BackgroundThe majority of influenza transmission occurs in homes, schools and workplaces, where many frequently touched communal items are situated. However the importance of transmission via fomites is unclear since few data exist on the survival of virus on commonly touched surfaces. We therefore measured the viability over time of two H1N1 influenza strains applied to a variety of materials commonly found in households and workplaces.Methodology and Principal FindingsInfluenza A/PuertoRico/8/34 (PR8) or A/Cambridge/AHO4/2009 (pandemic H1N1) viruses were inoculated onto a wide range of surfaces used in home and work environments, then sampled at set times following incubation at stabilised temperature and humidity. Virus genome was measured by RT-PCR; plaque assay (for PR8) or fluorescent focus formation (for pandemic H1N1) was used to assess the survival of viable virus.Conclusions/SignificanceThe genome of either virus could be detected on most surfaces 24 h after application with relatively little drop in copy number, with the exception of unsealed wood surfaces. In contrast, virus viability dropped much more rapidly. Live virus was recovered from most surfaces tested four hours after application and from some non-porous materials after nine hours, but had fallen below the level of detection from all surfaces at 24 h. We conclude that influenza A transmission via fomites is possible but unlikely to occur for long periods after surface contamination (unless re-inoculation occurs). In situations involving a high probability of influenza transmission, our data suggest a hierarchy of priorities for surface decontamination in the multi-surface environments of home and hospitals.

Purpose The aim of this work was to develop a quantitative, flow cytometric method for tracking the endolysosomal escape of a fluorescently labelled saporin toxin. Methods Flow cytometric measurements of fluorescent pulse width and height were used to track the endocytic uptake into Daudi cells of a fluorescently labelled saporin toxin and the saporin based immunotoxin, OKT10-SAP. Subsequently, measurement of changes in pulse width were used to investigate the effect of a triterpenoid saponin on the endolysosomal escape of internalised toxin into the cytosol. Live cell confocal microscopy was used to validate the flow cytometry data. Results Increased endolysosomal escape of saporin and OKT10-SAP was observed by confocal microscopy in cells treated with saponin. Fluorescent pulse width measurements were also able to detect and quantify escape more sensitively than confocal microscopy. Saponin induced endolysosomal escape could be abrogated by treatment with chloroquine, an inhibitor of endolysosomal acidification. Chloroquine abrogation of escape was also mirrored by a concomitant abrogation of cytotoxicity.Conclusions Poor endolysosomal escape is often a rate limiting step for the cytosolic delivery of protein toxins and other macromolecules. Pulse width analysis offers a simple method to semi-quantify the endolysosomal escape of this and similar molecules into the cytosol.

Triterpenoid saponins from Saponinum album (SA) significantly augment the cytotoxicity of saporin-based immunotoxins but the mechanism of augmentation is not fully understood. We investigated the effects of six small molecule pharmacological agents, which interfere with endocytic and other processes, on SA-mediated augmentation of saporin and saporin-based immunotoxins (ITs) directed against CD7, CD19, CD22 and CD38 on human lymphoma and leukaemia cell lines. Inhibition of clathrin-mediated endocytosis or endosomal acidification abolished the SA augmentation of saporin and of all four immunotoxins tested but the cytotoxicity of each IT or saporin alone was largely unaffected. The data support the hypothesis that endocytic processes are involved in the augmentative action of SA for saporin ITs targeted against a range of antigens expressed by leukaemia and lymphoma cells. In addition, the reactive oxygen species (ROS) scavenger tiron reduced the cytotoxicity of BU12-SAP and OKT10-SAP but had no effect on 4KB128-SAP or saporin cytotoxicity. Tiron also had no effect on SA-mediated augmentation of the saporin-based ITs or unconjugated saporin. These results suggest that ROS are not involved in the augmentation of saporin ITs and that ROS induction is target antigen-dependent and not directly due to the cytotoxic action of the toxin moiety.

Triterpenoid saponins augment the cytotoxicity of saporin based immunotoxins. It is postulated that this results from a saponin-mediated increase in the endolysosomal escape of the toxin to the cytosol, but this remains to be confirmed. To address this issue, we used a number of pharmacological inhibitors of endocytic processes as probes to investigate the role played by saponin in the endolysosomal escape of fluorescently labeled saporin and a saporin based immunotoxin targeted against CD38 on human lymphoma and leukemia cell lines. Endolysosomal escape of the toxin was measured by flow cytometric pulse shape analysis. These results were compared to the effects of the various inhibitors on the saponin-mediated augmentation of toxin and immunotoxin cytotoxicity. Inhibitors of clathrin-mediated endocytosis, micropinocytosis, and endosomal acidification abrogated the saponin-induced increase in the endolysosomal escape of the toxin into the cytosol, suggesting that these processes may be involved in the internalization of saponin to the same endolysosomal vesicle as the toxin. Alternatively, these processes may play a direct role in the mechanism by which saponin promotes toxin escape from the endolysosomal compartment to the cytosol. Correlation with the effects of these inhibitors on the augmentation of cytotoxicity provides additional evidence that endolysosomal escape is involved in driving augmentation.