Comparison of gamma and neutron radiation inactivation of influenza A virus.

Radiation inactivation of viral pathogens has potential application in sterilization and in the manufacture of biological reagents, including the production of non-infectious viral antigens. Viral inactivation by gamma radiation has been extensively investigated, but few direct comparisons to other qualities of radiation have been explored. Experiments were designed to examine direct radiation damage by both gamma photons (gamma) and neutrons (n) while minimizing methodological differences. Frozen samples of influenza A X31/H3N2 and PR8/H1N1 were exposed to gamma and n at doses between 0 and 15.6 kGy. Other experimental parameters, including dose-rate, were not varied. Virus titers were determined by tissue culture infectious dose (TCID(50)) and plaque forming unit (PFU) assays. D(10) values, kGy per log reduction, were calculated from these assays. PR8 D(10) values based on PFU assays were approximately 2 and 5 kGy for gamma and n exposures, respectively, and those based on TCID(50) were approximately 6 and 14 kGy. Similar results were obtained for the A/X31 strain. The data demonstrate that gamma was 2-3-fold more effective than n, with a relative biological effectiveness (RBE) range of 0.43-0.65. These neutron results are likely the first reported for a medically relevant virus. PAGE analysis of viral proteins and RNAs failed to show macromolecular damage. D(10) values were found to be similar to a broad summary of previously reported gamma inactivation values for other virus types. The dependence of the magnitudes of D(10) on titer assay in this study suggests that more than one titer method should be used to determine if complete inactivation has occurred.

Improvements in methods for calculating virus titer estimates from TCID50 and plaque assays.

Calculation of titer estimates and use of titer reduction assays are fundamental approaches used by virologists. Titer assays being biological assays and based on limiting dilution methods require good error control, both methodologically and analytically. The need for good statistical analysis is likely to become even greater as in clinical, manufacturing, as well as the research settings, improved analytical criteria, quality control, and assurance standards are adopted. Furthermore, increasingly, virus titer assays are based on high throughput methods, which generate continuous rather than traditional quantal data. Described here are two different weighted linear regression methods to determine TCID50 and PFU titers from CPE assays. The TCID50 analysis makes use of a generalized least squares approach using continuous colorimetric data. The plaque analysis makes use of weighted least squares forced through the origin using quantal plaque data generated by serial dilutions. Both methods are improvements in titer and error estimation compared to simpler calculation methods. These methods may have greatest value when lack of experimental material or costs of analysis precludes extensive replicate titer determinations but good estimates of titers and/or treatment differences are essential.

Characterization of influenza virus-induced death of J774.1 macrophages.

The mechanism and role of influenza virus (IV)-induced pathogenesis of macrophages during respiratory infection are ill defined. Reported here are findings on IV-induced cytopathic effects (CPEs) for an in vitro experimental system using the murine macrophage cell line J774.1. CPE was elicited by 0.2 or greater multiplicity of infection (m.o.i.). CPEs showed a lag of 6-8 h postinfection and occurred most rapidly between 6 and 12 h. J774.1 cells did not support productive IV replication, but immunofluorescence demonstrated that IV protein synthesis occurred. Light microscopy and DNA staining showed that after death cells had very condensed cytoplasm and nuclei. Cell remnants were surrounded by intact plasma membrane (PM) as demonstrated by exclusion of a membrane-impermeant dye. Time-lapse video microscopy recordings between 6 and 10 h postinfection showed sequential structural changes, including previously undescribed events. Notable changes were a rapid cytokinesis (zeiosis; "cell boiling"), followed by nuclear shrinkage, and an unusual transient blebbing of the PM. DNA fragmentation occurred after 12 h, producing a wide size range. UV-inactivated virus failed to induce CPEs, and CPE was blocked by amantadine. N-Acetylcysteine and pyrrolidine dithiocarbamate, but not other inhibitors of reactive oxygen intermediates, reduced or blocked the CPE. Most changes observed are those attributed to apoptotic processes rather than necrotic cell death. The kinetics and inhibitor effects suggest that IV infection and replication must be initiated to activate CPEs.

Evaluation of triple-band filters for quantitative epifluorescence microscopy.

The performance characteristics of two sets of triple-band epifluorescence filters have been evaluated for use with digitally enhanced fluorescence microscopy. Use of such filters, at most, requires movement of the excitation filter, while the dichroic and emission filters remain fixed, allowing multi-wavelength imaging to be performed on standard microscopes. The dyes appropriate for use with these particular filters include Texas Red (TR), Bodipy (BD), FITC and Cascade Blue (CB), four fluorophores now commonly conjugated to both immunochemical probes and other proteins and lipids of biological interest. Good spectral separation existed for most experimental conditions allowing accurate localization of the different fluorophores during multi-wavelength imaging. Anomalous responses were observed during near-UV excitation at high concentration for some dyes. Scanning spectrofluorometry demonstrated that concentration-dependent spectral shifts occurred, resulting in large increases in near-UV absorbance. Despite the complexity of concentration and dye-interaction effects, quantitative measurements of dye concentration could be made, even in regions of multiple dye co-localization. Therefore, multi-band pass filters are an additional valuable approach for performing quantitative fluorescence microscopic imaging.

Differences in dispersion of influenza virus lipids and proteins during fusion.

Digitally enhanced low-light-level fluorescence video microscopy and immunochemical staining were used to examine influenza virus envelope lipid and protein redistribution during pH-induced fusion. Video microscopy was performed using viruses labeled with either the lipid analogue octadecylrhodamine B (R18) or fluorescein isothiocyanate (FITC) covalently linked to envelope proteins. Viruses were bound to human red blood cells, and the pattern and intensity of fluorescence were monitored for 30 min while cell-virus complexes were perfused with pH 7.4 or 4.8 media at temperatures either above or below 20 degrees C. R18 showed complete redistribution and dequenching by 30 min at all incubation temperatures, confirming reports that viral fusion occurs at subphysiological temperatures. FITC-labeled protein showed spatial redistribution at 28 degrees C but no change at low temperature. Electron microscopy observations of immunochemical staining of viral proteins confirmed both that protein redistribution at 37 degrees C was slower than R18 and the failure of movement within 30 min at 16 degrees C. Video microscopy monitoring of RNA staining by acridine orange of virus-cell complexes showed redistribution to the RBCs at all temperatures but only after low pH-induced fusion. The results are consistent with differential dispersion of viral components into the RBC and the existence of relatively long-lived barriers to diffusion subsequent to fusion pore formation.

Transient domains induced by influenza haemagglutinin during membrane fusion.

During low pH-induced fusion of influenza virus with erythrocytes we have observed differential dispersion of viral lipid and haemagglutinin (HA) into the erythrocyte membrane, and viral RNA into the erythrocyte using fluorescence video microscopy. The movement of both viral lipid and HA from virus to cell was restricted during the initial stages of fusion relative to free diffusion. This indicates the existence of relatively long-lived barriers to diffusion subsequent to fusion pore formation. Fluorescence anisotropy of phospholipid analogues incorporated into the viral membrane decreased when the pH was lowered to levels required for optimum fusion. This indicates that the restricted motion of viral membrane components was not due to rigidification of membrane lipids. The movement of HA from the fusion site was also assessed by photosensitized labelling by means of a fluorescent substrate (NBD-taurine) passing through the band 3 sialoglycoprotein (the erythrocyte anion transporter). We also examined the flow of lipid and aqueous markers during fusion of HA-expressing cells with labelled erythrocytes. During this cell-cell fusion, movement of lipid between fusing membranes begins before the fusion pore is wide enough to allow diffusion of aqueous molecules (M(r) > 500). The data indicate that HA is capable of creating domains in the membrane and controlling continuity of aqueous compartments which are bounded by such domains.

Identification of riboflavin transport by MDCK cells using quantitative fluorescence video microscopy.

MDCK cells, when examined by low-light level video microscopy displayed an endogenous fluorescence with two differing patterns. A low intensity emission which was punctate and associated with cell organelles was observed with emission and excitation conditions generally used to observe either fluorescein (450-500 nm excitation/greater than 510 nm emission) or rhodamine (514 nm excitation/greater than 530 emission) type dyes. A second 5- to 10-fold brighter emission for 450-500 nm excitation was observed, which was unusual in that each cell appeared to be outlined. Evidence obtained from spectroscopy and from using culture media of altered composition supported the conclusion that the water-soluble vitamin riboflavin accumulated in the basolateral spaces and fluid-filled "domes" and was the source of this fluorescent emission. Quantitative measurements showed that exposure to cultures to 10 microM riboflavin resulted in accumulation in domes of 565 +/- 80 microM. The transport rate was calculated to be 189 +/- 30 pmol/min-cm2. One mM probenecid, a known inhibitor of riboflavin transport in vivo, reduced transport to 54% of control, while 10 mM nearly abolished the uptake. The results demonstrate that removal of riboflavin reduces MDCK cell fluorescence to levels compatible with low-light level imaging. Furthermore, these cells actively transport riboflavin and provide a new in vitro model for this process.

Observation of single influenza virus-cell fusion and measurement by fluorescence video microscopy.

We have used intensified video fluorescence microscopy and digital image processing to observe and quantitate influenza virus (A/PR8/34/H1N1) fusion to human erythrocyte membranes. Viruses labeled with the lipid probe octadecylrhodamine B (R18) were seen to undergo fluorescence dequenching and eventual disappearance after exposure to pH levels known to induce virus-cell membrane fusion. Quantitative intensity measurements of single individual particles were possible. From these fluorescence data it has been possible to calculate the fraction of R18 dye molecules transferred from the virus to the cell. The redistribution of the lipid probe upon fusion at pH 5.0 had a t1/2 of 46 s, longer than expected for a free-diffusion model. The R18 loss was approximately twice as fast at pH 5.0 as at pH 5.1. No obvious delay until the start of fluorescence dequenching was observed after the pH changes, suggesting that activation processes are faster than the time resolution, 1-5 s, of the current method.

Mechanism of ion transport by avian salt gland primary cell cultures.

Confluent sheets formed from primary culture of avian salt gland secretory cells exhibit a short-circuit current (Isc) in response to cholinergic and beta-adrenergic stimulation [Lowy, R. J., D. C. Dawson, and S. A. Ernst. Am J. Physiol. 249 (Cell Physiol. 18): C41-C47, 1985]. To establish the ionic basis for the Isc, transmural fluxes of 22Na and 36Cl were measured. Under short-circuit conditions there was little net flux of either ion in the absence of agonists. Addition of carbachol elevated net serosal-to-mucosal Cl flux to 1.71 mu eq.h-1.cm-2, whereas a smaller increase to 0.85 mu eq.h-1.cm-2 occurred with isoproterenol. Neither agonist altered net Na flux. The stimulated Isc accounted for 70% of the net Cl flux induced by carbachol and nearly 100% of that induced by isoproterenol. Replacement of Cl by gluconate or Na by choline abolished (carbachol) or greatly reduced (isoproterenol) the Isc, which could be restored in a dose-dependent fashion by ion restitution. Active ion transport was preferentially inhibited by basal (vs. apical) addition of ouabain, furosemide, or barium. The results provide evidence that cholinergic and beta-adrenergic agonists elicit active transmural Cl secretion. They further suggest that transport is dependent on the Na+-K+-adenosine-triphosphatase, a Na-Cl cotransport process, and a basal K conductance, all features of a secondary active Cl secretory mechanism.

Two K+ channel types, muscarinic agonist-activated and inwardly rectifying, in a Cl- secretory epithelium: the avian salt gland.

Patches of membrane on cells isolated from the nasal salt gland of the domestic duck typically contained two types of K+ channel. One was a large-conductance ("maxi") K+ channel which was activated by intracellular calcium and/or depolarizing membrane voltages, and the other was a smaller-conductance K+ channel which exhibited at least two conductance levels and displayed pronounced inward rectification. Barium blocked both channels, but tetraethylammonium chloride and quinidine selectively blocked the larger K+ channel. The large K+ channel did not appear to open under resting conditions but could be activated by application of the muscarinic agonist, carbachol. The smaller channels were open under resting conditions but the gating was not affected by carbachol. Both of these channels reside in the basolateral membranes of the Cl- secretory cells but they appear to play different roles in the life of the cell.

pH-dependent fusion of reconstituted vesicular stomatitis virus envelopes with Vero cells. Measurement by dequenching of fluorescence.

Reconstituted vesicular stomatitis virus (VSV) envelopes were formed by solubilization of the viral envelope with Triton X-100 followed by removal of detergent by direct addition of SM2 biobeads. We provide direct demonstration of fusion of reconstituted VSV with cells using fluorescent lipid and aqueous probes incorporated into the VSV virosomes during reconstitution. We show a direct comparison of the kinetics and pH profile of fusion with cells between reconstituted VSV and fluorescently labeled intact virus. With this preparation it is now possible to gain additional information about the role of cooperativity in viral protein-mediated fusion, and to permit construction of efficient vehicles for delivery of drugs and other materials into cells.

Activation of vesicular stomatitis virus fusion with cells by pretreatment at low pH.

Fusion of vesicular stomatitis virus (VSV) with Vero cells was measured after exposure of the virus to low pH under a variety of experimental conditions. The method of relief of fluorescence self-quenching of the probe octadecylrhodamine was used to monitor fusion. Incubation of the virus at pH 5.5 prior to binding to cells led to significant enhancement of fusion at the plasma membrane, whereas fusion via the endocytic pathway was inhibited. Fusion of pH 5.5-pretreated VSV showed a similar pH threshold for fusion as nontreated virus, and it was blocked by antibody to VSV G protein. Activation of VSV by pretreatment at low pH was only slightly dependent on temperature. In contrast, when VSV was first bound to target cells and subsequently exposed at 4 degrees C to the low pH, activation of the fusion process did not occur. The pH 5.5-mediated activation of VSV could be reversed by returning the pH to neutral in the absence of target membranes. The low pH pretreatment also led to aggregation of virus; large aggregates could be pelleted by low speed centrifugation and only the effects of the supernatant, which consist of single virions and/or microaggregates, were considered. The data were analyzed in the framework of an allosteric model according to which viral spike glycoproteins undergo a pH-dependent conformational transition to an active (fusion-competent) state. Based on that analysis we conclude that the conformational transition to the active state is rate-limiting for fusion and that the viral spike glycoproteins are fusion-competent only in their protonated form.

Vasoactive intestinal peptide stimulates ion transport in avian salt gland.

Avian salt glands are considered to be under the control of cholinergic nerve fibers. Here we report evidence that vasoactive intestinal peptide (VIP) also regulates ion transport. Nerve fibers stained immunocytochemically with anti-VIP were distributed throughout the tissue within the peritubular connective tissue and were in close proximity to the secretory tubules. VIP applied to primary cultures of the secretory cells elicited active ion transport as assayed by short-circuit current (Isc) analysis. The mucosal-to-serosal positive Isc was produced in a dose-dependent fashion [(EC50) = 3.1 X 10(-9) M], was potentiated by theophylline, and was inhibited by either ouabain or furosemide. This Isc was independent of activation by cholinergic agonists. VIP also increased ouabain-sensitive respiration 14-18% in acutely isolated cells from salt-stressed and unstressed animals. These data demonstrate for the first time that VIP is present in the avian salt gland and can act as a secretagogue by directly affecting the secretory cells. In addition, the results provide evidence for direct control of ion transport by an adenosine 3',5'-cyclic monophosphate-linked neurohormone in both adult unstressed and fully salt-stressed animals.

Beta-adrenergic stimulation of ion transport in primary cultures of avian salt glands.

Adrenergic stimulation of transmural ion transport was identified and characterized in primary cultures of avian salt gland. Adrenergic activation was mediated by beta-receptors since stimulation of the short-circuit current (Isc) was blocked by propranolol but not phentolamine. The Isc's elicited by isoproterenol, epinephrine, and norepinephrine were dose dependent, with respective EC50 values of 1.5 X 10(-8) M, 5.0 X 10(-6) M, and 1.1 X 10(-5) M. The apparent Ki for propranolol inhibition after isoproterenol stimulation was 7.5 X 10(-10) M. 8-Br cyclic AMP (8-Br cAMP) and forskolin-elicited Isc's that were insensitive to propranolol, were potentiated by theophylline, and inhibited by furosemide or ouabain. Isoproterenol also induced an increase in ouabain-sensitive respiration in acutely dispersed cells from salt-stressed juvenile or unstressed adult animals, but not in fully salt-stressed adults. The data indicate that, in addition to the well-established cholinergic receptors, beta-adrenergic receptors can control ion transport in these glands. Furthermore, the results suggest for the first time that an intracellular effector pathway involving cAMP is present.

Muscarinic receptor-stimulated Ca2+ signaling and inositol lipid metabolism in avian salt gland cells.

Activation of muscarinic cholinergic receptors was studied by measuring agonist-stimulated inositol lipid turnover and changes in [Ca2+]i in dissociated salt gland secretory cells. Carbachol stimulation of quin2-loaded cells results in a sustained 4-fold increase in [Ca2+]i, while incorporation of [32P]Pi into phosphatidylinositol (PI) and phosphatidate are similarly increased. [3H]Inositol phosphates, measured in the presence of Li+, increased 13-fold. The stimulated increment in [Ca2+]i required extracellular Ca2+, whereas [3H]inositol phosphate accumulation was independent of external Ca2+. Dose-response curves for carbachol-induced increments in [Ca2+]i, PI labeling, and labeled inositol phosphate release are similar, with EC50 values of 6, 4.5 and 8 microM, respectively. Dissociation constants for atropine vs. the quin2 and phospholipid responses are 0.59 +/- 0.3 nM and 0.48 +/- 0.28 nM, respectively. These cells thus provide a model system for the study of non-exocytotic secretion as a consequence of stimulated inositol lipid turnover.

Primary culture of duck salt gland. II. Neurohormonal stimulation of active transport.

Primary cultures of structurally polarized sheets of avian salt gland secretory cells were mounted in Lucite chambers for transmural electrophysiological analysis. Transmural resistance values increased during the first 3 days of culture to 293 +/- 35 omega X cm2 and then decreased slowly thereafter. There was little short-circuit current (Isc) in the absence of secretagogues. Serosal addition of either carbachol or epinephrine resulted in a Isc consistent with positive charge flow from mucosa to serosa, thus demonstrating that these cell layers were capable of active ion transport in response to either cholinergic or adrenergic neurohormonal stimulation. Serosal ouabain or furosemide abolished the response to either agonist, while theophylline enhanced the response. Receptor specificity for the electrical responses was shown by selective inhibition of carbachol- and epinephrine-induced Isc by atropine and propranolol, respectively. The results demonstrate that these primary epithelial cell cultures are capable of active ion transport and are sensitive to known inhibitors of secretory transport, and suggest that intracellular coupling mechanisms for hormonal control are retained in culture. These cultures should be useful for studying mechanisms of ion secretory transport and their regulatory control.

Primary culture of duck salt gland. I. Morphology of confluent cell layers.

Dissociated avian salt gland secretory cells were maintained in primary culture after plating on hydrated collagen gels. When seeded at 3 X 10(6) cells/cm2, confluent cell sheets formed within 2-3 days, whereas cultures seeded at lower densities formed a complex reticulum of cell aggregates, which remained nonconfluent even after 7 days. Scanning electron microscopy showed that the free surface of 3-day confluent cultures consisted of intermixed convex and flattened cell membranes with prominent junctional boundaries and abundant microvilli. Transmission electron microscopy indicated that these cultures were multilayers of 1-4 cells in thickness. The plasma membranes of the superficial cells were polarized into apical and basolateral regions displaying, respectively, microvilli and interdigitating lateral membrane folds. These membrane domains were separated by shallow occluding junctions, which consisted of both single strands and simple net-like arrays in freeze-fracture images. Underlying epithelial cells retained lateral membrane folds and formed desmosomal contacts with superficial and neighboring cells. These cultures, unlike the intact tissue, allow direct access to the apical and basolateral cell surfaces for electrophysiological analysis of transmural active ion transport.

Isolation and functional characterization of crustacean larval salt gland.

A batch method for isolating viable salt glands from the naupliar brine shrimp (Artemia salina) has been developed. This protocol produces a final preparation consisting of approximately 185 isolated salt glands, representing 1 X 10(4) secretory cells/g wet wt nauplii, with a final purity of 88%. Assays of cell integrity and function indicate good retention of in situ characteristics. Vital dye was excluded by 95% of the cells for at least 24 h. The O2 consumption rate was 22.7 nM O2 X min-1 X mg protein-1 and could be altered predictably by compounds known to affect oxidative phosphorylation and ion transport. The specific activity of the Na+-K+-ATPase in the salt gland, measured here for the first time, was 9.1 mM Pi X h-1 X mg protein-1. This is a substantial proportion of the body total, 17%, as expected for an active ion-transporting epithelium.

Morphology of isolated crustacean larval salt glands.

Larval salt glands isolated from the naupliar brine shrimp (Artemia salina) were examined using light microscopy and scanning and transmission electron microscopy. These methods demonstrated that most cellular and subcellular features of the in vitro organ compared favorably with those seen in vivo. This salt gland measures 130 micron in diameter and is comprised of 50-70 secretory cells, which are of a single epithelial cell type. Characteristic ultrastructural features that are well preserved include apical to basal cell polarity, apical plasma membrane projections, and the extent of the basolateral tubular labyrinth and its association with numerous mitochondria. Some features that have been altered are a decrease in cell-cell contact, separation of septate junctions, and expansion of tubular labyrinth lumens and mitochondrial cristae. Use of this preparation has allowed examination of the salt gland cell's hemocoelic surface for the first time and provided information about the ultrastructure of the tufts formed by the apical plasma membrane.