Potential pandemic risk of circulating swine H1N2 influenza viruses.

Influenza A viruses in swine have considerable genetic diversity and continue to pose a pandemic threat to humans due to a potential lack of population level immunity. Here we describe a pipeline to characterize and triage influenza viruses for their pandemic risk and examine the pandemic potential of two widespread swine origin viruses. Our analysis reveals that a panel of human sera collected from healthy adults in 2020 has no cross-reactive neutralizing antibodies against a α-H1 clade strain (α-swH1N2) but do against a γ-H1 clade strain. The α-swH1N2 virus replicates efficiently in human airway cultures and exhibits phenotypic signatures similar to the human H1N1 pandemic strain from 2009 (H1N1pdm09). Furthermore, α-swH1N2 is capable of efficient airborne transmission to both naïve ferrets and ferrets with prior seasonal influenza immunity. Ferrets with H1N1pdm09 pre-existing immunity show reduced α-swH1N2 viral shedding and less severe disease signs. Despite this, H1N1pdm09-immune ferrets that became infected via the air can still onward transmit α-swH1N2 with an efficiency of 50%. These results indicate that this α-swH1N2 strain has a higher pandemic potential, but a moderate level of impact since there is reduced replication fitness and pathology in animals with prior immunity.

Secondary infection with Streptococcus pneumoniae decreases influenza virus replication and is linked to severe disease.

Secondary bacterial infection is a common complication in severe influenza virus infections. During the H1N1 pandemic of 2009, increased mortality was observed among healthy young adults due to secondary bacterial pneumonia, one of the most frequent bacterial species being Streptococcus pneumoniae (Spn). Previous studies in mice and ferrets have suggested a synergistic relationship between Spn and influenza viruses. In this study, the ferret model was used to examine whether secondary Spn infection (strains BHN97 and D39) influence replication and airborne transmission of the 2009 pandemic H1N1 virus (H1N1pdm09). Secondary infection with Spn after H1N1pdm09 infection consistently resulted in a significant decrease in viral titers in the ferret nasal washes. While secondary Spn infection appeared to negatively impact influenza virus replication, animals precolonized with Spn were equally susceptible to H1N1pdm09 airborne transmission. In line with previous work, ferrets with preceding H1N1pdm09 and secondary Spn infection had increased bacterial loads and more severe clinical symptoms as compared to animals infected with H1N1pdm09 or Spn alone. Interestingly, the donor animals that displayed the most severe clinical symptoms had reduced airborne transmission of H1N1pdm09. Based on these data, we propose an asymmetrical relationship between these two pathogens, rather than a synergistic one, since secondary bacterial infection enhances Spn colonization and pathogenesis but decreases viral titers.

Three-Dimensional Simultaneous Imaging of Nucleic Acids and Proteins During Influenza Virus Infection in Single Cells Using Confocal Microscopy.

Three-dimensional imaging is a powerful tool for examining the spatial distribution of intracellular molecules like nucleic acids, proteins, and organelles in cells and tissues. Multicolor fluorescence imaging coupled with three-dimensional spatial information provide a platform to explore the relationship between different cellular features and molecules. We have previously developed a pipeline to study the intracellular localization of influenza virus genomic segments within an infected cell. Here, we describe the staining of multiple viral RNA segments in cells infected with influenza virus by combined fluorescence in situ hybridization (FISH) and immunofluorescence and quantification of colocalization between viral segments. This chapter will cover the acquisition and analysis of 3D images by the widely used laser scanning confocal microscope. These strategies can be applied to a wide range of biological processes and modified to examine colocalization of other cellular features.

Parallel evolution between genomic segments of seasonal human influenza viruses reveals RNA-RNA relationships.

The influenza A virus (IAV) genome consists of eight negative-sense viral RNA (vRNA) segments that are selectively assembled into progeny virus particles through RNA-RNA interactions. To explore putative intersegmental RNA-RNA relationships, we quantified similarity between phylogenetic trees comprising each vRNA segment from seasonal human IAV. Intersegmental tree similarity differed between subtype and lineage. While intersegmental relationships were largely conserved over time in H3N2 viruses, they diverged in H1N1 strains isolated before and after the 2009 pandemic. Surprisingly, intersegmental relationships were not driven solely by protein sequence, suggesting that IAV evolution could also be driven by RNA-RNA interactions. Finally, we used confocal microscopy to determine that colocalization of highly coevolved vRNA segments is enriched over other assembly intermediates at the nuclear periphery during productive viral infection. This study illustrates how putative RNA interactions underlying selective assembly of IAV can be interrogated with phylogenetics.

The viruses responsible for influenza evolve rapidly during infection. Changes typically emerge in two key ways: through random mutations in the genetic sequence of the virus, or by reassortment. Reassortment can occur when two or more strains infect the same cell. Once in a cell, viral particles 'open up' to release their genetic material so it can make copies of itself using the cell's machinery. The new copies of the genetic material of the virus are used to make new viral particles, which then envelop the genetic material and are released from the cell to infect other cells. If several strains of a virus infect the same cell, a new viral particle may pick up genetic segments from each of the infecting strains, creating a new strain via reassortment. Several factors are known to affect the success of the reassortment process. For example, if the new strain acquires a genetic defect that hinders its replication cycle, it is likely to die out quickly. Other times, this trading of genetic information can create a strain that is more resistant to the human immune system, allowing it to sweep across the globe and cause a deadly pandemic. However, a key part of the reassortment process that still remains unclear is how genome segments from two different influenza strains recognize each other before merging together to create hybrid daughter viruses. To explore this further, Jones et al. used a technique called fluorescence microscopy. They found that genome segments that evolved along similar paths were more likely to cluster in the same area inside infected cells, and therefore, more likely to be reassorted together into a new strain during assembly of daughter viruses. This suggests that assembly may guide the evolutionary path taken by individual genomic segments. Jones et al. also looked at the evolution of different genome segments collected from patients suffering from seasonal influenza, and found that these segments had a distinct evolutionary path to those in pandemic-causing strains. This research provides new insights into the role of reassortment in the evolution of influenza viruses during infection. In particular, it suggests that how the genome segments interact with one another may have a previously unknown and important role in guiding this evolution. These insights could be used to predict future reassortment events based on evolutionary relationships between influenza virus genomic segments, and may in the future be used as part of risk assessment tools to predict the emergence of new pandemic strains.

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.

Viral and host heterogeneity and their effects on the viral life cycle.

Traditionally, the viral replication cycle is envisioned as a single, well-defined loop with four major steps: attachment and entry into a target cell, replication of the viral genome, maturation of viral proteins and genome packaging into infectious progeny, and egress and dissemination to the next target cell. However, for many viruses, a growing body of evidence points towards extreme heterogeneity in each of these steps. In this Review, we reassess the major steps of the viral replication cycle by highlighting recent advances that show considerable variability during viral infection. First, we discuss heterogeneity in entry receptors, followed by a discussion on error-prone and low-fidelity polymerases and their impact on viral diversity. Next, we cover the implications of heterogeneity in genome packaging and assembly on virion morphology. Last, we explore alternative egress mechanisms, including tunnelling nanotubes and host microvesicles. In summary, we discuss the implications of viral phenotypic, morphological and genetic heterogeneity on pathogenesis and medicine. This Review highlights common themes and unique features that give nuance to the viral replication cycle.

Original antigenic sin priming of influenza virus hemagglutinin stalk antibodies.

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.

Ross River virus envelope glycans contribute to disease through activation of the host complement system.

Mannose binding lectin (MBL) generally plays a protective role during viral infection, yet MBL-mediated complement activation promotes Ross River virus (RRV)-induced inflammatory tissue destruction, contributing to arthritis and myositis. As MBL binds to carbohydrates, we hypothesized that N-linked glycans on the RRV envelope glycoproteins act as ligands for MBL. Using a panel of RRV mutants lacking the envelope N-linked glycans, we found that MBL deposition onto infected cells was dependent on the E2 glycans. Moreover, the glycan-deficient viruses exhibited reduced disease and tissue damage in a mouse model of RRV-induced myositis compared to wild-type RRV, despite similar viral load and inflammatory infiltrates within the skeletal muscle. Instead, the reduced disease induced by glycan-deficient viruses was linked to decreased MBL deposition and complement activation within inflamed tissues. These results demonstrate that the viral N-linked glycans promote MBL deposition and complement activation onto RRV-infected cells, contributing to the development of RRV-induced myositis.

Disruption of the Opal Stop Codon Attenuates Chikungunya Virus-Induced Arthritis and Pathology.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus responsible for several significant outbreaks of debilitating acute and chronic arthritis and arthralgia over the past decade. These include a recent outbreak in the Caribbean islands and the Americas that caused more than 1 million cases of viral arthralgia. Despite the major impact of CHIKV on global health, viral determinants that promote CHIKV-induced disease are incompletely understood. Most CHIKV strains contain a conserved opal stop codon at the end of the viral nsP3 gene. However, CHIKV strains that encode an arginine codon in place of the opal stop codon have been described, and deep-sequencing analysis of a CHIKV isolate from the Caribbean identified both arginine and opal variants within this strain. Therefore, we hypothesized that the introduction of the arginine mutation in place of the opal termination codon may influence CHIKV virulence. We tested this by introducing the arginine mutation into a well-characterized infectious clone of a CHIKV strain from Sri Lanka and designated this virus Opal524R. This mutation did not impair viral replication kinetics in vitro or in vivo Despite this, the Opal524R virus induced significantly less swelling, inflammation, and damage within the feet and ankles of infected mice. Further, we observed delayed induction of proinflammatory cytokines and chemokines, as well as reduced CD4+ T cell and NK cell recruitment compared to those in the parental strain. Therefore, the opal termination codon plays an important role in CHIKV pathogenesis, independently of effects on viral replication.IMPORTANCE Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes significant outbreaks of viral arthralgia. Studies with CHIKV and other alphaviruses demonstrated that the opal termination codon within nsP3 is highly conserved. However, some strains of CHIKV and other alphaviruses contain mutations in the opal termination codon. These mutations alter the virulence of related alphaviruses in mammalian and mosquito hosts. Here, we report that a clinical isolate of a CHIKV strain from the recent outbreak in the Caribbean islands contains a mixture of viruses encoding either the opal termination codon or an arginine mutation. Mutating the opal stop codon to an arginine residue attenuates CHIKV-induced disease in a mouse model. Compared to infection with the opal-containing parental virus, infection with the arginine mutant causes limited swelling and inflammation, as well as dampened recruitment of immune mediators of pathology, including CD4+ T cells and NK cells. We propose that the opal termination codon plays an essential role in the induction of severe CHIKV disease.

Association between Food Insecurity and Procurement Methods among People Living with HIV in a High Resource Setting.

OBJECTIVE: People living with HIV in high-resource settings suffer severe levels of food insecurity; however, limited evidence exists regarding dietary intake and sub-components that characterize food insecurity (i.e. food quantity, quality, safety or procurement) in this population. We examined the prevalence and characteristics of food insecurity among people living with HIV across British Columbia, Canada. DESIGN: This cross-sectional analysis was conducted within a national community-based research initiative. METHODS: Food security was measured using the Health Canada Household Food Security Scale Module. Logistic regression was used to determine key independent predictors of food insecurity, controlling for potential confounders. RESULTS: Of 262 participants, 192 (73%) reported food insecurity. Sub-components associated with food insecurity in bivariate analysis included: < RDI consumption of protein (p = 0.046); being sick from spoiled/unsafe food in the past six months (p = 0.010); and procurement of food using non-traditional methods (p <0.05). In multivariable analyses, factors significantly associated with food insecurity included: procurement of food using non-traditional methods [AOR = 11.11, 95% CI: 4.79-25.68, p = <0.001]; younger age [AOR = 0.92, 95% CI: 0.86-0.96, p = <0.001]; unstable housing [AOR = 4.46, 95% CI: 1.15-17.36, p = 0.031]; household gross annual income [AOR = 4.49, 95% CI: 1.74-11.60, p = 0.002]; and symptoms of depression [AOR = 2.73, 95% CI: 1.25-5.96, p = 0.012]. CONCLUSIONS: Food insecurity among people living with HIV in British Columbia is characterized by poor dietary quality and food procurement methods. Notably, participants who reported procuring in non-traditional manners were over 10 times more likely to be food insecure. These findings suggest a need for tailored food security and social support interventions in this setting.

Length-Dependent Formation of Transmembrane Pores by 310-Helical α-Aminoisobutyric Acid Foldamers.

The synthetic biology toolbox lacks extendable and conformationally controllable yet easy-to-synthesize building blocks that are long enough to span membranes. To meet this need, an iterative synthesis of α-aminoisobutyric acid (Aib) oligomers was used to create a library of homologous rigid-rod 310-helical foldamers, which have incrementally increasing lengths and functionalizable N- and C-termini. This library was used to probe the inter-relationship of foldamer length, self-association strength, and ionophoric ability, which is poorly understood. Although foldamer self-association in nonpolar chloroform increased with length, with a ∼ 14-fold increase in dimerization constant from Aib6 to Aib11, ionophoric activity in bilayers showed a stronger length dependence, with the observed rate constant for Aib11 ∼ 70-fold greater than that of Aib6. The strongest ionophoric activity was observed for foldamers with >10 Aib residues, which have end-to-end distances greater than the hydrophobic width of the bilayers used (∼ 2.8 nm); X-ray crystallography showed that Aib11 is 2.93 nm long. These studies suggest that being long enough to span the membrane is more important for good ionophoric activity than strong self-association in the bilayer. Planar bilayer conductance measurements showed that Aib11 and Aib13, but not Aib7, could form pores. This pore-forming behavior is strong evidence that Aibm (m ≥ 10) building blocks can span bilayers.

Helical peptaibol mimics are better ionophores when racemic than when enantiopure.

Helical peptide foldamers rich in α-aminoisobutyric acid (Aib) act as peptaibol-mimicking ionophores in the phospholipid bilayers of artificial vesicles. Racemic samples of these foldamers are more active than their enantiopure counterparts, which was attributed to differing propensities to form aggregates with crystal-like features in the bilayer.

Water-soluble, luminescent iridium(III)-ytterbium(III) complexes using dipyrido[3,2-a:2',3'-c]phenazine derivatives as bridging units.

Amino-substituted dipyrido[3,2-a:2',3'-c]phenazine (L(1)) and dimethyl-dipyrido[3,2-a:2',3'-c]phenazine (L(2)) have been investigated as: (i) chromophores in cyclen-based ligands for lanthanide(III) ions; (II) ancillary co-ligands in cyclometalated iridium(III) complexes; (III) bridging, linker units in covalently linked, water-soluble bimetallic lanthanide(III) iridium(III) hybrid complexes. The dipyrido[3,2-a:2',3'-c]phenazine (dppz) derivatives can act as sensitising chromophores (λ(ex) 400 nm) for Yb(III), resulting in characteristic near-IR emission at 950-1050 nm. The incorporation of dppz-type ligands into cyclometalated Ir(III) complexes of the general type [Ir(epqc)(2)(L(n))](PF(6)) (where epqc = ethylphenylquinoline carboxylate) gave luminescent species with solvent-sensitive emission properties. Steady state and time-resolved luminescence measurements on the water-soluble d-f hybrid species showed that Yb(III) can be sensitised using visible light.

Probing solution behaviour of metallosupramolecular complexes using pyrene fluorescence.

A new method for assessing the topology of metallosupramolecular assemblies using pyrene-appended ligands is reported. Two potentially tetradentate ligands containing one (L(1)) and two (L(2)) terminal pyrene moieties were synthesised and their complexes with Cu(+) and Cd(2+) were characterised. Photophysical measurements demonstrate that in [Cu(2)(L(1))(2)](2+), [CdL(1)](2+) and [Cu(2)(L(2))(2)](2+) the emission spectra are dominated by monomeric emission but in the cadmium complex of L(2) (where the pyrene units are in close proximity) a quenching of the luminescence coupled with weak emission at 540 nm is indicative of excimer formation.

Amino-anthraquinone chromophores functionalised with 3-picolyl units: structures, luminescence, DFT and their coordination chemistry with cationic Re(I) di-imine complexes.

The syntheses of four new ligands based upon 3-picolyl functionalised amino anthraquinone (AQ) chromophores are described via a one-pot reductive amination procedure giving the desired ligands L1-L4 (L1, 1-(3-picolylamino)anthracene-9,10-dione; L2, 1-hydroxy-4-(3-picolylamino)anthracene-9,10-dione; L3, 1,4-bis(3-picolylamino)anthracene-9,10-dione; L4, 1,5-bis(3-picolylamino)anthracene-9,10-dione). Each ligand was characterised in solution via(1)H and (13)C{(1)H} NMR, with three examples giving single crystal X-ray diffraction data. The structures confirmed the proposed formulations and also revealed the presence of intramolecular H-bonding between the quinone and secondary amine units. The electronic characteristics of the ligands were investigated using a combined experimental/theoretical approach, revealing that in each case absorption in the visible region constitutes significant charge transfer (CT) character, originating from N-(amine)-to-quinone transitions, and is solvent sensitive. Density functional theory (DFT) calculations also suggest that the position of amino-substitution at the AQ core influences the wavelength of the lowest energy feature, by modulation of the HOMO, rather than the LUMO energy. The coordination chemistry of the ligands was probed through reaction with fac-[Re(CO)(3)(di-imine)(MeCN)](BF(4)) where di-imine = 1,10-phenanthroline (phen) and 2,9-dimethyl-1,10-phenanthroline (dmp). Combined structural and spectroscopic studies confirmed that the ligands coordinate to Re(i) exclusively via the pyridyl units, however in the case of L3 only monometallic complexes were isolated. The optical properties of the complexes are dominated by AQ-centred (>425 nm) absorptions superimposed upon (1)MLCT features, as well as diimine-based intra-ligand (<350 nm) transitions. The luminescence properties of the complexes generally display dual emission, which was dependent upon the wavelength of sensitisation, with short-lived AQ fluorescence superimposed upon long-lived (3)MLCT phosphorescence.

Bimodal, dimetallic lanthanide complexes that bind to DNA: the nature of binding and its influence on water relaxivity.

UV-visible titrations, (1)H NMRD experiments and molecular docking studies show that emissive anthraquinone appended dimetallic lanthanide complexes bind to DNA. The strength of binding and the observed relaxivity behaviour depend on the nature of the substituted anthraquinone core.

Luminescent probes based on water-soluble, dual-emissive lanthanide complexes: metal ion-induced modulation of near-IR emission.

Responsive lanthanide complexes demonstrate differentiated modulated luminescence output upon exposure to metal di-cations in aqueous solution.

Purification-free synthesis of a highly efficient ruthenium dye complex for dye-sensitised solar cells (DSSCs).

A novel Ru(II) sensitiser A597 containing the 4,4'-dioctylamido-2,2'-bipyridine ancillary ligand is synthesised without the need for purification steps. It shows an irreversible oxidation at 0.92 V in the cyclic voltammogram and an absorbtion at 539 nm in the UV-vis spectrum corresponding to an (1)MLCT transition. Preliminary DFT calculations reveal that the HOMO is localised on the ruthenium metal centre and the thiocyanate ligands, whereas the LUMO is predominantly on the 4,4'-carboxy-2,2'-bipyridine ligand. The ruthenium complex exhibits a maximum power conversion efficiency (7.25%) compared with the known Z907 (8.32%) in dye-sensitised solar cells.

Sensitised near-IR lanthanide luminescence exploiting anthraquinone-derived chromophores: syntheses and spectroscopic properties.

The syntheses of two anthraquinone-derived tetraaza macrocyclic ligands (L1 via 1-amino-9,10-anthraquinone and L2 via 1-amino-4-hydroxy-9,10-anthraquinone) together with their corresponding LnIII complexes, Ln-L1/2 (Ln = NdIII, GdIII, ErIII, YbIII), are described. Both Ln-L1 (lambda(max) approximately 380 nm) and Ln-L2 (lambda(max) approximately 450 nm) complexes absorb in the visible region with good extinction coefficients (epsilon(vis) > 2 x 10(3) M-1 cm-1). Phosphorescence measurements on Gd-L1/2 at 77 K allowed the ligand-centred triplet states to be estimated at ca. 22000 cm-1 and 19800 cm-1 for Gd-L1 and Gd-L2 respectively. Steady state and time-resolved measurements showed that both chromophores sensitised NdIII, ErIII and YbIII ions, resulting in observable near-IR emission. Preliminary studies on the pH-dependent behaviour of the L2 derivatives demonstrated that deprotonation of the 4-hydroxyl group at pH 12 resulted in a significant bathochromic shift in the absorption profile, thus allowing sensitised near-IR emission utilising lambda(ex) = 575 nm.

Luminescent, water-soluble gold nanoparticles functionalised with 3MLCT emitting rhenium complexes.

Rhenium complexes functionalised with thioester-terminated alkyl chains can be attached to gold nanoparticles to yield water-soluble, visibly luminescent Re-GNP hybrid conjugates.