Pre-existing heterosubtypic immunity provides a barrier to airborne transmission of influenza viruses.

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.

Intracellular Colocalization of Influenza Viral RNA and Rab11A Is Dependent upon Microtubule Filaments.

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.

Pulse-shaping based two-photon FRET stoichiometry.

Förster Resonance Energy Transfer (FRET) based measurements that calculate the stoichiometry of intermolecular interactions in living cells have recently been demonstrated, where the technique utilizes selective one-photon excitation of donor and acceptor fluorophores to isolate the pure FRET signal. Here, we present work towards extending this FRET stoichiometry method to employ two-photon excitation using a pulse-shaping methodology. In pulse-shaping, frequency-dependent phases are applied to a broadband femtosecond laser pulse to tailor the two-photon excitation conditions to preferentially excite donor and acceptor fluorophores. We have also generalized the existing stoichiometry theory to account for additional cross-talk terms that are non-vanishing under two-photon excitation conditions. Using the generalized theory we demonstrate two-photon FRET stoichiometry in live COS-7 cells expressing fluorescent proteins mAmetrine as the donor and tdTomato as the acceptor.

Fundamental eigenmode of traveling-wave phase-sensitive optical parametric amplifier: experimental generation and verification.

We investigate experimentally the eigenmodes of a Gaussian-beam-pumped traveling-wave phase-sensitive optical parametric amplifier (PSA). By varying the waist of an input LG(00) signal mode, we show that PSA performance improves with increasing spatial overlap between the input and the theoretically predicted fundamental eigenmode. For optimum waist, we report amplification and deamplification markedly higher than those observed for the traditional case of signal waist=√2× (pump waist). Lastly, we demonstrate the generation and verification of the PSA fundamental eigenmode.

All-optical modulation of four-wave mixing in an Rb-filled photonic bandgap fiber.

We demonstrate efficient all-optical modulation using Rb vapor confined to a hollow-core photonic bandgap fiber. The intensity of a signal field participating in the four-wave-mixing process is modulated using a weak switching field. We observe 3 dB of attenuation in the signal field with only 3600 photons of switching energy, corresponding to 23 photons per atomic cross section lambda(2)/(2pi). Modulation bandwidths as high as 300 MHz are observed.

Quantitative live cell imaging reveals influenza virus manipulation of Rab11A transport through reduced dynein association.

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.

Nonlinear optics in hollow-core photonic bandgap fibers.

Hollow-core photonic-bandgap fibers provide a new geometry for the realization and enhancement of many nonlinear optical effects. Such fibers offer novel guidance and dispersion properties that provide an advantage over conventional fibers for various applications. In this review we summarize the nonlinear optics experiments that have been performed using these hollow-core fibers.

Generation of large alkali vapor densities inside bare hollow-core photonic band-gap fibers.

We demonstrate the ability to generate extremely large rubidium densities in uncoated hollow-core photonic band-gap fibers using light-induced atomic desorption. Once the fiber is exposed to Rb vapor for 1-2 weeks, and this atomic source is removed, the fiber yields large desorbable densities for an extended period of time. We show that optical depths greater than e(-1200) can be created within seconds. Our observed Rb densities are several orders of magnitude larger than any previously reported to be generated optically, and allow for the demonstration of a relatively easy-to-use fiber-based vapor cell capable of producing large optical depths without the need for thermal tuning.

Live Imaging of Influenza Viral Ribonucleoproteins Using Light-Sheet Microscopy.

Influenza viruses exhibit a complex life cycle that is still poorly understood. It involves independent replication of each of the eight segments that make up its genome and subsequent coordinated assembly as they egress from the host cell. Fast, time-resolved volumetric live cell imaging offers a powerful tool for understanding the various host mechanisms hijacked by the virus. Here, we describe the methods necessary for generating influenza viruses that carry a fluorescently tagged polymerase complex, infection of biologically relevant cells with these viruses, and finally protocols for live cell imaging and analysis.

Non-Uniform and Non-Random Binding of Nucleoprotein to Influenza A and B Viral RNA.

The genomes of influenza A and B viruses have eight, single-stranded RNA segments that exist in the form of a viral ribonucleoprotein complex in association with nucleoprotein (NP) and an RNA-dependent RNA polymerase complex. We previously used high-throughput RNA sequencing coupled with crosslinking immunoprecipitation (HITS-CLIP) to examine where NP binds to the viral RNA (vRNA) and demonstrated for two H1N1 strains that NP binds vRNA in a non-uniform, non-random manner. In this study, we expand on those initial observations and describe the NP-vRNA binding profile for a seasonal H3N2 and influenza B virus. We show that, similar to H1N1 strains, NP binds vRNA in a non-uniform and non-random manner. Each viral gene segment has a unique NP binding profile with areas that are enriched for NP association as well as free of NP-binding. Interestingly, NP-vRNA binding profiles have some conservation between influenza A viruses, H1N1 and H3N2, but no correlation was observed between influenza A and B viruses. Our study demonstrates the conserved nature of non-uniform NP binding within influenza viruses. Mapping of the NP-bound vRNA segments provides information on the flexible NP regions that may be involved in facilitating assembly.

Ultralow-power four-wave mixing with Rb in a hollow-core photonic band-gap fiber.

We demonstrate extremely efficient four-wave mixing with gains greater than 100 at microwatt pump powers and signal-to-idler conversion of 50% in Rb vapor confined to a hollow-core photonic band-gap fiber. We present a theoretical model that demonstrates such efficiency is consistent with the dimensions of the fiber and the optical depths attained. This is, to our knowledge, the largest four-wave mixing gain observed at such low total pump powers and the first demonstrated example of four-wave mixing in an alkali-metal vapor system with a large (approximately 30 MHz) ground state decoherence rate.

Low-light-level optical interactions with rubidium vapor in a photonic band-gap fiber.

We show that rubidium vapor can be produced within the core of a photonic band-gap fiber yielding an optical depth in excess of 2,000. Our technique for producing the vapor is based on coating the inner walls of the fiber core with organosilane and using light-induced atomic desorption to release Rb atoms into the core. As an initial demonstration of the potential of this system for supporting ultralow-level nonlinear optical interactions, we perform electromagnetically induced transparency with control-field powers in the nanowatt regime, which represents more than a 1,000-fold reduction from the power required for bulk, focused geometries.