Localization of viral and host RNA within soybean cyst nematode via fluorescence in situ hybridization

Nematode-infecting RNA viruses have recently been discovered via transcriptome sequencing. In soybean cyst nematode (SCN; Heterodera glycines), seven single-stranded RNA viruses have been identified from transcriptome data and experimentally confirmed with qRT-PCR and Sanger sequencing. Presently, there is still much unknown about the relationship between these viruses and the nematode host. In this study, we localize three viruses within the soybean cyst nematode: SCN socyvirus-1 (SbCNV-1), SCN nyami-like virus (NLV), and SCN bunya-like virus (BLV). To visually locate the viruses, whole-mount fluorescence in situ hybridization (FISH) methodology was developed for SCN pre-parasitic second-stage juveniles (ppJ2s). Two SCN populations with differing viral titers (LY1 and MM21) were used as a comparison for viral probe fluorescence intensity. Viral RNAs for all three viruses were abundant in cells throughout the SCN ppJ2 body of the high titer (LY1) population but absent within the majority of the intestinal tract. A significant reduction in viral fluorescence intensity was observed in a similar body pattern in ppJ2 of the low-titer (MM21) SCN, highlighting the specificity of the FISH method. As controls, viral RNAs were colocalized with host mRNA glyceraldehyde 3-phosphate dehydrogenase (GAPDH) for full body localization and a secretory ubiquitin protein (4G06) expressed specifically within the subventral esophageal glands. In addition, viral replication was confirmed in SCN eggs and ppJ2s via qRT-PCR detection of the anti-genomic RNA strands.