Efficacy of single pass UVC air treatment for the inactivation of coronavirus, MS2 coliphage and Staphylococcus aureus bioaerosols.

There is strong evidence that SARS-CoV-2 is spread predominantly by airborne transmission, with high viral loads released into the air as respiratory droplets and aerosols from the infected subject. The spread and persistence of SARS-CoV-2 in diverse indoor environments reinforces the urgent need to supplement distancing and PPE based approaches with effective engineering measures for microbial decontamination - thereby addressing the significant risk posed by aerosols. We hypothesized that a portable, single-pass UVC air treatment device (air flow 1254 L/min) could effectively inactivate bioaerosols containing bacterial and viral indicator organisms, and coronavirus without reliance on filtration technology, at reasonable scale. Robust experiments demonstrated UVC dose dependent inactivation of Staphylococcus aureus (UV rate constant (k) = 0.098 m2/J) and bacteriophage MS2, with up to 6-log MS2 reduction achieved in a single pass through the system (k = 0.119 m2/J). The inclusion of a PTFE diffuse reflector increased the effective UVC dose by up to 34% in comparison to a standard Al foil reflector (with identical lamp output), resulting in significant additional pathogen inactivation (1-log S. aureus and MS2, p < 0.001). Complete inactivation of bovine coronavirus bioaerosols was demonstrated through tissue culture infectivity (2.4-log reduction) and RT-qPCR analysis - confirming single pass UVC treatment to effectively deactivate coronavirus to the limit of detection of the culture-based method. Scenario-based modelling was used to investigate the reduction in risk of airborne person-to-person transmission based upon a single infected subject within the small room. Use of the system providing 5 air changes per hour was shown to significantly reduce airborne viral load and maintain low numbers of RNA copies when the infected subject remained in the room, reducing the risk of airborne pathogen transmission to other room users. We conclude that the application of single-pass UVC systems (without reliance on HEPA filtration) could play a critical role in reducing the risk of airborne pathogen transfer, including SARS-CoV2, in locations where adequate fresh air ventilation cannot be implemented.

Profiling host ANP32A splicing landscapes to predict influenza A virus polymerase adaptation.

Species' differences in cellular factors limit avian influenza A virus (IAV) zoonoses and human pandemics. The IAV polymerase, vPol, harbors evolutionary sites to overcome restriction and determines virulence. Here, we establish host ANP32A as a critical driver of selection, and identify host-specific ANP32A splicing landscapes that predict viral evolution. We find that avian species differentially express three ANP32A isoforms diverging in a vPol-promoting insert. ANP32As with shorter inserts interact poorly with vPol, are compromised in supporting avian-like IAV replication, and drive selection of mammalian-adaptive vPol sequences with distinct kinetics. By integrating selection data with multi-species ANP32A splice variant profiling, we develop a mathematical model to predict avian species potentially driving (swallow, magpie) or maintaining (goose, swan) mammalian-adaptive vPol signatures. Supporting these predictions, surveillance data confirm enrichment of several mammalian-adaptive vPol substitutions in magpie IAVs. Profiling host ANP32A splicing could enhance surveillance and eradication efforts against IAVs with pandemic potential.

Unexpected Functional Divergence of Bat Influenza Virus NS1 Proteins.

Recently, two influenza A virus (FLUAV) genomes were identified in Central and South American bats. These sequences exhibit notable divergence from classical FLUAV counterparts, and functionally, bat FLUAV glycoproteins lack canonical receptor binding and destroying activity. Nevertheless, other features that distinguish these viruses from classical FLUAVs have yet to be explored. Here, we studied the viral nonstructural protein NS1, a virulence factor that modulates host signaling to promote efficient propagation. Like all FLUAV NS1 proteins, bat FLUAV NS1s bind double-stranded RNA and act as interferon antagonists. Unexpectedly, we found that bat FLUAV NS1s are unique in being unable to bind host p85ß, a regulatory subunit of the cellular metabolism-regulating enzyme, phosphoinositide 3-kinase (PI3K). Furthermore, neither bat FLUAV NS1 alone nor infection with a chimeric bat FLUAV efficiently activates Akt, a PI3K effector. Structure-guided mutagenesis revealed that the bat FLUAV NS1-p85ß interaction can be reengineered (in a strain-specific manner) by changing two to four NS1 residues (96L, 99M, 100I, and 145T), thereby creating a hydrophobic patch. Notably, ameliorated p85ß-binding is insufficient for bat FLUAV NS1 to activate PI3K, and a chimeric bat FLUAV expressing NS1 with engineered hydrophobic patch mutations exhibits cell-type-dependent, but species-independent, propagation phenotypes. We hypothesize that bat FLUAV hijacking of PI3K in the natural bat host has been selected against, perhaps because genes in this metabolic pathway were differentially shaped by evolution to suit the unique energy use strategies of this flying mammal. These data expand our understanding of the enigmatic functional divergence between bat FLUAVs and classical mammalian and avian FLUAVs.IMPORTANCE The potential for novel influenza A viruses to establish infections in humans from animals is a source of continuous concern due to possible severe outbreaks or pandemics. The recent discovery of influenza A-like viruses in bats has raised questions over whether these entities could be a threat to humans. Understanding unique properties of the newly described bat influenza A-like viruses, such as their mechanisms to infect cells or how they manipulate host functions, is critical to assess their likelihood of causing disease. Here, we characterized the bat influenza A-like virus NS1 protein, a key virulence factor, and found unexpected functional divergence of this protein from counterparts in other influenza A viruses. Our study dissects the molecular changes required by bat influenza A-like virus NS1 to adopt classical influenza A virus properties and suggests consequences of bat influenza A-like virus infection, potential future evolutionary trajectories, and intriguing virus-host biology in bat species.

Novel Bat Influenza Virus NS1 Proteins Bind Double-Stranded RNA and Antagonize Host Innate Immunity.

We demonstrate that novel bat HL17NL10 and HL18NL11 influenza virus NS1 proteins are effective interferon antagonists but do not block general host gene expression. Solving the RNA-binding domain structures revealed the canonical NS1 symmetrical homodimer, and RNA binding required conserved basic residues in this domain. Interferon antagonism was strictly dependent on RNA binding, and chimeric bat influenza viruses expressing NS1s defective in this activity were highly attenuated in interferon-competent cells but not in cells unable to establish antiviral immunity.

Analysis of influenza A virus NS1 dimer interfaces in solution by pulse EPR distance measurements.

Pulsed electron-electron double resonance (PELDOR) is an electron paramagnetic resonance (EPR) spectroscopy technique for nanometer distance measurements between paramagnetic centers such as radicals. PELDOR has been recognized as a valuable tool to approach structural questions in biological systems. In this manuscript, we demonstrate the value of distance measurements for differentiating competing structural models on the dimerization of the effector domain (ED) of the non-structural protein 1 (NS1) of the influenza A virus. Our results show NS1 to be well amenable to nanometer distance measurements by EPR, yielding high quality data. In combination with mutants perturbing protein dimerization and in silico prediction based on crystal structures, we can exclude one of two potential dimerization interfaces. Furthermore, our results lead to a viable hypothesis of a NS1 ED:ED interface which is flexible through rotation around the vector interconnecting the two native cysteines. These results prove the high value of pulse EPR as a complementary method for structural biology.