MICROASSAY OF BLUETONGUE VIRUS FROM CULICOIDES VARIIPENNIS*
B, M. BANDO R. H, JONES
U. S. Department of Agriculture Agricultural Research Service
Arthropod-borne Animal Disease Research Laboratory Denver, Colorado
I. INTRODUCTION
Bluetongue is an arthropod-borne disease of domestic and wild ruminants and is biologically transmitted by the gnat, Culicoides variipennis. BTV has been isolated from a variety of animal tis- sues and insects by inoculation of ECE (Foster and Luedke, 1968).
The EAS is convenient and economical and has been used routinely by researchers at the Arthropod-borne Animal Disease Research Laboratory. However, studies of the genetic factors affecting oral susceptibility of C, variipennis to infection with BTV have created a need to assay large numbers of individual gnats; the EAS presently in use does not satisfy this increased demand.
Cell culture systems are not widely used to isolate BTV (Howell et al., 1970). However, a MAS that economizes on space,
•Abbreviations used: BTV, bluetongue virus; CPE, cytopathic effect; EAS, egg assay system; EBME, Eagle's basal minimal essen- tial medium in Earle1s balanced salt solution; ECE, embroyonating chicken eggs, E L D 5 0 , virus dose required to infect 50% of embryos inoculated; FBS, fetal bovine serum; IHM, insect-holding medium;
MAS, microassay system; TCID5Q, virus dose required to infect 50%
of cultures inoculated.
667
668 Β. M. BANDO AND R. H. JONES
reagents, and personnel would be an ideal replacement for the EAS.
The purpose of this study was to determine whether a cell cul- ture MAS would be as efficient in detecting BTV-infected gnats as the EAS and to compare the infection rate1 of gnats artificially provided a blood meal containing cell culture adapted BTV to the infection rate of gnats fed a blood meal containing ECE adapted BTV.
II. MATERIALS AND METHODS A. C. variipennis
Gnats were from two colonies of C, variipennis maintained at this laboratory. The Sonora (Texas) strain (000 line) has been maintained by Jones since 1957 without the addition of wild flies
(Jones et al., 1969). The Bruneau (Idaho) strain (036 line) was established in 1973.
B. Cell Cultures
BHK-21 (Clone 13)^ was subcultured twice weekly and was grown in 10% FBS, 10% tryptose phosphate broth, and 80% EBME at a pH of 7.2. Suspensions for microplates contained 100,000 cells/ml.
VERO^, African green monkey kidney, was subcultured once week- ly and was grown in 10% FBS and 90% 199E at a pH of 7.0. Suspen- sions for microplates contained 150,000 cells/ml. Antibiotics were added to growth media to obtain a final concentration of
1Infection rate, a measure of oral susceptibility, was deter- mined for each group of gnats and is the percentage of BTV-infect- ed gnats per group assayed.
2American Type Culture Collection, 12301 Park Lawn Drive, Rock- ville, Maryland 20852.
3Gorgas Memorial Institute - Middle America Research Unit, Box 2011, Balboa Heights, Canal Zone.
MICROASSAY OF BLUETONGUE VIRUS 669 gentamicin 50 mcg/ml, penicillin 200 units/ml, and Fungizone1 2
mcg/ml.
C. Egg Assay System
ECE were used to detect BTV in gnat samples and to prepare ECE adapted virus. Inoculations were made into the yolk sac of 11-day old ECE. Death pattern in eggs was the principal criteri- on used to determine whether gnats became infected with BTV.
D. Virus
2
BTV (62-45S) was subpassaged twice in BHK-21; final titer was 107·5TCID50/ml in BHK-21 MAS. To prepare ECE adapted BTVf we subpassaged 62-45S 8 times in ECE; final titer was 107·5ELD50/ml.
E. Artificial BTV-Blood Meal
Gnats, 1-4 days old, were provided a blood meal that con- sisted of 1 part cell culture or ECE adapted BTV and 9 parts de- fibrinated sheep blood (Jones and Foster, 1971). Blood-fed fe- male gnats (only the female is blood-sucking) were maintained on a sugar and water diet for 14 days to allow time for adequate virus multiplication; they were then killed and stored at -70°C until sample preparation. Individual gnats were disrupted by sonification in IHM (pH 7.0) that was 20% FBS and 80% EBME; genta- micin, penicillin, and Fungizone were added to obtain a final con- centration of 100 mcg/ml, 200 units/ml, and 4 mcg/ml, respective- ly. Prepared gnat samples were held at 4°C until assayed.
F. Microassay System
Rigid, plastic, 96-well, flat-bottomed microplates^ treated for cell culture were used. Each well received 50 pL of cell
^E. R. Squibb and Sons, Inc., Princeton, New Jersey 07540.
2Thomas L. Barber, USDA-ARS, Denver, Colorado 80225.
^Linbro Chemical Co., Los Angeles, California 90020.
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suspension and 50 uL of the appropriate growth medium; 4 wells of BHK-21 and VERO cell culture received 25 ]iL of sonified gnat sam- ple. To serve as a control, gnat-free IHM was added to 4 wells of each culture. Microplates were incubated at 35°C in a humidi- fied, 2% C 02 in air atmosphere. Monolayers were examined micro- scopically on the 5th, 6th, and 7th days for the development of CPE. Monolayers with 75% or greater CPE were scraped with a pipette, aspirated, and placed in a equal volume of buffered lac- tose peptone1. Cell cultures with CPE were confirmed as BTV- positive by an indirect immunofluorescent staining technique
(Jochim et al., 1974).
III. RESULTS AND DISCUSSION
Data in Table I, Test 1 showed that infection rates were similar for each assay system. Test 2 supported the results of Test 1 for the BHK-21 and VERO MAS. In all tests (Tables I and II) the BHK-21 MAS appeared to be able to detect more BTV-in- fected gnats than the VERO MAS regardless of the source of the blood meal.
Data in Table II, Test 1 confirmed that the infection rates obtained with each assay system were similar; however, the infec- tion rates for the 036 line gnats were typically higher than those for the 000 line. Unpublished laboratory data suggested that the 036 line had a greater oral susceptibility than the 000 line. Therefore, we expected the 036 line to have a higher in- fection rate than the 33% actually obtained when gnats were fed ECE adapted BTV and were assayed in EAS. An infection rate of 47% was obtained for the 036 line fed BHK-21 adapted BTV and as- sayed in BHK-21 MAS.
^Each liter of buffered lactose peptone (pH 7,2) contains 220 ml 0.15 M NaH2PO4'H20, 780 ml 0.15 M Na2HP04, 100 g lactose, and 2 g peptone. Final pH is 7.2.
MICROASSAY OF BLUETONGUE VIRUS 673 Our results indicated that the source of virus may have in-
fluenced the difference in the infection rates between the 000 and the 036 lines. When the infection rates for the 000 and 036 line gnats fed ECE adapted BTV are compared in all assay systems
(ECE, Test lf Table I), differences are 4%, 0%, and 16%, However, when infection rates for the 000 and 036 line gnats fed BHK-21 adapted BTV are compared in all assay systems (BHK-21, Test 1, Table I), differences are 27%, 23%f and 23%. This comparison of differences between Tables I and II for Test 1 suggests that the source of the BTV will affect the infection rate obtained in all the assay systems used. But differences in infection rates for the 000 line fed ECE or BHK-21 adapted BTV are 3% (Table I, Test 1) whereas differences for the 036 line were 10-20% (Table II, Test 1). Apparently, the source of virus affected the infection rates for the 036 line but not for the 000 line. The methods of virus preparation may also have influenced the number of gnats that are infected; the ECE adapted BTV was a whole embryo ex- tract containing extraneous protein but the BHK-21 adapted BTV contained very little extraneous protein.
In summary, the MAS apparently will be a suitable substitute for the EAS. Also the use of the BHK-21 MAS and BHK-21 adapted BTV infective blood meals may result in higher and more consistent infection rates.
ACKNOWLEDGMENTS
The authors gratefully acknowledge the technical assistance of Marlin Larson, Howard Rhodes, and Regina DeHerrera.
674 Â. M. B A N D O A N D R. H. JONES REFERENCES
Foster, N. M. and Luedke, A. J, Q.968). American J, Vet. Res. 29, 749-753.
Howell, P. G., Kumm, N. A. and Botha, M. J. (1970). Onderstpoort J. Vet. Res. 37, 59-66.
Jochim, M. M., Barber, T. L. and Bando, B. M. (1974). Proceedings of American Association of Veterinary Laboratory Diagnosticians, 17th Annual Meeting, 91-103.
Jones, R. H. and Foster, Ν. M. (1971). J. Med. Ent. 8, 499-501.
Jones, R. Ç., Potter, H. W. and Baker, S. K. (1969). J. Econ. Ent.
62, 1483-1486.
