SARS-CoV-2 RNA in exhaled air of hospitalized COVID-19 patients.

Knowledge about contagiousness is key to accurate management of hospitalized COVID-19 patients. Epidemiological studies suggest that in addition to transmission through droplets, aerogenic SARS-CoV-2 transmission contributes to the spread of infection. However, the presence of virus in exhaled air has not yet been sufficiently demonstrated. In pandemic situations low tech disposable and user-friendly bedside devices are required, while commercially available samplers are unsuitable for application in patients with respiratory distress. We included 49 hospitalized COVID-19 patients and used a disposable modular breath sampler to measure SARS-CoV-2 RNA load in exhaled air samples and compared these to SARS-CoV-2 RNA load of combined nasopharyngeal throat swabs and saliva. Exhaled air sampling using the modular breath sampler has proven feasible in a clinical COVID-19 setting and demonstrated viral detection in 25% of the patients.

Using human iPSC-derived kidney organoids to decipher SARS-CoV-2 pathology on single cell level.

We describe a protocol for single-cell RNA sequencing of SARS-CoV-2-infected human induced pluripotent stem cell (iPSC)-derived kidney organoids. After inoculation of kidney organoids with virus, we use mechanical and enzymatic disruption to obtain single cell suspensions. Next, we process the organoid-derived cells into sequencing-ready SARS-CoV-2-targeted libraries. Subsequent sequencing analysis reveals changes in kidney cells after virus infection. The protocol was designed for kidney organoids cultured in a 6-well transwell format but can be adapted to organoids with different organ backgrounds. For complete details on the use and execution of this protocol, please refer to Jansen et al. (2022).

SARS-CoV-2 infects the human kidney and drives fibrosis in kidney organoids.

Kidney failure is frequently observed during and after COVID-19, but it remains elusive whether this is a direct effect of the virus. Here, we report that SARS-CoV-2 directly infects kidney cells and is associated with increased tubule-interstitial kidney fibrosis in patient autopsy samples. To study direct effects of the virus on the kidney independent of systemic effects of COVID-19, we infected human-induced pluripotent stem-cell-derived kidney organoids with SARS-CoV-2. Single-cell RNA sequencing indicated injury and dedifferentiation of infected cells with activation of profibrotic signaling pathways. Importantly, SARS-CoV-2 infection also led to increased collagen 1 protein expression in organoids. A SARS-CoV-2 protease inhibitor was able to ameliorate the infection of kidney cells by SARS-CoV-2. Our results suggest that SARS-CoV-2 can directly infect kidney cells and induce cell injury with subsequent fibrosis. These data could explain both acute kidney injury in COVID-19 patients and the development of chronic kidney disease in long COVID.

Interferon gamma immunotherapy in five critically ill COVID-19 patients with impaired cellular immunity: A case series.

BACKGROUND: Prolonged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) shedding has been described in immunocompromised coronavirus disease 2019 (COVID-19) patients, resulting in protracted disease and poor outcome. Specific therapy to improve viral clearance and outcome for this group of patients is currently unavailable. METHODS: Five critically ill COVID-19 patients with severe defects in cellular immune responses, high SARS-CoV-2 viral RNA loads, and no respiratory improvement were treated with interferon gamma, 100 µg subcutaneously, thrice weekly. Bronchial secretion was collected every 48 h for routine diagnostic SARS-CoV-2 RT-PCR and viral culture. FINDINGS: Interferon gamma administration was followed by a rapid decline in SARS-CoV-2 load and a positive-to-negative viral culture conversion. Four patients recovered, and no signs of hyperinflammation were observed. CONCLUSIONS: Interferon gamma may be considered as adjuvant immunotherapy in a subset of immunocompromised COVID-19 patients. FUNDING: A.v.L. and R.v.C. are supported by National Institutes of Health (R01AI145781). G.J.O. and R.P.v.R. are supported by a VICI grant (016.VICI.170.090) from the Dutch Research Council (NWO). W.F.A. is supported by a clinical fellowship grant (9071561) of Netherlands Organization for Health Research and Development. M.G.N. is supported by an ERC advanced grant (833247) and a Spinoza grant of the Netherlands Organization for Scientific Research.

Berberine and Obatoclax Inhibit SARS-Cov-2 Replication in Primary Human Nasal Epithelial Cells In Vitro.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged as a new human pathogen in late 2019 and it has infected over 100 million people in less than a year. There is a clear need for effective antiviral drugs to complement current preventive measures, including vaccines. In this study, we demonstrate that berberine and obatoclax, two broad-spectrum antiviral compounds, are effective against multiple isolates of SARS-CoV-2. Berberine, a plant-derived alkaloid, inhibited SARS-CoV-2 at low micromolar concentrations and obatoclax, which was originally developed as an anti-apoptotic protein antagonist, was effective at sub-micromolar concentrations. Time-of-addition studies indicated that berberine acts on the late stage of the viral life cycle. In agreement, berberine mildly affected viral RNA synthesis, but it strongly reduced infectious viral titers, leading to an increase in the particle-to-pfu ratio. In contrast, obatoclax acted at the early stage of the infection, which is in line with its activity to neutralize the acidic environment in endosomes. We assessed infection of primary human nasal epithelial cells that were cultured on an air-liquid interface and found that SARS-CoV-2 infection induced and repressed expression of specific sets of cytokines and chemokines. Moreover, both obatoclax and berberine inhibited SARS-CoV-2 replication in these primary target cells. We propose berberine and obatoclax as potential antiviral drugs against SARS-CoV-2 that could be considered for further efficacy testing.