Interferon Receptor Knockout Mice Research Articles

Transmission of Schmallenberg Virus during Winter, Germany

Transmission of Schmallenberg Virus during Winter, Germany

The Role of Interleukin-6 in the Expression of PD-1 and PDL-1 on Central Nervous System Cells following Infection with Theiler's Murine Encephalomyelitis Virus

Infection with Theiler's murine encephalomyelitis virus (TMEV) in the central nervous system (CNS) of susceptible mice results in an immune-mediated demyelinating disease which is considered a relevant viral model of human multiple sclerosis. We previously demonstrated that the expression of positive costimulatory molecules (CD40, CD80, and CD86) is higher on the microglia of TMEV-resistant C57BL/6 (B6) mice than the microglia of TMEV-susceptible SJL/J (SJL) mice. In this study, we analyzed the expression levels of the negative costimulatory molecules PD-1 and PDL-1 in the CNS of TMEV-infected SJL mice and B6 mice. Our results indicated that TMEV infection induces the expression of both PD-1 and PDL-1 on microglia and macrophages in the CNS but not in the periphery. The expression of PD-1 only on CNS-infiltrating macrophages and not on resident microglia was considerably higher (>4-fold) in TMEV-infected SJL mice than TMEV-infected B6 mice. We further demonstrated that interleukn-6 (IL-6) is necessary to induce the maximal expression of PDL-1 but not PD-1 after TMEV infection using IL-6-deficient mice and IL-6-transgenic mice in conjunction with recombinant IL-6. In addition, cells from type I interferon (IFN) receptor knockout mice failed to upregulate PD-1 and PDL-1 expression after TMEV infection in vitro, indicating that type I IFN signaling is associated with the upregulation. However, other IFN signaling may also participate in the upregulation. Taken together, these results strongly suggest that the expression of PD-1 and PDL-1 in the CNS is primarily upregulated following TMEV infection via type I IFN signaling and the maximal expression of PDL-1 additionally requires IL-6 signaling.

Measles virus expressed Helicobacter pylori neutrophil-activating protein significantly enhances the immunogenicity of poor immunogens

Helicobacter pylori neutrophil-activating protein (NAP) is a toll-like receptor 2 (TLR2) agonist and potent immunomodulator inducing Th1-type immune response. Here we present data about characterization of the humoral immune response against NAP-tagged antigens, encoded by attenuated measles virus (MV) vector platform, in MV infection susceptible type I interferon receptor knockout and human CD46 transgenic (Ifnarko-CD46Ge) mice. Immunogenicity of MV expressing a full-length human immunoglobulin lambda light chain (MV-lambda) was compared to that of MV expressing lambda-NAP chimeric protein (MV-lambda-NAP). MV-lambda-NAP immunized Ifnarko-CD46Ge mice developed significantly higher (6–20-fold) anti-lambda ELISA titers as compared to the MV-lambda-immunized control animal group, indicating that covalently-linked NAP co-expression significantly enhanced lambda immunogenicity. In contrast, ELISA titers against MV antigens were not significantly different between the animals vaccinated with MV-lambda or MV-lambda-NAP. NAP-tagged antigen expression did not affect development of protective anti-measles immunity. Both MV-lambda and MV-lambda-NAP-immunized groups showed strong virus neutralization serum titers in plaque reduction microneutralization test. These results demonstrated that MV-encoded lambda-NAP is highly immunogenic as compared to the unmodified full-length lambda chain. Boost of immune response to poor immunogens using live vectors expressing NAP-tagged chimeric antigens is an attractive approach with potential application in immunoprophylaxis of infectious diseases and cancer immunotherapy.

Vascular leak ensues a vigorous proinflammatory cytokine response to Tacaribe arenavirus infection in AG129 mice

BackgroundTacaribe virus (TCRV) is a less biohazardous relative of the highly pathogenic clade B New World arenaviruses that cause viral hemorrhagic fever syndromes and require handling in maximum containment facilities not readily available to most researchers. AG129 type I and II interferon receptor knockout mice have been shown to be susceptible to TCRV infection, but the pathogenic mechanisms contributing to the lethal disease are unclear.MethodsTo gain insights into the pathogenesis of TCRV infection in AG129 mice, we assessed hematologic and cytokine responses during the course of infection, as well as changes in the permeability of the vascular endothelium. We also treated TCRV-challenged mice with MY-24, a compound that prevents mortality without affecting viral loads during the acute infection, and measured serum and tissue viral titers out to 40 days post-infection to determine whether the virus is ultimately cleared in recovering mice.ResultsWe found that the development of viremia and splenomegaly precedes an elevation in white blood cells and the detection of high levels of proinflammatory mediators known to destabilize the endothelial barrier, which likely contributes to the increased vascular permeability and weight loss that was observed several days prior to when the mice generally succumb to TCRV challenge. In surviving mice treated with MY-24, viremia and liver virus titers were not cleared until 2–3 weeks post-infection, after which the mice began to recover lost weight. Remarkably, substantial viral loads were still present in the lung, spleen, brain and kidney tissues at the conclusion of the study.ConclusionsOur findings suggest that vascular leak may be a contributing factor in the demise of TCRV-infected mice, as histopathologic findings are generally mild to moderate in nature, and as evidenced with MY-24 treatment, animals can survive in the face of high viral loads.

Myxomavirus-Derived Serpin Prolongs Survival and Reduces Inflammation and Hemorrhage in an Unrelated Lethal Mouse Viral Infection

Lethal viral infections produce widespread inflammation with vascular leak, clotting, and bleeding (disseminated intravascular coagulation [DIC]), organ failure, and high mortality. Serine proteases in clot-forming (thrombotic) and clot-dissolving (thrombolytic) cascades are activated by an inflammatory cytokine storm and also can induce systemic inflammation with loss of normal serine protease inhibitor (serpin) regulation. Myxomavirus secretes a potent anti-inflammatory serpin, Serp-1, that inhibits clotting factor X (fX) and thrombolytic tissue- and urokinase-type plasminogen activators (tPA and uPA) with anti-inflammatory activity in multiple animal models. Purified serpin significantly improved survival in a murine gammaherpesvirus 68 (MHV68) infection in gamma interferon receptor (IFN-γR) knockout mice, a model for lethal inflammatory vasculitis. Treatment of MHV68-infected mice with neuroserpin, a mammalian serpin that inhibits only tPA and uPA, was ineffective. Serp-1 reduced virus load, lung hemorrhage, and aortic, lung, and colon inflammation in MHV68-infected mice and also reduced virus load. Neuroserpin suppressed a wide range of immune spleen cell responses after MHV68 infection, while Serp-1 selectively increased CD11c(+) splenocytes (macrophage and dendritic cells) and reduced CD11b(+) tissue macrophages. Serp-1 altered gene expression for coagulation and inflammatory responses, whereas neuroserpin did not. Serp-1 treatment was assessed in a second viral infection, mouse-adapted Zaire ebolavirus in wild-type BALB/c mice, with improved survival and reduced tissue necrosis. In summary, treatment with this unique myxomavirus-derived serpin suppresses systemic serine protease and innate immune responses caused by unrelated lethal viral infections (both RNA and DNA viruses), providing a potential new therapeutic approach for treatment of lethal viral sepsis.

Hazara virus infection is lethal for adult type I interferon receptor-knockout mice and may act as a surrogate for infection with the human-pathogenic Crimean–Congo hemorrhagic fever virus

Hazara virus (HAZV) is closely related to the Crimean-Congo hemorrhagic fever virus (CCHFV). HAZV has not been reported to cause human disease; work with infectious material can be carried out at containment level (CL)-2. By contrast, CCHFV causes a haemorrhagic fever in humans and requires CL-4 facilities. A disease model of HAZV infection in mice deficient in the type I interferon receptor is reported in this study. Dose-response effects were seen with higher doses, resulting in a shorter time to death and earlier detection of viral loads in organs. The lowest dose of 10 p.f.u. was still lethal in over 50 % of the mice. Histopathological findings were identified in the liver, spleen and lymph nodes, with changes similar to a recent mouse model of CCHFV infection. The findings demonstrate that inoculation of mice with HAZV may act as a useful surrogate model for the testing of antiviral agents against CCHFV.

Immunogenicity and efficacy of chimeric dengue vaccine (DENVax) formulations in interferon-deficient AG129 mice.

Formulations of chimeric dengue vaccine (DENVax) viruses containing the pre-membrane (prM) and envelope (E) genes of serotypes 1-4 expressed in the context of the attenuated DENV-2 PDK-53 genome were tested for safety, immunogenicity and efficacy in interferon receptor knock-out mice (AG129). Monovalent formulations were safe and elicited robust neutralizing antibody responses to the homologous virus and only limited cross-reactivity to other serotypes. A single dose of monovalent DENVax-1, -2, or -3 vaccine provided eighty or greater percent protection against both wild-type (wt) DENV-1 (Mochizuki strain) and DENV-2 (New Guinea C strain) challenge viruses. A single dose of monovalent DENVax-4 also provided complete protection against wt DENV-1 challenge and significantly increased the survival times after challenge with wt DENV-2. In studies using tetravalent mixtures, DENVax ratios were identified that: (i) caused limited viremia, (ii) induced serotype-specific neutralizing antibodies to all four DENV serotypes with different hierarchies, and (iii) conferred full protection against clinical signs of disease following challenge with either wt DENV-1 or DENV-2 viruses. Overall, these data highlight the immunogenic profile of DENVax, a novel candidate tetravalent dengue vaccine and the advantage of sharing a common attenuated genomic backbone among the DENVax monovalent vaccines that confer protection against homologous or heterologous virus challenge.

Unique signatures of long noncoding RNA expression in response to virus infection and altered innate immune signaling.

Studies of the host response to virus infection typically focus on protein-coding genes. However, non-protein-coding RNAs (ncRNAs) are transcribed in mammalian cells, and the roles of many of these ncRNAs remain enigmas. Using next-generation sequencing, we performed a whole-transcriptome analysis of the host response to severe acute respiratory syndrome coronavirus (SARS-CoV) infection across four founder mouse strains of the Collaborative Cross. We observed differential expression of approximately 500 annotated, long ncRNAs and 1,000 nonannotated genomic regions during infection. Moreover, studies of a subset of these ncRNAs and genomic regions showed the following. (i) Most were similarly regulated in response to influenza virus infection. (ii) They had distinctive kinetic expression profiles in type I interferon receptor and STAT1 knockout mice during SARS-CoV infection, including unique signatures of ncRNA expression associated with lethal infection. (iii) Over 40% were similarly regulated in vitro in response to both influenza virus infection and interferon treatment. These findings represent the first discovery of the widespread differential expression of long ncRNAs in response to virus infection and suggest that ncRNAs are involved in regulating the host response, including innate immunity. At the same time, virus infection models provide a unique platform for studying the biology and regulation of ncRNAs.

Murine Gammaherpesvirus 68 LANA Is Essential for Virus Reactivation from Splenocytes but Not Long-Term Carriage of Viral Genome

ORF73, which encodes the latency-associated nuclear antigen (LANA), is a conserved gamma-2-herpesvirus gene. The murine gammaherpesvirus 68 (MHV68) LANA (mLANA) is critical for efficient virus replication and the establishment of latent infection following intranasal inoculation. To test whether the initial host immune response limits the capacity of mLANA-null virus to traffic to and establish latency in the spleen, we infected type I interferon receptor knockout (IFN-alpha/betaR(-/-)) mice via intranasal inoculation and observed the presence of viral genome-positive splenocytes at day 18 postinfection at approximately 10-fold-lower levels than in the genetically repaired marker rescue-infected mice. However, no mLANA-null virus reactivation from infected IFN-alpha/betaR(-/-) splenocytes was observed. To more thoroughly define a role of mLANA in MHV68 infection, we evaluated the capacity of an mLANA-null virus to establish and maintain infection apart from restriction in the lungs of immunocompetent mice. At day 18 following intraperitoneal infection of C57BL/6 mice, the mLANA-null virus was able to establish a chronic infection in the spleen albeit at a 5-fold-reduced level. However, as in IFN-alpha/betaR(-/-) mice, little or no virus reactivation could be detected from mLANA-null virus-infected splenocytes upon explant. An examination of peritoneal exudate cells (PECs) following intraperitoneal inoculation revealed nearly equivalent frequencies of PECs harboring the mLANA-null virus relative to the marker rescue virus. Furthermore, although significantly compromised, mLANA-null virus reactivation from PECs was detected upon explant. Notably, at later times postinfection, the frequency of mLANA-null genome-positive splenocytes was indistinguishable from that of marker rescue virus-infected animals. Analyses of viral genome-positive splenocytes revealed the absence of viral episomes in mLANA-null infected mice, suggesting that the viral genome is integrated or maintained in a linear state. Thus, these data provide the first evidence that a LANA homolog is directly involved in the formation and/or maintenance of an extrachromosomal viral episome in vivo, which is likely required for the reactivation of MHV68.

Crimean-Congo hemorrhagic fever virus infection is lethal for adult type I interferon receptor-knockout mice

Crimean-Congo hemorrhagic fever virus (CCHFV) poses a great threat to public health due to its high mortality, transmission and geographical distribution. To date, there is no vaccine or specific treatment available and the knowledge regarding its pathogenesis is highly limited. Using a small-animal model system, this study showed that adult mice missing the type I interferon (IFN) receptor (IFNAR(-/-)) were susceptible to CCHFV and developed an acute disease with fatal outcome. In contrast, infection of wild-type mice (129 Sv/Ew) was asymptomatic. Viral RNA was found in all analysed organs of the infected mice, but the amount of CCHFV RNA was significantly higher in the IFNAR(-/-) mice than in the wild-type mice. Furthermore, the liver of IFNAR(-/-) mice was enlarged significantly, showing that IFN is important for limiting virus spread and protecting against liver damage in mice.

A Live AttenuatedBordetella pertussisCandidate Vaccine Does Not Cause Disseminating Infection in Gamma Interferon Receptor Knockout Mice

Bordetella pertussis is the cause of whooping cough and responsible for 300,000 infant deaths per annum. Current vaccines require 6 months to confer optimal immunity on infants, the population at highest risk. Recently, an attenuated strain of B. pertussis (BPZE1) has been developed to be used as a low-cost, live, intranasal, single-dose vaccine for newborns. Preclinical proof of concept has been established; however, it is necessary to evaluate the safety of BPZE1, especially in immunodeficient models, prior to human clinical trials. Here, the preclinical safety of BPZE1 was examined in well-characterized murine models. Immunocompetent and gamma interferon (IFN-gamma) receptor knockout mice were challenged by aerosol with either virulent B. pertussis or BPZE1. The two strains colonized the lung at equal levels, but inflammation was associated with carriage of only virulent bacteria. Virulent bacteria disseminated to the liver of IFN-gamma receptor-deficient mice, resulting in atypical pathology. In contrast, attenuated BPZE1 did not disseminate in either immunocompetent or immunodeficient mice and did not induce atypical pathology. In neonatal challenge models, virulent B. pertussis infection resulted in significant mortality of both immunodeficient and immunocompetent mice, whereas no mortality was observed for any neonatal mice challenged with BPZE1. BPZE1 was shown to elicit strong IFN-gamma responses in mice, equivalent to those elicited by the virulent streptomycin-resistant B. pertussis Tohama I derivative BPSM, also inducing immunoglobulin G2a, a process requiring TH1 cytokines in mice. These data indicate that a live attenuated whooping cough vaccine candidate shows no signs of disseminating infection in preclinical models but rather evokes an immunological profile associated with optimal protection against disease.

Selective Ablation of Virion Host Shutoff Protein RNase Activity Attenuates Herpes Simplex Virus 2 in Mice

The virion host shutoff (vhs) protein of herpes simplex virus (HSV) has endoribonuclease activity and rapidly reduces protein synthesis in infected cells through mRNA degradation. Herpes simplex virus 1 (HSV-1) and HSV-2 vhs mutants are highly attenuated in vivo, but replication and virulence are largely restored to HSV-2 vhs mutants in the absence of a type I interferon (IFN) response. The role of vhs in pathogenesis and the hindrance of the type I IFN response have classically been examined with viruses that completely lack vhs or express a truncated vhs protein. To determine whether RNase activity is the principal mechanism of vhs-mediated type I IFN resistance and virulence, we constructed a HSV-2 point mutant that synthesizes full-length vhs protein lacking RNase activity (RNase(-) virus). Wild-type and mutant HSV-2 vhs proteins coimmunoprecipitated with VP16 and VP22. vhs protein bearing the point mutation was packaged into the virion as efficiently as the wild-type vhs protein. Like a mutant encoding truncated vhs, the RNase(-) virus showed IFN-dependent replication that was restricted compared with that of the wild-type virus. The RNase(-) virus was highly attenuated in wild-type mice infected intravaginally, with reduced mucosal replication, disease severity, and spread to the nervous system comparable to those of the vhs truncation mutant. Surprisingly, in alpha/beta interferon (IFN-alpha/beta) receptor knockout mice, the vhs RNase mutant was more attenuated than the vhs truncation mutant in terms of disease severity and virus titer in vaginal swabs and central nervous system samples, suggesting that non-enzymatically active vhs protein interferes with efficient virus replication. Our results indicate that vhs enzymatic activity plays a complex role in vhs-mediated type I IFN resistance during HSV-2 infection.

Brucella requires a functional Type IV secretion system to elicit innate immune responses in mice

The virB operon, encoding a Type IV secretion system (T4SS), is essential for intracellular survival and persistent infection by Brucella spp. To better understand the role of the T4SS in evading host defence mechanisms and establishing chronic infection, we compared transcriptional profiles of the host response to infection with wild-type and virB mutant Brucella strains. Analysis of gene expression profiles in murine splenocytes 3 days after inoculation with wild-type Brucella strains revealed an inflammatory response, with a prominent upregulation of genes induced by both type I and type II interferons. Real-time RT-PCR showed that a group of genes from these pathways were induced by day 3 post infection and declined to baseline levels by day 7. In contrast, neither of the two virB mutant strains elicited a proinflammatory gene expression profile, demonstrating that the T4SS was required to trigger this response. Infection studies using type I interferon receptor knockout mice showed that a lack of type I interferon signalling did not affect Brucella replication during the first 4 weeks of infection. Thus, induction of type I interferons does not appear to be an essential mechanism by which the T4SS promotes persistent infection by Brucella.

Targeting 2A Protease by RNA Interference Attenuates Coxsackieviral Cytopathogenicity and Promotes Survival in Highly Susceptible Mice

Enteroviridae such as coxsackievirus B3 (CVB3) are important infectious agents involved in viral heart disease, hepatitis, and pancreatitis, but no specific antiviral therapy is available. The aim of the present study was to evaluate the impact of RNA interference on viral replication, cytopathogenicity, and survival. Small interfering RNA (siRNA) molecules were designed against the viral 2A region (siRNA-2A), which is considered to be highly conserved and essential for both virus maturation and host cytopathogenicity. siRNA-2A exhibited a significant protective effect on cell viability mediated by marked inhibition of CVB3 gene expression and viral replication. In highly susceptible type I interferon receptor-knockout mice, siRNA-2A led to significant reduction of viral tissue titers, attenuated tissue damage, and prolonged survival. Repeated siRNA-2A transfection was associated with a further improvement of survival. Various control siRNA molecules had no protective effect in vitro or in vivo. RNA interference directed against the 2A protease encoding genomic region effectively confers intracellular immunity toward CVB3-mediated cell injury and improves survival, suggesting a potential role for RNA interference for future treatment options targeting enteroviral diseases.

Nitric Oxide Limits the Expansion of Antigen-Specific T Cells in Mice Infected with the Microfilariae ofBrugia pahangi

Infection of BALB/c mice with the microfilariae (Mf) of the filarial nematode Brugia pahangi results in an antigen-specific proliferative defect that is induced by high levels of NO. Using carboxyfluorescein diacetate succinimydl ester and cell surface labeling, it was possible to identify a population of antigen-specific T cells from Mf-infected BALB/c mice that expressed particularly high levels of CD4 (CD4(hi)). These cells proliferated in culture only when inducible NO synthase was inhibited and accounted for almost all of the antigen-specific proliferative response under those conditions. CD4(hi) cells also expressed high levels of CD44, consistent with their status as activated T cells. A similar population of CD4(hi) cells was observed in cultures from Mf-infected gamma interferon receptor knockout (IFN-gammaR(-/-)) mice. Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling staining revealed that the CD4(+) T cells from Mf-infected wild-type mice were preferentially susceptible to apoptosis compared to CD4(+) T cells from IFN-gammaR(-/-) mice. These studies suggest that the expansion of antigen-specific T cells in Mf-infected mice is limited by NO.

Ongoing viral replication is required for gammaherpesvirus 68-induced vascular damage.

The role of autoimmunity in large-vessel vasculitis in humans remains unclear. We have previously shown that infection of gamma interferon receptor knockout (IFN-gamma R(-/-)) mice with gammaherpesvirus 68 (gamma HV68) results in severe inflammation of the large elastic arteries that is pathologically similar to the lesions observed in Takayasu's arteritis, the nongranulomatous variant of temporal arteritis, and Kawasaki's disease (K. E. Weck et al., Nat. Med. 3:1346-1353, 1997). Here we define the mechanism of damage to the elastic arteries. We show that there is a persistent productive infection of the media of the large elastic vessels. In addition, we demonstrate that persistent virus replication is necessary for chronic arteritis, since antiviral therapy of mice with established disease resulted in increased survival, clearance of viral antigen from the media of the affected vessel, and dramatic amelioration of arteritic lesions. These data argue that ongoing virus replication, rather than autoimmunity, is the cause of gamma HV68-induced elastic arteritis.

Apoptotic regulation of T cells and absence of immune deficiency in virus-infected gamma interferon receptor knockout mice.

Acute viral infections often induce a transient period of immune deficiency in which the host's T cells fail to proliferate in response to T-cell mitogens and fail to make an antigen-specific memory recall response. This has been associated with the enhanced sensitivity of these highly activated T cells to undergo apoptosis, or activation-induced cell death (AICD), upon T-cell receptor ligation. Here we show that gamma interferon receptor-deficient (IFN-gamma R-/-) mice mount a T-cell response to lymphocytic choriomeningitis virus (LCMV) infection but fail to undergo the transient immune deficiency. Instead, their T cells were hyperproliferative and relatively, but not completely, resistant to AICD. The immune response returned to homeostasis, but with delayed kinetics, in parallel with delayed clearance of the virus. Wild-type mice receiving high doses of disseminating LCMV Clone 13 are known to undergo clonal exhaustion of their virus-specific cytotoxic T lymphocytes (CTL). To determine whether this process was mediated by AICD associated with IFN-gamma or with Fas-Fas ligand interactions, LCMV-specific precursor CTL frequencies were examined in LCMV Clone 13-infected IFN-gamma R-/- or lpr (Fas-deficient) mice. In both instances, viral persistence was established and CTL precursors were greatly eliminated. This finding indicates that clonal exhaustion of CTL does not require IFN-gamma or Fas, even though both molecules influence AICD and the transient immune deficiency seen in the LCMV infection.