Studies on SARS-CoV-2 viral persistence in a transgenic mouse model and the impact of supplier origin gut microbiomes on disease outcomes

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