Detection of Nora virus in infected <i>Drosophila melanogaster</i> heads without evidence of ubiquitous brain infection by smRNA FISH

<i>Drosophila melanogaster</i> Nora virus (DmNV), a positive-sense single stranded RNA virus related to picornaviruses. Given its genetic and structural similarity to neurotropic picornaviruses, such as poliovirus, we sought to determine whether DmNV could be found within the head and brain of <i>D. melanogaster</i>. RNA was extracted from heads of chronically DmNV-infected stocks, as well as from uninfected controls, and assayed using reverse transcription-polymerase chain reaction (RT-PCR) for DmNV <i>open reading frame 1</i> (<i>ORF1</i>). The results showed that DmNV genomic material can be isolated from the heads of DmNV-infected <i>D. melanogaster</i>, which suggests that the virus reaches the head during the course of infection. To determine whether DmNV infects the brain tissue itself, small-molecule RNA fluorescence <i>in situ</i> hybridization (smRNA FISH) experiments on whole brains dissected from DmNV-infected and uninfected <i>D. melanogaster</i> were done. The smRNA FISH detection method was validated by identifying DmNV RNA in gut tissue, but there was no evidence of DmNV localization in any brain specimens examined. These findings suggest an alternative explanation for why DmNV may be present in dissected head specimens. Additionally, we highlight the effectiveness of smRNA FISH as a highly specific and accessible method for detecting RNA viruses in Drosophila, offering an alternative to antibody-based or transgenic fluorescence approaches. Together, our results refine the understanding of DmNV tissue tropism and provide methodological insights for future studies using insect RNA viruses.