Longitudinal single cell atlas identifies complex temporal relationship between type I interferon response and COVID-19 severity.

Due to the paucity of longitudinal molecular studies of COVID-19, particularly those covering the early stages of infection (Days 1-8 symptom onset), our understanding of host response over the disease course is limited. We perform longitudinal single cell RNA-seq on 286 blood samples from 108 age- and sex-matched COVID-19 patients, including 73 with early samples. We examine discrete cell subtypes and continuous cell states longitudinally, and we identify upregulation of type I IFN-stimulated genes (ISGs) as the predominant early signature of subsequent worsening of symptoms, which we validate in an independent cohort and corroborate by plasma markers. However, ISG expression is dynamic in progressors, spiking early and then rapidly receding to the level of severity-matched non-progressors. In contrast, cross-sectional analysis shows that ISG expression is deficient and IFN suppressors such as SOCS3 are upregulated in severe and critical COVID-19. We validate the latter in four independent cohorts, and SOCS3 inhibition reduces SARS-CoV-2 replication in vitro. In summary, we identify complexity in type I IFN response to COVID-19, as well as a potential avenue for host-directed therapy.

Betacoronaviruses SARS-CoV-2 and HCoV-OC43 infections in IGROV-1 cell line require aryl hydrocarbon receptor.

The emergence of novel betacoronaviruses has posed significant financial and human health burdens, necessitating the development of appropriate tools to combat future outbreaks. In this study, we have characterized a human cell line, IGROV-1, as a robust tool to detect, propagate, and titrate betacoronaviruses SARS-CoV-2 and HCoV-OC43. IGROV-1 cells can be used for serological assays, antiviral drug testing, and isolating SARS-CoV-2 variants from patient samples. Using time-course transcriptomics, we confirmed that IGROV-1 cells exhibit a robust innate immune response upon SARS-CoV-2 infection, recapitulating the response previously observed in primary human nasal epithelial cells. We performed genome-wide CRISPR knockout genetic screens in IGROV-1 cells and identified Aryl hydrocarbon receptor (AHR) as a critical host dependency factor for both SARS-CoV-2 and HCoV-OC43. Using DiMNF, a small molecule inhibitor of AHR, we observed that the drug selectively inhibits HCoV-OC43 infection but not SARS-CoV-2. Transcriptomic analysis in primary normal human bronchial epithelial cells revealed that DiMNF blocks HCoV-OC43 infection via basal activation of innate immune responses. Our findings highlight the potential of IGROV-1 cells as a valuable diagnostic and research tool to combat betacoronavirus diseases.

Bat ASC2 suppresses inflammasomes and ameliorates inflammatory diseases.

Bats are special in their ability to live long and host many emerging viruses. Our previous studies showed that bats have altered inflammasomes, which are central players in aging and infection. However, the role of inflammasome signaling in combating inflammatory diseases remains poorly understood. Here, we report bat ASC2 as a potent negative regulator of inflammasomes. Bat ASC2 is highly expressed at both the mRNA and protein levels and is highly potent in inhibiting human and mouse inflammasomes. Transgenic expression of bat ASC2 in mice reduced the severity of peritonitis induced by gout crystals and ASC particles. Bat ASC2 also dampened inflammation induced by multiple viruses and reduced mortality of influenza A virus infection. Importantly, it also suppressed SARS-CoV-2-immune-complex-induced inflammasome activation. Four key residues were identified for the gain of function of bat ASC2. Our results demonstrate that bat ASC2 is an important negative regulator of inflammasomes with therapeutic potential in inflammatory diseases.

Population genomic analysis reveals distinct demographics and recent adaptation in the black flying fox (Pteropus alecto).

As the only mammalian group capable of powered flight, bats have many unique biological traits. Previous comparative genomic studies in bats have focused on long-term evolution. However, the micro-evolutionary processes driving recent evolution are largely under-explored. Using resequencing data from 50 black flying foxes (Pteropus alecto), one of the model species for bats, we find that black flying fox has much higher genetic diversity and lower levels of linkage disequilibrium than most of the mammalian species. Demographic inference reveals strong population fluctuations (>100 fold) coinciding with multiple historical events including the last glacial change and Toba super eruption, suggesting that the black flying fox is a very resilient species with strong recovery abilities. While long-term adaptation in the black flying fox is enriched in metabolic genes, recent adaptation in the black flying fox has a unique landscape where recently selected genes are not strongly enriched in any functional category. The demographic history and mode of adaptation suggest that black flying fox might be a well-adapted species with strong evolutionary resilience. Taken together, this study unravels a vibrant landscape of recent evolution for the black flying fox and sheds light on several unique evolutionary processes for bats comparing to other mammalian groups.

Single-cell transcriptome analysis of the in vivo response to viral infection in the cave nectar bat Eonycteris spelaea.

Bats are reservoir hosts of many zoonotic viruses with pandemic potential. We utilized single-cell transcriptome sequencing (scRNA-seq) to analyze the immune response in bat lungs upon in vivo infection with a double-stranded RNA virus, Pteropine orthoreovirus PRV3M. Bat neutrophils were distinguished by high basal IDO1 expression. NK cells and T cells were the most abundant immune cells in lung tissue. Three distinct CD8+ effector T cell populations could be delineated by differential expression of KLRB1, GFRA2, and DPP4. Select NK and T clusters increased expression of genes involved in T cell activation and effector function early after viral infection. Alveolar macrophages and classical monocytes drove antiviral interferon signaling. Infection expanded a CSF1R+ population expressing collagen-like genes, which became the predominant myeloid cell type post-infection. This work uncovers features relevant to viral disease tolerance in bats, lays a foundation for future experimental work, and serves as a resource for comparative immunology studies.

Human Nasal Epithelial Cells Sustain Persistent SARS-CoV-2 Infection In Vitro, despite Eliciting a Prolonged Antiviral Response.

The dynamics of SARS-CoV-2 infection in COVID-19 patients are highly variable, with a subset of patients demonstrating prolonged virus shedding, which poses a significant challenge for disease management and transmission control. In this study, the long-term dynamics of SARS-CoV-2 infection were investigated using a human well-differentiated nasal epithelial cell (NEC) model of infection. NECs were observed to release SARS-CoV-2 virus onto the apical surface for up to 28 days postinfection (dpi), further corroborated by viral antigen staining. Single-cell transcriptome sequencing (sc-seq) was utilized to explore the host response from infected NECs after short-term (3-dpi) and long-term (28-dpi) infection. We identified a unique population of cells harboring high viral loads present at both 3 and 28 dpi, characterized by expression of cell stress-related genes DDIT3 and ATF3 and enriched for genes involved in tumor necrosis factor alpha (TNF-α) signaling and apoptosis. Remarkably, this sc-seq analysis revealed an antiviral gene signature within all NEC cell types even at 28 dpi. We demonstrate increased replication of basal cells, absence of widespread cell death within the epithelial monolayer, and the ability of SARS-CoV-2 to replicate despite a continuous interferon response as factors likely contributing to SARS-CoV-2 persistence. This study provides a model system for development of therapeutics aimed at improving viral clearance in immunocompromised patients and implies a crucial role for immune cells in mediating viral clearance from infected epithelia. IMPORTANCE Increasing medical attention has been drawn to the persistence of symptoms (long-COVID syndrome) or live virus shedding from subsets of COVID-19 patients weeks to months after the initial onset of symptoms. In vitro approaches to model viral or symptom persistence are needed to fully dissect the complex and likely varied mechanisms underlying these clinical observations. We show that in vitro differentiated human NECs are persistently infected with SARS-CoV-2 for up to 28 dpi. This viral replication occurred despite the presence of an antiviral gene signature across all NEC cell types even at 28 dpi. This indicates that epithelial cell intrinsic antiviral responses are insufficient for the clearance of SARS-CoV-2, implying an essential role for tissue-resident and infiltrating immune cells for eventual viral clearance from infected airway tissue in COVID-19 patients.

Infection of human Nasal Epithelial Cells with SARS-CoV-2 and a 382-nt deletion isolate lacking ORF8 reveals similar viral kinetics and host transcriptional profiles.

The novel coronavirus SARS-CoV-2 is the causative agent of Coronavirus Disease 2019 (COVID-19), a global healthcare and economic catastrophe. Understanding of the host immune response to SARS-CoV-2 is still in its infancy. A 382-nt deletion strain lacking ORF8 (Δ382 herein) was isolated in Singapore in March 2020. Infection with Δ382 was associated with less severe disease in patients, compared to infection with wild-type SARS-CoV-2. Here, we established Nasal Epithelial cells (NECs) differentiated from healthy nasal-tissue derived stem cells as a suitable model for the ex-vivo study of SARS-CoV-2 mediated pathogenesis. Infection of NECs with either SARS-CoV-2 or Δ382 resulted in virus particles released exclusively from the apical side, with similar replication kinetics. Screening of a panel of 49 cytokines for basolateral secretion from infected NECs identified CXCL10 as the only cytokine significantly induced upon infection, at comparable levels in both wild-type and Δ382 infected cells. Transcriptome analysis revealed the temporal up-regulation of distinct gene subsets during infection, with anti-viral signaling pathways only detected at late time-points (72 hours post-infection, hpi). This immune response to SARS-CoV-2 was significantly attenuated when compared to infection with an influenza strain, H3N2, which elicited an inflammatory response within 8 hpi, and a greater magnitude of anti-viral gene up-regulation at late time-points. Remarkably, Δ382 induced a host transcriptional response nearly identical to that of wild-type SARS-CoV-2 at every post-infection time-point examined. In accordance with previous results, Δ382 infected cells showed an absence of transcripts mapping to ORF8, and conserved expression of other SARS-CoV-2 genes. Our findings shed light on the airway epithelial response to SARS-CoV-2 infection, and demonstrate a non-essential role for ORF8 in modulating host gene expression and cytokine production from infected cells.

Genetic Control over mtDNA and Its Relationship to Major Depressive Disorder.

Control over the number of mtDNA molecules per cell appears to be tightly regulated, but the mechanisms involved are largely unknown. Reversible alterations in the amount of mtDNA occur in response to stress suggesting that control over the amount of mtDNA is involved in stress-related diseases including major depressive disorder (MDD). Using low-coverage sequence data from 10,442 Chinese women to compute the normalized numbers of reads mapping to the mitochondrial genome as a proxy for the amount of mtDNA, we identified two loci that contribute to mtDNA levels: one within the TFAM gene on chromosome 10 (rs11006126, p value = 8.73 × 10(-28), variance explained = 1.90%) and one over the CDK6 gene on chromosome 7 (rs445, p value = 6.03 × 10(-16), variance explained = 0.50%). Both loci replicated in an independent cohort. CDK6 is thus a new molecule involved in the control of mtDNA. We identify increased rates of heteroplasmy in women with MDD, and show from an experimental paradigm using mice that the increase is likely due to stress. Furthermore, at least one heteroplasmic variant is significantly associated with changes in the amount of mtDNA (position 513, p value = 3.27 × 10(-9), variance explained = 0.48%) suggesting site-specific heteroplasmy as a possible link between stress and increase in amount of mtDNA. These findings indicate the involvement of mitochondrial genome copy number and sequence in an organism's response to stress.