All differential on the splicing front: Host alternative splicing alters the landscape of virus-host conflict.

Alternative RNA splicing is a co-transcriptional process that richly increases proteome diversity, and is dynamically regulated based on cell species, lineage, and activation state. Virus infection in vertebrate hosts results in rapid host transcriptome-wide changes, and regulation of alternative splicing can direct a combinatorial effect on the host transcriptome. There has been a recent increase in genome-wide studies evaluating host alternative splicing during viral infection, which integrates well with prior knowledge on viral interactions with host splicing proteins.

Alternative splicing liberates a cryptic cytoplasmic isoform of mitochondrial MECR that antagonizes influenza virus.

Viruses must balance their reliance on host cell machinery for replication while avoiding host defense. Influenza A viruses are zoonotic agents that frequently switch hosts, causing localized outbreaks with the potential for larger pandemics. The host range of influenza virus is limited by the need for successful interactions between the virus and cellular partners.

EPS8 Facilitates Uncoating of Influenza A Virus.

All viruses balance interactions between cellular machinery co-opted to support replication and host factors deployed to halt the infection. We use gene correlation analysis to perform an unbiased screen for host factors involved in influenza A virus (FLUAV) infection. Our screen identifies the cellular factor epidermal growth factor receptor pathway substrate 8 (EPS8) as the highest confidence pro-viral candidate.

ANP32B, or not to be, that is the question for influenza virus.

The evolutionary history of ANP32 proteins impacts how influenza virus jumps from birds to mammals.

Differential Splicing of ANP32A in Birds Alters Its Ability to Stimulate RNA Synthesis by Restricted Influenza Polymerase.

Adaptation of viruses to their hosts can result in specialization and a restricted host range. Species-specific polymorphisms in the influenza virus polymerase restrict its host range during transmission from birds to mammals. ANP32A was recently identified as a cellular co-factor affecting polymerase adaption and activity.

Influenza virus recruits host protein kinase C to control assembly and activity of its replication machinery.

Influenza virus expresses transcripts early in infection and transitions towards genome replication at later time points. This process requires de novo assembly of the viral replication machinery, large ribonucleoprotein complexes (RNPs) composed of the viral polymerase, genomic RNA and oligomeric nucleoprotein (NP). Despite the central role of RNPs during infection, the factors dictating where and when they assemble are poorly understood.

Mutations at highly conserved residues in influenza A(H1N1)pdm09 virus affect neuraminidase activity.

Influenza virus neuraminidase (NA) plays a pivotal role during viral growth since its sialidase activity allows the efficient release of nascent virions from infected cells. Consequently, mutations in the NA catalytic site affecting sialic acid (SA) cleavage may influence the biological properties of influenza viruses. This study reports two amino acid substitutions (N386K and P431S) in the NA of the influenza A(H1N1)pdm09 virus that emerged in 2009 in Mexico.

Replication-Competent Influenza A Viruses Expressing Reporter Genes.

Influenza A viruses (IAV) cause annual seasonal human respiratory disease epidemics. In addition, IAV have been implicated in occasional pandemics with inordinate health and economic consequences. Studying IAV, in vitro or in vivo, requires the use of laborious secondary methodologies to identify virus-infected cells.

Mutations Designed by Ensemble Defect to Misfold Conserved RNA Structures of Influenza A Segments 7 and 8 Affect Splicing and Attenuate Viral Replication in Cell Culture.

Influenza A virus is a significant public health threat, but little is understood about the viral RNA structure and function. Current vaccines and therapeutic options to control influenza A virus infections are mostly protein-centric and of limited effectiveness. Here, we report using an ensemble defect approach to design mutations to misfold regions of conserved mRNA structures in influenza A virus segments 7 and 8.

Replication-Competent Influenza A and B Viruses Expressing a Fluorescent Dynamic Timer Protein for In Vitro and In Vivo Studies.

Influenza A and B viruses (IAV and IBV, respectively) cause annual seasonal human respiratory disease epidemics. In addition, IAVs have been implicated in occasional pandemics with inordinate health and economic consequences. Studying influenza viruses in vitro or in vivo requires the use of laborious secondary methodologies to identify infected cells.

Development and applications of single-cycle infectious influenza A virus (sciIAV).

The diverse host range, high transmissibility, and rapid evolution of influenza A viruses justify the importance of containing pathogenic viruses studied in the laboratory. Other than physically or mechanically changing influenza A virus containment procedures, modifying the virus to only replicate for a single round of infection similarly ensures safety and consequently decreases the level of biosafety containment required to study highly pathogenic members in the virus family. This biological containment is more ideal because it is less apt to computer, machine, or human error.

Downregulating viral gene expression: codon usage bias manipulation for the generation of novel influenza A virus vaccines.

Vaccination represents the best option to protect humans against influenza virus. However, improving the effectiveness of current vaccines could better stifle the health burden caused by viral infection. Protein synthesis from individual genes can be downregulated by synthetically deoptimizing a gene's codon usage.

Competitive detection of influenza neutralizing antibodies using a novel bivalent fluorescence-based microneutralization assay (BiFMA).

Avian-derived influenza A zoonoses are closely monitored and may be an indication of virus strains with pandemic potential. Both successful vaccination and convalescence of influenza A virus in humans typically results in the induction of antibodies that can neutralize viral infection. To improve long-standing and new-generation methodologies for detection of neutralizing antibodies, we have employed a novel reporter-based approach that allows for multiple antigenic testing within a single sample.

An improved method for estimating antibody titers in microneutralization assay using green fluorescent protein.

Viruses that express reporter genes upon infection have been recently used to evaluate neutralizing antibody responses, where a lack of reporter expression indicates specific virus inhibition. The traditional model-based methods using standard outcome of percent neutralization could be applied to the data from the assays to estimate antibody titers. However, the data produced are sometimes irregular, which can yield meaningless outcomes of percent neutralization that do not fit the typical curves for immunoassays, making automated or semi-high throughput antibody titer estimation unreliable.

High-Affinity H7 Head and Stalk Domain-Specific Antibody Responses to an Inactivated Influenza H7N7 Vaccine After Priming With Live Attenuated Influenza Vaccine.

Recent studies have shown that live attenuated influenza vaccines (LAIVs) expressing avian influenza virus hemagglutinins (HAs) prime for strong protective antibody responses to an inactivated influenza vaccine (IIV) containing the HA. To better understand this priming effect, we compared H7 HA head and stalk domain-specific B-cell responses in H7N7 LAIV-primed subjects and non-H7-primed controls after a single dose of H7N7 IIV. As previously reported, H7N7 LAIV-primed subjects but not control subjects generated strong hemagglutination-inhibiting and neutralizing antibody responses to the H7N7 IIV.

Replication-competent influenza A viruses expressing a red fluorescent protein.

Like most animal viruses, studying influenza A in model systems requires secondary methodologies to identify infected cells. To circumvent this requirement, we describe the generation of replication-competent influenza A red fluorescent viruses. These influenza A viruses encode mCherry fused to the viral non-structural 1 (NS1) protein and display comparable growth kinetics to wild-type viruses in vitro.

Development of a mouse-adapted live attenuated influenza virus that permits in vivo analysis of enhancements to the safety of live attenuated influenza virus vaccine.

The live attenuated influenza virus vaccine (LAIV) is preferentially recommended for use in persons 2 through 49 years of age but has not been approved for children under 2 or asthmatics due to safety concerns. Therefore, increasing safety is desirable. Here we describe a murine LAIV with reduced pathogenicity that retains lethality at high doses and further demonstrate that we can enhance safety in vivo through mutations within NS1.

The specialized proresolving mediator 17-HDHA enhances the antibody-mediated immune response against influenza virus: a new class of adjuvant?

Influenza viruses remain a critical global health concern. More efficacious vaccines are needed to protect against influenza virus, yet few adjuvants are approved for routine use. Specialized proresolving mediators (SPMs) are powerful endogenous bioactive regulators of inflammation, with great clinical translational properties.

Induction of CD8 T cell heterologous protection by a single dose of single-cycle infectious influenza virus.

The effector functions of specific CD8 T cells are crucial in mediating influenza heterologous protection. However, new approaches for influenza vaccines that can trigger effective CD8 T cell responses have not been extensively explored. We report here the generation of single-cycle infectious influenza virus that lacks a functional hemagglutinin (HA) gene on an X31 genetic background and demonstrate its potential for triggering protective CD8 T cell immunity against heterologous influenza virus challenge.

Influenza A and B virus intertypic reassortment through compatible viral packaging signals.

Unlabelled: Influenza A and B viruses cocirculate in humans and together cause disease and seasonal epidemics. These two types of influenza viruses are evolutionarily divergent, and exchange of genetic segments inside coinfected cells occurs frequently within types but never between influenza A and B viruses. Possible mechanisms inhibiting the intertypic reassortment of genetic segments could be due to incompatible protein functions of segment homologs, a lack of processing of heterotypic segments by influenza virus RNA-dependent RNA polymerase, an inhibitory effect of viral proteins on heterotypic virus function, or an inability to specifically incorporate heterotypic segments into budding virions.

Influenza A virus attenuation by codon deoptimization of the NS gene for vaccine development.

Unlabelled: Influenza viral infection represents a serious public health problem that causes contagious respiratory disease, which is most effectively prevented through vaccination to reduce transmission and future infection. The nonstructural (NS) gene of influenza A virus encodes an mRNA transcript that is alternatively spliced to express two viral proteins, the nonstructural protein 1 (NS1) and the nuclear export protein (NEP). The importance of the NS gene of influenza A virus for viral replication and virulence has been well described and represents an attractive target to generate live attenuated influenza viruses with vaccine potential.

Use of bacteriophage particles displaying influenza virus hemagglutinin for the detection of hemagglutination-inhibition antibodies.

Bacteriophage lambda capsids provide a flexible molecular scaffold that can be engineered to display a wide range of exogenous proteins, including full-length viral glycoproteins produced in eukaryotic cells. One application for such particles lies in the detection of virus-specific antibodies, since they may obviate the need to work with infectious stocks of highly pathogenic or emerging viruses that can pose significant biosafety and biocontainment challenges. Bacteriophage lambda capsids were produced that displayed an insect-cell derived, recombinant H5 influenza virus hemagglutinin (HA) on their surface.

Crystallographic fragment screening and structure-based optimization yields a new class of influenza endonuclease inhibitors.

Seasonal and pandemic influenza viruses continue to be a leading global health concern. Emerging resistance to the current drugs and the variable efficacy of vaccines underscore the need for developing new flu drugs that will be broadly effective against wild-type and drug-resistant influenza strains. Here, we report the discovery and development of a class of inhibitors targeting the cap-snatching endonuclease activity of the viral polymerase.

Protection against lethal influenza with a viral mimic.

Despite countermeasures against influenza virus that prevent (vaccines) and treat (antivirals) infection, this upper respiratory tract human pathogen remains a global health burden, causing both seasonal epidemics and occasional pandemics. More potent and safe new vaccine technologies would contribute significantly to the battle against influenza and other respiratory infections. Using plasmid-based reverse genetics techniques, we have developed a single-cycle infectious influenza virus (sciIV) with immunoprotective potential.