Background and Aims: The genetic variability and population structure of Grapevine leafroll-associated virus 3 (GLRaV-3) have not already been studied in mid-Eurasia Iran. The investigations will contribute to developing efficient and durable control strategies of latent and graft-transmissible virus. Materials and Methods: During the winter of 2018-2019, a total of 229 dormant cutting samples were collected from 15 vineyards in West Azarbaijan province. All the samples were analyzed by serological and RT-PCR assays. The phylogenetic tree and two-dimensional nucleotide diversity plot were constructed using CP sequences by the SplitsTree4 v.4.12.6, and SDTv1.2 soft wares, respectively. Dnasp v.6.10.04 was used for genetic diversity and demographic analysis. We provide analyses of the codon usage and composition of GLRaV-3 based on 517 nucleotide sequences of the CP gene including 11 full CP sequences from Iran.  Results: Using serological assay 31.87% GLRaV-3 infection was detected. Neighbor-Net method analysis of the virus complete CP gene showed that the Iranian isolates fell into phylogroup I (GI) “as it is dominant in the rest of the world”. High haplotype diversities indicate the recent expansion of GLRaV-3. No clustering was found according to where the GLRaV-3 was isolated. Using dN/dS values it was found that the different populations of this virus is under negative (purifying) selection. The highest gene flow was determined between Europe and East Asia. Moderate or low genetic differentiation, and frequent gene flow (FST < 0.33 and Nm > 1) also confirmed with Ks*, Kst* Z, Z* and Snn statistics values. The frequency of amino acid coded by A/G ended optimal codon indicates the overlapping influences of natural selection and mutational pressure on the codon preferences in the CP gene. Codon bias of the CP gene was strongly affected by natural selection rather than mutation according to the effective number of codons-ENC vs. GC3s plot. Principal component plot analysis (PCA), illustrated the possible origin of GLRaV-3 in the Old World.  Conclusions: This analysis is the first demonstration of the population structuring of GLRaV-3 in mid-Eurasian Iran. Indeed, these consequences explain selectively driven codon bias in GLRaV-3 species; and reveal the potential importance of expression-mediated selection in shaping the genome evolution of this virus.

Grapevine viruses cause significant losses in the yield of grape. This study describes applying silver nanoparticles (AgNPs) to produce virus-free grapevine plants and compares it with chemo and thermotherapy. Preliminary molecular analysis proved the presence of Grapevine fanleaf virus (GFLV) and grapevine leafroll-associated virus-1 (GLRaV-1) in the ʻAsgariʼ, ʻPeykaniʼ, and ʻShahaniʼ cultivar samples, then single node explants were cultivated in the MS medium. Thermotherapy at 35 ± 1 ºC and cycles of 35/38 ± 1 ºC, chemotherapy with ribavirin 0, 20, 25, and 30 μg.ml-1 and using AgNPs at 0, 10, 15, and 20 ppm in medium and 40, 50, and 60 ppm sprayed during acclimatization stage were applied to obtain virus-free explants. The results indicated that using 20 ppm AgNPs in medium and AgNPs combined treatment (15 ppm AgNPs in medium and sprayed with 50 ppm AgNPs in the acclimatization stage) were the most effective treatments for the elimination of viruses. The best treatment led to 100% eradication of GLRaV-1 and 67% of GFLV in ʻAsgariʼ, 100% eradication of GLRaV-1 and GFLV in ʻPeykaniʼ and 100% eradication of GLRaV-1 and 67% of GFLV in ʻShahaniʼ. Furthermore, applying of AgNPs improved plant growth parameters, including plant height, which in infected plantlets was (18.06, 12.36, and 14.92 cm in ʻAsgariʼ, ʻPeykaniʼ, and ʻShahaniʼ, respectively) less than virus-free plantlets. Leaf number was 45, 34, and 27 in virus-free plantlets of ʻAsgariʼ, ʻPeykaniʼ, and ʻShahaniʼ, respectively, but in infected plantlets, it was 24.40, 19.80, and 12. Leaf area increased from 5.34, 5.50, and 5.94 cm2 in infected plantlets to 9.56, 11.43, and 12.33 cm2 in virus-free plantlets of ʻAsgariʼ, ʻPeykaniʼ, and ʻShahaniʼ, respectively. Complementary results proved that chlorophyll content in virus-free is significantly higher than in virus-infected plantlets, which explains and confirms the change in growth parameters after virus removal.