Generation of Attenuated Mutants of East Asian Passiflora Virus for Disease Management by Cross Protection

East Asian passiflora virus (EAPV) seriously affects passionfruit production in Taiwan and Vietnam. In this study, an infectious clone of the EAPV Taiwan strain (EAPV-TW) was constructed, and EAPV-TWnss, with an nss tag attached to its helper component-protease (HC-Pro), was generated for monitoring the virus. Four conserved motifs of EAPV-TW HC-Pro were manipulated to create single mutations of F I (simplified as I ), R I (I ), F L (L ), and E N (N ) and double mutations of I I , I L , I N , I L , I N , and L N . Four mutants, EAPV I I , I N , I L , and I N , infected and yellow passionfruit plants without conspicuous symptoms. Mutants EAPV I N and I N were stable after six passages in yellow passionfruit plants and expressed a zigzag pattern of accumulation dynamic, typical of beneficial protective viruses. An agroinfiltration assay indicated that the RNA silencing suppression capabilities of the four double mutated HC-Pros are significantly reduced. Mutant EAPV I N accumulated the highest level of the small interfering RNA at 10 days postinoculation (dpi) in plants, then dropped to background levels after 15 dpi. In both and yellow passionfruit plants, EAPV I N conferred complete cross protection (100%) against severe EAPV-TWnss, as defined by no severe symptoms and absence of the challenge virus, checked by Western blotting and reverse transcription PCR. Mutant EAPV I N provided high degrees of complete protection against EAPV-TWnss in yellow passionfruit plants (90%) but not in plants (0%). Both mutants showed complete protection (100%) against the Vietnam severe strain EAPV-GL1 in passionfruit plants. Thus, the mutants EAPV I N and I N have excellent potential for controlling EAPV in Taiwan and Vietnam. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.