Whole genome characterization of wisteria vein mosaic virus from Iran and its relationship to other members of bean common mosaic virus group

Abstract

To date, the complete genome of two wisteria vein mosaic virus (WVMV) has been sequenced worldwide. Here, the genomic sequence of WVMV isolated from Wisteria sinensis in Iran was determined for the first time, using deep RNA sequencing and RT-PCR followed by Sanger sequencing. The sequence was 9694 nucleotides in length; excluding the 3’-poly(A) tail and contained a single open reading frame of 9279 nucleotides encoding a large polyprotein of 3092 amino acids and predicted molecular weight of 35,368 KDa. The genome contained nine putative proteolytic cleavage sites and motifs conserved in homologous proteins of other potyviruses. Sequence analysis suggested that WVMV-Ir sequence shared 76.37–86.01% nucleotide (nt) identity and 82.45–91.91% amino acid (aa) identity with two other isolates (Beijing and JEBU-p) available in the GenBank, the highest with the Chinese isolate Beijing (86.01% nt identity, 91.91% aa identity). Sequence identities over most of the genome were within the range 80–86% and 85–95% at the nt and aa levels, respectively; however, high variability was observed in the 5’-UTR (51.62%), P1 (62.03% nt identity, 50.78% aa identity) and P3 (79.82%nt identity, 78.67% aa identity) regions, suggesting that Ir, Beijing, and JEBU-p are three different strains. These variabilities may be due to different mutation phenomena of a common ancestor virus or mutations caused by different selection pressures in different agro-ecological regions. The results of the phylogenetic analysis indicated that WVMV was most closely related to soybean mosaic virus and watermelon mosaic virus and less closely related to the zantedeschia mild mosaic virus and dasheen mosaic virus. In the greenhouse, WVMV-Ir caused severe symptoms in Phaseolus vulgaris, Vicia faba, W. sinensis, Chenopodium quinoa, C. amaranticolor, and Nicotiana benthamiana.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13205-021-02957-8.

Introduction

Genus Wisteria belongs to the third-largest family of the flowering plants, Fabaceae (Jiang et al. 2011). This family includes economically important crops grown for food, medicine, timber, and landscape. As with other flowering plants, W. sinensis is reported to be affected by several viral agents including wisteria vein mosaic virus (WVMV) (Clover et al. 2003), wisteria badnavirus 1 (WBV1) (Li et al. 2017), cucumber mosaic virus (CMV) (Milojević et al. 2016), and subterranean clover stunt virus (SCSV) (Grylls and Butler 1959). Wisteria mosaic disease, which is caused by WVMV (Clover et al. 2015; Liang et al. 2006) and CMV (Milojević et al. 2016), is an important disease of W. sinensis that reduces its quality and ornamental value by inducing chlorosis and mottling of the leaves. These viruses can be easily transmitted to propagules via vegetative propagation methods such as grafting, cuttings, and air layering, leading to the worldwide dispersal (Li et al. 2017). Considering that W. sinensis is an invasive perennial plant with extreme longevity, it might act as reservoir of these viruses (Milojević et al. 2016). WVMV is a member of the Potyvirus genus in the family Potyviridae, with flexuous filamentous particles of approximately 760 nm long (Bos 1970; Clover et al. 2003; Adams et al. 2012). It can be transmitted by aphids in a non-persistent manner as well as mechanical inoculation and no seed transmission has yet been reported for this (Bos 1996; Liang et al. 2006). The potyviral genome consists of a linear, single-stranded and positive-sense RNA of approximately 10 kb which has terminal untranslated regions (5’-VPg and a 3’-polyA tail) and, between them, a single open reading frame (ORF) which is translated into a large polyprotein (Adams et al. 2005a), and one overlapping ORF, termed PIPO (Chung et al. 2008). The polyprotein is hydrolyzed after translation into at least ten proteins by three virus-encoded proteinases (Adams et al. 2005a). WVMV has been reported in Wisteria spp. in Australia, China, the United States, and several European countries (Clover et al. 2003, 2015; Liang et al. 2004). The virus has a very narrow host range, naturally infecting only Wisteria spp. (W. sinensis, W. floribunda, and W. venusta) (Liang et al. 2006). To date, the complete genome sequence of only two WVMV isolates Beijing isolate (AY656816, from wisteria in China) and JEBU-p (MT603851, from soybean in South Korea) are available in the GenBank database. WVMV has been reported recently in Iran based on the partial sequence of CI (Al-Jaberi et al. 2018), CP, and 3’-untranslated region (3'-UTR) (Valouzi et al. 2020); however, so far, no studies have been done on the genomic features of this virus from Iran. Complete sequence data are essential for the understanding of genome structure, biological properties, diversity, and evolutionary relationships of a virus, for devising new control strategies and evaluating its risk (Moradi et al. 2017a). To achieve this objective, the small RNA deep-sequencing technology was employed for molecular identification and characterization of the virus, followed by biological characterization.

Materials and methods

Sampling and RNA isolation

In 2019, wisteria (W. sinensis cv. Prolific) plants in the landscape of Mashhad city, northeast of Iran, showed a high incidence of wisteria mosaic disease symptoms (Bos 1970; Clover et al. 2003; Liang et al. 2004), including vein clearing, mosaic, and chlorotic mottling on leaves. To determine the causal agent(s), total RNA was extracted from the five symptomatic leaf samples using SV Total RNA Isolation Kit (Promega, USA) following the manufacturer's instructions. Primarily detection was done by reverse-transcription followed by polymerase chain reaction (RT-PCR) using potyvirus degenerate primers CI (Ha et al. 2008), followed by Sanger sequencing. One positive sample with the most severe symptoms was selected for further investigation.

Library construction, sequencing, and RNA-Seq data analysis

Two cDNA libraries were prepared by Illumina TruSeq Stranded Total RNA with Ribo-Zero rRNA Removal kit (plant leaf) and sequenced using an Illumina NovaSeq 6000 (Macrogen, Seoul, South Korea) with 151 bp paired-end reads. To identify possible viruses, RNA Seq data were trimmed, and the quality control was checked. The cleaned raw transcript reads of one symptomatic plant were mapped to reads from one healthy-looking plant by CLC Genomics Workbench version 12 (CLC Bio, Qiagen). Unmapped reads were saved and used for viral genome de novo assembly using CLC Genomics Workbench (Qiagen). Virus contigs were determined by filtering of contigs. A nucleotide BLAST of obtained contigs was done using Geneious Prime software v. 2019.1.3 (Biomatters Ltd., Auckland, New Zealand).

Recombination and phylogenetic analyses

ORFs were predicted using the ORF finder function of the SnapGene software (GSL Biotech; http://www.snapgene.com). MUSCLE multiple sequence alignments, nucleotide and amino acid sequence identities between the isolates were carried out using the Geneious Prime (Biomatters).

The aligned nucleotide sequences (including three WVMV isolates and 47 other isolates of the bean common mosaic virus (BCMV) group) were examined for the presence of inter and intra-species recombination events using seven recombination detection methods implemented in the RDP4 software (Martin et al. 2015) with a Bonferroni corrected p value cut-off of 0.01. Only recombination events detected by at least four different methods, in combination with phylogenetic evidence for recombination, were evaluated significant. Sequence diversity analyses were executed using DnaSP6 (Rozas et al. 2017). Phylogenetic tree of the complete polyprotein amino acid sequences of WVMV and 16 other members of BCMV subgroup potyviruses (Table 1) was constructed using the maximum likelihood (ML) method based on the Jones–Thornton–Taylor (JTT) matrix-based model in MEGAX (Kumar et al. 2018) with 1000 bootstraps and 70% bootstrap threshold score. The genomic sequence of an isolate of onion yellow dwarf virus (OYDV) (GenBank accession no. AJ510223) was used as an outgroup as BLAST searches had shown it to be most closely and consistently related to those of BCMV group members. In addition, the CP gene sequences (462 nt of the N-terminal portion) of 10 WVMV isolates (one from this study and nine retrieved from GenBank) (Table 1) were used to determine the phylogenetic correlation and genetic diversity of WVMV populations throughout the world. The CP-based phylogenetic trees were constructed using the ML with Kimura 2-parameter (for nt sequences) and JTT (for amino acid sequences) models implemented in MEGAX.

Table 1.

Sources and GenBank accession numbers of virus sequences used in this study

Virus	Host	Country	Accession number	Genomic region
WVMV-Ir	Wisteria sinensis	Iran Mashhad	MN514947	Complete genome
WVMV-Beijing	Wisteria	China Beijing	AY656816	Complete genome
WVMV-JEBU-p	Soybean	South Korea	MT603851	Complete genome
WVMV-Australia	Wisteria	Australia	AF484549	CP
WVMV-WF03	Wisteria floribunda	Poland	DQ009883	CP
WVMV-BJ	Wisteria	China Beijing	AY519365	CP
WVMV-JW_2014	Wisteria	United Kingdom	KP161267	CP
WVMV-W7_2014	Wisteria brachybotrys	United Kingdom	KP161266	CP
WVMV-YZ	Wisteria sinensis	China	MK119780	CP
WVMV-SUX-1-HZ	Wisteria sinensis	China Hangzhou	KJ836282	CP
BCMNV-TM70	Phaseolus vulgaris	East Timor	KX302007	Complete genome
BCMNV-NL-3	Phaseolus vulgaris	USA Michigan	U19287	Complete genome
BCMNV-TN1	Phaseolus vulgaris	USA	HQ229995	Complete genome
BCMNV-K1	Phaseolus vulgaris	Kenya	MH169566	Complete genome
BCMV-RU1D	Common bean	USA	GQ219793	Complete genome
BCMV-Habin1	Glycine max	South Korea	KJ508092	Complete genome
BCMV-NKY019	Glycine max	China Jiangsu	KJ807818	Complete genome
BCMV-CD031	Glycine max	China Hubei	KM051430	Complete genome
ZYMV-BL-67	Pumpkin	USA	MK124612	Complete genome
ZYMV-Fars	Cucurbita pepo	Iran	JN183062	Complete genome
ZYMV-Kuchyna	Cucurbita pepo	Slovakia	DQ124239	Complete genome
ZYMV-Z-104	Cucurbita pepo	Italy	MK956829	Complete genome
SMV-413	Glycine max	USA	GU015011	Complete genome
SMV-NE-N1	Glycine max	China	KP710869	Complete genome
SMV-KY	Glycine max	USA	HQ845736	Complete genome
SMV-XFQ020	Glycine max	China Harbin	KP710878	Complete genome
WMV-CHI87-620	Zucchini	Chile	EU660580	Complete genome
WMV-RKG	Watermelon	India	KM597070	Complete genome
WMV-VE10-099	Cucumis anguria L	Venezuela	KC292915	Complete genome
WMV-Pg	Panax ginseng	China	KX926428	Complete genome
DsMV-T10	Amorphophallus paeoniifolius	India	KJ786965	Complete genome
DsMV-strain I	Colocasia esculenta	USA	KY242358	Complete genome
DsMV-Ug31	Xanthosoma sp.	Uganda	MG602235	Complete genome
DsMV-SdP	Pinellia pedatisecta	China	JX083210	Complete genome
CABMV-Z	Cowpea	Zimbabwe	AF348210	Complete genome
CABMV-RR3	Cowpea	India	KM597165	Complete genome
CABMV-BR1	Peanut	Brazil	HQ880242	Complete genome
CABMV-11 K	Passion fruit	Kenya	MH844588	Complete genome
PWV- SW8	Passiflora caerulea	Australia	KX577780	Complete genome
PWV-MU2	Passiflora caerulea	Australia	HQ122652	Complete genome
CLLV-strain E49	Calla lily	Taiwan	EF105299	Complete genome
CLLV- BM19	Calla lily	Taiwan	EF105298	Complete genome
VanMV-CI	Vanilla x tahitensis	Cook Islands	KX505964	Complete genome
EAPV-pt	passionfruit TN1	Taiwan	KY614052	Complete genome
EAPV- strain AO- isolate YW	Passiflora edulis x P. edulis f. flavicarpa	Japan	LC325839	Complete genome
EAPV-IB-dpd	passionfruit TN1	Taiwan	KT724930	Complete genome
EAPV-AO	Passionfruit Passiflora edulis x P. edulis f. flavicarpa	Japan	AB246773	Complete genome
FVY-Pan'an	Fritillaria thunbergii	China	NC_010954	Complete genome
HarMV-MD4-D	Hardenbergia comptoniana	Australia	KJ152157	Complete genome
HarMV-HarMV-57.1	Hardenbergia comptoniana	Australia	HQ161080	Complete genome
HarMV-MD3	Lupinus cosentinii	Australia	KJ152153	Complete genome
HarMV-VPK-1	Hardenbergia comptoniana	Australia	MF040762	Complete genome
IFBV- Asan	Impatiens walleriana hook	South Korea	KU981084	Complete genome
TeMV-PasFr	Passion fruit	China	MG944249	Complete genome
TeMV-Hanoi	Telosma crop	Viet Nam	DQ851493	Complete genome
ZaMMV-Australia	Alocasia sp.	Australia	KT729506	Complete genome
ZaMMV-TW	Zantedeschia spp.	Taiwan	AY626825	Complete genome
OYDV-Yuhang	Allium sativum	China Zhejiang	AJ510223	Complete genome

Note: The abbreviations of virus names used in this table stand for WVMV wisteria vein mosaic virus, BCMNV bean common mosaic necrosis virus, BCMV bean common mosaic virus, ZYMV zucchini yellow mosaic virus, SMV soybean mosaic virus, WMV watermelon mosaic virus, DsMV dasheen mosaic virus, CABMV cowpea aphid-borne mosaic virus, PWV passion fruit woodiness virus, VanMV vanilla mosaic virus, CLLV calla lily latent virus, EAPV East Asian passiflora virus, FVY fritillary virus Y, HarMV hardenbergia mosaic virus, IFBV impatiens flower break virus, TeMV telosma mosaic virus and ZaMMV zantedeschia mild mosaic virus. OYDV Onion yellow dwarf virus included as an outgroup. The letter(s) following a virus name represents an isolate or a strain of that virus

Biological assay

The isolate WVMV-Ir was serially inoculated by three passages of single local lesions on Chenopodium quinoa and then multiplied on it (Fig. S2). For this purpose, wisteria-infected leaf tissues were ground in five volumes of 50 mM phosphate buffer (pH 7) containing 2% polyvinylpyrrolidone and 0.05% sodium metabisulfate. The partial host range of the WVMV-Ir was considered by rub-inoculating sap prepared from infected C. quinoa plants onto carborandum-dusted leaves of W. sinensis, Chenopodium amaranticolor, Nicotiana benthamiana, N. glutinosa, N. tabacum cv. Samsun, N. tabacum cv. Xanthi, Phaseolus vulgaris, Vicia faba, Cucumis sativus, Datura stramonium, and Petunia hybrida. Three plants of each species were mechanically inoculated and one left uninoculated as control. The tested plants were maintained in a temperature-regulated insect-proof greenhouse. At 2 weeks post-inoculation, the plants were examined for symptoms and tested for WVMV infection by RT-PCR using WVMV-specific primers WVMVF1/WVMVR1 as described by Clover et al. (2003).

Results

RNA-Seq data assembly and RT-PCR assay

Illumina sequencer produced 5,99,62,621 reads for a total of 9,57,52,38,500 bp in infected wisteria plant. One long contig (9694 bp in length) was identified from the assembled non-plant reads which mapped to WVMV sequences in the GenBank. A total of 99,45,878 WVMV sequence reads mapped to the final contig obtained. RNA-seq result was confirmed by Sanger-based sequencing through genome walking using reverse-transcription PCR with potyvirus degenerate primers (for HC-Pro, CI, and NIb sequences) (Ha et al. 2008; Marie-Jeanne et al. 2000) and WVMV-specific primers (Clover et al. 2003). There was no evidence for the presence of other viruses in the NGS contigs (data not shown). The 5’-end of the genome was determined by 5’-RACE using the methods described by Liang et al. (2006). In addition, oligo (dT)18 was used as a reverse-transcription primer to characterize the 3’-end of the genome.

Genome organization of WVMV-Ir

The complete WVMV-Ir genomic RNA was found to be 9694 nt in size excluding the 3’- poly(A) tail. The 5’ and 3’-UTRs were 164 nt and 251 nt in length, respectively. The sequence has been deposited in the GenBank database under the accession number MN514947. Two highly conserved potyboxes, ‘a’ and ‘b’ (Turpen, 1989), and CAA repetitive sequences associated with translation enhancement (Gallie and Walbot 1992) were found in the 5’-UTR of WVMV-Ir. The 5’-UTR had a high content of AU (70.73%) and a low content of GC (29.27%) like values reported for other potyviruses (Moradi et al. 2017a). The 3’-UTR whose secondary structure might be involved in genome replication was AU-rich (61.35%) and contained TATA box-like sequences, as reported for other potyviruses (Moradi et al. 2017b). The genome contained a single large ORF which started with the AUG codon at nt position 165–167 and ended with a termination codon (UAA) at nt position 9441–9443 (Table S1). Upon expression, this large ORF (9279 nt) encodes a single large polyprotein (3092 aa, with the molecular mass of 35,368 KDa) which is processed by three self-encoded proteases (P1, HC-Pro and NIa-Pro) to yield ten following functional proteins: P1, HC-Pro, P3, 6K1, CI, 6K2, NIa-VPg, NIa-Pro, NIb, and CP (Table S1). It had a short overlapping ORF, PIPO, which can be expressed as a P3N-PIPO fusion product via transcriptional slippage of viral RNA-dependent RNA polymerase (Rodamilans et al. 2015). Like other reported members of the Potyviridae, there was a conserved G₁A₆ motif (nt 2940–2946) at the 5’-end of the PIPO ORF. The PIPO + 2 ORF started within a conserved G₁A₆ motif in the P3 cistron (position 2942) and terminated (position 3166) at the nearest in-frame UAA stop codon (positions 3167–3169), encoding an 8.92-kDa protein. Nine potential protease cleavage sites were identified based on recognition motifs for the viral proteases (Adams et al. 2005a) (Table S1). The order and relative positions of the motifs in the WVMV-Ir-encoded polyprotein was similar to that observed in other potyviruses as follows: the proteolytic domain, cysteine proteinase domain, RNA helicase domains, the nucleotide-binding motif, viral genome-linked protein (VPg), and RNA-dependent RNA polymerase (RdRp) motifs (Urcuqui-Inchima et al. 2001; Revers et al. 1999; Dougherty et al. 1993; Kadare´ and Haenni 1997; Adams et al. 2005a; Ferrer-Orta et al. 2015; Moradi et al. 2017a; Worrall et al. 2019; Liang et al. 2006), all of which were identified using CDD and InterProScan and through homology with other potyviruses. In addition, conserved KLSC, FRNK, PTK, and DAG motifs, associated with aphid transmission (Plisson et al. 2003; L´opez-Moya et al. 1999), were discovered in the HC-Pro and CP of WVMV-Ir.

Sequence comparison and phylogenetic analysis

The genomic sequence of WVMV-Ir shared identities of 76.37–86.01% and 82.45–91.91% with two other WVMV complete sequences (Beijing and JEBU-p) at the nucleotide (nt) and deduced amino acid (aa) levels, respectively (Table 2). The complete polyprotein sequence of WVMV-Ir displayed the highest nt (86.23%) and aa (91.91%) sequence identity with the Chinese isolate Beijing (AY656816). Haplotype diversity and nucleotide diversity for the polyprotein of all WVMV isolates were 1.000 ± 0.272 and 0.199 ± 0.059, respectively, indicating a high genetic diversity in WVMV population. No recombination event was detected in the WVMV-Ir and Beijing genomes with other members of BCMV group (data not shown). The sequence identities of each coding region of three WVMV isolates were within the range 58.91–91.19% (at nt level) and 45.91–97.63% (at aa level), respectively, suggesting that Ir, Beijing,and JEBU-p are three different strains. The average sequence identity in the 5’-UTRs was only 57.31%. In the 3’-UTRs, the average nt identity was 82.75%, indicating that the 3’-UTR was more conserved than the 5’-UTR. Among putative gene products of WVMV, the P1 and P3 were the most variable proteins (50.78 and 78.67%), while CI and NIa-VPg were the most conserved (94.47 and 92.62%) (Table S2).

Table 2.

Percentages of nucleotide (upper row) and amino acid (lower row) sequence identities between different genomic regions of the isolate Ir and representatives of the WVMV isolates Beijing and JEBU-p

Isolate	5’-UTR	P1	HC-Pro	P3	PIPO	6K1	CI	6K2	NIa-VPg	NIa-Pro	NIb	CP	3’-UTR	Genome	Polyprotein
Beijing	67.88	69.39 (59.94)	82.49 (91.47)	90.59 (93.37)	94.66 (93.33)	89.74 (96.15)	89.59 (97.63)	91.19 (94.34)	88.60 (97.37)	89.37 (96.30)	88.52 (96.52)	88.22 (96.81)	89.72	86.01	86.23 (91.91)
JEBU-p	48.99	58.91 (45.91)	76.81 (87.09)	74.64 (72.33)	83.11 (74.67)	79.49 (86.54)	81.49 (93.22)	81.13 (94.34)	80.35 (90.00)	80.52 (89.71)	79.56 (87.81)	76.88 (80.21)	77.56	76.37	76.78 (82.45)

Moreover, non-synonymous-to-synonymous nucleotide diversity ratio (ω) was obtained for each coding region smaller than 1, indicating that these regions are under negative selection (Table S2). As expected, BlastN and BlastX analyses showed that WVMV isolates were most closely related to BCMV group potyviruses as indicated by Gibbs and Ohshima (2010). The genomic sequence identities between WVMV isolates and other members of the BCMV group (Table 1) ranged from 58.36 to 73.48% (at nt level) and 58.70–81.18% (at aa level). The closest virus identified from genomic sequence analysis was soybean mosaic virus (SMV; 72.78% average nt identity, 80.15% average aa identity) followed by watermelon mosaic virus (WMV, 70.31% average nt identity, 76.90% aa identity). WVMV shared the lowest identities with zantedeschia mild mosaic virus (ZaMMV 59.56 average nt identity, 58.51% average aa identity) and dasheen mosaic virus (DsMV 59.11 average nt identity, 59.88% average aa identity) isolates.

The complete genomic sequences of three WVMV isolates and 47 other isolates of the BCMV group (see Table 1) were subjected to phylogenetic analyses, with one isolate of OYDV (AJ510223) as outgroup. A phylogeny inferred from the polyproteins encoded by these genomes revealed that there are at least 17 distinct lineages of potyviruses, which one of these is the WMMV lineage (Fig. 1). A similar phylogenetic tree was obtained from an alignment of the complete amino acid sequences of the same isolates (Fig. S1).

Fig. 1.

Phylogenetic tree of the complete polyprotein amino acid sequences of WVMV and 16 other members of BCMV subgroup potyviruses, rooted with the onion yellow dwarf virus (OYDV, AJ510223). The tree was generated from a multiple sequence alignment using the maximum likelihood method in MEGAX. The significance of each branch was evaluated by constructing 1000 trees in bootstrap analysis, and the bootstrap values (≥ 70) are shown above the horizontal line at each node. This analysis involved 51 amino acid sequences. The isolate WVMV-Ir (marked) was obtained in this study. All positions containing gaps and missing data were eliminated (complete deletion option). GenBank accession numbers for viruses were shown in Table 1

Comparison of the partial CP region (462 nt of the N-terminal portion) revealed that WVMV-Ir shared 76.88–99.13% nt identity (79.86–100% aa identity) with the nine other WVMV isolates whose partial sequences are only available in the GenBank (Table 1).

WVMV-Ir shared the lowest sequence identities with South Korean isolate JEBU-p (MT603851) (76.88% nt identity, 79.86% aa identity) followed by Chinese isolate SUX-1-HZ (KJ836282) (83.12% nt identity, 89.10% aa identity).

Iranian isolate shared the highest sequence identities with the United Kingdom isolate JW_2014 (KP161267) (99.13% nt identity, 100% aa identity) followed by W7_2014 (KP161266, from the UK) (98.92% nt, 100% aa), and WF03 (DQ009883, from Poland) (98.90% nt, 100% aa). In the phylogenetic tree analysis based on the nt and aa sequences of the partial CP, ten isolates were clustered into two groups and members of group I was divided into two subgroups (IA and IB). Group I included nine isolates from Iran, Australia, Poland, the United Kingdom, and China among which Chinese isolate SUX-1-HZ formed separate subgroup (IB). Group II consisted of just one isolate from South Korea (Fig. 2).

Fig. 2.

Maximum likelihood trees illustrating the phylogenetic relationships between the WVMV-Ir and other WVMV isolates available from the database. The phylogenetic trees were drawn from ClustalW generated multiple sequence alignments of 462 nucleotides (left) and 154 amino acids (right) of the N-terminal portion of CP gene. The isolate WVMV-Ir (marked) was obtained in this study. The sequence of OYDV (AJ510223) was used as an outgroup. Bootstrap percentages of clades, reported along the branches of the tree, derived from bootstrap-resampled data sets (1000 replications)

Biological properties

WVMV-Ir produced chlorotic spots, blotches, and leaf distortion on W. sinensis. This isolate infected N. benthamiana plants, systemically causing mild mosaic and vein clearing symptoms. C. quinoa and C. amaranticolor reacted to WVMV-Ir by the production of local chlorotic lesions in the inoculated leaves. In addition, systemic chlorotic lesions and vein clearing also observed on C. quinoa 14 days post-inoculation. Isolate WVMV-Ir caused vein banding and mosaic on Phaseolus vulgaris and systemic chlorotic spots on Vicia faba (Fig. S2). These results confirmed by RT-PCR using WVMV-specific primers WVMVF1/WVMVR1 as described earlier (Clover et al. 2003). The remaining six plant species did not produce any symptoms and WVMV was not detected in N. glutinosa, N. tabacum cv. Samsun, N. tabacum cv. Xanthi, Cucumis sativus, Datura stramonium, and Petunia hybrida by RT-PCR tests.

Discussion

The determination and analysis of the complete genomic RNA sequence of WVMV would be important to discriminate it from other closely related potyviruses as well as to improve our understanding of its evolutionary history and possibly use it in management strategies. In this study, the first complete genomic sequence and organization of WVMV-Ir from a naturally infected wisteria plant in the northeast of Iran is presented and compared to the previously determined sequences of WVMV, as well as to those of other members of the BCMV group. Our finding is in good agreement with the result of Liang et al. (2006), showing that WVMV is most closely related to SMV and WMV. Phylogenetic analysis based on the genomic and deduced amino acid sequences demonstrated that WVMV isolates are closely related to each other and form a cluster belonging to the BCMV group which was supported by high bootstrap values. However, Ir and Beijing (both isolated from wisteria) formed a separate sub-clade in both aa-based and nt-based phylogenetic trees.

A comparison of the JEBU-p isolate with others deposited in GenBank revealed a high level of genetic variability, throughout the genome analyzed. The genetic diversity of WVMV is mainly adapting to the different conditions. Although many environmental factors can exert evolutionary constraints on viruses, the host plant is believed to be one of the most important. As host-driven adaptation could affect the diversification of viral isolates (Garcı´a-Arenal et al. 2001; Gao et al. 2017), the high genetic variability observed in JEBU-p (isolated from soybean) could be explained, at least in part, by the ecological condition in which WVMV constantly accumulated the available variations to adapt to the host species. On the other hand, since the WVMVs from Iran and China were isolated from the same host, wisteria, it is possible that they have been under similar host adaptive selection.

Nucleotide and amino acid sequence identities between the complete polyprotein-coding genomic sequence and individual genes of WVMV isolates displayed the higher levels of identity for the amino acid sequences (Table 2) indicating that many of the nucleotide substitutions are silent. On the contrary, in P1, P3, and PIPO, the values of the amino acid sequence identity were lower than those estimated from nucleotide sequences, suggesting a prevalence of nonsynonymous mutations in these ORFs. These findings are in agreement with the previous studies showing that P1 and P3 exhibit higher variation than other potyviral proteins (Adams et al. 2005b; Moradi and Mehrvar 2019; Nigam et al. 2019). Collectively, these observations support a role for potyviral P1 and P3 in host adaptation and pathogenicity (Nigam et al. 2019).

Grouping of Iranian isolate in the CP-based phylogenetic tree of this study was in agreement with Valouzi et al. (2020) results, in which Iranian isolate with isolates from Europe and Oceania were clustered in World group.

According to the partial CP sequence identity analysis, WVMV-Ir has most closely resembled the UK and Polish isolates. It is possible that virus-infected plants were imported to Iran from Europe. Furthermore, this suggests that there may be numerous strains/isolates of WVMV in the country. However, the full-genome sequencing of these isolates is required to understand the genetic evolution of the virus. The phylogenetic trees based on the CP gene revealed a lack of clustering by the geographical position which further emphasizing the importance of the exchange and use of virus-free plant material in preventing the dissemination of the virus.

The result of the host range test using mechanical inoculation was in line with that of other studies (Clover et al. 2003; Liang et al. 2004; Naidu and Karthikeyan 2008). Among the tested plants, in addition to wisteria, bean, faba bean, and three weed species were infected with WVMV-Ir. These susceptible host plants could thus serve as potential reservoirs of WVMV and contribute to the off-season survival of the virus in the field. In addition, these plants can play as alternative hosts, and the occurrence/incidence of the virus in such plants should be studied in future studies.

Consistent with our previous report (Al Jaberi et al. 2018) infected plants were randomly distributed and were often situated adjacent to healthy plants. Accordingly, it seems likely that the disease is spread primarily through vegetative propagation organs instead of aphid or mechanical transmission.

This is the first comprehensive report of the complete genomic sequence and organization of WVMV from naturally infected wisteria plants in Iran using RNA deep sequencing, specific primers, and 5′ RACE. Our results provide valuable information on pathogenicity and the processes involved in virus evolution and epidemiology, some of which are crucial for designing reliable diagnostic tools and developing efficient and durable disease control strategies.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

MM conceived and designed the experiments. MSAJ did the experimental works. ZM and HRAI helped to conduct some laboratory experiments. ZM prepared the manuscript, performed data analysis, and interpretation. ZM, MM, MZ and MSAJ contributed to the interpretation and critical revision of the manuscript. All authors read and approved the final manuscript.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.