The discovery of novel coronaviruses in bat guano, Sarawak, Malaysian Borneo

PURPOSE: Virological surveillance was conducted in to determine the presence of bat CoVs in the bat population in Wind Cave Nature Reserve, Sarawak, Malaysian Borneo. METHODS & MATERIALS: Partial RNA dependent RNA polymerase (RdRp) gene sequences of 11 bat-CoV positive guano collected in Wind Cave Nature Reserve was analysed. Five pellets of fresh guano from insectivorous bats were collected on plastic tarp and pooled in 500uL of ice-cold viral transport medium. The 440-bp gene was amplified using family-wide hemi-nested RT-PCR for phylogenetic analysis. Amplicon sequences were analysed using Basic Local Alignment Search Tool software (BLAST) to determine their corresponding species. Sequence alignment was carried out without primer sequence and phylogenetic tree with Maximum Likelihood bootstrap-Bayesian inference value (ML(b)/PP(v)) support. RESULTS: The positive rate was 47.6% (10/21) with the distribution of 60% (n=6/10) and 40% (n=4/10) Alpha and Beta-coronaviruses respectively. BatCoVs identified in this study formed four novel monophyletic clades referred to as Borneo Alpha-1 and -2, and Borneo Beta-1 and -2. Borneo Alpha-1 and -2 viruses diverged to form a monophyletic clade with Decacovirus-1 and Decacovirus-2 respectively (83.0-85.1% and 80.7-81.0% nucleotide identity) with batCoV found in Sabah (NCBI Accession# KX284940). Borneo Beta-1 viruses are also closely related to BatCoV found in Sabah (KX284939) (86.5-87.5% nt identity), forming a monophyletic group with Sarbecovirus. Meanwhile, Borneo Beta-2 are closer to BatCoVs in Loas (MN312609) (89.7-90.4% nt identity), forming a monophyletic clade with Hibecovirus. It is worthy to note that viruses found in the Sarbecovirus subgenus such as SARS-CoV-1 and SARS-CoV-2 attributed to two zooneses with Public Health Emergency of International Concern (PHEIC) over the past two decades. However, Borneo Beta-1 viruses are distant to the RaTG13 (MN996532) (69.1-697% nt identity), the proposed progenitor of the pandemic SARS-CoV-2. CONCLUSION: The study has discovered both novel alpha and beta bat-coronaviruses in 40% of the guano sampled in Wind Cave Nature Reserve, Sarawak, East Malaysia based on the phylogeny of the partial RdRp gene sequence. The batCoVs are phylogenetically grouped into four clades via phylogeny confirms the presence of Borneo Beta-1 clade being monophyletic to the epidemic Sarbecoviruses.