Immunity to influenza viruses can be long-lived, but reinfections with antigenically distinct viral strains and subtypes are common. Reinfections can boost antibody responses against viral strains first encountered in childhood through a process termed “original antigenic sin.” It is unknown how initial childhood exposures affect the induction of antibodies against the hemagglutinin (HA) stalk domain of influenza viruses. This is an important consideration since broadly reactive HA stalk antibodies can protect against infection, and universal vaccine platforms are being developed to induce these antibodies. Here we show that experimentally infected ferrets and naturally infected humans establish strong “immunological imprints” against HA stalk antigens first encountered during primary influenza virus infections. We found that HA stalk antibodies are surprisingly boosted upon subsequent infections with antigenically distinct influenza A virus subtypes. Paradoxically, these heterosubtypic-boosted HA stalk antibodies do not bind efficiently to the boosting influenza virus strain. Our results demonstrate that an individual’s HA stalk antibody response is dependent on the specific subtype of influenza virus that they first encounter early in life. We propose that humans are susceptible to heterosubtypic influenza virus infections later in life since these viruses boost HA stalk antibodies that do not bind efficiently to the boosting antigen.

Assembly of infectious influenza A viruses (IAV) is a complex process involving transport from the nucleus to the plasma membrane. Rab11A-containing recycling endosomes have been identified as a platform for intracellular transport of viral RNA (vRNA). Here, using high spatiotemporal resolution light-sheet microscopy (~1.4 volumes/second, 330 nm isotropic resolution), we quantify Rab11A and vRNA movement in live cells during IAV infection and report that IAV infection decreases speed and increases arrest of Rab11A. Unexpectedly, infection with respiratory syncytial virus alters Rab11A motion in a manner opposite to IAV, suggesting that Rab11A is a common host component that is differentially manipulated by respiratory RNA viruses. Using two-color imaging we demonstrate co-transport of Rab11A and IAV vRNA in infected cells and provide direct evidence that vRNA-associated Rab11A have altered transport. The mechanism of altered Rab11A movement is likely related to a decrease in dynein motors bound to Rab11A vesicles during IAV infection.

Influenza A virus (IAV) consists of eight viral RNA (vRNA) segments that are replicated in the host cell nucleus and transported to the plasma membrane for packaging into progeny virions. We have previously proposed a model where subcomplexes of vRNA are exported from the nucleus and assembled en route to the plasma membrane. However, the role of host cytoskeletal proteins in the cytoplasmic assembly of IAV vRNA segments remains unknown. Previous studies have suggested that IAV vRNA segments are transported via Rab11A-containing recycling endosomes (RE) and use both microtubules (MT) and actin. Rab11A RE transport primarily along MT; therefore, investigation of the role of MT in vRNA assembly is warranted. We explored the role of MT in vRNA assembly and replication by using multiple IAV strains in various cell types, including primary human airway epithelial cells. We observed that Rab11A localization was altered in the presence of MT-depolymerizing drugs, but growth of IAV in all of the cell types tested was unchanged. Fluorescent in situ hybridization was performed to determine the role of MT in the assembly of multiple vRNA segments. Unexpectedly, we found that vRNA-vRNA association in cytoplasmic foci was independent of MT. Given the disparity of localization between Rab11A and vRNA segments in the absence of intact MT filaments, we analyzed the three-dimensional spatial relationship between Rab11A and vRNA in the cytoplasm of infected cells. We found that Rab11A and vRNA colocalization is dependent upon dynamic MT filaments. Taken together, our data suggest that cytoplasmic transport of influenza vRNA may include a Rab11A RE-independent mechanism.IMPORTANCE IAV infections cause a large public health burden through seasonal epidemics and sporadic pandemics. Pandemic IAVs emerge through reassortment of vRNA in animal or human hosts. Elucidation of the mechanism of intracellular dynamics of IAV assembly is necessary to understand reassortment. Our results describing the role of MT in vRNA transport and assembly expand upon previous studies characterizing vRNA assembly. This study is the first to assess the role of MT in influenza virus replication in human bronchial airway epithelial cells. In addition, we present novel data on the role of MT in facilitating the association between distinct vRNA segments. Interestingly, our results suggest that progressive assembly of vRNA segments may be cell type dependent and that vRNA may be transported through the cytoplasm without Rab11A RE in the absence of intact MT. These results enhance our understanding of vRNA assembly and the role of cytoskeletal proteins in that process.

Human-to-human transmission of influenza viruses is a serious public health threat, yet the precise role of immunity from previous infections on the susceptibility to airborne infection is still unknown. Using the ferret model, we examined the roles of exposure duration and heterosubtypic immunity on influenza transmission. We demonstrate that a 48 hour exposure is sufficient for efficient transmission of H1N1 and H3N2 viruses. To test pre-existing immunity, a gap of 8–12 weeks between primary and secondary infections was imposed to reduce innate responses and ensure robust infection of donor animals with heterosubtypic viruses. We found that pre-existing H3N2 immunity did not significantly block transmission of the 2009 H1N1pandemic (H1N1pdm09) virus to immune animals. Surprisingly, airborne transmission of seasonal H3N2 influenza strains was abrogated in recipient animals with H1N1pdm09 pre-existing immunity. This protection from natural infection with H3N2 virus was independent of neutralizing antibodies. Pre-existing immunity with influenza B virus did not block H3N2 virus transmission, indicating that the protection was likely driven by the adaptive immune response. We demonstrate that pre-existing immunity can impact susceptibility to heterologous influenza virus strains, and implicate a novel correlate of protection that can limit the spread of respiratory pathogens through the air.

Human-to-human transmission of influenza viruses is a serious public health threat, yet the precise role of immunity from previous infections on the susceptibility to airborne viruses is still unknown. Using human seasonal influenza viruses in a ferret model, we examined the roles of exposure duration and heterosubtypic immunity on influenza transmission. We found that airborne transmission of seasonal influenza strains is abrogated in recipient animals with pre-existing nonneutralizing immunity, indicating that transmissibility of a given influenza virus strain should be examined in the context of ferrets that are not immunologically naïve.

Airborne transmission of seasonal and pandemic influenza viruses is the reason for their epidemiological success and public health burden in humans. Efficient airborne transmission of the H1N1 influenza virus relies on the receptor specificity and pH of fusion of the surface glycoprotein hemagglutinin (HA). In this study, we examined the role of HA pH of fusion on transmissibility of a cell-culture-adapted H3N2 virus. Mutations in the HA head at positions 78 and 212 of A/Perth/16/2009 (H3N2), which were selected after cell culture adaptation, decreased the acid stability of the virus from pH 5.5 (WT) to pH 5.8 (mutant). In addition, the mutant H3N2 virus replicated to higher titers in cell culture but had reduced airborne transmission in the ferret model. These data demonstrate that, like H1N1 HA, the pH of fusion for H3N2 HA is a determinant of efficient airborne transmission. Surprisingly, noncoding regions of the NA segment can impact the pH of fusion of mutant viruses. Taken together, our data confirm that HA acid stability is an important characteristic of epidemiologically successful human influenza viruses and is influenced by HA/NA balance.

24The genome of Influenza A viruses consists of eight negative-sense RNA segments that 25 are bound by viral nucleoprotein (NP). We recently showed that NP binding is not uniform along 26 the segments but exhibits regions of enrichment as well as depletion. Furthermore, genome-wide 27 NP binding profiles are distinct even in strains with high sequence similarity, such as the two 28 H1N1 strains A/WSN/1933 and A/California/07/2009. Here, we performed interstrain segment 29 swapping experiments with segments of either high or low congruency in NP binding, which 30 suggested that a segment with a similar overall NP binding profile preserved replication fitness of 31 the resulting virus. Further sub-segmental swapping experiments demonstrated that NP binding 32 is affected by changes to the underlying nucleotide sequence, as NP peaks can either become 33 lost or appear de novo at mutated regions. Unexpectedly, these local nucleotide changes in one 34 segment not only affect NP binding in cis, but also impact the genome-wide NP binding profile on 35 other segments in a vRNA sequence-independent manner, suggesting that primary sequence 36 alone is not the sole determinant for NP association to vRNA. Moreover, we observed that sub-37 segmental mutations that affect NP binding profiles can result in reduced replication fitness, which 38 is caused by defects in vRNA packaging efficiency and an increase in semi-infectious particle 39 production. Taken together, our results indicate that the pattern of NP binding to vRNA is 40 important for efficient virus replication. 41 3 Author Summary 42 Each viral RNA (vRNA) segment is bound by the polymerase complex at the 5′ and 3′ 43 ends, while the remainder of the vRNA is coated non-uniformly and non-randomly by 44 nucleoprotein (NP). To explore the constraints of NP binding to vRNA, we used high-throughput 45 sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) of mutant H1N1 46 strains with exchanged vRNA sequences and observed that NP binding can be changed based 47 on vRNA sequence. The most striking observation of our study is that nucleotide changes in one 48 segment can have genome-wide effects on the NP binding profile of other segments. We refer to 49 this phenomenon as the 'butterfly effect' of influenza packaging. Our results provide an important 50 context in which to consider future studies regarding influenza packaging and assembly.51 4 Introduction 52The segmented nature of influenza A virus (IAV) genomes poses a logistical challenge for 53 viral replication, as all of the eight negative-sense single-stranded RNA segments must find their 54 way into a budding virion to give rise to an infectious particle [1, 2]. Following nuclear export, viral 55 ribonucleoprotein complexes (vRNP) containing newly synthesized viral RNA (vRNA) assemble 56 on recycling endosomes en route to the plasma membrane for packaging into virions [3, 4]. An 57 accumulating body of evidence suggests that the intracellular pre-assembly process of vRNP 58 trafficking is media...