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Recent developments in high-throughput sequencing (HTS) technologies and bioinformatics have drastically changed research in virology, especially for virus discovery. Indeed, proper monitoring of the viral population requires information on the different isolates circulating in the studied area. For this purpose, HTS has greatly facilitated the sequencing of new genomes of detected viruses and their comparison. However, bioinformatics analyses allowing reconstruction of genome sequences and detection of single nucleotide polymorphisms (SNPs) can potentially create bias and has not been widely addressed so far. Therefore, more knowledge is required on the limitations of predicting SNPs based on HTS-generated sequence samples. To address this issue, we compared the ability of 14 plant virology laboratories, each employing a different bioinformatics pipeline, to detect 21 variants of pepino mosaic virus (PepMV) in three samples through large-scale performance testing (PT) using three artificially designed datasets. To evaluate the impact of bioinformatics analyses, they were divided into three key steps: reads pre-processing, virus-isolate identification, and variant calling. Each step was evaluated independently through an original, PT design including discussion and validation between participants at each step. Overall, this work underlines key parameters influencing SNPs detection and proposes recommendations for reliable variant calling for plant viruses. The identification of the closest reference, mapping parameters and manual validation of the detection were recognized as the most impactful analysis steps for the success of the SNPs detections. Strategies to improve the prediction of SNPs are also discussed.

We investigated the presence of Hop stunt viroid (HSVd) in apple samples, showing dappling fruit symptoms, in the Maragheh area (Northwest Iran) by means of RT-PCR. The viroid was detected only in leaves collected from symptomatic trees and a 298bp amplicon (IR-Gala) was directly sequenced in both directions. Multiple sequence alignment and Blast analyzes revealed that IR-Gala isolate shares the highest identity with grapevine isolates from Brazil and China. Amongst Iranian isolates of HSVd available in the GenBank, this isolate had the highest identity with grapevine isolate of HSVd from Maragheh region. In Phylogenetic analysis by MrBayes, IR-Gala was clustered with grapevine isolates from Brazil, China and Iran and may suggest that HSVd-apple isolate could be originated from grapevine.

Pomegranate is an important crop in the Mediterranean Basin that can be affected by a range of pathogens. With the aim to better understand the impact of viral diseases on pomegranate, two leaf samples from Turkey showing virus-like symptoms such as chlorotic spots and oak-leaf patterns were subjected to high throughput sequencing (HTS). Data analysis indicated the presence of passiflora edulis symptomless virus (PeSV: genus Roymovirus, Potyviridae family) in these two pomegranate samples, consistent with the observation by electron microscopy of flexuous filamentous viral particles 760 to 780 nm long. Further analysis of HTS reads revealed the presence of five PeSV variants in one of the samples and another single variant in the other. PeSV occurrence was also identified from publicly available SRA pomegranate RNA-Seq transcriptomic data from India and China. The genome of these PeSV-pomegranate variants share 78.0–86.8% nucleotide identity with that of the reference isolate from passionfruit (MH379332). The presence of PeSV in pomegranate was confirmed by specific RT-PCR assays targeting either the coat protein (CP) or Nla-Pro genes in 37 cultivated and one ornamental pomegranate out of 133 samples collected from the Eastern Mediterranean region of Turkey. To our knowledge, this is the first application of HTS to assess virus occurrence in pomegranate and the first recognition of pomegranate as a new host for PeSV.

The improvements on the next generation sequencing or high-trough output technologies allowed the discovery of several unknown viruses in grapevines and also in other plants . The most important new emerging grapevine viruses were identified as Grapevine pinot gris virus (GPGV), Grapevine syrah virus 1 (GSyV-1), Grapevine red blotch-associated virus (GRBaV) and Grapevine roditis leaf discoloration virus (GRLDaV). The aim of the present study was to investigate the occurrence and characterization of these viruses in Tekirdag and Hatay viticulture production areas by PCR and DNA sequencing analyses. Totally 191 and 111 grapevine samples showing both virus-like symptoms and symptomless were collected from Tekirdağ and Hatay provinces, respectively. Among the tested samples GPGV and GSyV-1 were detected in both local and imported cultivars by the infection rate of 43.62 % and 1.04% in Tekirdağ , respectively. In Hatay provice, only GSyV-1 was detected by the infection rate of 0.9 % and all tested samples were negative for GPGV, GRBaV, GRLDaV. RT-PCR results showed that DNA fragments of 411 bp, 302 bp and 618 bp corresponding to the part of the coat protein (CP), movement protein (MP) and the replicase genes of GPGV were successfully amplified in Tekirdağ samples. All PCR products of GPGV were directly sequenced on both strands. All the nucleotide sequences of CP, MP and 5’ UTR and N-terminus of replicase genes shared the highest sequence identity with different GPGV isolates deposited in Genbank

Asma yaprak kıvırcıklık (grapevine leafroll) hastalığı geçmişten bu yana bağ virüs hastalıkları içerisinde ekonomik önem derecesi yüksek olan dünya çapında yayılım gösteren bir hastalıktır. Closterovirus cinsine ait virüs üyeleri içerisinde, Asma yaprak kıvırcıklık virüsü 1 (Grapevine leafroll-associated virus 1) (GLRaV-1) en eski olanıdır. Bağlarda önemli verim kayıplarına neden olan bu virüs, özellikle yapraklarda kıvrılma simptomu ile tanımlanır. Nar bitkisi, ekonomik değeri yüksek bitkiler arasında yer almakta olup henüz az sayıda virüs hastalıkları tanımlanmış ancak son yıllarda yürütülen çalışmalarda bu bitkinin de GLRaV-1’in konukçusu olabileceği bildirilmiştir. Bu çalışmanın amacı, bağ ve narlardan elde edilen GLRaV-1 izolatlarının genomik olarak kıyaslanmasıdır. Bu amaçla, 2014 yılında Hatay ve Niğde illerinden toplanan asma ve nar örneklerinden total RNA ve dsRNA izolasyonları yapılmış, virüs genomuna özgü 2 farklı bölgenin (hareket (movement) proteini (p24) ve ısı şok (heatshock) proteinini (HSP70h) çoğaltan primerler kullanılarak RT-PCR ile analizleri yapılmış elde edilen ürünler klonlanmış ve sekans analizleri yapılmıştır. Ayrıca Closterovirüs’ün HSP70h genine özgü dejenere primer kullanılarak DOP-PCR analizleri yapılmıştır. DNA dizi analizlerinin sonunda elde edilen kısmi genomun, BLAST analizleri yapıldıktan sonra filogenetik analizleri yapılmıştır. Bu çalışma sonunda, yeni bir konukçu olduğu düşünülen nardan izole edilen GLRaV-1 izolatlarının asma izolatları ile kıyaslamalı olarak analiz edilmesi sağlanmış ve izolatlar arası yüksek oranda benzerlik olduğu tespit edilmiştir.

High throughput sequencing of total RNA isolated from symptomatic leaves of a sweet cherry tree (Prunus avium cv. 0900 Ziraat) from Turkey identified a new member of the genus Robigovirus designated cherry virus Turkey (CVTR). The presence of the virus was confirmed by electron microscopy and overlapping RT-PCR for sequencing its whole-genome. The virus has a ssRNA genome of 8464 nucleotides which encodes five open reading frames (ORFs) and comprises two non-coding regions, 5′ UTR and 3′ UTR of 97 and 296 nt, respectively. Compared to the five most closely related robigoviruses, RdRp, TGB1, TGB2, TGB3 and CP share amino acid identities ranging from 43−53%, 44−60%, 39−43%, 38−44% and 45−50%, respectively. Unlike the four cherry robigoviruses, CVTR lacks ORFs 2a and 5a. Its genome organization is therefore more similar to African oil palm ringspot virus (AOPRV). Using specific primers, the presence of CVTR was confirmed in 15 sweet cherries and two sour cherries out of 156 tested samples collected from three regions in Turkey. Among them, five samples were showing slight chlorotic symptoms on the leaves. It seems that CVTR infects cherry trees with or without eliciting obvious symptoms, but these data should be confirmed by bioassays in woody and possible herbaceous hosts in future studies.