The increasing threat of epidemic and pandemic influenza underscore the need to better-understand the immune response to influenza virus infections and to better understand the factors that contribute to the clearance of virus without complications of immunopathology. A hallmark of the adaptive immune response to primary influenza virus infections is the induction of influenza-specific CD8+ T cell responses. These T cells target and kill influenza-infected epithelial cells in the airway, thereby clearing the virus and allowing recovery of the infected host. Recent reports demonstrated that CD8+ T cells express TNF-related apoptosis-inducing ligand (TRAIL) after influenza virus infection. While roles for perforin/granzyme and Fas:FasL interactions in clearing influenza virus infections had been established, little was known about the role of TRAIL in the CD8+ T cell responses to influenza virus infection. We hypothesized that influenza-specific CD8+ T cells would express TRAIL after influenza infection and could utilize TRAIL to induce the apoptosis of virally-infected cells. We discovered that CD8+ T cells do express TRAIL after influenza infection, and that this expression occurs in an influenza-specific fashion. Further, we demonstrated that these influenza-specific CD8+ T cells utilize this TRAIL to kill virally infected cells and protect the host from death, while T cells lacking TRAIL were unable to kill targets as efficiently and provided reduced protection. These data supported our hypothesis that CD8+ T cells utilize TRAIL to kill infected cells. Unexpectedly, when we increased the initial viral inoculum, the pulmonary cytotoxicity of T cells in TRAIL-/- mice was increased compared to those in TRAIL+/+ mice. Investigation of this phenomenon revealed that changes in cytotoxicity correlated not with changes in effector molecule expression on the T cells, but with increased recruitment of T cells to the lung. T cell recruitment to the lungs of TRAIL-/- mice was dependent on CCR5 and CXCR3, and likely the result of aberrant expression of MIG and MIP-1α in the lungs. Together, these data suggest that TRAIL expression contributes not only to T cell cytotoxicity, but also to the regulation of chemokine expression and associated cell recruitment after influenza virus infections. To confirm the relevance of our animal model to the study of human disease, we examined the potential role for TRAIL in the human immune response to infection. We determined that in vitro influenza infection stimulates upregulation of functional TRAIL on the surface of CD3+, CD14+, CD19+, and CD56+ PBMC populations. This expression was not caused by infection of the cells, but by interferon produced as a result of the infection. Infected (TRAIL-expressing) PBMCs killed influenza-infected lung epithelial cells, revealing that influenza infection sensitizes epithelial cells to TRAIL-induced apoptosis. Surprisingly, blocking TRAIL signaling, but not FasL signaling, was able to abrogate this killing of infected epithelial cells. Together, these data support a role for TRAIL in the human immune response to influenza virus infections. Considered as a whole, the data from these studies suggest an additional, previously-unappreciated mechanism by which CD8+ T cells can kill virally infected cells, TRAIL. They also suggest additional, previously-unappreciated roles for TRAIL in immune responses: in helping clear virally infected cells after infections and in helping control cytokine/chemokine expression, and thus the immune response, after virus infection.

Regulatory T cells (Tregs) suppress effector immune responses and have been implicated in promoting chronic viral infections. Their role during influenza infection and vaccination, however, is still unclear. Influenza is a major public health concern, claiming over 49,000 lives annually in the U.S. alone. Vaccination is the best approach for preventing disease but frequent mutations of immunogenic epitopes requires a new vaccine to be formulated and administered annually. This poses a challenge for vaccine manufacturing and may strain patient compliance. A universal influenza vaccine, which targets the highly conserved extracellular domain of the influenza matrix protein 2 (M2e), may circumvent this problem by generating cross-protective immunity. In this study, we tested the efficacy of the M2e universal vaccine in the ferret, and determined whether vaccination induces a Treg response after influenza infection. We found that vaccination promotes the development of M2e specific IgM and IgG antibodies after boosting. Upon challenge with A/Memphis H1N1, vaccinated ferrets exhibited a lower body temperature and reduced virus titer compared to non-vaccinated animals. Together these findings suggest that the M2e vaccine protects ferrets against influenza infection. In order to determine whether Tregs increase after vaccination in ferrets, we had to first clone and characterize genes involved with Treg phenotype and function including CD25, Foxp3, and IL-10. The reciprocal nature between Tregs and Th17 cells and their involvement during influenza infection prompted us to also clone ferret IL-17F. Using these sequences, we designed a qRT-PCR array to measure the expression of Foxp3, IL-10, and IL-17F in ferret tissue. We also identified cross-reactive antibodies against ferret CD8, CD25, and Foxp3 for use in FACS, western blot, and ICC. Using these tools, we found that vaccination significantly increased the expression of Foxp3 in the spleen. An increased percentage of Foxp3+ lymphocytes was detected in both the PBMCs and splenocytes of immunized animals. In contrast, IL-10 and IL-17F expression decreased significantly in both immunized and non-immunized ferrets compared with naïve animals. These studies suggest that the M2e influenza vaccine induces a regulatory T cell response in ferrets and protects against influenza infection. / acase@tulane.edu

Influenza virus is an enveloped virus with a negative sense single strand RNA. The viral surface is characterized by two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA)(Chen et al., 2007; Samuel, 2010). The HA subunit is responsible for the attachment of the virus to the host cell by binding to the sialic acid receptors. Influenza virus infection, occurring in the endosome of the host cell is a fusion dependent process (Daniels et al., 1983). Low pH inside the endosome facilitates the fusion process by triggering a major conformational change of HA. This conformational change exposes the fusion initiation region of the protein subsequently releasing a hydrophobic fusion peptide (which is otherwise buried inside the protein core). This hydrophobic peptide slips into the host cell membrane resulting in HA transiently being a part of the viral and cell membrane. The HA pulls the two membranes together, thus completing the fusion process and forming a clear passage for the release of the viral genetic material into the cytoplasm (Stevens et al., 2004; Ramalho-Santos and Pedroso De Lima, 1999; Carr and Kim, 1993). A 16 amino acid peptide sequence (Flufirvitide-3) derived from the fusion initiation region of the HA protein has shown effective inhibition of influenza virus infection. Plaque inhibition assays and animal studies show high efficacy of the peptide against the virus. However, the mechanism of action of this peptide is still unclear. We have extensively studied the ability of FF-3 to interact with and affect purified HA, pure lipid bilayers and whole viruses. Taken together, the results suggest a novel mechanism of action. / acase@tulane.edu

We have recently demonstrated in a model of influenza A virus (IAV) infection that the absence of specific pulmonary DC subsets, including plasmacytoid DC (pDC) and CD8a+ DC, from the lungs leads to a significant decrease in the number of virus-specific CD8 T cells. Reconstitution of the lungs with physiologic numbers of pDC or CD8a+ DC is able to restore the pulmonary IAV-specific CD8 T cell response to near normal levels via a mechanism that is dependent upon direct DC:T cell interactions, DC-expressed MHC I and the presence of viral antigen. Interestingly, however, this rescue is DC subset specific, as reconstitution with purified alveolar and airway DC or alveolar macrophages was unable to rescue the virus-specific CD8 T cell response. Following IAV infection there is an abundance of IAV antigen and MHC I expressing cells present in the lungs, including infected epithelial cells. Given this fact and the inability of all DC subsets to rescue the virus-specific CD8 T cell response, it suggested that there were additional, undefined requirements for pDC- and CD8a+ DC-mediated rescue of the T cell response in the lungs. Further, although it was known that the reduction in virus-specific CD8 T cells in the lungs was a result of increased T cell apoptosis, it remained unclear what pathways of apoptosis were contributing to the increased cell death, and what mechanism pulmonary DC subsets were utilizing to rescue this defect. Here, we demonstrate that in the absence of lung-resident DC subsets, virus-specific CD8 T cells undergo significantly increased levels of apoptosis via both extrinsic activation induced cell death and intrinsic activated cell-autonomous death pathways. Reconstitution of aDC depleted lungs with pulmonary pDC and CD8a+ DC promotes increased T cell expression of the pro-survival molecule Bcl-2 and hence, increased T cell survival and accumulation in the lungs. Our studies herein demonstrate that pulmonary DC subsets utilize a variety of mechanisms to promote the rescue of virus-specific CD8 T cells in the lungs. Blockade of the costimulatory molecules CD70, and in some cases, 4-1BBL and OX40L, ablates the pulmonary DC mediated rescue of CD8 T cell numbers in the lungs, suggesting that late costimulation is one essential mechanism that pulmonary DC use to regulate CD8 T cell immunity following IAV infection. Further, we demonstrate that the absence of DC following IAV infection results in significantly reduced levels of IL-15 in the lungs and that pulmonary DC-mediated rescue of virus-specific CD8 T cell responses in the lungs requires the trans-presentation of IL-15 via DC-expressed IL-15Ra. In addition to the role of pulmonary DC mediated costimulation and IL-15 trans-presentation, we further demonstrate a previously unrecognized role for viral antigen in regulating the accumulation of both pulmonary DC and virus-specific CD8 T cells in the lungs, suggesting that viral load can dictate the nature of the inflammatory environment in the lungs and thus, regulate the character of the ensuing IAV-specific immune response. Collectively, the results detailed here demonstrate a previously unrecognized role for pulmonary DC in regulating primary IAV-specific CD8 T cell immunity, and hence, promoting enhanced viral clearance and recovery from disease.

Detection of airborne influenza virus is needed in order to determine exposure and prevent and control infections. Few researchers have successfully detected airborne influenza virus in environmental settings with current bioaerosol samplers. Therefore, new sampling strategies should be considered to increase the likelihood of detection. This study compared four bioaerosol samplers in collection of airborne influenza virus – the SKC Biosampler, NIOSH Biosampler, Andersen N6 single-stage impactor containing a liquid media, and the newly developed Next Generation Inhalable Aerosol Sampler (NGIAS). Ten 30-minute laboratory trials were completed by aerosolizing active influenza virus (H1N1) in a bioaerosol chamber to compare the ability of four bioaerosol samplers to collect aerosolized virus. Samples were analyzed using RT-qPCR. The mean total virus particles per liter of sampled air (TVP) recovered with the NGIAS was significantly less than that measured by all other samplers (p < 0.001). The TVP recovered with the SKC Biosampler (111.41) and Andersen N6 sampler (102.36) was substantially larger than that recovered with the NIOSH Biosampler (58.59), however the difference in TVP between these samplers was not statistically significant (SKC – NIOSH p-value = 0.187 ; Andersen – NIOSH p-value = 0.297). Our results demonstrated that liquid based bioaerosol samplers recovered more TVP than dry collection samplers. The high flow rate sampler, the Andersen N6, did not collect more TVP, but had a lower limit of detection than other samplers. Furthermore, the SKC Biosampler collected the most TVP. Therefore, future investigators should design a liquid based personal bioaerosol sampler to maximize the likelihood of influenza virus detection.

Influenza causes thousands of illnesses and deaths annually in the United States. In part, this is a product of rapid changes in influenza genetics, resulting in different variants than a previous season. Influenza virus traverses landscapes by infecting susceptible hosts, thus allowing seasonal influenza to move great distances due to the mobility of humans who occupy diverse natural, social, and built environments. Using H3N2 influenza viral sequences from Minnesota in the 2012-2013 influenza season we explored relationships between the diversity of influenza genetics and the environments in which humans live. Landscape genetic methods were used to test for relationships between genetic diversity of influenza viruses with different concepts of distance separating the viruses in time and space. Additional analyses were used to identify relationships between influenza genetic evolution and socio-environmental characteristics of Minnesota zip code tabulation areas (ZCTAs) where those viruses were isolated. Influenza hospitalization data in Minnesota ZCTAs was also analyzed with spatial and statistical methods to compare differences and similarities between environmental features driving influenza genetic evolution and influenza morbidity. Findings indicated a complex genetic landscape with few significant correlations between genetic distance and other distance concepts. Elderly populations and populations without health insurance were found to be drivers of H3 hospitalizations. The synthesis of information from theses analyses can be used to inform our overall understanding of influenza diffusion and will allow for more targeted and effective public health prevention strategies.

With a case fatality rate higher than the 1918 Spanish Flu pandemic, H5N1 highly pathogenic avian influenza represents a threat to global public health. Efforts to identify locations with the greatest potential for pandemic emergence, as well as how the virus is spreading, may help minimize this threat. First detected in Egypt in 2006, H5N1 viruses have resulted in the deaths of millions of birds in both commercial and backyard poultry flocks, and more than 350 human infections, the most of any country, have been confirmed. Human outbreaks have been so far constrained by poor viral adaptation to non-avian hosts. There are two evolutionary mechanisms by which the H5N1 avian influenza virus could acquire pandemic potential: 1) via reassortment as a result of coinfection with another subtype (such as low pathogenic avian influenza H9N2); and/or 2) via antigenic drift and the accumulation of randomly occurring genetic changes found to improve viral fitness, herein called key substitutions (KS). Both mechanisms were investigated using geospatial methods including ecological niche modeling and hot spot analyses to predict locations with elevated potential for pandemic emergence. Using ecological niche modeling environmental, behavioral, and population characteristics of H5N1 and H9N2 niches within Egypt were identified, with niches differing markedly by subtype. Niche estimates were combined using raster overlay to estimate co-infection potential, with known occurrences used for validation. Co-infection was successfully predicted with high accuracy (area under the receiver operating characteristic (ROC) curve (AUC) 0.991). 41 distinct KS in H5N1 were detected in Egyptian isolates, including 17 not previously reported in Egypt. Phenotypic consequences of detected KS were varied, but the majority have been implicated in improving mammalian host adaptation and increasing virulence. Statistically significant spatial clustering of high KS rates was detected in the northwestern portion of the Nile River delta in the governorates of Alexandria and Beheira. To investigate how the virus spreads between poultry farms, landscape genetics techniques were employed. Viral genetic sequences were evaluated using phylogenetics to determine viral relatedness between samples, then distance models representing competing diffusion mechanisms were created using road networks and a least-cost path model designed to approximate wild waterbird travel using niche modeling and circuit theory. Spatial correlations were evaluated using Mantel tests, Mantel correlograms, and multiple regression of distance matrices within causal modeling and relative support frameworks. Samples from backyard farms were most strongly correlated with least cost path distances, implicating wild bird diffusion, while samples from commercial farms were most strongly correlated with road network distances, implicating human-mediated diffusion. Results were largely consistent across gene segments. Identifying areas at risk of co-infection can help target spaces for increased surveillance. Similarly, detecting spatial hot spots of KS highlight areas of concern for pandemic emergence from antigenic drift. Demonstration of different diffusion mechanisms by farm type should inform both surveillance and biosecurity practices. Knowledge of where to focus intervention efforts, both spatially and strategically, allows limited public health resources to be targeted most effectively. By detecting where in the country pandemic influenza is likely to emerge and identifying how the virus is spreading between farms, this work contributes to efforts to predict and prevent the next influenza pandemic.

Characterizing airborne influenza virus exposure is important for infection prevention and exposure control in health care and public settings. Detecting airborne influenza virus is important in assessing infection risk. The virus must also be protected from deterioration during aerosol sampling and long term storage. RNA preservation buffers (RNAPBs) may stabilize influenza virus after sampling and during storage. Bioaerosol samplers are used to collect airborne influenza virus, and many different types of samplers are available. The objectives of this experiment were to: 1) compare influenza virus concentrations across bioaerosol samplers; 2) compare the efficacy of RNAPB over Hanks Balanced Salt Solution (HBSS) as a sample collection media; and 3) determine whether RNAPB stabilizes viral particles stored over time. In this experiment we aerosolized active influenza virus (H1N1) in a bioaerosol chamber and compared sampling efficiencies using two different samplers: the SKC Biosampler and NIOSH Biosampler, and two different medias: Hanks Balanced Salt Solution (HBSS) and an RNAPB. Ten 15-minute experimental trials were completed. We also compared HBSS and RNAPB in terms of the maintenance of virus RNA integrity during storage at room temperatures. Samples were stored at room temperature for 1, 4, 7, and 14 days. Virus concentrations were measured and compared at each time point. Significant differences were found between sampler and media type - the SKC Biosampler collected a higher concentration of virus than the NIOSH Biosampler, and HBSS collected a higher concentration of virus than RNAPB. In storage at room temperature conditions, RNAPB maintained virus in concentrations significantly greater than in HBSS. The results of this experiment indicates that the SKC Biosampler should be used to characterize airborne influenza and that RNAPB should not be used as a sampling media but can be used to preserve samples if needed.

Tesis para optar al Grado de Magíster en Ciencias Animales y Veterinarias. / Los virus de Influenza tipo A representa una amenaza latente para la salud pública y para la salud animal. Debido a esto, y a raíz de la creciente preocupación por los focos de influenza aviar de alta patogenicidad que se registran en diferentes regiones del mundo, actualmente se lleva a cabo una vigilancia epidemiológica constante de estos virus por parte de diversas organizaciones de salud, monitoreando desde planteles comerciales hasta poblaciones de aves silvestres, especialmente de aquellas catalogadas como migratorias. Sin embargo, la información existente sobre estos virus en poblaciones silvestres en América del Sur aún es muy limitada. Las aves silvestres, especialmente aquellas relacionadas con ambientes acuáticos, son consideradas como reservorios de la gran mayoría de los subtipos de virus de influenza A conocidos y tienen el potencial de diseminarlos entre países e incluso continentes por medio de las rutas migratorias, debido a esto, resulta fundamental generar esta información epidemiológica. Este estudio, realizó un muestreo en los sectores Humedal Laguna Cartagena (HLC) y la Reserva Nacional Lago Peñuelas (RNLP), entre Septiembre de 2015 y Agosto de 2016. El objetivo de este estudio, fue la detección de virus de influenza A en muestras de heces de aves silvestres mediante rRT-PCR, determinando una relación entre la prevalencia del virus detectada y la estacionalidad de las comunidades de aves silvestres presentes durante el muestreo. Se recolectaron un total de 2.579 muestras ambientales (1.148 de HLC y 1.431 de la RNLP) y paralelamente, mediante la realización de censos se identificaron un total de 5.091 aves (3.815 en HLC y 1.276 en RNLP) pertenecientes a 46 especies, de las cuales 39 fueron clasificadas como residentes, 5 como visitantes de primavera-verano y 2 como visitantes de otoño-invierno. El análisis de las muestras indicó una prevalencia a VIA de un 0,4% (5/1.148) para el HLC y de un 1,04% (15/1.431) para la RNLP. Casi la totalidad de las muestras positivas fueron detectadas durante los meses de verano, siendo Diciembre y Enero los meses que presentaron los valores de prevalencia más altos, lo cual coincidió con una mayor presencia y abundancia de aves migratorias de origen boreal. Los factores de riesgo que presentaron asociación estadísticamente significativa fueron una abundancia de individuos baja (<200) (OR = 9,71; p = 0,0424) en el HLC y la estación del año, correspondiente a primavera-verano (OR 5,47 y p = 0,0288) en la RNLP. Los resultados de este estudio demuestran la presencia de virus de influenza A en los ecosistemas de ambos sitios y la asociación con la estacionalidad de las poblaciones de aves silvestres presentes en ellos / Influenza type A viruses represent a constant threat to both public and animal health. Therefore, and because of growing concern on high pathogenic avian influenza outbreaks around the world, health organizations maintain a strong epidemiological surveillance of this virus, monitoring from avian production systems till wild birds populations, especially the ones are catalogued as migratory birds. However, there is short information at moment about these types of viruses in wilds populations on South America. Wild birds, especially the ones are related with aquatic environment, are considered as reservoir of the most known influenza A virus and his subtypes, also they has the potential to disseminate the virus across countries or continents by migratory routes. Due to this, is extremely important generate this epidemiologic information. This Study, performed a sampling in Cartagena Lagoon Wetland y Peñuelas Lake National Reserve, between September 2015 and August 2016. The aim of this study was detect influenza A virus on samples feces of wild birds using rRT-PCR, determining a relationship between the prevalence of the virus and the seasonality of wild birds population. Were collected a number of 2.579 environmental samples (1.148 from HLC and 1.431 from RNLP), also it was possible by a census, identified 5.091 birds (3.815 in HLC and 1.276 in RNLP) belonging to 46 species, which one 39 were classified as residents birds, 5 as spring/summer visitors and 2 as an autumn/winter visitors. The analysis of the samples showed a prevalence to avian influenza virus of 0,4% (5/1.148) for HLC and a 1,04% (15/1.431) for RNLP. Almost all positive samples were detected in summer months, December and January showed the highest prevalence values, which coincided with a greater presence and abundance of boreal migratory birds. The risk factors that showed significant statistics association were low individuals abundance (<200) (OR = 9,71; p = 0,0424) in HLC and autumn/winter season (OR 5,47 y p = 0,0288) in RNLP / Financiamiento: Proyecto NIAID HHSN272201400006C.

Eine Influenza A Virus Infektion induziert die Expression zahlreicher Gene, einschließlich der TypI Interferone, die eine erste Abwehrlinie gegen virale Infektionen bilden. Hierbei ist IFNb das wichtigste Zytokin. IFNb wird durch einen multimeren Komplex, das Enhanceosom kontrolliert, das Bindungsstellen für die Transkriptionsfaktoren AP-1, NF-kB und IRF-3 in seiner Promotorsequenz besitzt. In früheren Arbeiten konnten wir zeigen, dass die Influenza Virus-induzierte AP-1 abhängige Genexpression über den JNK/SAPK-Signalweg erfolgt (Ehrhardt, 1999; Ludwig et al., 2001). Unter den, an DNA Elemente bindenden AP-1 Faktoren waren solche, die aufgrund von Phosphorylierung durch die JNKs reguliert werden, wie beispielsweise ATF-2. Weiterhin korrelierte die Induktion der AP-1 abhängigen Genexpression mit der starken Aktivierung von JNK und seiner upstream Regulatoren in permissiven Zellen. Die Virusmengen transfizierter und infizierter Zellen, in denen JNK inhibiert wurde, waren höher im Vergleich zu Virusmengen der Kontrollzellen. Demzufolge kann die Virus-induzierte Aktivierung von JNK und AP-1 nicht der Virusreplikation dienen, sondern gehört vielmehr zu einer antiviralen Immunantwort. Daten aus einem Virus-freien, auf Plasmiden basierenden vRNA Replikations-System deuten darauf hin, dass die JNK Aktivierung aus der Akkumulation viraler RNA resultiert. Entsprechend bewirkte die Infektion von Zellen mit einem Virus, dem das virale NS1 Protein fehlt, welches RNA binden und somit "wegfangen" kann, eine gesteigerte JNK Aktivität im Vergleich zu den Kontroll-Infektionen. Damit konnte das NS1 Protein als erstes virales Protein identifiziert werden, das der Virus- und dsRNA-induzierten Aktivierung des JNK/SAPK-Signalweges entgegen wirkt. Der Transkriptionsfaktor IRF-3 wird spezifisch infolge einer viralen Infektion aktiviert und ist daher ein potenter Kandidat, die schnelle und starke antivirale Genexpression zu regulieren. Infolge einer Influenza Virus Infektion wird IRF-3 phosphoryliert, wandert in den Kern und bindet dort an Promotoren, die die antivirale Genexpression steuern. Bislang sind die IRF-3 Kinase und zelluläre Signalwege, die eine IRF-3 Phosphorylierunge induzieren, unbekannt. Um in unserem Labor Signalmediatoren, die upstream von IRF-3 liegen, zu suchen, wurde ein IRF-3 responsives Promotor-Reportergen-Plasmid, aus dem IFNb Promotor stammend, konstruiert. Die kleine Rho-GTPase Rac1 wurde als erster nicht an RNA bindender, zellulärer Mediator identifiziert, der in die Influenza Virus-induzierte IRF-3 abhängige Genexpression involviert ist. Die Inaktivierung der Rho-GTPasen durch das spezifische Inhibitor Toxin B oder dominant negatives Rac1 resultierten in der Inhibierung der Virus- und dsRNA-induzierten IRF-3 Phosphorylierung und DNA Bindung, sowie der IRF-3 abhängigen Promotoraktivität, beispielsweise des IFNb Promotors. Damit konnten zwei wichtige Komponenten der Virus-induzierten Immunantwort identifiziert und charakterisiert werden. / Infection of cells with Influenza A virus induces the expression of a variety of genes, including the type I interferons which are a first line of defense against viral infections. IFNb, the most important cytokine, is controlled by a higher order complex, the enhanceosom, which contains binding sites for the transcription factors AP-1, NF-kB and IRF-3. We could show that the Influenza Virus induced AP-1 dependent gene expression occurs via the JNK/SAPK pathway (Ehrhardt, 1999; Ludwig et al., 2001). Among the AP-1 factors which were identified to bind their cognate DNA element during viral infection are those, that are regulated via phosphorylation by JNKs, such as ATF-2. Accordingly, the induction of AP-1 dependent gene expression correlates with a strong activation of JNK and its upstream activators MKK4 and 7 in permissive cells. Virus yields from transfected and infected cells in which JNK signaling was inhibited by different approaches were higher compared to the levels from control cells. Therefore we conclude that virus-induced activation of JNK and AP-1 is not exploited by the virus to support its replication but rather is required for the innate antiviral immune response. Data obtained with a virus-free plasmid-based vRNA replication system indicated that JNK activation is a cause of viral RNA accumulation during infection. This was supported by the observation, that infection of cells with a virus lacking viral NS1 protein, which is known to bind and to sequester RNA from cellular signaling intermediates, caused a strongly enhanced JNK activity compared to control infections. Furthermore, the NS1 protein was identified as the first viral protein that antagonizes virus- and dsRNA-induced activation of the stress response signaling pathway mediated through Jun N-terminal kinase. IRF-3 is specificially activated in response to viral infection and is therefore the most potent candidate to regulate the fast and strong antiviral gene expression. After an Influenza virus infection IRF-3 becomes phosphorylated and migrates to the nucleus where it binds to antiviral gene promoters. However, the IRF-3 kinase and the cellular signaling pathways leading to IRF-3 phosphorylation are unknown. To investigate signaling mediators upstream of IRF-3, we have constructed an IRF-3 responsive promoter-reporter gene plasmid derived from the IFNb promoter. The small Rho-GTPase Rac1 was identified as the first non-RNA binding cellular mediator involved in the Influenza virus-induced IRF-3 dependent gene expression. Inactivation of these Rho GTPases by the specific inhibitor toxin B or dominant negative Rac1 resulted in the inhibition of virus- and dsRNA-induced IRF-3 phosphorylation and DNA binding as well as of IRF-3 dependent promoter activity, e.g. of the IFNb promoter. Thus two important components of virus-mediated immune response were identified and characterised.