Influenza virus replication in cardiomyocytes drives heart dysfunction and fibrosis.

Cardiac dysfunction is a common complication of severe influenza virus infection, but whether this occurs due to direct infection of cardiac tissue or indirectly through systemic lung inflammation remains unclear. To test the etiology of this aspect of influenza disease, we generated a novel recombinant heart-attenuated influenza virus via genome incorporation of target sequences for miRNAs expressed in cardiomyocytes. Compared with control virus, mice infected with miR-targeted virus had significantly reduced heart viral titers, confirming cardiac attenuation of viral replication. However, this virus was fully replicative in the lungs and induced similar systemic inflammation and weight loss compared to control virus. The miR-targeted virus induced fewer cardiac conduction irregularities and significantly less fibrosis in mice lacking interferon-induced transmembrane protein 3 (IFITM3), which serve as a model for influenza-associated cardiac pathology. We conclude that robust virus replication in the heart is required for pathology, even when lung inflammation is severe.

Segment-specific kinetics of mRNA, cRNA and vRNA accumulation during influenza infection.

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.ImportanceInfluenza 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.

Cell type- and replication stage-specific influenza virus responses in vivo.

Influenza A viruses (IAVs) remain a significant global health burden. Activation of the innate immune response is important for controlling early virus replication and spread. It is unclear how early IAV replication events contribute to immune detection. Additionally, while many cell types in the lung can be infected, it is not known if all cell types contribute equally to establish the antiviral state in the host. Here, we use single-cycle influenza A viruses (scIAVs) to characterize the early immune response to IAV in vitro and in vivo. We found that the magnitude of virus replication contributes to antiviral gene expression within infected cells prior to the induction of a global response. We also developed a scIAV that is only capable of undergoing primary transcription, the earliest stage of virus replication. Using this tool, we uncovered replication stage-specific responses in vitro and in vivo. Using several innate immune receptor knockout cell lines, we identify RIG-I as the predominant antiviral detector of primary virus transcription and amplified replication in vitro. Through a Cre-inducible reporter mouse, we used scIAVs expressing Cre-recombinase to characterize cell type-specific responses in vivo. Individual cell types upregulate unique sets of antiviral genes in response to both primary virus transcription and amplified replication. We also identified antiviral genes that are only upregulated in response to direct infection. Altogether, these data offer insight into the early mechanisms of antiviral gene activation during influenza A infection.

Engineered Small-Molecule Control of Influenza A Virus Replication.

Influenza A virus (IAV) remains a global health concern despite the availability of a seasonal vaccine. It is difficult to predict which strains will circulate during influenza season, and therefore, it is extremely challenging to test novel vaccines in the human population. To overcome this obstacle, new vaccines must be tested in challenge studies. This approach poses significant safety problems, since current pharmacological interventions for IAV are poorly efficacious. New methods are needed to enhance the safety of these challenge studies. In this study, we have generated a virus expressing a small-molecule-assisted shutoff (SMASh) tag as a safety switch for IAV replication. The addition of the SMASh tag to an essential IAV protein allows for small-molecule-mediated inhibition of replication. Treatment with this drug controls the replication of a SMASh-tagged virus in vitro and in vivo This model for restriction of viral replication has potential for broad applications in vaccine studies, virotherapy, and basic virus research.IMPORTANCE Influenza A virus (IAV) causes significant morbidity and mortality annually worldwide, despite the availability of new formulations of the vaccine each season. There is a critical need to develop more-efficacious vaccines. However, testing novel vaccines in the human population in controlled studies is difficult due to the limited availability and efficacy of intervention strategies should the vaccine fail. There are also significant safety concerns for work with highly pathogenic IAV strains in the laboratory. Therefore, novel strategies are needed to improve the safety of vaccine studies and of research on highly pathogenic IAV. In this study, we developed an IAV strain engineered to contain a small-molecule-mediated safety switch. This tag, when attached to an essential viral protein, allows for the regulation of IAV replication in vitro and in vivo This strategy provides a platform for the regulation of virus replication without targeting viral proteins directly.