Pathogenesis of wild-type and leaderless foot-and-mouth disease virus in cattle.

Four calves were experimentally infected via aerosol with foot-and-mouth disease virus. Two were infected with a wild-type virus derived from a full-length infectious clone (A12-IC), and two were infected with a clone-derived virus lacking the leader gene (A12-LLV2), with euthanasia and tissue collection at 24 and 72 h postexposure (hpe). Clinical disease was apparent only in the animal given A12-IC and euthanized at 72 hpe. In situ hybridization revealed that the animal infected with A12-IC and euthanized at 24 hpe had abundant viral nucleic acid in the lung, present in clusters of positive cells in the respiratory bronchiolar epithelium and associated subepithelial regions. At 72 hpe in the A12-IC-infected calf, viral nucleic acid in the lung was present in interstitial areas, and in addition, viral nucleic acid was detectable in epithelial tissues around histologically apparent vesicles. In animals infected with A12-LLV2, viral nucleic acid was detectable in the lung at both 24 and 72 hpe, but staining revealed a more localized distribution with less nucleic acid than was found in animals given A12-IC. Therefore, it appears that after aerosol exposure to A12-IC, early replication is in the region of the lung, with subsequent dissemination to distal sites. In comparison, the A12-LLV2 virus is much less widely disseminated in the lung at 24 hpe, with no lesions or virus detectable in secondary sites at 72 hpe. The greatly reduced pathogenicity of A12-LLV2 may make it an excellent candidate for a modified live viral vaccine.     

Inhibition of L-deleted foot-and-mouth disease virus replication by alpha/beta interferon involves double-stranded RNA-dependent protein kinase

We previously demonstrated that the ability of foot-and-mouth disease virus (FMDV) to form plaques in cell culture is associated with the suppression of alpha/beta interferon (IFN-alpha/beta). In the present study, we used Escherichia coli-expressed porcine and bovine IFN-alpha or -beta individually to demonstrate that each was equally effective in inhibiting FMDV replication. The block in FMDV replication appeared to be at the level of protein translation, suggesting a role for double-stranded RNA-dependent protein kinase (PKR). In support of these findings, treatment of porcine and bovine cells with 2-aminopurine, an inhibitor of PKR, increased the yield of virus 8.8- and 11.2-fold, respectively, compared to that in untreated infected cells. In addition, results of FMDV infection in mouse embryonic fibroblast cells derived from gene knockout mice lacking the gene for RNase L(-/-) or PKR(-/-) or both indicated an important role for PKR in the inhibition of FMDV replication.     

Alpha/beta interferon protects against lethal West Nile virus infection by restricting cellular tropism and enhancing neuronal survival

West Nile virus (WNV) is a mosquito-borne flavivirus that is neurotropic in humans, birds, and other animals. While adaptive immunity plays an important role in preventing WNV spread to the central nervous system (CNS), little is known about how alpha/beta interferon (IFN-alpha/beta) protects against peripheral and CNS infection. In this study, we examine the virulence and tropism of WNV in IFN-alpha/beta receptor-deficient (IFN- alpha/betaR-/-) mice and primary neuronal cultures. IFN-alpha/betaR-/- mice were acutely susceptible to WNV infection through subcutaneous inoculation, with 100% mortality and a mean time to death (MTD) of 4.6 +/- 0.7 and 3.8+/- 0.5 days after infection with 10(0) and 10(2) PFU, respectively. In contrast, congenic wild-type 129Sv/Ev mice infected with 10(2) PFU showed 62% mortality and a MTD of 11.9 +/- 1.9 days. IFN-alpha/betaR-/- mice developed high viral loads by day 3 after infection in nearly all tissues assayed, including many that were not infected in wild-type mice. IFN-alpha/betaR-/- mice also demonstrated altered cellular tropism, with increased infection in macrophages, B cells, and T cells in the spleen. Additionally, treatment of primary wild-type neurons in vitro with IFN-beta either before or after infection increased neuronal survival independent of its effect on WNV replication. Collectively, our data suggest that IFN-alpha/beta controls WNV infection by restricting tropism and viral burden and by preventing death of infected neurons.     

Identification of a cell population that produces alpha/beta interferon in vitro and in vivo in response to noncytopathic bovine viral diarrhea virus

In vitro infection of bovine cells of many origins with the cytopathogenic bovine viral diarrhea virus (cpBVDV) results in the induction of alpha/beta interferon (IFN-alpha/beta), whereas noncytopathogenic BVDV (ncpBVDV) isolates have been shown not to induce IFN-alpha/beta in vitro. Similarly, cpBVDV induces IFN-alpha/beta in the early bovine fetus, but ncpBVDV does not. However, acute infection of naive cattle with ncpBVDV results in IFN-alpha/beta production. In this study, we identified and characterized a minor population of cells, present in lymph nodes that produce IFN-alpha in response to ncpBVDV. These cells expressed the myeloid markers CD14, CD11b, and CD172a but did not express CD4 and CD45RB. We also established that these cells produced IFN-alpha in the absence of detectable productive infection.     

Role of alpha/beta interferon in Venezuelan equine encephalitis virus pathogenesis: effect of an attenuating mutation in the 5' untranslated region

Venezuelan equine encephalitis virus (VEE) is an important equine and human pathogen of the Americas. In the adult mouse model, cDNA-derived, virulent V3000 inoculated subcutaneously (s.c.) causes high-titer peripheral replication followed by neuroinvasion and lethal encephalitis. A single change (G to A) at nucleotide 3 (nt 3) of the 5' untranslated region (UTR) of the V3000 genome resulted in a virus (V3043) that was avirulent in mice. The mechanism of attenuation by the V3043 mutation was studied in vivo and in vitro. Kinetic studies of virus spread in adult mice following s.c. inoculation showed that V3043 replication was reduced in peripheral organs compared to that of V3000, titers in serum also were lower, and V3043 was cleared more rapidly from the periphery than V3000. Because clearance of V3043 from serum began 1 to 2 days prior to clearance of V3000, we examined the involvement of alpha/beta interferon (IFN-alpha/beta) activity in VEE pathogenesis. In IFN-alpha/betaR(-/-) mice, the course of the wild-type disease was extremely rapid, with all animals dying within 48 h (average survival time of 30 h compared to 7.7 days in the wild-type mice). The mutant V3043 was as virulent as the wild type (100% mortality, average survival time of 30 h). Virus titers in serum, peripheral organs, and the brain were similar in V3000- and V3043-infected IFN-alpha/betaR(-/-) mice at all time points up until the death of the animals. Consistent with the in vivo data, the mutant virus exhibited reduced growth in vitro in several cell types except in cells that lacked a functional IFN-alpha/beta pathway. In cells derived from IFN-alpha/betaR(-/-) mice, the mutant virus showed no growth disadvantage compared to the wild-type virus, suggesting that IFN-alpha/beta plays a major role in the attenuation of V3043 compared to V3000. There were no differences in the induction of IFN-alpha/beta between V3000 and V3043, but the mutant virus was more sensitive than V3000 to the antiviral actions of IFN-alpha/beta in two separate in vitro assays, suggesting that the increased sensitivity to IFN-alpha/beta plays a major role in the in vivo attenuation of V3043.     

Alpha/beta interferon protects adult mice from fatal Sindbis virus infection and is an important determinant of cell and tissue tropism

Infection of adult 129 Sv/Ev mice with consensus Sindbis virus strain TR339 is subclinical due to an inherent restriction in early virus replication and viremic dissemination. By comparing the pathogenesis of TR339 in 129 Sv/Ev mice and alpha/beta interferon receptor null (IFN-alpha/betaR(-/-)) mice, we have assessed the contribution of IFN-alpha/beta in restricting virus replication and spread and in determining cell and tissue tropism. In adult 129 Sv/Ev mice, subcutaneous inoculation with 100 PFU of TR339 led to extremely low-level virus replication and viremia, with clearance under way by 96 h postinoculation (p.i.). In striking contrast, adult IFN-alpha/betaR(-/-) mice inoculated subcutaneously with 100 PFU of TR339 succumbed to the infection within 84 h. By 24 h p.i. a high-titer serum viremia had seeded infectious virus systemically, coincident with the systemic induction of the proinflammatory cytokines interleukin-12 (IL-12) p40, IFN-gamma, tumor necrosis factor alpha, and IL-6. Replicating virus was located in macrophage-dendritic cell (DC)-like cells at 24 h p.i. in the draining lymph node and in the splenic marginal zone. By 72 h p.i. virus replication was widespread in macrophage-DC-like cells in the spleen, liver, lung, thymus, and kidney and in fibroblast-connective tissue and periosteum, with sporadic neuroinvasion. IFN-alpha/beta-mediated restriction of TR339 infection was mimicked in vitro in peritoneal exudate cells from 129 Sv/Ev versus IFN-alpha/betaR(-/-) mice. Thus, IFN-alpha/beta protects the normal adult host from viral infection by rapidly conferring an antiviral state on otherwise permissive cell types, both locally and systemically. Ablation of the IFN-alpha/beta system alters the apparent cell and tissue tropism of the virus and renders macrophage-DC-lineage cells permissive to infection.     

Role of alpha/beta interferons in the attenuation and immunogenicity of recombinant bovine respiratory syncytial viruses lacking NS proteins

Alpha/beta interferons (IFN-alpha/beta) are not only a powerful first line of defense against pathogens but also have potent immunomodulatory activities. Many viruses have developed mechanisms of subverting the IFN system to enhance their virulence. Previous studies have demonstrated that the nonstructural (NS) genes of bovine respiratory syncytial virus (BRSV) counteract the antiviral effects of IFN-alpha/beta. Here we demonstrate that, in contrast to wild-type BRSVs, recombinant BRSVs (rBRSVs) lacking the NS proteins, and those lacking NS2 in particular, are strong inducers of IFN-alpha/beta in bovine nasal fibroblasts and bronchoalveolar macrophages. Furthermore, whereas the NS deletion mutants replicated to wild-type rBRSV levels in cells lacking a functional IFN-alpha/beta system, their replication was severely attenuated in IFN-competent cells and in young calves. These results suggest that the NS proteins block the induction of IFN-alpha/beta gene expression and thereby increase the virulence of BRSV. Despite their poor replication in the respiratory tract of young calves, prior infection with virus lacking either the NS1 or the NS2 protein induced serum antibodies and protection against challenge with virulent BRSV. The greater level of protection induced by the NS2, than by the NS1, deletion mutant, was associated with higher BRSV-specific antibody titers and greater priming of BRSV-specific, IFN-gamma-producing CD4(+) T cells. Since there were no detectable differences in the ability of these mutants to replicate in the bovine respiratory tract, the greater immunogenicity of the NS2 deletion mutant may be associated with the greater ability of this virus to induce IFN-alpha/beta.     

High resistance of human parainfluenza type 2 virus protein-expressing cells to the antiviral and anti-cell proliferative activities of alpha/beta interferons: cysteine-rich V-specific domain is required for high resistance to the interferons

Human parainfluenza type 2 virus (hPIV-2)-infected HeLa (HeLa-CA) cells and hPIV-2 V-expressing HeLa (HeLa-V) cells show high resistance to alpha/beta interferons (IFN-alpha/beta) irrespective of whether vesicular stomatitis virus or Sindbis virus is used as a challenge virus. When Sindbis virus is used, these cells show high susceptibility to human IFN-gamma. Furthermore, the multiplication of HeLa-V cells is not inhibited by IFN-alpha/beta. HeLa cells expressing the N-terminally truncated V protein show resistance to IFN-alpha/beta, showing that the IFN resistance determinant maps to the cysteine-rich V-specific domain. A complete defect of Stat2 is found in HeLa-CA and HeLa-V cells, whereas the levels of Stat1 expression are not significantly different among HeLa, HeLa-CA, HeLa-P, and HeLa-V cells, indicating that IFN-alpha/beta resistance of HeLa-CA and HeLa-V cells is due to a defect of Stat2. HeLa-SV41V cells show high resistance to all IFNs, and no expression of Stat1 can be detected. Stat2 mRNA is fully detected in HeLa-V cells. Stat2 was scarcely pulse-labeled in the HeLa-V cells, indicating that synthesis of Stat2 is suppressed or Stat2 is very rapidly degraded in HeLa-V cells. The V protein suppresses the in vitro translation of Stat2 mRNA more extensively than that of Stat1 mRNA. An extremely small amount of Stat2 can be detected in HeLa-V cells treated with proteasome inhibitors. The half-life of Stat2 is approximately 3.5 and 2 h in uninfected and hPIV-2-infected HeLa cells, respectively. This study shows that synthesis of Stat2 may be suppressed and Stat2 degradation is also enhanced in hPIV-2-infected HeLa and HeLa-V cells.     

Alpha and Gamma Interferons Inhibit Herpes Simplex Virus Type 1 Infection and Spread in Epidermal Cells after Axonal Transmission

The ability of alpha interferon (IFN-alpha) and IFN-gamma to inhibit transmission of herpes simplex virus type 1 (HSV-1) from neuronal axon to epidermal cells (ECs), and subsequent spread in these cells was investigated in an in vitro dual-chamber model consisting of human fetal dorsal root ganglia (DRG) innervating autologous skin explants and compared with direct HSV-1 infection of epidermal explants. After axonal transmission from HSV-1-infected DRG neurons, both the number and size of viral cytopathic plaques in ECs was significantly reduced by addition of recombinant IFN-gamma and IFN-alpha to ECs in the outer chamber in a concentration-dependent fashion. Inhibition was maximal when IFNs were added at the same time as the DRG were infected with HSV-1. The mean numbers of plaques were reduced by 52% by IFN-alpha, 36% by IFN-gamma, and by 62% when IFN-alpha and IFN-gamma were combined, and the mean plaque size was reduced by 64, 43, and 72%, respectively. Similar but less-inhibitory effects of both IFNs were observed after direct infection of EC explants, being maximal when IFNs were added simultaneously or 6 h before HSV-1 infection. These results show that both IFN-alpha and IFN-gamma can interfere with HSV-1 infection after axonal transmission and subsequent spread of HSV-1 in ECs by a direct antiviral effect. Therefore, both IFN-alpha and -gamma could contribute to the control of HSV-1 spread and shedding in a similar fashion in recurrent herpetic lesions.     

Alpha/beta interferons regulate murine gammaherpesvirus latent gene expression and reactivation from latency

Alpha/beta interferon (IFN-alpha/beta) protects the host from virus infection by inhibition of lytic virus replication in infected cells and modulation of the antiviral cell-mediated immune response. To determine whether IFN-alpha/beta also modulates the virus-host interaction during latent virus infection, we infected mice lacking the IFN-alpha/beta receptor (IFN-alpha/betaR(-/-)) and wild-type (wt; 129S2/SvPas) mice with murine gammaherpesvirus 68 (gammaHV68), a lymphotropic gamma-2-herpesvirus that establishes latent infection in B cells, macrophages, and dendritic cells. IFN-alpha/betaR(-/-) mice cleared low-dose intranasal gammaHV68 infection with wt kinetics and harbored essentially wt frequencies of latently infected cells in both peritoneum and spleen by 28 days postinfection. However, latent virus in peritoneal cells and splenocytes from IFN-alpha/betaR(-/-) mice reactivated ex vivo with >40-fold- and 5-fold-enhanced efficiency, respectively, compared to wt cells. Depletion of IFN-alpha/beta from wt mice during viral latency also significantly increased viral reactivation, demonstrating an antiviral function of IFN-alpha/beta during latency. Viral reactivation efficiency was temporally regulated in both wt and IFN-alpha/betaR(-/-) mice. The mechanism of IFN-alpha/betaR action was distinct from that of IFN-gammaR, since IFN-alpha/betaR(-/-) mice did not display persistent virus replication in vivo. Analysis of viral latent gene expression in vivo demonstrated specific upregulation of the latency-associated gene M2, which is required for efficient reactivation from latency, in IFN-alpha/betaR(-/-) splenocytes. These data demonstrate that an IFN-alpha/beta-induced pathway regulates gammaHV68 gene expression patterns during latent viral infection in vivo and that IFN-alpha/beta plays a critical role in inhibiting viral reactivation during latency.     

Bovine respiratory syncytial virus nonstructural proteins NS1 and NS2 cooperatively antagonize alpha/beta interferon-induced antiviral response

The functions of bovine respiratory syncytial virus (BRSV) nonstructural proteins NS1 and NS2 were studied by generation and analysis of recombinant BRSV carrying single and double gene deletions. Whereas in MDBK cells the lack of either or both NS genes resulted in a 5,000- to 10,000-fold reduction of virus titers, in Vero cells a moderate (10-fold) reduction was observed. Interestingly, cell culture supernatants from infected MDBK cells were able to restrain the growth of NS deletion mutants in Vero cells, suggesting the involvement of NS proteins in escape from cytokine-mediated host cell responses. The responsible factors in MDBK supernatants were identified as type I interferons by neutralization of the inhibitory effect with antibodies blocking the alpha interferon (IFN-alpha) receptor. Treatment of cells with recombinant universal IFN-alpha A/D or IFN-beta revealed severe inhibition of single and double deletion mutants, whereas growth of full-length BRSV was not greatly affected. Surprisingly, all NS deletion mutants were equally repressed, indicating an obligatory cooperation of NS1 and NS2 in antagonizing IFN-mediated antiviral mechanisms. To verify this finding, we generated recombinant rabies virus (rRV) expressing either NS1 or NS2 and determined their IFN sensitivity. In cells coinfected with NS1- and NS2-expressing rRVs, virus replication was resistant to doses of IFN which caused a 1,000-fold reduction of replication in cells infected with wild-type RV or with each of the NS-expressing rRVs alone. Thus, BRSV NS proteins have the potential to cooperatively protect an unrelated virus from IFN-alpha/beta mediated antiviral responses. Interestingly, BRSV NS proteins provided a more pronounced resistance to IFN in the bovine cell line MDBK than in cell lines of other origins, suggesting adaptation to host-specific antiviral responses. The findings described have a major impact on the design of live recombinant BRSV and HRSV vaccines.     

The role of alpha/beta and gamma interferons in development of immunity to influenza A virus in mice

During influenza virus infection innate and adaptive immune defenses are activated to eliminate the virus and thereby bring about recovery from illness. Both arms of the adaptive immune system, antibody neutralization of free virus and termination of intracellular virus replication by antiviral cytotoxic T cells (CTLs), play pivotal roles in virus elimination and protection from disease. Innate cytokine responses, such as alpha/beta interferon (IFN-alpha/beta) or IFN-gamma, can have roles in determining the rate of virus replication in the initial stages of infection and in shaping the initial inflammatory and downstream adaptive immune responses. The effect of these cytokines on the replication of pneumotropic influenza A virus in the respiratory tract and in the regulation of adaptive antiviral immunity was examined after intranasal infection of mice with null mutations in receptors for IFN-alpha/beta, IFN-gamma, and both IFNs. Virus titers in the lungs of mice unable to respond to IFNs were not significantly different from congenic controls for both primary and secondary infection. Likewise the mice were comparably susceptible to X31 (H3N2) influenza virus infection. No significant disruption to the development of normal antiviral CTL or antibody responses was observed. In contrast, mice bearing the disrupted IFN-alpha/beta receptor exhibited accelerated kinetics and significantly higher levels of neutralizing antibody activity during primary or secondary heterosubtypic influenza virus infection. Thus, these observations reveal no significant contribution for IFN-controlled pathways in shaping acute or memory T-cell responses to pneumotropic influenza virus infection but do indicate some role for IFN-alpha/beta in the regulation of antibody responses. Recognizing the pivotal role of CTLs and antibody in virus clearance, it is reasonable to assume a redundancy in IFN-mediated antiviral effects in pulmonary influenza. However, IFN-alpha/beta seems to be a valid factor in determining tissue tropism and replicative rates of highly virulent influenza virus strains as reported previously by others, and this aspect is discussed here.     

Antiviral protection by vesicular stomatitis virus-specific antibodies in alpha/beta interferon receptor-deficient mice.

The role of innate, alpha/beta interferon (IFN-alpha/beta)-dependent protection versus specific antibody-mediated protection against vesicular stomatitis virus (VSV) was evaluated in IFN-alpha/beta receptor-deficient mice (IFN-alpha/beta R0/0 mice). VSV is a close relative to rabies virus that causes neurological disease in mice. In contrast to normal mice, IFN-alpha/beta R0/0 mice were highly susceptible to infection with VSV because of ubiquitous high viral replication. Adoptive transfer experiments showed that neutralizing antibodies against the glycoprotein of VSV (VSV-G) protected these mice efficiently against systemic infection and against peripheral subcutaneous infection but protected only to a limited degree against intranasal infection with VSV. In contrast, VSV-specific T cells or antibodies specific for the nucleoprotein of VSV (VSV-N) were unable to protect IFN-alpha/beta R0/0 mice against VSV. These results demonstrate that mice are extremely sensitive to VSV if IFN-alpha/beta is not functional and that under these conditions, neutralizing antibody responses mediate efficient protection, but apparently only against extraneuronal infection.     

Differential induction of antiviral effects against West Nile virus in primary mouse macrophages derived from flavivirus-susceptible and congenic resistant mice by alpha/beta interferon and poly(I-C)

A cell model of primary macrophages isolated from the peritoneal cavity of flavivirus-susceptible and congenic resistant mice has been used to study the extent and kinetics of antiviral effects against West Nile virus upon priming with alpha/beta interferon (IFN-alpha/beta) or poly(I-C) (pIC). The pattern of flavivirus resistance expressed after priming of cells in this model was in good agreement with the pattern of flavivirus resistance described in the brains of the corresponding mouse strains. While priming with either IFN-alpha/beta or pIC completely blocked flavivirus replication in macrophages from resistant mice, it only transiently reduced flavivirus replication in macrophages from susceptible mice. It was only the combined pretreatment with IFN-alpha/beta and pIC that elicited strong antiviral responses that completely prevented flavivirus replication in macrophages from susceptible mice. Primary macrophages isolated from the blood of healthy human donors expressed a similar need for double-stranded RNA (dsRNA) cofactor in developing efficient antiviral responses against West Nile virus. These findings reveal that the inefficient IFN-alpha/beta-induced antiviral effects against flaviviruses in cells from susceptible hosts could be successfully complemented by an external dsRNA factor leading to the complete eradication of the virus. This treatment appears to compensate for the lack of an inborn resistance mechanism in cells from the susceptible host. Furthermore, it may also provide useful clues for the prevention and treatment of flavivirus infections.     

Role of interferon-gamma in inducing cytotoxicity of peripheral blood mononuclear leukocytes to bovine herpesvirus type 1 (BHV-1)-infected cells

In the present study, the possible role of interferon (IFN)-gamma on the induction of cytotoxic activity of peripheral blood mononuclear leukocytes (PBML) from BHV-1-immune cattle was investigated. Supernatants obtained from BHV-1-immune PBML, stimulated under conditions similar to those required to demonstrate cytotoxicity, contained an antiviral substance capable of inducing 2'-5'-oligoadenylate synthetase activity in MDBK cells and MHC class II antigen expression on epithelial cells. These supernatants also contained IFN-alpha, but were devoid of tumor necrosis factor and interleukin-2 biological activities. Further studies during primary infection and hyperimmunization with BHV-1 showed that IFN-gamma production and non-MHC-restricted cytotoxicity against BHV-1-infected targets always occurred concomitantly, suggesting that they represent an important part of the detectable CMI responses mounted against this virus. Furthermore, it was also demonstrated that cytotoxicity of PBML against BHV-1-infected cells was reduced with the addition of antibodies to bovine IFN-gamma to the cytotoxic assay. Bovine recombinant IFN-gamma was able to enhance the in vitro cytotoxic activity of PBML from immune cattle, but not from their nonimmune counterparts. This suggests that other factors, in addition to IFN-gamma, may be essential in the development of non-MHC-restricted cytotoxic responses during BHV-1 infection.     

Degradation of STAT1 and STAT2 by the V proteins of simian virus 5 and human parainfluenza virus type 2, respectively: consequences for virus replication in the presence of alpha/beta and gamma interferons

Human cell lines were isolated that express the V protein of either simian virus 5 (SV5) or human parainfluenza virus type 2 (hPIV2); the cell lines were termed 2f/SV5-V and 2f/PIV2-V, respectively. STAT1 was not detectable in 2f/SV5-V cells, and the cells failed to signal in response to either alpha/beta interferons (IFN-alpha and IFN-beta, or IFN-alpha/beta) or gamma interferon (IFN-gamma). In contrast, STAT2 was absent from 2f/PIV2-V cells, and IFN-alpha/beta but not IFN-gamma signaling was blocked in these cells. Treatment of both 2f/SV5-V and 2f/PIV2-V cells with a proteasome inhibitor allowed the respective STAT levels to accumulate at rates similar to those seen in 2fTGH cells, indicating that the V proteins target the STATs for proteasomal degradation. Infection with SV5 can lead to a complete loss of both phosphorylated and nonphosphorylated forms of STAT1 by 6 h postinfection. Since the turnover of STAT1 in uninfected cells is longer than 24 h, we conclude that degradation of STAT1 is the main mechanism by which SV5 blocks interferon (IFN) signaling. Pretreatment of 2fTGH cells with IFN-alpha severely inhibited both SV5 and hPIV2 protein synthesis. However, and in marked contrast, pretreatment of 2fTGH cells with IFN-gamma had little obvious effect on SV5 protein synthesis but did significantly reduce the replication of hPIV2. Pretreament with IFN-alpha or IFN-gamma did not induce an antiviral state in 2f/SV5-V cells, indicating either that the induction of an antiviral state is completely dependent on STAT signaling or that the V protein interferes with other, STAT-independent cell signaling pathways that may be induced by IFNs. Even though SV5 blocked IFN signaling, the addition of exogenous IFN-alpha to the culture medium of 2fTGH cells 12 h after a low-multiplicity infection with SV5 significantly reduced the subsequent cell-to-cell spread of virus. The significance of the results in terms of the strategy that these viruses have evolved to circumvent the IFN response is discussed.     

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.