Viral and Host Small RNA Response to SARS-CoV-2 Infection

After two years into the pandemic of the coronavirus disease 2019 (COVID-19), it remains unclear how the host RNA interference (RNAi) pathway and host miRNAs regulate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and impact the development of COVID-19. In this study, we profiled small RNAs in SARS-CoV-2-infected human ACE2-expressing HEK293T cells and observed dysregulated host small RNA groups, including specific host miRNAs that are altered in response to SARS-CoV-2 infection. By comparing dysregulated miRNAs in different SARS-CoV-2-infected samples, we identified miRNA-210-3p, miRNA-30-5p, and miR-146a/b as key host miRNAs that may be involved in SARS-CoV-2 infection. Furthermore, by comparing virally derived small RNAs (vsmRNAs) in different SARS-CoV-2-infected samples, we observed multiple hot spots in the viral genome that are prone to generating vsmRNAs, and their biogenesis can be dependent on the antiviral isoform of Dicer. Moreover, we investigated the biogenesis of a recently identified SARS-CoV-2 viral miRNA encoded by ORF7a and found that it is differentially expressed in different infected cell lines or in the same cell line with different viral doses. Our results demonstrate the involvement of both host small RNAs and vsmRNAs in SARS-CoV-2 infection and identify these small RNAs as potential targets for anti-COVID-19 therapeutic development. © 2022 by the authors.

Differential innate immune response and replication of SARS-CoV-2 in human diabetic corneal epithelial cells

Purpose : Diabetes predisposes an individual to severe COVID-19. Diabetic cornea is also known to have impaired wound healing, increasing the chances of infection. Earlier, we reported the ability of SARS-CoV-2 to infect conjunctival cells, and the presence of viral RNA and proteins was also detected in the corneas of COVID-19 donors. In this study, we evaluated the effect of diabetes on corneal innate immune response during SARS-CoV-2 infection and sought to determine the underlying mechanisms. Methods : Human primary corneolimbal epithelial cells (HCECs) were isolated from the corneas of three diabetic and three non-diabetic donors. In vitro studies were performed by infecting HCECs with SARS-CoV-2-USA-WA1/2020 strain at MOI 0.5. Viral replication was assessed by viral genome copy number. RNAseq analysis was performed to determine genes/pathways altered by diabetic vs non-diabetic HCECs. qPCR was used to assess the expression of innate inflammatory and antiviral genes. Western blot was performed to detect the protein expression of antiviral signaling molecules. Results : The primary HCECs were found permissive to SARS-CoV-2 infection, as evidenced by increased viral replication which peaked at day 3 p.i. along with an induction of pSTAT1. Interestingly, HCECs from diabetic cornea had higher viral RNA on all three days post-infection. SARS-CoV-2 infected HCECs exhibited induced expression of inflammatory genes and their levels were relatively higher in diabetic cells. RNA-seq analysis revealed significant differences in diabetic vs. non-diabetic SARS-CoV-2 infected cells with alteration in genes regulating viral response, inflammation, and injury. The most affected down-regulated genes are related to lipid metabolism, ferroptosis, and oxidative stress. Conclusions : Our study demonstrates increased SARS-CoV-2 replication and differential innate antiviral and inflammatory response in HCECs from diabetic corneas. These results indicate that diabetes is a potential risk for enhanced infectivity of SARS-CoV-2 for the ocular surface.

NEUTRALIZING ANTIBODY RESPONSE and TRANSPLACENTAL TRANSFER in COVID-19 in PREGNANCY

Background: There are limited data on how COVID-19 disease severity and vaccination throughout different trimesters in pregnancy impact maternal neutralizing antibody responses and transplacental transfer to the neonate at birth. Further characterization of the antibody response of in utero SARS-CoV-2 may inform vaccination schedules in pregnancy in order to optimize maternal and neonatal protection. Methods: The COVID-19 Outcomes in Mother-Infant Pairs (COMP) study is a longitudinal cohort of mother-infant dyads diagnosed with PCR-confirmed SARS-CoV-2 at any point during pregnancy. Maternal and cord sera from delivery, as well as infant sera collected at 24 hours of life, were analyzed by enzyme-linked immunosorbent assay (ELISA) for IgA, IgG, and IgM targeting receptor binding domain (RBD) of the SARS-CoV-2 spike protein. Neutralizing antibody (NAb) activity against the original L strain was evaluated in a subset of unvaccinated mother-infant dyads with evidence of IgG transfer or history of severe/critical COVID-19 in pregnancy. Results: Among 115 pregnant women, the NIH COVID-19 severity of illness categories were: 12% asymptomatic, 70% mild/moderate, 11% severe/critical disease, and 7% vaccinated prior to delivery following recovery. Fifty percent of the cohort was diagnosed in the 3rd trimester, and the median diagnosis date to delivery was 61.5 days (IQR 27.75-122.25). The majority (74%) of the cohort produced all three anti-SARS-CoV-2 isotypes, although 5% had no detectable antibody class. Transplacental transfer ratios increased with increasing duration between onset of infection and delivery (Figure 1, r2=0.17). Infant IgG levels (ng/mL) were the highest among neonates born to vaccinated mothers (Figure 1), and maternal IgG levels increased with disease severity, although vaccination elicited a comparable maternal antibody response to severe/critical disease (Figure 1). Among 50 maternal specimens, 80% demonstrated in vitro neutralization activity, and 52% of 33 neonatal specimens had NAb. Conclusion: While transplacental transfer of IgG was high with natural infection and correlates with increasing duration between onset of infection and delivery, only half of analyzed neonatal specimens demonstrated in vitro neutralization activity. Further research is needed to characterize the functionality and kinetics of both maternal and neonatal antibody responses elicited by in utero SARS-CoV-2 natural infection compared with COVID-19 vaccination.

Engineered extracellular vesicles antagonize SARS-CoV-2 infection by inhibiting mTOR signaling.

Effective treatment approaches for patients with COVID-19 remain limited and are neither curative nor widely applicable. Activated specialized tissue effector extracellular vesicles (ASTEX) derived from genetically-enhanced skin fibroblasts, exert disease-modifying bioactivity in vivo in models of heart and lung injury. Here we report that ASTEX antagonizes SARS-CoV-2 infection and its pathogenic sequelae. In human lung epithelial cells exposed to SARS-CoV-2, ASTEX is cytoprotective and antiviral. Transcriptomic analysis implicated the mammalian target of rapamycin (mTOR) pathway, as infected cells upregulated mTOR signaling and pre-exposure to ASTEX attenuated it. The implication of mTOR signaling was further confirmed using mTOR inhibition and activation, which increased and decreased viral load, respectively. Dissection of ASTEX cargo identifies miRs including miR-16 as potential inhibitors of mTOR signaling. The findings reveal a novel, dual mechanism of action for ASTEX as a therapeutic candidate for COVID-19, with synergistic antiviral and cytoprotective benefits.

SARS-CoV-2 infection of primary human lung epithelium for COVID-19 modeling and drug discovery

Coronavirus disease 2019 (COVID-19) is the latest respiratory pandemic resulting from zoonotic transmission of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2). Severe symptoms include viral pneumonia secondary to infection and inflammation of the lower respiratory tract, in some cases causing death. We developed primary human lung epithelial infection models to understand responses of proximal and distal lung epithelium to SARS-CoV-2 infection. Differentiated air-liquid interface cultures of proximal airway epithelium and 3D organoid cultures of alveolar epithelium were readily infected by SARS-CoV-2 leading to an epithelial cell-autonomous proinflammatory response. We validated the efficacy of selected candidate COVID-19 drugs confirming that Remdesivir strongly suppressed viral infection/replication. We provide a relevant platform for studying COVID-19 pathobiology and for rapid drug screening against SARS-CoV-2 and future emergent respiratory pathogens. One Sentence Summary: A novel infection model of the adult human lung epithelium serves as a platform for COVID-19 studies and drug discovery.

SARS-CoV-2 infects human corneal epithelium and elicits an antiviral immune response

Purpose : We recently reported the presence of SARS-CoV-2 RNA, Spike, and Envelope proteins in the corneas of COVID-19 donors. However, the presence of viral RNA and antigens does not necessarily equate to infection. In this study, using RNA-seq of COVID-19 corneal tissue, and SARS-CoV-2 infection of human corneal epithelial cultures, we now examined whether SARS-CoV-2 could replicate in the cornea and elicits an innate immune response. Methods : Eyes from healthy and COVID-19 donors were from the Eversight eye bank. The corneal tissue was used for IHC detection of SARS-CoV-2 by RNA-FISH. In another experiment, total RNA was extracted from corneas for RNA-seq analysis to identify genes/ pathways altered by infection. In vitro studies were performed by infecting primary human corneal epithelial cells (HCECs) from normal and diabetic donor corneas with SARS-CoV-2. Bioinformatics analysis was performed to determine the differential gene expression. qPCR was used to assess the expression of innate inflammatory and antiviral genes and to confirm RNA-seq data in corneal tissue and cells. Results : RNA-FISH analysis showed the presence of both positive and negative strands of SARS-CoV-2 viral RNA in the epithelium of COVID-19 donor corneas. This coincided with infiltration of CD45+ cells in the stroma and induced expression of inflammatory (IL-6, IL1β) and antiviral (ISG15, OAS2) genes. RNA-seq analysis revealed significant upregulation of genes involved in the viral response, inflammation, and injury along with induction of lncRNA XIST and TSIX involved in modulation of the immune response. The primary HCECs were found permissive to SARS-CoV-2 infection, as evidenced by increase viral replication which peaked at day 3 p.i. along with an induction of p-STAT1. Interestingly, HCECs from diabetic cornea had higher viral RNA on day 1 p.i. compared to non-diabetic cells. SARSCoV-2 infected HCECs also exhibited induced expression of antiviral innate response genes, which was elevated in diabetic donor cornea cells. Conclusions : Our study confirms the presence of replicating SARS-CoV-2 viral RNA and antigen in the cornea of COVID-19 affected donors resulting in the production of inflammatory mediators and recruitment of CD45+ immune cells to the cornea. Moreover, HCECs from diabetic corneas had increased SARS-CoV-2 replication and immune response, suggesting that diabetes is a potential risk for ocular transmission of COVID-19.

SARS-CoV-2 infection of primary human lung epithelium for COVID-19 modeling and drug discovery.

Coronavirus disease 2019 (COVID-19) is the latest respiratory pandemic resulting from zoonotic transmission of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2). Severe symptoms include viral pneumonia secondary to infection and inflammation of the lower respiratory tract, in some cases causing death. We developed primary human lung epithelial infection models to understand responses of proximal and distal lung epithelium to SARS-CoV-2 infection. Differentiated air-liquid interface cultures of proximal airway epithelium and 3D organoid cultures of alveolar epithelium were readily infected by SARS-CoV-2 leading to an epithelial cell-autonomous proinflammatory response. We validated the efficacy of selected candidate COVID-19 drugs confirming that Remdesivir strongly suppressed viral infection/replication. We provide a relevant platform for studying COVID-19 pathobiology and for rapid drug screening against SARS-CoV-2 and future emergent respiratory pathogens. ONE SENTENCE SUMMARY: A novel infection model of the adult human lung epithelium serves as a platform for COVID-19 studies and drug discovery.