Modification of the Helper Component Proteinase of Papaya Ringspot Virus Vietnam Isolate to Generate Attenuated Mutants for Disease Management by Cross Protection.

Papaya (Carica papaya) production is seriously limited by papaya ringspot virus (PRSV) worldwide, including in Vietnam. Control of PRSV by cross protection is dependent on the availability of effective mild strains. Here, an infectious cDNA clone was constructed from PRSV isolate TG5 from South Vietnam. Site-directed mutagenesis with point mutations on the essential motifs of the helper component proteinase (HC-Pro) was performed, with or without deleting five amino acids (d5) from its N-terminal region. Mutants TG-d5, TG-d5I7, and TG-d5L206 containing d5, d5 + F7I, and d5 + F206L, respectively, induced mild mottling followed by symptomless recovery on papaya and infected Chenopodium quinoa without lesion formation. Each mutant accumulated in papaya at reduced levels with a zigzag pattern and was stable beyond six monthly passages. The cross-protection effectiveness of the three mutants in papaya against TG5 was investigated, each with 60 plants from three independent trials. The results showed that each mutant provided complete protection (100%) against TG5, 1 month after the challenge inoculation, as verified by the lack of severe symptoms and lack of local lesions in C. quinoa. Further tests revealed that TG-d5I7 also confers high levels of protection against other severe PRSV isolates from South Vietnam, including isolates DN (97%) and ST2 (50%). However, TG-d5I7 is ineffective or less effective (0 to 33%) against seven other severe PRSV strains from different geographic origins, including the isolate HN from North Vietnam. Our results indicate that the protection by the three mutants is highly strain-specific and suitable for the control of PRSV in South Vietnam.

Generation of Mild Recombinants of Papaya Ringspot Virus to Minimize the Problem of Strain-Specific Cross-Protection.

Papaya ringspot virus (PRSV) causes severe damage to papaya (Carica papaya L.) and is the primary limiting factor for papaya production worldwide. A nitrous acid-induced mild strain, PRSV HA 5-1, derived from Hawaii strain HA, has been applied to control PRSV by cross-protection for decades. However, the problem of strain-specific protection hampers its application in Taiwan and other geographic regions outside Hawaii. Here, sequence comparison of the genomic sequence of HA 5-1 with that of HA revealed 69 nucleotide changes, resulting in 31 aa changes, of which 16 aa are structurally different. The multiple mutations of HA 5-1 are considered to result from nitrous acid induction because 86% of nucleotide changes are transition mutations. The stable HA 5-1 was used as a backbone to generate recombinants carrying individual 3' fragments of Vietnam severe strain TG5, including NIa, NIb, and CP3' regions, individually or in combination. Our results indicated that the best heterologous fragment for the recombinant is the region of CP3', with which symptom attenuation of the recombinant is like that of HA 5-1. This mild recombinant HA51/TG5-CP3' retained high levels of protection against the homologous HA in papaya plants and significantly increased the protection against the heterologous TG-5. Similarly, HA 5-1 recombinants carrying individual CP3' fragments from Thailand SMK, Taiwan YK, and Vietnam ST2 severe strains also significantly increase protection against the corresponding heterologous strains in papaya plants. Thus, our recombinant approach for mild strain generation is a fast and effective way to minimize the problem of strain-specific protection.