Cloning and sequence analysis of the coat protein gene of zucchini yellow mosaic potyvirus in the Philippines

The purpose of this study was to clone the coat protein gene of one Philippine isolate of zucchini yellow mosaic potyvirus (ZYMV). From a total of 30 field collected symptomatic squash leaf samples, one sample designated as Sq ZYMV, was putatively singly infected with ZYMV as assayed by ELISA and Western blotting. When the sample was assayed again by the same technique prior to the purification of the putative ZYMV, results have indicated that it was also infected with papaya ringspot potyvirus (PRSV). Furthermore, ELISA and Western blot data obtained after virus purification revealed that the purified virus consisted of PRSV and ZYMV. ZYMV coat protein gene (CP) gene specific primers were designated based on the multiple sequence alignments of nucleotide sequences of several ZYMV isolates downloaded from GenBank. To countercheck the specificity of the ZYMV primers, CP gene nucleotide sequences of several PRSV isolates were appended in the alignment. Specific primers for the PRSV CP gene were also designed for use in RT-PCR to confirm the presence of PRSV in the SqZYMV plant sample and in the purified virus preparation. When the oligo (dT) 12-18 primed cDNAs either from the SqZYMV plant sample or from the purified virus preparation were used as templates in PCR, both the full length ZYMV (837 bp) and portion of the PRSV (480 bp) coat protein gene were amplified using the ZYMV and the PRSV primers, respectively. This confirms the results obtained in ELISA and Western blot assays that the SqZYMV plant sample was doubly infected with PRSV and ZYMV and the resulting purified preparation contained both viruses. The amplified 837 bp band (approx) obtained by PCR amplification using the ZYMV primers was successfully cloned into a topoisomerase (TOPO) activated plasmid vector (PCR IL-TOPO) and the sequence of two representative clones was determined. Nucleotide sequence analysis revealed that the cloned PCR product also consisted of 837 nucleotides as reported for the coat protein gene of ZYMV isolates from other countries. The two clones differed from each other by only one nucleotide with clone 1 containing guanine (G) and clone 2 adenine (A) at the 12th position. The amino acid sequence of the the putative protein, consisting of 279 amino acids with a computed molecular weight of 31.35878 kDa, was conserved for both clones. Thirty-five BLAST matches of clones 1 and 2 corresponded to several isolates of the ZYMV CP gene with percent nucleotide identities ranging from 88.22-90.44. An E-value of 0 was obtained for each of the 35 alignments indicating that these results were significant. The nucleotide and the predicted amino acid sequences of the two clones were also compared with the corresponding sequences of the CP gene of ZYMV isolates from other countries. Overall, the two clones exhibited high sequence identities of 83-90% and 29-95% at the nucleotide and amino acid level, respectively, indicating that the DNA region amplified by RT-PCR is the ZYMV CP gene. This success would pave the way for further studies of the virus on its variation/distribution. The clones harboring the ZYMV CP gene can also be used as a first step towards determining whether or not CP mediated resistance will be successful under Philippine conditions. A more definite application of the results obtained in this study is the development of DNA probes for nucleic acid hybridization as well as PCR based techniques for the detection of ZYMV in squash and other cucurbits