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1473
Journal of General Virology (1991), 72, 1473-1477. Printed in Great Britain
Use of group-specific primers and the polymerase chain reaction for the
detection and identification of luteoviruses
Nancy L. Robertson, 1 Roy French 1. and Stewart M. Gray 2
1United States Department of Agriculture-Agricultural Research Service, Department of Plant Pathology, University of
Nebraska, Lincoln, Nebraska 68583 and 2United States Department of Agriculture-Agricultural Research Service,
Department of Plant Pathology, Cornell University, Ithaca, New York 14853, U.S.A.
A general diagnostic assay for a number of distinct
luteoviruses was developed using the polymerase chain
reaction (PCR) and restriction enzyme analysis. Two
minimally degenerate, group-specific primers were
derived from previously published RNA sequences of
three luteoviruses. This primer pair generated specific
PCR fragments of about 530 bp from extracts of plants
infected with potato leafroll virus, beet western yellows
virus, or New York barley yellow dwarf virus (BYDV)
serotypes MAV, PAV, RMV, RPV and SGV, which
span much of the respective viral coat protein gene.
Each virus was easily distinguished from the others by
restriction enzyme analysis of the amplified DNA
products. Samples from BYDV-infected oat and wheat
collected in Nebraska were identified as containing
PAV-like serotypes; micro-heterogeneity was detected
in several samples. This method provides a rapid,
sensitive and relatively inexpensive means of luteovirus
detection and identification. It is the first test capable of
simultaneously detecting all five BYDV serotypes.
Despite the fact that the aphid-transmitted luteoviruses
have long been known to cause destructive diseases in
numerous crops world-wide, these viruses still flourish in
their respective plant hosts. For example, the most
widely distributed virus disease of the Gramineae is
caused by five serotypes of barley yellow dwarf virus
(BYDV), designated MAV, PAV, RMV, RPV and SGV
(Casper, 1988). Numerous strains of beet western yellows
virus (BWYV) infect a wide range of economically
important crop plants and potato leafroll virus (PLRV)
occurs wherever potatoes are cultivated. Members of the
luteovirus group are serologically inter-related to varying
degrees and are subdivided into clusters on this basis
(Casper, 1988; Waterhouse et al., 1988). The biology of
luteoviruses complicates diagnosis and identification
because they are confined to phloem tissue, occur in low
concentrations and are not mechanically transmissible.
Luteovirus identification requires aphid transmission
studies (Casper, 1988; Waterhouse et al., 1988), serological assays with several polyclonal and/or monoclonal
antisera to the coat protein (Rochow & Carmichael,
1979; Diaco et al., 1986; D'Arcy et al., 1989; Forde,
1989; Martin & D'Arcy, 1990), or hybridization studies
with cDNA probes (Waterhouse et al., 1986; Habili et
aL, 1987; Arundel et aL, 1988; Falk et al., 1989; Fattouh
et al., 1990; Martin & D'Arcy, 1990). However, such
assays are limited in that several tests are required for
virus identification and they are either time-consuming
or use reagents (antisera or cDNA clones) which are
expensive to produce and are often not widely available.
These assays often yield inconclusive results and may not
detect luteovirus infection. Animal virologists have
recently used the polymerase chain reaction (PCR)
(Saiki et al., 1986, 1988) to detect viruses; the PCR
amplifies a specific genomic DNA fragment (Ou et al.,
1988) which is then analysed using restriction enzymes
(Torgersen et al., 1989). Here, we present a similar assay
for the detection and identification of a range of distinct
luteoviruses using appropriate group-specific primers.
The viruses included in this investigation fall into the
following clusters (Casper, 1988): (i) BWYV, BYDVRMV and BYDV-RPV, (ii) BYDV-MAV, BYDV-PAV
and BYDV-SGV and (iii) PLRV.
A number of group-specific primers were designed by
comparing the deduced amino acid sequences of the
conserved regions of the overlapping coat and 17K
proteins of PLRV, BWYV and BYDV-PAV. These
primers all amplified specific BYDV-PAV sequences (R.
French & N. L. Robertson, unpublished results), but a
primer pair amplifying the longest fragment was chosen
for this study. The upstream primer Lu 1 (5'
CCAGTGGTTRTGGTC Y), which is degenerate at
one position, corresponds to bases 2938 to 2952 of
BYDV-PAV (Miller et al., 1988), bases 3564 to 3578 of
BWYV (Veldt et al., 1988) and bases 3687 to 3701 of
PLRV (Van der Wilk et al., 1989). The downstream non-
0001-0088 © 1991 SGM
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degenerate primer Lu 4 (5' GTCTACCTATTTGG 3')
can pair with bases 3455 to 3468, 4084 to 4097 and 4207
to 4220 of BYDV-PAV, BWYV and PLRV, respectively.
The PCR products are predicted to be 531 bp long for
BYDV-PAV and 534 bp long for both BWYV and
PLRV.
Crude nucleic acid extracts were obtained from 0.5 g
leaf samples from oats (Arena byzantina cv. Coast Black)
infected with each of the five New York serotypes of
BYDV (MAV, PAV, RMV, RPV and SGV) by grinding
tissue with liquid nitrogen, adding 4 ml of buffer (0.1 Mglycine, pH 9.5, 0.1 M-NaC1, 10 mM-EDTA) and
emulsifying the extract with an equal volume of phenol.
After centrifugation, nucleic acids were precipitated
from the aqueous phase by addition of sodium acetate to
0.3 M and 2.5 vol. ethanol. The pellets were washed with
70% ethanol, vacuum-dried and resuspended in 100 lxl
distilled water or TE (10 mM-Tris-HCl pH 7.2, 1 mMEDTA). Healthy oat and wheat tissue, as well as
Nebraskan field samples of BYDV-infected wheat and
oat plants, and Physalis floridana infected with either
BWYV or PLRV, were all processed in the same manner
except that the PLRV RNA extraction procedure
contained an additional LiC1 precipitation step after
extraction with phenol. Lithium chloride (0.2 vol. ; 10 M)
was added to the aqueous phase and the solution was
incubated overnight at 4 °C. After centrifugation, the
resulting pellet was resuspended in 300 ~tl TE and
ethanol-precipitated as described above.
cDNA was synthesized by heating 1 ~tl nucleic acid
extract with 10 pmol primer Lu 4 in H20 (total volume
12.5 ttl) for 5 min at 95 °C, incubating at 40 °C for 10 min
and chilling on ice. An equal volume of 2 x reaction
buffer, containing either 0.5 units (U)Ad of avian
myeloblastosis virus (Boehringer Mannheim) or 10 UAd
of Moloney murine leukaemia virus (BRL) reverse
transcriptase, 4 mM each of dATP, dCTP, dGTP and
TTP, 100 mM-Tris-HCl pH 8.0, 10 mM-2- mercaptoethanol, 20 mm-MgCl2 and 140 mM-KCI, was then added
before incubating the reactions at 40 °C for 60 min. The
volume of the mixtures was increased to 50 ~tl with H20
after which they were boiled for 10 min.
DNA amplification was carried out in a 100 ~tl
reaction using 5 ttl of the cDNA preparation, 5 pmol
each of Lu 1 and Lu 4, 2.5 U Taq DNA polymerase
(Cetus) in reaction buffer provided with the enzyme
(10 x PCR buffer contains 100 mM-Tris-HC1 pH 8.3 at
25 °C, 500 mM-KCI, 15 mM-MgC12 and 0.01% w/v
gelatin) and 0.2 mM of each dNTP. Samples were overlaid
with 100 ~tl mineral oil and placed in a Perkin Elmer
Cetus thermal cycler programmed to give one cycle at
95 °C (1 min), 41 °C (2 min) and 72 °C (20 min), followed
by 40 cycles at 94 °C (1 min), 41 °C (1 min), 41 °C to
72 °C (3 min) and 72 °C (2 min), with a final cycle of
1
2
3
4
5
6
7
8
9
10
bp
625 - 492-369-246--
123-Fig. 1. Polyacrylamidegel electrophoresisof PCR products. Lanes 1
and 10, DNA sizemarkers(BRL);lane2, healthycontrol;lanes3 to 9,
PLRV, BWYV,and BYDVserotypesSGV, RPV, RMV, MAV and
PAV,
1
2
3
4
5
6
7
8
9
10
bp
-- 692
-- 585
-- 341
-- 263/258
--141
- - 105
--78/75
--46
--36
Fig. 2. Restrictionfragmentlength polymorphismof PCR products
fromluteoviruses.Sau3AI digestionswereanalysedon polyacrylamide
gels. Lanes 1 to 8, healthy control, PLRV, BWYV, and BYDV
serotypesSGV,RPV, RMV, MAVand PAV.Lane9, uncutPAVPCR
product. Lane 10, Sau3AI-cut pUC19 DNA size markers.
72 °C (10 min). The PCR products were visualized by
electrophoresis of 5 ~tl of the PCR reaction product on
10% polyacrylamide gels followed by staining with 0-15
~tg/ml ethidium bromide for 20 min. A major PCR
product of about 530 bp was present in samples from all
the luteoviruses, but not from healthy controls (Fig. 1) or
from plants infected with barley stripe mosaic virus or
brome mosaic virus (not shown). These results show that
primers Lu 1 and Lu 4 can specifically amplify sequences
from each of the luteoviruses tested.
To characterize the amplified DNAs from each of the
viruses, 5 ~tl aliquots of the crude PCR reaction products
were digested with Sau3AI and analysed on 10%
polyacrylamide gels. All five New York BYDV serotypes, PLRV and BWYV produced distinctive patterns
(Fig. 2). The BYDV serotype MAV and PAV, and
PLRV profiles were identical to those predicted from
published sequences (Rizzo & Gray, 1990; Miller et al.,
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1
bp
2
3
4
5
6
7
8
9
Probe
1
2
3
4
5
6
7
8
9
1475
10
11
MAV
692-585 - -
PAV
341 -263/258- -
RMV
141 - 105--
RPV
78/75--
SGV
46-36--
Fig. 3. Sau3AI restriction analysis of PCR products from BYDV field
samples. Lanes 1 to 3, Sau3AI-cut pUC19 DNA markers, and uncut
and digested PAV PCR product. Lanes 4 to 8, digested PCR product
from BYDV field isolates; lane 9, digested PCR product from healthy
oat leaves.
1988; Mayo et al., 1989; Van der Wilk et al., 1989; Keese
et al., 1990) even though the sequenced PAV-like isolate
and the P L R V isolates have different geographical
origins from the ones used here. The pattern obtained
with c D N A to a Nebraska BWYV isolate differed from
that predicted from the sequence of a European isolate
(Veidt et al., 1988). This is not surprising because unlike
various BYDV PAV-like isolates or P L R V isolates,
among which no serological differences have been
documented, great serological variability has been found
among BWYV isolates (Casper, 1988). The nucleotide
sequences of BYDV serotypes RPV, RMV and SGV
have not been published, but we found each of them to be
unique in this region, at least in terms of S a u 3 A I sites
(Fig. 2) as well as CviJI (Xia et al., 1987), HaelII, and
Hinfl sites (not shown). The S a u 3 A I patterns of most
Nebraska BYDV isolates were identical to that of BYDV
serotype PAV (Fig. 3), but several gave either a unique
pattern (e.g. isolate 2 in Fig. 3) or one that appeared to be
a mixture of the new and BYDV serotype PAV patterns
(e.g. isolate 5 in Fig. 3).
Typically, the background of host-specific P C R
products was more obvious in samples from luteovirusfree plants than in samples from luteovirus-infected
plants. This is particularly striking with the P C R
products shown in Fig. 2. The reasons for this are not
clear, but may be related to differences in rates of primer
depletion in reactions containing luteovirus c D N A
target molecules compared to those in reactions not
containing specific targets.
We were also interested in the degree of nucleic acid
similarity among the BYDV serotypes, based upon
hybridization of the 530 bp P C R products. To generate
Fig. 4. Southern blot of 530 bp PCR product from BYDV serotypes,
healthy oat control and BYDV-infectedfield isolates. The concentration of the PCR product differedamong the BYDV samples, but each
of the five blots contained an identical amount of any given sample.
Blots probed with RMV and SGV were exposedfive times as long as
other blots. Lanes 1 to 5, BYDV serotypes MAV, PAV, RMV, RPV
and SGV; lane 6, healthy plant control; lanes 7 to 11, field isolates
lto5.
radioactive probes (Schowalter & Sommer, 1989) that
included only the approximately 530 bp fragment for
each of the BYDV serotypes, 30 to 50 ~tl of each P C R
reaction was electrophoresed in a 2 ~ low melting point
agarose gel (Sea Plaque; F M C BioProducts), the D N A
band was cut out and purified (Maniatis et al., 1982), and
the dried pellet was resuspended in 20 ~tl TE. A 20 ~tl
P C R mix included 5 p.1 of the gel-purified DNA, 50 p.Ci
[ct-3zp]dCTP (3000 Ci/mmol), 0-2 mM each of dATP,
d G T P , and TTP, 5 pmol primers Lu 1 and Lu 4, 1 U Taq
D N A polymerase and P C R buffer (Cetus). The temperature/time regime was as previously described except that
the first 20 min annealing step was omitted and the
number of cycles was reduced to 30. After D N A
amplification, the layer of oil was removed by emulsifying with chloroform followed by brief centrifugation.
The radioactive probe was isolated by placing the
aqueous phase in a Millipore filter (10000 N M W L Filter
Unit), adding 100 ~tl distilled water and centrifuging at
6000 r.p.m, until 10 to 50 Ixl remained on the filter. The
cycle was repeated twice more to remove small fragments
and free [~-3"-P]dCTP. The remaining 10 ~tl of concentrated probe solution was then diluted with 100 ~tl
distilled water. Initially, 10 to 20 Ill of unpurified PCR
product of each of the five BYDV serotypes and five
BYDV field isolates was run on a 2 ~ agarose gel, stained
to ascertain the presence of the 530 bp band and then
blotted to Zeta-Probe blotting membranes according to
the supplier's instructions (Bio-Rad). Nucleic acids were
hybridized using 25 pl of the radioactive probe preparations under high stringency (0.5 M-NaH2PO4, 1 mMEDTA, 7~o SDS at 65 °C; washes in 40 mM-NaHzPO4 at
65 °C).
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As expected, Southern blots revealed that each of the
530 bp P C R products of the N Y - B Y D V serotypes
hybridized most strongly to itself (Fig: 4). BYDV
serotypes PAV, MAV, SGV and R P V showed limited
cross-hybridization with at least one other serotype. Only
D N A derived from serotype R M V did not crosshybridize with P C R products from any of the other
serotypes. Samples of the field isolates hybridized
strongly with both the serotype M A V and P A V probes,
but altogether their hybridization behaviour was most
similar to that of serotype PAV samples. Probes derived
from serotypes SGV and RPV also hybridized weakly to
samples of serotypes M A V and PAV, and all field
isolates. These results agree with the current placement
of serotypes MAV, PAV and SGV into one cluster, but
do not reveal any similarities between serotypes R M V
and RPV. Other studies have not found any relationship
between R M V and other BYDV serotypes (D'Arcy et al.,
1989; Fattouh et al., 1990).
Luteovirus group-specific primers were derived from
sequences conserved among sequenced isolates of
BYDV-PAV, BWYV and PLRV. Despite their relatively small size, Lu 1 (a 15-mer) and Lu 4 (a 14-met)
amplified relatively low levels of host-specific P C R
products under our reaction conditions. The primer pair
did, however, specifically amplify a 530 bp D N A
fragment from c D N A of total nucleic acid extracts from
plants infected with each of five N Y - B Y D V serotypes,
or several Nebraska BYDV, BWYV or P L R V isolates.
All seven viruses examined here could be differentiated
on the basis of restriction fragment length polymorphisms. Differences were found between some of the
BYDV field isolates which probably would have been
overlooked by methods of diagnosis in current usage.
Current methods of detecting and identifying luteoviruses depend on multiple tests with aphid species, antisera
or c D N A probes. The use of the P C R with a single
luteovirus primer pair described here allows diagnosis of
five BYDV serotypes, and BWYV and PLRV, by one
rapid and simple, yet sensitive, assay. This technique
clearly takes advantage of both conserved and variable
portions of a genomic region. The D N A fragment
produced by the P C R includes much of the coat protein
gene, which encodes those properties that determine
antigenicity and perhaps aphid vector specificity. This
P C R method yields results which correlate well with
previous insect transmission and serological studies.
Owing to its simplicity, the P C R provides an attractive
alternative to other diagnostic techniques. The 530 bp
c D N A fragments can provide a ready source of initial
sequence information for luteoviruses from which
c D N A s have not yet been made and cloned. These
sequence data could then be used to design virus-specific
P C R primers if desired. The primers and procedure
described here will be useful in the diagnosis of
luteovirus diseases and in epidemiological studies.
We gratefully acknowledge Rose Skopp, Nucleic Acid Synthesis
Core Facility, Center for Biotechnology, University of NebraskaLincoln, U.S.A., for synthesis of the oligonucleotide primers used in
this study. This is a cooperative investigation of the ARS, USDA and
the Nebraska Agricultural Station. Paper no. 9431, Journal Series,
Nebraska Agricultural Experiment Station.
References
ARUNDEL,P. H., VAN DE VELDE, R. & HOLLOWAY,P. J. (1988).
Optimal conditions for the use of eDNA probes to measure the
concentration of barley yellow dwarf virus in barley (Hordeum
vulgate). Journal of Virological Methods 19, 307-318.
CA,SPEW,R. (1988). Luteoviruses. In The Plant Viruses: Polyhedral
Virions with Monopartite RNA Genomes, vol. 3, pp. 235-258. Edited
by R. Koenig. New York: Plenum Press.
D'ARcY,C. J., TORRANCE,L. & MARTIN,R. R. (1989). Discrimination
among luteoviruses and their strains by monoelonal antibodies and
identification of common epitopes. Phytopathology 79, 869-873.
DIACO, R., LISTER,R. M., HILL, J. H. & DURAND,D. P. (1986).
Demonstration of serologicalrelationships among isolates of barley
yellow dwarf virus by using polyclonaland monoelonal antibodies.
Journal of General Virology 67, 353-362.
FALK, B. W., CHIN, L.-S. & DUFFUS,J. E. (1989). Complementary
DNA cloning and hybridization analysis of beet western yellows
luteovirus RNAs. Journal of General Virology 70, 1301-1309.
FATrOUH, F. A., UENG, P. P., KAWATA,E. E., BARBARA,B. A.,
LARKINS,B. A. & LISTER,R. i . (1990). Luteovirus relationships
assessed by cDNA clones from barley yellow dwarf viruses.
Phytopathology gO, 913-920.
FORDE, S. M. D. (1989). Strain differentiation of barley yellow dwarf
virus isolates using specific monoclonal antibodies in immunosorbent electron microscopy. Journal of Virological Methods 23,
313-320.
HABILI,N., MCINNES,J. L. & SYMONS,R. H. (1987). Nonradioactive
photobiotin-labelled DNA probes for the routine diagnosisof barley
yellow dwarf virus. Journal of Virological Methods 16, 225-237.
KEESE, P., MARTIN,R. R., KAWCHUK,L. M., WATERHOUSE,P. M. &
GERLACH,W. L. (1990). N ucleotide sequencesof an Australian and a
Canadian isolate of potato leafroll luteovirus and their relationships
with two European isolates. Journal of General Virology 71,719-724.
MANIATIS,T., FRITSCH,E. F. & SAMBROOK,J. (1982). Molecular
Cloning: A Laboratory Manual. New York: Cold Spring Harbor
Laboratory.
MARTIN, R. R. & D'ARCY, C. J. (1990). Relationships among
luteoviruses based on nucleic acid hybridization and serological
studies. Intercirology 31, 23-30.
MAYO,M. A., ROBINSON,D. J., JOLLY,C. A. & HYMAN,L. (1989).
Nucleotide sequence of potato leafroll luteovirus RNA. Journal of
General Virology 70, 1037-1051.
MILLER, W. A., WATERHOUSE,P. M. & GERLACH,W. L. (1988).
Sequence and organization of barley yellow dwarf virus genomic
RNA, Nucleic Acids Research 16, 6097-6111.
Ou, C.-Y., KWOK,S., MITCHELL,S. W., MACK,D., SNINSKY,J., KREBS,
J., FEORINO,P., WARFIELD,D. & SCHOCHETMAN,G. (1988). DNA
amplification for direct detection of HIV-1 DNA of peripheral blood
mononuclear cells. Science 239, 295-297.
RIZZO,T. M. & GRAY,S. M. (1990). Cloningand sequenceanalysisof a
cDNA encoding the capsid protein of the MAV isolate of barley
yellow dwarf virus. Nucleic Acids Research 18, 4625.
ROCHOW,W. F.& CARMICHAEL,L. E. (1979). Specificityamongyellow
dwarf viruses in enzyme immunosorbent assays. Virology 95,
415-420.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 03 Aug 2017 19:29:35
Short communication
SAmL R. K., BUGAWAN,T. L., HORN, G. T., MULLIS,K. B. & EHRLICH,
H. A. (1986). Analysis of enzymatically amplified beta-globulin and
HLA-DQ alpha DNA with allele-specific oligonucleotide probes.
Nature, London 324, 163-165.
SAIKI,R. K., GELFAND,G. H., STOFFEL,S., SCHARF,S. J., HIGUCHI,R.,
HORN, G. T., MULLIS, K. B. & EHRLICH, H. A. (1988). Primer
directed enzymatic amplification of DNA with a thermostable DNA
polymerase. Science 239, 487-491.
SCHOWALTER, D. B. & SOMMER, S. S. (1989). The generation of
radiolabelled DNA and RNA probes with polymerase chain
reaction. Analytical Biochemistry 177, 90-94.
TORGERSEN, H., SKERN, T. & BLAAS, D. (1989). Typing of human
rhinoviruses based upon sequence variations in the 5' non-coding
region. Journal of General Virology 70, 3111-3116.
VAN DER WILK, F. HUISMAN,M. J., CORNELISSEN,B. J. C., HUTrlNGA,
H. & GOLDBACH,R. (1989). Nucleotide sequence and organization of
potato leafroll virus genomic RNA. FEBS Letters 245, 51-56.
1477
VEIDT, I., LOT, H., LEISER, M., SCHEIDECKER, D., GUILLEY, H.,
RICHARDS, K. & JONARD, G. (1988). Nucleotide sequence of beet
western yellows virus RNA. Nucleic Acids Research 16, 9917-9932.
WATERHOUSE, P. M., GERLACH, W. L. & MILLER, W. A. (1986).
Serotype-specific and general luteovirus probes from cloned eDNA
sequences of barley yellow dwarf virus. Journal of General Virology
67, 1273-1281.
WATERHOUSE, P. M., GILDOW, F. E. & JOHNSTONE, G. R. (1988).
Luteovirus group. CMI/AAB Descriptions of Plant Viruses, no. 339.
XIA, Y., BURBANK,D. E., UHER, L., RABUSSAY,D. & VAN ETrEN, J. L.
(1987). IL-3A virus infection of a Chorella-like green alga induces a
DNA restriction endonuclease with novel sequence specificity.
Nucleic Acids Research 15, 6075~o090.
(Received 14 December 1990; Accepted 5 March 1991)
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