Identification and full-length sequencing of Zucchini yellow fleck virus and a novel Nepovirus from next-generation sequencing of mixed virus infections in cucumbers (Cucumis sativa) from Crete.

Cucumbers are susceptible to infections with many characterised virus species. In some cases, mixed virus infections occur and produce novel symptoms. In Crete, routine screening is carried out when virus infection is suspected, or novel symptoms are observed in the field. To identify the viruses associated with distinct symptoms observed in samples from commercial cucumber production areas in Crete we carried out high-throughput sequencing (HTS) from a pool of six samples. Following assembly and BLAST analysis we identified at least eight viruses based on similarity to published sequences. Two of these sequences represented novel, near-complete genomes of a putative new nepovirus and of zucchini yellow fleck virus (ZYFV), for which a complete genome sequence was not available at the time of the study. To confirm the HTS results, the six samples were screened for all identified viruses and their presence confirmed through Sanger sequencing of PCR products, verifying the HTS results. To confirm the full-length genomes of the nepovirus and ZYFV, complete genomes were amplified by PCR and the HTS data confirmed by Sanger sequencing. We have generated the complete genome of a novel nepovirus from cucumber as well as the first complete genome sequence of a cucumber-infecting ZYFV isolate from Crete. The nepovirus was mechanically transmissible to Nicotiana benthamiana and induced typical cytopathological modifications consistent with virus infection, as revealed by TEM studies. We propose to name this new virus cucumber nepovirus A (CuNVA).