Replication and infectivity of the single-embedded nuclear polyhedrosis virus, Baculovirus heliothis, in homologous cell lines

Three cell lines of Heliothis zea and one cell line of Heliothis virescens replicated the singleembedded, nuclear polyhedrosis virus (NPV) of H. Zea, (i.e., Baculovirus heliothis) with concomitant production of polyhedral inclusion bodies (PIB). Between 20 and 60% of the H. zea cells produced PIB, whereas only 3% of H. virescens cells were found to produce PIB. The H. zea cell lines produced 10 to 20 times more PIB than did the H. virescens cell line. The PIB from all cell lines produced typical symptoms of an NPV infection when bioassayed against larvae of H. zea. More than 99% of the total viral activity of the final whole culture was due to the PIB.     

A synchronous peroral technique for the bioassay of insect viruses

A relatively fast and simple peroral technique for the bioassay of insect viruses is described in which newly hatched larvae ingest a uniform volume of virus suspension. Three isolates of the Autographa californica nuclear polyhedrosis virus (NPV) and one isolate of the Heliothis zea NPV were used to test the procedure with Trichoplusia ni and H. zea larvae, respectively. Within-assay and between-assay variation was very low with coefficients of variation averaging 0.012 ± 0.006 and 0.20 ± 0.04 for time-mortality and dose-mortality tests, respectively. The synchronous uptake of virus removed the acquisition-time component of the LT₅₀ values while the constant volume improved the accuracy of LD₅₀ values. The procedure was shown to be suitable for a wide variety of lepidopterous species, including Spodoptera frugiperda, S. eridania, Estigmene acrea, Plutella xylostella, Choristoneura fumiferana, Ostrinia nubilalis, Plodia interpunctella, and Pieris rapae.     

Oncolytic measles viruses produced at different scales under serum‐free conditions

Measles virus (MV) with attenuated pathogenicity has potential as oncolytic agent. However, the clinical translation of this therapy concept has one major hurdle: the production of sufficient amounts of infectious oncolytic MV particles. The current study describes oncolytic MV production in Vero cells grown on microcarrier using serum‐free medium. The impact of the number of harvests, cell concentration at infection (CCI), multiplicity of infection (MOI), and temperature on MV production was determined in different production scales/systems (static T‐flasks, dynamic spinner, and bioreactor system) and modes (batch, repeated‐batch, and perfusion). Cell growth, metabolic, and production kinetics were analyzed. It was found that the number of harvests had the strongest positive impact on MV yield in each production scale, and that high temperatures affected MV yield adversely. Moderate MV titers were produced in T‐ and spinner flasks at 37°C (～10⁷ TCID₅₀ mL^?1, where TCID₅₀ is tissue culture infective doses 50%), but stirred tank reactor (STR) MV production at 37°C yielded up to 10 000‐fold lower MV titers. In contrast, at lower temperatures (32°C, 27°C), 1.4 × 10⁷ TCID₅₀ mL^?1 were achieved in the STR. Variations in MOI and CCI had almost no influence on MV production yield. The current study improves oncolytic MV production process understanding and identifies process bottlenecks for large‐scale production.     

Genetic variation and virulence of nucleopolyhedroviruses isolated worldwide from the heliothine pests Helicoverpa armigera, Helicoverpa zea, and Heliothis virescens

To assess the diversity and relationships of baculoviruses found in insects of the heliothine pest complex, a PCR-based method was used to classify 90 samples of nucleopolyhedrovirus (NPV; Baculoviridae: Alphabaculovirus) obtained worldwide from larvae of Heliothis virescens, Helicoverpa zea, and Helicoverpa armigera. Partial nucleotide sequencing and phylogenetic analysis of three highly conserved genes (lef-8, lef-9, and polh) indicated that 67 of these samples contained isolates of the H. zea-H. armigera single nucleopolyhedrovirus (Hz/HaSNPV) species group. Eighteen of the samples contained isolates of a multiple NPV from H. armigera, HearMNPV, and five of the samples contained isolates of Autographa californica MNPV (AcMNPV). Sequencing and analysis of an additional seven loci (orf5/orf5b, hr3-orf62, orf26, orf79, orf124/orf117a, orf42, and a part of the region between hr2 and hr3) in the Hz/HearSNPV isolates further classified these viruses into two groups of HearSNPV variants mostly from India and China and a third group of HzSNPV variants. Some of the samples contained isolates of more than one virus. In bioassays of a selection of isolates against H. zea, the commercially available Gemstar® isolate of HzSNPV killed larvae faster than most other Hz/HaSNPV and HearMNPV isolates. Gemstar® and two HearMNPV isolates exhibited significantly higher LC₅₀s than the Hz/HearSNPV isolates tested. This study expands significantly on what we know about the variation of heliothine NPV populations, provides novel information on the distinct groups in which these NPVs occur, and contributes to the knowledge required for improvement of heliothine baculoviruses as biological control agents.     

Apis iridescent virus and "clustering disease" of Apis cerana

NPV of Spodoptera littoralis was completely inactivated in vitro following 10 min of exposure to a temperature higher than 90°C, but survived 3 weeks at ?20°C. At pH 12, some 75% of the infectivity was lost. Measurable proteolysis in vitro of the polyhedral protein by a larval midgut extract could be obtained only when the pH of the reaction mixture was raised to an unnatural level of 10.5, the natural pH of the midgut content being 8.5 or 9.5 according to different authors. The plant growth retardant Phosfon synergized mortality caused by the NPV. The virus could be cross-transmitted to two congeneric species of Spodoptera (S. exigua and S. litura), but could not infect any of four tested species belonging to other genera of the Moctuid family.     

Thermal behavior of bovine β-trypsin at physiological temperature range

The effects of temperature on the steady-state kinetics of β-trypsin hydrolysis of α-Ntosyl-l-arginine methyl ester (k_cat, K_m) and its inhibition by phenylguanidinium ion (K_i) were studied in the temperature range 27–37 °C, at 1 °C intervals, pH 8.0. Within this temperature range inhibition of β-trypsin by phenylguanidine was strictly competitive. The Eyring and van't Hoff plots were nonlinear; interpretation of the data was based on two possible alternatives: in the first, there occurs a thermal transition centered at 31 °C, characterized by ΔH° = 42.2 ± 8.7 kcal/mol and ΔS ° = 138 ± 29 e.u. According to the second interpretation the phenomenon would be determined by a large value of Δ Cp; its value was estimated to be ΔCp = ?7192 cal/deg · mol. A decision as to what interpretation is more adequate must wait until further experimental information is obtained.     

Two subpopulations of α1-adrenergic receptors with high and low affinity for agonists: Short-term exposure to agonists reduced the high-affinity sites

Short-term receptor regulation by agonists is a well-known phenomenon for a number of receptors, including β-adrenergic receptors, and has been associated with receptor changes revealed by radioligand binding. In the present study, we investigated the rapid changes in α₁-adrenergic receptors induced by agonists. α₁-receptors were studied on DDT₁ MF-2 smooth muscle cells (DDT₁-MF-2 cells) by specific [³H]prazosin binding. In competition binding on membranes and on intact cells at 4°C or at 37°C in 1-min assays, agonists competed for a single class of sites with relatively high affinity. By contrast, in equilibrium binding at 37°C on intact cells agonists competed with two receptor forms (high- and low-affinity). We quantified the receptors in the high-affinity form by measuring the [³H]prazosin binding inhibited by 20 μM norepinephrine (this concentration selectively saturated the high-affinity sites). The low-affinity sites were measured by subtracting the binding of [³H]prazosin to the high-affinity sites from the total specific binding. High-affinity receptors were 85% of the total sites in binding experiments at 4°C, but only 30% at 37°C. On DDT₁-MF-2 cells preequilibrated with [³H]prazosin at 4°C, and then shifted to 37°C for a few minutes, norepinephrine selectively reduced the high-affinity sites by 30%. We suggest that at 4°C it is the native form of α₁-receptors that is measured, with most of the sites in the high-affinity form, while during incubation at 37°C the norepinephrine present in the binding assay converts most of the receptors to an apparent low-affinity form, so that they are no longer recognized by 20 μM norepinephrine. The nature of this low-affinity form was further investigated. On DDT₁-MF-2 cells preincubated with the agonist and then extensively washed at 4°C (to maintain the receptor changes induced by the agonist) the number of receptors recognized by [³H]prazosin at 4°C was reduced by 38%. After fragmentation of the cells, the number of receptors measured at 4°C was the same in control and norepinephrine-treated cells, suggesting that the disruption of cellular integrity might expose the receptors which are probably sequestered after agonist treatment. In conclusion, the appearance of the low affinity for agonists at 37°C may be due to the agonist-induced sequestration of α₁-adrenergic receptors, resulting in a limited accessibility to hydrophilic ligands.     

Binding of [3H]batrachotoxinin A-20-α-benzoate and [3h]saxitoxin to receptor sites associated with sodium channels in trout brain synaptoneurosomes

1. [³H]Batrachotoxinin A-20-α-benzoate ([³H]BTX-b) and [³H]saxitoxin ([³H]STX), radioligands that bind to distinct sites on the voltage-sensitive sodium channel, were bound specifically to saturable sites in rainbow trout (Oncorhynchus mykiss) brain synaptoneurosomes.2. Specific [³H]BTX-B binding was temperature dependent with highest levels of specific [³H]BTX-B binding observed at 7°C. Specific binding was inversely correlated with assay temperature at temperatures above 7°C.3. Saturating concentrations of scorpion (Leiurus quinquestriatus) venom (ScV) stimulated specific [³H]BTX-B binding at 27°C, but not at 7°C. The dihydropyrazole insecticide RH 3421 inhibited specific [³H]BTX-B binding at 7°C but had no effect on specific binding at 27°C. The sodium channel activators veratridine and aconitine and the local anesthetic dibucaine inhibited specific [³H]BTX-B binding at both 7°C and 27°C.4. Displacement experiments in the presence of ScV at 27°C gave an equilibrium dissociation constant (K_d) for [³H]BTX-B of 710 nM and a maximal binding capacity (B_max) of 11.3 pmol/mg protein. Kinetic experiments established the rates of association (1.17 × 10⁵min⁻¹ nM⁻¹) and dissociation (0.0514min⁻¹) of the ligand-receptor complex.5. The binding of [³H]STX reached apparent saturation at 7.5 nM. Scatchard analysis of the saturation data indicated a K_d of 3.8nM and a B_max of 1.9 pmol/mg protein.6. These studies provide evidence for high affinity, saturable binding sites for [³H]BTX-B and [³H]STX in trout brain preparations. Whereas certain neurotoxins modified the specific binding of [³H]BTX-B in trout brain synaptoneurosomes in a predictable fashion, other compounds known to affect specific [³H]BTX-B binding in mammalian brain preparations had no effect on specific [³H]BTX-B binding in the trout.     

The replication of a cytoplasmic polyhedrosis virus from Chrysodeixis eriosoma (Lepidoptera: Noctuidae) in Spodoptera frugiperda cells

A cytoplasmic polyhedrosis virus (CPV) from Chrysodeixis eriosoma (Lepidoptera: Noctuidae) replicated in Spodoptera frugiperda cells. Low rates of infection were achieved, even at high multiplicities of infection and TCID₅₀ assays showed that there was negligible release of virus particles from infected cells. In an infected focus assay, based on formation of PIB, the dose-response data demonstrated that a single particle could initiate infection. No loss of infectivity occurred in virus preparations stored at 4°, ?20°, or ?90°C, but infectivity of virus stored at 20°C declined sharply. A small isometric virus contaminant was present in some CPV preparations and its interaction with the CPV is discussed. Limited CPV infection was achieved in Trichoplusia ni cells, but attempts to infect Aedes aegypti cells were unsuccessful.     

Bioassay of mosquito iridescent virus of Aedes taeniorhynchus in cell cultures of Aedes aegypti.

A bioassay of mosquito iridescent virus (MIV) of Aedes taeniorhynchus was developed using cell cultures of Aedes aegypti. The dilution end point technique was based on the occurrence of cytopathic effects which were optimum at 31°C. Peleg's A. aegypti cell line was more sensitive and reliable than Singh's A. aegypti cell line for infectivity titration of the “R” and “T” strains of MIV. The highest tissue culture infectivity dose 50s (TCID₅₀) were elicited by virion:cell ratios of approximately 10. TCID₅₀ titers were significantly reduced by virus neutralization with either homologous or heterologous antiserum to either RMIV or TMIV. The virus propagated in either cell line was not infectious to A. taeniorhynchus larvae, or to the respective cells from which the virus was produced. All plaque assay attempts were unsuccessful.     

Temperature-sensitive mechanism regulating diapause in Heliothis zea

Pupal diapause in Heliothis zea is regulated by a temperature-sensitive mechanism which prevents ecdysone production despite the release of prothoracicotropic hormone. To determine how this mechanism functioned, donor prothoracic glands were implanted into prothoracic gland-ablated hosts to test their ability to produce ecdysone in a diapause-sustaining temperature of 19°C. Results of these experiments ruled out the possibility that ecdysis production was regulated by the nervous system or by a mechanism intrinsic to the prothoracic glands, and suggested that a humoral factor was required for diapause termination.Haemolymph injection experiments supported this humoral factor hypothesis, i.e. haemolymph from non-diapausing donor pupae terminated diapause in hosts maintained at 19°C, whereas haemolymph from diapausing donor pupae had no such effect. These findings indicate that the temperature-sensitive mechanism regulating H. zea diapause functions by controlling the availability of a humoral factor necessary for ecdysone production by the prothoracic glands.     

In vitro culture of Plasmodium yoelii blood stages

Lewis-Hughes P. H. and Howell M. J. 1984. In vitro culture of Plasmodium yoelii blood stages. International Journal for Parasitology14: 447–451. Plasmodium yoelii infected reticulocytes were cultured for 72 h at either 37 or 20°C in MEM (Eagle's modification) medium containing, in addition, glucose, para-aminobenzoic acid and 5% foetal calf serum, buffered at pH 7.3 with sodium bicarbonate/ HEPES and maintained under 10% CO₂ in air. Red blood cell numbers were more stable at 20°C than at 37°C. Culture at both temperatures resulted in an increase in parasitaemia of the reticulocyte population over the initial 36 h at 37°C and for at least 72 h at 20°C. The effects of different temperatures appeared to be related to the continued presence of target cells. Parasites were not detected after 72 h culture at 37°C, but persisted for up to 120 h at 20°C. Increasing parasitaemia at both temperatures was associated with changes in the numbers of some parasite development types. Early falls in schizont numbers were associated with an increase in the numbers of ring forms. Trophozoite numbers tended to remain constant throughout the culture period. Viability of parasites cultured for 36 h was confirmed by their infectivity to CBA mice. In addition, parasites progressively incorporated H³-leucine into TCA-precipitable material over the initial 36 h of culture.     

Effect of ammonia and urea treatments on digestibility and nitrogen content of dehydrated lucerne

Dehydrated lucerne of low (L: 0.53), normal (N: 0.55) and high (H: 0.73) in vivo dry matter (DM) digestibility were treated with ammonia or urea to study the effects on in situ and pepsin-cellulase DM digestibilities, water solubility and nitrogen content (Experiments 1, 2, 4) and on cell wall composition and degradability (Experiment 3). (1) N lucerne was treated with 30 g NH₃ kg⁻¹ DM for 1 to 12 weeks at 30°C and 2 to 6 days at 80°C; (2) L, N and H lucerne were treated with increasing ammonia levels: 15 to 100 g kg⁻¹ DM for 3 weeks at 30°C and 4 days at 80°C; (3) L, N and H lucerne were treated with 60 g NH₃ kg⁻¹ DM for 3 weeks at 30°C and 4 days at 80°C; (4) L, N and H lucerne were treated with 60 g urea kg⁻¹ DM without addition of urease for 3 and 6 weeks at 30°C. All treatments were carried out at 40% humidity.In situ and pepsin-cellulase DM digestibilities increased significantly (P < 0.05) with the duration of treatment (up to 3 weeks at 30°C and 4 days at 80°C) and with the level of ammonia (P < 0.01) (up to 30 g kg⁻¹ DM). The greatest improvements (similar at both temperatures) were for L, N and H of 7.3, 7.2 and 3.9 points for in situ and of 10.6, 11.3 and 6.3 points for cellulase digestibilities, respectively. Water solubility also increased with duration of treatment and level of ammonia (P < 0.01) and was greater at 80°C than at 30°C. Urea treatment significantly improved (P < 0.01) digestibilities and water solubility but the doubling of treatment duration had no influence. The degree of ureolysis was only 50 to 60%. Ammonia and urea treatments considerably increased (P < 0.01) nitrogen content.Treatment with 60 g NH₃ kg⁻¹ DM induced a decrease in ethanol insoluble residue content, which was significant (P < 0.01 for L and N, P < 0.05 for H) at 80°C but not at 30°C, and was greater for L and N than for H (about 12 and 5 points, respectively). This decrease was essentially due to solubilisation of hemicelluloses (− 15%) and uronic acids (− 26%). Thus, at 30°C, the chemical solubility of the cell wall was lower than at 80°C for the same total increase in microbial degradation. This result indicates that other phenomena are involved, such as an increase in cell wall porosity and consequently improved accessibility of cell wall polysaccharides to glycolytic enzymes.     

Effect of temperature and humidity on the development ofPhytoseiulus Persimilis and its ability to regulate populations ofTetranychus urticae [Acarina: Phytoseiidae. Tetranychidae]

The development ofPhytoseiulus persimilis Athias-Henriot and the effectiveness of it as a predator ofTetranychus urticae (Koch) were studied at constant temperatures of 15°, 18°, 21°, 24° and 27°C (humidity fluctuations from 60% to 90% R.H.) and at constant humidities of 40% and 80% R.H. at the temperatures 21° and 27°. Optimal temperature for time of development was 27° (at 60%–85% R.H.). A high reduction in egg vitality was recorded at 40% R. H. and 27% At 21° the egg vitality was only slightly lower at 40% R.H. than at 80% R.H. The predator gave control ofT. urticae at temperatures from 15° to 27° (humidity fluctuation from 60%–90% R.H.), and the most rapid and efficient control was obtained at 27° (60%–85% R.H.). The predator did not give sufficient control ofT. urticae at 27° and 40% R.H. At 21° control ofT. urticae was obtained at both 40% and 80% R.H., but the prey population was reduced faster at 80% R.H. than at 40% R.H.     

Binding of [3H] γ-Aminobutyric Acid and [3H]Muscimol in Purified Rat Brain Synaptic Plasma Membranes and the Effects of Bicuculline

Abstract: The binding of [³H] γ-aminobutyric acid ([³H]GABA) and [³H]muscimol has been studied in purified synaptic plasma membrane (SPM) preparations from rat brain. Scatchard analysis of specific binding (defined as that displaced by 100 μMγ-aminobutyrate) indicated that the binding of both radiolabelled ligands was best described by a two component Langmuir adsorption isotherm. The apparent K_D and B_max values for [³H]GABA at 4°C were K_D1, 20 nM; K_D2,165 nM; B_max1, 0.48 pmol;B_max2, 6.0 pmol. mg^?1; for [³H]muscimol at 4°C they were: K_D1, 1.75 nM; K_D2, 17.5 nM; B_maxl, 0.84 pmol. mg^?1; B_max2, 4.8 pmol.mg^?1; and for [³H]muscimol at 37°C they were: K_D1, 7.0 nM; K_m, 60 nM; B_max], 0.5 pmol-mg^?1; B_max2, 7.2 pmol-mg¹. Under the experimental conditions used, the similar B_milx values for [³H]GABA and [³H]muscimol binding to the SPM preparations suggests that the high- and low-affinity components for the two radiolabeled ligands are identical. The effects of the GAB A antagonist bicuculline on the binding of [³H]muscimol at 4^CC and 37°C were studied. At 4°C, antagonism of muscimol binding appeared to be competitive at the high-affinity site but noncompetitive at the low-affinity site. At 37°C, antagonism was again competitive at the high-affinity site but was of a mixed competitive/noncompetitive nature at the low-affinity site. Assuming that binding to the high-affinity site is associated with the pharmacological actions of bicuculline, the apparent K_D values obtained suggest a pA₂ value of 5.3 against [³H]muscimol at 4°C and 37°C. This figure is in good agreement with several estimates of the potency of bicuculline based on pharmacological measurements. Results from displacement studies using [³H]GABA and [³H]muscimol suggest that [³H]GABA might be a more satisfactory ligand than [³H]muscimol in GABA radioreceptor assays.     

Effect of temperature and ionic environment on the specific binding of 3H(—)sulpiride to membranes from different rat brain regions

Sulpiride is an antipsychotic drug endowed with the properties of a dopamine antagonist. The failure of sulpiride to inhibit neostriatal dopamine stimulated adenylate cyclase activity indicated that this drug is a selective D₂ receptor antagonist. In this study we used a novel synthesized ²H(—)sulpiride with very high specific activity (72 Ci/mol) and characterized the temperature sensitivity of the binding sites labeled by this compound. Kinetic analysis of ³H(—)sulpiride binding in rat striatum showed unstable behavior when incubation was performed at 37 or 30°C. However when experiments were carried out at 15 or 10°C, binding reached a stable steady-state within 10 min. Scatchard analysis of binding isotherms obtained at 10°C showed a 5-fold increase in the maximum number of binding sites and a decrease in K_d values to one-third those obtained at 37°C. Pharmacological characterization of the binding sites labeled by ³H(—)sulpiride at 10°C showed a greater affinity for antagonists but not for agonists than 37°C. Under both experimental condition, ³H(—)sulpiride binding sites were Na⁺ and GTP-sensitive. The temperature sensitive binding phenomenon appeared to be area specific. ³H(—)sulpiride binding sites in tissues other than from striatum were influenced less or not at all by changes in incubation temperature.     

Mass production and storage of Vairimorpha necatrix (protozoa: Microsporida)

Mass production and storage methods were evaluated for maximization of spores of Vairimorpha necatrix, a promising protozoan for microbial control due to its virulence and prolificity in lepidopterous pests. In vivo spore production was at a maximum when 3rd instar Heliothis zea were exposed to 6.6 spores/mm² of artificial diet surface and reared for 15 days. Approximately 1.67 × 10¹⁰ spores/larva were produced, or ca. 1 × 10¹⁰ spores/larva after partial purification of the spores by homogenization of the larvae in water, filtration, and centrifugation. The spores were inactivated by relatively short exposures to several chemicals which were tested to counteract contamination of the diet surface by fungi in the spore inoculum. Spores of V. necatrix were stored at refrigerated and freezing temperatures for up to 2 years and bioassayed periodically with 2nd instar H. zea. Spores lost little infectivity after 23 months at 6°C if they were stored in a purified water suspension plus antibiotic, but they were noninfective after 18 months at 6°C if stored in host tissue. Storage at ?15°C caused little loss of infectivity whether the spores were stored in water and glycerine, in host tissue, or after lyophilization. The spores withstood lyophilization in host cadavers better than in purified water suspension. Samples of a dry V. necatrix-corn meal formulation, which was prepared for field efficacy tests and stored at ?15° and 6°C, were highly infective after 9 months. Large numbers of V. necatrix spores can thus be produced and later made available for microbial control field trials with little loss of infectivity.     

Benzodiazepine receptors: temperature dependence of [3H]flunitrazepam binding.

The characteristics of [³H]flunitrazepam binding to brain specific benzodiazepine receptors were determined at varying temperatures. The rates at which [³H]flunitrazepam associated with and dissociated from benzodiazepine receptors increased with increasing temperatures. The dissociation constant (K_D) also increased with increases in temperature. The (K_D) determined by Scatchard analyses of saturation isotherms showed a similar change with changes in temperature. The maximal binding capacity (Bmax) did not change with changes in temperature. The inhibitory constants of several benzodiazepines to inhibit [³H]flunitrazepam binding to brain were also higher at 37°C than at 0°C, suggesting that the binding affinity of all benzodiazepines to brain benzodiazepine receptors is lower at 37°C than at 0°C. Van't Hoff analysis of [³H]flunitrazepam binding to brain at different temperatures reveals two linear components to this relationship.     

A hemagglutinating antigen from a nuclear polyhedrosis virus of Spodoptera frugiperda

The 100,000g supernatant from an alkaline dissolution of polyhedra isolated from an NPV of Spodoptera frugiperda was found to agglutinate adult chicken erythrocytes in a pH range of 5.5–6.9. Optimal conditions for active hemagglutination and hemagglutination-inhibition, with antisera prepared against polyhedron protein, occurred at pH 5.8 with an incubation temperature of 37°C and a solublization time of 45 min at pH 11.2 Minimum quantities of antigen detectable were at 2–4 μg/ml of protein.     

Growth at 37 degrees C of the EGs strain of the amoeboflagellate Naegleria gruberi containing viruslike particles. I. Nuclear changes

Naegleria gruberi amoebae, EG_s strain, containing viruslike particles (VLP) were grown at temperatures of 21° and 37°C. At 21°C, the amoebae displayed the morphological structures associated with development of the VLP's. At 37°C, however, gross morphological modifications and new structures appeared. When amoebae were at 37°C for less than 12 hr, nuclei were found to have a larger number of VLP's than amoebae at 21°C. Exposure of the amoebae to the higher temperature for 12–24 hr resulted in a scarcity of particles. Large bundles of microtubulelike fibrils were present in the nucleoplasm of amoebae at 37°C, and, in addition, the nuclei showed degenerative modifications. The fibrillar changes were not due to the elevated temperature alone since a substrain of EG_s (=EG_B) not infected with VLP's exhibited no nuclear modifications. It is assumed that the elevated temperature accelerated and enhanced a lytic effect of the VLP's upon the cells.