ABSTRACT
Human induced pluripotent stem cell (iPSC) derived intestinal organoids (HIOs) represent an inexhaustible cellular resource that could serve as a valuable tool to study viral infections such as SARS-CoV-2 as well as other enteric viruses that infect the intestinal epithelium. Intestinal symptoms of COVID-19 are present in a significant number of patients, and include nausea, diarrhea, and viral RNA shedding in feces. Using this platform we found that SARS-CoV-2 productively infects both proximally and distally patterned HIOs, leading to the release of infectious viral particles while stimulating a robust transcriptomic response, including a significant upregulation of interferon-related genes that appeared to be conserved across multiple epithelial cell types. These findings illuminate a potential inflammatory epithelial-specific signature that may contribute to both the multisystemic nature of COVID- 19 as well as its highly variable clinical presentation. We are now expanding our studies to investigate the role of Ebola virus infection in intestinal epithelial injury.
ABSTRACT
Rationale There is a lack of knowledge of how CFTR-deficient airway epithelium intrinsically responds to SARS-CoV-2. Though prior work has demonstrated altered CF airway expression of viral entry factors, it is unknown whether these alterations are protective and whether they reflect host genetic variation or secondary response of chronic inflammation. We address this gap by infecting induced pluripotent stem cell (iPSC)-derived airways from CF patients and syngeneic CFTR-corrected controls with SARS-CoV-2 and assessing differential susceptibility to infection and inflammatory and anti-viral response. MethodsCF (F508del homozygous) and syngeneic CFTR-corrected (CRISPR-Cas9) iPSC- were differentiated into airway epithelium cultured at airliquid interface (ALI) by a directed differentiation protocol that generates a pure population of major and rare airway cell-types. After 21 days in ALI culture, the iPSC-airway were infected with either mock or SARS-CoV-2 (isolate USA-WA1/2020) with MOI of 4, and harvested at 0, 1, 3 days post infection (dpi) for RT-PCR and immune-stainingResultsBoth CF and CFTR-corrected iPSC-airway express viral entry factors of ACE2 and TMPRSS2, and are permissive to SARS-CoV-2 infection. CF iPSC-airway exhibited significantly increase in SARS-CoV-2 nucleocapsid protein (N) transcript at 1 dpi, accompanied by increases in IFN2, RSAD2, and CXCL10 at 3 dpi, compared to its CFTR-corrected counter-part. There are no baseline significant differences in ACE2, TMPRSS2, TP63, NGFR, MUC5B, MUC5AC, SCGB1A1, FOXJ1, FOXI1 expression between CF and CFTR-corrected iPSC-airway before SARS-CoV-2 infection. ConclusionsOur preliminary studies indicate increased early SARS-CoV-2 infection in CFTR-deficient epithelium with accompanied subsequent rise in anti-viral and inflammatory response compared to its genetically controlled CFTR-corrected counterpart. Future studies are aimed at assessing differential CF epithelial kinetics of SARS-CoV-2 viral entry and replication, morphological changes, global transcriptomic response, and how treatment with CFTRmodulator would alter the epithelial response. Ultimately, we aim to establish a reductionist, physiologically relevant model system that is coupled with gene-editing technology to study intrinsic CF epithelial response to SARS-CoV-2, which would generate insights to aid practice guidelines for CF patients, and open future directions to evaluate gene-specific mechanisms of airway response to pathogens. (Figure Presented).
ABSTRACT
Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been associated with immune hyperactivation and high levels of proinflammatory cytokines. Extensive lung infiltration by CD169+ inflammatory monocytes and presence of activated CD169+ alveolar macrophages suggest monocyte/macrophages are key drivers of severe morbidity and mortality. In this study, we determined whether CD169 mediated ACE2-independent SARS-CoV-2 entry and restricted viral genome replication in macrophages triggers pro-inflammatory cytokine expression. Methods: Monocyte-derived macrophages (MDMs) and PMA-differentiated THP-1 macrophages engineered to constitutively express CD169, ACE2, or CD169 and ACE2 were infected with USA-WA1/2020/SARS-CoV-2 isolate with or without Remdesivir pre-treatment. To identify mechanism of innate immune activation, nucleic acid sensing pathways were selectively depleted in CD169+ macrophages. Extent of viral genomic (gRNA) and sub-genomic (sgRNA) expression and induction of pro-inflammatory cytokines was determined by qRT-PCR and single molecule RNA FISH analysis. Viral protein expression and infectious virus particle production was determined by immunofluorescence analysis and TCID50. Results: While productive virus infection (viral protein expression and infectious virus particle release) was only observed in ACE2+ macrophages, SARS-CoV-2 N or S expression and infectious virus production was not observed in CD169+ macrophages. Co-expression of ACE2 and CD169 significantly enhanced infectious virus production and spread. Interestingly, smFISH and RT-qPCR analysis revealed CD169+ cells express cytosolic negative-strand gRNA and positive strand sgRNA. Importantly, CD169-mediated SARS-CoV-2 infection of macrophages and expression of viral mRNAs led to induction of pro-inflammatory cytokines, IL-6, TNFα, and IL-1β, despite lack of viral protein expression in CD169+ macrophages. Pre-treatment with Remdesivir blocked de novo expression of viral mRNAs and induction of inflammatory cytokines in CD169-dependent infection of macrophages. Furthermore, knockdown of cytosolic RLRs (RIG-I and MDA-5) or MAVS significantly attenuated inflammatory cytokine expression in CD169+ macrophages, confirming that nucleic acid sensing of restricted cytosolic viral mRNA expression in macrophages triggers innate immune activation. Conclusion: These results suggest that restricted SARS-CoV-2 infection of CD169+ macrophages contributes to COVID-19-associated hyperinflammatory cytokine response.
ABSTRACT
Initial recommendations for surface disinfection to prevent SARS-CoV-2 transmission were developed using previous evidence from potential surrogates. To the best of our knowledge, no appropriate surrogate for SARS-CoV-2 has been identified or confirmed for chlorine and antimicrobial surface disinfection. We completed a study to evaluate the efficacy of two hypothesized antimicrobial surfaces, and four chlorine solutions on nonporous and porous surfaces, against SARS-CoV-2 and three potential SARS-CoV-2 surrogates [coronavirus mouse hepatitis virus (MHV) and bacteriophages Phi6 and MS2], to identify a BSL-1 or BSL-2 virus to use in future studies. We found SARS-CoV-2 can be reduced >4 log10 on porous and nonporous surfaces within 30 s upon exposure to 0.5% NaOCl. The results indicate coronavirus MHV-GFP is inactivated faster than SARS-CoV-2 (MHV-GFP >= 6.08 log(10);SARS-CoV-2 = 0.66 log(10) at 30 s with 0.05% NaOCl on steel) and MS2 is inactivated more slowly. Phi6 is inactivated like SARS-CoV-2, and we propose Phi6 as a slightly conservative surrogate for SARS-CoV-2 chlorine disinfection. Additionally, disinfection of bacteriophages on wood was challenging, and exposure to antimicrobial surfaces had no disinfection efficacy as tested. We recommend using 0.5% chlorine on surfaces for a minimum of 30 s of contact to disinfect SARS-CoV-2 and recommend additional research on Phi6 disinfection with varied surfaces and conditions.
ABSTRACT
The coronavirus disease 2019 (COVID-19) pandemic has created an urgent need for therapeutics that inhibit the SARS-COV-2 virus and suppress the fulminant inflammation characteristic of advanced illness. Here, we describe the anti-COVID-19 potential of PTC299, an orally bioavailable compound that is a potent inhibitor of dihydroorotate dehydrogenase (DHODH), the rate-limiting enzyme of the de novo pyrimidine nucleotide biosynthesis pathway. In tissue culture, PTC299 manifests robust, dose-dependent, and DHODH-dependent inhibition of SARS-COV-2 replication (EC50 range, 2.0-31.6 nM) with a selectivity index >3,800. PTC299 also blocked replication of other RNA viruses, including Ebola virus. Consistent with known DHODH requirements for immunomodulatory cytokine production, PTC299 inhibited the production of interleukin (IL)-6, IL-17A (also called IL-17), IL-17 F, and vascular endothelial growth factor (VEGF) in tissue culture models. The combination of anti-SARS-CoV-2 activity, cytokine inhibitory activity, and previously established favorable pharmacokinetic and human safety profiles render PTC299 a promising therapeutic for COVID-19.