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Short Communication |
1 Unité de Virologie et Immunologie Moléculaires, INRA, 78350 Jouy-en-Josas, France
2 UMR1225, École nationale vétérinaire de Toulouse (ENVT), 31076 Toulouse, France
Correspondence
Jean-François Eléouët
jean-francois.eleouet{at}jouy.inra.fr
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ABSTRACT |
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)-mediated antiviral response for efficient replication in a host-specific manner. Mice models have been developed for experimental infection with human, but not bovine, respiratory syncytial virus strains. Here, it is shown that BRSV can replicate efficiently on primary cell cultures derived from type I IFN receptor-deficient, but not from wild-type IFN-competent, mice. However, BRSV infection was not enhanced in mice devoid of the type I IFN receptor. These results show that type I IFN is a major host-range determinant for infection at the cellular level, but that other factors control virus replication and pathology in vivo. Published online ahead of print on 4 May 2006 as DOI 10.1099/vir.0.82091-0.
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; Collins et al., 2001). HRSV and BRSV are the major cause of bronchiolitis and pneumonia in infants and calves, resulting in substantial economic losses. They belong to the genus Pneumovirus, family Paramyxoviridae, and are enveloped viruses with a single-, negative-strand RNA genome of about 15 kb that encodes 11 proteins.
Alpha/beta interferons (IFN-/) represent the first line of host defence against viruses, as both IFN- and IFN- trigger immediate innate antiviral activities (Conzelmann, 2005). All members of the IFN-/ family have a single common receptor, IFN-/R, composed of two chains (IFNAR1 and IFNAR2). Binding of IFN-/ to its receptor results in a signalling cascade leading to the formation of a protein complex (STAT1, STAT2 and IRF9) named IFN-stimulated factor 3 (ISGF3) that translocates to the nucleus. ISGF3 binds to DNA sequences (IFN-stimulated response elements or ISRE) present in the promoters of hundreds of genes and promotes their transcription. The sensors triggering IFN production include members of the Toll-like receptor (TLR) family present at the cell surface or in endosomal compartments, namely TLR3, TLR4, TLR7, TLR8 and TLR9 (Jefferies & Fitzgerald, 2005). Signalling through TLRs leads to IRF3, IRF7 and NF-
B activation, factors controlling IFN-/ gene transcription (Moynagh, 2005).
Respiratory syncytial virus (RSV) has been reported to be a poor inducer of IFN-/ and partially resistant to its antiviral activity, indicating that this virus has evolved strategies to overcome the IFN system in order to establish a productive infection (Conzelmann, 2005). However, the capacity of HRSV to inhibit production of IFN- in epithelial cells varies among virus strains (Schlender et al., 2005). Recombinant deletion mutants of RSV demonstrated that the two non-structural proteins NS1 and NS2 cooperatively suppress the induction of IFN-/ and the IFN-mediated antiviral response (Schlender et al., 2000; Bossert et al., 2003; Spann et al., 2004). It has been determined that NS1 and NS2 block activation of IRF3 in infected cells (Bossert et al., 2003) and that NS2 downregulates STAT2 expression (Ramaswamy et al., 2004, 2006; Lo et al., 2005). Furthermore, the host range of RSV infection depends upon the ability of NS1 and NS2 to neutralize the IFN-/ response of the host cell (Bossert & Conzelmann, 2002; Valarcher et al., 2003). NS proteins of BRSV might have evolved with the bovine host to optimally counteract bovine-cell antiviral responses.
HRSV replicates at low levels in the respiratory tract of rodents and these animals do not exhibit overt respiratory-tract disease, even if weight loss is observed when high titres of virus are used for inoculation. However the absence of a functional type I IFN pathway does not result in increased virus replication in vivo, indicating that IFN is not the sole host range-limiting factor (Johnson et al., 2005). Previous trials to infect mice with BRSV have been poorly reported, probably because of the inefficiency of this infection. However, recently, Almeida et al. (2004) have detected BRSV replication at low levels after experimental inoculation of wild-type BALB/c mice. We therefore wondered whether BRSV replication in mice could depend on its ability to escape from the host IFN response.
To address this issue, we took advantage of mice deficient in a functional type I IFN receptor (Müller et al., 1994). We derived primary cells from type 1 IFN receptor-competent (IFN-/R+/+) or -deficient (IFN-/R–/–) mice. IFN-/R–/– and IFN-/R+/+ cells were isolated from either lung or muscle tissue of 1-day-old pups and passaged once in Eagle's minimal essential medium (Gibco) containing 10 % gamma-irradiated fetal calf serum before BRSV infection. Primary cell cultures were plated on six-well plates and infected with BRSV at an m.o.i. of 10–2 for 2 h at 37 °C with the BRSV Maryland strain grown on bovine turbinate cells. We monitored the propagation of BRSV on IFN-/R–/– or IFN-/R+/+ cells by immunostaining with anti-F mAbs (Serotec) and a secondary fluorescein isothiocyanate-conjugated anti-mouse Ig (P.A.R.I.S.). Mock- or BRSV-infected cells were fixed with paraformaldehyde (4 %) at 0, 4, 8 and 12 days post-infection. As shown in Fig. 1(a), BRSV propagated on IFN-/R–/– mouse-derived lung cells as efficiently as on bovine turbinate cells, but no virus was detected on IFN-/R+/+ cells. At 8 days post-infection, about 50 % of the cell monolayer was infected, and all of the cells were infected at 12 days post-infection. The formation of syncytia was revealed by staining cell nuclei with the fluorescent dye Hoechst 33342 (Fig. 1a). Viral titres were determined to be 1.5±0.5x103 p.f.u. ml–1 at 8 days post-infection with IFN-/R–/– cells, which are equivalent to viral titres obtained with bovine-cell infections (data not shown). Only traces of virus were detected in IFN-/R+/+ cells infected with BRSV at 8 days post-infection or later. Similar results were obtained when using cells derived from muscles (data not shown) and when using two other BRSV strains, A2Gelfi and W2, that were isolated from calves (Valarcher et al., 2000) [results obtained with A2 strain are shown in Fig. 1(b)]. All of the virus strains used in this study were able to replicate on Madin–Darby bovine kidney (MDBK) cells (Fig. 1b), which are IFN-competent (Schlender et al., 2000; Bossert & Conzelmann, 2002).
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/R+/+ cells, it is expected that BRSV NS proteins are unable to block either IFN-/ synthesis in murine cells or IFN-induced antiviral pathways. Cells derived from IFN-/R–/– mice have lost the capacity to respond to IFN-induced stimuli, but might be still able to synthesize IFN-/. We thus analysed the secretion of type I IFN in IFN-/R–/– cells infected with BRSV. Type I IFN was monitored by a biological assay (Riffault et al., 1996). As shown in Table 1, IFN was detected in the supernatants of IFN-/R–/– cells infected with BRSV at 8 and 12 days post-infection, but not in IFN-/R+/+ infected cells. These results showed clearly that BRSV was unable to inhibit IFN synthesis in murine cells after infection. Besides, BRSV induced a higher level of IFN-/ in lung primary-cell cultures, where it also replicated more rapidly than in muscle primary-cell culture. These findings point towards a positive correlation between virus replication and IFN-/ induction.
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/R–/– cells infected with BRSV, showing that BRSV was unable to block IFN synthesis in these cells. The NS1 and NS2 proteins were shown to block the synthesis of IFN (Schlender et al., 2000; Spann et al., 2004). Our results suggest strongly that bovine NS1 and NS2 proteins are unable to interact with murine cellular factors involved in the IFN-activation pathway. The resulting establishment of antiviral mechanisms could explain, at least in part, the poor ability of BRSV to infect mice.
To investigate the factors limiting virus infection in mice, we first aimed to select an appropriate BRSV strain. For that purpose, we compared in BALB/c mice the Maryland BRSV strain with a field strain, 3761, isolated from infected calves and never passaged in cell culture (G. Meyer, personal communication). Mice were infected intranasally with 104 p.f.u. Maryland or 500 p.f.u. isolate 3761 at day 0 and sacrificed at various time points up to 23 days post-infection. Weight and clinical symptoms (ruffled fur) were recorded daily. Virus replication in lung was determined by using semi-quantitative RT-PCR to amplify the nucleoprotein gene [primer sequences as described by Boxus et al. (2005)] on RNA samples normalized for their -actin mRNA content. We did not observe symptoms of disease or weight loss, and recorded little (if any) inflammatory infiltrate in bronchoalveolar lavage (data not shown). However, semi-quantitative PCR showed some level of virus replication in lung, peaking at day 5 after infection and disappearing after 8 days. Interestingly, isolate 3761 replicated more strongly and for a longer period of time (up to day 12) than the Maryland strain (data not shown). We therefore decided to infect IFN-/R+/+ and IFN-/R–/– mice with the BRSV 3761 field isolate. We performed two independent experiments with four mice per genetic status and time point. Neither the level of virus replication nor the kinetics of replication was altered significantly by the absence of a functional IFN-signalling pathway (Fig. 2). Besides, both IFN-/R+/+ and IFN-/R–/– mice failed to show clinical symptoms (data not shown). By using the same mouse strains, similar findings were obtained previously with HRSV (Johnson et al., 2005). These and our data point to more complex mechanisms than type I IFN controlling virus replication and pathology in vivo.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Bossert, B. & Conzelmann, K.-K. (2002). Respiratory syncytial virus (RSV) nonstructural (NS) proteins as host range determinants: a chimeric bovine RSV with NS genes from human RSV is attenuated in interferon-competent bovine cells. J Virol 76, 4287–4293.
Bossert, B., Marozin, S. & Conzelmann, K.-K. (2003). Nonstructural proteins NS1 and NS2 of bovine respiratory syncytial virus block activation of interferon regulatory factor 3. J Virol 77, 8661–8668.
Boxus, M., Letellier, C. & Kerkhofs, P. (2005). Real time RT-PCR for the detection and quantitation of bovine respiratory syncytial virus. J Virol Methods 125, 125–130.[CrossRef][Medline]
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Riffault, S., Eloranta, M. L., Carrat, C., Sandberg, K., Charley, B. & Alm, G. (1996). Herpes simplex virus induces appearance of interferon-alpha/beta-producing cells and partially interferon-alpha/beta-dependent accumulation of leukocytes in murine regional lymph nodes. J Interferon Cytokine Res 16, 1007–1014.[Medline]
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Schlender, J., Hornung, V., Finke, S. & 7 other authors (2005). Inhibition of Toll-like receptor 7- and 9-mediated alpha/beta interferon production in human plasmacytoid dendritic cells by respiratory syncytial virus and measles virus. J Virol 79, 5507–5515.
Spann, K. M., Tran, K. C., Chi, B., Rabin, R. L. & Collins, P. L. (2004). Suppression of the induction of alpha, beta, and lambda interferons by the NS1 and NS2 proteins of human respiratory syncytial virus in human epithelial cells and macrophages [corrected]. J Virol 78, 4363–4369.
Valarcher, J.-F., Schelcher, F. & Bourhy, H. (2000). Evolution of bovine respiratory syncytial virus. J Virol 74, 10714–10728.
Valarcher, J.-F., Furze, J., Wyld, S., Cook, R., Conzelmann, K.-K. & Taylor, G. (2003). Role of alpha/beta interferons in the attenuation and immunogenicity of recombinant bovine respiratory syncytial viruses lacking NS proteins. J Virol 77, 8426–8439.
Received 31 March 2006;
accepted 2 May 2006.
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