Evaluation of bioaerosol samplers for the detection and quantification of influenza virus from artificial aerosols and influenza virus-infected ferrets.

BACKGROUND: Bioaerosol sampling devices are necessary for the characterization of infectious bioaerosols emitted by naturally-infected hosts with acute respiratory virus infections. Assessment of these devices under multiple experimental conditions will provide insight for device use. OBJECTIVES: The primary objective of this study was to assess and compare bioaerosol sampling devices using a) an in vitro, environmentally-controlled artificial bioaerosol system at a range of different RH conditions and b) an in vivo bioaerosol system of influenza virus-infected ferrets under controlled environmental conditions. Secondarily, we also sought to examine the impact of NSAIDs on bioaerosol emission in influenza virus-infected ferrets to address its potential as a determinant of bioaerosol emission. METHODS: We examined the performance of low and moderate volume bioaerosol samplers for the collection of viral RNA and infectious influenza virus in vitroand in vivo using artificial bioaerosols and the ferret model of influenza virus infection. The following samplers were tested: the polytetrafluoroethylene filter (PTFE filter), the 2-stage National Institute of Occupational Safety and Health cyclone sampler (NIOSH cyclone sampler) and the 6-stage viable Andersen impactor (Andersen impactor). RESULTS: The PTFE filter and NIOSH cyclone sampler collected similar amounts of viral RNA and infectious virus from artificially-generated aerosols under a range of relative humidities (RH). Using the ferret model, the PTFE filter, NIOSH cyclone sampler and the Andersen impactor collected up to 3.66 log10 copies of RNA/L air, 3.84 log10 copies of RNA/L air and 6.09 log10 copies of RNA/L air respectively at peak recovery. Infectious virus was recovered from the PTFE filter and NIOSH cyclone samplers on the peak day of viral RNA recovery. CONCLUSION: The PTFE filter and NIOSH cyclone sampler are useful for influenza virus RNA and infectious virus collection and may be considered for clinical and environmental settings.

Bioaerosols and Transmission, a Diverse and Growing Community of Practice.

The transmission of infectious microbes via bioaerosols is of significant concern for both human and animal health. However, gaps in our understanding of respiratory pathogen transmission and methodological heterogeneity persist. New developments have enabled progress in this domain, and one of the major turning points has been the recognition that cross-disciplinary collaborations across spheres of human and animal health, microbiology, biophysics, engineering, aerobiology, infection control, public health, occupational health, and industrial hygiene are essential. Collaborative initiatives support advances in topics such as bioaerosol behavior, dispersion models, risk assessment, risk/exposure effects, and mitigation strategies in clinical, experimental, agricultural, and other field settings. There is a need to enhance the knowledge translation for researchers, stakeholders, and private partners to support a growing network of individuals and agencies to achieve common goals to mitigate inter- and intra-species pathogen transmission via bioaerosols.

The Pollution Particulate Concentrator (PoPCon): A platform to investigate the effects of particulate air pollutants on viral infectivity.

Anthropogenic air pollution is ubiquitous in urban areas worldwide. Microorganisms such as bacteria and fungi in addition to other biological matter like endotoxins and spores comingle with particulate matter (PM) air pollutants but have rarely been considered in air pollution research. Microorganisms may be influenced by interactions with ambient particles in matrices such as soil and dust leading to the inhibition or enhancement of viability and environmental stability (e.g. tolerance to variation in seasonality, temperature, humidity, etc.). Similar effects of airborne particles on microbes are plausible; however, to our knowledge the influence of PM on airborne microbes has remained largely unexamined. In the case of microbial agents of communicable disease, such as viruses, the potential for interactions with pollution may have public health implications. Here we describe an experimental platform to study aerosol-aerosol interactions between PM2.5 particulate from urban air and artificially generated viral bioaerosol. Preliminary studies using this platform have revealed interactions between PM2.5 and the enveloped bacteriophage Φ6 that reduce infectivity of the bacteriophage by 44% compared to a control exposed only to HEPA-filtered air. Co-aerosolization and aging of concentrated PM2.5 with Φ6 in combination with ΦX174 (a non-enveloped bacteriophage) showed a similar trend in reduction of Φ6 infectivity but revealed an antithetical enhancement of ΦX174 infectivity compared to control exposures in HEPA-filtered air. Ongoing investigations are needed to understand the nature of interactions between bioaerosols and PM2.5 particles.

Single molecule fluorescence study of the Bacillus thuringiensis toxin Cry1Aa reveals tetramerization.

Pore-forming toxins constitute a class of potent virulence factors that attack their host membrane in a two- or three-step mechanism. After binding to the membrane, often aided by specific receptors, they form pores in the membrane. Pore formation either unfolds a cytolytic activity in itself or provides a pathway to introduce enzymes into the cells that act upon intracellular proteins. The elucidation of the pore-forming mechanism of many of these toxins represents a major research challenge. As the toxins often refold after entering the membrane, their structure in the membrane is unknown, and key questions such as the stoichiometry of individual pores and their mechanism of oligomerization remain unanswered. In this study, we used single subunit counting based on fluorescence spectroscopy to explore the oligomerization process of the Cry1Aa toxin of Bacillus thuringiensis. Purified Cry1Aa toxin molecules labeled at different positions in the pore-forming domain were inserted into supported lipid bilayers, and the photobleaching steps of single fluorophores in the fluorescence time traces were counted to determine the number of subunits of each oligomer. We found that toxin oligomerization is a highly dynamic process that occurs in the membrane and that tetramers represent the final form of the toxins in a lipid bilayer environment.

Rapid topology probing using fluorescence spectroscopy in planar lipid bilayer: the pore-forming mechanism of the toxin Cry1Aa of Bacillus thuringiensis.

Pore-forming toxins, many of which are pathogenic to humans, are highly dynamic proteins that adopt a different conformation in aqueous solution than in the lipid environment of the host membrane. Consequently, their crystal structures obtained in aqueous environment do not reflect the active conformation in the membrane, making it difficult to deduce the molecular determinants responsible for pore formation. To obtain structural information directly in the membrane, we introduce a fluorescence technique to probe the native topology of pore-forming toxins in planar lipid bilayers and follow their movement during pore formation. Using a Förster resonance energy transfer (FRET) approach between site-directedly labeled proteins and an absorbing compound (dipicrylamine) in the membrane, we simultaneously recorded the electrical current and fluorescence emission in horizontal planar lipid bilayers formed in plastic chips. With this system, we mapped the topology of the pore-forming domain of Cry1Aa, a biological pesticide from Bacillus thuringiensis, by determining the location of the loops between its seven α helices. We found that the majority of the toxins initially traverse from the cis to the trans leaflet of the membrane. Comparing the topologies of Cry1Aa in the active and inactive state in order to identify the pore-forming mechanism, we established that only the α3-α4 hairpin translocates through the membrane from the trans to the cis leaflet, whereas all other positions remained constant. As toxins are highly dynamic proteins, populations that differ in conformation might be present simultaneously. To test the presence of different populations, we designed double-FRET experiments, where a single donor interacts with two acceptors with very different kinetics (dipicrylamine and oxonol). Due to the nonlinear response of FRET and the dynamic change of the acceptor distribution, we can deduce the distribution of the acceptors in the membrane from the time course of the donor fluorescence. We found that Cry1Aa is present on both membrane leaflets.

Ca2+-dependent ATP release from A549 cells involves synergistic autocrine stimulation by coreleased uridine nucleotides.

Extracellular ATP is a potent surfactant secretagogue but its origin in the alveolus, its mechanism(s) of release and its regulatory pathways remain unknown. Previously, we showed that hypotonic swelling of alveolar A549 cells induces Ca(2+)-dependent secretion of several adenosine and uridine nucleotides, implicating regulated exocytosis. In this study, we examined sources of Ca(2+) for the elevation of intracellular Ca(2+) concentration ([Ca(2+)](i)) evoked by acute 50% hypotonic stress and the role of autocrine purinergic signalling in Ca(2+)-dependent ATP release. We found that ATP release does not directly involve Ca(2+) influx from extracellular spaces, but depends entirely on Ca(2+) mobilization from intracellular stores. The [Ca(2+)](i) response consisted of slowly rising elevation, representing mobilization from thapsigargin (TG)-insensitive stores and a superimposed rapid spike due to Ca(2+) release from TG-sensitive endoplasmic reticulum (ER) Ca(2+) stores. The latter could be abolished by hydrolysis of extracellular triphospho- and diphosphonucleotides with apyrase; blocking P2Y(2)/P2Y(6) receptors of A549 cells with suramin; blocking UDP receptors (P2Y(6)) with pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid (PPADS); emptying TG-sensitive stores downstream with TG or caffeine in Ca(2+)-free extracellular solution; or blocking the Ca(2+)-release inositol 1,4,5-triphosphate receptor channel of the ER with 2-aminoethyldiphenylborinate. These data demonstrate that the rapid [Ca(2+)](i) spike results from the autocrine stimulation of IP(3)/Ca(2+)-coupled P2Y, predominantly P2Y(6), receptors, accounting for approximately 70% of total Ca(2+)-dependent ATP release evoked by hypotonic shock. Our study reveals a novel paradigm in which stress-induced ATP release from alveolar cells is amplified by the synergistic autocrine/paracrine action of coreleased uridine and adenosine nucleotides. We suggest that a similar mechanism of purinergic signal propagation operates in other cell types.

Membrane reserves and hypotonic cell swelling.

To accommodate expanding volume (V) during hyposmotic swelling, animal cells change their shape and increase surface area (SA) by drawing extra membrane from surface and intracellular reserves. The relative contributions of these processes, sources and extent of membrane reserves are not well defined. In this study, the SA and V of single substrate-attached A549, 16HBE14o(-), CHO and NIH 3T3 cells were evaluated by reconstructing cell three-dimensional topology based on conventional light microscopic images acquired simultaneously from two perpendicular directions. The size of SA reserves was determined by swelling cells in extreme 98% hypotonic (approximately 6 mOsm) solution until membrane rupture; all cell types examined demonstrated surprisingly large membrane reserves and could increase their SA 3.6 +/- 0.2-fold and V 10.7 +/- 1.5-fold. Blocking exocytosis (by N-ethylmaleimide or 10 degrees C) reduced SA and V increases of A549 cells to 1.7 +/- 0.3-fold and 4.4 +/- 0.9-fold, respectively. Interestingly, blocking exocytosis did not affect SA and V changes during moderate swelling in 50% hypotonicity. Thus, mammalian cells accommodate moderate (<2-fold) V increases mainly by shape changes and by drawing membrane from preexisting surface reserves, while significant endomembrane insertion is observed only during extreme swelling. Large membrane reserves may provide a simple mechanism to maintain membrane tension below the lytic level during various cellular processes or acute mechanical perturbations and may explain the difficulty in activating mechanogated channels in mammalian cells.