Longitudinal monitoring in Cambodia suggests higher circulation of alpha and betacoronaviruses in juvenile and immature bats of three species

Abstract

Recent studies suggest that coronaviruses circulate widely in Southeast Asian bat species and that the progenitors of the SARS-Cov-2 virus could have originated in rhinolophid bats in the region. Our objective was to assess the diversity and circulation patterns of coronavirus in several bat species in Southeast Asia. We undertook monthly live-capture sessions and sampling in Cambodia over 17 months to cover all phases of the annual reproduction cycle of bats and test specifically the association between their age and CoV infection status. We additionally examined current information on the reproductive phenology of Rhinolophus and other bat species presently known to occur in mainland southeast China, Vietnam, Laos and Cambodia. Results from our longitudinal monitoring (573 bats belonging to 8 species) showed an overall proportion of positive PCR tests for CoV of 4.2% (24/573) in cave-dwelling bats from Kampot and 4.75% (22/463) in flying-foxes from Kandal. Phylogenetic analysis showed that the PCR amplicon sequences of CoVs (n = 46) obtained clustered in Alphacoronavirus and Betacoronavirus. Interestingly, Hipposideros larvatus sensu lato harbored viruses from both genera. Our results suggest an association between positive detections of coronaviruses and juvenile and immature bats in Cambodia (OR = 3.24 [1.46-7.76], p = 0.005). Since the limited data presently available from literature review indicates that reproduction is largely synchronized among rhinolophid and hipposiderid bats in our study region, particularly in its more seasonal portions (above 16° N), this may lead to seasonal patterns in CoV circulation. Overall, our study suggests that surveillance of CoV in insectivorous bat species in Southeast Asia, including SARS-CoV-related coronaviruses in rhinolophid bats, could be targeted from June to October for species exhibiting high proportions of juveniles and immatures during these months. It also highlights the need to develop long-term longitudinal surveys of bats and improve our understanding of their ecology in the region, for both biodiversity conservation and public health reasons.

Figure 1

Map of Cambodia showing the two study sites (generated with QGIS 3.18, www.qgis.org).

Figure 2

Results of the longitudinal sampling of three bat species for coronaviruses. The first graph shows estimation of the coronavirus prevalence (with CI95) for Pteropus lylei at the second study site in Kandal province. The second and third graph show the same for Hipposideros larvatus s.l. and Eonycteris spelaea (respectively) at the first study site in Kampot province. The light grey areas show periods when pregnant females were captured, whereas dark grey areas show periods when lactating females were captured.

Figure 3

Phylogenetic tree of (A) alphaCoV and (B) betaCoV partial RdRp gene with modified primers. The sequences detected in Cambodia are marked by red triangles for strains in the current study and black diamonds for strains previously described by Lacroix et al.. The tree was built using the maximum likelihood method based on the GTR + G4 + I model. The robustness of nodes was assessed with 1000 bootstrap replicates. Bootstrap values < 70 are not shown. The GenBank accession numbers of CoV detected in this study and included in the phylogenetic analysis are: MW507190–MW507207 and MW507209–MW507235.

Figure 3

Phylogenetic tree of (A) alphaCoV and (B) betaCoV partial RdRp gene with modified primers. The sequences detected in Cambodia are marked by red triangles for strains in the current study and black diamonds for strains previously described by Lacroix et al.. The tree was built using the maximum likelihood method based on the GTR + G4 + I model. The robustness of nodes was assessed with 1000 bootstrap replicates. Bootstrap values < 70 are not shown. The GenBank accession numbers of CoV detected in this study and included in the phylogenetic analysis are: MW507190–MW507207 and MW507209–MW507235.

Figure 4

Summary of available data on the reproductive phenology of Rhinolophus, Hipposideros and Eonycteris species in mainland southeast China, Vietnam, Laos and Cambodia. The chart shows the number of species for which a reproduction phase has been observed in a given month of the year for the northern (above 16° N) and southern (below 16° N) portions of the region. Each cell is shaded proportionally according to the number of species for which data were retrieved during the literature review, whereas superscript figures refer to the number of studies for which reproductive data were available for the genus in a given month.