Identification and characterization of two novel RNA viruses in the pine wood nematode Bursaphelenchus xylophilus

Abstract The pine wood nematode (PWN) Bursaphelenchus xylophilus is a devastating plant pathogen responsible for pine wilt disease. With the development of high-throughput sequencing technology, the virome of important pathogen organisms have been extensively studied. Despite their ecological significance, the virome of nematodes remains largely unexplored. Our study leverages high-throughput sequencing to uncover two novel RNA viruses within the transcriptome of PWN, thereby expanding the currently recognized variety of RNA viruses within this organism. These viruses, possessing positive RNA genomes, are distantly related to the families of Totiviridae and Partitiviridae . Based on analyses of the complete sequences of these novel RNA viruses acquired through rapid amplification of cDNA ends (RACE), they were designated Bursaphelenchus xylophilus victoei-like virus 1 (BxVLv1) and Bursaphelenchus xylophilus gammapartitivirus 1 (BxGv1). An examination of virus-derived small interfering RNA revealed the significant accumulation of the diminutive RNAs originating from BxVLv1 and BxGv1, indicating that the viruses replicated in PWN. BxVLv1 and BxGv1 were stably expressed at all life stages in the PWN strain C14-5. The expression levels were higher in the egg stage, suggesting vertical transmission through eggs. The prevalence of viruses in 39 parasitic nematode strains from various pine trees analyzed by RT-PCR revealed the presence of BxVLv1 only in the weakly virulent strain C14-5 and the presence of BxGv1 in the highly virulent strains J28 and YNZT, in addition to their presence in C14-5. The findings represent the first description of viral genomes in the PWN, emphasizing the need for comprehensive investigations of nematode-associated viruses to understand their implications on nematode fitness and pathogenicity mechanisms.