JOURNAL

OF INVERTEBRATE

PATHOLOGY

Host-Dependent

57,

3142 (1991)

Variation of Bluetongue

Virus Neutralization

FRANZISKA B. GRIEDERAND KEVIN T. SCHULTZ' Department of Pathobiological

Sciences, School of Veterinary Medicine, Madison, Wisconsin 53706

University of Wisconsin-Madison,

Received December 11, 1989; accepted April 10, 1990 To determine if neutralizing epitopes of Bluetongue virus (BTV) 17 are host dependent, e.g., that monoclonal antibodies (mAb) to Bluetongue virus 17 (BTV 17) differ in their ability to neutralize BTV infectivity in insect versus mammalian cells, a panel of neutralizing mAb was developed. The relative neutralizing titer of eight mAb for BTV 17 infectivity in mammalian versus insect target cells was determined. Four mAb differed in their relative neutralization titer when assayed on mammalian target cells as compared to insect target cells. These findings suggest that different epitopes involved in neutralization might be important in virus infectivity and neutralization in mammalian versus insect target cells. To determine which viral protein(s) these mAb bind, the specificity of the mAb was determined by radioimmunoprecipitations. Five BTV 17 neutralizing mAb bound to the major outer coat protein P2, a 9%kDa protein, whereas the BTV protein(s) bound by the other three neutralizing mAb could not be determined. The potential role of the two BTV outer coat proteins in infection of mammalian and insect host cells is discussed. o WI Academic

Press, Inc.

Bluetongue virus; arbovirus; virus neutralization; (mammalian) cells; monoclonal antibodies. KEY

WORDS:

INTRODUCTION

invertebrate ceils: vertebrate

ogy of BTV (Fenner et al., 1987; King and Alders, 1985). It is responsible for the amplification of the virus during the extrinsic incubation period, provides the main mode of horizontal viral transmission, and contributes to the spread of the disease. The pathogenesis and virogenesis of BTV in the insect host versus mammalian host are clearly different (Chandler et al., 1985). While insects become infected orally by feeding on an infected mammal, mammals cannot be infected by the oral route. The major route of infection in the mammalian host is through the skin by biting arthropods. Additionally, mechanism of viral spread in the body, invasion of virus in secondary organs, and virus shedding are distinct in the two hosts. The difference in pathogenesis in insects versus mammals might be a result of different sites on a single viral protein or on multiple viral proteins that are involved in the initiation of BTV infection. In fact, such involvement of different protein sites (epitopes) in hostdependent neutralization has recently been described in another arbovirus, Lacrosse virus (Grady and Kinch, 1985). A monoclo-

Bluetongue virus (BTV) is an arthropodtransmitted Orbivirus in the family Reoviridae with an obligatory extrinsic incubation period in the insect host (arbovirus). The virus is composed of 10 segments of double-stranded RNA which code for seven structural and three nonstructural proteins (Huismans et al., 1983; Verwoerd and Huismans, 1972). The viral proteins, P2 and P5, form the diffuse outer coat of BTV and are referred to as the major and the minor outer coat protein, respectively. The major outer coat protein has been associated with serotype restriction, viral attachment in mammalian cells, and induction of neutralizing antibodies. The function of the minor outer coat protein is unknown, but recently it has been shown that this protein contributes to virus neutralization (Cowley and Gorman, 1989; Merrens et al., 1989). The five other structural BTV proteins form the inner core of the virus. The insect vector plays an important role in the natural life cycle and the epidemiol’ To whom correspondence should be addressed. 31

0022-201 l/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

38

GRIEDER

AND

nal antibody (mAb), specific for the Gl glycoprotein of Lacrosse virus, neutralized viral infectivity in mammalian cells, whereas a second mAb neutralized viral infectivity in insect cells, but neither mAb had virusneutralizing activity in both cell lines. This observation demonstrates that mAb can differ in their ability to neutralize virus infectivity on mammalian or insect cells, i.e., that neutralization may be host dependent. To determine if this observation is a phenomenon of other arboviruses, a panel of neutralizing mAb to BTV serotype 17 (BTV 17) was produced. These mAb were used in neutralization assays to evaluate their virus neutralization titers in vertebrate (mammalian) and invertebrate (insect) target cells. Additionally, the mAb were used in radioimmunoprecipitations combined with polyacrylamide gel electrophoresis to determine their binding specificity for BTV proteins. MATERIALS

AND METHODS

Animals. Four male and 12 female Balb/c BJ mice, kept in an isolated, closed colony, were bred at monthly intervals to produce the mice used in the study. The colony was free of murine hepatitis virus and minute virus of mice. Cells and virus. Bluetongue virus serotype 17 (a gift provided by Drs. Jochim and Luedke, ABADRL, Agricultural Research Service, Department of Agriculture Laboratory, Laramie, Wyoming, which is identical to the ATCC strain VR875) was propagated as previously described except that Madin-Darby-Bovine Kidney (MDBK) cells were used rather than BHK-21 cells (Ghalib et al., 1985). Certain experiments required that the stock virus was partially purified by Huebschle’s (1980) modification of the procedure first described by Verwoerd (1969). The virus which was employed in all the experiments was grown in MDBK cells. The mammalian cells were grown in sterile-filtered Eagle’s minimum essential medium (MEM) supplemented with 8% heat-inactivated fetal bovine serum (FBS) and sodium pyruvate. At the time of these

SCHULTZ

experiments, no in vitro cell line had been established from the natural insect host of BTV, Culicoides variipennis. Therefore, in preliminary experiments, using a panel of insect cell lines, two insect cell lines were found to be permissive for BTV infection: C6/36 cells (from Aedes albopictus, identical to the ATCC strain No. CCL 126 and MAT cells (from Aedes triseriutus, kindly provided by Dr. T. Mather, University of Wisconsin-Madison) were grown in MEM containing 5% FBS. MAT cells were maintained in L15 medium supplemented with 1% tryptone broth and 5% FBS. Both insect cell lines were cultured at 28°C under CO, exclusion. It should be noted that the titer of virus on both insect and mammalian target cells was the same. Hybridomu production. Hybridomas were produced as previously described by Appleton and Letchworth (1983). Hybridoma colonies were tested for neutralizing activity by a standard virus neutralization assay. Colonies with neutralizing activity were expanded and cloned by limited dilution. Hybridomas were recloned by limited dilution three times and until all subclones showed the same immunoglobulin class. Neutralization test on mammalian and insect cells. Two insect cell lines (C6/36 and

MAT) and two mammalian cell lines (MDBK and Vero m) were grown to high density in 96-well tissue culture plates as test cells. Serial twofold dilutions of heatinactivated hybridoma ascites containing the different neutralizing mAb were incubated with an equal volume of MEM containing 100 tissue culture infective doses 50 (TCID,,) of BTV 17 for 1 hr at 4°C. An equal aliquot of the hybridoma ascitesvirus mixture was then transferred onto either test insect or mammalian cell cultures and incubated for 72 hr at 28” or 37”C, respectively. At this point, the cells were lysed and the cell lysate as well as supernatants of either the insect cell cultures or the mammalian cell cultures were transferred onto indicator 96-well tissue culture plate containing a confluent monolayer of

HOST-SPECIES

DEPENDENT

MDBK cells and incubated at 37°C for 72 hr. This step was included because BTV does not lyse insect cells and the virus titer therefore cannot be determined on insect cells by plaque formation. The indicator plates were then stained with crystal violet and evaluated for viral growth. The virus neutralization titers were determined as the greatest dilution that completely neutralized 100 TCIDso of BTV 17. Labeling ofviralproteins. Bluetongue virus 17 proteins were biosynthetically labeled with [35S]methionine. Briefly, following infection of monolayer of MDBK cells with plaque-cloned BTV 17 at a multiplicity of infection of three to five for 6 hr, the culture supernatant was removed, and the monolayer was washed twice with methionine-free Dulbecco’s modified Eagles medium (DMEM) and incubated for 30 min with this same medium. The cell monolayer was then incubated with [35S]methionine containing DMEM for a subsequent 90-min period. The radioactive supernatant was decanted and the cells were incubated with DMEM with L-methionine for 30 min. The cells were lysed with 3 ml of NET buffer (0.15 M NaCl, 0.04 M EDTA, 0.04 M Tris, pH 7.0, 2 mM phenylmethylsulfonyl fluoride, containing 0.03% NP-40, and 0.5% bovine serum albumin). The lysate was centrifuged at 100,OOOgat 4°C for 30 min to remove cellular debris and was then preabsorbed to staphylococcus protein A-Sepharose beads coated with rabbit anti-mouse Ab. Radioimmunoprecipitation. The radioimmunoprecipitations were performed in a modified version as previously described (Grubman et al., 1983). Briefly, 1 ml of hybridoma culture supernatant from an antiBTV 17 neutralizing clone was incubated at 4°C for several hours with rabbit antimouse antibody previously bound to protein A-Sepharose beads. Following three wash steps with NET washing solution (0.15 M NaCl, 5 mM Tris, 0.05% Triton X100), approximately 5 X lo5 countslmin of radiolabeled cell lysate was added to the antibody/bead mixture, incubated for 2 hr,

BTV

39

NEUTRALIZATION

and then washed three times. Immune complexes were disrupted by heating the beads in the presence of sodium dodecyl sulfate and 2-mercaptoethanol and analyzed by polyacrylamide gel electrophoresis (Laemmli, 1970). Molecular weight estimation was determined by comparison with standard molecular weight markers, Additionally, the migration of the precipitated proteins was compared to proteins precipitated with other anti-BTV 17 antibodies. These were anti-BTV 17 mAb specific for the outer protein (P2) and for proteins P7 and P9, respectively. The gels were fixed and stained in 10% trichloroacetic acid containing 0.1% Coomassie brilliant blue for 2 hr at room temperature and diffusion destained in 10% glacial acetic acid and 40% methanol. The gels were exposed to radiograph film. RESULTS

A panel of eight neutralizing BTV 17 mAb was produced in two different fusions. The neutralizing titers of the BTV-specific mAb were determined as the dilution of mAb which neutralized 100 TCID,, of plaque-cloned expanded BTV 17 in both mammalian and insect cells. Results are summarized in Table 1. The virus preparation used had the same infectivity for both insect and mammalian cells. Neutralization titers of the mAb ranged from 1:400 to 1:64,000 when assayed in mammalian and insect target cells. Three different patterns were observed when the neutralizing titers of the mAb on mammalian cells were compared to the neutralizing titers on insect cells: (1) Four mAb (82C8, 83(38,92F8, and 83E8) neutralized the virus in mammalian target cells as effectively, e.g., within one dilution, as in insect target cells. (2) Three mAb (83B2, 91E2, and 94GlO) neutralized the virus in mammalian target cells with a lower titer as compared to the neutralization titer in insect cells. The neutralization titer on the insect cells was at least fourfold higher than the titers on the mammalian cells. For example, mAb 94GlO had a neutralization titer of 400 when

40

GRIEDER AND TABLE

1

SCHULTZ

I). This protein was presumed to be the major outer coat protein, because mAb 6C2A (provided by Dr. Letchworth, University of Wisconsin-Madison) precipitated a comparable protein (lane 4, Fig. 1) which has been C6/36 MAT ppt P2” shown to be the major outer coat protein mAb MDBK (Appleton and Letchworth, 1983). Addi82F5 400 800 + 8006 + 83C8 1,600 3,200 3,200 tional proteins were precipitated but were 92F8 32,000 32,000 + 64,000 the result of nonspecific binding because + 83E8 16,000 16,000 32,000 they appeared at the same or higher inten+ 83B2 64,000 64.000 4,000 sity in the control precipitation (lane 2, Fig. 16,000 91E2 2,000 8,000 1). The identity and the molecular weight of 94GlO 400 3.200 6,400 8,000 32,000 9469 2,000 the viral proteins were determined by comparison to known molecular weight stana Results of the immunoprecipitations are listed as dards electrophoresed on the same gel and + for precipitation of BTV 17 major outer coat protein P2 and - for no precipitation of any BTV proteins in by comparison to the protein precipitations several repeats of the experiment. with mAb to other known proteins. Precip’ Neutralization titers of the mAb were determined itations with mouse ascites fluid containing as the reciprocal of the greatest dilution of mouse asa mAb specific for P7 of BTV 17 (clone cites fluid which completely neutralized 100 TCIDjO of 7DSC, lane 5, Fig. 1) or a mAb specific for BTV 17. P9 of BTV 17 (clone 8A3B, lane 6, Fig. 1) resulted in protein precipitations of 45 and tested on mammalian cells as compared to a titer of 3200 when the identical mAb preparation was tested on insect cells. (3) One mAb (9469) neutralized the virus with a different titer in mammalian and insect target cells, as well as a different neutralization titer in the two insect cell lines. These differences in neutralization were observed in three repeats of the experiment. The possibility that the differences were a result of differences between cell lines rather than of cell type (i.e., vertebrate versus invertebrate) was excluded by using different mammlian cell lines (MDBK and Vero m) for the modified virus neutralization test. There were no differences in neutralization titers for all eight mAb observed in the two mammalian cell lines (data not shown). AdFIG. 1. Autoradiography of [35S]methionine-Iabeled ditionally, the cells were lysed to determine Bluetongue virus 17 proteins immunoprecipitated by if virus-cell association during viral replicaneutralizing anti-Bluetongue virus 17 monoclonal antion might be important in this assay, but no tibodies (anti-BTV 17 NMAb). Lane 1, anti-BTV 17 change in the neutralization titer was ob- NMAb 82F; lane 2, Ig Cl control antibody; lane 3, antiBTV 17 NMAb 83C8; lane 4, anti-BTV 17 P2 NMAb served. 6C2A; lane 5, anti-BTV 17 P7 NMAb 7DSC; lane 6, The mAb specificity for viral proteins anti-BTV 17 P9 NMAb 8A3B; and lane 7, anti-BTV 17 was determined by radioimmunoprecipitarabbit typing serum. Bluetongue virus proteins P2, PS, tion. Two mAb, 82F5 and 83C8, precipiP6, F7, and P9 are labeled with their relative molecular tated a 9%kDa protein (lanes 1 and 3, Fig. masses (kDa). NEUTRALIZATION TITERSOF MONOCLONAL ANTIBODIES ON INSECT (C6/36, MAT) AND MAMMALIAN (MDBK) CELLS AND IMMUNOPRECIPITATION RESULTS

HOST-SPECIES

DEPENDENT

43 kDa, respectively. Additionally, rabbit anti-BTV 17 typing serum precipitated at least five viral-associated proteins: P2 (98 kDa), P5 (66 kDa), P6 (52 kDa), P7 (45 kDa), and P9 (43 kDa) (lane 7, Fig. 1). Three other mAb (83B2, 83E8, and 92F8) precipitated a 98-kDa protein, the BTV 17 major outer coat protein (data not shown). DISCUSSION

In the present study, eight neutralizing mAb produced against BTV 17 were described. To determine if there were differences in the neutralization of viral infectivity for insect cells versus mammalian cells, a virus neutralization test was performed on both insect and mammalian target cells, and the neutralization titers of the different mAb were compared. All mAb which had positive neutralization titers for BTV infectivity in mammalian cells also had positive neutralizing activity for BTV infectivity in the two insect cell lines, C6/36 and MAT. To evaluate differences in neutralization of viral infectivity in mammalian cells versus insect cells, sets of identical ascites fluid dilutions containing the different mAb were used in neutralization assays in both mammalian and insect target cells. Although all eight mAb neutralized BTV infectivity in both insect and mammalian cells, differences in the ability of the same mAb neutralized the virus with a higher titer when the target cell was of insect origin as compared to the neutralization titer in mammalian cells. One explanation for the differences in the neutralizing activity is that the mAb bind to different viral proteins. Therefore, immunoprecipitations were performed to determine the binding specificity of the mAb. Five of the mAb precipitated the 98-kDa major outer coat protein, while three mAb failed to precipitate any detectable BTV protein in several repeats of the immunoprecipitation. It should be noted that the epitopes bound by these mAb are conformationally dependent as was recently dem-

BTV

NEUTRALIZATION

41

onstrated by Western blot analysis (Grieder and Schultz, 1989). The three neutralizing mAb that failed to precipitate the major outer coat protein might bind to the minor outer coat protein, P5, or to a combination of both the major and the minor outer coat protein. Repeated attempts to crosslink Ab to viral proteins gave inconclusive results. We have topographically mapped the epitopes bound by these mAb and have identified five separate epitopes. As would be predicted by the differences in both neutralization and immunoprecipitation, mAb 83B2 and 91E2 bind distinct epitopes (Grieder and Schultz, 1990). These observations support the possibility that some of the mAb bind to quaternary protein structures composed of noncontiguous protein sequences of separate BTV outer coat proteins. Recently, studies using reassortants of two different BTV serotypes provided further evidence that conformational determinants of both outer coat proteins (P2 and P5) interact with neutralizing Ab to form neutralizing epitopes (Cowley and Gorman, 1989; Mertens et al., 1989). The differences then in the neutralizing activity of these mAb may be a result of different roles played by the two BTV outer coat protein, P2lVP3 and P5, in infectivity in insect target cells versus mammalian target cells. Despite the lack of knowledge about the mechanism of BTV neutralization, it is possible that both the major and the minor outer coat protein contribute neutralizing determinants in both the mammalian and insect cells. In this case, mAb that bind to P5 and/or interfere with the P5 interaction with insect cells may have an increased neutralizing effect because the minor outer coat protein may have a unique function in the infectivity in insect cells. A similar situation has been observed in another arbovirus (e.g., Lacrosse virus) where the small envelope glycoprotein appears to be important for the attachment of the virus to insect target cells and mosquito midgut epithelium cells. The larger envelope glycoprotein is the viral attachment

42

GRIEDER

AND

protein to mammalian cells (Ludwig et al., 1989). The two outer coat proteins of BTV may also have similar functions. In conclusion, differences in neutralization activity were observed when a panel of neutralizing mAb was used to neutralize BTV infectivity on mammalian target cells versus insect target cells. Studies are in progress to determine the basis of these differences. ACKNOWLEDGMENTS The authors thank Dr. G. Letchworth for assisting in the initiation of these studies as well as supplying monoclonal reagents and Jocelyn Penner for technical assistance. This work was supported by the U.S. Department of Agriculture Grants 85CRSR-2-2632 and 87-CRSR-2-3172 and the School of Veterinary Medicine, University of Wisconsin, Madison.

REFERENCES APPLETON, A. J.. AND LETCHWORTH, G. J. 1983. Monoclonal antibody analysis of serotype-restricted and unrestricted Bluetongue virus antigenic determinants. Virology, 124, 286-299. CHANDLER, L. J., BALLINGER, M. E., JONES. R. H.. AND BEATY, B. J. 1985. The virogenesis of Bluetongue virus in Culicoides variipennis. In “Bluetongue and related Orbiviruses” (T. L. Barber and M. M. Jochim, Eds.), pp. 245-253. A. R. Liss., New York. COWLEY, J. A., AND GORMAN, B. M. 1989. Crossneutralization of genetic reassortants of Bluetongue virus serotypes 20 and 21. Vet. Microbial. 19, 37-51. FENNER, F., BACHMAN, P. A., GIBBS, E. P. J., MURPHY, F. A., STUDDERT, M. J., AND WHITE, D. 0. 1987. In “Veterinary Virology,” pp. 583-586. Academic Press, Orlando. GHALIB, H. W., CHERRINGTON, J. M., AND OSBURN, B. I. 1985. Virological, clinical, and serological response of sheep infected with tissue culture adapted Bluetongue virus serotype 10, 11, 13, and 17. Vet. Microbiol.

10, 178-188.

GRADY, L. J., AND KINCH,

W. 1985. Two monoclonal

SCHULTZ

antibodies against La Crosse virus show hostdependent neutralizing activity. J. Gen. Virol., 66, 2773-2776. GREIDER. F. B., AND SCHULTZ, K. T. 1989. Conformationally dependent epitopes of Bluetongue virus neutralizing antigen. Viral Immunol. 2, 17-24. GREIDER, F. B., AND SCHULTZ, K. T. 1990. Antigenic topography of neutralizing antigen of Bluetongue virus. Microb. Pathol., in press. GRUBMAN, M. J., APPLETON, A. J., AND LETCHWORTH, G. J. 1983. Identification of Bluetongue virus type 17 genome segments coding for polypeptides associated with viral neutralizing and intergroup reactivity. Virology, 313, 355-366. HUEBSCHLE, 0. J. B. 1980. Bluetongue, virus hemagglutinin and its inhibition by specific sera. Arch. Virol., 64, 133-140. HUISMANS, H., VAN DER WALT, N. T., AND CLOETE, M. 1983. The biochemical and immunological characterization of Bluetongue virus outer capsid polypeptides. In “Double-Stranded RNA viruses” (R. W. Compans and D. H. L. Bishop, Eds.), pp. 165-172. Elsevier, New York. KING, B. M., AND ALDERS, M. A. 1985. Morphology of Bluetongue virus-infected Aedes albopictus (C6/ 36) ceil culture. In “Bluetongue Virus and Related Orbiviruses” (T. L. Barber and M. M. Jochim, Eds.), pp. 289-294. A. R. Liss, New York. LAEMMLI. U. K. 1970. Cleavage of the structural protein during the assembly of Bacteriophage T4, Nature (London),

277, 680-685.

LUDWIG, G. V., CHRISTENSEN, B. M., YUILL. T. M., AND SCHULTZ, K. T. 1989. Enzyme processing of Lacrosse virus glycoprotein Gl: A Bunyavirusvector infection model. Virology, 171, 108-l 13. MERTENS, P. P. L., PEDLEY, S., COWLEY, J., BURROUGHS, J. N., CORTEYN, A. H., JECCO, M. H., JENNING, D. H., AND GORMAN, B. M. 1989. Analysis of the role of Bluetongue virus outer proteins VP2 and VP5 in determination of rural serotypes. Virology, VERWOERD,

170, 561-565.

D. W. 1969. Purification and characterization of Bluetongue virus. Virology, 38, 133-312. VERWOERD, D. W., ELS, H.. AND HUISMANS, H. 1972. Structure of the Bluetongue virus capsid. J. Viral.,

10, 783-794.

Host-dependent variation of bluetongue virus neutralization.

To determine if neutralizing epitopes of Bluetongue virus (BTV) 17 are host dependent, e.g., that monoclonal antibodies (mAb) to Bluetongue virus 17 (...
724KB Sizes 0 Downloads 0 Views