تعیین توالی کامل RNA2 یک جدایه از ویروس موزاییک کدو (SqMV) از استان خراسان جنوبی در ایران

Introduction: Squash mosaic virus (SqMV) is a member of the genus Comovirus in the family Comoviridae. It is a seedborne and beetle-transmitted virus infecting most plants in the genera Cucurbita and Cucumis. Like other comoviruses, SqMV 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 genomes contain a poly (A) tail at the 3'-terminus and the genome-linked viral protein (VPg) attached to the 5'-end. It has been frequently reported in North and South America and Japan. Isolates from different regions have been extensively characterized and based on agar double-diffusion serological tests and host reactions, those isolates had been classified into different serogroups and biotypes. SqMV, which causes a serious disease of cucurbits, is transmitted by beetles and plant-plant contact and is readily seedborne in cucurbits. Fifteen species including, Cowpea mosaic virus (CPMV; type member), Andean potato mottle virus (APMoV), Bean pod mottle virus (BPMV), Cowpea severe mosaic virus (CPSMV), and Red clover mottle virus (RCMV) are distinguished in the genus Comovirus. SqMV has been reported frequently from North and South America, and less often from Australia, Israel, and Japan. Nelson and Knuhtsen have extensively characterized SqMV isolates from the United States and Puerto Rico biologically and serologically. They reported that SqMV isolates in the western hemisphere could be classified into six biotypes based on host reactions, but only into two serotypes (group I and II) by agar double-diffusion serological tests.
Materials and Methods: Plant material and virus isolates
In order to verify the presence of this virus in Southern Khorasan (Tabas) province of Iran and characterize some molecular aspects of it, 62 symptomatic leaf samples showing mosaic, vein clearing and distortion were collected from melon fields in growing season of 2015. RT-PCR reactions were used as a molecular method for the virus detection. The symptomatic leaves samples were used for RNA extraction using Rneasy mini Kit (Qiagen) and the extracted RNA were used directly or stored at minus 70 oC.

RT-PCR and sequencing

An amplification of the expected size of 1900bp for SqMV coat protein gene was obtained followed by specific primers for complete genome sequencing of RNA2. The SqMV total RNA2 was sequenced.
Results and Discussion: In spring and summer 2015, a survey was carried out in southern Khorasan province of Iran which were under the cultivation of melon (Tabas) in order to assess the current situation of SqMV. The 62 gathered samples were tested for the presence of SqMV. The symptoms were mostly mosaic, vein clearing and distortion. RT-PCR was developed using three pairs of specific primers targeting SqMV total poly protein and RNA2. The detection method was validated with melon plants sampled in fields known to be infested by this virus. The RT-PCR method also allowed SqMV to be detected in fruit and leaf samples. The RNA2 of Tabas isolate was 3271 nt in length, excluding the 3'terminal poly (A) tail. This sequence encoded a single ORF starting at AUG121 and terminating at UAG3148. This isolate translates a polyprotein containing 1009 amino acids with 110.99 KDa. The results showed that Iranian isolate RNA2 poly protein shared the highest nucleotide (88.45%) and amino acid (90.59%) identity with an Australia isolate (MF166754) and the lowest identity with a Japan isolate (NC_003800) 86.82% and Spanish isolate (KP223324) 86.36% in amino acid and nucleotide level, respectively.
Conclusion: The results illustrated that Iranian isolate RNA2 poly protein shared the highest nucleotide (88.45%) and amino acid (90.59%) identity with an Australia isolate (MF166754) and the lowest identity with a Japan isolate (NC_003800) 86.82% and Spanish isolate (KP223324) 86.36% in amino acid and nucleotide level, respectively. Phylogenetic analysis of SqMV RNA2 nucleotide sequences showed that Khorasan isolate was clustered with China (EU421060), Arizona (AF059532) and Spanish isolate in one subgroup.
This is consistent with the observation of Nelson &Knuhtsen (1973) for six biotypes, on the basis of symptoms and host range, but only two serological groups, one of which (group I) contained all the variability in host range and symptomatology. Hu et al. (1993) reported typing H-SqMV as a member of SqMV serogroup I on the basis of biological rather than serological comparisons, and L-SqMV was typed to serogroup I by Alvarez & Campbell (1978). Given that other stdies have encountered difficulties in reproducing the expected phenotypes on watermelon, the latter explanations are most likely. In comoviruses, RNA-2 encodes the two CPs and the MP. Thus, amino acid sequence differences found in the putative polyprotein of our RNA-2 clones may be expected to result in possible variations in serological response. However, both RNAs may function in determining host range and symptomatology and a precise correlation of our hybridization groups with the serological groupings has not yet been established.