The interferon (IFN) response is the first line of defense against viral infections, and the majority of viruses have developed different strategies to counteract IFN responses in order to ensure their survival in an infected host. In this study, the abilities to inhibit IFN signaling of two closely related West Nile viruses, the New York 99 strain (NY99) and Kunjin virus (KUN), strain MRM61C, were analyzed using reporter plasmid assays, as well as immunofluorescence and Western blot analyses. We have demonstrated that infections with both NY99 and KUN, as well as transient or stable transfections with their replicon RNAs, inhibited the signaling of both alpha/beta IFN (IFN-␣/␤) and gamma IFN (IFN-␥) by blocking the phosphorylation of STAT1 and its translocation to the nucleus. In addition, the phosphorylation of STAT2 and its translocation to the nucleus were also blocked by KUN, NY99, and their replicons in response to treatment with IFN-␣. IFN-␣ signaling and STAT2 translocation to the nucleus was inhibited when the KUN nonstructural proteins NS2A, NS2B, NS3, NS4A, and NS4B, but not NS1 and NS5, were expressed individually from the pcDNA3 vector. The results clearly demonstrate that both NY99 and KUN inhibit IFN signaling by preventing STAT1 and STAT2 phosphorylation and identify nonstructural proteins responsible for this inhibition.The interferons (IFNs) are a large family of multifunctional secreted cytokines involved in antiviral defense, cell growth regulation, and immune activation. IFNs are produced by the majority of cells and include 14 different species of alpha IFN (IFN-␣) and one species of beta IFN (IFN-␤); these IFNs are involved primarily in antiviral and antiproliferative responses (7,16,17,28). Gamma IFN (IFN-␥) is IFN that is usually produced by specific cells of the immune system, including CD8 ϩ T cells, and has potent antiviral and immunomodulating activities (7,16,17,28). The binding of IFNs to corresponding receptors on cell surfaces triggers a cascade of different signaling pathways that eventually lead to the transcriptional activation of a large number of IFN-stimulated genes (ISGs), which can establish antiviral, antiproliferative, and/or immunoregulatory states in host cells. The best-studied IFN signaling pathways are based on IFN receptor-Janus Kinase (JAK)/signal transducer and activator of transcription (STAT) activation (7, 16). The binding of IFN-␣ and IFN-␤ to the IFN-␣/␤ receptor, which consists of IFNAR1 and IFNAR2 molecules, leads to the activation of JAK1 and Tyk-2 kinases via tyrosine phosphorylation. Activated Tyk-2 phosphorylates IFNAR1, which then serves as a binding site for STAT2. STAT2 is then phosphorylated by Tyk-2 and serves as a binding site for STAT1, which is subsequently phosphorylated by JAK1. The phosphorylated STAT2-STAT1 heterodimers then dissociate from the receptor and associate with p48/IRF-9 to form an ISGF3 complex that translocates to the nucleus, where it initiates the transcription of ISGs via binding to the IFN-stimulated response element (ISRE...

Overexpression of TNF contributes to pathogenesis of multiple autoimmune diseases, accounting for a remarkable success of anti-TNF therapy. TNF is produced by a variety of cell types, and it can play either a beneficial or a deleterious role. In particular, in autoimmunity pathogenic TNF may be derived from restricted cellular sources. In this study we evaluated the feasibility of cell-type-restricted TNF inhibition in vivo. To this end, we engineered MYSTI (Myeloid-Specific TNF Inhibitor)-a recombinant bispecific antibody that binds to the F4/80 surface molecule on myeloid cells and to human TNF (hTNF). In macrophage cultures derived from TNF humanized mice MYSTI could capture the secreted hTNF, limiting its bioavailability. Additionally, as evaluated in TNF humanized mice, MYSTI was superior to an otherwise analogous systemic TNF inhibitor in protecting mice from lethal LPS/D-Galactosamine-induced hepatotoxicity. Our results suggest a novel and more specific approach to inhibiting TNF in pathologies primarily driven by macrophage-derived TNF.anti-cytokine therapy | TNF | bispecific antibody | autoimmunity | humanized mice

We have recently developed a new personal sampler and demonstrated its feasibility for detection of viable airborne microorganisms including bacteria, fungi and viruses. To accelerate the time-consuming analytical procedure involving 2-5 days of biological testing, we employed a real-time PCR protocol in conjunction with the personal sampler for collection of airborne viruses. The advantage of this approach is that if the presence of a particular pathogen in the air is detected by the PCR, the remaining collecting liquid can be further analysed by more time-consuming biological methods to estimate the number of airborne infectious/live microorganisms. As sampling of bioaerosols in natural environments is likely to be associated with substantial contamination by a range of microorganisms commonly existing in an ambient air, an investigation of the specificity of detection by targeted PCR analysis is required. Here we present the results of the study on the detection of Influenza virus in the ambient air contaminated with high concentrations of bacteria and fungi using real-time PCR protocol. The combined sampling PCR detection method was found to be fully feasible for the rapid ( approximately 2.5 h) and highly specific (no cross-reactivity) identification of the labile airborne virus in the air containing elevated concentrations of other microorganisms.

An attenuated Australian strain of West Nile virus (WNV), Kunjin (KUN), shares ~98% amino acid homology with the pathogenic New York 99 NY99 strain (NY99). To investigate the viral factors involved in NY99 virulence we generated an infectious cDNA clone of the WNV NY99 4132 isolate from which virus was recovered and was shown to be indistinguishable from the parental isolate. We then introduced the regions of the NY99 non-structural (NS) proteins and/or untranslated regions (UTRs) into the KUN backbone. Chimeric KUN viruses containing NY99 5′UTR and the parts of NS coding region were more virulent in mice than parental KUN virus. Chimeric NY99 viruses, containing KUN NS2A protein with alanine 30 to proline substitution were significantly less cytopathic in cells and less virulent in mice. Our results identify the 5′UTR and NS proteins as WNV virulence determinants and confirm a role for the NS2A in WNV cytopathicity and virulence.