A portion of RNA 3 of Citrus variegation virus (CVV), comprising part of the intergenic region and the coat protein (CP) gene from eight viral isolates, was amplified by RT-PCR and cloned. The clones were compared for intra and inter-isolate variations by single-strand conformation polymorphism analysis. Some of the results were compared with sequence data previously obtained. The test discriminated between clones differing in as little as 3.2% of the nucleotides. Most isolates included several variants, in some cases with a predominant pattern, which, however, could no longer be recognised in new RT-PCR products obtained 13 months later. This procedure can there-fore be used to identify and detect variations between CVV isolates. It is rapid, inexpensive and may reduce the amount of sequencing needed for comparing viral isolates

Azeddoug, H.

Bennani, B.

Nolasco, Gustavo

Zemzami, M.

A portion of RNA 3 of Citrus variegation virus (CVV), comprising part of the intergenic region and the
coat protein (CP) gene from eight viral isolates, was amplified by RT-PCR and cloned. The clones were compared for
intra and inter-isolate variations by single-strand conformation polymorphism analysis. Some of the results were
compared with sequence data previously obtained. The test discriminated between clones differing in as little as
3.2% of the nucleotides. Most isolates included several variants, in some cases with a predominant pattern, which,
however, could no longer be recognised in new RT-PCR products obtained 13 months later. This procedure can therefore
be used to identify and detect variations between CVV isolates. It is rapid, inexpensive and may reduce the
amount of sequencing needed for comparing viral isolates

Nolasco, G.

Firenze University Press: E-Journals

188 Phytopathologia Mediterranea
Phytopathol. Mediterr. (2002) 41, 188–192
Introduction
Citrus variegation virus (CVV) is a widespread
virus (Bennani et al., 2002) that has not been much
studied. It is a member of the ilarvirus group of
the Bromoviridae and has a tripartite single-
stranded RNA genome. Infected citrus trees exhibit
a range of symptoms, which are usually mild on
oranges and mandarins but may be severe on cit-
ron and lemon trees; on these yield reduction and
fruit malformation may also occur. Two types of
Variability of a portion of RNA 3 containing the coat protein gene
of Citrus variegation virus (CVV) using single-strand
conformation polymorphism (SSCP)
BAHIA BENNANI1, 2, MUSTAPHA ZEMZAMI2, GUSTAVO NOLASCO3 and HOUSSINE AZEDDOUG1
1Université Hassan II, Faculté des Sciences Aïn Chock, Laboratoire de biochimie,
B.P.  Maârif, 20100 Casablanca Morocco
2Direction des Domaines Agricoles, Domaine UCP Maamora, Km 11, Route P.le No. 1 Hussaïne Salé 10000, Morocco
3Universidade do Algarve/ FERN, 8000 Faro, Portugal
Summary. A portion of RNA 3 of Citrus variegation virus (CVV), comprising part of the intergenic region and the
coat protein (CP) gene from eight viral isolates, was amplified by RT-PCR and cloned. The clones were compared for
intra and inter-isolate variations by single-strand conformation polymorphism analysis. Some of the results were
compared with sequence data previously obtained. The test discriminated between clones differing in as little as
3.2% of the nucleotides. Most isolates included several variants, in some cases with a predominant pattern, which,
however, could no longer be recognised in new RT-PCR products obtained 13 months later. This procedure can there-
fore be used to identify and detect variations between CVV isolates. It is rapid, inexpensive and may reduce the
amount of sequencing needed for comparing viral isolates.
Key words: Citrus viruses, infectious variegation-crinkly leaf, molecular variability, RT-PCR, SSCP.
Corresponding author: H. Azeddoug
E-mail: h_azeddoug@hotmail.com
Fax: +212 22 230674
symptoms, attributed to different strains of the
virus are commonly distinguished: infectious vari-
egation, characterised by crinkling of the leaves
associated with chlorosis, and crinkly leaf, which
includes warping, pocketing and crinkling but with-
out variegation or reduction in leaf size (Desjardin
and Bové, 1980). Detection and characterisation
of CVV in the field is by ELISA and more recently,
at the molecular level, by RT-PCR and sequencing
(Bennani et al., 2002). In this study, the coat pro-
tein gene and part of the intergenic region were
cloned and sequenced to compare several CVV iso-
lates with different geographic origin and biologi-
cal properties. Nucleotide sequencing is the most
accurate procedure for strain identification, but it
is too expensive and time consuming for routine
189Vol. 41, No. 3, December 2002
Molecular variability in Citrus variegation virus
purposes. Single-strand conformational polymor-
phism (SSCP) analysis (Orita et al., 1989) appears
an attractive alternative due to its suitability and
has been used to assess the variability of several
viruses. In SSCP analysis, double-stranded DNA
is heat-denatured in the presence of formamide and
both DNA strands are separated by electrophore-
sis in non-denaturing polyacrylamide gel. Under
these conditions, part of the single-stranded DNA
acquires a globular conformation that depends on
the existence of self-complementary regions. The
occurrence of mutations in these regions strongly
affects the single-strand conformation, resulting in
electrophoretic mobility shifts.
SSCP has been used for different purposes, such
as: evaluating sequence heterogeneity among iso-
lates of hepatitis B (Yap et al., 1992) and C (Enomo-
to et al., 1994) viruses; assigning beet necrotic yel-
low vein virus isolates to a known strain group
(Koenig et al., 1995); differentiating CTV isolates
on the basis of their genetic variability (Rubio et
al., 1996; Ayllon et al., 1999); studying the popula-
tion structure of CTV (Kong et al., 2000); and char-
acterising different viroids (Palacio and Duran-
Vila, 1999). Recently, fluorescent SSCP has also
been used to detect variants of avocado sunblotch
viroid (Schnell et al., 2001).
In this paper SSCP was used to study intra and
inter-isolate variability of CVV and results were
compared with data recently obtained.
Materials and methods
Virus isolates
Virus isolates from different geographical are-
as were those used in previous studies (Bennani et
al., 2002) and were maintained in citrus seedlings
grown in insect-proof greenhouses. They included:
isolates CVV-1 (Garnsey et al., 1968) and CVV-2
(Garnsey et al., 1984), obtained from USDA, ARS,
Orlando FL, USA; CVV-E1234, characterised by
Grant (Desjardins and Bové, 1980), obtained from
the Station de San Giuliano, Corsica, France; CVV-
IV400 from IVIA, Spain (Duran-Vila et al., 1988);
CVV-81A65 from Corsica; CVV-CL903 from Spain
(Desjardins and Bové, 1980); CVV-3 from a field
near Marrakech, Marocco; and CVV-ES86 obtained
from a citrus repository in Portugal (Bennani et
al., 2002). Virus-free citrus was included as a
healthy control.
cDNA synthesis, RNA amplification and cloning
Total nucleic acid of each isolate was extracted
from 0.2 g of fresh bark tissue by a lithium-chlo-
ride-based protocol as described in an earlier study
(Bennani et al., 2002). CVVa and CVVb primers
(Bennani et al., 2002), designed from conserved
regions of ilarvirus subgroup 2 (except Citrus leaf
rugose virus (CiLRV)) were used to amplify the full-
length coat protein (CP) gene and 63 nt of the inter-
genic region upstream of this gene. Amplification
was carried out with 2 µl of RNA and RT-PCR mix-
es (25 µl final volume) containing 10 mM Tris (pH
8.8), 50 mM KCl, 2 mM MgCl2, 400 nM each of the
primers, 0.4 mM of each dNTP, 0.08% NP-40, 0.5U
Taq DNA polymerase (MBI-Fermentas, Vilnius,
Lithuania), 5U RNAguard (Amersham Pharmacia,
Buckingamshire, UK) and 6U MuLV reverse tran-
scriptase (Perkin Elmer, Wellesley, MA, USA). The
amplification conditions were: 30 min at 39°C, 2
min at 94°C, followed by 35 cycles at 92°C for 30s,
56°C for 45s and 72°C for 1 min and finally 5 min
at 72°C. The amplified products were separated
by 1% agarose gel electrophoresis. RT-PCR prod-
ucts of the expected size were cloned into the
EcoRV-cut PGEMT-Easy vector system I (Prome-
ga, Madison, WI, USA) and transformed into E.
coli INVα F’ cells (Invitrogen, S. Giuliano Milanese,
Italy). For all isolates, positive clones were identi-
fied by direct PCR amplification from the white
colonies using primers CVVa/CVVb (Bennani et al.,
2002). The plasmid DNA was purified using a Wiz-
ard plus SV miniprep Kit (Promega) according to
manufacturer’s instructions. The identity of the
insert was confirmed by digestion with EcoRI.
SSCP analysis
Fifty-one clones of 63 nt intergenic region up-
stream of the CP gene, and the entire CP gene ob-
tained from eight isolates were compared by SSCP.
SSCP analysis was done on PCR products am-
plified from recombinant E. coli colonies or, in some
experiments, directly from RT-PCR products. For
SSCP analysis, 1 to 3 µl of the amplified products
was mixed with 10 µl of denaturing buffer (19.6
mM EDTA, pH 8, in formamide containing a min-
imal amount of bromophenol blue), denatured for
5 min at 90°C, and loaded on a non-denaturing 8%
polyacrylamyde gel. After running for 3 h at 200 V,
4°C, the gels were stained with silver nitrate (Ori-
ta et al., 1989).
190 Phytopathologia Mediterranea
B. Bennani et al.
Sequence comparison
The sequences compared were those of clones
CVV-1-U, CVV-2-L, CVV-3-O, CVV-E1234-11, CVV-
E1234-13, CVV-IV400-12, CVV-IV400-13, CVV-
81A65-12, CVV-81A65-27, CVV-CL903-15, CVV-
CL903-21 and CVV-ES86, with accession numbers
AF434918, AF434917, AF434916, AF434915,
AF434914, AF434922, AF434921, AF434911,
AF434912, AF434920, AF434919 and AF434913
respectively.
Results
SSCP analysis of the amplicon
Amplification from the plasmids and from RNA
isolates in all cases yielded a single fragment of
718 bp, encompassing the CP gene and 63 nt of the
intergenic region upstream of this gene. Electro-
phoresis used to compare the RT-PCR products of
different isolates and the 51 clones by SSCP, as
described in Materials and methods, yielded very
reproducible patterns when repeated analysis was
done (data not shown). All SSCP results are illus-
trated in Fig. 1.
The SSCP patterns obtained directly from RT-
PCR products amplified with primers CVVa–CVVb
(Fig. 1-I) differed between most isolates, except
those for ES86, E1234 and CVV3. Usually the pat-
terns consisted of only two conspicuous bands, sug-
gesting that the isolates were closely related vari-
ants. However, when SSCP analysis was carried
out with the CVVa–CVVb PCR product amplified
directly from transformed E. coli colonies, some
isolates consisted of more than one variant. Iso-
lates 81A65 (Fig. 1F), CVV3 (Fig. 1C), CVV1(Fig.
1A) and CVV2 (Fig. 1B) each produced a predomi-
nant pattern that was common to the majority of
that isolate’s clones. On the other hand, only about
50% of the clones from isolates IV400 (Fig. 1D) and
CL903 (Fig. 1G) yielded similar patterns. The pat-
terns of clones IV400-13, CVV1-U and ES86 were
very similar among each other (Fig. 1H).
Fig. 1. SSCP analysis of PCR and RT-PCR products obtained with primers CVVa and CVVb. Panels A to G, direct
PCR on isolates CVV-1, CVV-2, CVV-3, CVV-IV400, CVV-E1234, CVV-81A65 and CVV-CL903 respectively, carried
out with E.coli colonies harbouring the RT-PCR product. Sequenced clones are presented in lanes A6 (CVV-1-U), B7
(CVV-2-L), C4 (CVV-3-O), D1 (IV400-12), D2 (IV400-13), E1 (E1234-11), E2 (E1234-13), F1 (81A65-12), F8 (81A65-
27), G5 (CL903-15) and G6 (CL903-21). Panel H, side-by-side comparison of SSCP patterns from some sequenced
clones: CVV-81A65-12, CVV-IV400-13, CVV-IV400-12, CVV-ES86, CVV-CL903-15, CVV1-U, CVV2-L, CVV-E1234-
13 and CVV-E1234-11 (lanes 1 to 9 respectively). Panel I, SSCP patterns of RT-PCR products obtained from plants
infected with isolates CVV-IV400, CVV-E1234, CVV-81A65, CVV-ES86, CVV-2, CVV-1, and CVV-CL903 (lanes 1 to 7
respectively).
191Vol. 41, No. 3, December 2002
Molecular variability in Citrus variegation virus
Nucleotide differences between clones
Nucleotide sequence homology of clones previ-
ously sequenced (Bennani et al., 2002) and ob-
tained with the program Clustal (Thompson et al.,
1994) is summarised in Table 1. Differences in the
nucleotide sequence were not always correlated
with differences in electrophoretic mobility. When
sequences of the very similar clones IV400-13,
CVV1-U and ES86 were compared, they differed
from each other by 14 to 22 bp. All clones differ-
ing by more than 23 bp yielded different patterns.
Conversely, in the case of clones that differed only
for 3 nt different patterns were produced by
IV400-12 and E1234-11, and by CVV3 and IV400-
13, and identical patterns by 81A65-12 and 81A65-
27. In summary, any two amplicons producing
different patterns differed by at least 3 bp (0.4%),
while amplicons with similar patterns always dif-
fered by less than 23 bp (3.2%).
Discussion
SSCP analysis of several clones of each CVV
isolate showed that some of the isolates (e.g.
CVV1, CVV2, CVV3, 81A65) consisted of one pre-
dominant variant. In these cases the pattern of
the predominant variant was recognised in the
SSCP pattern of the RT-PCR products from which
the clones were obtained. To have an idea of the
accuracy of this indication, sequences of two
clones from 81A65 having the same pattern were
compared and a total of 3 nucleotide differences
were found. This suggests that SSCP gives a fair
approximation of intra-isolate variability. On the
other hand, patterns from clones obtained from
isolates CL903, E1234 and IV400 produced dif-
ferent patterns, suggesting that each of these iso-
lates consisted of several variants, typical of a
viral quasi-species. However, in these cases there
was no straightforward correspondence between
the SSCP patterns of the clones and the sequence
of the RT-PCR products which yielded them. It
was expected that the SSCP pattern of the RT-
PCR product would be similar to the most fre-
quent pattern of the clones or would be the same
as the individual patterns, but this was not the
case. SSCP analysis also did not reveal any par-
ticular feature of the RT-PCR patterns that could
be associated with either infectious variegation
or crinkly leaf symptoms. Although conditions
were far from those usually considered ideal for
SSCP, in that short PCR products (Sheffield et
al., 1993) were used, it was possible to discrimi-
nate among clones differing by at least 3.2% of
their nucleotide sequence. Other Authors (Rubio
et al., 1996) using products of approximately the
same length, were able to distinguish clones dif-
fering by 1.4%. Field isolates of CVV consist of a
large number of CP gene variants. Under the
conditions used, SSCP of RT-PCR products ap-
peared not to be adequate to reveal the sequence
variants that compose a particular isolate. The
SSCP of individual clones should be used for that
purpose.
Table 1. Homology percentage and number of nucleotide differences (in bold type) among 12 clones in the region
considered. U17389 was obtained from GenBank.
       Seq U17389 IV400-12 IV400-13 CVV1-U CVV2-L CVV3-O E1234-11 E1234-13 CL903-15 CL903-21 ES86 81A65-27 81A65-12
U17389 100 98.3 98.3 99.7 97.4 98.1 97.9 97.7 95.6 97.9 97.2 94.7 94.5
IV400-12 11 100 98.3 98 97.4 98.1 99.5 97.7 95.1 97.9 98.6 94.1 94.0
IV400-13 12 12 100 98 99.1 99.5 97.9 99.1 95.4 99.3 97.4 94.4 94.2
CVV1-U 2 14 14 100 97.2 97.9 97.6 97.4 95.4 97.6 96.9 94.4 94.2
CVV2-L 18 17 7 19 100 98.7 97 98.6 95.1 99 96.6 94 93.8
CVV3-O 13 12 3 15 10 100 97.7 99 95.2 99.1 97 94.2 94.1
E1234-11 14 3 15 16 21 16 100 97.3 94.7 97.4 98.7 94 93.8
E1234-13 16 16 4 18 10 7 19 100 95.9 99 96.6 94.9 94.8
CL903-15 31 33 31 33 35 32 38 29 100 95.2 94.2 98 97.9
CL903-21 15 15 5 17 7 6 18 7 33 100 96.7 94.2 94.1
ES86 22 10 18 22 24 21 9 24 41 23 100 93.5 93.4
81A65-27 38 42 32 40 43 40 43 36 14 41 46 100 99.5
81A65-12 39 43 41 42 44 42 43 36 14 41 47 3 100
192 Phytopathologia Mediterranea
B. Bennani et al.
Acknowledgements
This research was in part supported by a grant
PRAXIS XXI-3/3.3/HORT/2159-95 provided by FCT
(Portugal) and by bi-lateral scientific and techno-
logical cooperation between ICCTI (Portugal) and
CNCPRST (Morocco).
Literature cited
Ayllon M.A., L. Rubio, A. Moya, J. Guerri and P. Moreno,
1999. The haplotype distribution of two genes of citrus
tristeza virus is altered after host change or aphid trans-
mission. Virology 255(1), 32–39.
Bennani B., C. Mendes, M. Zemzami, H. Azeddoug and G.
Nolasco, 2002. Citrus variegation virus: Molecular var-
iability of a portion of the RNA 3 containing the coat
protein gene and design of primers for RT-PCR detec-
tion. European Journal of Plant Pathology 108(2), 155–
162.
Desjardins P.R. and J.M. Bové, 1980. Infectious variega-
tion and crinkly leaf. In: Description and Illustration of
Virus and Virus-like Diseases of Citrus. A collection of
colour slides. Proceedings of the International Organi-
sation of Citrus Virologists (J.M. Bové, R. Vogel, ed.),
Vol. 2, IRFA, 1–9.
Duran-Vila N., M. Cambra, J.A. Pina, J.F. Ballester and L.
Navarro, 1988. Virus content and growth patterns of
callus cultured in vitro from healthy and virus infected
Citrus species. Proceedings of the International Organ-
isation of Citrus Virologists 9, 310–313.
Enomoto N., M. Kurosaki, Y. Tanaka, F. Marumo  and C.
Sato, 1994. Fluctuation of hepatitis C virus quasispe-
cies in persistent infection and interferon treatment
revealed by single-strand conformation polymorphism
analysis. Journal of General Virology 75, 1361–1369.
Garnsey S.M., 1968. Additional non-citrus hosts for the
Florida isolate for citrus variegation virus. Phytopathol-
ogy 58, 1433–1434.
Garnsey S.M., N. Baksh, M. Davio and J.P. Agostini, 1984.
A mild isolate of Citrus Variegation Virus found in Flor-
ida Citrus. Proceedings of the International Organisa-
Accepted for publication: August 27, 2002
tion of Citrus Virologists 9, 188–195.
Hall T.C., 1979. Transfer RNA-like structures in viral ge-
nomes. International Review of Cytology 60, 1–26.
Koenig R., P. Lüddecke and A.M. Haeberle, 1995. Detec-
tion of beet necrotic yellow vein virus strains, variants
and mixed infections by examining single-strand con-
formation polymorphisms of immunocapture RT-PCR
products. Journal of General Virology 76, 2051–2055.
Kong P., L. Rubio, M. Polek and  B.W. Falk, 2000. Popula-
tion structure and genetic diversity within California
Citrus tristeza virus (CTV) isolates. Virus Genes 21(3),
139–145.
Orita M., H. Iwahana, K. Kanazawa and T. Sekia, 1989.
Detection of polymorphism of human DNA by gel elec-
trophoresis as single strand conformation polymor-
phism, Proceedings of the National Academy of Scienc-
es of the USA 86, 2766–2770.
Palacio A. and N. Duran-Vila, 1999. Single-strand confor-
mation polymorphism (SSCP) analysis as a tool for vi-
roid characterization. Journal of Virological Methods
77(1), 27–36.
Rubio L., M.A. Ayllon, J. Guerri, H. Pappu, C. Niblett and
P. Moreno, 1996. Differentiation of citrus tristeza clos-
terovirus (CTV) isolates by single-strand conformation
polymorphism analysis of the coat protein gene. Annals
of Applied Biology 129, 479–489.
Schnell R.J., C.T. Olano and D.N. Kuhn, 2001. Detection of
avocado sunblotch viroid variants using fluorescent sin-
gle-strand conformation polymorphism analysis. Elec-
trophoresis 22(3), 427–432.
Sheffield V.C., J.S. Beck, A.E. Kwitek,  D.W Sandstrom  and
E.M. Stone, 1993. The sensitivity of single-strand con-
formation polymorphism analysis for the detection of
single base substitutions. Genomics 16, 325–332.
Thompson J.D., D.G. Higgins and T.J. Gibson, 1994. CLUS-
TAL W: improving the sensitivity of progressive multi-
ple sequence alignment through sequence weighting,
position specific gap penalties and weight matrix choice.
Nucleic Acids Research 22(22), 4673–4680.
Yap E.P.H., Y-M.D. Lo, K. Cooper, K.A. Fleming and J.O.D.
McGee, 1992. Exclusion of false-positive PCR viral di-
agnosis by single-strand conformation polymorphism.
The Lancet 340, 736.


Variability of a portion of RNA 3 containing the coat protein gene of Citrus variegation virus (CVV) using single-strand conformation polymorphism (SSCP)

B. Bennani

G. Nolasco

H. Azeddoug

M. Zemzami

Directory of Open Access Journals

188 Phytopathologia MediterraneaPhytopathol. Mediterr. (2002) 41, 188–192IntroductionCitrus variegation virus (CVV) is a widespreadvirus (Bennani et al., 2002) that has not been muchstudied. It is a member of the ilarvirus group ofthe Bromoviridae and has a tripartite single-stranded RNA genome. Infected citrus trees exhibita range of symptoms, which are usually mild onoranges and mandarins but may be severe on cit-ron and lemon trees; on these yield reduction andfruit malformation may also occur. Two types ofVariability of a portion of RNA 3 containing the coat protein geneof Citrus variegation virus (CVV) using single-strandconformation polymorphism (SSCP)BAHIA BENNANI1, 2, MUSTAPHA ZEMZAMI2, GUSTAVO NOLASCO3 and HOUSSINE AZEDDOUG11Université Hassan II, Faculté des Sciences Aïn Chock, Laboratoire de biochimie,B.P.  Maârif, 20100 Casablanca Morocco2Direction des Domaines Agricoles, Domaine UCP Maamora, Km 11, Route P.le No. 1 Hussaïne Salé 10000, Morocco3Universidade do Algarve/ FERN, 8000 Faro, PortugalSummary. A portion of RNA 3 of Citrus variegation virus (CVV), comprising part of the intergenic region and thecoat protein (CP) gene from eight viral isolates, was amplified by RT-PCR and cloned. The clones were compared forintra and inter-isolate variations by single-strand conformation polymorphism analysis. Some of the results werecompared with sequence data previously obtained. The test discriminated between clones differing in as little as3.2% of the nucleotides. Most isolates included several variants, in some cases with a predominant pattern, which,however, could no longer be recognised in new RT-PCR products obtained 13 months later. This procedure can there-fore be used to identify and detect variations between CVV isolates. It is rapid, inexpensive and may reduce theamount of sequencing needed for comparing viral isolates.Key words: Citrus viruses, infectious variegation-crinkly leaf, molecular variability, RT-PCR, SSCP.Corresponding author: H. AzeddougE-mail: h_azeddoug@hotmail.comFax: +212 22 230674symptoms, attributed to different strains of thevirus are commonly distinguished: infectious vari-egation, characterised by crinkling of the leavesassociated with chlorosis, and crinkly leaf, whichincludes warping, pocketing and crinkling but with-out variegation or reduction in leaf size (Desjardinand Bové, 1980). Detection and characterisationof CVV in the field is by ELISA and more recently,at the molecular level, by RT-PCR and sequencing(Bennani et al., 2002). In this study, the coat pro-tein gene and part of the intergenic region werecloned and sequenced to compare several CVV iso-lates with different geographic origin and biologi-cal properties. Nucleotide sequencing is the mostaccurate procedure for strain identification, but itis too expensive and time consuming for routine189Vol. 41, No. 3, December 2002Molecular variability in Citrus variegation viruspurposes. Single-strand conformational polymor-phism (SSCP) analysis (Orita et al., 1989) appearsan attractive alternative due to its suitability andhas been used to assess the variability of severalviruses. In SSCP analysis, double-stranded DNAis heat-denatured in the presence of formamide andboth DNA strands are separated by electrophore-sis in non-denaturing polyacrylamide gel. Underthese conditions, part of the single-stranded DNAacquires a globular conformation that depends onthe existence of self-complementary regions. Theoccurrence of mutations in these regions stronglyaffects the single-strand conformation, resulting inelectrophoretic mobility shifts.SSCP has been used for different purposes, suchas: evaluating sequence heterogeneity among iso-lates of hepatitis B (Yap et al., 1992) and C (Enomo-to et al., 1994) viruses; assigning beet necrotic yel-low vein virus isolates to a known strain group(Koenig et al., 1995); differentiating CTV isolateson the basis of their genetic variability (Rubio etal., 1996; Ayllon et al., 1999); studying the popula-tion structure of CTV (Kong et al., 2000); and char-acterising different viroids (Palacio and Duran-Vila, 1999). Recently, fluorescent SSCP has alsobeen used to detect variants of avocado sunblotchviroid (Schnell et al., 2001).In this paper SSCP was used to study intra andinter-isolate variability of CVV and results werecompared with data recently obtained.Materials and methodsVirus isolatesVirus isolates from different geographical are-as were those used in previous studies (Bennani etal., 2002) and were maintained in citrus seedlingsgrown in insect-proof greenhouses. They included:isolates CVV-1 (Garnsey et al., 1968) and CVV-2(Garnsey et al., 1984), obtained from USDA, ARS,Orlando FL, USA; CVV-E1234, characterised byGrant (Desjardins and Bové, 1980), obtained fromthe Station de San Giuliano, Corsica, France; CVV-IV400 from IVIA, Spain (Duran-Vila et al., 1988);CVV-81A65 from Corsica; CVV-CL903 from Spain(Desjardins and Bové, 1980); CVV-3 from a fieldnear Marrakech, Marocco; and CVV-ES86 obtainedfrom a citrus repository in Portugal (Bennani etal., 2002). Virus-free citrus was included as ahealthy control.cDNA synthesis, RNA amplification and cloningTotal nucleic acid of each isolate was extractedfrom 0.2 g of fresh bark tissue by a lithium-chlo-ride-based protocol as described in an earlier study(Bennani et al., 2002). CVVa and CVVb primers(Bennani et al., 2002), designed from conservedregions of ilarvirus subgroup 2 (except Citrus leafrugose virus (CiLRV)) were used to amplify the full-length coat protein (CP) gene and 63 nt of the inter-genic region upstream of this gene. Amplificationwas carried out with 2 µl of RNA and RT-PCR mix-es (25 µl final volume) containing 10 mM Tris (pH8.8), 50 mM KCl, 2 mM MgCl2, 400 nM each of theprimers, 0.4 mM of each dNTP, 0.08% NP-40, 0.5UTaq DNA polymerase (MBI-Fermentas, Vilnius,Lithuania), 5U RNAguard (Amersham Pharmacia,Buckingamshire, UK) and 6U MuLV reverse tran-scriptase (Perkin Elmer, Wellesley, MA, USA). Theamplification conditions were: 30 min at 39°C, 2min at 94°C, followed by 35 cycles at 92°C for 30s,56°C for 45s and 72°C for 1 min and finally 5 minat 72°C. The amplified products were separatedby 1% agarose gel electrophoresis. RT-PCR prod-ucts of the expected size were cloned into theEcoRV-cut PGEMT-Easy vector system I (Prome-ga, Madison, WI, USA) and transformed into E.coli INVα F’ cells (Invitrogen, S. Giuliano Milanese,Italy). For all isolates, positive clones were identi-fied by direct PCR amplification from the whitecolonies using primers CVVa/CVVb (Bennani et al.,2002). The plasmid DNA was purified using a Wiz-ard plus SV miniprep Kit (Promega) according tomanufacturer’s instructions. The identity of theinsert was confirmed by digestion with EcoRI.SSCP analysisFifty-one clones of 63 nt intergenic region up-stream of the CP gene, and the entire CP gene ob-tained from eight isolates were compared by SSCP.SSCP analysis was done on PCR products am-plified from recombinant E. coli colonies or, in someexperiments, directly from RT-PCR products. ForSSCP analysis, 1 to 3 µl of the amplified productswas mixed with 10 µl of denaturing buffer (19.6mM EDTA, pH 8, in formamide containing a min-imal amount of bromophenol blue), denatured for5 min at 90°C, and loaded on a non-denaturing 8%polyacrylamyde gel. After running for 3 h at 200 V,4°C, the gels were stained with silver nitrate (Ori-ta et al., 1989).190 Phytopathologia MediterraneaB. Bennani et al.Sequence comparisonThe sequences compared were those of clonesCVV-1-U, CVV-2-L, CVV-3-O, CVV-E1234-11, CVV-E1234-13, CVV-IV400-12, CVV-IV400-13, CVV-81A65-12, CVV-81A65-27, CVV-CL903-15, CVV-CL903-21 and CVV-ES86, with accession numbersAF434918, AF434917, AF434916, AF434915,AF434914, AF434922, AF434921, AF434911,AF434912, AF434920, AF434919 and AF434913respectively.ResultsSSCP analysis of the ampliconAmplification from the plasmids and from RNAisolates in all cases yielded a single fragment of718 bp, encompassing the CP gene and 63 nt of theintergenic region upstream of this gene. Electro-phoresis used to compare the RT-PCR products ofdifferent isolates and the 51 clones by SSCP, asdescribed in Materials and methods, yielded veryreproducible patterns when repeated analysis wasdone (data not shown). All SSCP results are illus-trated in Fig. 1.The SSCP patterns obtained directly from RT-PCR products amplified with primers CVVa–CVVb(Fig. 1-I) differed between most isolates, exceptthose for ES86, E1234 and CVV3. Usually the pat-terns consisted of only two conspicuous bands, sug-gesting that the isolates were closely related vari-ants. However, when SSCP analysis was carriedout with the CVVa–CVVb PCR product amplifieddirectly from transformed E. coli colonies, someisolates consisted of more than one variant. Iso-lates 81A65 (Fig. 1F), CVV3 (Fig. 1C), CVV1(Fig.1A) and CVV2 (Fig. 1B) each produced a predomi-nant pattern that was common to the majority ofthat isolate’s clones. On the other hand, only about50% of the clones from isolates IV400 (Fig. 1D) andCL903 (Fig. 1G) yielded similar patterns. The pat-terns of clones IV400-13, CVV1-U and ES86 werevery similar among each other (Fig. 1H).Fig. 1. SSCP analysis of PCR and RT-PCR products obtained with primers CVVa and CVVb. Panels A to G, directPCR on isolates CVV-1, CVV-2, CVV-3, CVV-IV400, CVV-E1234, CVV-81A65 and CVV-CL903 respectively, carriedout with E.coli colonies harbouring the RT-PCR product. Sequenced clones are presented in lanes A6 (CVV-1-U), B7(CVV-2-L), C4 (CVV-3-O), D1 (IV400-12), D2 (IV400-13), E1 (E1234-11), E2 (E1234-13), F1 (81A65-12), F8 (81A65-27), G5 (CL903-15) and G6 (CL903-21). Panel H, side-by-side comparison of SSCP patterns from some sequencedclones: CVV-81A65-12, CVV-IV400-13, CVV-IV400-12, CVV-ES86, CVV-CL903-15, CVV1-U, CVV2-L, CVV-E1234-13 and CVV-E1234-11 (lanes 1 to 9 respectively). Panel I, SSCP patterns of RT-PCR products obtained from plantsinfected with isolates CVV-IV400, CVV-E1234, CVV-81A65, CVV-ES86, CVV-2, CVV-1, and CVV-CL903 (lanes 1 to 7respectively).191Vol. 41, No. 3, December 2002Molecular variability in Citrus variegation virusNucleotide differences between clonesNucleotide sequence homology of clones previ-ously sequenced (Bennani et al., 2002) and ob-tained with the program Clustal (Thompson et al.,1994) is summarised in Table 1. Differences in thenucleotide sequence were not always correlatedwith differences in electrophoretic mobility. Whensequences of the very similar clones IV400-13,CVV1-U and ES86 were compared, they differedfrom each other by 14 to 22 bp. All clones differ-ing by more than 23 bp yielded different patterns.Conversely, in the case of clones that differed onlyfor 3 nt different patterns were produced byIV400-12 and E1234-11, and by CVV3 and IV400-13, and identical patterns by 81A65-12 and 81A65-27. In summary, any two amplicons producingdifferent patterns differed by at least 3 bp (0.4%),while amplicons with similar patterns always dif-fered by less than 23 bp (3.2%).DiscussionSSCP analysis of several clones of each CVVisolate showed that some of the isolates (e.g.CVV1, CVV2, CVV3, 81A65) consisted of one pre-dominant variant. In these cases the pattern ofthe predominant variant was recognised in theSSCP pattern of the RT-PCR products from whichthe clones were obtained. To have an idea of theaccuracy of this indication, sequences of twoclones from 81A65 having the same pattern werecompared and a total of 3 nucleotide differenceswere found. This suggests that SSCP gives a fairapproximation of intra-isolate variability. On theother hand, patterns from clones obtained fromisolates CL903, E1234 and IV400 produced dif-ferent patterns, suggesting that each of these iso-lates consisted of several variants, typical of aviral quasi-species. However, in these cases therewas no straightforward correspondence betweenthe SSCP patterns of the clones and the sequenceof the RT-PCR products which yielded them. Itwas expected that the SSCP pattern of the RT-PCR product would be similar to the most fre-quent pattern of the clones or would be the sameas the individual patterns, but this was not thecase. SSCP analysis also did not reveal any par-ticular feature of the RT-PCR patterns that couldbe associated with either infectious variegationor crinkly leaf symptoms. Although conditionswere far from those usually considered ideal forSSCP, in that short PCR products (Sheffield etal., 1993) were used, it was possible to discrimi-nate among clones differing by at least 3.2% oftheir nucleotide sequence. Other Authors (Rubioet al., 1996) using products of approximately thesame length, were able to distinguish clones dif-fering by 1.4%. Field isolates of CVV consist of alarge number of CP gene variants. Under theconditions used, SSCP of RT-PCR products ap-peared not to be adequate to reveal the sequencevariants that compose a particular isolate. TheSSCP of individual clones should be used for thatpurpose.Table 1. Homology percentage and number of nucleotide differences (in bold type) among 12 clones in the regionconsidered. U17389 was obtained from GenBank.       Seq U17389 IV400-12 IV400-13 CVV1-U CVV2-L CVV3-O E1234-11 E1234-13 CL903-15 CL903-21 ES86 81A65-27 81A65-12U17389 100 98.3 98.3 99.7 97.4 98.1 97.9 97.7 95.6 97.9 97.2 94.7 94.5IV400-12 11 100 98.3 98 97.4 98.1 99.5 97.7 95.1 97.9 98.6 94.1 94.0IV400-13 12 12 100 98 99.1 99.5 97.9 99.1 95.4 99.3 97.4 94.4 94.2CVV1-U 2 14 14 100 97.2 97.9 97.6 97.4 95.4 97.6 96.9 94.4 94.2CVV2-L 18 17 7 19 100 98.7 97 98.6 95.1 99 96.6 94 93.8CVV3-O 13 12 3 15 10 100 97.7 99 95.2 99.1 97 94.2 94.1E1234-11 14 3 15 16 21 16 100 97.3 94.7 97.4 98.7 94 93.8E1234-13 16 16 4 18 10 7 19 100 95.9 99 96.6 94.9 94.8CL903-15 31 33 31 33 35 32 38 29 100 95.2 94.2 98 97.9CL903-21 15 15 5 17 7 6 18 7 33 100 96.7 94.2 94.1ES86 22 10 18 22 24 21 9 24 41 23 100 93.5 93.481A65-27 38 42 32 40 43 40 43 36 14 41 46 100 99.581A65-12 39 43 41 42 44 42 43 36 14 41 47 3 100192 Phytopathologia MediterraneaB. Bennani et al.AcknowledgementsThis research was in part supported by a grantPRAXIS XXI-3/3.3/HORT/2159-95 provided by FCT(Portugal) and by bi-lateral scientific and techno-logical cooperation between ICCTI (Portugal) andCNCPRST (Morocco).Literature citedAyllon M.A., L. Rubio, A. Moya, J. Guerri and P. Moreno,1999. The haplotype distribution of two genes of citrustristeza virus is altered after host change or aphid trans-mission. Virology 255(1), 32–39.Bennani B., C. Mendes, M. Zemzami, H. Azeddoug and G.Nolasco, 2002. Citrus variegation virus: Molecular var-iability of a portion of the RNA 3 containing the coatprotein gene and design of primers for RT-PCR detec-tion. European Journal of Plant Pathology 108(2), 155–162.Desjardins P.R. and J.M. Bové, 1980. Infectious variega-tion and crinkly leaf. In: Description and Illustration ofVirus and Virus-like Diseases of Citrus. A collection ofcolour slides. Proceedings of the International Organi-sation of Citrus Virologists (J.M. Bové, R. Vogel, ed.),Vol. 2, IRFA, 1–9.Duran-Vila N., M. Cambra, J.A. Pina, J.F. Ballester and L.Navarro, 1988. Virus content and growth patterns ofcallus cultured in vitro from healthy and virus infectedCitrus species. Proceedings of the International Organ-isation of Citrus Virologists 9, 310–313.Enomoto N., M. Kurosaki, Y. Tanaka, F. Marumo  and C.Sato, 1994. Fluctuation of hepatitis C virus quasispe-cies in persistent infection and interferon treatmentrevealed by single-strand conformation polymorphismanalysis. Journal of General Virology 75, 1361–1369.Garnsey S.M., 1968. Additional non-citrus hosts for theFlorida isolate for citrus variegation virus. Phytopathol-ogy 58, 1433–1434.Garnsey S.M., N. Baksh, M. Davio and J.P. Agostini, 1984.A mild isolate of Citrus Variegation Virus found in Flor-ida Citrus. Proceedings of the International Organisa-Accepted for publication: August 27, 2002tion of Citrus Virologists 9, 188–195.Hall T.C., 1979. Transfer RNA-like structures in viral ge-nomes. International Review of Cytology 60, 1–26.Koenig R., P. Lüddecke and A.M. Haeberle, 1995. Detec-tion of beet necrotic yellow vein virus strains, variantsand mixed infections by examining single-strand con-formation polymorphisms of immunocapture RT-PCRproducts. Journal of General Virology 76, 2051–2055.Kong P., L. Rubio, M. Polek and  B.W. Falk, 2000. Popula-tion structure and genetic diversity within CaliforniaCitrus tristeza virus (CTV) isolates. Virus Genes 21(3),139–145.Orita M., H. Iwahana, K. Kanazawa and T. Sekia, 1989.Detection of polymorphism of human DNA by gel elec-trophoresis as single strand conformation polymor-phism, Proceedings of the National Academy of Scienc-es of the USA 86, 2766–2770.Palacio A. and N. Duran-Vila, 1999. Single-strand confor-mation polymorphism (SSCP) analysis as a tool for vi-roid characterization. Journal of Virological Methods77(1), 27–36.Rubio L., M.A. Ayllon, J. Guerri, H. Pappu, C. Niblett andP. Moreno, 1996. Differentiation of citrus tristeza clos-terovirus (CTV) isolates by single-strand conformationpolymorphism analysis of the coat protein gene. Annalsof Applied Biology 129, 479–489.Schnell R.J., C.T. Olano and D.N. Kuhn, 2001. Detection ofavocado sunblotch viroid variants using fluorescent sin-gle-strand conformation polymorphism analysis. Elec-trophoresis 22(3), 427–432.Sheffield V.C., J.S. Beck, A.E. Kwitek,  D.W Sandstrom  andE.M. Stone, 1993. The sensitivity of single-strand con-formation polymorphism analysis for the detection ofsingle base substitutions. Genomics 16, 325–332.Thompson J.D., D.G. Higgins and T.J. Gibson, 1994. CLUS-TAL W: improving the sensitivity of progressive multi-ple sequence alignment through sequence weighting,position specific gap penalties and weight matrix choice.Nucleic Acids Research 22(22), 4673–4680.Yap E.P.H., Y-M.D. Lo, K. Cooper, K.A. Fleming and J.O.D.McGee, 1992. Exclusion of false-positive PCR viral di-agnosis by single-strand conformation polymorphism.The Lancet 340, 736.

Sapientia

Universidade do Algarve

http://www.fupress.net/index.php/pm/article/download/1679/1614

Variability of a portion of RNA 3 containing the coat protein gene of Citrus variegation virus (CVV) using single-strand conformation polymorphism (SSCP)

Abstract

Similar works

Full text

Available Versions

Firenze University Press: E-Journals

Directory of Open Access Journals

Sapientia

Universidade do Algarve