First Report and Complete Genome Characterization of Cherry Virus A and Little Cherry Virus 1 from Russia

Abstract

Virus diseases affect the yield and fruit quality and shorten the productive life of stone fruits (Prunus spp. in the family Rosaceae). Of over fifty known viruses infecting these crops, cherry virus A (CVA) is among the most common, and little cherry virus 1 (LChV1) is one of the most economically important. Using high-throughput sequencing, full-length genomes of CVA and LChV1 isolates, found on interspecies hybrids in the Prunus collection of the Nikita Botanical Gardens, Russia, were sequenced, assembled, and characterized. CVA was found in the P. cerasifera × P. armeniaca hybrid and in phylogenetic analysis clustered with non-cherry virus isolates. The LChV1 isolate Stepnoe was detected in ((P. cerasifera Ehrh. × P. armeniaca L.) × P. brigantiaca Vill.) trihybrid suggesting that both P. cerasifera and P. brigantiaca potentially can be the LChV1 hosts. The isolate Stepnoe was most closely related to the Greece isolate G15_3 from sweet cherry, sharing 77.3% identity at the nucleotide level. Possibly, the highly divergent Russian isolate represents one more phylogroup of this virus. This is the first report of CVA and LChV1 from Russia, expanding the information on their geographical distribution and genetic diversity.

Full text

Citation: Chirkov, S.; Sheveleva, A.;
Tsygankova, S.; Slobodova, N.;
Sharko, F.; Petrova, K.; Mitrofanova, I.
First Report and Complete Genome
Characterization of Cherry Virus A
and Little Cherry Virus 1 from Russia.
Plants 2023,12, 3295. https://
doi.org/10.3390/plants12183295
Academic Editors: Igor Koloniuk and
Jana Fránová
Received: 27 July 2023
Revised: 11 September 2023
Accepted: 12 September 2023
Published: 18 September 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
plants
Communication
First Report and Complete Genome Characterization of Cherry
Virus A and Little Cherry Virus 1 from Russia
Sergei Chirkov 1, * , Anna Sheveleva 1, Svetlana Tsygankova 2, Natalia Slobodova 2,3, Fedor Sharko 2,4 ,
Kristina Petrova 2,5 and Irina Mitrofanova 6
1Department of Virology, Faculty of Biology, Lomonosov Moscow State University, 119234 Moscow, Russia;
anncsh@yandex.ru
2National Research Center “Kurchatov Institute”, 123182 Moscow, Russia;
svetlana.tsygankova@gmail.com (S.T.); nv.slobodova@gmail.com (N.S.); fedosic@gmail.com (F.S.);
petrova.k.o@yandex.ru (K.P.)
3Faculty of Biology and Biotechnology, HSE University, 101000 Moscow, Russia
4Federal Research Center “Fundamentals of Biotechnology”, Russian Academy of Sciences,
119071 Moscow, Russia
5Research Center for Medical Genetics, 115552 Moscow, Russia
6Tsitsin Main Botanical Garden of Russian Academy of Sciences, 127276 Moscow, Russia;
irimitrofanova@yandex.ru
*Correspondence: s-chirkov1@yandex.ru
Abstract:
Virus diseases affect the yield and fruit quality and shorten the productive life of stone fruits
(Prunus spp. in the family Rosaceae). Of over fifty known viruses infecting these crops, cherry virus A
(CVA) is among the most common, and little cherry virus 1 (LChV1) is one of the most economically
important. Using high-throughput sequencing, full-length genomes of CVA and LChV1 isolates,
found on interspecies hybrids in the Prunus collection of the Nikita Botanical Gardens, Russia, were
sequenced, assembled, and characterized. CVA was found in the P. cerasifera
×
P. armeniaca hybrid
and in phylogenetic analysis clustered with non-cherry virus isolates. The LChV1 isolate Stepnoe was
detected in ((P. cerasifera Ehrh.
×
P. armeniaca L.)
×
P. brigantiaca Vill.) trihybrid suggesting that both P.
cerasifera and P. brigantiaca potentially can be the LChV1 hosts. The isolate Stepnoe was most closely
related to the Greece isolate G15_3 from sweet cherry, sharing 77.3% identity at the nucleotide level.
Possibly, the highly divergent Russian isolate represents one more phylogroup of this virus. This is
the first report of CVA and LChV1 from Russia, expanding the information on their geographical
distribution and genetic diversity.
Keywords:
Prunus spp.; virome; high-throughput sequencing; cherry virus A; little cherry virus 1;
full-length genomes
1. Introduction
All cultivated stone fruits (Prunus spp. in the family Rosaceae) are of great economic
significance. Viruses affect the yield and fruit quality and shorten the productive life of the
infected trees. The vegetative propagation that is widely used in the stone fruits cultivation
contributes to the spread of viruses from plant to plant and into new regions with infected
plant material. Over fifty viruses infecting these crops were discovered. In recent years,
this list was continuously expanded due to the use of high-throughput sequencing (HTS)
to study fruit tree viromes [
1
–
4
]. Cherry virus A (CVA) is among the most common Prunus
viruses, and little cherry virus 1 (LChV1) is one of the most economically important [5,6].
CVA is a member of the genus Capillovirus in the family Betaflexiviridae [
7
]. The virus
genome is single-stranded positive-sense polyadenylated RNA of about 7.5 kb with two
open reading frames (ORFs). ORF1 encodes a polyprotein containing replication-associated
proteins and the coat protein (CP). ORF2 is nested within ORF1 in a different reading frame
Plants 2023,12, 3295. https://doi.org/10.3390/plants12183295 https://www.mdpi.com/journal/plants

Plants 2023,12, 3295 2 of 10
and encodes the movement protein (MP). ORF1 is flanked with the 5
0
- and 3
0
-non-coding
regions (NCR).
CVA is widely distributed in cherry-growing regions worldwide and was also detected
in non-cherry hosts such as peach (P. persica), apricot (P. armeniaca), plum (P. domestica),
myrobalan (P. cerasifera), Japanese (Chinese) plum (P. salicina), flowering cherry (P. serrulata),
and Japanese apricot (P. mume). CVA is transmitted from plant to plant by grafting and
vegetative propagation; no vector is known [2,4,5].
In cherry, CVA infection alone is usually latent and no disease can be related to the
virus. However, in mixed infection with other viruses CVA can modulate the symptoms
severity and affect scion/rootstock compatibility [
8
,
9
]. In contrast, symptoms of vein
clearing, chlorosis, necrosis and mosaic were observed on CVA-infected apricot and my-
robalan [
10
–
12
]. Although some of them may be potentially attributed to a mixed infection,
no other viruses were detected by HTS in the CVA-infected apricot displaying vein-clearing
symptoms [10].
LChV1 is a member of the genus Velarivirus in the family Closteroviridae [
13
]. The
LChV1 genome is single-stranded positive-sense RNA of 16–17 kb and includes eight
ORFs. The 5
0
-end is likely capped and the 3
0
-NCR is not polyadenylated and does not
organize in a tRNA-like structure. Overlapping ORF1a and ORF1b encode replicase and
RNA-dependent RNA polymerase (RdRp) and are translated as a polyprotein containing
methyltransferase (MET), helicase (HEL), and RNA-dependent RNA polymerase (RdRp)
domains. ORF2 encodes a small hydrophobic protein with a transmembrane domain.
ORF3 encodes a homologue of the cellular HSP70h heat shock protein. ORF4 encodes a
p61 protein with some similarity with the cellular HSP90. ORF5 and ORF6 code for the CP
and minor capsid protein (CPm), respectively. Both CPs encapsidate the genome and are
necessary for the cell-to-cell virus movement. ORF7 and ORF8 encode putative p21 and
p27 proteins with no known functions [14–17].
LChV1 is a phloem-limited and graft-transmissible pathogen. There is no known insect
vector for LChV1, so the virus spread among plants occurs mainly through vegetative
propagation of infected plant material. The LChV1 hosts sweet and sour cherry and less
frequently other Prunus species such as almond, apricot, peach, plum, and flowering
cherry [
18
–
23
]. No clear symptoms are usually observed in non-cherry hosts. In contrast,
in cherry LChV1 is associated with little cherry disease, which is distributed worldwide
and manifested in the decreasing yield and fruit quality. Fruit symptoms occurring in
susceptible sweet cherry cultivars include reduction in fruit size, color and taste while other
cultivars can be symptomless. LChV1 is also responsible for Kwanzan stunting syndrome
and Shirofugen stunt disease of flowering cherry [24,25].
The large collection of peach, nectarine, apricot, almond, plum, sweet and sour cherry
genotypes, both local and introduced from North America, Southern Europe, and Central
Asia, is maintained in the Nikita Botanical Gardens (NBG), Yalta, Russia [
26
]. An easy
hybridization between Prunus species contributes to this gene pool, which is constantly
being updated, expanded and exploited in breeding and biotechnological work. Many
Prunus cultivars originated from this collection are distributed across the southern regions
of Russia. Monitoring of viral diseases using various detection tools is important to limit
the further spread of viruses. The phytosanitary status of this germplasm collection was
studied using metatranscriptomic analysis of trees displaying virus-like symptoms on the
leaves. The reads related to CVA and LChV1 were generated by HTS in two samples from
Prunus interspecies hybrids.
The objectives of this work were sequencing, assembly, and characterization of com-
plete genomes of the Russian CVA and LChV1 isolates and their comparison with known
isolates of these viruses available in GenBank.

Plants 2023,12, 3295 3 of 10
2. Results
2.1. Plant Material and Virus Detection
Two twelve-year-old Prunus trees displaying virus-like symptoms on the leaves
(Figure 1) were selected for the metatranscriptomic analysis. These samples were named as
follows: PTC from (P. cerasifera Ehrh.
×
P. armeniaca L. cultivar Shlor Tsiran) hybrid, grafted
on a wild apricot, and Stepnoe from ((P. cerasifera Ehrh.
×
P. armeniaca L.)
×
P. brigantiaca
Vill.) hybrid, grafted on P. cerasifera.
Plants 2023, 12, x FOR PEER REVIEW 3 of 11
2. Results
2.1. Plant Material and Virus Detection
Two twelve-year-old Prunus trees displaying virus-like symptoms on the leaves (Fig-
ure 1) were selected for the metatranscriptomic analysis. These samples were named as
follows: PTC from (P. cerasifera Ehrh. × P. armeniaca L. cultivar Shlor Tsiran) hybrid, grafted
on a wild apricot, and Stepnoe from ((P. cerasifera Ehrh. × P. armeniaca L.) × P. brigantiaсa
Vill.) hybrid, grafted on P. cerasifera.
Figure 1. Virus-like symptoms on the leaves of Prunus trees infected with cherry virus A (PTC) and
lile cherry virus 1 (Stepnoe).
The reverse transcription–polymerase chain reaction (RT-PCR) products of the ex-
pected sizes of 837 and 518 base pairs (bps) were obtained when analyzing total RNA from
these specimens (Figure S1). Their sequencing by the Sanger method confirmed CVA and
LChV1 in the corresponding samples. Thus, the HTS results were corroborated by the RT-
PCR assay.
2.2. HTS Results
The reads related to CVA and LChV1 were generated from the samples PTC and
Stepnoe, respectively (Table 1). They covered the assembled virus-specific contigs with an
average depth of coverage 400× to 720×. In addition, reads related to prunus necrotic ring-
spot virus (PNRSV) were revealed in the sample Stepnoe.
Table 1. Results of high-throughput sequencing of Prunus samples.
Sample Number of Clean Reads a Per a Sample Virus Detected Number of Virus-Specific Reads b
Stepnoe 2,903,382 Little cherry virus 1 27,359
PTC 2,250,077 Cherry virus A 21,341
a Pair-ended reads of 250 nucleotides. b Determined using Bowtie2 v.2.4.4.
2.3. Characterization of the CVA Genome
One contig 7426 nucleotides (nt) length was most closely related (99.5% identity) to
the genome of the CVA isolates 19SP013 (MZ291922) and 13TF169_N11 (KY510919) from
Canada as well as Ruzyne (ON088603) and WK (LN879388) from the Czech Republic and
Australia, respectively. This contig covered the genomes of these isolates nearly com-
pletely and seemed to represent a new full-length CVA genome. With other CVA isolates,
for which complete genomes were retrieved from GenBank, the isolate PTC shared 81 to
99% identity. Typically, of the genus Capillovirus, two ORFs were identified in the PTC
genome. ORF1 of 7029 nt encodes a polyprotein of 2342 amino acid (aa) residues and ORF2
in a different reading frame 1392 nt long encodes the MP of 463 aa. MET, HEL, RdRp, MP
and CP domains were mapped at positions 227–1153, 2552–3388, 3947–4900, 5506–6012,
and 6632–7105, respectively. The 5′- and 3′-NCRs (excluding the poly (A) tail) consisted of
Figure 1.
Virus-like symptoms on the leaves of Prunus trees infected with cherry virus A (PTC) and
little cherry virus 1 (Stepnoe).
The reverse transcription–polymerase chain reaction (RT-PCR) products of the ex-
pected sizes of 837 and 518 base pairs (bps) were obtained when analyzing total RNA from
these specimens (Figure S1). Their sequencing by the Sanger method confirmed CVA and
LChV1 in the corresponding samples. Thus, the HTS results were corroborated by the
RT-PCR assay.
2.2. HTS Results
The reads related to CVA and LChV1 were generated from the samples PTC and
Stepnoe, respectively (Table 1). They covered the assembled virus-specific contigs with
an average depth of coverage 400
×
to 720
×
. In addition, reads related to prunus necrotic
ringspot virus (PNRSV) were revealed in the sample Stepnoe.
Table 1. Results of high-throughput sequencing of Prunus samples.
Sample Number of Clean Reads aPer a Sample Virus Detected Number of Virus-Specific Reads b
Stepnoe 2,903,382 Little cherry virus 1 27,359
PTC 2,250,077 Cherry virus A 21,341
aPair-ended reads of 250 nucleotides. bDetermined using Bowtie2 v.2.4.4.
2.3. Characterization of the CVA Genome
One contig 7426 nucleotides (nt) length was most closely related (99.5% identity) to
the genome of the CVA isolates 19SP013 (MZ291922) and 13TF169_N11 (KY510919) from
Canada as well as Ruzyne (ON088603) and WK (LN879388) from the Czech Republic and
Australia, respectively. This contig covered the genomes of these isolates nearly completely
and seemed to represent a new full-length CVA genome. With other CVA isolates, for
which complete genomes were retrieved from GenBank, the isolate PTC shared 81 to 99%
identity. Typically, of the genus Capillovirus, two ORFs were identified in the PTC genome.
ORF1 of 7029 nt encodes a polyprotein of 2342 amino acid (aa) residues and ORF2 in a
different reading frame 1392 nt long encodes the MP of 463 aa. MET, HEL, RdRp, MP and
CP domains were mapped at positions 227–1153, 2552–3388, 3947–4900, 5506–6012, and
6632–7105, respectively. The 5
0
- and 3
0
-NCRs (excluding the poly (A) tail) consisted of 100
and 297 nt, respectively. The sequences of the 5
0
-terminal part of the MP gene, determined

Plants 2023,12, 3295 4 of 10
by HTS and Sanger sequencing, were identical. A near-complete genome of the Russian
CVA isolate PTC was deposited in GenBank under accession number OQ865368. No other
contigs showed similarity with any known virus available in GenBank.
Phylogenetic analysis of all available full-length CVA genomes (n= 127, accessed on
April 2023) showed that most isolates were clustered in several phylogroups (Figure 2).
The PTC was assigned to the clade formed by two Canadian (MZ291922, KY510919),
Czech (ON088603), and Australian (LN879388) isolates, thus confirming the results of
sequence identity analysis.
Plants 2023, 12, x FOR PEER REVIEW 4 of 11
100 and 297 nt, respectively. The sequences of the 5′-terminal part of the MP gene, deter-
mined by HTS and Sanger sequencing, were identical. A near-complete genome of the
Russian CVA isolate PTC was deposited in GenBank under accession number OQ865368.
No other contigs showed similarity with any known virus available in GenBank.
Phylogenetic analysis of all available full-length CVA genomes (n = 127, accessed on
April 2023) showed that most isolates were clustered in several phylogroups (Figure 2).
The PTC was assigned to the clade formed by two Canadian (MZ291922, KY510919),
Czech (ON088603), and Australian (LN879388) isolates, thus confirming the results of se-
quence identity analysis.
Figure 2.
Phylogenetic analysis of cherry virus A (CVA) complete genomes conducted in MEGA7 [
27
].
The evolutionary history was inferred using the neighbor-joining method. The evolutionary distances
were computed using the Kimura 2-parameter model. The accession numbers of isolates in GenBank
are shown next to the end of branches. Bootstrap values (>85%) from 1000 replicates are shown next
to the corresponding nodes. Non-cherry isolates are highlighted in green. Russian CVA isolate is
indicated with a black circle (
•
). Pink filled clade unites cherry isolates potentially recognized with
CVA-specific primers designed in this work. The scale bar indicates the number of substitutions per
nucleotide. The black triangle means the condensed clade.
Both this and the sister clade mainly consist of the isolates from non-cherry hosts such
as P. armeniaca (LC523018, KY510873, LC125634), P. mume (KY286055, KY445749, KY510874),

Plants 2023,12, 3295 5 of 10
P. cerasifera (ON088603, LN879388), and P. salicina (KY510919). Existence of a discrete cluster
consisting of non-cherry isolates was in line with the previous results [
9
,
12
,
28
]. At the same
time, a number of CVA isolates from plum (LC523016) and apricot (LC422952, LC523017,
KY510876, KY510875, KY510880—the latter two are in the condensed clades) from Canada,
Australia and India clustered with cherry isolates.
CVA is known to be a genetically diverse virus and identity between complete genomes
ranges 79% to 99% [
4
,
12
,
28
]. Given the considerable variability of CVA, virus-specific
primers based on the full-length genome of the Russian isolate PTC were designed in
this work. PCR with these primers enabled confirmation of the HTS results (Figure S1).
In addition, in silico analysis of the alignment of the complete CVA genomes showed
that, although non-universal, these primers would potentially recognize most (if not all)
non-cherry isolates as well as cherry isolates grouped in the neighboring clade (highlighted
in pink fill in Figure 2). Zero or one mismatch between forward and reverse primers and
targeted genome sequences were found. At the same time, non-cherry isolates from other
clades showed several mismatches with both forward and reverse primers that can crucially
affect primer binding.
2.4. Characterization of the LChV1 Genome
BLASTn showed that a contig of 16,930 nt was most closely related (77.3% identity)
to the genome of the Greece LChV1 isolate G15_3 (LN794218) from sweet cherry [
29
]
and covered it nearly completely. Apparently, this contig represented the new full-length
LChV1 genome.
Typically, for the genus Velarivirus, eight ORFs were identified in the genome of the
Russian isolate Stepnoe. ORF1a and ORF1b overlapped due to the ribosomal slippage at
position 6931. All other ORFs were separated from one another by non-coding intergenic
sequences ranging 1 to 171 nt in length. The MET, HEL, and RdRp motifs were predicted
in the ORF1a/ORF1b-encoded polyprotein at positions 679–993, 2006–2263, and 2477–2744,
respectively. CDD search showed that the product encoded by ORF4 was a viral homolog
of the heat shock protein HSP90.
The ORFs of the isolates Stepnoe and G15_3 were compared (Table 2). The ORF finder
showed that ORF2, ORF4, and ORF6 to ORF8 of two isolates had the same length. The
variability was rather randomly distributed along the genomes. ORF2 and ORF8 were most
closely and, correspondingly, most distantly related on both the nt and aa levels. In ORF5,
ORF6, and ORF8 the differences were more pronounced at the aa level, suggesting that
some mutations were non-synonymous. In the isolate G15_3, ORF2 and ORF3 overlapped
by eight nt, while in the isolate Stepnoe they were separated by an intergenic region of 4 nt in
length. ORF3 and ORF5 of the isolate Stepnoe were shorter then their G15_3 counterparts,
since in the latter the starts of the translation were shifted 12 nt upstream. Several indels were
also detected when comparing these isolates. The Stepnoe ORF1a/b was shorter due to a 15
nt deletion of about six hundred nt upstream the MET domain. The intergenic region between
ORF4 and ORF5 was shorter in the G15_3 due to a ten nt deletion. Although genomes of both
isolates were coterminal, the G15_3 30-NCR was shorter due to a 56 nt deletion.
Table 2.
Comparison of open reading frames (ORF) of the little cherry virus 1 isolates Stepnoe and
G15_3.
ORF Name ORF Length (Nucleotide (nt)/Amino Acid (aa)) Identity (nt/aa), %
Stepnoe G15_3
ORF1a 6891/2296 6906/2301 77.5/82.4
ORF1b 1549/515 1549/515 82.9/92.8
ORF2 96/31 96/31 84.4/87.1
ORF3 1656/551 1668/555 79.4/85.7

Plants 2023,12, 3295 6 of 10
Table 2. Cont.
ORF Name ORF Length (Nucleotide (nt)/Amino Acid (aa)) Identity (nt/aa), %
Stepnoe G15_3
ORF4 1554/517 1554/517 77.4/79.5
ORF5 1215/404 1227/408 74.6/74.4
ORF6 1989/662 1989/662 74.5/72.3
ORF7 696/231 696/231 78.3/86.0
ORF8 720/239 720/239 73.4/73.2
The genome regions of the isolate Stepnoe, which differed most strongly from the
G15_3, were re-sequenced by the Sanger method using primers designed according to
the full-length genome sequence of the Russian LChV1 isolate (Table S1). The sequences
determined by the Sanger method were identical to those obtained by HTS. All indels and
mismatches were confirmed by Sanger sequencing. The full-length genome of the Russian
LChV1 isolate Stepnoe was deposited in GenBank under accession number OR260412.
Phylogenetic analysis of all available full-length LChV1 genomes (n= 38, accessed on
April 2023) showed that most isolates were clustered into five distinct groups (Figure 3).
The isolate Stepnoe was assigned to the group V formed by two Greece isolates from sweet
cherry [
29
], confirming the BLASTn results. The intragroup sequence identities ranged
from 92.3% (group II) to 96.0% (group I). The complete genomes of the isolates G15_3 and
C118-Iso1 in the group V shared 99.3% identity. The average intergroup identity was 76.8%.
Plants 2023, 12, x FOR PEER REVIEW 7 of 11
Figure 3. Phylogenetic analysis of lile cherry virus 1 complete genome sequences. The tree was
reconstructed using the neighbor-joining algorithm implemented in MEGA7. Bootstrap values
(from 1000 replicates) are indicated next to the corresponding nodes as a percentage (>75%). Each
of the five phylogroups (I–V) is joined by brackets. The accession numbers and names of isolates are
shown at the end of branches. Russian isolate Stepnoe is highlighted with a black circle (●).
3. Discussion
Both CVA and LChV1 were found on many stone fruit crops worldwide, but have
never been revealed in Russia. In this study CVA and LChV1 were first reported from
Russia, expanding the information on their geographical distribution. The viruses were
detected by metagenomic HTS and confirmed using RT-PCR assay (Figure S1).
CVA was found in the P. cerasifera × P. armeniaca interspecies hybrid showing virus-
like symptoms on the leaves (Figure 1). This is consistent with the data that both myroba-
lan and apricot can be infected with CVA [10–12,30]. However, the observed symptoms
differed from those previously described on these cultures, suggesting that they may
Figure 3. Phylogenetic analysis of little cherry virus 1 complete genome sequences. The tree was

Plants 2023,12, 3295 7 of 10
reconstructed using the neighbor-joining algorithm implemented in MEGA7. Bootstrap
values (from 1000 replicates) are indicated next to the corresponding nodes as a percentage
(>75%). Each of the five phylogroups (I–V) is joined by brackets. The accession numbers and
names of isolates are shown at the end of branches. Russian isolate Stepnoe is highlighted
with a black circle (•).
3. Discussion
Both CVA and LChV1 were found on many stone fruit crops worldwide, but have
never been revealed in Russia. In this study CVA and LChV1 were first reported from
Russia, expanding the information on their geographical distribution. The viruses were
detected by metagenomic HTS and confirmed using RT-PCR assay (Figure S1).
CVA was found in the P. cerasifera
×
P. armeniaca interspecies hybrid showing virus-like
symptoms on the leaves (Figure 1). This is consistent with the data that both myrobalan and
apricot can be infected with CVA [
10
–
12
,
30
]. However, the observed symptoms differed
from those previously described on these cultures, suggesting that they may represent a
special response of this Prunus genotype to the CVA infection. Mixed infection with an
unidentified virus is unlikely because no other viruses were detected in this tree by HTS.
LChV1 was detected in the ((P. cerasifera Ehrh.
×
P. armeniaca L.)
×
P. brigantiaca Vill.)
trihybrid. This suggests that myrobalan (P. cerasifera) and alpen plum (P. brigantiaca) can
be potentially infected with this virus. Apricot is known to be the LChV1 host [
18
,
19
].
Interestingly, only mild mosaic symptoms were observed on the leaves of this tree despite
it also seeming to be infected with PNRSV.
CVA and LChV1 were found in Prunus trees grafted on a wild apricot and P. cerasifera,
respectively. Since no insect vector is known for either virus, these trees were most likely
infected through the rootstocks, which were no longer available and could not be tested for
the viruses.
HTS technology can detect viruses by metagenomic analysis of infected plants and
allows assembly of the complete virus genomes. Using this approach, the full-length
genomes of the Russian CVA and LChV1 isolates were sequenced. The genomes of both
viruses were shown to be typical for the Capillovirus and Velarivirus genera. Their positions
among other isolates were determined using sequence identity and phylogenetic analyses.
Phylogeny of all available full-length CVA genomes showed that, in agreement with
the previous data [
4
,
9
,
12
,
28
,
31
], most isolates were clustered in several distinct phylogroups.
Existence of a discrete phylogenetic group of non-cherry isolates is proposed to stem from
the agricultural practice as cherry species are rarely grafted on non-cherry species thus
preventing transmission of CVA isolates between these groups of hosts [
9
]. It seems logical
that the isolate PTC from the interspecies hybrid of apricot and cherry plum was grouped
together with non-cherry isolates.
Phylogenetic analysis of all available full-length LChV1 genomes showed that most
isolates were clustered into five phylogroups (Figure 3). This result was in compliance with
the previous phylogenetic data obtained by analyzing a smaller number of the complete
LChV1 genomes [
4
,
22
,
29
]. The isolate Stepnoe was clustered with two Greece isolates from
sweet cherry [
29
], thus expanding this divergent group. At the same time, the intragroup
diversity among LChV1 isolates is known to be relatively low (3.3% to 7.4% at the nt level),
whereas intergroup diversity is higher (15% to 39%) [
29
]. The intragroup complete genome
identities calculated in this work ranged 92.3% to 96.0%, while the average intergroup
identity was 76.8%. The isolate Stepnoe differed from its closest relative G13_3 considerably
(Table 2). Their complete genomes shared only 77.3% identity. This obviously exceeds the
level of intragroup variability usual for LChV1. Thus, the differences between the isolates
G15_3 and Stepnoe are more correlated with intergroup diversity. It cannot be ruled out
that the highly divergent isolate Stepnoe is the only representative of one more phylogroup
of this virus so far. In this regard it is worth noting that the isolate Kyoto-2 (MG934545)
was also the only member of the phylogroup G5 until recently [
22
,
29
]. However, the
phylogenetic analysis of all currently available, full-length LChV1 genomes performed

Plants 2023,12, 3295 8 of 10
in this work showed that the Kyoto-2 is a member of the group III composed from new
isolates, which have been sequenced in the very last years.
In conclusion, trees infected with CVA and LChV1 were revealed in the NBG stone
fruits collection. The full-length genomes of these viruses were sequenced for the first time
in Russia and their positions among other CVA and LChV1 isolates from different hosts
and geographical locations were determined. The prevalence of CVA and LChV1 in the
collection and their genetic diversity have yet to be studied.
4. Materials and Methods
4.1. Sampling
Leaves displaying virus-like symptoms were gathered in the Prunus germplasm collec-
tion of the NBG in August of 2020. Individual samples composed of four to six symptomatic
leaves were taken from each selected tree. The bagged samples were delivered to the virol-
ogy department of Lomonosov Moscow State University and stored at 4
◦
C until used for
the total RNA extraction.
4.2. High-Throughput Sequencing (HTS)
Total RNA was extracted from fresh leaves using the cetyltrimethylammonium bromide
(CTAB)-based protocol [
32
]. DNA libraries were synthesized using the TruSeq Stranded Total
RNA Library Prep Plant kit (Illumina, San Diego, CA, USA) and sequenced on the Illumina
MiSeq platform. Raw pair-ended reads of 250 bps were subjected to quality filtering and
adapter removal using FastQC v.0.11.9 and Trim Galore v.0.6.5 (https://www.bioinformatics.
babraham.ac.uk/projects/trim_galore (accessed on 19 September 2021)) using default param-
eters. Contigs were assembled de novo using the metaSPAdes program version 3.15 [
33
].
Virus-related contigs were identified by a BLASTn (https://blast.ncbi.nlm.nih.gov/Blast.cgi)
against the GenBank nucleotide collection (accessed on 19 December 2022). The clean
reads were mapped to the contigs using Bowtie2 v.2.4.4 [
34
]. The raw reads were de-
posited in the NCBI Sequence Read Archive (SRA) (https://www.ncbi.nlm.nih.gov/sra/
PRJNA966926 (accessed on 19 September 2021)). The full-length genomes of the Russian CVA
and LChV1 isolates were deposited in GenBank under accession numbers OQ865368 and
OR260412, respectively.
4.3. Sequence Analyses
To analyze the whole genomes of the Russian CVA and LChV1 isolates, the available
sequences of these viruses were retrieved from GenBank. Multiple alignments of nt sequences,
calculation of sequence identities, and phylogenetic analysis were performed in MEGA7 [
27
].
Phylogenetic trees were reconstructed using the neighbor-joining method and the Kimura
2-parameter model. ORFs in the complete virus genomes were identified using the ORF finder
(https://ncbi.nlm.nih.gov/orffinder (accessed on 19 December 2022)). Conserved domains in
virus proteins were mapped using the Conserved Domain Database (CDD, https://ncbi.
nlm.nih.gov/Structure/cdd/wrpsb.cgi (accessed on 19 December 2022)).
4.4. Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
Total RNA, extracted as described above (see Section 4.2), was used as the template
for the RT-PCR assay of CVA and LChV1. Random hexamer primers and Moloney murine
leukemia virus (MMLV) reverse transcriptase (Evrogen, Moscow, Russia) were used for the
first-strand cDNA synthesis.
For the CVA detection, PCR was conducted using proof-reading Encyclo DNA poly-
merase (Evrogen) and primers CVA-F1 (5
0
-AGAATCAGGCTCTGTCTTAGGT-3
0
) and CVA-
R1 (5
0
-TCTAGCTCTTGTTGAGCGTGGT-3
0
). These primers were designed based on the
full-length genomic RNA of the Russian CVA isolate and amplified the 5
0
-terminal half of
the MP gene generating a PCR product of 837 bps. The cycling conditions were 94
◦
C for
3 min, 35 cycles of 94
◦
C for 30 s, 54
◦
C for 30 s, 72
◦
C for 1 min, and a final extension at
72 ◦C for 10 min.

Plants 2023,12, 3295 9 of 10
For the LChV1 detection, PCR was also conducted using Encyclo DNA polymerase
(Evrogen) and primers lchv-F5 (5
0
-AGCTATACGTGTGAACGAGAGA-3
0
) and lchv-R5
(5
0
-ATCATCGCCAATGTCTAAGGCA-3
0
), which amplified the genome region encom-
passing the 3
0
-end of ORF4, the 5
0
-end of ORF5 and intergenic sequence between them
(positions 11,735 to 12,253 in the genome of the isolate Stepnoe), generating a PCR product
of 518 bps. These primers were designed based on the full-length genome RNA of the
Russian LChV1 isolate. The cycling conditions were 94
◦
C for 3 min, 35 cycles of 94
◦
C for
30 s, 52 ◦C for 30 s, 72 ◦C for 40 s, and a final extension at 72 ◦C for 7 min.
Total RNAs from Prunus plants, which were shown to be CVA- and LChV1-free
by HTS, were negative controls. Amplicons were analyzed by 1.5% (w/v) agarose gel
electrophoresis, visualized by ethidium bromide staining, and photographed under the
gel documentation system MultiDoc-It (Analytik Jena US LLC, Upland, CA, USA). PCR
products were purified from agarose gel using the BC022 Cleanup Standard kit (Evrogen)
and directly sequenced using Evrogen facilities.
Supplementary Materials:
The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/plants12183295/s1, Figure S1: Agarose gel electrophoresis of
PCR products generated by RT-PCR assay of PTC and Stepnoe samples using cherry virus A (CVA)-
and little cherry virus 1 (LChV1)-specific primers developed in this work (lanes 1 and 2, respectively).
The arrows to the right to the picture indicate PCR products of the corresponding size. Lane 3—isolate
PTC tested with LChV1-specific primers. Lane 4—isolate Stepnoe tested with CVA-specific primers.
M—GeneRuler 100 bp Plus DNA Ladder (Thermo Scientific, Waltham, MA, USA). Table S1: Primers
developed for the Sanger sequencing of little cherry virus 1 genome regions.
Author Contributions:
Conceptualization, S.C. and I.M.; methodology, A.S., S.T. and N.S.; software,
F.S. and N.S.; validation, A.S., S.T., N.S., F.S., K.P. and I.M.; investigation, S.C., A.S., N.S. and K.P.;
writing—original draft preparation, S.C.; writing—review and editing, A.S., S.T., N.S., F.S., K.P. and
I.M.; visualization, A.S. and F.S.; supervision, I.M.; funding acquisition, S.C. All authors have read
and agreed to the published version of the manuscript.
Funding: This research was funded by the Russian Science Foundation, grant number 23-16-00032.
Data Availability Statement:
Sequencing data were deposited in SRA and GenBank, and their
accession numbers are provided within the article.
Acknowledgments:
We thank Lubov Lukicheva and Valentina Gorina, the supervisors of the Nikita
Botanical Garden Prunus germplasm collection, for their assistance during collection survey. The
authors thank Alexey Agranovsky for helpful discussions.
Conflicts of Interest: The authors declare no conflict of interest.
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