Journal of General Virology (2011), 92, 2664–2673
DOI 10.1099/vir.0.034686-0
Bell pepper endornavirus: molecular and biological
properties, and occurrence in the genus Capsicum
Ryo Okada,1 Eri Kiyota,1 Sead Sabanadzovic,2 Hiromitsu Moriyama,1
Toshiyuki Fukuhara,1 Prasenjit Saha,3 Marilyn J. Roossinck,3 Ake Severin4
and Rodrigo A. Valverde5
Correspondence
Rodrigo A. Valverde
[email protected]
1
Laboratory of Molecular and Cellular Biology, Faculty of Agriculture, Tokyo University of Agriculture
and Technology, Tokyo 183-8509, Japan
2
Department of Biochemistry, Molecular Biology, Entomology and Plant Pathology,
Mississippi State University, Mississippi State, MS 39762, USA
3
Plant Biology Division, The Samuel Roberts Noble Foundation, Ardmore, OK 73402, USA
4
Laboratoire de Physiologie Vegetale, Universite de Cocody-Abidjan, UFR Biosciences, 22 BP,
582 Abidjan 22, Côte d’Ivoire
5
Department of Plant Pathology & Crop Physiology, Louisiana State University Agricultural Center,
Baton Rouge, LA 70803, USA
Received 31 May 2011
Accepted 19 July 2011
Bell peppers (Capsicum annuum) harbour a large dsRNA virus. The linear genome (14.7 kbp) of
two isolates from Japanese and USA bell pepper cultivars were completely sequenced and
compared. They shared extensive sequence identity and contained a single, long ORF encoding a
4815 aa protein. This polyprotein contained conserved motifs of putative viral methyltransferase
(MTR), helicase 1 (Hel-1), UDP-glycosyltransferase and RNA-dependent RNA polymerase. This
unique arrangement of conserved domains has not been reported in any of the known
endornaviruses. Hence this virus, for which the name Bell pepper endornavirus (BPEV) is
proposed, is a distinct species in the genus Endornavirus (family Endornaviridae). The BPEVencoded polyprotein contains a cysteine-rich region between the MTR and Hel-1 domains, with
conserved CXCC motifs shared among several endornaviruses, suggesting an additional
functional domain. In agreement with general endornavirus features, BPEV contains a nick in the
positive-strand RNA molecule. The virus was detected in all bell pepper cultivars tested and
transmitted through seed but not by graft inoculations. Analysis of dsRNA patterns and RT-PCR
using degenerate primers revealed putative variants of BPEV, or closely related species, infecting
other C. annuum genotypes and three other Capsicum species (C. baccatum, C. chinense and
C. frutescens).
INTRODUCTION
Endornaviruses have a single linear dsRNA genome (9.8–
17.6 kbp), infect plants, fungi and oomycetes and are
transmitted at a high rate through seeds and spores
(Fukuhara et al., 2006; Gibbs et al., 2000). They infect
economically important crops, such as rice (Moriyama
et al., 1995), French bean (Wakarchuk & Hamilton, 1990),
broad bean (Pfeiffer, 1998), barley (Zabalgogeazcoa &
Gildow, 1992), cucurbits (Coutts, 2005) and pepper
The GenBank/EMBL/DDBJ accession numbers for the bell pepper
endornavirus sequences reported in this paper are: AB597230,
JN019858, JN019859, JN019860, JN019861, JN019862 and
JN019863.
Three supplementary figures are available with the online version of this
paper.
2664
(Valverde & Gutierrez, 2007), as well as some plantpathogenic fungi and the oomycete Phytophthora (Hacker
et al., 2005). The presence of endornaviruses in phenotypically normal plants is a unique feature of particular
genotypes of these crops. The full sequence of the genome
of approved or putative Endornavirus species from cultivated rice [Oryza sativa endornavirus (OSV)] (Moriyama
et al., 1995), wild rice [Oryza rufipogon endornavirus (ORV)]
(Moriyama et al., 1999), broad bean [Vicia faba endornavirus
(VFV)] (Pfeiffer, 1998), Phytophthora spp. [Phytophthora
endornavirus 1 (PEV-1)] (Hacker et al., 2005), Helicobasidium mompa [Helicobasidium mompa endornavirus 1
(HmEV-1)] (Osaki et al., 2006), Gremmeniella abietina
[Gremmeniella abietina type B RNA virus XL (GaBRV-XL)]
(Tuomivirta et al., 2009) and Tuber aestivum [Tuber
aestivum endornavirus (TaEV) (Stielow et al., 2011) have
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An endornavirus from bell pepper
been reported. Moreover, endornavirus-like dsRNAs have
been isolated from bottle gourd (Lagenaria siceraria),
Malabar spinach (Basella alba) and eel grass (Zostera
marina) (Fukuhara et al., 2006).
Endornaviruses encode a single polypeptide, which is
presumed to be processed by virus-encoded proteinases.
Genomes of all completely sequenced endornaviruses
contain conserved motifs of a viral RNA-dependent RNA
polymerase (RdRp_2, pfam00978), similar to the alpha-like
virus superfamily of positive-stranded RNA viruses (Gibbs
et al., 2000).
Peppers (Capsicum spp.) are perennial plants native to
South America and today they are cultivated throughout
the world for their nutritional and medicinal value and for
use as a cooking condiment (DeWitt & Bosland, 1996). A
dsRNA was reported from tissue extracts of apparently
healthy bell pepper (Capsicum annuum) cultivars (Valverde
et al., 1990b). Partial sequence information suggested that
the dsRNA constitutes the genome of an endornavirus
(Valverde & Gutierrez, 2007).
In this investigation, we report the full nucleotide
sequence, genome organization and some biological
properties of the putative endornavirus, tentatively designated Bell pepper endornavirus (BPEV), isolated from Yolo
Wonder (YW) bell pepper reported by Valverde &
Gutierrez (2007) and an isolate from Kyousuzu (KS) bell
pepper from Japan. Furthermore, we analysed a number of
additional pepper cultivars for endornavirus-sized dsRNAs
and we report its occurrence in other C. annuum genotypes
and three other Capsicum species. Partial sequence analysis
of the RdRp(s) from additional pepper isolates suggests
that its evolution is congruent with that of its host.
RESULTS AND DISCUSSION
Screening Capsicum spp. and cultivars for BPEV
BPEV dsRNA was detected in all seven bell pepper cultivars
from Japan. Testing for BPEV dsRNA in Capsicum
genotypes from the USA, including bell peppers and other
horticultural types, resulted in 40 additional positive
genotypes, shown in Table 1. BPEV was detected in all
tested bell peppers and Capsicum baccatum lines. Some
C. annuum, Capsicum chinense and Capsicum frutescens
genotypes were BPEV-positive while others were negative
(Tables 1 and 2). A sample of PAGE results is shown in
Fig. 1. Several dsRNAs with a lower molecular mass than
BPEV were associated with some Capsicum genotypes. The
majority of these dsRNAs were determined to constitute
the genome of plant partitiviruses (Sabanadzovic &
Valverde, 2011). BPEV-KS dsRNA was detected in pollen,
callus and cultured cells and the relative concentration was
similar to that of the dsRNA in leaf tissue (data not
shown), unlike OSV and ORV that were reported to be
tenfold more concentrated in pollen than in the leaf tissue
of rice (Moriyama et al., 1999). BPEV appears to be
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common among domesticated Capsicum species, particularly in cultivated peppers (C. annuum, C. frutescens and C.
chinense) (Valverde & Fontenot, 1991) and in C. baccatum.
These closely related species share a mutual ancestral gene
pool (Moscone et al., 2007). One can speculate that the
gene pool from which the domesticated peppers originated
included pepper genotypes that were infected with BPEV.
Nevertheless the origin of BPEV in pepper remains unclear.
Two lines of the most primitive Capsicum species tested in
this study (Capsicum chacoense) were free of BPEV. In
addition, five accessions of C. annuum var. glabriusculum,
also known as chiltepepin, were negative. This pepper is
eaten by indigenous peoples in Mexico, and is thought by
some to be an ancestor of many cultivated peppers.
As in previous investigations, all tested bell pepper
contained BPEV (Valverde & Fontenot, 1991; Valverde
et al., 1990b). Bell pepper cultivars have a narrow gene
pool, and, while making crosses to develop new cultivars,
breeders may have inadvertently introduced and spread
BPEV among bell peppers. Since Japanese bell pepper
breeders have used Capsicum germ plasm from the
Americas to develop Japanese cultivars, it was not
surprising to find that bell pepper cultivars from Japan
were also infected with BPEV.
Occurrence of BPEV in bell pepper seedlings
The level of vertical transmission of BPEV is higher
through the ovule than through pollen, thus it is very high
when both parents are infected (Valverde & Gutierrez,
2007). Screening of seedlings from two bell pepper
cultivars, Yolo Wonder (YW) and Marengo (MR), with
both parents being BPEV-infected, resulted in 50 of 50 and
136 of 137 positive plants, respectively. These results
confirm that high rates of vertical transmission are
obtained when both parents are virus carriers. Screening
of seedlings of three other bell pepper cultivars, originating
from seeds purchased at a local store, resulted in different
percentages of BPEV infections. A high proportion of
seedling of KS (47 of 49) and California Wonder (CW, 36
of 36) were infected by BPEV, indicating that both parents
contained the virus. In contrast, only 8 of 28 seedlings
of Capsicum annuum L. ‘Kyounami’ (KY) tested BPEV
positive, suggesting that the virus was not present in one of
the parents.
In agreement with the general properties of endornaviruses, the BPEV-free and BPEV-infected lines of MR
pepper were phenotypically undistinguishable, revealing
a lack of visible effect on the host. Furthermore, in
preliminary experiments, co-infections of BPEV with four
acute ssRNA viruses [pepper mild mottle virus, potato
virus Y (PVY), cucumber mosaic virus and tomato spotted
wilt virus] in MR pepper did not have a visible effect on the
symptoms caused by these viruses when compared with
single infections of the BPEV-free MR line by any of these
viruses (not shown).
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R. Okada and others
Table 1. Capsicum spp. and cultivars that tested positive for bell pepper endornavirus by RT-PCR and/or agarose gel
electrophoresis (GE) analyses
+, Positive;
NT,
not tested; LSU, Louisiana State University; AVRDC, Asian Vegetable Research and Development Center.
Capsicum species
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
annuum
annuum
annuum
annuum
annuum
annuum
annuum
annuum
annuum
annuum
annuum
annuum
annuum
annuum
annuum
annuum
annuum
annuum
annuum
annuum
annuum
frutescens
frutescens
frutescens
frutescens
baccatum
baccatum
baccatum
baccatum
baccatum
baccatum
baccatum
baccatum
baccatum
baccatum
baccatum
baccatum
baccatum
baccatum
baccatum
chinense
chinense
chinense
chinense
chinense
chinense
chinense
Type
Cultivar or PI line
Origin
GE
RT-PCR
Bell
Bell
Bell
Bell
Bell
Bell
Bell
Bell
Bell
Bell
Bell
Bell
Bell
Bell
Bell
Bell
Bell
Bell
Pimento
Cayenne
Cayenne
Tabasco
Tabasco
Tabasco
Tabasco
Aji
Aji
Aji
Aji
Aji
Aji
Aji
Aji
Aji
Aji
Aji
Aji
Aji
Aji
Aji
Habanero
Habanero
Habanero
Habanero
Habanero
Habanero
Habanero
California Wonder
Yolo Wonder
Kyousuzu
Kyounami
Kyoumidori
Ace
Suigyokunigou
High Green
Jumbo Colour
Marengo
Avelar
Casca Dura
King Arthur
VR-4
Magda
Bonnie’s Green Bell,
Red Bell
Chocolate Beauty Sweet
Pimento Sweet
Cayenne Long Red Thick
Super Cayenne
Greenleaf
LSU
PI 159239
PI 193470
Monk’s Hat
PI 238061
PI 633752
PI 260549
PI 215699
PI 260590
PI 260543
PI 441589
PI 257135
PI 337524
PI 337522
C00754 (AVRDC)
C01218 (AVRDC)
C01527 (AVRDC)
C01300 (AVRDC)
PI 159236
PI 315008
PI 315023
PI 315024
PI 273426
C00943 (AVRDC)
C00949 (AVRDC)
USA
USA
Japan
Japan
Japan
Japan
Japan
Japan
Japan
USA
USA
Brazil
USA
USA
USA
USA
USA
USA
USA
USA
USA
USA
USA
USA
Ethiopia
USA
Bolivia
Paraguay
Peru
Peru
Peru
Brazil
Brazil
Ecuador
USA
Argentina
Argentina
Egypt
USA
Germany
USA
Peru
Peru
Peru
USA
Peru
England
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Attempts to transmit BPEV
None of the BPEV-free MR scions grafted onto BPEVinfected rootstocks tested positive for BPEV. ELISA
detection of PVY in the scions confirmed successful grafts.
2666
NT
NT
NT
NT
NT
+
+
+
+
+
+
NT
+
NT
NT
+
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
+
NT
+
NT
NT
+
NT
The presence of PVY in the rootstocks and subsequent
systemic infection did not aid the movement of BPEV from
the rootstocks into the scions. The failure to transmit
BPEV by grafting supports the idea that endornaviruses
lack systemic movement. Infection of the BPEV-infected
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Journal of General Virology 92
An endornavirus from bell pepper
Table 2. Capsicum spp. and cultivars that tested negative for bell pepper endornavirus by PAGE analyses
Capsicum species
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
annuum
annuum
annuum
annuum
annuum
annuum
annuum
annuum
annuum
annuum
annuum var.
annuum var.
annuum var.
annuum var.
annuum var.
chacoense
chacoense
chinense
frutescens
frutescens
frutescens
frutescens
frutescens
pubescens
pubescens
pubescens
pubescens
pubescens
glabriusculum
glabriusculum
glabriusculum
glabriusculum
glabriusculum
Type
Cayenne
Unknown
Unknown
Serrano
Jalapeño
Unknown
Yellow wax
Yellow wax
Serrano
Cayenne
Chiltepin
Chiltepin
Chiltepin
Chiltepin
Chiltepin
Unknown
Unknown
Habanero
Tabasco
Tabasco
Tabasco
Tabasco
Tabasco
Rocoto
Rocoto
Rocoto
Rocoto
Rocoto
Cultivar or PI line
Country of origin
Thai Cluster
Pico de Pájaro
MI-2
Criollo de Morelos
Jaloro
PBC-535
Sweet Banana
Rio Grande Gold
Hidalgo
Thai Cluster
PI 224405
PI 310488
PI 511887
PI 631135
PI 632932
PI 260429
PI 260435
PI 152225
PI 368084
PI 193470
PI 439502
PI 441650
PI 593924
C00640
C00778
PI 593639
PI 593630
Grif 1614
Thailand
Honduras
Sri Lanka
Mexico
USA
Taiwan
USA
USA
USA
Thailand
Mexico
Mexico
Mexico
Guatemala
Guatemala
Argentina
Bolivia
USA
Malaysia
Ethiopia
Costa Rica
Brazil
Ecuador
Guatemala
Guatemala
Guatemala
Ecuador
Mexico
rootstock by PVY did not aid transmission. Similar results
were reported for a partitivirus infecting Jalapeño M
pepper (Valverde & Gutierrez, 2008).
Nucleotide sequence analysis of BPEV-YW and
BPEV-KS
Fig. 1. Polyacrylamide gel electrophoresis (5 % acrylamide) of bell
pepper endornavirus dsRNA from C. annuum YW (lane 1) and
related endornavirus dsRNAs infecting various Capsicum species;
lane 2, C. frutescens LSU Tabasco; lane 3, C. chinense PI
315023; lane, 4, C. chinense PI 159236; lane 5, C. chinense PI
273426; lane 6, C. chinense PI315008; lane 7, C. chinense PI
C00943; lane 8; C. baccatum PI 260590; lane 9, dsRNAs
extracted from Nicotiana benthamiana infected with grapevine
leafroll-associated virus 2 (GLRaV-2); lane 10, dsRNA-free C.
annuum. Arrow indicates endorna-like dsRNAs. Sizes of visible
GLRaV-2 dsRNAs are indicated.
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The complete sequences of BPEV-KS (14 727 nt) and
BPEV-YW (14 728 nt) were determined. The two genomes
were very similar, except for the presence of a guanine at
the 59 end in the case of BPEV-YW. They shared 88.2 %
identical nucleotide sequences in the whole genome, with
highly conserved 59 and 39 UTRs, and only single
nucleotide differences in each of the UTRs, which underscores their functional importance in virus replication.
A single ORF was found on the plus strand of both BPEVKS and BPEV-YW, starting at nucleotide 225 in the case of
BPEV-KS (nt 226 in the case of BPEV-YW) and ending at
nucleotide 14 670, which could encode a polyprotein of
4815 aa of with an estimated molecular mass of 545 kDa
(Fig. 2a). The genome size and protein-encoding strategy
are typical of endornaviruses. The putative protein
products of BPEV-KS and BPEV-YW shared 92 % identical amino acid residues (96 % similarity). Nevertheless,
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R. Okada and others
0
(a) (+)
1
5
10
15 kbp
880
14 727
(_)
14 673
225
1
Hel-1
MTR
RdRp
UGT
14 727
4815 aa
(b) 100
90
Identity or similarity (%)
80
70
60
50
Identity
40
Similarity
30
I
II
III
20
4561_4600
4761_4800
4161_4200
4361_4400
3761_3800
3961_4000
3361_3400
3561_3600
2961_3000
3161_3200
2561_2600
2761_2800
2161_2200
2361_2400
1761_1800
1961_2000
1361_1400
1561_1600
961_1000
1161_1200
561_600
761_800
0
161_200
361_400
10
Position of amino acids
Fig. 2. (a) Schematic representation of the genome organization of BPEV with key nucleotide and amino acid references,
including the position of the nick in the coding plus strand (+). The box represents the large ORF, whereas lines depict UTRs.
MTR, Viral methyltransferase; Hel-1, viral helicase 1; UGT, UDP-glycosyltransferase; RdRp, viral RNA-dependent RNA
polymerase. (b) Graphic representation of pairwise comparison of amino acid sequences (every 40 aa) encoded by the large
ORFs of BPEV isolates KS and YW. Note the presence of three variable regions between the two polyproteins (denoted as I, II
and III). The pairwise comparison was performed with a FASTA homology and similarity search of protein versus protein with
GENETYX. The graph was generated with EXCEL by manually counting the similarity and identity of each 40 amino acid region.
detailed analyses revealed three variable regions in the two
polyproteins (Fig. 2b). Interestingly, this pattern roughly
corresponds to regions of low similarity between ORV
and OSV (Moriyama et al., 1999). Considering that the
genomes of these two viral isolates shared all their major
characteristics, further discussion is based on the BPEV-KS
genome data (except when otherwise stated).
The estimated 59 UTR of BPEV-KS was 225 nt long (226 nt
in the case of BPEV-YW). Although two AUG codons were
found at nucleotides 18–21 and 121–123, in silico analyses
showed that these two codons were probably not utilized
owing to their unfavourable initiation context. Compared
with the consensus sequence for plants AA(G)CAAUGGC (Lütcke et al., 1987), the third AUG codon
(AGUGAUGGC) of BPEV is in the most favourable
context for translation initiation and is assumed to be
the starting codon for the translation of a long ORF. The 39
end of BPEV consists of a 57 nt-long UTR, ending in the
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unique sequence GGGGAGGGAGGGCCCCCCCCCCCC
and lacking a poly(A) tail.
A BLAST search using the BPEV sequence detected
conserved domains of a putative viral methyltransferase
(MTR), RNA helicase 1 (Hel-1), UDP-glucose–glycosyltransferase (UGT) and an RdRp (Fig. 2a). The BPEV
genome organization is unique since it is the only
endornavirus to contain these four domains (Roossinck,
et al., 2011).
The search in the protein family (Pfam) database (Finn
et al., 2010) revealed the presence of a highly conserved
MTR (pfam01660) domain between amino acids 288 and
482 with an expect (E) value of 1.1610210. Similar results
were obtained in analyses performed with the conserved
domain database (CDD) database (Marchler-Bauer et al.,
2007). Multiple-sequence alignment of the putative MTR
region of BPEV and several ssRNA viruses belonging to the
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Journal of General Virology 92
An endornavirus from bell pepper
alpha-like superfamily showed the conserved motifs I–IV
of the ‘Sindbis-like’ supergroup (Rozanov et al., 1992). The
nearest relative of this motif is the MTR domain from a
tobamovirus, Sunn hemp mosaic virus; other relatives are
related to tymoviruses. The invariant amino acid residues
for MTR activity, a histidine in motif I, the DXXRArg
signature in motif II and a tyrosine in motif IV, are found
in BPEV.
suggested that the hypovirulence caused by HmEV-1 might
be due to the UGT, as the cellular sterol UGT gene of the
host fungus, which is essential for pathogenesis in plantpathogenic fungi, might be repressed by gene silencing.
A putative Hel-1 domain was found between amino acids
1342 and 1584 and contained conserved motifs from I to
VI (Koonin et al., 1993). A BLAST search of this region
against the Pfam database produced significant matches
with the consensus sequences of the viral superfamily 1
helicase (pfam01443; E51.7610210). This type of helicase
is found in many ssRNA viruses (Roossinck, et al., 2011).
The RdRp_2 domain encoded by BPEV is located in the
carboxy-terminal region of the ORF and contained
conserved motifs I–IV (Poch et al., 1989). It shares
similarity with the RdRp domains encoded by other
endornaviruses (E values ranging from 8610221 to
5610271) and was slightly more related to PEV-1 (43 %
identity, 63 % similarity) than to endornaviruses reported
from plants (40–42 % identity, 55–57 % similarity)
(Supplementary Fig. S1, available in JGV Online). The
nearest non-endornavirus relatives are members of genera
Ampelovirus and Closterovirus in the family Closteroviridae.
The presence of a region with significant similarity (E
values from 961024 to 5610216) to cellular UDPglycosyltranferases is located between amino acids 3049
and 3362, and is in a similar location to glycosyltranserase
domains found in other endornaviruses (Fig. 3a). The
UGT-like region in the BPEV genome showed greatest
similarity to motifs A and B, which were reported by
Hacker et al. (2005) for PEV-1 and are present in some
other endornaviruses, hypoviruses and cellular UGTs.
While no direct effect of endornaviruses on plants has
been reported (except for VFV being associated with the
cytoplasmic male sterility; Pfeiffer, 1998), the fungal
endornavirus HmEV-1 reduces the virulence of the host
fungus, H. mompa (Ikeda et al., 2003). Osaki et al. (2006)
An additional putative domain of approximately 300 aa
was identified by in silico analyses of BPEV-encoded
polyproteins. This domain, located downstream of the
MTR, had significant matches with corresponding regions
in other endornaviruses (E values ranged between 561024
in the case of OSV to 1610215 in the case of HmEV-1).
The carboxy-terminal part of this putative product was
identified as a ‘cysteine-rich region’ (CRR), and is a
candidate for a protease domain in PEV-1 (Hacker et al.,
2005) and in GaBRV-XL (Tuomivirta et al., 2009). In
particular, the region between amino acids 700 and 777 was
rich in cysteine residues (22 %) and characterized by the
presence of four CXCC motifs. Two of these CXCC motifs
(motifs 1 and 3), along with two histidine residues, were
Fig. 3. (a) Multiple alignments of conserved motifs A and B of putative UDP glycotransferases encoded by endornaviruses.
Positions of these motifs in each polyprotein are indicated along with numbers of amino acids between motifs A and B. (b)
Cysteine-rich region (CRR) present in BPEV and in several approved or putative species of the genus Endornavirus. Note the
presence of several CXCC motifs (white lettering on a black background). Motifs conserved in all examined sequences are
indicated by a solid line while those present only in selected viruses are indicated by a dotted line (motifs 2 and 4). Two
conserved histidine residues are also shown (grey background). Asterisks indicate conserved residues in all proteins used for
comparison. Colons indicate conserved substitutions.
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R. Okada and others
highly conserved in BPEV and several other recognized
and putative species of the genus Endornavirus, while ORV
and OSV contained only conserved motifs 1 and 2 (Fig. 3b
and not shown). Although the importance of this region,
shared among endornaviruses, is not yet clear, its conservation suggests an important role in the life cycle of
endornaviruses. The large endornavirus-encoded polyprotein has been assumed to be processed by viral proteinase(s).
This region could be a candidate region for a protease
function. However, the importance and function of the CRR
region is yet to be experimentally demonstrated.
RT-PCR
with BPEV, these results suggest a rather inefficient vertical
transmission of the virus in certain genotypes. Curiously,
two lines of the most primitive Capsicum species (C.
chacoense), five of the non-cultivated C. annuum var.
glabriusculum (Chiltepin pepper) and five of C. pubescens
(Rocoto) were free of BPEV. The nucleotide-sequence
diversity found among various isolates of BPEV from
different Capsicum species may reflect how long this virus
has been associated with these hosts.
Detection and characterization of the nick in the
sequence of BPEV-KS
RT-PCR products (381 bp) were generated from the
following Capsicum species: C. annuum YW, KS, Cayenne
Long Red Thick and Super Cayenne; C. chinense lines
C00943, PI 159236 and PI 315023; and C. frutescens
Tabasco-LSU and Greenleaf Tabasco. Sequence analysis
showed variation among them. Sequences of multiple
clones generated from the same specimen were uniform
(99–100 % amino acid identity), whereas amino acid
variation of up to 5 % was found among genotypes of
the same Capsicum species (e.g. maximum level of
differences between clones generated from YW and from
Super Cayenne pepper). The same level of amino acid
diversity was observed among clones generated from C.
chinense and C. frutescens. However, the most significant
differences in amino acid variation (up to 16 %) were
observed between YW and C. chinense. The level of identity
between BPEV and orthologous sequences of the endornavirus amplified from C. chinense and C. frutescens (84–
85 %) was lower than identities between ORV and OSV
(94 %) in the same region. This suggests that the
endornaviruses infecting these two species could be
distinct, BPEV-related species.
Purified BPEV-KS was subjected to electrophoresis on a
0.8 % denaturing agarose gel, blotted onto a membrane
and hybridized using three cDNA probes located between
nucleotides 805 and 1135 (Probe 1, Cads577), nucleotides
906 and 1528 (Probe 2, Cads532) and nucleotides 1251 and
1704 (Probe 3, Cads759-59) from the 59-end of the plus
strand of the dsRNA. These probes detected both (plus and
minus) strands, which were separated by denaturing
agarose gel electrophoresis. Approximately 14 and 15 kb
RNA fragments were detected with all probes (Fig. 5). A
fragment of approximately 1 kb was detected with probe 1
but not with probe 3. With probe 2, the 1 kb fragment was
faintly detected. These results indicate that BPEV-KS has a
nick in the plus strand between nucleotides 800 and 1100.
Eighteen clones were obtained (by using 59 RACE) to
determine the position of the nick on the plus strand
(Supplementary Fig. S2, available in JGV Online). Three
independently obtained clones indicated a nick in the plus
strand at nucleotide 880. Of the remaining clones, nicks
were detected at nucleotides 931, 937, 940, 941, 946, 954 or
962, 1183, 1185 and 1187 or 1199. These results suggest
that BPEV-KS has a nick in the plus strand at nucleotide
880, but it is likely that this virus has several nicks in the
Sequence differences among RT-PCR products generated
from different Capsicum species mirrors the taxonomic
relationships of the hosts. Phylogenetic analysis using the
RdRp region of the various endornavirus sequences (Fig. 4)
shows a tree that is congruent with what is known about
relationships in the Capsicum group, based on isozyme
data (McLeod et al., 1982). These closely related species
share a mutual ancestral gene pool (Moscone et al., 2007).
In the case of C. frutescens and C. chinense, some have
argued that they should be combined into one species
owing to their minimal differences and easy interbreeding
(Pickersgill, 1966, 1971). This supports our suggestion of
host–virus relationship and that BPEV was present in the
common ancestor of these three Capsicum species and coevolved with its hosts. Nevertheless, the origin of BPEV in
pepper remains unknown and could be elucidated by
testing more domesticated genotypes and wild Capsicum
species. Not all lines of C. frutescens and C. chinense showed
evidence of an endornavirus, and many C. annuum
cultivars also lack the virus (Table 2). Assuming that the
common ancestor of these Capsicum species was infected
Fig. 4. Bayesian phylogenetic analysis of the RdRp regions of
putative endornaviruses isolated from several Capsicum spp. and
cultivars. Analysis is as described in the text. Relative branch
lengths are shown above the lines, support for branches is shown
at the nodes.
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Journal of General Virology 92
An endornavirus from bell pepper
greenhouse with day/night temperatures of about 25/18 uC, respectively, and 16 h days.
Extraction, purification and analysis of dsRNA from Capsicum
spp. Leaf tissues were pulverized in a mortar and pestle after being
Fig. 5. Detection of the nick in the coding strand of BPEV-KS by
Northern-blot analysis. BPEV-KS dsRNA was resolved by
electrophoresis in a denaturing 0.8 % agarose gel, transferred to
a membrane and probed with probes Cads577 (corresponding to
nucleotides 805—1135), Cads532 (corresponding to nucleotides
906–1528) and Cads759-59 (corresponding to nt 1251–1704).
While two bands of 14 and 15 kb were detected with all three
probes, a band of 1 kb was clearly detected only when probed
with probe Cads577.
plus strand, as reported for PEV-1 and OSV (Hacker et al.,
2005; Fukuhara et al., 1995).
The biological properties, unique genomic organization
and phylogeny indicate that the dsRNAs isolated from the
bell pepper cultivars KS and YW represent two closely
related isolates of a distinct and novel species of the genus
Endornavirus (family Endornaviridae), for which the name
Bell pepper endornavirus (BPEV) is proposed.
METHODS
Plant materials. Bell pepper plants Kyousuzu (KS), Kyounami
(KN), Kyoumidori and Ace were from TAKII Seed Company;
Suigyokunigou from Sakata Seed; High Green from Tohoku Seed;
and Jumbo Colour Bell Pepper from Kaneko Seeds. Seed of KS, KN
and bell pepper California Wonder (CW) were purchased from
commercial stores in Japan. Bell peppers Red Bell, Bonnie’s Green
Bell, Pimiento Sweet and Chocolate Beauty Sweet were purchased as
young plants from commercial garden stores in Ardmore, Oklahoma,
USA, and Marengo (MR) seeds were from Asgrow Seed. The source of
Yolo Wonder (YW) and all other C. annuum genotypes and Capsicum
species used in this investigation were reported in previous
publications (Valverde et al., 1990b; Valverde & Fontenot, 1991) or
were obtained from L. L. Black (Louisiana State University, Louisiana,
USA). Plant introductions (PI) and accessions that included
selections from different geographical locations of C. annuum, C.
baccatum, C. chacoense, C. chinense, C. frutescens and C. pubescens
were provided by R. L. Jarret (USDA-ARS, Griffin, Georgia, USA) and
L. M. Engle (AVRDC, Taiwan). Seed used in some experiments was
increased by self-pollination. All plants and seedlings were grown in a
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frozen in liquid nitrogen, and total nucleic acids were extracted by
using 26 STE buffer (200 mM NaCl, 20 mM Tris/HCl and 2 mM
EDTA, pH 8.0). Total nucleic acids were further purified with phenol
and subjected to agarose gel electrophoresis. Alternatively, dsRNA was
fractionated from total nucleic acid extracts by column chromatography on CF-11 cellulose (Whatman), as described by Morris &
Dodds (1979) or as modified by Valverde et al. (1990a), Márquez et al.
(2007) and Roossinck et al. (2010). The dsRNAs from all other C.
annuum genotypes and Capsicum species were extracted from 3.5 g of
leaf tissue and analysed on 5 % polyacrylamide gels. For each
Capsicum genotype and Capsicum species, foliar samples from at least
three plants were tested. In the case of KS pepper, dsRNA was also
extracted from pollen, callus and cultured cells. The nature of the
purified nucleic acids was ascertained by digestion with RNase-free
DNase I (Promega), followed by selective treatments with DNase-free
RNase A in high (26 SSC) and low (0.26 SSC) salt conditions (206
SSC is 3 M sodium chloride, 0.3 M sodium citrate). Preparations
were further treated with Proteinase K (Sigma) followed by phenol/
chloroform purification and ethanol precipitation.
Occurrence of BPEV in bell pepper seedlings. Seeds from KS,
KN, CW, YW and MR bell peppers were planted and 8-week-old
plants screened for BPEV by RT-PCR or by electrophoretic analyses
of dsRNA. A total of 49, 28, 36, 50 and 137 plants of KS, KN, CW, YW
and MR, respectively, were screened. A single MR plant was found
free of BPEV and was self-pollinated in order to generate a virus-free
line for further use in graft-transmission experiments. Seed of CW,
YW and MR originated from self-pollinated BPEV-infected plants
while seed of KS and KN were purchased from commercial stores, and
thus with no knowledge of the parental status in regard to BPEV
infections.
Attempts to transmit BPEV. BPEV-infected MR was used as
rootstock and the BPEV-free line as the scion in graft inoculation
experiments. A total of eight rootstocks were used. Four were infected
with PVY, which was mechanically inoculated to the plants when they
were 6 weeks old. One month after the PVY inoculation, the stems of
all plants were cut and scions from the BPEV-free line graft were
inoculated (Supplementary Fig. S3, available in JGV Online). Six
weeks after grafting, foliar samples were taken and tested for BPEV by
electrophoresis and RT-PCR. Scions grafted on PVY inoculated plants
were tested for PVY by ELISA.
Cloning, sequencing and sequence analyses. BPEV dsRNA from
KS (BPEV-KS) and YW (BPEV-YW) were gel purified, heat
denatured and reverse transcribed with random hexadeoxynucleotide
primers (TaKaRa). Generated cDNA fragments were cloned into
pUC109 (TaKaRa) or pGEM-T Easy plasmid (Promega) and the
resulting recombinant plasmids were transferred into Escherichia coli
DH5a competent cells. DNAs from selected colonies were sequenced
by automated sequence analysis using a capillary sequencer, 3130xl
Genetic Analyzer (Applied Biosystems). After computer-assisted
analysis of the initial sequence data, specific primers were designed
in order to fill the gaps between adjacent clones by RT-PCR. Each
nucleotide in both genomes (BPEV-KS and -YW) was sequenced
from multiple clones from independent experiments in order to
ensure at least fivefold coverage.
For determination of the sequence of the 39 end of BPEV-KS, firststrand cDNAs were synthesized by using 59-end-phosphorylated RT
primer and single-stranded cDNAs were circularized or concatamers
were formed at 16 h and at 15 uC by using RNA Ligase (TaKaRa).
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R. Okada and others
Circular cDNAs or concatamers were amplified by PCR. The 39 viral
end of BPEV-YW was determined by the method described by
Lambden et al. (1992), involving the ligation of a 59-phosphorylated/
39-amino-blocked oligonucleotide (primer 1) to the target dsRNAs,
followed by cDNA synthesis primed by a complementary oligonucleotide (primer 2) and amplification of the 39 end with a virusspecific primer and primer 2. For determination of the sequence of
the 59 end of BPEV-KS, cDNAs were amplified by PCR after adding a
homopolymeric tail at the 39-end of the single-stranded cDNAs by
terminal deoxynucleotidyltransferase (TaKaRa). Amplification products were cloned and sequenced by using M13 RV-N (59TGTGGAATTGTGAGCGG-39) and M13 reverse primers (TaKaRa).
For MR pepper samples, dsRNA was isolated and converted to cDNA,
followed by multiplexing and sequence analysis on a 454/GS FLX
system (Roche), as described previously (Roossinck et al., 2010).
Amino acid sequences were assembled and analysed using GENETYX
version 9 (GENETYX) and/or LASERGENE (DNASTAR). Multiple sequence alignments were conducted by using CLUSTAL W (Thompson
et al., 1994), and alignments were edited manually using MESQUITE
(version 2.74) (Maddison & Maddison, 2010). Phylogenetic analyses
were done using MRBAYES (Huelsenbeck & Ronquist, 2001),
implemented via a plug-in for GENEIOUS. VFV (GenBank accession
# CAA04392) was used as an out-group. Rate matrix was set to a
Poisson distribution with a gamma rate variation. Burn-in was
100 000 and total chain length was 1 100 000. Branch lengths were
unconstrained. PhyML (Guindon & Gascuel, 2003) and PAUP* version
4.0 beta 4b10 (Swofford, 2002) were used to confirm the tree topology
(not shown).
Molecular hybridization. For Northern blot analysis, 100 ng of
BPEV-KS dsRNA was separated on a 0.8 % agarose MOPS gel with
6 % formaldehyde and transferred to a nylon Zeta-Probe membrane
(Bio-Rad) by capillary blotting (Moriyama et al., 1999). After UV
cross-linking and prehybridization in hybrisolution (250 mM phosphate buffer, pH 7.2, 1 mM EDTA, 7 % SDS, 1 % BSA), blots were
hybridized for 16 h at 65 uC in the same solution with 32P-labelled
DNA-probes specific for BPEV-clones Cads577, Cads532 and
Cads759-59. Probes were synthesized with a random prime labelling
system, BcaBEST Labeling kit (TaKaRa). Membranes were washed
twice at 65 uC with 40 mM phosphate buffer containing 5 % SDS for
1 h and twice at 65 uC with 40 mM phosphate buffer, pH 7.2,
containing 1 % SDS for 1 h. Hybridization signals were detected with
a BAS 1500 system (Fujifilm). Images taken by the BAS 1500 system
were prepared with IMAGE GAUGE (Fujifilm).
RT-PCR. To confirm the presence of BPEV in C. annuum genotypes
and to investigate its occurrence in three other Capsicum species,
selected genotypes and species that yielded BPEV-like dsRNAs by gel
electrophoresis were subjected to RT-PCR using a pair of degenerate
primers: Endor-F (59-AAGSGAGAATWATHGTRTGGCA-39) and
Endor-R (59-CTAGWGCKGTBGTAGCTTGWCC-39), which were
designed to amplify a 381 nt fragment of the RdRp of plant
endornaviruses.
Aliquots of dsRNAs were heat denatured at 95 uC for 5 min, cooled
rapidly on ice and used for RT-PCR detection. RT-PCR was
performed using an Access one-tube, two-enzyme system RT-PCR
System (Promega), following the manufacturer instructions. The
following PCR conditions were applied for BPEV detection: (i) initial
denaturation at 94 uC for 2 min, (ii) denaturation for 1 min at 94 uC,
annealing for 45 s at 50 uC, extension for 45 s at 70 uC (40 cycles) and
(iii) final extension for 5 min at 70 uC. PCR products were cloned by
using a pGEM-T Easy Vector System. For each dsRNA sample, three
recombinant plasmids were sequenced and the amino acid sequences
derived were compared with sequences in the GenBank database by
using BLAST.
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ACKNOWLEDGEMENTS
We wish to thank R. Jarret (USDA/ARS, Griffin, Georgia, USA)
for providing seeds of Capsicum species; Guoan Shen (Noble
Foundation) and Dina Gutierrez (Louisiana State University, USA)
for helping with some experiments; Dr Tomohide Natsuaki
(Utsunomiya University) for helpful comments; Estación Experimental ‘La Mayora’, CSIC, Spain for providing facilities for some
experiments. Approved for publication as journal article no. 12047 of
the Mississippi Agricultural and Forestry Experiment Station, MSU.
REFERENCES
Coutts, R. H. A. (2005). First report of an endornavirus in the
Cucurbitaceae. Virus Genes 31, 361–362.
DeWitt, D. & Bosland, P. W. (1996). Peppers of the World. Berkeley,
CA: Ten Speed Press.
Finn, R. D., Mistry, J., Tate, J., Coggill, P., Heger, A., Pollington, J. E.,
Gavin, O. L., Gunasekaran, P., Ceric, G. & other authors (2010). The
Pfam protein families database. Nucleic Acids Res 38 (Database issue),
D211–D222.
Fukuhara, T., Moriyama, H. & Nitta, T. (1995). The unusual structure
of a novel RNA replicon in rice. J Biol Chem 270, 18147–18149.
Fukuhara, T., Koga, R., Aoki, N., Yuki, C., Yamamoto, N., Oyama, N.,
Udagawa, T., Horiuchi, H., Miyazaki, S. & other authors (2006). The
wide distribution of endornaviruses, large double-stranded RNA
replicons with plasmid-like properties. Arch Virol 151, 995–1002.
Gibbs, M. J., Koga, R., Moriyama, H., Pfeiffer, P. & Fukuhara, T.
(2000). Phylogenetic analysis of some large double-stranded RNA
replicons from plants suggests they evolved from a defective singlestranded RNA virus. J Gen Virol 81, 227–233.
Guindon, S. & Gascuel, O. (2003). A simple, fast, and accurate
algorithm to estimate large phylogenies by maximum likelihood. Syst
Biol 52, 696–704.
Hacker, C. V., Brasier, C. M. & Buck, K. W. (2005). A double-stranded
RNA from a Phytophthora species is related to the plant endornaviruses and contains a putative UDP glycosyltransferase gene. J Gen
Virol 86, 1561–1570.
Huelsenbeck, J. P. & Ronquist, F. (2001). MRBAYES: Bayesian inference
of phylogenetic trees. Bioinformatics 17, 754–755.
Ikeda, K., Nakamura, H. & Matsumoto, N. (2003). Hypovirulent
strain of the violet root rot fungus Helicobasidium mompa. J Gen Plant
Pathol 69, 385–390.
Koonin, E. V., Dolja, V. V. & Morris, T. J. (1993). Evolution and
taxonomy of positive-strand RNA viruses: implications of comparative analysis of amino acid sequences. Crit Rev Biochem Mol Biol 28,
375–430.
Lambden, P. R., Cooke, S. J., Caul, E. O. & Clarke, I. N. (1992).
Cloning of noncultivatable human rotavirus by single primer
amplification. J Virol 66, 1817–1822.
Lütcke, H. A., Chow, K. C., Mickel, F. S., Moss, K. A., Kern, H. F. &
Scheele, G. A. (1987). Selection of AUG initiation codons differs in
plants and animals. EMBO J 6, 43–48.
Maddison, W. P. & Maddison, D. R. (2010). Mesquite: a modular
system for evolutionary analysis. Version 2.74. http://mesquiteproject.
org
Marchler-Bauer, A., Anderson, J. B., Derbyshire, M. K., DeWeeseScott, C., Gonzales, N. R., Gwadz, M., Hao, L., He, S., Hurwitz, D. I. &
other authors (2007). CDD: a conserved domain database for
interactive domain family analysis. Nucleic Acids Res 35 (Database
issue), D237–D240.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Fri, 16 Jun 2017 18:00:38
Journal of General Virology 92
An endornavirus from bell pepper
Márquez, L. M., Redman, R. S., Rodrı́guez, R. J. & Roossinck, M. J.
(2007). A virus in a fungus in a plant: three-way symbiosis required
massively parallel pyrosequencing to understand virus ecology. Mol
Ecol 19 (Suppl. 1), 81–88.
for thermal tolerance. Science 315, 513–515.
Rozanov, M. N., Koonin, E. V. & Gorbalenya, A. E. (1992).
McLeod, M. J., Guttman, S. I. & Eshbaugh, W. H. (1982). Early
Moriyama, H., Nitta, T. & Fukuhara, T. (1995). Double-stranded RNA
Conservation of the putative methyltransferase domain: a hallmark
of the ‘Sindbis-like’ supergroup of positive-strand RNA viruses. J Gen
Virol 73, 2129–2134.
in rice: a novel RNA replicon in plants. Mol Gen Genet 248, 364–369.
Sabanadzovic, S. & Valverde, R. A. (2011). Properties and detection
Moriyama, H., Horiuchi, H., Koga, R. & Fukuhara, T. (1999).
of two cryptoviruses from pepper (Capsicum annuum). Virus Genes
43, 307–312.
evolution of chili peppers (Capsicum). Econ Bot 36, 361–368.
Molecular characterization of two endogenous double-stranded
RNAs in rice and their inheritance by interspecific hybrids. J Biol
Chem 274, 6882–6888.
Morris, T. J. & Dodds, J. A. (1979). Isolation and analysis of double-
Stielow, B., Klenk, H.-P. & Menzel, W. (2011). Complete genome
sequence of the first endornavirus from the ascocarp of the
ectomycorrhizal fungus Tuber aestivum Vittad. Arch Virol 156, 343–345.
PAUP*: Phylogenetic analysis using parsimony
(and other methods), version 4. Sunderland, MA: Sinauer Associates.
stranded RNA from virus-infected plant and fungal tissue.
Phytopathology 69, 854–858.
Swofford, D. L. (2002).
Moscone, E. A., Scaldaferro, M. A., Grabiele, M, Cecchini, N. M.,
Sánchez Garcia, Y., Jarret, R., Daviña, J. R., Ducasse, D. A., Barboza,
G. E. & Eherendorfer, F. (2007). The evolution of chili peppers
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994).
CLUSTAL W:
improving the sensitivity of progressive multiple sequence alignment
through sequence weighting, position-specific gap penalties and
weight matrix choice. Nucleic Acids Res 22, 4673–4680.
(Capsicum – Solanaceae): a cytogenetic perspective. Acta Hortic 745,
137–170.
Tuomivirta, T. T., Kaitera, J. & Hantula, J. (2009). A novel putative virus of
Osaki, H., Nakamura, H., Sasaki, A., Matsumoto, N. & Yoshida, K.
(2006). An endornavirus from a hypovirulent strain of the violet root
Gremmeniella abietina type B (Ascomycota: Helotiaceae) has a composite
genome with endornavirus affinities. J Gen Virol 90, 2299–2305.
rot fungus, Helicobasidium mompa. Virus Res 118, 143–149.
Valverde, R. A. & Fontenot, J. F. (1991). Variation in double-stranded
Pfeiffer, P. (1998). Nucleotide sequence, genetic organization and
ribonucleic acid among pepper cultivars. J Am Soc Hortic Sci 116,
903–905.
expression strategy of the double-stranded RNA associated with the
‘447’ cytoplasmic male sterility trait in Vicia faba. J Gen Virol 79,
2349–2358.
Pickersgill, B. (1966). The variability and relationships of Capsicum
chinense Jacq. PhD dissertation, Indiana University, Bloomington,
Indiana, USA.
Pickersgill, B. (1971). Relationships between weedy and cultivated
Valverde, R. A. & Gutierrez, D. L. (2007). Transmission of a dsRNA in
bell pepper and evidence that it consists of the genome of an
endornavirus. Virus Genes 35, 399–403.
Valverde, R. A. & Gutierrez, D. L. (2008). Molecular and biological
properties of a putative partitivirus from Jalapeño pepper (Capsicum
annuum L.). Rev Mex Fitopatol 26, 1–6.
forms in some species of chili peppers (genus Capsicum). Evolution
25, 683–691.
Valverde, R. A., Nameth, S. T. & Jordan, R. L. (1990a). Analysis of
Poch, O., Sauvaget, I., Delarue, M. & Tordo, N. (1989). Identification
Valverde, R. A., Nameth, S., Abdalla, O., Al-Musa, O., Desjardins,
P. R. & Dodds, J. A. (1990b). Indigenous double-stranded RNA from
of four conserved motifs among the RNA-dependent polymerase
encoding elements. EMBO J 8, 3867–3874.
Roossinck, M. J., Sabanadzovic, S., Okada, R. & Valverde, R. A.
(2011). The remarkable evolutionary history of endornaviruses.
J Gen Virol 92, 2674–2678.
Roossinck, M. J., Saha, P., Wiley, G. B., Quan, J., White, J. D., Lai, H.,
Chavarrı́a, F., Shen, G. & Roe, B. A. (2010). Ecogenomics: using
http://vir.sgmjournals.org
double-stranded RNA for plant virus diagnosis. Plant Dis 74, 255–258.
pepper (Capsicum annum). Plant Sci 67, 195–201.
Wakarchuk, D. A. & Hamilton, R. I. (1990). Partial nucleotide
sequence from enigmatic dsRNAs in Phaseolus vulgaris. Plant Mol Biol
14, 637–639.
Zabalgogeazcoa, I. A. & Gildow, F. E. (1992). Double-stranded
ribonucleic acid in ‘Barsoy’ Barley. Plant Sci 83, 187–194.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Fri, 16 Jun 2017 18:00:38
2673