PSMD12-Mediated M1 Ubiquitination of Influenza A Virus at K102 Regulates Viral Replication.

The M1 of influenza A virus (IAV) is important for the virus life cycle, especially for the assembly and budding of viruses, which is a multistep process that requires host factors. Identifying novel host proteins that interact with M1 and understanding their functions in IAV replication are of great interest in antiviral drug development. In this study, we identified 19 host proteins in DF1 cells suspected to interact with the M1 protein of an H5N6 virus through immunoprecipitation (IP)/mass spectrometry. Among them, PSMD12, a 26S proteasome regulatory subunit, was shown to interact with influenza M1, acting as a positive host factor in IAV replication in avian and human cells. The data showed that PSMD12 promoted K63-linked ubiquitination of M1 at the K102 site. H5N6 and PR8 with an M1-K102 site mutant displayed a significantly weaker replication ability than the wild-type viruses. Mechanistically, PSMD12 promoted M1-M2 virus-like particle (VLP) release, and an M1-K102 mutation disrupted the formation of supernatant M1-M2 VLPs. An H5N6 M1-K102 site mutation or knockdown PSMD12 disrupted the budding release of the virus in chicken embryo fibroblast (CEF) cells, which was confirmed by transmission electron microscopy. Further study confirmed that M1-K102 site mutation significantly affected the virulence of H5N6 and PR8 viruses in mice. In conclusion, we report the novel host factor PSMD12 which affects the replication of influenza virus by mediating K63-linked ubiquitination of M1 at K102. These findings provide novel insight into the interactions between IAV and host cells, while suggesting an important target for anti-influenza virus drug research. IMPORTANCE M1 is proposed to play multiple biologically important roles in the life cycle of IAV, which relies largely on host factors. This study is the first one to identify that PSMD12 interacts with M1, mediates K63-linked ubiquitination of M1 at the K102 site, and thus positively regulates influenza virus proliferation. PSMD12 promoted M1-M2 VLP egress, and an M1-K102 mutation affected the M1-M2 VLP formation. Furthermore, we demonstrate the importance of this site to the morphology and budding of influenza viruses by obtaining mutant viruses, and the M1 ubiquitination regulator PSMD12 has a similar function to the M1 K102 mutation in regulating virus release and virus morphology. Additionally, we confirm the reduced virulence of H5N6 and PR8 (H1N1) viruses carrying the M1-K102 site mutation in mice. These findings provide novel insights into IAV interactions with host cells and suggest a valid and highly conserved candidate target for antiviral drug development.

Neutralizing antibody PR8-23 targets the footprint of the sialoglycan receptor binding site of H1N1 hemagglutinin.

Influenza virus cause seasonal influenza epidemic and seriously sporadic influenza pandemic outbreaks. Hemagglutinin (HA) is an important target in the therapeutic treatment and diagnostic detection of the influenza virus. Variation in the sialic acid receptor binding site leads to strain-specific binding and results in different binding modes to the host receptors. Here, we evaluated the neutralizing activity and hemagglutination inhibition activity of a prepared murine anti-H1N1 monoclonal antibody PR8-23. Then we identified the epitope peptide of antibody PR8-23 by phage display technique from phage display peptide libraries. The identified epitope, 63-IAPLQLGKCNIA-74, containing two α-helix and two ß-fold located at the footprint of the sialoglycan receptor on the RBS in the globular head domain of HA. It broads the growing arsenal of motifs for the amino acids on the globular head domain of HA in sialic acid receptor binding site and neutralizing antibody production.

The soft palate is an important site of adaptation for transmissible influenza viruses.

Influenza A viruses pose a major public health threat by causing seasonal epidemics and sporadic pandemics. Their epidemiological success relies on airborne transmission from person to person; however, the viral properties governing airborne transmission of influenza A viruses are complex. Influenza A virus infection is mediated via binding of the viral haemagglutinin (HA) to terminally attached α2,3 or α2,6 sialic acids on cell surface glycoproteins. Human influenza A viruses preferentially bind α2,6-linked sialic acids whereas avian influenza A viruses bind α2,3-linked sialic acids on complex glycans on airway epithelial cells. Historically, influenza A viruses with preferential association with α2,3-linked sialic acids have not been transmitted efficiently by the airborne route in ferrets. Here we observe efficient airborne transmission of a 2009 pandemic H1N1 (H1N1pdm) virus (A/California/07/2009) engineered to preferentially bind α2,3-linked sialic acids. Airborne transmission was associated with rapid selection of virus with a change at a single HA site that conferred binding to long-chain α2,6-linked sialic acids, without loss of α2,3-linked sialic acid binding. The transmissible virus emerged in experimentally infected ferrets within 24 hours after infection and was remarkably enriched in the soft palate, where long-chain α2,6-linked sialic acids predominate on the nasopharyngeal surface. Notably, presence of long-chain α2,6-linked sialic acids is conserved in ferret, pig and human soft palate. Using a loss-of-function approach with this one virus, we demonstrate that the ferret soft palate, a tissue not normally sampled in animal models of influenza, rapidly selects for transmissible influenza A viruses with human receptor (α2,6-linked sialic acids) preference.

Monoclonal Antibody Targeting the HA191/199 Region of H1N1 Influenza Virus Mediates the Damage of Neural Cells.

Vaccination is the most effective mean of preventing influenza virus infections. However, vaccination-induced adverse reactions of the nervous system, the causes of which are unknown, lead to concerns on the safety of influenza A vaccine. In this study, we used flow cytometry, cell ELISA, and immunofluorescence to find that H1-84 monoclonal antibody (mAb) against the191/199 region of the H1N1 influenza virus hemagglutinin (HA) protein binds to neural cells and mediates cell damage. Using molecular simulation software, such as PyMOL and PDB viewer, we demonstrated that the HA191/199 region maintains the overall structure of the HA head. Since the HA191/199 region cannot be removed from the HA structure, it has to be altered via introducing point mutations by site-directed mutagenesis. This will provide an innovative theoretical support for the subsequent modification the influenza A vaccine for increasing its safety.

Influenza hemagglutinin membrane anchor.

Viruses with membranes fuse them with cellular membranes, to transfer their genomes into cells at the beginning of infection. For Influenza virus, the membrane glycoprotein involved in fusion is the hemagglutinin (HA), the 3D structure of which is known from X-ray crystallographic studies. The soluble ectodomain fragments used in these studies lacked the "membrane anchor" portion of the molecule. Since this region has a role in membrane fusion, we have determined its structure by analyzing the intact, full-length molecule in a detergent micelle, using cryo-EM. We have also compared the structures of full-length HA-detergent micelles with full-length HA-Fab complex detergent micelles, to describe an infectivity-neutralizing monoclonal Fab that binds near the ectodomain membrane anchor junction. We determine a high-resolution HA structure which compares favorably in detail with the structure of the ectodomain seen by X-ray crystallography; we detect, clearly, all five carbohydrate side chains of HA; and we find that the ectodomain is joined to the membrane anchor by flexible, eight-residue-long, linkers. The linkers extend into the detergent micelle to join a central triple-helical structure that is a major component of the membrane anchor.

Defining the sizes of airborne particles that mediate influenza transmission in ferrets.

Epidemics and pandemics of influenza are characterized by rapid global spread mediated by non-mutually exclusive transmission modes. The relative significance between contact, droplet, and airborne transmission is yet to be defined, a knowledge gap for implementing evidence-based infection control measures. We devised a transmission chamber that separates virus-laden particles by size and determined the particle sizes mediating transmission of influenza among ferrets through the air. Ferret-to-ferret transmission was mediated by airborne particles larger than 1.5 µm, consistent with the quantity and size of virus-laden particles released by the donors. Onward transmission by donors was most efficient before fever onset and may continue for 5 days after inoculation. Multiple virus gene segments enhanced the transmissibility of a swine influenza virus among ferrets by increasing the release of virus-laden particles into the air. We provide direct experimental evidence of influenza transmission via droplets and fine droplet nuclei, albeit at different efficiency.

Receptor binding by H10 influenza viruses.

H10N8 follows H7N9 and H5N1 as the latest in a line of avian influenza viruses that cause serious disease in humans and have become a threat to public health. Since December 2013, three human cases of H10N8 infection have been reported, two of whom are known to have died. To gather evidence relating to the epidemic potential of H10 we have determined the structure of the haemagglutinin of a previously isolated avian H10 virus and we present here results relating especially to its receptor-binding properties, as these are likely to be major determinants of virus transmissibility. Our results show, first, that the H10 virus possesses high avidity for human receptors and second, from the crystal structure of the complex formed by avian H10 haemagglutinin with human receptor, it is clear that the conformation of the bound receptor has characteristics of both the 1918 H1N1 pandemic virus and the human H7 viruses isolated from patients in 2013 (ref. 3). We conclude that avian H10N8 virus has sufficient avidity for human receptors to account for its infection of humans but that its preference for avian receptors should make avian-receptor-rich human airway mucins an effective block to widespread infection. In terms of surveillance, particular attention will be paid to the detection of mutations in the receptor-binding site of the H10 haemagglutinin that decrease its avidity for avian receptor, and could enable it to be more readily transmitted between humans.

Intact Glycopeptide Analysis of Influenza A/H1N1/09 Neuraminidase Revealing the Effects of Host and Glycosite Location on Site-Specific Glycan Structures.

Influenza H1N1 virus has posed a serious threat to human health. The glycosylation of neuraminidase (NA) could affect the infectivity and virulence of the influenza virus, but detailed site-specific glycosylation information of NA is still missing. In this study, intact glycopeptide analysis is performed on an influenza NA (A/H1N1/California/2009) that is expressed in human 293T and insect Hi-5 cells. The data indicate that three of four potential N-linked glycosylation sites are glycosylated, including one partial glycosylation site from both cell lines. The NA expressed in human cells has more complex glycans than that of insect cells, suggesting the importance of selecting an appropriate expression system for the production of functional glycoproteins. Different types of glycans are identified from different glycosites of NA expressed in human cells, which implies the site-dependence of glycosylation on NA. This study provides valuable information for the research of influenza virus as well as the functions of viral protein glycosylation.

Gold Nanoparticles with Ligand/Zwitterion Hybrid Layer for Individual Counting of Influenza A H1N1 Subtype Using Resistive Pulse Sensing.

Resistive pulse sensing (RPS) is an analytical technique for detecting particles with nano- to micrometer diameters, such as proteins, viruses, and bacteria. RPS is a promising tool for diagnosis as it can analyze the characteristics of target particles individually from ion current blockades as pulse waveforms. However, it is difficult to discriminate analog targets because RPS merely provides physical information such as size, shape, concentration, and charge density of the analyte. Influenza A virus, which is 80-120 nm in diameter, has various subtypes, demonstrating the diversity of virus characteristics. For example, highly pathogenic avian influenza infections in humans are recognized as an emerging infectious disease with high mortality rates compared with human influenza viruses. Distinguishing human from avian influenza using their differing biological characteristics would be challenging using RPS. To develop a highly selective diagnostic system for infectious diseases, we combined RPS with molecular recognition. Gold nanoparticles (GNPs) that have human influenza A (H1N1 subtype) virus-specific sialic acid receptors on the surface were prepared as a virus label for RPS analysis. A sulfobetaine and sialic acid (ligand) hybrid surface was formed on the GNPs for the suppression of nonspecific interaction. The results show a size change of viruses derived from specific interactions with GNPs. In contrast, no size shift was observed when nonspecific sialic acid receptor-immobilized GNPs were used. Detection of viruses by individual particle counting could be a new facet of diagnosis.

The interaction of cellular protein ANP32A with influenza A virus polymerase component PB2 promotes vRNA synthesis.

The subunits PA, PB1, and PB2 of influenza A virus RNA polymerase are essential for efficient viral RNA synthesis and virus replication because of their role in recruiting multiple nuclear proteins. ANP32A is an acidic leucine-rich nuclear phosphoprotein 32 (ANP32) family member and a crucial cellular protein that determines the species specificity of the influenza virus RNA polymerase activity. However, how ANP32A modulates polymerase activity remains largely unknown. In this study, we showed that viral RNA synthesis was increased in A549 cells overexpressing ANP32A and decreased after treatment with ANP32A RNAi. This decrease in RNA synthesis was reversed by rescued ANP32A expression. The results of docking modeling, co-immunoprecipitation, and yeast two-hybrid assays showed that PB2 was the only subunit of the three that interacted with ANP32A. The C-terminal portion of ANP32A and the middle domains (residues 307-534) of PB2 were required for PB2-ANP32A interaction. Glu189 and Glu196 in ANP32A and Gly450 and Gln447 in PB2 were essential for interaction between ANP32A and PB2. These residues were located in conserved regions of the ANP32A or PB2 protein sequences. These data suggest that ANP32A is recruited to the polymerase through direct interaction with PB2 via critical amino acid residue interactions and promotes viral RNA synthesis. Our findings might provide new insights into the molecular mechanisms underlying influenza virus RNA synthesis and replication in infected human cells.

Carbohydrate-Based Polymer Brushes Prevent Viral Adsorption on Electrostatically Heterogeneous Interfaces.

Chemical heterogeneity on biomaterial surfaces can transform its interfacial properties, rendering nanoscale heterogeneity profoundly consequential during bioadhesion. To examine the role played by chemical heterogeneity in the adsorption of viruses on synthetic surfaces, a range of novel coatings is developed wherein a tunable mixture of electrostatic tethers for viral binding, and carbohydrate brushes, bearing pendant α-mannose, ß-galactose, or ß-glucose groups, is incorporated. The effects of binding site density, brush composition, and brush architecture on viral adsorption, with the goal of formulating design specifications for virus-resistant coatings are experimentally evaluated. It is concluded that virus-coating interactions are shaped by the interplay between brush architecture and binding site density, after quantifying the adsorption of adenoviruses, influenza, and fibrinogen on a library of carbohydrate brushes co-immobilized with different ratios of binding sites. These insights will be of utility in guiding the design of polymer coatings in realistic settings where they will be populated with defects.

Equipping Inner Central Components of Influenza A Virus with Quantum Dots.

Influenza A virus (IAV), a risk to public health, is enveloped and contains viral ribonucleoprotein (vRNP) complexes, where vRNP complexes are central to every aspect of the IAV life cycle. Labeling both the vRNP complexes and viral envelope with quantum dots (QDs) is conducive to achieving global long-term tracking of a single IAV infecting host cell, which has potential to provide valuable information for revealing mechanisms of IAV infection. However, even though some strategies for labeling of the viral envelope with QDs have been developed, there are few strategies for coupling of QDs to the vRNP complexes inside IAV so far. Herein, we devised a convenient electroporation-based strategy, coupled with antibody binding, to transfer green QDs-labeled nucleoprotein antibodies (GQDs-NPAb) into H1N1 and achieved the labeling of vRNP complexes with QDs [H1N1(GQDs)]. Under the optimal condition of 20 nM GQDs-NPAb and a single pulse with 20 ms duration and 750 V/cm pulse intensity, the actual efficiency of labeling is ca. 34% and H1N1(GQDs) can retain 93% infectivity. Then, dual labeling of H1N1 was realized by labeling the envelope of H1N1(GQDs) with red QDs (RQDs) via a mild and efficient hydrazine-aldehyde-based strategy. At the optimal RQDs concentration of 5 nM, the actual efficiency of dual labeling can reach to 11% and the dual-labeled H1N1 can retain 93% infectivity. Because of the similar components and structure of different IAV subtypes, this dual-labeling strategy is applicable to other subtypes of IAV, e.g., H9N2.

Host Cellular Protein TRAPPC6AΔ Interacts with Influenza A Virus M2 Protein and Regulates Viral Propagation by Modulating M2 Trafficking.

Influenza A virus (IAV) matrix protein 2 (M2) plays multiple roles in the early and late phases of viral infection. Once synthesized, M2 is translocated to the endoplasmic reticulum (ER), travels to the Golgi apparatus, and is sorted at the trans-Golgi network (TGN) for transport to the apical plasma membrane, where it functions in virus budding. We hypothesized that M2 trafficking along with its secretory pathway must be finely regulated, and host factors could be involved in this process. However, no studies examining the role of host factors in M2 posttranslational transport have been reported. Here, we used a yeast two-hybrid (Y2H) system to screen for host proteins that interact with the M2 protein and identified transport protein particle complex 6A (TRAPPC6A) as a potential binding partner. We found that both TRAPPC6A and its N-terminal internal-deletion isoform, TRAPPC6A delta (TRAPPC6AΔ), interact with M2. Truncation and mutation analyses showed that the highly conserved leucine residue at position 96 of M2 is critical for mediating this interaction. The role of TRAPPC6AΔ in the viral life cycle was investigated by the knockdown of endogenous TRAPPC6AΔ with small interfering RNA (siRNA) and by generating a recombinant virus that was unable to interact with TRAPPC6A/TRAPPC6AΔ. The results indicated that TRAPPC6AΔ, through its interaction with M2, slows M2 trafficking to the apical plasma membrane, favors viral replication in vitro, and positively modulates virus virulence in mice. IMPORTANCE: The influenza A virus M2 protein regulates the trafficking of not only other proteins but also itself along the secretory pathway. However, the host factors involved in the regulation of the posttranslational transport of M2 are largely unknown. In this study, we identified TRAPPC6A and its N-terminal internal-deletion isoform, TRAPPC6AΔ, as interacting partners of M2. We found that the leucine (L) residue at position 96 of M2 is critical for mediating this interaction, which leads us to propose that the high level of conservation of 96L is a consequence of M2 adaptation to its interacting host factor TRAPPC6A/TRAPPC6AΔ. Importantly, we discovered that TRAPPC6AΔ can positively regulate viral replication in vitro by modulating M2 trafficking to the plasma membrane.

Identification of different hemagglutinin isoforms of influenza A virus H1N1.

RATIONALE: Influenza A viruses (IAVs) are still a threat to human health and life. The process of virus infection involves a series of biological regulations, such as signal transduction, that may be closely linked with the function of glycoproteins. However, the number and level of glycoproteins is low compared with other proteins in the whole protein pool. METHODS: Viruses obtained from chicken embryos were purified by sucrose gradient centrifugation. PNGase F enzyme was then used to remove the glycan modification, followed by two-dimensional electrophoresis (2DE) to separate the hemagglutinin1 (HA1) glycoprotein. In-gel digestion was used to obtain peptides that were detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). RESULTS: Remarkably, we found five isoforms of HA1 with the same molecular weight but different isoelectric points. Furthermore, HA1 treatment with PNGase F enzyme changed all but one protein spot from 2DE, indicating that the different HA1 isoforms in 2DE were a result of different glycosylation modifications. CONCLUSIONS: The difference in isoelectric points of these HA1 isoforms was caused by glycan modification. This method provides a new approach for the study of glycosylation of the proteome for viruses or any other organisms.

Evolution and dynamics of the pandemic H1N1 influenza hemagglutinin protein from 2009 to 2017.

The emergence of swine-origin pandemic H1N1 (pH1N1) in 2009 invigorated extensive surveillance programs worldwide which have resulted in the deposition of large numbers of H1N1 sequences to Genbank. In the present study, we report on global evolution and dynamics of the pandemic H1N1 influenza Hemagglutinin (HA) protein in viruses isolated from three different continents (North America, Europe and Asia) during the period between April 2009 until April 2017. Close to 2000 HA full protein sequences were downloaded from the Influenza Research Database of the NCBI and analyzed using DNAStar to run an alignment, the web-based NetNglyc to predict N-Glycosylation sites and finally, the BEAST software package to calculate evolution and substitution rates. Our analysis improves upon other published papers in that we report on frequencies, dynamics and impact of HA mutations in pH1N1 viruses isolated from three continents during the past decade, as well as the evolution rate and site-specific selection pressures. Sequence based analysis demonstrated substantial changes in the HA protein over the last decade. Results showed that the HA gene is under negative selection (P value; HA= -2.253). The evolution rates varied among the three continents ranging from 2.36 × 10-3 in Europe to 3.18 × 10-3 in Asia. Mutations were detected at higher frequency and faster rate at the antigenic sites surrounding the receptor-binding domain (RBD), in particular, in the Sa and Sb sites. Mutations were either gradually accumulated to become fixed in currently circulating strains (D114N, S179N, S202T, S220T, I233T, K300E and E391K) or dynamic in terms of appearance and disappearance, both spatially and temporally (A203T, N458K and E508G). Some of the reported mutations have been shown to increase infection severity (D239G/N; globular head), enhance HA binding affinity to its receptor (S200P and S202T; RBD), or have deleterious effect on HA function (N458K and E508G; stem region). The continuous accumulation of mutations at the Sa site led to the gradual acquisition of glycosylation at residue 179 starting from 2015, which became a dominant feature in all strains isolated in the following years. In addition to sharing common amino acid substitutions (e.g. S179N in HA head and E516K in HA stem) with previous seasonal strains, the pattern of glycosylation acquisition/loss at 177 and 179 positions on the globular head, which are prominent features of immune escape, implicate that pH1N1 might follow a similar evolution trend as the SC1918 pandemic virus.

Computational analysis of the effect of polymerase acidic (PA) gene mutation F35L in the 2009 pandemic influenza A (H1N1) virus on binding aspects of mononucleotides in the endonuclease domain.

An F35L mutation in the N-terminal domain of the polymerase acidic protein (PA-Nter), which contains the active site of the endonuclease, has been reported to result in higher polymerase activity in mouse-adapted strains of the 2009 pandemic influenza A H1N1 virus. We modeled wild and mutant complexes of uridine 5'-monophosphate (UMP) as the endonuclease substrate and performed molecular dynamics simulations. The results demonstrated that the F35L mutation could result in a changed orientation of a helix containing active site residues and improve the ligand affinity in the mutant strain. This study suggests a molecular mechanism of enhanced polymerase activity.

Cross-neutralization of influenza A viruses mediated by a single antibody loop.

Immune recognition of protein antigens relies on the combined interaction of multiple antibody loops, which provide a fairly large footprint and constrain the size and shape of protein surfaces that can be targeted. Single protein loops can mediate extremely high-affinity binding, but it is unclear whether such a mechanism is available to antibodies. Here we report the isolation and characterization of an antibody called C05, which neutralizes strains from multiple subtypes of influenza A virus, including H1, H2 and H3. X-ray and electron microscopy structures show that C05 recognizes conserved elements of the receptor-binding site on the haemagglutinin surface glycoprotein. Recognition of the haemagglutinin receptor-binding site is dominated by a single heavy-chain complementarity-determining region 3 loop, with minor contacts from heavy-chain complementarity-determining region 1, and is sufficient to achieve nanomolar binding with a minimal footprint. Thus, binding predominantly with a single loop can allow antibodies to target small, conserved functional sites on otherwise hypervariable antigens.

Aggregation of Influenza A Virus Nuclear Export Protein.

Influenza A virus nuclear export protein (NEP) plays an important role in the viral life cycle. Recombinant NEP proteins containing (His)6-tag at either N- or C-terminus were obtained by heterologous expression in Escherichia coli cells and their high propensity for aggregation was demonstrated. Dynamic light scattering technique was used to study the kinetics and properties of NEP aggregation in solutions under different conditions (pH, ionic strength, presence of low-molecular-weight additives and organic solvents). Using atomic force microscopy, the predominance of spherical aggregates in all examined NEP preparations was shown, with some amyloid-like structures being observed in the case of NEP-C protein. A number of structure prediction programs were used to identify aggregation-prone regions in the NEP structure. All-atom molecular dynamics simulations indicate a high rate of NEP molecule aggregation and reveal the regions preferentially involved in the intermolecular contacts that are located at the edges of the rod-like protein molecule. Our results suggest that NEP aggregation is determined by different types of interactions and represents an intrinsic property of the protein that appears to be necessary for its functioning in vivo.

Development of screening method for intranasal influenza vaccine and adjuvant safety in preclinical study.

Recently, many vaccine adjuvants have been developed; however, most of the newly developed adjuvants have been dropped out of preclinical and clinical trials owing to their unexpected toxicity. Thus, the development of highly quantitative and comparable screening methods for evaluating adjuvant safety is needed. In a previous study, we identified specific biomarkers for evaluating the safety of an intranasal influenza vaccine with CpG K3 adjuvant by comparing biomarker expression. We hypothesized that these biomarkers might be useful for screening newly developed adjuvant safety. We compared the expression of biomarkers in mouse lungs by the intranasal administration of 4 types of adjuvants: Alum, Pam3CSK4, NanoSiO2, and DMXAA with subvirion influenza vaccine. The control adjuvant alum did not show any significant increase in biomarker expression or preclinical parameters; however, NanoSiO2 and Pam3CSK4 increased the expression of biomarkers, such as Timp1 and Csf1. DMXAA at 300 µg induced the expression of over 80% of biomarkers. Hierarchical clustering analysis showed that 300 µg DMXAA was classified in the toxicity reference whole-particle influenza vaccine cluster. FACS analysis to confirm specific phenotypes that the number of T cells decreased in DMXAA-treated mouse lungs. Thus, our biomarkers are useful for initial adjuvant safety and toxicity screening.

Single-Step Detection of the Influenza Virus Hemagglutinin Using Bacterially-Produced Quenchbodies.

We have successfully generated a Quenchbody that enables the detection of the influenza virus hemagglutinin (HA), in a simple and convenient manner. By two-site labeling of the bacterially-produced anti-HA Fab with ATTO520, its fluorescence intensity was increased to 4.4-fold, in the presence of a nanomolar concentration of H1N1 HA. Our results indicate the potential use of this Quenchbody, as a sensor for the simple in situ detection of influenza A virus.