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 (MLb/PPv) 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

Ecological surveillance of bat coronaviruses in Sarawak, Malaysian Borneo

Objective: Coronaviruses (CoVs) are natural commensals of bats. Two subgenera, namely Sarbecoviruses and Merbecoviruses have a high zoonotic potential and have been associated with three separate spillover events in the past 2 decades, making surveillance of bat-CoVs crucial for the prevention of the next epidemic. The study was aimed to elucidate the presence of coronavirus in fresh bat guano sampled from Wind Cave Nature Reserve (WCNR) in Sarawak, Malaysian Borneo. Samples collected were placed into viral transport medium, transported on ice within the collection day, and preserved at − 80 °C. Nucleic acid was extracted using the column method and screened using consensus PCR primers targeting the RNA-dependent RNA polymerase (RdRp) gene. Amplicons were sequenced bidi‑ rectionally using the Sanger method. Phylogenetic tree with maximum-likelihood bootstrap and Bayesian posterior probability were constructed. Results: CoV-RNA was detected in ten specimens (47.6%, n =21). Six alphacoronavirus and four betacoronaviruses were identified. The bat-CoVs can be phylogenetically grouped into four novel clades which are closely related to Decacovirus-1 and Decacovirus-2, Sarbecovirus, and an unclassified CoV. CoVs lineages unique to the Island of Borneo were discovered in Sarawak, Malaysia, with one of them closely related to Sarbecovirus. All of them are distant from currently known human coronaviruses. Keywords: Bat, Cave, Coronavirus, Guano, Sarawa

Mitochondrial DNA diversity of the long-tailed macaque (Macaca fascicularis) from the northern region of Peninsular Malaysia

We examined the genetic diversity of 64 long-tailed macaques from the northern states of Peninsular Malaysia covering the states of Perlis and Kedah including the Langkawi Island using the complete control region (CR) segment of the mitochondrial DNA. Standard genetic diversity including nucleotide diversity, haplotype diversity and genetic divergence were calculated. Moderate nucleotide diversity (π = 0.021) was observed which is higher than a previous study on the Penang M. fascicularis population. Twenty-three haplotypes were detected with haplotype diversity, h of 0.936. Haplotype sharing was observed among Langkawi and Perlis macaques indicating historical connection between the island and the mainland. Phylogenetic trees constructed grouped the samples into 4 groups without any obvious populations structuring

A brief review of Bornean banded langur Presbytis chrysomelas (Müller, 1838) of Sarawak

The critically endangered Bornean banded langur (Presbytis chrysomelas) is exclusively found in Borneo. This species is classified among the world's 25 most endangered primate species. The geographical locations and distribution patterns of P. chrysomelas remain inadequately understood, and their taxonomic classification remains unclear. Although it was once abundant in Sarawak, the distribution of P. chrysomelas has become one of the most restricted among all Presbytis species. Presently, documented sightings of P. chrysomelas span across five specific locations in Sarawak: Samunsam Wildlife Sanctuary, Tanjung Datu National Park, Gunung Pueh National Park, Similajau National Park, and Maludam National Park. Thus, the purpose of this review is to present previous studies conducted on P. chrysomelas in Sarawak, elucidating its taxonomy, characteristics, distributions, important sites, research gaps, threats, and recommendations for further studies. Immediate conservation efforts and attention from the primatologist, relevant authorities and stakeholders are needed to prevent the localized extinction of P. chrysomelas

A Review of Primate Research and Conservation in Sarawak, Malaysia

Sarawak is home to a diverse array of primate species in Malaysia, i.e., 14 species from five families. Over the years, many studies on primates’ behavior, ecology and conservation have been conducted in Sarawak. Here, we provide a comprehensive overview of previous primate studies in Sarawak from 1960 to 2022 and discuss primates' threats, conservation status, and initiatives. Despite the vast number of these primate studies, the majority of them only focus on proboscis monkeys (n=24) and orangutans (n=21) and lack comprehensive studies that cover all primate species in Sarawak. Studies on ecology and natural history were the most prevalent for all Sarawak primate genera (n=51), accounting for more than half of publications. Thus, it advocates a more holistic approach to fill the knowledge gaps and meet conservation needs. Conservation efforts are urgently needed to protect primates currently threatened by deforestation, habitat fragmentation, hunting, and illegal trade. However, conservation efforts may be restrained by the limited information on primates in Sarawak. To preserve the primates in Sarawak, a robust protection strategy that synergizes the participation of government authorities, non-government organizations, and local communities needs to be develope

Phylogeography of the Sunda pangolin, Manis javanica: Implications for taxonomy, conservation management and wildlife forensics

The Sunda pangolin (Manis javanica) is the most widely distributed Asian pangolin species, occurring across much of Southeast Asia and in southern China. It is classified as Critically Endangered and is one of the most trafficked mammals in the world, which not only negatively impacts wild Sunda pangolin populations but also poses a potential disease risk to other species, including humans and livestock. Here, we aimed to investigate the species' phylogeography across its distribution to improve our understanding of the species' evolutionary history, elucidate any taxonomic uncertainties and enhance the species' conservation genetic management and potential wildlife forensics applications. We sequenced mtDNA genomes from 23 wild Sunda pangolins of known provenance originating from Malaysia to fill sampling gaps in previous studies, particularly in Borneo. To conduct phylogenetic and population genetic analyses of Sunda pangolins across their range, we integrated these newly generated mitochondrial genomes with previously generated mtDNA and nuclear DNA data sets (RAD‐seq SNP data). We identified an evolutionarily distinct mtDNA lineage in north Borneo, estimated to be ~1.6 million years divergent from lineages in west/south Borneo and the mainland, comparable to the divergence time from the Palawan pangolin. There appeared to be mitonuclear discordance, with no apparent genetic structure across Borneo based on analysis of nuclear SNPs. These findings are consistent with the ‘out of Borneo hypothesis’, whereby Sunda pangolins diversified in Borneo before subsequently migrating throughout Sundaland, and/or a secondary contact scenario between mainland and Borneo. We have elucidated possible taxonomic issues in the Sunda/Palawan pangolin complex and highlight the critical need for additional georeferenced samples to accurately apportion its range‐wide genetic variation into appropriate taxonomic and conservation units. Additionally, these data have improved forensic identification testing involving these species and permit the implementation of geographic provenance testing in some scenarios

Ecological surveillance of bat coronaviruses in Sarawak, Malaysian Borneo

Objective: Coronaviruses (CoVs) are natural commensals of bats. Two subgenera, namely Sarbecoviruses and Merbecoviruses have a high zoonotic potential and have been associated with three separate spillover events in the past 2 decades, making surveillance of bat-CoVs crucial for the prevention of the next epidemic. The study was aimed to elucidate the presence of coronavirus in fresh bat guano sampled from Wind Cave Nature Reserve (WCNR) in Sarawak, Malaysian Borneo. Samples collected were placed into viral transport medium, transported on ice within the collection day, and preserved at − 80 °C. Nucleic acid was extracted using the column method and screened using consensus PCR primers targeting the RNA-dependent RNA polymerase (RdRp) gene. Amplicons were sequenced bidi‑ rectionally using the Sanger method. Phylogenetic tree with maximum-likelihood bootstrap and Bayesian posterior probability were constructed. Results: CoV-RNA was detected in ten specimens (47.6%, n =21). Six alphacoronavirus and four beta coronaviruses were identifed. The bat-CoVs can be phylogenetically grouped into four novel clades which are closely related to Decacovirus-1 and Decacovirus-2, Sarbeco virus, and an unclassifed CoV. CoVs lineages unique to the Island of Borneo were discovered in Sarawak, Malaysia, with one of them closely related to Sarbecovirus. All of them are distant from currently known human coronaviruses

A brief review of Bornean banded langur Presbytis chrysomelas (Müller, 1838) of Sarawak

The critically endangered Bornean banded langur (Presbytis chrysomelas) is exclusively found in Borneo. This species is classified among the world's 25 most endangered primate species. The geographical locations and distribution patterns of P. chrysomelas remain inadequately understood, and their taxonomic classification remains unclear. Although it was once abundant in Sarawak, the distribution of P. chrysomelas has become one of the most restricted among all Presbytis species. Presently, documented sightings of P. chrysomelas span across five specific locations in Sarawak: Samunsam Wildlife Sanctuary, Tanjung Datu National Park, Gunung Pueh National Park, Similajau National Park, and Maludam National Park. Thus, the purpose of this review is to present previous studies conducted on P. chrysomelas in Sarawak, elucidating its taxonomy, characteristics, distributions, important sites, research gaps, threats, and recommendations for further studies. Immediate conservation efforts and attention from the primatologist, relevant authorities and stakeholders are needed to prevent the localized extinction of P. chrysomelas

Preliminary assessment of group composition and activity pattern of the critically endangered Bornean Banded Langur Presbytis chrysomelas chrysomelas in Tanjung Datu National Park

The Bornean banded langur (Presbytis chrysomelas chrysomelas) is critically endangered species primarily found in Sarawak, Malaysia. Albeit this species is in peril, the ecology knowledge of this endemic species of Borneo is still scarce. Thus, a rapid survey employing total count and scan sampling method was conducted between July to August 2023 at Tanjung Datu National Park (TDNP), Sarawak to observe the social interaction of species with the environment. The behaviour of langur was recorded by employing scanning sampling method at 10 minutes intervals. This study sought to provide preliminary data on behavioural ecology of the Bornean banded langur within the national park. During the survey, three groups (consisting two to seven individuals) and a solitary male Bornean banded langur were recorded. The langurs were observed in both dipterocarp forests and coastal forests within the park. The daily activities of the Bornean banded langurs in TDNP were predominantly resting (31%), moving (29%), feeding (26%), vocalizing (14%), but not engaging in other social activities such as grooming, playing and mating. Knowing the behavioural ecological status as well as understanding ecology by identifying the activity pattern of langur is essential to government authorities and pertinent stakeholders to implement conservation strategies for the Bornean banded langur and their habitats