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In early 2024, a clade 2.3.4.4b high pathogenic H5N1 avian influenza virus was detected in dairy cows and humans in the United States. Since then, it has spread to herds in at least 13 states and caused symptomatic disease in at least fifteen people. To facilitate rapid testing of existing and novel countermeasures, here, we report the development of an H5N1 viral reverse genetic system, its use to produce fluorescent and bioluminescent variant strains, and their utility in high-throughput evaluation of antiviral interventions., Competing Interests: The authors declare no conflict of interest.

Seasonal influenza vaccines provide mostly strain-specific protection due to the elicitation of antibody responses focused on evolutionarily plastic antigenic sites in the hemagglutinin head domain. To direct the humoral response toward more conserved epitopes, we generated an influenza virus particle where the full-length hemagglutinin protein was replaced with a membrane-anchored, "headless" variant while retaining the normal complement of other viral structural proteins such as the neuraminidase as well as viral RNAs. We found that a single administration of a headless virus particle-based vaccine elicited high titers of antibodies that recognized more conserved epitopes on the major viral glycoproteins. Furthermore, the vaccine could elicit these responses even in the presence of pre-existing, hemagglutinin (HA) head-focused influenza immunity. Importantly, these antibody responses mediated protective, but non-neutralizing functions such as neuraminidase inhibition and antibody-dependent cellular cytotoxicity. Additionally, we show the vaccine can provide protection from homologous and heterologous challenges in mouse models of severe influenza without any measurable HA head-directed antibody responses. Thus, headless hemagglutinin containing viral particles may represent a tool to drive the types of antibody responses predicted to increase influenza vaccine breadth and durability.IMPORTANCECurrent seasonal influenza vaccines provide incomplete protection from disease. This is partially the result of the antibody response being directed toward parts of the virus that are tolerant of mutations. Redirecting the immune response to more conserved regions of the virus has been a central strategy of next-generation vaccine designs and approaches. Here, we develop and test a vaccine based on a modified influenza virus particle that expresses a partially deleted hemagglutinin protein along with the other viral structural proteins. We demonstrate this vaccine elicits antibodies that recognize the more conserved viral epitopes of the hemagglutinin stalk and neuraminidase protein to facilitate protection against influenza viruses despite a lack of classical viral neutralization activity., Competing Interests: Duke University has filed for intellectual property protection of the vaccine production approaches and antigen designs described in this work.

Influenza viruses cause substantial morbidity and mortality every year despite seasonal vaccination. mRNA-based vaccines have the potential to elicit more protective immune responses, but for maximal breadth and durability, it is desirable to deliver both the viral hemagglutinin and neuraminidase glycoproteins. Delivering multiple antigens individually, however, complicates manufacturing and increases cost, thus it would be beneficial to express both proteins from a single mRNA. Here, we develop an mRNA genetic configuration that allows the simultaneous expression of unmodified, full-length NA and HA proteins from a single open reading frame. We apply this approach to glycoproteins from contemporary influenza A and B viruses and, after vaccination, observe high levels of functional antibodies and protection from disease in female mouse and male ferret challenge models. This approach may further efforts to utilize mRNA technology to improve seasonal vaccine efficacy by efficiently delivering multiple viral antigens simultaneously and in their native state., (© 2024. The Author(s).)

Coronaviruses (CoVs) encode nonstructural proteins (nsps) 1-16, which assemble to form replication-transcription complexes that function in viral RNA synthesis. All CoVs encode a proofreading 3'-5' exoribonuclease (ExoN) in nsp14 (nsp14-ExoN) that mediates proofreading and high-fidelity replication and is critical for other roles in replication and pathogenesis. The in vitro enzymatic activity of nsp14 ExoN is enhanced in the presence of the cofactor nsp10. We introduced alanine substitutions in nsp14 of murine hepatitis virus (MHV) at the nsp14-10 interface and recovered mutant viruses with a range of impairments in replication and in vitro biochemical exonuclease activity. Two of these substitutions, nsp14 K7A and D8A, had impairments intermediate between WT-MHV nsp14 and the known ExoN(-) D89A/E91A nsp14 catalytic inactivation mutant. All introduced nsp14-10 interface alanine substitutions impaired in vitro exonuclease activity. Passage of the K7A and D8A mutant viruses selected second-site non-synonymous mutations in nsp14 associated with improved mutant virus replication and exonuclease activity. These results confirm the essential role of the nsp14-nsp10 interaction for efficient enzymatic activity and virus replication, identify proximal and long-distance determinants of nsp14-nsp10 interaction, and support targeting the nsp14-10 interface for viral inhibition and attenuation., Importance: Coronavirus replication requires assembly of a replication transcription complex composed of nonstructural proteins (nsp), including polymerase, helicase, exonuclease, capping enzymes, and non-enzymatic cofactors. The coronavirus nsp14 exoribonuclease mediates several functions in the viral life cycle including genomic and subgenomic RNA synthesis, RNA recombination, RNA proofreading and high-fidelity replication, and native resistance to many nucleoside analogs. The nsp-14 exonuclease activity in vitro requires the non-enzymatic co-factor nsp10, but the determinants and importance the nsp14-10 interactions during viral replication have not been defined. Here we show that for the coronavirus murine hepatitis virus, nsp14 residues at the nsp14-10 interface are essential for efficient viral replication and in vitro exonuclease activity. These results shed new light on the requirements for protein interactions within the coronavirus replication transcription complex, and they may reveal novel non active-site targets for virus inhibition and attenuation.

A central signal that marshals host defense against many infections is the lymphocyte-derived cytokine interferon-gamma (IFNγ). The IFNγ receptor is expressed on most human cells and its activation leads to the expression of antimicrobial proteins that execute diverse cell-autonomous immune programs. One such immune program consists of the sequential detection, ubiquitylation, and destruction of intracellular pathogens. Recently, the IFNγ-inducible ubiquitin E3 ligase RNF213 was identified as a pivotal mediator of such a defense axis. RNF213 provides host protection against viral, bacterial, and protozoan pathogens. To establish infections, potentially susceptible intracellular pathogens must have evolved mechanisms that subdue RNF213-controlled cell-autonomous immunity. In support of this hypothesis, we demonstrate here that a causative agent of bacillary dysentery, Shigella flexneri , uses the type III secretion system (T3SS) effector IpaH1.4 to induce the degradation of RNF213. S. flexneri mutants lacking IpaH1.4 expression are bound and ubiquitylated by RNF213 in the cytosol of IFNγ-primed host cells. Linear (M1-) and lysine-linked ubiquitin is conjugated to bacteria by RNF213 independent of the linear ubiquitin chain assembly complex (LUBAC). We find that ubiquitylation of S. flexneri is insufficient to kill intracellular bacteria, suggesting that S. flexneri employs additional virulence factors to escape from host defenses that operate downstream from RNF213-driven ubiquitylation. In brief, this study identified the bacterial IpaH1.4 protein as a direct inhibitor of mammalian RNF213 and highlights evasion of RNF213-driven immunity as a characteristic of the human-tropic pathogen Shigella .

The CRISPR-Cas13 system has been proposed as an alternative treatment of viral infections. However, for this approach to be adopted as an antiviral, it must be optimized until levels of efficacy rival or exceed the performance of conventional approaches. To take steps toward this goal, we evaluated the influenza viral RNA degradation patterns resulting from the binding and enzymatic activity of mRNA-encoded LbuCas13a and two crRNAs from a prior study, targeting PB2 genomic and messenger RNA. We found that the genome targeting guide has the potential for significantly higher potency than originally detected, because degradation of the genomic RNA is not uniform across the PB2 segment, but it is augmented in proximity to the Cas13 binding site. The PB2 genome targeting guide exhibited high levels (>1 log) of RNA degradation when delivered 24 hours post-infection in vitro and maintained that level of degradation over time, with increasing multiplicity of infection (MOI), and across modern influenza H1N1 and H3N2 strains. Chemical modifications to guides with potent LbuCas13a function, resulted in nebulizer delivered efficacy (>1-2 log reduction in viral titer) in a hamster model of influenza (Influenza A/H1N1/California/04/09) infection given prophylactically or as a treatment (post-infection). Maximum efficacy was achieved with two doses, when administered both pre- and post-infection. This work provides evidence that mRNA-encoded Cas13a can effectively mitigate Influenza A infections opening the door to the development of a programmable approach to treating multiple respiratory infections., Competing Interests: D.V. and P.J.S. have a patent applied for regarding the polymer used in this work. D.V., C.Z. and P.J.S. have a provisional patent filed for the guides described in this work, and are co-founders of Tether Therapeutics. The terms of these arrangements have been reviewed and approved by Emory University in accordance with its conflict-of-interest policies. All other authors declare no competing interests., (Copyright: © 2024 Chaves et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Current seasonal influenza virus vaccines induce responses primarily against immunodominant but highly plastic epitopes in the globular head of the hemagglutinin (HA) glycoprotein. Because of viral antigenic drift at these sites, vaccines need to be updated and readministered annually. To increase the breadth of influenza vaccine-mediated protection, we developed an antigenically complex mixture of recombinant HAs designed to redirect immune responses to more conserved domains of the protein. Vaccine-induced antibodies were disproportionally redistributed to the more conserved stalk of the HA without hindering, and in some cases improving, antibody responses against the head domain. These improved responses led to increased protection against homologous and heterologous viral challenges in both mice and ferrets compared with conventional vaccine approaches. Thus, antigenically complex protein mixtures can at least partially overcome HA head domain antigenic immunodominance and may represent a step toward a more universal influenza vaccine.

The innate immune system features a web of interacting pathways that require exquisite regulation. To identify novel nodes in this immune landscape, we conducted a gain-of-function, genome-wide CRISPR activation screen with influenza A virus. We identified both appreciated and novel antiviral genes, including Jade family PHD zinc finger 3 (JADE3) a protein involved in directing the histone acetyltransferase histone acetyltransferase binding to ORC1 complex to modify chromatin and regulate transcription. JADE3 is both necessary and sufficient to restrict influenza A virus infection. Our results suggest a distinct function for JADE3 as expression of the closely related paralogs JADE1 and JADE2 does not confer resistance to influenza A virus infection. JADE3 is required for both constitutive and inducible expression of the well-characterized antiviral gene interferon-induced transmembrane protein 3 (IFITM3). Furthermore, we find JADE3 activates the NF-kB signaling pathway, which is required for the promotion of IFITM3 expression by JADE3. Therefore, we propose JADE3 activates an antiviral genetic program involving NF-kB-dependent IFITM3 expression to restrict influenza A virus infection., Competing Interests: Conflict of interest The authors declare that they have no conflict of interests with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Negative-stranded RNA viruses are a large group of viruses that encode their genomes in RNA across multiple segments in an orientation antisense to messenger RNA. Their members infect broad ranges of hosts, and there are a number of notable human pathogens. Here, we examine the development of reverse genetic systems as applied to these virus families, emphasizing conserved approaches illustrated by some of the prominent members that cause significant human disease. We also describe the utility of their genetic systems in the development of reporter strains of the viruses and some biological insights made possible by their use. To conclude the review, we highlight some possible future uses of reporter viruses that not only will increase our basic understanding of how these viruses replicate and cause disease but also could inform the development of new approaches to therapeutically intervene.

Although genetically modified mouse models have long been a powerful tool for microbiology research, the manipulation of the mouse genome is expensive, time consuming, and has historically remained the domain of dedicated animal facilities. The recent use of in vivo clustered regularly interspaced short palindromic repeats (CRISPR)-based editing technology has been reported to reduce the expertise, cost, and time required to generate novel mouse lines; it has remained unclear, however, if this new technology could meaningfully alter experimental timelines. Here, we report the optimization of an in oviduct murine genetic manipulation technique for use by microbiologists. We use this approach to generate a series of knockout mice and detail a protocol using an influenza A virus infection model to test the preliminary importance of a host factor in as short as 11 weeks (with a fully backcrossed knockout line in ~22 weeks) from initiation of the study. Broader use of this approach by the microbiology community will allow for more efficient, and rapid, definition of novel pathogenic mechanisms in vivo . IMPORTANCE Clustered regularly interspaced short palindromic repeats (CRISPR)-based technologies have already begun to revolutionize biomedical science. An emerging application of this technology is in the development of genetically modified model organisms to study the mechanisms underlying infectious disease. Here, we describe a protocol using an in vivo CRISPR-based approach that can be used to test the importance of a candidate host factor for microbial pathogenesis in less than 3 months and before complete establishment of a new mouse line. Adoption of this approach by the broader microbiology community will help to decrease the resources and time required to understand how pathogens cause disease which will ultimately speed up the development of new clinical interventions and therapies.

The ultimate success of a viral infection at the cellular level is determined by the number of progeny virions produced. However, most single-cell studies of infection quantify the expression of viral transcripts and proteins, rather than the amount of progeny virions released from infected cells. Here, we overcome this limitation by simultaneously measuring transcription and progeny production from single influenza virus-infected cells by embedding nucleotide barcodes in the viral genome. We find that viral transcription and progeny production are poorly correlated in single cells. The cells that transcribe the most viral mRNA do not produce the most viral progeny and often represent aberrant infections that fail to express the influenza NS gene. However, only some of the discrepancy between transcription and progeny production can be explained by viral gene absence or mutations: there is also a wide range of progeny production among cells infected by complete unmutated virions. Overall, our results show that viral transcription is a relatively poor predictor of an infected cell's contribution to the progeny population., Competing Interests: DB, BD, AB, NH No competing interests declared, AG Inventor on Fred Hutch licensed patents related to viral deep mutational scanning. The patent number is 62/692,398, JB consults or has recently consulted with Apriori Bio, Merck, Moderna, or Oncorus on topics related to viruses and their evolution. Inventor on Fred Hutch licensed patents related to viraldeep mutational scanning. The patent number is 62/692,398, (© 2023, Bacsik et al.)

Multiple coronaviruses (CoVs) can cause respiratory diseases in humans. While prophylactic vaccines designed to prevent infection are available for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), incomplete vaccine efficacy, vaccine hesitancy, and the threat of other pathogenic CoVs for which vaccines do not exist have highlighted the need for effective antiviral therapies. While antiviral compounds targeting the viral polymerase and protease are already in clinical use, their sensitivity to potential resistance mutations as well as their breadth against the full range of human and preemergent CoVs remain incompletely defined. To begin to fill that gap in knowledge, we report here the development of an improved, noninfectious, cell-based fluorescent assay with high sensitivity and low background that reports on the activity of viral proteases, which are key drug targets. We demonstrate that the assay is compatible with not only the SARS-CoV-2 M pro protein but also orthologues from a range of human and nonhuman CoVs as well as clinically reported SARS-CoV-2 drug-resistant M pro variants. We then use this assay to define the breadth of activity of two clinically used protease inhibitors, nirmatrelvir and ensitrelvir. Continued use of this assay will help define the strengths and limitations of current therapies and may also facilitate the development of next-generation protease inhibitors that are broadly active against both currently circulating and preemergent CoVs. IMPORTANCE Coronaviruses (CoVs) are important human pathogens with the ability to cause global pandemics. Working in concert with vaccines, antivirals specifically limit viral disease in people who are actively infected. Antiviral compounds that target CoV proteases are already in clinical use; their efficacy against variant proteases and preemergent zoonotic CoVs, however, remains incompletely defined. Here, we report an improved, noninfectious, and highly sensitive fluorescent method of defining the sensitivity of CoV proteases to small molecule inhibitors. We use this approach to assay the activity of current antiviral therapies against clinically reported SARS-CoV-2 protease mutants and a panel of highly diverse CoV proteases. Additionally, we show this system is adaptable to other structurally nonrelated viral proteases. In the future, this assay can be used to not only better define the strengths and limitations of current therapies but also help develop new, broadly acting inhibitors that more broadly target viral families., Competing Interests: Some authors have IP on viral protease reporter technology.

During pandemics, individuals exhibit differences in risk and clinical outcomes. Here, we developed single-cell high-throughput human in vitro susceptibility testing (scHi-HOST), a method for rapidly identifying genetic variants that confer resistance and susceptibility. We applied this method to influenza A virus (IAV), the cause of four pandemics since the start of the 20 th century. scHi-HOST leverages single-cell RNA sequencing (scRNA-seq) to simultaneously assign genetic identity to cells in mixed infections of cell lines of European, African, and Asian origin, reveal associated genetic variants for viral burden, and identify expression quantitative trait loci. Integration of scHi-HOST with human challenge and experimental validation demonstrated that a missense variant in endoplasmic reticulum aminopeptidase 1 ( ERAP1 ; rs27895) increased IAV burden in cells and human volunteers. rs27895 exhibits population differentiation, likely contributing to greater permissivity of cells from African populations to IAV. scHi-HOST is a broadly applicable method and resource for decoding infectious-disease genetics., Competing Interests: DECLARATION OF INTERESTS The authors declare no competing interests.

Communicable respiratory viral infections pose both epidemic and pandemic threats and broad-spectrum antiviral strategies could improve preparedness for these events. To discover host antiviral restriction factors that may act as suitable targets for the development of host-directed antiviral therapies, we here conduct a whole-genome CRISPR activation screen with influenza B virus (IBV). A top hit from our screen, beta-1,3-glucuronyltransferase 1 (B3GAT1), effectively blocks IBV infection. Subsequent studies reveal that B3GAT1 activity prevents cell surface sialic acid expression. Due to this mechanism of action, B3GAT1 expression broadly restricts infection with viruses that require sialic acid for entry, including Victoria and Yamagata lineage IBVs, H1N1/H3N2 influenza A viruses (IAVs), and the unrelated enterovirus D68. To understand the potential utility of B3GAT1 induction as an antiviral strategy in vivo, we specifically express B3GAT1 in the murine respiratory epithelium and find that overexpression is not only well-tolerated, but also protects female mice from a lethal viral challenge with multiple influenza viruses, including a pandemic-like H1N1 IAV. Thus, B3GAT1 may represent a host-directed broad-spectrum antiviral target with utility against clinically relevant respiratory viruses., (© 2022. The Author(s).)

Multiple coronaviruses have emerged independently in the past 20 years that cause lethal human diseases. Although vaccine development targeting these viruses has been accelerated substantially, there remain patients requiring treatment who cannot be vaccinated or who experience breakthrough infections. Understanding the common host factors necessary for the life cycles of coronaviruses may reveal conserved therapeutic targets. Here, we used the known substrate specificities of mammalian protein kinases to deconvolute the sequence of phosphorylation events mediated by three host protein kinase families (SRPK, GSK-3, and CK1) that coordinately phosphorylate a cluster of serine and threonine residues in the viral N protein, which is required for viral replication. We also showed that loss or inhibition of SRPK1/2, which we propose initiates the N protein phosphorylation cascade, compromised the viral replication cycle. Because these phosphorylation sites are highly conserved across coronaviruses, inhibitors of these protein kinases not only may have therapeutic potential against COVID-19 but also may be broadly useful against coronavirus-mediated diseases.

The cellular fate after infection with human coronaviruses (HCoVs) is typically death. Previous data suggest, however, that the transcriptional state of an individual cell may sometimes allow additional outcomes of infection. Here, to probe the range of interactions a permissive cell type can have with a HCoV, we perform a CRISPR activation screen with HCoV-229E. The screen identified the transcription factor ZBTB7A, which strongly promotes cell survival after infection. Rather than suppressing viral infection, ZBTB7A upregulation allows the virus to induce a persistent infection and homeostatic state with the cell. We also find that control of oxidative stress is a primary driver of cellular survival during HCoV-229E infection. These data illustrate that, in addition to the nature of the infecting virus and the type of cell that it encounters, the cellular gene expression profile prior to infection can affect the eventual fate., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

In vitro tissue models hold great promise for modeling diseases and drug responses. Here, we used emulsion microfluidics to form micro-organospheres (MOSs), which are droplet-encapsulated miniature three-dimensional (3D) tissue models that can be established rapidly from patient tissues or cells. MOSs retain key biological features and responses to chemo-, targeted, and radiation therapies compared with organoids. The small size and large surface-to-volume ratio of MOSs enable various applications including quantitative assessment of nutrient dependence, pathogen-host interaction for anti-viral drug screening, and a rapid potency assay for chimeric antigen receptor (CAR)-T therapy. An automated MOS imaging pipeline combined with machine learning overcomes plating variation, distinguishes tumorspheres from stroma, differentiates cytostatic versus cytotoxic drug effects, and captures resistant clones and heterogeneity in drug response. This pipeline is capable of robust assessments of drug response at individual-tumorsphere resolution and provides a rapid and high-throughput therapeutic profiling platform for precision medicine., Competing Interests: Conflicts of interest X.S., D.H., and H.C. are co-founders of Xilis, Inc. X.S. left Duke and joined Terasaki Institute and Xilis on November 9, 2021. H.C. is also a member of the board of directors of Roche. H.C.’s full disclosure is given at https://www.uu.nl/staff/JCClevers/. Z.W. recently left Duke University and joined Xilis, Inc. as a full-time employee. Patents WO2020242594, US 2021/0285054, and US 2022/006279 are related to this work., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Vaccines targeting SARS-CoV-2 have been shown to be highly effective; however, the breadth against emerging variants and the longevity of protection remains unclear. Postimmunization boosting has been shown to be beneficial for disease protection, and as new variants continue to emerge, periodic (and perhaps annual) vaccination will likely be recommended. New seasonal influenza virus vaccines currently need to be developed every year due to continual antigenic drift, an undertaking made possible by a robust global vaccine production and distribution infrastructure. To create a seasonal combination vaccine targeting both influenza viruses and SARS-CoV-2 that is also amenable to frequent reformulation, we have developed an influenza A virus (IAV) genetic platform that allows the incorporation of an immunogenic domain of the SARS-CoV-2 spike (S) protein onto IAV particles. Vaccination with this combination vaccine elicited neutralizing antibodies and provided protection from lethal challenge with both pathogens in mice. This approach may allow the leveraging of established influenza vaccine infrastructure to generate a cost-effective and scalable seasonal vaccine solution for both influenza and coronaviruses. IMPORTANCE The rapid emergence of SARS-CoV-2 variants since the onset of the pandemic has highlighted the need for both periodic vaccination "boosts" and a platform that can be rapidly reformulated to manufacture new vaccines. In this work, we report an approach that can utilize current influenza vaccine manufacturing infrastructure to generate combination vaccines capable of protecting from both influenza virus- and SARS-CoV-2-induced disease. The production of a combined influenza/SARS-CoV-2 vaccine may represent a practical solution to boost immunity to these important respiratory viruses without the increased cost and administration burden of multiple independent vaccines.

Influenza virus infections are thought to be initiated in a small number of cells; however, the heterogeneity across the cellular responses of the epithelial cells during establishment of disease is incompletely understood. Here, we used an H1N1 influenza virus encoding a fluorescent reporter gene, a cell lineage-labeling transgenic mouse line, and single-cell RNA sequencing to explore the range of responses in a susceptible epithelial cell population during an acute influenza A virus (IAV) infection. Focusing on multiciliated cells, we identified a subpopulation that basally expresses interferon-stimulated genes (ISGs), which we hypothesize may be important for the early response to infection. We subsequently found that a population of infected ciliated cells produce most of the ciliated cell-derived inflammatory cytokines, and nearly all bystander ciliated cells induce a broadly antiviral state. From these data together, we propose that variable preexisting gene expression patterns in the initial cells targeted by the virus may ultimately affect the establishment of viral disease. IMPORTANCE Influenza A virus poses a significant threat to public health, and each year, millions of people in the United States alone are exposed to the virus. We do not currently, however, fully understand why some individuals clear the infection asymptomatically and others become severely ill. Understanding how these divergent phenotypes arise could eventually be leveraged to design therapeutics that prevent severe disease. As a first step toward understanding these different infection states, we used a technology that allowed us to determine how thousands of individual murine lung epithelial cells behaved before and during IAV infection. We found that small subsets of epithelial cells exhibited an antiviral state prior to infection, and similarly, some cells made high levels of inflammatory cytokines during infection. We propose that different ratios of these individual cellular responses may contribute to the broader antiviral state of the lung and may ultimately affect disease severity.

The productive replication of human influenza viruses is almost exclusively restricted to cells in the respiratory tract. However, a key aspect of the host response to viral infection is the production of inflammatory cytokines and chemokines that are not similarly tissue restricted. As such, circulating inflammatory mediators, as well as the resulting activated immune cells, can induce damage throughout the body, particularly in individuals with underlying conditions. As a result, more holistic experimental approaches are required to fully understand the pathogenesis and scope of influenza virus-induced disease. This review summarizes what is known about some of the most well-appreciated nonrespiratory tract sites of influenza virus-induced disease, including neurological, cardiovascular, gastrointestinal, muscular and fetal developmental phenotypes. In the context of this discussion, we describe the in vivo experimental systems currently being used to study nonrespiratory symptoms. Finally, we highlight important future questions and potential models that can be used for a more complete understanding of influenza virus-induced disease., (© 2022 Federation of European Biochemical Societies.)

Influenza viruses are simultaneously supported and antagonized by factors within the host cell. This close relationship is the theoretical basis for future antivirals that target the host rather than the virus itself, a concept termed host-directed therapeutics. Genetic screening has led to the identification of host factors capable of modulating influenza virus infections, and these factors represent candidate targets for host-directed antiviral strategies. Despite advances in understanding host targets, however, there are currently no host-directed interventions for influenza viruses in clinical use. In this brief review, we discuss some host factors identified in knockout/knockdown and overexpression screens that could potentially be targeted as host-directed influenza intervention strategies. We further comment on the feasibility of changing gene expression in the respiratory tract with RNA delivery vectors and transient CRISPR-mediated gene targeting., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Sex hormones, such as estrogen and testosterone, are steroid compounds with well-characterized effects on the coordination and development of vertebrate reproductive systems. Since their discovery, however, it has become clear that these "sex hormones" also regulate/influence a broad range of biological functions. In this review, we will summarize some current findings on how estrogens interact with and regulate inflammation and immunity. Specifically, we will focus on describing the mechanisms by which estrogens alter immune pathway activation, the impact of these changes during infection and the development of long-term immunity, and how different types of estrogens and their respective concentrations mediate these outcomes.

Influenza A viruses encode their genomes across eight, negative sense RNA segments. The six largest segments produce mRNA transcripts that do not generally splice; however, the two smallest segments are actively spliced to produce the essential viral proteins NEP and M2. Thus, viral utilization of RNA splicing effectively expands the viral coding capacity without increasing the number of genomic segments. As a first step towards understanding why splicing is not more broadly utilized across genomic segments, we designed and inserted an artificial intron into the normally nonsplicing NA segment. This insertion was tolerated and, although viral mRNAs were incompletely spliced, we observed only minor effects on viral fitness. To take advantage of the unspliced viral RNAs, we encoded a reporter luciferase gene in frame with the viral ORF such that when the intron was not removed the reporter protein would be produced. This approach, which we also show can be applied to the NP encoding segment and in different viral genetic backgrounds, led to high levels of reporter protein expression with minimal effects on the kinetics of viral replication or the ability to cause disease in experimentally infected animals. These data together show that the influenza viral genome is more tolerant of splicing than previously appreciated and this knowledge can be leveraged to develop viral genetic platforms with utility for biotechnology applications., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: Duke University has filed for intellectual property protection for the technology described in this report.

There is an urgent need for antiviral agents that treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We screened a library of 1900 clinically safe drugs against OC43, a human beta coronavirus that causes the common cold, and evaluated the top hits against SARS-CoV-2. Twenty drugs significantly inhibited replication of both viruses in cultured human cells. Eight of these drugs inhibited the activity of the SARS-CoV-2 main protease, 3CLpro, with the most potent being masitinib, an orally bioavailable tyrosine kinase inhibitor. X-ray crystallography and biochemistry show that masitinib acts as a competitive inhibitor of 3CLpro. Mice infected with SARS-CoV-2 and then treated with masitinib showed >200-fold reduction in viral titers in the lungs and nose, as well as reduced lung inflammation. Masitinib was also effective in vitro against all tested variants of concern (B.1.1.7, B.1.351, and P.1)., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The type I interferon (IFN) response is an important component of the innate immune response to viral infection. Precise control of IFN responses is critical because insufficient expression of IFN-stimulated genes (ISGs) can lead to a failure to restrict viral spread, whereas excessive ISG activation can result in IFN-related pathologies. Although both positive and negative regulatory factors control the magnitude and duration of IFN signaling, it is also appreciated that several ISGs regulate aspects of the IFN response themselves. In this study, we performed a CRISPR activation screen to identify previously unknown regulators of the type I IFN response. We identified the strongly induced ISG encoding ETS variant transcription factor 7 (ETV7) as a negative regulator of the type I IFN response. However, ETV7 did not uniformly suppress ISG transcription. Instead, ETV7 preferentially targeted a subset of antiviral ISGs that were particularly important for IFN-mediated control of influenza viruses. Together, our data assign a function for ETV7 as an IFN response regulator and also identify ETV7 as a potential therapeutic target to increase innate antiviral responses and enhance IFN-based antiviral therapies., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Antiviral therapeutics are a front-line defense against virally induced diseases. Because viruses frequently mutate to escape direct inhibition of viral proteins, there is interest in targeting the host proteins that the virus must co-opt to complete its replication cycle. However, a detailed understanding of the interactions between the virus and the host cell is necessary in order to facilitate development of host-directed therapeutics. As a first step, we performed a genome-wide loss of function screen using the alphacoronavirus HCoV-229E to better define the interactions between coronaviruses and host factors. We report the identification and validation of an ER-resident host protein, TMEM41B, as an essential host factor for not only HCoV-229E but also genetically distinct coronaviruses including the pandemic betacoronavirus SARS-CoV-2. We show that the protein is required at an early, but post-receptor engagement, stage of the viral lifecycle. Further, mechanistic studies revealed that although the protein was not enriched at replication complexes, it likely contributes to viral replication complex formation via mobilization of cholesterol and other lipids to facilitate host membrane expansion and curvature. Continued study of TMEM41B and the development of approaches to prevent its function may lead to broad spectrum anti-coronavirus therapeutics., Competing Interests: The authors have declared that no competing interests exist.

The development of improved and universal anti-influenza vaccines would represent a major advance in the protection of human health. In order to facilitate the development of such vaccines, understanding how viral proteins can contribute to protection from disease is critical. Much of the previous work to address these questions relied on reductionist systems (i.e. vaccinating with individual proteins or VLPs that contain only a few viral proteins); thus we have an incomplete understanding of how immunity to different subsets of viral proteins contribute to protection. Here, we report the development of a platform in which a single viral protein can be deleted from an authentic viral particle that retains the remaining full complement of structural proteins and viral RNA. As a first study with this system, we chose to delete the major IAV antigen, the hemagglutinin protein, to evaluate how the other components of the viral particle contribute en masse to protection from influenza disease. Our results show that while anti-HA immunity plays a major role in protection from challenge with a vaccine-matched strain, the contributions from other structural proteins were the major drivers of protection against highly antigenically drifted, homosubtypic strains. This work highlights the importance of evaluating the inclusion of non-HA viral proteins in the development of broadly efficacious and long-lasting influenza vaccines. Importance Influenza virus vaccines currently afford short-term protection from viruses that are closely related to the vaccine strains. There is currently much effort to develop improved, next-generation influenza vaccines that elicit broader and longer-lasting protection. While the hemagglutinin protein is the major viral antigen, in this work, we developed an approach with which to evaluate the contributions of the non-hemagglutinin proteins to vaccine mediated protection. Our results indicate that other structural proteins together may help to mediate broad antiviral protection and should be considered in the development of more universal influenza vaccines., (Copyright © 2021 American Society for Microbiology.)

Type I interferon (IFN) signaling in fetal tissues causes developmental abnormalities and fetal demise. Although pathogens that infect fetal tissues can induce birth defects through the local production of type I IFN, it remains unknown why systemic IFN generated during maternal infections only rarely causes fetal developmental defects. Here, we report that activation of the guanine nucleotide-binding protein-coupled estrogen receptor 1 (GPER1) during pregnancy is both necessary and sufficient to suppress IFN signaling and does so disproportionately in reproductive and fetal tissues. Inactivation of GPER1 in mice halted fetal development and promoted fetal demise, but only in the context of maternal inflammation. Thus, GPER1 is a central regulator of IFN signaling during pregnancy that allows dynamic antiviral responses in maternal tissues while also preserving fetal health., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Coronavirus infection causes diffuse alveolar damage leading to acute respiratory distress syndrome. The absence of ex vivo models of human alveolar epithelium is hindering an understanding of coronavirus disease 2019 (COVID-19) pathogenesis. Here, we report a feeder-free, scalable, chemically defined, and modular alveolosphere culture system for the propagation and differentiation of human alveolar type 2 cells/pneumocytes derived from primary lung tissue. Cultured pneumocytes express the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor angiotensin-converting enzyme receptor type-2 (ACE2) and can be infected with virus. Transcriptome and histological analysis of infected alveolospheres mirror features of COVID-19 lungs, including emergence of interferon (IFN)-mediated inflammatory responses, loss of surfactant proteins, and apoptosis. Treatment of alveolospheres with IFNs recapitulates features of virus infection, including cell death. In contrast, alveolospheres pretreated with low-dose IFNs show a reduction in viral replication, suggesting the prophylactic effectiveness of IFNs against SARS-CoV-2. Human stem cell-based alveolospheres, thus, provide novel insights into COVID-19 pathogenesis and can serve as a model for understanding human respiratory diseases., Competing Interests: Declaration of Interests A patent application (PCT/US20/53158) related to this work has been filed. H.K. and P.R.T. are listed as co-inventors on this application. P.R.T. serves as a consultant for Cellarity and Surrozen., (Copyright © 2020 Elsevier Inc. All rights reserved.)

In late 2019, a human coronavirus, now known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged, likely from a zoonotic reservoir. This virus causes COVID-19, has infected millions of people, and has led to hundreds of thousands of deaths across the globe. While the best interventions to control and ultimately stop the pandemic are prophylactic vaccines, antiviral therapeutics are important to limit morbidity and mortality in those already infected. At this time, only one FDA-approved anti-SARS-CoV-2 antiviral drug, remdesivir, is available, and unfortunately, its efficacy appears to be limited. Thus, the identification of new and efficacious antivirals is of the highest importance. In order to facilitate rapid drug discovery, flexible, sensitive, and high-throughput screening methods are required. With respect to drug targets, most attention is focused on either the viral RNA-dependent RNA polymerase or the main viral protease, 3CL pro 3CL pro is an attractive target for antiviral therapeutics, as it is essential for processing newly translated viral proteins and the viral life cycle cannot be completed without protease activity. In this work, we report a new assay to identify inhibitors of 3CL pro Our reporter is based on a green fluorescent protein (GFP)-derived protein that fluoresces only after cleavage by 3CL pro This experimentally optimized reporter assay allows for antiviral drug screening in human cell culture at biosafety level 2 (BSL2) with high-throughput compatible protocols. Using this screening approach in combination with existing drug libraries may lead to the rapid identification of novel antivirals to suppress SARS-CoV-2 replication and spread. IMPORTANCE The COVID-19 pandemic has already led to more than 700,000 deaths and innumerable changes to daily life worldwide. Along with development of a vaccine, identification of effective antivirals to treat infected patients is of the highest importance. However, rapid drug discovery requires efficient methods to identify novel compounds that can inhibit the virus. In this work, we present a method for identifying inhibitors of the SARS-CoV-2 main protease, 3CL pro This reporter-based assay allows for antiviral drug screening in human cell culture at biosafety level 2 (BSL2) with high-throughput compatible sample processing and analysis. This assay may help identify novel antivirals to control the COVID-19 pandemic., (Copyright © 2020 American Society for Microbiology.)

Influenza viruses initiate infection in the upper respiratory tract (URT), but early viral tropism and the importance of cell-type-specific antiviral responses in this tissue remain incompletely understood. By infecting transgenic lox-stop-lox reporter mice with a Cre-recombinase-expressing influenza B virus, we identify olfactory sensory neurons (OSNs) as a major viral cell target in the URT. These cells become infected, then eliminate the virus and survive in the host post-resolution of infection. OSN responses to infection are characterized by a strong induction of interferon-stimulated genes and more rapid clearance of viral protein relative to other cells in the epithelium. We speculate that this cell-type-specific response likely serves to protect the central nervous system from infection. More broadly, these results highlight the importance of evaluating antiviral responses across different cell types, even those within the same tissue, to more fully understand the mechanisms of viral disease., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

The coronavirus disease 2019 (COVID-19) pandemic, caused by the SARS-CoV-2 virus, has highlighted the need for antiviral approaches that can target emerging viruses with no effective vaccines or pharmaceuticals. Here, we demonstrate a CRISPR-Cas13-based strategy, PAC-MAN (prophylactic antiviral CRISPR in human cells), for viral inhibition that can effectively degrade RNA from SARS-CoV-2 sequences and live influenza A virus (IAV) in human lung epithelial cells. We designed and screened CRISPR RNAs (crRNAs) targeting conserved viral regions and identified functional crRNAs targeting SARS-CoV-2. This approach effectively reduced H1N1 IAV load in respiratory epithelial cells. Our bioinformatic analysis showed that a group of only six crRNAs can target more than 90% of all coronaviruses. With the development of a safe and effective system for respiratory tract delivery, PAC-MAN has the potential to become an important pan-coronavirus inhibition strategy., Competing Interests: Declaration of Interests The authors have filed provisional patents via Stanford University related to this work., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Influenza A viruses (IAVs) encode their genome across eight, negative sense RNA segments. During viral assembly, the failure to package all eight segments, or packaging a mutated segment, renders the resulting virion incompletely infectious. It is known that the accumulation of these defective particles can limit viral disease by interfering with the spread of fully infectious particles. In order to harness this phenomenon therapeutically, we defined which viral packaging signals were amenable to duplication and developed a viral genetic platform which produced replication competent IAVs that require up to two additional artificial genome segments for full infectivity. The modified and artificial genome segments propagated by this approach are capable of acting as "decoy" segments that, when packaged by coinfecting wild-type viruses, lead to the production of non-infectious viral particles. Although IAVs which require 10 genomic segments for full infectivity are able to replicate themselves and spread in vivo, their genomic modifications render them avirulent in mice. Administration of these viruses, both prophylactically and therapeutically, was able to rescue animals from a lethal influenza virus challenge. Together, our results show that replicating IAVs designed to propagate and spread defective genomic segments represent a potent anti-influenza biological therapy that can target the conserved process of particle assembly to limit viral disease., Competing Interests: The authors have declared that no competing interests exist.

Despite the cytopathic nature of influenza A virus (IAV) replication, we recently reported that a subset of lung epithelial club cells is able to intrinsically clear the virus and survive infection. However, the mechanisms that drive cell survival during a normally lytic infection remained unclear. Using a loss-of-function screening approach, we discovered that the DNA mismatch repair (MMR) pathway is essential for club cell survival of IAV infection. Repair of virally induced oxidative damage by the DNA MMR pathway not only allowed cell survival of infection, but also facilitated host gene transcription, including the expression of antiviral and stress response genes. Enhanced viral suppression of the DNA MMR pathway prevented club cell survival and increased the severity of viral disease in vivo. Altogether, these results identify previously unappreciated roles for DNA MMR as a central modulator of cellular fate and a contributor to the innate antiviral response, which together control influenza viral disease severity.

Influenza A virus (IAV) is a seasonal pathogen with the potential to cause devastating pandemics. IAV infects multiple epithelial cell subsets in the respiratory tract, eliciting damage to the lungs. Clearance of IAV is primarily dependent on CD8+ T cells, which must balance control of the infection with immunopathology. Using a virus expressing Cre recombinase to permanently label infected cells in a Cre-inducible reporter mouse, we previously discovered infected club cells that survive both lytic virus replication and CD8+ T cell-mediated clearance. In this study, we demonstrate that ciliated epithelial cells, type I and type II alveolar cells can also become survivor cells. Survivor cells are stable in the lung long-term and demonstrate enhanced proliferation compared to uninfected cells. When we investigated how survivor cells evade CD8+ T cell killing we observed that survivor cells upregulated the inhibitory ligand PD-L1, but survivor cells did not use PD-L1 to evade CD8+ T cell killing. Instead our data suggest that survivor cells are not inherently resistant to CD8+ T cell killing, but instead no longer present IAV antigen and cannot be detected by CD8+ T cells. Finally, we evaluate the failure of CD8+ T cells to kill these previously infected cells. This work demonstrates that additional cell types can survive IAV infection and that these cells robustly proliferate and are stable long term. By sparing previously infected cells, the adaptive immune system may be minimizing pathology associated with IAV infection., Competing Interests: The authors have declared that no competing interests exist.

Influenza B viruses (IBVs) are major contributors to total human influenza disease, responsible for ~1/3 of all infections. These viruses, however, are relatively less studied than the related influenza A viruses (IAVs). While it has historically been assumed that the viral biology and mechanisms of pathogenesis for all influenza viruses were highly similar, studies have shown that IBVs possess unique characteristics. Relative to IAV, IBV encodes distinct viral proteins, displays a different mutational rate, has unique patterns of tropism, and elicits different immune responses. More work is therefore required to define the mechanisms of IBV pathogenesis. One valuable approach to characterize mechanisms of microbial disease is the use of genetically modified pathogens that harbor exogenous reporter genes. Over the last few years, IBV reporter viruses have been developed and used to provide new insights into the host response to infection, viral spread, and the testing of antiviral therapeutics. In this review, we will highlight the history and study of IBVs with particular emphasis on the use of genetically modified viruses and discuss some remaining gaps in knowledge that can be addressed using reporter expressing IBVs.

The mitochondrial antiviral signaling protein (MAVS) orchestrates host antiviral innate immune response to RNA virus infection. However, how MAVS signaling is controlled to eradicate virus while preventing self-destructive inflammation remains obscure. Here, we show that protein geranylgeranylation, a posttranslational lipid modification of proteins, limits MAVS-mediated immune signaling by targeting Rho family small guanosine triphosphatase Rac1 into the mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) at the mitochondria-ER junction. Protein geranylgeranylation and subsequent palmitoylation promote Rac1 translocation into MAMs upon viral infection. MAM-localized Rac1 limits MAVS' interaction with E3 ligase Trim31 and hence inhibits MAVS ubiquitination, aggregation, and activation. Rac1 also facilitates the recruitment of caspase-8 and cFLIP L to the MAVS signalosome and the subsequent cleavage of Ripk1 that terminates MAVS signaling. Consistently, mice with myeloid deficiency of protein geranylgeranylation showed improved survival upon influenza A virus infection. Our work revealed a critical role of protein geranylgeranylation in regulating antiviral innate immune response.

Influenza B virus (IBV) is an acute, respiratory RNA virus that has been assumed to induce the eventual death of all infected cells. We and others have shown however, that infection with apparently cytopathic viruses does not necessarily lead to cell death; some cells can intrinsically clear the virus and persist in the host long-term. To determine if any cells can survive direct IBV infection, we here generate a recombinant IBV capable of activating a host-cell reporter to permanently label all infected cells. Using this system, we demonstrate that IBV infection leads to the formation of a survivor cell population in the proximal airways that are ciliated-like, but transcriptionally and phenotypically distinct from both actively infected and bystander ciliated cells. We also show that survivor cells are critical to maintain respiratory barrier function. These results highlight a host response pathway that preserves the epithelium to limit the severity of IBV disease.

Influenza A and B viruses can continuously evade humoral immune responses by developing mutations in the globular head of the hemagglutinin (HA) that prevent antibody binding. However, the influenza B virus HA over time displays less antigenic variation despite being functionally and structurally similar to the influenza A virus HA. To determine if the influenza B virus HA is under constraints that limit its antigenic variation, we performed a transposon screen to compare the mutational tolerance of the currently circulating influenza A virus HAs (H1 and H3 subtypes) and influenza B virus HAs (B/Victoria87 and B/Yamagata88 antigenic lineages). A library of insertional mutants for each HA was generated and deep sequenced after passaging to determine where insertions were tolerated in replicating viruses. The head domains of both viruses tolerated transposon mutagenesis, but the influenza A virus head was more tolerant to insertions than the influenza B virus head domain. Furthermore, all five of the known antigenic sites of the influenza A virus HA were tolerant of 15 nucleotide insertions, while insertions were detected in only two of the four antigenic sites in the influenza B virus head domain. Our analysis demonstrated that the influenza B virus HA is inherently less tolerant of transposon-mediated insertions than the influenza A virus HA. The reduced insertional tolerance of the influenza B virus HA may reveal genetic restrictions resulting in a lower capacity for antigenic evolution. IMPORTANCE Influenza viruses cause seasonal epidemics and result in significant human morbidity and mortality. Influenza viruses persist in the human population through generating mutations in the hemagglutinin head domain that prevent antibody recognition. Despite the similar selective pressures on influenza A and B viruses, influenza A virus displays a higher rate and breadth of antigenic variability than influenza B virus. A transposon mutagenesis screen was used to examine if the reduced antigenic variability of influenza B virus was due to inherent differences in mutational tolerance. This study demonstrates that the influenza A virus head domain and the individual antigenic sites targeted by humoral responses are more tolerant to insertions than those of influenza B virus. This finding sheds light on the genetic factors controlling the antigenic evolution of influenza viruses., (Copyright © 2018 American Society for Microbiology.)

Influenza viruses infect approximately 20% of the global population annually, resulting in hundreds of thousands of deaths. While there are Food and Drug Administration (FDA) approved antiviral drugs for combating the disease, vaccination remains the best strategy for preventing infection. Due to the rapid mutation rate of influenza viruses, vaccine formulations need to be updated every year to provide adequate protection. In recent years, a great amount of effort has been focused on the development of a universal vaccine capable of eliciting broadly protective immunity. While universal influenza vaccines clearly have the best potential to provide long-lasting protection against influenza viruses, the timeline for their development, as well as the true universality of protection they afford, remains uncertain. In an attempt to reduce influenza disease burden while universal vaccines are developed and tested, many groups are working on a variety of strategies to improve the efficacy of the standard seasonal vaccine. This review will highlight the different techniques and technologies that have been, or are being, developed to improve the seasonal vaccination efforts against influenza viruses., Competing Interests: Duke University has filed for protection of the intellectual property utilizing dual-HA influenza viruses as vaccines described in this review.

Many viral RNAs are modified by methylation of the N 6 position of adenosine (m 6 A). m 6 A is thought to regulate RNA splicing, stability, translation, and secondary structure. Influenza A virus (IAV) expresses m 6 A-modified RNAs, but the effects of m 6 A on this segmented RNA virus remain unclear. We demonstrate that global inhibition of m 6 A addition inhibits IAV gene expression and replication. In contrast, overexpression of the cellular m 6 A "reader" protein YTHDF2 increases IAV gene expression and replication. To address whether m 6 A residues modulate IAV RNA function in cis, we mapped m 6 A residues on the IAV plus (mRNA) and minus (vRNA) strands and used synonymous mutations to ablate m 6 A on both strands of the hemagglutinin (HA) segment. These mutations inhibited HA mRNA and protein expression while leaving other IAV mRNAs and proteins unaffected, and they also resulted in reduced IAV pathogenicity in mice. Thus, m 6 A residues in IAV transcripts enhance viral gene expression., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Influenza A virus (IAV) is a pathogen that poses significant risks to human health. It is therefore critical to develop strategies to prevent influenza disease. Many loss-of-function screens have been performed to identify the host proteins required for viral infection. However, there has been no systematic screen to identify the host factors that, when overexpressed, are sufficient to prevent infection. In this study, we used CRISPR/dCas9 activation technology to perform a genome-wide overexpression screen to identify IAV restriction factors. The major hit from our screen, B4GALNT2, showed inhibitory activity against influenza viruses with an α2,3-linked sialic acid receptor preference. B4GALNT2 overexpression prevented the infection of every avian influenza virus strain tested, including the H5, H9, and H7 subtypes, which have previously caused disease in humans. Thus, we have used CRISPR/dCas9 activation technology to identify a factor that can abolish infection by avian influenza viruses., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Influenza virus vaccine production is currently limited by the ability to grow circulating human strains in chicken eggs or in cell culture. To facilitate cost-effective growth, vaccine strains are serially passaged under production conditions, which frequently results in mutations of the major antigenic protein, the viral hemagglutinin (HA). Human vaccination with an antigenically drifted strain is known to contribute to poor vaccine efficacy. To address this problem, we developed a replication-competent influenza A virus (IAV) with an artificial genomic organization that allowed the incorporation of two independent and functional HA proteins with different growth requirements onto the same virion. Vaccination with these viruses induced protective immunity against both strains from which the HA proteins were derived, and the magnitude of the response was as high as or higher than vaccination with either of the monovalent parental strains alone. Dual-HA viruses also displayed remarkable antigenic stability; even when using an HA protein known to be highly unstable during growth in eggs, we observed high-titer virus amplification without a single adaptive mutation. Thus, the viral genomic design described in this work can be used to grow influenza virus vaccines to high titers without introducing antigenic mutations. IMPORTANCE Influenza A virus (IAV) is a major public health threat, and vaccination is currently the best available strategy to prevent infection. While there have been many advances in influenza vaccine production, the fact that we cannot predict the growth characteristics of a given strain under vaccine production conditions a priori introduces fundamental uncertainty into the process. Clinically relevant IAV strains frequently grow poorly under vaccine conditions, and this poor growth can result in the delay of vaccine production or the exchange of the recommended strain for one with favorable growth properties. Even in strains that grow to high titers, adaptive mutations in the antigenic protein hemagglutinin (HA) that make it antigenically dissimilar to the circulating strain are common. The genomic restructuring of the influenza virus described in this work offers a solution to the problem of uncertain or unstable growth of IAV during vaccine production., (Copyright © 2017 Harding et al.)