Infection, Genetics and Evolution 44 (2016) 510–513 Contents lists available at ScienceDirect Infection, Genetics and Evolution j ourna l homepage: www.e lsev ie r .com/ locate /meeg id Short communication Genetic diversity of bats coronaviruses in the Atlantic Forest hotspot biome, Brazil Luiz Gustavo Bentim Góes a,⁎, Angélica Cristine de Almeida Campos a, Cristiano de Carvalho b, Guilherme Ambar c, Luzia Helena Queiroz b, Ariovaldo Pereira Cruz-Neto c, Muhammad Munir d, Edison Luiz Durigon a a Universidade de São Paulo (USP), Dept. Microbiologia, Instituto de Ciências Biomédicas (ICB), Sao Paulo, SP, Brazil b Universidade Estadual Paulista (UNESP), Dept. de Apoio, Produção e Saúde Animal, Faculdade de Medicina Veterinária de Araçatuba, Araçatuba, SP, Brazil c Universidade Estadual Paulista (UNESP), Dept. Zoologia, Instituto de Biociências, Rio Claro, SP, Brazil d The Pirbright Institute, Woking, Surrey, United Kingdom ⁎ Corresponding author. E-mail addresses: lgbgoes@usp.br (L.G.B. Góes), campo criscar@fmva.unesp.br (C. Carvalho), guilhermeambar@gm lhqueiroz@fmva.unesp.br (L.H. Queiroz), ariovaldopcruz@ muhammad.munir@pirbright.ac.uk (M. Munir), eldurigo@ http://dx.doi.org/10.1016/j.meegid.2016.07.034 1567-1348/© 2016 Elsevier B.V. All rights reserved. a b s t r a c t a r t i c l e i n f o Article history: Received 3 May 2016 Received in revised form 22 July 2016 Accepted 25 July 2016 Available online 26 July 2016 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. © 2016 Elsevier B.V. All rights reserved. 1. Introduction 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 vary- ing 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, theβ-CoVs can fur- ther be subdivided into four clades: A to D (Drexler et al., 2014). All CoVs that can potentially infect human were originated from an- imal reservoirs, and four of such CoVs are believe to be transmitted to sac@usp.br (A.C.A. Campos), ail.com (G. Ambar), gmail.com (A.P. Cruz-Neto), usp.br (E.L. Durigon). 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 previ- ous 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 possi- ble 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 collect- ed from Iguaçu National Park, and from two distinct disturbed land- scape sites, respectively, and provided by our collaborators of the Zoology Department at UNESP, Rio Claro-SP. All activities were http://crossmark.crossref.org/dialog/?doi=10.1016/j.meegid.2016.07.034&domain=pdf http://dx.doi.org/10.1016/j.meegid.2016.07.034 mailto:eldurigo@usp.br Journal logo http://dx.doi.org/10.1016/j.meegid.2016.07.034 Unlabelled image http://www.sciencedirect.com/science/journal/ www.elsevier.com/locate/meegid Table 1 Bat species tested for CoV RNA in different ecological regions of Brazil. Species No of samples tested/no positive (%) Sex - no of samples tested/no positive (%) Location - no of samples tested/no CoV positive (%) Bats family Bats diet types Male Female Undist. Forest Fragm. Forest Urban area Artibeus fimbriatus 3/0 3/0 0/0 3/0 0/0 0/0 Phyllostomidae Frugivorous Artibeus lituratus 126/3 (2.4%) 79/0 44/3 (6.8%) 60/2 (3.3%) 60/1 (1.6%) 6/0 Phyllostomidae Frugivorous Artibeus obscurus 1/0 1/0 0/0 0/0 0/0 1/0 Phyllostomidae Frugivorous Artibeus planirostris 4/0 4/0 0/0 2/0 1/0 1/0 Phyllostomidae Frugivorous Carollia perspicillata 44/3 (6.8%) 30/2 (6.7%) 14/1 (7.1%) 12/0 32/3 (9.4%) 0/0 Phyllostomidae Frugivorous Desmodus rotundus 10/0 8/0 2/0 0/0 10a/0 0/0 Phyllostomidae Hematophagous Eptesicus furinalis 5/0 3/0 2/0 0/0 0/0 5/0 Vespertilionidae Insectivorous Eumops glaucinus 27/1 (3.7%) 11/0 16/1 (6.2%) 0/0 0/0 27/1 (3.7%) Molossidae Insectivorous Glossophaga soricina 3/1 (33.3%) 2/1 (50%) 1/0 0/0 0/0 3/1 (33.3%) Phyllostomidae Nectivorous Lasiurus cinereus 1/0 1/0 0/0 0/0 0/0 1/0 Vespertilionidae Insectivorous Molossus molossus 80/0 42/0 38/0 0/0 0/0 80/0 Molossidae Insectivorous Molossus rufus 56/2 (3.6%) 37/1 (2.7%) 19/1 (5.2%) 0/0 0/0 56/2 (3.6%) Molossidae Insectivorous Myotis nigricans 8/1 (12.5%) 7/1 (14.3%) 1/0 0/0 0/0 8/1 (12.5%) Vespertilionidae Insectivorous Myotis riparius 1/1 (100%) 1/0 1/1 (100%) 0/0 0/0 1/1 (100%) Vespertilionidae Insectivorous Platyrrhinus lineatus 3/0 1/0 2/0 0/0 0/0 3/0 Phyllostomidae Frugivorous Sturnira lilium 28/3 (10.7%) 19/0 9/3 (33.3%) 22/2 (9.1%) 6/1 (16.6%) 0/0 Phyllostomidae Frugivorous Vampyressa pusilla 1/0 1/0 0/0 0/0 1/1 (100%) 0/0 Phyllostomidae Frugivorous Total 401/15 (3.7%) 251/5 (2%) 149/10 (6.7%) 99/4 (4%) 110/5 (4.5%) 192/6 (3.1%) Undist. Forest (Undisturbed Forest), Fragm. Forest (Fragmented Forest); bold font indicates species positive for CoV RNA. a Sample obtained from rural area. 511L.G.B. Góes et al. / Infection, Genetics and Evolution 44 (2016) 510–513 authorized and approved by theEthic Committee of Institute of Biomed- ical Research from University of São Paulo (56-18-03/2014) and bats species were identified based on the morphological characteristics in- cluding 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 Ar- chive 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 specieswith distinct diet habit, demonstrating amarked potential of CoVs distribution among bat species in AFB that harbours 9% of world's bat diversity. All CoVs-positive bat species were geographical distribut- ed 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 13α-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 geographi- cally distant regions. This relation can be exemplified by the high simi- larity 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%) andweremost closely re- lated 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 ofα-CoVs lineage 1 species from bats and other animal species. However, extensive evolutionary studies on complete genome sequences of these iso- lates are required to provide information on the virus origin and divergence. Out of two β-CoVs, the virus from Eumops glaucinus clusteredwithin MERS-CoV containing lineage C. The second β-CoV detected in Artibeus lituratus shownhighest similarity (96.4%)with CoV reported fromCosta 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 humanMERS-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. Itwas not possible to rule out any event of virus trans- mission to human, however, this highlight the potential of domesticated animals in virus transmission and disease dynamics at the virus-animal- human 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 re- lationship between lineage Cβ-CoVs in Asia, Africa and Europe, cumula- tive 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). 512 L.G.B. Góes et al. / Infection, Genetics and Evolution 44 (2016) 510–513 Image of Fig. 1 513L.G.B. Góes et al. / Infection, Genetics and Evolution 44 (2016) 510–513 2. Conclusions Wepresent a great diversity of CoV genotypes and clusters in Brazilian bats, highlighting a biogeographic distribution of bats CoVs in the re- gion. 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 humanbeing. 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. Acknowledgements This study was supported by FAPESP (São Paulo Research Founda- tion) process number 2013/11006-0 and British Council grant number 172710323. 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Discovery of seven novel mamma- lian 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. J. Virol. 86, 3995–4008. –q) in the Atlantic Forest Biome from two adjacent Paraná and São Paulo states, Brazil. B: ilies were bold and underlined). Phylogenetic tree was adapted from Simmons (2005). C: . Brazilian sequences reported in this study aremarkedwith arrows. D: A higher resolution filled square. Originalmap available in http://mapas.sosma.org.br fromAtlas of theAtlantic doi:10.1016/j.meegid.2016.07.034 doi:10.1016/j.meegid.2016.07.034 http://refhub.elsevier.com/S1567-1348(16)30324-0/rf0005 http://refhub.elsevier.com/S1567-1348(16)30324-0/rf0005 http://refhub.elsevier.com/S1567-1348(16)30324-0/rf0010 http://refhub.elsevier.com/S1567-1348(16)30324-0/rf0010 http://refhub.elsevier.com/S1567-1348(16)30324-0/rf0015 http://refhub.elsevier.com/S1567-1348(16)30324-0/rf0015 http://refhub.elsevier.com/S1567-1348(16)30324-0/rf0020 http://refhub.elsevier.com/S1567-1348(16)30324-0/rf0020 http://refhub.elsevier.com/S1567-1348(16)30324-0/rf0025 http://refhub.elsevier.com/S1567-1348(16)30324-0/rf0025 http://refhub.elsevier.com/S1567-1348(16)30324-0/rf0030 http://refhub.elsevier.com/S1567-1348(16)30324-0/rf0030 http://refhub.elsevier.com/S1567-1348(16)30324-0/rf0030 http://refhub.elsevier.com/S1567-1348(16)30324-0/rf0035 http://refhub.elsevier.com/S1567-1348(16)30324-0/rf0040 http://refhub.elsevier.com/S1567-1348(16)30324-0/rf0045 http://refhub.elsevier.com/S1567-1348(16)30324-0/rf0045 http://refhub.elsevier.com/S1567-1348(16)30324-0/rf0050 http://refhub.elsevier.com/S1567-1348(16)30324-0/rf0050 http://refhub.elsevier.com/S1567-1348(16)30324-0/rf0055 http://refhub.elsevier.com/S1567-1348(16)30324-0/rf0055 http://refhub.elsevier.com/S1567-1348(16)30324-0/rf0060 http://refhub.elsevier.com/S1567-1348(16)30324-0/rf0060 http://refhub.elsevier.com/S1567-1348(16)30324-0/rf0065 http://refhub.elsevier.com/S1567-1348(16)30324-0/rf0065 http://refhub.elsevier.com/S1567-1348(16)30324-0/rf0065 http://www.ictvonline.org/virusTaxonomy.asp http://www.ictvonline.org/virusTaxonomy.asp http://refhub.elsevier.com/S1567-1348(16)30324-0/rf0075 http://refhub.elsevier.com/S1567-1348(16)30324-0/rf0075 http://refhub.elsevier.com/S1567-1348(16)30324-0/rf0080 http://refhub.elsevier.com/S1567-1348(16)30324-0/rf0080 http://refhub.elsevier.com/S1567-1348(16)30324-0/rf0085 http://refhub.elsevier.com/S1567-1348(16)30324-0/rf0085 http://refhub.elsevier.com/S1567-1348(16)30324-0/rf0090 http://refhub.elsevier.com/S1567-1348(16)30324-0/rf0090 http://refhub.elsevier.com/S1567-1348(16)30324-0/rf0095 http://refhub.elsevier.com/S1567-1348(16)30324-0/rf0095 http://refhub.elsevier.com/S1567-1348(16)30324-0/rf0095 http://refhub.elsevier.com/S1567-1348(16)30324-0/rf0100 http://refhub.elsevier.com/S1567-1348(16)30324-0/rf0100 http://refhub.elsevier.com/S1567-1348(16)30324-0/rf0100 http://refhub.elsevier.com/S1567-1348(16)30324-0/rf0105 http://refhub.elsevier.com/S1567-1348(16)30324-0/rf0110 http://refhub.elsevier.com/S1567-1348(16)30324-0/rf0110 http://refhub.elsevier.com/S1567-1348(16)30324-0/rf0115 http://refhub.elsevier.com/S1567-1348(16)30324-0/rf0115 http://refhub.elsevier.com/S1567-1348(16)30324-0/rf0120 http://refhub.elsevier.com/S1567-1348(16)30324-0/rf0120 http://refhub.elsevier.com/S1567-1348(16)30324-0/rf0120 http://refhub.elsevier.com/S1567-1348(16)30324-0/rf0120 http://refhub.elsevier.com/S1567-1348(16)30324-0/rf0120 http://mapas.sosma.org.br Genetic diversity of bats coronaviruses in the Atlantic Forest hotspot biome, Brazil 1. Introduction 2. Conclusions Acknowledgements References