Generation of a Bivalent Recombinant Vaccine Conferring Dual Protection Against Potyvirus and Orthotospovirus in Cucurbits

Climate warming has facilitated the expansion of insect vectors and plant viral pathogens, leading to increased incidence of viral diseases in crops. Cucurbit crops, including cucumber (<i>Cucumis sativus</i>), melon (<i>Cucumis melo</i>), squash (<i>Cucurbita pepo</i>), and watermelon (<i>Citrullus lanatus</i>), are of major economic importance worldwide, but their production is severely threatened by viral infections. Among the most damaging viruses are zucchini yellow mosaic virus (ZYMV; genus <i>Potyvirus</i>), transmitted by aphids, and melon yellow spot virus (MYSV; genus <i>Orthotospovirus</i>), transmitted by thrips, both of which cause significant yield losses in Asia, including Taiwan. Previously, an attenuated ZYMV mutant, ZAC, was shown to confer effective cross-protection against ZYMV in several cucurbit species. In the present study, we engineered a recombinant virus, ZAC-MYnp, by inserting the nucleocapsid protein (NP) open reading frame of MYSV into the ZAC genome. ZAC-MYnp retained the attenuated phenotype of ZAC and remained effective in protecting against ZYMV infection, with protection rates of 70.4% and 87.0% in zucchini and muskmelon plants, respectively. In addition, under both mechanical and thrips-mediated challenge conditions, ZAC-MYnp significantly reduced MYSV symptom severity in muskmelon, with a protection rate of 66.7% and a protective efficacy of 79.0%, respectively. These results demonstrate that ZAC-derived recombinant viruses can function as a bivalent viral vaccine, offering dual protection against an aphid-borne potyvirus and a thrips-borne orthotospovirus. Our study highlights the feasibility of using a bivalent recombinant vaccine to manage two distinct insect-borne viruses in cucurbit crops.