Chronic lung disease is often accompanied by disabling extrapulmonary symptoms, notably skeletal muscle dysfunction and atrophy. Moreover, the severity of respiratory symptoms correlates with decreased muscle mass and in turn lowered physical activity and survival rates. Previous models of muscle atrophy in chronic lung disease often modeled COPD and relied on cigarette smoke exposure and LPS-stimulation, but these conditions independently affect skeletal muscle even without accompanying lung disease. Moreover, there is an emerging and pressing need to understand the extrapulmonary manifestations of long-term post-viral lung disease (PVLD) as found in Covid-19. Here, we examine the development of skeletal muscle dysfunction in the setting of chronic pulmonary disease using a mouse model of PVLD caused by infection due to the natural pathogen Sendai virus. We identify a significant decrease in myofiber size when PVLD is maximal at 49 d after infection. We find no change in the relative types of myofibers, but the greatest decrease in fiber size is localized to fast-twitch type IIB myofibers based on myosin heavy chain immunostaining. Remarkably, all biomarkers of myocyte protein synthesis and degradation (total RNA, ribosomal abundance, and ubiquitin-proteasome expression) were stable throughout the acute infectious illness and chronic post-viral disease process. Together, the results demonstrate a distinct pattern of skeletal muscle dysfunction in a mouse model of long-term PVLD. The findings thereby provide new insight into prolonged limitations in exercise capacity in patients with chronic lung disease after viral infections and perhaps other types of lung injury.

The resurgence of influenza and continued circulation of SARS-CoV-2 raise the question of how these viruses interact in a co-exposed host. Here we studied virus-virus and host-virus interactions during influenza A virus (IAV) -SARS-CoV-2 coinfection using differentiated cultures of the human airway epithelium. Coexposure to IAV enhanced the tissue antiviral response during SARS-CoV-2 infection and suppressed SARS-CoV-2 replication. Oseltamivir, an antiviral targeting influenza, reduced IAV replication during coinfection but also reduced the antiviral response and paradoxically restored SARS-CoV-2 replication. These results highlight the importance of diagnosing coinfections and compel further study of how coinfections impact the outcome of antiviral therapy.

Persistent and uncontrolled SARS-CoV-2 replication in immunocompromised individuals has been observed and may be a contributing source of novel viral variants that continue to drive the pandemic. Importantly, the effects of immunodeficiency associated with chronic HIV infection on COVID-19 disease and viral persistence have not been directly addressed in a controlled setting. Here we conducted a pilot study wherein two pigtail macaques (PTM) chronically infected with SIVmac239 were exposed to SARS-CoV-2 and monitored for six weeks for clinical disease, viral replication, and viral evolution, and compared to our previously published cohort of SIV-naïve PTM infected with SARS-CoV-2. At the time of SARS-CoV-2 infection, one PTM had minimal to no detectable CD4+ T cells in gut, blood, or bronchoalveolar lavage (BAL), while the other PTM harbored a small population of CD4+ T cells in all compartments. Clinical signs were not observed in either PTM; however, the more immunocompromised PTM exhibited a progressive increase in pulmonary infiltrating monocytes throughout SARS-CoV-2 infection. Single-cell RNA sequencing (scRNAseq) of the infiltrating monocytes revealed a less activated/inert phenotype. Neither SIV-infected PTM mounted detectable anti-SARS-CoV-2 T cell responses in blood or BAL, nor anti-SARS-CoV-2 neutralizing antibodies. Interestingly, despite the diminished cellular and humoral immune responses, SARS-CoV-2 viral kinetics and evolution were indistinguishable from SIV-naïve PTM in all sampled mucosal sites (nasal, oral, and rectal), with clearance of virus by 3-4 weeks post infection. SIV-induced immunodeficiency significantly impacted immune responses to SARS-CoV-2 but did not alter disease progression, viral kinetics or evolution in the PTM model. SIV-induced immunodeficiency alone may not be sufficient to drive the emergence of novel viral variants.

The germicidal properties of short wavelength ultraviolet C (UVC) light are well established and used to inactivate many viruses and other microbes. However, much less is known about germicidal effects of terrestrial solar UV light, confined exclusively to wavelengths in the UVA and UVB regions. Here, we have explored the sensitivity of the human coronaviruses HCoV-NL63 and SARS-CoV-2 to solar-simulated full spectrum ultraviolet light (sUV) delivered at environmentally relevant doses. First, HCoV-NL63 coronavirus inactivation by sUV-exposure was confirmed employing ( i ) viral plaque assays, ( ii ) RT-qPCR detection of viral genome replication, and ( iii ) infection-induced stress response gene expression array analysis. Next, a detailed dose-response relationship of SARS-CoV-2 coronavirus inactivation by sUV was elucidated, suggesting a half maximal suppression of viral infectivity at low sUV doses. Likewise, extended sUV exposure of SARS-CoV-2 blocked cellular infection as revealed by plaque assay and stress response gene expression array analysis. Moreover, comparative (HCoV-NL63 versus SARS-CoV-2) single gene expression analysis by RT-qPCR confirmed that sUV exposure blocks coronavirus-induced redox, inflammatory, and proteotoxic stress responses. Based on our findings, we estimate that solar ground level full spectrum UV light impairs coronavirus infectivity at environmentally relevant doses. Given the urgency and global scale of the unfolding SARS-CoV-2 pandemic, these prototype data suggest feasibility of solar UV-induced viral inactivation, an observation deserving further molecular exploration in more relevant exposure models.

The COVID-19 pandemic remains uncontrolled despite the rapid rollout of safe and effective SARS-CoV-2 vaccines, underscoring the need to develop highly effective antivirals. In the setting of waning immunity from infection and vaccination, breakthrough infections are becoming increasingly common and treatment options remain limited. Additionally, the emergence of SARS-CoV-2 variants of concern with their potential to escape therapeutic monoclonal antibodies emphasizes the need to develop second-generation oral antivirals targeting highly conserved viral proteins that can be rapidly deployed to outpatients. Here, we demonstrate the in vitro antiviral activity and in vivo therapeutic efficacy of GS-621763, an orally bioavailable prodrug of GS-441524, the parental nucleoside of remdesivir, which targets the highly conserved RNA-dependent RNA polymerase. GS-621763 exhibited significant antiviral activity in lung cell lines and two different human primary lung cell culture systems. The dose-proportional pharmacokinetic profile observed after oral administration of GS-621763 translated to dose-dependent antiviral activity in mice infected with SARS-CoV-2. Therapeutic GS-621763 significantly reduced viral load, lung pathology, and improved pulmonary function in COVID-19 mouse model. A direct comparison of GS-621763 with molnupiravir, an oral nucleoside analog antiviral currently in human clinical trial, proved both drugs to be similarly efficacious. These data demonstrate that therapy with oral prodrugs of remdesivir can significantly improve outcomes in SARS-CoV-2 infected mice. Thus, GS-621763 supports the exploration of GS-441524 oral prodrugs for the treatment of COVID-19 in humans.

The airway epithelium is composed of diverse cell types with specialized functions that mediate homeostasis and protect against respiratory pathogens. Human airway epithelial cultures at air-liquid interface (HAE) are a physiologically relevant in vitro model of this heterogeneous tissue, enabling numerous studies of airway disease 1â€"7 . HAE cultures are classically derived from primary epithelial cells, the relatively limited passage capacity of which can limit experimental methods and study designs. BCi-NS1.1, a previously described and widely used basal cell line engineered to express hTERT, exhibits extended passage lifespan while retaining capacity for differentiation to HAE 5 . However, gene expression and innate immune function in HAE derived from BCi-NS1.1 versus primary cells have not been fully characterized. Here, combining single cell RNA-Seq (scRNA-Seq), immunohistochemistry, and functional experimentation, we confirm at high resolution that BCi-NS1.1 and primary HAE cultures are largely similar in morphology, cell type composition, and overall transcriptional patterns. While we observed cell-type specific expression differences of several interferon stimulated genes in BCi-NS1.1 HAE cultures, we did not observe significant differences in susceptibility to infection with influenza A virus and Staphylococcus aureus . Taken together, our results further support BCi-NS1.1-derived HAE cultures as a valuable tool for the study of airway infectious disease.

In an effort to identify therapeutic intervention strategies for the treatment of COVID-19, we have investigated a selection of FDA-approved small molecules and biologics that are commonly used to treat other human diseases. A investigation into 18 small molecules and 3 biologics was conducted in cell culture and the impact of treatment on viral titer was quantified by plaque assay. The investigation identified 4 FDA-approved small molecules, Maraviroc, FTY720 (Fingolimod), Atorvastatin and Nitazoxanide that were able to inhibit SARS-CoV-2 infection. Confocal microscopy with over expressed S-protein demonstrated that Maraviroc reduced the extent of S-protein mediated cell fusion as observed by fewer multinucleate cells in the context of drug-treatment. Mathematical modeling of drug-dependent viral multiplication dynamics revealed that prolonged drug treatment will exert an exponential decrease in viral load in a multicellular/tissue environment. Taken together, the data demonstrate that Maraviroc, Fingolimod, Atorvastatin and Nitazoxanide inhibit SARS-CoV-2 in cell culture.

Wolbachia pipientis (= Wolbachia ) has promise as a tool to suppress virus transmission by Aedes aegypti mosquitoes. However, Wolbachia can have variable effects on mosquito-borne viruses. This variation remains poorly characterized, yet the multimodal effects of Wolbachia on diverse pathogens could have important implications for public health. Here, we examine the effects of somatic infection with two strains of Wolbachia ( w AlbB and w Mel) on the alphaviruses Sindbis virus (SINV), O'nyong-nyong virus (ONNV), and Mayaro virus (MAYV) in Ae. aegypti . We found variable effects of Wolbachia including enhancement and suppression of viral infections, with some effects depending on Wolbachia strain. Both w AlbB- and w Mel-infected mosquitoes showed enhancement of SINV infection rates one week post-infection, with w AlbB-infected mosquitoes also having higher viral titers than controls. Infection rates with ONNV were low across all treatments and no significant effects of Wolbachia were observed. The effects of Wolbachia on MAYV infections were strikingly strain-specific; w Mel strongly blocked MAYV infections and suppressed viral titers, while w AlbB did not influence MAYV infection. The variable effects of Wolbachia on vector competence underscore the importance of further research into how this bacterium impacts the virome of wild mosquitoes including the emergent human pathogens they transmit.

Many poxviruses are significant human and animal pathogens, including viruses that cause smallpox and mpox. Identification of inhibitors of poxvirus replication is critical for drug development to manage poxvirus threats. Here we tested two compounds, nucleoside trifluridine and nucleotide adefovir dipivoxil, for antiviral activities against vaccinia virus (VACV) and mpox virus (MPXV) in physiologically relevant primary human fibroblasts. Both trifluridine and adefovir dipivoxil potently inhibited replication of VACV and MPXV (MA001 2022 isolate) in a plaque assay. Upon further characterization, they both conferred high potency in inhibiting VACV replication with half maximal effective concentrations (EC 50 ) at low nanomolar levels in our recently developed assay based on a recombinant VACV secreted Gaussia luciferase. Our results further validated that the recombinant VACV with Gaussia luciferase secretion is a highly reliable, rapid, non-disruptive, and simple reporter tool for identification and chracterization of poxvirus inhibitors. Both compounds inhibited VACV DNA replication and downstream viral gene expression. Given that both compounds are FDA-approved drugs, and trifluridine is used to treat ocular vaccinia in medical practice due to its antiviral activity, our results suggest that it holds great promise to further test trifluridine and adefovir dipivoxil for countering poxvirus infection, including mpox.

Autophagy is a critical modulator of pathogen invasion response in vertebrates and invertebrates. However, how it affects mosquito-borne viral pathogens that significantly burden public health remains underexplored. To address this gap, we use a genetic approach to activate macroautophagy/autophagy in the yellow fever mosquito ( Aedes aegypti ), infected with a recombinant Sindbis virus (SINV) expressing an autophagy activator. We first demonstrate a 17- amino acid peptide derived from the Ae. aegypti autophagy-related protein 6 (ATG-6/beclin-1-like protein) is sufficient to induce autophagy in C6/36 mosquito cells, as marked by lipidation of ATG- 8 and puncta formation. Next, we engineered a recombinant SINV expressing this bioactive beclin- 1-like peptide and used it to infect and induce autophagy in adult mosquitoes. We find that modulation of autophagy using this recombinant SINV negatively regulated production of infectious viruses. The results from this study improve our understanding of the role of autophagy in arboviruses in invertebrate hosts and also highlight the potential for the autophagy pathway to be exploited for arboviral control.

The SARS-CoV-2 pandemic, and the likelihood of future coronavirus pandemics, has rendered our understanding of coronavirus biology more essential than ever. Small molecule chemical probes offer to both reveal novel aspects of virus replication and to serve as leads for antiviral therapeutic development. The RNA-biased amiloride scaffold was recently tuned to target a viral RNA structure critical for translation in enterovirus 71, ultimately uncovering a novel mechanism to modulate positive-sense RNA viral translation and replication. Analysis of CoV RNA genomes reveal many conserved RNA structures in the 5'-UTR and proximal region critical for viral translation and replication, including several containing bulge-like secondary structures suitable for small molecule targeting. Following phylogenetic conservation analysis of this region, we screened an amiloride-based small molecule library against a less virulent human coronavirus, OC43, to identify lead ligands. Amilorides inhibited OC43 replication as seen in viral plaque assays. Select amilorides also potently inhibited replication competent SARS-CoV-2 as evident in the decreased levels of cell free virions in cell culture supernatants of treated cells. Reporter screens confirmed the importance of RNA structures in the 5'-end of the viral genome for small molecule activity. Finally, NMR chemical shift perturbation studies of the first six stem loops of the 5'-end revealed specific amiloride interactions with stem loops 4, 5a, and 6, all of which contain bulge like structures and were predicted to be strongly bound by the lead amilorides in retrospective docking studies. Taken together, the use of multiple orthogonal approaches allowed us to identify the first small molecules aimed at targeting RNA structures within the 5'-UTR and proximal region of the CoV genome. These molecules will serve as chemical probes to further understand CoV RNA biology and can pave the way for the development of specific CoV RNA-targeted antivirals.

Infections with respiratory viruses can spread via liquid droplets and aerosols, and cause diseases such as influenza and COVID-19. Face masks and other personal protective equipment (PPE) can act as barriers that prevent the spread of respiratory droplets containing these viruses. However, influenza A viruses and coronaviruses are stable for hours on various materials, which makes frequent and correct disposal of these PPE important. Metal ions embedded into PPE may inactivate respiratory viruses, but confounding factors such as absorption of viruses make measuring and optimizing the inactivation characteristics difficult. Here we used polyamide 6.6 (PA66) fibers that had zinc ions embedded during the polymerisation process and systematically investigated if these fibers can absorb and inactivate pandemic SARS-CoV-2 and influenza A virus H1N1. We find that these viruses are readily absorbed by PA66 fabrics and inactivated by zinc ions embedded into this fabric. The inactivation rate (pfu·gram-1·min-1) exceeds the number of active virus particles expelled by a cough and supports a wide range of viral loads. Moreover, we found that the zinc content and the virus inactivating property of the fabric remain stable over 50 standardized washes. Overall, these results provide new insight into the development of "pathogen-free" PPE and better protection against RNA virus spread.

SARS-CoV-2 non-structural protein Nsp14 is a highly conserved enzyme necessary for viral replication. Nsp14 forms a stable complex with non-structural protein Nsp10 and exhibits exoribonuclease and N7-methyltransferase activities. Protein-interactome studies identified human sirtuin 5 (SIRT5) as a putative binding partner of Nsp14. SIRT5 is an NAD-dependent protein deacylase critical for cellular metabolism that removes succinyl and malonyl groups from lysine residues. Here we investigated the nature of this interaction and the role of SIRT5 during SARS-CoV-2 infection. We showed that SIRT5 stably interacts with Nsp14, but not with Nsp10, suggesting that SIRT5 and Nsp10 are parts of separate complexes. We found that SIRT5 catalytic domain is necessary for the interaction with Nsp14, but that Nsp14 does not appear to be directly deacylated by SIRT5. Furthermore, knock-out of SIRT5 or treatment with specific SIRT5 inhibitors reduced SARS-CoV-2 viral levels in cell-culture experiments. SIRT5 knock-out cells expressed higher basal levels of innate immunity markers and mounted a stronger antiviral response. Our results indicate that SIRT5 is a proviral factor necessary for efficient viral replication, which opens novel avenues for therapeutic interventions.

Based on genome-scale loss-of-function screens we discovered that Topoisomerase III-ß (TOP3B), a human topoisomerase that acts on DNA and RNA, is required for yellow fever virus and dengue virus-2 replication. Remarkably, we found that TOP3B is required for efficient replication of all positive-sense-single stranded RNA viruses tested, including SARS-CoV-2. While there are no drugs that specifically inhibit this topoisomerase, we posit that TOP3B is an attractive anti-viral target.

We report the first Human Immune System (HIS)-humanized mouse model ("DRAGA": HLA-A2.HLA-DR4.Rag1KO.IL-2RγcKO.NOD) for COVID-19 research. This mouse is reconstituted with human cord blood-derived, HLA-matched hematopoietic stem cells. It engrafts human epi/endothelial cells expressing the human ACE2 receptor for SARS-CoV-2 and TMPRSS2 serine protease co-localized on lung epithelia. HIS-DRAGA mice sustained SARS-CoV-2 infection, showing deteriorated clinical condition, replicating virus in the lungs, and human-like lung immunopathology including T-cell infiltrates, microthrombi and pulmonary sequelae. Among T-cell infiltrates, lung-resident (CD103+) CD8+ T cells were sequestered in epithelial (CD326+) lung niches and secreted granzyme B and perforin, indicating cytotoxic potential. Infected mice also developed antibodies against the SARS-CoV-2 viral proteins. Hence, HIS-DRAGA mice showed unique advantages as a surrogate in vivo human model for studying SARS-CoV-2 immunopathology and for testing the safety and efficacy of candidate vaccines and therapeutics.

Secondary bacterial infections can exacerbate SARS-CoV-2 infection, but their prevalence and impact remain poorly understood. Here, we established that a mild to moderate SARS-CoV-2 infection increased the risk of pneumococcal coinfection in a time-dependent, but sexindependent, manner in the transgenic K18-hACE mouse model of COVID-19. Bacterial coinfection was not established at 3 d post-virus, but increased lethality was observed when the bacteria was initiated at 5 or 7 d post-virus infection (pvi). Bacterial outgrowth was accompanied by neutrophilia in the groups coinfected at 7 d pvi and reductions in B cells, T cells, IL-6, IL-15, IL-18, and LIF were present in groups coinfected at 5 d pvi. However, viral burden, lung pathology, cytokines, chemokines, and immune cell activation were largely unchanged after bacterial coinfection. Examining surviving animals more than a week after infection resolution suggested that immune cell activation remained high and was exacerbated in the lungs of coinfected animals compared with SARS-CoV-2 infection alone. These data suggest that SARS-CoV-2 increases susceptibility and pathogenicity to bacterial coinfection, and further studies are needed to understand and combat disease associated with bacterial pneumonia in COVID-19 patients.

The newly emerged SARS-CoV-2 caused a global pandemic with astonishing mortality and morbidity. The mechanisms underpinning its highly infectious nature remain poorly understood. We report here that SARS-CoV-2 exploits cellular CTP synthetase 1 (CTPS1) to promote CTP synthesis and suppress interferon (IFN) induction. Screening a SARS-CoV-2 expression library identified ORF7b and ORF8 that suppressed IFN induction via inducing the deamidation of interferon regulatory factor 3 (IRF3). Deamidated IRF3 fails to bind the promoters of classic IRF3-responsible genes, thus muting IFN induction. Conversely, a shRNA-mediated screen focused on cellular glutamine amidotransferases corroborated that CTPS1 deamidates IRF3 to inhibit IFN induction. Functionally, ORF7b and ORF8 activate CTPS1 to promote de novo CTP synthesis while shutting down IFN induction. De novo synthesis of small-molecule inhibitors of CTPS1 enabled CTP depletion and IFN induction in SARS-CoV-2 infection, thus impeding SARS-CoV-2 replication. Our work uncovers a strategy that a viral pathogen couples immune evasion to metabolic activation to fuel viral replication. Inhibition of the cellular CTPS1 offers an attractive means for developing antiviral therapy that would be resistant to SARS-CoV-2 mutation.

There is an urgent need for vaccines and therapeutics to prevent and treat COVID-19. Rapid SARS-CoV-2 countermeasure development is contingent on the availability of robust, scalable, and readily deployable surrogate viral assays to screen antiviral humoral responses, and define correlates of immune protection, and to down-select candidate antivirals. Here, we describe a highly infectious recombinant vesicular stomatitis virus bearing the SARS-CoV-2 spike glycoprotein S as its sole entry glycoprotein that closely resembles the authentic agent in its entry-related properties. We show that the neutralizing activities of a large panel of COVID-19 convalescent sera can be assessed in high-throughput fluorescent reporter assay with rVSV-SARS-CoV-2 S and that neutralization of the rVSV and authentic SARS-CoV-2 by spike-specific antibodies in these antisera is highly correlated. Our findings underscore the utility of rVSV-SARS-CoV-2 S for the development of spike-specific vaccines and therapeutics and for mechanistic studies of viral entry and its inhibition.

SARS-CoV2, the etiologic agent of COVID-19, uses ACE2 as a cell entry receptor. Soluble ACE2 has been shown to have neutralizing antiviral activity but has a short half-life and no active transport mechanism from the circulation into the alveolar spaces of the lung. To overcome this, we constructed an ACE2-human IgG1 fusion protein with mutations in the catalytic domain of ACE2. This fusion protein contained a LALA mutation that abrogates Fcrγ binding, but retains FcRN binding to prolong the half-life, as well as achieve therapeutic concentrations in the lung lavage. Interestingly, a mutation in the catalytic domain of ACE2, MDR504, completely abrogated catalytic activity, but significantly increased binding to SARS-CoV2 spike protein in vitro. This feature correlated with more potent viral neutralization in a plaque assay. Parental administration of the protein showed stable serum concentrations with a serum half-life of ~ 145 hours with excellent bioavailability in the epithelial lining fluid of the lung. Prophylactic administration of MDR504 significantly attenuated SARS-CoV2 infection in a murine model. These data support that the MDR504 hACE2-Fc is an excellent candidate for pre or post-exposure prophylaxis or treatment of COVID-19.

A key feature of the mammalian innate immune response to viral infection is the transcriptional induction of interferon (IFN) genes, which encode for secreted proteins that prime the antiviral response and limit viral replication and dissemination. A hallmark of severe COVID-19 disease caused by SARS-CoV-2 is the low presence of IFN proteins in patient serum despite elevated levels of IFN -encoding mRNAs, indicative of post-transcriptional inhibition of IFN protein production. Herein, we show SARS-CoV-2 infection limits type I and type III IFN biogenesis by preventing the release of mRNA from their sites of transcription and/or triggering their nuclear degradation. In addition, SARS-CoV-2 infection inhibits nuclear-cytoplasmic transport of IFN mRNAs as a consequence of widespread cytosolic mRNA degradation mediated by both activation of the host antiviral endoribonuclease, RNase L, and by the SARS-CoV-2 protein, Nsp1. These findings argue that inhibition of host and/or viral Nsp1-mediated mRNA decay, as well as IFN treatments, may reduce viral-associated pathogenesis by promoting the innate immune response.