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Annually, roughly 2.5 billion people are at risk for dengue virus (DENV) infection, and the incidence of infection has increased 30-fold since its discovery in the 1900s. At present, there are no globally licensed antiviral treatments or vaccines that protect against all four of the DENV serotypes. The NIAID Live Attenuated Tetravalent Vaccine (LATV) dengue vaccine candidate is composed of variants of three DENV serotypes attenuated by a 30 nucleotide (Δ30) deletion in the 3′ untranslated region and a fourth component that is a chimeric virus in which the prM and E genes of DENV-2 replace those of DENV-4 on the rDEN4Δ30 backbone. The vaccine candidate encodes the non-structural proteins of DENV-1, DENV-3, and DENV-4, which could be of critical importance in the presentation of DENV-specific epitopes in a manner that facilitates antigen presentation and confers higher protection. Our findings demonstrate that the attenuation mechanism (Δ30) resulted in decreased viral infectivity and replication for each vaccine virus in monocyte-derived dendritic cells but were able to generate a robust innate immune response. When tested as monovalent viruses, DEN-4Δ30 displayed the most immunogenic profile. In addition, we found that the tetravalent DENV formulation induced a significantly greater innate immune response than the trivalent formulation. We demonstrate that the presence of two components with a DENV-4Δ30 backbone is necessary for the induction of RANTES, CD40, IP-10, and Type I IFN by the tetravalent formulation. Finally, we found that the DEN-4Δ30 backbone in the DENV-2 component of the vaccine enhanced its antigenic properties, as evidenced by enhanced ability to induce IP-10 and IFNα2 in monocyte-derived dendritic cells. In sum, our study shows that the Δ30 and Δ30/Δ31 mutations attenuate the DENV vaccine strains in terms of replication and infectivity while still allowing the induction of a robust innate immune response.

Dengue virus (DENV) is the mosquito-borne virus of greatest human health concern. There are four serotypes of DENV (1-4) that co-circulate in endemic areas. Each serotype of DENV is individually capable of causing the full spectrum of disease, ranging from self-resolving dengue fever to the more severe dengue haemorrhagic fever (DHF) or dengue shock syndrome (DSS). Based on data published by the CDC, one in four people who become infected with dengue will become ill. Of those that do develop symptomology, the symptoms can range from mild to severe. Symptoms can vary from rash, ocular aches and pains to more intense symptoms in the manifestation of severe dengue. Roughly, 1 in 20 people who become ill will develop severe dengue, which can result in shock, internal bleeding and death. There is currently no specific treatment for dengue and only one licensed vaccine (Dengvaxia) for children 9 through 16 years of age in just a few countries. Despite its licensure for clinical use, Dengvaxia has performed with low efficacy in children and dengue naïve individuals and critically has resulted in increased risk of developing severe dengue in young, vaccinated recipients. Currently, there are various novel strategies for the development of a dengue vaccine. In this review we have conducted a detailed overview of the DENV vaccine landscape, focusing on nine vaccines in the pipeline to provide a comprehensive overview of the most state-of-the-art developments in strategies for vaccines against DENV.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evades antiviral immunity through the expression of viral proteins that block detection, signaling, interferon (IFN) induction, and IFN-stimulated gene (ISG) expression. Weak induction of type I IFNs is associated with a hyperinflammatory response in patients that develop severe COVID-19. Here we uncover a role for cellular nucleic acid-binding protein (CNBP) in restricting SARS-CoV-2. Typically, CNBP resides in the cytosol and, in response to RNA sensing pathways, undergoes phosphorylation, nuclear translocation, and IFNβ enhancer DNA binding to turn on IFNβ gene transcription. In SARS-CoV-2-infected cells CNBP coordinates IFNβ gene transcription. In addition, CNBP binds SARS-CoV-2 viral RNA directly. CNBP competes with the nucleocapsid (N) protein and prevents viral RNA and nucleocapsid protein from undergoing liquid-liquid phase separation (LLPS) forming condensates critical for viral replication. Consequently, cells and animals lacking CNBP have higher viral loads and CNBP-deficient mice succumb rapidly to infection. Altogether, these findings identify CNBP as a key antiviral factor for SARS-CoV-2, functioning both as a regulator of antiviral IFN gene expression and a cell intrinsic restriction factor that disrupts LLPS to limit viral replication and spread.

BackgroundBetter understanding of the association between characteristics of patients hospitalized with coronavirus disease 2019 (COVID-19) and outcome is needed to further improve upon patient management.MethodsImmunophenotyping Assessment in a COVID-19 Cohort (IMPACC) is a prospective, observational study of 1164 patients from 20 hospitals across the United States. Disease severity was assessed using a 7-point ordinal scale based on degree of respiratory illness. Patients were prospectively surveyed for 1 year after discharge for post-acute sequalae of COVID-19 (PASC) through quarterly surveys. Demographics, comorbidities, radiographic findings, clinical laboratory values, SARS-CoV-2 PCR and serology were captured over a 28-day period. Multivariable logistic regression was performed.FindingsThe median age was 59 years (interquartile range [IQR] 20); 711 (61%) were men; overall mortality was 14%, and 228 (20%) required invasive mechanical ventilation. Unsupervised clustering of ordinal score over time revealed distinct disease course trajectories. Risk factors associated with prolonged hospitalization or death by day 28 included age ≥ 65 years (odds ratio [OR], 2.01; 95% CI 1.28-3.17), Hispanic ethnicity (OR, 1.71; 95% CI 1.13-2.57), elevated baseline creatinine (OR 2.80; 95% CI 1.63- 4.80) or troponin (OR 1.89; 95% 1.03-3.47), baseline lymphopenia (OR 2.19; 95% CI 1.61-2.97), presence of infiltrate by chest imaging (OR 3.16; 95% CI 1.96-5.10), and high SARS-CoV2 viral load (OR 1.53; 95% CI 1.17-2.00). Fatal cases had the lowest ratio of SARS-CoV-2 antibody to viral load levels compared to other trajectories over time (p=0.001). 589 survivors (51%) completed at least one survey at follow-up with 305 (52%) having at least one symptom consistent with PASC, most commonly dyspnea (56% among symptomatic patients). Female sex was the only associated risk factor for PASC.InterpretationIntegration of PCR cycle threshold, and antibody values with demographics, comorbidities, and laboratory/radiographic findings identified risk factors for 28-day outcome severity, though only female sex was associated with PASC. Longitudinal clinical phenotyping offers important insights, and provides a framework for immunophenotyping for acute and long COVID-19.FundingNIH.

A well-tolerated and cost-effective oral drug that blocks SARS-CoV-2 growth and dissemination would be a major advance in the global effort to reduce COVID-19 morbidity and mortality. Here, we show that the oral FDA-approved drug nitazoxanide (NTZ) significantly inhibits SARS-CoV-2 viral replication and infection in different primate and human cell models including stem cell-derived human alveolar epithelial type 2 cells. Furthermore, NTZ synergizes with remdesivir, and it broadly inhibits growth of SARS-CoV-2 variants B.1.351 (beta), P.1 (gamma), and B.1617.2 (delta) and viral syncytia formation driven by their spike proteins. Strikingly, oral NTZ treatment of Syrian hamsters significantly inhibits SARS-CoV-2-driven weight loss, inflammation, and viral dissemination and syncytia formation in the lungs. These studies show that NTZ is a novel host-directed therapeutic that broadly inhibits SARS-CoV-2 dissemination and pathogenesis in human and hamster physiological models, which supports further testing and optimization of NTZ-based therapy for SARS-CoV-2 infection alone and in combination with antiviral drugs. ispartof: bioRxiv ispartof: location:United States status: Published online

The global emergence of novel pathogenic viruses presents an important challenge for research, as high biosafety levels are required to process samples. While inactivation of infectious agents facilitates the use of less stringent safety conditions, its effect on other biological entities of interest present in the sample is generally unknown. Here, we analyzed the effect of five inactivation methods (heat, ethanol, formaldehyde, psoralen, and TRIzol) on microbiome composition and diversity in samples collected from four different body sites (gut, nasal, oral, and skin) and compared them against untreated samples from the same tissues. We performed 16S rRNA gene sequencing and estimated abundance and diversity of bacterial taxa present in all samples. Nasal and skin samples were the most affected by inactivation, with ethanol and TRIzol inducing the largest changes in composition, and heat, formaldehyde, TRIzol, and psoralen inducing the largest changes in diversity. Oral and stool microbiomes were more robust to inactivation, with no significant changes in diversity and only moderate changes in composition. Firmicutes was the taxonomic group least affected by inactivation, while Bacteroidetes had a notable enrichment in nasal samples and moderate enrichment in fecal and oral samples. Actinobacteria were more notably depleted in fecal and skin samples, and Proteobacteria exhibited a more variable behavior depending on sample type and inactivation method. Overall, our results demonstrate that inactivation methods can alter the microbiome in a tissue-specific manner and that careful consideration should be given to the choice of method based on the sample type under study. IMPORTANCE Understanding how viral infections impact and are modulated by the microbiome is an important problem in basic research but is also of high clinical relevance under the current pandemic. To facilitate the study of interactions between microbial communities and pathogenic viruses under safe conditions, the infectious agent is generally inactivated prior to processing samples. The effect of this inactivation process in the microbiome is, however, unknown. Further, it is unclear whether biases introduced by inactivation methods are dependent on the sample type under study. Estimating the magnitude and nature of the changes induced by different methods in samples collected from various body sites thus provides important information for current and future studies that require inactivation of pathogenic agents.

Mosquitoes are known as important vectors of many arthropod-borne (arbo)viruses causing disease in humans. These include dengue (DENV) and Zika (ZIKV) viruses. The exogenous small interfering (si)RNA (exo-siRNA) pathway is believed to be the main antiviral defense in arthropods, including mosquitoes. During infection, double-stranded RNAs that form during viral replication and infection are cleaved by the enzyme Dicer 2 (Dcr2) into virus-specific 21 nt vsiRNAs, which are subsequently loaded into Argonaute 2 (Ago2). Ago2 then targets and subsequently cleaves complementary RNA sequences, resulting in degradation of the target viral RNA. Although various studies using silencing approaches have supported the antiviral activity of the exo-siRNA pathway in mosquitoes, and despite strong similarities between the siRNA pathway in the Drosophila melanogaster model and mosquitoes, important questions remain unanswered. The antiviral activity of Ago2 against different arboviruses has been previously demonstrated. However, silencing of Ago2 had no effect on ZIKV replication, whereas Dcr2 knockout enhanced its replication. These findings raise the question as to the role of Ago2 and Dcr2 in the control of arboviruses from different viral families in mosquitoes. Using a newly established Ago2 knockout cell line, alongside the previously reported Dcr2 knockout cell line, we investigated the impact these proteins have on the modulation of different arboviral infections. Infection of Ago2 knockout cell line with alpha- and bunyaviruses resulted in an increase of viral replication, but not in the case of ZIKV. Analysis of small RNA sequencing data in the Ago2 knockout cells revealed a lack of methylated siRNAs from different sources, such as acute and persistently infecting viruses-, TE- and transcriptome-derived RNAs. The results confirmed the importance of the exo-siRNA pathway in the defense against arboviruses, but highlights variability in its response to different viruses and the impact the siRNA pathway proteins have in controlling viral replication. Moreover, this established Ago2 knockout cell line can be used for functional Ago2 studies, as well as research on the interplay between the RNAi pathways.

Zika and dengue virus (ZIKV and DENV) are two flaviviruses responsible for important vector-borne emerging infectious diseases. While there have been multiple DENV epidemics in the last decades, there have been fewer documented epidemics caused by ZIKV until recent years. Thus, our current knowledge about the biology of ZIKV, the disease, and the immune responses in humans is limited. Here, we used mass cytometry (CyTOF) to perform a detailed characterization of the innate immune responses elicited by ZIKV and DENV in human peripheral blood mononuclear cells (PBMCs) from healthy donors infected ex vivo. We found that ZIKV and DENV exposure of human PBMCs induces global phenotypic changes in myeloid cells, characterized mainly by upregulation of costimulatory molecules (CD86 and CD40), CD38, and the type I interferon-inducible protein CD169, a marker for phagocytic function and cross-priming potential in myeloid cells. We also found that ZIKV induces expansion of nonclassical monocytes in cell culture. The analysis of the phenotype of the three monocyte subtypes (classical, intermediate, and nonclassical) at the single-cell level identified differences in their expression of CD86, CD38, CXCL8, and CXCL10 during ZIKV and DENV infection. Overall, using CyTOF, we found that ex vivo infections of PBMCs with ZIKV and DENV reproduced many aspects of the profile found in blood from patients in previously described cohort studies, which highlights the suitability of this system for the study of the human host responses to these viruses. IMPORTANCE Zika and dengue viruses are emergent arboviruses of great public health impact. Both viruses are responsible for important diseases, yet there is currently no vaccine or specific treatment available. Immune cells play critical roles in the virus cycle as well as in the innate and adaptive immune response elicited in the host; therefore, it is critical to understand the changes induced by virus infection in peripheral blood mononuclear cells (PBMCs). In this study, we used a model of ex vivo infection of PBMCs and CyTOF technology to profile the early innate immune changes induced by Zika virus and dengue virus in blood.

The past decade has provided critical information about the cytoplasmic innate immune sensing pathway of cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING). These discoveries have broadened our understanding of the interconnectedness of the cGAS-STING pathway with autophagy, programmed cell death, Rig-I-like receptor (RLR) signaling, DNA independent interferon induction, and how this pathway responds to RNA virus infection. These advances highlight how multiple families of RNA viruses are restricted by and in turn have mechanisms to inhibit cGAS-STING dependent type-I interferon (IFN-I) induction. Here we review recent discoveries of how and why the cGAS-STING pathway responds to infection with RNA viruses, novel findings of RNA viral antagonism of the cGAS-STING innate immune sensing pathway, and attempt to provide context for a shift in thinking as to how critical this DNA sensing pathway is for the restriction of a wide range of RNA viruses.

Dengue virus (DENV) is a mosquito-borne virus that infects upward of 300 million people annually and has the potential to cause fatal hemorrhagic fever and shock. While the parameters contributing to dengue immunopathogenesis remain unclear, the collapse of redox homeostasis and the damage induced by oxidative stress have been correlated with the development of inflammation and progression toward the more severe forms of disease. In the present study, we demonstrate that the accumulation of reactive oxygen species (ROS) late after DENV infection (>24 hpi) resulted from a disruption in the balance between oxidative stress and the nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent antioxidant response. The DENV NS2B3 protease complex strategically targeted Nrf2 for degradation in a proteolysis-independent manner; NS2B3 licensed Nrf2 for lysosomal degradation. Impairment of the Nrf2 regulator by the NS2B3 complex inhibited the antioxidant gene network and contributed to the progressive increase in ROS levels, along with increased virus replication and inflammatory or apoptotic gene expression. By 24 hpi, when increased levels of ROS and antiviral proteins were observed, it appeared that the proviral effect of ROS overcame the antiviral effects of the interferon (IFN) response. Overall, these studies demonstrate that DENV infection disrupts the regulatory interplay between DENV-induced stress responses, Nrf2 antioxidant signaling, and the host antiviral immune response, thus exacerbating oxidative stress and inflammation in DENV infection. IMPORTANCE Dengue virus (DENV) is a mosquito-borne pathogen that threatens 2.5 billion people in more than 100 countries annually. Dengue infection induces a spectrum of clinical symptoms, ranging from classical dengue fever to severe dengue hemorrhagic fever or dengue shock syndrome; however, the complexities of DENV immunopathogenesis remain controversial. Previous studies have reported the importance of the transcription factor Nrf2 in the control of redox homeostasis and antiviral/inflammatory or death responses to DENV. Importantly, the production of reactive oxygen species and the subsequent stress response have been linked to the development of inflammation and progression toward the more severe forms of the disease. Here, we demonstrate that DENV uses the NS2B3 protease complex to strategically target Nrf2 for degradation, leading to a progressive increase in oxidative stress, inflammation, and cell death in infected cells. This study underlines the pivotal role of the Nrf2 regulatory network in the context of DENV infection.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus known to cause epidemics resulting in predominantly symptomatic infections, which in rare cases cause long term debilitating arthritis and arthralgia. Significant progress has been made in understanding the roles of canonical RNA sensing pathways in the host recognition of CHIKV; however, less is known regarding antagonism of CHIKV by cytosolic DNA sensing pathways like that of cyclic GMP-AMP synthase (cGAS) and Stimulator of Interferon Genes (STING). With the use of cGAS or STING null cells we demonstrate that the pathway restricts CHIKV replication in fibroblasts and immune cells. We show that DNA accumulates in the cytoplasm of infected cells and that CHIKV blocks DNA dependent IFN-β transcription. This antagonism of DNA sensing is via an early autophagy-mediated degradation of cGAS and expression of the CHIKV capsid protein is sufficient to induce cGAS degradation. Furthermore, we identify an interaction of CHIKV nsP1 with STING and map the interaction to 23 residues in the cytosolic loop of the adaptor protein. This interaction stabilizes the viral protein and increases the level of palmitoylated nsP1 in cells. Together, this work supports previous publications highlighting the relevance of the cGAS-STING pathway in the early detection of (+)ssRNA viruses and provides direct evidence that CHIKV interacts with and antagonizes cGAS-STING signaling., Author summary Chikungunya virus (CHIKV) is an emerging vector-borne virus which causes outbreaks that spread within populations quickly. The virus has been shown to be highly susceptible to type-I interferon signaling and can block this antiviral pathway. No work has identified if CHIKV is able to block the induction of type-I IFN by antagonizing cytosolic DNA sensing. Here we show that CHIKV induces the accumulation of DNA in the cytoplasm of infected cells and that the virus blocks cGAS-STING dependent innate immune sensing via degradation of cGAS. This work provides the first description of CHIKV mediated inhibition of DNA dependent innate immune sensing and expands upon our previous works by identifying and characterizing another RNA virus family able to counteract cGAS-STING mediated DNA sensing.

Background The mosquito Aedes aegypti is a major vector for the arthropod-borne viruses (arboviruses) chikungunya, dengue, yellow fever and Zika viruses. Vector immune responses pose a major barrier to arboviral transmission, and transgenic insects with altered immunity have been proposed as tools for reducing the global public health impact of arboviral diseases. However, a better understanding of virus-immune interactions is needed to progress the development of such transgenic insects. Although the NF-κB-regulated Toll and ‘immunodeficiency’ (Imd) pathways are increasingly thought to be antiviral, relevant pattern recognition receptors (PRRs) and pathogen-associated molecular patterns (PAMPs) remain poorly characterised in A. aegypti. Methodology/Principle findings We developed novel RT-qPCR and luciferase reporter assays to measure induction of the Toll and Imd pathways in the commonly used A. aegypti-derived Aag2 cell line. We thus determined that the Toll pathway is not inducible by exogenous stimulation with bacterial, viral or fungal stimuli in Aag2 cells under our experimental conditions. We used our Imd pathway-specific assays to demonstrate that the viral dsRNA mimic poly(I:C) is sensed by the Imd pathway, likely through intracellular and extracellular PRRs. The Imd pathway was also induced during infection with the model insect-specific virus cricket paralysis virus (CrPV). Conclusions/Significance Our demonstration that a general PAMP shared by many arboviruses is sensed by the Imd pathway paves the way for future studies to determine how viral RNA is sensed by mosquito PRRs at a molecular level. Our data also suggest that studies measuring inducible immune pathway activation through antimicrobial peptide (AMP) expression in Aag2 cells should be interpreted cautiously given that the Toll pathway is not responsive under all experimental conditions. With no antiviral therapies and few effective vaccines available to treat arboviral diseases, our findings provide new insights relevant to the development of transgenic mosquitoes as a means of reducing arbovirus transmission., Author summary The mosquito Aedes aegypti, found globally across the tropics and subtropics, transmits viruses with a significant global public health impact, including chikungunya, dengue, yellow fever and Zika viruses. There are no antiviral drugs to treat these diseases and few effective vaccines. One way of reducing the global burden of mosquito-borne diseases would be to develop genetically modified mosquitoes unable to transmit viruses. One approach would be to alter the mosquitoes’ immune system to allow them to better fight viral infections. To do so, we first need to understand how viruses are detected by the mosquito immune system. We developed new methods of measuring immune responses in laboratory-cultured mosquito cells and used them to show that one specific arm of the immune system, called the ‘Imd pathway’, can detect the RNA that constitutes the genome of mosquito-borne viruses. These findings pave the way for future immune studies that could inform the development of transmission-incompetent mosquitoes. We also found that another arm of the immune system, called the ‘Toll pathway’, is not functional under any experimental conditions used in this study. This finding has implications for how different laboratories interpret data from these particular cultured cells.

Influenza A viruses (IAVs) cause seasonal epidemics which result in an important health and economic burden. Wild aquatic birds are the natural host of IAV. However, IAV can infect diverse hosts, including humans, domestic poultry, pigs, and others. IAVs circulating in animals occasionally cross the species barrier, infecting humans, which results in mild to very severe disease. In some cases, these viruses can acquire the ability to be transmitted among humans and initiate a pandemic. The nonstructural 1 (NS1) protein of IAV is an important antagonist of the innate immune response. In this study, using recombinant viruses and primary human cells, we show that NS1 proteins from human and avian hosts show intrinsic differences in the modulation of the innate immunity in human dendritic cells and epithelial cells, as well as different cellular localization dynamics in infected cells., The influenza A virus (IAV) nonstructural protein 1 (NS1) contributes to disease pathogenesis through the inhibition of host innate immune responses. Dendritic cells (DCs) release interferons (IFNs) and proinflammatory cytokines and promote adaptive immunity upon viral infection. In order to characterize the strain-specific effects of IAV NS1 on human DC activation, we infected human DCs with a panel of recombinant viruses with the same backbone (A/Puerto Rico/08/1934) expressing different NS1 proteins from human and avian origin. We found that these viruses induced a clearly distinct phenotype in DCs. Specifically, viruses expressing NS1 from human IAV (either H1N1 or H3N2) induced higher levels of expression of type I (IFN-α and IFN-β) and type III (IFN-λ1 to IFNλ3) IFNs than viruses expressing avian IAV NS1 proteins (H5N1, H7N9, and H7N2), but the differences observed in the expression levels of proinflammatory cytokines like tumor necrosis factor alpha (TNF-α) or interleukin-6 (IL-6) were not significant. In addition, using imaging flow cytometry, we found that human and avian NS1 proteins segregate based on their subcellular trafficking dynamics, which might be associated with the different innate immune profile induced in DCs by viruses expressing those NS1 proteins. Innate immune responses induced by our panel of IAV recombinant viruses were also characterized in normal human bronchial epithelial cells, and the results were consistent with those in DCs. Altogether, our results reveal an increased ability of NS1 from avian viruses to antagonize innate immune responses in human primary cells compared to the ability of NS1 from human viruses, which could contribute to the severe disease induced by avian IAV in humans. IMPORTANCE Influenza A viruses (IAVs) cause seasonal epidemics which result in an important health and economic burden. Wild aquatic birds are the natural host of IAV. However, IAV can infect diverse hosts, including humans, domestic poultry, pigs, and others. IAVs circulating in animals occasionally cross the species barrier, infecting humans, which results in mild to very severe disease. In some cases, these viruses can acquire the ability to be transmitted among humans and initiate a pandemic. The nonstructural 1 (NS1) protein of IAV is an important antagonist of the innate immune response. In this study, using recombinant viruses and primary human cells, we show that NS1 proteins from human and avian hosts show intrinsic differences in the modulation of the innate immunity in human dendritic cells and epithelial cells, as well as different cellular localization dynamics in infected cells.

Influenza A virus (IAV) is a respiratory pathogen of high importance to public health. Annual epidemics of seasonal IAV infections in humans are a significant public health and economic burden. IAV also causes sporadic pandemics, which can have devastating effects. The main target cells for IAV replication are epithelial cells in the respiratory epithelium. The cellular innate immune responses induced in these cells upon infection are critical for defense against the virus, and therefore, it is important to understand the complex interactions between the virus and the host cells. In this study, we investigated the innate immune response to IAV in the respiratory epithelium at the single-cell level, providing a better understanding on how a population of epithelial cells functions as a complex system to orchestrate the response to virus infection and how the virus counteracts this system., Early interactions of influenza A virus (IAV) with respiratory epithelium might determine the outcome of infection. The study of global cellular innate immune responses often masks multiple aspects of the mechanisms by which populations of cells work as organized and heterogeneous systems to defeat virus infection, and how the virus counteracts these systems. In this study, we experimentally dissected the dynamics of IAV and human epithelial respiratory cell interaction during early infection at the single-cell level. We found that the number of viruses infecting a cell (multiplicity of infection [MOI]) influences the magnitude of virus antagonism of the host innate antiviral response. Infections performed at high MOIs resulted in increased viral gene expression per cell and stronger antagonist effect than infections at low MOIs. In addition, single-cell patterns of expression of interferons (IFN) and IFN-stimulated genes (ISGs) provided important insights into the contributions of the infected and bystander cells to the innate immune responses during infection. Specifically, the expression of multiple ISGs was lower in infected than in bystander cells. In contrast with other IFNs, IFN lambda 1 (IFNL1) showed a widespread pattern of expression, suggesting a different cell-to-cell propagation mechanism more reliant on paracrine signaling. Finally, we measured the dynamics of the antiviral response in primary human epithelial cells, which highlighted the importance of early innate immune responses at inhibiting virus spread. IMPORTANCE Influenza A virus (IAV) is a respiratory pathogen of high importance to public health. Annual epidemics of seasonal IAV infections in humans are a significant public health and economic burden. IAV also causes sporadic pandemics, which can have devastating effects. The main target cells for IAV replication are epithelial cells in the respiratory epithelium. The cellular innate immune responses induced in these cells upon infection are critical for defense against the virus, and therefore, it is important to understand the complex interactions between the virus and the host cells. In this study, we investigated the innate immune response to IAV in the respiratory epithelium at the single-cell level, providing a better understanding on how a population of epithelial cells functions as a complex system to orchestrate the response to virus infection and how the virus counteracts this system.

Which ideas will shape the microbiology of the future? How will they do so? In our new series of “mSphere of Influence” commentaries, we ask rising young microbiologists to tell us just that. We focus on these young scientists because they are uniquely capable of seeing the scientific literature

BackgroundAedes aegypti is a vector mosquito of major public health importance, transmitting arthropod-borne viruses (arboviruses) such as chikungunya, dengue, yellow fever and Zika viruses. Wild mosquito populations are persistently infected at high prevalence with insect-specific viruses that do not replicate in vertebrate hosts. In experimental settings, acute infections with insect-specific viruses have been shown to modulate arbovirus infection and transmission in Ae. aegypti and other vector mosquitoes. However, the impact of persistent insect-specific virus infections that more closely mimic the situation in nature has not been investigated extensively. Cell lines are useful models for studying virus-host interactions, however the available Ae. aegypti cell lines are poorly defined and heterogenous cultures.Methodology/Principle FindingsWe generated single cell-derived clonal cell lines from the commonly used Ae. aegypti cell line Aag2. Two of the fourteen Aag2-derived clonal cell lines generated harboured markedly and consistently reduced levels of the insect-specific bunyavirus Phasi Charoen-like virus (PCLV) known to persistently infect Aag2 cells. In contrast to studies with acute insect-specific virus infections in cell culture and in vivo, we found that pre-existing persistent PCLV infection had no major impact on the replication of the flaviviruses dengue virus and Zika virus, the alphavirus Sindbis virus, or the rhabdovirus vesicular stomatitis virus. We also performed a detailed characterisation of the morphology, transfection efficiency and immune status of our Aag2-derived clonal cell lines, and have made a clone that we term Aag2-AF5 available to the research community as a well-defined cell culture model for arbovirus-vector interaction studies.Conclusions/SignificanceOur findings highlight the need for further in vivo studies that more closely recapitulate natural arbovirus transmission settings in which arboviruses encounter mosquitoes harbouring persistent rather than acute insect-specific virus infections. Furthermore, we provide the well-characterised Aag2-derived clonal cell line as a valuable resource to the arbovirus research community.AUTHOR SUMMARYMosquito-borne viruses usually only infect humans through the bite of a mosquito that carries the virus. Viruses transmitted by the ‘yellow fever mosquito’ Aedes aegypti, including dengue virus, Zika virus, yellow fever virus and chikungunya virus, are causing an ever-increasing number of human disease cases globally. Mosquito-borne viruses have to infect and replicate inside the mosquito before they are transmitted to humans, and the presence of other infectious agents can change the efficiency of virus transmission. Mosquitoes are known to be infected with ‘insect-specific viruses’ that only infect mosquitoes and cannot cause human disease. We have shown here that in laboratory cell cultures derived from the Aedes aegypti mosquito, pre-existing infection with an insect-specific virus called Phasi Charoen-like virus does not affect the infection and growth of the mosquito-borne viruses dengue virus, Zika virus, Sindbis virus or vesicular stomatitis virus. Compared to previous research, our research is more reflective of conditions that mosquito-borne viruses encounter in nature, and our results provide important new insights into whether and how insect-specific viruses affect mosquito-borne virus transmission. Ultimately, this information could inform ongoing research into whether insect-specific viruses could be used to prevent the transmission of mosquito-borne viruses to reduce global disease burdens.

Background Aedes aegypti is a vector mosquito of major public health importance, transmitting arthropod-borne viruses (arboviruses) such as chikungunya, dengue, yellow fever and Zika viruses. Wild mosquito populations are persistently infected at high prevalence with insect-specific viruses that do not replicate in vertebrate hosts. In experimental settings, acute infections with insect-specific viruses have been shown to modulate arbovirus infection and transmission in Ae. aegypti and other vector mosquitoes. However, the impact of persistent insect-specific virus infections, which arboviruses encounter more commonly in nature, has not been investigated extensively. Cell lines are useful models for studying virus-host interactions, however the available Ae. aegypti cell lines are poorly defined and heterogenous cultures. Methodology/Principle findings We generated single cell-derived clonal cell lines from the commonly used Ae. aegypti cell line Aag2. Two of the fourteen Aag2-derived clonal cell lines generated harboured markedly and consistently reduced levels of the insect-specific bunyavirus Phasi Charoen-like virus (PCLV) known to persistently infect Aag2 cells. In contrast to studies with acute insect-specific virus infections in cell culture and in vivo, we found that pre-existing persistent PCLV infection had no major impact on the replication of the flaviviruses dengue virus and Zika virus, the alphavirus Sindbis virus, or the rhabdovirus vesicular stomatitis virus. We also performed a detailed characterisation of the morphology, transfection efficiency and immune status of our Aag2-derived clonal cell lines, and have made a clone that we term Aag2-AF5 available to the research community as a well-defined cell culture model for arbovirus-vector interaction studies. Conclusions/Significance Our findings highlight the need for further in vivo studies that more closely recapitulate natural arbovirus transmission settings in which arboviruses encounter mosquitoes harbouring persistent rather than acute insect-specific virus infections. Furthermore, we provide the well-characterised Aag2-derived clonal cell line as a valuable resource to the arbovirus research community.

At the beginning of 2017, mSphere initiated an experiment in peer review called mSphereDirect ([1][1]). At that time, we sensed the desire among authors to have more control over the peer review process, which had become slow and rigid, with some even arguing that peer review is “broken.”

SummaryType I interferons (IFN-I) are essential antiviral cytokines produced upon microbial infection. IFN-I elicits this activity through the upregulation of hundreds of IFN-I-stimulated genes (ISGs). The full breadth of ISG induction demands activation of a number of cellular factors including the IκB kinase epsilon (IKKε). However, the mechanism of IKKε activation upon IFN receptor signaling has remained elusive. Here we show that TRIM6, a member of the E3-ubiquitin ligase tripartite motif (TRIM) family of proteins, interacted with IKKε and promoted induction of IKKε-dependent ISGs. TRIM6 and the E2-ubiquitin conjugase UbE2K cooperated in the synthesis of unanchored K48-linked polyubiquitin chains, which activated IKKε for subsequent STAT1 phosphorylation. Our work attributes a previously unrecognized activating role of K48-linked unanchored polyubiquitin chains in kinase activation and identifies the UbE2K-TRIM6-ubiquitin axis as critical for IFN signaling and antiviral response.

Flaviviruses are arthropod-borne RNA viruses that have been used extensively to study host antiviral responses. Often selected just to represent standard single-stranded positive-sense RNA viruses in early studies, the Flavivirus genus over time has taught us how truly unique it is in its remarkable ability to target not just the RNA sensory pathways but also the cytosolic DNA sensing system for its successful replication inside the host cell. This review summarizes the main developments on the unexpected antagonistic strategies utilized by different flaviviruses, with RNA genomes, against the host cyclic GAMP synthase (cGAS)/stimulator of interferon genes (STING) cytosolic DNA sensing pathway in mammalian systems. On the basis of the recent advancements on this topic, we hypothesize that the mechanisms of viral sensing and innate immunity are much more fluid than what we had anticipated, and both viral and host factors will continue to be found as important factors contributing to the host innate immune system in the future.