Journal of General Virology (2006), 87, 3113–3117
Short
Communication
DOI 10.1099/vir.0.82121-0
Molecular characterization of the largest mycovirallike double-stranded RNAs associated with
Amasya cherry disease, a disease of presumed
fungal aetiology
Z. Kozlakidis,1 L. Covelli,2,3 F. Di Serio,4 A. Citir,5 S. Açıkgöz,6
C. Hernández,2 A. Ragozzino,3 R. Flores2 and R. H. A. Coutts1
Correspondence
R. H. A. Coutts
[email protected]
1
Division of Biology, Faculty of Natural Sciences, Sir Alexander Fleming Building, Imperial
College London, Imperial College Road, London SW7 2AZ, UK
2
Instituto de Biologı́a Molecular y Celular de Plantas (UPV-CSIC), Universidad Politécnica de
Valencia, 46022 Valencia, Spain
3
Dipartimento di Arboricoltura, Botanica e Patologia Vegetale, Università di Napoli,
80055 Portici, Italy
4
Istituto di Virologia Vegetale del CNR, Sezione di Bari, 70126 Bari, Italy
5
Tekirdag Ziraat Fakültesi, Trakya Universitesi, 59030 Tekirdag, Turkey
6
Adnan Menderes University, Agricultural Faculty, Plant Pathology Department, 09100 Aydin,
Turkey
Received 10 April 2006
Accepted 5 June 2006
The sequence of the four large (L) double-stranded RNAs (dsRNAs) associated with Amasya
cherry disease (ACD), which has a presumed fungal aetiology, is reported. ACD L dsRNAs 1
(5121 bp) and 2 (5047 bp) potentially encode proteins of 1628 and 1620 aa, respectively, that
are 37 % identical and of unknown function. ACD L dsRNAs 3 (4458 bp) and 4 (4303 bp)
potentially encode proteins that are 68 % identical and contain the eight motifs conserved in
RNA-dependent RNA polymerases (RdRp) of dsRNA mycoviruses, having highest similarity with
those of members of the family Totiviridae. Both terminal regions share extensive conservation in all
four RNAs, suggesting a functional relationship between them. As ACD L dsRNAs 1 and 2 do
not encode RdRps, both are probably replicated by those from either ACD L dsRNA 3 or 4. Partial
characterization of the equivalent L dsRNAs 3 and 4 associated with cherry chlorotic rusty spot
revealed essentially identical sequences.
In previous publications, we illustrated that two cherry
diseases, cherry chlorotic rusty spot (CCRS) from Italy and
Amasya cherry disease (ACD) from Turkey, are two disorders that are likely to be of fungal aetiology, have very
similar symptoms and are consistently associated with a
complex profile of 10–12 double-stranded RNAs (dsRNAs),
presumably of viral origin, that are absent from healthylooking trees (Açıkgöz et al., 1994; Di Serio et al., 1996, 1998;
Coutts et al., 2004; Covelli et al., 2004). We reported that
four of them comprised the genome of a novel species in
the genus Chrysovirus (Covelli et al., 2004; Ghabrial et al.,
2005a), whilst another two comprise the genome of a novel
species in the genus Partitivirus (Coutts et al., 2004; Ghabrial
The GenBank/EMBL/DDBJ accession numbers for the sequences
reported in this paper are AM085136, AM085137, AM085134 and
AM085135 for full-length ACD-associated large dsRNAs 1, 2, 3 and 4,
respectively, and AM181139–AM181141 and AM181142 for partiallength CCRS-associated large dsRNAs 3 and 4, respectively.
0008-2121 G 2006 SGM
et al., 2005b), two genera in which all species infect fungi. In
support of the mycoviral character of these six dsRNAs,
mycelial structures have been observed in tissues affected
by both diseases (Alioto et al., 2003). The precise nature of
the putative fungus remains unknown because attempts to
culture it have failed (A. Ragozzino, unpublished results);
alternative approaches to tackle this question, based on the
characterization of internal transcribed spacer regions, are
in progress (G. Geraci, personal communication).
To verify that other ACD- and CCRS-associated dsRNAs
also have properties consistent with those expected for
mycoviruses and to get an insight into the taxonomic group
that they belong to, here we report the complete sequence
for the four largest ACD-associated dsRNAs and the partial
sequence for two of the equivalent CCRS-associated dsRNAs.
By using a range of randomly primed cDNA clones generated from individual gel-purified dsRNAs and sequencing,
we were able to show that the two largest dsRNAs (Fig. 1)
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 31 Jul 2017 21:15:07
Printed in Great Britain
3113
Z. Kozlakidis and others
are in fact both pairs of similarly sized dsRNAs that we have
nominated large (L) 1–4 in this investigation.
The L dsRNAs 1–4 were isolated from sweet cherry leaves
collected in Turkey and Italy exhibiting typical symptoms
of ACD and CCRS, respectively; this material was the same
as used in previous studies (Coutts et al., 2004; Covelli et al.,
2004). After separation by electrophoresis on polyacrylamide or agarose gels, they were used either collectively or
individually for reverse transcription, PCR amplification,
cloning and sequencing as described previously (Coutts
et al., 2004; Covelli et al., 2004). The cDNA clones of ACD
L dsRNAs 1–4 were obtained by random priming of methyl
mercuric hydroxide-denatured dsRNA, with further DNA
manipulations being performed according to standard
protocols (Sambrook & Russell, 2001). For the synthesis
of additional cDNAs covering their complete sequence,
purified ACD L dsRNAs 1–4 were denatured with methyl
mercuric hydroxide and subjected to a single-primer,
genome-walking RT-PCR protocol developed previously
to sequence plant virus RNAs (Livieratos et al., 1999). An
RNA ligase-mediated (RLM)-RACE PCR procedure was
used to determine the 59- and 39-terminal sequences of the
dsRNAs (Coutts & Livieratos, 2003). The sequences of
the equivalent CCRS-associated dsRNAs were obtained by
the single-primer method as described previously (Covelli
et al., 2004). Nucleotide and amino acid sequences were
manipulated (Devereux et al., 1984), aligned by using
CLUSTAL_X (Thompson et al., 1997) and drawn by using the
MEGA program v. 3 (Kumar et al., 2004).
The complete sequences of ACD-associated L dsRNAs 1–4
were respectively 5121, 5047, 4458 and 4303 bp in length
and each included one open reading frame (ORF) potentially encoding proteins of 1628, 1620, 1363 and 1294 aa
with molecular masses of approximately 178 920, 179 218,
151 860 and 143 120 Da, respectively. For ACD L dsRNAs
3 and 4, the C-terminal regions of the predicted proteins
(residues 720–1100 and 720–1099, respectively) contained
the eight conserved motifs characteristic of RNA-dependent
RNA polymerases (RdRps) of dsRNA viruses infecting
simple eukaryotes (Bruenn, 1993) (Fig. 2a). Multiple alignment of these regions (Fig. 2a) and assembly of a phylogenetic tree (Fig. 2b) revealed that ACD L dsRNAs 3 and
4 were most similar to the RdRp regions of the totiviruses
Saccharomyces cerevisiae virus L-A (ScV-L1) and Ustilago
maydis virus H1 (UmV-H1). Further analysis of the putative RdRp regions encoded by ACD L dsRNAs 3 and 4
with the program BLASTP (http://www.ncbi.nlm.nih.gov)
revealed similarities between them and the ankyrin-repeat
proteins of viral origin (data not shown), but the significance of this observation is unclear.
Fig. 1. Agarose-gel electrophoresis (1 % in
TAE) of dsRNA-rich preparations from ACDaffected leaves (lane 2) and control preparations obtained under the same conditions
from an asymptomatic cherry tree (lane 3),
compared with DNAs of known molecular
size indicated to the left in kbp (lane 1). The
four L dsRNAs were derived from the upper
two dsRNA species seen on the gel in lane
2. The four genomic dsRNAs of a chrysovirus and the two genomic dsRNAs of a
partitivirus, characterized previously (Coutts
et al., 2004; Covelli et al., 2004), are also
indicated.
3114
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 31 Jul 2017 21:15:07
Journal of General Virology 87
http://vir.sgmjournals.org
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 31 Jul 2017 21:15:07
Characterization of Amasya cherry disease large dsRNAs
3115
Fig. 2. Relationships between the ACD-associated large (L) dsRNAs 3 and 4 and some representative species of three families of dsRNA viruses. (a) Multiple alignment of the region containing the
eight motifs conserved in RdRps of dsRNA viruses. Abbreviations precede virus names and
GenBank accession numbers are also shown. Family Totiviridae: genus Totivirus: UmV-H1 (Ustilago
maydis virus H1), ScV-L1 (Saccharomyces cerevisiae virus L-A), ACD-L 4 (Amasya cherry disease
large dsRNA 4), ACD-L 3 (Amasya cherry disease large dsRNA 3), Hv190SV (Helminthosporium
victoriae virus 190S); genus Leishmaniavirus: LRV1-1 (Leishmania RNA virus 1-1); genus
Giardiavirus: TVV (Trichomonas vaginalis virus), GLV (Giardia lamblia virus). Family Chrysoviridae:
genus Chrysovirus: PcV (Penicillium chrysogenum virus), Hv145SV (Helminthosporium victoriae
virus 145SV), ACD-CV (Amasya cherry disease-associated chrysovirus). Family Partitiviridae: genus
Partitivirus: AhV-2H (Atkinsonella hypoxylon virus 2H), RhsV 717 (Rhizoctonia solani virus 717),
FpV1 (Fusarium poae virus 1), ACD-PV (Amasya cherry disease-associated partitivirus dsRNA 1);
genus Alphacryptovirus: BCV-3 (Beet cryptic virus 3). Numbers at the top refer to the eight motifs
conserved in RdRps of dsRNA viruses of lower eukaryotes (Bruenn, 1993). Numbers of residues
between the conserved motifs are indicated. Asterisks in the consensus sequence denote identical
amino acids and capital letters denote those found in at least five sequences. (b) Unrooted phylogenetic tree based on the neighbour-joining method with a 10 000 replicate bootstrap search.
Bootstrap values (expressed as percentages) are indicated at the branch points.
Z. Kozlakidis and others
As both ACD L dsRNAs 3 and 4 potentially encode proteins that show similarities to the conserved motifs of RdRps
of several totiviruses and share nucleotide and amino acid
identities of 67 and 68 %, respectively, both can therefore
be regarded tentatively as two novel species of the family
Totiviridae. However, in contrast with most members of
this family, whose genomic dsRNAs contain two ORFs –
the first, ORF A, encoding the coat protein (CP) and the
second, ORF B, the RdRp – those from ACD L dsRNAs 3 and
4 potentially encode a single protein, in the C-terminal
region of which are found the eight motifs conserved in
RdRps. In the genus Totivirus, UmV-H1 is related phylogenetically to L dsRNAs 3 and 4 (Fig. 2b), but whether
ACD L dsRNAs 3 and 4 have similar properties, e.g. UmVH1 dsRNA is translated into a single polyprotein that is then
processed to the CP and the RdRp by a (viral) papain-like
protease (Kang et al., 2001), is not known. Whilst ACD L
dsRNAs 3 and 4 might have a similar expression strategy, a
search of amino acid motifs similar to those present in viral
proteases failed to identify any in the proteins predicted
from their sequences. BLASTP analysis of the N-terminal
regions of proteins predicted from the sequences of ACD L
dsRNAs 3 and 4 did not reveal any significant similarity with
CPs of other totiviruses. Additional comparative analysis, using the programs LALIGN (http://www.ch.embnet.org/
software/LALIGN_form.html) and SCANPROSITE (http://
www.expasy.org/prosite), of these N-terminal regions with
the N-terminal regions of the single ORF of UmV-H1 and
ScV-L1 fusion protein (Wickner et al., 2005) and the global
database, respectively, revealed scattered amino acid identity
of between 19 and 32 % for the totiviruses and the pattern
M–X(6)–P–D–X(3)–T between aa 637 and 649, also
present in the CPs of some other viruses, but absent from
totivirus CPs characterized thus far (data not shown).
Whether the N-terminal regions of proteins predicted from
the sequences of ACD L dsRNAs 3 and 4 encode CPs is thus
far unknown.
For ACD L dsRNAs 1 and 2, the predicted proteins were
37 % identical to one another, contained no recognizable
motifs and had no similarity to any other proteins found
in global databases. However, the two dsRNAs appear to be
related to ACD L dsRNAs 3 and 4 (see below).
The lengths of the 59-untranslated regions (59 UTRs) flanking the single ORFs of the ACD-associated L dsRNAs 1–4
were 35, 35, 59 and 73 nt, respectively. The lengths of the 39
UTRs of the same molecules were 199, 149, 307 and 345 nt,
respectively. The complementary strands did not contain
ORFs of a minimal size compatible with a functional protein (data not shown). The 59-terminal 10 nt were almost
identical in all four dsRNAs and there was also significant, although lower, conservation in the sequences of the
39 UTRs (Fig. 3). As there is significant identity in the 59terminal sequences and the 39 UTRs of ACD L dsRNAs 1–4
(Fig. 3), and ACD L dsRNAs 1 and 2 do not apparently
encode RdRps, both may be replicated by the RdRp of either
ACD L dsRNAs 3 or 4 and be part of a multipartite genome.
The possibility that ACD L dsRNAs 1 or 2 may be satellite
viruses or satellite dsRNAs, dependent functionally on either
ACD L dsRNAs 3 or 4, appears less likely. The absence of any
similarity of the proteins predicted from the sequences of
either ACD L dsRNAs 1 or 2 to any other protein makes any
further speculation very difficult. However, it is unlikely that
ACD L dsRNAs 1 and 2 are associated functionally with
other viruses found in ACD-diseased tissue because the
UTRs of both dsRNAs have no sequence similarity to the
dsRNAs of the two previously characterized viruses, both of
which are complete in genomic RNA content (Coutts et al.,
2004; Covelli et al., 2004).
Only partially complete nucleotide sequences for two of
the CCRS-associated L dsRNAs, 3 and 4 (1840 and
3557 nt, respectively), were determined and visual inspection of the dsRNAs by PAGE revealed that L dsRNA 2 might
not always be present in Italian samples (results not shown).
However, as the partial sequences of CCRS-associated
L dsRNAs 3 and 4 were 98 and 97 % identical to the
equivalent ACD-associated L dsRNAs, respectively, it is
reasonable to assume that the CCRS-associated L dsRNAs
3 and 4 are variants of ACD L dsRNAs 3 and 4, described
fully here.
It is now generally accepted and verified by sequencing that
the incidence of multiple viruses in single fungal isolates is
a relatively common occurrence. This has been reported for
mitoviruses in Ophiostoma novo-ulmi, infecting Dutch elm
(Hong et al., 1998), for two viruses belonging to the families
Totiviridae and Chrysoviridae found in a single isolate of
Helminthosporium victoriae that infects oats (Ghabrial et al.,
2002), for several viruses belonging to different families in
Helicobasidium mompa, which infects a wide range of
plants (Nomura et al., 2003; Osaki et al., 2004, 2005), and for
three unrelated viruses in Gremmeniella abietina, infecting
coniferous trees (Tuomivirta & Hantula, 2005). However,
the complexity of viruses associated with the fungus or fungi
presumed responsible for ACD and CCRS is unprecedented
and appears to include representatives of at least three fungal
Fig. 3. Comparison of the 59- and 39-untranslated terminal regions of the coding strands of the four ACD large dsRNAs.
Identical nucleotides have a black background, whereas those conserved in two or three of the dsRNAs are shaded.
3116
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 31 Jul 2017 21:15:07
Journal of General Virology 87
Characterization of Amasya cherry disease large dsRNAs
dsRNA virus families (Coutts et al., 2004; Covelli et al.,
2004; this study). Whilst we can report the presence of two
new viruses, ACD L dsRNAs 3 and 4, that are related to the
family Totiviridae, until their mode of replication and their
relationships with ACD L dsRNAs 1 and 2 are known, we
consider it prudent to nominate them as tentative novel
members of this family.
Acknowledgements
Ghabrial, S. A., Soldevila, A. I. & Havens, W. M. (2002). Mole-
cular genetics of the viruses infecting the plant pathogenic fungus
Helminthosporium victoriae. In dsRNA Genetic Elements: Concepts
and Applications in Agriculture, Forestry, and Medicine, pp. 213–236.
Edited by S. M. Tavantzis. Boca Raton, FL: CRC Press.
Ghabrial, S. A., Jiang, D. & Castón, J. R. (2005a). Chrysoviridae. In
Eighth Report of the International Committee on Taxonomy of Viruses,
pp. 591–595. Edited by C. M. Fauquet, M. A. Mayo, J. Maniloff,
U. Desselberger & L. A. Ball. London: Elsevier/Academic Press.
Ghabrial, S. A., Buck, K. W., Hillman, B. I. & Milne, R. G. (2005b).
Work in the laboratories of R. F. and A. R. was partially supported
by the Generalidad Valenciana (Spain) and the Ministero delle
Politiche Agricole e Forestali (Italy), respectively. Z. K. was supported
by a bursary from the Faculty of Natural Sciences, Imperial College
London, UK. We thank Dr D. Alioto for detailed information
about the fungus-like organism found in CCRS and ACD
symptomatic leaves. During this work, L. C. was the recipient of
predoctoral fellowships from the Marie Curie Program
(European Union) and from the Università ‘Federico II’ di Napoli
(Italy).
Partitiviridae. In Eighth Report of the International Committee on
Taxonomy of Viruses, pp. 581–590. Edited by C. M. Fauquet, M. A.
Mayo, J. Maniloff, U. Desselberger & L. A. Ball. London: Elsevier/
Academic Press.
Hong, Y., Cole, T. E., Brasier, C. M. & Buck, K. W. (1998).
Evolutionary relationships among putative RNA-dependent RNA
polymerases encoded by a mitochondrial virus-like RNA in the
Dutch elm disease fungus, Ophiostoma novo-ulmi, by other viruses
and virus-like RNAs and by the Arabidopsis mitochondrial genome.
Virology 246, 158–169.
Kang, J.-G., Wu, J.-C., Bruenn, J. A. & Park, C.-M. (2001). The H1
References
Açıkgöz, S., Doken, M. T., Citir, A. & Bostan, H. (1994). The analysis
of double stranded (ds) RNA for the diagnosis of the causal agent of
Amasya cherry disease in Turkey. In EMBO-INRA Workshop Plant
and Viruses: Partners in Pathogenicity, Grignon, abstract 36.
double-stranded RNA genome of Ustilago maydis virus-H1 encodes a
polyprotein that contains structural motifs for capsid polypeptide,
papain-like protease, and RNA-dependent RNA polymerase. Virus
Res 76, 183–189.
Kumar, S., Tamura, K. & Nei, M. (2004). MEGA3: integrated software
for molecular evolutionary genetics analysis and sequence alignment.
Brief Bioinform 5, 150–163.
Alioto, D., Zaccaria, F., Covelli, L., Di Serio, F., Ragozzino, A. & Milne,
R. G. (2003). Light and electron microscope observations on chlorotic
Livieratos, I. C., Avgelis, A. D. & Coutts, R. H. A. (1999). Molecular
rusty spot, a disorder of cherry in Italy. J Plant Pathol 85, 215–218.
characterization of the cucurbit yellow stunting disorder virus coat
protein gene. Phytopathology 89, 1050–1055.
Bruenn, J. A. (1993). A closely related group of RNA-dependent
RNA polymerases from double-stranded RNA viruses. Nucleic Acids
Res 21, 5667–5669.
Nomura, K., Osaki, H., Iwanami, T., Matsumoto, N. & Ohtsu, Y.
(2003). Cloning and characterization of a totivirus double-stranded
Coutts, R. H. A. & Livieratos, I. C. (2003). A rapid method for
RNA from the plant pathogenic fungus, Helicobasidium mompa
Tanaka. Virus Genes 26, 219–226.
sequencing the 59- and 39-termini of double-stranded RNA viral
templates using RLM-RACE. J Phytopathol 151, 525–527.
Coutts, R. H. A., Covelli, L., Di Serio, F., Citir, A., Açıkgöz, S.,
Hernández, C., Ragozzino, A. & Flores, R. (2004). Cherry chlorotic
rusty spot and Amasya cherry diseases are associated with a complex
pattern of mycoviral-like double-stranded RNAs. II. Characterization
of a new species in the genus Partitivirus. J Gen Virol 85, 3399–3403.
Covelli, L., Coutts, R. H. A., Di Serio, F., Citir, A., Açıkgöz, S.,
Hernández, C., Ragozzino, A. & Flores, R. (2004). Cherry chlorotic
rusty spot and Amasya cherry diseases are associated with a complex
pattern of mycoviral-like double-stranded RNAs. I. Characterization
of a new species in the genus Chrysovirus. J Gen Virol 85, 3389–3397.
Osaki, H., Nomura, K., Matsumoto, N. & Ohtsu, Y. (2004). Charac-
terization of double-stranded RNA elements in the violet root rot
fungus Helicobasidium mompa. Mycol Res 108, 635–640.
Osaki, H., Nakamura, H., Nomura, K., Matsumoto, N. & Yoshida, K.
(2005). Nucleotide sequence of a mitochondrial RNA virus from the
plant pathogenic fungus, Helicobasidium mompa Tanaka. Virus Res
107, 39–46.
Sambrook, J. & Russell, D. (2001). Molecular Cloning: a Laboratory
Manual, 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor
Laboratory.
Devereux, J., Haeberli, P. & Smithies, O. (1984). A comprehensive
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. &
Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible
set of sequence analysis programs for the VAX. Nucleic Acids Res 12,
387–395.
strategies for multiple sequence alignment aided by quality analysis
tools. Nucleic Acids Res 25, 4876–4882.
Di Serio, F., Flores, R. & Ragozzino, A. (1996). Cherry chlorotic
Tuomivirta, T. T. & Hantula, J. (2005). Three unrelated viruses
rusty spot: description of a virus-like disease from cherry and studies
on its etiological agent. Plant Dis 80, 1203–1206.
occur in a single isolate of Gremmeniella abietina var. abietina type
A. Virus Res 110, 31–39.
Di Serio, F., Flores, R., Alioto, D. & Ragozzino, A. (1998). Both the
Wickner, R. B., Wang, C. C. & Patterson, J. L. (2005). Totiviridae. In
small circular RNAs and the double-stranded RNAs associated with
the chlorotic rusty spot disease of sweet cherry are also found in sour
cherry with similar symptoms. Acta Hortic 472, 291–297.
Eighth Report of the International Committee on Taxonomy of Viruses,
pp. 571–580. Edited by C. M. Fauquet, M. A. Mayo, J. Maniloff,
U. Desselberger & A. L. Ball. London: Elsevier/Academic Press.
http://vir.sgmjournals.org
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 31 Jul 2017 21:15:07
3117