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INTRODUCTION: Nosocomial transmission of SARS-CoV-2 has been a major feature of the COVID-19 pandemic. Evidence suggests patients can auto-emit aerosols containing viable viruses, these aerosols could be further propagated when patients undergo certain treatments including continuous positive airway pressure (PAP) therapy. Our aim was to assess i) the degree of viable virus propagated from PAP circuit mask leak, ii) the efficacy of a ventilated plastic canopy to mitigate virus propagation. METHOD(S): Bacteriophage PhiX174 (108 copies.mL-1) was nebulised into a custom PAP circuit. Mask leak was systematically varied at the mask interface. Plates containing Escherichia coli host quantified viable virus (via plaque forming unit) settling on surfaces around the room. The efficacy of a low-cost ventilated headboard created from a tarpaulin hood and a high efficiency particulate air (HEPA) filter was tested. RESULT(S): Mask leak was associated with virus contamination in a dose-dependent manner (chi2=58.24, df=4, p<0.001). Moderate mask leak (>=21 L.min-1) was associated with virus counts equivalent to using PAP with a vented mask. The highest frequency of viruses was detected on surfaces 1 m away, however, viable viruses were recorded up to 3.86 m from the source. A plastic hood with HEPA filtration significantly reduced viable viruses on all plates. HEPA exchange rates >=170 m3.hr-1 eradicated all evidence of virus contamination. CONCLUSION(S): Mask leak from PAP may be a major source of environmental contamination and nosocomial spread of infectious respiratory diseases. Subclinical mask leak levels should be treated as an infectious risk. Low-cost patient hoods with HEPA filtration are an effective countermeasure.Copyright ©ERS 2020.

INTRODUCTION: Nosocomial transmission of SARS-CoV-2 has been a major feature of the COVID-19 pandemic. Evidence suggests patients can auto-emit aerosols containing viable viruses, these aerosols could be further propagated when patients undergo certain treatments including continuous positive airway pressure (PAP) therapy. Our aim was to assess i) the degree of viable virus propagated from PAP circuit mask leak, ii) the efficacy of a ventilated plastic canopy to mitigate virus propagation. METHOD(S): Bacteriophage PhiX174 (108 copies.mL-1) was nebulised into a custom PAP circuit. Mask leak was systematically varied at the mask interface. Plates containing Escherichia coli host quantified viable virus (via plaque forming unit) settling on surfaces around the room. The efficacy of a low-cost ventilated headboard created from a tarpaulin hood and a high efficiency particulate air (HEPA) filter was tested. RESULT(S): Mask leak was associated with virus contamination in a dose-dependent manner (chi2=58.24, df=4, p<0.001). Moderate mask leak (>=21 L.min-1) was associated with virus counts equivalent to using PAP with a vented mask. The highest frequency of viruses was detected on surfaces 1 m away, however, viable viruses were recorded up to 3.86 m from the source. A plastic hood with HEPA filtration significantly reduced viable viruses on all plates. HEPA exchange rates >=170 m3.hr-1 eradicated all evidence of virus contamination. CONCLUSION(S): Mask leak from PAP may be a major source of environmental contamination and nosocomial spread of infectious respiratory diseases. Subclinical mask leak levels should be treated as an infectious risk. Low-cost patient hoods with HEPA filtration are an effective countermeasure.Copyright ©ERS 2020.

Viruses are a significant threat to both human health and the economy, and there is an urgent need for novel anti-viral drugs and vaccines. High-resolution viral structures inform our understanding of the virosphere, and inspire novel therapies. Here we present a method of obtaining such structural information that avoids potentially disruptive handling, by collecting diffraction data from intact infected cells. We identify a suitable combination of cell type and virus to accumulate particles in the cells, establish a suitable time point where most cells contain virus condensates and use electron microscopy to demonstrate that these are ordered crystalline arrays of empty capsids. We then use an X-ray free electron laser to provide extremely bright illumination of sub-micron intracellular condensates of bacteriophage phiX174 inside living Escherichia coli at room temperature. We have been able to collect low resolution diffraction data. Despite the limited resolution and completeness of these initial data, due to a far from optimal experimental setup, we have used novel methodology to determine a putative space group, unit cell dimensions, particle packing and likely maturation state of the particles.

Viruses are a significant threat to both human health and the economy, and there is an urgent need for novel anti-viral drugs and vaccines. High-resolution viral structures inform our understanding of the virosphere, and inspire novel therapies. Here we present a method of obtaining such structural information that avoids potentially disruptive handling, by collecting diffraction data from intact infected cells. We identify a suitable combination of cell type and virus to accumulate particles in the cells, establish a suitable time point where most cells contain virus condensates and use electron microscopy to demonstrate that these are ordered crystalline arrays of empty capsids. We then use an X-ray free electron laser to provide extremely bright illumination of sub-micron intracellular condensates of bacteriophage phiX174 inside living Escherichia coli at room temperature. We have been able to collect low resolution diffraction data. Despite the limited resolution and completeness of these initial data, due to a far from optimal experimental setup, we have used novel methodology to determine a putative space group, unit cell dimensions, particle packing and likely maturation state of the particles.

The mechanism of DNA ejection, viral assembly and evolution are related to the structure of bacteriophage phi X174. The F protein forms a T = 1 capsid whose major folding motif is the eight-stranded antiparallel beta barrel found in many other icosahedral viruses. Groups of 5 G proteins form 12 dominating spikes that enclose a hydrophilic channel containing some diffuse electron density. Each G protein is a tight beta barrel with its strands running radially outwards and with a topology similar to that of the F protein. The 12 'pilot' H proteins per virion may be partially located in the putative ion channel. The small, basic J protein is associated with the DNA and is situated in an interior cleft of the F protein. Tentatively, there are three regions of partially ordered DNA structure

The three-dimensional structure of bacteriophage phi X174 (phi X174) was determined to approximately 2.6 nm resolution from images of frozen-hydrated 114 S particles. The outer surface of phi X174 is characterized by several prominent features: (i) 12 mushroom-shaped caps (approximately 7.1 nm wide x 3.8 nm high) are situated at each of the vertices of the icosahedral virion and extend to a maximum radius of 16.8 nm; (ii) a "collar" of density surrounds the base of each apical cap; and (iii) 20 conical protrusions (approximately 2.3 nm high) lie along the three-fold symmetry axes. The caps have a pentagonal morphology composed of five globular "subunits" and appear to be loosely connected to the underlying capsid. The distribution of the four gene products present in virions (60 copies each of gpF, gpG, and gpJ, and 12 copies of gpH), and the single-stranded DNA (ssDNA) genome cannot be directly discerned in the reconstructed density map, although plausible assignments can be made on the basis of solvent-excluded volume estimates and previous biochemical data. Thus, gpG accounts for most of the mass in the caps; gpH, a presumed cap protein, cannot be identified in part due to the symmetry-averaging procedures, but may be partially located within the interior of the capsid; and gpF and gpJ make up the remainder of the capsid. The genome appears to be less densely packaged inside the capsid compared to many dsDNA viruses whose nucleic acid is arranged in a liquid-crystalline state.

The mechanism of DNA ejection, viral assembly and evolution are related to the structure of bacteriophage phi X174. The F protein forms a T = 1 capsid whose major folding motif is the eight-stranded antiparallel beta barrel found in many other icosahedral viruses. Groups of 5 G proteins form 12 dominating spikes that enclose a hydrophilic channel containing some diffuse electron density. Each G protein is a tight beta barrel with its strands running radially outwards and with a topology similar to that of the F protein. The 12 'pilot' H proteins per virion may be partially located in the putative ion channel. The small, basic J protein is associated with the DNA and is situated in an interior cleft of the F protein. Tentatively, there are three regions of partially ordered DNA structure

The three-dimensional structure of bacteriophage phi X174 (phi X174) was determined to approximately 2.6 nm resolution from images of frozen-hydrated 114 S particles. The outer surface of phi X174 is characterized by several prominent features: (i) 12 mushroom-shaped caps (approximately 7.1 nm wide x 3.8 nm high) are situated at each of the vertices of the icosahedral virion and extend to a maximum radius of 16.8 nm; (ii) a "collar" of density surrounds the base of each apical cap; and (iii) 20 conical protrusions (approximately 2.3 nm high) lie along the three-fold symmetry axes. The caps have a pentagonal morphology composed of five globular "subunits" and appear to be loosely connected to the underlying capsid. The distribution of the four gene products present in virions (60 copies each of gpF, gpG, and gpJ, and 12 copies of gpH), and the single-stranded DNA (ssDNA) genome cannot be directly discerned in the reconstructed density map, although plausible assignments can be made on the basis of solvent-excluded volume estimates and previous biochemical data. Thus, gpG accounts for most of the mass in the caps; gpH, a presumed cap protein, cannot be identified in part due to the symmetry-averaging procedures, but may be partially located within the interior of the capsid; and gpF and gpJ make up the remainder of the capsid. The genome appears to be less densely packaged inside the capsid compared to many dsDNA viruses whose nucleic acid is arranged in a liquid-crystalline state.