide circulation in non-domestic felids (kennedy et al., 2002 (kennedy et al., , 2003 , with fip cases being reported in servals (felis serval) (juan-salles et al., 1997) , cheetah (acinonyx jubatus) (kennedy et al., 2001b) , mountain lion (puma concolor) (stephenson et al., 2013) , and european wildcat (felis silvestris) (watt et al., 1993) . divergent alphacoronavirus-1 viruses were detected in chinese ferret badger (nyctereutes procyonoides) and raccoon dog (melogale moschata) (dong et al., 2007) . the same study reported the identification in asian leopard cat (prionailurus bengalensis) and chinese ferret badger of an unclassified cov, which was closely related to gammacoronaviruses in most parts of the genome, whereas the s gene displayed the highest sequence identity to alphacoronaviruses (dong et al., 2007) . with the discovery of deltacoronaviruses, these viruses were later included in this novel genus along with avian and porcine strains (woo et al., 2009; wang et al., 2014) . some domestic and wild carnivores are also susceptible to sars-cov infection. while the potential natural reservoirs are horseshoe bats, sars-like cov strains were found to be widespread in masked palm civets (paguma larvata) and raccoon dogs, which were suspected to be intermediate hosts (guan et al., 2003) . full-genomic comparative analysis has shown that sars-like covs isolated from palm civets are under strong selective pressure and are genetically most closely related to sars-cov strains infecting humans early in the outbreaks (song et al., 2005) . sequence analysis of the sars-cov-like virus in masked palm civets indicated that they were highly homologous to human sars-cov with nucleotide identity over 99.6 %, indicating the virus has not been circulating in the population of masked palm civets for a very long time (shi and hu, 2008) . a chinese ferret-badger (melogale moschata) was found to have neutralising antibodies against sars-cov (guan et al., 2003) , whereas sars-cov rna was detected in naturally infected cats and red foxes (vulpes vulpes), but not in domestic dogs . there was, however, a single dog testing positive for sars-cov (https://apps.who.int/iris/bitstream/handle/10665/ 70863/who_cds_csr_gar_2003.11_eng.pdf). among carnivores, sars-cov-2 is able to infect cats, ferrets and, at a lesser extent, dogs . in 2008, a highly divergent cov, tentatively named sw1, was discovered a deceased beluga wale (delphinapterus leucas) with pneumonia and hepatic necrosis (mihindukulasuriya et al., 2008) . the virus was only distantly related to ibv, so that it now represents the prototype of the single mammalian cov species belonging to the genus gammacoronavirus, namely beluga wale coronavirus sw1 (bwcov-sw1) (subgenus cegacovirus). few years later, related gammacoronaviruses were retrieved from faecal samples of three indo-pacific bottlenose dolphins (tursiops aduncus), which were named bottlenose dolphin cov (bdcov) hku22. comparative genome analysis showed that bdcov-hku22 and bwcov-sw1 have similar genome characteristics and structures, displaying a 98 % nucleotide sequence identity each to other (woo et al., 2014) . a novel betacoronavirus distantly related to mers-cov was detected in the faeces of european hedgehogs (erinaceus europaeus), an insectivorous mammal belonging to a related order of chiroptera, from germany. the virus was tentatively referred to as erinaceus cov (ericov) (corman et al., 2014b) and covs found in hedgehogs in france, england and italy had an identity from 92% to 98 % with the ericov (monchatreleroy et al., 2017; saldanha et al., 2019; delogu et al., 2020) . these hedgehog covs are are now included in a unique species, hedgehog coronavirus 1 (subgenus merbecovirus). the virus was not associated to any form of disease, so that western european hedgehog is a reservoir host of ericov in the absence of apparent disease, suggesting that hedgehogs in addition to bats may contribute to the evolution of merbecovirus (saldanha et al., 2019) . a slightly divergent merbecovirus was later found in amur hedgehogs (erinaceus amurensis) in china and was poposed as a prototype of a separate species, namely erinaceus amurensis hedgehog coronavirus hku31 (ea-hedcov hku31) . a novel coronavirus, named wénchéng shrew coronavirus (wesv) was detected in shrews (suncus murinus) in china . wesv is highly divergent from other alphacoronaviruses, exhibiting less than 71.1 % amino acid similarity to any known members of the genus alphacoronavirus in the coronavirus-wide conserved domains of the replicase polyprotein pp1ab and less than 61.3 % amino acid similarity to the other three coronavirus genera. however, taking into account the current ictv criteria, wesv is sufficiently divergent to be considered a distinct member of the genus alphacoronavirus, but not a new genus of the subfamily orthocornavirinae . covs have been known in veterinary medicine since many decades; some of these viruses, such as ibv, swine enteric covs, bcov and mustelid covs, can cause diseases that have a great impact on the farm industry. other covs, namely fipv, frscv and mhv, cause severe disease in companion (cats, ferrets) or laboratory (mice) animals. animal covs are paradigmatic on how covs evolve through accumulation of point mutations and homologous (and heterologous) recombination, generating different genotypes and pathotypes. these virus variants may have different antigenic properties, escaping the host immunity induced by vaccines, as is the case of ibv. alternatively, they may have a different tissue tropism in the same host that can increase or decrease the virus pathogenicity, as observed for the virus pairs fecv/fipv or frecv/frscv and tgev/prcov, respectively. in other circumstances, the cov evolution may result in the switch of the host range from one animal species to another one or from animals to humans. the former event is well documented in veterinary medicine, with a plethora of viruses being originated from ibv and bcov that adapted to different animal species. however, the most interesting scenario is the jumping and further adaptation of an animal cov to humans. there is increasing evidence that all hcovs currently known recognise an animal origin, with bat or rodent covs being the most probable ancestors. in most instances, it was suggested that other mammals served as intermediate hosts prior to final adaptation to humans, i.e., alpacas and cattle for the low-pathogenic hcov-229e and hcov-oc43, respectively, and wild carnivores and dromedary camels for the high-pathogenic sars-cov and mers-cov, respectively. other two hcovs, namely hcov-nl63 and hcov-hku1, were likely derived from bats and rodents, respectively, but whether this transmission required an intermediate mammalian host is presently unknown. the origin of sars-cov-2 should be zoonotic, since highly related sequences were detected in bats, but a definitive intermediate host has been not identified so far. what should we expect from the current pandemic? when hcov-oc43 crossed the species barrier to infect humans from domestic livestock around 1890, an epidemic of respiratory infection was recorded. even though, several years later, influenza was suspected to be the cause of it, in that pandemic involvement of central nervous system was more pronounced than in other influenza outbreaks. this evidence is further supported by molecular studies claiming that the most recent common ancestor of bcov and hcov-oc43 emerged around 1890 (vijgen et al., 2005) and by the fact that hcov-oc43 can be neuroinvasive (arbour et al., 2000) . likely, hcov-oc43 crossed species to infect dogs becoming established in this species as crcov . a similar scenario could be observed with sars-cov-2 with dogs and, at a greater extent, cats. apparently, cats represent, within the domestic animals which have been experimentally infected, the host, together with ferrets, which is able to sustain more efficiently sars-cov-2 replication . furthermore, based on structural studies and biochemical experiments, sars-cov-2 seems to have an rbd that binds with high affinity to ace2 also from ferrets and cats (andersen et al., 2020) . reasonably, a full comprehension of the animal cov molecular evolution, host range and pathobiology is beneficial to better understand the mechanism driving the emergence and adaptation to humans of zoonotic covs. the present review has highlighted that in the last 18 years, also thanks to the availability of novel sequencing technologies, we have witnessed a large number of novel covs being discovered in a large number of animals. truth to be told, it was difficult for us to summarise, in this single review, all covs detected in animals and the tight interaction existing between them and human covs. among animals, it is evident that bats are the group of mammals that harbor the largest number of covs and that many other animal covs recognise their ancestors in bat covs. in an excellent review (cui et al., 2019) written by the group coordinated by dr. zheng-li shi of the wuhan institute of virology, hubei, (china), city infamously known for being the epicenter and origin of the covid-19 outbreak, authors stated that "...given the prevalence and great genetic diversity of bat sars-rcovs, their close coexistence and the frequent recombination of covs, it is expected that novel variants will emerge in the future". this forecasting statement was not surprising to coronavirologists and it was not, importantly, surprising to those scientists that daily deal with the plethora of viruses existing at the human/animal health interface. although scientists were well aware of this hazard, no substantial actions were taken forward the limitations of strict and repeated contacts between humans and wildlife. indeed, whereas biological mechanisms underlying viral evolution are not under human control, social and cultural habits can be modified accordingly through a deep and pounding informative campaigns. if to the human habits we sum the impact of modern agricultural practices and urbanization and the decrease of vital space for wildlife, it is quite easy to understand that, if countermeasures are not taken, we will face novel serious health emergencies of animal origin in the following years with tremendous social and economic impact on our lives. as clearly demonstrated by the sars-cov-2 emergence, covs are the main characters of this intricate puzzle characterised by the interactions of viral biological mechanisms and human habits. our review was reasonably prepared also to highlight (once more!) how covs originate, evolve, jump, mutate and infect their host. could have the current covid-19 outbreak been avoided? answering this question is not relevant now, but actions to avoid the next viral spillover from animals to humans is certainly a priority. this task needs to be coupled with massive genomic surveillance in wild animals not limited to covs. massive sequencing of sars-cov-2 strains detected in humans and covs of wildlife will help further assess the origin of this novel human pandemic and plan future measures able to reduce the risk of emergence of new cov spillover events. however, additional tasks should be provisionally addressed in order to reduce the risk of future cov pandemic like the current one. these include: i) prevention of animal-to-human infections through a ban of the wet markets and a more friendly management of the environment; ii) studies on cov-host interactions to be performed both in vitro (cell cultures, ex-vivo explants of the respiratory tract) and in vivo (animals susceptible to sars-cov-2 infection); iii) development of new anticoronaviral drugs and evaluation of their efficacy in cell cultures and animal models. feline infectious peritonitis. abcd guidelines on prevention and management inoculation of goats, sheep, and horses with mers-cov does not result in productive viral shedding dampened nlrp3-mediated inflammation in bats and implications for a special viral reservoir host new chimeric porcine coronavirus in swine feces bovine-like coronaviruses isolated from four species of captive wild ruminants are homologous to bovine coronaviruses, based on complete genomic sequences 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supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.vetmic.2020.108693.