Surface Contamination of Reusable Respirators and Face Shields During Care of Critically Ill COVID-19 Patients.

BACKGROUND: With the emergence of SARS-CoV-2, healthcare workers (HCW) have relied on reusable personal protective equipment (PPE), including respirators and face shields (FSs). The effectiveness of decontamination procedures outside experimental settings is unclear. We examined the prevalence of surface contamination on reusable PPE used by HCWs at a hospital incorporating daily centralized decontamination and post-use wiping by sampling for common pathogens. METHOD: Samples were collected from HCWs' CleanSpace Halo respirator face masks (FMs) and FSs at the start of shift, immediately after use, and after cleaning with disinfecting wipes. Samples were analyzed for pathogens using the Applied Biosystems™ TaqPath™ COVID-19 Combo Kit and ThermoFisher TaqMan Array Card. Patient charts were reviewed for clinical correlation. FINDINGS: Of the 89 samples, 51 from FMs and 38 from FSs, none tested positive for SARS-CoV-2, despite 58 being obtained from PPE used in the care of patients with COVID-19, many with recent aerosol-generating procedures. Four samples tested positive (4.5%) for Staphylococcus aureus, two each from FMs and FSs. FMs that tested positive were not worn concurrently with FSs that tested positive. The FM and FS samples testing positive were worn in the care of patients without diagnosed S. aureus infection. No FMs tested positive following wipe-based disinfection, but both positive FS samples were found after disinfection wiping. CONCLUSION/APPLICATION TO PRACTICE: Contamination of reusable PPE appears uncommon, especially with SARS-CoV-2, when regular decontamination programs are in place. The rare presence of S. aureus highlights the importance of doffing procedures and hand hygiene by HCW to prevent surface contamination.

Highly stable, antiviral, antibacterial cotton textiles via molecular engineering.

Cotton textiles are ubiquitous in daily life and are also one of the primary mediums for transmitting viruses and bacteria. Conventional approaches to fabricating antiviral and antibacterial textiles generally load functional additives onto the surface of the fabric and/or their microfibres. However, such modifications are susceptible to deterioration after long-term use due to leaching of the additives. Here we show a different method to impregnate copper ions into the cellulose matrix to form a copper ion-textile (Cu-IT), in which the copper ions strongly coordinate with the oxygen-containing polar functional groups (for example, hydroxyl) of the cellulose chains. The Cu-IT displays high antiviral and antibacterial performance against tobacco mosaic virus and influenza A virus, and Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa and Bacillus subtilis bacteria due to the antimicrobial properties of copper. Furthermore, the strong coordination bonding of copper ions with the hydroxyl functionalities endows the Cu-IT with excellent air/water retainability and superior mechanical stability, which can meet daily use and resist repeated washing. This method to fabricate Cu-IT is cost-effective, ecofriendly and highly scalable, and this textile appears very promising for use in household products, public facilities and medical settings.

Real-time ultra-sensitive detection of SARS-CoV-2 by quasi-freestanding epitaxial graphene-based biosensor.

We report the rapid detection of SARS-CoV-2 in infected patients (mid-turbinate swabs and exhaled breath aerosol samples) in concentrations as low as 60 copies/mL of the virus in seconds by electrical transduction of the SARS-CoV-2 S1 spike protein antigen via SARS-CoV-2 S1 spike protein antibodies immobilized on bilayer quasi-freestanding epitaxial graphene without gate or signal amplification. The sensor demonstrates the spike protein antigen detection in a concentration as low as 1 ag/mL. The heterostructure of the SARS-CoV-2 antibody/graphene-based sensor is developed through a simple and low-cost fabrication technique. Furthermore, sensors integrated into a portable testing unit distinguished B.1.1.7 variant positive samples from infected patients (mid-turbinate swabs and saliva samples, 4000-8000 copies/mL) with a response time of as fast as 0.6 s. The sensor is reusable, allowing for reimmobilization of the crosslinker and antibodies on the biosensor after desorption of biomarkers by NaCl solution or heat treatment above 40 °C.

Plasma Membrane Anchoring and Gag:Gag Multimerization on Viral RNA Are Critical Properties of HIV-1 Gag Required To Mediate Efficient Genome Packaging.

Human immunodeficiency virus type 1 (HIV-1) Gag selects and packages the HIV RNA genome during virus assembly. However, HIV-1 RNA constitutes only a small fraction of the cellular RNA. Although Gag exhibits a slight preference to viral RNA, most of the cytoplasmic Gag proteins are associated with cellular RNAs. Thus, it is not understood how HIV-1 achieves highly efficient genome packaging. We hypothesize that besides RNA binding, other properties of Gag are important for genome packaging. Many Gag mutants have assembly defects that preclude analysis of their effects on genome packaging. To bypass this challenge, we established complementation systems that separate the particle-assembling and RNA-binding functions of Gag: we used a set of Gag proteins to drive particle assembly and an RNA-binding Gag to package HIV-1 RNA. We have developed two types of RNA-binding Gag in which packaging is mediated by the authentic nucleocapsid (NC) domain or by a nonviral RNA-binding domain. We found that in both cases, mutations that affect the multimerization or plasma membrane anchoring properties of Gag reduce or abolish RNA packaging. These mutant Gag can coassemble into particles but cannot package the RNA genome efficiently. Our findings indicate that HIV-1 RNA packaging occurs at the plasma membrane and RNA-binding Gag needs to multimerize on RNA to encapsidate the viral genome. IMPORTANCE To generate infectious virions, HIV-1 must package its full-length RNA as the genome during particle assembly. HIV-1 Gag:RNA interactions mediate genome packaging, but the mechanism remains unclear. Only a minor portion of the cellular RNA is HIV-1 RNA, and most of the RNAs associated with cytoplasmic Gag are cellular RNAs. However, >94% of the HIV-1 virions contain viral RNA genome. We posited that, besides RNA binding, other properties of Gag contribute to genome packaging. Using two complementation systems, we examined features of Gag that are important for genome packaging. We found that the capacities for Gag to multimerize and to anchor at the plasma membrane are critical for genome packaging. Our results revealed that Gag needs to multimerize on viral RNA at the plasma membrane in order to package RNA genome.

Viral RNA and Infectious Influenza Virus on Mobile Phones of Patients With Influenza in Hong Kong and the United States.

Mobile phones are among the most highly touched personal objects. As part of a broader study on the contribution of fomites to influenza transmission, between 2017 and 2019, we swabbed mobile phones from 138 patients with influenza in 2 locations. Influenza viral RNA detection rates were 23% (23 of 99 phones) and 36% (14 of 39) in Hong Kong and Maryland, respectively. In Hong Kong, infectious influenza virus was recovered from 3 of 23 mobile phones which had influenza viral RNA detected. Mobile phone influenza contamination was positively associated with upper respiratory tract viral load and negatively associated with age. Cleaning personal objects of patients with influenza should be recommended, and individuals should avoid sharing objects with these patients.

Analysis of SARS-CoV-2 antibodies in COVID-19 convalescent blood using a coronavirus antigen microarray.

The current practice for diagnosis of COVID-19, based on SARS-CoV-2 PCR testing of pharyngeal or respiratory specimens in a symptomatic patient at high epidemiologic risk, likely underestimates the true prevalence of infection. Serologic methods can more accurately estimate the disease burden by detecting infections missed by the limited testing performed to date. Here, we describe the validation of a coronavirus antigen microarray containing immunologically significant antigens from SARS-CoV-2, in addition to SARS-CoV, MERS-CoV, common human coronavirus strains, and other common respiratory viruses. A comparison of antibody profiles detected on the array from control sera collected prior to the SARS-CoV-2 pandemic versus convalescent blood specimens from virologically confirmed COVID-19 cases demonstrates near complete discrimination of these two groups, with improved performance from use of antigen combinations that include both spike protein and nucleoprotein. This array can be used as a diagnostic tool, as an epidemiologic tool to more accurately estimate the disease burden of COVID-19, and as a research tool to correlate antibody responses with clinical outcomes.

Specific Guanosines in the HIV-2 Leader RNA are Essential for Efficient Viral Genome Packaging.

HIV-2, a human pathogen that causes acquired immunodeficiency syndrome, is distinct from the more prevalent HIV-1 in several features including its evolutionary history and certain aspects of viral replication. Like other retroviruses, HIV-2 packages two copies of full-length viral RNA during virus assembly and efficient genome encapsidation is mediated by the viral protein Gag. We sought to define cis-acting elements in the HIV-2 genome that are important for the encapsidation of full-length RNA into viral particles. Based on previous studies of murine leukemia virus and HIV-1, we hypothesized that unpaired guanosines in the 5' untranslated region (UTR) play an important role in Gag:RNA interactions leading to genome packaging. To test our hypothesis, we targeted 18 guanosines located in 9 sites within the HIV-2 5' UTR and performed substitution analyses. We found that mutating as few as three guanosines significantly reduce RNA packaging efficiency. However, not all guanosines examined have the same effect; instead, a hierarchical order exists wherein a primary site, a secondary site, and three tertiary sites are identified. Additionally, there are functional overlaps in these sites and mutations of more than one site can act synergistically to cause genome packaging defects. These studies demonstrate the importance of specific guanosines in HIV-2 5'UTR in mediating genome packaging. Our results also demonstrate an interchangeable and hierarchical nature of guanosine-containing sites, which was not previously established, thereby revealing key insights into the replication mechanisms of HIV-2.

Analysis of SARS-CoV-2 Antibodies in COVID-19 Convalescent Blood using a Coronavirus Antigen Microarray.

The current practice for diagnosis of COVID-19, based on SARS-CoV-2 PCR testing of pharyngeal or respiratory specimens in a symptomatic patient at high epidemiologic risk, likely underestimates the true prevalence of infection. Serologic methods can more accurately estimate the disease burden by detecting infections missed by the limited testing performed to date. Here, we describe the validation of a coronavirus antigen microarray containing immunologically significant antigens from SARS-CoV-2, in addition to SARS-CoV, MERS-CoV, common human coronavirus strains, and other common respiratory viruses. A comparison of antibody profiles detected on the array from control sera collected prior to the SARS-CoV-2 pandemic versus convalescent blood specimens from virologically confirmed COVID-19 cases demonstrates near complete discrimination of these two groups, with improved performance from use of antigen combinations that include both spike protein and nucleoprotein. This array can be used as a diagnostic tool, as an epidemiologic tool to more accurately estimate the disease burden of COVID-19, and as a research tool to correlate antibody responses with clinical outcomes.

Analysis of Serologic Cross-Reactivity Between Common Human Coronaviruses and SARS-CoV-2 Using Coronavirus Antigen Microarray.

The current practice for diagnosis of SARS-CoV-2 infection relies on PCR testing of nasopharyngeal or respiratory specimens in a symptomatic patient at high epidemiologic risk. This testing strategy likely underestimates the true prevalence of infection, creating the need for serologic methods to detect infections missed by the limited testing to date. Here, we describe the development of a coronavirus antigen microarray containing immunologically significant antigens from SARS-CoV-2, in addition to SARS-CoV, MERS-CoV, common human coronavirus strains, and other common respiratory viruses. A preliminary study of human sera collected prior to the SARS-CoV-2 pandemic demonstrates overall high IgG reactivity to common human coronaviruses and low IgG reactivity to epidemic coronaviruses including SARS-CoV-2, with some cross-reactivity of conserved antigenic domains including S2 domain of spike protein and nucleocapsid protein. This array can be used to answer outstanding questions regarding SARS-CoV-2 infection, including whether baseline serology for other coronaviruses impacts disease course, how the antibody response to infection develops over time, and what antigens would be optimal for vaccine development.

Ventilation and laboratory confirmed acute respiratory infection (ARI) rates in college residence halls in College Park, Maryland.

Strategies to protect building occupants from the risk of acute respiratory infection (ARI) need to consider ventilation for its ability to dilute and remove indoor bioaerosols. Prior studies have described an association of increased self-reported colds and influenza-like symptoms with low ventilation but have not combined rigorous characterization of ventilation with assessment of laboratory confirmed infections. We report a study designed to fill this gap. We followed laboratory confirmed ARI rates and measured CO2 concentrations for four months during the winter-spring of 2018 in two campus residence halls: (1) a high ventilation building (HVB) with a dedicated outdoor air system that supplies 100% of outside air to each dormitory room, and (2) a low ventilation building (LVB) that relies on infiltration as ventilation. We enrolled 11 volunteers for a total of 522 person-days in the HVB and 109 volunteers for 6069 person-days in the LVB, and tested upper-respiratory swabs from symptomatic cases and their close contacts for the presence of 44 pathogens using a molecular assay. We observed one ARI case in the HVB (0.70/person-year) and 47 in the LVB (2.83/person-year). Simultaneously, 154 CO2 sensors distributed primarily in the dormitory rooms collected 668,390 useful data points from over 1 million recorded data points. Average and standard deviation of CO2 concentrations were 1230 ppm and 408 ppm in the HVB, and 1492 ppm and 837 ppm in the LVB, respectively. Importantly, this study developed and calibrated multi-zone models for the HVB with 229 zones and 983 airflow paths, and for the LVB with 529 zones and 1836 airflow paths by using a subset of CO2 data for model calibration. The models were used to calculate ventilation rates in the two buildings and potential for viral aerosol migration between rooms in the LVB. With doors and windows closed, the average ventilation rate was 12 L/s in the HVB dormitory rooms and 4 L/s in the LVB dormitory rooms. As a result, residents had on average 6.6 L/(s person) of outside air in the HVB and 2.3 L/(s person) in the LVB. LVB rooms located at the leeward side of the building had smaller average ventilation rates, as well as a somewhat higher ARI incidence rate and average CO2 concentrations when compared to those values in the rooms located at the windward side of the building. Average ventilation rates in twenty LVB dormitory rooms increased from 2.3 L/s to 7.5 L/s by opening windows, 3.6 L/s by opening doors, and 8.8 L/s by opening both windows and doors. Therefore, opening both windows and doors in the LVB dormitory rooms can increase ventilation rates to the levels comparable to those in the HVB. But it can also have a negative effect on thermal comfort due to low outdoor temperatures. Simulation results identified an aerobiologic pathway from a room occupied by an index case of influenza A to a room occupied by a possible secondary case.

Novel spirothiazamenthane inhibitors of the influenza A M2 proton channel.

The development of treatments for influenza that inhibit the M2 proton channel without being susceptible to the widespread resistance mechanisms associated with the adamantanes is an ongoing challenge. Using a yeast high-throughput yeast growth restoration assay designed to identify M2 channel inhibitors, a single screening hit was uncovered. This compound (3), whose structure was incorrectly identified in the literature, is an inhibitor with similar potency to amantadine against WT M2. A library of derivatives of 3 was prepared and activity against WT M2 and the two principal mutant strains (V27A and S31N) was assessed in the yeast assay. The best compounds were further evaluated in an antiviral plaque reduction assay using engineered WT, V27A and S31N M2 influenza A strains with otherwise identical genetic background. Compound 63 was found to inhibit all three virus strains in this cell based antiviral assay at micromolar concentrations, possibly through a mechanism other than M2 inhibition.

Mechanisms of Action of Novel Influenza A/M2 Viroporin Inhibitors Derived from Hexamethylene Amiloride.

The increasing prevalence of influenza viruses with resistance to approved antivirals highlights the need for new anti-influenza therapeutics. Here we describe the functional properties of hexamethylene amiloride (HMA)-derived compounds that inhibit the wild-type and adamantane-resistant forms of the influenza A M2 ion channel. For example, 6-(azepan-1-yl)-N-carbamimidoylnicotinamide ( 9: ) inhibits amantadine-sensitive M2 currents with 3- to 6-fold greater potency than amantadine or HMA (IC50 = 0.2 vs. 0.6 and 1.3 µM, respectively). Compound 9: competes with amantadine for M2 inhibition, and molecular docking simulations suggest that 9: binds at site(s) that overlap with amantadine binding. In addition, tert-butyl 4'-(carbamimidoylcarbamoyl)-2',3-dinitro-[1,1'-biphenyl]-4-carboxylate ( 27: ) acts both on adamantane-sensitive and a resistant M2 variant encoding a serine to asparagine 31 mutation (S31N) with improved efficacy over amantadine and HMA (IC50 = 0.6 µM and 4.4 µM, respectively). Whereas 9: inhibited in vitro replication of influenza virus encoding wild-type M2 (EC50 = 2.3 µM), both 27: and tert-butyl 4'-(carbamimidoylcarbamoyl)-2',3-dinitro-[1,1'-biphenyl]-4-carboxylate ( 26: ) preferentially inhibited viruses encoding M2(S31N) (respective EC50 = 18.0 and 1.5 µM). This finding indicates that HMA derivatives can be designed to inhibit viruses with resistance to amantadine. Our study highlights the potential of HMA derivatives as inhibitors of drug-resistant influenza M2 ion channels.

HIV-1 RNA genome dimerizes on the plasma membrane in the presence of Gag protein.

Retroviruses package a dimeric genome comprising two copies of the viral RNA. Each RNA contains all of the genetic information for viral replication. Packaging a dimeric genome allows the recovery of genetic information from damaged RNA genomes during DNA synthesis and promotes frequent recombination to increase diversity in the viral population. Therefore, the strategy of packaging dimeric RNA affects viral replication and viral evolution. Although its biological importance is appreciated, very little is known about the genome dimerization process. HIV-1 RNA genomes dimerize before packaging into virions, and RNA interacts with the viral structural protein Gag in the cytoplasm. Thus, it is often hypothesized that RNAs dimerize in the cytoplasm and the RNA-Gag complex is transported to the plasma membrane for virus assembly. In this report, we tagged HIV-1 RNAs with fluorescent proteins, via interactions of RNA-binding proteins and motifs in the RNA genomes, and studied their behavior at the plasma membrane by using total internal reflection fluorescence microscopy. We showed that HIV-1 RNAs dimerize not in the cytoplasm but on the plasma membrane. Dynamic interactions occur among HIV-1 RNAs, and stabilization of the RNA dimer requires Gag protein. Dimerization often occurs at an early stage of the virus assembly process. Furthermore, the dimerization process is probably mediated by the interactions of two RNA-Gag complexes, rather than two RNAs. These findings advance the current understanding of HIV-1 assembly and reveal important insights into viral replication mechanisms.