ORF8 and ORF3b antibodies are accurate serological markers of early and late SARS-CoV-2 infection.

The SARS-CoV-2 virus emerged in December 2019 and has caused a worldwide pandemic due to the lack of any pre-existing immunity. Accurate serology testing is urgently needed to help diagnose infection, determine past exposure of populations and assess the response to a future vaccine. The landscape of antibody responses to SARS-CoV-2 is unknown. In this study, we utilized the luciferase immunoprecipitation system to assess the antibody responses to 15 different SARS-CoV-2 antigens in patients with COVID-19. We identified new targets of the immune response to SARS-CoV-2 and show that nucleocapsid, open reading frame (ORF)8 and ORF3b elicit the strongest specific antibody responses. ORF8 and ORF3b antibodies, taken together as a cluster of points, identified 96.5% of COVID-19 samples at early and late time points of disease with 99.5% specificity. Our findings could be used to develop second-generation diagnostic tests to improve serological assays for COVID-19 and are important in understanding pathogenicity.

Author Correction: ORF8 and ORF3b antibodies are accurate serological markers of early and late SARS-CoV-2 infection.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Pathogenesis and transmission of SARS-CoV-2 in golden hamsters.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel coronavirus with high nucleotide identity to SARS-CoV and to SARS-related coronaviruses that have been detected in horseshoe bats, has spread across the world and had a global effect on healthcare systems and economies1,2. A suitable small animal model is needed to support the development of vaccines and therapies. Here we report the pathogenesis and transmissibility of SARS-CoV-2 in golden (Syrian) hamsters (Mesocricetus auratus). Immunohistochemistry assay demonstrated the presence of viral antigens in nasal mucosa, bronchial epithelial cells and areas of lung consolidation on days 2 and 5 after inoculation with SARS-CoV-2, followed by rapid viral clearance and pneumocyte hyperplasia at 7 days after inoculation. We also found viral antigens in epithelial cells of the duodenum, and detected viral RNA in faeces. Notably, SARS-CoV-2 was transmitted efficiently from inoculated hamsters to naive hamsters by direct contact and via aerosols. Transmission via fomites in soiled cages was not as efficient. Although viral RNA was continuously detected in the nasal washes of inoculated hamsters for 14 days, the communicable period was short and correlated with the detection of infectious virus but not viral RNA. Inoculated and naturally infected hamsters showed apparent weight loss on days 6-7 post-inoculation or post-contact; all hamsters returned to their original weight within 14 days and developed neutralizing antibodies. Our results suggest that features associated with SARS-CoV-2 infection in golden hamsters resemble those found in humans with mild SARS-CoV-2 infections.

Genetic diversity of astroviruses detected in wild aquatic birds in Hong Kong.

Wild waterfowl serve as a reservoir of some astroviruses. Fecal samples from wild waterfowl collected at Hong Kong's Marshes were tested using pan-astrovirus reverse transcription-PCR. Positive samples underwent subsequent host identification using DNA barcoding. Based on deduced partial sequences, noteworthy samples from three astrovirus groups (mammalian, avian and unclassified astroviruses) were further analyzed by next-generation sequencing. One sample of Avastrovirus 4 clade, MP22-196, had a nearly complete genome identified. The results of ORF2 phylogenetic analysis and genetic distance analysis indicate that Avastrovirus 4 is classified as a distinct subclade within Avastrovirus. MP22-196 has typical astrovirus genome characteristics. The unique characteristics and potential differences of this genome, compared to other avian astrovirus sequences, involve the identification of a modified sgRNA sequence situated near the ORF2 start codon, which precedes the ORF1b stop codon. Additionally, the 3' UTR of MP22-196 is shorter than other avian astroviruses. This study expands our understanding of the Avastrovirus 4 clade.

Evaluation of RT-qPCR Primer-Probe Sets to Inform Public Health Interventions Based on COVID-19 Sewage Tests.

Sewage surveillance is increasingly employed as a supplementary tool for COVID-19 control. Experiences learnt from large-scale trials could guide better interpretation of the sewage data for public health interventions. Here, we compared the performance of seven commonly used primer-probe sets in RT-qPCR and evaluated the usefulness in the sewage surveillance program in Hong Kong. All selected primer-probe sets reliably detected SARS-CoV-2 in pure water at 7 copies per µL. Sewage matrix did not influence RT-qPCR determination of SARS-CoV-2 concentrated from a small-volume sewage (30 mL) but introduced inhibitory impacts on a large-volume sewage (920 mL) with a ΔCt of 0.2-10.8. Diagnostic performance evaluation in finding COVID-19 cases showed that N1 was the best single primer-probe set, while the ORF1ab set is not recommended. Sewage surveillance using the N1 set for over 3200 samples effectively caught the outbreak trend and, importantly, had a 56% sensitivity and a 96% specificity in uncovering the signal sources from new cases and/or convalescent patients in the community. Our study paves the way for selecting detection primer-probe sets in wider applications in responding to the COVID-19 pandemic.

SARS-CoV-2 Virus Culture and Subgenomic RNA for Respiratory Specimens from Patients with Mild Coronavirus Disease.

We investigated 68 respiratory specimens from 35 coronavirus disease patients in Hong Kong, of whom 32 had mild disease. We found that severe acute respiratory syndrome coronavirus 2 and subgenomic RNA were rarely detectable beyond 8 days after onset of illness. However, virus RNA was detectable for many weeks by reverse transcription PCR.

Effect of moist heat reprocessing of N95 respirators on SARS-CoV-2 inactivation and respirator function.

BACKGROUND: Unprecedented demand for N95 respirators during the coronavirus disease 2019 (COVID-19) pandemic has led to a global shortage of these masks. We validated a rapidly applicable, low-cost decontamination protocol in compliance with regulatory standards to enable the safe reuse of N95 respirators. METHODS: We inoculated 4 common models of N95 respirators with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and evaluated viral inactivation after disinfection for 60 minutes at 70°C and 0% relative humidity. Similarly, we evaluated thermal disinfection at 0% to 70% relative humidity for masks inoculated with Escherichia coli. We assessed masks subjected to multiple cycles of thermal disinfection for structural integrity using scanning electron microscopy and for protective functions using standards of the United States National Institute for Occupational Safety and Health for particle filtration efficiency, breathing resistance and respirator fit. RESULTS: A single heat treatment rendered SARS-CoV-2 undetectable in all mask samples. Compared with untreated inoculated control masks, E. coli cultures at 24 hours were virtually undetectable from masks treated at 70°C and 50% relative humidity (optical density at 600 nm wavelength, 0.02 ± 0.02 v. 2.77 ± 0.09, p < 0.001), but contamination persisted for masks treated at lower relative humidity. After 10 disinfection cycles, masks maintained fibre diameters similar to untreated masks and continued to meet standards for fit, filtration efficiency and breathing resistance. INTERPRETATION: Thermal disinfection successfully decontaminated N95 respirators without impairing structural integrity or function. This process could be used in hospitals and long-term care facilities with commonly available equipment to mitigate the depletion of N95 masks.

Circulation of Influenza A(H5N8) Virus, Saudi Arabia.

Highly pathogenic avian influenza A(H5N8) viruses have been detected in several continents. However, limited viral sequence data are available from countries in the Middle East. We report full-genome analyses of highly pathogenic H5N8 viruses recently detected in different provinces in Saudi Arabia.

Recombinant influenza virus with a pandemic H2N2 polymerase complex has a higher adaptive potential than one with seasonal H2N2 polymerase complex.

The reassortment of influenza viral gene segments plays a key role in the genesis of pandemic strains. All of the last three pandemic viruses contained reassorted polymerase complexes with subunits derived from animal viruses, suggesting that the acquisition of a reassorted polymerase complex might have a role in generating these pandemic viruses. Here, we studied polymerase activities of the pandemic H2N2, seasonal H2N2 and pandemic H3N2 viruses. We observed that the viral ribonucleoprotein (vRNP) of pandemic H2N2 virus has a highly robust activity. The polymerase activity of seasonal H2N2 viruses, however, was much reduced. We further identified three mutations (PB2-I114V, PB1-S261N and PA-D383N) responsible for the reduced activity. To determine the potential impact of viral polymerase activity on the viral life cycle, recombinant H3N2 viruses carrying pandemic and seasonal H2N2 vRNP were studied in cell cultures supplemented with oseltamivir carboxylate and tested for their abilities to develop adaptive or resistant mutations. It was found that the recombinant virus with pandemic H2N2 vRNP was more capable of restoring the viral fitness than the one with seasonal vRNP. These results suggest that a robust vRNP is advantageous to influenza virus to cope with a new selection pressure.

Lack of middle East respiratory syndrome coronavirus transmission from infected camels.

To determine risk for Middle East respiratory syndrome coronavirus transmission from camels to humans, we tested serum from 191 persons with various levels of exposure to an infected dromedary herd. We found no serologic evidence of human infection, suggesting that zoonotic transmission of this virus from dromedaries is rare.

Désinfection à la chaleur humide des respirateurs N95, inactivation du SRAS-CoV-2 et effets sur les propriétés des respirateurs.

CONTEXTE: La demande sans précédent de respirateurs N95 durant la pandémie de maladie à coronavirus 2019 (COVID-19) a entraîné une pénurie mondiale. Nous avons validé un protocole de décontamination rapide et économique répondant aux normes réglementaires afin de permettre la réutilisation sûre de ce type de masque. MÉTHODES: Nous avons contaminé 4 modèles courants de respirateurs N95 avec le coronavirus du syndrome respiratoire aigu sévère 2 (SRAS-CoV-2) et avons évalué l'inactivation virale après une désinfection de 60 minutes à 70 °C et à une humidité relative de 0 %. De même, nous avons étudié l'efficacité de la désinfection thermique, à une humidité relative allant de 0 % à 70 %, de masques contaminés à Escherichia coli. Enfin, nous avons examiné des masques soumis à de multiples cycles de désinfection thermique: nous avons évalué leur intégrité structurelle à l'aide d'un microscope à balayage, et leurs propriétés protectrices au moyen des normes du National Institute for Occupational Safety and Health des États-Unis relatives à la filtration particulaire, à la résistance respiratoire et à l'ajustement. RÉSULTATS: Une seule désinfection thermique a suffi pour que le SRAS-CoV-2 ne soit plus décelable sur les masques étudiés. En ce qui concerne les masques contaminés à E. coli, une culture de 24 heures a révélé que la bactérie n'était pratiquement plus décelable sur les masques désinfectés à 70 °C et à une humidité relative de 50 %, contrairement aux masques non désinfectés (densité optique à une longueur d'onde de 600 nm : 0,02 ± 0,02 contre 2,77 ± 0,09; p < 0,001), mais qu'elle persistait sur les masques traités à une humidité relative moindre. Les masques ayant subi 10 cycles de désinfection avaient toujours des fibres de diamètre semblable à celui des fibres des masques non traités, et ils répondaient encore aux normes d'ajustement, de filtration et de résistance respiratoire. INTERPRÉTATION: La désinfection thermique a réussi à décontaminer les respirateurs N95 sans compromettre leur intégrité structurelle ni modifier leurs propriétés. Elle pourrait se faire dans les hôpitaux et les établissements de soins de longue durée avec de l'équipement facilement accessible, ce qui réduirait la pénurie de N95.

Stability of SARS-CoV-2 on Commercial Aircraft Interior Surfaces with Implications for Effective Control Measures.

BACKGROUND: The COVID-19 pandemic from 2019 to 2022 devastated many aspects of life and the economy, with the commercial aviation industry being no exception. One of the major concerns during the pandemic was the degree to which the internal aircraft environment contributed to virus transmission between humans and, in particular, the stability of SARS-CoV-2 on contact surfaces in the aircraft cabin interior. METHOD: In this study, the stability of various major strains of SARS-CoV-2 on interior aircraft surfaces was evaluated using the TCID50 assessment. RESULTS: In contrast to terrestrial materials, SARS-CoV-2 was naturally less stable on common contact points in the aircraft interior, and, over a 4 h time period, there was a 90% reduction in culturable virus. Antiviral and surface coatings were extremely effective at mitigating the persistence of the virus on surfaces; however, their benefit was diminished by regular cleaning and were ineffective after 56 days of regular use and cleaning. Finally, successive strains of SARS-CoV-2 have not evolved to be more resilient to survival on aircraft surfaces. CONCLUSIONS: We conclude that the mitigation strategies for SARS-CoV-2 on interior aircraft surfaces are more than sufficient, and epidemiological evidence over the past three years has not found that surface spread is a major route of transmission.

Effect of Surface Porosity on SARS-CoV-2 Fomite Infectivity.

Previous reports indicated the low stability of severe actute respiratory syndrome coronovirus 2 (SARS-CoV-2) on various porous surfaces, but the role of porosity was unclear because there was no direct comparison between porous and nonporous solids of the same chemistry. Through comparing pairs of solids with very similar chemistry, we find that porosity is important: porous glass has a much lower infectivity than nonporous glass. However, porosity is not sufficient to lower infectivity; permeability, which is the ability of a liquid to move through a material, is the important parameter. We show this by comparing a pair of porous CuO coatings where the pores are accessible in one case and inaccessible in the other case. When the pores are inaccessible, the infectivity remains similar to that for nonporous solids. Thus, for both glass and CuO, it is the access to porosity that decreases the infectivity of extracted liquid droplets. Having established the importance of permeability, there is the open question of the mechanism of changing the infectivity of SARS-CoV-2. Several hypotheses are possible, such as increasing the difficulty of extracting the virus from the solid, changing the drying time, increasing the surface area of active ingredient, etc. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) measurements show that less viral DNA is extracted from a permeable surface, suggesting that the virus becomes trapped in the pores. Finally, we consider the effect of drying. We show that permeability and the water contact angle on the solid have effects on the drying time of a contaminated droplet, which may in turn affect infectivity.

Transparent Anti-SARS-CoV-2 and Antibacterial Silver Oxide Coatings.

Transparent antimicrobial coatings can maintain the aesthetic appeal of surfaces and the functionality of a touch-screen while adding the benefit of reducing disease transmission. We fabricated an antimicrobial coating of silver oxide particles in a silicate matrix on glass. The matrix was grown by a modified Stöber sol-gel process with vapor-phase water and ammonia. A coating on glass with 2.4 mg of Ag2O per mm2 caused a reduction of 99.3% of SARS-CoV-2 and >99.5% of Pseudomonas aeruginosa, Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus compared to the uncoated glass after 1 h. We envisage that screen protectors with transparent antimicrobial coatings will find particular application to communal touch-screens, such as in supermarkets and other check-out or check-in facilities where a number of individuals utilize the same touch-screen in a short interval.

Comparison of virus concentration methods and RNA extraction methods for SARS-CoV-2 wastewater surveillance.

Wastewater surveillance is a promising tool for population-level monitoring of the spread of infectious diseases, such as the coronavirus disease 2019 (COVID-19). Different from clinical specimens, viruses in community-scale wastewater samples need to be concentrated before detection because viral RNA is highly diluted. The present study evaluated eleven different virus concentration methods for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in wastewater. First, eight concentration methods of different principles were compared using spiked wastewater at a starting volume of 30 mL. Ultracentrifugation was the most effective method with a viral recovery efficiency of 25 ± 6%. The second-best option, AlCl3 precipitation method, yielded a lower recovery efficiency, only approximately half that of the ultracentrifugation method. Second, the potential of increasing method sensitivity was explored using three concentration methods starting with a larger volume of 1000 mL. Although ultracentrifugation using a large volume outperformed the other two large-volume methods, it only yielded a comparable method sensitivity as the ultracentrifugation using a small volume (30 mL). Thus, ultracentrifugation using less volume of wastewater is more preferable considering the sample processing throughput. Third, a comparison of two viral RNA extraction methods showed that the lysis-buffer-based extraction method resulted in higher viral recovery efficiencies, with cycle threshold (Ct) values 0.9-4.2 lower than those obtained for the acid-guanidinium-phenol-based method using spiked samples. These results were further confirmed by using positive wastewater samples concentrated by ultracentrifugation and extracted separately by the two viral RNA extraction methods. In summary, concentration using ultracentrifugation followed by the lysis buffer-based extraction method enables sensitive and robust detection of SARS-CoV-2 for wastewater surveillance.

A human pluripotent stem cell-based model of SARS-CoV-2 infection reveals an ACE2-independent inflammatory activation of vascular endothelial cells through TLR4.

To date, the direct causative mechanism of SARS-CoV-2-induced endotheliitis remains unclear. Here, we report that human ECs barely express surface ACE2, and ECs express less intracellular ACE2 than non-ECs of the lungs. We ectopically expressed ACE2 in hESC-ECs to model SARS-CoV-2 infection. ACE2-deficient ECs are resistant to the infection but are more activated than ACE2-expressing ones. The virus directly induces endothelial activation by increasing monocyte adhesion, NO production, and enhanced phosphorylation of p38 mitogen-associated protein kinase (MAPK), NF-κB, and eNOS in ACE2-expressing and -deficient ECs. ACE2-deficient ECs respond to SARS-CoV-2 through TLR4 as treatment with its antagonist inhibits p38 MAPK/NF-κB/ interleukin-1ß (IL-1ß) activation after viral exposure. Genome-wide, single-cell RNA-seq analyses further confirm activation of the TLR4/MAPK14/RELA/IL-1ß axis in circulating ECs of mild and severe COVID-19 patients. Circulating ECs could serve as biomarkers for indicating patients with endotheliitis. Together, our findings support a direct role for SARS-CoV-2 in mediating endothelial inflammation in an ACE2-dependent or -independent manner.

Cupric Oxide Coating That Rapidly Reduces Infection by SARS-CoV-2 via Solids.

The ongoing COVID-19 pandemic has created a need for coatings that reduce infection from SARS-CoV-2 via surfaces. Such a coating could be used on common touch surfaces (e.g., door handles and railings) to reduce both disease transmission and fear of touching objects. Herein, we describe the design, fabrication, and testing of a cupric oxide anti-SARS-CoV-2 coating. Rapid loss of infectivity is an important design criterion, so a porous hydrophilic coating was created to allow rapid infiltration of aqueous solutions into the coating where diffusion distances to the cupric oxide surface are short and the surface area is large. The coating was deposited onto glass from a dispersion of cuprous oxide in ethanol and then thermally treated at 700 °C for 2 h to produce a CuO coating that is ≈30 µm thick. The heat treatment oxidized the cuprous oxide to cupric oxide and sintered the particles into a robust film. The SARS-CoV-2 infectivity from the CuO film was reduced by 99.8% in 30 min and 99.9% in 1 h compared to that from glass. The coating remained hydrophilic for at least 5 months, and there was no significant change in the cross-hatch test of robustness after exposure to 70% ethanol or 3 wt % bleach.

Reduction of Infectivity of SARS-CoV-2 by Zinc Oxide Coatings.

We developed antimicrobial coatings from ZnO particles that reduce the infectivity of SARS-CoV-2 suspensions by >99.9% in 1 h. The advantage of a coating is that it can be applied to a variety of objects, e.g., hand rails and door knobs, to hinder the spread of disease. Two porous coatings were prepared: one from submicrometer zinc oxide particles bound with silica menisci and the other from zinc oxide tetrapods bound with polyurethane. Experiments on glass coatings show that infectivity depends on porosity for hydrophilic materials, wherein aqueous droplets are imbibed into the pores.

SARS-CoV-2 virus transfers to skin through contact with contaminated solids.

Transfer of SARS-CoV-2 from solids to fingers is one step in infection via contaminated solids, and the possibility of infection from this route has driven calls for increased frequency of handwashing during the COVID-19 pandemic. To analyze this route of infection, we measured the percentage of SARS-CoV-2 that was transferred from a solid to an artificial finger. A droplet of SARS-CoV-2 suspension (1 µL) was placed on a solid, and then artificial skin was briefly pressed against the solid with a light force (3 N). Transfer from a variety of solids was detected, and transfer from the non-porous solids, glass, stainless steel, and Teflon, was substantial when the droplet was still wet. The viral titer for the finger was 13-16% or 0.8-0.9 log less than for the input droplet. Transfer still occurred after the droplet evaporated, but was smaller, 3-9%. We found a lower level of transfer from porous solids but did not find a significant effect of solid wettability for non-porous solids.

Transparent and Sprayable Surface Coatings that Kill Drug-Resistant Bacteria Within Minutes and Inactivate SARS-CoV-2 Virus.

Antimicrobial coatings are one method to reduce the spread of microbial diseases. Transparent coatings preserve the visual properties of surfaces and are strictly necessary for applications such as antimicrobial cell phone screens. This work describes transparent coatings that inactivate microbes within minutes. The coatings are based on a polydopamine (PDA) adhesive, which has the useful property that the monomer can be sprayed, and then the monomer polymerizes in a conformal film at room temperature. Two coatings are described (1) a coating where PDA is deposited first and then a thin layer of copper is grown on the PDA by electroless deposition (PDA/Cu) and (2) a coating where a suspension of Cu2O particles in a PDA solution is deposited in a single step (PDA/Cu2O). In the second coating, PDA menisci bind Cu2O particles to the solid surface. Both coatings are transparent and are highly efficient in inactivating microbes. PDA/Cu kills >99.99% of Pseudomonas aeruginosa and 99.18% of methicillin-resistant Staphylococcus aureus (MRSA) in only 10 min and inactivates 99.98% of SARS-CoV-2 virus in 1 h. PDA/Cu2O kills 99.94% of P. aeruginosa and 96.82% of MRSA within 10 min and inactivates 99.88% of SARS-CoV-2 in 1 h.