Lambda interferon is the predominant interferon induced by influenza A virus infection in vivo.

The type I alpha/beta interferons (IFN-α/ß) are known to play an important role in host defense against influenza A virus infection, but we have now discovered that the recently identified type III IFNs (IFN-λ) constitute the major response to intranasal infection with this virus. Type III IFNs were present at much higher levels than type I IFNs in the lungs of infected mice, and the enhanced susceptibility of STAT2-/- animals demonstrated that only signaling through the IFN-α/ß or IFN-λ pathways was sufficient to mediate protection. This finding offers a possible explanation for the similar levels of antiviral protection found in wild-type (WT) mice and in animals lacking a functional type I IFN receptor (IFNAR-/-) but also argues that our current understanding of type III IFN induction is incomplete. While murine IFN-λ production is thought to depend on signaling through the type I IFN receptor, we demonstrate that intranasal influenza A virus infection leads to the robust type III IFN induction in the lungs of both WT and IFNAR-/- mice. This is consistent with previous studies showing that IFNAR-mediated protection is redundant for mucosal influenza virus infection and with data showing that the type III IFN receptor is expressed primarily by epithelial cells. However, the overlapping effects of these two cytokine families are limited by their differential receptor expression, with a requirement for IFN-α/ß signaling in combating systemic disease.

Methods used to study respiratory virus infection.

This unit describes protocols for infecting the mouse respiratory tract, and assaying virus replication and host response in the lung. Respiratory infections are the leading cause of acute illness worldwide, affecting mostly infants and children in developing countries. The purpose of this unit is to provide a basic strategy and protocols to study the pathogenesis and immunology of respiratory virus infection using the mouse as an animal model. The procedures include: (1) basic techniques for mouse infection, tissue sampling, and preservation, (2) determination of viral titers, isolation and analysis of lymphocytes and dendritic cells using flow-cytometry, and (3) lung histology, immunohistochemistry, and in situ hybridization.

Influenza A virus inhibits alveolar fluid clearance in BALB/c mice.

RATIONALE: Pulmonary infections can impair alveolar fluid clearance (AFC), contributing to formation of lung edema. Effects of influenza A virus (IAV) on AFC are unknown. OBJECTIVES: To determine effects of IAV infection on AFC, and to identify intercellular signaling mechanisms underlying influenza-mediated inhibition of AFC. METHODS: BALB/c mice were infected intranasally with influenza A/WSN/33 (10,000 or 2,500 focus-forming units per mouse). AFC was measured in anesthetized, ventilated mice by instilling 5% bovine serum albumin into the dependent lung. MEASUREMENTS AND MAIN RESULTS: Infection with high-dose IAV resulted in a steady decline in arterial oxygen saturation and increased lung water content. AFC was significantly inhibited starting 1 hour after infection, and remained suppressed through Day 6. AFC inhibition at early time points (1-4 h after infection) did not require viral replication, whereas AFC inhibition later in infection was replication-dependent. Low-dose IAV infection impaired AFC for 10 days, but induced only mild hypoxemia. High-dose IAV infection increased bronchoalveolar lavage fluid ATP and UTP levels. Impaired AFC at Day 2 resulted primarily from reduced amiloride-sensitive AFC, mediated by increased activation of the pyrimidine-P2Y purinergic receptor axis. However, an additional component of AFC impairment was due to activation of A(1) adenosine receptors and stimulation of increased cystic fibrosis transmembrane regulator-mediated anion secretion. Finally, IAV-mediated inhibition of AFC at Day 2 could be reversed by addition of beta-adrenergic agonists to the AFC instillate. CONCLUSIONS: AFC inhibition may be an important feature of early IAV infection. Its blockade may reduce the severity of pulmonary edema and hypoxemia associated with influenza pneumonia.

Differential type I interferon induction by respiratory syncytial virus and influenza a virus in vivo.

Type I interferon (IFN) induction is an immediate response to virus infection, and very high levels of these cytokines are produced when the Toll-like receptors (TLRs) expressed at high levels by plasmacytoid dendritic cells (pDCs) are triggered by viral nucleic acids. Unlike many RNA viruses, respiratory syncytial virus (RSV) does not appear to activate pDCs through their TLRs and it is not clear how this difference affects IFN-alpha/beta induction in vivo. In this study, we investigated type I IFN production triggered by RSV or influenza A virus infection of BALB/c mice and found that while both viruses induced IFN-alpha/beta production by pDCs in vitro, only influenza virus infection could stimulate type I IFN synthesis by pDCs in vivo. In situ hybridization studies demonstrated that the infected respiratory epithelium was a major source of IFN-alpha/beta in response to either infection, but in pDC-depleted animals only type I IFN induction by influenza virus was impaired.

Type I interferon inhibition and dendritic cell activation during gammaherpesvirus respiratory infection.

The respiratory tract is a major mucosal site for microorganism entry into the body, and type I interferon (IFN) and dendritic cells constitute a first line of defense against viral infections. We have analyzed the interaction between a model DNA virus, plasmacytoid dendritic cells, and type I IFN during lung infection of mice. Our data show that murine gammaherpesvirus 68 (gammaHV68) inhibits type I IFN secretion by dendritic cells and that plasmacytoid dendritic cells are necessary for conventional dendritic cell maturation in response to gammaHV68. Following gammaHV68 intranasal inoculation, the local and systemic IFN-alpha/beta response is below detectable levels, and plasmacytoid dendritic cells are activated and recruited into the lung with a tissue distribution that differs from that of conventional dendritic cells. Our results suggest that plasmacytoid dendritic cells and type I IFN have important but independent roles during the early response to a respiratory gammaHV68 infection. gammaHV68 infection inhibits type I IFN production by dendritic cells and is a poor inducer of IFN-alpha/beta in vivo, which may serve as an immune evasion strategy.

Protection against respiratory syncytial virus by a recombinant Newcastle disease virus vector.

Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract disease in infants and the elderly, but no safe and effective RSV vaccine is yet available. For reasons that are not well understood, RSV is only weakly immunogenic, and reinfection occurs throughout life. This has complicated the search for an effective live attenuated viral vaccine, and past trials with inactivated virus preparations have led to enhanced immunopathology following natural infection. We have tested the hypothesis that weak stimulation of innate immunity by RSV correlates with ineffective adaptive responses by asking whether expression of the fusion glycoprotein of RSV by Newcastle disease virus (NDV) would stimulate a more robust immune response to RSV than primary RSV infection. NDV is a potent inducer of both alpha/beta interferon (IFN-alpha/beta) production and dendritic cell maturation, while RSV is not. When a recombinant NDV expressing the RSV fusion glycoprotein was administered to BALB/c mice, they were protected from RSV challenge, and this protection correlated with a robust anti-F CD8+ T-cell response. The effectiveness of this vaccine construct reflects the differential abilities of NDV and RSV to promote dendritic cell maturation and is retained even in the absence of a functional IFN-alpha/beta receptor.

Construction and characterization of deltaretrovirus indicator cell lines.

The deltaretroviruses, which include bovine leukemia virus (BLV) and human T-cell leukemia virus types 1 and 2 (HTLV-1 and HTLV-2), replicate poorly in culture and the molecular details of their life cycles are limited. To facilitate the analysis of virus replication, mammalian cell lines were created with the long terminal repeats (LTRs) of each virus driving expression of the enhanced green fluorescent protein gene (egfp). The BLGFP, H1GFP and H2GFP cell lines detect virus infection by the expression of GFP via the transactivation of the LTR via the Tax protein of BLV, HTLV-1 or HTLV-2, respectively. GFP expression was measured by flow cytometry, yielding sensitive and rapid detection of virus infectivity. Interestingly, we observed that the Tax proteins of HTLV-1 and HTLV-2 could transactivate the BLV LTR at levels that were comparable to that of BLV Tax. In contrast, the BLV Tax showed low levels of transactivation in H1GFP and H2GFP cells. HTLV-1 and HTLV-2 Tax proteins efficiently transactivated both the HTLV-1 and HTLV-2 LTRs. Finally, spinoculation of BLV resulted in only a two-fold increase in viral titer.