The emergence and spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in significant global morbidity, mortality, and societal disruption. A better understanding of ...virus-host interactions may potentiate therapeutic insights toward limiting this infection. Here we investigated the dynamics of the systemic response to SARS-CoV-2 in hamsters by histological analysis and transcriptional profiling. Infection resulted in consistently high levels of virus in the upper and lower respiratory tracts and sporadic occurrence in other distal tissues. A longitudinal cohort revealed a wave of inflammation, including a type I interferon (IFN-I) response, that was evident in all tissues regardless of viral presence but was insufficient to prevent disease progression. Bolstering the antiviral response with intranasal administration of recombinant IFN-I reduced viral disease, prevented transmission, and lowered inflammation in vivo. This study defines the systemic host response to SARS-CoV-2 infection and supports use of intranasal IFN-I as an effective means of early treatment.
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•Generation of a transcriptional atlas of SARS-CoV-2 infection in hamsters•Infection and transmission can be initiated by respiratory or ocular exposure•Systemic inflammation occurs despite little productive replication in distal tissues•Intranasal IFN-I administered pre- or post-virus challenge reduces disease burden
The host response to SARS-CoV-2 results in significant inflammation. To understand this biology, Hoagland et al. utilize infected hamsters to elucidate transcriptional footprints across tissues longitudinally, showing an inflammatory response beyond the site of acute replication. Local administration of IFN-I reduces virus load and improves immune infiltrate.
The rapid repurposing of antivirals is particularly pressing during pandemics. However, rapid assays for assessing candidate drugs typically involve in vitro screens and cell lines that do not ...recapitulate human physiology at the tissue and organ levels. Here we show that a microfluidic bronchial-airway-on-a-chip lined by highly differentiated human bronchial-airway epithelium and pulmonary endothelium can model viral infection, strain-dependent virulence, cytokine production and the recruitment of circulating immune cells. In airway chips infected with influenza A, the co-administration of nafamostat with oseltamivir doubled the treatment-time window for oseltamivir. In chips infected with pseudotyped severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), clinically relevant doses of the antimalarial drug amodiaquine inhibited infection but clinical doses of hydroxychloroquine and other antiviral drugs that inhibit the entry of pseudotyped SARS-CoV-2 in cell lines under static conditions did not. We also show that amodiaquine showed substantial prophylactic and therapeutic activities in hamsters challenged with native SARS-CoV-2. The human airway-on-a-chip may accelerate the identification of therapeutics and prophylactics with repurposing potential.
The host response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can result in prolonged pathologies collectively referred to as post-acute sequalae of COVID-19 (PASC) or ...long COVID. To better understand the mechanism underlying long COVID biology, we compared the short- and long-term systemic responses in the golden hamster after either SARS-CoV-2 or influenza A virus (IAV) infection. Results demonstrated that SARS-CoV-2 exceeded IAV in its capacity to cause permanent injury to the lung and kidney and uniquely affected the olfactory bulb (OB) and olfactory epithelium (OE). Despite a lack of detectable infectious virus, the OB and OE demonstrated myeloid and T cell activation, proinflammatory cytokine production, and an interferon response that correlated with behavioral changes extending a month after viral clearance. These sustained transcriptional changes could also be corroborated from tissue isolated from individuals who recovered from COVID-19. These data highlight a molecular mechanism for persistent COVID-19 symptomology and provide a small animal model to explore future therapeutics.
Abstract
Many neural sex differences are differences in the number of neurons of a particular phenotype. For example, male rodents have more calbindin-expressing neurons in the medial preoptic area ...(mPOA) and bed nucleus of the stria terminalis (BNST), and females have more neurons expressing estrogen receptor alpha (ERα) and kisspeptin in the ventromedial nucleus of the hypothalamus (VMH) and the anteroventral periventricular nucleus (AVPV), respectively. These sex differences depend on neonatal exposure to testosterone, but the underlying molecular mechanisms are unknown. DNA methylation is important for cell phenotype differentiation throughout the developing organism. We hypothesized that testosterone causes sex differences in neurochemical phenotype via changes in DNA methylation, and tested this by inhibiting DNA methylation neonatally in male and female mice, and in females given a masculinizing dose of testosterone. Neonatal testosterone treatment masculinized calbindin, ERα and kisspeptin cell number of females at weaning. Inhibiting DNA methylation with zebularine increased calbindin cell number only in control females, thus eliminating sex differences in calbindin in the mPOA and BNST. Zebularine also reduced the sex difference in ERα cell number in the VMH, in this case by increasing ERα neuron number in males and testosterone-treated females. In contrast, the neonatal inhibition of DNA methylation had no effect on kisspeptin cell number. We conclude that testosterone normally increases the number of calbindin cells and reduces ERα cells in males through orchestrated changes in DNA methylation, contributing to, or causing, the sex differences in both cell types.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19) pandemic, drastically modifies infected cells to optimize virus ...replication. One such modification is the activation of the host p38 mitogen-activated protein kinase (MAPK) pathway, which plays a major role in inflammatory cytokine production, a hallmark of severe COVID-19. We previously demonstrated that inhibition of p38/MAPK activity in SARS-CoV-2-infected cells reduced both cytokine production and viral replication. Here, we combined quantitative genetic screening, genomics, proteomics, and phosphoproteomics to better understand mechanisms underlying the dependence of SARS-CoV-2 on the p38 pathway. We found that p38β is a critical host factor for SARS-CoV-2 replication in multiple relevant cell lines and that it functions at a step after viral mRNA expression. We identified putative host and viral p38β substrates in the context of SARS-CoV-2 infection and found that most host substrates have intrinsic antiviral activities. Taken together, this study reveals a unique proviral function for p38β and supports exploring p38β inhibitor development as a strategy toward creating a new class of COVID-19 therapies. IMPORTANCE SARS-CoV-2 is the causative agent of the COVID-19 pandemic that has claimed millions of lives since its emergence in 2019. SARS-CoV-2 infection of human cells requires the activity of several cellular pathways for successful replication. One such pathway, the p38 MAPK pathway, is required for virus replication and disease pathogenesis. Here, we applied systems biology approaches to understand how MAPK pathways benefit SARS-CoV-2 replication to inform the development of novel COVID-19 drug therapies.
Although largely confined to the airways, SARS-CoV-2 infection has been associated with sensory abnormalities that manifest in both acute and chronic phenotypes. To gain insight on the molecular ...basis of these sensory abnormalities, we used the golden hamster model to characterize and compare the effects of infection with SARS-CoV-2 and influenza A virus (IAV) on the sensory nervous system. We detected SARS-CoV-2 transcripts but no infectious material in the cervical and thoracic spinal cord and dorsal root ganglia (DRGs) within the first 24 hours of intranasal virus infection. SARS-CoV-2-infected hamsters exhibited mechanical hypersensitivity that was milder but prolonged compared with that observed in IAV-infected hamsters. RNA sequencing analysis of thoracic DRGs 1 to 4 days after infection suggested perturbations in predominantly neuronal signaling in SARS-CoV-2-infected animals as opposed to type I interferon signaling in IAV-infected animals. Later, 31 days after infection, a neuropathic transcriptome emerged in thoracic DRGs from SARS-CoV-2-infected animals, which coincided with SARS-CoV-2-specific mechanical hypersensitivity. These data revealed potential targets for pain management, including the RNA binding protein ILF3, which was validated in murine pain models. This work elucidates transcriptomic signatures in the DRGs triggered by SARS-CoV-2 that may underlie both short- and long-term sensory abnormalities.
Clinical presentations that develop in response to infection result from interactions between the pathogen and host defenses. SARS-CoV-2, the etiologic agent of COVID-19, directly antagonizes these ...defenses, leading to delayed immune engagement in the lungs that materializes only as cells succumb to infection and are phagocytosed. Leveraging the golden hamster model of COVID-19, we sought to understand the dynamics between SARS-CoV-2 infection in the airways and the systemic host response that ensues. We found that early SARS-CoV-2 replication was largely confined to the respiratory tract and olfactory system and, to a lesser extent, the heart and gastrointestinal tract but generated a host antiviral response in every organ as a result of circulating type I and III interferons. Moreover, we showed that diminishing the response in the airways by immunosuppression or administration of SARS-CoV-2 intravenously resulted in decreased immune priming, viremia, and increased viral tropism, including productive infection of the liver, kidney, spleen, and brain. Last, we showed that productive infection of the airways was required for mounting an effective and system-wide antiviral response. Together, these data illustrate how COVID-19 can result in diverse clinical presentations in which disease outcomes can be a by-product of the speed and strength of immune engagement. These studies provide additional evidence for the mechanistic basis of the diverse clinical presentations of COVID-19 and highlight the ability of the respiratory tract to generate a systemic immune defense after pathogen recognition.
Genetically engineered mouse models (GEMMs) help us to understand human pathologies and develop new therapies, yet faithfully recapitulating human diseases in mice is challenging. Advances in ...genomics have highlighted the importance of non-coding regulatory genome sequences, which control spatiotemporal gene expression patterns and splicing in many human diseases
. Including regulatory extensive genomic regions, which requires large-scale genome engineering, should enhance the quality of disease modelling. Existing methods set limits on the size and efficiency of DNA delivery, hampering the routine creation of highly informative models that we call genomically rewritten and tailored GEMMs (GREAT-GEMMs). Here we describe 'mammalian switching antibiotic resistance markers progressively for integration' (mSwAP-In), a method for efficient genome rewriting in mouse embryonic stem cells. We demonstrate the use of mSwAP-In for iterative genome rewriting of up to 115 kb of a tailored Trp53 locus, as well as for humanization of mice using 116 kb and 180 kb human ACE2 loci. The ACE2 model recapitulated human ACE2 expression patterns and splicing, and notably, presented milder symptoms when challenged with SARS-CoV-2 compared with the existing K18-hACE2 model, thus representing a more human-like model of infection. Finally, we demonstrated serial genome writing by humanizing mouse Tmprss2 biallelically in the ACE2 GREAT-GEMM, highlighting the versatility of mSwAP-In in genome writing.
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