J. gen. Virol. (1988), 69, 983-990. Printed in Great Britain
983
Key words: MAbs/ MS V/serological relationships
Characterization of Maize Streak Virus Isolates from Different Plant
Species by Polyclonal and Monoclonal Antibodies
By E. L. D E K K E R , 1. M. S. P I N N E R , 2 P. G. M A R K H A M 2 AND
M. H. V. V A N R E G E N M O R T E L 1
1Laboratoire d'Immunochimie, Institut de Biologie Mol~culaire et Cellulaire du CNRS,
15, rue Descartes, 67084 Strasbourg COdex, France and 2John Innes Institute, Colney Lane,
Norwich N R 4 7UH, U.K.
(Accepted 2 February 1988)
SUMMARY
Rabbit polyclonal antisera raised against six different isolates of maize streak virus
(MSV) were used to determine the serological relationships among a range of MSV
isolates from different plant species originating from various African countries and
from Vanuatu. Their reactions indicated a great serological diversity among these
isolates. Fifteen monoclonal antibodies (MAbs) raised against a Nigerian maize
isolate, MSV-M(N)M, were characterized according to their reactivity with the various
MSV isolates in indirect ELISA. All the MAbs reacted with the virus both in an
antigen-coated plate and in a double antibody sandwich ELISA. None of the MAbs
reacted with all isolates, and some isolates most distantly related to MSV-M(N)M
according to results obtained with polyclonal antisera were not recognized by any of the
MAbs. Some isolates which were shown to be closely related using the polyclonal
antisera could be distinguished by several of the MAbs. The results obtained both with
the polyclonal antisera and the MAbs suggested that several of the isolates were a
mixture of a number of different serotypes.
INTRODUCTION
Maize streak geminivirus (MSV) is an economically significant pathogen in maize, cereals
and sugar-cane throughout Africa (Damsteegt, 1983; Rose, 1978) and has also been isolated
from grasses such as Coix spp., Panicum spp., Paspalum spp. and Setaria spp. in Africa (Storey &
McClean, 1930; Rose, 1978), and from Digitaria in Vanuatu (Dollet et al., 1986). This virus has
twin quasi-icosahedral particles (18 × 30 nm) with a coat protein of Mr 26000. The genome is
composed of a single 2.7 kb single-stranded circular DNA (Mullineaux et al., 1984). It is
transmitted by leafhoppers of the genus Cicadulina (Rose, 1978; Van Rensburg, 1981).
MSV has been reported to be serologically unrelated to any of the other leafhoppertransmitted geminiviruses (Roberts et al., 1984). Serological relationships have been found
between many isolates of MSV from widely separated geographical locations and from different
host plants, using a polyclonal antiserum to a Nigerian isolate, M(N)M (Pinner et al., 1988).
In this paper we describe the production and use, in ELISA, of polyclonal antisera against
several MSV isolates to determine the degree of serological cross-reactivity between the
individual viruses. We also report the production of a panel of monoclonal antibodies (MAbs)
raised against the M(N)M isolate of MSV and their reactions with a range of MSV isolates from
different plant species and countries. A number of MAbs enabled the detection of antigenic
differences between some of the isolates which were not apparent using polyclonal antisera.
METHODS
Virus isolates. The isolates used in this study have been described fully in a previous paper (Pinner et al., 1988)
and the same isolate designation has been used here (Table 1). The code used for each isolate was composedof a
combinationof letters indicative of: the host in which the isolate was found, countryof origin (in parentheses), the
0000-8161 © 1988 SGM
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984
E. L. D E K K E R A N D O T H E R S
T a b l e 1. Host, origin a n d nomenclature o f isolates o f M S V
Isolate
Country
of origin
Received*
in host
Code
Maize
Maize
Maize
Maize
Maize
Maize
Maize
Maize
Maize
Maize
Setaria
Setaria
Panicum
Panicum
Panicum
Coix
Sugar-cane
Sugar-cane
Digitaria
Nigeria
Nigeria
Ghana
Kenya
Kenya
South Africa
Mauritius
Mauritius
Mauritius
Reunion
Burundi
Rwanda
Nigeria
Kenya
Kenya
Mauritius
Mauritius
South Africa
Vanuatu
Maize
Maize
Maize
Maize
Maize
Maize
Sugar-cane
Coix
Paspalum
Maize
Setaria
Setaria
Panicum
Panicum
Panicum
Sugar-cane
Sugar-cane
Sugar-cane
Digitaria
M(N)M
M(N)M-M 1
M(G)M
M(K)M-FI,F2
M(K)M-M99
M(SA)M
M(M)S
M(M)C
M(M)Ps
M(Re)M
St(B)St
St(R)St
P(N)P
P(K)P-M
P(K)P-B
C(M)S
S(M)S
S(SA)S
D(V)D
* The names of those who supplied the isolates are as given in Pinner et aL (1988).
host in which the isolate was originally received and the current experimental host, if other than host in which the
isolate was received. For example, M(N)M denotes a maize isolate (from Nigeria), currently in maize; M(M)CM
denotes a maize isolate (from Mauritius), received in Coix, currently in maize. The countries from which isolates
were obtained, with their code in parentheses, were: Burundi (B), Ghana (G), Kenya (K), Mauritius (M), Nigeria
(N), Reunion (Re), Rwanda (R), South Africa (SA) and Vanuatu (V). The maize isolate from Reunion, received in
maize, was obtained from M. Peterschmitt (MontpeUier, France) and was designated M(Re)M. When more than
one isolate was received from a country these were designated by a hyphenated strain number, e.g. M(K)M-M99.
The mild isolate M(N)M-MI was derived from the severe M(N)M isolate in 1984 by repeated transmission by
individual insects that had been fed on M(N)M-infected plants to healthy maize plants or, more frequently,
following repeated cycles of freezing and thawing of a virus [M(N)M] preparation. This isolate was semi-purified
from plants when it was first obtained in 1984 [M(N)M-M1 84], as well as from plants in 1987 after having been
passaged every 2 to 3 months by insects from maize to maize [M(N)M-M1 87] (Pinner et al., 1988).
Graminaceous plants used to maintain isolates, together with the abbreviations used in the isolate codes, were:
Zea mays, M; Coix laeryma-jobi L., C; Saceharum officinarum L. (sugar-cane), S; Panieum species, P; Paspalum
conjugatum Bergius, Ps; Digitaria sanguinalis, D; Setaria species, St.
All African isolates were leafhopper-transmitted to maize using Cicadulina mbila (Naud6) and were purified
both from their original host and from maize. The isolate of digitaria streak virus (Dollet et al., 1986) from
Vanuatu, D(V)D, received in D. sanguinalis, was not transmissible by any of the Cicadulina species available
(Pinner et aL, 1988) and was therefore purified from Digitaria only.
M(N)M, purified as described by Boulton et al., (1984), was used for immunization, testing of mouse antisera
and initial screening of hybridomas. For use in comparative ELISA, all isolates were semi-purified preparations
(Pinner et al., 1988) from maize and, where appropriate, from their original host.
Polyclonal antisera. Groups of isolates that appeared to be serologically very similar, on the basis of results
obtained using a polyclonal antiserum to M(N)M, were further analysed using antisera obtained from rabbits
immunized with semi-purified preparations of the isolates St(R)St, D(V)D, P(N)P and P(K)P-B.
Hybridomaproduction and selection. Three 6-week old BALB/c mice were immunized subcutaneously with 50 ~tg
purified M(N)M, emulsified in complete Freund's adjuvant, per mouse. This was followed by an intramuscular
and then an intraperitoneal injection with the same amount of virus emulsified in incomplete Freund's adjuvant at
2 week intervals. The two mice giving the best response in double antibody sandwich (DAS) and in antigen-coated
plate (ACP) ELISA were rested for 6 weeks and given two booster injections with 10 ixg virus in saline on days 4
and 3 before fusion. Cell fusion was performed using a modified version of the technique described by Fazekas de
St. Groth & Scheidegger (1980). Spleen cells were mixed with non-secreting PAI cells (Stocker et al., 1982) in a
1-2:1 ratio and co-pelleted. Fusion was carried out using 50% polyethylene glycol (PEG; Mr 4000; Merck). The
cells were then distributed into 24-well Falcon tissue culture plates preseeded with 1 ml HAT medium (RPMI 1640
supplemented with 10~ Foetal calf serum and HAT; Gibco) containing 5 x 104 macrophages/ml, at a
concentration of 5 x 105 cells per well. The plates were then incubated at 37 °C in a 7 ~ CO2 incubator. The next
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Serology o f maize streak virus
985
day, 1 ml of HAT medium was added to each well. Half of the culture medium was replaced with fresh HAT
medium on days 5, 8 and 11 after fusion. Screening of the culture medium for the presence of specific antibody was
started 14 to 19 days after fusion. Positive cultures were cloned by limiting dilution of about 0-5 cells/well in 96-well
Falcon plates, using 5 x 104 macrophages/well as feeder cells.
ELISA procedures. Wells containing growing hybridoma clones were screened for the presence of specific
antibodies to M(N)M in both a DAS and ACP indirect ELISA. The ACP ELISAs were performed by coating the
plates with M(N)M at l gg/ml in phosphate-buffered saline (PBS) pH 7.4. The DAS ELISA tests were carried out
using rabbit IgG raised against M(N)M as a first antibody, at 2 txg/ml in 0.05 M-carbonate buffer pH 9-6, followed
by incubation with the antigen at 1 ~tg/ml in PBS-T (PBS containing 0-05~ Tween 20). In both methods the
remaining binding sites on the plastic plates were then saturated by incubation with 1~ bovine serum albumin
(BSA) in PBS-T. PBS-T was used as the diluting buffer in the subsequent steps. Hybridoma culture supernatant
fluids were diluted 1:10 and a sheep anti-mouse IgG (H + L)-phosphatase conjugate (Biosys, France) was used as
the detecting antibody. The bound conjugate was detected by addition of the substrate, p-nitrophenyl phosphate in
0-1 M-diethanolamine buffer pH 9.8. A Titertek Multiskan photometer (Flow Laboratories) was used to measure
the A405' Absorbance values were considered positive if they exceeded that of the buffer control without virus by a
factor of 2.
Isotyping of MAbs was carried out by indirect ACP ELISA, with purified homologous virus-coated plates,
using subclass-specific rabbit anti-mouse antisera (Nordic, Tilburg, The Netherlands) and a goat anti-rabbit Igalkaline phosphatase conjugate.
Determination of SDl values between MSV isolates. The isolates were tested in parallel in an indirect ELISA
against a twofold dilution series of the individual rabbit antisera. The degree of serological cross-reactivity was
expressed by a serological differentiation index (SDI), calculated by comparing the antiserum dilutions that lead to
an absorbance value of 1.0 at 405 nm for the homologous and heterologous viruses (Jaegle & Van Regenmortel,
1985). SDI values were expressed as log2 titre differences and were reproducible within +0.5 log2 units. The
ELISA procedure used was as described above, substituting the sheep-anti mouse Ig-phosphatase conjugate with
a goat anti-rabbit Ig-phosphatase conjugate to detect bound antibody. Where possible, the average SDI values
were calculated from reciprocal ELISA experiments. Healthy plant sap and 1~ BSA were used as negative
controls.
Comparisons of MSV isolatesusing MAbs. Culture supernatants from all hybridoma clones were tested in parallel
against all MSV isolates semi-purified from maize and from their original host. The reactivity with the different
isolates was determined by indirect ACP ELISA for all MAbs by using twofold serial dilutions of the culture
supernatant. Replicate tests were performed to determine the dilution step required to obtain an absorbance
reading of 1.0 at 405 nm. When a MAb had to be used at a twofold lower dilution in order to obtain an absorbance
of 1.0, compared to When it was used against M(N)M, the isolate was considered to be serologically distinct.
Isolates considered to be serologically equal to M(N)M never varied by more than half a dilution step to obtain an
absorbance of 1-0, compared to when the MAb was used against M(N)M.
RESULTS
Serological cross-reactivities between M S V isolates
T h e individual polyclonal antisera reacted in E L I S A w i t h all isolates to v a r y i n g degrees.
T h e r e was no reaction with h e a l t h y plant sap nor w i t h the buffer control. T h e degree of a n t i g e n i c
relationship b e t w e e n different isolates, expressed as a v e r a g e S D I values for different pairs o f
M S V isolates, are presented in T a b l e 2.
Maize isolates
All m a i z e isolates, except for M ( N ) M - M 1 84 and M ( M ) C , were indistinguishable f r o m
M ( N ) M (as indicated by S D I values of 0), using the a n t i s e r u m to M ( N ) M . T h e m i l d isolate
purified in 1987, M ( N ) M - M 1 87, and one o f the M a u r i t i u s isolates purified f r o m maize,
M ( M ) C M , could not be distinguished f r o m M ( N ) M or the o t h e r m a i z e isolates. M ( N ) M - M 1 84
and M ( M ) C , h o w e v e r , were s h o w n to be only distantly related to M ( N ) M .
Setaria isolates
W h e n purified f r o m Setar&, the Setaria isolates St(B)St and St(R)St w e r e n o t distinguishable
by any of the antisera, either f r o m e a c h o t h e r or f r o m m a i z e isolates. H o w e v e r , w h e n these
isolates were purified f r o m m a i z e [St(B)StM and St(R)StM] t h e y were distinct b o t h f r o m e a c h
other and f r o m St(B)St, St(R)St and M ( N ) M .
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986
E. L . D E K K E R
AND
OTHERS
Table 2. Serological differenth~tion indicesfor pairs of MSV isolates determined using ant&era to
M(N)M, St(R)St, D(IOD, P(K)P-B and P(N)P
Isolate
<
~
~
~
:~
'~
~
~
Antiserum
~
~
~
~
"~
r~
r~
M(N)M
St(R)St
D(V)D
P(K)P-B
P(N)P
5-0
0
2-8
2-6
2.8
0
0 2.6
0
2-0
2-8 2-8 - 2.6 - -
3-6 0
~"
~
~
~
~" ~
~
z
N
0
0
2-8
2.6
4.0
4.1
---
N
2-8 2.6 2.6 4.0 2.6 2.8 2-6 3.6 6.3
3.1
5.7
2-8 1.2 2.6
2-6 - 6.2
6-6
2.6 3.5
3.5 - 3.4 3.1
3.5 4
3.4 0 1.1 - 0 3-3 3.1
3.5 2.6 1-1 5.1 1.5
* M(G)M, M(K)M-F1,F2, M99, M(SA)M, M(M)S, M(M)SM, M(M)CM, M(M)Ps, M(M)PsM and M(Re)M.
t Assay not performed
Digitaria isolate
The D(V)D antiserum reacted more strongly with the Digitaria isolate than with any of the
other isolates. D(V)D was readily distinguishable from all other isolates against which antisera
were raised. The S D I values obtained with the D(V)D antiserum indicate that D(V)D is most
distantly related to both sugarcane isolates.
Coix isolate
C(M)S was different from all other isolates tested, being most closely related to St(R)St.
Panicum isolates
All Panicum isolates cross-reacted poorly with the antisera to M ( N ) M , St(R)St and D(V)D.
Equally, both Panicum antisera gave weak cross-reactions with all non-Panicum isolates. Strong
cross-reactions were obtained with all Panicum isolates except P(K)PM-B. This K e n y a n isolate
purified from maize differed from P(K)P-B and P(N)P by S D I values of 3-4 and 5.1,
respectively.
Sugar-cane isolates
SDI values ranging from 3.1 to 6.6 obtained using the different antisera indicated that both
sugar-cane isolates were very distinct from the other isolates. Using antisera to both M ( N ) M and
St(R)St, the Mauritius isolate S(M)S was found to be the most distantly related to these two
isolates and was readily distinguishable from the South African isolate S(SA)S.
Production of MAbs
The antisera of the two mice used for the fusion had titres of 5 × 10 -5 and 10 -6 for M ( N ) M in
both A C P and D A S indirect ELISA. Fifteen stable hybridoma clones secreting M A b s to
M ( N ) M were generated, and reacted both in D A S and in A C P ELISAs.
Reactivity of MAbs with MSV isolates
The M A b s could be divided into five groups (A to E) according to their ability to react with
the different MSV isolates and by the strength of their reactions: group A, M A b s 11 and 12;
group B, M A b 13; group C, M A b s 3 and 10; group D, M A b s 1, 2, 5, 8, 14 and 15 ; group E, M A b s
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Serology of maize streak virus
987
T a b l e 3. Reactivity of MAbs raised against M S V - M ( N ) M with other maize isolates (a), the
S e t a r i a isolates (b), the P a n i c u m isolates (c), and the Coix, sugar-cane and D i g i t a r i a isolates (d)
Isolate~"
&
MAb*(a) r
M(G)M
M(N)M-M1 84 M(N)M-M1 87M(M)cM(M)SM(M)Ps M(M)CM M(M)SM M(M)PsM
M(K)M-F1,FE
M(K)M-M99
M(SA)M
M(Re)M
A
=~
O¶
--§
0
B
=
0
--
0
C
D
E
=
=
=
0
0
0
----
0
0
0
St(B)St
=
St(R)St
=
(b)
A
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
St(B)StM St(R)StM
0
0
B
=
0
0
0
C
D
E
=
=
=
0
=
=
0
0
0
-0
0
A
P(K)P-B
0
P(N)P
0
l
~
D
E
.
=
(c)
(d)
P(K)P-M
0
.
P(K)PM-B P(N)PM P(K)PM-M
0
0
0
.
.
.
.
.
=
=
A
S(M)S
0
S(SA)S
--
D(V)D
0
B
0
0
=
C
D
E
0
0
0
0
0
0
=
=
=
.
.
.
.
--
C(M)S
=
=
=
C(M)SM S(M)SM S(SA)SM
=
0
--
--
--
0
0
=
0
-=
0
0
0
0
0
0
0
* The MAbs were assigned to five different groups according to their reaction pattern with the isolates (see
text).
t The code used for each isolate is defined in Methods.
;~ Equal binding to that observed with M(N)M.
§ Lower binding than that observed with M(N)M.
¶ No binding.
4, 6, 7 a n d 9 ( T a b l e 3). N o n e o f t h e M A b s g e n e r a t e d w a s specific for a n e p i t o p e c o m m o n to all
isolates tested. F u r t h e r m o r e , isolates M ( M ) C , M ( N ) M - M 1 84, S t ( B ) S t M , S ( M ) S a n d S ( M ) S M
w e r e n o t r e c o g n i z e d b y a n y of t h e M A b s .
Maize isolates
All M A b s r e a c t e d s t r o n g l y w i t h t h e m a i z e isolates e x c e p t M ( M ) C anti M ( N ) M - M 1 84 ( T a b l e
3 a). M ( M ) C , a m a i z e isolate i n Coix, w a s serologically d i s t i n c t f r o m M ( N ) M w h e n e x t r a c t e d
f r o m Coix, w h e r e a s it w a s i n d i s t i n g u i s h a b l e f r o m M ( N ) M w h e n p u r i f i e d f r o m m a i z e
[ M ( M ) C M ] . All t h e M A b s w e r e able to d i s t i n g u i s h b e t w e e n M ( N ) M a n d M ( N ) M - M 1 87 a n d
b e t w e e n M ( N ) M - M 1 8 7 a n d M ( N ) M - M 1 84.
S e t a r i a isolates
St(B)St w a s r e c o g n i z e d b y all M A b s to t h e s a m e d e g r e e as M ( N ) M ( T a b l e 3b). M A b s in
g r o u p s B a n d C c o u l d d i s t i n g u i s h b e t w e e n b o t h M ( N ) M a n d S t ( R ) S t a n d b e t w e e n b o t h Setaria
isolates b y r e a c t i n g w i t h a n e p i t o p e t h a t w a s n o t p r e s e n t o n St(R)St. O n l y M A b s in g r o u p C
c r o s s - r e a c t e d w i t h S t ( R ) S t M , a l t h o u g h to a lesser degree.
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E.L.
DEKKER AND OTHERS
Panicum isolates
Each of the MAbs, except for MAbs 11 and l 2, reacted with all Panicum isolates, confirming
the results that were obtained with the polyclonal antisera, which showed that the coat proteins
of these isolates are antigenically very similar. MAb 13 (group B) reacted with P(K)P-B to the
same degree as with M(N)M; this MAb could be used to distinguish between these two isolates
and both P(N)P and P(K)P-M due to a lesser reaction with the two latter isolates. MAbs in
group C could distinguish between P(K)P-B and P(N)P and P(K)P-M, indicating that M(N)M
shares an epitope with both P(K)P-B and P(N)P that is modified on the coat protein of
P(K)P-M.
The reactivity of the different MAbs with P(N)P and P(K)P-M was identical, regardless of
the host used for their propagation, whereas P(K)P-B was less well recognized by MAbs in
groups B, C and E when purified from maize.
Coix isolate
The Coix isolate was shown, using MAbs in group A, to have at least one epitope in common
with M(N)M, St(B)St and St(R)St. The results obtained with MAbs in group D indicate that
C(M)S shares at least one epitope with M(N)M, both Setaria isolates and with D(V)D.
Sugar-cane isolates
The sugar-cane isolate from Mauritius, S(M)S, was not recognized by any of the MAbs. MAbs
in group A reacted with a modified epitope on the coat proteins of the South African isolates
without distinguishing between S(SA)S and S(SA)SM.
Digitaria isolate
All MAbs, except for those in group A, reacted with D(V)D, indicating that M(N)M and
D(V)D have several epitopes in common. In addition, D(V)D was shown to share epitopes with
both Setaria isolates, several Panicum isolates and the Coix isolate.
DISCUSSION
Previous work using polyclonal rabbit antisera has shown that no serological relationship
exists between the coat proteins of the whitefly- and the leafhopper-transmitted geminiviruses
(Roberts et al., 1984). Whereas strong relationships were detected between several whiteflytransmitted viruses in immunosorbent electron microscopy, no antigenic relationship was
detected between MSV and other leafhopper-transmitted geminiviruses such as beet curly top
virus and wheat dwarf virus (Roberts et al., 1984). As these three leafhopper-transmitted viruses
differ in their vector specificity, this suggests that the coat protein of these viruses may have an
important role in vector transmission.
Comparisons of the nucleotide sequences of M(N)M (Mullineaux et al., 1984) with M(K)MM99 (Howell, 1984) and D(V)D (Donson et al., 1987) showed that there was considerable
similarity in their coat protein genes (i.e. a DNA sequence homology of 9 9 ~ and 64~
respectively). The total amino acid homology of the coat proteins of D(V)D with M(N)M is
80 ~ ; this is reflected in their serological relatedness as observed in tests using polyclonal rabbit
antisera to D(V)D (Dollet et al., 1986) and to M(N)M (Pinner et al., 1988) and to other isolates of
MSV, included in this work. This suggests that, as in the case of tobamoviruses, the degree of
sequence homology in the coat proteins is correlated with serological relatedness (Altschuh et al.,
1985; Van Regenmortel, 1986).
In this study, further information on the serological relationships between coat proteins of
MSV isolates has been obtained. The results obtained using polyclonal antisera show that
although large variations exist, all isolates are serologically related to each other. The MAbs that
have been obtained were used to give detailed information, at the epitope level, on the
serological relationship between (M(N)M and the other isolates and also between the individual
isolates themselves. Most maize isolates that cause severe symptoms were shown to be
indistinguishable from M(N)M, indicating the presence of highly conserved regions in their
coat proteins.
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989
None of the MAbs reacted with either M(M)C or M(N)M-M 1 84. This confirmed the results
obtained using the rabbit polyclonal antiserum to M(N)M, which showed that M(N)M is
serologically very distinct from both M(M)C and M(N)M-M1 84, thus indicating that these
isolates do not have many epitopes in common with M(N)M. It would therefore be expected that
when M(N)M was used as the immunogen, few, if any, MAbs were likely to be obtained that
would react with either of these two isolates. None of the MAbs showed any cross-reactivity
with isolates that differed from M(N)M by an SDI value greater than 4.0. Cross-reactivity
studies in the tobamovirus group have shown that MAbs are generally unable to detect an
antigenic relationship between two viruses separated by an SDI value of 4.0 (Briand et al., 1982;
Dekker et al., 1987).
An isolate (-M1) of M(N)M, first derived and isolated in 1984, produced very mild symptoms
and was serologically very different from M(N)M. From 1984 to 1987, M(N)M-M1 was
continually passaged (every 2 to 3 months) in maize, using leafhopper vectors, during which
time the symptoms became more severe (Pinner et al., 1988). M(N)M-M1 87 was apparently
serologically related to M(N)M but distinct from M(N)M-M1 84. These results indicate that
M(N)M is a mixture of at least two different serotypes that are distinct in the symptoms they
cause. The difference in reactivity observed with M(N)M-M1 84 and M(N)M-M1 87 may have
been because M(N)M-M 1 84 was composed of a mixed population of a mild and a severe isolate,
with the latter increasing in relative proportion with successive passages through maize. When
used in ELISA, both isolates would be competing for binding space, resulting in a reduced
concentration of the severe isolate M(N)M and hence a reduced antibody reaction. Whereas no
distinction could be made using a polyclonal antiserum, possibly due to the presence of
antibodies specific for common epitopes, the MAbs could distinguish between M(N)M and
M(N)M-M 1 87.
The Setaria isolate from Rwanda, St(R)St, could be distinguished from the isolate from
Burundi, St(B)St, and from M(N)M using MAbs in groups B and C. This distinction could not
be made using the polyclonal antisera thereby illustrating the superior discriminatory power of
MAbs.
Each of the MAbs, with the exception of MAbs 11 and 12, reacted with all Panicum isolates,
confirming the results obtained using the polyclonal antisera which demonstrated that these
isolates are antigenically closely related. M(M)C, St(R)St, St(B)St, P(K)P-B and P(K)P-M
reacted differently, according to the host from which they were purified; this was probably
because they were mixed populations, with one phenotype multiplying faster than the other(s),
depending on their host plant. Previous data (Pinner et al., 1988) showed that symptoms caused
by M(N)M, M(M)C, St(B)St and P(K)P-B were mixed.
The results obtained with the polyclonal mouse antisera, and the fact that all MAbs reacted
equally well in ACP and DAS ELISA, suggests that the MSV virions are stable at pH 7.4 and
readily adsorb to the plastic surface in ACP ELISA. This is in contrast to the finding that intact
tobacco mosaic virus particles (at 1 ~tg/ml) do not appear to become adsorbed to the solid phase
in ELISA at pH 9.6 (Dore et al., 1988).
We would like to thank R. Tees (Sandoz, Basel, Switzerland) for excellent technical advice on hybridoma
technology, M. Boulton for the M ( N ) M antigen which was used for the initial screening of the hybridomas and J.
Cook for technical assistance in raising the polyclonal antisera. This work was carried out under The Plant Pests
(Great Britain) Order, 1980, Licence Nos. P H F 49-139, P H F 49-140 and P H F 49-41.
REFERENCES
ALTSCHUH, D., AL MOUDALLAL,Z., BRIAND, J. P. & VAN REGENMORTEL,M. H. V. (1985). Immunochemical studies of
tobacco mosaic virus. VI. Attempts to localize viral epitopes with monoclonal antibodies. Molecular
Immunology 22, 329-337.
BOULTON, M. L, MARKHAM,P. G. & DAVIES, J. W. (1984). Nucleic acid hybridisation techniques for the detection of
plant pathogens in insect vectors. In Proceedingsof the British CropProtection Conference- Pests and Diseases,
Brighton, 1984, pp. 181-186.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 31 Jul 2017 17:08:19
990
E. L . D E K K E R
AND
OTHERS
BRIAND, J. P., AL MOUDALLAL,Z. & VAN REGENMORTEL,M. H. V. (1982). Serological differentiation of tobamoviruses
by m e a n s of monoclonal antibodies. Journal of Virological Methods 5, 293-300.
DAMSTEEGT,V. O. (1983). Maize streak virus: I. Host range and vulnerability of maize germplasm. Plant Disease67,
734-737.
DEKKER, E. L., DORE, 1., PORTA, C. & VAN REGENMORTEL,M. H. V. (1987). Conformational specificity of monoclonal
antibodies used in the diagnosis of tomato mosaic virus. Archives of Virology 94, 191-203.
DOLLET, M., ACCOTTO,G.-P., LISA, V., MENISSIER, J. & BOCCARDO,G. (1986). A geminivirus, serologically related to
maize streak virus, from Digitaria sanguinalis from Vanuatu. Journal of General Virology 67, 933-937.
DONSON, J., ACCOTTO,G.-P., BOULTON,M. I., MULLINEAUX,P. M. & DAVIES,J. W. (1987). The nucleotide sequence of a
geminivirus from Digitaria sanguinalis. Virology 161, 160-169.
DORE, I., WEISS, E., ALTSCHUH,D. & VAN REGENMORTEL,M. H. V. (1988). Visualization by electron microscopy of the
location of tobacco mosaic virus epitopes reacting with monoclonal antibodies in enzyme immunoassay.
Virology 162, 179-189.
FAZEKAS DE ST. GROTH, S. & SCHEIDEGGER, D. (1980). Production of monoclonal antibodies: strategy and tactics.
Journal of Immunological Methods 35, 1-21.
HOWELL, S. H. (1984). Physical structure and genetic organisation of the genome of maize streak virus ( K e n y a n
isolate). Nucleic Acids Research 12, 7359-7375.
JAEGLE, M. & VAN REGENMORTEL, M. H. V. (1985). Use of ELISA for measuring the extent of serological crossreactivity between plant viruses. Journal of Virological Methods l l , 189-198.
MULLINEAUX, P. M., DONSON, J., MORRIS-KRSINICH,B. A. M., BOULTON,M. I. & DAVIES, J. W. (1984). The nucleotide
sequence of maize streak virus D N A . EMBO Journal 3, 3063-3068.
PINNER, M. S., MARKHAM,P. G., MARKHAM,R. H. & DEKKER, E. L. (1988). Characterisation of maize streak virus.
Description of strains and symptoms. Plant Pathology 37 (in press).
ROBERTS, I. M., ROBINSON, D. J. & HARRISON, B. D. (1984). Serological relationships and genome homologies among
geminiviruses. Journal of General Virology 65, 1723-1730.
ROSE, O. J. W. (1978). Epidemiology of maize streak virus. Annual Review of Entomology 23, 259-282.
STOCKER, J. W., FOSTER, H. K., MIGGIANO, V., STAHLI, C., STAIGER, G., TABACS, B. & STAEI-IELIN, T. H. (19821.
Generation of two new myeloma cell lines " P A I " and " P A I - O " for hybridoma production. Research
Disclosure 217, 155-157.
STOREY, H. H. & McCLEAN,A. P. D. (1930). The transmission of streak disease between maize, sugarcane and wild
grasses. Annals of Applied Biology 17, 691-719.
VAN REGENMORTEL, M. H. V. (1986). Tobacco mosaic virus. Antigenic structure. In The Plant Viruses, vol. 2: The
Rod-Shaped Plant Viruses, p.99. Edited by M. H. V. Van Regenmortel & H. Fraenkel-Conrat. New York &
London: Plenum Press.
VAN RENSBURG,G. D. J. (1981). Effect of plant age at the time of infection with maize streak virus on yield of maize.
Phytophylactica 13, 197-198.
(Received 18 November 1987)
Downloaded from www.microbiologyresearch.org by
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On: Mon, 31 Jul 2017 17:08:19