Advertisement

Microbial Ecology

, Volume 79, Issue 1, pp 203–212 | Cite as

Alphacoronavirus Detection in Lungs, Liver, and Intestines of Bats from Brazil

  • Cíntia BittarEmail author
  • Rafael Rahal Guaragna Machado
  • Manuela Tosi Comelis
  • Larissa Mayumi Bueno
  • Mateus Rodrigues Beguelini
  • Eliana Morielle-Versute
  • Maurício Lacerda Nogueira
  • Paula Rahal
Host Microbe Interactions

Abstract

Bats are flying mammals distributed worldwide known to host several types of Coronavirus (CoV). Since they were reported as the probable source of spillover of highly pathogenic CoV into the human population, investigating the circulation of this virus in bats around the world became of great importance. We analyzed samples from 103 bats from two distinct regions in Brazil. Coronavirus from the Alphacoronavirus genus was detected in 12 animals, 11 from São José do Rio Preto—SP region and 1 from Barreiras—BA region, resulting in a prevalence of 17.18% and 2.56% respectively. The virus was detected not only in intestines but also in lungs and liver. Phylogenetic analysis based on nsP12 genomic region suggests that the sequences group according to host family and sampling location. Studies on the circulation of these viruses in bats remain important to understand the ecology and evolutionary relationship of these pathogens.

Keywords

Bats Coronavirus Alphacoronavirus Phylogeny 

Notes

Funding

This work was funded by FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico). Grant numbers: FAPESP 2015/09704-6 and CNPq 165802/2015-4.

Compliance with Ethical Standards

This research project was approved by the Ethics Committee on the Use of Animals of the Institute of Biosciences, Letters and Exact Sciences (CEUA/IBILCE-Protocol 135/2016) (Information stated in the Methods section under the headline Samples).

Conflict of Interest

The authors declare that they have no conflict of interest.

Ethical Approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted. This article does not contain any studies with human participants performed by any of the authors.

Supplementary material

248_2019_1391_MOESM1_ESM.xlsx (9 kb)
Online Resource 1: GenBank accession number of the sequences from Dataset 1, including the sequences from this study. (XLSX 9 kb)
248_2019_1391_MOESM2_ESM.xlsx (16 kb)
Online Resource 2: GenBank accession number, host family and species, date and place of host sampling of the sequences from Dataset 2. (XLSX 16 kb)
248_2019_1391_MOESM3_ESM.xlsx (13 kb)
Online Resource 3: Information on sex, family, species and organs of all animals collected in this study. (XLSX 13 kb)
248_2019_1391_MOESM4_ESM.pdf (977 kb)
Online Resource 4: Results of statistical test of association between traits and phylogeny done on BaTS software. A. Statistical analysis of the association of host family and phylogenetic clustering; B. Statistical analysis of the association of location and phylogenetic clustering. Parsimony Score (PS); Association Index (AI); Monophyletic Clade (MC). Values of p ≤ 0.01 were considered significant. (PDF 976 kb)

References

  1. 1.
    Adams MJ, Carstens EB (2012) Ratification vote on taxonomic proposals to the International Committee on Taxonomy of Viruses (2012). Arch Virol 157:1411–1422.  https://doi.org/10.1007/s00705-012-1299-6 CrossRefPubMedGoogle Scholar
  2. 2.
    Weiss SR, Navas-Martin S (2005) Coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus. Microbiol Mol Biol Rev: MMBR 69:635–664.  https://doi.org/10.1128/MMBR.69.4.635-664.2005 CrossRefPubMedGoogle Scholar
  3. 3.
    Coleman CM, Frieman MB (2014) Coronaviruses: important emerging human pathogens. J Virol 88:5209–5212.  https://doi.org/10.1128/JVI.03488-13 CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Zaki AM, van Boheemen S, Bestebroer TM, Osterhaus AD, Fouchier RA (2012) Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia. N Engl J Med 367:1814–1820.  https://doi.org/10.1056/NEJMoa1211721 CrossRefGoogle Scholar
  5. 5.
    Drosten C, Gunther S, Preiser W, van der Werf S, Brodt HR, Becker S, Rabenau H, Panning M, Kolesnikova L, Fouchier RA, Berger A, Burguiere AM, Cinatl J, Eickmann M, Escriou N, Grywna K, Kramme S, Manuguerra JC, Muller S, Rickerts V, Sturmer M, Vieth S, Klenk HD, Osterhaus AD, Schmitz H, Doerr HW (2003) Identification of a novel coronavirus in patients with severe acute respiratory syndrome. N Engl J Med 348:1967–1976.  https://doi.org/10.1056/NEJMoa030747 CrossRefPubMedGoogle Scholar
  6. 6.
    Feldhamer GA, Drickamer LC, Vessey SH, Merritt JF, Krajewski C (2005) Mammalogy: adaptation, diversity, ecology. Johns Hopkins University Press, BaltimoreGoogle Scholar
  7. 7.
    Nogueira MR, IPd L, Moratelli R, Tavares VC, Gregorin R, Peracchi AL (2014) Checklist of Brazilian bats, with comments on original records. Check List 10:808–821.  https://doi.org/10.15560/10.4.808 CrossRefGoogle Scholar
  8. 8.
    Wilson DERD (2005) Mammal species of the world: a taxonomic and geographic reference. Johns Hopkins University Press, BaltimoreGoogle Scholar
  9. 9.
    Luis AD, Hayman DT, O'Shea TJ, Cryan PM, Gilbert AT, Pulliam JR, Mills JN, Timonin ME, Willis CK, Cunningham AA, Fooks AR, Rupprecht CE, Wood JL, Webb CT (2013) A comparison of bats and rodents as reservoirs of zoonotic viruses: are bats special? Proc Biol Sci 280:20122753.  https://doi.org/10.1098/rspb.2012.2753rspb.2012 CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Calisher CH, Childs JE, Field HE, Holmes KV, Schountz T (2006) Bats: important reservoir hosts of emerging viruses. Clin Microbiol Rev 19:531–545.  https://doi.org/10.1128/CMR.00017-06 CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Drexler JF, Gloza-Rausch F, Glende J, Corman VM, Muth D, Goettsche M, Seebens A, Niedrig M, Pfefferle S, Yordanov S, Zhelyazkov L, Hermanns U, Vallo P, Lukashev A, Muller MA, Deng H, Herrler G, Drosten C (2010) 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. J Virol 84:11336–11349.  https://doi.org/10.1128/JVI.00650-10JVI.00650-10 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Drexler JF, Corman VM, Wegner T, Tateno AF, Zerbinati RM, Gloza-Rausch F, Seebens A, Muller MA, Drosten C (2011) Amplification of emerging viruses in a bat colony. Emerg Infect Dis 17:449–456.  https://doi.org/10.3201/eid1703.100526 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Gloza-Rausch F, Ipsen A, Seebens A, Gottsche M, Panning M, Drexler JF, Petersen N, Annan A, Grywna K, Muller M, Pfefferle S, Drosten C (2008) Detection and prevalence patterns of group I coronaviruses in bats, northern Germany. Emerg Infect Dis 14:626–631.  https://doi.org/10.3201/eid1404.071439 CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Pfefferle S, Oppong S, Drexler JF, Gloza-Rausch F, Ipsen A, Seebens A, Muller MA, Annan A, Vallo P, Adu-Sarkodie Y, Kruppa TF, Drosten C (2009) Distant relatives of severe acute respiratory syndrome coronavirus and close relatives of human coronavirus 229E in bats, Ghana. Emerg Infect Dis 15:1377–1384.  https://doi.org/10.3201/eid1509.090224 CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Poon LL, Chu DK, Chan KH, Wong OK, Ellis TM, Leung YH, Lau SK, Woo PC, Suen KY, Yuen KY, Guan Y, Peiris JS (2005) Identification of a novel coronavirus in bats. J Virol 79:2001–2009.  https://doi.org/10.1128/JVI.79.4.2001-2009.2005 CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Chu DK, Poon LL, Chan KH, Chen H, Guan Y, Yuen KY, Peiris JS (2006) Coronaviruses in bent-winged bats (Miniopterus spp.). J Gen Virol 87:2461–2466.  https://doi.org/10.1099/vir.0.82203-0 CrossRefPubMedGoogle Scholar
  17. 17.
    Tang XC, Zhang JX, Zhang SY, Wang P, Fan XH, Li LF, Li G, Dong BQ, Liu W, Cheung CL, Xu KM, Song WJ, Vijaykrishna D, Poon LL, Peiris JS, Smith GJ, Chen H, Guan Y (2006) Prevalence and genetic diversity of coronaviruses in bats from China. J Virol 80:7481–7490.  https://doi.org/10.1128/JVI.00697-06 CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Tong S, Conrardy C, Ruone S, Kuzmin IV, Guo X, Tao Y, Niezgoda M, Haynes L, Agwanda B, Breiman RF, Anderson LJ, Rupprecht CE (2009) Detection of novel SARS-like and other coronaviruses in bats from Kenya. Emerg Infect Dis 15:482–485.  https://doi.org/10.3201/eid1503.081013 CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Quan PL, Firth C, Street C, Henriquez JA, Petrosov A, Tashmukhamedova A, Hutchison SK, Egholm M, Osinubi MO, Niezgoda M, Ogunkoya AB, Briese T, Rupprecht CE, Lipkin WI (2010) Identification of a severe acute respiratory syndrome coronavirus-like virus in a leaf-nosed bat in Nigeria. mBio 1.  https://doi.org/10.1128/mBio.00208-10e00208
  20. 20.
    Anthony SJ, Ojeda-Flores R, Rico-Chavez O, Navarrete-Macias I, Zambrana-Torrelio CM, Rostal MK, Epstein JH, Tipps T, Liang E, Sanchez-Leon M, Sotomayor-Bonilla J, Aguirre AA, Avila-Flores R, Medellin RA, Goldstein T, Suzan G, Daszak P, Lipkin WI (2013) Coronaviruses in bats from Mexico. J Gen Virol 94:1028–1038.  https://doi.org/10.1099/vir.0.049759-0 CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Goes LG, Ruvalcaba SG, Campos AA, Queiroz LH, de Carvalho C, Jerez JA, Durigon EL, Davalos LI, Dominguez SR (2013) Novel bat coronaviruses, Brazil and Mexico. Emerg Infect Dis 19:1711–1713.  https://doi.org/10.3201/eid1910.130525 CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Goes LGB, Campos ACA, Carvalho C, Ambar G, Queiroz LH, Cruz-Neto AP, Munir M, Durigon EL (2016) Genetic diversity of bats coronaviruses in the Atlantic Forest hotspot biome, Brazil. Infect Genet Evol 44:510–513.  https://doi.org/10.1016/j.meegid.2016.07.034 CrossRefPubMedGoogle Scholar
  23. 23.
    Corman VM, Rasche A, Diallo TD, Cottontail VM, Stocker A, Souza BF, Correa JI, Carneiro AJ, Franke CR, Nagy M, Metz M, Knornschild M, Kalko EK, Ghanem SJ, Morales KD, Salsamendi E, Spinola M, Herrler G, Voigt CC, Tschapka M, Drosten C, Drexler JF (2013) Highly diversified coronaviruses in neotropical bats. J Gen Virol 94:1984–1994.  https://doi.org/10.1099/vir.0.054841-0 CrossRefPubMedGoogle Scholar
  24. 24.
    Asano KM, Hora AS, Scheffer KC, Fahl WO, Iamamoto K, Mori E, Brandao PE (2016) Alphacoronavirus in urban Molossidae and Phyllostomidae bats, Brazil. Virol J 13:110.  https://doi.org/10.1186/s12985-016-0569-4 CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Carrington CV, Foster JE, Zhu HC, Zhang JX, Smith GJ, Thompson N, Auguste AJ, Ramkissoon V, Adesiyun AA, Guan Y (2008) Detection and phylogenetic analysis of group 1 coronaviruses in South American bats. Emerg Infect Dis 14:1890–1893.  https://doi.org/10.3201/eid1412.080642 CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Hu B, Ge X, Wang LF, Shi Z (2015) Bat origin of human coronaviruses. Virol J 12:221.  https://doi.org/10.1186/s12985-015-0422-1 CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    NIH (2011) Guide for the care and use of laboratory animals. In: CftUotGftCaUoL (ed) Animals. National Academies Press (US), WashingtonGoogle Scholar
  28. 28.
    Vizotto LD, Taddei VA (1973) Chave para determinação de quirópteros brasileiros. Gráfica Francal, São José do Rio PretoGoogle Scholar
  29. 29.
    Miranda JMD, Bernardi IP, Passos FC (2006) A new species of Eptesicus (Mammalia: Chiroptera: Vespertilionidae) from the Atlantic Forest, Brazil. Zootaxa:57–68Google Scholar
  30. 30.
    LaVal RK (1973) A revision of the Neotropical bats of the genus Myotis. Natural History Museum, Los Angeles County , Los AngelesGoogle Scholar
  31. 31.
    Chu DK, Leung CY, Gilbert M, Joyner PH, Ng EM, Tse TM, Guan Y, Peiris JS, Poon LL (2011) Avian coronavirus in wild aquatic birds. J Virol 85:12815–12820.  https://doi.org/10.1128/JVI.05838-11JVI.05838-11 CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A 74:5463–5467CrossRefGoogle Scholar
  33. 33.
    Ewing B, Green P (1998) Base-calling of automated sequencer traces using phred. II. Error probabilities. Genome Res 8:186–194CrossRefGoogle Scholar
  34. 34.
    Huang X, Madan A (1999) CAP3: a DNA sequence assembly program. Genome Res 9:868–877CrossRefGoogle Scholar
  35. 35.
    Edgar RC (2004) MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC bioinformatics 5:113.  https://doi.org/10.1186/1471-2105-5-113 CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32:1792–1797.  https://doi.org/10.1093/nar/gkh340 CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Gouy M, Guindon S, Gascuel O (2010) SeaView version 4: a multiplatform graphical user interface for sequence alignment and phylogenetic tree building. Mol Biol Evol 27:221–224.  https://doi.org/10.1093/molbev/msp259 CrossRefPubMedGoogle Scholar
  38. 38.
    Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic acids symposium Oxford University PressGoogle Scholar
  39. 39.
    Drummond AJ, Suchard MA, Xie D, Rambaut A (2012) Bayesian phylogenetics with BEAUti and the BEAST 1.7. Mol Biol Evol 29:1969–1973 doi: 10.1093/molbev/mss075mss075 [pii] CrossRefGoogle Scholar
  40. 40.
    Darriba D, Taboada GL, Doallo R, Posada D (2012) jModelTest 2: more models, new heuristics and parallel computing. Nat Methods 9:772.  https://doi.org/10.1038/nmeth.2109nmeth CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Letunic I, Bork P (2007) Interactive Tree Of Life (iTOL): an online tool for phylogenetic tree display and annotation. Bioinformatics 23:127–128.  https://doi.org/10.1093/bioinformatics/btl529 CrossRefPubMedGoogle Scholar
  42. 42.
    Letunic I, Bork P (2016) Interactive tree of life (iTOL) v3: an online tool for the display and annotation of phylogenetic and other trees. Nucleic Acids Res 44:W242–W245.  https://doi.org/10.1093/nar/gkw290 CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Letunic I, Bork P (2011) Interactive Tree Of Life v2: online annotation and display of phylogenetic trees made easy. Nucleic Acids Res 39:W475–W478.  https://doi.org/10.1093/nar/gkr201 CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Parker J, Rambaut A, Pybus OG (2008) Correlating viral phenotypes with phylogeny: accounting for phylogenetic uncertainty. Infect Genet Evol 8:239–246.  https://doi.org/10.1016/j.meegid.2007.08.001 CrossRefPubMedGoogle Scholar
  45. 45.
    QGIS (2018) QGIS Development Team - QGIS Geographic Information System. Open Source Geospatial Foundation ProjectGoogle Scholar
  46. 46.
    IBGE (2007) IBGE - Instituto Brasileiro de Geografia e Estatística - bases cartográficas. https://mapas.ibge.gov.br/bases-e-referenciais/bases-cartograficas.html. Accessed 2018
  47. 47.
    Lau SK, Woo PC, Li KS, Huang Y, Wang M, Lam CS, Xu H, Guo R, Chan KH, Zheng BJ, Yuen KY (2007) Complete genome sequence of bat coronavirus HKU2 from Chinese horseshoe bats revealed a much smaller spike gene with a different evolutionary lineage from the rest of the genome. Virology 367:428–439.  https://doi.org/10.1016/j.virol.2007.06.009 CrossRefPubMedGoogle Scholar
  48. 48.
    August TA, Mathews F, Nunn MA (2012) Alphacoronavirus detected in bats in the United Kingdom. Vector Borne Zoonotic Dis 12:530–533.  https://doi.org/10.1089/vbz.2011.0829 CrossRefPubMedGoogle Scholar
  49. 49.
    Goffard A, Demanche C, Arthur L, Pincon C, Michaux J, Dubuisson J (2015) Alphacoronaviruses detected in French bats are phylogeographically linked to coronaviruses of European bats. Viruses 7:6279–6290 doi: 10.3390/v7122937v7122937 [pii] CrossRefGoogle Scholar
  50. 50.
    Osborne C, Cryan PM, O'Shea TJ, Oko LM, Ndaluka C, Calisher CH, Berglund AD, Klavetter ML, Bowen RA, Holmes KV, Dominguez SR (2011) Alphacoronaviruses in New World bats: prevalence, persistence, phylogeny, and potential for interaction with humans. PLoS One 6:e19156.  https://doi.org/10.1371/journal.pone.0019156PONE-D-11-00052 CrossRefPubMedPubMedCentralGoogle Scholar
  51. 51.
    Lelli D, Papetti A, Sabelli C, Rosti E, Moreno A, Boniotti MB (2013) Detection of coronaviruses in bats of various species in Italy. Viruses 5:2679–2689.  https://doi.org/10.3390/v5112679v5112679 CrossRefPubMedPubMedCentralGoogle Scholar
  52. 52.
    Dominguez SR, O'Shea TJ, Oko LM, Holmes KV (2007) Detection of group 1 coronaviruses in bats in North America. Emerg Infect Dis 13:1295–1300.  https://doi.org/10.3201/eid1309.070491 CrossRefPubMedPubMedCentralGoogle Scholar
  53. 53.
    Wacharapluesadee S, Duengkae P, Rodpan A, Kaewpom T, Maneeorn P, Kanchanasaka B, Yingsakmongkon S, Sittidetboripat N, Chareesaen C, Khlangsap N, Pidthong A, Leadprathom K, Ghai S, Epstein JH, Daszak P, Olival KJ, Blair PJ, Callahan MV, Hemachudha T (2015) Diversity of coronavirus in bats from Eastern Thailand. Virol J 12:57.  https://doi.org/10.1186/s12985-015-0289-110.1186/s12985-015-0289-1 CrossRefPubMedPubMedCentralGoogle Scholar
  54. 54.
    Anthony SJ, Johnson CK, Greig DJ, Kramer S, Che X, Wells H, Hicks AL, Joly DO, Wolfe ND, Daszak P, Karesh W, Lipkin WI, Morse SS, Mazet JAK, Goldstein T (2017) Global patterns in coronavirus diversity. Virus Evol 3:vex012.  https://doi.org/10.1093/ve/ CrossRefPubMedPubMedCentralGoogle Scholar
  55. 55.
    Luo Y, Li B, Jiang RD, Hu BJ, Luo DS, Zhu GJ, Hu B, Liu HZ, Zhang YZ, Yang XL, Shi ZL (2018) Longitudinal surveillance of Betacoronaviruses in fruit bats in Yunnan Province, China during 2009-2016. Virol Sin 33:87–95.  https://doi.org/10.1007/s12250-018-0017-2 CrossRefPubMedPubMedCentralGoogle Scholar
  56. 56.
    Hu D, Zhu C, Wang Y, Ai L, Yang L, Ye F, Ding C, Chen J, He B, Zhu J, Qian H, Xu W, Feng Y, Tan W, Wang C (2017) Virome analysis for identification of novel mammalian viruses in bats from Southeast China. Sci Rep 7:10917.  https://doi.org/10.1038/s41598-017-11384-w CrossRefPubMedPubMedCentralGoogle Scholar
  57. 57.
    Watanabe S, Masangkay JS, Nagata N, Morikawa S, Mizutani T, Fukushi S, Alviola P, Omatsu T, Ueda N, Iha K, Taniguchi S, Fujii H, Tsuda S, Endoh M, Kato K, Tohya Y, Kyuwa S, Yoshikawa Y, Akashi H (2010) Bat coronaviruses and experimental infection of bats, the Philippines. Emerg Infect Dis 16:1217–1223.  https://doi.org/10.3201/eid1608.100208 CrossRefPubMedPubMedCentralGoogle Scholar
  58. 58.
    Hutterer RIT, Meyer-Cords C, Rodrigues L (2005) Bat migrations in Europe: a review of banding data and literature. Federal Agency for Nature Conservation, BonnGoogle Scholar
  59. 59.
    Adams RA, Pedersen SC 2013 Bat evolution, ecology, and conservationGoogle Scholar
  60. 60.
    Arnone IS, Trajano E, Pulchério-Leite A, Passos FC (2016) Long-distance movement by a great fruit-eating bat, Artibeus lituratus (Olfers, 1818), in southeastern Brazil (Chiroptera, Phyllostomidae): evidence for migration in Neotropical bats? Biota Neotropica 16.  https://doi.org/10.1590/1676-0611-BN-2015-0026

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Cíntia Bittar
    • 1
    Email author
  • Rafael Rahal Guaragna Machado
    • 1
  • Manuela Tosi Comelis
    • 1
  • Larissa Mayumi Bueno
    • 1
  • Mateus Rodrigues Beguelini
    • 1
    • 2
  • Eliana Morielle-Versute
    • 1
  • Maurício Lacerda Nogueira
    • 3
  • Paula Rahal
    • 1
  1. 1.Instituto de Biociências, Letras e Ciências Exatas (IBILCE)Universidade Estadual Paulista “Júlio de Mesquita Filho” (UNESP) - Campus de São José do Rio PretoSão Jose do Rio PretoBrazil
  2. 2.Centro das Ciências Biológicas e da Saúde (CCBS)Universidade Federal do Oeste da Bahia (UFOB)BarreirasBrazil
  3. 3.Faculdade de Medicina de São José do Rio Preto (FAMERP)Sao Jose do Rio PretoBrazil

Personalised recommendations