Genetic analyses of Rhinolophus species identified as reservoirs of viruses closely related to SARS-CoV-2
Until now, SCoV2rCs have been found in four bat species of the genus Rhinolophus: R. acuminatus, R. affinis, R. malayanus, and R. shameli. The haplotype networks constructed using CO1 sequences of these four species are shown in Fig. 3. A star-like genetic pattern, characterized by one dominant haplotype and several satellite haplotypes was found for the two bat species endemic to Southeast Asia, i.e. R. acuminatus and R. shameli.
Figure 3 Haplotype networks based on CO1 sequences of the four Rhinolophus species found positive for viruses closely related to SARS-CoV-2 (SCoV2rCs). The networks were constructed with the median joining method available in PopART 1.513 and modified under Adobe Illustrator CS6 (version 16.0). The codes used for the countries are the following: B (Myanmar), C (Cambodia), Ch (China), I (Indonesia), L (Laos), M (Malaysia), T (Thailand), and V (Vietnam). Colours indicate the geographic origin of haplotypes according to Fig. 2 (see online supplementary Table S1). The circles indicate haplotypes separated by at least one mutation. The black lines on the branches show the number of mutations ≥ 2. Black circles represent missing haplotypes. Circle size is proportional to the number of haplotypes. Haplogroups separated by more than seven mutations (pairwise nucleotide distances > 1%) are highlighted by dotted lines. The red arrows show the positions of the nine bats found positive for SCoV2rCs.
In the network of R. acuminatus, the most common haplotype (named Rac1 in online supplementary Table S1) was found in northern Cambodia, southern Laos, eastern Thailand and southern Vietnam, indicating recent gene flow among these populations. Since a virus related to SARS-CoV-2 (91.8% of genome identity), named RacCS203, was detected in five R. acuminatus bats caught in eastern Thailand in June 20206, the genetic pattern obtained for this species suggests that viruses closely related to RacCS203 may have circulated in most southern regions of mainland Southeast Asia. In contrast, R. acuminatus bats collected in Borneo (M5) showed a divergent haplotype (separated by 12 mutations; haplogroup II), suggesting that the South China Sea between mainland Southeast Asia and Borneo constitutes a barrier to gene flow. Isolated populations of R. acuminatus described in northern Myanmar, Indonesia (Java and Sumatra) and the Philippines14 should be further studied.
The network of R. shameli shows a typical star-like pattern, the most common haplotype (named Rsh1 in online supplementary Table S1) being detected in northern Cambodia and Laos. Since a virus related to SARS-CoV-2 (93.1% of genome identity), named RshSTT200, was recently discovered in two R. shameli bats collected in northern Cambodia in December 20107, the genetic pattern obtained for this species suggests that viruses closely related to RshSTT200 may have circulated, at least in the zone between northern Cambodia and central Laos. The bats sampled south to the Tonle Sap lake (n = 4; southern Cambodia and Vietnamese island of Phu Quoc) were found to be genetically isolated from northern populations (four mutations). However, further sampling in the south is required to confirm this result, as it may reveal CO1 sequences identical to the haplotypes detected in the north.
For the two species distributed in both China and Southeast Asia, i.e. R. affinis and R. malayanus, the genetic patterns are more complex with different haplogroups showing more than 1% of nucleotide divergence. In the network of R. affinis, there are three major haplogroups (named I, II and III in Fig. 3) separated by a minimum of seven mutations. The results are therefore in agreement with those previously published using CO1 and D-loop mitochondrial sequences15. The CO1 haplotypes detected in the localities sampled in southern China (ch1, ch4, ch5) are distantly related to the single haplotype available for central China (ch6), but they are also found in Laos, northern and central Vietnam, northern Thailand and northeastern Myanmar. This result suggests recent gene flow between populations from southern Yunnan and those from northern mainland Southeast Asia. Since a virus related to SARS-CoV-2 (96.2% of genome identity), named RaTG13, was detected in one R. affinis bat captured in southern Yunnan in 20131, the genetic pattern obtained for this species suggests that viruses closely related to RaTG13 may have circulated in the zone comprising southern Yunnan and northern mainland Southeast Asia.
In the network of R. malayanus, there are four major haplogroups (named I, II, III and IV in Fig. 3) separated by a minimum of seven mutations. The CO1 haplotypes detected in the localities sampled in southern China (ch2 and ch3) were also found in northern Laos (L1 and L3), suggesting recent gene flow between populations from these two countries. Since a virus related to SARS-CoV-2 (93.7% of genome identity), named RmYN02, was recently isolated from one R. malayanus bat collected in southern Yunnan in June 20195, the genetic pattern obtained for this species suggests that viruses closely related to RmYN02 may have circulated, at least between southern Yunnan and northern Laos. In contrast, the bats sampled in Myanmar were found to be genetically isolated from other geographic populations (haplogroup II in Fig. 3).