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Little is known about the amount and infectiousness of influenza virus shed into exhaled breath. This contributes to uncertainty about the importance of airborne influenza transmission. We screened 355 symptomatic volunteers with acute respiratory illness and report 142 cases with confirmed influenza infection who provided 218 paired nasopharyngeal (NP) and 30-minute breath samples (coarse >5-µm and fine ≤5-µm fractions) on days 1-3 after symptom onset. We assessed viral RNA copy number for all samples and cultured NP swabs and fine aerosols. We recovered infectious virus from 52 (39%) of the fine aerosols and 150 (89%) of the NP swabs with valid cultures. The geometric mean RNA copy numbers were 3.8 × 104/30-minutes fine-, 1.2 × 104/30-minutes coarse-aerosol sample, and 8.2 × 108 per NP swab. Fine- and coarse-aerosol viral RNA were positively associated with body mass index and number of coughs and negatively associated with increasing days since symptom onset in adjusted models. Fine-aerosol viral RNA was also positively associated with having influenza vaccination for both the current and prior season. NP swab viral RNA was positively associated with upper respiratory symptoms and negatively associated with age but was not significantly associated with fine- or coarse-aerosol viral RNA or their predictors. Sneezing was rare, and sneezing and coughing were not necessary for infectious aerosol generation. Our observations suggest that influenza infection in the upper and lower airways are compartmentalized and independent.

Influenza A virus (IAV) lacks the enzyme for adding 5' caps to its RNAs and snatches the 5' ends of host capped RNAs to prime transcription. Neither the preference of the host RNA sequences snatched nor the effect of cap-snatching on host processes is completely defined. Previous studies of influenza cap-snatching used poly(A)-selected RNAs from infected cells or relied on annotated host genes to define the snatched host RNAs, and thus lack details on many noncoding host RNAs including snRNAs, snoRNAs, and promoter-associated capped small (cs)RNAs, which are made by "paused" Pol II during transcription initiation. In this study, we used a nonbiased technique, CapSeq, to identify host and viral-capped RNAs including nonpolyadenylated RNAs in the same samples, and investigated the substrate-product correlation between the host RNAs and the viral RNAs. We demonstrated that noncoding host RNAs, particularly U1 and U2, are the preferred cap-snatching source over mRNAs or pre-mRNAs. We also found that csRNAs are highly snatched by IAV. Because the functions of csRNAs remain mostly unknown, especially in somatic cells, our finding reveals that csRNAs at least play roles in the process of IAV infection. Our findings support a model where nascent RNAs including csRNAs are the preferred targets for cap-snatching by IAV and raise questions about how IAV might use snatching preferences to modulate host-mRNA splicing and transcription.

Influenza virus inflicts a heavy death toll annually and resistance to existing antiviral drugs has generated interest in the development of agents with novel mechanisms of action. Favipiravir is an antiviral drug that acts by increasing the genome-wide mutation rate of influenza A virus (IAV). Potential synergistic benefits of combining oseltamivir and favipiravir have been demonstrated in animal models of influenza, but the population-level effects of combining the drugs are unknown. In order to elucidate the underlying evolutionary processes at play, we performed genome-wide sequencing of IAV experimental populations subjected to serial pass-aging in vitro under a combined protocol of oseltamivir and favipiravir. We describe the interplay between mutation, selection, and genetic drift that ultimately culminates in population extinction. In particular, selective sweeps around oseltamivir resistance mutations reduce genome-wide variation while deleterious mutations hitchhike to fixation given the increased mutational load generated by favipiravir. This latter effect reduces viral fitness and accelerates extinction compared with IAV populations treated with favipiravir alone, but risks spreading both established and newly emerging mutations, including possible drug resistance mutations, if transmission occurs before the viral populations are eradicated. [ABSTRACT FROM AUTHOR]

Influenza A virus (IAV) has a segmented genome that allows for the exchange of genome segments between different strains. This reassortment accelerates evolution by breaking linkage, helping IAV cross species barriers to potentially create highly virulent strains. Challenges associated with monitoring the process of reassortment in molecular detail have limited our understanding of its evolutionary implications. We applied a novel deep sequencing approach with quantitative analysis to assess the in vitro temporal evolution of genomic reassortment in IAV. The combination of H1N1 and H3N2 strains reproducibly generated a new H1N2 strain with the hemagglutinin and nucleoprotein segments originating from H1N1 and the remaining six segments from H3N2. By deep sequencing the entire viral genome, we monitored the evolution of reassortment, quantifying the relative abundance of all IAV genome segments from the two parent strains over time and measuring the selection coefficients of the reassorting segments. Additionally, we observed several mutations coemerging with reassortment that were not found during passaging of pure parental IAV strains. Our results demonstrate how reassortment of the segmented genome can accelerate viral evolution in IAV, potentially enabled by the emergence of a small number of individual mutations. [ABSTRACT FROM AUTHOR]

Many oseltamivir resistance mutations exhibit fitness defects in the absence of drug pressure that hinders their propagation in hosts. Secondary permissive mutations can rescue fitness defects and facilitate the segregation of resistance mutations in viral populations. Previous studies have identified a panel of permissive or compensatory mutations in neuraminidase (NA) that restore the growth defect of the predominant oseltamivir resistance mutation (H275Y) in H1N1 influenza A virus. In prior work, we identified a hyperactive mutation (Y276F) that increased NA activity by approximately 70%. While Y276F had not been previously identified as a permissive mutation, we hypothesized that Y276F may counteract the defects caused by H275Y by buffering its reduced NA expression and enzyme activity. In this study, we measured the relative fitness, NA activity, and surface expression, as well as sensitivity to oseltamivir, for several oseltamivir resistance mutations, including H275Y in the wild-type and Y276F genetic background. Our results demonstrate that Y276F selectively rescues the fitness defect of H275Y by restoring its NA surface expression and enzymatic activity, elucidating the local compensatory structural impacts of Y276F on the adjacent H275Y. [ABSTRACT FROM AUTHOR]

Influenza viruses pose severe public health threats globally. Influenza viruses are extensively pleomorphic, in shape, size, and organization of viral proteins. Analysis of influenza morphology and ultrastructure can help elucidate viral structure-function relationships and aid in therapeutics and vaccine development. While cryo-electron tomography (cryoET) can depict the 3D organization of pleomorphic influenza, the low signal-to-noise ratio inherent to cryoET and viral heterogeneity have precluded detailed characterization of influenza viruses. In this report, we leveraged convolutional neural networks and cryoET to characterize the morphological architecture of the A/Puerto Rico/8/34 (H1N1) influenza strain. Our pipeline improved the throughput of cryoET analysis and accurately identified viral components within tomograms. Using this approach, we successfully characterized influenza morphology, glycoprotein density, and conducted subtomogram averaging of influenza glycoproteins. Application of this processing pipeline can aid in the structural characterization of not only influenza viruses, but other pleomorphic viruses and infected cells. [Display omitted] • Pleomorphic influenza A virus visualization using CryoET • Trained convolutional neural network pipeline to rapidly identify viral proteins • Differences in viral morphologies do not affect inter-glycoprotein spacings • Rapid particle picking helps determine in situ influenza HA structure at 12.9 Å Detailed structural investigations of influenza viruses have been difficult due to their extensive pleomorphism. Huang et al. use cryo-electron tomography and convolutional neural networks to segment influenza structural proteins, characterize virion morphology, determine inter-glycoprotein spacing, and conduct subtomogram averaging of influenza glycoproteins. [ABSTRACT FROM AUTHOR]