Comparative In silico Analysis of Partial Coat Protein Gene Sequence of Zucchini Yellow Mosaic Virus Infecting Summer Squash (Cucurbita pepo L.) Isolated From India

Zucchini yellow mosaic virus (ZYMV) generally produces symptoms like mosaic, yellowing, shoe stringing of leaves, fruits and seed deformations and stunting of plants [1] and infection at early stage of the crop could cause as much as 94 per cent reduction of marketable fruits of summer squash. ZYMV In this study, the partial coat protein gene sequence of ZYMV of Indian isolate was determined and compared with 67 other isolates of ZYMV at both genomic and proteomic level because a better knowledge of the virus characterization and tracing its phylogeny provide a major contribution to understand the complexity and epidemiology of the pathogen and hence management of the disease.


Introduction
Zucchini yellow mosaic virus (ZYMV) generally produces symptoms like mosaic, yellowing, shoe stringing of leaves, fruits and seed deformations and stunting of plants [1] and infection at early stage of the crop could cause as much as 94 per cent reduction of marketable fruits of summer squash. ZYMV In this study, the partial coat protein gene sequence of ZYMV of Indian isolate was determined and compared with 67 other isolates of ZYMV at both genomic and proteomic level because a better knowledge of the virus characterization and tracing its phylogeny provide a major contribution to understand the complexity and epidemiology of the pathogen and hence management of the disease.

Collection of samples and maintenance of the virus isolate
Tender leaves of summer squash plants showing symptoms of ZYMV were collected from the hill state of Himachal Pradesh located in the North Western Himalayan regions and the virus cultures were maintained on healthy seedlings of summer squash variety "Australian Dark Green" by mechanical sap inoculation under insect proof glass house conditions.

Serological Indexing of the virus
Serological detection and identification of virus was carried out by following enzyme linked immunosorbant assay (ELISA). Commercially available immunoreagents (BIOREBA -AG Switzerland) were used and protocols of suppliers of ELISA kits were used. The presence of potyvirus in experimental test plants was checked by DAC ELISA and the ZYMV presence was detected by DAS ELISA.

RNA extraction and cDNA synthesis
The RNA of the test virus was extracted from the experimental test plants using the RNA easy Plant Mini Kit (Qiagen). The RNA was used as a template for cDNA synthesis by using specific Oligonucleotide primer p9502 shown in Table 1.

Amplification of cDNA using specific primers
For further amplification of cDNA, PCR was carried out in a thermocycler (Applied Biosystem, USA) using specific primers p9502 (as reverse primer) and CPUP (as forward primer) shown in Table 1. PCR was run for 40 cycles and final elongation was carried out at 72º C for 10 minutes.

Sequencing and sequence analysis
Sequencing using both reverse and forward primers was carried out [2] and the sequence so obtained was translated using EXPASY (Expert Protein Analysis System) tool. Nucleotide and protein sequence data was analyzed using BLAST program at NCBI website whereas Clustal W was performed for multiple sequence alignment of the test sequence with other 67 isolates of ZYMV available in NCBI database. Phylograms

Abstract
Zucchini yellow mosaic virus (ZYMV; Family: Potyviridae, Genus: Potyvirus) is a serious virus infecting summer squash causing severe damage to both crop and ornamental cucurbit crop production. In the present study, molecular characterization of ZYMV (at genomic and proteomic level) infecting summer squash was carried out and cDNA of approximately 700bp was amplified. The PCR amplified product was further sequenced and analyzed. The sequence of partial coat protein of 154 nucleotides of Indian isolate of zucchini yellow mosaic virus (ZYMV) was determined and translated to proteins. Later, the sequence was submitted to NCBI and has got accession no. GU144796 with protein id ACZ36948. In BLASTN analysis, nucleotide test sequence showed 91% homology with D13914 (sequence from USA), whereas, protein test sequence was 75.9% homologous in BLASTP analysis with a number of protein sequences present in the database. The alignment score of test sequence with 67 other isolates of ZYMV retrieved from NCBI database was highest for USA among varied countries and lowest for China in case of nucleotides and Korea in case of proteins. Phylogenetic analysis revealed similarity of the test virus sequence with a USA ZYMV CP (D13914) and similarity of the partial polyprotein sequence with that of Japan (BAE75935). Conserved domain of the test virus was found to show homology with the potyvirus coat protein domain alignment collection (pfam00767). Computational restriction digestion revealed that 22 different restriction enzymes restrict present isolate of ZYMV. Secondary structures for polyprotein of the test virus was predicted which inferred dominance of alpha (α) helix in the protein sequence. and phylogenetic trees analysis was carried out using maximum likelihood (ML), maximum parsimony (MP), neighbor joining (NJ) and unweighted pair group mathematical averages (UPGMA) methods using phylip 3.68 and EXOME TM . Restriction enzyme map was constructed (http://tools.neb.com/NEBcutter2/index.php) and conserved domain search (http://www.ncbi.nlm.nih.gov/structure/ cdd/wrpsb.cgi) and secondary structure prediction of proteins was carried out (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/ NPSA_server.html). Amino acid content calculation was performed to know more about the protein test sequence.

Results
ZYMV isolate of India was collected from naturally infected summer squash plants shown in Figure 9  Fresh leaves from infected and healthy plants were collected and then used for RNA isolation. Presence of RNA was checked by running it on 0.8% agarose gel under 100 V potential difference for 1.5 hours in TAE buffer ( Figure 3). The RNA was reverse transcribed into cDNA (complementary DNA) with RT-PCR. This RT-PCR was followed by amplification of the cDNA with PCR. Amplified PCR product ~700bp obtained from the sample was subjected to agarose gel electrophoresis using 100 bp DNA ladder (Banglore Genei) as molecular weight marker ( Figure 4).

Nucleotide sequence accession number
The nucleotide sequence reported in this paper has been deposited in the GenBank database under accession number GU144796 at NCBI.

Sequence
No. of Bases Designated as

BLAST
The nucleotide and protein sequence of Indian isolate was analyzed using Blast program available at NCBI with test virus coat protein gene sequences from all around the world, available in the database of NCBI.

BLASTN and BLASTP analysis
In BLASTN analysis at NCBI, nucleotide test sequence showed 91% homology with D13914 (sequence from USA), and 90% homology with isolates ZYMV C-16 (DQ645729) and TW-NT1 (AF127933 Multiple sequence alignment of selected nucleotide and protein sequences of zucchini yellow mosaic virus with that of Indian isolate was performed using CLUSTAL W program [3] available online at European Bioinformatics institute (EBI) (http://www.ebi.ac.uk/) and similarly, country wise CLUSTAL W alongwith query nucleotide and protein sequence was also performed and these CLUSTAL W outputs were then used in (phylip 3.68 and EXOME ™ software) bioinformatic tools for constructing phylograms and phylogenetic trees.
Phylograms (using phylip 3.68) and phylogenetic trees (using EXOME ™ ) analysis was carried out using the test sequence (nucleotide ad protein) with other isolates of ZYMV from eight different countries retrieved from NCBI. And these studies revealed similarity of the test nucleotide with D13914 ZYMV isolate of USA, while in case of proteins the test protein found to be most similar to Japanese sequence of BAE75935. 64 phylograms (32 for nucleotide and 32 for protein) and 64 phylogenetic trees (32 for nucleotide and 32 for protein) were constructed using phylip 3.68 and EXOME ™ , respectively. On phylogenetic studies, in case of nucleotide sequences the test sequence showed common ancestry with DQ925447 (Australian), AJ420020 (Austrian), AB188115 and AB188116 (Japanese), AJ429071 (Korean), AF127933 (Taiwanese), D13914 (USA among varied countries) isolates of ZYMV whereas, in case of proteins the test protein sequence showed common ancestry with ABL0422 (Australian), CAD12315 (Austrian), CAD31036 and CAD31056 (Hungarian), BAE75935 and BAD74201 (Japanese) isolates of ZYMV.
Conserved domain search [4] of the test isolate was found with 3e-14E-value and a bit score of 71.92 (Figure 7). Further Conserved Domain Architecture Retrieval Tool (CDART) [5] was employed to perform similarity search for conserved domain in the test isolate with the NCBI Entrez Protein Database based on domain architecture (Figure 8).
Restriction enzyme maps for one cutter (Table 3 and 4), two cutter (Table 5) and three cutter ( Table 6) restriction enzymes were constructed for the nucleotide query sequence using online web tool (NEB cutter 2.0). Secondary structure of protein was predicted with the help of various methods like PREDATOR, MLRC and SOPMA at Network Protein Sequence Analysis, (NPS@webserver) ( Table 7).
Similarly amino acid content of the protein was calculated using Network Protein Sequence Analysis, (NPS@webserver). Total residue mass for protein of Indian isolate was 22.7kDa with 0.73 cm 3 .g -1 mean specific volume and it was found that Alanine was in maximum percentage in the sequence (Table 8).

Discussion
In recent years, diseases caused by plant viruses have become a significant limiting factor in the sustainable production of vegetables, Color key for alignment score of nucleotide query sequence Indian isolate with other nucleotide sequences present in the database using BLASTN analysis (Pink lines show the similarity of the query sequence is more than 80%.

Figure 6:
Color key for alignment score of protein query sequence Indian isolate with other protein sequences present in the database using BLASTP analysis (Green lines show the similarity of the query sequence is not more than 80%.  ornamentals and fruit crops. Cucurbitaceous crops including summer squash (Cucurbita pepo L.) have been reported to be infected by more than 30 viruses under natural conditions in different parts of the world [6]. Crop failure due to debilitating viruses creates significant financial loss throughout the world, mainly in developing countries. Therefore, successful crop management strategies require improved scientific understanding about the viruses. For this purpose, sequencing of virus genome is prerequisite. Sequencing of the virus genome and its in silico analysis helps in generation of information about evolution of the virus and subsequently designing reliable management strategies against the virus.

List of domain hits
In the present studies, partial CP gene sequence of Indian isolate of ZYMV compared with other 67 isolates of ZYMV at both genomic and proteomic level to see its evolutionary behavior.  Under present investigations a Reverse Transcription-Polymerase Chain Reaction assay using Potyvirus group specific primers for the test virus isolate amplified a product of ~700 bp in nucleic acid extracts (RNA) from the plants collected from District Una Himachal Pradesh. The Polymerase chain Reaction (PCR) and Reverse transcription-PCR are powerful tools for highly sensitive detection of plant viruses with DNA and RNA genomes. There have been many reports to detect plant viruses using RT-PCR. Lately, in 2007, detection of ZYMV using RT-   PCR was carried out in C. sativus L. and Cucumis melo L. in Poland [10]. Auger et al.
[8] identified a strain of ZYMV on squash by means of DAS ELISA and PCR using ZYMV specific primers ZY-2 and ZY-3 and a segment of 1186 bp was amplified and sequenced.
As given in results, the partial CP sequence of ZYMV (Indian isolate) was determined, which was found to be 154 nucleotides long. Prieto had also sequenced a fragment of 395 bp in length from the 3' portion of CP gene of Chilean isolate of ZYMV. In the present case however only 154 nucleotide long DNA was amplified confirming only partial amplification and sequencing of the CP gene.

Conclusion
On sequence comparison, current studies revealed close relation of the test isolate of zucchini yellow mosaic virus with USA sequence of ZYMV coat protein. As the parent crop (zucchini) is of Central American origin [11] and the relationship of the present isolate with that of USA is not surprising. It is however indicating that the virus may    have been imported into India along with the crop long ago and evolved slowly into the present isolate.

Impact of the Study
This study will provide better knowledge of the virus and its phyllogenetic relationship aimed at management of the disease caused by Zucchini yellow mosaic virus.
This study will give a flip to management of the viral disease.