Rationale : Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infections in children, for which no specific treatment or vaccine is currently available. We have previously shown that RSV induces reactive oxygen species in cultured cells and oxidative injury in the lungs of experimentally infected mice. The mechanism(s) of RSV-induced oxidative stress in vivo is not known. Objectives : To measure changes of lung antioxidant enzymes expression/activity and activation of NF-E2-related factor 2 (Nrf2), a transcription factor that regulates detoxifying and antioxidant enzyme gene expression, in mice and in infants with naturally acquired RSV infection. Methods : Superoxide dismutase 1 (SOD 1), SOD 2, SOD 3, catalase, glutathione peroxidase, and glutathione S-transferase, as well as Nrf2 expression, were measured in murine bronchoalveolar lavage, cell extracts of conductive airways, and/or in human nasopharyngeal secretions by Western blot and two-dimensional gel electrophoresis. Antioxidant enzyme activity and markers of oxidative cell injury were measured in either murine bronchoalveolar lavage or nasopharyngeal secretions by colorimetric/immunoassays. Measurements and Main Results : RSV infection induced a significant decrease in the expression and/or activity of SOD, catalase, glutathione S-transferase, and glutathione peroxidase in murine lungs and in the airways of children with severe bronchiolitis. Markers of oxidative damage correlated with severity of clinical illness in RSVinfected infants. Nrf2 expression was also significantly reduced in the lungs of viral-infected mice. Conclusions : RSV infection induces significant down-regulation of the airway antioxidant system in vivo, likely resulting in lung oxidative damage. Modulation of oxidative stress may pave the way toward important advances in the therapeutic approach of RSV-induced acute lung disease.

Human metapneumovirus (hMPV), a leading cause of respiratory tract infections in infants, encodes a small hydrophobic (SH) protein of unknown function. Here we show that infection of plasmacytoid dendritic cells (pDCs) with a recombinant virus lacking SH expression (rhMPV-ΔSH) enhanced the secretion of type I interferons (IFNs), which required TLR7 and MyD88 expression. HMPV SH protein inhibited TLR7/MyD88/TRAF6 signaling leading to IFN gene transcription, identifying a novel mechanism by which paramyxovirus SH proteins modulate innate immune responses.

RATIONALE: Human metapneumovirus (hMPV) is a leading cause of upper and lower respiratory tract infections in infants, elderly and immunocompromised patients. hMPVencodes a small hydrophobic (SH) protein thought to be important in immunopathogenesis, although the exact function is unknown. Plasmocytoid dendritic cells represent an important source of IFN produced upon entry of bacterial and viral pathogens. METHODS: Human pDCs were infected with recombinant hMPV, either wild type (rhMPV-WT) or lacking SH protein expression (rhMPV-DSH), followed by measurement of type I IFN in the supernatants by ELISA. IFN secretion was also measured from spleen pDCs from TLR7-/mice following hMPV infection. HEK293 cells, which stably express TLR7, were transfected with a luciferase-tagged IFN-a4 promoter, MyD88, IKK-a and either TRAF6 or TRAF3 expression plasmids in the presence of SH expression plasmid or empty vector, and treated with recombinant IFN-a to induce TLR7 expression. RESULTS: Increased production of IFN-a and b by rhMPV-DSH infected pDCs vs rhMPV-WT infected pDCs suggests that SH protein inhibits type I IFN production in these cells. Compared to wt mice, neither TLR7-/-or MyD88-/-mice produced type I IFNs after hMPV infection. SH expression inhibited TRAF6-, but not TRAF3-dependent, IFN-a4 promoter activation, suggesting that SH targets TRAF6 to inhibit TLR7-induced IFN production. CONCLUSIONS: HMPV SH protein inhibits TLR7/MyD88/TRAF6 signaling leading to IFN gene transcription, identifying a novel mechanism by which hPMV SH protein modulates innate immune responses.

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