Comparison of cytokine profiles induced by nonlethal and lethal doses of influenza A virus in mice.

Influenza viruses are among the most common human pathogens and are responsible for causing extensive seasonal morbidity and mortality. To investigate the immunological factors associated with severe influenza infection, the immune responses in mice infected with nonlethal (LD0) doses of A/PR/8/34 (H1N1) influenza virus were compared with those of mice infected with a lethal dose (LD100) of the virus. The virus titer and activation of retinoic acid-inducible gene (RIG)-I-like receptor signaling pathways were similar in the mice infected with LD0 and LD100 at 2 days post-infection; however, mice infected with LD100 exhibited a greater abundance of cytokines and a more diverse cytokine profile. Infection with LD100 induced the expression of the following factors: Interleukins (ILs), IL-4, IL-7, IL-10, IL-11, IL-12p40, IL-13 and IL-15; inflammatory chemokines, C-C motif chemokine ligand (CCL)2, CCL3/4, CCL12, CCL17, CCL19; and lung injury-associated cytokines, leptin, leukaemia inhibitory factor, macrophage colony stimulating factor, pentraxin (PTX)2 and PTX3, WNT1-inducible-signaling pathway protein 1, matrix metallopeptidase (MMP)-2, MMP-3, proprotein convertase subtilisin/kexin type 9, and T cell immunoglobulin and mucin domain. Switching in macrophage polarization from M1 to M2 was evidenced by the increase in M2 markers, including arginase-1 (Arg1) and early growth response protein 2 (Egr2), in the lungs of mice infected with LD100. Since IL-12 and interferon-γ are the major T helper (Th)1 cytokines, increased expression of interferon regulatory factor 4, IL-4, IL-10 and IL-13 promoted the differentiation of naïve CD4+ T cells into Th2 cells. In conclusion, the present study identified key cytokines involved in the pathogenicity of influenza infection, and demonstrated that lethal influenza virus infection induces a mixed Th1/Th2 response.

Transforming Activity of Murine Herpesvirus 68 Putative Growth Factor Is Related to the Ability to Change Cytoskeletal Structure.

Murine herpesvirus 68 (MHV-68) can transform cells in vitro and in vivo. We investigated putative murine herpesvirus growth factors (MHGFs) obtained by the separation of cell-free media from MHV-68-transformed cells on an FPLC Sephadex G15 column. The transforming activity of the MHGFA fraction was related to depolymerization of actin, disruption of the microtubule network, and punctate-reticular changes of the Golgi. The MHGFW fraction had only repressing activity on the transformed phenotype. Incubation of MRC-5 cells with MHGFW resulted in reticular changes of the Golgi apparatus, minor depolymerization of actin filaments, and no detectable changes of the microtubule network. Reorganization of the actin cytoskeleton is associated with oncogenesis. Further study of the MHGFs from herpesviruses and proteins responsible for changes in the organization of the cytoskeleton could give insight into the cell transformation mechanism and oncogenesis.

Synergic and antagonistic effect of small hairpin RNAs targeting the NS gene of the influenza A virus in cells and mice.

In the present study, we demonstrate the effect of individual and mixtures of shRNAs targeting the NS gene to treat an established infection of influenza A virus (IAV). We prepared 10 shRNAs targeting the NS gene of the IAV, and these shRNAs were tested individually or in mixtures 16h after infection. Our results revealed: (i) shRNA targeting the NS1 transcript decreased the virus titre up to 21% (P<0.01), (ii) shRNA targeting NEP transcript did not influence the replication of IAV in the infected cells; (iii) a mixture of shRNAs targeting the NS1 transcript was less effective than the individual shRNAs and decreased the virus titre up to 42% in vitro; (iv) a mixture of individually inactive shRNAs targeting the NEP transcript significantly inhibited the replication of IAV in vitro; (v) the activities of the individual shRNAs in vivo predominantly corresponded to their activities in vitro; (vi) a synergistic effect of the shRNAs was observed in vivo; and (vii) a shRNA targeting the region common to both the NS1 and NEP transcripts, shNS593, exhibited the strongest inhibition and reduced the virus titre up to 16.4% in vitro, prolonged the survival of the mice by three days and abolished the protective effect of other shRNAs in vivo. shRNAs inhibited influenza virus infection in a gene-specific manner. NS1 mRNA was significantly reduced in lungs treated with shRNAs and the levels of RIG-1, IFN-α, IFN-ß and IFN-γ mRNAs shRNAs were not altered.

Influenza A virus replication is inhibited in IFN-λ2 and IFN-λ3 transfected or stimulated cells.

Interferons lambda (IFN-λ) are the most recently defined members of the class III cytokine family. To investigate whether IFN-λ2 and IFN-λ3 displayed antiviral activity against influenza A virus (IAV), a number of cell lines induced with IFNs - as well as two established cell lines (A549-IFN-λ2 and A549-IFN-λ3) - were infected with IAV. Our results indicate that IFN-λ2 has statistically significant antiviral activity in A549-IFN-λ2 (P=0.0028) although less so than IFN-λ3, which reduced viral titer to 10% (P<0.0001). The reverse was observed for cells treated with IFNs, with IFN-λ2-treated A549 cells inhibiting IAV infection more efficiently than IFN-λ3-treated A549 cells. The antiviral effect on IFN-stimulated cells was most apparent on Vero cells (compared with MDCK and HeLa). Both IFNs significantly inhibited IAV replication and inhibition was observed in a dose-dependent manner, with an optimal IFN concentration of 20 ng/ml. IFN-λ2 was more potent than IFN-λ3 on Vero cells while IFN-λ3 appeared more efficient than IFN-λ2 on MDCK and HeLa cells.