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Diagram illustrating the potential mechanism of BmNPV infection in the 306 strain. The three-color modules represent distinct signaling pathways: NF-kB (depicted in purple), MAPK (illustrated in green), and JAK/STAT (highlighted in pink). Genes outlined in red signify upregulation, while those outlined in green denote downregulation. Genes outlined in black represent expression levels of uncertainty. Illustrations were generated using BioRender. [Display omitted] • Integration of third-generation and second-generation high-throughput sequencing technologies for precise identification and analysis of alternative splicing (AS) patterns. • Identification of five pivotal genes (Dsclp, LOC692903, LOC101743583, LOC101742498, LOC101743809) associated with the response to BmNPV infection. • Discovery of novel transcripts, expanding the current understanding beyond what is available in SilkBase. The silkworm, Bombyx mori, stands out as one of the few economically valuable insects within the realm of model organisms. However, Bombyx mori nucleopolyhedrovirus (BmNPV) poses a significant threat, decreasing the quality and quantity of silkworm cocoons. Over the past few decades, a multitude of researchers has delved into the mechanisms that underlie silkworm resistance to BmNPV, employing diverse methodologies and approaching the problem from various angles. Despite this extensive research, the role of alternative splicing (AS) in the silkworm's response to BmNPV infection has been largely unexplored. This study leveraged both third-generation (Oxford Nanopore Technologies) and second-generation (Illumina) high-throughput sequencing technologies to meticulously identify and analyze AS patterns in the context of BmNPV response, utilizing two distinct silkworm strains—the susceptible strain 306 and the resistant strain NB. Consequently, we identified five crucial genes (Dsclp , LOC692903, LOC101743583, LOC101742498, LOC101743809) that are linked to the response to BmNPV infection through AS and differential expression. Additionally, a thorough comparative analysis was conducted on their diverse transcriptomic expression profiles, including alternative polyadenylation, simple sequence repeats, and transcription factors. [ABSTRACT FROM AUTHOR]