Influenza A virus drives significant morbidity and mortality in humans and livestock. Annual circulation of the virus in livestock and waterfowl contributes to severe economic disruption and increases the risk of zoonotic transmission of novel strains into the human population, where there is no preexisting immunity. Seasonal vaccinations in humans help prevent infection and can reduce symptoms when infection does occur. However, current vaccination regimens available for livestock are limited in part due to safety concerns regarding reassortment/recombination with circulating strains. Therefore, inactivated vaccines are used instead of the more immunostimulatory live attenuated vaccines. MicroRNAs (miRNAs) have been used previously to generate attenuated influenza A viruses for use as a vaccine. Here, we systematically targeted individual influenza gene mRNAs using the same miRNA to determine the segment(s) that yields maximal attenuation potential. This analysis demonstrated that targeting of NP mRNA most efficiently ablates replication. We further increased the plasticity of miRNA-mediated attenuation of influenza A virus by exploiting a miRNA, miR-21, that is ubiquitously expressed across influenza-susceptible hosts. In order to construct this targeted virus, we used CRISPR/Cas9 to eliminate the universally expressed miR-21 from MDCK cells. miR-21-targeted viruses were attenuated in human, mouse, canine, and avian cells and drove protective immunity in mice. This strategy has the potential to enhance the safety of live attenuated vaccines in humans and zoonotic reservoirs. Influenza A virus circulates annually in both avian and human populations, causing significant morbidity, mortality, and economic burden. High incidence of zoonotic infections greatly increases the potential for transmission to humans, where no preexisting immunity or vaccine exists. There is a critical need for new vaccine strategies to combat emerging influenza outbreaks. MicroRNAs were used previously to attenuate influenza A viruses. We propose the development of a novel platform to produce live attenuated vaccines that are highly customizable, efficacious across a broad species range, and exhibit enhanced safety over traditional vaccination methods. This strategy exploits a microRNA that is expressed abundantly in influenza virus-susceptible hosts. By eliminating this ubiquitous microRNA from a cell line, targeted viruses that are attenuated across susceptible strains can be generated. This approach greatly increases the plasticity of the microRNA targeting approach and enhances vaccine safety.

Influenza A virus (IAV) is a segmented negative-sense RNA virus and is the cause of major epidemics and pandemics. The replication of IAV is complex, involving the production of three distinct RNA species; mRNA, cRNA, and vRNA for all eight genome segments. While understanding IAV replication kinetics is important for drug development and improving vaccine production, current methods for studying IAV kinetics has been limited by the ability to detect all three different RNA species in a scalable manner. Here we report the development of a novel pipeline using total stranded RNA-Seq, which we named Influenza Virus Enumerator of RNA Transcripts (InVERT), that allows for the simultaneous quantification of all three RNA species produced by IAV. Using InVERT, we provide a full landscape of the IAV replication kinetics and found that different groups of viral genes follow different kinetics. The segments coding for RNA-dependent RNA Polymerase (RdRP) produced more vRNA than mRNA while some other segments (NP, NS, HA) consistently made more mRNA than vRNA. vRNA expression levels did not correlate with cRNA expression, suggesting complex regulation of vRNA synthesis. Furthermore, by studying the kinetics of a virus lacking the capacity to generate new polymerase complexes, we found evidence that further supports the model that cRNA synthesis requires newly synthesized RdRP and that incoming RdRP can only generate mRNA. Overall, InVERT is a powerful tool for quantifying IAV RNA species to elucidate key features of IAV replication. Importance Influenza A virus (IAV) is a respiratory pathogen that has caused significant mortality throughout history and remains a global threat to human health. Although much is known about IAV replication, the regulation of IAV replication dynamics is not completely understood. This is due in part to both technical limitations and the complexity of the virus replication, which has a segmented genome and produces three distinct RNA species for each gene segment. We developed a new approach that allows the methodical study of IAV replication kinetics, shedding light on many interesting features of IAV replication biology. This study advances our understanding of the kinetics of IAV replication and will help to facilitate future research in the field.

Many pathogens encode proteases that serve to antagonize the host immune system. In particular, viruses with a positive-sense single-stranded RNA genome [(+)ssRNA], including picornaviruses, flaviviruses, and coronaviruses, encode proteases that are not only required for processing viral polyproteins into functional units but also manipulate crucial host cellular processes through their proteolytic activity. Because these proteases must cleave numerous polyprotein sites as well as diverse host targets, evolution of these viral proteases is expected to be highly constrained. However, despite this strong evolutionary constraint, mounting evidence suggests that viral proteases such as picornavirus 3C, flavivirus NS3, and coronavirus 3CL, are engaged in molecular ‘arms races’ with their targeted host factors, resulting in host- and virus-specific determinants of protease cleavage. In cases where protease-mediated cleavage results in host immune inactivation, recurrent host gene evolution can result in avoidance of cleavage by viral proteases. In other cases, such as recently described examples in NLRP1 and CARD8, hosts have evolved ‘tripwire’ sequences that mimic protease cleavage sites and activate an immune response upon cleavage. In both cases, host evolution may be responsible for driving viral protease evolution, helping explain why viral proteases and polyprotein sites are divergent among related viruses despite such strong evolutionary constraint. Importantly, these evolutionary conflicts result in diverse protease-host interactions even within closely related host and viral species, thereby contributing to host range, zoonotic potential, and pathogenicity of viral infection. Such examples highlight the importance of examining viral protease-host interactions through an evolutionary lens.

Influenza virus has a broad cellular tropism in the respiratory tract. Infected epithelial cells sense the infection and initiate an antiviral response. To define the antiviral response at the earliest stages of infection we used a series of single-cycle reporter viruses. These viral probes demonstrated cells in vivo harbor a range in magnitude of virus replication. Transcriptional profiling of cells supporting different levels of replication revealed tiers of IFN-stimulated gene expression. Uninfected cells and cells with blunted replication expressed a distinct and potentially protective antiviral signature, while cells with high replication expressed a unique reserve set of antiviral genes. Finally, we used these single-cycle reporter viruses to determine the antiviral landscape during virus spread, which unveiled disparate protection of epithelial cell subsets mediated by IFN in vivo. Together these results highlight the complexity of virus-host interactions within the infected lung and suggest that magnitude and round of replication tune the antiviral response.

Resident memory T cells (T RM) in the lung are vital for heterologous protection against influenza A virus (IAV). Environmental factors are necessary to establish lung T RM ; however, the role of T cell-intrinsic factors like TCR signal strength have not been elucidated. In this study, we investigated the impact of TCR signal strength on the generation and maintenance of lung T RM after IAV infection. We inserted high-and low-affinity OT-I epitopes into IAV and infected mice after transfer of OT-I T cells. We uncovered a bias in T RM formation in the lung elicited by lower affinity TCR stimulation. TCR affinity did not impact the overall phenotype or long-term maintenance of lung T RM. Overall, these findings demonstrate that T RM formation is negatively correlated with increased TCR signal strength. Lower affinity cells may have an advantage in forming T RM to ensure diversity in the Ag-specific repertoire in tissues.