Identification of genes involved in the host response to neurovirulent alphavirus infection

Single-amino-acid mutations in Sindbis virus proteins can convert clinically silent encephalitis into uniformly lethal disease. However, little is known about the host gene response during avirulent and virulent central nervous system (CNS) infections. To identify candidate host genes that modulate alphavirus neurovirulence, we utilized GeneChip Expression analysis to compare CNS gene expression in mice infected with two strains of Sindbis virus that differ by one amino acid in the E2 envelope glycoprotein. Infection with Sindbis virus, dsTE12H (E2-55 HIS), resulted in 100% mortality in 10-day-old mice, whereas no disease was observed in mice infected with dsTE12Q (E2-55 GLN). dsTE12H, compared with dsTE12Q, replicated to higher titers in mouse brain and induced more CNS apoptosis. Infection with the neurovirulent dsTE12H strain was associated with both a greater number of host genes with increased expression and greater changes in levels of host gene expression than was infection with the nonvirulent dsTE12Q strain. In particular, dsTE12H infection resulted in greater increases in the levels of mRNAs encoding chemokines, proteins involved in antigen presentation and protein degradation, complement proteins, interferon-regulated proteins, and mitochondrial proteins. At least some of these increases may be beneficial for the host, as evidenced by the demonstration that enforced expression of the antiapoptotic mitochondrial protein peripheral benzodiazepine receptor (PBR) protects neonatal mice against lethal Sindbis virus infection. Thus, our findings identify specific host genes that may play a role in the host protective or pathologic response to neurovirulent Sindbis virus infection.     

Alphavirus-induced apoptosis in mouse brains correlates with neurovirulence.

Sindbis virus induces apoptotic cell death in cultured cell lines, raising the possibility that apoptosis of infected neurons and other target cells in vivo may contribute to the resulting disease and mortality. To investigate the role of apoptosis in Sindbis virus pathogenesis, infected mouse brains were assayed by the in situ terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling technique and for DNA ladder formation. Infection with recombinant Sindbis virus strain 633 resulted in widespread apoptosis in newborn mouse brains and spinal cords, but few apoptotic cells were observed following infection of 2-week-old animals. This finding correlates with the age-dependent mortality observed in mice. The more neurovirulent virus TE, which differs from 633 by a single amino acid in the E2 glycoprotein, induced significant apoptosis in brains and spinal cords of 2-week-old animals, consistent with its ability to cause fatal disease in older animals. Double-labeling experiments demonstrated that the apoptotic cells were also infected with Sindbis virus. Thus, Sindbis virus-induced apoptosis appears to be a result of virus infection and is likely to reflect pathogenic mechanisms for other viruses.     

Molecular analysis of neurovirulent strains of Sindbis virus that evolve during persistent infection of scid mice.

To understand the role of tissue-specific adaptation and antibody-induced selectional pressures in the evolution of neurovirulent viruses, we analyzed three strains of Sindbis virus isolated from the brains of persistently infected scid mice and four strains of Sindbis virus isolated from the brains of scid mice with viral reactivation following immune serum treatment. For each viral isolate, we tested neurovirulence in weanling BALB/c mice and sequenced regions of the E2 and E1 envelope glycoprotein genes that are known to contain important determinants of Sindbis virus neurovirulence. One strain isolated from a persistently infected scid mouse and two strains isolated from scid mice with viral reactivation were neurovirulent, resulting in mortality in 80 to 100% of weanling BALB/c mice. All three neurovirulent strains contained an A-->U change at nucleotide 8795, which predicts a Gln-->His substitution at E2 amino acid position 55. No nucleotide changes were detected in the other sequenced regions of the E2 and E1 envelope glycoprotein genes or in the avirulent isolates. Our findings indicate that tissue-specific adaptations, rather than antibody-induced selectional pressures, are a critical determinant of the evolution of neurovirulent strains of Sindbis virus and provide evidence that E2 His-55 is an important neuroadaptive mutation that confers neurovirulence properties on Sindbis virus.     

Effect of E2 envelope glycoprotein cytoplasmic domain mutations on Sindbis virus pathogenesis.

The cytoplasmic domain of the E2 envelope glycoprotein is important in Sindbis virus assembly, but little is known about its role in the pathogenesis of Sindbis virus encephalitis. To investigate its role in viral pathogenesis, we constructed six recombinant viruses containing site mutations in the E2 cytoplasmic domain, using the neurovirulent background strain, TE12. Our findings demonstrate that the E2 cytoplasmic domain is a determinant of Sindbis virus growth and neurovirulence in suckling mice as well as persistent infection in weanling scid mice. They also suggest that the tyrosine, serine, or threonine residues are not essential for replication in mouse brain or anti-E2 monoclonal antibody-mediated restriction of Sindbis virus replication.     

Long term intraparenchymal Ig secretion after acute viral encephalitis in mice.

Oligoclonal bands in the cerebrospinal fluid indicate intrathecal synthesis of Ig of restricted heterogeneity and are associated with a number of central nervous system inflammatory diseases. To gain better insight into the persistence of oligoclonal bands found in the central nervous system we studied mice infected with Sindbis virus (SV), a RNA virus that causes an acute, nonfatal encephalitis in mice. SV was inoculated intracerebrally into weanling mice and brains and spleens were harvested at various time points long after the acute encephalitis had resolved. A modified enzyme-linked immunoassay was used to study cultured B cells separated from the brain and spleen for their Ig isotype expression and specificity for SV. We used the polymerase chain reaction technique to detect SV RNA in brain. Three mo after inoculation 47% of the B cells found in brain are secreting antibody specific for SV structural proteins. By 1 yr 62% are SV specific. B cells secreting IgG2a predominate. Polymerase chain reaction data indicate that despite complete clearance of infectious virus by 7 days SV RNA is still present in brain at least 6 mo after infection. The data indicate that B cells in brain secrete antibody to SV long after the acute encephalitis has resolved. The persistence of SV RNA suggests that viral protein may continue to be made, providing the impetus for the continued presence of SV-specific B cells in the brain.     

Age-dependent pathogenesis of murine gammaherpesvirus 68 infection of the central nervous system

Gammaherpesvirus infection of the central nervous system (CNS) has been linked to various neurological diseases, including meningitis, encephalitis, and multiple sclerosis. However, little is known about the interactions between the virus and the CNS in vitro or in vivo. Murine gammaherpesvirus 68 (MHV-68 or _γHV-68) is genetically related and biologically similar to human gammaherpesviruses, thereby providing a tractable animal model system in which to study both viral pathogenesis and replication. In the present study, we show the successful infection of cultured neuronal cells, microglia, and astrocytes with MHV-68 to various extents. Upon intracerebroventricular injection of a recombinant virus (MHV-68/LacZ) into 4–5-week-old and 9–10-week-old mice, the 4–5-week-old mice displayed high mortality within 5–7 days, while the majority of the 9–10-week-old mice survived until the end of the experimental period. Until a peak at 3–4 days post-infection, viral DNA replication and gene expression were similar in the brains of both mouse groups, but only the 9–10-week-old mice were able to subdue viral DNA replication and gene expression after 5 days post-infection. Pro-inflammatory cytokine mRNAs of tumor necrosis factor-α, interleukin 1β, and interleukin 6 were highly induced in the brains of the 4–5-week-old mice, suggesting their possible contributions as neurotoxic factors in the agedependent control of MHV-68 replication of the CNS. These authors contributed equally to this work.     

Identification of interferon-stimulated gene 15 as an antiviral molecule during Sindbis virus infection in vivo

The innate immune response, and in particular the alpha/beta interferon (IFN-alpha/beta) system, plays a critical role in the control of viral infections. Interferons alpha and beta exert their antiviral effects through the induction of hundreds of interferon-induced (or -stimulated) genes (ISGs). While several of these ISGs have characterized antiviral functions, their actions alone do not explain all of the effects mediated by IFN-alpha/beta. To identify additional IFN-induced antiviral molecules, we utilized a recombinant chimeric Sindbis virus to express selected ISGs in IFN-alpha/beta receptor (IFN-alpha/betaR)(-/-) mice and looked for attenuation of Sindbis virus infection. Using this approach, we identified a ubiquitin homolog, interferon-stimulated gene 15 (ISG15), as having antiviral activity. ISG15 expression protected against Sindbis virus-induced lethality and decreased Sindbis virus replication in multiple organs without inhibiting the spread of virus throughout the host. We establish that, much like ubiquitin, ISG15 requires its C-terminal LRLRGG motif to form intracellular conjugates. Finally, we demonstrate that ISG15's LRLRGG motif is also required for its antiviral activity. We conclude that ISG15 can be directly antiviral.     

Apoptosis in the Mouse Central Nervous System in Response to Infection with Mouse-Neurovirulent Dengue Viruses

Apoptosis has been suggested as a mechanism by which dengue (DEN) virus infection may cause neuronal cell death (P. Desprès, M. Flamand, P.-E. Ceccaldi, and V. Deubel, J. Virol. 70:4090–4096, 1996). In this study, we investigated whether apoptotic cell death occurred in the central nervous system (CNS) of neonatal mice inoculated intracerebrally with DEN virus. We showed that serial passage of a wild-type human isolate of DEN virus in mouse brains selected highly neurovirulent variants which replicated more efficiently in the CNS. Infection of newborn mice with these neurovirulent variants produced fatal encephalitis within 10 days after inoculation. Virus-induced cell death and oligonucleosomal DNA fragmentation were observed in mouse brain tissue by day 9. Infected mouse brain tissue was assayed for apoptosis by in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and for virus replication by immunostaining of viral antigens and in situ hybridization. Apoptotic cell death and DEN virus replication were restricted to the neurons of the cortical and hippocampal regions. Thus, DEN virus-induced apoptosis in the CNS was a direct result of virus infection. In the murine neuronal cell line Neuro 2a, neuroadapted DEN virus variants showed infection patterns similar to those of the parental strain. However, DEN virus-induced apoptosis in these cells was more pronounced after infection with the neurovirulent variants than after infection with the parental strain.     

The interferon-inducible gene viperin restricts West Nile virus pathogenesis

Type I interferon (IFN) signaling coordinates an early antiviral program in infected and uninfected cells by inducing IFN-stimulated genes (ISGs) that modulate viral entry, replication, and assembly. However, the specific antiviral functions in vivo of most ISGs remain unknown. Here, we examined the contribution of the ISG viperin to the control of West Nile virus (WNV) in genetically deficient cells and mice. While modest increases in levels of WNV replication were observed for primary viperin(-/-) macrophages and dendritic cells, no appreciable differences were detected in deficient embryonic cortical neurons or fibroblasts. In comparison, viperin(-/-) adult mice infected with WNV via the subcutaneous or intracranial route showed increased lethality and/or enhanced viral replication in central nervous system (CNS) tissues. In the CNS, viperin expression was induced in both WNV-infected and adjacent uninfected cells, including activated leukocytes at the site of infection. Our experiments suggest that viperin restricts the infection of WNV in a tissue- and cell-type-specific manner and may be an important ISG for controlling viral infections that cause CNS disease.     

Protection against Fatal Sindbis Virus Encephalitis by Beclin, a Novel Bcl-2-Interacting Protein

bcl-2, the prototypic cellular antiapoptotic gene, decreases Sindbis virus replication and Sindbis virus-induced apoptosis in mouse brains, resulting in protection against lethal encephalitis. To investigate potential mechanisms by which Bcl-2 protects against central nervous system Sindbis virus infection, we performed a yeast two-hybrid screen to identify Bcl-2-interacting gene products in an adult mouse brain library. We identified a novel 60-kDa coiled-coil protein, Beclin, which we confirmed interacts with Bcl-2 in mammalian cells, using fluorescence resonance energy transfer microscopy. To examine the role of Beclin in Sindbis virus pathogenesis, we constructed recombinant Sindbis virus chimeras that express full-length human Beclin (SIN/beclin), Beclin lacking the putative Bcl-2-binding domain (SIN/beclinΔBcl-2BD), or Beclin containing a premature stop codon near the 5′ terminus (SIN/beclinstop). The survival of mice infected with SIN/beclin was significantly higher (71%) than the survival of mice infected with SIN/beclinΔBcl-2BD (9%) or SIN/beclinstop (7%) (P < 0.001). The brains of mice infected with SIN/beclin had fewer Sindbis virus RNA-positive cells, fewer apoptotic cells, and lower viral titers than the brains of mice infected with SIN/beclinΔBcl-2BD or SIN/beclinstop. These findings demonstrate that Beclin is a novel Bcl-2-interacting cellular protein that may play a role in antiviral host defense.     

Interferon-stimulated gene ISG12b2 is localized to the inner mitochondrial membrane and mediates virus-induced cell death

Interferons (IFNs) are crucial for host defence against viruses. Many IFN-stimulated genes (ISGs) induced by viral infection exert antiviral effects. Microarray analysis of gene expression induced in liver tissues of mice on dengue virus (DENV) infection has led to identification of the ISG gene ISG12b2. ISG12b2 is also dramatically induced on DENV infection of Hepa 1-6 cells (mouse hepatoma cell line). Here, we performed biochemical and functional analyses of ISG12b2. We demonstrate that ISG12b2 is an inner mitochondrial membrane (IMM) protein containing a cleavable mitochondrial targeting sequence and multiple transmembrane segments. Overexpression of ISG12b2 in Hepa 1-6 induced release of cytochrome c from mitochondria, disruption of the mitochondrial membrane potential, and activation of caspase-9, caspase-3, and caspase-8. Treatment of ISG12b2-overexpressing Hepa 1-6 with inhibitors of pan-caspase, caspase-9, or caspase-3, but not caspase-8, reduced apoptotic cell death, suggesting that ISG12b2 activates the intrinsic apoptotic pathway. Of particular interest, we further demonstrated that ISG12b2 formed oligomers, and that ISG12b2 was able to mediate apoptosis through both Bax/Bak-dependent and Bax/Bak-independent pathways. Our study demonstrates that the ISG12b2 is a novel IMM protein induced by IFNs and regulates mitochondria-mediated apoptosis during viral infection.     

Apoptosis in Alphavirus Encephalitis

Sindbis virus causes acute encephalitis in mice and serves as a useful model for encephalitic alphaviruses that infect humans. The outcome of infection is determined by whether infected neurons are resistant to virus-induced programmed cell death or activate their apoptotic pathway. The host immune response may also cause death of infected neurons. Determinants of neuronal apoptosis include the maturity of the neuron, the virulence of the infecting virus and the cellular immune response to infection. In many situations viral and cellular factors that decrease virus replication also decrease apoptosis. Antiviral antibody can downregulate virus replication in surviving neurons without affecting cell viability. Other innate and induced host immune responses can alter the outcome of infection without a change in virus production. Failure to induce apoptosis in infected neurons leads to long-term persistence of small amounts of viral RNA in the nervous system of infected mice despite the clearance of infectious virus. The molecular mechanisms that govern these pathogenesis factors are beginning to be elucidated.     

BCL-2 and BAX protect adult mice from lethal Sindbis virus infection but do not protect spinal cord motor neurons or prevent paralysis

Cellular proteins that regulate apoptotic cell death can modulate the outcome of Sindbis virus (SV) encephalitis in mice. Both endogenous and overexpressed BCL-2 and BAX proteins protect newborn mice from fatal SV infection by blocking apoptosis in infected neurons. To determine the effects of these cellular factors on the course of infection in older animals, a more neurovirulent SV vector (dsNSV) was constructed from a viral strain that causes both prominent spinal cord infection with hind-limb paralysis and death in weanling mice. This vector has allowed assessment of the effects of BCL-2 and BAX on both mortality and paralysis in these hosts. Similar to newborn hosts, weanling mice infected with dsNSV encoding BCL-2 or BAX survived better than animals infected with control viruses. This finding indicates that BCL-2 and BAX both protect neurons that mediate host survival. Neither cellular factor, however, could suppress the development of hind-limb paralysis or prevent the degeneration of motor neurons in the lumbar spinal cord. Infection of BAX knockout mice with dsNSV demonstrated that endogenous BAX also enhances the survival of animals but has no effect on paralysis. These findings for the spinal cord are consistent with earlier data showing that dying lumbar motor neurons do not exhibit an apoptotic morphology. Thus, divergent cell death pathways are activated in different target populations of neurons during neurovirulent SV infection of weanling mice.     

Glucocortiocoid Treatment of MCMV Infected Newborn Mice Attenuates CNS Inflammation and Limits Deficits in Cerebellar Development

Infection of the developing fetus with human cytomegalovirus (HCMV) is a major cause of central nervous system disease in infants and children; however, mechanism(s) of disease associated with this intrauterine infection remain poorly understood. Utilizing a mouse model of HCMV infection of the developing CNS, we have shown that peripheral inoculation of newborn mice with murine CMV (MCMV) results in CNS infection and developmental abnormalities that recapitulate key features of the human infection. In this model, animals exhibit decreased granule neuron precursor cell (GNPC) proliferation and altered morphogenesis of the cerebellar cortex. Deficits in cerebellar cortical development are symmetric and global even though infection of the CNS results in a non-necrotizing encephalitis characterized by widely scattered foci of virus-infected cells with mononuclear cell infiltrates. These findings suggested that inflammation induced by MCMV infection could underlie deficits in CNS development. We investigated the contribution of host inflammatory responses to abnormal cerebellar development by modulating inflammatory responses in infected mice with glucocorticoids. Treatment of infected animals with glucocorticoids decreased activation of CNS mononuclear cells and expression of inflammatory cytokines (TNF-α, IFN-β and IFNγ) in the CNS while minimally impacting CNS virus replication. Glucocorticoid treatment also limited morphogenic abnormalities and normalized the expression of developmentally regulated genes within the cerebellum. Importantly, GNPC proliferation deficits were normalized in MCMV infected mice following glucocorticoid treatment. Our findings argue that host inflammatory responses to MCMV infection contribute to deficits in CNS development in MCMV infected mice and suggest that similar mechanisms of disease could be responsible for the abnormal CNS development in human infants infected in-utero with HCMV.     

Viral determinants of age-dependent virulence of Sindbis virus for mice.

Many alphaviruses cause more severe disease in young animals than in older animals. The age-dependent resistance to severe disease is determined primarily by maturation of the host, but strains of virus can be selected that overcome the increased resistance of mature animals. Sindbis virus (SV) strain AR339 causes fatal encephalitis in newborn mice and nonfatal encephalitis in weanling mice, whereas NSV, a neuroadapted strain of SV, causes fatal encephalitis in weanling as well as newborn mice. We have previously shown that the E2 glycoprotein of NSV contained His-55, whereas AR339 E2 had Gln-55 (S. Lustig, A. C. Jackson, C. S. Hahn, D. E. Griffin, E. G. Strauss, and J. H. Strauss, J. Virol. 62:2329-2336, 1988) and that SV with E2 containing Gly-172 was more virulent for newborn mice than SV with E2 containing Arg-172 (P. C. Tucker and D. E. Griffin, J. Virol. 65:1551-1557, 1991). Here we tested the virulence for both newborn and older mice of SV containing a number of different amino acids at E2 position 55 (His, Gln, Lys, Arg, Glu, Gly) in combination with both Gly-172 and Arg-172. All the viruses were virulent for newborn mice, but the residues at both 55 and 172 influenced the virulence of the virus, and there were differences in virulence observed among the various viruses. However, only viruses with His-55 were fully virulent for 14-day-old mice, and this virulence was independent of the residue at position 172. Virus with Lys-55 was virulent for 7-day-old mice, although slightly attenuated relative to His-55. Viruses with His-55 grew more rapidly and to higher titer in the brains of 7- and 14-day-old mice, in N18 neuroblastoma cells, and in BHK cells. Our data suggest that His-55 is important for neurovirulence in older mice and acts by increasing the efficiency of virus replication.     

Herpes simplex virus US3 protein kinase regulates virus-induced apoptosis in olfactory and vomeronasal chemosensory neurons in vivo

A role for the US3 protein kinase of herpes simplex virus (HSV) in regulating virus-induced neuronal apoptosis was investigated in an experimental mouse system, in which wild-type HSV invades the central nervous system (CNS) via the olfactory and vomeronasal systems upon intranasal infection. Wild-type HSV-2 strain 186 infected a fraction of olfactory and vomeronasal chemosensory neurons without inducing apoptosis and was transmitted to the CNS, precipitating lethal encephalitis. In sharp contrast, an US3-disrupted mutant, L1BR1, induced neuronal apoptosis in these peripheral conduits upon infection, blocking viral transmission to the CNS and causing no signs of disease. An US3-repaired mutant, L1B(-)11, behaved similarly to the wild-type virus. Only 5 p.f.u. of L1BR1 was sufficient to compromise mice when the mutant virus was introduced directly into the olfactory bulb, a viral entry site of the CNS. These results suggest that the US3 protein kinase of HSV regulates virus-induced neuronal apoptosis in peripheral conduits and determines the neuroinvasive phenotype of HSV. Furthermore, virus-induced neuronal apoptosis of peripheral nervous system cells may be a protective host response that blocks viral transmission to the CNS.     

Caspase 3-dependent cell death of neurons contributes to the pathogenesis of West Nile virus encephalitis

West Nile virus (WNV) is a neurotropic, arthropod-borne flavivirus that has become a significant global cause of viral encephalitis. To examine the mechanisms of WNV-induced neuronal death and the importance of apoptosis in pathogenesis, we evaluated the role of a key apoptotic regulator, caspase 3. WNV infection induced caspase 3 activation and apoptosis in the brains of wild-type mice. Notably, congenic caspase 3(-/-) mice were more resistant to lethal WNV infection, although there were no significant differences in the tissue viral burdens or the kinetics of viral spread. Instead, decreased neuronal death was observed in the cerebral cortices, brain stems, and cerebella of caspase 3(-/-) mice. Analogously, primary central nervous system (CNS)-derived neurons demonstrated caspase 3 activation and apoptosis after WNV infection, and treatment with caspase inhibitors or a genetic deficiency in caspase 3 significantly decreased virus-induced death. These studies establish that caspase 3-dependent apoptosis contributes to the pathogenesis of lethal WNV encephalitis and suggest possible novel therapeutic targets to restrict CNS injury.     

Infection with street strain rabies virus induces modulation of the microRNA profile of the mouse brain

ABSTRACT: BACKGROUND: Rabies virus (RABV) causes a fatal infection of the central nervous systems (CNS) of warm-blooded animals. Once the clinical symptoms develop, rabies is almost invariably fatal. The mechanism of RABV pathogenesis remains poorly understood. Recent studies have shown that microRNA (miRNA) plays an important role in the pathogenesis of viral infections. Our recent findings have revealed that infection with laboratory-fixed rabies virus strain can induce modulation of the microRNA profile of mouse brains. However, no previous report has evaluated the miRNA expression profile of mouse brains infected with RABV street strain. RESULTS: The results of microarray analysis show that miRNA expression becomes modulated in the brains of mice infected with street RABV. Quantitative real-time PCR assay of the differentially expressed miRNAs confirmed the results of microarray assay. Functional analysis showed the differentially expressed miRNAs to be involved in many immune-related signaling pathways, such as the Jak-STAT signaling pathway, the MAPK signaling pathway, cytokine-cytokine receptor interactions, and Fc gamma R-mediated phagocytosis. The predicted expression levels of the target genes of these modulated miRNAs were found to be correlated with gene expression as measured by DNA microarray and qRT-PCR. CONCLUSION: RABV causes significant changes in the miRNA expression profiles of infected mouse brains. Predicted target genes of the differentially expression miRNAs are associated with host immune response, which may provide important information for investigation of RABV pathogenesis and therapeutic method.