Phylogenetic analysis of two Ukrainian isolates of Wheat streak mosaic virus

The study of molecular characteristics and, in particular, the nucleotide (nt) and amino acid (aa) sequences of the viral genomes, is necessary to know the changes in their geographical range, phylogenetic relationships, viruses’ evolution, and their emergence as new epidemics. Aim. Phylogenetic analysis of the coat protein ( CP ) gene sequences of two new Ukrainian isolates of Wheat streak mosaic virus : Ukraine-Mal-18 and Ukraine-Ep-18. Results. The nucleotide sequences of 676 nt region of the CP gene of two Ukrainian WSMV isolates were compared with the sequences of 72 WSMV isolates/strains from GenBank. The phylogenetic analysis showed that the Ukrainian WSMV isolates cluster with the clade B or WSMV-ΔE isolates (originating from Europe and Asia) and have a typical for this clade triplet deletion at the position 8412-8414 nt in the CP gene. The isolate Ukraine-Mal-18 has the highest level of the sequence identity (93.5 %-95.9 % nt and 93.6-95.0 % aa) with the clade B isolates. The Ukraine-Ep-18 isolate shares 89.2 %-91.4 % (nt) and 88.6-87.1 % (аа) identity with the clade B isolates. Additionally, both Ukrainian WSMV isolates have a number of unique aa substitutions in the central CP gene domain. Conclusions. Ukrainian WSMV isolates belong to the clade B. But Ukraine-Mal-18 and Ukraine-Ep-18 have some differences from other members of the clade: i) a higher divergence compared to other B isolates (the Ukraine-Mal-18 has 12 aa substitutions, the Ukraine-Ep-18 has 25 aa substitutions, where as the other clade B isolates have no more than 2 aa substitutions); ii) have aa substitution identical with the B1 non-crop isolates of this virus, many aa substitutions are in the same motifs as the substitutions of B1 grass WSMV isolates.


Introduction
In recent years, not only the number and prevalence of cereal viruses have increased expressively, but also their economic significance [1,2]. Wheat streak mosaic virus (WSMV) is the most harmful and widespread virus of cereals in all wheat grown areas in the world. WSMV can cause significant yield losses of wheat -up to 60 % [3] and in some cases -up to 100 %. That is why a detail investigation of WSMV is warranted. WSMV is spread and detected in USA, Canada, Argentina, Australia, Asia, and Europe. In Europe, WSMV was found in Romania, Austria, Czech Republic, France, Hungary, Turkey, Italy, Poland, Russia, Slovakia, and Ukraine. In Lithuania and Germany, this virus was first detected only in 2013 [4,5]. The WSMV hosts are plant species of the family Poaceae. The main of them are: wheat, barley, maize, oat and many other grasses [6][7][8]. These species are reservoirs of the virus and its vector.
The study of molecular characteristics and in particular, the nucleotide sequences of the genome, provides an opportunity to follow the virus phylogenetic relationships with others, to elucidate its evolutionary history and prospects. Furthermore, based on these data it is possible to predict the changes and emergence of new properties in the strains or isolates circulating in a specific area.
Based on the coat protein (CP) gene sequences, WSMV isolates have been divided into four clades, named A-D [4,9,10].
Clade A represents one isolate from Mexico, known as El-Batán. Clade B or WSMV-ΔE includes Asian and European isolates [4,11]. B isolates have a deletion of triplet codon GCA (Glycine amino acid) in the CP gene sequence [11]. Except the CP gene, the differences between isolates from different clades were revealed in the putative protein P1/helper-component-proteinase (HC-Pro) protease cleavage site [4,12].
Recently, Singh & Kundu [8] proposed a new clade B1 for the WSMV isolates infecting grasses. This clade includes Czech isolates: ar1 detected in Agropyron repens (KY419572), pp1 from Phleum pratense (KY419573), and pp2 from Poa pratensis (KY419574). It was shown that these grass WSMV isolates are more similar to the B isolates rather than to A and D isolates. Moreover, the Czech grass isolates are characterized by the presence of a triplet deletion at the position 8412-8414 nt in the CP gene as well as the clade B isolates. On the other hand, the B1 isolates were slightly different from previously described the WSMV isolates from Czech Republic and had several aa substitutions located in the central domain of the CP gene [8].
Clade D includes isolates from USA, Canada, Argentina, Australia, Canada, Turkey (AF454455), and Iran (EU914917) [13,14]. Interestingly, some USA WSMVs have Gly deletion in the CP gene as well as B isolates [14]. The clade D isolates are divided into four subclades, D1-D4 [15,16]. This clade also includes WSMV described as isolate Ger (Ac. No AJ889242). Isolate Ger actually represents the strain PV57 from North America which had been maintained in the virus collection of the former Institute for Resistance Research and Pathogen Diagnostics in Aschersleben, Germany [4].
Analysis of the WSMV whole genomes showed that the clustering of some of them into different clades is based not only on geographical origin. Isolate Saadat-Shahr (Ac. No EU914918) is in the clade B, isolate with Ac. No AF454458 is in the clade C, and one more Iranian isolate Naghadeh (Ac. No EU914917) has high genetic similarity with the clade D isolates [4]. Similar situation is with Turkish isolates. Whereas the isolate Turkei (Ac. No FJ606886) clustered with European isolates from the clade B, another Turkish isolates Turkey1 (Ac. No AF454455) and Turkey2 (Ac. No AF454457) clustered with isolates from the clade D [17]. Isolate Agdia (FJ695510) from Czech Republic is also grouped with the clade D isolates.
Noteworthy, considerable attention is paid to the study of wheat viruses in Ukraine, in particular, WMSV. The WSMV` investigation was started in 1968 [18]. In the 1990s the disease was marked with different intensity on winter wheat crops in many regions [19]. Further, study on the WSMV circulation in Ukraine has shown that the virus is distributed in the central and eastern regions of the country. Later, an increase in the impact of climate change on the manifestation and circulation of this virus has been marked [3,20]. Monitoring of wheat crops in the Poltava, Kyiv and Kharkiv regions of Ukraine in the last years has shown minor damage with WSMV. Earlier, some molecular properties of Poltava isolate of WSMV were investigated [3]. In 2018, we noted the streak symptoms in the fields of winter wheat in the Kharkiv region. So, this study describes the phylogenetic analysis of the CP gene sequences of two new Ukrainian isolates of Wheat streak mosaic virus.

Materials and Methods
Samples collection. Winter wheat leaf samples with streak symptoms were collected in May 2018 in the fields of Kharkiv region (east part of Ukraine).
Enzyme-linked immunosorbent assay. Identification of the viruses in sap of wheat leaves was performed by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). Specific antibodies against Wheat streak mosaic virus (WSMV) and Barley yellow mosaic virus (BYDV-PAV) (Loewe, Germany) were used. Antigen samples were prepared by grinding leaf tissue in PBS buffer, pH 7.4, at the ratio 1:2 (w/V). Leaf samples from healthy wheat were also included as negative controls. Positive controls were commercial (Loewe, Germany). The results were recorded on Termo Labsystems Opsis MR reader (USA) with Dynex Revelation Quicklink software at the wavelength of 405 nm. Samples were considered positive when their absorbance values at 405 nm were at least three times higher than those of negative controls [21].
RNA extraction, RT-PCR and sequencing. Total RNA was extracted from fresh leaves using RNeasy Plant Mini kit (Qiagen, Great Britain) following the manufacturer's instructions. Two-step RT-PCR was performed. The reverse transcription was performed using RevertAid Reverse Transcriptase (Thermo Scientific, USA) according to the manufacturer's instructions. Amplification was performed using thermocycler (Genetic research instrumentation LTD, Great Britain). WSMV CP-specific primers were used: WS-8166F 5′ GAGAGCAATACTGCGTGTACG 3′ and WS-8909R 5′ GCATAATGGCTCGAAGTGATG 3′ [22]. DNA product of 750 bp was amplified. Amplification was performed in 12.5 µl of Dream Taq PCR Master Mix (2x) buffer (containing Dream Taq DNA polymerase, 2x Dream Taq buffer, 0.4 mmol/l of each dNTP and 4 mmol/l of MgCl 2 ), 7.5 µl of nucleasefree water, 1 µl of each primer (10 µmol/l), and 3 µl of cDNA. The temperature regime for amplification reactions was as follows: initial denaturation for 3 min at 95°C, followed by 35 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 1 min. The final extension was at 72°C for 10 min. PCR products were separated on a 1.5 % agarose gel with DNA markers MassRuler DNA Ladder Mix ready-to-use (SM 0311, Thermo Scientific, USA), stained with ethidium bromide, and visualized under UV light. The PCR products were purified from the agarose gel using a QIAquick Gel Extraction Kit (Qiagen, Great Britain). Sequencing of the purified amplified DNA fragments was carried out with the 3130 Genetic Analyzer (Applied Biosystems, USA).
Phylogenetic analysis. Sequences of Ukrainian WSMV isolates were compared with WSMV sequences in the NCBI database with the BLAST program (http:// www.ncbi.nlm. him.gov). WSMV isolates used in this study are listed in Table 1. Nucleotide and amino acid sequences were aligned using Clustal W in MEGA 7 (http://www.megasoftware.net/). Phylogenetic tree for the part of coat protein gene of 2 Ukrainian WSMV isolates and 72 WSMV isolates from different countries was constructed by the Neighbor-Joining method [23] using the best-fitting model (Jukes -Cantor). To check the reliability of the constructed tree we used bootstrap test with 1000 bootstrap replications. Multiple alignments of the coat protein amino acid sequences (225 аа) of WSMV isolates were performed by BioEdit program.

Results and Discussion
In 2018, winter wheat plants сv. Malynivka and Epokha Odeska with WSMV-like mild and severe streak symptoms were found near Kharkiv in the east part of Ukraine (Fig.1).
DAS-ELISA and RT-PCR showed that both wheat samples were infected with WSMV. BYDV-PAV antigens were not detected. These two samples were taken for this study. The WSMV isolate from wheat cv. Malynivka was Phylogenetic analysis showed that Ukrainian WSMV isolates with all European and Asian wheat isolates (except isolate Agdia from Czech Republic, Ger from Germany, Turkey 1 from Tukey and Iranian isolate Naghadeh) are clustered into the clade B (Fig. 2).
Ukrainian isolates, like all isolates of the B and B1 clades, have a triplet deletion at the position 8412-8414 nt in the CP gene in comparison to isolates from the clade D.
Comparative analysis of aa sequences of Ukrainian isolates with the isolates from clades B and B1 revealed significant differences. Thus, the Ukrainian isolate Ukraine-Mal-18 has 12 aa substitutions in the studied region of the CP gene and Ukraine-Ep-18 has 25 aa substitutions, whereas all other isolates of group B have 0 to 2 aa substitutions (Fig. 3).
It is necessary to mention that the substitution А→G at position aa 94 is identical among the Ukrainian and B1 isolates (Fig. 3). However, all other substitutions revealed in putative CP amino acid sequences are unique. Additionally, amino acid substitutions were observed at positions aa 112-116 (F.LFL motif in the central region of CP gene). None of WSMV isolates were characterized by such mutations in this region of the CP gene. Noteworthy, many aa substitutions are in the same motifs or near them like substitutions of the B1 grass WSMV isolates (Fig. 3). These are motifs «№1» (TVESC, 100-104 aa), «№4» (NE.RY.EDPVVFY, 135-147аа) that were previously found in the non-crop WSMV isolates of the B1 clade [8]. Also, the aa sequence of the Ukraine-Ep-18 isolate revealed more aa substitutions in the N-terminal domain of the CP gene, compared with other WSMV isolates (Fig. 3).

Conclusions
In our study we revealed that Ukrainian WSMV isolates are clustered into the clade B or WSMV-ΔE. The Ukrainian isolates, like other isolates of the B and B1 clades, have a triplet deletion at the position 8412-8414 nt in the CP gene sequence which led to the absence of Glycine amino acid. Noteworthy, the Ukraine-Ep-18 and Ukraine-Mal-18 isolates are the most divergent among the WSMV-ΔE isolates based on amino acid CP sequence. Besides, it was shown that the Ukrainian wheat WSMV isolates have identical aa substitution (А→G, 94аа) with the B1 non-crop isolates of this virus. Also, many aa substitutions are in the same motifs or near them (motif «№1» or TVESC at positions aa 100-104 and motif «№4» or NE.RY.EDPVVFY at positions aa 135-147) that were previously found in the non-crop WSMV isolates of the B1 clade [8]. Also, more aa substitutions in the CP sequence of the Ukraine-Ep-18 isolate were revealed in the N-terminal domain of the CP gene, than all the WSMV isolates taken to our study. It is known that tritimoviruses N-terminal domain of the CP gene is less conserved than the central and C-terminal regions [24]. Also, it was found that major aa variations between the wheat and grasses WSMVs were in the N-terminal region, namely in the motifs 3 and 4 [8]. However, in the wheat isolate Ukraine-Ep-18 we revealed aa substitutions in the motif 4, in comparison to other European wheat WSMVs. Prendeville et al. [25] suggest that mutations in BYDV from wild grass populations can be connected with the virulence factors and can act as positive fitness elements in wild plants. Recently, it has been revealed that the N-terminal region of tritimoviral CP is involved in the host-and strain-specific longdistance movement [26]. Perhaps, aa substitutions in this CP region revealed by us for the isolate Ukraine-Ep-18 led to more severe symptoms, compared with the Ukraine-Mal-18 isolate but this requires additional research.