Repeated transfer of small RNA virus populations leading to balanced fitness with infrequent stochastic drift

The population dynamics of RNA viruses have an important influence on fitness variation and, in consequence, on the adaptative potential and virulence of this ubiquitous group of pathogens. Earlier work with vesicular stomatitis virus showed that large population transfers were reproducibly associated with fitness increases, whereas repeated transfers from plaque to plaque (genetic bottlenecks) lead to losses in fitness. We demonstrate here that repeated five-plaque to five-plaque passage series yield long-term fitness stability, except for occasional stochastic fitness jumps. Repeated five-plaque passages regularly alternating with two consecutive large population transmissions did not cause fitness losses, but did limit the size of fitness gains that would otherwise have occurred. These results underscore the profound effects of bottleneck transmissions in virus evolution.     

Modification of oligosaccharide-lipid synthesis and protein glycosylation in glucose-deprived cells

Incubation of vesicular stomatitis virus-infected glucose-starved baby hamster kidney cells with [³⁵S]methionine results in the synthesis of all viral proteins. However, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and tryptic peptide mapping, the G protein is abnormally glycosylated. Metabolic labeling of the oligosaccharide-lipid precursors with [³H]mannose for 15 min, followed by Chromatographic and enzymatic analysis, indicates that the radiolabeled lipid-linked oligosaccharides are devoid of glucose in contrast to the glucosylated oligosaccharide-lipids synthesized by cells grown in the presence of glucose. Also, in contrast to control cells, examination of the glycopeptide fraction reveals the presence of [³H]mannose-labeled glycopeptides which are resistant to erado-β-N-acetylglucos-aminidase H and are smaller in size than glycopeptides from mature vesicular stomatitis virus. In order to observe these effects, a minimum time of 5 h of glucose deprivation is necessary and the addition of 55 μm glucose or mannose to the medium reverses these effects. These results indicate that vesicular stomatitis virus-infected BHK cells deprived of glucose are unable to glucosylate the oligosaccharide-lipid intermediates and, consequently, are unable to glycosylate the G protein normally.     

Immune response to human influenza virus hemagglutinin expressed in Escherichia coli

Cloned DNA fragments coding for parts of strain WSN (H1N1) influenza virus hemagglutinin (HA) were fused to a bacterial leader DNA derived from the Escherichia coli trp operon. Fusion proteins produced consisted of 190 amino acids of trpLE' protein at the amino terminus, and HA amino acids, either 1-308, 1-396, or 1-548 (complete HA), at the carboxyl terminus. These proteins were expressed at high levels (10-20% of total protein) in E. coli starved for tryptophan. A CNBr fragment (HA1-211) was derived from HA-308. Each of the proteins was purified and used for immunizing mice and rabbits. The antibody produced was shown to bind to (i) the HA fusion proteins, (ii) detergent-treated viral HA, (iii) HA, on intact virions, and (iv) the HA on the surface of cells infected with influenza virus. This shows that the HA fusion proteins expressed in bacteria can elicit antibodies that recognize at least some determinants of the native viral HA, and probably could lead to development of an anti-influenza vaccine.     

Evidence that plasma membrane electrical potential is required for vesicular stomatitis virus infection of MDCK cells: A study using fluorescence measurements through polycarbonate supports

Summary We used fluorescence microscopy of Madin-Darby Canine Kidney (MDCK) cells grown on polycarbonate filters to study a possible link between plasma membrane electrical potential (_pm) and infectivity of vesicular stomatitis virus (VSV). Complete substitution of K⁺ for extracellular Na⁺blocks VSV infection of MDCK cells as well as baby hamster kidney (BHK) cells. When we independently perfused the apical and basal-lateral surfaces of high resistance monolayers, high K⁺ inhibited VSV infection of MDCK cells only when applied to the basal-lateral side; high K⁺ applied apically had no effect on VSV infection. This morphological specificity correlates with a large decrease in _pm of MDCK cells when high K⁺ buffer is perfused across the basal-lateral surface. Depolarization of the plasma membrane by 130 mm basal K⁺ causes a sustained increase of cytosol pH in MDCK cells from 7.3 to 7.5 as reported by the fluorescent dye BCECF. Depolarization also causes a transient increase of cytosol Ca²⁺ from 70 to 300 nm as reported by the dye Fura-2. Neither increase could explain the block of VSV infectivity by plasma membrane depolarization. One alternative hypothesis is that _pm facilitates membrane translocation of viral macromolecules as previously described for colicins, mitochondrial import proteins, and proteins secreted by Escherichia coli.We thank Kenneth Spring for many helpful discussions concerning fluorescence digitized imaging systems, James Russell for his collaboration in the design of our imaging system, Herbert Chase for suggestions on dye loading into MDCK cells, and Manfred Schubert and George Harmison for providing expertise on VSV.     

Monensin blocks endocytosis of vesicular stomatitis virus

Monensin inhibits the infection of mouse cells by Vesicular Stomatitis Virus (VSV). At low drug concentrations (0.5 μM), endocytosis of VSV is inhibited whereas viral binding is unaffected. Monensin may be useful for analyzing the internalization of other viruses as well as soluble ligands.     

Antimetabolic activities of 2-fluoro-L-histidine.

2-Fluoro-L-Histidine inhibits protein synthesis in various cell cultures, as measured by ³H-leucine incorporation. This histidine analog also inhibits the cytopathogenicity of a number of RNA and DNA viruses in primary and continuous cell cultures; it blocks the transformation of normal mouse (MO) cells by murine sarcoma virus, and partially suppresses the release of murine leukemia virus by a continuously infected mouse cell line (JLSV5). In human skin fibroblasts, it reduces the interferon-inducing capacity of poly(I)·poly(C). Inhibition of cell protein synthesis may be the common cause of the various effects. 4-Fluoro-L-histidine is essentially inert in all of the test systems examined.     

Permeability properties of the membrane of vesicular stomatitis virions

Observations of the light-scattering properties of several enveloped viruses indicate that virions (vesicular stomatitis, SV5 and influenza), in common with other membrane systems, are osmotically active, responding to NaCl gradients by swelling in hypo-osmolar solutions and shrinking in hyperosmolar solutions. The permeability barrier responsible for this osmotic response in vesicular stomatitis virions was modified both by protease treatment to remove the viral glycoprotein and by treatment with the polyene antibiotic filipin, an agent known to interact with cholesterol in liposomes and membranes. Filipin altered the kinetic and equilibrium permeability behavior of virions but the extent of leakage of osmotic shocking agent was less than that in lecithin/cholesterol and lecithin/ergosterol liposomes and in ergosterol-containing ciliary membranes. Negative-staining electron microscopy revealed that filipin treatment caused structural changes in the viral membrane. Intact virions exhibited appreciably larger responses to osmotic change than did protease-treated virus particles. Thus, the osmotic barrier in intact vesicular stomatitis virions may not be exclusively lipid in nature.     

口腔雾化药物吸入联合小儿双金清热口服液治疗疱疹性口炎的临床疗效

目的:研究口腔雾化药物吸入联合小儿双金清热口服液治疗疱疹性口炎的临床疗效。方法:选择2012年5月至2014年5月在我院接受治疗的疱疹性口炎患者122例实施研究。根据数字法随机分成观察组及对照组各61例,观察组给予口腔雾化药物吸入以及小儿双金清热口服液进行治疗,对照组单纯给予小儿双金清热口服液进行治疗,对比两组治疗后的疗效、相关康复指标以及治疗后的不良反应。结果:观察组的总有效率是98.36%,显著高于对照组的88.52%,差异有统计学意义(P0.05)。观察组的热退时间和疱疹消退时间以及康复时间均分别显著少于对照组,差异均有统计学意义(均P0.05)。观察组治疗后的总不良反应发生率与对照组相比,差异无统计学意义(P0.05)。结论:利用口腔雾化药物吸入联合小儿双金清热口服液对疱疹性口炎患儿进行治疗,效果较好,安全性更高,值得在临床上推广应用。     

Efficacy of antifungal agents in tissue conditioners in treating candidiasis

Chronic atrophic candidiasis is prevalent in up to 72% of institutionalized geriatric populations and is causally associated with Candida albicans. Topical antifungal treatments are difficult to implement in some geriatric patients due to cognitive impairment, reduced motor dexterity and memory loss. Objective: This in vitro study incorporated antifungal agents into tissue conditioners to investigate the effectiveness of this method of drug delivery. Design: Combinations of nystatin, fluconazole, itraconazole and Coe Soft, Viscogel, Fitt were tested at 1, 3, 5, 7, 9 and 11wt/wt%, with and without sterilized saliva. 6 mm diameter cores were punched in Sabouraud plates pre-grown with standardized C. albicans. Antifungal agents plus tissue conditioner mixtures were injected into each core. Inhibition diameters were measured for 14 days. Results: Cores with only tissue conditioners acted as negative control and showed no significant inhibition activity (ANOVA, p>0.05). Peak activity was between 65 to 89 hours; followed by a plateau. Itraconazole had greater fungicidal activity than fluconazole; while nystatin was found to have the least fungicidal activity (ANOVA. p<0.05). The most effective concentration for nearly all combinations was 5%wt/wt (ANOVA, p<0.05). Specimens with saliva showed greater antifungal activity than those without (t-test. p<0.001). Itraconazole altered the physical properties of Viscogel hence this combination is not recommended for clinical use. Conclusion: The treatment of chronic atrophic candidiasis by incorporation of antifungal drugs into tissue conditioners is efficacious. 5% wt/wt itraconazole mixed with Coe Soft or Fitt is recommended for clinical study where compliance of patient or care giver cannot be relied upon. Peak antifungal activity at 3 days suggests that mixtures prepared for clinical study may be replaced soon after this time for maximum effectiveness.