JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 1975, p. 241-242 Copyright 0 1975 American Society for Microbiology

Vol. 1, No. 2 Printed in U.S.A.

Plaque Assay for Rabies Serogroup Viruses in Vero Cells SONJA M. BUCKLEY*

AND

G. H. TIGNOR

Yale Arbovirus Research Unit, Department of Epidemiology and Public Health, Medicine, New Haven, Connecticut 06510

Yale University School of

Received for publication 4 November 1974

Plaque formation in Vero cells has been induced with seven rabies serogroup viruses either cocultivated as infected BHK-21 or Aedes albopictus cells, or directly cultivated as infected mouse brain.

Viruses of the rabies serogroup (5, 6) are usually difficult to propagate directly in cell culture under agar overlay without prior adaptation (3). Comparison of field strains by the plaque reduction neutralization test has thus been impracticable. In this laboratory, however, plaques have been induced in Vero cells under agar overlay containing neutral red and diethylaminoethyldextran with use of material passed only once in vitro-derived either from Vero cells infected

kotonkan, from arthropods, multiplied only in A. albopictus cells; and the other five strains, all from vertebrates, multiplied only in BHK-21 cells. None of the isolates multiplied in Singh's A. aegypti cells. Plaque formation in Vero cells under agar overlay was subsequently obtained either by direct cultivation of mouse-brain virus or by cocultivation of infected BHK-21 or A. albopictus cells (Table 2). Under the cocultivation method used (1), infected cells were dis-

TABLE 1. Results of testing mouse-brain preparations of rabies serogroup viruses for plaque formation in Vero cells and ability to infect BHK-21 and Singh's Aedes albopictus and Aedes aegypti cells Virus

Rabies

Lagos bat Mokola Obodhiang Kotonkan Nigerian horse Duvenhage

Multiplicationa

7Mouse passage Strain level

CVS TRVL 5843 Prototype IbAn 27377 SudAr 1154-64 IbAr 23380 Prototype

Prototype

(Ves (Vero)

202+ 3 15

0 + + + 0 0

8 3 9 25 3

a As determined by subinoculation of undiluted mice by the intracerebral route.

0 0

BHK-21

+ + + + 0 0 + +

A.

A.

albopictus

aegypti

0 0 0 +

0 0 0 0

+

0 0 0 0

+ 0 0

fluid phase or combined fluid and cell phases into 2-day-old

with Mokola virus (7) or from Singh's Aedes albopictus cells infected with Obodhiang or kotonkan virus (1). We report here the development of a plaque assay for rabies serogroup viruses in Vero cells. When mouse-brain preparations of the seven viruses (eight strains) listed in Table 1 were tested for in vitro characteristics, only three, rabies (TRVL 5843), Lagos bat, and Mokola, contained plaque-producing viral progenies capable of initiating serial plaque passages in Vero cells. Mokola, isolated from shrews (insectivores), multiplied in both BHK-21 and Singh's A. albopictus cells; Obodhiang and 241

sociated nonenzymatically with a rubber policeman and a small amount (1 to 2 ml per 106 cells) of diluent consisting of 0.75% bovine albumin in phosphate-buffered saline, pH 7.2, containing diethylaminoethyl-dextran; the cell suspension, after additional dispersal by pipetting, was inoculated into plaque bottles and cocultivated with Vero cells under agar overlay (4, 8). Welldefined, readily countable plaques, each produced by one infectious virion, appeared within 6 to 10 days (Table 2). Plaque-derived clones were obtained by randomly selecting progenies of well-separated plaques and plaque-passaging them four to seven times; cloned stocks were

J. CLIN. MICRC)BIOL.

NOTES

242

TABLE 2. Plaque-passaging of rabies serogroup viruses in Vero cells Maxi-

J Virus

Passage historVa

Cocultl ocut1

[No. of No. of' plaques passages plaque vation examined

Plaque Diameter (mm) PPVPh from one appearing on day (day 21)

mum no.

plaque

BHK-21' Rabies, CVS Rabies, TRVL 5843 3 MB 15 MB Lagos bat 8MB Mokola 3 MB, 1 Ades albopictus Obodhiang 9 MB, 1 A. albopictus Kotonkan 25 MB, 2 BHK-21 Nigerian horse 3 MB, 1 BHK-21 Duvenhage

+ NDd ND ND + 4-

+

20 39 30 8 8 5

50+ 50+

5 4

4 4

4 4 7 7

> 200 > 200 > 200 > 10.000 38 94 > 200 > 200

3 3 8 7 7 10 5 4 6 2 8 2 63

6 10

MB = mouse brain. h PPVP = plaque-producing viral progenies. c This rabies CVS strain was kindly supplied by F. A. Murphy as a BHK-21 adapted strain. It was given one additional passage in BHK-21 cells in our laboratory. d ND = cocultivation not done. a

prepared in Vero cells or in mice. Development of plaques was specifically inhibited by hyperimmune mouse ascitic fluids or sera prepared against the parent strains. In addition, cloned viruses were reidentified by complement-fixation (2) or mouse neutralization test, or by

fluorescent-antibody technique. Plaque-derived clones of Lagos bat, Obodhiang, and kotonkan viruses were less virulent for mice than their parent strains. Cocultivation of infected rodent or mosquito cells with either BHK-21 or Vero cells under fluid medium induced a cytopathic effect that ranged from moderate (2 to 3+) to severe (4+); this was repeated in serial successive passages. The plaque assay in Vero cells has already proved of value in the selection of variants of rabies serogroup viruses. This work was supported by The Rockefeller Foundation and by Public Health Service grant 1-PO1-AI-11132 from the National Institute of Allergy and Infectious Diseases. We thank Charles Mullen, Elinor Gilson, Mildred Malhoit, and Katherine Moreno for excellent technical assistance.

LITERATURE CITED 1. Buckley, S. M. 1973. Singh's Aedes albopictus cell cultures as helper cells for the adaptation of Obodhiang and kotonkan viruses of the rabies serogroup to some vertebrate cell cultures. Appl. Microbiol. 25:695-696. 2. Casals, J. 1967. Immunological techniques for animal viruses, p. 113. In K. Maramorosch and H. Koprowski (eds.), Methods in virology. Academic Press Inc., New York. 3. Clark, H. F. 1972. Growth and attenuation of rabies virus in cell cultures of reptilian origin. Proc. Soc. Exp. Biol. Med. 139:1317-1324. 4. Karabatsos, N. 1969. Characterization of viruses isolated from bats. Am. J. Trop. Med. Hyg. 18:803-810. 5. Kemp, G. E., V. H. Lee, D. L. Moore, R. E. Shope, 0. R. Causey, and F. A. Murphy. 1973. Kotonkan, a new rhabdovirus related to Mokola virus of the rabies serogroup. Am. J. Epidemiol. 98:43-49. 6. Shope, R. E., F. A. Murphy, A. K. Harrison, 0. R. Causey, G. E. Kemp, D. I. H. Simpson, and D. L. Moore. 1970. Two African viruses serologically and morphologically related to rabies virus. J. Virol. 6:690-692. 7. Shope, R. E., and G. H. Tignor. 1971. Rabies and serologically related viruses from Africa, p. 45-48. In Y. Nagano and F. M. Davenport (ed.). Rabies. University of Tokyo Press, Tokyo. 8. Simizu, B., J. S. Rhim, and N. H. Wiebenga. 1967. Characterization of the Tacaribe group of arboviruses. I. Propagation and plaque assay of Tacaribe virus in a line of African green monkey kidney cells (Vero). Proc. Soc. Exp. Biol. Med. 125:119-123.

Plaque assay for rabies serogroup viruses in vero cells.

JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 1975, p. 241-242 Copyright 0 1975 American Society for Microbiology Vol. 1, No. 2 Printed in U.S.A. Plaque As...
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