Optimisations and Challenges Involved in the Creation of Various Bioluminescent and Fluorescent Influenza A Virus Strains for In Vitro and In Vivo Applications

Bioluminescent and fluorescent influenza A viruses offer new opportunities to study influenza virus replication, tropism and pathogenesis. To date, several influenza A reporter viruses have been described. These strategies typically focused on a single reporter gene (either bioluminescent or fluorescent) in a single virus backbone. However, whilst bioluminescence is suited to in vivo imaging, fluorescent viruses are more appropriate for microscopy. Therefore, the idea l reporter virus varies depending on the experiment in question, and it is important that any reporter virus strategy can be adapted accordingly. Herein, a strategy was developed to create five different reporter viruses in a single virus backbone. Specifically, enhanced green fluorescent protein (eGFP), far-red fluorescent protein (fRFP), near-infrared fluorescent protein (iRFP), Gaussia luciferase (gLUC) and firefly luciferase (fLUC) were inserted into the PA gene segment of A/PR/8/34 (H1N1). This study provides a comprehensive characterisation of the effects of different reporter genes on influenza virus replication and reporter activity. In vivo reporter gene expression, in lung tissues, was only detected for eGFP, fRFP and gLUC expressing viruses. In vitro, the eGFP-expressing virus displayed the best reporter stability and could be used for correlative light electron microscopy (CLEM). This strategy was then used to create eGFP-expressing viruses consisting entirely of pandemic H1N1, highly pathogenic avian influenza (HPAI) H5N1 and H7N9. The HPAI H5N1 eGFP-expressing virus infected mice and reporter gene expression was detected, in lung tissues, in vivo. Thus, this study provides new tools and insights for the creation of bioluminescent and fluorescent influenza A reporter viruses.     

PDlim2 selectively interacts with the PDZ binding motif of highly pathogenic avian H5N1 influenza A virus NS1

The multi-functional NS1 protein of influenza A virus is a viral virulence determining factor. The last four residues at the C-terminus of NS1 constitute a type I PDZ domain binding motif (PBM). Avian influenza viruses currently in circulation carry an NS1 PBM with consensus sequence ESEV, whereas human influenza viruses bear an NS1 PBM with consensus sequence RSKV or RSEV. The PBM sequence of the influenza A virus NS1 is reported to contribute to high viral pathogenicity in animal studies. Here, we report the identification of PDlim2 as a novel binding target of the highly pathogenic avian influenza virus H5N1 strain with an NS1 PBM of ESEV (A/Chicken/Henan/12/2004/H5N1, HN12-NS1) by yeast two-hybrid screening. The interaction was confirmed by in vitro GST pull-down assays, as well as by in vivo mammalian two-hybrid assays and bimolecular fluorescence complementation assays. The binding was also confirmed to be mediated by the interaction of the PDlim2 PDZ domain with the NS1 PBM motif. Interestingly, our assays showed that PDlim2 bound specifically with HN12-NS1, but exhibited no binding to NS1 from a human influenza H1N1 virus bearing an RSEV PBM (A/Puerto Rico/8/34/H1N1, PR8-NS1). A crystal structure of the PDlim2 PDZ domain fused with the C-terminal hexapeptide from HN12-NS1, together with GST pull-down assays on PDlim2 mutants, reveals that residues Arg16 and Lys31 of PDlim2 are critical for the binding between PDlim2 and HN12-NS1. The identification of a selective binding target of HN12-NS1 (ESEV), but not PR8-NS1 (RSEV), enables us to propose a structural mechanism for the interaction between NS1 PBM and PDlim2 or other PDZ-containing proteins.     

A Versatile Vector for In Vivo Monitoring of Type I Interferon Induction and Signaling

Development of reporter systems for in vivo examination of IFN-β induction or signaling of type I interferon (IFN-I) pathways is of great interest in order to characterize biological responses to different inducers such as viral infections. Several reporter mice have been developed to monitor the induction of both pathways in response to different agonists. However, alternative strategies that do not require transgenic mice breeding have to date not been reported. In addition, detection of these pathways in vivo in animal species other than mice has not yet been addressed. Herein we describe a simple method based on the use of an adeno-associated viral vector (AAV8-3xIRF-ISRE-Luc) containing an IFN-β induction and signaling-sensitive promoter sequence controlling the expression of the reporter gene luciferase. This vector is valid for monitoring IFN-I responses in vivo elicited by diverse stimuli in different organs. Intravenous administration of the vector in C57BL/6 mice and Syrian hamsters was able to detect activation of the IFN pathway in the liver upon systemic treatment with different pro-inflammatory agents and infection with Newcastle disease virus (NDV). In addition, intranasal instillation of AAV8-3xIRF-ISRE-Luc showed a rapid and transient IFN-I response in the respiratory tract of mice infected with the influenza A/PR8/34 virus lacking the NS1 protein. In comparison, this response was delayed and exacerbated in mice infected with influenza A/PR/8 wild type virus. In conclusion, the AAV8-3xIRF-ISRE-Luc vector offers the possibility of detecting IFN-I activation in response to different stimuli and in different animal models with no need for reporter transgenic animals.     

Differential Targeting of Viral Components by CD4+ versus CD8+ T Lymphocytes in Dengue Virus Infection

Dengue virus (DENV) is the principal arthropod-borne viral pathogen afflicting human populations. While repertoires of antibodies to DENV have been linked to protection or enhanced infection, the role of T lymphocytes in these processes remains poorly defined. This study provides a comprehensive overview of CD4⁺ and CD8⁺ T cell epitope reactivities against the DENV 2 proteome in adult patients experiencing secondary DENV infection. Dengue virus-specific T cell responses directed against an overlapping 15mer peptide library spanning the DENV 2 proteome were analyzed ex vivo by enzyme-linked immunosorbent spot assay, and recognition of individual peptides was further characterized in specific T cell lines. Thirty novel T cell epitopes were identified, 9 of which are CD4⁺ and 21 are CD8⁺ T cell epitopes. We observe that whereas CD8⁺ T cell epitopes preferentially target nonstructural proteins (NS3 and NS5), CD4⁺ epitopes are skewed toward recognition of viral components that are also targeted by B lymphocytes (envelope, capsid, and NS1). Consistently, a large proportion of dengue virus-specific CD4⁺ T cells have phenotypic characteristics of circulating follicular helper T cells (CXCR5 expression and production of interleukin-21 or gamma interferon), suggesting that they are interacting with B cells in vivo. This study shows that during a dengue virus infection, the protein targets of human CD4⁺ and CD8⁺ T cells are largely distinct, thus highlighting key differences in the immunodominance of DENV proteins for these two cell types. This has important implications for our understanding of how the two arms of the human adaptive immune system are differentially targeted and employed as part of our response to DENV infection.     

Recombinant Influenza Virus Carrying the Respiratory Syncytial Virus (RSV) F85-93 CTL Epitope Reduces RSV Replication in Mice

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in infants worldwide. Despite decades of research, there is still no registered vaccine available for this major pathogen. We investigated the protective efficacy of a recombinant influenza virus, PR8/NA-F_85–93, that carries the RSV CD8⁺ T cell epitope F_85–93 in its neuraminidase stalk. F_85–93-specific cytotoxic T lymphocytes (CTLs) were induced in mice after a single intranasal immunization with PR8/NA-F_85-93 virus, and these CTLs provided a significant reduction in the lung viral load upon a subsequent challenge with RSV. To avoid influenza-induced morbidity, we treated mice with matrix protein 2 (M2e)-specific monoclonal antibodies before PR8/NA-F_85-93 virus infection. Treatment with anti-M2e antibodies reduced the infiltration of immune cells in the lungs upon PR8/NA-F_85-93 infection, whereas the formation of inducible bronchus-associated lymphoid tissue was not affected. Moreover, this treatment prevented body weight loss yet still permitted the induction of RSV F-specific T cell responses and significantly reduced RSV replication upon challenge. These results demonstrate that it is possible to take advantage of the infection-permissive protection of M2e-specific antibodies against influenza A virus to induce heterologous CD8⁺ T cell-mediated immunity by an influenza A virus vector expressing the RSV F_85-93 epitope.     

Transfectant Influenza A Viruses with Long Deletions in the NS1 Protein Grow Efficiently in Vero Cells

We established a reverse genetics system for the nonstructural (NS) gene segment of influenza A virus. This system is based on the use of the temperature-sensitive (ts) reassortant virus 25A-1. The 25A-1 virus contains the NS gene from influenza A/Leningrad/134/57 virus and the remaining gene segments from A/Puerto Rico (PR)/8/34 virus. This particular gene constellation was found to be responsible for the ts phenotype. For reverse genetics of the NS gene, a plasmid-derived NS gene from influenza A/PR/8/34 virus was ribonucleoprotein transfected into cells that were previously infected with the 25A-1 virus. Two subsequent passages of the transfection supernatant at 40°C selected viruses containing the transfected NS gene derived from A/PR/8/34 virus. The high efficiency of the selection process permitted the rescue of transfectant viruses with large deletions of the C-terminal part of the NS1 protein. Viable transfectant viruses containing the N-terminal 124, 80, or 38 amino acids of the NS1 protein were obtained. Whereas all deletion mutants grew to high titers in Vero cells, growth on Madin-Darby canine kidney (MDCK) cells and replication in mice decreased with increasing length of the deletions. In Vero cells expression levels of viral proteins of the deletion mutants were similar to those of the wild type. In contrast, in MDCK cells the level of the M1 protein was significantly reduced for the deletion mutants.     

Visualizing the Beta Interferon Response in Mice during Infection with Influenza A Viruses Expressing or Lacking Nonstructural Protein 1

The innate host defense against influenza virus is largely dependent on the type I interferon (IFN) system. However, surprisingly little is known about the cellular source of IFN in the infected lung. To clarify this question, we employed a reporter mouse that contains the firefly luciferase gene in place of the IFN-β-coding region. IFN-β-producing cells were identified either by simultaneous immunostaining of lungs for luciferase and cellular markers or by generating conditional reporter mice that express luciferase exclusively in defined cell types. Two different strains of influenza A virus were employed that either do or do not code for nonstructural protein 1 (NS1), which strongly suppresses innate immune responses of infected cells. We found that epithelial cells and lung macrophages, which represent the prime host cells for influenza viruses, showed vigorous IFN-β responses which, however, were severely reduced and delayed if the infecting virus was able to produce NS1. Interestingly, CD11c⁺ cell populations that were either expressing or lacking macrophage markers produced the bulk of IFN-β at 48 h after infection with wild-type influenza A virus. Our results demonstrate that the virus-encoded IFN-antagonistic factor NS1 disarms specifically epithelial cells and lung macrophages, which otherwise would serve as main mediators of the early response against infection by influenza virus.     

Human T Lymphotropic Virus Type 1 SU Residue 195 Plays a Role in Determining the Preferential CD4+ T Cell Immortalization/Transformation Tropism

Human T lymphotropic virus type 1 (HTLV-1) mainly causes adult T cell leukemia and predominantly immortalizes/transforms CD4⁺ T cells in culture. HTLV-2 is aleukemic and predominantly immortalizes/transforms CD8⁺ T cells in culture. We have shown previously that the viral envelope is the genetic determinant of the differential T cell tropism in culture. The surface component (SU) of the HTLV-1 envelope is responsible for binding to the cellular receptors for entry. Here, we dissect the HTLV-1 SU further to identify key domains that are involved in determining the immortalization tropism. We generated HTLV-1 envelope recombinant virus containing the HTLV-2 SU domain. HTLV-1/SU2 was capable of infecting and immortalizing freshly isolated peripheral blood mononuclear cells in culture. HTLV-1/SU2 shifted the CD4⁺ T cell immortalization tropism of wild-type HTLV-1 (wtHTLV-1) to a CD8⁺ T cell preference. Furthermore, a single amino acid substitution, N195D, in HTLV-1 SU (Ach.195) resulted in a shift to a CD8⁺ T cell immortalization tropism preference. Longitudinal phenotyping analyses of the in vitro transformation process revealed that CD4⁺ T cells emerged as the predominant population by week 5 in wtHTLV-1 cultures, while CD8⁺ T cells emerged as the predominant population by weeks 4 and 7 in wtHTLV-2 and Ach.195 cultures, respectively. Our results indicate that SU domain independently influences the preferential T cell immortalization tropism irrespective of the envelope counterpart transmembrane (TM) domain. We further showed that asparagine at position 195 in HTLV-1 SU is involved in determining this CD4⁺ T cell immortalization tropism. The slower emergence of the CD8⁺ T cell predominance in Ach.195-infected cultures suggests that other residues/domains contribute to this tropism preference.     

Low Seroprevalent Species D Adenovirus Vectors as Influenza Vaccines

Seasonal and pandemic influenza remains a constant threat. While standard influenza vaccines have great utility, the need for improved vaccine technologies have been brought to light by the 2009 swine flu pandemic, highly pathogenic avian influenza infections, and the most recent early and widespread influenza activity. Species C adenoviruses based on serotype 5 (AD5) are potent vehicles for gene-based vaccination. While potent, most humans are already immune to this virus. In this study, low seroprevalent species D adenoviruses Ad26, 28, and 48 were cloned and modified to express the influenza virus A/PR/8/34 hemagglutinin gene for vaccine studies. When studied in vivo, these species D Ad vectors performed quite differently as compared to species C Ad vectors depending on the route of immunization. By intramuscular injection, species D vaccines were markedly weaker than species C vaccines. In contrast, the species D vaccines were equally efficient as species C when delivered mucosally by the intranasal route. Intranasal adenovirus vaccine doses as low as 10⁸ virus particles per mouse induced complete protection against a stringent lethal challenge dose of influenza. These data support translation of species D adenoviruses as mucosal vaccines and highlight the fundamental effects of differences in virus tropism on vaccine applications.     

HLA-A*11:01-restricted CD8+ T cell immunity against influenza A and influenza B viruses in Indigenous and non-Indigenous people

HLA-A*11:01 is one of the most prevalent human leukocyte antigens (HLAs), especially in East Asian and Oceanian populations. It is also highly expressed in Indigenous people who are at high risk of severe influenza disease. As CD8⁺ T cells can provide broadly cross-reactive immunity to distinct influenza strains and subtypes, including influenza A, B and C viruses, understanding CD8⁺ T cell immunity to influenza viruses across prominent HLA types is needed to rationally design a universal influenza vaccine and generate protective immunity especially for high-risk populations. As only a handful of HLA-A*11:01-restricted CD8⁺ T cell epitopes have been described for influenza A viruses (IAVs) and epitopes for influenza B viruses (IBVs) were still unknown, we embarked on an epitope discovery study to define a CD8⁺ T cell landscape for HLA-A*11:01-expressing Indigenous and non-Indigenous Australian people. Using mass-spectrometry, we identified IAV- and IBV-derived peptides presented by HLA-A*11:01 during infection. 79 IAV and 57 IBV peptides were subsequently screened for immunogenicity in vitro with peripheral blood mononuclear cells from HLA-A*11:01-expressing Indigenous and non-Indigenous Australian donors. CD8⁺ T cell immunogenicity screening revealed two immunogenic IAV epitopes (A11/PB2_320-331 and A11/PB2_323-331) and the first HLA-A*11:01-restricted IBV epitopes (A11/M_41-49, A11/NS1_186-195 and A11/NP_511-520). The immunogenic IAV- and IBV-derived peptides were >90% conserved among their respective influenza viruses. Identification of novel immunogenic HLA-A*11:01-restricted CD8⁺ T cell epitopes has implications for understanding how CD8⁺ T cell immunity is generated towards IAVs and IBVs. These findings can inform the development of rationally designed, broadly cross-reactive influenza vaccines to ensure protection from severe influenza disease in HLA-A*11:01-expressing individuals.     

Multi-dose Romidepsin Reactivates Replication Competent SIV in Post-antiretroviral Rhesus Macaque Controllers

Viruses that persist despite seemingly effective antiretroviral treatment (ART) and can reinitiate infection if treatment is stopped preclude definitive treatment of HIV-1 infected individuals, requiring lifelong ART. Among strategies proposed for targeting these viral reservoirs, the premise of the “shock and kill” strategy is to induce expression of latent proviruses [for example with histone deacetylase inhibitors (HDACis)] resulting in elimination of the affected cells through viral cytolysis or immune clearance mechanisms. Yet, ex vivo studies reported that HDACis have variable efficacy for reactivating latent proviruses, and hinder immune functions. We developed a nonhuman primate model of post-treatment control of SIV through early and prolonged administration of ART and performed in vivo reactivation experiments in controller RMs, evaluating the ability of the HDACi romidepsin (RMD) to reactivate SIV and the impact of RMD treatment on SIV-specific T cell responses. Ten RMs were IV-infected with a SIVsmmFTq transmitted-founder infectious molecular clone. Four RMs received conventional ART for >9 months, starting from 65 days post-infection. SIVsmmFTq plasma viremia was robustly controlled to <10 SIV RNA copies/mL with ART, without viral blips. At ART cessation, initial rebound viremia to ~10⁶ copies/mL was followed by a decline to < 10 copies/mL, suggesting effective immune control. Three post-treatment controller RMs received three doses of RMD every 35–50 days, followed by in vivo experimental depletion of CD8⁺ cells using monoclonal antibody M-T807R1. RMD was well-tolerated and resulted in a rapid and massive surge in T cell activation, as well as significant virus rebounds (~10⁴ copies/ml) peaking at 5–12 days post-treatment. CD8⁺ cell depletion resulted in a more robust viral rebound (10⁷ copies/ml) that was controlled upon CD8⁺ T cell recovery. Our results show that RMD can reactivate SIV in vivo in the setting of post-ART viral control. Comparison of the patterns of virus rebound after RMD administration and CD8⁺ cell depletion suggested that RMD impact on T cells is only transient and does not irreversibly alter the ability of SIV-specific T cells to control the reactivated virus.     

Coadministration of Hedera helix L. Extract Enabled Mice to Overcome Insufficient Protection against Influenza A/PR/8 Virus Infection under Suboptimal Treatment with Oseltamivir

Several anti-influenza drugs that reduce disease manifestation exist, and although these drugs provide clinical benefits in infected patients, their efficacy is limited by the emergence of drug-resistant influenza viruses. In the current study, we assessed the therapeutic strategy of enhancing the antiviral efficacy of an existing neuraminidase inhibitor, oseltamivir, by coadministering with the leaf extract from Hedera helix L, commonly known as ivy. Ivy extract has anti-inflammatory, antibacterial, antifungal, and antihelminthic properties. In the present study, we investigated its potential antiviral properties against influenza A/PR/8 (PR8) virus in a mouse model with suboptimal oseltamivir that mimics a poor clinical response to antiviral drug treatment. Suboptimal oseltamivir resulted in insufficient protection against PR8 infection. Oral administration of ivy extract with suboptimal oseltamivir increased the antiviral activity of oseltamivir. Ivy extract and its compounds, particularly hedrasaponin F, significantly reduced the cytopathic effect in PR8-infected A549 cells in the presence of oseltamivir. Compared with oseltamivir treatment alone, coadministration of the fraction of ivy extract that contained the highest proportion of hedrasaponin F with oseltamivir decreased pulmonary inflammation in PR8-infected mice. Inflammatory cytokines and chemokines, including tumor necrosis factor-alpha and chemokine (C-C motif) ligand 2, were reduced by treatment with oseltamivir and the fraction of ivy extract. Analysis of inflammatory cell infiltration in the bronchial alveolar of PR8-infected mice revealed that CD11b⁺Ly6G⁺ and CD11b⁺Ly6C^int cells were recruited after virus infection; coadministration of the ivy extract fraction with oseltamivir reduced infiltration of these inflammatory cells. In a model of suboptimal oseltamivir treatment, coadministration of ivy extract fraction that includes hedrasaponin F increased protection against PR8 infection that could be explained by its antiviral and anti-inflammatory activities.     

Human Cytotoxic T Lymphocytes Directed to Seasonal Influenza A Viruses Cross-React with the Newly Emerging H7N9 Virus

In February 2013, zoonotic transmission of a novel influenza A virus of the H7N9 subtype was reported in China. Although at present no sustained human-to-human transmission has been reported, a pandemic outbreak of this H7N9 virus is feared. Since neutralizing antibodies to the hemagglutinin (HA) globular head domain of the virus are virtually absent in the human population, there is interest in identifying other correlates of protection, such as cross-reactive CD8⁺ T cells (cytotoxic T lymphocytes [CTLs]) elicited during seasonal influenza A virus infections. These virus-specific CD8⁺ T cells are known to recognize conserved internal proteins of influenza A viruses predominantly, but it is unknown to what extent they cross-react with the newly emerging H7N9 virus. Here, we assessed the cross-reactivity of seasonal H3N2 and H1N1 and pandemic H1N1 influenza A virus-specific polyclonal CD8⁺ T cells, obtained from HLA-typed study subjects, with the novel H7N9 virus. The cross-reactivity of CD8⁺ T cells to H7N9 variants of known influenza A virus epitopes and H7N9 virus-infected cells was determined by their gamma interferon (IFN-γ) response and lytic activity. It was concluded that, apart from recognition of individual H7N9 variant epitopes, CD8⁺ T cells to seasonal influenza viruses display considerable cross-reactivity with the novel H7N9 virus. The presence of these cross-reactive CD8⁺ T cells may afford some protection against infection with the new virus.     

Filament-Producing Mutants of Influenza A/Puerto Rico/8/1934 (H1N1) Virus Have Higher Neuraminidase Activities than the Spherical Wild-Type

Influenza virus exhibits two morphologies – spherical and filamentous. Strains that have been grown extensively in laboratory substrates are comprised predominantly of spherical virions while clinical or low passage isolates produce a mixture of spheres and filamentous virions of varying lengths. The filamentous morphology can be lost upon continued passage in embryonated chicken eggs, a common laboratory substrate for influenza viruses. The fact that the filamentous morphology is maintained in nature but lost in favor of a spherical morphology in ovo suggests that filaments confer a selective advantage within the infected host that is not necessary for growth in laboratory substrates. Indeed, we have recently shown that filament-producing variant viruses are selected upon passage of the spherical laboratory strain A/Puerto Rico/8/1934 (H1N1) [PR8] in guinea pigs. Toward determining the nature of the selective advantage conferred by filaments, we sought to identify functional differences between spherical and filamentous particles. We compared the wild-type PR8 virus to two previously characterized recombinant PR8 viruses in which single point mutations within M1 confer a filamentous morphology. Our results indicate that these filamentous PR8 mutants have higher neuraminidase activities than the spherical PR8 virus. Conversely, no differences were observed in HAU:PFU or HAU:RNA ratios, binding avidity, sensitivity to immune serum in hemagglutination inhibition assays, or virion stability at elevated temperatures. Based on these results, we propose that the pleomorphic nature of influenza virus particles is important for the optimization of neuraminidase functions in vivo.     

Induction and Role of Indoleamine 2,3 Dioxygenase in Mouse Models of Influenza A Virus Infection

Influenza infection stimulates protective host immune responses but paradoxically enhances lung indoleamine 2,3 dioxygenase (IDO) activity, an enzyme that suppresses helper/effector T cells and activates Foxp3-lineage regulatory CD4 T cells (Tregs). Influenza A/PR/8/34 (PR8) infection stimulated rapid elevation of IDO activity in lungs and lung-draining mediastinal lymph nodes (msLNs). Mice lacking intact IDO1 genes (IDO1-KO mice) exhibited significantly lower morbidity after sub-lethal PR8 infection, and genetic or pharmacologic IDO ablation led to much faster recovery after virus clearance. More robust influenza-specific effector CD8 T cell responses manifested in lungs of PR8-infected IDO1-KO mice, though virus clearance rates were unaffected by IDO ablation. Similar outcomes manifested in mice infected with a less virulent influenza A strain (X31). IDO induction in X31-infected lungs was dependent on IFN type II (IFNγ) signaling and was restricted to non-hematopoietic cells, while redundant IFN type 1 or type II signaling induced IDO exclusively in hematopoietic cells from msLNs. Memory T cells generated in X31-primed IDO1-KO mice protected mice from subsequent challenge with lethal doses of PR8 (100×LD₅₀). However recall T cell responses were less robust in lung interstitial tissues, and classic dominance of TCR Vβ8.3 chain usage amongst memory CD8⁺ T cells specific for influenza nucleoprotein (NP₃₆₆) did not manifest in IDO1-KO mice. Thus, influenza induced IDO activity in lungs enhanced morbidity, slowed recovery, restrained effector T cell responses in lungs and shaped memory T cell repertoire generation, but did not attenuate virus clearance during primary influenza A infection.     

NADPH Oxidase 1 Is Associated with Altered Host Survival and T Cell Phenotypes after Influenza A Virus Infection in Mice

The role of the reactive oxygen species-producing NADPH oxidase family of enzymes in the pathology of influenza A virus infection remains enigmatic. Previous reports implicated NADPH oxidase 2 in influenza A virus-induced inflammation. In contrast, NADPH oxidase 1 (Nox1) was reported to decrease inflammation in mice within 7 days post-influenza A virus infection. However, the effect of NADPH oxidase 1 on lethality and adaptive immunity after influenza A virus challenge has not been explored. Here we report improved survival and decreased morbidity in mice with catalytically inactive NADPH oxidase 1 (Nox1*^/Y) compared with controls after challenge with A/PR/8/34 influenza A virus. While changes in lung inflammation were not obvious between Nox1*^/Y and control mice, we observed alterations in the T cell response to influenza A virus by day 15 post-infection, including increased interleukin-7 receptor-expressing virus-specific CD8⁺ T cells in lungs and draining lymph nodes of Nox1*^/Y, and increased cytokine-producing T cells in lungs and spleen. Furthermore, a greater percentage of conventional and interstitial dendritic cells from Nox1*^/Y draining lymph nodes expressed the co-stimulatory ligand CD40 within 6 days post-infection. Results indicate that NADPH oxidase 1 modulates the innate and adaptive cellular immune response to influenza virus infection, while also playing a role in host survival. Results suggest that NADPH oxidase 1 inhibitors may be beneficial as adjunct therapeutics during acute influenza infection.     

A Single Amino Acid Substitution in the Novel H7N9 Influenza A Virus NS1 Protein Increases CPSF30 Binding and Virulence

Although an effective interferon antagonist in human and avian cells, the novel H7N9 influenza virus NS1 protein is defective at inhibiting CPSF30. An I106M substitution in H7N9 NS1 can restore CPSF30 binding together with the ability to block host gene expression. Furthermore, a recombinant virus expressing H7N9 NS1-I106M replicates to higher titers in vivo, and is subtly more virulent, than the parental virus. Natural polymorphisms in H7N9 NS1 that enhance CPSF30 binding may be cause for concern.     

A straightforward experimental approach to expression, purification, refolding, and enzymatic analysis of recombinant dengue virus NS2B(H)-NS3pro protease

Dengue virus threatens around 2.5 billion people worldwide; about 50 million become infected every year, and yet no vaccine or drug is available for prevention and/or treatment. The flaviviral NS2B-NS3pro complex is indispensable for flaviviral replication and is considered to be an important drug target. The aim of this study was to develop a simple and generally applicable experimental strategy to construct, purify, and assay a highly active recombinant NS2B(H)-NS3pro complex that would be useful for high-throughput screening of potential inhibitors. The sequence of NS2B(H)-NS3pro was generated by overlap extension PCR (SOE-PCR) and cloned into the pTrcHisA vector. Hexahistidine-tagged NS2B(H)-NS3pro complex was expressed in E. coli predominantly as insoluble protein and purified to >95% purity by single-step immobilized metal affinity chromatography. SDS-PAGE followed by immunoblotting of the purified enzyme demonstrated the presence of the NS2B(H)-NS3pro precursor and its autocleavage products, NS3pro and NS2B(H), as 37, 21, and 10 kDa bands, respectively. Kinetic parameters, K _m, k _cat, and k _cat/K _m for the fluorophore-linked protease model substrate Ac-nKRR-amc were obtained using inner-filter effect correction. The kinetic parameters K _m, k _cat, and k _cat/K _m for Ac-nKRR-amc substrate were 100 μM, 0.112 s^?1, and 1120 M^?1·s^?1, respectively. A simplified procedure for the cloning, overexpression, and purification of the NS2B(H)-NS3pro complex was applied, and a highly active recombinant NS2B(H)-NS3pro complex was obtained that could be useful for the design of high-throughput assays aimed at flaviviral inhibitor discovery.     

Imaging and characterizing influenza A virus mRNA transport in living cells

The mechanisms of influenza A virus mRNA intracellular transport are still not clearly understood. Here, we visualized the distribution and transport of influenza A virus mRNA in living cells using molecular beacon (MB) technology. Confocal-FRAP measurements determined that the transport of influenza A virus intronless mRNA, in both nucleus and cytoplasm, was energy dependent, being similar to that of Poly(A)⁺ RNA. Drug inhibition studies in living cells revealed that the export of influenza A virus mRNA is independent of the CRM1 pathway, while the function of RNA polymerase II (RNAP-II) may be needed. In addition, viral NS1 protein and cellular TAP protein were found associated with influenza A virus mRNA in the cell nucleus. These findings characterize influenza A virus mRNA transport in living cells and suggest that influenza A virus mRNA may be exported from the nucleus by the cellular TAP/p15 pathway with NS1 protein and RNAP-II participation.