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Smoke generated by cautery devices used during surgery may contain infective particles and may cause transmission of airborne viruses. This study determines whether live viruses are present in surgical smoke and evaluates the effectiveness of several proposed methods of removal so as to improve safety of healthcare workers.

Background Multiple severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) superspreading events suggest that aerosols play an important role in driving the coronavirus disease 2019 (COVID-19) pandemic. To better understand how airborne SARS-CoV-2 transmission occurs, we sought to determine viral loads within coarse (>5 μm) and fine (≤5 μm) respiratory aerosols produced when breathing, talking, and singing. Methods Using a G-II exhaled breath collector, we measured viral RNA in coarse and fine respiratory aerosols emitted by COVID-19 patients during 30 minutes of breathing, 15 minutes of talking, and 15 minutes of singing. Results Thirteen participants (59%) emitted detectable levels of SARS-CoV-2 RNA in respiratory aerosols, including 3 asymptomatic and 1 presymptomatic patient. Viral loads ranged from 63–5821 N gene copies per expiratory activity per participant, with high person-to-person variation. Patients earlier in illness were more likely to emit detectable RNA. Two participants, sampled on day 3 of illness, accounted for 52% of total viral load. Overall, 94% of SARS-CoV-2 RNA copies were emitted by talking and singing. Interestingly, 7 participants emitted more virus from talking than singing. Overall, fine aerosols constituted 85% of the viral load detected in our study. Virus cultures were negative. Conclusions Fine aerosols produced by talking and singing contain more SARS-CoV-2 copies than coarse aerosols and may play a significant role in SARS-CoV-2 transmission. Exposure to fine aerosols, especially indoors, should be mitigated. Isolating viable SARS-CoV-2 from respiratory aerosol samples remains challenging; whether this can be more easily accomplished for emerging SARS-CoV-2 variants is an urgent enquiry necessitating larger-scale studies.

SARS-CoV-2 is a newly identified member of the coronavirus family that has caused the Coronavirus disease 2019 (COVID-19) pandemic. This rapidly evolving and unrelenting SARS-CoV-2 has disrupted the lives and livelihoods of millions worldwide. As of 23 August 2021, a total of 211,373,303 COVID-19 cases have been confirmed globally with a death toll of 4,424,341. A strong understanding of the infection pathway of SARS-CoV-2, and how our immune system responds to the virus is highly pertinent for guiding the development and improvement of effective treatments. In this review, we discuss the current understanding of neutralising antibodies (NAbs) and their implications in clinical practice. The aspects include the pathophysiology of the immune response, particularly humoral adaptive immunity and the roles of NAbs from B cells in infection clearance. We summarise the onset and persistence of IgA, IgM and IgG antibodies, and we explore their roles in neutralising SARS-CoV-2, their persistence in convalescent individuals, and in reinfection. Furthermore, we also review the applications of neutralising antibodies in the clinical setting—from predictors of disease severity to serological testing to vaccinations, and finally in therapeutics such as convalescent plasma infusion.

Background The increasing implementation of transcatheter aortic valve implantation (TAVI) in Australia warrants real-world data on the prevalence and outcomes of these patients. The aim of this study is to describe trends in case-volumes of TAVI in New South Wales (NSW), Australia and associated mortality outcomes. Methods From the Centre of Health Record Linkage registry, all NSW residents who underwent TAVI between 5 June 2013 and 30 June 2018 were identified. Cause-specific mortality was tracked from the statewide death registry. Temporal trends in case-volumes between 2013 and 2018 were assessed by linear regression. Binary logistic regression was used to compare differences in in-hospital and 30-day mortality, while Cox proportional hazards regression was used to compare mortality beyond 30 days. Results Case-volumes increased from 30 in 2013 to 345 by 2017. The cohort comprised 1,098 persons (mean[±SD] age: 83.3±7.7 yrs). Cumulative in-hospital, 180-day and at end-of-study (mean: 1.8±1.2 yrs) all-cause mortality were 1.3% (n=14), 4.9% (n=54) and 20.3% (n=224) respectively. Heart failure (14.3%, n=2), myocardial infarction (14.3%, n=2), and sepsis (14.3%, n=2) were the primary causes of in-hospital death. Post-discharge, sepsis (25.2%, n=53) was the main cause-specific death, while combined cardiovascular deaths accounted for 46% (n=97), mostly from heart failure (n=35). Heart failure, chronic kidney disease, and requirement for ventilation post-TAVI were independent predictors of in-hospital death and at 180 days. TAVI procedure in low-volume public centres was a predictor of mortality at 180 days. Conclusion The number of TAVI procedures increased 10-fold between 2013 and 2017 state-wide, with mortality rates comparable to international cohorts at short and medium-term follow-up. Pre-existing comorbidities and site-specific caseloads may be important determinants of outcome, emphasising the importance of appropriate patient selection and treating centre.

Haiyu Hong,1– 3,* Kai Sen Tan,3– 6,* Yan Yan,3,7,8,* Fenghong Chen,2,* Hsiao Hui Ong,3,5 Yukei Oo,4 Jing Liu,3,5 Yew Kwang Ong,3,9 Mark Thong,3,9 Richard Sugrue,10 Vincent T Chow,4,5 De Yun Wang3,5 1Allergy Center, Department of Otolaryngology, The Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai, People’s Republic of China; 2Otorhinolaryngology Hospital, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, People’s Republic of China; 3Department of Otolaryngology, Yong Loo Lin School of Medicine, National University of Singapore, National University Health System, Singapore; 4Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, National University Health System, Singapore; 5NUHS Infectious Diseases Translational Research Program, Yong Loo Lin School of Medicine, National University of Singapore, Singapore; 6Biosafety level 3 Core Facility, Yong Loo Lin School of Medicine, National University Health System, National University of Singapore, Singapore; 7Guangdong Provincial Key Laboratory of Biomedical and Guangdong Engineering Research Center of Molecular Imaging, The Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai, People’s Republic of China; 8Central Laboratory, The Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai, People’s Republic of China; 9Department of Otolaryngology Head & Neck Surgery, National University Hospital, National University Health System, Singapore; 10School of Biological Sciences, Nanyang Technological University, Singapore*These authors contributed equally to this workCorrespondence: Vincent T ChowDepartment of Microbiology and Immunology, National University of Singapore, 5 Science Drive 2, Singapore, 117545, SingaporeEmail micctk@nus.edu.sgDe Yun WangDepartment of Otolaryngology, National University of Singapore, National University Health System, 1E Kent Ridge Road, Singapore, 119228, SingaporeEmail entwdy@nus.edu.sgBackground: Epithelial cytokines including IL-25, IL-33 and thymic stromal lymphopoietin (TLSP) are recently established as drivers of type 2 chronic inflammatory diseases such as chronic rhinosinusitis with nasal polyps (CRSwNP). Here, we further confirmed the increased expression of IL-25 in CRSwNP and investigated potential contributors of IL-25 in CRSwNP epithelium.Methods: Sixty CRSwNP, 25 CRSsNP and 15 healthy control tissues were examined for IL-25 expression and for the accompanying type 2 inflammatory cytokines. We then tested different respiratory virus infections on human nasal epithelial cells (hNECs) for their ability to trigger IL-25 expression. In addition, we subjected hNECs generated from CRSwNP tissues to pretreatment with recombinant interferon-alpha (IFN-α) prior to viral infection to evaluate IFN effects on IL-25 induction.Results: We confirmed that significantly enhanced levels of IL-25 were observed in CRSwNP tissues, and that IL-25 expression correlated with type 2 inflammatory cytokine expression. In vitro, we observed significantly elevated IL-25 in hNECs infected with influenza A virus as early as 24 hours post-infection (hpi), regardless of tissue origin, and IL-25 correlated positively with viral load. While other respiratory viruses exhibited increasing trends of IL-25, these were not significant at the time-points tested. IFN-α treatment of CRSwNP epithelium was found to exert bimodal effects, ie IFN-α treatment alone induced moderate IL-25 expression, whereas IFN-α pretreatment of hNECs before influenza infection significantly diminished IL-25 induction by active influenza virus infection.Conclusion: We have authenticated the observation of elevated IL-25 in CRSwNP, which is correlated with type 2 inflammatory cytokines. Notably, we identified influenza virus infection as a potential contributor of IL-25 in both control and CRSwNP epithelium during active infection. This IL-25 induction can be abated by IFN-α pretreatment which ameliorated active influenza infection.Trial Registration: Chictr.org.cn ChiCTR-BON-16010179, Registered 18 December 2016, http://www.chictr.org.cn/showproj.aspx?proj=17331. The authors agree on the sharing of deidentified participant data where it pertains to request directly related to the data in this article when contacted (Haiyu Hong; honghy@mail.sysu.edu.cn).Keywords: chronic rhinosinusitis with nasal polyps, interferon-alpha, interleukin 25, respiratory viruses, influenza virus, type 2 inflammation

Excessive neutrophil influx, their released neutrophil extracellular traps (NETs), and extracellular histones are associated with disease severity in influenza-infected patients. Neutrophil chemokine receptor CXC chemokine receptor 2 (CXCR2) is a critical target for suppressing neutrophilic inflammation. Herein, temporal dynamics of neutrophil activity and NETosis were investigated to determine the optimal timing of treatment with the CXCR2 antagonist, SCH527123 (2-hydroxy-N,N-dimethyl-3-[2-([(R)-1-(5-methyl-furan-2-yl)-propyl]amino)-3,4-dioxo-cyclobut-1-enylamino]-benzamide), and its efficacy together with antiviral agent, oseltamivir, was tested in murine and piglet influenza-pneumonia models. SCH527123 plus oseltamivir markedly improved survival of mice infected with lethal influenza, and diminished lung pathology in swine-influenza-infected piglets. Mechanistically, addition of SCH527123 in the combination treatment attenuated neutrophil influx, NETosis, in both mice and piglets. Furthermore, neutrophils isolated from influenza-infected mice showed greater susceptibility to NETotic death when stimulated with a CXCR2 ligand, IL-8. In addition, CXCR2 stimulation induced nuclear translocation of neutrophil elastase, and enhanced citrullination of histones that triggers chromatin decondensation during NET formation. Studies on temporal dynamics of neutrophils and NETs during influenza thus provide important insights into the optimal timing of CXCR2 antagonist treatment for attenuating neutrophil-mediated lung pathology. These findings reveal that pharmacologic treatment with CXCR2 antagonist together with an antiviral agent could significantly ameliorate morbidity and mortality in virulent and sublethal influenza infections.

Background Aortic valve surgery (AVS) is the gold standard treatment for symptomatic aortic valve (AV) disease patients. We report the temporal trends in the incidence of patients requiring isolated AVS in an unselected statewide population and their mortality outcomes over 17-years. Methods Patients were identified from the New South Wales, Australia, Admitted-Patient-Data-Collection registry between 1-July-2001 and 31-December-2018. Annual case-volumes and survival outcomes, adjusted for age, sex, referral source, endocarditis, concomitant coronary-artery-bypass-grafting, comorbidities including atrial fibrillation, hypertension and Charlson comorbidity index, were compared across calendar years. Results The study cohort comprised 16436 patients who underwent isolated AVS (mean age: 72.2 ± 11.3y; 67.5% males). Annual case-volume increased from 768 to 1048 cases between 2002 and 2017 (r2 = 0.82; p Conclusion The volume of AVS has increased progressively over time and has been associated with increased use of bioprosthetic valves and markedly improved 30-day and 1-year survival.

Background Contemporary Australian epidemiological data on acute pulmonary embolism (PE) are lacking. Objectives To determine the admission rates of acute PE in Australia, and to assess the temporal trends in short- and medium-term mortality following acute PE. Methods Retrospective population-linkage study of all New South Wales residents admitted with a primary diagnosis of PE between January 1, 2002 and December 31, 2018 using data from the Centre for Health Record Linkage databases. Main outcome measures included temporal trends in total PE admissions and all-cause mortality at prespecified time points up to 1 year, stratified by gender. Results There were 61,607 total PE admissions between 2002 and 2018 (mean ± standard deviation: 3,624 ± 429 admissions per annum; 50.42 ± 3.70 admissions per 100,000 persons per annum). The mean admission rate per annum was higher for females than for males (54.85 ± 3.65 vs. 44.91 ± 4.34 admissions per 100,000 persons per annum, respectively) and remained relatively stable for both genders throughout the study period. The main study cohort, limited to index PE admission only, comprised 46,382 persons (mean age: 64.6 ± 17.3 years; 44.4% males). The cumulative in-hospital, 30-day, 3-month, and 1-year mortality rates were 3.7, 5.6, 9.6, and 16.8%, respectively. When compared with 2002 as the reference year, there was a significant reduction in in-hospital (odds ratio [OR] = 0.34; 95% confidence interval [CI] = 0.25–0.46), 30-day (OR = 0.58, 95% CI = 0.46–0.73), and 1-year (hazard ratio = 0.74, 95% CI = 0.66–0.84) (all p Conclusion Mortality following PE has improved with reductions observed in both short- and medium-term follow-ups between 2002 and 2018 with greater reductions in females despite their higher admission rates over time.

Influenza remains one of the most prevalent viruses circulating amongst humans and has resulted in several pandemics. The prevention and control of H3N2 influenza is complicated by its propensity for evolution, which leads to vaccine mismatch and reduced vaccine efficacies. This study employed the strategy of serial passaging to compare the evolution of the human seasonal influenza strain A/Singapore/G2-31.1/2014(H3N2) in MDCK-SIAT1 versus primary chick embryo fibroblast (CEF) cells. Genetic analysis of the HA, NS1, NA, and PB1 gene segments by Sanger sequencing revealed the presence of specific mutations and a repertoire of viral quasispecies following serial passaging. Most quasispecies were also found in PB1, which exhibited consistently high transversion-to-transition ratios in all five MDCK-SIAT1 passages. Most notably, passage 5 virus harbored the D457G substitution in the HA2 subunit, while passage 3 virus acquired K53Q and Q69H mutations in PB1-F2. An A971 variant leading to a non-synonymous R316Q substitution in PB1 was also identified in MDCK-SIAT1 passages 2 and 4. With an increasing number of passages, the proportion of D457G mutations progressively increased and was associated with larger virus plaque sizes. However, microneutralization assays revealed no significant differences in the neutralizing antibody profiles of human-influenza-immune serum samples against pre-passaged virus and passage 5 virus. In contrast, viable virus was only detected in passage 1 of CEF cells, which gave rise to multiple viral RNA quasispecies. Our findings highlight that serial passaging is able to drive differential adaptation of H3N2 influenza in different host species and may alter viral virulence. More studies are warranted to elucidate the complex relationships between H3N2 virus evolution, viral virulence changes, and low vaccine efficacy.

Clinical features and outcomes of patients with hypoalbuminaemia in acute pulmonary embolism (PE) have never been studied. The present study investigated the incidence and determined the prognostic significance of hypoalbuminaemia in patients with confirmed acute PE.From a dedicated tertiary-referral centre database involving 1,426 consecutive patients admitted with confirmed PE (2000-2012), 1,032 patients had serum albumin assessed on admission (day-1). Patients were stratified into hypoalbuminaemia (35 g/L) or normal serum albumin (≥35 g/L). Multivariable logistic and Cox proportional-hazards regression methods were used to assess 30-day and 90-day all-cause mortality.Hypoalbuminaemia was present in 160 (15.5%) patients at day-1 and was associated with higher mean (±SD) heart rate (94.4 ± 21.8 vs 87.8 ± 21.5 bpm), lower systolic blood pressure (131.0 ± 24.7 vs 142.3 ± 24.7 mmHg), lower arterial oxyhaemoglobin saturation (93.3 ± 6.1% vs 95.6 ± 4.0%), lower day-1 serum sodium (137.0 ± 4.7 vs 138.8 ± 3.8 mmol/L) and haemoglobin levels (114.4 ± 20.7 vs 131.5 ± 18.7 g/L). Patients with hypoalbuminaemia had higher incidence of malignancy (44.4% vs 18.8%) and chronic renal disease (9.4% vs 5.2%), and at admission were less likely to be taking aspirin/clopidogrel (19.3% vs 27.7%) and more likely to be using enoxaparin (6.7% vs 3.0%). During a mean follow-up of 5.0 ± 4.0 years, patients with hypoalbuminaemia had higher 30-day (16.3% vs 3.6%) and 90-day (26.3% vs 6.2%) mortality. Multivariable analyses showed hypoalbuminaemia independently predicted both 30-day (odds ratio 2.57, 95% confidence interval [CI] 1.03-6.41) and 90-day (hazard ratio 2.42 95% CI 1.38-4.22) mortality.Hypoalbuminaemia is an independent predictor of mortality following PE and may improve risk stratification of patients in risk prediction models.

Summary The family of Suppressor of Cytokine Signalling (SOCS) proteins plays pivotal roles in cytokine and immune regulation. Despite their key roles, little attention has been given to the SOCS family as compared to other feedback regulators. To date, SOCS proteins have been found to be exploited by viruses such as herpes simplex virus (HSV), hepatitis B virus (HBV), hepatitis C virus (HCV), Zika virus, respiratory syncytial virus (RSV), Ebola virus, influenza A virus (IAV) and SARS‐CoV, just to name a few. The hijacking and subsequent upregulation of the SOCS proteins upon viral infection, suppress the associated JAK‐STAT signalling activities, thereby reducing the host antiviral response and promoting viral replication. Two SOCS protein family members, SOCS1 and SOCS3 are well‐studied and their roles in the JAK‐STAT signalling pathway are defined as attenuating interferon (IFN) signalling upon viral infection. The upregulation of SOCS protein by SARS‐CoV during the early stages of infection implies strong similarity with SARS‐CoV‐2, given their closely related genomic organisation. Thus, this review aims to outline the plausibility of SOCS protein inhibitors as a potential therapeutic regimen for COVID‐19 patients. We also discuss the antagonists against SOCS protein to offer an overview on the previous ‘successes’ of SOCS protein inhibition in various viral infections that may portray possible clues for COVID‐19 disease management.

BackgroundMultiple SARS-CoV-2 superspreading events suggest that aerosols play an important role in driving the COVID-19 pandemic. However, the detailed roles of coarse (>5μm) and fine (≤5μm) respiratory aerosols produced when breathing, talking, and singing are not well-understood.MethodsUsing a G-II exhaled breath collector, we measured viral RNA in coarse and fine respiratory aerosols emitted by COVID-19 patients during 30 minutes of breathing, 15 minutes of talking, and 15 minutes of singing.ResultsAmong the 22 study participants, 13 (59%) emitted detectable levels of SARS-CoV-2 RNA in respiratory aerosols, including 3 asymptomatic patients and 1 presymptomatic patient. Viral loads ranged from 63–5,821 N gene copies per expiratory activity per patient. Patients earlier in illness were more likely to emit detectable RNA, and loads differed significantly between breathing, talking, and singing. The largest proportion of SARS-CoV-2 RNA copies was emitted by singing (53%), followed by talking (41%) and breathing (6%). Overall, fine aerosols constituted 85% of the viral load detected in our study. Virus cultures were negative.ConclusionsFine aerosols produced by talking and singing contain more SARS-CoV-2 copies than coarse aerosols and may play a significant role in the transmission of SARS-CoV-2. Exposure to fine aerosols should be mitigated, especially in indoor environments where airborne transmission of SARS-CoV-2 is likely to occur. Isolating viable SARS-CoV-2 from respiratory aerosol samples remains challenging, and whether this can be more easily accomplished for emerging SARS-CoV-2 variants is an important enquiry for future studies.Key PointsWe sampled respiratory aerosols emitted by COVID-19 patients and discovered that fine aerosols (≤5μm) generated during talking and singing contain more SARS-CoV-2 copies than coarse aerosols (>5μm) and may play a significant role in the transmission of SARS-CoV-2.

Background: This study compared the genomes of influenza viruses that caused mild infections among outpatients and severe infections among hospitalized patients in Singapore, and characterized their molecular evolution and receptor-binding specificity. Methods: The complete genomes of influenza A/H1N1, A/H3N2 and B viruses that caused mild infections among outpatients and severe infections among inpatients in Singapore during 2012–2015 were sequenced and characterized. Using various bioinformatics approaches, we elucidated their evolutionary, mutational and structural patterns against the background of global and vaccine strains. Results: The phylogenetic trees of the 8 gene segments revealed that the outpatient and inpatient strains overlapped with representative global and vaccine strains. We observed a cluster of inpatients with A/H3N2 strains that were closely related to vaccine strain A/Texas/50/2012(H3N2). Several protein sites could accurately discriminate between outpatient versus inpatient strains, with site 221 in neuraminidase (NA) achieving the highest accuracy for A/H3N2. Interestingly, amino acid residues of inpatient but not outpatient isolates at those sites generally matched the corresponding residues in vaccine strains, except at site 145 of hemagglutinin (HA). This would be especially relevant for future surveillance of A/H3N2 strains in relation to their antigenicity and virulence. Furthermore, we observed a trend in which the HA proteins of influenza A/H3N2 and A/H1N1 exhibited enhanced ability to bind both avian and human host cell receptors. In contrast, the binding ability to each receptor was relatively stable for the HA of influenza B. Conclusions: Overall, our findings extend our understanding of the molecular and structural evolution of influenza virus strains in Singapore within the global context of these dynamic viruses. Keywords: Influenza, A/H1N1 viruses, A/H3N2 viruses, Influenza B, Evolution, Mutations, Receptor binding, Severity, Singapore

Enterovirus 71 (EV-A71) is a human pathogen that does not naturally infect rodent cells. However, virus strains that productively infect rodent cells could be produced by serial passage in a rodent cell line. The resulting viruses are useful tools to identify molecular determinants of adaptation to a new host species. We adapted an EV-A71 clinical isolate (EV71:BS) in Chinese hamster ovary (CHO-K1) cells and sequenced the genome of the resulting virus. We explored the contribution of viral capsid mutations that may have contributed to the CHO-adapted phenotype by introducing the mutations surrounding the receptor-binding canyon region into the genome of EV71:BS by reverse genetics tools. We subsequently assessed whether the resulting mutant viruses productively infected CHO-K1 cells. Contrary to previous reports in the literature, our findings demonstrate that a single amino acid substitution in capsid VP2149 K→I is not sufficient for the resulting mutant virus to infect CHO-K1 cells. Moreover, a mutant virus harbouring other capsid mutations we evaluated (VP1 K98→E, E145→A, and L169→F) also did not infect CHO-K1 cells. These findings strongly suggest that cooperation of various adaptive mutations in the capsid is necessary to enable successful EV-A71 infection of a different host species.

Background Cell-surface mucins are expressed in apical epithelial cells of the respiratory tract, and contribute a crucial part of the innate immune system. Despite anti-inflammatory or antiviral functions being revealed for certain cell-surface mucins such as MUC1, the roles of other mucins are still poorly understood, especially in viral infections. Methods To further identify mucins significant in influenza infection, we screened the expression of mucins in human nasal epithelial cells infected by H3N2 influenza A virus. Results We found that the expression of MUC15 was significantly upregulated upon infection, and specific only to active infection. While MUC15 did not interact with virus particles or reduce viral replication directly, positive correlations were observed between MUC15 and inflammatory factors in response to viral infection. Given that the upregulation of MUC15 was only triggered late into infection when immune factors (including cytokines, chemokines, EGFR and phosphorylated ERK) started to peak and plateau, MUC15 may potentially serve an immunomodulatory function later during influenza viral infection. Conclusions Our study revealed that MUC15 was one of the few cell-surface mucins induced during influenza infection. While MUC15 did not interact directly with influenza virus, we showed that its increase coincides with the peak of immune activation and thus MUC15 may serve an immunomodulatory role during influenza infection. Electronic supplementary material The online version of this article (10.1186/s12879-019-4213-y) contains supplementary material, which is available to authorized users.

The recent COVID-19 pandemic has highlighted the urgency to develop effective antiviral therapies against the disease. Murine hepatitis virus (MHV) is a coronavirus that infects mice and shares some sequence identity to SARS-CoV-2. Both viruses belong to the Betacoronavirus genus, and MHV thus serves as a useful and safe surrogate model for SARS-CoV-2 infections. Clinical trials have indicated that remdesivir is a potentially promising antiviral drug against COVID-19. Using anin vitromodel of MHV infection of RAW264.7 macrophages, the safety and efficacy of monotherapy of remdesivir, chloroquine, ivermectin, and doxycycline were investigated. Of the four drugs tested, remdesivir monotherapy exerted the strongest inhibition of live virus and viral RNA replication of about 2-log10and 1-log10, respectively (at 6 µM). Ivermectin treatment showed the highest selectivity index. Combination drug therapy was also evaluated using remdesivir (6 µM) together with chloroquine (15 µM), ivermectin (2 µM) or doxycycline (15 µM) – above their IC50 values and at high macrophage cell viability of over 95%. The combination of remdesivir and ivermectin exhibited highly potent synergism by achieving significant reductions of about 7-log10of live virus and 2.5-log10of viral RNA in infected macrophages. This combination also resulted in the lowest cytokine levels of IL-6, TNF-α, and leukemia inhibitory factor. The next best synergistic combination was remdesivir with doxycycline, which decreased levels of live virus by ~3-log10and viral RNA by ~1.5-log10. These results warrant further studies to explore the mechanisms of action of the combination therapy, as well as futurein vivoexperiments and clinical trials for the treatment of SARS-CoV-2 infection.

The ongoing COVID-19 pandemic is a clear and present threat to global public health. Research into how the causative SARS-CoV-2 virus together with its individual constituent genes and proteins interact with target host cells can facilitate the development of improved strategies to manage the acute and long-term complications of COVID-19. In this study, to better understand the biological roles of critical SARS-CoV-2 proteins, we determined and compared the host transcriptomic responses of the HL-CZ human pro-monocytic cell line upon transfection with key viral genes encoding the spike S1 subunit, S2 subunit, nucleocapsid protein (NP), NSP15 (endoribonuclease), and NSP16 (2′-O-ribose-methyltransferase). RNA sequencing followed by gene set enrichment analysis and other bioinformatics tools revealed that host genes associated with topologically incorrect protein, virus receptor activity, heat shock protein binding, endoplasmic reticulum stress, antigen processing and presentation were up-regulated in the presence of viral spike S1 expression. With spike S2 expression, pro-monocytic genes associated with the interferon-gamma-mediated signaling pathway, regulation of phosphatidylinositol 3-kinase activity, adipocytokine signaling pathway, and insulin signaling pathway were down-regulated, whereas those associated with cytokine-mediated signaling were up-regulated. The expression of NSP15 induced the up-regulation of genes associated with neutrophil degranulation, neutrophil-mediated immunity, oxidative phosphorylation, prion disease, and pathways of neurodegeneration. The expression of NSP16 resulted in the down-regulation of genes associated with S-adenosylmethionine-dependent methyltransferase activity. The expression of NP down-regulated genes associated with positive regulation of neurogenesis, nervous system development, and heart development. Taken together, the complex transcriptomic alterations arising from these viral-host gene interactions offer useful insights into host genes and their pathways that potentially contribute to SARS-CoV-2 pathogenesis.

The early interactions between the nasal epithelial layer and the innate immune cells during viral infections remains an under-explored area. The significance of innate immunity signaling in viral infections has increased substantially as patients with respiratory infections who exhibit high innate T cell activation show a better disease outcome. Hence, dissecting these early innate immune interactions allows the elucidation of the processes that govern them and may facilitate the development of potential therapeutic targets and strategies for dampening or even preventing early progression of viral infections. This protocol details a versatile model that can be used to study early crosstalk, interactions, and activation of innate immune cells from factors secreted by virally infected airway epithelial cells. Using an H3N2 influenza virus (A/Aichi/2/1968) as the representative virus model, innate cell activation of co-cultured peripheral blood mononuclear cells (PBMCs) has been analyzed using flow cytometry to investigate the subsets of cells that are activated by the soluble factors released from the epithelium in response to the viral infection. The results demonstrate the gating strategy for differentiating the subsets of cells and reveal the clear differences between the activated populations of PBMCs and their crosstalk with the control and infected epithelium. The activated subsets can then be further analyzed to determine their functions as well as molecular changes specific to the cells. Findings from such a crosstalk investigation may uncover factors that are important for the activation of vital innate cell populations, which are beneficial in controlling and suppressing the progression of viral infection. Furthermore, these factors can be universally applied to different viral diseases, especially to newly emerging viruses, to dampen the impact of such viruses when they first circulate in naïve human populations.

Importance Three-dimensionally printed nasopharyngeal swabs (3DP swabs) have been used to mitigate swab shortages during the coronavirus disease 2019 (COVID-19) pandemic. Clinical validation for diagnostic accuracy and consistency, as well as patient acceptability, is crucial to evaluate the swab’s performance. Objective To determine the accuracy and acceptability of the 3DP swab for identifying severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Design, Setting, and Participants A diagnostic study was conducted from May to July 2020 at 2 tertiary care centers in Singapore with different reference swabs (FLOQSwab [COPAN Diagnostics] or Dacron swab [Deltalab]) and swab processing techniques (wet or dry) to evaluate the performance of the 3DP swab compared with traditional, standard-of-care nasopharyngeal swabs used in health care institutions. The participants were patients with COVID-19 in the first 2 weeks of illness and controls with acute respiratory illness with negative test results for SARS-CoV-2. Paired nasopharyngeal swabs were obtained from the same nostril and tested for SARS-CoV-2 by reverse-transcriptase polymerase chain reaction. The sequence of swabs was randomized based on odd and even participant numbers. Main Outcomes and Measures Primary outcome measures were overall agreement (OA), positive percentage agreement (PPA), and negative percentage agreement of the 3DP swab compared with reference swabs. Secondary outcome measures were the correlation of cycle threshold (Ct) values of both swabs. Results The mean (SD) age of participants was 45.4 (13.1) years, and most participants were men (87 of 89 [97.8%]), in keeping with the epidemiology of the COVID-19 pandemic in Singapore. A total of 79 patients with COVID-19 and 10 controls were recruited. Among the patients with COVID-19, the overall agreement and PPA of the 3DP swab was 91.1% and 93.5%, respectively, compared with reference swabs. The PPA was 100% for patients with COVID-19 who were tested within the first week of illness. All controls tested negative. The reverse-transcriptase polymerase chain reaction Ct values for theORF1abandE-genetargets showed a strong correlation (intraclass correlations coefficient, 0.869-0.920) between the 3DP and reference swab on independent testing at each institution despite differences in sample processing. Discordant results for both gene targets were observed only at high Ct values. Conclusions and Relevance In this diagnostic study of 79 patients with COVID-19 and 10 controls, the 3DP swab performed accurately and consistently across health care institutions and could help mitigate strained resources in the escalating COVID-19 pandemic.