Molecular Identification and Characterization of Bat-borne Viruses from Pakistan

Abstract

The spread of zoonotic diseases that significantly affect humans has brought much attention to viruses circulating in animals, which have the potential to cross barriers. Bats remain reservoirs for highly contagious and pathogenic viral families, including the Coronaviridae, Filoviridae, Paramyxoviruses, and Rhabdoviridae. Spillover of viral species (SARS-CoV, MERS-CoV, and SARS-CoV2) from bats (as possible potential reservoirs) have been reported to cause the worst outbreaks. Hence, early detection of viral species of pandemic potential in bats is crucial. The results of the current study have been divided into three parts. Part A: We detected beta coronaviruses in the studied bat population, performed the evolutionary and amino acid substitution analyses, and analyzed the 3-dimensional protein structure for the viral RdRp. Beta coronaviruses analysis based on RdRp partial gene sequencing suggests that the studied viral strains are closely related to MERS-CoVlike viruses as they exhibit close structure similarities (with few substitutions) and also observed a substitution in highly conserved SDD in the palm subdomain of motif C to ADD when compared with earlier reported viral strains. The current study reported the coronavirus isolates from Pakistan for the first time in bats and submitted them to GenBank under accession numbers OM103587, OM103588, OM103589, and OM103590. Part B: We further investigated the bat-virome for detecting Orthomyxoviruses because bats serve as potential reservoir hosts for unique influenza A virus subtypes. The analysis is based on Influenza A viruses' highly conserved region (partial M gene). The phylogenetic analysis of the studied viral strains suggests they are closely related to Influenza A viruses. We further performed the analysis of amino acid sequence substitutions, and the current study sequences were submitted to GenBank under the accession numbers PP151243, PP151244, PP151245, PP151246, PP151247, PP151248, PP151249, and PP151250. We detected the bats-associated influenza A viruses for the first time in Pakistan. Part C: As we know, there is very limited information about the genetic identification of bat species from Pakistan. In addition, there are around 1400 bat species (order Chiroptera), which pose a challenge for morphology-based identification of the specific species. In this study, we optimized experiments for non-invasive molecular identification of bat species from different regions of Pakistan using markers for two different xiv mitochondrial genes (cytochrome b and cytochrome c oxidase I). The current study identified four Pipistrellus javanicus and seven Pteropus giganteus species. Evolutionary analysis of Pipistrellus javanicus sequences clustered with a sequence previously reported sequences from Bangalore, India (Accession numbers: OL513430 and OL513431) and a joint cluster with Guizhou, China (Accession numbers: ON640725 and ON640724). The sequences submitted to GenBank under accessions numbers PP209547, PP209548, PP209549, and PP209550. A phylogenetic analysis of Pteropus giganteus was made and clustered with previously reported sequences from Kushtia and Tangail, Bangladesh (Accession numbers: OP856810 and MH544064). These sequences were submitted to GenBank under accession numbers PP209540, PP209541, PP209542, PP209543, PP209544, PP209545, and PP209546. This finding highlights the importance of molecular tools in uncovering cryptic diversity and underscores the need for further taxonomic research on bats in Pakistan. Future studies should incorporate additional genetic markers, morphological examinations, and ecological data to definitively characterize these potential new species and contribute to a more comprehensive understanding of Pakistan's bat fauna. Virus characterization in bats has been prioritized in tropical regions. Despite increased efforts, significant variations exist in the worldwide geographical ranges and approaches. Consequently, there are substantial knowledge gaps in understanding bat viromes and how they could play a role in the spread of disease. Based on the present findings, we recommend extensive scale screening of bat virome across the country to find other potential pathogenic viral species.