Infection of Aedes aegypti cells with mosquito iridescent virus

JOURNAL

OF INVERTEBRATE

Infection

23, 255-258

PATHOLOGY

of Aedes

aegypti

(1974)

cells

with

Mosquito

Iridescent

Virus

Mosquito iridescent virus (MIV) of the were used for inoculation before there was appreciable contact inhibition. The ‘T’ black salt marsh mosquito, Aedes taenioand ‘R’ (RMIV, strains of MIV rhynchus, (T. B. Clark, W. R. Kellen, and (TMIV) P. T. M. Lum, J. Invertebr. Pathol. 7, (J. F. Matta and R. E. Lowe, J. Invertebr. Pathol. 16, 38-41, 1970), were puri,fied ac519-521, 1965) has received recent attention as a potentially useful pathogen for the cording to the procedure of G. W. Wagner, microbial control of mosquitoes and as a J. D. Paschke, W. R. Campbell, and S. R. model for the icosahedral cytoplasmic de- Webb (Virology 52, 72-80, 1973) then passed through a filter with a 0.65 pm pore oxyriboviruses (D. B. Stoltz, J. Ultrastruct. Res. 43, 58-74, 1973). Substantial evidence size (Millipore Corp., Bedford, Mass.). has accumulated regarding the pathology Aliquots of 0.1 ml of the virus, suspended of the disease in infected larvae as well as in M and M salt solution, were added dibiochemical and biophysical characterizarectly to the existing 5 ml media covering tion of the virions. However, progress has the cells to give a virion/cell ratio of 300; been impeded by the low incidence of infec- control cells were inoculated with an equal tion in laboratory inoculated mosquitoes. volume of diluent. The cells were incubated The infection appears independent of dose at 21”, 25”, 31”, and 35°C and inspected and a suitable bioassay has not been develdaily by light microscopy for the occuroped. Efforts to infect other invertebrates rence of cytopathogenic effects (CPE). as an alternate source of MIV have only No CPE was evident at 21°C and the strengthened the evidence for the specificity evidence for a reproducible CPE at 25°C of the virus to A. taeniorhynchus (D. B. was inconclusive. However, at 31” or 35°C Woodard and H. C. Chapman, J. Invertebr. a characteristic CPE was exhibited. The PathoZ. 11, 296-301, 1968). Studies were CPE caused by either TMIV or RMIV in initiated to develop a reliable bioassay pro- Singh’s A. aegypti cells was initially manicedure in vitro. This report presents evi- fest at 2.5 days as a slight granular appeardence that MIV will infect Aedes aegypti ance and increased vacuolization of the cells in continuous culture. cytoplasm followed by a tendency for adjaTwo cell lines derived from A. aegypti cent cells to form small clusters. Incubation (K. R. P. Singh, Curr. Sci. 36, 506-508, beyond 3 days resulted in the formation of 1967; J. Peleg, Virology 35, 617-619, 1968) tenuous cytoplasmic filaments, frequently were maintained on M and M medium containing refractile ,bodies, that adhered (J. Mitsuhashi and K. Maramorosch, Con- to surrounding cell clusters or individual trib. Boyce Thompson Inst. 22, 435-436, cells (Fig. la). In contrast, the control cells 1964) containing 100 units/ml of penicillin, continued development and established a 100 pgjml of streptomycin, and 0.25 pg/ml confluent monolayer (Fig. lb). At 5 days of amphotericin B. Approximately 5 X lo5 the inoculated cells became more enlarged, cells were transferred to tissue culture refractile, and rounded followed by abnorflasks with 25 cm” available substrate (Falmal detachment from the substrate. By 7-8 con Plastics, Oxnard, Cal.) and allowed to days after virus inoculation most cells were grow for 2-3 days at 25°C. Cell monolayers floating with only small groups of normal255 Copyright All rights

@ 1974 by Academic Press, Inc. of reproduction in any form reserved.

NOTES

FIG.

with x300.

1. Singh’s TMIV and

Aedes aegypti cells incubation at 31°C

(a) for

showing 3 days;

look :ing cells adhering to the substrate. The cant ;rol cells at this time had begun to grow in st:veral layers. c iontrol and MIV inoculated cells were

cytopathogenic (b) control

cells

effects after inocu latic 3n of the same cell lir le,

prepared for electron microscopy by fixation in situ with 2.57% gluteraldel bd le followed by 0.5% 0~0~; both were buff ered and washed with M and M salt sol ution.

257

NOTES

FIG. 2. Electron micograph of Singh’s Aedes negypti cells inoculated with TMIV and incubated at 31°C for 2.5 days. V, virus; VS, virogenic stroma; N, nucleus; P, plasma membrane protrusion.

After staining overnight in 0.5% aqueous uranyl acetate, the cells were embedded according to the method of H. J. Luft (J. Biophys. Biochem. Cytol. 9, 409-414, 1961). Ultrathin sections were stained with lead citrate and examined with a Phillips EM 201 operating at 60 kv. Cells incubated at 31% for 2.5 days with either RMIV or TMIV contained virions in a virogenic stroma which was relatively void of cytoplasmic organelles (Fig. 2). In addition to these foci of infection the virions were found in proximity to the

plasma membrane of numerous vacuoles and the external surface of the cell. Protrusions of the plasma membrane, homologous to the cytoplasmic filaments seen by light microscopy, were more predominant in the inoculated cells. In additional contrast to the control cells, chromatin material of infected cells concentrated near the periphery of the nuclear envelope and formed a dense ring. Peleg’s A. aegypti cells exhibited CPE characteristics similar to Singh’s A. aegypti cells with both strains of MIV. However,

258

NOTES

the early occurrence of cytoplasmic filaments (2-3 days) could not be consistently used as a criterion for infection in this more elongate cell type. Electron microscopic observations of Peleg’s cells incubated for 2.5 days at 31% with either TMIV or RMIV were also similar.

part by the U.S. Public Grant AI-09972.

Health Service Research

8. R. WEBB J. D. PASCHKE G. W. WAGNER W. R. CAMPBELL Department of Entomology

Purdue University The authors gratefully acknowledge the technical assistance of Ms. Ramona Lambert in carrying out this study. The investigation was supported in

West La/aye t te, Indiana 4790?’ Received May 31, 1973