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Serological relationships of an iridescent virus (type 25) recently isolated from Tipula sp. with two other iridescent viruses (types 2 and 22)
VIROLOGY
81, 309-316 (1977)
Serological Relationships Isolated from Tipula R. M. ELLIOTT, N.E.R.C.,
Unit
of Invertebrate 5 South
of an Iridescent Virus (Type 25) Recently sp. with Two Other Iridescent Viruses (Types 2 and 22) THELMA
Virology, and Department Parks Road, Oxford, OX1 Accepted
The isolation, (IV), type 25, different from Simulium sp. neutralization, and immune pruinosa was
LESCO’M’,
April
AND
D. C. KELLY
of Forestry, 3RB, England
University
of Oxford,
29,1977
purification, and serological properties of a 130-nm iridescent virus from Tipula sp. is reported. The polypeptide profile of this isolate was that of another small Dipterous iridescent virus -IV22 isolated from However, these viruses were indistinguishable when compared by serum gel immunodiffusion, complement fixation, tube immunoprecipitation, electron microscopy. Iridescent virus type 2 isolated from Sericesthis shown to be distinct from IV22 and IV25 by all of these techniques.
sent a number of different viruses. In the United Kingdom small iridescent viruses (i.e., 130 nm in size) have only been identified from Tip&a and Simulium species. In this paper we report the isolation and purification of a new Tip&a iridescent virus, which we have designated type 25 (IV25). It was of interest to compare this virus with a Simulium isolate, IV22, found 13 months previously (Batson et al., 1976) to determine whether they were the same virus, especially as both were from the larvae of Diptera living in an aqueous environment. Iridescent virus type 2 was included in these studies as an internal standard. The techniques used to discriminate between the viruses were neutralization, gel immunodiffusion, complement fixation, immunoprecipitation, and immune electron microscopy. This afforded the opportunity to evaluate the usefulness of a variety of serological procedures in the field of insect virology. The structural proteins were compared by polyacrylamide gel electrophoresis.
INTRODUCTION
Iridescent viruses (IV) are icosahedral cytoplasmic DNA-containing viruses which have been isolated from a variety of invertebrate host species having a wide geographical distribution. Tinsley and Kelly (1970) suggested a provisional classification scheme whereby isolates are listed in numerical sequence according to their date of isolation. Under this scheme 27 types are now recognized (D. C. Kelly, unpublished) but only a few of these have been extensively investigated (for review, see Kelly and Robertson, 1973). Serological studies have shown that iridescent virus type 1 (IVl, ex Tip&a paludosa; Xeros, 1954) and type 2 (IV2, ex Sericesthis pruinosa; Steinhaus and Leutenegger, 1963) are related but not identical on the basis of complement fixation, gel immunodiffision, and immunoprecipitation (Day and Mercer, 1964; Cunningham and Tinsley, 1968; Glitz et al., 1968). However, it has been reported that a number of isolates from Tip&a species (i.e., “type 1” virus) have different polypeptide profiles (J. B. Carter, personal communication), and Cunningham and Tinsley (1968) commented that serological differences existed between Tipula viruses grown in a variety of hosts. Therefore it is conceivable that the isolates classed as “type 1” may repre-
MATERIALS
AND
METHODS
Reagents
Acrylamide and sodium dodecyl sulfate (SDS; especially pure grade) were purchased from British Drug Houses Ltd., 309
Copyright All rights
0 1977 by Academic Press, Inc. of reoroduction in any form reserved.
ISSN
0042-6822
310
ELLIOTT.
LESCOTT,
Poole, Dorset, England; N,N’-methylenebisacrylamide from Eastern Organic Chemicals, Rochester, New York, molecular weight standards for slab gel calibration from Sigma Chemical Co., London. Lyophilized guinea pig complement, horse hemolytic serum, and sheep red blood cells in Alsever’s solution were obtained from Wellcome Laboratories, Beckenham, Kent, England. Freund’s complete and incomplete adjuvants were from Difco La-a~tories, East Molesey, Surrey, EngVirus
Production
and Purification
Iridescent virus (IV) type 25 was isolated from an infected Tipulid larva found in September, 1975 in Guildford, Surrey, England. IV type 2 and IV type 22 were obtained from the virus collection maintained in the Unit of Invertebrate VirolOgYThe viruses were grown in late instar larvae of the greater wax moth, Galleria mellonella; for all experimental work, viruses which had been passaged twice in this host were used. The larvae were sacrificed 18 days after infection by freezing at -20”. Purification of the viruses was based on the method of Kelly and Tinsley (1972). Infected larvae were thawed in a small volume of 0.1 M phosphate buffer (pH 7.2) and ground with a pestle and mortar. The slurry was spun for 10 min at 2600 g on an MSE bench centrifuge, and the precipitate and floating lipid layer were discarded. The supernatant was pelleted through a 40% w/v sucrose cushion at 30,000 g for 90 min, and the pellet was allowed to resuspend in a small amount of buffer overnight. The suspension was centrifuged at 35,000 g for 1 hr on a 5-40% w/v sucrose gradient, and the virus bands were pooled and diluted. The virus was centrifuged to equilibrium for 15 hr at 25,000 g on a 3065% w/v sucrose gradient. The purified virus was pelleted at 20,OOOg for 45 min, and the pellet was resuspended in 0.1 M phosphate. Throughout the purification schedule the temperature was maintained at 4”, and the purified virus suspensions were stored at -20”. The protein content of the virus suspen-
AND
KELLY
sion was estimated by the Folin method (Lowry et al., 1951), using bovine serum albumin as a standard. Preparation
of Antisera
Rabbits were bled from the marginal ear vein to provide preimmune sera before the start of active hyperimmunization. One milliliter of virus suspension containing 500 pg of viral protein was emulsified with 1 ml of Freund’s complete adjuvant, and injected intramuscularly, half into each leg. The same procedure was followed 7 and 14 days later, except that Freund’s incomplete adjuvant was used. The rabbits were bled from the marginal- ear vein weekly for 3 weeks after the final inoculation, and terminally from the aorta at week 4. The sera were divided into small aliquots and stored at -20”. Electron
Microscopy
Sections of the infected Tipulid larva were prepared as described by Kelly and Tinsley (19741, and examined in an AEI EMGB electron microscope at an accelerating voltage of 60 kV. SDS-Polyacrylamide
Gel Electrophoresis
Proteins were analyzed on 18cm-long 12.5% polyacrylamide slab gels, using the buffer system described by Laemmli (1970). Samples for electrophoresis were boiled for 2 min in 1 M Tris-HCl, pH 6.8, 2% w/v SDS, 2% v/v 2-mercaptoethanol, 10% v/v glycerol, and 0.01% bromophenol blue. For molecular weight determinations standards of /3-galactosidase (132,000), transferin (90,000), bovine serum albumin (69,000), ovalbumin (45,000), lactic dehydrogenase (35,000), carbonic anhydrase cY-chymotrypsinogen (25,000), (31,000), myoglobin (16,900), and cytochrome c (12,400) were electrophoresed alongside the virus samples. The positions of the proteins were detected by staining with Coomassie blue. Titration
of Viruses
The viruses were titrated by the TCID, assay as described for an Oryctes virus (Kelly, 1976) using a Spodoptera frugiperda cell line (J. L. Vaughn, unpub-
SEROLOGY
OF
THREE
lished). These cells were grown at 28”, but were placed at 21” following infection. Serological
Tests
Neutralization in Spodoptera fiugiperda cells. The method of constant virus
was employed. One hundred microliters of virus suspension containing 500 TCID,, were mixed with 100 ~1 of antiserum dilutions (doubling dilutions from 1:20 to 1:2560) and allowed to react overnight at 4”. Ten-microliter aliquots of these mixtures were applied to their respective wells in microtiter plates, and the cells were examined for cytopathic effect 4-7 days after inoculation. Viruses and antisera were sterilized by passing through a 0.22 pm Millipore filter, pretreated with fetal bovine serum to prevent excessive loss of material due to adsorption by the filter. Gel diffusion. Basically the procedure was that described by Cunningham and Tinsley (1968). The virus suspensions contained 500 pg/ml of protein, and lines of precipitation were stained with 0.1% naphthalene black in 7% acetic acid. The optimal antiserum dilution was found by titrating doubling dilutions of antiserum against the homologous virus suspension, and this dilution was used in the comparative studies. Complement fixation. The microtiter technique of Sever (1962) was used, employing 25~1 volumes. The optimal concentrations of virus suspension contained 25 pg/ml of protein, and 3 HC,, of complement were used. (One unit of complement, HC&,-,, is the dilution of complement giving 50% lysis of red blood cells at the optimal sensitizing concentration of hemolytic serum). Immunoprecipitation in free buffer. The method of Cunningham and Tinsley (1968) was followed. Immune electron microscopy. The procedure was based on that of Almeida and Waterson (1969). Two hundred microliters of virus suspension (20 pg/ml of protein), 100 ~1 of antiserum dilution (l:lO, 1:25, 1:50, or l:lOO), and 700 ~1 of 0.1 M phosphate buffer were thoroughly mixed, reacted for 1 hr at 37”, and left overnight at 4”. Immune complexes were pelleted by
IRIDESCENT
VIRUSES
311
centrifuging at 50,000 g for 30 min, and resuspended in 100 ~1 of buffer. Samples were negatively stained with 2% phosphotungstic acid (pH 6.5) and examined in the electron microscope. RESULTS
Electron
Microscopy
The diseased Tipulid larva appeared vividly blue when dissected and an iridescent virus infection was suspected. Thin sections of the fat body revealed many icosahedral virus particles about 130 nm in diameter which were observed only in the cytoplasm and not in the nucleus (Fig. la); microcrystals of virus were also seen (Fig. lb). Figure lc shows a thin section of an IVBB-infected fat body of the Simulium sp; the virus particles were similar in appearance to those of IV25, but no microcrystals of virus were detected. Polyacrylamide
Gel Electrophoresis
The polypeptide profiles of the three viruses are different, but IV22 and IV25 have similar but not identical profiles, and these are markedly different from that of IV2 (Fig. 2). Between 22 and 25 polypeptides were resolved on the gels; their molecular weights ranged from about 10,000 to 145,000 (Table 1). The major structural protein had a molecular weight of 53,00054,000. However, IV type 2 appeared to possess two polypeptides at this position on the gel with molecular weights of 52.7 and 54.8 x 103. Iridescent virus types 22 and 25 had large amounts of small polypeptide, approximately 13,000-14,000 in molecular weight; type 2 did not have large quantities of this protein. Neutralization
Test
The results of the test are given in Table 2. In the homologous systems good protection was given by the antisera. Iridescent virus types 22 and 25 appeared to be very similar, as the heterologous antiserum protection was within one dilution of the homologous antiserum. Type 2 virus was quite distinct from types 22 and 25. Preimmune rabbit serum afforded no protection in this test.
312
ELLIOTT,
LESCOTT,
FIGURE
AND
1
KELLY
SEROLOGY
OF
THREE
IRIDESCENT
313
VIRUSES
Gel Immunodiffusion
Complement
The homologous type 2 reaction produced at least three lines of precipitation (Fig. 3a) whilst the homologous IV22 and IV25 reactions showed two lines (Figs. 3b, c). Both lines were identical to the two viruses. There appeared to be one line of identity between IV2 and the other viruses when reacted with type 25 antiserum (Fig. 3~). It was not therefore nossible to distinguish IV22 from IV25 by ihis technique.
Table 3 shows the results of the complement fixation test with the viruses. The homologous systems gave high titers of 1:2560. In the heterologous tests iridescent virus types 22 and 25 appeared almost identical, but they were quite distinct from IV2.
22225
MW
Fixation
Immunoprecipitation
The results of immunoprecipitation
2
22
-VP -VP
31 28
(Ta-
25
-VP
39
,e -VP
34
-VP
28
18%-VP
-VP -VP
40 33
-VP
28
VP
25
21
FIG. 2. The polypeptides of iridescent virus types 2, 22, and 25 resolved on a 12.5% polyacrylamide slab gel. The left-hand side shows the three viruses directly compared, together with molecular weight markers: A, transferin; B, bovine serum albumin; C, ovalbumin; D, lactic dehydrogenase; E, carbonic anhydrase; F, a-chymotrypsinogen; G, inyoglobin; H, cytochrome c. The right-hand side shows the individual polypeptides labeled according to Table 1.
FIG. 1. (a) Fat body of T&.&a sp. infected in the cytoplasm. Bar, 1 pm. (b) Microcrystal Simulium sp. infected with IV type 22. Bar,
with IV type 25. Note that the virus particles are only present of IV25 in fat body of ZXpula sp. Bar, 1 pm. (c) Fat body of 1 pm.
314
ELLIOTT,
LESCOTT,
AND
TABLE MOLECULAR Iridescent
virus
Protein VP VP VP VP VP VP VP VP VP VP VP VP VP VP VP VP VP VP VP VP VP VP
type
MW
137 128 119 115 111 105 98 96 88 80 65 55 53 48 31 28 21 18 16 14 13 12
a Calculated
WEIGHTS
137.4 128.1 118.8 115.1 111.3 105.0 97.5 95.5 88.2 79.9 64.8 54.8 52.7 48.0 31.1 27.9 21.0 17.7 15.8 14.2 12.9 11.7
from
OF THE PROTEINS
2
Iridescent
x 1O-3” 2 ” k k k t + + + + + + 2 + + + + 5 + + ? +
Protein
9.3 3.5 3.9 5.3 5.3 1.6 3.3 2.9 3.2 3.7 3.3 2.4 1.9 1.3 2.8 2.3 2.1 0.7 0.8 0.7 0.4 1.1
a minimum
VP VP VP VP VP VP VP VP VP VP VP VP VP VP VP VP VP VP VP VP VP VP VP VP VP of five
144 131 121 116 109 102 95 92 85 73 67 59 54 50 39 34 28 21 20 18 16 15 13 12 11
determinations,
ble 4) concur closely with those of the complement fixation test. Type 2 virus reacted strongly with its homologous antiserum, but appreciably less so with the antisera to types 22 and 25. Iridescent virus types 22 and 25 appeared to be closely related, there being only one dilution between the homologous and hetecologous precipitating titers. These viruses were quite different (three or four dilutions) from type 2 virus. Immune Electron Microscopy The results of this technique did not give clear-cut results (Table 5). It appeared that type 2 virus was more distantly related to types 22 and 25 than these viruses were to each other, thereby agreeing with the other methods employed. DISCUSSION
We have not been able to distinguish iridescent virus types 22 and 25 by the serological procedures employed in this study. The internal control, IV2, was readily discriminated from the other two vi-
KELLY
1
OF IRIDESCENT virus
type
MW
Vmus
TYPES 2, 22, AND 25
22
Iridescent
x 1O-3 o
144.3 131.1 121.1 115.9 109.2 101.5 95.0 91.7 85.1 72.8 66.9 58.8 53.7 50.4 39.4 33.6 27.9 21.4 19.7 17.5 15.8 14.8 13.3 11.5 11.0
+ + ? + + ? 2 2 ? ? + + k + + r + k ? r 2 + + + k
Protein
3.7 5.5 4.4 4.3 4.9 4.7 1.3 4.8 1.2 1.4 0.2 3.5 2.5 2.4 0.8 1.8 0.7 1.2 0.2 0.7 1.2 1.2 1.4 1.2 2.0
? standard
virus
VP VP VP VP VP VP VP VP VP VP VP VP VP VP VP VP VP VP VP VP VP VP VP VP VP
type
MW
145 135 126 120 113 109 104 97 87 72 65 60 53 51 47 40 33 28 25 21 19 17 15 14 13
144.5 134.9 125.9 119.6 113.2 109.0 104.3 97.1 86.8 72.4 65.3 60.0 52.8 50.6 47.4 39.8 33.0 27.9 25.0 20.9 18.9 17.0 15.2 13.7 12.6
25
x 1O-3 = 2 2 2 k 2 2 + + % k 5 e f + + + + k + + ? + + k +
1.3 2.8 2.8 1.5 0.2 0.7 1.5 3.1 3.8 3.9 2.4 2.0 1.6 1.2 1.7 1.1 1.9 1.5 2.0 1.2 0.8 0.6 1.0 0.7 1.0
deviation. TABLE
2
RECIPROCAL NEUTRALIZING TITERS OF ANTISERA TO IRIDESCENT VIRUS TYPES 2, 22, AND 25 WHEN COMPARED BY SERUM NEUTRALIZATION IN Spodoptera frugiperdu CELLS Virus
IV2 IV22 IV25
Antiserum IV2
IV22
2560 40 40
20 1280 640
IV25 40 640 640
ruses by all the methods described. The viruses used in this study were produced in the same host insect in order to minimize differences that could be due to hosts of different species, possibly by the incorporation of host antigens in the virus (Cunningham and Tinsley, 1968). The viruses were purified by both rate and equilibrium centrifugation on sucrose gradients, and antisera were raised to those pure preparations. The polypeptide profiles of the viruses were obviously different, though this was
SEROLOGY
OF
FIG. 3. Comparison of iridescent virus the central well. (a) IV type 2 antiserum;
TABLE
THREE
IV2 IV22 IV25 a Results shown serum dilutions.
3
Antiserum IV2
IV22
IV25
2560 320 320
640 2560 2560
320 1280 2560
as 50% end-point
TABLE COMPARISON OF IIZIDESCENT 25 BY IYMUNOPRECIPITATION Virus
rum
reciprocal
anti-
4 VIRUS TYPES 2, 22, AND IN FREE BUFFER” Antiserum
IV2 IV2 IV22 IV25
IV22
128 16 32
16 256 128
(1 Results shown as the highest dilution at which precipitation was observed. TABLE
IV25 32 128 256 of antise-
5
COMPARISON OF IRIDESCENT VIRUS TYPES 2, 22, AND 25 BY IMMUNE ELECTRON MICROSCOPY” Antiserum Virus IV2 IV2 IV22 IV25 rum
100
IV22 10 50 25
a Results shown as the highest dilution at which agglutination was observed.
VIRUSES
types 2, 22, and 25 by gel immunodiffusion. The antiserum (b) IV type 22 antiserum; (cl IV type 25 antiserum.
COMPARI~XJN OF IRIDESCENT VIRUS TYPES 2, 22, AND 25 BY THE COMPLEMENT FIXATION TESTO Virus
IRIDESCENT
IV25
not reflected by the serology of IV22 and IV25. An earlier study on the polypeptides of IV type 2, using a phosphate gel system (Kelly and Tinsley, 1972) gave molecular
315
is in
weight estimates somewhat different from those presented in this paper. We have greater confidence in the estimates given here because more standards covering a wider range of molecular weights were used to calibrate the gel. Our results are significantly different from the data of Krell and Lee (1974) on a Tip&a isolate. The serological results indicated that IV type 2 was distantly related to the Tipula isolate, IV25. This is in agreement with the data of Cunningham and Tinsley (1968) who compared an undesignated Tipula iridescent virus isolate with IV2 on the basis of gel immunodiffusion, immunoprecipitation, and complement fixation. It is possible that such relationships could be due to polyamines present in the virus particles, though using a sensitive thinlayer chromatographic technique (Elliott and Kelly, 1977) we have been unable to demonstrate polyamines in iridescent viruses; this result concurs with the studies of Glitz et al. (1968) on a Tipula iridescent virus. The neutralization test is the most specific of serological procedures employed to discriminate between viruses, and has the advantage of requiring small quantities of virus; however, there are no reports of its use to compare ,different insect viruses. This technique was apparently ins&Xciently sensitive to distinguish IV types 22 and 25, indicating that these viruses must be very closely related; IV2 was clearly distinct by this method. Gel immunodiffusion, complement fixation, and immunoprecipitation gave results comparable to those of the neutralization test, and thus
316
ELLIOTT,
LESCOTT,
were considered to be as useful as neutralization in this instance. Although these methods are more extravagant with the antigen this is not a great problem when dealing with iridescent viruses. When employed as a quantitative technique immune electron microscopy was the least satisfactory of the methods, and was extremely laborious. It may, however, be useful as a diagnostic tool in insect virolWYWe now have in our laboratory an iridescent virus from Tip& sp. of known history which we will use for future studies, but as yet we cannot distinguish this isolate from IV22 by serological procedures. However, preliminary results from work in this unit on developing a plaque assay system for insect viruses have shown that IV22 and IV25 produce plaques of significantly different sizes (D. A. Brown and K. A. Harrap, unpublished observations). ACKNOWLEDGMENTS We gratefully acknowledge the help of Margaret Arnold with the electron microscopy. Part of this work was submitted by R. M. Elliott in partial fulfillment for the degree of Bachelor of Science in Microbiology at the University of Surrey, Guildford, Surrey, England, and Dr. M. Butler of the University is thanked for his advice and constructive criticism. R. M. Elliott was in receipt of a Science Research Council postgraduate studentship. REFERENCES J. D., and WATERSON, A. P. The morphology of virus-antibody interaction. Adv. Virus Res. 15, 307-338 (1969). BATSON, B. S., JOHNSTON, M. R. L., ARNOLD, M. K., and KELLY, D. C. An iridescent virus from SimuRum sp. (Diptera:Simuliidae) in Wales. J. Znvertebr. Pathol. 27, 133-135 (1976). CUNNINGHAM, J. C., and TINSLEY, T. W. A serological comparison of some iridescent non-occluded ALMEIDA,
AND KELLY
insect viruses. J. Gen. Virol. 3, 1-8 (1968). DAY, M. F., and MERCER, E. H. Properties of an iridescent virus from the beetle Sericesthis pruinom. Aust. J. Biol. Sci. 17, 892-902 (1964). ELLIOTT, R. M., and KELLY, D. C. The polyamine content of a nuclear polyhedrosis virus from Spodoptera littoralis. Virology 76, 472-474 (1977). GLITZ, D. G., HILLS, G. J., and RIVERS, C. F. A comparison of the Tipula and Sericesthis iridescent viruses. J. Gem. ViroZ. 3, 209-220 (1968). KELLY, D. C. “Oryctes” virus replication: electron microscopic observations on infected moth and mosquito cells. Virology 69, 596-606 (1976). KELLY, D. C., and ROBERTSON, J. S. Icosahedral cytoplasmic deoxyriboviruses. J. Gen. Virol. 20, 17-41 (1973). KELLY, D. C., and TINSLEY, T. W. The proteins of iridescent virus types 2 and 6. J. Invertebr. Pathol. 19, 273-275 (1972). KELLY, D. C., and TINSLEY, T. W. Iridescent virus replication: a microscope study of Aeo!es aegypti and Antherea eucalypti cells in culture infected with iridescent virus type 2 and 6. Microbios 9,7593 (1974). KRELL, P., and LEE, P. E. Polypeptides in Tip&a iridescent virus (TIV) and in TIV-infected hemocytos of Galleria mellonella (L) larvae. Virology 60, 315-326 (1974). LAEMMLI, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-685 (1970). LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275 (1951). SEVER, J. L. Application of a microtechnique to viral serological investigations. J. Zmmunol. 88, 320329 (1962). STEINHAUS, E. A., and LEUTENEGGER, R. Icosahedral virus from a scarabeid (Sericesthis). J. Insect Pathol. 5, 266-270 (1963). TINSLEY, T. W., and KELLY, D. C. An interim nomenclature system for the iridescent group of insect viruses. J. Znvertebr. Pathol. 16, 470-472 (1970). XEROS, N. A second virus disease of the leatherjacket Tipulapaludosa. Nature (London) 174,562563 (1954).
81, 309-316 (1977)
Serological Relationships Isolated from Tipula R. M. ELLIOTT, N.E.R.C.,
Unit
of Invertebrate 5 South
of an Iridescent Virus (Type 25) Recently sp. with Two Other Iridescent Viruses (Types 2 and 22) THELMA
Virology, and Department Parks Road, Oxford, OX1 Accepted
The isolation, (IV), type 25, different from Simulium sp. neutralization, and immune pruinosa was
LESCO’M’,
April
AND
D. C. KELLY
of Forestry, 3RB, England
University
of Oxford,
29,1977
purification, and serological properties of a 130-nm iridescent virus from Tipula sp. is reported. The polypeptide profile of this isolate was that of another small Dipterous iridescent virus -IV22 isolated from However, these viruses were indistinguishable when compared by serum gel immunodiffusion, complement fixation, tube immunoprecipitation, electron microscopy. Iridescent virus type 2 isolated from Sericesthis shown to be distinct from IV22 and IV25 by all of these techniques.
sent a number of different viruses. In the United Kingdom small iridescent viruses (i.e., 130 nm in size) have only been identified from Tip&a and Simulium species. In this paper we report the isolation and purification of a new Tip&a iridescent virus, which we have designated type 25 (IV25). It was of interest to compare this virus with a Simulium isolate, IV22, found 13 months previously (Batson et al., 1976) to determine whether they were the same virus, especially as both were from the larvae of Diptera living in an aqueous environment. Iridescent virus type 2 was included in these studies as an internal standard. The techniques used to discriminate between the viruses were neutralization, gel immunodiffusion, complement fixation, immunoprecipitation, and immune electron microscopy. This afforded the opportunity to evaluate the usefulness of a variety of serological procedures in the field of insect virology. The structural proteins were compared by polyacrylamide gel electrophoresis.
INTRODUCTION
Iridescent viruses (IV) are icosahedral cytoplasmic DNA-containing viruses which have been isolated from a variety of invertebrate host species having a wide geographical distribution. Tinsley and Kelly (1970) suggested a provisional classification scheme whereby isolates are listed in numerical sequence according to their date of isolation. Under this scheme 27 types are now recognized (D. C. Kelly, unpublished) but only a few of these have been extensively investigated (for review, see Kelly and Robertson, 1973). Serological studies have shown that iridescent virus type 1 (IVl, ex Tip&a paludosa; Xeros, 1954) and type 2 (IV2, ex Sericesthis pruinosa; Steinhaus and Leutenegger, 1963) are related but not identical on the basis of complement fixation, gel immunodiffision, and immunoprecipitation (Day and Mercer, 1964; Cunningham and Tinsley, 1968; Glitz et al., 1968). However, it has been reported that a number of isolates from Tip&a species (i.e., “type 1” virus) have different polypeptide profiles (J. B. Carter, personal communication), and Cunningham and Tinsley (1968) commented that serological differences existed between Tipula viruses grown in a variety of hosts. Therefore it is conceivable that the isolates classed as “type 1” may repre-
MATERIALS
AND
METHODS
Reagents
Acrylamide and sodium dodecyl sulfate (SDS; especially pure grade) were purchased from British Drug Houses Ltd., 309
Copyright All rights
0 1977 by Academic Press, Inc. of reoroduction in any form reserved.
ISSN
0042-6822
310
ELLIOTT.
LESCOTT,
Poole, Dorset, England; N,N’-methylenebisacrylamide from Eastern Organic Chemicals, Rochester, New York, molecular weight standards for slab gel calibration from Sigma Chemical Co., London. Lyophilized guinea pig complement, horse hemolytic serum, and sheep red blood cells in Alsever’s solution were obtained from Wellcome Laboratories, Beckenham, Kent, England. Freund’s complete and incomplete adjuvants were from Difco La-a~tories, East Molesey, Surrey, EngVirus
Production
and Purification
Iridescent virus (IV) type 25 was isolated from an infected Tipulid larva found in September, 1975 in Guildford, Surrey, England. IV type 2 and IV type 22 were obtained from the virus collection maintained in the Unit of Invertebrate VirolOgYThe viruses were grown in late instar larvae of the greater wax moth, Galleria mellonella; for all experimental work, viruses which had been passaged twice in this host were used. The larvae were sacrificed 18 days after infection by freezing at -20”. Purification of the viruses was based on the method of Kelly and Tinsley (1972). Infected larvae were thawed in a small volume of 0.1 M phosphate buffer (pH 7.2) and ground with a pestle and mortar. The slurry was spun for 10 min at 2600 g on an MSE bench centrifuge, and the precipitate and floating lipid layer were discarded. The supernatant was pelleted through a 40% w/v sucrose cushion at 30,000 g for 90 min, and the pellet was allowed to resuspend in a small amount of buffer overnight. The suspension was centrifuged at 35,000 g for 1 hr on a 5-40% w/v sucrose gradient, and the virus bands were pooled and diluted. The virus was centrifuged to equilibrium for 15 hr at 25,000 g on a 3065% w/v sucrose gradient. The purified virus was pelleted at 20,OOOg for 45 min, and the pellet was resuspended in 0.1 M phosphate. Throughout the purification schedule the temperature was maintained at 4”, and the purified virus suspensions were stored at -20”. The protein content of the virus suspen-
AND
KELLY
sion was estimated by the Folin method (Lowry et al., 1951), using bovine serum albumin as a standard. Preparation
of Antisera
Rabbits were bled from the marginal ear vein to provide preimmune sera before the start of active hyperimmunization. One milliliter of virus suspension containing 500 pg of viral protein was emulsified with 1 ml of Freund’s complete adjuvant, and injected intramuscularly, half into each leg. The same procedure was followed 7 and 14 days later, except that Freund’s incomplete adjuvant was used. The rabbits were bled from the marginal- ear vein weekly for 3 weeks after the final inoculation, and terminally from the aorta at week 4. The sera were divided into small aliquots and stored at -20”. Electron
Microscopy
Sections of the infected Tipulid larva were prepared as described by Kelly and Tinsley (19741, and examined in an AEI EMGB electron microscope at an accelerating voltage of 60 kV. SDS-Polyacrylamide
Gel Electrophoresis
Proteins were analyzed on 18cm-long 12.5% polyacrylamide slab gels, using the buffer system described by Laemmli (1970). Samples for electrophoresis were boiled for 2 min in 1 M Tris-HCl, pH 6.8, 2% w/v SDS, 2% v/v 2-mercaptoethanol, 10% v/v glycerol, and 0.01% bromophenol blue. For molecular weight determinations standards of /3-galactosidase (132,000), transferin (90,000), bovine serum albumin (69,000), ovalbumin (45,000), lactic dehydrogenase (35,000), carbonic anhydrase cY-chymotrypsinogen (25,000), (31,000), myoglobin (16,900), and cytochrome c (12,400) were electrophoresed alongside the virus samples. The positions of the proteins were detected by staining with Coomassie blue. Titration
of Viruses
The viruses were titrated by the TCID, assay as described for an Oryctes virus (Kelly, 1976) using a Spodoptera frugiperda cell line (J. L. Vaughn, unpub-
SEROLOGY
OF
THREE
lished). These cells were grown at 28”, but were placed at 21” following infection. Serological
Tests
Neutralization in Spodoptera fiugiperda cells. The method of constant virus
was employed. One hundred microliters of virus suspension containing 500 TCID,, were mixed with 100 ~1 of antiserum dilutions (doubling dilutions from 1:20 to 1:2560) and allowed to react overnight at 4”. Ten-microliter aliquots of these mixtures were applied to their respective wells in microtiter plates, and the cells were examined for cytopathic effect 4-7 days after inoculation. Viruses and antisera were sterilized by passing through a 0.22 pm Millipore filter, pretreated with fetal bovine serum to prevent excessive loss of material due to adsorption by the filter. Gel diffusion. Basically the procedure was that described by Cunningham and Tinsley (1968). The virus suspensions contained 500 pg/ml of protein, and lines of precipitation were stained with 0.1% naphthalene black in 7% acetic acid. The optimal antiserum dilution was found by titrating doubling dilutions of antiserum against the homologous virus suspension, and this dilution was used in the comparative studies. Complement fixation. The microtiter technique of Sever (1962) was used, employing 25~1 volumes. The optimal concentrations of virus suspension contained 25 pg/ml of protein, and 3 HC,, of complement were used. (One unit of complement, HC&,-,, is the dilution of complement giving 50% lysis of red blood cells at the optimal sensitizing concentration of hemolytic serum). Immunoprecipitation in free buffer. The method of Cunningham and Tinsley (1968) was followed. Immune electron microscopy. The procedure was based on that of Almeida and Waterson (1969). Two hundred microliters of virus suspension (20 pg/ml of protein), 100 ~1 of antiserum dilution (l:lO, 1:25, 1:50, or l:lOO), and 700 ~1 of 0.1 M phosphate buffer were thoroughly mixed, reacted for 1 hr at 37”, and left overnight at 4”. Immune complexes were pelleted by
IRIDESCENT
VIRUSES
311
centrifuging at 50,000 g for 30 min, and resuspended in 100 ~1 of buffer. Samples were negatively stained with 2% phosphotungstic acid (pH 6.5) and examined in the electron microscope. RESULTS
Electron
Microscopy
The diseased Tipulid larva appeared vividly blue when dissected and an iridescent virus infection was suspected. Thin sections of the fat body revealed many icosahedral virus particles about 130 nm in diameter which were observed only in the cytoplasm and not in the nucleus (Fig. la); microcrystals of virus were also seen (Fig. lb). Figure lc shows a thin section of an IVBB-infected fat body of the Simulium sp; the virus particles were similar in appearance to those of IV25, but no microcrystals of virus were detected. Polyacrylamide
Gel Electrophoresis
The polypeptide profiles of the three viruses are different, but IV22 and IV25 have similar but not identical profiles, and these are markedly different from that of IV2 (Fig. 2). Between 22 and 25 polypeptides were resolved on the gels; their molecular weights ranged from about 10,000 to 145,000 (Table 1). The major structural protein had a molecular weight of 53,00054,000. However, IV type 2 appeared to possess two polypeptides at this position on the gel with molecular weights of 52.7 and 54.8 x 103. Iridescent virus types 22 and 25 had large amounts of small polypeptide, approximately 13,000-14,000 in molecular weight; type 2 did not have large quantities of this protein. Neutralization
Test
The results of the test are given in Table 2. In the homologous systems good protection was given by the antisera. Iridescent virus types 22 and 25 appeared to be very similar, as the heterologous antiserum protection was within one dilution of the homologous antiserum. Type 2 virus was quite distinct from types 22 and 25. Preimmune rabbit serum afforded no protection in this test.
312
ELLIOTT,
LESCOTT,
FIGURE
AND
1
KELLY
SEROLOGY
OF
THREE
IRIDESCENT
313
VIRUSES
Gel Immunodiffusion
Complement
The homologous type 2 reaction produced at least three lines of precipitation (Fig. 3a) whilst the homologous IV22 and IV25 reactions showed two lines (Figs. 3b, c). Both lines were identical to the two viruses. There appeared to be one line of identity between IV2 and the other viruses when reacted with type 25 antiserum (Fig. 3~). It was not therefore nossible to distinguish IV22 from IV25 by ihis technique.
Table 3 shows the results of the complement fixation test with the viruses. The homologous systems gave high titers of 1:2560. In the heterologous tests iridescent virus types 22 and 25 appeared almost identical, but they were quite distinct from IV2.
22225
MW
Fixation
Immunoprecipitation
The results of immunoprecipitation
2
22
-VP -VP
31 28
(Ta-
25
-VP
39
,e -VP
34
-VP
28
18%-VP
-VP -VP
40 33
-VP
28
VP
25
21
FIG. 2. The polypeptides of iridescent virus types 2, 22, and 25 resolved on a 12.5% polyacrylamide slab gel. The left-hand side shows the three viruses directly compared, together with molecular weight markers: A, transferin; B, bovine serum albumin; C, ovalbumin; D, lactic dehydrogenase; E, carbonic anhydrase; F, a-chymotrypsinogen; G, inyoglobin; H, cytochrome c. The right-hand side shows the individual polypeptides labeled according to Table 1.
FIG. 1. (a) Fat body of T&.&a sp. infected in the cytoplasm. Bar, 1 pm. (b) Microcrystal Simulium sp. infected with IV type 22. Bar,
with IV type 25. Note that the virus particles are only present of IV25 in fat body of ZXpula sp. Bar, 1 pm. (c) Fat body of 1 pm.
314
ELLIOTT,
LESCOTT,
AND
TABLE MOLECULAR Iridescent
virus
Protein VP VP VP VP VP VP VP VP VP VP VP VP VP VP VP VP VP VP VP VP VP VP
type
MW
137 128 119 115 111 105 98 96 88 80 65 55 53 48 31 28 21 18 16 14 13 12
a Calculated
WEIGHTS
137.4 128.1 118.8 115.1 111.3 105.0 97.5 95.5 88.2 79.9 64.8 54.8 52.7 48.0 31.1 27.9 21.0 17.7 15.8 14.2 12.9 11.7
from
OF THE PROTEINS
2
Iridescent
x 1O-3” 2 ” k k k t + + + + + + 2 + + + + 5 + + ? +
Protein
9.3 3.5 3.9 5.3 5.3 1.6 3.3 2.9 3.2 3.7 3.3 2.4 1.9 1.3 2.8 2.3 2.1 0.7 0.8 0.7 0.4 1.1
a minimum
VP VP VP VP VP VP VP VP VP VP VP VP VP VP VP VP VP VP VP VP VP VP VP VP VP of five
144 131 121 116 109 102 95 92 85 73 67 59 54 50 39 34 28 21 20 18 16 15 13 12 11
determinations,
ble 4) concur closely with those of the complement fixation test. Type 2 virus reacted strongly with its homologous antiserum, but appreciably less so with the antisera to types 22 and 25. Iridescent virus types 22 and 25 appeared to be closely related, there being only one dilution between the homologous and hetecologous precipitating titers. These viruses were quite different (three or four dilutions) from type 2 virus. Immune Electron Microscopy The results of this technique did not give clear-cut results (Table 5). It appeared that type 2 virus was more distantly related to types 22 and 25 than these viruses were to each other, thereby agreeing with the other methods employed. DISCUSSION
We have not been able to distinguish iridescent virus types 22 and 25 by the serological procedures employed in this study. The internal control, IV2, was readily discriminated from the other two vi-
KELLY
1
OF IRIDESCENT virus
type
MW
Vmus
TYPES 2, 22, AND 25
22
Iridescent
x 1O-3 o
144.3 131.1 121.1 115.9 109.2 101.5 95.0 91.7 85.1 72.8 66.9 58.8 53.7 50.4 39.4 33.6 27.9 21.4 19.7 17.5 15.8 14.8 13.3 11.5 11.0
+ + ? + + ? 2 2 ? ? + + k + + r + k ? r 2 + + + k
Protein
3.7 5.5 4.4 4.3 4.9 4.7 1.3 4.8 1.2 1.4 0.2 3.5 2.5 2.4 0.8 1.8 0.7 1.2 0.2 0.7 1.2 1.2 1.4 1.2 2.0
? standard
virus
VP VP VP VP VP VP VP VP VP VP VP VP VP VP VP VP VP VP VP VP VP VP VP VP VP
type
MW
145 135 126 120 113 109 104 97 87 72 65 60 53 51 47 40 33 28 25 21 19 17 15 14 13
144.5 134.9 125.9 119.6 113.2 109.0 104.3 97.1 86.8 72.4 65.3 60.0 52.8 50.6 47.4 39.8 33.0 27.9 25.0 20.9 18.9 17.0 15.2 13.7 12.6
25
x 1O-3 = 2 2 2 k 2 2 + + % k 5 e f + + + + k + + ? + + k +
1.3 2.8 2.8 1.5 0.2 0.7 1.5 3.1 3.8 3.9 2.4 2.0 1.6 1.2 1.7 1.1 1.9 1.5 2.0 1.2 0.8 0.6 1.0 0.7 1.0
deviation. TABLE
2
RECIPROCAL NEUTRALIZING TITERS OF ANTISERA TO IRIDESCENT VIRUS TYPES 2, 22, AND 25 WHEN COMPARED BY SERUM NEUTRALIZATION IN Spodoptera frugiperdu CELLS Virus
IV2 IV22 IV25
Antiserum IV2
IV22
2560 40 40
20 1280 640
IV25 40 640 640
ruses by all the methods described. The viruses used in this study were produced in the same host insect in order to minimize differences that could be due to hosts of different species, possibly by the incorporation of host antigens in the virus (Cunningham and Tinsley, 1968). The viruses were purified by both rate and equilibrium centrifugation on sucrose gradients, and antisera were raised to those pure preparations. The polypeptide profiles of the viruses were obviously different, though this was
SEROLOGY
OF
FIG. 3. Comparison of iridescent virus the central well. (a) IV type 2 antiserum;
TABLE
THREE
IV2 IV22 IV25 a Results shown serum dilutions.
3
Antiserum IV2
IV22
IV25
2560 320 320
640 2560 2560
320 1280 2560
as 50% end-point
TABLE COMPARISON OF IIZIDESCENT 25 BY IYMUNOPRECIPITATION Virus
rum
reciprocal
anti-
4 VIRUS TYPES 2, 22, AND IN FREE BUFFER” Antiserum
IV2 IV2 IV22 IV25
IV22
128 16 32
16 256 128
(1 Results shown as the highest dilution at which precipitation was observed. TABLE
IV25 32 128 256 of antise-
5
COMPARISON OF IRIDESCENT VIRUS TYPES 2, 22, AND 25 BY IMMUNE ELECTRON MICROSCOPY” Antiserum Virus IV2 IV2 IV22 IV25 rum
100
IV22 10 50 25
a Results shown as the highest dilution at which agglutination was observed.
VIRUSES
types 2, 22, and 25 by gel immunodiffusion. The antiserum (b) IV type 22 antiserum; (cl IV type 25 antiserum.
COMPARI~XJN OF IRIDESCENT VIRUS TYPES 2, 22, AND 25 BY THE COMPLEMENT FIXATION TESTO Virus
IRIDESCENT
IV25
not reflected by the serology of IV22 and IV25. An earlier study on the polypeptides of IV type 2, using a phosphate gel system (Kelly and Tinsley, 1972) gave molecular
315
is in
weight estimates somewhat different from those presented in this paper. We have greater confidence in the estimates given here because more standards covering a wider range of molecular weights were used to calibrate the gel. Our results are significantly different from the data of Krell and Lee (1974) on a Tip&a isolate. The serological results indicated that IV type 2 was distantly related to the Tipula isolate, IV25. This is in agreement with the data of Cunningham and Tinsley (1968) who compared an undesignated Tipula iridescent virus isolate with IV2 on the basis of gel immunodiffusion, immunoprecipitation, and complement fixation. It is possible that such relationships could be due to polyamines present in the virus particles, though using a sensitive thinlayer chromatographic technique (Elliott and Kelly, 1977) we have been unable to demonstrate polyamines in iridescent viruses; this result concurs with the studies of Glitz et al. (1968) on a Tipula iridescent virus. The neutralization test is the most specific of serological procedures employed to discriminate between viruses, and has the advantage of requiring small quantities of virus; however, there are no reports of its use to compare ,different insect viruses. This technique was apparently ins&Xciently sensitive to distinguish IV types 22 and 25, indicating that these viruses must be very closely related; IV2 was clearly distinct by this method. Gel immunodiffusion, complement fixation, and immunoprecipitation gave results comparable to those of the neutralization test, and thus
316
ELLIOTT,
LESCOTT,
were considered to be as useful as neutralization in this instance. Although these methods are more extravagant with the antigen this is not a great problem when dealing with iridescent viruses. When employed as a quantitative technique immune electron microscopy was the least satisfactory of the methods, and was extremely laborious. It may, however, be useful as a diagnostic tool in insect virolWYWe now have in our laboratory an iridescent virus from Tip& sp. of known history which we will use for future studies, but as yet we cannot distinguish this isolate from IV22 by serological procedures. However, preliminary results from work in this unit on developing a plaque assay system for insect viruses have shown that IV22 and IV25 produce plaques of significantly different sizes (D. A. Brown and K. A. Harrap, unpublished observations). ACKNOWLEDGMENTS We gratefully acknowledge the help of Margaret Arnold with the electron microscopy. Part of this work was submitted by R. M. Elliott in partial fulfillment for the degree of Bachelor of Science in Microbiology at the University of Surrey, Guildford, Surrey, England, and Dr. M. Butler of the University is thanked for his advice and constructive criticism. R. M. Elliott was in receipt of a Science Research Council postgraduate studentship. REFERENCES J. D., and WATERSON, A. P. The morphology of virus-antibody interaction. Adv. Virus Res. 15, 307-338 (1969). BATSON, B. S., JOHNSTON, M. R. L., ARNOLD, M. K., and KELLY, D. C. An iridescent virus from SimuRum sp. (Diptera:Simuliidae) in Wales. J. Znvertebr. Pathol. 27, 133-135 (1976). CUNNINGHAM, J. C., and TINSLEY, T. W. A serological comparison of some iridescent non-occluded ALMEIDA,
AND KELLY
insect viruses. J. Gen. Virol. 3, 1-8 (1968). DAY, M. F., and MERCER, E. H. Properties of an iridescent virus from the beetle Sericesthis pruinom. Aust. J. Biol. Sci. 17, 892-902 (1964). ELLIOTT, R. M., and KELLY, D. C. The polyamine content of a nuclear polyhedrosis virus from Spodoptera littoralis. Virology 76, 472-474 (1977). GLITZ, D. G., HILLS, G. J., and RIVERS, C. F. A comparison of the Tipula and Sericesthis iridescent viruses. J. Gem. ViroZ. 3, 209-220 (1968). KELLY, D. C. “Oryctes” virus replication: electron microscopic observations on infected moth and mosquito cells. Virology 69, 596-606 (1976). KELLY, D. C., and ROBERTSON, J. S. Icosahedral cytoplasmic deoxyriboviruses. J. Gen. Virol. 20, 17-41 (1973). KELLY, D. C., and TINSLEY, T. W. The proteins of iridescent virus types 2 and 6. J. Invertebr. Pathol. 19, 273-275 (1972). KELLY, D. C., and TINSLEY, T. W. Iridescent virus replication: a microscope study of Aeo!es aegypti and Antherea eucalypti cells in culture infected with iridescent virus type 2 and 6. Microbios 9,7593 (1974). KRELL, P., and LEE, P. E. Polypeptides in Tip&a iridescent virus (TIV) and in TIV-infected hemocytos of Galleria mellonella (L) larvae. Virology 60, 315-326 (1974). LAEMMLI, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-685 (1970). LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275 (1951). SEVER, J. L. Application of a microtechnique to viral serological investigations. J. Zmmunol. 88, 320329 (1962). STEINHAUS, E. A., and LEUTENEGGER, R. Icosahedral virus from a scarabeid (Sericesthis). J. Insect Pathol. 5, 266-270 (1963). TINSLEY, T. W., and KELLY, D. C. An interim nomenclature system for the iridescent group of insect viruses. J. Znvertebr. Pathol. 16, 470-472 (1970). XEROS, N. A second virus disease of the leatherjacket Tipulapaludosa. Nature (London) 174,562563 (1954).