a total of 114 bats collected from their natural habitats from december 2006 through july 2007 ( 8 species from 10 locations ; table ) were euthanized after being deeply anesthetized with 2% xylazine and 10% ketamine administered subcutaneously ( in some cases after sedation with co2 ) . the bats were then taxonomically classified on the basis of morphology , and the carcasses were stored at 70c until used . for sampling , the carcasses were thawed at 4c for 34 hours , then oropharyngeal and anal samples were taken with dacron - tipped swabs that were then placed in rnalater ( ambion , austin , tx , usa ) and stored at 20c until used . briefly , viral rna was extracted from swabs by using the qiaamp viral rna minikit ( qiagen , westburg , the netherlands ) and used as the template for reverse transcription primers based on the rdrp gene , conserved for all known coronaviruses , were then used for rt - pcr detection . the rdrp pcr products were gel purified by using the qiaquick pcr purification kit ( qiagen ) and sequenced to confirm virus species . rna from samples positive for coronavirus was then used for cdna synthesis by using random hexamer , gene - specific , and oligo(dt ) primers . the rdrp gene and 1b open reading frames , including the helicase ( hel ) domain , were then sequenced , also as previously described ( 4 ) . sequences derived from this study were deposited in genbank ( accession nos . eu769557 and eu769558 ) . sequences were aligned with previously published cov sequences from genbank by using clustalx ( http://bips.u-strasbg.fr/fr/documentation/clustalx ) then manually aligned by using the se - al program ( http://tree.bio.ed.ac.uk/software/seal ) . the genbank accession numbers of all sequences used are noted in the taxon names in figures 1 and 2 . the rdrp sequences were trimmed to equal length , which created 2 datasets of 780 bp ( n = 40 ) and 378 bp ( n = 45 ) . the latter included bt - cov sequences from north america and germany that were too short to be included in the first dataset . a third dataset comprised an alignment of the hel domain ( n = 46 ) trimmed to 1,797 bp . maximum likelihood ( ml ) phylogenies were inferred under a general time reversible ( gtr + 4 + i ) model , which was identified as the best - fit model of nucleotide substitution using modeltest version 3.7 ( 10 ) . bootstrapping was performed to assess the robustness of tree topologies by using 1,000 replicate neighbor - joining ( nj ) trees under the ml substitution model . all analyses were performed with paup * version 4.0b ( sinauer associates , inc . , sunderland , ma , usa ) . maximum likelihood trees of coronaviruses based on a ) 780-bp fragment of the rna - dependent rna polymerase gene and b ) 1,797 bp of the helicase ( hel ) domain of open reading frame 1b . trees were inferred under the general time reversible ( gtr + 4 + i ) model by using paup * version 4.0b ( sinauer associates , inc . , sunderland , ma , usa ) . previously defined phylogenetic groups and a putative novel group ( 10 ) are delineated by the bars on the right . the numbering of these groups is as described in the eighth report of the international committee on taxonomy of viruses with the alternative grouping proposed by tang et al . maximum likelihood tree of coronaviruses based on 378-bp fragment of the rna - dependent rna polymerase gene . the tree was inferred under the general time reversible ( gtr + 4 + i ) by using paup * version 4.0b ( sinauer associates , inc . , trinidadian bat coronavirus ( bt - cov ) sequences are highlighted in red and north american bt - cov in blue . previously defined phylogenetic groups and a putative novel group ( 10 ) are delineated by the bars on the right . the numbering of these groups is as described in the eighth report of the international committee on taxonomy of viruses with the alternative grouping proposed by tang et al . bootstrap support values for groups 1a , 1b , 2a  c , 3 , and the lineage containing trinidadian bt - covs are shown . cov rna was detected in 1 of 21 glossophaga soricina and 1 of 5 carollia perspicillata bats tested . the latter , designated bt - cov / trinidad/1fy2b , was an adult male from the area of fyzabad , and virus was detected in anal and oropharyngeal swabs . the infected g. soricina specimen ( bt - cov / trinidad/1co7b ) was an adult female from the area of couva , and the virus was detected in the anal swab only . a total of 3,905 bp of rdrp from bt - cov / tri/1co7b and 5,160 bp from bt - cov / tri/1fy2b were sequenced , in addition to 1,782 bp from the hel domain of both samples ( full sequence data not shown ) . across the shared 3,798-bp region of rdrp , divergence was 28.7% at the nucleotide level and 15.5% at the amino acid level . similarly , the hel domains of the 2 viruses showed 27.3% divergence at the nucleotide level and 13.3% at the amino acid level . ml phylogenies inferred from the rdrp genes of 40 viruses ( 780 bp ) and the hel domains of 46 viruses ( 1,797 bp ) are shown in figure 1 . in both cases , the 5 groups proposed by tang et al . ( 4 ) on the basis of nj trees , and the lineage containing the recently reported novel cov sequences ( 11 ) , were strongly supported with bootstrap values > 95% in all cases . in each case the trinidadian sequences clustered with group 1 covs within a clade containing all other group 1 bat and human covs as well as porcine cov . to determine the phylogenetic relationship between the trinidadian bt - covs and north american bt - covs , for which only relatively short sequences for rdrp were available , we inferred a second rdrp ml phylogeny based on a 378-bp fragment ( figure 2 ) . when this shorter fragment was used , 5 groups were again defined , but bootstrap support for the putative group 5 ( 4 ) was lower . the level of divergence between trinidadian sequences was notably higher than among the north american ( 6 ) and german sequences ( 7 ) . our detection of rna from group 1 covs in trinidadian bats shows that bt - covs have a wider distribution than previously suspected and is added support for bats as the original host species for these viruses . group 1 covs form 2 well - supported clades designated 1a and 1b ( 12 ) . the trinidadian bt - cov clustered within the latter clade , which contains all other group 1 bt - covs , including those from germany and north america , and the 3 known group 1 human covs associated with respiratory illness ( 1315 ) . despite the geographic proximity of the bats from which the trinidadian bt - cov sequences were derived  couva and fyzabad are 28 km ( 17 miles ) apart , and trinidad has an area of only 4,769 km ( 1,864 square miles)they are relatively highly divergent . this divergence might reflect virus adaptations to different host species ; however , more data would be needed to confirm this . given the mobility of bats , the possibility of the viruses having different geographic origins ( perhaps even from outside trinidad ) can not be ruled out . further work on cov diversity in trinidad and the rest of the americas , as well as on the ecology and behavior of susceptible bat species , is needed to understand the origins , evolution , and dispersal of these viruses .
bat coronaviruses ( bt - covs ) are thought to be the precursors of severe acute respiratory syndrome coronavirus . 
 we detected bt - covs in 2 bat species from trinidad . 
 phylogenetic analysis of the rna - dependent rna polymerase gene and helicase confirmed them as group 1 coronaviruses .