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 F₈I (simplified as I₈), R₁₈₁I (I₁₈₁), F₂₀₆L (L₂₀₆), and E₃₉₇N (N₃₉₇) and double mutations of I₈I₁₈₁, I₈L₂₀₆, I₈N₃₉₇, I₁₈₁L₂₀₆, I₁₈₁N₃₉₇, and L₂₀₆N₃₉₇. Four mutants, EAPV I₈I₁₈₁, I₈N₃₉₇, I₁₈₁L₂₀₆, and I₁₈₁N₃₉₇, infected Nicotiana benthamiana and yellow passionfruit plants without conspicuous symptoms. Mutants EAPV I₁₈₁N₃₉₇ and I₈N₃₉₇ 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 I₁₈₁N₃₉₇ accumulated the highest level of the small interfering RNA at 10 days postinoculation (dpi) in N. benthamiana plants, then dropped to background levels after 15 dpi. In both N. benthamiana and yellow passionfruit plants, EAPV I₁₈₁N₃₉₇ 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 I₈N₃₉₇ provided high degrees of complete protection against EAPV-TWnss in yellow passionfruit plants (90%) but not in N. benthamiana plants (0%). Both mutants showed complete protection (100%) against the Vietnam severe strain EAPV-GL1 in passionfruit plants. Thus, the mutants EAPV I₁₈₁N₃₉₇ and I₈N₃₉₇ have excellent potential for controlling EAPV in Taiwan and Vietnam.

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