various barley yellow dwarf luteoviruses from singly ... some mixed infections of other barley yellow dwarf ..... gels by using glyoxal and acridine orange.
211
Journal of General Virology (1990), 71, 211-217. Printed in Great Britain
Direct detection of transcapsidated barley yellow dwarf luteoviruses in doubly infected plants Rebecca Creamert and Bryce W. Falk* Department of Plant Pathology, University of California, Davis, California 95616, U.S.A.
A novel immunohybridization assay was used to analyse the virion capsid proteins and nucleic acids of various barley yellow dwarf luteoviruses from singly and doubly infected plants. Plants singly infected with New York MAV or RPV contained only viruses indistinguishable from the parental types, but plants doubly infected with these viruses contained transcapsidated virions (MAV RNA in RPV protein capsids). The presence of transcapsidated virions was positively correlated with altered aphid transmission characteristics of MAV from the same plants. Virions with
phenotypically mixed capsids (chimeric capsids containing subunits from both co-infecting viruses) were not detected in these plants using heterologous and homologous ELISAs. Transcapsidated virions were detected in mixed infections of California and New York MAV and RPV isolates and, more epidemiologically significantly, in natural doubly infected field plants. In addition, evidence for two-way transcapsidation was obtained using immunohybridization and aphid transmission studies from mixed infections of New York RPV and PAV isolates.
Introduction
aphid vector specificity, which was first observed by Rochow (1970) in mixed infections of laboratory New York (NY) cultures of RPV and MAV. NY-MAV was efficiently transmitted by R. padi from plants infected by both NY-RPV and NY-MAV, but not from plants infected only with NY-MAV. Rochow proposed two mechanisms to explain the altered aphid vector specificity: transcapsidation and phenotypic mixing (Rochow, 1970, 1977). Transcapsidation denotes the encapsidation of the genomic RNA of one virus within a capsid composed of protein subunits from the other virus. Phenotypic mixing denotes viral RNA encapsidation by a virion capsid composed of protein subunits from both co-infecting viruses. Experiments in which specific antisera were used to block aphid transmission from double infections of NY-MAV and NY-RPV supported the theory of transcapsidation and provided evidence discounting phenotypic mixing (Rochow & Muller, 1975; Rochow, 1982). However, recent data suggest that phenotypic mixing occurs in some mixed infections of other barley yellow dwarf luteoviruses, such as PAV and MAV (Hu et al., 1988). The previous evidence suggesting that transcapsidated virions occurred in doubly infected plants is somewhat indirect and is limited to laboratory virus isolates in a single species of plant. We report here the detection of transcapsidated virions by use of an immunohybridization assay which analyses both the virion nucleic acid and the protein capsid. Our work shows that transcapsidation is a general phenomenon, occurring in several
The barley yellow dwarf luteoviruses BYDV cause important economic losses in cereals world-wide (Burnett, 1984). These low titre, phloem-limited viruses, for the most part, exhibit extreme virus and aphid vector specificity (Rochow, 1969; Johnson & Rochow, 1972), which is thought to be partly determined by recognition between the virion capsid and specific receptors in the aphid gut and salivary glands (Gildow, 1987). The five best characterized of these viruses were named originally on the basis of their aphid transmission specificity. RPV is transmitted by Rhopalosiphum padi L., MAV by Sitobion (formerly Macrosiphum) avenae F., SGV by Schizaphus graminum Rond. and RMV by R. maidis Fitch. The fifth virus in this group, PAV, exhibits a broader vector range and is readily transmitted by S. avenae, R. padi and S. graminum (Rochow, 1969; Johnson & Rochow, 1972). These five viruses can also be separated on the basis of their serological and nucleic acid properties (Waterhouse et al., 1986; Gildow et al., 1983; Rochow & Carmichael, 1979; Hsu et al., 1984). Mixed infections of these viruses are common in field plants (Rochow, 1979) and can cause a synergistic increase in symptom severity in the infected host plant (Baltenberger et al., 1987). Another possible result of these mixed infections is an apparent broadening of t Present address: Department of Plant Pathology, Ohio State University, OARDC, Wooster, Ohio 44691-4096, U.S.A. 0000-9079 © 1990 SGM
212
R. Creamer and B. W. F a l k
p l a n t species, w i t h several different b a r l e y yellow d w a r f luteoviruses. M o r e i m p o r t a n t l y , by using t h e i m m u n o h y b r i d i z a t i o n assay, we were a b l e to d e t e c t t r a n s c a p s i d a t e d virions in n a t u r a l l y i n f e c t e d field plants. T h i s p r o v i d e s strong e v i d e n c e t h a t t r a n s c a p s i d a t i o n occurs r e a d i l y in the field a n d m a y p l a y a n i m p o r t a n t role in the ecology o f b a r l e y yellow d w a r f luteoviruses.
Methods Virus and vector manipulation. NY isolates of MAV, PAV and RPV, their antisera and NY aphid clones of R. padi and S. avenae were as described by Creamer & Falk (1989). The California (CA) isolates, CARPV-4 and CA-MAV-4, were isolated from oats (Avenafatua L.) and goat grass [Aegilops juvenalis (Thell.) Eig.], respectively. All aphid colonies were maintained and aphid transmissions performed as described by Creamer & Falk (1989) using A. sativa L. California Red oats. Monoclonal antibodies (MAbs) made against NY-RPV (RPV-I), NY-MAV (MAV-1) and NY-PAV (MAV-3) (Hsu et al., 1984) were gifts of Dr H. T. Hsu (USDA, Beltsville, Md., U.S.A.). Recombinant eDNA clones to NY-RPV (pRPV29) and NY-MAV (pMAV 14+) (Barbara et al., 1987) were obtained from Dr Richard Lister (Purdue University, Lafayette, Ind., U.S.A.). A recombinant eDNA clone made to an Australian isolate ofPAV (pPA8) (Miller et al., 1988) was obtained from Dr W. L. Gerlach (CSIRO, Canberra, Australia). Serological analysis. Infections were analysed by double antibody sandwich (DAS) ELISA as before (Creamer & Falk, 1989). Immulon II plates (Dynatech) were coated with Protein A-purified IgG at 2.5 ~tg/ml, or with ascites fluid containing MAbs at 1/2500 dilution for MAV-1 and MAV-3, or 1/5000 dilution for RPV-1. Plates were blocked with PBST (0.02 M-sodium phosphate pH7.2, 0.05% Tween-20) containing 2% bovine serum albumin. Polyclonal IgGs conjugated with alkaline phosphatase (Sigma) were added at 0.6 mg/ml for colour development. Results were assessed spectrophotometricallyat 405 nm with either a Titertek Multiskan MC (Flow Laboratories) or a V-MAX (Molecular Devices) ELISA plate reader. Sap was extracted from plant samples (1.0 g) with a sap expressor (Pidemont Tool and Die) diluted in 0.05 M-sodium phosphate buffer pH 7.0 (1:4, w/v) and clarified with chloroform (1:1, v/v). Paired replicated samples (200 ml) were placed in antibody-coatedwells. After an overnight incubation at 4 °C, the plates were washed four times with PBST. MAb-trapped antigens were analysed by adding alkaline phosphatase-conjugated antibodies and substrate as previously described (Creamer & Falk, 1989). Immunohybridization analysis. Immunohybridizations were done by coating microtitre plates with MAbs, preparing samples and washing plates as in ELISAs. Nucleic acid hybridization analysis of MAbtrapped samples was done by adding 200 p.l of 1 x sodium/potassium phosphate buffer (5 x buffer is 0-06 r~-Na2HPO4, 0.04 M-KH2PO4, pH 7.0, 0.1 M disodium/EDTA) to each microtitre well. Plates were then heated at 80 °C for 3 min and immediately cooled on ice. The samples (200 ~tl) were removed from wells and applied to 0-45 ~tm nitrocellulose membranes (Schleicher and Schuell) presoaked in 20 x SSC (i.e. 0-3 M-trisodium citrate, 3"0 M-sodium chloride), using a BioDot apparatus (Bio-Rad). Blots were baked for 1 to 2 h at 80 °C and subjected to hybridization analysis with 3zp-labelled recombinant DNA probes (pRPV29, pPA8, or pMAV14+). Caesium chloride gradient-purified plasmids were labelled with [~-3zPldCTP using a nick translation kit (Amersham). Prehybridiza-
tions, hybridizations, washes and autoradiography were done as before (Creamer & Falk, 1989). The quality of the RNA recovered from immunologically trapped virions was determined by denaturing agarose gel electrophoresis and Northern blot hybridization with 32p-labelled pRPV29. The RNA solution removed from eight microtitre plate wells was combined and extracted twice with phenol :chloroform (2 : 1). The RNA was ethanolprecipitated, resuspended in sterile distilled water and glyoxylated (McMaster & Carmichael, 1977). The RNA was analysed by electrophoresis for 1.5 h at 100 V in a 10cm x 6.5 cm x 3 mm 1% agarose gel, using a mini-gel apparatus (Bio-Rad). The RNA size ladder (Bethesda Research Laboratories) used as a standard was stained with toluidine blue. Nucleic acids in sample lanes were transferred by capillary blotting overnight to Nytran membranes (Schleicher and Schuell) in 20 x SSC and analysed by Northern hybridization. Prehybridizations, hybridizations and washes were done as described by Creamer & Falk (1989).
Results A p h i d transm&sion N Y - M A V was efficiently t r a n s m i t t e d b y R. padi f r o m plants doubly infected with NY-MAV and NY-RPV, b u t not f r o m p l a n t s singly i n f e c t e d w i t h N Y - M A V ( T a b l e 1). P l a n t s i n f e c t e d singly w i t h N Y - M A V were sources for efficient t r a n s m i s s i o n b y S. avenae, w h e r e a s p l a n t s infected w i t h N Y - R P V were sources for efficient t r a n s m i s s i o n by R. padi. N o t r a n s m i s s i o n o f N Y - R P V b y S. avenae was o b t a i n e d f r o m singly i n f e c t e d plants, o r those i n f e c t e d w i t h N Y - R P V a n d N Y - M A V . M i x e d a n d single infections o f N Y isolates o f R P V a n d P A V were also a n a l y s e d by a p h i d t r a n s m i s s i o n to d e t e r m i n e w h e t h e r an a l t e r a t i o n in a p h i d v e c t o r specificity was d e t e c t a b l e f r o m these d o u b l y i n f e c t e d plants. N Y - R P V a n d N Y - P A V were t r a n s m i t t e d t o g e t h e r b y S. avenae f r o m g r e e n h o u s e m i x e d infections, w i t h a 13% efficiency ( T a b l e 2). N Y - P A V was t r a n s m i t t e d alone to 53 % o f the plants, S. avenae d i d not t r a n s m i t N Y - R P V f r o m p l a n t s i n f e c t e d only w i t h N Y - R P V . Serological analysis P l a n t s singly a n d d o u b l y infected w i t h N Y - M A V a n d N Y - R P V , or w i t h N Y - P A V a n d N Y - R P V were a n a lysed by D A S E L I S A in w h i c h M A b s were used to t r a p the virions a n d e n z y m e - l i n k e d r a b b i t p o l y c l o n a l a n t i b o dies were used as d e t e c t i n g a n t i b o d i e s . O n l y the h o m o l o g o u s a n t i b o d y c o m b i n a t i o n s for a g i v e n b a r l e y yellow d w a r f virus s e r o t y p e gave p o s i t i v e reactions, p r o v i d i n g e v i d e n c e t h a t v i r i o n c a p s i d s f r o m the d o u b l y i n f e c t e d p l a n t s were n o t p h e n o t y p i c a l l y m i x e d (Tables 3 a n d 5). S p e c t r o p h o t o m e t r i c intensities o f r e a c t i o n s for singly a n d d o u b l y i n f e c t e d p l a n t s were similar. I m m u n o h y b r i d i z a t i o n was used to a n a l y s e the c o m p o sition o f virus p a r t i c l e s in single a n d m i x e d infections o f
Detection o f transcapsidated viruses
T a b l e 1. Aphid transmission analysis o f barley yellow dwarf luteoviruses from single and mixed infections with N Y - M A V and N Y - R P V
Source
Aphid*
Virus transmittedt
NY-RPV + NY-MAV NY-RPV NY-MAV NY-MAV Non-viruliferous aphids Non-inoculated oats
R. padi R. padi S. avenae R. padi~ R. padi
MAV + RPV RPV MAY
--
--
No. plants infested/ tested 19/20 20/20 20/20 0/4 0/4 0/4
---
* Transmissions were done using California Red oats and 10 aphids per plant. t Viruses transmitted were identified by DAS ELISA. :~ Additional transmission experiments with NY-MAV using R. padi gave no transmission to 30 test plants.
T a b l e 2. Alteration in aphid transmission specificity from mixed infections o f N Y - R P V
and N Y - P A V barley yellow dwarf luteoviruses in California Red oats Source
Aphid*
Virus transmittedt
PAV + RPV PAV + RPV PAV + RPV PAV + RPV PAV PAV RPV RPV Healthy oats Healthy oats
S. avenae S. avenae R. padi R. padi S. avenae R. padi S. avenae R. padi S. avenae R. padi
PAV + RPV PAV PAV + RPV PAV PAV PAV
Frequency (~)~ 12/92 (13) 49/92 (53) 56/89§ (63) 29/89 (33) 11/20 (55) 21/22 (95) 0/21 (0) 21/21 (100) 0/20 (0) 0/20 (0)
-RPV
---
* Transmissions were done using approximately 10 aphids per plant. t Infections were confirmed by DAS ELISA. J; The ratio represents the number of infected plants/total number of plants tested. § No transmission of RPV alone by R. padi was achieved in these experiments from the PAV-RPV mixed infections.
T a b l e 3. D A S E L I S A analysis from single and mixed infections o f N Y - M A V and N Y R P V barley yellow dwarf luteoviruses DAS ELISA t Source* RPV + MAV RPV MAV Non-inoculated oats
Coating antibody MAV Detecting antibody MAV
RPV RPV
MAV RPV
RPV MAV
No. plants tested
0-81.~ 0-17 0-98 0-16
0-38 0-39 0-04 0.04
0-08 0-04 0.09 0.04
0-19 0-19 0-16 0.13
12 7 7 5
* Source plants were those shown in Table 1. t MAb ascites fluids were used to coat plates at 1:2500 (MAV-1) or 1:5000 (RPV-1). Alkaline phosphatase-conjugated Protein A-purified rabbit polyclonal immunoglobulins were used to detect antibodies at 2.0 lxg/ml. :~ Mean absorbance at 405 nm for duplicate wells from all plants tested, with a possible absorbance range of 0.00 to 2.00.
213
214
R. Creamer and B. IV. Falk
NY-RPV and NY-MAV (Fig. 1). The MAV-specific and RPV-specific cDNA probes gave positive reactions for samples trapped by the homologous MAbs either from plants infected singly with the homologous viruses, or from plants infected with both viruses. In addition, the MAV-specific probe reacted positively with samples from doubly infected plants which were trapped with the RPV-specific MAb. The converse reaction, RPVspecific probe analysis of MAV-specific MAb-trapped samples from the doubly infected plants, was negative. Results obtained using mixtures of sap from plants singly infected with NY-MAV and NY-RPV were identical to those obtained when testing only singly infected plants. These results suggest that doubly infected plants contain, in addition to progeny virions of composition identical to the parental viruses, one-way transcapsidated virions consisting of NY-MAV RNA in NY-RPV capsids. No evidence was obtained for virions composed of NY-RPV R N A in a NY-MAV capsid. Altered transmissibility of NY-MAV by both S. avenae and R. padi was always obtained only from plants which yielded positive immunohybridization reactions for the presence of transcapsidated virions. When we evaluated the quality of the viral R N A recovered from immunologically trapped RPV virions by gel electrophoresis and Northern blot hybridization, RNA of approximately 6 kb was recovered (Fig. 2). This suggested that the RNA was not significantly degraded into fragments smaller than the full length during the virion disruption phase of the immunohybridization assay.
pMAV MAV RPV
pRPV MAV RPV
MAb
To evaluate possible effects of plant species and/or virus isolate on transcapsidation interactions, experiments were done using combinations of NY and CA MAV and RPV isolates in oats and barley (Hordeum vulgare Kombar). We isolated transcapsidated virions from doubly infected oats by using the immunohybridization assay and both CA-MAV and NY-MAV were transmitted by R. padi from the doubly infected plants, which were positive for transcapsidated virions by immunohybridization. These results were identical whether the doubly infected plants contained CA-RPV or NY-RPV (Table 4). Similar results were obtained using barley (data not shown). An alteration in transmissibility of NY-RPV by S. avenae was noted from mixed infections of NY-RPV and NY-PAV, so we also attempted to use the immunohybridization assay to analyse plants doubly infected with these two viruses for the presence of transcapsidated virions. In contrast to the results predicted by aphid transmission data, no positive immunohybridization reactions for NY-RPV R N A were obtained from mixed infections of NY-RPV and NY-PAV using MAV-3
RPV
RNA STDS
--7"5 --4"4
o
• ::
MAV + RPV Mixed infections
--2"4 MAV Control RPV Control ~1.4 Check oats MAV + RPV Control Fig. l. Immunohybridization analysis of single and mixed infections with NY-MAV and NY-RPV. M A V + RPV control represents extracts of singly infected plants processed separately and combined. MAb ascites fluid was used to coat microtitre plates at 1:2500 (MAV) or 1:5000 (RPV). Recombinant cDNA clones (pRPV29, pRPV, and pMAVI4, pMAV) were 32P-labelled for hybridization analysis. Negative samples were from uninfected ('check') oats.
Fig. 2. Size analysis of viral RNA recovered from MAb RPV-1trapped NY-RPV virions. RNA was recovered by beating antibodytrapped samples at 80 °C. Glyoxylated RNA was then subjected to electrophoresis for 1.5 h at 100 V in a 1~ agarose gel. RNA standards (RNA STDS) were visualized by staining with toluidine blue, while NY-RPV RNA (RPV) was transferred overnight to Nytran membranes and analysed by hybridization with 32p-labelled pRPV29. Numbers on the right show sizes (kb) of standard RNAs.
Detection o f transcapsidated viruses
T a b l e 4. Breakdown in aphid transmission specificity and immunohybridization detection of transcapsidated virions from mixed infections of CA and N Y RP V and MA V barley yellow dwarf luteoviruses A* Immunohybridizationt
Source
Aphid
Transmission
NY-RPV+NY-MAV NY-RPV+CA-MAV CA-RPV+NY-MAV CA-RPV+CA-MAV NY-RPV CA-RPV
R. padi R. padi R. padi R. padi R. padi R. padi S. avenae R. padi S. avenae R. padi S. avenae R. padi S. avenae
5/5 5/5 5/5 5/5 5/5 5/5 2/5 0/5 5/5 0/5 5/5 0/5 0/5 0/5
NY-MAV CA-MAV Check oats Check oats
--
Clone
pMAV
pRPV
B
MAb
M
R
M
R
Transmission~
+ + + +
+ + + +
-
+ + + + + + +
20/20 20/20 18/19 19/20 4/4 4/4 1/10
+
4/4
+
4/4 0/4 0/4 0/4
* Transmissions were from source plants to California Red ('check') oats using 10 aphids of the species shown per plant. The ratio represents the number of infected plants/total number of plants tested. Each plant from transmission A was tested by immunohybridization and ELISA. t MAb ascites fluids used to coat microtitre plates and trap antigens at 1:2500 (M; MAV-1) or 1:5000 (R; RPV-I). Samples trapped with the corresponding monoclonal antibody were then probed using the cDNA clone shown. A + indicates the sample was positive for the MAb/cDNA clone combination given and a - indicates it was negative. :~ Each doubly infected plant from transmissions in A was used as a source for a confirmatory transmission (B) using the aphid species shown in A to at least four plants. These plants were checked by ELISA and the ratios show the plants that contained the same viruses as in A.
T a b l e 5. Detection of transcapsidated virions by immunohybridization and D A S ELISA from mixed infections of N Y - R P V and NY-PA V barley yellow dwarf luteoviruses Immunohybridizationt DAS ELISA¢ Source* Trial 1 PAV + RPV RPV PAV Non-inoculated oats Trial 2 PAV + RPV RPV PAV Non-inoculated barley
Clone
pPA8
pRPV
MAb
P
P
R
-
+
-
+
-
+
-
+
R
+ + + -
+ + + -
Coating antibody RPV Detecting antibody RPV
PAV PAV
RPV PAV
PAV RPV
0.20 0.24 0.05 0-08
0.36 0-05 0-28 0.13
0-09 0-12 0.08 0.13
0.16 0.05 0.11 0-06
1-83 1.92 0.69 0.57
1.25 0.54 1.06 0-43
0.19 0.16 0-10 0.08
0-58 0-40 0.56 0.31
* Infected source plants were California Red oats (trial 1) or Kombar barley (trial 2). t MAb ascites fluids were used to coat plates and trap antigens, at dilutions of 1:2500 (P; PAV) or 1:5000 (R; RPV). Recombinant cDNA clones pPA8 for PAV or pRPV for RPV were labelled with [32p]dCTP by nick translation and used as probes to analyse antibody-trapped samples. :~ Coating antibodies were the MAbs used in immunohybridization analysis. Detecting antibodies were alkaline phosphataseconjugated, Protein A-purified rabbit polyclonal immunoglobulins at 2-0 mg/ml. Mean absorbance at 405 nm of duplicate wells with a possible absorbance range of 0-00 to 2.00 is shown.
215
216
R. Creamer and B. W. Falk
MAb (which reacts to PAV) and pRPV29. However, the pPA8 cDNA clone hybridized with NY-PAV RNA recovered from RPV-1 MAb-trapped virions (Table 5), suggesting transcapsidation of NY-PAV RNA by NYRPV capsid protein. No positive hybridization reactions were obtained for NY-PAV RNA from plants infected only with NY-PAV when RPV-1 MAb was the trapping antibody. When immunohybridizations were done using RPV-1 MAb and pRPV29, positive hybridizations for NY-RPV RNA were obtained from plants infected with NY-RPV and NY-RPV plus NY-PAV. Immunohybridization analysis of mixtures of sap from NY-PAV and NY-RPV singly infected plants gave reactions indistinguishable from those of singly infected plants assayed separately. Thus, only the transcapsidation of NY-PAV RNA by NY-RPV capsid protein was found by immunohybridization, whereas the converse conformation, i.e. transcapsidation of NY-RPV RNA by NYPAV capsid protein, was expected from the aphid transmission data. Field isolate analysis
Immunohybridization was used to look for transcapsidated virions in field plants found to be naturally infected with isolates of MAV and RPV, or PAV and RPV. We obtained positive reactions for MAV-RPV interactions, similar to those described earlier, from two doubly infected field plants, one an oat plant and one a barley plant. Transcapsidated virions were also detected in naturally occurring mixed infections of PAV and RPV pRPV29 RPV PAV
pPA8 RPV PAV
MAb PAV+ RPV Mixed infections PAV+ RPV Control Check oats PAV Control RPV Control
Fig. 3. Immunohybridizationdetection of transcapsidated virions fromtwo naturalmixedinfectionsof RPV and PAV isolates.The first and secondrows of PAV + RPV mixedinfectionsrepresent extracts from Ae. ovata and Ae. cylindrica, respectively.PAV + RPV control represents extractsof singlyinfectedplants processedseparatelyand combined. Monoclonal antibody ascites fluids were used to coat microtitreplates at 1:5000(RPV-1, whichreacts with RPV)or 1:2500 (MAV-3,whichreactswithPAV). RecombinantcDNA clones(pPA8 and pRPV29) were 32p-labelled and used for hybridizationanalysis. Control plants were greenhouse-grownoats infectedwith NY isolates of PAV or RPV, or non-inoculated('check') oats.
from field-collected samples of A. sativa, Ae. ovata and Ae. cylindrica (Fig. 3). Again, only signals for PAV RNA in RPV capsids were obtained.
Discussion Several previous studies have suggested that structural interactions between plant viruses can affect the aphid transmissibility of progeny virions from mixed infections (Rochow, 1970; Falk et al., 1979, Waterhouse & Murant, 1983). The data presented here suggest that immunohybridization provides a means for detecting heterologously encapsidated RNAs in doubly infected plants. Our results demonstrated that one-way transcapsidation occurred in mixed infections of MAV and RPV isolates in both greenhouse and field plants. Only MAV RNA was found in the heterologous coat protein by immunohybridization, and doubly infected plants that showed alterations in aphid vector specificity were always found to contain transcapsidated virions by immunohybridization analyses. Similar interpretations as to one-way transcapsidation were made by Rochow (1970) in his analysis of laboratory mixed infections. We found evidence suggesting two-way transcapsidation in mixed infections by PAV and RPV. S. avenae transmitted NY-RPV (most likely as virions composed of NY-RPV RNA and NY-PAV capsid protein) from mixed infections of NY-RPV and NY-PAV. NY-RPV was not transmitted by S. avenae from singly infected plants. This is the first report of altered transmission specificity for NY-RPV with S. avenae. The efficiency of this transmission was only 13%, much lower than the altered transmission efficiency for NY-MAV from mixed infections of NY-MAV plus NY-RPV. Failure to detect (by immunohybridization) the PAVRPV transcapsidated virions predicted by the altered aphid transmission results might be explained by the low titre of these transcapsidated virions in the doubly infected plant. The low transmission efficiency suggests that the transcapsidation event may be rare. An alternative explanation for the failure to detect RPV RNA transcapsidated in PAV capsids by immunohybridization could be the instability of NY-RPV RNA within a NY-PAV capsid and virions may not survive the extraction and detection methods used in the immunohybridization assay. Immunohybridization analysis of the RPV-PAV mixed infections suggested that transcapsidated virions of PAV RNA and RPV capsid were present. However, we were unable to confirm the presence of such particles by aphid transmission because NY-RPV and NY-PAV are both efficiently transmitted by R. padi and evidence for structural interactions between these BYDV types would
Detection of transcapsidated viruses
not be easily detected by a qualitative breakdown in aphid vector specificity. We found no evidence suggesting that phenotypic mixing occurred between MAV and RPV, or PAV and RPV in mixed infections. This is consistent with earlier results of serological blocking experiments (Rochow & Muller, 1975). However, recent work has suggested that phenotypic mixing can occur between NY-MAV and NY-PAV viruses in mixed infections (Hu et al., 1988), These authors speculated that phenotypic mixing probably occurs among closely related viruses. MAV and PAV are in the same taxonomic subgroup and RPV is in a distinct subgroup. All of the pioneering work suggesting transcapsidation of MAV and RPV was done with the NY laboratory isolates and all experiments were performed in controlled conditions using Coast Black oats (Rochow & Muller, 1975). Our work shows that transcapsidation also occurs in other hosts and with other BYDV isolates. Also, by using immunohybridization analysis, we were able to establish for the first time that transcapsidation occurs in natural mixed infections, which are often reported (Rochow, 1979). Although this is not surprising, it has important epidemiological significance, because it shows that transcapsidation can expand the vector range of a given luteovirus. We thank Dr James Duffus for the aphid clones, BYDV isolates and antisera, Dr H. T. Hsu for the BYDV Mabs and Dr R. M. Lister and Dr W. L. Gerlach for the BYDV cDNA clones. This work was supported by a Jastro Shields research fellowship to R. Creamer and a USDA grant (86-CRSR-2-2935) to B. Falk.
References BALTENBERGER,D. E., OHM, H. W. & FOSTER, J. E. (1987). Reactions of oat, barley, and wheat to infection with barley yellow dwarf virus isolates. Crop Science 27, 195-198. BARBARA, D. J., KAWATA, E. E., UENG, P. P., LISTER, R. M. & LARKINS, B. A. (1987). Production of cDNA clones from the MAV isolate of barley yellow dwarf virus. Journal of General Virology 68, 2419-2427. BURNE'rr, P. A. (1984). Preface. In Barley Yellow Dwarf, A Proceedings of the Workshop, pp. 6-13. Mexico City: Centro Internacional de Mejoramiento de Maiz y Trigo.
217
CREAMER, R. & FALK, B. W. (1989). Characterization of a nonspecifically aphid transmitted CA-RPV isolate of barley yellow dwarf virus. Phytopathology 76, 942-946. FALK, B. W., DUFFUS, J. E. & MORRIS, T. J. (1979). Transmission, host range, and serological properties of the viruses that cause lettuce speckles disease. Phytopathology 69, 612-617. GILDOW, F. E. (1987). Virus-membrane interactions involved in circulative transmission of luteoviruses by aphids. Current Topics in Vector Research 4, 93-120. GILDOW, F. E., BALLINGER, M. E. & ROCnOW, W. F. (i983). Identification of double stranded RNAs associated with barley dwarf infection of oats. Phytopathology 73, 1570-1572. Hsu, H. T., AEBIG, J. & ROCHOW, W. F. (1984). Differences among monoclonal antibodies to barley yellow dwarf viruses. Phytopathology 74, 600-605. Hu, J. S., RocI~OW, W. F., PALUKAITIS, P. & DIETERT, R. R. (1988). Phenotypic mixing: mechanisms of dependent transmission for two related isolates of barley yellow dwarf virus. Phytopathology 78, 1326-1330. JOHNSON, R. A. & ROCHOW, W. F. (1972). An isolate of barley yellow dwarf virus transmitted specifically by Schizaphis graminum. Phytopathology 62, 921-925. McMASTER, G. K. & CARMICHAEL,G. C. (1977). Analysis of singleand double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange. Proceedings of the National Academy of Sciences, U.S.A. 74, 4835-4838. MILLER, W. A., WATERHOUSE, P. M. & GERLACH, W. L. (1988). Nucleotide sequence and genome organization of barley yellow dwarf virus. Nucleic Acids Research 16, 6097-7010. ROCHOW, W. F. (1969). Biological properties of four isolates of barley yellow dwarf virus. Phytopathology 59, 1580-1589. ROCHOW, W. F. (1970). Barley yellow dwarf virus: phenotypic mixing and vector specificity. Science 22, 875-878. ROCHOW, W. F. (1977). Dependent virus transmission from mixed infections. In Aphids as Virus Vectors. vol. 24 pp. 253-273. Edited by K. F. Harris & K. Maramorosch. New York: Academic Press. RocIaow, W. F. (1979). Field variants of barley yellow dwarf virus: detection and fluctuation during twenty years. Phytopathology 69, 655-660. Rocnow, W. F. (1982). Dependent transmission by aphids of barley yellow dwarf luteoviruses from mixed infections. Phytopathology 72, 302 305. RocrIow, W. F. & CARMICHAEL,L. E. (1979). Specificity among barley yellow dwarf viruses in enzyme immunosorbent assays. Virology 95, 415-420. Rocnow, W. F. & MULLER, I. (1975). Use of aphids injected with virus-specific antiserum for study of plant viruses that circulate in vectors. Virology 63, 282-286. WATERnOUSE, P. M. & MURAYr, A. F. (1983). Further evidence on the nature of the dependence of carrot mottle virus on carrot red leaf virus for transmission by aphids. Annals of Applied Biology 103, 455-464. WATERHOUSE, P. M., GERLACH, W. L. & MILLER, W. A. (1986). Serotype-specific and general luteovirus probes from cloned cDNA sequences of barley yellow dwarf virus. Journal of General Virology 67, 1273 1281.
(Received 18 May 1989; Accepted 11 September 1989)
