Replication of a cytoplasmic polyhedrosis virus in an established cell line of Trichoplusia ni cells

VIROLOGY

69,

584-586

Replication

(1974)

of a Cytoplasmic Cell

ROBERT

Line

R. GRANADOS,

Polyhedrosis of Trichoplusia W. J. MCCARTHY,

Virus

in an

Established

ni Cells AND

M. NAUGHTON

Boyce Thompson Znstitute for Plant Research, Yonkers, New York 10701 Accepted February 81, 1974 The complete replication of a cytoplasmic polyhedrosis virus in an established line of the insect Trichoplusia ,ni was demonstrated. Virus produced in cultures infectious to T. ni larvae.

Grace (1) reported on the development of a cytoplasmic polyhedrosis virus (CPV) in primary ovarian cell cultures of Antheraea eucalypti inoculated with a nuclear polyhedrosis virus of Bombyx mori. He also reported a spontaneous appearance of CPV in cultures of Antheraea cells maintained in vitro for approximately 10 months. Infection was attributed to a CPV which was apparently latent in Antheraea ovarian cells. Vago and Bergoin (2) found that inoculum prepared from intestines of Lymantria dispar infected with a CPV was capable of infecting primary ovarian Lymantria cultures. Recently, Sohi et al. (3) demonstrated that primary ovarian and tracheal cultures of B. mori could be infected with free virions of CPV from Malacosoma disstria. The infection of an established cell line of Aedes aegypti by CPV was first reported by Kawarabata and Hayashi (4). The inoculum contained virions with [6-3H]uridine labeled viral RNA. Measurements of incorporation of [6-3H]uridine into intact virus purified from inoculated cell cultures indicated that the virus contained newly synthesized RNA. Apparently “normal” replication was not achieved since polyhedra were not observed in the inoculated cultures. We present here, light and electron microscopic evidence on the complete replication of a CPV in an established insect cell line. CPV used in these studies had been isolated from the cabbage looper, 7% cl)-plusla ni, by Vail et al. (5). The cell line 584 Copyright All rights

0 1974 by Academic Press, Inc. of reproduction in any form reserved.

cell was

established from ovarian cells of T. ni was obtained from Dr. W. Fred Hink? Ohio State University, Columbus, OH, and grown under conditions described previously (6). Infection of tissue cultures was init,iated using extracts from diseased intestines from T. ni larvae or virions of CPV chemically released from polyhedra as inoculum. Intestines dissected from diseased larvae were rinsed twice in a penicillin-streptomycin solution (10,000 U penicillin and 10,000 pg streptomycin/ml) and once in Rinaldini’s balanced salt solution (pH 7.5). The rinsed intestines were then placed in a Dounce tissue grinder with growth medium (1 ml medium/intestine) and homogenized. The homogenate was centrifuged at 200 g for 15 min and the infectious supernatant was filtered through a 0.45 pm Millipore filter. Free virions of CPV were chemically released from polyhedra by the methods of Hayashi and Bird (7) and pelleted at 75,000 g for 90 min. The virus pellet was resuspended in 0.01 1M phosphate buffer, pH 7.5, diluted 1:lO with growth medium, and passed through a 0.45 pm filter. Cells were grown as monolayers of loosely attached cells in 30 ml plastic tissue culture flasks (Falcon Plastics) containing 5.0 ml growth medium. Cells were inoculated immediately following subculture from 4-dayold stock cultures. One ml of inoculum was added per flask containing 4 ml of medium. The final concentration of cells was approximately 1.5 X lo5 cells/ml. Twenty-

FIG. 1. Phase contrast photograph of cultured cells of Trichoplusia ni infected with CPV 5 days after inoculation. Note polyhedra (arrows) in cell cytoplasm. N = nucleus. X 1200. FIG. 2. Infected cells of T. ni 7 days after inoculation containing numerous polyhedra (arrows) in the cytoplasm. N = nucleus. X 1200. FIG. 3. Portion of a cultured cell of T. ni showing a virogenic stroma 0%) containing numerous electron dense CPV virions. Polyhedra (P) are formed at the periphery of the virogenic stroma. X 15,000. Insert: higher magnification of virions in a virogenic stroma. A dense core surrounded by a less dense coat can be seen in the virions. X 110,000. 585

586

SHORT

COMMUNICATIONS

four hours after inoculation the inoculum was removed, 5 ml of fresh medium was added, and the flasks incubated at 25-26”. To prepare infected cells for electron microscopy, the cells were detached from the flask bottom by mechanical agitation, pelleted at 200 g for 10 min, ,and fixed with 3 % glutaraldehyde in 0.01 M cacodylate buffer, pH 7.2. The fixed cells were processed by methods described previously (8). Both extracts from diseased intestines and virions released from polyhedra were infectious. The first evidence of infection was observed by phase-contrast microscopy 3640 hr after inoculation. There was an increased accumulation of refractive granules in the cell cytoplasm concomitant with a slight enlargement of the cell. By 48 hr typical polyhedra could be discerned. Cells infected for 5 and 7 days are shown in Figs. 1 and 2. The percentage of infected cells varied and usually ranged from 1 to 40 %. Infection of cell cultures with CPV was confirmed by electron microscopy (Fig. 3). The development of the virus in cell cultures appeared similar to that of other CPV cytopathological studies (3). Large masses of electron-dense virogenic stroma containing virions appeared in the cytoplasm (Fig. 3). Virions were occluded in spherical or cuboidal-shaped polyhedra which formed at the periphery of the stroma. Approximately 60-70 % of the polyhedra formed in vitro were cuboidal-shaped and the remainder were spherical. In infected larvae the polyhedra were predominantly spherical and less than 1% were cuboidal. Thus it appears that the in vitro conditions favor the formation of cuboidal-shaped polyhedra. Grace (1) also noted that the CPV polyhedra in Antherueu cell cultures were mainly cuboidal in shape while diseased Antheruea larvae had fewer cuboidal and more hexagonal-shaped polyhedra. The susceptibility of the original host to the CPV produced in tissue culture was de-

termined by isolating polyhedra from infected cultures and feeding 2nd and 3rd instar T. ni larvae grown in 1 oz plastic cups containing artificial diet. Fifty microliters of the polyhedral suspension were layered on the surface of the artificial diet at a dose of 70 polyhedra/mm2 of diet surface area. Sixteen of 24 larvae fed polyhedra became infected while 14 control larvae remained healthy. Infected cell cultures were homogenized, centrifuged at 200 g for 15 min, and the supernatant fraction was used to serially passage the virus to healthy cell cultures. The virus was passaged twice. The virus was not serially passaged by using cell-free medium obtained from cultures that were infected 5-7 days previously. Lysis of infected cells was not commonly observed in infected cultures and this may explain the noninfectious nature of the cell-free medium. These studies have demonstrated that a T. ni cell line will support the complete replication of a CPV and that virus from cultures is infectious to T. ni larvae. REFERENCES 1. GRACE, 9. VAGO,

T. D. C., Virology

C.,

AND

BERGOIN,

l&33-42 (1962). M., Entomophaga

8,

253-261 (1963). S. S., BIRD, F. T., AND HAYASHI, Y., Proceedings of the International Colloquim on Insect Pathology IV.” pp. 340-351, Society for Invertebrate Pathology, College Park, Maryland, 1970. 4. KAWARABATA, T., AND HAYASHI, Y., J. In3. SOHI,

vertebr. Pathok 19, 414-415 (1972). 6. VAIL, P. V., MILLER, T., AND HALL, I. Znvertebr. Pathol. 9,436441 (1967). 6. HINK, W. F., Nature 226,466-467 (1970). 7. HAYASHI, Y., AND BIRD, F. T., Can. J. biol. 16, 695-701 (1970). 8. GRANADOS, R. R., “Proceedings of the national Colloquim on Znvertebrate

M., J.

MicroZnter-

Tissue Culture III (1971).” pp. 491-499, Slovak Academy of Sciences, Bratislava, Czechoslovakia, 1973.