Generation of an attenuated mutant of papaya ringspot virus with broad-spectrum protection against different geographic strains

Papaya ringspot virus (PRSV) devastates papaya production worldwide, including Thailand. Cross protection is considered an effective approach to counter the threat. In this study, an infectious cDNA clone was constructed from the PRSV-SMK isolate collected from Samut Sakhon, Thailand. An attenuated mutant SMK-d5 was generated by the deletion of 5 aa residues (d5, 2nd-6th aa) in the N-terminal region of the helper component protease (HC-Pro) of SMK. The mutant SMK-d5 infected papaya, horn melon, and quinoa host plants without discernible symptoms. Furthermore, the accumulation dynamics of SMK-d5 in papaya plants displayed a zigzag pattern, a characteristic of a beneficial mild strain. The high stability of PRSV SMK-d5 was confirmed after 6 passages over 3 months on horn melon plants. The SMK-d5-infected papaya plants retained SMK-d5 without alteration of the deletion mutation up to 2 years under greenhouse conditions. Cross-protection tests were performed under greenhouse conditions, with a total of 45 plants from three independent tests. Challenge inoculation by different geographic isolates was conducted 3 weeks after the protective inoculation. Up to 35 days, SMK-d5 provided 100% protection against the parental SMK isolate, and the protection rates remained over 80% against phylogenetically close PRSV isolates from Taiwan, Indonesia, and South Vietnam, which belong to Asia clade. Conversely, protection against phylogenetically distant isolates from Hawaii and Mexico was only 61.2% and 37.2%, respectively. Full protection against SMK was observed for 60 days. Thus, we consider that SMK-d5 has great potential for controlling PRSV by cross protection in Thailand and other Asian countries.