SARS-CoV-2 impairs interferon production via NSP2-induced repression of mRNA translation

Viruses evade the innate immune response by suppressing the production or activity of cytokines such as type I interferons (IFNs). Here we report the discovery of a novel mechanism by which the SARS-CoV-2 virus co-opts an intrinsic cellular machinery to suppress the production of the key immunostimulatory cytokine IFN-{beta}. We reveal that the SARS-CoV-2 encoded Non-Structural Protein 2 (NSP2) directly interacts with the cellular GIGYF2 protein. This interaction enhances the binding of GIGYF2 to the mRNA cap-binding protein 4EHP, thereby repressing the translation of the Ifnb1 mRNA. Depletion of GIGYF2 or 4EHP significantly enhances IFN-{beta} production, leading to reduced viral infection. Our findings reveal a new target for rescuing the antiviral innate immune response to SARS-CoV-2 and other RNA viruses.

An allele-specific primer extension assay to quantify the proportion of B.1.1.7-specific SARS-CoV-2 RNA in wastewater

The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has claimed millions of lives to date. Antigenic drift has resulted in viral variants with putatively greater transmissibility, virulence, or both. Early and near real-time detection of these variants of concern (VOC) and the ability to accurately follow their incidence and prevalence in communities is wanting. Wastewater-based epidemiology (WBE), which uses nucleic acid amplification tests to detect viral fragments, is a faithful proxy of COVID-19 incidence and prevalence, and thus offers the potential to monitor VOC viral load in a given population. Here, we describe and validate a primer extension PCR strategy targeting a signature mutation in the N gene of SARS-CoV-2. This allows quantification of the proportional expression of B.1.1.7 versus non-B.1.1.7 alleles in wastewater without the need to employ quantitative RT-PCR standard curves. We show that the wastewater B.1.1.7 profile correlates with its clinical counterpart and benefits from a near real-time and facile data collection and reporting pipeline. This assay can be quickly implemented within a current SARS-CoV-2 WBE framework with minimal cost; allowing early and contemporaneous estimates of B.1.1.7 community transmission prior to, or in lieu of, clinical screening and identification. Our study demonstrates that this strategy can provide public health units with an additional and much needed tool to rapidly triangulate VOC incidence/prevalence with high sensitivity and lineage specificity.

Catching a resurgence: Increase in SARS-CoV-2 viral RNA identified in wastewater 48 hours before COVID-19 clinical tests and 96 hours before hospitalizations

Curtailing the Spring 2020 COVID-19 surge required sweeping and stringent interventions by governments across the world. Wastewater-based COVID-19 epidemiology programs have been initiated in many countries to provide public health agencies with a complementary disease tracking metric and facile surveillance tool. However, their efficacy in prospectively capturing resurgence following a period of low prevalence is unclear. In this study, the SARS-CoV-2 viral signal was measured in primary clarified sludge harvested every two days at the City of Ottawas water resource recovery facility during the summer of 2020, when clinical testing recorded daily percent positivity below 1%. In late July, increases of >400% in normalized SARS-CoV-2 RNA signal in wastewater were identified 48 hours prior to reported >300% increases in positive cases that were retrospectively attributed to community-acquired infections. During this resurgence period, SARS-CoV-2 RNA signal in wastewater preceded the reported >160% increase in community hospitalizations by approximately 96 hours. This study supports wastewater-based COVID-19 surveillance of populations in augmenting the efficacy of diagnostic testing, which can suffer from sampling biases or timely reporting as in the case of hospitalization census.

Quantitative analysis of SARS-CoV-2 RNA from wastewater solids in communities with low COVID-19 incidence and prevalence

In the absence of an effective vaccine to prevent COVID-19 it is important to be able to track community infections to inform public health interventions aimed at reducing the spread and therefore reduce pressures on health-care units, improve health outcomes and reduce economic uncertainty. Wastewater surveillance has rapidly emerged as a potential tool to effectively monitor community infections for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), through measuring trends of viral RNA signal in wastewater systems. In this study SARS-CoV-2 viral RNA N1 and N2 genes are quantified in solids collected from influent post grit solids (PGS) and primary clarified sludge (PCS) in two water resource recovery facilities (WRRF) serving Canadas national capital region, i.e., the City of Ottawa, ON (pop. {approx} 1.1M) and the City of Gatineau, QC (pop. {approx} 280K). PCS samples show signal inhibition using RT-ddPCR compared to RT-qPCR, with PGS samples showing similar quantifiable concentrations of RNA using both assays. RT-qPCR shows higher frequency of detection of N1 and N2 genes in PCS (92.7, 90.6%) as compared to PGS samples (79.2, 82.3%). Sampling of PCS may therefore be an effective approach for SARS-CoV-2 viral quantification, especially during periods of declining and low COVID-19 incidence in the community. The pepper mild mottle virus (PMMV) is determined to have a less variable RNA signal in PCS over a three month period for two WRRFs, regardless of environmental conditions, compared to Bacteroides 16S rRNA or human eukaryotic 18S rRNA, making PMMV a potentially useful biomarker for normalization of SARS-CoV-2 signal. PMMV-normalized PCS RNA signal from WRRFs of two cities correlated with the regional public health epidemiological metrics, identifying PCS normalized to a fecal indicator (PMMV) as a potentially effective tool for monitoring trends during decreasing and low-incidence of infection of SARS-Cov-2 in communities.

Identification of SARS-CoV2-mediated suppression of NRF2 signaling reveals a potent antiviral and anti-inflammatory activity of 4-octyl-itaconate and dimethyl fumarate

Antiviral strategies to inhibit Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2) and the pathogenic consequences of COVID-19 are urgently required. Here we demonstrate that the NRF2 anti-oxidant gene expression pathway is suppressed in biopsies obtained from COVID-19 patients. Further, we uncover that NRF2 agonists 4-octyl-itaconate (4-OI) and the clinically approved dimethyl fumarate (DMF) induce a cellular anti-viral program, which potently inhibits replication of SARS-CoV2 across cell lines. The anti-viral program extended to inhibit the replication of several other pathogenic viruses including Herpes Simplex Virus-1 and-2, Vaccinia virus, and Zika virus through a type I interferon (IFN)-independent mechanism. In addition, induction of NRF2 by 4-OI and DMF limited host inflammatory responses to SARS-CoV2 infection associated with airway COVID-19 pathology. In conclusion, NRF2 agonists 4-OI and DMF induce a distinct IFN-independent antiviral program that is broadly effective in limiting virus replication and suppressing the pro-inflammatory responses of human pathogenic viruses, including SARS-CoV2. One Sentence SummaryNRF2 agonists 4-octyl-itaconate (4-OI) and dimethyl fumarate inhibited SARS-CoV2 replication and virus-induced inflammatory responses, as well as replication of other human pathogenic viruses.