This study assesses the effects of two distinct, murine gut microbiome profiles on acute and post-acute outcomes of SARS-CoV-2 infection and highlights the utility of running wheels for monitoring health status in infectious disease studies and their potential as a behavioral assay in high containment settings.
Abstract
Post acute sequelae of COVID-19 (PASC) remains a persistent public health threat and continues to affect people worldwide, frequently disrupting daily life and negatively impacting the economy. Effective therapeutics are necessary to alleviate the distress of those currently suffering from PASC. During our study in determining viral clearance time in SARS-CoV-2 infected B6.Cg-Tg(K18-ACE2)2Prlmn/J (K18-hACE2) mice, we discovered that these mice remain persistently infected with viral RNA present in their lungs, brains, and fecal pellets up to 16 weeks post-inoculation. We hypothesized that the persistent viral RNA was a result of viral replication occurring within tissue reservoirs. To test this hypothesis, we treated surviving K18-hACE2 mice with either nirmatrelvir (SARS-CoV-2 replication inhibitor) or dexamethasone. Our results indicated that neither nirmatrelvir nor dexamethasone had a significant impact on viral RNA levels in the lung, suggesting that persistent viral RNA was not due to low levels of replication occurring in reservoirs. This study provides valuable insight into the potential mechanisms underlying the persistence of viral RNA in previously infected individuals. Infectious disease studies utilizing animal models housed under Animal Biosafety Level-3 (ABSL-3) containment present significant challenges for routine health monitoring. Standard cage-side health checks are subjective, can be time consuming, and require in person observations. Here, we propose a method of remotely and non-invasively monitoring voluntary running activity in SARS-CoV-2 infected K18-hACE2 mice in ABSL-3 containment. We demonstrate that running wheels could detect signs of illness earlier (4 days post-infection) than cage-side observers (5 days post-infection). Furthermore, surviving K18-hACE2 mice did not show evidence of fatigue or post-exertional malaise based on their voluntary running activity. These findings highlight the utility of running wheels for monitoring health status in infectious disease studies and their potential as a behavioral assay in high containment settings. The composition and diversity of the gut microbiome (GM) and the metabolites they produce have been shown to play an important role in human health and disease. COVID-19 has been shown to significantly alter the normal gut microbiome, resulting in a loss of bacterial richness and diversity, but it is unclear how the gut microbiome itself affects SARS-CoV-2 disease outcomes. The goal of this study was to assess the effects of two distinct, murine gut microbiome profiles on acute and post-acute outcomes of SARS-CoV-2 infection. We hypothesized that K18-hACE2 mice with a more diverse and rich gut microbiome profile (GMHigh) would be less susceptible to disease and have fewer markers of inflammation in tissues within the acute and post-acute phase of disease compared to mice with a less diverse and rich gut microbiome profile (GMLow). Age was the primary risk factor of disease, where older mice were more susceptible to disease than younger mice. Cytokine and chemokine analysis revealed that GMHigh animals had higher levels of inflammatory markers in their lungs during both the acute and recovery phase of infection. When it came to the impact of sex, in general, males had higher levels of cytokines and chemokines in both the lung and the brain at 14 days post-infection. Additionally, GMLow animals had higher relative abundances of Ruminococcaceae, Anaerovoracaceae, Clostridia_UCG-014, Peptococcacaeae, Acholeplasmataceae, and Erysipelotrichaceae compared to GMHigh animals. This study provides valuable insight into the role of GM composition, age, and sex in COVID-19 susceptibility and recovery. Interindividual variability in responses to dexamethasone is well documented and has largely been attributed to genetic factors, however, the gut microbiome is increasingly recognized as a potential contributor to this variability. Current evidence suggests that the gut microbiome can affect the pharmacokinetics and pharmacodynamics of orally delivered drugs. The goal of this study was to assess the impact of two murine gut microbiomes (GMLow and GMHigh) on the outcomes of a high-dose oral DEX protocol. We observed that GMHigh DEX treated mice had a significantly lower survival rate than the other experimental groups and that all DEX treated animals became septicemic. We also observed multiple coccoid structures arranged in a linear chain adherent to the jejunal epithelium only in GMHigh DEX animals. This study demonstrates that high-dose DEX treatment can induce sepsis, likely from bacterial gut translocation, and that the GM composition influences survival rate and the jejunal microbiome composition. Collectively, these findings demonstrate that K18-hACE2 mice are useful in studying multiple facets of COVID-19. The persistence of viral RNA in this model enables investigation of its effects on the immune system, evaluation of treatment impacts on viral persistence, and assessment of symptom development associated with prolonged viral RNA presence. Although we did not observe evidence of fatigue or post-exertional malaise, it remains possible that mice exhibit other features associated with long COVID. Additionally, this model can be used to examine the role of the gut microbiome in COVID-19 outcomes, as well as its influence on drug responses, which may help inform therapeutic strategies
SARS-CoV-2 persistence has been proposed as a potential contributor to the pathogenesis of long COVID, with reservoir tissues potentially serving as sites for viral persistence, intra-host evolution, and intermittent viral shedding. Here, we used experimentally infected Syrian hamsters to investigate long-term SARS-CoV-2 persistence across tissues, viral infectivity, and associated immunological and metabolic alterations. Syrian hamsters (Mesocricetus auratus) were intranasally infected with a SARS-CoV-2 parental strain or Gamma and Delta variants and monitored for up to one year, with samples collected at 3, 15, 30, 90, 150, and 365 days post-infection (dpi). During the acute phase, infected animals exhibited significant weight loss, viral shedding, and marked pulmonary inflammation, accompanied by increased expression of pro-inflammatory cytokines at 3 dpi. Infection was confirmed by seroconversion, with sustained IgG responses and low-titer neutralizing antibodies against Omicron. Viral nucleoprotein was detected in multiple tissues up to 365 dpi, while RT-qPCR identified persistent low-level viral RNA in the lungs, brain, spleen, and thymus throughout the observation period, without evidence of productive viral replication. Immune gene expression displayed organ-specific temporal patterns: acute pulmonary inflammation transitioned into broad late-stage suppression, except for sustained TGF-β expression; the brain exhibited a late chemokine signature at 365 dpi; and the thymus showed a delayed immune activation peak at 150 dpi, particularly in Delta-infected animals. Metabolomic profiling revealed a shared acute-phase metabolic signature across variants that largely resolved by 365 dpi, whereas Delta-infected animals retained distinct residual metabolic alterations. Collectively, these findings establish a model of long-term SARS-CoV-2 tissue persistence characterized by organ-specific immune and metabolic signatures, providing a platform to investigate mechanisms underlying post-acute sequelae and evaluate potential therapeutic strategies.
Thais Melquiades de Lima, Carlos Eduardo Capelini Eli Lopes, Maria Vitoria Oliveira de Souza et al.· bioRxiv· 0 citations
Over 11 million people in the US are affected by long-COVID syndrome, or post-acute sequelae of SARS-CoV-2 infection (PASC), occurring in 10% of those infected. Despite advances, PASC studies remain largely observational due to the lack of a good animal model and the disease’s heterogeneity. It is therefore imperative to discover the underlying causes of PASC and develop effective treatments. Recent studies showed that SARS-CoV-2 Spike RNA can persist in the colon for>2 years after infection. Given the frequency of microbial dysbiosis and T cell hyperactivation in PASC, we reasoned that a gut viral reservoir may cause T cell dysfunction by altering the microbial metabolome.
To investigate viral persistence, we acquired colon biopsy patients from the University of Chicago colonoscopy clinic and conducted RNAscope targeting SARS-CoV-2 Orf1ab RNA. On the same patient’s stool, we did metabolomic and metagenomic analysis to investigate differences in metabolites from PASC patients. Subsequently, we probed how exposure to PASC metabolites affected T cell activation and metabolism by conducting flow cytometry experiments on T cells exposed to autologous or long COVID metabolites.
Using colon biopsy samples from colonoscopy patients, we found evidence of SARS-CoV-2 Orf1ab RNA persistence in patients with persistent symptoms after COVID-19. We also found PASC patient stool was enriched in pro-inflammatory tryptophan metabolites. After TCR stimulation, T cells exposed to long COVID metabolites upregulated the inhibitory marker PD1 while increasing fatty acid synthesis/OXPHOS and reducing fatty acid oxidation, demonstrating that long COVID metabolites reduced the ability of T cells to activate metabolic programs leading to functional impairment.
Taken together, our data show that viral persistence may be a frequent occurrence linked to long COVID symptoms and may play a role in promoting a dysregulated inflammatory state by altering the metabolite microenvironment of the gut.
Center for Intestinal Inflammatory Diseases (C-IID) P30 DK042086
Mucosal and Regional Immunology (MUC)
Daniel Carroll, L. Visvabharathy, Jennifer Lopez et al.· Journal of Immunology· 0 citations
Several reports have shown disparity in male and female responses to SARS-CoV-2 infection. Males often develop move severe acute infection, while the incidence of females developing long-COVID out-numbers males. Commensal microbes play a vital role in shaping both the mucosal and systemic immune response to pathogens. Using germ-free mice, we investigated whether commensal microbes play a role in male and female host susceptibility to SARS-Cov-2 infection.
Age-matched male and female germ-free (GF) and control specific pathogen free (SPF) C57BL/6 mice were intranasally infected with the mouse-adapted SARS-CoV-2 MA10. Body weight, temperature, and behavior scores were monitored to assess the severity of disease. Innate responses in the lungs and peripheral blood were determined by flow cytometry analysis of innate subsets, viral load in lung tissues, and the profile of host mRNA responses.
Male and female GF mice differentially responded to SARS-CoV-2 infection. Thus, female GF mice also showed no change in body temperature, while the infected male GF mice displayed the characteristic drop in body temperature seen in SPF C57BL/6 mice. However, neither male nor female GF mice lost weight after infection. Blood collected three days post infection, showed higher numbers of CD11b+CD11c+ and CD11b+Ly6G+ cells in male GF compared to female GF mice. Further, the lungs of male GF mice contained higher titers of live SARS-CoV-2 MA10 virus than female GF mice four days post infection. Ongoing scRNAseq analysis of male and female GF mice lungs are expected to identify mechanisms underlying these differences.
Our results suggest that commensal microbes-dependent and -independent mechanisms contribute to the difference of outcome after SARS-CoV-2 infection of males and females. Cellular and molecular mechanisms underlying these differences are being investigated.
NIH P01AI175399
Mucosal and Regional Immunology (MUC)
R. Woodfint, Douglas Natoce, Sungjun Yoo et al.· Journal of Immunology· 0 citations
In the wake of the SARS-CoV-2 pandemic, most people have experienced at least one respiratory viral infection and many endured severe disease, yet the long term immune consequences remain unclear. We hypothesized that severe infection episode durably reshapes circulating immunity and accelerates age-like change.
To enable within-animal longitudinal inference, we focused on blood so the same mice could be profiled over time. Wild type mice received sublethal influenza A virus (IAV) or mock, and whole blood single-cell RNA-seq of PBMCs was performed at 2, 4, 8, and 14 months post infection from the same animals.
Severe IAV was associated with a progressive rise in the neutrophil-to-lymphoid ratio (NLR) from 2-14 months versus mock, with concomitant declines in naïve B and T cells. A GZMK+ CD8 T-cell subset increased in frequency and activation state in infected mice. These features paralleled aging like myeloid bias programs, consistent with infection accelerated immunosenescence.
Ongoing analyses use batch aware mixed effects models and gene set scoring to define cell-state programs linked to NLR and GZMK+ CD8 expansion. Our findings suggest that severe respiratory viral insult can imprint durable remodeling of circulating immunity, identifying the specific transcriptional signatures and upstream mediators may reveal targets to restore immune balance and resilience during age.
n/a
Viral Immunology (VIR)
A. Chaudhuri, H. Narasimhan, Gislane de Almeida Santos et al.· Journal of Immunology· 0 citations
Coronavirus disease 2019 (COVID-19) is an acute respiratory disease caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Impaired and dysregulated host immunities, such as impaired coordination and disruption of CD4/CD8 T-cell-mediated virus-specific adaptive immune responses to SARS-CoV-2, have been hypothesized as age-related risk factors in COVID-19 disease severity. However, the characteristics and heterogeneity of CD4/CD8 T cell subsets that respond to SARS-CoV-2 remain elusive. In the present study, we focused on investigating those subsets and there role in COVID-19 infection.
We established an age-dependent COVID-19 model by infecting middle-age mice (7-8 months old) or young mice (6-8 weeks old) with 100 or 500pfu of a mouse adapted SARS-CoV-2 virus (SARS2-N501YMA30). Lung, spleen tissues and blood harvested at 60 and 90 days post infection (dpi) were subjected to flow-cytometry and CyTOF for CD4/CD8 T cell profiling. Data was analyzed using FlowJo_v10.10.0 software.
Only middle-age mice showed an average weight loss of 10% and 20% in low (100pfu) and high (500pfu) viral dose infected group respectively. Immune profiling revealed increase in the CD11b, CD11c expressing CD4/CD8 T cell subsets in the infected mice. These infection-prompted subsets increased significantly in middle-age mice and were found to contribute in effector functions as well as consist T cell-mediated immune memory, characterized by higher Granzyme B and Perforin expression.
This study characterizes CD11b, CD11c expressing CD4/CD8 T cell subsets with potent antiviral effects, which is in accord with the reported data of association of CD11b and CD11c T cell with increased cytotoxic activity in other viral infections like Influenza, RSV. Further analysis of these immune cells is underway to better understand their roles in driving protective or pathogenic immune responses upon SARS-CoV-2 infection in mice.
Functional Microbiomics, Inflammation and Pathogenicity- COBRE pilot grant (OGMB220226F1) (JZ); UofL starting package (F1256, F1260) (JZ)
Viral Immunology (VIR)
Divyasha Saxena, Jian Zheng· Journal of Immunology· 0 citations
The ability of respiratory viruses to exploit host immune responses to promote transmission is a defining feature of pandemics. SARS-CoV-2 remains a major global public health threat because of its persistent evolution and capacity to counteract evolving immune defenses. Although the immune evasion properties of the SARS-CoV-2 Spike protein are well characterized, the contributions of other viral proteins to transmission remain poorly understood. Here, we used the infant mouse model to define the role of the accessory protein ORF8 in SARS-CoV-2 spread. We demonstrate that ORF8 supports efficient upper respiratory tract (URT) infection, infectious virus shedding, and host-to-host transmission. Mice infected with a recombinant SARS-CoV-2 strain lacking ORF8 (rΔORF8) had less infectious virus recovered from URT tissues and nasal secretions and transmitted less efficiently than mice infected with the isogenic ancestral strain rWA-1, which contains an intact ORF8. Recombinant viruses encoding naturally occurring ORF8 mutations exhibited distinct transmission phenotypes, with ORF8-deficient viruses resembling rΔORF8. Infection with ORF8-sufficient viruses induced greater macrophage recruitment, inflammatory cytokine production, and type-I interferon (IFN-I) signaling programs than ORF8-deficient viruses. Intranasal IFNβ supplementation partially restored URT shedding by rΔORF8-infected mice and rescued transmission to contacts, whereas blockade of the type I interferon receptor (IFNAR) in rWA-1-infected index mice reduced contact infection and transmission. Together, these findings demonstrate that ORF8 promotes SARS-CoV-2 transmission by engaging an IFN-I-associated inflammatory and secretory program in the URT that supports virus shedding from the infected host. These data identify ORF8 as a viral determinant of host mucosal responses that promote contagiousness. Importance Efficient host-to-host transmission underlies the success of respiratory viruses. Although SARS-CoV-2 research has largely focused on the Spike protein, accessory proteins can also shape viral fitness and spread. We previously identified ORF8 as a determinant of SARS-CoV-2 transmission. Here we show that ORF8 promotes SARS-CoV-2 infectious viral shedding and transmission by engaging IFN-I-associated inflammatory and secretory responses in the URT. These findings reveal how a SARS-CoV-2 accessory protein can exploit mucosal antiviral responses to increase host contagiousness.
G. Ciabattoni, Stacey Bartlett, M. McGrath et al.· bioRxiv· 0 citations