Department of Plant Pathology, National Chung Hsing University, Taichung, Taiwan, ROC

Background and objectives
Papaya ringspot virus (PRSV), a member of the (+)ssRNA potyviruses, is the major limiting factor for growing papaya in tropical and subtropical areas. An attenuated mutant PRSV HA 5-1 that infects papaya without conspicuous symptoms was previously obtained by nitrous acid induction from the severe parental strain HA, originating from Hawaii. It has been used successfully for control of PRSV by cross-protection on a large scale in Hawaii and Taiwan. However, strain-specific protection limits its application in Taiwan and other geographic areas [1]. In this study, the genomic region of HA 5-1 responsible for attenuation of the virus was identified and the differences between HA and HA 5-1 in this particular region were analysed. The genomic segment was further transferred to a Taiwan severe strain for generation of an attenuated strain for control of Taiwan PRSV by cross-protection.

Materials and methods
Infectious in vitro transcripts of HA 5-1, driven by a T3 promoter, were constructed similar to that of the parental strain HA [2]. Various virus hybrids between the severe parental virus and its mild mutant were generated by recombination at the cDNA level. Symptoms on papaya induced by the recombinant hybrids were examined under greenhouse conditions. The genomic region responsible for the mildness of HA 5-1 was identified and further sequenced. The nucleotide and amino-acid differences of this particular region between HA and HA 5-1 were analysed. This genomic segment was further transferred to a newly constructed in vitro infectious cDNA clone of a Taiwan severe strain PRSV, YK, to demolish its pathogenicity. The pathogenicity of the hybrid on papaya and its cross-protection effectiveness against PRSV YK were evaluated under greenhouse conditions.

Results and conclusions
The genomic region responsible for attenuation of HA 5-1 was located at nucleotide positions 950-3261, including the C-terminal half of the P1 gene, the compete HC-Pro gene, and the N-terminal region of the P3 gene. Analysis of the nucleotide sequences between HA and HA 5-1 revealed that 17 nucleotides and nine amino acids are different in this region. Among these changes, six amino acids are critical which may affect the conformation of the corresponding proteins and their functions. In order to modify the local severe strain of Taiwan PRSV, YK, an in vitro infectious cDNA clone, driven by a T3 promoter, was also constructed. The genomic region of nucleotides 606-3526 of the YK strain was replaced with the corresponding region of HA 5-1 at the cDNA level. Similar to HA 5-1, this hybrid virus demolished pathogenicity and induced symptomless infection on papaya. Under greenhouse conditions, the genetically engineered attenuated hybrid provided a higher degree of protection in papaya against the Taiwan severe strain than HA 5-1. Our results indicated that the identified genomic region of HA 5-1 can be transferred to another strain of PRSV to create an attenuated hybrid, to avoid the problem of strain-specific cross protection.

1. Yeh SD, Gonsalves D, 1994. Advances in Disease Vector Research 10, 237-257.
2. Chiang CH, Yeh SD, 1997. Botanical Bulletin of Academia Sinica 38, 153-163.