First Molecular Evidence of Sacbrood Virus in Indian Honey Bee (Apis cerana indica F.) Colonies of Kerala, India

Abstract

Aim: The study aimed to isolate, characterize and identify the pathogenic entity responsible for viral disease in infected brood of Apis cerana indica colonies of different locations of Kerala. Study Design: Purposive sampling of infected brood samples, RT-PCR, sequencing and phylogenetic analysis. Place and Duration of Study: Collection of infected brood through purposive sampling was done from the Indian bee apiaries of native beekeepers under the authority of Department of Agricultural Entomology, Kerala Agricultural University, College of Agriculture, Vellayani. Isolation and molecular characterization studies were performed at AICRP on Honey Bees and Pollinators during the period from February 2024 to March 2025. Methodology: The study involved a purposive sampling of virus infected larvae/pupae from the Indian bee apiaries of Kerala based on the appearance of symptoms. Further, samples were subjected to isolation and characterization using molecular techniques (RNA isolation, RT-PCR and Sequencing). Phylogenetic analysis was performed to realize the evolutionary relationship of the isolates. Results: The purposive sampling for virus disease incidence resulted in four sample collections (AYI270724, PCK221225, KOL271024, CTPM270425) based on the symptoms from different Indian bee apiaries of various districts (Kollam, Kottayam, Kasargod & Malappuram) of Kerala. The characteristic symptoms observed were in the late larval to pupal stages, the brood cells were uncapped with the head of the pupa oriented upwards exhibiting a sac-like appearance and retarded development. Molecular characterization of four RNA isolates (AYI01, PCK03, KOL01, CTPM01) and RT-PCR using primers specific to ‘Polyprotein gene of SBV genome’ resulted in only one isolate (CTPM01) Chattipparamba from Malappuram produced amplicons (~450-480 bp), while no amplification was observed from the other three isolates (AYI01, PCK03, KOL01).  Sequencing of the PCR product revealed that the virus isolate (CTPM01) showed close homology to sacbrood virus isolate II10, Indian sacbrood virus (AcSBV-India-II10) infecting Apis cerana indica with sequence identity 97.69% (NCBI accession number- PX055611.1.). Conclusion: Molecular characterization of virus infected brood samples revealed that the key pathogenic agent is the Sacbrood virus. The isolate from Malappuram showed close sequence homology to Indian sacbrood viral strains, expanding the known geographic distribution of this virus in Indian honey bee populations. These findings highlight the first molecular evidence of Sacbrood virus infection in the Indian honey bee colonies of Kerala. This further strengthens the understanding of epidemiology of sacbrood virus in Kerala and forms a foundation for future research and effective management strategies to protect Indian bee colonies of Kerala.