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Induction of a nonoccluded baculovirus persistently infecting Heliothis zea cells by Heliothis armigera and Trichoplusia ni nuclear polyhedrosis viruses
VIROLOGY
112, 1’74-189
Induction
(1981)
of a Nonoccluded Baculovirus zea Cells by Heliothis armigera Nuclear Polyhedrosis
D. C. KELLY,l
Natural
THELMA ANNE
Environment
Persistently Infecting Heliofhis and Trichoplusia ni Viruses
LESCOTT, M. D. AYRES, DOROTHY COUTTS, AND K. A. HARRAP
Research Oxfwd
Council, Institute OX1 SUB, United
Accepted
December
of Virologl/, Kingdom
5 South
Parks
CAREY,
Road,
30, 1980
A nonoccluded singly enveloped baculovirus (baculovirus X) persistently infects Heliothis zea (IMC-HZ-l) cells in culture. Singly enveloped nuclear polyhedrosis viruses from H. zea and Heliothis amnigera, and multiply enveloped nuclear polyhedrosis viruses from Trichoplusia ni, Spodoptera frugiperda, and Spodoptera littoralis were all found to induce baculovirus X. Experiments are reported which use metabolic inhibitors and inactivated inducing virus to show that it is probable that a structural component of the virus, most likely a protein, is responsible for inducing baculovirus X. The persistent virus is induced to replicate by uv-inactivated virus but not by heat-inactivated inducing virus. The virus is not induced to replicate by a number of metabolic inhibitors in the absence of an inducing virus. Inhibition of transcription and translation prevents the induction of the persistent virus by an inducing virus. Inhibition of DNA replication has no effect on the induction of the virus. This suggests that the persistent virus pencme is present in abundance in all cells.
the Heliothis spp. viruses. With the exception of the phenomenon described in this paper, we have consistently failed to demonstrate any interaction of these two viruses with any invertebrate cell line as assessed by optical and electron microscopy or analysis of “infected” cell polypeptides (D. C. Kelly and T. Lescott, unpublished observations). The attempted infection of a H. xea cell line (IMC-HZ-l: Hink and Ignoffo, 1970) with the two Heliothis spp. viruses did, however, produce a marked cytopathic effect and this could be correlated with the appearance of singly enveloped baculovirus-like particles. However, as this paper demonstrates, there is no serological relationship between the Heliothis spp. viruses initiating infection and the virus subsequently produced in these cells. Further experiments reported here show that a virus present in the cells was being stimulated to replicate actively. The morphogenesis of the persistent virus
There are few convincing reports that singly enveloped “occluded” baculoviruses (both nuclear polyhedrosis and granulosis) will replicate in cultured invertebrate cells (Granados, 1976) although Oryctes virus, a “nonoccluded” baculovirus (Payne, 1974), replicates readily in certain invertebrate cell lines (Kelly, 1976). We have attempted to infect a variety of cell lines with viruses from Heliothis armigera and Heliothis xea as part of an investigation of the basic biological and biochemical properties of singly enveloped nuclear polyhedrosis viruses from Heliothis spp. (Kelly et al., 1980; K. A. Harrap and D. Carey, unpublished observations). This could provide an opportunity to compare the strategy of replication of these viruses with the multiply enveloped nuclear polyhedrosis viruses and could provide a basis for in vitro safety testing procedures for ‘To whom dressed.
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004%6822/81/090174-16$02.00/O Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.
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174
INDUCTION
OF A BACULOVIRUS
in H. xea cells differed in certain respects from that of other baculoviruses and we report these observations. Since the H. xea and H. armigera viruses fail to replicate in cell culture we also chose a multiply enveloped nuclear polyhedrosis virus of Trichopdusia ni (whose replication has been defined in earlier papers: Kelly and Lescott, 1981; Kelly, 1981a, b) to superinfeet the H. xea cells. This virus had two advantages. First, we could titrate the virus by plaque assay (Brown et al., 1978; Kelly and Lescott, 1981) and so estimate the multiplicity of infection. Second, the multiply enveloped nature of the virus readily distinguishes it morphologically from the virus present in the H. xea cells. Granados et al. (1978) showed that the IMC-HZ-1 cell line contained baculoviruslike particles which they designated HZ1. The infection was said to be persistent though features that would confirm this were not described. They were able to demonstrate that the virus replicated efficiently in T. ni TN-268 cells (Hink, 1970) and we have consequently chosen these cells to titrate the persistent virus. This paper reports basic observations on the persistence of the virus in the IMCHZ-l cell line, the induction of the virus by nuclear polyhedrosis viruses, and preliminary studies on the nature of the stimulus to induce. MATERIALS
Cells. The following
AND
METHODS
cell lines were used:
H. xea (IMC-HZ-l) (Hink and Ignoffo, 1970), Tl ni (Hink, 1970), Spodoptera frugiperda (lPLB-SF-21AE) (Vaughn et al., 1977), Schneider’s line 1 (Drosophila melanogaster (Schneider, 1972; kindly supplied by P. D. Scotti), Aedes albopictus (Singh, :1967), baby hamster kidney (BHK21) (Stoker and MacPherson, 1961), fat head minnow (FHM) (Gravel1 and Malsberger, 1965), and Xenopus laevis cells (XTC-2) (Pudney et al., 1973). The vertebrate cells were grown as described by Elliott et al. (1979) and the invertebrate cells were grown in BML TClO medium (Gardinler and Stockdale, 1975) or MM
BY NPV
175
medium (Mitsuhashi and Maramorosch, 1964) in the case of A. albopictus cells. Viruses. The following viruses were used: T. ni, S. frugiperda, and Spodoptera littoralis multiply enveloped nuclear polyhedrosis viruses (Bud and Kelly, 1980; Harrap et al., 1977); H. armigera and H. zea singly enveloped nuclear polyhedrosis viruses (Kelly et al., 1980); and iridescent virus type 22 (Batson et al., 1976). The baculoviruses were grown in their homologous insect host with the exception of the T. ni nuclear polyhedrosis virus which was grown in S. frugiperda cells. Iridescent virus type 22 was grown in Galleria mellonella larvae. Titration of the viruses. T. ni nuclear polyhedrosis virus and iridescent virus were plaque assayed, or assayed by TCIDBO assay, as previously described (Kelly, 1976; Brown et al., 1978). The persistent virus was titrated in T. ni cells also by TCIDBO assay. Inactivation of virus. Virus (lo8 PFU/ ml of BML/TClOO) was heat inactivated at 60, 70, 80, and 90” in a water bath for 30 min, reducing the titre to 10’ PFU/ml at 60” and less at higher temperatures. Virus (lo8 PFU/ml of TClOO was uv-inactivated with a germicidal uv tube (maximum emission 254 nm) as previously described (Kelly and Dimmock, 1974). Treatment for 5 min reduced the titre to 10’ PFU/ml, and less at 10,15, and 20 min. Haemolymph-derived “nonoccluded ” virus. Infectious “nonoccluded” nuclear polyhedrosis virus was obtained from larval haemolymph as a potent source of inoculum. H. armigera, H. zea, S.frugiperda, and S. littoralis larvae were infected (per OS) with polyhedra of their homologous virus. Haemolymph was collected by piercing a proleg and collecting the haemolymph (approx 50 ~1) in 1 ml of BML/ TClOO on ice. The haemolymph was sonicated for 2 min and the debris collected by centrifugation at 100 g for 10 min. Production of antisera to Heliothis armigera nuclear polyhedrosis virus. Virus particle antiserum was produced as described by Kelly et al. (1980).
176
KELLY
Enzyme-linked immunosorbent assays. These were performed as previously described (Kelly et al., 19’78). Fluorescent antibody staining. These tests were performed essentially as described by Kelly and Dimmock (1974). Polyacrylamide gel electrophoresis of intracellular proteins. Cells were radiolabelled with [35S]methionine, usually for 1 hr, by adding 25 &i in 2 ml of methioninedeficient medium to a 25-cm flask of cells containing 3 X lo5 cells. The methioninedeficient medium contained 2% dialysed calf serum, basal TClOO medium minus methionine, and tryptose phosphate broth. The cells were washed in ice-cold PBS, scraped into 250 ~1 of 2% (w/v) SDS, 2% (v/v) 2-mercaptoethanol, 10% (v/v) glycerol in 1.0 M Tris-HCl, pH 6.8, and immediately dissociated by boiling for 2 min. The extracts (approx 50 ~1) were analysed on 12.0% polyacrylamide-SDS gels as previously described (Elliott and Kelly, 1980; Kelly and Lescott, 1981). Electron microscopy. Cells were processed for transmission electron microscopy of thin sections as previously described (Kelly, 1976). Materials. [35S]Methionine (3000 Ci/ mmol) was purchased from the Radiochemical Centre Ltd., Amersham, U. K. Caffeine, mitomycin C, actinomycin D, cycloheximide, hydroxyurea, iododeoxyuridine, bromodeoxyuridine, ethidium bromide, canavanine, and azetidine were all purchased from the Sigma Chemical Company (London) Ltd. RESULTS
Interaction of Heliothis armigera and Heliothis xea Nuclear Polyhedrosis Viruses with Heliothis xea Cells These experiments were designed originally to isolate these two viruses in cell culture. Haemolymph-derived “nonoccluded” virus was added in arbitrary amounts to determine the amount of virus required to produce a cytopathic effect in all cells. Cells inoculated with this amount of virus were sampled 5, 9, 15, and 22 hr
ET
AL.
after infection for electron microscopy and “infected cell-specific polypeptides” (ICSP). The cytopathic effect produced by the Heliothis spp. nuclear polyhedrosis viruses in H. zea cells was one of cell rounding with accompanying granularity and enlargement. No polyhedra were visible. Electron micrographs of H. arm&era nuclear polyhedrosis virus-“infected” H. xea cells are shown in Figs. 1 and 2. Similar observations were made on H. xea nuclear polyhedrosis virus-infected cells. The first morphological change observed in these cells was a condensation of the cell chromatin (Fig. 1A). Subsequently rodshaped particles, 380 nm long and 80 nm wide, were observed within the nucleus associated with a virogenic stroma (Fig. 1B). By 15 hr after infection all cells showed signs of “infection” although there was a certain amount of asynchrony in the apparent morphological stages of development. The virogenic stroma appears as an electron-translucent area which is associated with virus particles. Generally virus particles were scattered peripheral to this central stroma adjacent to the nuclear membrane, though it was not uncommon to detect aggregates of particles within the stroma as well. Figure 2 shows a number of nuclei containing virus. A frequent feature characteristic of virus development was the diffuse ill-defined nature of the nuclear membrane. Like other baculoviruses the particles appeared to be enveloped de novo in the nucleus. Virus was not observed budding at the nuclear or plasma membranes and release appeared to be by total cell lysis and then intracytoplasmic and extracellular virus was observed. Figure 2 (inset) shows detail of an aggregate of virus particles adjacent to a diffuse nuclear membrane. Such aggregates, with particles aligned with each other, were common. Neither occlusion of virus particles into polyhedra nor the presence of polyhedra was detected. Originally we considered that the nuclear polyhedrosis virus of H. armigera was replicating in the cells. We were, however, unable to confirm this either by testing for the production of viral antigens
FIG. 1. Heliothis zea cells “infected” with Heliothis armigera singly enveloped drosis virus (A) 5 hr after infection and (B) 12 hr after infection. Note the condensed early in infection (A). Subsequently singly enveloped virus particles occur peripheral translucent virogenic stroma (B). Bar = 2 km.
177
nuclear polyhecell chromatin to an electron-
178
KELLY
ET AL.
FIG. 2. Heliothis zea cells “infected” with Heliothis drosis virus 15 hr after infection. Note the presence electron-translucent virogenic stroma, and the apparent brane. Bar = 2 pm. Inset shows detail of an aggregate
using fluorescent antibody staining or by enzyme-linked immunosorbent assays. The “infected” cells gave consistently negative results whereas the tests performed on haemocytes taken from H. armigera NPVinfected H. armigera larvae were positive. In addition we were unable to plaque assay the H. armigera virus in H. xea cells; and
armigera singly enveloped nuclear polyheof virus particles in just the nucleus, the dissipated appearance of the nuclear memof nonoccluded virus.
titration of the virus by TCIDSO assay failed to give titres greater than lo3 TCIDSO units/ml although to produce the cpe observed the effective titre must be around lo7 infectious units/ml (assuming, as the evidence suggests, one cycle of “replication” occurs). Finally the alteration in polypeptide synthesis in “infected” cells
INDUCTION
OF A BACULOVIRUS
ABCDEFGHI
BY NPV JKLMNO
FIG. 3. Polypeptide synthesis in Heliothis zea cells “infected” with Heliothis armigera nuclear polyhedrosis virus. Cells were pulse labelled for 1 hr with [%]methionine at the times indicated. A and 0 are uninfected controls; B, cells 1 hpi; C, cells 2 hpi; D, cells 3 hpi; E, cells 4 hpi; F, cells 5 hpi; G, cells 6 hpi; H, cells 7 hpi; I, cells 8 hpi; J, cells 9 hpi; K, cells 10 hpi; L, cells 11 hpi; M, cells 12 hpi; N, cells 13 hpi.
(Fig. 3) showed that no “infected” cell-specific polypeptides had relative migrations similar to those of H. armigera NPV (Kelly et al., 1980). Consequently, we considered that we must be inducing a virus present in the H. xea cells. Figure 4 shows a comparison of the polypeptides of this virus and H. armigera NPV. Examination of control cells either untreated or mock-infected with haemolymph from healthy insect species (H. armigera, H. xea, S. frugiperda, or S. littoralis) failed to show the presence of the virus. This occurred in three experiments ((but see later section on persistence).
Interaction of Trichoplusia ni and Other Multiply Enveloped Nuclear Polyhedrosis Viruses with Heliothis xea Cells “Infection” of the H. zea cells with 50 PFU/cell of T. ni multiply enveloped nuclear polyhedrosis virus produced a cytopathic effect similar to that produced by the Heliothis NPVs. Examination of the cells at 9, 15, and 22 hr after “infection” (times at which (Y, @, y, 6 polypeptide phases of T. ni NPV protein induction occur in S. frugiperda cells (Kelly and Lescott, 1981)) showed an induction of singly enveloped rods with a morphogenesis and appearance similar to that described for
180
KELLY
ET AL.
the virus induced by the Heliothis viruses. Figure 5C shows the appearance of such cells. “Infection” of H. zea cells with haemolymph-derived S. frugiperda and S. littoral& viruses also created the same phenomenon (Figs. 5A, B). In all cases the H. xea cells showed evidence of singly enveloped rods, no multiply enveloped virus, and no polyhedra. As such it is probable that the nonoccluded virus is induced by the multiply enveloped viruses which in turn fail to replicate themselves. Analysis of the polypeptides synthesised in cells “infected” with T. ni NPV showed that the polypeptides induced were similar to those detected in H. armigera NPV-“infected” cells and that no polypeptides typical of the T. ni NPV-infected cell-specific polypeptides were detected (Fig. 6).
Persistent Nature of the Nonoccluded rus Found in Heliothis xea Cells
Vi-
Since the nonoccluded virus was consistently inducible in the H. zea cells it was probable that the virus existed in the cells in a latent or persistent state. Careful examination of cells processed for electron microscopy during (and indeed prior to) this project showed that cells containing the virus could be detected. The percentage of cells showing the overt presence of the virus varied from less than 0.1 to 25% (Table 1). Titration of the virus on various passage levels of the cells also showed considerable variation in the amount of virus present in the cells (Table 2). The presence of virus in variable amounts on prolonged passage is typical of a persistent virus infection.
Superi$ection of Heliothis xea Cells with the Homologous Nonoccluded Virus Addition of 50 TCIDsO of the nonoceluded virus of H. xea cells subsequently referred to as baculovirus X obtained from noninduced cells to the H. zea cells resulted in the production of a cytopathic effect, an increased production of baculovirus X in the tissue culture fluid, and the induction of baculovirus X-infected
FIG.
4. Polypeptides contained by (A) Heliothis nuclear polyhedrosis virus particles and (B) baculovirus X virus particles. The polypeptides were run on 12.5% polyacrylamide gels and stained with Coomassie blue. The polypeptides are identified by estimates of their molecular weight. Minor polypeptides in the baculovirus X preparation are probably host cell polypeptides and these are unlabelled.
armigera
cell-specific polypeptides similar to those observed when T. ni or Heliothis spp. nuclear polyhedrosis viruses were added (data not shown).
Effect of Drugs on the Abundance of Baculovirus X in Heliothis xea Cells Since a number of nuclear polyhedrosis viruses were able to induce the presence of baculovirus X in all Heliothis cells we
INDUCTION
OF A BACULOVIRUS
attempted to induce the virus with each of a number of drugs. Different culture cells were treated for 16 hr with each of the folllowing, 0.5 mM hydroxyurea, 1.5 mM caffeine, 20 pg/ml bromodeoxyuridine, 1 pg/ml ethidium bromide, 1 pg/ml mitomycin C, and 1 pg/ml cycloheximide. The treated cells were examined by phasecontrast microscopy and transmission electron microscopy of thin sections, by examination of infected cell-specific polypeptides and by titration of baculovirus X in T. ni cells, at 72 and 120 hr after treatment. N-o treatment succeeded in producing any evidence of increased levels of virus in t’he cell cultures.
Effect of Iridescent Virus Type 22 on Baculovirus X Induction Iridescent virus type 22 is an “icosahedral cytoplasmic deoxyribovirus” (Batson et al., 1976) which infects a broad spectrum of invertebrate cells in culture (T. Lescott and D. C. Kelly, unpublished observations). Infection of H. xea cells with 50 PFU per cell of this virus failed to stimulate the growth of baculovirus X assessed by electron microscopy, titration of the virus in T. ni cells, and induction of infected cell-specific polypeptides typical of baculovirus X. The iridescent virus itself also failed to replicate.
Induction of Baculovirus X in the Presence of Metabolic Inhibitors A variety of metabolic inhibitors were used to examine the general macromolecular synthesis in baculovirus X induction by T. ni nuclear polyhedrosis virus. No induction occurred when transcription of viral DNA was inhibited by actinomycin D (10 p&/ml). Cells were treated 0 to 5, 0 to 9,1 to 5,2 to 5, and 3 to 5 hr and sampled (where appropriate) at 5, 7, 9, 12, 18, and 22 hr after infection. Cells blocked with actinomycin D later than 5 hr after infection showed evidence of induction. This showed that transcription of a genome is required for induction to occur. Cells were treated from 0 to 7 hr with cycloheximide
BY NPV
181
(50 pg/ml) to prevent translation, the drug was washed out, and sampling was done immediately and at 9,10, 11,12, 15 and 22 hr after infection. Evidence of gradual induction delayed 8 hr was found (data not shown). This showed that messenger responsible for the synthesis of protein involved in baculovirus X induction did not accumulate in cycloheximide-treated cells although it was presumably able to do so after release from the block. Cycloheximide alone failed to induce the virus. Azetidine and canavanine, amino acid analogues of proline and hydroxyproline, respectively, can be incorporated into proteins to render them nonfunctional. H. zea cells were treated with 12 mM azetidine and 12 mM canavanine and infected with T. ni nuclear polyhedrosis virus, and it was found that induction of baculovirus X polypeptides did not occur, when samples were taken 5, 9, 15 and 22 hpi (Fig. 7). These concentrations of azetidine and canavanine interfere with T. ni nuclear polyhedrosis ICSP induction presumably by rendering the ICSP induced nonfunctional (Kelly and Lescott, 1981). When DNA replication was inhibited by using hydroxyurea (800 pg/ml) or cytosine arabinoside (50 pug/ml), concentrations which prevent T. ni nuclear polyhedrosis virus-specific DNA synthesis and late protein synthesis (Kelly and Lescott, 1981; H. M. Bud and D. C. Kelly, unpublished observations), baculovirus X ICSP production appeared to be normal. Samples were taken at 3,6,9,12,18, and 24 hr after infection and the drugs were present throughout (data not shown).
Induction of Baculovirus X by Heat- and uv-Inactivated Trichoplusia ni Nuclear Polyhedrosis Virus Virus inactivated by heat to denature primarily the virus structural; proteins, and by uv light to denature primarily the genome, were used to test the dependence of baculovirus X induction on structural components of the inducing virus. As shown in Fig. 8 virus inactivated by uv light was still able to induce baculovirus X ICSP, whereas virus inactivated by
KELLY
182
ET
heating at 70” or greater did not. Virus heat inactivated at 60” induced normal baculovirus X ICSP. Both uv- and heat-inactivated virus failed to induce virus-specific polypeptide synthesis in S. frugiperda cells in culture (data not shown).
AL.
infecting cells including variability of titre, and numbers of cells overtly showing the presence of virus, on cell passage. The origin of the virus is not clear and this has been extensively discussed by Granados and co-workers (1978). The presence of baculovirus X appeared to make the cells refractile to the replication of the superinfecting nuclear polyhedrosis viruses, and no evidence of T. ni nuclear polyhedrosis virus macromolecular synthesis was obtained. Baculoviruses generally have a narrow cell culture range for replication (Knudson and Buckley, 1977) and so the refractile nature of the H. zea cells may not be directly attributable to the presence of baculovirus X. The cells were also refractile to iridescent virus type 22 which has a broad experimental host cell range and this is the first invertebrate cell line we have discovered which is nonpermissive for an iridescent virus. The morphogenesis of baculovirus X on induction is different from that of the inducing baculoviruses in a number of respects. The morphology of baculovirus X in thin section was typical of a singly enveloped virus but was larger than most baculoviruses which have a size of 70 x 280 nm when singly enveloped (Harrap and Payne, 1979). We have been unable to define clearly how nuclear polyhedrosis viruses trigger the induction of baculovirus X in cell culture. Both viruses contain DNA and so it is difficult to discriminate with drugs between the two viruses when they replicate in cells. However, blocking DNA synthesis in H. xea cells allowed normal induction which could indicate two things. First, the contribution by the T. ni virus was an early event because if the virus were to replicate at all it would proceed no further than a synthesis with hydroxyurea or p synthesis with cytosine arabinoside (Kelly and Lescott, 1981). Second, it is probable that baculovirus X DNA exists (possibly
Induction of Baculovirus X by Trichoplusia ni Nuclear Polyhedrosis Virus in the Presence of Ethidium Bromide From earlier experiments it was tentatively concluded that the baculovirus X genome exists in all cells prior to induction. Ethidium bromide has a high affinity for supercoiled DNA (Gale et al., 1972), and since baculovirus DNA occurs in the supercoiled form in virus particles (Summers and Anderson, 1973), inhibition or suppression of induction may be due to an interaction with the viral DNA in the nucleus. Ethidium bromide (1 pg/ml) suppresses the induction of baculovirus X by T. ni nuclear polyhedrosis virus (data not shown). DISCUSSION
Our results show that a virus which apparently infects a H. zea cell line in a persistent manner can be efficiently induced to replicate by a number of singly and multiply enveloped nuclear polyhedrosis viruses. The virus is presumed to be a baculovirus because it possesses the general morphological appearance of a nonoceluded baculovirus (i.e., an enveloped rodshaped virus devoid of polyhedron protein), contains DNA (D. C. Kelly, unpublished observations), and replicates in the cell nucleus. The virus apparently does not become occluded within polyhedra and in this respect resembles the class C group of baculoviruses (Matthews, 1979) which includes the Oryctes viruses (Monsarrat et al., 1973) and the braconid baculoviruses (Stoltz and Vinson, 1979). Baculovirus X had a number of characteristics typical of a virus persistently -
FIG. 5. Electron micrographs of Heliothis zea cells “infected” with three multiply enveloped nuclear polyhedrosis viruses from (A) Spodoptera littoralis, (B) Spodopteru fmgiperda, and (C) Trichoplusia ni. In each case a singly enveloped nonoccluded virus was induced. Bar = 2 pm.
INDUCTION
OF A BACULOVIRUS
BY NPV
183
184
KELLY A
B
C
D
E
ET
AL. F
G
H
I
J
FIG. 6. Polypeptide synthesis in (A to E) Heliothis zea cells and (F to J) Spodoptera frugiperda cells infected with Trichoplusia ni nuclear polyhedrosis virus. Cells were pulse labelled with [%]methionine for 1 hr in uninfected control cells (A and F) and 5 hr (B and G), 9 hr (C and H), 15 hr (D and I), and 22 hr (E and J) after infection. The polypeptides induced in H. zea cells resemble those induced by H. amigera virus in H. zea cells.
in a proviral state) in reasonable quantities prior to induction. In addition, treatment of cells with ethidium bromide, which binds with high affinity to supercoiled DNA, prevents induction of baculovirus X, suggesting that the baculovirus X genome is present in a supercoiled state. A similar suppression of induction of C-type virus by a chemical inducer (iododeoxyuridine) occurs when cells are treated with ethidium bromide (Avery and Levy, 1979). Treatment of cells with actinomycin D
shows that transcription of either the cell genome, the baculovirus X genome, or the inducing T. ni baculovirus genome is required for induction to occur. However uv light inactivation of T. ni virus does not affect induction and so, if inactivation of any “inducing genes” occurs at the same rate as total inactivation, it is unlikely that transcription of the inducing virus is responsible for the induction. Ultraviolet light inactivation of cells prevents induction (T. Lescott and D. C. Kelly, unpub-
INDUCTION TABLE
1
VARIATIONIN PROPORTIONOF Heliothis CONTAINING PASSAGE
NONOCCLUDED
Passage
data”
01.12.77 11.01.78 09.02.78 07.03.78 14.05.78 14.06.78 22.06.78 29.06.78 14.12.78
OF A BACULOVIRUS
BACULOVIRUS
zea CELLS ON CELL
Percentage cells positive* 0.1 0.1 0.2 0.3 1.0 5.0 25.0 1.0 5.0
” The date cells were sampled for electron microscopy. The cells were subcultured weekly and continuously. “The proportion of cells showing virus by randomly selecting thin sections of cells and scoring.
lished observations), but this observation does not clarify the point of initial transcription. Treatment of cells with cycloheximide did not permit baculovirus X induction by T. ni nuclear polyhedrosis virus; and induction of baculovirus X ICSP proceeded with timing equivalent to a normal induction on reversal of the cycloheximide block. This indicates that protein synthesis and messenger RNA synthesis from the cell/baculovirus X genome is also required. for induction to occur. No amplification of baculovirus X messenger RNA occurred during the cycloheximide block because on release from the block no increased baculovirus X ICSP synthesis was detected. Azetidine and canavanine also inhibited the induction of baculovirus X ICSP, indicating that a functional protein is required for the induction of baculovirus X after Trichoplusia ni virus “infection.” These observations demonstrate that functional protein synthesis is required before baculovirus X induction occurs. We can obtain no evidence of the synthesis of T. ni nuclear polyhedrosis virus (YICSP synthesis in H. zea cells, and this coupled with the observation that heat-in-
185
BY NPV
activated (presumably structural protein impaired) virus failed to induce baculovirus X indicates that the inducing virus probably interacts with baculovirus X or a genome product whose synthesis is prevented by direct or indirect inhibition of protein synthesis. In other words, an inducing virus structural protein may interact with a gene product which represses baculovirus X replication. Interestingly the transition from early to late baculovirus X ICSP occurs in cells in which DNA synthesis is blocked with hydroxyurea or cytosine arabinoside. This means that the polypeptides indicated as early (Fig. 5) are merely indicators of time of appearance and not related to DNA synthesis. In addition, only two temporal phases of baculovirus X ICSP induction are detected compared to four with the T. ni virus. Preliminary experiments using the more sensitive two-dimensional gel electrophoresis of ICSP (O’Farrell, 1975) have failed to detect any additional baculovirus X ICSP, or indeed any T. ni ICSP (D. C. Kelly and M. D. Ayres, unpublished observations). The results that we have obtained indicating that the persistent virus is induced in all cells without replication of viral DNA suggest that the genome of the persistent virus is present in abundance in all cells.
TABLE TITRES
2
OF NONOCCLUDED BACULOVIRUS PRESENT Heliothis zea CELLS ON CELL PASSAGE Passage
date”
Titre’
15.12.78 03.01.79 10.01.79 17.01.79 23.01.79 12.02.79 19.02.79 03.03.79 a The date cells were sampled. cated cell extracts was titrated. b TCIDso units per lo6 cells.
IN
2 2 4 2 1 2 3 2 The
x x x x x x x x
lo3 lo3 lo5 lo3 lo* lo7 lo5 lo3
virus
in soni-
KELLY
186 A
B
C
D
E
ET AL. F
G
H
I
J
FIG. 7. Polypeptides synthesised in Heliothis zea cells “infected” with Trichoplusia ni nuclear polyhedrosis virus in cells treated with azetidine (12 mM) (A to E) and canavanine (12 mM) (F to J). (A and F) Control cells; (B and G) cells 5 hpi; (C and H) cells 9 hpi; (D and I) cells 15 hpi; (E and J) cells 22 hpi. Induction of polypeptide typical of baeulovirus X stimulation is barely detected 22 hpi.
The general phenomenon of induction of a persistent virus in an insect cell line is of interest because it mimicks in part the situation found in some insects where infection of a particular insect species by a nuclear polyhedrosis virus from a het-
erologous insect species results in the test insect succumbing to a disease caused by a virus typical of the homologous species (Longworth and Cunningham, 1968; Jurkovicova, 1979; McKinley et al., 1981). Baculoviruses are used commercially as bio-
INDUCTION A
B
C
D
OF A BACULOVIRUS E
F
BY NPV G
H
I
J
FIG. 8. Induction of polypeptides in Heliothis zea cells “infected” with heat-inactivated and uvinactivated Ttichoplusia ni nuclear polyhedrosis virus. Cells were infected with virus and sampled with a 1-hr pulse of [%]methionine 15 hpi. (A) Virus heat inactivated at 60”; (B) virus heat inactivated at 70”; (C) virus heat inactivated at 80”; (D) virus heat inactivated at 90”; (E) uninfected control cells; (F) live virus; (G) virus uv inactivated for 5 min; (H) virus uv inactivated for 10 min; (I) virus uv inactivated for 15 min; (J) virus uv inactivated for 20 min.
logical control agents, and the activation of a persistent (possibly latent) virus by a viral insecticide raises problems about the safe and responsible use of these viruses and the interpretation of epidemiological studies of a given virus. The dem-
on&ration that a comparable situation exists in a cell culture model system means that the contribution made by the inducing virus to initiate the replication of the induced virus may be studied. Such work is now in progress and particular empha-
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KELLY
sis is being placed on the state and location of the baculovirus X genome in the cell, and the replication of this virus in other cell lines. REFERENCES AVERY, R. J., and LEVY, J. A. (1979). The effect of ethidium bromide on C type virus production and induction. Virology 95, 277-284. BATSON, B. S., JOHNSTON, M. R. L., ARNOLD, M. K., and KELLY, D. C. (1976). An iridescent virus from Simulium sp. (Diptera: Simuliidae) in Wales. J. Znwertebr. Pathol. 27, 133-135. BROWN, D. A., LESCOTT, T., HARRAP, K. A., and KELLY, D. C. ( 1978). The replication and titration of iridescent virus type 22 in Spodopterafrugiperda cells. J. Gen. Viral. 38, 175-178. BUD, H. M., and KELLY, D. C. (1980). Nuclear polyhedrosis virus DNA is infectious. Microbiologica 33, 177-180. ELLIOTT, R. M., ARNOLD, M. K., and KELLY, D. C. (1979). The replication of frog virus 3 in an amphibian ceil line fromxenopus laevis. J. Gen. W-01. 44, 89-98. ELLIOTT, R. M., and KELLY, D. C. (1980). Frog virus 3 replication: Induction and intracellular distribution of polypeptides in infected cells. J. Virol. 33, 28-51. GALE, E. F., CUNLIFFE, E., REYNOLDS, P. E., RICHMOND, M. H., and WAREING, M. J. (1972). “The molecular basis of antibiotic action.” Wiley, London. GARDINER, G. R., and STOCKDALE, H. (1975). TWO tissue culture media for production of lepidopteran cells and nuclear polyhedrosis viruses. J. Znvertebr. Pathol. 25, 303-370. GRANADOS, R. R. (1976). Infection and replication of insect pathogenic viruses in tissue culture. Advan. Virus Res. 20, 189-236. GRANADOS, R. R., NGUYEN, T., and CATO, B. (1978). An insect cell line persistently infected with a baculovirus like particle. Zntervirology 10, 309-317. GRAVELL, M., and MALSBERGER, R. G. (1965). A permanent cell line from the fat head minnow (Pimephales promelas). Ann. N. Y. Acad. Sci. 126,55565. HARRAP, K. A., and PAYNE, C. C. (1979). The structural properties and identificaiton of insect viruses. Advan. Virus Res. 25, 273-355. HARRAP, K. A., PAYNE, C. C., and ROBERTSON, J. S. (1977). The properties of three baculoviruses from closely related hosts. Virology 79, 14-31. HINK, W. F. (1970). Establishment of a new cell line from the cabbage looper, Trichoplusia ni. Nature (London) 226, 466-467.
ET AL. HINK, W. F., and IGNOFFO, C. M. (1970). Establishment of a new cell line (IMC-HZ-l) from ovaries of cotton bollworm moths, Heliothis zea (Boddie). Ezp. Cell Res. 60, 307-309. JURKOVICOVA, M. (1979). Activation of latent virus infections in larvae of Adozophyes orana (Lepidoptera:tortricidae) and Baruthra brassicae (Lepidoptera:noctuidae) by foreign polyhedra. J. Znvertebr. Pathol. 34, 213-223. KELLY, D. C. (1976). ‘Oryctes’ virus replication: Electron microscopic observations on infected moth and mosquito cells. Virology 69, 599-606. KELLY, D. C. (1981a). Baculovirus replication: Electron microscopy of the sequence of infection of Trichoplusia ni nuclear polyhedrosis virus in Spodoptera frugiperda cells. J. Gen. Virol. 52,209-219. KELLY, D. C. (1981b). Baculovirus replication: Stimulation of thymidine kinase and DNA polymerase activities in Spodoptera frugiperda cells infected with Trichoplwia ni nuclear polyhedrosis virus. J. Gen. Virol. 52, 313-319. KELLY, D. C., BROWN, D. A., ROBERTSON, J. S., and HARRAP, K. A. (1980). Biochemical biophysical and serological properties of two singly enveloped nuclear polyhedrosis viruses from Heliothis armigera and Heliothis zea. Microbiologica 3, 319-331. KELLY, D. C., and DIMMOCK, N. J. (1974). Fowl plague virus replication in mammalian cell-avian erythrocyte heterokaryons: Studies concerning the actinomycin D and ultraviolet light sensitive phase in influenza virus replication. Virology 61,210-222. KELLY, D. C., EDWARDS, M. L., EVANS, H. F., and ROBERTSON, J. S. (1978). The use of enzyme-linked immunosorbent assay to detect a nuclear polyhedrosis virus in Heliothis armigera larvae. J. Gen. W-01. 40, 465-469. KELLY, D. C., and LESCOTT, T. (1981). Baculovirus replication: Protein synthesis in Spodoptera frugiperda cells infected with Trichoplusia ni nuclear polyhedrosis virus. Microbiologica 4, 35-57. KNUDSON, D. L., and BUCKLEY, S. M. (1977). Invertebrate cell culture methods for the study of invertebrate-associated animal viruses. Methods Virol. 6, 323-391. LONGWORTH, J. F., and CUNNINGHAM, J. C. (1968). The activation of occult nuclear polyhedrosis viruses by foreign nuclear polyhedra. J. Znvertebr. Pathol. 10,361-367. MATTHEWS, R. E. F. (1979). Classification and nomenclature of viruses. Intervirology 12,129-296. MCKINLEY, D. J., BROWN, D. A., PAYNE, C. C., and HARRAP, K. A. (1981). Cross-infectivity and activation studies with four baculoviruses. Entomophaga, 26, 79-90. MITSUHASHI, J., and MARAMOROSCH, K. (1964). Leafhopper tissue culture: embryonic, nymphal
INDUCTION and imaginal tissues from aseptic Boyce Thompson Inst. 22,435-460.
insects.
OF A BACULOVIRUS Contrib.
MONSARRA.T, P., MEYNADIER, G., CROIZIER, G., and VAGO, C. (1973). Recherches cytopathologiques sur une maladie virale du coleoptere Oryctes rhinoceros. C.R. Acad. Sci. Paris Ser. D 277,1413-1415. O’FARRELL, P. H. (1975). High resolution two dimensional electrophoresis of proteins. J. Biol. Chem. 250, 4007-4021. PAYNE, C. C. (1974). The isolation and characterisation of a virus from &y&es rhinoceros. J. Gen. Viral. 25, 105-116. PUDNEY, M., VARMA, M. G. R., and LEAKE, C. Establishment of a cell line from the South clawed toad, Xenopus laevis. Experientia 467. SCHNEIDER, I. (1972). Cell lines derived from
J. (1973). African 29,466late em-
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bryonic stages of Drosophila melanogaster. J. Embryol. Exp. Morphol. 27,353-365. SINGH, K. R. P. (1967). Cell cultures derived from larvae of Aedes albopictus (Skuse) and Aedes aegypti (L.). Curr. Sci. 36, 506-508. STOKER, M. G. P., and MACPHERSON, I. A. (1961). Studies on the transformation of hamster cells by polyoma virus in vitro. Virology 14, 359-370. STOLTZ, D.,and VINSON, S. B. (1979). Viruses and parasitism in insects. Advan. Virus Res. 24, 125-172. SUMMERS, M. D., and ANDERSON, D. L. (1973). Characterisation of nuclear polyhedrosis virus DNA. J. Viral. 12, 1336-1346. VAUGHN, J. L., GOODWIN, R. H., THOMPKINS, G. J., and MCCAWLEY, P. (1977). The establishment of two cell lines from the insect Spodoptera frugiperda (Lepidoptera: Noctuidae). In Vitro 13, 213217.
112, 1’74-189
Induction
(1981)
of a Nonoccluded Baculovirus zea Cells by Heliothis armigera Nuclear Polyhedrosis
D. C. KELLY,l
Natural
THELMA ANNE
Environment
Persistently Infecting Heliofhis and Trichoplusia ni Viruses
LESCOTT, M. D. AYRES, DOROTHY COUTTS, AND K. A. HARRAP
Research Oxfwd
Council, Institute OX1 SUB, United
Accepted
December
of Virologl/, Kingdom
5 South
Parks
CAREY,
Road,
30, 1980
A nonoccluded singly enveloped baculovirus (baculovirus X) persistently infects Heliothis zea (IMC-HZ-l) cells in culture. Singly enveloped nuclear polyhedrosis viruses from H. zea and Heliothis amnigera, and multiply enveloped nuclear polyhedrosis viruses from Trichoplusia ni, Spodoptera frugiperda, and Spodoptera littoralis were all found to induce baculovirus X. Experiments are reported which use metabolic inhibitors and inactivated inducing virus to show that it is probable that a structural component of the virus, most likely a protein, is responsible for inducing baculovirus X. The persistent virus is induced to replicate by uv-inactivated virus but not by heat-inactivated inducing virus. The virus is not induced to replicate by a number of metabolic inhibitors in the absence of an inducing virus. Inhibition of transcription and translation prevents the induction of the persistent virus by an inducing virus. Inhibition of DNA replication has no effect on the induction of the virus. This suggests that the persistent virus pencme is present in abundance in all cells.
the Heliothis spp. viruses. With the exception of the phenomenon described in this paper, we have consistently failed to demonstrate any interaction of these two viruses with any invertebrate cell line as assessed by optical and electron microscopy or analysis of “infected” cell polypeptides (D. C. Kelly and T. Lescott, unpublished observations). The attempted infection of a H. xea cell line (IMC-HZ-l: Hink and Ignoffo, 1970) with the two Heliothis spp. viruses did, however, produce a marked cytopathic effect and this could be correlated with the appearance of singly enveloped baculovirus-like particles. However, as this paper demonstrates, there is no serological relationship between the Heliothis spp. viruses initiating infection and the virus subsequently produced in these cells. Further experiments reported here show that a virus present in the cells was being stimulated to replicate actively. The morphogenesis of the persistent virus
There are few convincing reports that singly enveloped “occluded” baculoviruses (both nuclear polyhedrosis and granulosis) will replicate in cultured invertebrate cells (Granados, 1976) although Oryctes virus, a “nonoccluded” baculovirus (Payne, 1974), replicates readily in certain invertebrate cell lines (Kelly, 1976). We have attempted to infect a variety of cell lines with viruses from Heliothis armigera and Heliothis xea as part of an investigation of the basic biological and biochemical properties of singly enveloped nuclear polyhedrosis viruses from Heliothis spp. (Kelly et al., 1980; K. A. Harrap and D. Carey, unpublished observations). This could provide an opportunity to compare the strategy of replication of these viruses with the multiply enveloped nuclear polyhedrosis viruses and could provide a basis for in vitro safety testing procedures for ‘To whom dressed.
requests
for
reprints
004%6822/81/090174-16$02.00/O Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.
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be
ad-
174
INDUCTION
OF A BACULOVIRUS
in H. xea cells differed in certain respects from that of other baculoviruses and we report these observations. Since the H. xea and H. armigera viruses fail to replicate in cell culture we also chose a multiply enveloped nuclear polyhedrosis virus of Trichopdusia ni (whose replication has been defined in earlier papers: Kelly and Lescott, 1981; Kelly, 1981a, b) to superinfeet the H. xea cells. This virus had two advantages. First, we could titrate the virus by plaque assay (Brown et al., 1978; Kelly and Lescott, 1981) and so estimate the multiplicity of infection. Second, the multiply enveloped nature of the virus readily distinguishes it morphologically from the virus present in the H. xea cells. Granados et al. (1978) showed that the IMC-HZ-1 cell line contained baculoviruslike particles which they designated HZ1. The infection was said to be persistent though features that would confirm this were not described. They were able to demonstrate that the virus replicated efficiently in T. ni TN-268 cells (Hink, 1970) and we have consequently chosen these cells to titrate the persistent virus. This paper reports basic observations on the persistence of the virus in the IMCHZ-l cell line, the induction of the virus by nuclear polyhedrosis viruses, and preliminary studies on the nature of the stimulus to induce. MATERIALS
Cells. The following
AND
METHODS
cell lines were used:
H. xea (IMC-HZ-l) (Hink and Ignoffo, 1970), Tl ni (Hink, 1970), Spodoptera frugiperda (lPLB-SF-21AE) (Vaughn et al., 1977), Schneider’s line 1 (Drosophila melanogaster (Schneider, 1972; kindly supplied by P. D. Scotti), Aedes albopictus (Singh, :1967), baby hamster kidney (BHK21) (Stoker and MacPherson, 1961), fat head minnow (FHM) (Gravel1 and Malsberger, 1965), and Xenopus laevis cells (XTC-2) (Pudney et al., 1973). The vertebrate cells were grown as described by Elliott et al. (1979) and the invertebrate cells were grown in BML TClO medium (Gardinler and Stockdale, 1975) or MM
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175
medium (Mitsuhashi and Maramorosch, 1964) in the case of A. albopictus cells. Viruses. The following viruses were used: T. ni, S. frugiperda, and Spodoptera littoralis multiply enveloped nuclear polyhedrosis viruses (Bud and Kelly, 1980; Harrap et al., 1977); H. armigera and H. zea singly enveloped nuclear polyhedrosis viruses (Kelly et al., 1980); and iridescent virus type 22 (Batson et al., 1976). The baculoviruses were grown in their homologous insect host with the exception of the T. ni nuclear polyhedrosis virus which was grown in S. frugiperda cells. Iridescent virus type 22 was grown in Galleria mellonella larvae. Titration of the viruses. T. ni nuclear polyhedrosis virus and iridescent virus were plaque assayed, or assayed by TCIDBO assay, as previously described (Kelly, 1976; Brown et al., 1978). The persistent virus was titrated in T. ni cells also by TCIDBO assay. Inactivation of virus. Virus (lo8 PFU/ ml of BML/TClOO) was heat inactivated at 60, 70, 80, and 90” in a water bath for 30 min, reducing the titre to 10’ PFU/ml at 60” and less at higher temperatures. Virus (lo8 PFU/ml of TClOO was uv-inactivated with a germicidal uv tube (maximum emission 254 nm) as previously described (Kelly and Dimmock, 1974). Treatment for 5 min reduced the titre to 10’ PFU/ml, and less at 10,15, and 20 min. Haemolymph-derived “nonoccluded ” virus. Infectious “nonoccluded” nuclear polyhedrosis virus was obtained from larval haemolymph as a potent source of inoculum. H. armigera, H. zea, S.frugiperda, and S. littoralis larvae were infected (per OS) with polyhedra of their homologous virus. Haemolymph was collected by piercing a proleg and collecting the haemolymph (approx 50 ~1) in 1 ml of BML/ TClOO on ice. The haemolymph was sonicated for 2 min and the debris collected by centrifugation at 100 g for 10 min. Production of antisera to Heliothis armigera nuclear polyhedrosis virus. Virus particle antiserum was produced as described by Kelly et al. (1980).
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KELLY
Enzyme-linked immunosorbent assays. These were performed as previously described (Kelly et al., 19’78). Fluorescent antibody staining. These tests were performed essentially as described by Kelly and Dimmock (1974). Polyacrylamide gel electrophoresis of intracellular proteins. Cells were radiolabelled with [35S]methionine, usually for 1 hr, by adding 25 &i in 2 ml of methioninedeficient medium to a 25-cm flask of cells containing 3 X lo5 cells. The methioninedeficient medium contained 2% dialysed calf serum, basal TClOO medium minus methionine, and tryptose phosphate broth. The cells were washed in ice-cold PBS, scraped into 250 ~1 of 2% (w/v) SDS, 2% (v/v) 2-mercaptoethanol, 10% (v/v) glycerol in 1.0 M Tris-HCl, pH 6.8, and immediately dissociated by boiling for 2 min. The extracts (approx 50 ~1) were analysed on 12.0% polyacrylamide-SDS gels as previously described (Elliott and Kelly, 1980; Kelly and Lescott, 1981). Electron microscopy. Cells were processed for transmission electron microscopy of thin sections as previously described (Kelly, 1976). Materials. [35S]Methionine (3000 Ci/ mmol) was purchased from the Radiochemical Centre Ltd., Amersham, U. K. Caffeine, mitomycin C, actinomycin D, cycloheximide, hydroxyurea, iododeoxyuridine, bromodeoxyuridine, ethidium bromide, canavanine, and azetidine were all purchased from the Sigma Chemical Company (London) Ltd. RESULTS
Interaction of Heliothis armigera and Heliothis xea Nuclear Polyhedrosis Viruses with Heliothis xea Cells These experiments were designed originally to isolate these two viruses in cell culture. Haemolymph-derived “nonoccluded” virus was added in arbitrary amounts to determine the amount of virus required to produce a cytopathic effect in all cells. Cells inoculated with this amount of virus were sampled 5, 9, 15, and 22 hr
ET
AL.
after infection for electron microscopy and “infected cell-specific polypeptides” (ICSP). The cytopathic effect produced by the Heliothis spp. nuclear polyhedrosis viruses in H. zea cells was one of cell rounding with accompanying granularity and enlargement. No polyhedra were visible. Electron micrographs of H. arm&era nuclear polyhedrosis virus-“infected” H. xea cells are shown in Figs. 1 and 2. Similar observations were made on H. xea nuclear polyhedrosis virus-infected cells. The first morphological change observed in these cells was a condensation of the cell chromatin (Fig. 1A). Subsequently rodshaped particles, 380 nm long and 80 nm wide, were observed within the nucleus associated with a virogenic stroma (Fig. 1B). By 15 hr after infection all cells showed signs of “infection” although there was a certain amount of asynchrony in the apparent morphological stages of development. The virogenic stroma appears as an electron-translucent area which is associated with virus particles. Generally virus particles were scattered peripheral to this central stroma adjacent to the nuclear membrane, though it was not uncommon to detect aggregates of particles within the stroma as well. Figure 2 shows a number of nuclei containing virus. A frequent feature characteristic of virus development was the diffuse ill-defined nature of the nuclear membrane. Like other baculoviruses the particles appeared to be enveloped de novo in the nucleus. Virus was not observed budding at the nuclear or plasma membranes and release appeared to be by total cell lysis and then intracytoplasmic and extracellular virus was observed. Figure 2 (inset) shows detail of an aggregate of virus particles adjacent to a diffuse nuclear membrane. Such aggregates, with particles aligned with each other, were common. Neither occlusion of virus particles into polyhedra nor the presence of polyhedra was detected. Originally we considered that the nuclear polyhedrosis virus of H. armigera was replicating in the cells. We were, however, unable to confirm this either by testing for the production of viral antigens
FIG. 1. Heliothis zea cells “infected” with Heliothis armigera singly enveloped drosis virus (A) 5 hr after infection and (B) 12 hr after infection. Note the condensed early in infection (A). Subsequently singly enveloped virus particles occur peripheral translucent virogenic stroma (B). Bar = 2 km.
177
nuclear polyhecell chromatin to an electron-
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KELLY
ET AL.
FIG. 2. Heliothis zea cells “infected” with Heliothis drosis virus 15 hr after infection. Note the presence electron-translucent virogenic stroma, and the apparent brane. Bar = 2 pm. Inset shows detail of an aggregate
using fluorescent antibody staining or by enzyme-linked immunosorbent assays. The “infected” cells gave consistently negative results whereas the tests performed on haemocytes taken from H. armigera NPVinfected H. armigera larvae were positive. In addition we were unable to plaque assay the H. armigera virus in H. xea cells; and
armigera singly enveloped nuclear polyheof virus particles in just the nucleus, the dissipated appearance of the nuclear memof nonoccluded virus.
titration of the virus by TCIDSO assay failed to give titres greater than lo3 TCIDSO units/ml although to produce the cpe observed the effective titre must be around lo7 infectious units/ml (assuming, as the evidence suggests, one cycle of “replication” occurs). Finally the alteration in polypeptide synthesis in “infected” cells
INDUCTION
OF A BACULOVIRUS
ABCDEFGHI
BY NPV JKLMNO
FIG. 3. Polypeptide synthesis in Heliothis zea cells “infected” with Heliothis armigera nuclear polyhedrosis virus. Cells were pulse labelled for 1 hr with [%]methionine at the times indicated. A and 0 are uninfected controls; B, cells 1 hpi; C, cells 2 hpi; D, cells 3 hpi; E, cells 4 hpi; F, cells 5 hpi; G, cells 6 hpi; H, cells 7 hpi; I, cells 8 hpi; J, cells 9 hpi; K, cells 10 hpi; L, cells 11 hpi; M, cells 12 hpi; N, cells 13 hpi.
(Fig. 3) showed that no “infected” cell-specific polypeptides had relative migrations similar to those of H. armigera NPV (Kelly et al., 1980). Consequently, we considered that we must be inducing a virus present in the H. xea cells. Figure 4 shows a comparison of the polypeptides of this virus and H. armigera NPV. Examination of control cells either untreated or mock-infected with haemolymph from healthy insect species (H. armigera, H. xea, S. frugiperda, or S. littoralis) failed to show the presence of the virus. This occurred in three experiments ((but see later section on persistence).
Interaction of Trichoplusia ni and Other Multiply Enveloped Nuclear Polyhedrosis Viruses with Heliothis xea Cells “Infection” of the H. zea cells with 50 PFU/cell of T. ni multiply enveloped nuclear polyhedrosis virus produced a cytopathic effect similar to that produced by the Heliothis NPVs. Examination of the cells at 9, 15, and 22 hr after “infection” (times at which (Y, @, y, 6 polypeptide phases of T. ni NPV protein induction occur in S. frugiperda cells (Kelly and Lescott, 1981)) showed an induction of singly enveloped rods with a morphogenesis and appearance similar to that described for
180
KELLY
ET AL.
the virus induced by the Heliothis viruses. Figure 5C shows the appearance of such cells. “Infection” of H. zea cells with haemolymph-derived S. frugiperda and S. littoral& viruses also created the same phenomenon (Figs. 5A, B). In all cases the H. xea cells showed evidence of singly enveloped rods, no multiply enveloped virus, and no polyhedra. As such it is probable that the nonoccluded virus is induced by the multiply enveloped viruses which in turn fail to replicate themselves. Analysis of the polypeptides synthesised in cells “infected” with T. ni NPV showed that the polypeptides induced were similar to those detected in H. armigera NPV-“infected” cells and that no polypeptides typical of the T. ni NPV-infected cell-specific polypeptides were detected (Fig. 6).
Persistent Nature of the Nonoccluded rus Found in Heliothis xea Cells
Vi-
Since the nonoccluded virus was consistently inducible in the H. zea cells it was probable that the virus existed in the cells in a latent or persistent state. Careful examination of cells processed for electron microscopy during (and indeed prior to) this project showed that cells containing the virus could be detected. The percentage of cells showing the overt presence of the virus varied from less than 0.1 to 25% (Table 1). Titration of the virus on various passage levels of the cells also showed considerable variation in the amount of virus present in the cells (Table 2). The presence of virus in variable amounts on prolonged passage is typical of a persistent virus infection.
Superi$ection of Heliothis xea Cells with the Homologous Nonoccluded Virus Addition of 50 TCIDsO of the nonoceluded virus of H. xea cells subsequently referred to as baculovirus X obtained from noninduced cells to the H. zea cells resulted in the production of a cytopathic effect, an increased production of baculovirus X in the tissue culture fluid, and the induction of baculovirus X-infected
FIG.
4. Polypeptides contained by (A) Heliothis nuclear polyhedrosis virus particles and (B) baculovirus X virus particles. The polypeptides were run on 12.5% polyacrylamide gels and stained with Coomassie blue. The polypeptides are identified by estimates of their molecular weight. Minor polypeptides in the baculovirus X preparation are probably host cell polypeptides and these are unlabelled.
armigera
cell-specific polypeptides similar to those observed when T. ni or Heliothis spp. nuclear polyhedrosis viruses were added (data not shown).
Effect of Drugs on the Abundance of Baculovirus X in Heliothis xea Cells Since a number of nuclear polyhedrosis viruses were able to induce the presence of baculovirus X in all Heliothis cells we
INDUCTION
OF A BACULOVIRUS
attempted to induce the virus with each of a number of drugs. Different culture cells were treated for 16 hr with each of the folllowing, 0.5 mM hydroxyurea, 1.5 mM caffeine, 20 pg/ml bromodeoxyuridine, 1 pg/ml ethidium bromide, 1 pg/ml mitomycin C, and 1 pg/ml cycloheximide. The treated cells were examined by phasecontrast microscopy and transmission electron microscopy of thin sections, by examination of infected cell-specific polypeptides and by titration of baculovirus X in T. ni cells, at 72 and 120 hr after treatment. N-o treatment succeeded in producing any evidence of increased levels of virus in t’he cell cultures.
Effect of Iridescent Virus Type 22 on Baculovirus X Induction Iridescent virus type 22 is an “icosahedral cytoplasmic deoxyribovirus” (Batson et al., 1976) which infects a broad spectrum of invertebrate cells in culture (T. Lescott and D. C. Kelly, unpublished observations). Infection of H. xea cells with 50 PFU per cell of this virus failed to stimulate the growth of baculovirus X assessed by electron microscopy, titration of the virus in T. ni cells, and induction of infected cell-specific polypeptides typical of baculovirus X. The iridescent virus itself also failed to replicate.
Induction of Baculovirus X in the Presence of Metabolic Inhibitors A variety of metabolic inhibitors were used to examine the general macromolecular synthesis in baculovirus X induction by T. ni nuclear polyhedrosis virus. No induction occurred when transcription of viral DNA was inhibited by actinomycin D (10 p&/ml). Cells were treated 0 to 5, 0 to 9,1 to 5,2 to 5, and 3 to 5 hr and sampled (where appropriate) at 5, 7, 9, 12, 18, and 22 hr after infection. Cells blocked with actinomycin D later than 5 hr after infection showed evidence of induction. This showed that transcription of a genome is required for induction to occur. Cells were treated from 0 to 7 hr with cycloheximide
BY NPV
181
(50 pg/ml) to prevent translation, the drug was washed out, and sampling was done immediately and at 9,10, 11,12, 15 and 22 hr after infection. Evidence of gradual induction delayed 8 hr was found (data not shown). This showed that messenger responsible for the synthesis of protein involved in baculovirus X induction did not accumulate in cycloheximide-treated cells although it was presumably able to do so after release from the block. Cycloheximide alone failed to induce the virus. Azetidine and canavanine, amino acid analogues of proline and hydroxyproline, respectively, can be incorporated into proteins to render them nonfunctional. H. zea cells were treated with 12 mM azetidine and 12 mM canavanine and infected with T. ni nuclear polyhedrosis virus, and it was found that induction of baculovirus X polypeptides did not occur, when samples were taken 5, 9, 15 and 22 hpi (Fig. 7). These concentrations of azetidine and canavanine interfere with T. ni nuclear polyhedrosis ICSP induction presumably by rendering the ICSP induced nonfunctional (Kelly and Lescott, 1981). When DNA replication was inhibited by using hydroxyurea (800 pg/ml) or cytosine arabinoside (50 pug/ml), concentrations which prevent T. ni nuclear polyhedrosis virus-specific DNA synthesis and late protein synthesis (Kelly and Lescott, 1981; H. M. Bud and D. C. Kelly, unpublished observations), baculovirus X ICSP production appeared to be normal. Samples were taken at 3,6,9,12,18, and 24 hr after infection and the drugs were present throughout (data not shown).
Induction of Baculovirus X by Heat- and uv-Inactivated Trichoplusia ni Nuclear Polyhedrosis Virus Virus inactivated by heat to denature primarily the virus structural; proteins, and by uv light to denature primarily the genome, were used to test the dependence of baculovirus X induction on structural components of the inducing virus. As shown in Fig. 8 virus inactivated by uv light was still able to induce baculovirus X ICSP, whereas virus inactivated by
KELLY
182
ET
heating at 70” or greater did not. Virus heat inactivated at 60” induced normal baculovirus X ICSP. Both uv- and heat-inactivated virus failed to induce virus-specific polypeptide synthesis in S. frugiperda cells in culture (data not shown).
AL.
infecting cells including variability of titre, and numbers of cells overtly showing the presence of virus, on cell passage. The origin of the virus is not clear and this has been extensively discussed by Granados and co-workers (1978). The presence of baculovirus X appeared to make the cells refractile to the replication of the superinfecting nuclear polyhedrosis viruses, and no evidence of T. ni nuclear polyhedrosis virus macromolecular synthesis was obtained. Baculoviruses generally have a narrow cell culture range for replication (Knudson and Buckley, 1977) and so the refractile nature of the H. zea cells may not be directly attributable to the presence of baculovirus X. The cells were also refractile to iridescent virus type 22 which has a broad experimental host cell range and this is the first invertebrate cell line we have discovered which is nonpermissive for an iridescent virus. The morphogenesis of baculovirus X on induction is different from that of the inducing baculoviruses in a number of respects. The morphology of baculovirus X in thin section was typical of a singly enveloped virus but was larger than most baculoviruses which have a size of 70 x 280 nm when singly enveloped (Harrap and Payne, 1979). We have been unable to define clearly how nuclear polyhedrosis viruses trigger the induction of baculovirus X in cell culture. Both viruses contain DNA and so it is difficult to discriminate with drugs between the two viruses when they replicate in cells. However, blocking DNA synthesis in H. xea cells allowed normal induction which could indicate two things. First, the contribution by the T. ni virus was an early event because if the virus were to replicate at all it would proceed no further than a synthesis with hydroxyurea or p synthesis with cytosine arabinoside (Kelly and Lescott, 1981). Second, it is probable that baculovirus X DNA exists (possibly
Induction of Baculovirus X by Trichoplusia ni Nuclear Polyhedrosis Virus in the Presence of Ethidium Bromide From earlier experiments it was tentatively concluded that the baculovirus X genome exists in all cells prior to induction. Ethidium bromide has a high affinity for supercoiled DNA (Gale et al., 1972), and since baculovirus DNA occurs in the supercoiled form in virus particles (Summers and Anderson, 1973), inhibition or suppression of induction may be due to an interaction with the viral DNA in the nucleus. Ethidium bromide (1 pg/ml) suppresses the induction of baculovirus X by T. ni nuclear polyhedrosis virus (data not shown). DISCUSSION
Our results show that a virus which apparently infects a H. zea cell line in a persistent manner can be efficiently induced to replicate by a number of singly and multiply enveloped nuclear polyhedrosis viruses. The virus is presumed to be a baculovirus because it possesses the general morphological appearance of a nonoceluded baculovirus (i.e., an enveloped rodshaped virus devoid of polyhedron protein), contains DNA (D. C. Kelly, unpublished observations), and replicates in the cell nucleus. The virus apparently does not become occluded within polyhedra and in this respect resembles the class C group of baculoviruses (Matthews, 1979) which includes the Oryctes viruses (Monsarrat et al., 1973) and the braconid baculoviruses (Stoltz and Vinson, 1979). Baculovirus X had a number of characteristics typical of a virus persistently -
FIG. 5. Electron micrographs of Heliothis zea cells “infected” with three multiply enveloped nuclear polyhedrosis viruses from (A) Spodoptera littoralis, (B) Spodopteru fmgiperda, and (C) Trichoplusia ni. In each case a singly enveloped nonoccluded virus was induced. Bar = 2 pm.
INDUCTION
OF A BACULOVIRUS
BY NPV
183
184
KELLY A
B
C
D
E
ET
AL. F
G
H
I
J
FIG. 6. Polypeptide synthesis in (A to E) Heliothis zea cells and (F to J) Spodoptera frugiperda cells infected with Trichoplusia ni nuclear polyhedrosis virus. Cells were pulse labelled with [%]methionine for 1 hr in uninfected control cells (A and F) and 5 hr (B and G), 9 hr (C and H), 15 hr (D and I), and 22 hr (E and J) after infection. The polypeptides induced in H. zea cells resemble those induced by H. amigera virus in H. zea cells.
in a proviral state) in reasonable quantities prior to induction. In addition, treatment of cells with ethidium bromide, which binds with high affinity to supercoiled DNA, prevents induction of baculovirus X, suggesting that the baculovirus X genome is present in a supercoiled state. A similar suppression of induction of C-type virus by a chemical inducer (iododeoxyuridine) occurs when cells are treated with ethidium bromide (Avery and Levy, 1979). Treatment of cells with actinomycin D
shows that transcription of either the cell genome, the baculovirus X genome, or the inducing T. ni baculovirus genome is required for induction to occur. However uv light inactivation of T. ni virus does not affect induction and so, if inactivation of any “inducing genes” occurs at the same rate as total inactivation, it is unlikely that transcription of the inducing virus is responsible for the induction. Ultraviolet light inactivation of cells prevents induction (T. Lescott and D. C. Kelly, unpub-
INDUCTION TABLE
1
VARIATIONIN PROPORTIONOF Heliothis CONTAINING PASSAGE
NONOCCLUDED
Passage
data”
01.12.77 11.01.78 09.02.78 07.03.78 14.05.78 14.06.78 22.06.78 29.06.78 14.12.78
OF A BACULOVIRUS
BACULOVIRUS
zea CELLS ON CELL
Percentage cells positive* 0.1 0.1 0.2 0.3 1.0 5.0 25.0 1.0 5.0
” The date cells were sampled for electron microscopy. The cells were subcultured weekly and continuously. “The proportion of cells showing virus by randomly selecting thin sections of cells and scoring.
lished observations), but this observation does not clarify the point of initial transcription. Treatment of cells with cycloheximide did not permit baculovirus X induction by T. ni nuclear polyhedrosis virus; and induction of baculovirus X ICSP proceeded with timing equivalent to a normal induction on reversal of the cycloheximide block. This indicates that protein synthesis and messenger RNA synthesis from the cell/baculovirus X genome is also required. for induction to occur. No amplification of baculovirus X messenger RNA occurred during the cycloheximide block because on release from the block no increased baculovirus X ICSP synthesis was detected. Azetidine and canavanine also inhibited the induction of baculovirus X ICSP, indicating that a functional protein is required for the induction of baculovirus X after Trichoplusia ni virus “infection.” These observations demonstrate that functional protein synthesis is required before baculovirus X induction occurs. We can obtain no evidence of the synthesis of T. ni nuclear polyhedrosis virus (YICSP synthesis in H. zea cells, and this coupled with the observation that heat-in-
185
BY NPV
activated (presumably structural protein impaired) virus failed to induce baculovirus X indicates that the inducing virus probably interacts with baculovirus X or a genome product whose synthesis is prevented by direct or indirect inhibition of protein synthesis. In other words, an inducing virus structural protein may interact with a gene product which represses baculovirus X replication. Interestingly the transition from early to late baculovirus X ICSP occurs in cells in which DNA synthesis is blocked with hydroxyurea or cytosine arabinoside. This means that the polypeptides indicated as early (Fig. 5) are merely indicators of time of appearance and not related to DNA synthesis. In addition, only two temporal phases of baculovirus X ICSP induction are detected compared to four with the T. ni virus. Preliminary experiments using the more sensitive two-dimensional gel electrophoresis of ICSP (O’Farrell, 1975) have failed to detect any additional baculovirus X ICSP, or indeed any T. ni ICSP (D. C. Kelly and M. D. Ayres, unpublished observations). The results that we have obtained indicating that the persistent virus is induced in all cells without replication of viral DNA suggest that the genome of the persistent virus is present in abundance in all cells.
TABLE TITRES
2
OF NONOCCLUDED BACULOVIRUS PRESENT Heliothis zea CELLS ON CELL PASSAGE Passage
date”
Titre’
15.12.78 03.01.79 10.01.79 17.01.79 23.01.79 12.02.79 19.02.79 03.03.79 a The date cells were sampled. cated cell extracts was titrated. b TCIDso units per lo6 cells.
IN
2 2 4 2 1 2 3 2 The
x x x x x x x x
lo3 lo3 lo5 lo3 lo* lo7 lo5 lo3
virus
in soni-
KELLY
186 A
B
C
D
E
ET AL. F
G
H
I
J
FIG. 7. Polypeptides synthesised in Heliothis zea cells “infected” with Trichoplusia ni nuclear polyhedrosis virus in cells treated with azetidine (12 mM) (A to E) and canavanine (12 mM) (F to J). (A and F) Control cells; (B and G) cells 5 hpi; (C and H) cells 9 hpi; (D and I) cells 15 hpi; (E and J) cells 22 hpi. Induction of polypeptide typical of baeulovirus X stimulation is barely detected 22 hpi.
The general phenomenon of induction of a persistent virus in an insect cell line is of interest because it mimicks in part the situation found in some insects where infection of a particular insect species by a nuclear polyhedrosis virus from a het-
erologous insect species results in the test insect succumbing to a disease caused by a virus typical of the homologous species (Longworth and Cunningham, 1968; Jurkovicova, 1979; McKinley et al., 1981). Baculoviruses are used commercially as bio-
INDUCTION A
B
C
D
OF A BACULOVIRUS E
F
BY NPV G
H
I
J
FIG. 8. Induction of polypeptides in Heliothis zea cells “infected” with heat-inactivated and uvinactivated Ttichoplusia ni nuclear polyhedrosis virus. Cells were infected with virus and sampled with a 1-hr pulse of [%]methionine 15 hpi. (A) Virus heat inactivated at 60”; (B) virus heat inactivated at 70”; (C) virus heat inactivated at 80”; (D) virus heat inactivated at 90”; (E) uninfected control cells; (F) live virus; (G) virus uv inactivated for 5 min; (H) virus uv inactivated for 10 min; (I) virus uv inactivated for 15 min; (J) virus uv inactivated for 20 min.
logical control agents, and the activation of a persistent (possibly latent) virus by a viral insecticide raises problems about the safe and responsible use of these viruses and the interpretation of epidemiological studies of a given virus. The dem-
on&ration that a comparable situation exists in a cell culture model system means that the contribution made by the inducing virus to initiate the replication of the induced virus may be studied. Such work is now in progress and particular empha-
188
KELLY
sis is being placed on the state and location of the baculovirus X genome in the cell, and the replication of this virus in other cell lines. REFERENCES AVERY, R. J., and LEVY, J. A. (1979). The effect of ethidium bromide on C type virus production and induction. Virology 95, 277-284. BATSON, B. S., JOHNSTON, M. R. L., ARNOLD, M. K., and KELLY, D. C. (1976). An iridescent virus from Simulium sp. (Diptera: Simuliidae) in Wales. J. Znwertebr. Pathol. 27, 133-135. BROWN, D. A., LESCOTT, T., HARRAP, K. A., and KELLY, D. C. ( 1978). The replication and titration of iridescent virus type 22 in Spodopterafrugiperda cells. J. Gen. Viral. 38, 175-178. BUD, H. M., and KELLY, D. C. (1980). Nuclear polyhedrosis virus DNA is infectious. Microbiologica 33, 177-180. ELLIOTT, R. M., ARNOLD, M. K., and KELLY, D. C. (1979). The replication of frog virus 3 in an amphibian ceil line fromxenopus laevis. J. Gen. W-01. 44, 89-98. ELLIOTT, R. M., and KELLY, D. C. (1980). Frog virus 3 replication: Induction and intracellular distribution of polypeptides in infected cells. J. Virol. 33, 28-51. GALE, E. F., CUNLIFFE, E., REYNOLDS, P. E., RICHMOND, M. H., and WAREING, M. J. (1972). “The molecular basis of antibiotic action.” Wiley, London. GARDINER, G. R., and STOCKDALE, H. (1975). TWO tissue culture media for production of lepidopteran cells and nuclear polyhedrosis viruses. J. Znvertebr. Pathol. 25, 303-370. GRANADOS, R. R. (1976). Infection and replication of insect pathogenic viruses in tissue culture. Advan. Virus Res. 20, 189-236. GRANADOS, R. R., NGUYEN, T., and CATO, B. (1978). An insect cell line persistently infected with a baculovirus like particle. Zntervirology 10, 309-317. GRAVELL, M., and MALSBERGER, R. G. (1965). A permanent cell line from the fat head minnow (Pimephales promelas). Ann. N. Y. Acad. Sci. 126,55565. HARRAP, K. A., and PAYNE, C. C. (1979). The structural properties and identificaiton of insect viruses. Advan. Virus Res. 25, 273-355. HARRAP, K. A., PAYNE, C. C., and ROBERTSON, J. S. (1977). The properties of three baculoviruses from closely related hosts. Virology 79, 14-31. HINK, W. F. (1970). Establishment of a new cell line from the cabbage looper, Trichoplusia ni. Nature (London) 226, 466-467.
ET AL. HINK, W. F., and IGNOFFO, C. M. (1970). Establishment of a new cell line (IMC-HZ-l) from ovaries of cotton bollworm moths, Heliothis zea (Boddie). Ezp. Cell Res. 60, 307-309. JURKOVICOVA, M. (1979). Activation of latent virus infections in larvae of Adozophyes orana (Lepidoptera:tortricidae) and Baruthra brassicae (Lepidoptera:noctuidae) by foreign polyhedra. J. Znvertebr. Pathol. 34, 213-223. KELLY, D. C. (1976). ‘Oryctes’ virus replication: Electron microscopic observations on infected moth and mosquito cells. Virology 69, 599-606. KELLY, D. C. (1981a). Baculovirus replication: Electron microscopy of the sequence of infection of Trichoplusia ni nuclear polyhedrosis virus in Spodoptera frugiperda cells. J. Gen. Virol. 52,209-219. KELLY, D. C. (1981b). Baculovirus replication: Stimulation of thymidine kinase and DNA polymerase activities in Spodoptera frugiperda cells infected with Trichoplwia ni nuclear polyhedrosis virus. J. Gen. Virol. 52, 313-319. KELLY, D. C., BROWN, D. A., ROBERTSON, J. S., and HARRAP, K. A. (1980). Biochemical biophysical and serological properties of two singly enveloped nuclear polyhedrosis viruses from Heliothis armigera and Heliothis zea. Microbiologica 3, 319-331. KELLY, D. C., and DIMMOCK, N. J. (1974). Fowl plague virus replication in mammalian cell-avian erythrocyte heterokaryons: Studies concerning the actinomycin D and ultraviolet light sensitive phase in influenza virus replication. Virology 61,210-222. KELLY, D. C., EDWARDS, M. L., EVANS, H. F., and ROBERTSON, J. S. (1978). The use of enzyme-linked immunosorbent assay to detect a nuclear polyhedrosis virus in Heliothis armigera larvae. J. Gen. W-01. 40, 465-469. KELLY, D. C., and LESCOTT, T. (1981). Baculovirus replication: Protein synthesis in Spodoptera frugiperda cells infected with Trichoplusia ni nuclear polyhedrosis virus. Microbiologica 4, 35-57. KNUDSON, D. L., and BUCKLEY, S. M. (1977). Invertebrate cell culture methods for the study of invertebrate-associated animal viruses. Methods Virol. 6, 323-391. LONGWORTH, J. F., and CUNNINGHAM, J. C. (1968). The activation of occult nuclear polyhedrosis viruses by foreign nuclear polyhedra. J. Znvertebr. Pathol. 10,361-367. MATTHEWS, R. E. F. (1979). Classification and nomenclature of viruses. Intervirology 12,129-296. MCKINLEY, D. J., BROWN, D. A., PAYNE, C. C., and HARRAP, K. A. (1981). Cross-infectivity and activation studies with four baculoviruses. Entomophaga, 26, 79-90. MITSUHASHI, J., and MARAMOROSCH, K. (1964). Leafhopper tissue culture: embryonic, nymphal
INDUCTION and imaginal tissues from aseptic Boyce Thompson Inst. 22,435-460.
insects.
OF A BACULOVIRUS Contrib.
MONSARRA.T, P., MEYNADIER, G., CROIZIER, G., and VAGO, C. (1973). Recherches cytopathologiques sur une maladie virale du coleoptere Oryctes rhinoceros. C.R. Acad. Sci. Paris Ser. D 277,1413-1415. O’FARRELL, P. H. (1975). High resolution two dimensional electrophoresis of proteins. J. Biol. Chem. 250, 4007-4021. PAYNE, C. C. (1974). The isolation and characterisation of a virus from &y&es rhinoceros. J. Gen. Viral. 25, 105-116. PUDNEY, M., VARMA, M. G. R., and LEAKE, C. Establishment of a cell line from the South clawed toad, Xenopus laevis. Experientia 467. SCHNEIDER, I. (1972). Cell lines derived from
J. (1973). African 29,466late em-
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189
bryonic stages of Drosophila melanogaster. J. Embryol. Exp. Morphol. 27,353-365. SINGH, K. R. P. (1967). Cell cultures derived from larvae of Aedes albopictus (Skuse) and Aedes aegypti (L.). Curr. Sci. 36, 506-508. STOKER, M. G. P., and MACPHERSON, I. A. (1961). Studies on the transformation of hamster cells by polyoma virus in vitro. Virology 14, 359-370. STOLTZ, D.,and VINSON, S. B. (1979). Viruses and parasitism in insects. Advan. Virus Res. 24, 125-172. SUMMERS, M. D., and ANDERSON, D. L. (1973). Characterisation of nuclear polyhedrosis virus DNA. J. Viral. 12, 1336-1346. VAUGHN, J. L., GOODWIN, R. H., THOMPKINS, G. J., and MCCAWLEY, P. (1977). The establishment of two cell lines from the insect Spodoptera frugiperda (Lepidoptera: Noctuidae). In Vitro 13, 213217.