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Erschienen in: Virology Journal 1/2023

Open Access 01.12.2023 | Brief Report

Snake River alfalfa virus, a persistent virus infecting alfalfa (Medicago sativa L.) in Washington State, USA

verfasst von: Olga A. Postnikova, Brian M. Irish, Jonathan Eisenback, Lev G. Nemchinov

Erschienen in: Virology Journal | Ausgabe 1/2023

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Abstract

Here we report an occurrence of Snake River alfalfa virus (SRAV) in Washington state, USA. SRAV was recently identified in alfalfa (Medicago sativa L.) plants and western flower thrips in south-central Idaho and proposed to be a first flavi-like virus identified in a plant host. We argue that the SRAV, based on its prevalence in alfalfa plants, readily detectable dsRNA, genome structure, presence in alfalfa seeds, and seed-mediated transmission is a persistent new virus distantly resembling members of the family Endornaviridae.
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Supplementary Information

The online version contains supplementary material available at https://​doi.​org/​10.​1186/​s12985-023-01991-7.

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Abkürzungen
SRAV
Snake River alfalfa virus
RdRp
RNA-dependent RNA polymerase
HTS
High-throughput sequencing
HMM
Hidden Markov Models
RACE
Rapid Amplification of cDNA Ends
RT-PCR
Reverse transcription-polymerase chain reaction

Main text

Snake River alfalfa virus (SRAV) was recently identified from alfalfa plants and thrips Frankliniella occidentalis collected in the Minidoka and Twin Falls counties of Idaho, USA [1]. Based on the genome structure and phylogeny of its RNA-dependent RNA polymerase (RdRp), the authors hypothesized that SRAV is the first flavi-like virus identified in a plant host [1]. The SRAV polyprotein, however, contained no predicted helicase domain found in all flaviviruses.
To date, no occurrences of SRAV have been reported in alfalfa or on different hosts from other locations. In this work, applying high-throughput sequencing (HTS), we detected SRAV in 50 individual alfalfa plant samples collected from 10 commercial fields in Grant County, WA. Plants used for RNA extraction displayed a diverse symptomatology that occasionally correlated with the symptoms allegedly reported for SRAV, such as yellowing and vein clearing (Fig. 1), [1]. These symptoms, however, were likely due to the presence of multiple co-infecting pathogens in the same plants.
Total RNA was extracted using Promega Maxwell® RSC Plant RNA Kit (Promega Corp., Fitchburg, WI). Library preparation was performed with Illumina TruSeq Stranded Total RNA with Ribo-Zero kit (Illumina Inc., San Diego, CA), and the sequencing platform used was HiseqX10 (PE150) (Omega Biosciences, Norcross, GA). Bioinformatic pipeline included adapter trimming followed by de-novo assembly of reads, unmapped to M. sativa genome and M. truncatula mitochondrion genomes using SPAdes [7] in HMM-guided mode. Phylogenetic analysis was performed with MEGA software [6] using Maximum Likelihood method and bootstrap analysis of 1000 replicates. Conserved RdRp domains for multiple alignment were obtained using InterPro tool (https://​www.​ebi.​ac.​uk/​interpro/​).
All 50 unique alfalfa plant samples contained viral reads, varying in quantity from 46 to 71,267 (Table 1). Total number of reads mapping to the SRAV genome was 1,017,715 with an approximate length of each read 150 nt (Table 1). Several contigs apparently representing the complete genome of the virus were assembled de novo. The sequences varied slightly in length and contained small number of SNPs, indicating a presence of different genetic variants of the virus. Nevertheless, the sequence identity when compared to the reference genome (ON669064) in all cases remained > 99%.
Table 1
Occurrence of SRAV-related reads in 10 commercial alfalfa fields (five samples per field) of Grant County, Washington State
Sample ID
Length
Covered %
Covered bases
Total viral reads
Reads/sample
Yield (Mb)
% ≥ Q30
A2-1
11,872
100
11,872
8079
31,990,204
4831
94.56
A1-4
11,872
99.9832
11,870
27,925
37,707,294
5694
94.54
A4-3
11,872
99.9663
11,868
22,147
39,187,338
5917
94.75
B8-4
11,872
99.9579
11,867
15,093
45,330,070
6845
95.26
A3-4
11,872
99.9326
11,864
44,605
44,468,798
6715
94.77
A3-5
11,872
99.7473
11,842
32,167
34,259,318
5173
94.76
B8-2
11,872
99.7136
11,838
47,175
44,581,834
6732
95.21
A5-5
11,872
99.6715
11,833
21,219
45,938,534
6937
94.76
B6-5
11,872
99.6631
11,832
13,815
44,882,040
6777
95.29
A5-4
11,872
99.6462
11,830
8513
34,368,240
5190
94.66
B8-5
11,872
99.621
11,827
53,101
37,624,098
5681
95
A1-2
11,872
99.5788
11,822
28,724
42,968,950
6488
94.74
A3-1
11,872
99.5115
11,814
33,926
42,255,734
6381
94.78
A2-2
11,872
99.4862
11,811
71,267
37,337,248
5638
94.71
B6-1
11,872
99.4862
11,811
17,265
47,716,504
7205
93.95
B7-5
11,872
99.4525
11,807
25,870
31,743,182
4793
95.13
A1-5
11,872
99.4441
11,806
20,408
46,898,746
7082
94.9
B6-4
11,872
99.4104
11,802
24,605
36,660,924
5536
95.13
B7-4
11,872
99.402
11,801
29,217
39,153,470
5912
95.18
B8-1
11,872
99.402
11,801
44,383
33,674,988
5085
94.86
A1-3
11,872
99.3851
11,799
14,704
34,965,248
5280
94.65
A2-5
11,872
99.3851
11,799
27,777
50,933,376
7691
94.69
A4-4
11,872
99.3851
11,799
20,253
31,710,774
4788
94.62
A5-2
11,872
99.3851
11,799
14,048
42,018,256
6345
94.7
B10-4
11,872
99.3851
11,799
16,305
35,022,936
5288
95.1
B8-3
11,872
99.3851
11,799
37,761
36,163,008
5461
95.34
B9-3
11,872
99.3851
11,799
26,815
41,588,106
6280
95.12
B6-3
11,872
99.3767
11,798
30,672
43,616,596
6586
95.3
B9-4
11,872
99.3767
11,798
10,475
50,653,058
7649
95.3
A2-3
11,872
99.3683
11,797
64,184
39,012,034
5891
94.7
A4-1
11,872
99.3683
11,797
12,057
35,547,054
5368
94.6
A5-3
11,872
99.3683
11,797
7016
50,538,150
7631
94.98
B6-2
11,872
99.3683
11,797
8332
55,187,384
8333
95.25
B7-1
11,872
99.3683
11,797
39,608
35,432,658
5350
95.2
B7-3
11,872
99.3683
11,797
28,372
45,878,018
6928
95.27
B9-2
11,872
99.3683
11,797
12,085
43,347,806
6546
95.11
A2-4
11,872
99.3514
11,795
19,539
32,282,770
4875
94.76
A4-5
11,872
99.343
11,794
4425
32,902,614
4968
94.63
B7-2
11,872
99.343
11,794
10,734
35,619,196
5378
95.05
B10-5
11,872
98.8376
11,734
9244
35,500,288
5361
95.15
B9-5
11,872
98.5428
11,699
10,684
37,603,012
5678
95.42
A3-2
11,872
98.0374
11,639
1288
39,786,654
6008
94.56
A3-3
11,872
96.6139
11,470
1035
40,166,166
6065
94.71
A1-1
11,872
85.3858
10,137
392
49,277,406
7441
94.78
B10-3
11,872
41.9643
4982
83
41,582,718
6279
95.19
A5-1
11,872
40.0775
4758
94
40,943,890
6183
94.85
B10-2
11,872
38.0475
4517
73
33,886,868
5117
94.98
B9-1
11,872
30.4835
3619
59
35,593,490
5375
95.07
B10-1
11,872
26.9794
3203
46
37,715,730
5695
95.36
A4-2
11,872
22.7257
2698
51
36,605,156
5527
94.69
One of the de novo-assembled contigs recovered from the individual library and sample containing 8,079 viral reads, was 11,811 nt in length and had 100% base coverage with the reference, thus representing a complete genome of the virus. It was 7 nt longer at the 5' end than ON669064, which was confirmed by 5' RACE using SMARTer® RACE 5'/3' Kit (Takara Bio USA, Inc., San Jose, CA). The 3’ end of the sequence was 59 nt longer than that of the reference ON669064 and matched another isolate of the virus, SRAV_ALF1071, found in GenBank under accession number ON669090.1 [1]. Application of the 3'RACE also showed that the virus has ~ 30-long 3′-terminal poly(A) tract, which is absent in all members of the family Flaviviridae ([14]; https://​ictv.​global/​report/​chapter/​flaviviridae/​flaviviridae).
At the nucleotide level, the SRAV-WA1 (for Washington State) was 99.8% identical to the reference genome ON669064 with 18 SNPs between the two, therefore depicting an isolate of the same virus. The genome of SRAV-WA1 encoded a single 3,835 amino acid (aa) polyprotein 99.9% identical to the reference. BLASTP and PSI-BLAST searchers of the SRAV-WA1 polyprotein against the GenBank database identified no related sequences except existing SRAV submissions. In silico analyses of the viral polyprotein using Pfam, InterPro and CDD databases revealed the presence of the conserved RdRp domain (3220–3478 aa, E-value = 1.43E-21, InterPro). No other domains were reliably identified. A weak relation to the superfamily of trypsin-like serine proteases (E-value = 1.01e-03) was found in the 1855–1910 aa region of the polyprotein when Superfamily database (https://​supfam.​org) was used to detect protein sequence similarities.
The results obtained by HTS were validated by RT-PCR with two sets of primers in three technical replicates using SuperScript III One-Step RT-PCR System (Thermo Fisher Scientific, Waltham, MA). One set was ANPV_3 derived from Dahan et al. [1], and another set of primers was designed in this work: LN1036-F, GGGAGAACCAGGAAACTGTTAG and LN1037-R, CTGTCGCATAGTCCGCTTATT. RT-PCR using both primers pairs produced correct amplicons, while no products were generated from control samples in which no SRAV-related HTS reads were found (Fig. 2). The amplicons were sequenced and validated to be SRAV.
The omnipresence of the virus in all analyzed samples indicated the possibility of persistent infection like those caused by partitiviruses and endornaviruses [16]. Considering resemblance in the size and structure of the genome, we compared SRAV to endornaviruses. Viruses in this family infect plants, fungi, and Oomycetes, and are generally associated with symptomless infections and no pathogenic effects [3]. They have a linear genome of 10 to 17 kbp in length, that encodes a polyprotein ranging from 3,217 to 5,825 aa [3, 16, 19]. Notably, several known endornaviruses, same as SRAV, lack helicase domain [16, 19]. While members of the Endornaviridae family were often reported as double-stranded RNA viruses [3, 16], current ICTV classification describes them as single-stranded, positive-sense RNA genomes that have been characterized using replicative dsRNAs forms [19].
Phylogenetic analysis using the polyproteins of SRAV, different viruses of the family Endornaviridae, and members of the family Flaviviridae, placed both SRAV isolates within Endornaviridae, although SRAV isolates formed a separate cluster (Additional File 1). When we performed phylogenetic analysis using InterPro-extracted RdRp domains of the Endornaviridae and Flaviviridae (3,204–3,462 aa in SRAV-WA1), SRAV clustered with the former as well, again forming a distinct grouping (Additional File 2). It is worth noting, however, that SRAV placement was not consistent, pointing to potentially incorrect phylogenies or an irreproducibility in maximum likelihood inference and [18].
When we used Sequence Demarcation Tool program that allows classification of virus sequences based on sequence pairwise identity (SDTv1.2; [10]), it showed low similarities of both polyprotein and RdRp of SRAV with those of endornaviruses and flaviviruses (Additional Files 3 and 4).
Since plant endornaviruses are transmitted through seeds via the gametes [8, 13, 19], we decided to test seeds of several alfalfa cultivars for the presence of the virus by RT-PCR. Seeds were scarified with concentrated H2SO4, surface-sterilized with 70% ethanol, and rinsed with sterile water [11]. Total RNA was extracted with Takara Plant and Fungal RNA isolation kit (Takara Bio, San Jose, CA) and used in RT-PCR with primers LN1036/37. RT-PCRs with five out of six tested seed samples derived from different alfalfa cultivars (Maverick, SW-9215, SW-8421, SW-9720, and CUF101) were virus-positive, indicating a high rate of seed infection (Fig. 2). Resultant amplicons were sequenced and confirmed to be SRAV-WA1. Seeds of one cultivar, Regency SY, were RT-PCR-negative (not shown). To additionally confirm seed transmission of the virus, leaves of the germinated seedlings were randomly checked by RT-PCR one week after germination. Except for Regency SY, seedlings of other tested cultivars were positive for SRAV-WA1 (not shown). These experiments demonstrated localization of SRAV-WA1 in the internal parts of the seed, likely in the embryo. They also showed a high rate of seed infection by the virus, and its efficient vertical transmission via seeds, thus confirming persistent nature of the virus [15].
One of the characteristic features of all endornaviruses is readily detectable viral replicative form, double-stranded RNAs (dsRNAs), that accumulates in the host tissues in high quantities [19]. To extract dsRNA from leaves of the SRAV-WA1-infected alfalfa plants, we followed the protocol of Khankhum et al. [5]. Agarose gel electrophoresis showed the presence of dsRNA of the approximately correct size corresponding to that predicted by de novo assembly of the HTS reads (Fig. 3).
Additionally, given that many endornavirus RNAs have a site-specific discontinuity (nick) on 5’ terminus of the coding strand, we have attempted, but failed, to determine its presence in the genome of SRAV-WA1 using 5'RACE approach [13].
Dahan et al. [1] detected SRAV in western flower thrips and suggested a possible role for the insect in virus transmission. However, thrips are known to transmit tospoviruses and plant viruses in the Ilarvirus, Carmovirus, Sobemovirus and Machlomovirus genera [4]. Based on our data, SRAV, analogously to vertically transmitted endornaviruses [8, 9, 13, 19], is also transmitted by seeds. Although alfalfa is one of the primary hosts for western flower thrips (and other species) and acquiring the SRAV during feeding cannot be excluded, transmission of the virus by thrips would require additional experimental confirmation.
Other viruses found in samples infected with SRAV-WA1 included alfalfa mosaic virus, pea streak virus, lucerne transient streak virus, bean leaf roll virus, partitiviruses, and amalgavirus. Fungal and bacterial pathogens, described in alfalfa, like Alternaria spp., Bipolaris spp., Stemphylium spp., Fusarium spp., Pseudomonas spp., Erwinia spp. etc. were also detected. These findings suggested that traditional Koch’s postulate of “one microbe—one disease” should be broadened into the principle of a pathobiome, when disease symptoms are attributed to a diverse community of pathogenic organisms within the plant, rather than to a single infectious agent [20].
Overall, our research confirmed association of SRAV with alfalfa and, for the first time, identified an extensive occurrence of this virus in Washington State. The importance of this work also relies on the hypothesis that placement of SRAV within the flavi-like lineage, as suggested by Dahan et al. [1], may not be entirely accurate. Prevalence of the virus in alfalfa plants, its genome organization, seed-mediated transmission, presence of the easily detectable dsRNA and, although partly, phylogenetic reconstruction, suggest that SRAV is a persistent virus possessing some features characteristic for endornaviruses.
However, the low percent identity of SRAV with endornaviruses and flaviviruses, absence of the poly (C) and presence of the poly (A) tract at the 3’ terminus of the genome, and lack of the site-specific nick at the 5’ end, indicate that SRAV may represent an entirely new taxonomic group of persistent viruses that does not belong to either of the two families. Altogether, more data are needed to assess taxonomy, biology, and economic importance of the virus.

Acknowledgements

We thank Jonathan Shao for useful discussions.

Declarations

Not applicable. All authors consent to the publication of the manuscript.

Competing interests

The authors declare no competing interests.
Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creativecommons.​org/​licenses/​by/​4.​0/​. The Creative Commons Public Domain Dedication waiver (http://​creativecommons.​org/​publicdomain/​zero/​1.​0/​) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Literatur
1.
Zurück zum Zitat Dahan J, Wolf YI, Orellana GE, Wenninger EJ, Koonin EV, Karasev AV. A novel flavi-like virus in Alfalfa (Medicago sativa L.) crops along the Snake River Valley. Viruses. 2022;14:1320.CrossRefPubMedPubMedCentral Dahan J, Wolf YI, Orellana GE, Wenninger EJ, Koonin EV, Karasev AV. A novel flavi-like virus in Alfalfa (Medicago sativa L.) crops along the Snake River Valley. Viruses. 2022;14:1320.CrossRefPubMedPubMedCentral
4.
Zurück zum Zitat Jones DR. Plant viruses transmitted by thrips. Eur J Plant Pathol. 2005;113:119–57.CrossRef Jones DR. Plant viruses transmitted by thrips. Eur J Plant Pathol. 2005;113:119–57.CrossRef
5.
Zurück zum Zitat Khankhum S, Escalante C, de Rodrigues SE, Valverde RA. Extraction and electrophoretic analysis of large dsRNAs from desiccated plant tissues infected with plant viruses and biotrophic fungi. Eur J Plant Pathol. 2017;147:431–41.CrossRef Khankhum S, Escalante C, de Rodrigues SE, Valverde RA. Extraction and electrophoretic analysis of large dsRNAs from desiccated plant tissues infected with plant viruses and biotrophic fungi. Eur J Plant Pathol. 2017;147:431–41.CrossRef
6.
Zurück zum Zitat Kumar S, Stecher G, Li M, Knyaz C, Tamura K. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol. 2018;35:1547–9.CrossRefPubMedPubMedCentral Kumar S, Stecher G, Li M, Knyaz C, Tamura K. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol. 2018;35:1547–9.CrossRefPubMedPubMedCentral
8.
Zurück zum Zitat Moriyama H, Kanaya K, Wang JZ, Nitta T, Fukuhara T. Stringently and developmentally regulated levels of a cytoplasmic double-stranded RNA and its high-efficiency transmission via egg and pollen in rice. Plant Mol Biol. 1996;31:713–9.CrossRefPubMed Moriyama H, Kanaya K, Wang JZ, Nitta T, Fukuhara T. Stringently and developmentally regulated levels of a cytoplasmic double-stranded RNA and its high-efficiency transmission via egg and pollen in rice. Plant Mol Biol. 1996;31:713–9.CrossRefPubMed
9.
Zurück zum Zitat Moriyama H, Horiuchi H, Nitta T, Fukuhara T. Unusual inheritance of evolutionarily-related double-stranded RNAs in interspecific hybrid between rice plants Oryza sativa and Oryza rufipogon. Plant Mol Biol. 1999;39:1127–36.CrossRefPubMed Moriyama H, Horiuchi H, Nitta T, Fukuhara T. Unusual inheritance of evolutionarily-related double-stranded RNAs in interspecific hybrid between rice plants Oryza sativa and Oryza rufipogon. Plant Mol Biol. 1999;39:1127–36.CrossRefPubMed
12.
Zurück zum Zitat Okada R, Kiyota E, Sabanadzovic S, Moriyama H, Fukuhara T, Saha P, Roossinck MJ, Severin A, Valverde RA. Bell pepper endornavirus: molecular and biological properties, and occurrence in the genus Capsicum. J Gen Virol. 2011;92:2664–73.CrossRefPubMed Okada R, Kiyota E, Sabanadzovic S, Moriyama H, Fukuhara T, Saha P, Roossinck MJ, Severin A, Valverde RA. Bell pepper endornavirus: molecular and biological properties, and occurrence in the genus Capsicum. J Gen Virol. 2011;92:2664–73.CrossRefPubMed
13.
Zurück zum Zitat Okada R, Yong CK, Valverde RA, Sabanadzovic S, Aoki N, Hotate S, Kiyota E, Moriyama H, Fukuhara T. Molecular characterization of two evolutionarily distinct endornaviruses co-infecting common bean (Phaseolus vulgaris). J Gen Virol. 2013;94:220–9.CrossRefPubMed Okada R, Yong CK, Valverde RA, Sabanadzovic S, Aoki N, Hotate S, Kiyota E, Moriyama H, Fukuhara T. Molecular characterization of two evolutionarily distinct endornaviruses co-infecting common bean (Phaseolus vulgaris). J Gen Virol. 2013;94:220–9.CrossRefPubMed
14.
Zurück zum Zitat Payne S. Family Flaviviridae. In: Viruses, from understanding to investigation. Elsevier Inc. 2017. p. 130–9. Payne S. Family Flaviviridae. In: Viruses, from understanding to investigation. Elsevier Inc. 2017. p. 130–9.
18.
Zurück zum Zitat Shen XX, Li Y, Hittinger CT, Chen XX, Rokas A. An investigation of irreproducibility in maximum likelihood phylogenetic inference. Nature Comm. 2020;11:6096.CrossRef Shen XX, Li Y, Hittinger CT, Chen XX, Rokas A. An investigation of irreproducibility in maximum likelihood phylogenetic inference. Nature Comm. 2020;11:6096.CrossRef
Metadaten
Titel
Snake River alfalfa virus, a persistent virus infecting alfalfa (Medicago sativa L.) in Washington State, USA
verfasst von
Olga A. Postnikova
Brian M. Irish
Jonathan Eisenback
Lev G. Nemchinov
Publikationsdatum
01.12.2023
Verlag
BioMed Central
Erschienen in
Virology Journal / Ausgabe 1/2023
Elektronische ISSN: 1743-422X
DOI
https://doi.org/10.1186/s12985-023-01991-7

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