Rhinovirus C replication is associated with the endoplasmic reticulum and triggers cytopathic effects in an in vitro model of human airway epithelium

The clinical impact of rhinovirus C (RV-C) is well-documented; yet, the viral life cycle remains poorly defined. Thus, we characterized RV-C15 replication at the single-cell level and its impact on the human airway epithelium (HAE) using a physiologically-relevant in vitro model. RV-C15 replication was restricted to ciliated cells where viral RNA levels peaked at 12 hours post-infection (hpi), correlating with elevated titers in the apical compartment at 24hpi. Notably, infection was associated with a loss of polarized expression of the RV-C receptor, cadherin-related family member 3. Visualization of double-stranded RNA (dsRNA) during RV-C15 replication revealed two distinct replication complex arrangements within the cell, likely corresponding to different time points in infection. To further define RV-C15 replication sites, we analyzed the expression and colocalization of giantin, phosphatidylinositol-4-phosphate, and calnexin with dsRNA. Despite observing Golgi fragmentation by immunofluorescence during RV-C15 infection as previously reported for other RVs, a high ratio of calnexin-dsRNA colocalization implicated the endoplasmic reticulum as the primary site for RV-C15 replication in HAE. RV-C15 infection was also associated with elevated stimulator of interferon genes (STING) expression and the induction of incomplete autophagy, a mechanism used by other RVs to facilitate non-lytic release of progeny virions. Notably, genetic depletion of STING in HAE attenuated RV-C15 and -A16 (but not -B14) replication, corroborating a previously proposed proviral role for STING in some RV infections. Finally, RV-C15 infection resulted in a temporary loss in epithelial barrier integrity and the translocation of tight junction proteins while a reduction in mucociliary clearance indicated cytopathic effects on epithelial function. Together, our findings identify both shared and unique features of RV-C replication compared to related rhinoviruses and define the impact of RV-C on both epithelial cell organization and tissue functionality–aspects of infection that may contribute to pathogenesis in vivo.

Pre-clinical evaluation of antiviral activity of nitazoxanide against Sars-CoV-2

To address the emergence of SARS-CoV-2, multiple clinical trials in humans were rapidly started, including those involving an oral treatment by nitazoxanide, despite no or limited pre-clinical evidence of antiviral efficacy. In this work, we present a complete pre-clinical evaluation of the antiviral activity of nitazoxanide against SARS-CoV-2. First, we confirmed the in vitro efficacy of nitazoxanide and tizoxanide (its active metabolite) against SARS-CoV-2. Then, we demonstrated nitazoxanide activity in a reconstructed bronchial human airway epithelium model. In a SARS-CoV-2 virus challenge model in hamsters, oral and intranasal treatment with nitazoxanide failed to impair viral replication in commonly affected organs. We hypothesized that this could be due to insufficient diffusion of the drug into organs of interest. Indeed, our pharmacokinetic study confirmed that concentrations of tizoxanide in organs of interest were always below the in vitro EC50. These preclinical results suggest, if directly applicable to humans, that the standard formulation and dosage of nitazoxanide is not effective in providing antiviral therapy for Covid-19.

Nonspecific binding of common anti-CFTR antibodies in ciliated cells of human airway epithelium

There is evidence that the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel is highly expressed at the apical pole of ciliated cells in human bronchial epithelium (HBE), however recent studies have detected little CFTR mRNA in those cells. To understand this discrepancy we immunostained well differentiated primary HBE cells using CFTR antibodies. We confirmed apical immunofluorescence in ciliated cells and quantified the covariance of the fluorescence signals and that of an antibody against the ciliary marker centrin-2 using image cross-correlation spectroscopy (ICCS). Super-resolution stimulated emission depletion (STED) imaging localized the immunofluorescence in distinct clusters at the bases of the cilia. However, similar apical fluorescence was observed when the monoclonal CFTR antibodies 596, 528 and 769 were used to immunostain ciliated cells expressing F508del-CFTR, or cells lacking CFTR due to a Class I mutation. A BLAST search using the CFTR epitope identified a similar amino acid sequence in the ciliary protein rootletin X1. Its expression level correlated with the intensity of immunostaining by CFTR antibodies and it was detected by 596 antibody after transfection into CFBE cells. These results may explain the high apparent expression of CFTR in ciliated cells and reports of anomalous apical immunofluorescence in well differentiated cells that express F508del-CFTR.

Oral prodrug of remdesivir parent GS-441524 is efficacious against SARS-CoV-2 in ferrets

Remdesivir is an antiviral approved for COVID-19 treatment, but its wider use is limited by intravenous delivery. An orally bioavailable remdesivir analog may boost therapeutic benefit by facilitating early administration to non-hospitalized patients. This study characterizes the anti-SARS-CoV-2 efficacy of GS-621763, an oral prodrug of remdesivir parent nucleoside GS-441524. Both GS-621763 and GS-441524 inhibit SARS-CoV-2, including variants of concern (VOC) in cell culture and human airway epithelium organoids. Oral GS-621763 is efficiently converted to plasma metabolite GS-441524, and in lungs to the triphosphate metabolite identical to that generated by remdesivir, demonstrating a consistent mechanism of activity. Twice-daily oral administration of 10 mg/kg GS-621763 reduces SARS-CoV-2 burden to near-undetectable levels in ferrets. When dosed therapeutically against VOC P.1 gamma γ, oral GS-621763 blocks virus replication and prevents transmission to untreated contact animals. These results demonstrate therapeutic efficacy of a much-needed orally bioavailable analog of remdesivir in a relevant animal model of SARS-CoV-2 infection.

Early nasal type I IFN immunity against SARS-CoV-2 is compromised in patients with autoantibodies against type I IFNs

IFN-I and IFN-III immunity in the nasal mucosa is poorly characterized during SARS-CoV-2 infection. We analyze the nasal IFN-I/III signature, namely the expression of ISGF-3–dependent IFN-stimulated genes, in mildly symptomatic COVID-19 patients and show its correlation with serum IFN-α2 levels, which peak at symptom onset and return to baseline from day 10 onward. Moreover, the nasal IFN-I/III signature correlates with the nasopharyngeal viral load and is associated with the presence of infectious viruses. By contrast, we observe low nasal IFN-I/III scores despite high nasal viral loads in a subset of critically ill COVID-19 patients, which correlates with the presence of autoantibodies (auto-Abs) against IFN-I in both blood and nasopharyngeal mucosa. In addition, functional assays in a reconstituted human airway epithelium model of SARS-CoV-2 infection confirm the role of such auto-Abs in abrogating the antiviral effects of IFN-I, but not those of IFN-III. Thus, IFN-I auto-Abs may compromise not only systemic but also local antiviral IFN-I immunity at the early stages of SARS-CoV-2 infection.