A Cross Sectional Sampling Reveals Novel Coronaviruses in Bat Populations of Georgia

Abstract

Mammal-associated coronaviruses have a long evolutionary history across global bat populations, which makes them prone to be the most likely ancestral origins of coronavirus-associated epidemics and pandemics globally. Limited coronavirus research has occurred at the junction of Europe and Asia, thereby investigations in Georgia are critical to complete the coronavirus diversity map in the region. We conducted a cross-sectional coronavirus survey in bat populations at eight locations of Georgia, from July to October of 2014. We tested 188 anal swab samples, remains of previous pathogen discovery studies, for the presence of coronaviruses using end-point pan-coronavirus RT-PCR assays. Samples positive for a 440 bp amplicon were Sanger sequenced to infer coronavirus subgenus or species through phylogenetic reconstructions. Overall, we found a 24.5% positive rate, with 10.1% for Alphacoronavirus and 14.4% for Betacoronavirus. Albeit R. euryale, R. ferrumequinum, M. blythii and M. emarginatus were found infected with both CoV genera, we could not rule out CoV co-infection due to limitation of the sequencing method used and sample availability. Based on phylogenetic inferences and genetic distances at nucleotide and amino acid levels, we found one putative new subgenus and three new species of Alphacoronavirus, and two new species of Betacoronavirus.

Keywords: alphacoronavirus; bats; betacoronavirus; coronavirus; eastern europe; georgia; phylogeny.

Figure 1

Geographic location and relative proportion of bat species collected per collection site. Red circles indicate the approximate location of collection site. Pie charts indicate the relative proportion of each bat species collected per site. Size of the pie charts indicates the relative number of samples collected per site. Asterisks within the pie indicate whether individuals of that species tested positive for alpha (red) and/or beta CoVs (blue). Lack of asterisks indicates no CoV were detected in a bat species and/or site. Color codes for each bat species represented in the pie charts are indicated in the lower part of the figure.

Figure 2

Unrooted Bayesian phylogenetic tree for AlphaCoVs generated with a partial informative 411 bp fragment of the RdRp. Representative BetaCoV taxa were used in the alignment to demonstrate consistent segregation between both genera. Values at nodes represent branch support values expressed as Bayesian posterior probabilities. Scale bar indicates branch lengths. Highlighted nodes are robustly supported with colored bars indicating relevant branches encompassing taxa pertaining to a given subgenus on the left. Bars on the right indicate taxa pertaining to recognized coronavirus species according to the most recent ICTV classification scheme. Taxa highlighted in orange on the right side of the tree indicate coronavirus sequences obtained from Georgian bats. Dark red bars indicate unclassified coronavirus subgenera or species identified herein. Taxa names for all reference sequences in the tree start with their respective GenBank accession number. Animal silhouettes on the extreme right indicate the animal species from which reference sequences were recovered.

Figure 3

Unrooted Bayesian phylogenetic tree for beta CoVs generated with a partial informative 411 bp fragment of the RdRp. Representative BetaCoV taxa were used in the alignment to demonstrate consistent segregation between both genera. Values at nodes represent branch support values expressed as Bayesian posterior probabilities. Scale bar indicates branch lengths. Highlighted nodes are robustly supported with colored bars indicating relevant branches encompassing taxa pertaining to a given subgenus on the left. Bars on the right indicate taxa pertaining to recognized CoV species according to the most recent ICTV classification scheme. Taxa highlighted in orange on the right side of the tree indicate CoV sequences obtained from Georgian bats, and the dark blue bars indicate unclassified CoV species. Taxa names for all reference sequences in the tree start with their respective GenBank accession number. Animal silhouettes on the extreme right indicate the animal species from which reference sequences were recovered.

Figure 4

Distribution maps for bat species found to be CoV positive in this study compared with the geographic range of other bat species presenting highly similar CoVs across Eurasia as observed in our phylogenetic reconstructions. (A) depicts the geographic range of bat species with highly similar AlphaCoV obtained from Georgia with those reported in other countries across Eurasia. (B) depicts the geographic range of bat species with highly similar BetaCoV obtained from Georgia with those reported in other countries across Eurasia.

Figure 4

Distribution maps for bat species found to be CoV positive in this study compared with the geographic range of other bat species presenting highly similar CoVs across Eurasia as observed in our phylogenetic reconstructions. (A) depicts the geographic range of bat species with highly similar AlphaCoV obtained from Georgia with those reported in other countries across Eurasia. (B) depicts the geographic range of bat species with highly similar BetaCoV obtained from Georgia with those reported in other countries across Eurasia.