J. gen. Virol. (1969), 5, 545-546
With I plate
545
Printed in Great Britain
Plaque Formation by an Arbovirus in a Mosquito Cell Line
(Accepted 23 June 1969)
Established lines of insect cells have been obtained only recently and their use in
arbovirus studies is only beginning (Suitor, I966; Converse & Nagle, 1967; Rehacek,
1968; Singh & Paul, 1968a, b; Banerjce & Singh, 1968; Yunker & Cory, 1968). Most
arbovirus growth reported in insect cell lines does not cause concomitant cytopathic
effect (CPE) and plaque formation has not been reported before.
The cell line employed in this study originated from tissues of the mosquito A~des
albopictus (Singh, 1967). The cells were cultured in a manner identical to that described
by Singh (1967) and complete monolayers were obtained about 5 days after subculture. Because this line contains cell types of various sizes, a single cloning was made
in order to isolate the smallest cell type. Small coverslips were placed in Petri dishes
and dilutions of cell suspensions added. After incubation, coverslips containing single
colonies were transferred to tubes in order to increase the cell population. Although
this method still led to cell types of different morphologies, one such 'clone' was used
for these initial tests of plaque formation.
Singh & Paul (1968a) reported appearance of CPE when cells of A~des albopictus
were infected with a mouse-brain-adapted strain of Japanese encephalitis virus. In the
present work, the ocT-541 (35-24) strain of Japanese encephalitis virus (Rohitayodhin
& Hammon, 1962) adapted to growth at 24 ° was used. It was obtained from the
American Type Culture Collection in its 39th tissue culture passage and from this
was prepared a virus pool in primary hamster kidney cells cultured at 28 °. The pool had
a TCD5o of lO7 for both hamster kidney and A(des albopictus cells when titrated
under liquid medium.
The following method was used for plaque formation. Monolayers in T-15 glass
flasks of the cloned Agdes albopictus cells at about 6 days old were washed once with
phosphate buffered saline, pH 7"4, containing 0"75 % bovine plasma albumin. Virus
dilutions, made in the same solution, were added (0.2 ml. volume for T-I5 glass
flasks), and the virus allowed to absorb while the flasks were rocked slowly for 2 hr at
28 °. The flasks were then rinsed twice in phosphate buffered saline + bovine albumin
and the monolayers overlayered with z ml. overlay medium at 43 °. The overlay
consisted of equal volumes of 2 % Agarose (Seakem) in distilled water and doublestrength Mitsuhashi & Maramorosch (M & M) medium, prepared as described by
Singh (1967) except that only 8% foetal bovine serum (FBS) was used: the final
concentrations were 1% Agarose, 4 % F.B.S, in single-strength M & M medium.
After incubation for I week at 28 °, 2 mI. of a second over layer containing I/3O,OOO
neutral red was added. Plaques were evident within another 24 hr, but were clearer at
72 hr. The plaques had irregular outlines, but were of similar size (about 4 mm.
diameter: Plate I). Plaque titres were about lO times less than the TCD5o in hamster
kidney or Agdes albopietus cells in liquid medium, but were very reproducible. The
plaque content was directly proportional to the virus concentration over the range
showing clearly countable plaques. The plaque number was reduced when the virus
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Journal of General Virology, Vol. 5, No. 4
Plate I
tl
•
_
,~2~':
¸
•
Monolayers of A~des albopictus cells in T-6o flasks showing plaques due to Japanese encephalitis
virus, strain ocw-541 (35-z4) on left. Uninfected, control cells on right.
(Facing p. 545)
E. C. SUITOR, JUN.
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546
Short communications
was neutralized b y i n c u b a t i o n in ascitic fluid f r o m mice i m m u n e to J a p a n e s e encephalitis
virus (I h r at r o o m t e m p e r a t u r e ) before i n o c u l a t i o n o f the cells.
T h e fact t h a t the cell line used can be cultured c o n t i n u o u s l y a n d t h a t the p l a q u e s
f o r m e d in the present system can be easily c o u n t e d m a k e s it a p o t e n t i a l l y p o w e r f u l new
t o o l for q u a n t i t a t i v e assay o f arboviruses in insect cells.
I gratefully a c k n o w l e d g e the able technical assistance o f Miss F. P a u l a n d A. V.
V o r n d a m , Lt, U S N R .
T h e opinions a n d statements in this p a p e r are those o f the a u t h o r a n d are n o t to be
u n d e r s t o o d as official o r reflecting the views o f the D e p a r t m e n t o f the N a v y o r the
N a v a l service at large.
F r o m the B u r e a u o f M e d i c i n e a n d Surgery, D e p a r t m e n t o f the N a v y , R e s e a r c h
T a s k M R o o 5 . 09 . ooo6.
T h e late E. C.
Naval Medical Research Institute
National Naval Medical Center
Bethesda, M a r y l a n d 2oo14, U.S.A.
SUITOR,JUN.*
REFERENCES
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line infected with arboviruses. Indian J. reed Res. 56, 812.
Co~rcERSE,J. L. & NAGLE,S. C., JLrN. (1967). Multiplication of yellow fever virus in insect tissue cell
cultures. J. ViroL i, lO96.
REnACEK, J. (I968). The growth of arboviruses in mosquito cells in vitro. Acta virol. Prague i2, 24I.
RonrrAvODrm% S. & HA~ON, W. McD. (1962). Studies on Japanese B encephalitis virus vaccines
from tissue culture. II. Development of an attenuated strain of virus. J. Immun. 89, 589.
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aegypti (L). Curt. Sci. 36, 5o6.
SrNGH, K. R. P. & PAUL, S. D. (I968a). Multiplication of arboviruses in cell lines from Aedes albopictus and Aedes aegyptL Curr. Sci. 37, 65.
SING~I,K. R. P. & PAUL, S. D. (1968b). Susceptibility of Aedes albopictus and Aedes aegypti cell lines
to infection by arbo and other viruses. Indian J. reed. Res. 56, 815.
SUITOR, E. C. Jtm. (1966). Growth of Japanese encephalitis virus in Grace's continuous line of moth
cells. Virology 3o, 143.
Ytr~CER, C. E. & CORV, J. (I968). Infection of Grace's Antheraea cells with arboviruses. Am. J. trop.
Med. Hyg. x7, 889.
(Received 17 February 1969)
* Reprints may be obtained from The Director, Department of Microbiology at the Naval Medical
Research Institute.
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