Promotion of alpha/beta interferon induction during in vivo viral infection through alpha/beta interferon receptor/STAT1 system-dependent and -independent pathways

Viruses and viral components can be potent inducers of alpha/beta interferons (IFN-alpha/beta). In culture, IFN-alpha/beta prime for their own expression, in response to viruses, through interferon regulatory factor 7 (IRF-7) induction. The studies presented here evaluated the requirements for functional IFN receptors and the IFN signaling molecule STAT1 in IFN-alpha/beta induction during infections of mice with lymphocytic choriomeningitis virus (LCMV). At 24 h after infection, levels of induced IFN-alpha/beta in serum were reduced 90 to 95% in IFN-alpha/beta receptor-deficient (IFN-alpha/betaR(-/-)) and STAT1(-/-) mice compared to those in wild-type mice. However, at 48 h, these mice showed elevated expression in the serum whereas IFN-alpha/beta levels were still reduced >75% in IFN-alpha/betagammaR(-/-) mice even though the viral burden was heavy. Levels of IFN-beta, IFN-alpha4, and non-IFN-alpha4 subtype mRNA expression correlated with IFN-alpha/beta bioactivity, and all IFN-alpha/beta subtypes were coincidentally detectable. IRF-7 mRNA was induced under conditions of IFN-alpha/beta production, including late production in IFN-alpha/betaR(-/-) mice. These data demonstrate that the presence of the virus alone is not sufficient to induce IFN-alpha/beta during LCMV infection in vivo. Instead, autocrine amplification through the IFN-alpha/betaR is necessary for optimal induction. In the absence of a functional IFN-alpha/betaR, however, alternative mechanisms, independent of STAT1 but requiring a functional IFN-gammaR, take over.     

ISG15 is critical in the control of Chikungunya virus infection independent of UbE1L mediated conjugation

Chikungunya virus (CHIKV) is a re-emerging alphavirus that has caused significant disease in the Indian Ocean region since 2005. During this outbreak, in addition to fever, rash and arthritis, severe cases of CHIKV infection have been observed in infants. Challenging the notion that the innate immune response in infants is immature or defective, we demonstrate that both human infants and neonatal mice generate a robust type I interferon (IFN) response during CHIKV infection that contributes to, but is insufficient for, the complete control of infection. To characterize the mechanism by which type I IFNs control CHIKV infection, we evaluated the role of ISG15 and defined it as a central player in the host response, as neonatal mice lacking ISG15 were profoundly susceptible to CHIKV infection. Surprisingly, UbE1L^−/− mice, which lack the ISG15 E1 enzyme and therefore are unable to form ISG15 conjugates, displayed no increase in lethality following CHIKV infection, thus pointing to a non-classical role for ISG15. No differences in viral loads were observed between wild-type (WT) and ISG15^−/− mice, however, a dramatic increase in proinflammatory cytokines and chemokines was observed in ISG15^−/− mice, suggesting that the innate immune response to CHIKV contributes to their lethality. This study provides new insight into the control of CHIKV infection, and establishes a new model for how ISG15 functions as an immunomodulatory molecule in the blunting of potentially pathologic levels of innate effector molecules during the host response to viral infection.     

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.     

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.     

Development of a next-generation chikungunya virus vaccine based on the HydroVax platform

Chikungunya virus (CHIKV) is an emerging/re-emerging mosquito-borne pathogen responsible for explosive epidemics of febrile illness characterized by debilitating polyarthralgia and the risk of lethal infection among the most severe cases. Despite the public health risk posed by CHIKV, no vaccine is currently available. Using a site-directed hydrogen peroxide-based inactivation approach, we developed a new CHIKV vaccine, HydroVax-CHIKV. This vaccine technology was compared to other common virus inactivation approaches including β-propiolactone (BPL), formaldehyde, heat, and ultraviolet (UV) irradiation. Heat, UV, and BPL were efficient at inactivating CHIKV-181/25 but caused substantial damage to neutralizing epitopes and failed to induce high-titer neutralizing antibodies in vaccinated mice. HydroVax-CHIKV and formaldehyde-inactivated CHIKV retained intact neutralizing epitopes similar to live virus controls but the HydroVax-CHIKV approach demonstrated a more rapid rate of virus inactivation. HydroVax-CHIKV vaccination induced high neutralizing responses to homologous and heterologous CHIKV clades as well as to other alphaviruses including Mayaro virus, O’nyong’nyong virus, and Una virus. Following heterologous infection with CHIKV-SL15649, HydroVax-CHIKV-immunized mice were protected against viremia, CHIKV-associated arthritic disease, and lethal CHIKV infection by an antibody-dependent mechanism. In contrast, animals vaccinated with Heat- or UV-inactivated virus showed no protection against viremia in addition to demonstrating significantly exacerbated CD4⁺ T cell-mediated footpad swelling after CHIKV infection. Together, these results demonstrate the risks associated with using suboptimal inactivation methods that fail to elicit protective neutralizing antibody responses and show that HydroVax-CHIKV represents a promising new vaccine candidate for prevention of CHIKV-associated disease.     

Herpes simplex virus type 2 virion host shutoff protein regulates alpha/beta interferon but not adaptive immune responses during primary infection in vivo

The herpes simplex virus (HSV) virion host shutoff (vhs) protein, the product of the UL41 (vhs) gene, is an important determinant of HSV virulence. vhs has been implicated in HSV interference with host antiviral immune responses, down-regulating expression of major histocompatibility complex molecules to help HSV evade host adaptive immunity. The severe attenuation of vhs-deficient viruses in vivo could reflect their inability to escape immune detection. To test this hypothesis, BALB/c or congenic SCID mice were infected intravaginally (i.vag.) with the HSV type 2 (HSV-2) vhs null mutant 333d41 or the vhs rescue virus 333d41(R). vhs-deficient virus remained severely attenuated in SCID mice compared with rescue virus, indicating that vhs regulation of adaptive immune responses does not influence HSV pathogenesis during acute infection. Innate antiviral effectors remain intact in SCID mice; prominent among these is alpha/beta interferon (IFN-alpha/beta). The attenuation of HSV-2 vhs mutants could reflect their failure to suppress IFN-alpha/beta-mediated antiviral activity. To test this hypothesis, 129 and congenic IFN-alpha/beta receptor-deficient (IFN-alpha/betaR(-/-)) mice were infected i.vag. with wild-type virus, vhs null mutants 333-vhsB or 333d41, or the vhs rescue virus 333d41(R). Whereas vhs-deficient viruses showed greatly reduced replication in the genital mucosa of 129 mice compared with wild-type or vhs rescue viruses, they were restored to nearly wild-type levels of replication in IFN-alpha/betaR(-/-) mice over the first 2 days postinfection. Only wild-type and vhs rescue viruses caused severe genital disease and hind limb paralysis in 129 mice, but infection of IFN-alpha/betaR(-/-) mice restored the virulence of vhs-deficient viruses. vhs-deficient viruses replicated as vigorously as wild-type and rescue viruses in the nervous systems of IFN-alpha/betaR(-/-) mice. Restoration was specific for the vhs mutation, because thymidine kinase-deficient HSV-2 did not regain virulence or the capacity to replicate in the nervous systems of IFN-alpha/betaR(-/-) mice. Furthermore, the defect in the IFN-alpha/beta response was required for restoration of vhs-deficient virus replication and virulence, but the IFN-alpha/beta-stimulated protein kinase R pathway was not involved. Finally, vhs of HSV-2 has a unique capacity to interfere with the IFN-alpha/beta response in vivo, because an HSV-1 vhs null mutant did not recover replication and virulence after i.vag. inoculation into IFN-alpha/betaR(-/-) mice. These results indicate that vhs plays an important role early in HSV-2 pathogenesis in vivo by interfering with the IFN-alpha/beta-mediated antiviral response.     

Inhibition of Interferons by Ectromelia Virus

Ectromelia virus (EV) is an orthopoxvirus (OPV) that causes mousepox, a severe disease of laboratory mice. Mousepox is a useful model of OPV infection because EV is likely to be a natural mouse pathogen, unlike its close relatives vaccinia virus (VV) and variola virus. Several studies have highlighted the importance of mouse interferons (IFNs) in resistance to and recovery from EV infection, but little is known of the anti-IFN strategies encoded by the virus itself. We have determined that 12 distinct strains and isolates of EV encode soluble, secreted receptors for IFN-gamma (vIFN-gammaR) and IFN-alpha/beta (vIFN-alpha/betaR) that are homologous to those identified in other OPVs. We demonstrate for the first time that the EV vIFN-gammaR has the unique ability to inhibit the biological activity of mouse IFN-gamma. The EV vIFN-alpha/betaR was a potent inhibitor of human and mouse IFN-alpha and human IFN-beta but, surprisingly, was unable to inhibit mouse IFN-beta. The replication of all of the EVs included in our study and of cowpox virus was more resistant than VV to the antiviral effects induced in mouse L-929 cells by IFN-alpha/beta and IFN-gamma. Sequencing studies showed that this EV resistance is likely to be partly mediated by the double-stranded-RNA-binding protein encoded by an intact EV homolog of the VV E3L gene. The absence of a functional K3L gene, which encodes a viral eIF-2alpha homolog, in EV suggests that the virus encodes a novel mechanism to counteract the IFN response. These findings will facilitate future studies of the role of viral anti-IFN strategies in mousepox pathogenesis. Their significance in the light of earlier data on the role of IFNs in mousepox is discussed.     

The cell biology of Chikungunya virus infection

Chikungunya virus (CHIKV) infection causes a disease which appears to affect multiple cell types and tissues. The acute phase is manifested by a non-fatal febrile illness, polyarthralgia and maculopapular rashes in adults, but with recurrent arthralgia that may linger for months during convalescence. The issue of cellular and tissue tropism of CHIKV has elicited interest primarily because of this lingering incapacitating chronic joint pain, as well as clear encephalopathy in severe cases among neonates during the re-emergence of the virus in recent epidemics. The principle cell types productively infected by CHIKV are skin fibroblasts, epithelial cells and lymphoid tissues. There is controversy as to whether CHIKV productively infects haematopoietic cells and neurones/glia. CHIKV infection triggers rapid and robust innate immune responses which quickly clears the acute phase infection. However, significant acute as well as chronic infection of less obvious cell types, such as monocytes, neurones/glia or even CNS neural progenitors may conceivably occur. There is therefore a need to ascertain the full range potential of CHIKV tropism, fully understand the cellular responses triggered during the acute the convalescent phases, and explore possible cell types that might be the source of chronic problems associated with CHIKV infection.     

TNF-α Acts as an Immunoregulator in the Mouse Brain by Reducing the Incidence of Severe Disease Following Japanese Encephalitis Virus Infection

Japanese encephalitis virus (JEV) causes acute central nervous system (CNS) disease in humans, in whom the clinical symptoms vary from febrile illness to meningitis and encephalitis. However, the mechanism of severe encephalitis has not been fully elucidated. In this study, using a mouse model, we investigated the pathogenetic mechanisms that correlate with fatal JEV infection. Following extraneural infection with the JaOArS982 strain of JEV, infected mice exhibited clinical signs ranging from mild to fatal outcome. Comparison of the pathogenetic response between severe and mild cases of JaOArS982-infected mice revealed increased levels of TNF-α in the brains of severe cases. However, unexpectedly, the mortality rate of TNF-α KO mice was significantly increased compared with that of WT mice, indicating that TNF-α plays a protective role against fatal infection. Interestingly, there were no significant differences of viral load in the CNS between WT and TNF-α KO mice. However, exaggerated inflammatory responses were observed in the CNS of TNF-α KO mice. Although these observations were also obtained in IL-10 KO mice, the mortality and enhanced inflammatory responses were more pronounced in TNF-α KO mice. Our findings therefore provide the first evidence that TNF-α has an immunoregulatory effect on pro-inflammatory cytokines in the CNS during JEV infection and consequently protects the animals from fatal disease. Thus, we propose that the increased level of TNF-α in severe cases was the result of severe disease, and secondly that immunopathological effects contribute to severe neuronal degeneration resulting in fatal disease. In future, further elucidation of the immunoregulatory mechanism of TNF-α will be an important priority to enable the development of effective treatment strategies for Japanese encephalitis.     

Identification of Novel Compounds Inhibiting Chikungunya Virus-Induced Cell Death by High Throughput Screening of a Kinase Inhibitor Library

Chikungunya virus (CHIKV) is a mosquito-borne arthrogenic alphavirus that causes acute febrile illness in humans accompanied by joint pains and in many cases, persistent arthralgia lasting weeks to years. The re-emergence of CHIKV has resulted in numerous outbreaks in the eastern hemisphere, and threatens to expand in the foreseeable future. Unfortunately, no effective treatment is currently available. The present study reports the use of resazurin in a cell-based high-throughput assay, and an image-based high-content assay to identify and characterize inhibitors of CHIKV-infection in vitro. CHIKV is a highly cytopathic virus that rapidly kills infected cells. Thus, cell viability of HuH-7 cells infected with CHIKV in the presence of compounds was determined by measuring metabolic reduction of resazurin to identify inhibitors of CHIKV-associated cell death. A kinase inhibitor library of 4,000 compounds was screened against CHIKV infection of HuH-7 cells using the resazurin reduction assay, and the cell toxicity was also measured in non-infected cells. Seventy-two compounds showing ≥50% inhibition property against CHIKV at 10 µM were selected as primary hits. Four compounds having a benzofuran core scaffold (CND0335, CND0364, CND0366 and CND0415), one pyrrolopyridine (CND0545) and one thiazol-carboxamide (CND3514) inhibited CHIKV-associated cell death in a dose-dependent manner, with EC₅₀ values between 2.2 µM and 7.1 µM. Based on image analysis, these 6 hit compounds did not inhibit CHIKV replication in the host cell. However, CHIKV-infected cells manifested less prominent apoptotic blebs typical of CHIKV cytopathic effect compared with the control infection. Moreover, treatment with these compounds reduced viral titers in the medium of CHIKV-infected cells by up to 100-fold. In conclusion, this cell-based high-throughput screening assay using resazurin, combined with the image-based high content assay approach identified compounds against CHIKV having a novel antiviral activity - inhibition of virus-induced CPE - likely by targeting kinases involved in apoptosis.     

Establishment of hepatitis C virus replicon cell lines possessing interferon-resistant phenotype

To clarify the mechanism underlying resistance to interferon (IFN) by the hepatitis C virus (HCV) in patients with chronic hepatitis, we attempted to develop an IFN-resistant HCV replicon from the IFN-sensitive 50-1 replicon established previously. By treating 50-1 replicon cells with a prolonged low-dose treatment of IFN-alpha and then transfecting the total RNA derived from the IFN-alpha-treated replicon cells, we successfully obtained four clones (named 1, 3, 4, and 5) of HCV replicon cells that survived against IFN-alpha (200 IU/ml). These cloned cells were further treated with IFN-alpha or IFN-beta (increased gradually to 2000 or 1000 IU/ml, respectively). This led to four replicon cell lines (alphaR series) possessing the IFN-alpha-resistant phenotype and four replicon cell lines (betaR series) possessing the IFN-beta-resistant phenotype. Furthermore, we obtained an additional replicon cell line (alphaRmix) possessing the IFN-alpha-resistant phenotype by two rounds of prolonged treatment with IFN-alpha and RNA transfection as mentioned above. Characterization of these obtained HCV replicon cell lines revealed that the betaR series were highly resistant to both IFN-alpha and IFN-beta, although the alphaR series containing alphaRmix were only partially resistant to both IFN-alpha and IFN-beta. Genetic analysis of these HCV replicons found one common amino acid substitution in the NS4B and several additional amino acid substitutions in the NS5A of the betaR series, suggesting that these genetic alterations are involved in the IFN resistance of these HCV replicons. These newly established HCV replicon cell lines possessing IFN-resistant phenotypes are the first useful tools for understanding the mechanisms by which HCV acquires IFN resistance in vivo.     

Chronic Joint Disease Caused by Persistent Chikungunya Virus Infection Is Controlled by the Adaptive Immune Response

Chikungunya virus (CHIKV) is a reemerging mosquito-borne pathogen that causes incapacitating disease in humans characterized by intense joint pain that can persist for weeks, months, or even years. Although there is some evidence of persistent CHIKV infection in humans suffering from chronic rheumatologic disease symptoms, little is known about chronic disease pathogenesis, and no specific therapies exist for acute or chronic CHIKV disease. To investigate mechanisms of chronic CHIKV-induced disease, we utilized a mouse model and defined the duration of CHIKV infection in tissues and the associated histopathological changes. Although CHIKV RNA was readily detectable in a variety of tissues very early after infection, CHIKV RNA persisted specifically in joint-associated tissues for at least 16 weeks. Inoculation of Rag1^−/− mice, which lack T and B cells, resulted in higher viral levels in a variety of tissues, suggesting that adaptive immunity controls the tissue specificity and persistence of CHIKV infection. The presence of CHIKV RNA in tissues of wild-type and Rag1^−/− mice was associated with histopathological evidence of synovitis, arthritis, and tendonitis; thus, CHIKV-induced persistent arthritis is not mediated primarily by adaptive immune responses. Finally, we show that prophylactic administration of CHIKV-specific monoclonal antibodies prevented the establishment of CHIKV persistence, whereas therapeutic administration had tissue-specific efficacy. These findings suggest that chronic musculoskeletal tissue pathology is caused by persistent CHIKV infection and controlled by adaptive immune responses. Our results have significant implications for the development of strategies to mitigate the disease burden associated with CHIKV infection in humans.     

Immunotherapy with CpG oligonucleotides and antibodies to TNF-alpha rescues neonatal mice from lethal arenavirus-induced meningoencephalitis

Viral encephalitides are life-threatening diseases in neonates partly due to the irreversible damage inflammation causes to the CNS. This study explored the role of proinflammatory cytokines in the balance between controlling viral replication and eliciting pathologic immune responses in nonlytic viral encephalitis. We show that neonatal mice challenged with arenavirus Tacaribe (TCRV) develop a meningoencephalitis characterized by high IFN-gamma and TNF-alpha levels and mild T cell infiltration. Neutralization of the TNF-alpha using mAb was associated with lower chemokine expression, reduced T cell infiltration, and lower levels of IFN-gamma, and TNF-alpha in the CNS and led to 100% survival. Moreover, treatment with Abs to TNF-alpha improved mobility and increased survival even after the mice developed bilateral hind limb paralysis. Of note, animals treated with anti-TNF-alpha Abs alone did not clear the virus despite generating Abs to TCRV. Direct activation of the innate immune response using CpG oligodeoxynucleotides in combination with anti-TNF-alpha Abs resulted in 100% survival and complete viral clearance. To our knowledge, this is the first demonstration of the use of innate immune modulators plus Abs to TNF-alpha as therapeutics for a lethal neurotropic viral infection.     

Nodular Inflammatory Foci Are Sites of T Cell Priming and Control of Murine Cytomegalovirus Infection in the Neonatal Lung

Neonates, including mice and humans, are highly susceptible to cytomegalovirus (CMV) infection. However, many aspects of neonatal CMV infections such as viral cell tropism, spatio-temporal distribution of the pathogen as well as genesis of antiviral immunity are unknown. With the use of reporter mutants of the murine cytomegalovirus (MCMV) we identified the lung as a primary target of mucosal infection in neonatal mice. Comparative analysis of neonatal and adult mice revealed a delayed control of virus replication in the neonatal lung mucosa explaining the pronounced systemic infection and disease in neonates. This phenomenon was supplemented by a delayed expansion of CD8⁺ T cell clones recognizing the viral protein M45 in neonates. We detected viral infection at the single-cell level and observed myeloid cells forming “nodular inflammatory foci” (NIF) in the neonatal lung. Co-localization of infected cells within NIFs was associated with their disruption and clearance of the infection. By 2-photon microscopy, we characterized how neonatal antigen-presenting cells (APC) interacted with T cells and induced mature adaptive immune responses within such NIFs. We thus define NIFs of the neonatal lung as niches for prolonged MCMV replication and T cell priming but also as sites of infection control.     

Hematogenous vertical transmission of herpes simplex virus type 1 in mice

Herpes simplex virus type 1 (HSV-1) is a neurotropic virus that causes severe disease and death in newborn humans but, to date, it remains unclear how neonatal infection occurs. We show here that the vertical transmission of HSV-1 in mice is mainly hematogenous and involves the colonization of the neonate central nervous system (CNS). HSV-1 DNA was mainly detected in the blood and CNS of the offspring born to latently infected mothers; no significant differences were seen between the viral DNA concentrations in the blood of these mothers and their female progeny (either neonate or adult). The administration of acyclovir during gestation reduced or eliminated both the maternal and the neonatal viral DNA in the blood. Embryo transfer was performed to ensure (as far as possible) that only vertical hematogenous infection took place. Immunohistochemical analysis detected viral proteins in the encephalon of the offspring. Immunofluorescence studies provided immunoreactive evidence of HSV-1 proteins in the neurons of the hippocampus and showed that these viruses can molecularly reactivate after hyperthermia. Neonatal HSV-1 infection therefore appears to be mainly caused by hematogenous vertical transmission, and the viruses that colonize the offspring CNS are capable of molecular reactivation after a period of latency.     

Poly (I:C), an agonist of toll-like receptor-3, inhibits replication of the Chikungunya virus in BEAS-2B cells

ABSTRACT: BACKGROUND: Double-stranded RNA (dsRNA) and its mimic, polyinosinic acid: polycytidylic acid [Poly (I:C)], are recognized by toll-like receptor 3 (TLR3) and induce interferon (IFN)-beta in many cell types. Poly (I:C) is the most potent IFN inducer. In in vivo mouse studies, intraperitoneal injection of Poly (I:C) elicited IFN-alpha/beta production and natural killer (NK) cells activation. The TLR3 pathway is suggested to contribute to innate immune responses against many viruses, including influenza virus, respiratory syncytial virus, herpes simplex virus 2, and murine cytomegalovirus. In Chikungunya virus (CHIKV) infection, the viruses are cleared within 7-10 days postinfection before adaptive immune responses emerge. The innate immune response is important for CHIKV clearance. RESULTS: The effects of Poly (I:C) on the replication of CHIKV in human bronchial epithelial cells, BEAS-2B, were studied. Poly (I:C) suppressed cytopathic effects (CPE) induced by CHIKV infection in BEAS-2B cells in the presence of Poly (I:C) and inhibited the replication of CHIKV in the cells. The virus titers of Poly (I:C)-treated cells were much lower compared with those of untreated cells. CHIKV infection and Poly (I:C) treatment of BEAS-2B cells induced the production of IFN-beta and increased the expression of anti-viral genes, including IFN-alpha, IFN-beta, MxA, and OAS. Both Poly (I:C) and CHIKV infection upregulate the expression of TLR3 in BEAS-2B cells. CONCLUSIONS: CHIKV is sensitive to innate immune response induced by Poly (I:C). The inhibition of CHIKV replication by Poly (I:C) may be through the induction of TLR3, which triggers the production of IFNs and other anti-viral genes. The innate immune response is important to clear CHIKV in infected cells.     

IFN-alpha/beta signaling is required for polarization of cytokine responses toward a protective type 1 pattern during experimental cryptococcosis

The antiviral activities of type I IFNs have long been established. However, comparatively little is known of their role in defenses against nonviral pathogens. We examined here the effects of type I IFNs on host resistance against the model pathogenic yeast Cryptococcus neoformans. After intratracheal or i.v. challenge with this fungus, most mice lacking either the IFN-alpha/beta receptor (IFN-alpha/betaR) or IFN-beta died from unrestrained pneumonia and encephalitis, while all wild-type controls survived. The pulmonary immune response of IFN-alpha/betaR-/- mice was characterized by increased expression of IL-4, IL-13, and IL-10, decreased expression of TNF-alpha, IFN-gamma, inducible NO synthetase, and CXCL10, and similar levels of IL-12 mRNA, compared with wild-type controls. Histopathological analysis showed eosinophilic infiltrates in the lungs of IFN-alpha/betaR-/- mice, although this change was less extensive than that observed in similarly infected IFN-gammaR-deficient animals. Type I IFN responses could not be detected in the lung after intratracheal challenge. However, small, but statistically significant, elevations in IFN-beta levels were measured in the supernatants of bone marrow-derived macrophages or dendritic cells infected with C. neoformans. Our data demonstrate that type I IFN signaling is required for polarization of cytokine responses toward a protective type I pattern during cryptococcal infection.