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 in which 16 aa are structurally different. The multiple mutations of HA 5-1 are considered resulting from nitrous-acid induction since 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 the 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.