Amplification and Spread of Viruses in a Growing Plaque

The two-dimensional propagation of viruses through a "lawn" of receptive hosts, commonly called plaque growth, reflects the dynamics of interactions between viruses and host cells. Here we treat the amplification of viruses during plaque growth as a reaction-diffusion system, where interactions among the virus, uninfected host cells, and virus-producing host-virus complexes are accounted for using rates of viral adsorption to and desorption from the host-cell surface, rates of reproduction and release of progeny viruses by lysis of the host, and by the coupling of these reactions with diffusion of free virus within the agar support. Numerical solution of the system shows the development of a traveling wave of reproducing viruses, where the velocity of the wave is governed by the kinetic and diffusion parameters. The model has been applied to predict the propagation velocity of a bacteriophage plaque. Different mechanisms may account for the dependence of this velocity on the host density during early stages of a growing plaque. The model provides a means to explore how changes in the virus-host interactions may be manifest in a growing plaque.     

Evolution of bacteriophage T7 in a growing plaque.

The emergence of mutants during the 10(9)-fold amplification of a bacteriophage was spatially resolved in a growing plaque. When wild-type phage T7 was grown on an Escherichia coli host which expressed an essential early enzyme of the phage infection cycle, the T7 RNA polymerase, mutant phage relying on this enzyme appeared in 10(8) phage replications and outgrew the wild type. Spatial resolution of the selection process was achieved by analyzing stab samples taken along a plaque radius. Different mutants were selected at different rates along different radii of the plaque, based on host range and restriction patterns of the isolates. The mutants deleted up to 11% of their genomes, including the gene for their own RNA polymerase. They gained an advantage over the wild type by replicating more efficiently, as determined by one-step growth cultures.     

Replication of viruses in a growing plaque: a reaction-diffusion model.

An understanding of the viral replication process commonly referred to as "plaque growth" is developed in the context of a reaction-diffusion model. The interactions among three components: the virus, the healthy host, and the infected host are represented using rates of viral adsorption and desorption to the cell surface, replication and release by host lysis, and diffusion. The solution to the full model reveals a maximum in the dependence of the velocity of viral propagation on its equilibrium adsorption constant, suggesting that conditions can be chosen where viruses which adsorb poorly to their hosts will replicate faster in plaques than those which adsorb well. Analytic expressions for the propagation velocity as a function of the kinetic and diffusion parameters are presented for the limiting cases of equilibrated adsorption, slow adsorption, fast adsorption, and large virus yields. Hindered diffusion at high host concentrations must be included for quantitative agreement with experimental data.     

Loading history determines the velocity of actin-network growth

Directional polymerization of actin filaments in branched networks is one of the most powerful force-generating systems in eukaryotic cells. Growth of densely cross-linked actin networks drives cell crawling, intracellular transport of vesicles and organelles, and movement of intracellular pathogens such as Listeria monocytogenes. Using a modified atomic force microscope (AFM), we obtained force-velocity (Fv) measurements of growing actin networks in vitro until network elongation ceased at the stall force. We found that the growth velocity of a branched actin network against increasing forces is load-independent over a wide range of forces before a convex decline to stall. Surprisingly, when force was decreased on a growing network, the velocity increased to a value greater than the previous velocity, such that two or more stable growth velocities can exist at a single load. These results demonstrate that a single Fv relationship does not capture the complete behaviour of this system, unlike other molecular motors in cells, because the growth velocity depends on loading history rather than solely on the instantaneous load.     

Plasmid stability and alpha-amylase production in batch and continuous cultures of Bacillus subtilis TN106[pAT5

Cell growth and enzyme (alpha-amylase) production characteristics of Bacillus subtilis TN106 containing the recombinant plasmid pAT5 are investigated in batch and continuous cultures using a defined medium with glucose as the limiting nutrient. The batch culture studies demonstrate that the recombinant plasmid, reported earlier(1) to be stably maintained in the host, suffers from segregational and structural instabilities. The structural instability of this strain occurred during culture storage and can be eliminated in bioreactor experiments by using a modified inoculum preparation procedure. Such elimination allows an unbiased investigation of segregational instability via continuous culture studies. Such studies conducted with this fast growing microorganism, in the absence of antibiotic selection pressure, indicate a very efficient glucose utilization (very low residual glucose concentrations) over a wide range of dilution rates (0.16 h(-1) - 0.94 h(-1)). The nearly time-invariant and low residual glucose concentrations at each such dilution rate enable convenient estimation of growth parameters of the host and recombinant cells and frequency of segregational instability from transients in the resulting mixed cultures. The specific alpha-amylase activity exhibits an inverse relationship to the specific growth rate of recombinant cells. The growth of recombinant cells is not affected by the presence of antibiotic (kanamycin). The growth advantage of host cells over recombinant cells diminishes with increasing dilution rate.     

Flotation characteristics of cyanobacterium Anabaena flos-aquae for gas vesicle production

Cyanobacterium Anabaena flos-aquae was cultivated in photobioreactors for production of intracellular gas vesicles (GVs), as potential oxygen microcarriers. Natural flotation of the buoyant culture was investigated as a potential means of cell harvesting, because filtration and centrifugation tended to destroy the vesicles. Best flotation was found with actively growing culture and when conducted in the dark. The flotation-related cell properties, including the specific GV content, vesicle-collapsed filament density, and intracellular carbohydrate content, were measured to understand the phenomena. During the batch culture, the specific GV content remained relatively constant at 370 microL/(g dry cells) but the filament density (ranging 1.02 to 1.08 g/cm3) showed a decrease-then-increase profile. The increase began when the growth slowed down because of the reduced light availability at high cell concentrations. The dark flotation was studied with both actively growing (mu approximately 0.2 day-1) and stationary-phase cultures. The specific GV content of the stationary-phase culture remained relatively constant while that of the growing culture increased slightly. The intracellular carbohydrate content of the growing culture decreased much faster and more significantly, from 57 to 10 mg/(g dry cells) in 相似文献   

Effects of pyrrol-carboxylic acid derivatives on the growth of coliphages.

Effects of 14 pyrrol-carboxylic acid derivatives and analogues (PY-compounds) on the growth of coliphage MS2 using E. coli E102 (Hfr) as the host were measured by the agar double-layer method. Enlargements of plaque size were observed with 7 PY-compounds but increase in plaque numbers was not induced. These enlargements of plaque size were specific to RNA coliphages MS2, GA and qbeta and not found with DNA coliphages delta AC and T4. Furthermore, the interaction between PY-compound PY-10 and the coliphage MS2 was dependent on the host bacterium (indicator strain). When E102 (Hfr) was used, the enlargement was marked, in the case of substrain W1895 (Hfr) it was less, while in the case of substrain W6 (F+) it was undetectable. The one-step growth of the phage MS2 and the production of intracellular phage MS2 were little affected by the PY-compound PY-10. However, the rate of one-step growth was increased in the early stage after infection. Accordingly, the enlargements of plaque size by the PY-compounds might be correlated with an increase in rate of release of phage particles.     

CELL CYCLE DEPENDENT VIRUS PRODUCTION IN MARINE PHYTOPLANKTON1

In this study we investigated virus production in two marine phytoplankton species and how it relates to the host's cell cycle. Phaeocystis pouchetii (Hariot) Lagerheim and Pyramimonas orientalis McFadden, Hill & Wetherby, growing synchronously in batch cultures, were infected with their respective viruses (PpV and PoV) at four different stages in the cell cycle and the production of free virus was then measured for 30 h. The virus production in P. orientalis infected with PoV depended on the time of infection, whereas no such relation was found for P. pouchetii infected with PpV. The P. orientalis cultures infected at the end of the dark period and at the beginning of the light period produced three times more virus than those infected in the middle of the light period and eight times more virus than those infected at the beginning of the dark period. The latent periods for PpV and PoV were 12–14 h and 18–20 h, respectively, and in both cases were independent of the host cell cycle. The differences in virus production may be attributed to light or cell cycle dependent regulation of host infection, metabolism, or burst size. Regardless of the mechanism, these differences may be related to differences in the ecological strategies of the hosts and their ability to form blooms.     

Initial phase of infection with Tyzzer's organism in cultured mouse hepatocytes

The initial phase of infection with Tyzzer's organisms in cultured mouse hepatocytes was observed using indirect immunofluorescence (IF) and a plaque assay. The organisms adhered poorly to aldehyde- or acetone-fixed cells, but once adhered to host cells, whether methanol-fixed or unfixed, they were not removed by methanol or acetone. By the IF as well as the plaque assay, both of which discriminated intra- and extracellularly located organisms, the number of cell-associated organisms increased linearly up to 3 h post-inoculation. The number of intra-cellular organisms increased rapidly in the first 1 h, followed by linear increase at a much lower rate. Similar results were obtained by the plaque assay.     

Virucidal effect of peppermint oil on the enveloped viruses herpes simplex virus type 1 and type 2 in vitro

The virucidal effect of peppermint oil, the essential oil of Mentha piperita, against herpes simplex virus was examined. The inhibitory activity against herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2) was tested in vitro on RC-37 cells using a plaque reduction assay. The 50% inhibitory concentration (IC50) of peppermint oil for herpes simplex virus plaque formation was determined at 0.002% and 0.0008% for HSV-1 and HSV-2, respectively. Peppermint oil exhibited high levels of virucidal activity against HSV-1 and HSV-2 in viral suspension tests. At noncytotoxic concentrations of the oil, plaque formation was significantly reduced by 82% and 92% for HSV-1 and HSV-2, respectively. Higher concentrations of peppermint oil reduced viral titers of both herpesviruses by more than 90%. A clearly time-dependent activity could be demonstrated, after 3 h of incubation of herpes simplex virus with peppermint oil an antiviral activity of about 99% could be demonstrated. In order to determine the mode of antiviral action of the essential oil, peppermint oil was added at different times to the cells or viruses during infection. Both herpesviruses were significantly inhibited when herpes simplex virus was pretreated with the essential oil prior to adsorption. These results indicate that peppermint oil affected the virus before adsorption, but not after penetration into the host cell. Thus this essential oil is capable to exert a direct virucidal effect on HSV. Peppermint oil is also active against an acyclovir resistant strain of HSV-1 (HSV-1-ACV(res)), plaque formation was significantly reduced by 99%. Considering the lipophilic nature of the oil which enables it to penetrate the skin, peppermint oil might be suitable for topical therapeutic use as virucidal agent in recurrent herpes infection.     

Increase in nitrate uptake by soybean plants during interruption of the dark period with low intensity light

Diurnal patterns of net NO⁻₃ uptake by nonnodulated soybean [ Glycine max (L.) Merr. cv. Ransom] plants growing in flowing hydroponic culture at 26 and 16°C root temperatures were measured at hourly intervals during alternate days of a 12-day growth period. Ion chromatography was used to determine removal of NO⁻₃ from the culture solution. Day and night periods of 9 and 15 h were used during growth. The night period included two 6-h dark periods and an intervening 3-h period of night interruption by incandescent lamps to effect a long-day photoperiod and repress floral initiation. At both root temperatures, the average specific rates of NO⁻₃ uptake were twice as great during the night interruption period as during the day period; they were greater during the day period than during the dark periods; and they were greater during the dark period immediately following the day period than during the later dark period that followed the night interruption. While these average patterns were repetitious among days, measured rates of uptake varied hourly and included intervals of net efflux scattered through the day period and more frequently through the 2 dark periods. Root temperature did not affect the average daily specific rates of uptake or the qualitative relationships among day, dark and night interruption periods of the diurnal cycle.     

Allocation of photoassimilated carbon into major algal metabolite fractions: Variation between two diatom species isolated from the Weddell Sea (Antarctica)

Distribution of photoassimilated carbon into major metabolite classes differed between two Antarctic diatom species, Nitzschia curta and a small unicellular Chaetoceros sp.. Time course uptake studies (over 54 h) revealed that ¹⁴C allocation appeared to be equilibrated after approximately 8 h at light saturated photosynthesis. During short term dark periods (6 h), polysaccharides as well as low-molecular-weight compounds were catabolised to sustain protein synthesis in the dark, whilst lipid reserves were not mobilised for this process. Experiments with these two species were conducted at 0 and -1.5°C, although no difference in the distribution of radiolabel was measured between the two temperatures. It is hypothesised that under near-optimal conditions fast growing species are characterised by a high carbon turnover associated with a rapid flow of newly assimilated carbon into polymeric compound classes. On the other hand, slower growing species (such as N. curta) may store a significant amount of surplus carbon in the low-molecular-weight metabolite fraction. Species specific preferences were observed when comparing the accumulation of radiolabel into the lipid pools.     

Effect of hyperprolactinemia on luteinizing hormone and prolactin secretion assessed using the reverse hemolytic plaque assay

Hyperprolactinemia (hyperPRL) frequently suppresses luteinizing hormone (LH) and endogenous rat prolactin (rPRL) secretion under a variety of experimental circumstances. Several lines of evidence suggest that elevated prolactin (PRL) may act at the hypothalamic-pituitary axis to inhibit pituitary hormone secretion. The goal of this study was to determine whether hyperPRL, achieved by administration of ovine PRL (oPRL), influences LH and rPRL secretion as assessed by the reverse hemolytic plaque assay. Young Sprague-Dawley rats were ovariectomized on Day 0 and were treated with oPRL (4 mg/kg body weight, 3 times/day) beginning at 0900 h on Day 4. They were killed at 1000 h on Day 6, anterior pituitaries were collected, and cells were dispersed and prepared for the reverse hemolytic plaque assay. We analyzed mean plaque area by using a computerized image analysis system and determined the percentage of plaque-forming cells by counting the number of plaques compared to the total number of cells. HyperPRL decreases the percentage of LH plaque-forming cells under basal conditions. Although the mean LH plaque area was the same in vehicle-treated and oPRL-treated rats under basal and gonadotropin-releasing hormone-stimulated conditions, hyperPRL altered the frequency distribution of different-sized plaques under basal conditions. It appears that hyperPRL shifts the distribution of different-sized plaques such that there are more small plaques and no plaques of the largest size classes. Basal and thyrotropin-releasing hormone-induced rPRL release from single lactotropes, as measured by mean plaque area and the percentage of plaque-forming cells, is lower in lactotropes from hyperPRL rats than in controls after 1 h, but not 2 h, of incubation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of growth and photosynthesis by Oscillatoria agardhii grown with a light/dark cycle

Abstract The cyanobacterium Oscillatoria agardhii was grown in turbidostat cultures with the light energy supply in either the continuous mode or in the pulsed mode (8/16 h light/dark (L/D) cycle). The light irradiance value used was sufficient to allow the maximal growth rate to be attained, when supplied continuously. Adaptation of O. agardhii to the L/D cycle was characterized by an increase in pigment content and photosynthetic performance, accompanied by a decrease in growth rate. This mode of adaptation resembled the adaptation of O. agardhii to continuous low light intensities. It is suggested that in this case the L/D cycle provokes this adaptation in order to allow the cells to accumulate carbohydrate rapidly during the light period. This was attributed to the storage of polyglucose, which served as a carbon and energy source for growth in the dark. The utilization of polyglucose in the dark was able to sustain the synthesis of all other cell components at the same rate as when cells were growing in the light. The growth yield in the dark, whilst metabolizing internally stored polyglucose, was 0.52 g cell C/g polyglucose C, or 0.62 g cell dry weight/g polyglucose. Although in the pulsed mode there is a 66% loss in light irradiance per 24 h when compared with a continuous light regime, the growth rate of the cyanobacteria grown in the pulsed mode was only 35% lower than the growth rate of a culture grown in continuous light. This can be explained by a high growth yield in the dark and by increased CO₂ fixation rates in the light of cells grown in the pulsed mode.     

Physiological characterisation of Colletotrichum gloeosporioides,the incitant of anthracnose disease of noni in India

Ten isolates of Colletotrichum gloeosporioides were collected from different noni growing areas of Tamil Nadu, Karnataka and Kerala in India and their pathogenicity was proved under glass house conditions. Effect of different pH levels, temperature, light intensity and media were tested against the growth of C. gloeosporioides under in vitro. The results indicated that the growth of C. gloeosporioides was maximum in pH range of 6.50–7.00 and temperature range of 25–30°C. Exposure of the fungus to alternate cycles of 12 h light and 12 h darkness resulted in the maximum mycelial growth of C. gloeosporioides compared to the 24 h exposure to continuous light and 24 h exposure to continuous dark. Among the different media tested, host leaf extract medium supported significantly the maximum growth of all the 10 isolates of C. gloeosporioides followed by potato dextrose agar. Further, the strains were found to vary morphologically between the isolates under the study.     

Simultaneous Phytochrome-controlled Promotion and Inhibition of Arginine Decarboxylase Activity in Buds and Epicotyls of Etiolated Peas

The specific activity of arginine decarboxylase (ADC; l-arginine carboxylase; EC 4.1.1.19) rises steadily over an 8 hour experimental period in the growing buds and subapical epicotyl internodes of 6-day-old totally etiolated pea seedlings. Treatment with red light (R) completely annuls this rise in epicotyls but increases it in buds, thus paralleling the opposite effects of R on the growth of these two organs. Far red light (FR) reverses both effects of R on ADC and is, in turn, reversed by R, indicating phytochrome control. Effects in both organs are clearly seen within 2 hours. By 6 hours after R, the post-irradiation rise in ADC specific activity in buds is 3 times greater than that of the dark controls. Over the same period, ADC specific activity in epicotyls is inhibited by 56% relative to dark controls, reflecting zero net change after R and a continued rise in the dark. Cycloheximide inhibits the rise in ADC activity in both rapidly growing organs (epicotyls in dark and buds after R) but is without effect in both slower growing organs. Actinomycin D inhibits only in dark grown epicotyls, whereas chloramphenicol produces no inhibition in any system tested.ADC is the first enzyme to show a two-way, organ-specific response to phytochrome conversion from Pr to Pfr. This finding is discussed in relation to the growing evidence that polyamines formed from arginine may be important growth regulators in plants, as well as in microbial and animal cells.     

Temperature inducible protochlorophyllide reduction in darkness in a pigment mutant of Scenedesmus obliquus

Accumulation of chlorophyll and protochlorophyllide (PChlide) was followed during beterotrophic growth of the pigment mutant C-2A' of Scenedesmus obliquus L. in the darkness at 30 and 20°C. At 30°C the cells remained yellow with accumulation of protochlorophyllide, whereas they became green at 20°C with only traces of protochlorophyllide. The capacity of mutant cells to reduce PChlide to chlorophyllide (Chlide) in the dark with or without addition of 5-aminolevulinic acid as measured in isolated membranes, was high in cells grown at 20°C but negligible at 30°C. The high capacity to reduce PChlide created in cells growing at 20°C was only slightly diminished by exposure of cells to 38°C for 3 h. Mechanisms of temperature-sensitive chlorosis in algae and higher plants are discussed in relation to the results with pigment mutant C-2A' of Scenedesmus obliquus . It is assumed that either an activator of NADPH protochlorophyllide oxidoreductase (EC 1.6.99.1) or a different enzyme system can be activated by lower temperature as by light.     

Computer simulations of atherosclerotic plaque growth in coronary arteries

A three dimensional mathematical model with a linear plaque growth function was developed to investigate the geometrical adaptation of atherosclerotic plaques in coronary arteries and study the influences of flow wall shear stress (WSS), blood viscosity and the inlet flow rate on the growth of atherosclerotic plaques using computational plaque growth simulations. The simulation results indicated that the plaque wall thickness at the neck of the stenosis increased at a decreasing rate in the atherosclerosis progression. The simulation results also showed a strong dependence of the plaque wall thickness increase on the blood viscosity and the inlet flow rate. The progression rate in a coronary artery was lower with a higher inlet velocity flow rate and higher with a smaller value of the blood viscosity.