Dengue virus (DENV) is a mosquito-transmitted flavivirus that circulates globally as four distinct serotypes and poses a substantial threat to public health. There are an estimated ~96 million symptomatic infections yearly, including severe cases of dengue fever, underscoring the urgency of identifying effective therapeutics targeting all four serotypes. Nucleoside analogs, which mimic endogenous nucleosides to inhibit viral RNA replication, offer a promising strategy for broad-spectrum antiviral development. Here, we conducted a high-throughput screen of 1,101 nucleoside analogs against DENV serotype 2 (DENV2) in a panel of human cell models, including human epithelial cells, hepatocytes, and fibroblasts. Candidates that were active against DENV2 were screened against all four serotypes. Since flaviviruses including West Nile virus and Zika virus are also important human pathogens, we screened these compounds for activity and identified compounds that were broadly active in these cellular and viral models. We further evaluated antivirals in primary human keratinocytes and fibroblasts, which are early targets of mosquito-transmitted DENV infection. From this screen, we identified 23 nucleoside analogs with broad antiviral activity against DENV and focused on two purine analogs UPGNUC255 and UPGNUC558, that demonstrated potent pan-flaviviral activity achieving >10-fold viral load reduction across all four DENV serotypes and other flaviviruses across cell models. Mechanistic studies revealed that both compounds target the viral RNA-dependent RNA polymerase (RdRp) domain of NS5. Resistance to UPGNUC558 was associated with a conserved S604T substitution, conferring cross-resistance to other 2'C-substituted nucleoside analogs. Resistance to UPGNUC255 was linked to a previously unknown R355Q mutation, located near the catalytic GDD motif of RdRp. These findings highlight UPGNUC255 and UPGNUC558 as promising leads for the development of broad-spectrum antiviral agents against flaviviruses.

Homeostatic control of interferon (IFN) signaling is essential, as dysregulation leads to poor outcomes. While the stimuli-dependent induction of IFNs is well understood, less is known about how IFN mRNAs are maintained at low levels basally or rapidly returned to baseline post-stimulation. We find that selective post-transcriptional control of type I and type III IFN mRNAs by the RNA-binding specificity factor tristetraprolin (TTP) and mRNA decay machinery contributes to homeostatic control by negatively regulating IFN mRNAs at steady state and post-stimulation. Loss of this pathway leads to increased poly(A) tail length, stability, and translation. This increases expression of IFNs, potently blocking severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in vitro and in vivo. A human TTP variant associated with autoimmunity shows decreased TTP levels and increased IFN signaling, further demonstrating that regulation by this pathway impacts disease. Prolonged IFN signaling driven by transient loss of mRNA decay primes cells, inducing innate memory and providing long-term antiviral protection.

SARS-CoV-2 emerged, and continues to evolve, to efficiently infect humans worldwide. SARS-CoV-2 evades early innate recognition, interferon signaling occurring only in bystander cells. How the virus continues to evolve in the face of innate responses has important consequences, but the pathways involved are incompletely understood. Here, we find that autophagy genes regulate innate immune signaling, impacting the basal set point of interferons and, thus, permissivity to infection. Mechanistically, autophagy (mitophagy) genes negatively regulate MAVS, and this low basal level of MAVS is efficiently antagonized by SARS-CoV-2 ORF9b, blocking interferon activation in infected cells. However, loss of autophagy increased MAVS and overcomes ORF9b-mediated antagonism. This has driven the evolution of SARS-CoV-2 to express more ORF9b, allowing SARS-CoV-2 to replicate under conditions of increased MAVS signaling. Altogether, we find a critical role of mitophagy in the regulation of innate immunity and uncover an evolutionary trajectory of SARS-CoV-2 ORF9b to overcome host defenses.

Upon infection, viruses alter the proteome, creating a hospitable environment for infection. Cells respond to limit viral replication, including through protein regulation by post-translational modifications. We use mass spectrometry to define proteome alterations during West Nile virus (WNV) infection. Our studies identify upregulation of HERPUD1, which restricts WNV replication through a mechanism independent of its role in endoplasmic reticulum (ER)-associated degradation (ERAD). We also identify modifications on viral proteins, including a WNV NS3 phosphorylation site that impacts viral replication. Finally, we reveal activation of two host kinases with antiviral activity. We identify phosphorylation at S108 of AMPKβ1, a non-catalytic subunit that regulates activity of the AMPK complex. We also show activation of PAK2 by phosphorylation at S141, which restricts translation of the viral genome. This work contributes to our understanding of the interplay between host and virus while providing a resource to define the changes to the proteome that regulate viral infection.

Rift Valley fever virus (RVFV) is an encephalitic bunyavirus that can infect neurons in the brain. There are no approved therapeutics that can protect from RVFV encephalitis. Innate immunity, the first line of defense against infection, canonically antagonizes viruses through interferon signaling. We found that interferons did not efficiently protect primary cortical neurons from RVFV, unlike other cell types. To identify alternative neuronal antiviral pathways, we screened innate immune ligands and discovered that the TLR2 ligand Pam3CSK4 inhibited RVFV infection, and other bunyaviruses. Mechanistically, we found that Pam3CSK4 blocks viral fusion, independent of TLR2. In a mouse model of RVFV encephalitis, Pam3CSK4 treatment protected animals from infection and mortality. Overall, Pam3CSK4 is a bunyavirus fusion inhibitor active in primary neurons and the brain, representing a new approach toward the development of treatments for encephalitic bunyavirus infections.

Rubella is a highly contagious viral infection that usually causes a mild disease in children and adults. However, infection during pregnancy can result in a fetal or newborn death or congenital rubella syndrome (CRS), a constellation of permanent birth defects including cataracts, heart defects, and sensorineural deafness. The live-attenuated rubella vaccine has been highly effective, with the Americas declared free of endemic rubella transmission in 2015. However, rubella remains a significant problem worldwide and the leading cause of vaccine-preventable birth defects globally. Thus, elimination of rubella and CRS is a goal of the World Health Organization. No specific therapeutics are approved for the rubella virus. Therefore, we set out to identify whether existing small molecules may be repurposed for use against rubella virus infection. Thus, we performed a high-throughput screen for small molecules active against rubella virus in human respiratory cells and identified two nucleoside analogs, NM107 and AT-527, with potent antiviral activity. Furthermore, we found that combining these nucleoside analogs with inhibitors of host nucleoside biosynthesis had synergistic antiviral activity. These studies open the door to new potential approaches to treat rubella infections.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused millions of deaths, posing a substantial threat to global public health. Viruses evolve different strategies to antagonize or evade host immune responses. While ectopic expression of SARS-CoV-2 accessory protein ORF6 blocks interferon (IFN) production and downstream IFN signaling, the role of ORF6 in IFN signaling during bona fide viral infection of respiratory cells is unclear. By comparing wild-type (WT) and ORF6-deleted (ΔORF6) SARS-CoV-2 infection and IFN signaling in respiratory cells, we found that ΔORF6 SARS-CoV-2 replicates more efficiently than WT virus and, thus, stimulates more robust immune signaling. Loss of ORF6 does not alter innate signaling in infected cells: both WT and ΔORF6 virus induce delayed IFN responses only in bystander cells. Moreover, expression of ORF6 in the context of SARS-CoV-2 infection has no effect on Sendai virus-stimulated IFN induction: robust translocation of IRF3 is observed in both SARS-CoV-2 infected and bystander cells. Furthermore, IFN pretreatment potently blocks WT and ΔORF6 virus replication similarly, and both viruses fail to suppress the induction of interferon-stimulated genes (ISGs) upon IFN-β treatment. However, upon treatment with IFN-β, only bystander cells induce STAT1 translocation during infection with WT virus, whereas ΔORF6 virus-infected cells now show translocation. This suggests that under conditions of high IFN activation, ORF6 can attenuate STAT1 activation. These data provide evidence that ORF6 is not sufficient to antagonize IFN production or IFN signaling in SARS-CoV-2-infected respiratory cells but may impact the efficacy of therapeutics that stimulate innate immune pathways. IMPORTANCE Previous studies identified several SARS-CoV-2 proteins, including ORF6, that antagonize host innate immune responses in the context of overexpression of viral proteins in non-respiratory cells. We set out to determine the role of ORF6 in IFN responses during SARS-CoV-2 infection of respiratory cells. Using a deletion strain, we observed no reduction of infection and no difference in evasion of IFN signaling, with responses limited to bystander cells. Moreover, stimulation of Sendai virus-induced IFN production or IFN-β-stimulated ISG expression was comparable between SARS-CoV-2 virus and SARS-CoV-2 lacking ORF6 virus, suggesting that ORF6 is not sufficient to counteract IFN induction or IFN signaling during viral infection.

SARS-CoV-2 emerged, and is evolving to efficiently infect humans worldwide. SARS-CoV-2 evades early innate recognition, interferon signaling activated only in bystander cells. This balance of innate activation and viral evasion has important consequences, but the pathways involved are incompletely understood. Here we find that autophagy genes regulate innate immune signaling, impacting the basal set point of interferons, and thus permissivity to infection. Mechanistically, autophagy genes negatively regulate MAVS, and this low basal level of MAVS is efficiently antagonized by SARS-CoV-2 ORF9b, blocking interferon activation in infected cells. However, upon loss of autophagy increased MAVS overcomes ORF9b-mediated antagonism suppressing infection. This has led to the evolution of SARS-CoV-2 variants to express higher levels of ORF9b, allowing SARS-CoV-2 to replicate under conditions of increased MAVS signaling. Altogether, we find a critical role of autophagy in the regulation of innate immunity and uncover an evolutionary trajectory of SARS-CoV-2 ORF9b to overcome host defenses.

Arthropod-borne viruses, including the alphavirus chikungunya virus (CHIKV), cause acute disease in millions of people and utilize potent mechanisms to antagonize and circumvent innate immune pathways including the type I interferon (IFN) pathway. In response, hosts have evolved antiviral counterdefense strategies that remain incompletely understood. Recent studies have found that long noncoding RNAs (lncRNAs) regulate classical innate immune pathways; how lncRNAs contribute to additional antiviral counterdefenses remains unclear. Using high-throughput genetic screening, we identified a cytoplasmic antiviral lncRNA that we named antiviral lncRNA prohibiting human alphaviruses (ALPHA), which is transcriptionally induced by alphaviruses and functions independently of IFN to inhibit the replication of CHIKV and its closest relative, O’nyong’nyong virus (ONNV), but not other viruses. Furthermore, we showed that ALPHA interacts with CHIKV genomic RNA and restrains viral RNA replication. Together, our findings reveal that ALPHA and potentially other lncRNAs can mediate non-canonical antiviral immune responses against specific viruses.

The SARS-CoV-2 virus has infected more than 261 million people and has led to more than 5 million deaths in the past year and a half 1 ( https://www.who.org/ ). Individuals with SARS-CoV-2 infection typically develop mild-to-severe flu-like symptoms, whereas infection of a subset of individuals leads to severe-to-fatal clinical outcomes 2 . Although vaccines have been rapidly developed to combat SARS-CoV-2, there has been a dearth of antiviral therapeutics. There is an urgent need for therapeutics, which has been amplified by the emerging threats of variants that may evade vaccines. Large-scale efforts are underway to identify antiviral drugs. Here we screened approximately 18,000 drugs for antiviral activity using live virus infection in human respiratory cells and validated 122 drugs with antiviral activity and selectivity against SARS-CoV-2. Among these candidates are 16 nucleoside analogues, the largest category of clinically used antivirals. This included the antivirals remdesivir and molnupiravir, which have been approved for use in COVID-19. RNA viruses rely on a high supply of nucleoside triphosphates from the host to efficiently replicate, and we identified a panel of host nucleoside biosynthesis inhibitors as antiviral. Moreover, we found that combining pyrimidine biosynthesis inhibitors with antiviral nucleoside analogues synergistically inhibits SARS-CoV-2 infection in vitro and in vivo against emerging strains of SARS-CoV-2, suggesting a clinical path forward.

The investigation of the 2019-2020 E-cigarette or Vaping Product Use-Associated Lung Injury (EVALI) outbreak in New York State provided a unique opportunity to examine the formulations and chemical components found in clandestine cannabis-containing e-liquids. In this EVALI investigation, it was determined that an unusually high proportion (16%) of the cannabis e-liquids analyzed contained significant levels of ∆8-tetrahydrocannabinol (∆8-THC). Although not thought to be the causative agent in the outbreak, the manufacturing origin of vaping e-liquids containing large concentrations of ∆8-THC was of great interest, since high ∆8-THC concentrations are not observed in the extracts of common cannabis strains. A principal component analysis of multiple cannabinoid concentrations revealed clusters of similar or identical ∆8-THC-containing products. This technique may be useful in identifying common manufacturing sources in this and future investigations. Several possible manufacturing methods to enrich ∆8-THC appear in literature and are discussed based on their likelihood as sources of this cannabinoid in these samples from the EVALI investigation. The presence of high levels of ∆8-THC in numerous illicit vaping products may implicate cannabidiol, which is readily available at low cost, as its synthetic precursor.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic, resulting millions of infections and deaths with few effective interventions available. Here, we demonstrate that SARS-CoV-2 evades interferon (IFN) activation in respiratory epithelial cells, resulting in a delayed response in bystander cells. Since pretreatment with IFNs can block viral infection, we reasoned that pharmacological activation of innate immune pathways could control SARS-CoV-2 infection. To identify potent antiviral innate immune agonists, we screened a panel of 75 microbial ligands that activate diverse signaling pathways and identified cyclic dinucleotides (CDNs), canonical STING agonists, as antiviral. Since CDNs have poor bioavailability, we tested the small molecule STING agonist diABZI, and found that it potently inhibits SARS-CoV-2 infection of diverse strains including variants of concern (B.1.351) by transiently stimulating IFN signaling. Importantly, diABZI restricts viral replication in primary human bronchial epithelial cells and in mice in vivo. Our study provides evidence that activation of STING may represent a promising therapeutic strategy to control SARS-CoV-2.

The ongoing COVID-19 pandemic has highlighted the dearth of approved drugs to treat viral infections, with only ∼90 FDA approved drugs against human viral pathogens. To identify drugs that can block SARS-CoV-2 replication, extensive drug screening to repurpose approved drugs is underway. Here, we screened ∼18,000 drugs for antiviral activity using live virus infection in human respiratory cells. Dose-response studies validate 122 drugs with antiviral activity and selectivity against SARS-CoV-2. Amongst these drug candidates are 16 nucleoside analogs, the largest category of clinically used antivirals. This included the antiviral Remdesivir approved for use in COVID-19, and the nucleoside Molnupirivir, which is undergoing clinical trials. RNA viruses rely on a high supply of nucleoside triphosphates from the host to efficiently replicate, and we identified a panel of host nucleoside biosynthesis inhibitors as antiviral, and we found that combining pyrimidine biosynthesis inhibitors with antiviral nucleoside analogs synergistically inhibits SARS-CoV-2 infection in vitro and in vivo suggesting a clinical path forward.

Enteric pathogens overcome barrier immunity within the intestinal environment that includes the endogenous flora. The microbiota produces diverse ligands, and the full spectrum of microbial products that are sensed by the epithelium and prime protective immunity is unknown. Using Drosophila, we find that the gut presents a high barrier to infection, which is partially due to signals from the microbiota, as loss of the microbiota enhances oral viral infection. We report cyclic dinucleotide (CDN) feeding is sufficient to protect microbiota-deficient flies from enhanced oral infection, suggesting that bacterial-derived CDNs induce immunity. Mechanistically, we find CDN protection is dSTING- and dTBK1-dependent, leading to NF-kB-dependent gene expression. Furthermore, we identify the apical nucleoside transporter, CNT2, as required for oral CDN protection. Altogether, our studies define a role for bacterial products in priming immune defenses in the gut.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a devastating global pandemic, infecting over 43 million people and claiming over 1 million lives, with these numbers increasing daily. Therefore, there is urgent need to understand the molecular mechanisms governing SARS-CoV-2 pathogenesis, immune evasion, and disease progression. Here, we show that SARS-CoV-2 can block IRF3 and NF-κB activation early during virus infection. We also identify that the SARS-CoV-2 viral proteins NSP1 and NSP13 can block interferon activation via distinct mechanisms. NSP1 antagonizes interferon signaling by suppressing host mRNA translation, while NSP13 downregulates interferon and NF-κB promoter signaling by limiting TBK1 and IRF3 activation, as phospho-TBK1 and phospho-IRF3 protein levels are reduced with increasing levels of NSP13 protein expression. NSP13 can also reduce NF-κB activation by both limiting NF-κB phosphorylation and nuclear translocation. Last, we also show that NSP13 binds to TBK1 and downregulates IFIT1 protein expression. Collectively, these data illustrate that SARS-CoV-2 bypasses multiple innate immune activation pathways through distinct mechanisms.

E-cigarette or vaping product use-associated lung injury (EVALI) is a serious pulmonary condition that is associated with the extended use of certain vaping products. EVALI was first characterized in the summer of 2019 and has since been reported in all 50 U.S. states. From August 2019 through June 2021, the New York State Department of Health has reported more than 197 confirmed cases emanating from all regions of the state. The Wadsworth Center at the New York State Department of Heath received vaping cartridges recovered from EVALI patients for chemical analysis of their contents. Untargeted analytical methods using gas chromatography-mass spectrometry and liquid chromatography-high-resolution mass spectrometry as well as targeted analyses for a variety of analytes including cannabinoids, pesticides, vitamin E acetate (VEA) and mycotoxins were used to characterize the composition of the vaping fluids and several commercial vaping fluid additives. From the analyses of the 284 e-cigarette devices recovered from patients, 82 were found to be nicotine-containing pods, and 202 devices containing cannabis oil, apparently from unauthorized or black-market dealers. The fluids from the cannabis-oil cartridges tended to have lower levels of THCs (Δ9-tetrahydrocannabinol + Δ8-tetrahydrocannabinol) and total cannabinoids compared with those of commercially produced formulations and contained significant levels of diluents including VEA, medium-chain triglycerides, polyethylene glycol, and castor oil. VEA was the diluent most frequently detected, which was present in 132 (65.3%) of the vaping fluids that contained cannabis oil. When present, VEA ranged from 2.0 to 67.8% of the total mass of the oil with a mean content of 37.0%. In some cases, two or three diluents were detected in the same sample. The ratio of VEA to THCs varied widely, from 0.07 to 5.34. VEA and specifically the high ratios of VEA to THCs in black-market vaping fluids may be causative in EVALI. The safety of additional components and additives that are present in vaping fluids are likewise of concern.

There is an urgent need for antivirals to treat the newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To identify new candidates, we screen a repurposing library of ∼3,000 drugs. Screening in Vero cells finds few antivirals, while screening in human Huh7.5 cells validates 23 diverse antiviral drugs. Extending our studies to lung epithelial cells, we find that there are major differences in drug sensitivity and entry pathways used by SARS-CoV-2 in these cells. Entry in lung epithelial Calu-3 cells is pH independent and requires TMPRSS2, while entry in Vero and Huh7.5 cells requires low pH and triggering by acid-dependent endosomal proteases. Moreover, we find nine drugs are antiviral in respiratory cells, seven of which have been used in humans, and three are US Food and Drug Administration (FDA) approved, including cyclosporine. We find that the antiviral activity of cyclosporine is targeting Cyclophilin rather than calcineurin, revealing essential host targets that have the potential for rapid clinical implementation.

Beginning in June of 2019, there was a marked increase in reported cases of serious pulmonary injury associated with vaping. The condition, referred to as e-cigarette or vaping product use-associated lung injury (EVALI), does not appear to involve an infectious agent; rather, a chemical adulterant or contaminant in vaping fluids is suspected. In August of 2019, the Wadsworth Center began receiving vaporizer cartridges recovered from patients with EVALI for analysis. Having no a priori information of what might be in the cartridges, we employed untargeted analyses using gas chromatography-mass spectrometry and high-resolution mass spectrometry to identify components of concern. Additionally, we employed targeted analyses used for New York medical marijuana products. Here, we report on the analyses of 38 samples from the first 10 New York cases of EVALI for which we obtained cartridges. The illicit fluids had relatively low cannabinoid content, sometimes with unusual Δ9-/Δ8-tetrahydrocannabinol ratios, sometimes containing pesticides and many containing diluents. A notable diluent was α-tocopheryl acetate (vitamin E acetate; VEA), which was found in 64% of the cannabinoid-containing fluids. To investigate potential sources of the VEA, we analyzed six commercial cannabis-oil diluents/thickeners. Three were found to be >95% VEA, two were found to be primarily squalane, and one was primarily α-bisabolol. The cause(s) of EVALI is unknown. VEA and squalane are components of some personal care products; however, there is growing concern that vaping large amounts of these compounds is not safe.