Multiple Influenza Virus Research Articles

Modeling influenza seasonality in the tropics and subtropics.

Climate drivers such as humidity and temperature may play a key role in influenza seasonal transmission dynamics. Such a relationship has been well defined for temperate regions. However, to date no models capable of capturing the diverse seasonal pattern in tropical and subtropical climates exist. In addition, multiple influenza viruses could cocirculate and shape epidemic dynamics. Here we construct seven mechanistic epidemic models to test the effect of two major climate drivers (humidity and temperature) and multi-strain co-circulation on influenza transmission in Hong Kong, an influenza epidemic center located in the subtropics. Based on model fit to long-term influenza surveillance data from 1998 to 2018, we found that a simple model incorporating the effect of both humidity and temperature best recreated the influenza epidemic patterns observed in Hong Kong. The model quantifies a bimodal effect of absolute humidity on influenza transmission where both low and very high humidity levels facilitate transmission quadratically; the model also quantifies the monotonic but nonlinear relationship with temperature. In addition, model results suggest that, at the population level, a shorter immunity period can approximate the co-circulation of influenza virus (sub)types. The basic reproductive number R0 estimated by the best-fit model is also consistent with laboratory influenza survival and transmission studies under various combinations of humidity and temperature levels. Overall, our study has developed a simple mechanistic model capable of quantifying the impact of climate drivers on influenza transmission in (sub)tropical regions. This model can be applied to improve influenza forecasting in the (sub)tropics in the future.

Progress and Challenge in Computational Identification of Influenza Virus Reassortment.

Genomic reassortment is an important evolutionary mechanism for influenza viruses. In this process, the novel viruses acquire new characteristics by the exchange of the intact gene segments among multiple influenza virus genomes, which may cause flu endemics and epidemics within or even across hosts. Due to the safety and ethical limitations of the experimental studies on influenza virus reassortment, numerous computational researches on the influenza virus reassortment have been done with the explosion of the influenza virus genomic data. A great amount of computational methods and bioinformatics databases were developed to facilitate the identification of influenza virus reassortments. In this review, we summarized the progress and challenge of the bioinformatics research on influenza virus reassortment, which can guide the researchers to investigate the influenza virus reassortment events reasonably and provide valuable insight to develop the related computational identification tools.

Broadly Protective CD8+ T Cell Immunity to Highly Conserved Epitopes Elicited by Heat Shock Protein gp96-Adjuvanted Influenza Monovalent Split Vaccine.

Currently, immunization with inactivated influenza virus vaccines is the most prevalent method to prevent infections. However, licensed influenza vaccines provide only strain-specific protection and need to be updated and administered yearly; thus, new vaccines that provide broad protection against multiple influenza virus subtypes are required. In this study, we demonstrated that intradermal immunization with gp96-adjuvanted seasonal influenza monovalent H1N1 split vaccine could induce cross-protection against both group 1 and group 2 influenza A viruses in BALB/c mouse models. Vaccination in the presence of gp96 induced an apparently stronger antigen-specific T cell response than split vaccine alone. Immunization with the gp96-adjuvanted vaccine also elicited an apparent cross-reactive CD8+ T cell response that targeted the conserved epitopes across different influenza virus strains. These cross-reactive CD8+ T cells might be recalled from a pool of memory cells established after vaccination and recruited from extrapulmonary sites to facilitate viral clearance. Of note, six highly conserved CD8+ T epitopes from the viral structural proteins hemagglutinin (HA), M1, nucleoprotein (NP), and PB1 were identified to play a synergistic role in gp96-mediated cross-protection. Comparative analysis showed that most of conservative epitope-specific cytotoxic T lymphocytes (CTLs) apparently induced by heterologous virus infection were also activated by gp96-adjuvanted vaccine, thus resulting in broader protective CD8+ T cell responses. Our results demonstrated the advantage of adding gp96 to an existing seasonal influenza vaccine to improve its ability to provide better cross-protection.IMPORTANCE Owing to continuous mutations in hemagglutinin (HA) or neuraminidase (NA) or recombination of the gene segments between different strains, influenza viruses can escape the immune responses developed by vaccination. Thus, new strategies aimed to efficiently activate immune response that targets to conserved regions among different influenza viruses are urgently needed in designing broad-spectrum influenza vaccine. Heat shock protein gp96 is currently the only natural T cell adjuvant with special ability to cross-present coupled antigen to major histocompatibility complex class I (MHC-I) molecule and activate the downstream antigen-specific CTL response. In this study, we demonstrated the advantages of adding gp96 to monovalent split influenza virus vaccine to improve its ability to provide cross-protection in the BALB/c mouse model and proved that a gp96-activated cross-reactive CTL response is indispensable in our vaccine strategy. Due to its unique adjuvant properties, gp96 might be a promising adjuvant for designing new broad-spectrum influenza vaccines.

Skin vaccination with dissolvable microneedle patches incorporating influenza neuraminidase and flagellin protein nanoparticles induces broad immune protection against multiple influenza viruses.

We generated self-adjuvanted protein nanoparticles of conserved influenza antigens and immunized mice via skin vaccination with dissolvable microneedle patches (MNPs) to increase the strength and breadth of immune responses. We produced M2e nanoparticles via ethanol desolvation, and double-layered NA1/M2e (shell/core), NA1-FliC/M2e, NA2/M2e, and NA2-FliC/M2e protein nanoparticles by chemically crosslinking influenza NA and flagellin (FliC) onto the surfaces of the M2e nanoparticles. The resulting nanoparticles retained FliC TLR5 innate signaling activity and significantly increased antigen-uptake and dendritic cell maturation in vitro. We incorporated the nanoparticles into MNPs for skin vaccination in mice. The nanoparticle MNPs significantly increased M2e and NA-specific antibody levels, the numbers of germinal center B cells, and IL-4 positive splenocytes. Double-layered nanoparticle MNP skin vaccination protected mice against homologous and heterosubtypic influenza viruses. Our results demonstrated that MNP skin vaccination of NA-FliC/M2e nanoparticles could be developed into a standalone or synergistic component of a universal influenza vaccine strategy.

CACO-2 cells: A continuous cell line with sensitive and broad-spectrum utility for respiratory virus culture

BackgroundPrimary rhesus monkey kidney cells (RhMK) can be used for the detection of respiratory viruses, including influenza and parainfluenza. The human colon adeno-carcinoma cell line, CACO-2, has been previously used for the growth of multiple influenza viruses, including seasonal, novel and avian lineages. ObjectiveWe compared CACO-2, Madin-Darby Canine Kidney (MDCK), and RhMK cells for the isolation of viruses from patients presenting with influenza like-illness (ILI). Study designNasopharyngeal specimens from patients with ILI in primary care settings were processed for conventional viral culture in MDCK, RhMK, and CACO-2. Cells were examined microscopically for cytopathic effect (CPE) and confirmatory testing included immunofluorescent antigen (IFA) detection and real-time RT-PCR. Additionally, 16 specimens positive for respiratory syncytial virus (RSV) by PCR were inoculated on CACO-2 cells. Statistical analysis was done using Chi-square test with IBM Statistical Program. ResultsOf 1031 respiratory specimens inoculated, viruses were isolated and confirmed from 331 (32.1 %) in MDCK cells, 304 (29.5 %) in RhMk cells, and 433 (42.0 %) in CACO-2 cells. These included influenza A/(H1N1)pdm09, influenza A(H3N2), influenza B, parainfluenza virus (PIV) types 1, 2, and 3, human coronavirus 229E (CoV-229E), human adenovirus (HAdV), herpes simplex virus 1 (HSV 1), and enterovirus (EV). Influenza A viruses grew best in the CACO-2 cell line. Time to observation of CPE was similar for all three cell types but unlike RhMK and MDCK cells, virus-specific morphological changes were indistinguishable in CACO-2 cells. None of the 16 specimens positive for RSV by PCR grew on CACO-2 cells. ConclusionsThe overall respiratory virus culture isolation rate in CACO-2 cells was significantly higher than that in RhMK or MDCK cells (p < 0.05). CACO-2 cells also supported the growth of some viruses that did not grow in either RhMK or MDCK cells. Except for RSV, CACO-2 cells provide a worthwhile addition to culture algorithms for respiratory specimens.

Next generation methodology for updating HA vaccines against emerging human seasonal influenza A(H3N2) viruses

While vaccines remain the best tool for preventing influenza virus infections, they have demonstrated low to moderate effectiveness in recent years. Seasonal influenza vaccines typically consist of wild-type influenza A and B viruses that are limited in their ability to elicit protective immune responses against co-circulating influenza virus variant strains. Improved influenza virus vaccines need to elicit protective immune responses against multiple influenza virus drift variants within each season. Broadly reactive vaccine candidates potentially provide a solution to this problem, but their efficacy may begin to wane as influenza viruses naturally mutate through processes that mediates drift. Thus, it is necessary to develop a method that commercial vaccine manufacturers can use to update broadly reactive vaccine antigens to better protect against future and currently circulating viral variants. Building upon the COBRA technology, nine next-generation H3N2 influenza hemagglutinin (HA) vaccines were designed using a next generation algorithm and design methodology. These next-generation broadly reactive COBRA H3 HA vaccines were superior to wild-type HA vaccines at eliciting antibodies with high HAI activity against a panel of historical and co-circulating H3N2 influenza viruses isolated over the last 15 years, as well as the ability to neutralize future emerging H3N2 isolates.

Computationally Optimized Broadly Reactive H2 HA Influenza Vaccines Elicited Broadly Cross-Reactive Antibodies and Protected Mice from Viral Challenges.

Influenza viruses have caused numerous pandemics throughout human history. The 1957 influenza pandemic was initiated by an H2N2 influenza virus. This H2N2 influenza virus was the result of a reassortment event between a circulating H2N2 avian virus and the seasonal H1N1 viruses in humans. Previously, our group has demonstrated the effectiveness of hemagglutinin (HA) antigens derived using computationally optimized broadly reactive antigen (COBRA) methodology against H1N1, H3N2, and H5N1 viruses. Using the COBRA methodology, H2 HA COBRA antigens were designed using sequences from H2N2 viruses isolated from humans in the 1950s and 1960s, as well as H2Nx viruses isolated from avian and mammalian species between the 1950s and 2016. In this study, the effectiveness of H2 COBRA HA antigens (Z1, Z3, Z5, and Z7) was evaluated in DBA/2J mice and compared to that of wild-type H2 HA antigens. The COBRA HA vaccines elicited neutralizing antibodies to the majority of viruses in our H2 HA panel and across all three clades as measured by hemagglutination inhibition (HAI) and neutralization assays. Comparatively, several wild-type HA vaccines elicited antibodies against a majority of the viruses in the H2 HA panel. DBA/2J mice vaccinated with COBRA vaccines showed increase survival for all three viral challenges compared to the wild-type H2 vaccines. In particular, the Z1 COBRA is a promising candidate for future work toward a pandemic H2 influenza vaccine.IMPORTANCE H2N2 influenza has caused at least one pandemic in the past. Given that individuals born after 1968 have not been exposed to H2N2 influenza viruses, a future pandemic caused by H2 influenza is likely. An effective H2 influenza vaccine would need to elicit broadly cross-reactive antibodies to multiple H2 influenza viruses. Choosing a wild-type virus to create a vaccine may elicit a narrow immune response and not protect against multiple H2 influenza viruses. COBRA H2 HA vaccines were developed and evaluated in mice along with wild-type H2 HA vaccines. Multiple COBRA H2 HA vaccines protected mice from all three viral challenges and produced broadly cross-reactive neutralizing antibodies to H2 influenza viruses.

Adjuvanted H5N1 influenza vaccine enhances both cross-reactive memory B cell and strain-specific naive B cell responses in humans

There is a need for improved influenza vaccines. In this study we compared the antibody responses in humans after vaccination with an AS03-adjuvanted versus nonadjuvanted H5N1 avian influenza virus inactivated vaccine. Healthy young adults received two doses of either formulation 3 wk apart. We found that AS03 significantly enhanced H5 hemagglutinin (HA)-specific plasmablast and antibody responses compared to the nonadjuvanted vaccine. Plasmablast response after the first immunization was exclusively directed to the conserved HA stem region and came from memory B cells. Monoclonal antibodies (mAbs) derived from these plasmablasts had high levels of somatic hypermutation (SHM) and recognized the HA stem region of multiple influenza virus subtypes. Second immunization induced a plasmablast response to the highly variable HA head region. mAbs derived from these plasmablasts exhibited minimal SHM (naive B cell origin) and largely recognized the HA head region of the immunizing H5N1 strain. Interestingly, the antibody response to H5 HA stem region was much lower after the second immunization, and this suppression was most likely due to blocking of these epitopes by stem-specific antibodies induced by the first immunization. Taken together, these findings show that an adjuvanted influenza vaccine can substantially increase antibody responses in humans by effectively recruiting preexisting memory B cells as well as naive B cells into the response. In addition, we show that high levels of preexisting antibody can have a negative effect on boosting. These findings have implications toward the development of a universal influenza vaccine.

Pulmonary endothelium-derived PD-L1 induced by the H9N2 avian influenza virus inhibits the immune response of T cells

BackgroundThe PD-1/PD-L1 pathway is an inhibitory signaling pathway that maintains the balance between the immune response and immunotolerance, and its overactivation in cancer and viral infections inhibits T cell function. The target cells of various viruses, microvascular endothelial cells (MECs) have been shown to be key regulatory points in immune regulation and virion diffusion in vivo during infection with multiple influenza virus subtypes. Furthermore, avian influenza virus (AIV) infection can induce immunosuppression by causing imbalances in immune responses and immune organ damage. Thus, the aim of this study was to investigate whether the H9N2 virus inhibited the immune function of T cells that migrated across MECs by upregulating PD-L1 expression on MECs.MethodsThe susceptibility of rat pulmonary microvascular endothelial cells (RPMECs) to the H9N2 virus was evaluated by a plaque-forming assay and immunofluorescence staining. Then, we quantified the mRNA and protein levels of PD-L1 in RPMECs induced by H9N2 virus infection using quantitative real-time PCR and flow cytometry. The interaction between the activated T cells and RPMECs infected with the H9N2 virus was revealed using a coculture system. The effect of endothelial-derived PD-L1 on T cell function was investigated by using ELISA and flow cytometry with or without a PD-L1-specific antibody.ResultsSurface staining and the plaque-forming assay showed that the H9N2 virus infected and replicated in RPMECs. Both the PD-L1 mRNA level and PD-L1 protein level were upregulated in RPMECs infected with the H9N2 virus. H9N2 virus-induced PD-L1 expression significantly reduced the secretions of IL-2, IFN-γ and granzyme B and perforin expression in T cells. The above data were significantly increased after treatment with an anti-PD-L1 antibody, confirming the above mentioned findings. In addition, the induction of PD-L1 expression decreased the proliferative capacity of the cocultured T cells but did not affect the apoptosis rate of T cells.ConclusionsTaken together, the results suggest that the H9N2 virus is able to inhibit the T cell immune response by upregulating PD-L1 expression in pulmonary microvascular endothelial cells.

Potential for elimination of SAR-CoV-2 through vaccination as inspired by elimination of multiple influenza viruses through natural pandemics or mass vaccination.

The ongoing pandemic of coronavirus disease 2019 (COVID‐19) caused by the novel virus severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) has claimed many lives worldwide. To combat the pandemic, multiple types of vaccines are under development with unprecedented rapidity. Theoretically, future vaccination against COVID‐19 may fall into long‐term costly guerrilla warfare between SARS‐CoV‐2 and humans. Elimination of SARS‐CoV‐2 through vaccination to avoid the potential long‐term costly guerrilla warfare, if possible, is highly desired and worth intensive consideration. Human influenza pandemics emerging in 1957, 1968, and 2009 established strong global herd immunity and led to the elimination of three human influenza viruses, which circulated worldwide for years before the pandemics. Moreover, both clade 7.2 of subtype H5 highly pathogenic avian influenza virus and subtype H7N9 avian influenza virus circulated in poultry in China for years, and they have been virtually eliminated through mass vaccination in recent years. These facts suggest that the rapid establishment of global herd immunity through mass vaccination using an appropriate vaccine could eliminate SARS‐CoV‐2. The coming 2 years are a golden time for elimination through vaccination, which requires tremendous national and international collaboration. This review also prioritizes the efficacy of vaccines for COVID‐19 and elucidates the importance of the development of more live vaccines for COVID‐19.

Co-circulation of multiple reassortant H6 subtype avian influenza viruses in wild birds in eastern China, 2016\u20132017

BackgroundH6 subtype influenza viruses were prevalent in domestic poultry and wild birds, which also could pose potential threat to humans. However, little is known about the prevalence of H6 subtype viruses in wild birds in eastern China, a crucial stopover or wintering site for migratory wild birds along the East Asian-Australasian Flyway.MethodsDuring the routine surveillance in 2016–2017, H6 subtype AIVs positive samples were identified, and the representative strains were selected for further sequence and phylogenetic analysis and the pathogenicity in mice were evaluated.ResultsAmong the 30 H6 positive samples, there were at least four subtypes H6N1, H6N2, H6N5 and H6N8 co-circulated in Shanghai, China. Genetic analysis showed the 8 representative isolates shared homology with different AIV sub-lineages isolated from domestic ducks or wild birds in different countries along the East Asian-Australasian flyways, and were classified into 7 new genotypes. The pathogenicity to mice showed that these H6 viruses could replicate efficiently in the lungs without prior adaptation, but could not cause mice death.ConclusionsEight novel strains belonged to H6N1, H6N2, H6N5 and H6N8 subtypes were isolated. Phylogenetic analyses revealed multiple origins of internal genes indicative of robust reassortment events and frequent wild birds-poultry interaction encouraging the evolution and emergence of new genotypes. The pathogenicity to mammals should be closely monitored to prevent the emergence of novel pandemic viruses.

Heterosubtypic Protection Induced by a Live Attenuated Influenza Virus Vaccine Expressing Galactose-α-1,3-Galactose Epitopes in Infected Cells.

Anti-galactose-α-1,3-galactose (anti-α-Gal) antibody is naturally expressed at a high level in humans. It constitutes about 1% of immunoglobulins found in human blood. Here, we designed a live attenuated influenza virus vaccine that can generate α-Gal epitopes in infected cells in order to facilitate opsonization of infected cells, thereby enhancing vaccine-induced immune responses. In the presence of normal human sera, cells infected with this mutant can enhance phagocytosis of human macrophages and cytotoxicity of NK cells in vitro Using a knockout mouse strain that allows expression of anti-α-Gal antibody in vivo, we showed that this strategy can increase vaccine immunogenicity and the breadth of protection. This vaccine can induce 100% protection against a lethal heterosubtypic group 1 (H5) or group 2 (mouse-adapted H3) influenza virus challenge in the mouse model. In contrast, its heterosubtypic protective effect in wild-type or knockout mice that do not have anti-α-Gal antibody expression is only partial, demonstrating that the enhanced vaccine-induced protection requires anti-α-Gal antibody upon vaccination. Anti-α-Gal-expressing knockout mice immunized with this vaccine produce robust humoral and cell-mediated responses upon a lethal virus challenge. This vaccine can stimulate CD11blo/- pulmonary dendritic cells, which are known to be crucial for clearance of influenza virus. Our approach provides a novel strategy for developing next-generation influenza virus vaccines.IMPORTANCE Influenza A viruses have multiple HA subtypes that are antigenically diverse. Classical influenza virus vaccines are subtype specific, and they cannot induce satisfactory heterosubtypic immunity against multiple influenza virus subtypes. Here, we developed a live attenuated H1N1 influenza virus vaccine that allows the expression of α-Gal epitopes by infected cells. Anti-α-Gal antibody is naturally produced by humans. In the presence of this antibody, human cells infected with this experimental vaccine virus can enhance several antibody-mediated immune responses in vitro Importantly, mice expressing anti-α-Gal antibody in vivo can be fully protected by this H1N1 vaccine against a lethal H5 or H3 virus challenge. Our work demonstrates a new strategy for using a single influenza virus strain to induce broadly cross-reactive immune responses against different influenza virus subtypes.

Application of volumetric absorptive microsampling (VAMS) to measure multidimensional anti-influenza IgG antibodies by the mPlex-Flu assay.

Introduction: Recently, volumetric absorptive microsampling (VAMS) has been used for accurate sampling of a fixed peripheral blood volume (10 µL) on a volumetric swab, and long-term sample storage. The mPlex-Flu assay is a novel, high-throughput assay that simultaneously measures the concentration of antibodies against the hemagglutinin (HA) proteins from multiple influenza virus strains with ≤5 µL of serum. Here we describe combining these two methods to measure multidimensional anti-influenza IgG activity in whole blood samples collected by a finger stick and VAMS, with correction for serum volume based on simultaneous hemoglobin measurement. Methods: We compared capillary blood samples obtained from a finger stick using a VAMS device with serum samples collected by traditional phlebotomy from 20 subjects, with the influenza antibody profiles measured by the mPlex-Flu assay. Results: We found that results with the two sampling methods were highly correlated within subjects and across all influenza strains (mean R 2 = 0.9470). Adjustment for serum volume, based on hemaglobin measurement, was used to estimate serum volume of samples and improved the accuracy. IgG measurements were stable over 3 weeks when VAMS samples were stored at room temperature or transported using a variety of shipping methods. Additionally, when volunteers performed finger-stick VAMS at-home by themselves, the comparison results of anti-HA antibody concentrations were highly consistent with sampling performed by study personnel on-site (R 2 = 0.9496). Conclusions: This novel approach can provide a simple, accurate, and low-cost means for monitoring the IgG anti-influenza HA antibody responses in large population studies and clinical trials.

Rapid and simple colorimetric detection of multiple influenza viruses infecting humans using a reverse transcriptional loop-mediated isothermal amplification (RT-LAMP) diagnostic platform

BackgroundIn addition to seasonal influenza viruses recently circulating in humans, avian influenza viruses (AIVs) of H5N1, H5N6 and H7N9 subtypes have also emerged and demonstrated human infection abilities with high mortality rates. Although influenza viral infections are usually diagnosed using viral isolation and serological/molecular analyses, the cost, accessibility, and availability of these methods may limit their utility in various settings. The objective of this study was to develop and optimized a multiplex detection system for most influenza viruses currently infecting humans.MethodsWe developed and optimized a multiplex detection system for most influenza viruses currently infecting humans including two type B (both Victoria lineages and Yamagata lineages), H1N1, H3N2, H5N1, H5N6, and H7N9 using Reverse Transcriptional Loop-mediated Isothermal Amplification (RT-LAMP) technology coupled with a one-pot colorimetric visualization system to facilitate direct determination of results without additional steps. We also evaluated this multiplex RT-LAMP for clinical use using a total of 135 clinical and spiked samples (91 influenza viruses and 44 other human infectious viruses).ResultsWe achieved rapid detection of seasonal influenza viruses (H1N1, H3N2, and Type B) and avian influenza viruses (H5N1, H5N6, H5N8 and H7N9) within an hour. The assay could detect influenza viruses with high sensitivity (i.e., from 100 to 0.1 viral genome copies), comparable to conventional RT-PCR-based approaches which would typically take several hours and require expensive equipment. This assay was capable of specifically detecting each influenza virus (Type B, H1N1, H3N2, H5N1, H5N6, H5N8 and H7N9) without cross-reactivity with other subtypes of AIVs or other human infectious viruses. Furthermore, 91 clinical and spiked samples confirmed by qRT-PCR were also detected by this multiplex RT-LAMP with 98.9% agreement. It was more sensitive than one-step RT-PCR approach (92.3%).ConclusionsResults of this study suggest that our multiplex RT-LAMP assay may provide a rapid, sensitive, cost-effective, and reliable diagnostic method for identifying recent influenza viruses infecting humans, especially in locations without access to large platforms or sophisticated equipment.

Multiple Influenza Virus Infections in 4 Consecutive Epidemiological Seasons: A Retrospective Study in Children and Adolescents.

BackgroundRecent observations provide evidence for group-specific immunity toward influenza A infections and raise the question of how often we can get the flu.MethodsWe retrospectively analyzed 2308 cases of children and adolescents with clinically manifested influenza and a positive PCR-test during the last 4 epidemiological seasons (2014–15 through 2017–18).ResultsIn the 2015–16 epidemiological season, almost 12% of patients had experienced an influenza infection during the previous season; in the 2016–17 season, more than 14% had at least 1 infection during the previous 2 seasons, and in 2017–18 season, over 18% had 1 or more infections during the previous 3 seasons. The majority of these repetitive infections occurred in children between 3–8 years of age. 29 patients experienced 3 or 4 infections during these seasons, whereas 38 children had 2 influenza episodes within the same season. Epidemiological pattern of circulating viral strains changed yearly; however, we identified 5 patients with confirmed influenza B infections during the 2014–15 and 2017–18 seasons, when only subtype Yamagata was circulating in Austria.ConclusionsRepetitive influenza infections in consecutive epidemiological seasons occurred quite frequently in children and adolescents. Observations like ours contribute to a better understanding of the immunity against influenza virus infections and could have implications for future vaccination strategies.

Spirostaphylotrichin X from a Marine-Derived Fungus as an Anti-influenza Agent Targeting RNA Polymerase PB2.

A new spirocyclic γ-lactam, named spirostaphylotrichin X (1), and three related known spirostaphylotrichins (2-4) were isolated from the marine-derived fungus Cochliobolus lunatus SCSIO41401. Their structures were determined by spectroscopic analyses. Spirostaphylotrichin X (1) displayed obvious inhibitory activities against multiple influenza virus strains, with IC50 values from 1.2 to 5.5 μM. Investigation of the mechanism showed that 1 inhibited viral polymerase activity and interfered with the production of progeny viral RNA. Homogeneous time-resolved fluorescence, surface plasmon resonance assays, and a molecular docking study revealed that 1 could inhibit polymerase PB2 protein activity by binding to the highly conserved region of the cap-binding domain of PB2. These results suggest that 1 inhibits the replication of influenza A virus by interfering with the activity of PB2 protein and that 1 represents a new type of potential lead compound for the development of anti-influenza therapeutics.

Broadly Reactive Human Monoclonal Antibodies Elicited following Pandemic H1N1 Influenza Virus Exposure Protect Mice against Highly Pathogenic H5N1 Challenge.

Broadly cross-reactive antibodies (Abs) that recognize conserved epitopes within the influenza virus hemagglutinin (HA) stalk domain are of particular interest for their potential use as therapeutic and prophylactic agents against multiple influenza virus subtypes, including zoonotic virus strains. Here, we characterized four human HA stalk-reactive monoclonal antibodies (MAbs) for their binding breadth and affinity, in vitro neutralization capacity, and in vivo protective potential against an highly pathogenic avian influenza virus. The monoclonal antibodies were isolated from individuals shortly following infection with (70-1F02 and 1009-3B05) or vaccination against (05-2G02 and 09-3A01) A(H1N1)pdm09. Three of the MAbs bound HAs from multiple strains of group 1 viruses, and one MAb, 05-2G02, bound to both group 1 and group 2 influenza A virus HAs. All four antibodies prophylactically protected mice against a lethal challenge with the highly pathogenic A/Vietnam/1203/04 (H5N1) strain. Two MAbs, 70-1F02 and 09-3A01, were further tested for their therapeutic efficacy against the same strain and showed good efficacy in this setting as well. One MAb, 70-1F02, cocrystallized with H5 HA and showed heavy-chain-only interactions similar to those seen with the previously described CR6261 anti-stalk antibody. Finally, we show that antibodies that compete with these MAbs are prevalent in serum from an individual recently infected with the A(H1N1)pdm09 virus. The antibodies described here can be developed into broad-spectrum antiviral therapeutics that could be used to combat infections by zoonotic or emerging pandemic influenza viruses.IMPORTANCE The rise in zoonotic infections of humans by emerging influenza viruses is a worldwide public health concern. The majority of recent zoonotic human influenza cases were caused by H7N9 and H5Nx viruses and were associated with high morbidity and mortality. In addition, seasonal influenza viruses are estimated to cause up to 650,000 deaths annually worldwide. Currently available antiviral treatment options include only neuraminidase inhibitors, but some influenza viruses are naturally resistant to these drugs, and others quickly develop resistance-conferring mutations. Alternative therapeutics are urgently needed. Broadly protective antibodies that target the conserved "stalk" domain of the hemagglutinin represent potential potent antiviral prophylactic and therapeutic agents that can assist pandemic preparedness. Here, we describe four human monoclonal antibodies that target conserved regions of influenza HA and characterize their binding spectrum as well as their protective capacity in prophylactic and therapeutic settings against a lethal challenge with a zoonotic influenza virus.

Overarching Immunodominance Patterns and Substantial Diversity in Specificity and Functionality in the Circulating Human Influenza A and B Virus-Specific CD4+ T-Cell Repertoire.

There is limited information on the antigen specificity and functional potential of the influenza virus-specific CD4+ T-cell repertoire in humans. Here, enzyme-linked immunospot assays were used to examine circulating CD4+ T-cell specificities for influenza virus directly ex vivo in healthy adults. Our studies revealed CD4+ T-cell reactivity to multiple influenza virus proteins, including hemagglutinins, neuraminidases, M1 proteins, and nucleoproteins. Unexpectedly, the immunodominance hierarchies and functional potential of cells reactive toward influenza A virus were distinct from those toward influenza B virus. We also identified influenza virus-specific cells producing granzyme B. Our findings revealed individual and virus-specific patterns that may differentially poise humans to respond to infection or vaccination.

Migratory birds in southern Brazil are a source of multiple avian influenza virus subtypes.

BackgroundThere is insufficient knowledge about the relation of avian influenza virus (AIV) to migratory birds in South America. Accordingly, we studied samples obtained over a 4‐year period (2009‐2012) from wild birds at a major wintering site in southern Brazil.MethodsWe obtained 1212 oropharyngeal/cloacal samples from wild birds at Lagoa do Peixe National Park and screened them for influenza A virus by RT‐PCR amplification of the matrix gene. Virus isolates were subjected to genomic sequencing and antigenic characterization.ResultsForty‐eight samples of 1212 (3.96%) contained detectable influenza virus RNA. Partial viral sequences were obtained from 12 of these samples, showing the presence of H2N2 (1), H6Nx (1), H6N1 (8), H9N2 (1), and H12N5 (1) viruses. As H6 viruses predominated, we generated complete genomes from all 9 H6 viruses. Phylogenetic analyses showed that they were most similar to viruses of South American lineage. The H6N1 viruses caused no disease signs in infected ferrets and, despite genetic differences, were antigenically similar to North American isolates.ConclusionsLagoa do Peixe National Park is a source of multiple AIV subtypes, with the levels of influenza virus in birds being highest at the end of their wintering period in this region. H6N1 viruses were the predominant subtype identified. These viruses were more similar to viruses of South American lineage than to those of North American lineage.

Rapid detection and subtyping of multiple influenza viruses on a microfluidic chip integrated with controllable micro-magnetic field

Influenza viruses have threatened animals and public health systems continuously. Moreover, there are many subtypes of influenza viruses, which have brought great difficulties to the classification of influenza viruses during any influenza outbreak. So it is crucial to develop a rapid and accurate method for detecting and subtyping influenza viruses. In this work, we reported a rapid method for simultaneously detecting and subtyping multiple influenza viruses (H1N1, H3N2 and H9N2) based on nucleic acid hybridization on a microfluidic chip integrated with controllable micro-magnetic field. H1N1, H3N2 and H9N2 could be simultaneously detected in 80min with detection limits about 0.21nM, 0.16nM, 0.12nM in order. Moreover, the sample and reagent consumption was as low as only 3μL. The results indicated that this approach possessed fast analysis and high specificity. Therefore, it is expected to be used to simultaneously subtype and detect multiple targets, and may provide a powerful technique platform for the rapid detection and subtyping analysis of influenza viruses.