Pathogenesis Of Influenza Virus Infection Research Articles

Experimental Animal Models for Influenza/Flu Virus Vaccine Development

Objectives: The objective of this review is to explore the different animal models used in the development of Influenza / Flu Virus Vaccine as well as some important guidelines adhered to it.
 Methods: Laboratory animal models are widely used in the preclinical evaluation of potential vaccines and antiviral compounds to investigate the safety and efficacy of the vaccine or compound in preventing or moderating infection, disease, or secondary transmission. Animal models must also represent humans in terms of similarity of clinical signs, histopathological changes, virus growth kinetics, or transmission. Common animal models for influenza virus vaccine testing include mice, ferrets, and non-human primates. These models help researchers assess vaccine efficacy, immune response, and potential side effects before advancing to human trials. Animal models play a crucial role in the development and testing of influenza vaccines, as they help researchers examine potential side effects and adverse reactions, evaluate the efficacy of vaccines, study the immune response elicited by vaccines, understand the pathogenesis of influenza virus infection, and predict the potential impact of emerging strains. They also help test various vaccine platforms and delivery methods, and compare the effectiveness of different vaccine formulations in animals to identify promising candidates for further human trials.
 Results: We have discussed in detail the various animal models such as Chick embryo model for the development of influenza virus vaccine and mice, ferrets, guinea pig and non-human primates etc. used for the evaluation of safety and therapeutic effectiveness.
 Conclusion: As we have discussed various animal models, their merits, demerits etc. for the evaluation of safety and effectiveness of influenza virus vaccine and expect that these will help for the young researchers to carry out their vaccine research using suitable animal models.
 Keywords: influenza virus, vaccine development, mouse models, ferret model, guinea pig model, cotton rat model

The Enigma of Lymphocyte Apoptosis in the Response to Influenza Virus Infection

In the pathogenesis of influenza virus infection, lymphocyte apoptosis as a part of the infection and/or the immune response to the virus can be somewhat puzzling. The percentage of human T lymphocytes within the peripheral blood mononuclear cell population that becomes apoptotic greatly exceeds the percentage that are infected after exposure to the virus, consistent with substantial apoptosis of bystander T lymphocytes. Studies reveal an important role of viral neuraminidase expression by co-cultured monocyte/macrophages in induction of apoptosis, including that of uninfected bystander lymphocytes. Despite these observations, it is a reasonable perspective to recognize that the development of lymphocyte apoptosis during the response to infection does not preclude a successful immune response and recovery of the infected host in the great majority of cases. Further investigation is clearly warranted to understand its role in the pathogenesis of influenza virus infection for human subjects.

Potential Role of Superoxide Dismutase 3 (SOD3) in Resistance to Influenza A Virus Infection.

Influenza A virus infection induces the production of excessive reactive oxygen species (ROS). Overproduction of ROS can overwhelm the antioxidant defense system, leading to increasing intensive oxidative stress. However, antioxidant defense against oxidative damage induced by influenza A virus infection, and in particular the significance of the SOD3 response in the pathogenesis of influenza virus infection, has not been well characterized. Here, we investigated the potential role of SOD3 in resistance to influenza A virus infection. In this study, SOD3, as an important antioxidant enzyme, was shown to be highly elevated in A549 cells following influenza A virus infection. Furthermore, inhibition of SOD3 impacted viral replication and virulence. We found that SOD3 disrupts IAV replication by impairing the synthesis of vRNA, whereas it did not affect viral ribonucleoprotein nuclear export. In addition, overexpression of SOD3 greatly reduced the levels of ROS caused by influenza A virus infection, regulated the inflammatory response to virus infection by inhibiting the phosphorylation of p65 of the NF-κB signaling pathway, and inhibited virus-induced apoptosis to a certain extent. Taken together, these findings indicate that SOD3 is actively involved in influenza A virus replication. Pharmacological modulation or targeting of SOD3 may pave the way for a novel therapeutic approach to combating influenza A virus infection.

The Roles of Ubiquitination in Pathogenesis of Influenza Virus Infection.

The ubiquitin system denotes a potent post-translational modification machinery that is capable of activation or deactivation of target proteins through reversible linkage of a single ubiquitin or ubiquitin chains. Ubiquitination regulates major cellular functions such as protein degradation, trafficking and signaling pathways, innate immune response, antiviral defense, and virus replication. The RNA sensor RIG-I ubiquitination is specifically induced by influenza A virus (IAV) to activate type I IFN production. Influenza virus modulates the activity of major antiviral proteins in the host cell to complete its full life cycle. Its structural and non-structural proteins, matrix proteins and the polymerase complex can regulate host immunity and antiviral response. The polymerase PB1-F2 of mutated 1918 IAV, adapts a novel IFN antagonist function by sending the DDX3 into proteasomal degradation. Ultimately the fate of virus is determined by the outcome of interplay between viral components and host antiviral proteins and ubiquitination has a central role in the encounter of virus and its host cell.

Preventive Effect of a Polyphenol-Rich Extract from Geranium sanguineum L. on Hepatic Drug Metabolism in Influenza Infected Mice

The decreased hepatic drug metabolism (predominately first phase) is one of the essential reasons for numerous side effects and for increased drug toxicity during influenza virus infection (IVI). The present study aims to investigate some mechanisms of the preventive effect of a standardized polyphenol complex from the medicinal plant Geranium sanguineum L. (PPhC) (10 mg/kg nasally). A verified experimental model of IVI A/Aichi/2/68 (H3N2) (4.5 lg LD50) in male ICR (Institute of Cancer Research, USA) mice was used. Changes in hepatic monooxygenase activities as well as nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome C reductase activity and cytochtome P450 content were studied on days 2, 6, 9, 21 of the infection together with thiobarbituric acid reactive substances in the liver supernatant. Our data clearly demonstrates that IVI affects all components of the electronic chain of cytochrome P-450. N-demethylases and hydroxylases as well as the activity of cytochrome C reductase and cytochtome P-450 content were decreased in the course of the virus infection. This implies that free radicals play an important role not only in the pathogenesis of IVI, but also in the modulation of the hepatic monooxygenase activity. This is also consistent with the established polyphenol complex PPhC from the medicinal plant Geranium sanguineum L. preventive effect against increased thiobarbituric acid reactive substances (TBARS)-levels. PPhC restored most of the monooxygenase activities that were inhibited in IVI animals, even over the control levels, probably via multiple mechanisms that may entail antioxidant activity and selective antiviral and protein-binding effects. In contrast to infected animals, in healthy mice, PPhC showed moderate reversible inhibitory effect on hepatic monooxygenase activities.

H9N2 swine influenza virus infection-induced damage is mediated by TRPM2 channels in mouse pulmonary microvascular endothelial cells

Oxidative stress is implicated in the pathogenesis of influenza virus infection. Increasing evidences show that transient receptor potential melastatin 2 (TRPM2), a Ca2+-permeable non-selective cation channel, plays an important role in the pathomechanism of reactive oxygen species (ROS)-coupled diseases. The present study investigated the role of TRPM2 in pulmonary microvascular endothelial cells (PMVECs) during H9N2 influenza virus infection. We knocked down TRPM2 in PMVECs using TRPM2 shRNA lentiviral particles. Subsequently, we utilized enzyme-linked immunosorbent assay and flow cytometry to compare ROS levels, DNA damage, mitochondrial integrity, apoptosis, and inflammatory factors between control and TRPM2-knockdown PMVECs following H9N2 influenza virus infection. Inhibition of TRPM2 channels reduced H9N2 virus-induced intracellular ROS production, decreased DNA damage, and inhibited H9N2-induced cellular apoptosis. This study shows that the inhibition of TRPM2 channels may protect PMVECs from the damage caused by H9N2 virus infection. Our results highlight the importance of TRPM2 in modulating ROS production, apoptosis, mitochondrial dysfunction, cytokine expression, and DNA damage in H9N2 virus-infected PMVECs, and suggest that TRPM2 may be a potential antiviral target.

The Influenza Virus H5N1 Infection Can Induce ROS Production for Viral Replication and Host Cell Death in A549 Cells Modulated by Human Cu/Zn Superoxide Dismutase (SOD1) Overexpression.

Highly pathogenic H5N1 infections are often accompanied by excessive pro-inflammatory response, high viral titer, and apoptosis; as such, the efficient control of these infections poses a great challenge. The pathogenesis of influenza virus infection is also related to oxidative stress. However, the role of endogenic genes with antioxidant effect in the control of influenza viruses, especially H5N1 viruses, should be further investigated. In this study, the H5N1 infection in lung epithelial cells decreased Cu/Zn superoxide dismutase (SOD1) expression at mRNA and protein levels. Forced SOD1 expression significantly inhibited the H5N1-induced increase in reactive oxygen species, decreased pro-inflammatory response, prevented p65 and p38 phosphorylation, and impeded viral ribonucleoprotein nuclear export and viral replication. The SOD1 overexpression also rescued H5N1-induced cellular apoptosis and alleviated H5N1-caused mitochondrial dysfunction. Therefore, this study described the role of SOD1 in the replication of H5N1 influenza virus and emphasized the relevance of this enzyme in the control of H5N1 replication in epithelial cells. Pharmacological modulation or targeting SOD1 may open a new way to fight H5N1 influenza virus.

Influenza virus and endothelial cells: a species specific relationship.

Influenza A virus (IAV) infection is an important cause of respiratory disease in humans. The original reservoirs of IAV are wild waterfowl and shorebirds, where virus infection causes limited, if any, disease. Both in humans and in wild waterbirds, epithelial cells are the main target of infection. However, influenza virus can spread from wild bird species to terrestrial poultry. Here, the virus can evolve into highly pathogenic avian influenza (HPAI). Part of this evolution involves increased viral tropism for endothelial cells. HPAI virus infections not only cause severe disease in chickens and other terrestrial poultry species but can also spread to humans and back to wild bird populations. Here, we review the role of the endothelium in the pathogenesis of influenza virus infection in wild birds, terrestrial poultry and humans with a particular focus on HPAI viruses. We demonstrate that whilst the endothelium is an important target of virus infection in terrestrial poultry and some wild bird species, in humans the endothelium is more important in controlling the local inflammatory milieu. Thus, the endothelium plays an important, but species-specific, role in the pathogenesis of influenza virus infection.

Expression profile and histological distribution of IFITM1 and IFITM3 during H9N2 avian influenza virus infection in BALB/c mice.

The H9N2 avian influenza virus is a pandemic threat which has repeatedly caused infection in humans and shows enhanced replication and transmission in mice. Previous reports showed that host factors, the interferon-inducible transmembrane (IFITM) protein, can block the replication of pathogens and affect their pathogenesis. BALB/c mice are routine laboratory animals used in influenza virus research, but the effects of H9N2 influenza virus on tissue distribution and expression pattern of IFITM in these mice are unknown. Here, we investigated the expression patterns and tissue distribution of IFITM1 and IFITM3 in BALB/c mice by infection with H9N2 AIV strains with only a PB2 residue 627 difference. The results showed that the expression patterns of ITITM1 and IFITM3 differ in various tissues of BALB/c mice at different time points after infection. IFITM1 and IFITM3 showed cell- and tissue-specific distribution in the lung, heart, liver, spleen, kidney and brain. Notably, the epithelial and neuronal cells all expressed the proteins of IFITM1 and IFITM3. Our results provide the first look at differences in IFITM1 and IFITM3 expression patterns in BALB/c mice infected by H9N2 influenza viruses. This will enhance research on the interaction between AIV and host and further will elucidate the pathogenesis of influenza virus infection based on the interferon-inducible transmembrane (IFITM) protein.Electronic supplementary materialThe online version of this article (doi:10.1007/s00430-014-0361-2) contains supplementary material, which is available to authorized users.

Mapping the innate signaling cascade essential for cytokine storm during influenza virus infection

During pathogenic influenza virus infection, robust cytokine production (cytokine storm), excessive inflammatory infiltrates, and virus-induced tissue destruction all contribute to morbidity and mortality. Earlier we reported that modulation of sphingosine-1-phosphate-1 receptor (S1P1R) signaling provided a chemically tractable approach for the effective blunting of cytokine storm, leading to the improvement of clinical and survival outcomes. Here, we show that S1P1R agonist treatment suppresses global cytokine amplification. Importantly, S1P1R agonist treatment was able to blunt cytokine/chemokine production and innate immune cell recruitment in the lung independently of endosomal and cytosolic innate sensing pathways. S1P1R signaling suppression of cytokine amplification was independent of multiple innate signaling adaptor pathways for myeloid differentiation primary response gene 88 (MyD88) and IFN-β promoter stimulator-1 signaling, indicating a common pathway inhibition of cytokine storm. We identify the MyD88 adaptor molecule as responsible for the majority of cytokine amplification observed following influenza virus challenge.

Microarray analysis of microRNA expression in peripheral blood mononuclear cells of critically ill patients with influenza A (H1N1).

BackgroundWith concerns about the disastrous health and economic consequences caused by the influenza pandemic, comprehensively understanding the global host response to influenza virus infection is urgent. The role of microRNA (miRNA) has recently been highlighted in pathogen-host interactions. However, the precise role of miRNAs in the pathogenesis of influenza virus infection in humans, especially in critically ill patients is still unclear.MethodsWe identified cellular miRNAs involved in the host response to influenza virus infection by performing comprehensive miRNA profiling in peripheral blood mononuclear cells (PBMCs) from critically ill patients with swine-origin influenza pandemic H1N1 (2009) virus infection via miRNA microarray and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) assays. Receiver operator characteristic (ROC) curve analysis was conducted and area under the ROC curve (AUC) was calculated to evaluate the diagnostic accuracy of severe H1N1 influenza virus infection. Furthermore, an integrative network of miRNA-mediated host-influenza virus protein interactions was constructed by integrating the predicted and validated miRNA-gene interaction data with influenza virus and host-protein-protein interaction information using Cytoscape software. Moreover, several hub genes in the network were selected and validated by qRT-PCR.ResultsForty-one significantly differentially expressed miRNAs were found by miRNA microarray; nine were selected and validated by qRT-PCR. QRT-PCR assay and ROC curve analyses revealed that miR-31, miR-29a and miR-148a all had significant potential diagnostic value for critically ill patients infected with H1N1 influenza virus, which yielded AUC of 0.9510, 0.8951 and 0.8811, respectively. We subsequently constructed an integrative network of miRNA-mediated host-influenza virus protein interactions, wherein we found that miRNAs are involved in regulating important pathways, such as mitogen-activated protein kinase signaling pathway, epidermal growth factor receptor signaling pathway, and Toll-like receptor signaling pathway, during influenza virus infection. Some of differentially expressed miRNAs via in silico analysis targeted mRNAs of several key genes in these pathways. The mRNA expression level of tumor protein T53 and transforming growth factor beta receptor 1 were found significantly reduced in critically ill patients, whereas the expression of Janus kinase 2, caspase 3 apoptosis-related cysteine peptidase, interleukin 10, and myxovirus resistance 1 were extremely increased in critically ill patients.ConclusionsOur data suggest that the dysregulation of miRNAs in the PBMCs of H1N1 critically ill patients can regulate a number of key genes in the major signaling pathways associated with influenza virus infection. These differentially expressed miRNAs could be potential therapeutic targets or biomarkers for severe influenza virus infection.

Consensus QSAR model for identifying novel H5N1 inhibitors

Due to the importance of neuraminidase in the pathogenesis of influenza virus infection, it has been regarded as the most important drug target for the treatment of influenza. Resistance to currently available drugs and new findings related to structure of the protein requires novel neuraminidase 1 (N1) inhibitors. In this study, a consensus QSAR model with defined applicability domain (AD) was developed using published N1 inhibitors. The consensus model was validated using an external validation set. The model achieved high sensitivity, specificity, and overall accuracy along with low false positive rate (FPR) and false discovery rate (FDR). The performance of model on the external validation set and training set were comparable, thus it was unlikely to be overfitted. The low FPR and low FDR will increase its accuracy in screening large chemical libraries. Screening of ZINC library resulted in 64,772 compounds as probable N1 inhibitors, while 173,674 compounds were defined to be outside the AD of the consensus model. The advantage of the current model is that it was developed using a large and diverse dataset and has a defined AD which prevents its use on compounds that it is not capable of predicting. The consensus model developed in this study is made available via the free software, PaDEL-DDPredictor.

Plasmacytoid dendritic cell depletion leads to an enhanced mononuclear phagocyte response in lungs of mice with lethal influenza virus infection

Plasmacytoid dendritic cells (pDCs) have been implicated both in the control and pathogenesis of influenza virus infection. We demonstrate that pDC depletion has marked effects on the response of mononuclear phagocytes, including conventional DCs (cDCs) and macrophages, to lethal influenza virus infection. Infection of mice lacking pDCs through antibody-mediated depletion resulted in substantially increased accumulation of mononuclear phagocytes and their progenitors in lungs compared to non-treated controls. pDC ablation resulted in a 5- to 35-fold enhancement of intracellular TNF-α and IL-6 production from inflammatory cDCs and exudate macrophages. Purified pulmonary cDCs and macrophages cultured from pDC-depleted mice produced significantly elevated levels of pro-inflammatory cytokines and chemokines compared to pDC-intact counterparts. Elimination of pDCs resulted in decreased lung IFN-α production and an immediate and transient reduction in lung virus burden but did not impact disease outcome. These data reveal a suppressive effect of pDCs on the inflammatory response to influenza virus infection in the lung.

Implication of nitric oxide in the pathogenesis of influenza virus infection

Influenza continues to be a significant threat to global public health. Even annual vaccination fails to prevent complicated illness and death. The findings in various studies suggest that severe disease is attributed to pulmonary complications – primary viral and secondary bacterial pneumonia. A number of investigations on influenza virus infection in the mouse model demonstrate that overproduction of free radicals, including nitric oxide (NO), contributes to the development of pneumonia during influenza virus infection. The role of NO on influenza virus infection in mice and differences between inducible nitric oxide synthase activation in mouse and human systems are discussed in this review. The biosynthesis of NO, mechanisms for regulation of NO production, nitrative stress during pathogenesis of various microbial infections are overviewed as well.

N-terminal region of the PB1-F2 protein is responsible for increased expression of influenza A viral protein PB1

Influenza A virus (IAV) PB1-F2 protein is encoded by an alternative reading frame (+1) within the PB1 gene. PB1-F2 has been shown to contribute to the pathogenesis of influenza virus infection as well as to the secondary bacterial infection. More recently has been shown that PB1-F2 protein may regulate a viral RNA (vRNA) polymerase activity by the interaction with PB1 protein. We proved that PB1-F2 protein increased the level of expression of PB1 protein and vRNA in the infected cells. Moreover, we demonstrated that a higher level of vRNA expression resulted in the increase of expression of multiple viral proteins, including NP, M1, and NS1. Finally, we used plasmids expressing N-terminal (1-50 aa) or C-terminal (51-87 aa) region of the PB1-F2 molecule for transfection of MDCK cells co-infected with influenza A/Puerto Rico/8/34 (H1N1) virus deficient in the PB1-F2 protein expression (PR8ΔPB1-F2). These experiments clearly showed that N-terminal region of PB1-F2 protein was responsible for the increase in PB1 protein expression. C-terminal region of PB1-F2 protein had no effect. Thus, we have identified the important function for N-terminal region of PB1-F2 protein.

Preventive effect of polyphenols from geranium sanguineum l on hepatic drug metabolism in influenza infected mice.

Event Abstract Back to Event Preventive effect of polyphenols from geranium sanguineum l on hepatic drug metabolism in influenza infected mice. Lyubka Tantcheva1, Eleonora Encheva2*, Silvyia Abarova2, Julia Serkedjieva3, Iliana Dimova1 and Elitza Pavlova4 1 Institute of Neurobiology, Bulgaria 2 Medical University-Sofia, Bulgaria 3 Bulgarian Academy of Sciences, Institute of Microbiology, Bulgaria 4 St Kliment Ohridsky Sofia University, Bulgaria Introduction: Inhibited first phase of drug metabolism is a main reason for numerous side effects and increased drug toxicity, observed in the course of influenza virus infection (IVI). Medical plant Geranium Sanguineum L. (GSL), spread in the Balkans, demonstrated significant antioxidant and antiviral activity. The aim of the study is to evaluate the preventive effect of polyphenol complex (PPC) from GSL on the oxidative drug metabolism in IVI. Methods: The experimental model of IVI (A/Aichi/2/68/(H3N2)(4.5 lg LD50) is developed in male albino ICR mice. PPC is applied nasally (10 mg/kg). In liver 9 000 g supernatant thiobarbituric acid reactive substances (TBARS) and antioxidant total activity (ATA) as well as monooxygenase N-demethylase activity (ethylmorphyne, amidopyrine and analgin) and hydroxylase activity (aniline and p-nitrophenol), NADPH- cytochrtome P-450 reductase and cytochrome P-450 content are determined. Student-Fisher t-test and correlation coefficients are used for statistics. Results: High correlation between increased TBARS and decreased monooxygenase activity (p< 0.001) is found in infected animals. It suggests that activation of free radicals can be one of the main reasons for unspecific monooxygenase inhibition, because all components of cytochrome P-450 system are inhibited (mostly the on the 6th and 9th days). PPC pretreatment demonstrates significant preventive effect against increased levels of TBRS and decreased ATA. Drug metabolism in IVI mice is restored by PPC near to healthy controls. Conclusion: TBARS probably play important role not only in the pathogenesis of IVI, but also in modulation of hepatic monooxygenase activity. All inhibited P-450 monooxygenase activities are significantly restored by PPC pretreatment. The complex mechanism of PPC-protective effect obviously combines several biological activities- antioxidant activity, selective antiviral and protein-bounding effect. In contrast to infected animals, PPC has medium pro-oxidant and weak inhibiting effect on monooxygenases in healthy animals, probably related to its protein–binding effect on membrane proteins and enzymes. Keywords: influenza infection, drug metabolism, cyt P-450 monooxygenases, Polyphenols, Geranium Sanguineum L. Conference: 8th Southeast European Congress on Xenobiotic Metabolism and Toxicity - XEMET 2010, Thessaloniki, Greece, 1 Oct - 5 Oct, 2010. Presentation Type: Poster Topic: Xenobiotic metabolism Citation: Tantcheva L, Encheva E, Abarova S, Serkedjieva J, Dimova I and Pavlova E (2010). Preventive effect of polyphenols from geranium sanguineum l on hepatic drug metabolism in influenza infected mice.. Front. Pharmacol. Conference Abstract: 8th Southeast European Congress on Xenobiotic Metabolism and Toxicity - XEMET 2010. doi: 10.3389/conf.fphar.2010.60.00203 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 28 Oct 2010; Published Online: 04 Nov 2010. * Correspondence: Dr. Eleonora Encheva, Medical University-Sofia, Sofia, Bulgaria, eleonora.encheva@medfac.acad.bg Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Lyubka Tantcheva Eleonora Encheva Silvyia Abarova Julia Serkedjieva Iliana Dimova Elitza Pavlova Google Lyubka Tantcheva Eleonora Encheva Silvyia Abarova Julia Serkedjieva Iliana Dimova Elitza Pavlova Google Scholar Lyubka Tantcheva Eleonora Encheva Silvyia Abarova Julia Serkedjieva Iliana Dimova Elitza Pavlova PubMed Lyubka Tantcheva Eleonora Encheva Silvyia Abarova Julia Serkedjieva Iliana Dimova Elitza Pavlova Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Influenza A virus matrix protein 1 interacts with hTFIIIC102-s, a short isoform of the polypeptide 3 subunit of human general transcription factor IIIC

Influenza A virus matrix protein 1 (M1) is a multifunctional protein that plays important roles during replication, assembly and budding of the virus. To search for intracellular protein components that interact with M1 protein and explore the potential roles of these interactions in the pathogenesis of influenza virus infection, 11 independent proteins, including hTFIIIC102-s protein, encoding a short isoform of the TFIIIC102 subunit of the human TFIIIC transcription factor, were screened from a human cell cDNA library using a yeast two-hybrid technique. The interaction between M1 protein and hTFIIIC102-s was studied in more detail. Mapping assays showed that the N-terminal globular region (amino acids 1-164) of the M1 protein and the five tandem tetratricopeptide repeats (TPR1-5, amino acids 149-362) in hTFIIIC102-s were necessary for the interaction. The interaction was confirmed by both glutathione-S-transferase (GST) pull-down assays and coimmunoprecipitation assays. In addition, coexpression of hTFIIIC102-s with M1 in HeLa cells inhibited the translocation of M1 into the nucleus. Taken together, the present data indicate that hTFIIIC102-s can interact with the structural M1 protein of the influenza virus, which provides a novel clue toward further understanding of the roles of M1 protein in the interactions between influenza virus and host cells.

Influenza A Virus PB1-F2 Protein Contributes to Viral Pathogenesis in Mice

The influenza virus PB1-F2 protein is a novel protein previously shown to be involved in induction of cell death. Here we characterize the expression and the function of the protein within the context of influenza viral infection in tissue culture and a mouse model. We show that the C-terminal region of the protein can be expressed from a downstream initiation codon and is capable of interaction with the full-length protein. Using this knowledge, we generated influenza viruses knocked out for the expression of PB1-F2 protein and its downstream truncation products. Knocking out the PB1-F2 protein had no effect on viral replication in tissue culture but diminished virus pathogenicity and mortality in mice. The viruses replicated to similar levels in mouse lungs by day 3 postinfection, suggesting that the knockout did not impair viral replication. However, while the PB1-F2 knockout viruses were cleared after day 5, the wild-type viruses were detectable in mouse lungs until day 7, implying that expression of PB1-F2 resulted in delayed clearance of the viruses by the host immune system. Based on our findings and on the fact that the PB1 genomic segment was always newly introduced into some pandemic influenza viruses of the last century, we speculate that the PB1-F2 protein plays an important role in pathogenesis of influenza virus infection and may be an important contributor to pathogenicity of pandemic influenza viruses.

Neuraminidase as a target for drugs for the treatment of influenza

Neuraminidase, or sialidase, is a surface glycoprotein that possesses enzymatic activity essential for viral replication in both influenza A and B anuses. To date, 9 neuraminidase subtypes (N1-N9) have been identified from influenza A viruses and only 1 subtype has been identified from influenza B viruses. Because of the importance of this enzyme in the pathogenesis of influenza virus infection and the close correspondence of the conserved residues of the active sites from all influenza neuraminidases, the enzyme structure has been successfully used for the design of neuraminidase inhibitors, which would be effective for various influenza viruses. The neuraminidase inhibitors zanamivir and oseltamivir are now commonly used for the therapy of influenza virus infections with minimal adverse effects, and they can also be used for the prophylaxis of influenza, especially when vaccination is unsuitable or ineffective. Up to now, the data on neuraminidase inhibitors suggest that the enzyme is an attractive target for the discovery of new antiinfluenza drugs.

Inhibition of Influenza-Virus-Induced Apoptosis in Chorion Cells of Human Fetal Membranes by Nordihydroguaiaretic Acid

Objectives: It has been postulated that the pathogenesis of influenza virus infection involves not only the virus-proliferation-mediated apoptotic cell death in infected cells, but also a direct reactive oxygen species (ROS)-induced cellular injury in the infected organs. We examined effects of an antioxidant, nordihydroguaiaretic acid (NDGA), on apoptosis induction and viral proliferation. Subsequently, the results were compared with those of pyrrolidine dithiocarbamate (PDTC), another antioxidant. Methods: The levels of ROS production were measured with 2′,7′-dichlorofluorescein diacetate; apoptosis induction and viral proliferation were analyzed by DNA fragmentation and plaque-forming assays, respectively. Results: The treatment of infected cells with NDGA inhibited ROS overproduction, apoptotic DNA fragmentation and virus proliferation. The maximum inhibition against DNA fragmentation (76%) was observed with 500 µM NDGA. The antiviral activity of NDGA against influenza virus was more potent than that of PDTC. Conclusions: The present study, therefore, suggests for the first time that NDGA, a known antioxidant reagent, inhibits the induction of apoptosis in human fetal membrane chorion cells infected with influenza virus through the more potent antiviral activity than that of PDTC.