Comparative genomics highlights the virulence and evolutionary trajectory of white spot syndrome virus

White spot syndrome virus (WSSV) poses a significant threat to shrimp aquaculture, leading to substantial economic losses. This study aims to evaluate the virulence and evolution of recent WSSV outbreaks in Japan. Shrimp infected with WSSV were collected from Okinawa, Miyakojima and Miyazaki prefectures, yielding a total of seven isolates. Through injection and immersion tests, the lethal dose 50% endpoints were determined. Genomic analysis revealed isolates with sizes ranging from 288 to 299 kbp, sharing similar to 99% nucleotide identity with the reference genome (CN01: NC_003225.3). Variant analysis identified 1197 forms, primarily single- nucleotide polymorphisms, with Miyakojima isolates displaying the highest diversity. Frameshift mutations, notably in ORFs such as wsv006, wsv011, wsv091 and wsv403, were observed across all isolates. Phylogenetic analysis indicated clustering of Miyakojima isolates, suggesting similar outbreak intensities. Furthermore, isolates exhibited smaller genomic sizes compared with the reference genome, indicating ongoing WSSV evolution. Notably, a high frameshift mutation in wsv403, a viral E3 ubiquitin ligase, implies its potential role in the observed outbreaks, particularly in Miyakojima. This study addresses the research question regarding the virulence and evolutionary dynamics of WSSV outbreaks, proposing a hypothesis that genetic variations contribute to the severity and spread of WSSV in shrimp aquaculture.