Identification and Molecular Analysis of Squash mosaic virus in Khorasan Razavi, Southern Khorasan and Mazandaran Provinces

Squash mosaic virus (SqMV) is a seed-borne, beetle transmitted Comovirus, infecting a wide range of cucurbits. This virus has a bipartite positive-strand RNA genome consisting of RNA1 and RNA2, which are separately encapsidated in isometric particles of 28 nm in diameter. The RNA-1-encoded polyprotein yields the viral polymerase, a helicase and a protease, as well as a protease cofactor and the genome-linked viral protein (VPg), while the RNA-2-encoded polyproteins yield the movement protein, large capsid protein and small capsid protein. In this virus, the gene for the LCP (41 kDa) and SCP (22 kDa) was mapped by in vitro translation studies to RNA-2 (contained within the M component), which is in concordance with established information on CPMV and the other comoviruses. While the nucleotide sequence encoding the CP genes in one SqMV isolate (designated here as H-SqMV) has been determined, the nature and extent of sequence divergence between isolates have not been examined.
Material and Samples, RT-PCR, cloning and sequencing: In order to identify Squash mosaic virus in squash, melon and watermelon fields in Khorasan Razavi, Southern Khorasan and Mazandaran provinces, 176 plant samples were collected from different areas during 2013 and 2014 growing seasons. The obtained sequences were compared with another source of SqMV in the GenBank. Using DNAMAN version 8 software, a phylogenetic tree was drawn and the nucleotide and amino acid sequence similarity percentages were determined. Total RNA was extracted from fresh leaves using Total RNA Isolation Kit (Denazist, Iran) following the manufacturer’s instructions. The First cDNA strand was synthesized using antisense primer (SqMV-CPR) and Moloney murine leukemia virus (MMuLV) reverse transcriptase (Parstoos, Iran). 3-5 microliters of purified RNA was mixed with reverse transcription mixture (50 mM Tris-HCl pH 8.3, 50 mM KCl, 4 mM MgCl2, 10 mM dithiothreitol, 1 mM of each dNTP, 200 units of MMuLV reverse transcriptase) and incubated at 42 C for 60. The complete length of the coat proteins (CPL, CPS) gene was amplified using specific primers SqMV-CPF by Taq master mix red (Amplicon, Denmark). These primers amplify the complete CPL and CPS genes of SqMV with 1900 bp length. PCR products were run on 1% agarose gel, purified from the gel using the Denazist Gel Extraction Kit (Denazist, Iran). The purified products were ligated into a pTg19 vector (Vivantis, Malaysia) according to the manufacturer's protocol. Plasmids were transformed into Escherichia coli strain DH5a, then Recombinant plasmids were purified from bacterial cells using Plasmid DNA isolation Kit (Denazist, Iran). Finally, the purified recombinant plasmids were subjected to sequencing bidirectional using pUC-M13 universal primers by Macrogen Inc. (Seoul, South Korea). Consensus sequences were verified using the BLAST program in NCBI database.
Squash mosaic virus (SqMV) is a member of the genus Comovirus in the family Secoviridae. It is a seed-borne and beetle-transmitted virus infecting most plants in the genera Cucurbita and Cucumis. In order to study and determine the distribution of Squash mosaic virus in cucurbit fields of three Iranian provinces (Khorasan Razavi, Southern Khorasan and Mazandaran) a number of 176 samples were collected from these provinces during 2013 and 2014 growing seasons. Ten out of 176 samples were infected by the virus which is indicating that distribution of the virus in Iran is low. Most samples showed symptoms of Squash mosaic virus and the virus was mostly isolated from melons. As cucurbitaceous seeds have been imported from other countries it can be assumed that possibly the virus come alongside infected seeds and then spread by other means such as vectors (Beetle). According to our findings in this study, all Squash mosaic virus isolates divided into two main groups (I and II). Group I is also subdivided into two subgroups A and B. In Group IA, Japanese, melon and Kimble isolates of SqMV are classified with Iranian Jovein and Boshrouyeh 1, 2, 3 isolates. Group IB includes Chinese (CH99 / 211), Arizona, Czech Republic (Tas-1) and one Iranian SqMV isolates (Tabas). In Group II, Iranian SqMV isolates of Khoosf, Birjand and Qayen are located with Spanish isolate (RZ). Based on the comparison of the coat protein gene sequences the highest similarities were 98% and 99.8% between Iranian SqMV isolates and Spanish and Japanese isolates respectively. The lowest similarities were 84.1% and 94% between Iranian Birjand and Boshrouyeh-3 SqMV isolates and Chinese isolates respectively.