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Purification and antigenicity of three isolates of barley yellow dwarf virus
46, 117-126
VIHOLOGY
(1971)
Purification
and
Antigenicity
Barley
Yellow
W. F. ROCHOW,2
of Three Dwarf
Isolates
of
Virus’
A. I. E. AAPOLA; MYRON L. E. CARMICHAEL5
K. BRAKKE:
AND
of Plant University of
Department
Pathology, Nebraska,
Cornell Lincoln, Cornell
University, Nebraska University,
Ithaca, New York 14850; Platlt Pathology Departme)lt, 68508; and J7eterinary Virus Research Institute, Ithaca, Mew York 14860
Accepted
May
24, 1971
An isolate (RPV) of barley yellow dwarf virus transmitted specifically by Rhopalosiphuwt padi and an isolate (PAV) transmitted nonspecifically by both R. padi and Macrosiphum avenae were purified by procedures previously found satisfactory for another isolate (MAV) transmitted specifically by M. avenae. As with MAV, infectivity of RPV and PAV samples removed from sucrose gradient columns was associated with a dense polyhedral particle about 30 nm in diameter in shadowed preparations. No differences in sedimentation rate (sedimentation coefficient 115-118 S) among the 3 virus isolates were detected in parallel sucrose gradient centrifugation tests. Temperature at which source plants were grown was an important factor affecting results of purification. Preparations made from oats grown at 15”-20” contained about 2-5 times more virus than did preparations made from plants grown at temperatures above 25“. Coast Black oats (Avena byzantina) was the best virus source among 5 host plant species tested. Preparations of the PAV isolate made from plants harvested 2-3 weeks after inoculation contained about 4 times as much virus as did preparations from plants harvested after 5-S weeks. Thorough extraction of virus from infected tissue was another critical step in the purification procedure. The average yield of virus (per 1000 g of source tissue) was 106,79, and 20 pg, respectively, for MAV, RPV, and PAV. Accumulation of frozen crude concentrates for each of the 3 virus isolates during a period of about 2 years made possible the production of final virus preparations for use in development of specific antisera. A highly reactive antiserum specific for each of t.he 3 virus isolates w&9 obtained by injecting 20&350 pg of purified virus into single rabbits in a series of 3 injections. Serological comparisons of the 3 virus isolates, based on a type of infectivity neutralization that involves serological blocking of virus transmission by aphids, suggested that the MAV and PAV isolates are relat,ed, but that RPV is distinct from the other 2 isolates. 1 Cooperative Investigation, Plant Science Research Division, Agricultural Research Service, U.S. Department of Agriculture; Cornell University Agricultural Experiment Stat.ion; Nebraska Agricultural Experiment Station; and Cornell University Veterinary Virus Research Institute. Supported in part by NSF grant GB-21013 to Cornell University, and by NSF grant GB-8283 to the University of Nebraska. Published with the approval of the Director as Paper No. 3062, Journal Series, Nebraska Agricultural Experiment Station, Project No. 21-12. 2 Research Plant Pathologist, Plant Science Re-
search Division, Agricultural Research Service, U.S. Department of Agriculture; and Professor of Plant Pathology, Cornell University. 3 Formerly, Graduate Assistant, Department of Plant Pathology, Cornell University. Present atldress : Irrigated Agriculture Research and I:sterlsion Center, Presser, Washington 99350. 4 Chemist,, Plant Science Research Divisioll, Agricult,ural Research Service, U.S. Department. of Agriculture; and Professor of Plant Pathology, Universit,y of Nebraska. 5 Professor of Virology, Veterinary \‘ims I:{:search Instit,ute, Cornell Universit J-. 117
118
ROCHOW INTRODUCTION
Progress in purification studies has been slow and difficult for that small but economically important group of persistent or circulative aphid-transmitted plant viruses that have not been transmitted mechanically to plants. Limiting factors in such studies include the difficulties of bioassay, the low concentration of virus extracted from infected plants or from viruliferous aphids, and the instability of some of the viruses. Three representatives of the group, which have many features in common, are potato leaf roll virus (Arai et al., 1969; Peters, 1967; Peters and Van Loon, 1968; Kojima el al., 1969), beet western yellows virus (Duffus, 1969; Duffus and Gold, 1969), and barley yellow dwarf virus (BYDV), the subject of this report. Purification of the MAV isolate of BYDV was described previously (Rochow and Brakke, 1964). Here we report studies on purification and identification of the infectious particle for 2 additional isolates, RPV and PAV. All 3 isolates were originally differentiated on the basis of virus-vector relationships (Rochow, 1969). We also report here on factors that affect purification of BYDV, on production of antisera, and on some serological comparisons among 3 isolates of the virus. A more detailed study of relationships among the 3 isolates appears separately (Aapola and Rochow, 1971). MATERIALS
AND
METHODS
Virus-free aphids were maintained in isolated rea,ring rooms on caged barley plants (Hordeum vulgare L.) under special precautions (Rochow, 1959; Rochow and Brakke, 1964). Mucrosiphum avenue (Fabricius), the English grain aphid, and Rhopalosiphum padi (Linnaeus), the oat bird-cherry aphid, were the 2 speciesused regularly. Occasionally additional aphid species were used in some comparative tests. At least 30 aphids from each group were always tested as controls in every experiment. The biological properties of the 3 isolates of BYDV have recently been described (Rochow, 1969). One isolate (PAV) is transmitted nonspecifically by both R. pa& and M. avenue, another isolate (RPV) is transmitted specifically by R. pa&, and the third
ET AL.
isolate (MAV) is transmitted specifically by M. avenue. The virus isolates were maintained by serial transmissions at intervals of 5-6 weeks to Coast Black oats (Avena byzuntina K. Koch). Comparative tests with 4 aphid specieswere made at the time of each transfer to make certain that no major change in the relative vector specificity of the isolate occurred during these studies (Rochow, 1969). Coast Black oats were test plants as well as source plants for virus purification work, unless specified otherwise. Plants were grown in 4-inch clay pots of composted, steam-sterilized soil. Unless stated otherwise, the buffer used in all studies was 0.1 M neutral potassium phosphate; it will be referred to simply asbuffer. Infectivity of virus preparations was estimated by membrane-feeding or injection methods previously described (Rochow, 1969,197O). Virus concentration in the preparations was estimated by comparing the height of peaks of analytical density-gradient scanning patterns obtained from an ISCOG density-gradient fractionator with those obtained with known concentrations of a purified preparation of southern bean mosaic virus (Aapola and Rochow, 1971). Unless stated otherwise, all gradients were prepared 48 hrs before use with 4,7,7, and 7 ml of 100, 200,300, and 400 mg of sucroseper milliliter, respectively, dissolved in buffer. Rate zonal centrifugation was for 3 hrs at 23,000 rpm at about 9” in the SW 25.1 rotor of the Spinco Model L centrifuge.6 Our current routine procedure for producing preparations of BYDV involves aphid inoculation of 3 or 4 seedlingsin each of 90 pots of Coast Black oats, growing the inoculated plants in a greenhouse for 4-6 weeks, harvesting entire shoots (rarely, also roots), and cutting the harvested plants into sections about 4 cm long for storage of the tissue in plastic bags in a freezer at about -20’. When 4-8 such bags, each containing 5002000 g of tissue, have been accumulated for a virus isolate, the tissue is chopped while still 6 Mention of a trademark name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the U.S. Department of Agriculture, and does not imply its approval to the exclusion of other products also available.
BARLEY
YELLOW
DWARF
partly frozen in a Hobart6 food cutter. The chopped tissue is then ground in a Hobart6 type D fruit juice extractor, which separates the juice from the residue. In lots of 450 ml the crude juice is mixed with 50 ml of 1.0 M neutral potassium phosphate buffer for clarification with chloroform and n-amyl alcohol (Rochow and Brakke, 1964). The tissue residue is soaked in cold 0.1 M buffer and ground again in the fruit juice extractor. In lots of 500 ml the juice from this second extraction is also clarified. The twice-ground residue is soaked again in buffer, packed into a plastic bag, and refrozen for further extractions at a later date when several kilograms of such tissue have been accumulated. The several liters of clarified juice are then centrifuged for 4 hrs in a 30 rotor of a SpincoG Model L centrifuge. Pellets are resuspended in a small amount of buffer with the aid of a ground glass homogenizer, and the concentrated preparation is centrifuged at 4800 rpm for 15 min in a Servall SP6 centrifuge at 4”. The pellet from t-his low-speed centrifugation is washed several times with buffer, and the superna.tants are combined to make a concentrate about 20-fold relative to the original volume of clarified juice. Such concentrates are then stored in 25 or 50-ml bottles in a freezer. When final virus preparations are needed for an experiment, the frozen concentrates are removed from the freezer, t’hawed, and used to make the required virus preparation by further cycles of differential and sucrose gradient centrifugation. RESULTS
Purification of RPV and PAV Concentrated preparations of the RPV or PAV isolates were made from frozen oat tissue by chloroform clarification and differential centrifugation as previously described for MAV (Rochow and Brakke, 1964). Correlation of virus particles and infectivity was st.udied by assay of samples removed from density-gradient centrifugation columns (Table 1). F’or density-gradient centrifugation, 1.0 ml of a virus preparation was layered on one of three sucrosegradient’s, 1.0 ml of a, comparable preparation made from healthy oats was layered on a second, and 1.0 ml of a purified preparation of southern
VIRUS
PURIFICATION
119
bean mosaic virus (containing 0.1 mg of virus) was layered on a third. When a visible zone was seen in tubes containing RPV or PAV, its location coincided with, or was slightly lower t)han, that of the visible zone in the tube containing only southern bean mosaic virus. In experiments where a visible zone did not occur for either isolate of BYDV, the location of the corresponding southern bean mosaic virus zone was used as the basis for sampling. Samples were removed from the virus zone, from areas above and below the zone, and from the gradient, containing the preparation from healthy oats. at a location corresponding to the dept’h of the virus zone. About 0.2 ml of each of the 4 samples from an experiment was shipped t.o Lincoln, Nebraska, for electron microscopy, and the remaining portion of each sample was used in Ithaca for infectivity assays b? the membrane-feeding method. In some experiments t,he cernrifuged sucrose gradient. columns were scanned and sampleswere colected by means of the ISCOG density-gradient fractionator. Results of 11 separate experiments, in volving both rate zonal and equilibrium zonal centrifugation, were similar to results previously obtained for RIAV (Rochow and Brakke, 1964). Infectivity of both the RPV and PAV isolates was associatedwith a dense polyhedral particle about 30 nm in diameter in shadowed preparations (Table 1j. Rot.11 RPV and PAV appeared similar in sedimentation and morphology to the previously described hIAV isolate. Sedimentation of the t
120
ROCHOW TABLE
ET AL. 1
RELATION BETWEEN PARTICLES COUNTED IN ELECTRON MICROGRAPHS AND INFECTIVITY OF SAMPLES FOR Two ISOLATES OF BARLEY YELLOW DWARF VIRUS (BYDV) REMOVED FROM RATE-Z• NAL (RZ) OR EQUILIBRIUM-Z• NAL (EZ) DENSITY-GRADIENT COLUMNS
BYDV isolate and kind of colum@
Buffer used to dissolve sucrose
RPV RZ
0.05 M borate, pH 8.0
PAV RZ
0.1 M phosphate, pH 7.0
RPV EZ
0.1 M phosphate, pH 7.0
PAV EZ
0.1 M phosphate, pH 7.0
Sample identification and cm below meniscusb A B C H A B C H A B c H A B c H
1.2 1.7 2.7 1.7 1.2 1.7 2.7 1.7 1.9 2.4 2.9 2.4 2.0 2.5 3.5 2.5
Relative number particles per sample” 2 397, 124, 31 8 1 3 12, 3, 0.6 0.08 0.03 0.01 48, 21, 5, 0.5 8 0.004 0.8 97, 24, 4, 1 1 1
Infectivity of sampled
7/g,l/9 9/g,g/g,819 71% o/g o/g o/g,l/9 7/g, w-4 o/g 01% 9/g, 7/g, o/g o/g, 8/g, 2/g, 019
3/g, 31% l/9 o/9 o/g ‘J/9, 6/g, 6/g 71% 319 o/g, o/g g/g, 5/g, 2/g 2/g, 119
a RZ gradient columns were prepared 48 hr before use with 4, 7, 7, 7 ml of 100, 200, 300, and 400 mg sucrose per milliliter, respectively, dissolved in the buffer indicated. EZ columns were similarly prepared with 0.9 ml each of solutions containing 300,400, 500, and 600 mg of sucrose per milliliter. b Samples A, B, and C were from tubes containing the virus preparation; sample H was from tube containing the control preparation made from healthy plants. Centrifugation wm for 3 hr at 23,000 rpm at about 9” in the SW 25 rotor of the Spinco centrifuge for RZ tubes, and for 5 hr at 36,009 rpm at about 9” in the SW 39 rotor for EZ tubes. c Specimens were shadowed from three directions, and the particles were counted on the screen of the RCA EMU3G at 105,000 X . Numbers given are averagesper 3 X 4-inch area at this magnification. Particles were counted on at least 25 randomly selected areas when they were numerous. When there were only a few particles per 3 X 4-inch area, the number per 3-inch traverse across a grid opening was counted and divided by 75. At least 7 such traverses were counted for each such sample. Two grids were examined per sample. The first number in each series is for an undiluted sample and subsequent numbers represent successive B-fold dilutions. d Number of plants that became infected over number infested with 10 aphids (R. pa&) that had fed through membranes for about 18 hr on sample indicated before start of 5-day inoculation test feeding period on oat seedlings. First fraction is test of 1:5 dilution, successive fractions are tests on serial B-fold dilutions of sample indicated. None of 36 plants infested with about 360 control aphids became infected.
ples was collected from the virus zone area of such centrifuged gradients, bioassays of the samplesshowed an even distribution of both virus isolates within the zone area. Since the sedimentation rate of MAV is about 115118 S (Rochow and Brakke, 1964), that of PAV and RPV is similar. Factors That Affect PuriJication Temperature at which source plants are grown is one of the major factors that in-
fluence yield of virus in purified preparations for all isolates of BYDV we have studied. Generally, virus yields have consistently been lower from plants grown in the greenhouse during hot summer months than from plants grown at other times of the year. Similar results occurred in controlled experiments. For example, in 3 separate comparisons Coast Black oats were inoculated with the PAV isolate and grown in growth chambers at 15’ or at 32”. An average of 14 pg of
BARLEY
YELLOW
DWARF
PAV was obtained from 100 ml of crude juice in preparations from plants grown at 15”; comparable preparations from plants grown at 32’ contained an average of 2 pg of PAV. In a comparison with the RPV isolate, a preparation made from plants grown in a growth chamber at 21” contained about 25 % more virus than did :i comparable preparation made from plant’s grown in the greenhouse, where fluctuat,ing bemperatures averaged about 15” higher than those in the growth chamber. Preparations of the MAV isolate from plants grown in growth chambers at 15’ , 21”, or 27” contained 10, S, and 5 pg of virus, respectively. We have not yet found a better host than Coast Black oats for production of virus. In some tests with the PAV isolate Coast Black oats were compared wit,11 2 varieties of barley (Horcleunz vulgare L.), a single variety of wheat (T&cum aostivum L.) and of rye (Se&e cereule L.), and several varieties of corn (Zea mays L.). In each of 13 separate test,s, when oats were compared with one of the other source plants, yields of the PAV isolate from oats were at least double that of virus yields from the other source plants. The interval between inoculation of oats and harvest for use as source plant,s can affect yields, especially for the PAV isolate. In 3 separate experiments plants were harvested and frozen 2,5, or S weeks after inoculat#ion. An average of 12 /*g of PAV was obtained from 100 ml of crude juice from plants harvested after 2 weeks, but comparable preparations from those harvested after 5 or 8 weeks contained only about 3 pg of PAV. Careful comparisons of this factor have not been made for the other isolates of BYDV, but observations suggest that the interval is less critical for the vector-specific isolates (MAV and RPV), which cause milder symptoms and less necrosis than does PAV. Various attempts to increase virus yields by treatment of plants with nutrients or with growth hormones have not been successful. Extraction of virus from infected tissue is a critical step in the purification procedure, as would be expected for a tissue-specific virus, such as BYDV, that affects plants by causing collapse of phloem tissue. An example of the import,ance of extraction is an
VIRUS
PCRIFICATIOK
121
experiment in which 4204 g of frozen, 11.1Vinfected tissue \yas processed once t,hrougll the fruit, juice extractor. Some of the crud0 juice obtained was used to make a prepsrntion concentrated loo-fold in volume. Thr> tissue from this single extraction was t.htw soaked in buffer, stored in a freezer for I:! days, thawed, and reground to prqtare :t second lot of crude juice. This juice \~XS used to make another preparation concentratthtl loo-fold for comparison with the one made from the original juice. The preparation nlade from the first extraction contained only half as much MAV as did t,hat ma& from the reground tissue. In another esperiment 5619 g of ?tL4V-infected tissue was used to make 8 comparable prepa&ions which were assayed by analytical sucrose density-gradient centrifugation. The first preparat,ion was made from juice obtained h\ one grinding in the extractor, the second from juice combined after two grindings, and the third from juice obtained from tissue ground twice in the juice extractor and once in a commercial Waring Blendor. The :3 final preparations contained 23, 41, and 46 pg of MAV, respectively. In tests l\ith small lots of P,4V-infected tissue, virus preparations made by extraction in the Waring Blendor” contained an average of 16 pg of PAV; parallel preparations made by extraction in small meat grinders cont,ained an average of 12 fig of PAV. Thorough tests of alternate possibilities have not been carried out for all steps of thch purification procedure, but results to tlatcb support use of current procedures. l“or example, in an experiment with MAT; a direct comparison was made among six different, clarification methods. In addition to the regularly-used chloroform procedure, treatments included clarification onlv by centrifugation, heating of the crude juice for IO min at 50”, treatment. with charcoal, trentment with calcium phosphate (formed in situ), and temporary acidification (pH l..i) of the juice. Teshs of the six final ~wpar:rtions showed that the one made from juice clarified by chloroform had more virus p:utitles, fewer impurities, and more infectivit>than did any of the comparable prepar:~tiolts. Similarly, nttempts to find a huffrr bettel
122
ROCHOW
than 0.1 M neutral potassium phosphate have so far been unsuccessful. One undesirable feature of the current purification procedure is the dependence on high-speed centrifugation for the initial concentration of virus. The low virus concentration and the large volume of clarified juice that must be processedmake centrifugation a limiting step in the procedure. Preliminary tests have been made of other methods of concentrating the virus from clarified juice. Precipitation by ethanol has been efficient and useful, especially for RPV, in some experiments. For routine use, however, precipitation by ethanol is not desirable because of the difficulty in resuspending virus and because of the large amounts of nonvirus material also precipitated. Preliminary tests with polyethylene glycol (PEG) (Hebert, 1963) have shown some promise. For example, when MAV was initially concentrated by mixing 48 g of PEG (Carbowax 6000)6 with 600 ml of clarified juice, a final preparation containing 16 pg/ml of MAV resulted from 1 cycle of differential centrifugation. A comparable preparation made by the standard procedure of 2 cycles of differential centrifugation yielded 30 pg/ml of virus. In comparison with the control, the preparation made by the PEG method had lessvirus, but the facts that it also contained fewer impurities and was made more easily and quickly suggest that’ further tests with PEG would be worthwhile. In the original studies on the MAV isolate yields of virus were only about 25-50 fig from 1 liter of plant juice. By careful attention to the factors just discussed,we have been able to increase yields over the original amounts, but they are still very low (Table 2). Results are usually most consistent with the MAV isolate, and the highest single yield to date, nearly 200 pg per 1000 g of source tissue, or 216 pg per liter of juice, has been obtained with MAV (Table 2). Yields of PAV have consistently been lowest, perhaps because much of the tissue for PAV came from plants harvested beyond the optimum interval of 2-3 weeks. The average yield of virus for MAV, RPV, and PAV in experiments during the past 3 years was 106, 79, and 20 pg, respectively, per 1000 g of source tissue.
ET
AL.
Storage of Concentrated Virus Preparations Comparative studies on preparations of different BYDV isolates are limited by the low yield of virus. To offset this difficulty, crude virus concentrates are stored in a freezer, and processedto make a final preparation just before virus is needed for an experiment. Tests were made on the adequacy of this procedure. In one experiment separate preparations of RPV and MAV were made by chloroform clarification and one cycle of differential centrifugation. Each preparation was concentrated about 200-fold relative to the original volume of clarified juice. A sample was removed for immediate assay, and the remaining preparation was divided among TABLE
2
YIELDS OF THREE ISOLATES OF BARLEY YELLOW DWARF VIRUS (BYDV) IN PREPARATIONS MADE FROM INFECTED OATS Estimates of ue of virus in final preparation .
BYDV isolate
MAV
RPV
PAV
Source tissue extracted kc)
4026 4098 5474 4544 2752 5173 3902 9378 4033 3586 7692 3771 3101 5452 3111 6249 9192 6785 3582
Per liter clarified juicea
134 98 108 216 106 133 164 123 84 43 103 97 135 55 16 4 31 40 23
”
Per 1000 g of source tissue 96 89
82 198 84 87 121 72 57 36 67 85 113 41 15 3 14 25 20
a In most cases the clarified juice included that obtained from the first extraction of the frozen source tissue together with that obtained from the second grinding of the tissue after it had been soaked in cold 0.1 M neutral potassium phosphate buffer.
BARLEY
YELLOW
DWARF
small tubes for st,orage in a freezer. The nonfrozen sample of each isolate was diluted in 20 “% sucrose and used in bioassays made by t,he membrane-feeding method. During the following year, at, 4-week intervals, a tube for each isolate was removed from the freezer, thawed, and diluted in 20 % buffered sucrose for similar bioassays. Although variat,ion occurred among results of 13 assays made for MAV, no t,rend boward loss of virus act,ivitg \vas evident. P’or example, the numbers of plants that became infected over t’he numbers infested with aphids in assays of three successive lo-fold dilutions of the original preparation were 9/9, 619, and l/9. The corresponding values for the assay made on similar dilut,ions of a frozen sample after 50 weeks of storage were 12/l’, 5,/12, and 4/l?. None of 21 plants infested as controls in t,hese 2 assays became infected. Somewhat more variation occurred among the 13 bioassays for RPV, but, no loss of infect,ivit,y was evident, for this virus isolate. In other experiments 25-50 ml of preparations concentrat,ed about %O-fold were stored in the freezer. At various intervals comparable samples if-ere removed from the freezer to make final virus preparations. When yields of these preparations were compared by analytical sucrose density-gradient centrifugation, suitability of the procedure was confirmed. Yields of nIAV in 3 final preparaCons stored for 28, 47, and 65 weeks were 140, 104, and 119 ~g, respectively. In another comparison with the MAV isolate, yields were 140, $2, and I:%$ pg after storage for 63, 79, and 112 weeks, respectively. For RPV one series of tests yielded X4, 84, and 56 pg of virus after storage for 51, 98, and 131 weeks, respectively. Because storage of such concentrates has so man>- advantages and because no serious disadvantages were encountered in these and ot’her t’ests, the procedure has become st,andard in our studies of BYDV. Preparation
of’ Antisera
The antigens for immunization of rabbits were preparations of the MAV, RPV, and PL4V isolates of BYDV and a comparable concent,rate made from healthy oats. Each ureuaration. concentrated about, ,%M-fold ~.. 1 1
VIRUS
I z:i
PURIFICATION
in a final volume of about 6 ml, was made from frozen concent,rates t,he day before each of 3 injections. About 0.2 ml of each prepaw t,ion was used for bioassay to det,ermine vect,or specificity of the preparations act,uallJ inject)ed into the rabbits. All tests (Table :3) confirmed the identity of each of the 3 virus isolat,es and showed the absence of virus from the control preparations. All preparations were centrifuged on sucrose gradient,s (rate zonal, as in Table 1). Some of the gradient columns were scanned with the IMXP drnsity-gradient fractionator to estimate virus concentration; from other tubes the virus zones were collected directly by hypodermic syringe. Virus collect,ed from t,he densitygradient columns was used for injection into rabbit,s either by mixing with Freund’s complete adjuvant or with st>erile distilled water. Tot,al amounts of virus inject,ed into each rabbit were about 345 pg for MAV, 271 pg for RI’V, and 1SS pg for PAV. Approximat,elg one-third of the tot,al was used for each of 3 injections. About’ :<5 liters of crude juice were used t’o make t’hexe preparations, which had been accumulated as frozen wncentrat,es during t,hr previous 2 years. TABLIS INFECTIVITY
3
OF FOUR KINDS USED TO IMMCNIZE
Preparation from plants infected by YXUS isolate shown
RPV MAV PAV None (healthy oats) Aphid cont,rols
OF PREPARATIONS RAHBITS
Transmissiona in membrane-feeding or injection assays in parallel tests with two aphid species Membrane
Injection
R. padi
Af. nwnae
R. podi
M. mmae
25/27 O/27 18/27 O/27
o/27 27/27 11127 O/27
g/g O/18 12/18 - --
O/18 9/9 6/1X
O/27
O/27
O/18
O/18
D Numerator is number of plants that became infected; denominat,or is number infested wit#h 10 aphids that had fed through membranes for about 18 hr at 15”, or number that were infected with 5 aphids each injected with about 0.02 ~1 of preparation shown. In both kinds of assays, inoculated test feeding was 5 days at 21”. Data are combined results of a separate test on each of 3 preparations made at the bime rabbits were injected.
124
ROCHOW
Two rabbits were injected with each of the 4 purified preparations, but only serum from 1 rabbit was used in the work with each of the 4 sera described here and elsewhere (Aapola and Rochow, 1971). About 6 ml of each antigen preparation from the densitygradient zones was mixed with 2 ml of Freund’s complete adjuvant. About 2 ml of the emulsion was then injected into each hind leg of two rabbits. The second injection was carried out in the same way 1 week after the first. For the final injection, 4 weeks after the first, preparations from the sucrose gradients were mixed with an equal volume of sterile distilled water for injection through the marginal ear vein. Terminal bleeding was carried out 2 weeks after the final injection. Yields of serum for each animal varied from 19 to 40 ml. Sera were stored in a freezer.
ET AL.
no transmission occurred after incubation with the homologous antiserum (Table 4). Transmission of MAV by M. avenue was reduced following incubation with the PAV antiserum, and transmission of PAV by R. padi was reduced following incubation with the MAV antiserum, but other heterologous combinations had no pronounced effect. These data suggest that MAV and PAV are related, but that RPV is distinct from the other two. A more thorough study of the relationship among the 3 virus isolates (Aapola and Rochow, 1971), based on in vivo interactions and use of 2 additional serological techniques, supports this relationship among the 3 virus isolates and demonstrates that the antisera are highly reactive. TABLE REACTION
Comparison of Virus Isolates by Serological Blocking of Aphid Transmission
The 3 isolates of BYDV were compared by using the antisera in a type of infectivity neutralization test that has proved useful for circulative aphid-transmitted viruses (Gold and Duffus, 1967; Rochow and Ball, 1967). The test is based on incubation of virus with antiserum, allowing aphids to feed through membranes on the mixture, and determining whether or not the aphids can transmit virus after the acquisition feeding. Previous tests by this procedure have suggested that MAV and RPV are serologically distinct (Rochow and Ball, 1967; Rochow, 1970). Each of the 4 antisera was mixed with a concentrate of one of the virus isolates, incubated at 37” for 30 min, often stored overnight at 4’, combined with an equal volume of 40 % sucrose in buffer, and used in membrane feeding assays. In some tests, the virus-antiserum mixture was centrifuged before sucrose was added, but results were the same if centrifugation was omitted. Aphids were allowed to feed through stretched ParafilmG on the buffered antigenantibody mixture for about 18 hr at 15” and then transferred to Coast Black oat seedlings (10 aphids per seedling) for a 5-day inoculation test feeding period. In all tests, except one with the highest concentration of MAV,
DWARF ANTISERA BLOCKING
Virus
isolate
OF THREE VIRUS AS
(Irg/mlY
MAV
RPV
PAV
0.4 0.8 2.0 4.0 4.0 9.0 4.0 5.0 10.0 12.9
OF BARLEY
WITH EXH DETERMINED
OF APHID Virus gti$
4
ISOLATES
YELLOW
OF FOUR SPECIFIC BY SEROLOGICAL
TRANSMISSION
OF VIRUS
Transmissionb following incubation of virus with antiserum or control shown MAV serum
RPV serum
0 0 0 0 0 2 5 4 7 6 0 1 1
8 9 9 9 9 9 0 0 0 0 7 7 8
PAV Serum
0 0 2 3 2 8 2 1 6 5 0 0 0
H.O. serum
aphid control
5 9 9 8 9 9 4 7 9 6 5 8 8
0 0 0 0 0 0 0 0 0 0 0 0 0
a Virus concentration of preparation used; concentration actually fed upon by aphids was s that shown because virus was first mixed with an equal volume of antiserum (diluted 1:5), and this mixture was later added to an equal amount of 40% sucrose for aphid feeding. In 3 experiments virus concentration was not known. b Number of plants that became infected out of 9 infested with 10 aphids (M. awelzae for MAV; R. pa& for RPV and PAV) that had fed through membranes for about 18 hr at 15’ on mixture indi. cated before start of 5-day inoculation test feeding period on oat seedlings.
BtlRLEY
YELLOW
DWARF
DISCUSSION
A distinctive feature for purification of BYDV is the minute amount of virus obtained from infected plants in comparison with yields of most other plant viruses that have been purified. Although we have been able to increase yields of BYDV over those of earlier experiments by altering factors that affect purificat,ion, any major increase in yields probabl>* will have to await discovery of better source plants or of methods to alter the restriction of virus Do phloem tissue and the disint’egrabion of t,his tissue as infection proceeds. The stability of BYDV and t,he use of frozen crude concentrates partly compensates for the low amounts of virus obtained from infected plants. The polyhedral virus particles observed in shadowed preparations of RPV and PAV were about 30 nm in diameter, a value previously reported for MAV (Rochow and Brakke, 1964). *Jensen (1969) has observed BYDV particles about 24 nm in diameter in thin sections of host tissue and H. W. Israel (unpublished data) has found the particles of several isolates to be about 20 nm in negat,ivel?, stained virus preparations. As Jensen (1969) has point’ed out’, these discrepancies probably are merely reflections of differences among the methods used to prepare specimens for electron microscopy. In addition t’o the 3 isolates of BYDV investigated here 2 additional variants have been encountered in our st,udies of field-collected plants. One is the R;\IV isolate transmitted specifically by K. maidis (Fitch), the biological properties of which have been described (Rochon-, 1969). The other variant was recovered from several plants collected in the field in Xew York during the summer of 1969. It appears to be similar to the vector specific variant previously described by Gill (1969) and is transmitted specifically by Schizaphis qraminunl (Rondani) . Preliminary purification experiments n-it,11these 2 isolates have shown that the same procedures can be used for them as for the 3 isolates studied here, but that yields of virus are even lower than t,hose obtained for the I’IZV isolate. Current evidence for the relationship among the 3 isolates of BYDV (Aapola and
VIRUS
12.3
PURIFICATION
Rochow, 1971) suggests that MAV and PAV are related but distinct from RPV. The similarity in sedimentation and morphology for all 3 isolates does not, conflict with the evidence hhat RPV is different from the other t,wo becausedata are not exhaustive on either point and neither criterion is especiallluseful in determining virus relationships. Moreover, preliminary data from ultraviolet absorption spectra of RI’V and MAV have also shown the 2 isolates to be different,. The A 260/280 for MAV wa’sabout 1.92 and that for RPV was a,bout 1.72 in some studies at Lincoln. ACKNOWLEDGMENTS We thank and providing
Irmgard general
Muller for injecting technical assistance.
aphids
REFERENCES AAI>oL.~, A. I. E., and RocH~~, W. F. (1971). Relationships among three isolates of barley yellow dwarf virus. VGology 46, 127-141. ARAI, K., DOI, Y., YOR.\, K., and Asuu~mn, H. (1969). Electron microscopy of the potato leafroll virus in leaves of three kinds of host plants and the partial purification of the virus. .I~TL. Phytopathol. Sot. Japan 35, lo--Xi. DUFFUS, J. E. (1969). Membrane feeding used in determining the properties of beet western yellows virus. Phytopathology 59, 1668-1669. DUFFUS, J. E., and GOLU, A. H. (1969). Membrane feeding and infectivity neut,ralization used in a serological comparison of potato leaf roll and beet western yellows viruses. I’irology :1’i, 150 153. GILL, C. C. (1969). Annual variation in strains of barley yellow dwarf virus in Manitoba, and 1 he occurrence of greenbug-specific isolates. Cnr,. .r. Bot. 47, 1277-1283. GOLD, A. H., and DUFFUS, J. E. (1967). Infect,ivitJ neutralization-a serological method as applied to persistent viruses of beets. T’irol0g.q 31, 308.. 313. HERERT, T. T. (1963). Precipitation of plant viruses by polyethylene glycol. Phytopathology 53,362. JENSEN, 8. G. (1969). Occurrence of virus particles in the phloem tissue of BYDV-infected barley. Virology 38, 83-91. KOJIM.~, M., SHIK.~T~, E., S~UG.~\~~.~Z, M., and MURAYMA, D. (1969). Purification and elect ran microscopy of potato leafroll virus. T’irolog!/ 30, 162-174.
126
ROCHOW
D. (1967). The purification of potato leafroll virus from its vector Myzus persicae. Viralogy 31, 46-54. PETERS, D., and VAN LOON, L. C. (1968). Transmission of potato leafroll virus by aphids after feeding on virus preparations from aphids and plants. VGoZogy 35, 597-609. ROCHOW, W. F. (1959). Transmission of strains of barley yellow dwarf virus by two aphid species. PETERS,
Phytopathology ROCHOW, W.
49, 744-748.
F. (1969). Biological
properties
of
ET
AL.
four isolates of barley yellow dwarf virus. Phyto59, 1580-1589. W. F. (1970). Barley yellow dwarf virus: Phenotypic mixing and vector specificity. Science 167, 875878. ROCHOW, W. F., and BALL, E. M. (1967). Serological blocking of aphid transmission of barley yellow dwarf virus. Virology 33, 359-362. ROCHOW, W. F., and BRAKKE, M. K. (1964). Purification of barley yellow dwarf virus. Vi’irology 24, 31&322. pathology ROCHOW,
VIHOLOGY
(1971)
Purification
and
Antigenicity
Barley
Yellow
W. F. ROCHOW,2
of Three Dwarf
Isolates
of
Virus’
A. I. E. AAPOLA; MYRON L. E. CARMICHAEL5
K. BRAKKE:
AND
of Plant University of
Department
Pathology, Nebraska,
Cornell Lincoln, Cornell
University, Nebraska University,
Ithaca, New York 14850; Platlt Pathology Departme)lt, 68508; and J7eterinary Virus Research Institute, Ithaca, Mew York 14860
Accepted
May
24, 1971
An isolate (RPV) of barley yellow dwarf virus transmitted specifically by Rhopalosiphuwt padi and an isolate (PAV) transmitted nonspecifically by both R. padi and Macrosiphum avenae were purified by procedures previously found satisfactory for another isolate (MAV) transmitted specifically by M. avenae. As with MAV, infectivity of RPV and PAV samples removed from sucrose gradient columns was associated with a dense polyhedral particle about 30 nm in diameter in shadowed preparations. No differences in sedimentation rate (sedimentation coefficient 115-118 S) among the 3 virus isolates were detected in parallel sucrose gradient centrifugation tests. Temperature at which source plants were grown was an important factor affecting results of purification. Preparations made from oats grown at 15”-20” contained about 2-5 times more virus than did preparations made from plants grown at temperatures above 25“. Coast Black oats (Avena byzantina) was the best virus source among 5 host plant species tested. Preparations of the PAV isolate made from plants harvested 2-3 weeks after inoculation contained about 4 times as much virus as did preparations from plants harvested after 5-S weeks. Thorough extraction of virus from infected tissue was another critical step in the purification procedure. The average yield of virus (per 1000 g of source tissue) was 106,79, and 20 pg, respectively, for MAV, RPV, and PAV. Accumulation of frozen crude concentrates for each of the 3 virus isolates during a period of about 2 years made possible the production of final virus preparations for use in development of specific antisera. A highly reactive antiserum specific for each of t.he 3 virus isolates w&9 obtained by injecting 20&350 pg of purified virus into single rabbits in a series of 3 injections. Serological comparisons of the 3 virus isolates, based on a type of infectivity neutralization that involves serological blocking of virus transmission by aphids, suggested that the MAV and PAV isolates are relat,ed, but that RPV is distinct from the other 2 isolates. 1 Cooperative Investigation, Plant Science Research Division, Agricultural Research Service, U.S. Department of Agriculture; Cornell University Agricultural Experiment Stat.ion; Nebraska Agricultural Experiment Station; and Cornell University Veterinary Virus Research Institute. Supported in part by NSF grant GB-21013 to Cornell University, and by NSF grant GB-8283 to the University of Nebraska. Published with the approval of the Director as Paper No. 3062, Journal Series, Nebraska Agricultural Experiment Station, Project No. 21-12. 2 Research Plant Pathologist, Plant Science Re-
search Division, Agricultural Research Service, U.S. Department of Agriculture; and Professor of Plant Pathology, Cornell University. 3 Formerly, Graduate Assistant, Department of Plant Pathology, Cornell University. Present atldress : Irrigated Agriculture Research and I:sterlsion Center, Presser, Washington 99350. 4 Chemist,, Plant Science Research Divisioll, Agricult,ural Research Service, U.S. Department. of Agriculture; and Professor of Plant Pathology, Universit,y of Nebraska. 5 Professor of Virology, Veterinary \‘ims I:{:search Instit,ute, Cornell Universit J-. 117
118
ROCHOW INTRODUCTION
Progress in purification studies has been slow and difficult for that small but economically important group of persistent or circulative aphid-transmitted plant viruses that have not been transmitted mechanically to plants. Limiting factors in such studies include the difficulties of bioassay, the low concentration of virus extracted from infected plants or from viruliferous aphids, and the instability of some of the viruses. Three representatives of the group, which have many features in common, are potato leaf roll virus (Arai et al., 1969; Peters, 1967; Peters and Van Loon, 1968; Kojima el al., 1969), beet western yellows virus (Duffus, 1969; Duffus and Gold, 1969), and barley yellow dwarf virus (BYDV), the subject of this report. Purification of the MAV isolate of BYDV was described previously (Rochow and Brakke, 1964). Here we report studies on purification and identification of the infectious particle for 2 additional isolates, RPV and PAV. All 3 isolates were originally differentiated on the basis of virus-vector relationships (Rochow, 1969). We also report here on factors that affect purification of BYDV, on production of antisera, and on some serological comparisons among 3 isolates of the virus. A more detailed study of relationships among the 3 isolates appears separately (Aapola and Rochow, 1971). MATERIALS
AND
METHODS
Virus-free aphids were maintained in isolated rea,ring rooms on caged barley plants (Hordeum vulgare L.) under special precautions (Rochow, 1959; Rochow and Brakke, 1964). Mucrosiphum avenue (Fabricius), the English grain aphid, and Rhopalosiphum padi (Linnaeus), the oat bird-cherry aphid, were the 2 speciesused regularly. Occasionally additional aphid species were used in some comparative tests. At least 30 aphids from each group were always tested as controls in every experiment. The biological properties of the 3 isolates of BYDV have recently been described (Rochow, 1969). One isolate (PAV) is transmitted nonspecifically by both R. pa& and M. avenue, another isolate (RPV) is transmitted specifically by R. pa&, and the third
ET AL.
isolate (MAV) is transmitted specifically by M. avenue. The virus isolates were maintained by serial transmissions at intervals of 5-6 weeks to Coast Black oats (Avena byzuntina K. Koch). Comparative tests with 4 aphid specieswere made at the time of each transfer to make certain that no major change in the relative vector specificity of the isolate occurred during these studies (Rochow, 1969). Coast Black oats were test plants as well as source plants for virus purification work, unless specified otherwise. Plants were grown in 4-inch clay pots of composted, steam-sterilized soil. Unless stated otherwise, the buffer used in all studies was 0.1 M neutral potassium phosphate; it will be referred to simply asbuffer. Infectivity of virus preparations was estimated by membrane-feeding or injection methods previously described (Rochow, 1969,197O). Virus concentration in the preparations was estimated by comparing the height of peaks of analytical density-gradient scanning patterns obtained from an ISCOG density-gradient fractionator with those obtained with known concentrations of a purified preparation of southern bean mosaic virus (Aapola and Rochow, 1971). Unless stated otherwise, all gradients were prepared 48 hrs before use with 4,7,7, and 7 ml of 100, 200,300, and 400 mg of sucroseper milliliter, respectively, dissolved in buffer. Rate zonal centrifugation was for 3 hrs at 23,000 rpm at about 9” in the SW 25.1 rotor of the Spinco Model L centrifuge.6 Our current routine procedure for producing preparations of BYDV involves aphid inoculation of 3 or 4 seedlingsin each of 90 pots of Coast Black oats, growing the inoculated plants in a greenhouse for 4-6 weeks, harvesting entire shoots (rarely, also roots), and cutting the harvested plants into sections about 4 cm long for storage of the tissue in plastic bags in a freezer at about -20’. When 4-8 such bags, each containing 5002000 g of tissue, have been accumulated for a virus isolate, the tissue is chopped while still 6 Mention of a trademark name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the U.S. Department of Agriculture, and does not imply its approval to the exclusion of other products also available.
BARLEY
YELLOW
DWARF
partly frozen in a Hobart6 food cutter. The chopped tissue is then ground in a Hobart6 type D fruit juice extractor, which separates the juice from the residue. In lots of 450 ml the crude juice is mixed with 50 ml of 1.0 M neutral potassium phosphate buffer for clarification with chloroform and n-amyl alcohol (Rochow and Brakke, 1964). The tissue residue is soaked in cold 0.1 M buffer and ground again in the fruit juice extractor. In lots of 500 ml the juice from this second extraction is also clarified. The twice-ground residue is soaked again in buffer, packed into a plastic bag, and refrozen for further extractions at a later date when several kilograms of such tissue have been accumulated. The several liters of clarified juice are then centrifuged for 4 hrs in a 30 rotor of a SpincoG Model L centrifuge. Pellets are resuspended in a small amount of buffer with the aid of a ground glass homogenizer, and the concentrated preparation is centrifuged at 4800 rpm for 15 min in a Servall SP6 centrifuge at 4”. The pellet from t-his low-speed centrifugation is washed several times with buffer, and the superna.tants are combined to make a concentrate about 20-fold relative to the original volume of clarified juice. Such concentrates are then stored in 25 or 50-ml bottles in a freezer. When final virus preparations are needed for an experiment, the frozen concentrates are removed from the freezer, t’hawed, and used to make the required virus preparation by further cycles of differential and sucrose gradient centrifugation. RESULTS
Purification of RPV and PAV Concentrated preparations of the RPV or PAV isolates were made from frozen oat tissue by chloroform clarification and differential centrifugation as previously described for MAV (Rochow and Brakke, 1964). Correlation of virus particles and infectivity was st.udied by assay of samples removed from density-gradient centrifugation columns (Table 1). F’or density-gradient centrifugation, 1.0 ml of a virus preparation was layered on one of three sucrosegradient’s, 1.0 ml of a, comparable preparation made from healthy oats was layered on a second, and 1.0 ml of a purified preparation of southern
VIRUS
PURIFICATION
119
bean mosaic virus (containing 0.1 mg of virus) was layered on a third. When a visible zone was seen in tubes containing RPV or PAV, its location coincided with, or was slightly lower t)han, that of the visible zone in the tube containing only southern bean mosaic virus. In experiments where a visible zone did not occur for either isolate of BYDV, the location of the corresponding southern bean mosaic virus zone was used as the basis for sampling. Samples were removed from the virus zone, from areas above and below the zone, and from the gradient, containing the preparation from healthy oats. at a location corresponding to the dept’h of the virus zone. About 0.2 ml of each of the 4 samples from an experiment was shipped t.o Lincoln, Nebraska, for electron microscopy, and the remaining portion of each sample was used in Ithaca for infectivity assays b? the membrane-feeding method. In some experiments t,he cernrifuged sucrose gradient. columns were scanned and sampleswere colected by means of the ISCOG density-gradient fractionator. Results of 11 separate experiments, in volving both rate zonal and equilibrium zonal centrifugation, were similar to results previously obtained for RIAV (Rochow and Brakke, 1964). Infectivity of both the RPV and PAV isolates was associatedwith a dense polyhedral particle about 30 nm in diameter in shadowed preparations (Table 1j. Rot.11 RPV and PAV appeared similar in sedimentation and morphology to the previously described hIAV isolate. Sedimentation of the t
120
ROCHOW TABLE
ET AL. 1
RELATION BETWEEN PARTICLES COUNTED IN ELECTRON MICROGRAPHS AND INFECTIVITY OF SAMPLES FOR Two ISOLATES OF BARLEY YELLOW DWARF VIRUS (BYDV) REMOVED FROM RATE-Z• NAL (RZ) OR EQUILIBRIUM-Z• NAL (EZ) DENSITY-GRADIENT COLUMNS
BYDV isolate and kind of colum@
Buffer used to dissolve sucrose
RPV RZ
0.05 M borate, pH 8.0
PAV RZ
0.1 M phosphate, pH 7.0
RPV EZ
0.1 M phosphate, pH 7.0
PAV EZ
0.1 M phosphate, pH 7.0
Sample identification and cm below meniscusb A B C H A B C H A B c H A B c H
1.2 1.7 2.7 1.7 1.2 1.7 2.7 1.7 1.9 2.4 2.9 2.4 2.0 2.5 3.5 2.5
Relative number particles per sample” 2 397, 124, 31 8 1 3 12, 3, 0.6 0.08 0.03 0.01 48, 21, 5, 0.5 8 0.004 0.8 97, 24, 4, 1 1 1
Infectivity of sampled
7/g,l/9 9/g,g/g,819 71% o/g o/g o/g,l/9 7/g, w-4 o/g 01% 9/g, 7/g, o/g o/g, 8/g, 2/g, 019
3/g, 31% l/9 o/9 o/g ‘J/9, 6/g, 6/g 71% 319 o/g, o/g g/g, 5/g, 2/g 2/g, 119
a RZ gradient columns were prepared 48 hr before use with 4, 7, 7, 7 ml of 100, 200, 300, and 400 mg sucrose per milliliter, respectively, dissolved in the buffer indicated. EZ columns were similarly prepared with 0.9 ml each of solutions containing 300,400, 500, and 600 mg of sucrose per milliliter. b Samples A, B, and C were from tubes containing the virus preparation; sample H was from tube containing the control preparation made from healthy plants. Centrifugation wm for 3 hr at 23,000 rpm at about 9” in the SW 25 rotor of the Spinco centrifuge for RZ tubes, and for 5 hr at 36,009 rpm at about 9” in the SW 39 rotor for EZ tubes. c Specimens were shadowed from three directions, and the particles were counted on the screen of the RCA EMU3G at 105,000 X . Numbers given are averagesper 3 X 4-inch area at this magnification. Particles were counted on at least 25 randomly selected areas when they were numerous. When there were only a few particles per 3 X 4-inch area, the number per 3-inch traverse across a grid opening was counted and divided by 75. At least 7 such traverses were counted for each such sample. Two grids were examined per sample. The first number in each series is for an undiluted sample and subsequent numbers represent successive B-fold dilutions. d Number of plants that became infected over number infested with 10 aphids (R. pa&) that had fed through membranes for about 18 hr on sample indicated before start of 5-day inoculation test feeding period on oat seedlings. First fraction is test of 1:5 dilution, successive fractions are tests on serial B-fold dilutions of sample indicated. None of 36 plants infested with about 360 control aphids became infected.
ples was collected from the virus zone area of such centrifuged gradients, bioassays of the samplesshowed an even distribution of both virus isolates within the zone area. Since the sedimentation rate of MAV is about 115118 S (Rochow and Brakke, 1964), that of PAV and RPV is similar. Factors That Affect PuriJication Temperature at which source plants are grown is one of the major factors that in-
fluence yield of virus in purified preparations for all isolates of BYDV we have studied. Generally, virus yields have consistently been lower from plants grown in the greenhouse during hot summer months than from plants grown at other times of the year. Similar results occurred in controlled experiments. For example, in 3 separate comparisons Coast Black oats were inoculated with the PAV isolate and grown in growth chambers at 15’ or at 32”. An average of 14 pg of
BARLEY
YELLOW
DWARF
PAV was obtained from 100 ml of crude juice in preparations from plants grown at 15”; comparable preparations from plants grown at 32’ contained an average of 2 pg of PAV. In a comparison with the RPV isolate, a preparation made from plants grown in a growth chamber at 21” contained about 25 % more virus than did :i comparable preparation made from plant’s grown in the greenhouse, where fluctuat,ing bemperatures averaged about 15” higher than those in the growth chamber. Preparations of the MAV isolate from plants grown in growth chambers at 15’ , 21”, or 27” contained 10, S, and 5 pg of virus, respectively. We have not yet found a better host than Coast Black oats for production of virus. In some tests with the PAV isolate Coast Black oats were compared wit,11 2 varieties of barley (Horcleunz vulgare L.), a single variety of wheat (T&cum aostivum L.) and of rye (Se&e cereule L.), and several varieties of corn (Zea mays L.). In each of 13 separate test,s, when oats were compared with one of the other source plants, yields of the PAV isolate from oats were at least double that of virus yields from the other source plants. The interval between inoculation of oats and harvest for use as source plant,s can affect yields, especially for the PAV isolate. In 3 separate experiments plants were harvested and frozen 2,5, or S weeks after inoculat#ion. An average of 12 /*g of PAV was obtained from 100 ml of crude juice from plants harvested after 2 weeks, but comparable preparations from those harvested after 5 or 8 weeks contained only about 3 pg of PAV. Careful comparisons of this factor have not been made for the other isolates of BYDV, but observations suggest that the interval is less critical for the vector-specific isolates (MAV and RPV), which cause milder symptoms and less necrosis than does PAV. Various attempts to increase virus yields by treatment of plants with nutrients or with growth hormones have not been successful. Extraction of virus from infected tissue is a critical step in the purification procedure, as would be expected for a tissue-specific virus, such as BYDV, that affects plants by causing collapse of phloem tissue. An example of the import,ance of extraction is an
VIRUS
PCRIFICATIOK
121
experiment in which 4204 g of frozen, 11.1Vinfected tissue \yas processed once t,hrougll the fruit, juice extractor. Some of the crud0 juice obtained was used to make a prepsrntion concentrated loo-fold in volume. Thr> tissue from this single extraction was t.htw soaked in buffer, stored in a freezer for I:! days, thawed, and reground to prqtare :t second lot of crude juice. This juice \~XS used to make another preparation concentratthtl loo-fold for comparison with the one made from the original juice. The preparation nlade from the first extraction contained only half as much MAV as did t,hat ma& from the reground tissue. In another esperiment 5619 g of ?tL4V-infected tissue was used to make 8 comparable prepa&ions which were assayed by analytical sucrose density-gradient centrifugation. The first preparat,ion was made from juice obtained h\ one grinding in the extractor, the second from juice combined after two grindings, and the third from juice obtained from tissue ground twice in the juice extractor and once in a commercial Waring Blendor. The :3 final preparations contained 23, 41, and 46 pg of MAV, respectively. In tests l\ith small lots of P,4V-infected tissue, virus preparations made by extraction in the Waring Blendor” contained an average of 16 pg of PAV; parallel preparations made by extraction in small meat grinders cont,ained an average of 12 fig of PAV. Thorough tests of alternate possibilities have not been carried out for all steps of thch purification procedure, but results to tlatcb support use of current procedures. l“or example, in an experiment with MAT; a direct comparison was made among six different, clarification methods. In addition to the regularly-used chloroform procedure, treatments included clarification onlv by centrifugation, heating of the crude juice for IO min at 50”, treatment. with charcoal, trentment with calcium phosphate (formed in situ), and temporary acidification (pH l..i) of the juice. Teshs of the six final ~wpar:rtions showed that the one made from juice clarified by chloroform had more virus p:utitles, fewer impurities, and more infectivit>than did any of the comparable prepar:~tiolts. Similarly, nttempts to find a huffrr bettel
122
ROCHOW
than 0.1 M neutral potassium phosphate have so far been unsuccessful. One undesirable feature of the current purification procedure is the dependence on high-speed centrifugation for the initial concentration of virus. The low virus concentration and the large volume of clarified juice that must be processedmake centrifugation a limiting step in the procedure. Preliminary tests have been made of other methods of concentrating the virus from clarified juice. Precipitation by ethanol has been efficient and useful, especially for RPV, in some experiments. For routine use, however, precipitation by ethanol is not desirable because of the difficulty in resuspending virus and because of the large amounts of nonvirus material also precipitated. Preliminary tests with polyethylene glycol (PEG) (Hebert, 1963) have shown some promise. For example, when MAV was initially concentrated by mixing 48 g of PEG (Carbowax 6000)6 with 600 ml of clarified juice, a final preparation containing 16 pg/ml of MAV resulted from 1 cycle of differential centrifugation. A comparable preparation made by the standard procedure of 2 cycles of differential centrifugation yielded 30 pg/ml of virus. In comparison with the control, the preparation made by the PEG method had lessvirus, but the facts that it also contained fewer impurities and was made more easily and quickly suggest that’ further tests with PEG would be worthwhile. In the original studies on the MAV isolate yields of virus were only about 25-50 fig from 1 liter of plant juice. By careful attention to the factors just discussed,we have been able to increase yields over the original amounts, but they are still very low (Table 2). Results are usually most consistent with the MAV isolate, and the highest single yield to date, nearly 200 pg per 1000 g of source tissue, or 216 pg per liter of juice, has been obtained with MAV (Table 2). Yields of PAV have consistently been lowest, perhaps because much of the tissue for PAV came from plants harvested beyond the optimum interval of 2-3 weeks. The average yield of virus for MAV, RPV, and PAV in experiments during the past 3 years was 106, 79, and 20 pg, respectively, per 1000 g of source tissue.
ET
AL.
Storage of Concentrated Virus Preparations Comparative studies on preparations of different BYDV isolates are limited by the low yield of virus. To offset this difficulty, crude virus concentrates are stored in a freezer, and processedto make a final preparation just before virus is needed for an experiment. Tests were made on the adequacy of this procedure. In one experiment separate preparations of RPV and MAV were made by chloroform clarification and one cycle of differential centrifugation. Each preparation was concentrated about 200-fold relative to the original volume of clarified juice. A sample was removed for immediate assay, and the remaining preparation was divided among TABLE
2
YIELDS OF THREE ISOLATES OF BARLEY YELLOW DWARF VIRUS (BYDV) IN PREPARATIONS MADE FROM INFECTED OATS Estimates of ue of virus in final preparation .
BYDV isolate
MAV
RPV
PAV
Source tissue extracted kc)
4026 4098 5474 4544 2752 5173 3902 9378 4033 3586 7692 3771 3101 5452 3111 6249 9192 6785 3582
Per liter clarified juicea
134 98 108 216 106 133 164 123 84 43 103 97 135 55 16 4 31 40 23
”
Per 1000 g of source tissue 96 89
82 198 84 87 121 72 57 36 67 85 113 41 15 3 14 25 20
a In most cases the clarified juice included that obtained from the first extraction of the frozen source tissue together with that obtained from the second grinding of the tissue after it had been soaked in cold 0.1 M neutral potassium phosphate buffer.
BARLEY
YELLOW
DWARF
small tubes for st,orage in a freezer. The nonfrozen sample of each isolate was diluted in 20 “% sucrose and used in bioassays made by t,he membrane-feeding method. During the following year, at, 4-week intervals, a tube for each isolate was removed from the freezer, thawed, and diluted in 20 % buffered sucrose for similar bioassays. Although variat,ion occurred among results of 13 assays made for MAV, no t,rend boward loss of virus act,ivitg \vas evident. P’or example, the numbers of plants that became infected over t’he numbers infested with aphids in assays of three successive lo-fold dilutions of the original preparation were 9/9, 619, and l/9. The corresponding values for the assay made on similar dilut,ions of a frozen sample after 50 weeks of storage were 12/l’, 5,/12, and 4/l?. None of 21 plants infested as controls in t,hese 2 assays became infected. Somewhat more variation occurred among the 13 bioassays for RPV, but, no loss of infect,ivit,y was evident, for this virus isolate. In other experiments 25-50 ml of preparations concentrat,ed about %O-fold were stored in the freezer. At various intervals comparable samples if-ere removed from the freezer to make final virus preparations. When yields of these preparations were compared by analytical sucrose density-gradient centrifugation, suitability of the procedure was confirmed. Yields of nIAV in 3 final preparaCons stored for 28, 47, and 65 weeks were 140, 104, and 119 ~g, respectively. In another comparison with the MAV isolate, yields were 140, $2, and I:%$ pg after storage for 63, 79, and 112 weeks, respectively. For RPV one series of tests yielded X4, 84, and 56 pg of virus after storage for 51, 98, and 131 weeks, respectively. Because storage of such concentrates has so man>- advantages and because no serious disadvantages were encountered in these and ot’her t’ests, the procedure has become st,andard in our studies of BYDV. Preparation
of’ Antisera
The antigens for immunization of rabbits were preparations of the MAV, RPV, and PL4V isolates of BYDV and a comparable concent,rate made from healthy oats. Each ureuaration. concentrated about, ,%M-fold ~.. 1 1
VIRUS
I z:i
PURIFICATION
in a final volume of about 6 ml, was made from frozen concent,rates t,he day before each of 3 injections. About 0.2 ml of each prepaw t,ion was used for bioassay to det,ermine vect,or specificity of the preparations act,uallJ inject)ed into the rabbits. All tests (Table :3) confirmed the identity of each of the 3 virus isolat,es and showed the absence of virus from the control preparations. All preparations were centrifuged on sucrose gradient,s (rate zonal, as in Table 1). Some of the gradient columns were scanned with the IMXP drnsity-gradient fractionator to estimate virus concentration; from other tubes the virus zones were collected directly by hypodermic syringe. Virus collect,ed from t,he densitygradient columns was used for injection into rabbit,s either by mixing with Freund’s complete adjuvant or with st>erile distilled water. Tot,al amounts of virus inject,ed into each rabbit were about 345 pg for MAV, 271 pg for RI’V, and 1SS pg for PAV. Approximat,elg one-third of the tot,al was used for each of 3 injections. About’ :<5 liters of crude juice were used t’o make t’hexe preparations, which had been accumulated as frozen wncentrat,es during t,hr previous 2 years. TABLIS INFECTIVITY
3
OF FOUR KINDS USED TO IMMCNIZE
Preparation from plants infected by YXUS isolate shown
RPV MAV PAV None (healthy oats) Aphid cont,rols
OF PREPARATIONS RAHBITS
Transmissiona in membrane-feeding or injection assays in parallel tests with two aphid species Membrane
Injection
R. padi
Af. nwnae
R. podi
M. mmae
25/27 O/27 18/27 O/27
o/27 27/27 11127 O/27
g/g O/18 12/18 - --
O/18 9/9 6/1X
O/27
O/27
O/18
O/18
D Numerator is number of plants that became infected; denominat,or is number infested wit#h 10 aphids that had fed through membranes for about 18 hr at 15”, or number that were infected with 5 aphids each injected with about 0.02 ~1 of preparation shown. In both kinds of assays, inoculated test feeding was 5 days at 21”. Data are combined results of a separate test on each of 3 preparations made at the bime rabbits were injected.
124
ROCHOW
Two rabbits were injected with each of the 4 purified preparations, but only serum from 1 rabbit was used in the work with each of the 4 sera described here and elsewhere (Aapola and Rochow, 1971). About 6 ml of each antigen preparation from the densitygradient zones was mixed with 2 ml of Freund’s complete adjuvant. About 2 ml of the emulsion was then injected into each hind leg of two rabbits. The second injection was carried out in the same way 1 week after the first. For the final injection, 4 weeks after the first, preparations from the sucrose gradients were mixed with an equal volume of sterile distilled water for injection through the marginal ear vein. Terminal bleeding was carried out 2 weeks after the final injection. Yields of serum for each animal varied from 19 to 40 ml. Sera were stored in a freezer.
ET AL.
no transmission occurred after incubation with the homologous antiserum (Table 4). Transmission of MAV by M. avenue was reduced following incubation with the PAV antiserum, and transmission of PAV by R. padi was reduced following incubation with the MAV antiserum, but other heterologous combinations had no pronounced effect. These data suggest that MAV and PAV are related, but that RPV is distinct from the other two. A more thorough study of the relationship among the 3 virus isolates (Aapola and Rochow, 1971), based on in vivo interactions and use of 2 additional serological techniques, supports this relationship among the 3 virus isolates and demonstrates that the antisera are highly reactive. TABLE REACTION
Comparison of Virus Isolates by Serological Blocking of Aphid Transmission
The 3 isolates of BYDV were compared by using the antisera in a type of infectivity neutralization test that has proved useful for circulative aphid-transmitted viruses (Gold and Duffus, 1967; Rochow and Ball, 1967). The test is based on incubation of virus with antiserum, allowing aphids to feed through membranes on the mixture, and determining whether or not the aphids can transmit virus after the acquisition feeding. Previous tests by this procedure have suggested that MAV and RPV are serologically distinct (Rochow and Ball, 1967; Rochow, 1970). Each of the 4 antisera was mixed with a concentrate of one of the virus isolates, incubated at 37” for 30 min, often stored overnight at 4’, combined with an equal volume of 40 % sucrose in buffer, and used in membrane feeding assays. In some tests, the virus-antiserum mixture was centrifuged before sucrose was added, but results were the same if centrifugation was omitted. Aphids were allowed to feed through stretched ParafilmG on the buffered antigenantibody mixture for about 18 hr at 15” and then transferred to Coast Black oat seedlings (10 aphids per seedling) for a 5-day inoculation test feeding period. In all tests, except one with the highest concentration of MAV,
DWARF ANTISERA BLOCKING
Virus
isolate
OF THREE VIRUS AS
(Irg/mlY
MAV
RPV
PAV
0.4 0.8 2.0 4.0 4.0 9.0 4.0 5.0 10.0 12.9
OF BARLEY
WITH EXH DETERMINED
OF APHID Virus gti$
4
ISOLATES
YELLOW
OF FOUR SPECIFIC BY SEROLOGICAL
TRANSMISSION
OF VIRUS
Transmissionb following incubation of virus with antiserum or control shown MAV serum
RPV serum
0 0 0 0 0 2 5 4 7 6 0 1 1
8 9 9 9 9 9 0 0 0 0 7 7 8
PAV Serum
0 0 2 3 2 8 2 1 6 5 0 0 0
H.O. serum
aphid control
5 9 9 8 9 9 4 7 9 6 5 8 8
0 0 0 0 0 0 0 0 0 0 0 0 0
a Virus concentration of preparation used; concentration actually fed upon by aphids was s that shown because virus was first mixed with an equal volume of antiserum (diluted 1:5), and this mixture was later added to an equal amount of 40% sucrose for aphid feeding. In 3 experiments virus concentration was not known. b Number of plants that became infected out of 9 infested with 10 aphids (M. awelzae for MAV; R. pa& for RPV and PAV) that had fed through membranes for about 18 hr at 15’ on mixture indi. cated before start of 5-day inoculation test feeding period on oat seedlings.
BtlRLEY
YELLOW
DWARF
DISCUSSION
A distinctive feature for purification of BYDV is the minute amount of virus obtained from infected plants in comparison with yields of most other plant viruses that have been purified. Although we have been able to increase yields of BYDV over those of earlier experiments by altering factors that affect purificat,ion, any major increase in yields probabl>* will have to await discovery of better source plants or of methods to alter the restriction of virus Do phloem tissue and the disint’egrabion of t,his tissue as infection proceeds. The stability of BYDV and t,he use of frozen crude concentrates partly compensates for the low amounts of virus obtained from infected plants. The polyhedral virus particles observed in shadowed preparations of RPV and PAV were about 30 nm in diameter, a value previously reported for MAV (Rochow and Brakke, 1964). *Jensen (1969) has observed BYDV particles about 24 nm in diameter in thin sections of host tissue and H. W. Israel (unpublished data) has found the particles of several isolates to be about 20 nm in negat,ivel?, stained virus preparations. As Jensen (1969) has point’ed out’, these discrepancies probably are merely reflections of differences among the methods used to prepare specimens for electron microscopy. In addition t’o the 3 isolates of BYDV investigated here 2 additional variants have been encountered in our st,udies of field-collected plants. One is the R;\IV isolate transmitted specifically by K. maidis (Fitch), the biological properties of which have been described (Rochon-, 1969). The other variant was recovered from several plants collected in the field in Xew York during the summer of 1969. It appears to be similar to the vector specific variant previously described by Gill (1969) and is transmitted specifically by Schizaphis qraminunl (Rondani) . Preliminary purification experiments n-it,11these 2 isolates have shown that the same procedures can be used for them as for the 3 isolates studied here, but that yields of virus are even lower than t,hose obtained for the I’IZV isolate. Current evidence for the relationship among the 3 isolates of BYDV (Aapola and
VIRUS
12.3
PURIFICATION
Rochow, 1971) suggests that MAV and PAV are related but distinct from RPV. The similarity in sedimentation and morphology for all 3 isolates does not, conflict with the evidence hhat RPV is different from the other t,wo becausedata are not exhaustive on either point and neither criterion is especiallluseful in determining virus relationships. Moreover, preliminary data from ultraviolet absorption spectra of RI’V and MAV have also shown the 2 isolates to be different,. The A 260/280 for MAV wa’sabout 1.92 and that for RPV was a,bout 1.72 in some studies at Lincoln. ACKNOWLEDGMENTS We thank and providing
Irmgard general
Muller for injecting technical assistance.
aphids
REFERENCES AAI>oL.~, A. I. E., and RocH~~, W. F. (1971). Relationships among three isolates of barley yellow dwarf virus. VGology 46, 127-141. ARAI, K., DOI, Y., YOR.\, K., and Asuu~mn, H. (1969). Electron microscopy of the potato leafroll virus in leaves of three kinds of host plants and the partial purification of the virus. .I~TL. Phytopathol. Sot. Japan 35, lo--Xi. DUFFUS, J. E. (1969). Membrane feeding used in determining the properties of beet western yellows virus. Phytopathology 59, 1668-1669. DUFFUS, J. E., and GOLU, A. H. (1969). Membrane feeding and infectivity neut,ralization used in a serological comparison of potato leaf roll and beet western yellows viruses. I’irology :1’i, 150 153. GILL, C. C. (1969). Annual variation in strains of barley yellow dwarf virus in Manitoba, and 1 he occurrence of greenbug-specific isolates. Cnr,. .r. Bot. 47, 1277-1283. GOLD, A. H., and DUFFUS, J. E. (1967). Infect,ivitJ neutralization-a serological method as applied to persistent viruses of beets. T’irol0g.q 31, 308.. 313. HERERT, T. T. (1963). Precipitation of plant viruses by polyethylene glycol. Phytopathology 53,362. JENSEN, 8. G. (1969). Occurrence of virus particles in the phloem tissue of BYDV-infected barley. Virology 38, 83-91. KOJIM.~, M., SHIK.~T~, E., S~UG.~\~~.~Z, M., and MURAYMA, D. (1969). Purification and elect ran microscopy of potato leafroll virus. T’irolog!/ 30, 162-174.
126
ROCHOW
D. (1967). The purification of potato leafroll virus from its vector Myzus persicae. Viralogy 31, 46-54. PETERS, D., and VAN LOON, L. C. (1968). Transmission of potato leafroll virus by aphids after feeding on virus preparations from aphids and plants. VGoZogy 35, 597-609. ROCHOW, W. F. (1959). Transmission of strains of barley yellow dwarf virus by two aphid species. PETERS,
Phytopathology ROCHOW, W.
49, 744-748.
F. (1969). Biological
properties
of
ET
AL.
four isolates of barley yellow dwarf virus. Phyto59, 1580-1589. W. F. (1970). Barley yellow dwarf virus: Phenotypic mixing and vector specificity. Science 167, 875878. ROCHOW, W. F., and BALL, E. M. (1967). Serological blocking of aphid transmission of barley yellow dwarf virus. Virology 33, 359-362. ROCHOW, W. F., and BRAKKE, M. K. (1964). Purification of barley yellow dwarf virus. Vi’irology 24, 31&322. pathology ROCHOW,