A Functional Role for Neutralizing Antibodies in Borna Disease: Influence on Virus Tropism outside the Central Nervous System

Borna disease virus (BDV) is a negative-strand RNA virus that infects the central nervous systems (CNS) of warm-blooded animals and causes disturbances of movement and behavior. The basis for neurotropism remains poorly understood; however, the observation that the distribution of infectious virus in immunocompetent rats is different from that in immunoincompetent rats indicates a role for the immune system in BDV tropism: whereas in immunocompetent rats virus is restricted to the central, peripheral, and autonomic nervous systems, immunoincompetent rats also have virus in nonneural tissues. In an effort to examine the influence of the humoral immune response on BDV pathogenesis, we examined the effects of passive immunization with neutralizing antiserum in immunoincompetent rats. Serum transfer into immunoincompetent rats did not prevent persistent CNS infection but did result in restriction of virus to neural tissues. These results indicate that neutralizing antibodies may play a role in preventing generalized infection with BDV.     

Raccoon poxvirus recombinants expressing the rabies virus nucleoprotein protect mice against lethal rabies virus infection.

Raccoon poxvirus (RCN) recombinants expressing the rabies virus internal structural nucleoprotein (RCN-N) protected A/WySnJ mice against a lethal challenge with street rabies virus (SRV). Maximum survival was achieved following vaccination by tail scratch and footpad (FP) SRV challenge. RCN-N-vaccinated mice inoculated in the FP with SRV were resistant to infection for at least 54 weeks postvaccination. Protection was also elicited by RCN recombinants expressing the rabies virus glycoprotein (RCN-G). Vaccination with RCN-G evoked rabies virus neutralizing antibody. Rabies virus neutralizing antibody was not detected in RCN-N-vaccinated mice prior to or following SRV infection. Radioimmunoprecipitation assays showed that sera from RCN-N-vaccinated mice which survived SRV infection did not contain antibody to SRV structural protein G, M, or NS. The mechanism(s) of N-induced resistance appears to correlate with the failure of peripherally inoculated SRV to enter the central nervous system (CNS). Support for this correlation with resistance was documented by the observations that SRV-inoculated RCN-N-vaccinated mice did not develop clinical signs of CNS rabies virus infection, infectious SRV was not detected in the spinal cord or brain following FP challenge, and all RCN-N-vaccinated mice died following direct intracranial infection of the CNS with SRV. These results suggest that factors other than anti-G neutralizing antibody are important in resistance to rabies virus and that the N protein should be considered for incorporation with the G protein in recombinant vaccines.     

Mechanisms of central nervous system viral persistence: the critical role of antibody and B cells.

Contributions of humoral and cellular immunity in controlling neurotropic mouse hepatitis virus persistence within the CNS were determined in B cell-deficient J(H)D and syngeneic H-2(d) B cell+ Ab-deficient mice. Virus clearance followed similar kinetics in all mice, confirming initial control of virus replication by cellular immunity. Nevertheless, virus reemerged within the CNS of all Ab-deficient mice. In contrast to diminished T cell responses in H-2(b) B cell-deficient muMT mice, the absence of B cells or Ab in the H-2(d) mice did not compromise expansion, recruitment into the CNS, or function of virus-specific CD4+ and CD8+ T cells. The lack of B cells and lymphoid architecture thus appears to manifest itself on T cell responses in a genetically biased manner. Increasing viral load did not enhance frequencies or effector function of virus-specific T cells within the CNS, indicating down-regulation of T cell responses. Although an Ab-independent antiviral function of B cells was not evident during acute infection, the presence of B cells altered CNS cellular tropism during viral recrudescence. Reemerging virus localized almost exclusively to oligodendroglia in B cell+ Ab-deficient mice, whereas it also replicated in astrocytes in B cell-deficient mice. Altered tropism coincided with distinct regulation of CNS virus-specific CD4+ T cells. These data conclusively demonstrate that the Ab component of humoral immunity is critical in preventing virus reactivation within CNS glial cells. B cells themselves may also play a subtle role in modulating pathogenesis by influencing tropism.     

Cysteinyl leukotrienes as novel host factors facilitating Cryptococcus neoformans penetration into the brain

Cryptococcus neoformas infection of the central nervous system (CNS) continues to be an important cause of mortality and morbidity, and a major contributing factor is our incomplete knowledge of the pathogenesis of this disease. Here, we provide the first direct evidence that C. neoformans exploits host cysteinyl leukotrienes (LTs), formed via LT biosynthetic pathways involving cytosolic phospholipase A₂α (cPLA₂α) and 5‐lipoxygenase (5‐LO) and acting via cysteinyl leukotriene type 1 receptor (CysLT1), for penetration of the blood–brain barrier. Gene deletion of cPLA₂α and 5‐LO and pharmacological inhibition of cPLA₂α, 5‐LO and CysLT1 were effective in preventing C. neoformans penetration of the blood–brain barrier in vitro and in vivo. A CysLT1 antagonist enhanced the efficacy of an anti‐fungal agent in therapy of C. neoformans CNS infection in mice. These findings demonstrate that host cysteinyl LTs, dependent on the actions of cPLA₂α and 5‐LO, promote C. neoformans penetration of the blood–brain barrier and represent novel targets for elucidating the pathogenesis and therapeutic development of C. neoformans CNS infection.     

Systemic and brain macrophage infections in relation to the development of simian immunodeficiency virus encephalitis

The brains of individuals with lentiviral-associated encephalitis contain an abundance of infected and activated macrophages. It has been hypothesized that encephalitis develops when increased numbers of infected monocytes traffic into the central nervous system (CNS) during the end stages of immunosuppression. The relationships between the infection of brain and systemic macrophages and circulating monocytes and the development of lentiviral encephalitis are unknown. We longitudinally examined the extent of monocyte/macrophage infection in blood and lymph nodes of pigtailed macaques that did or did not develop simian immunodeficiency virus encephalitis (SIVE). Compared to levels in macaques that did not develop SIVE, more ex vivo virus production was detected from monocyte-derived macrophages and nonadherent peripheral blood mononuclear cells (PBMCs) from macaques that did develop SIVE. Prior to death, there was an increase in the number of circulating PBMCs following a rise in cerebrospinal fluid viral load in macaques that did develop SIVE but not in nonencephalitic macaques. At necropsy, macaques with SIVE had more infected macrophages in peripheral organs, with the exception of lymph nodes. T cells and NK cells with cytotoxic potential were more abundant in brains with encephalitis; however, T-cell and NK-cell infiltration in SIVE and human immunodeficiency virus encephalitis was more modest than that observed in classical acute herpes simplex virus encephalitis. These findings support the hypothesis that inherent differences in host systemic and CNS monocyte/macrophage viral production are associated with the development of encephalitis.     

In vivo effects of coronavirus-specific T cell clones: DTH inducer cells prevent a lethal infection but do not inhibit virus replication

A lethal infection by neurotropic JHM strain of mouse hepatitis virus was prevented by the local adoptive transfer of three virus-specific Lyt-1+2-, L3T4+ T cell clones. The transfer of Lyt-1+ T cells specific for an unrelated antigen (hen egg lysozyme) did not protect. Protection required I region compatibility between the T cells and the recipients, and was reversed either by irradiation of the cells before transfer or by pretreatment of the recipients with cyclophosphamide. Adoptive transfer prevented death due to JHM virus infection but did not result in altered antiviral immunoglobulin synthesis or the suppression of viral replication in the central nervous system (CNS). The data presented implicate a local DTH response in the protection of the host from lethal infection of the CNS by a neurotropic virus, but clearly imply that other antiviral effector mechanisms are necessary for the suppression of viral replication.     

Epitope-tagged L* protein of Theiler's murine encephalomyelitis virus is expressed in the central nervous system in the acute phase of infection

TO subgroup strains of Theiler's murine encephalomyelitis virus (TMEV) synthesize L* protein from an alternative initiation codon. We first demonstrated L* expression in the central nervous system (CNS) of TMEV-infected mice during the acute phase of infection by immunoprecipitation and immunoblotting with anti-L* antibody. In addition, we generated mutant viruses which synthesize FLAG or 3xFLAG epitope-tagged L* protein. With a mutant virus expressing 3xFLAG epitope-tagged L*, designated DA/3xFLAGL*, we investigated L* in the CNS in the acute phase of infection. DA/3xFLAGL* did not change the virus tropism in comparison with wild-type virus, and L* was clearly identified in the CNS in both susceptible and resistant strains of mice. Double immunolabeling studies showed that L* is colocalized with TMEV polyprotein and exclusively expressed in neurons.     

Presence of Virus Neutralizing Antibodies in Cerebral Spinal Fluid Correlates with Non-Lethal Rabies in Dogs

Background

Rabies is traditionally considered a uniformly fatal disease after onset of clinical manifestations. However, increasing evidence indicates that non-lethal infection as well as recovery from flaccid paralysis and encephalitis occurs in laboratory animals as well as humans.

Methodology/Principal Findings

Non-lethal rabies infection in dogs experimentally infected with wild type dog rabies virus (RABV, wt DRV-Mexico) correlates with the presence of high level of virus neutralizing antibodies (VNA) in the cerebral spinal fluid (CSF) and mild immune cell accumulation in the central nervous system (CNS). By contrast, dogs that succumbed to rabies showed only little or no VNA in the serum or in the CSF and severe inflammation in the CNS. Dogs vaccinated with a rabies vaccine showed no clinical signs of rabies and survived challenge with a lethal dose of wild-type DRV. VNA was detected in the serum, but not in the CSF of immunized dogs. Thus the presence of VNA is critical for inhibiting virus spread within the CNS and eventually clearing the virus from the CNS.

Conclusions/Significance

Non-lethal infection with wt RABV correlates with the presence of VNA in the CNS. Therefore production of VNA within the CNS or invasion of VNA from the periphery into the CNS via compromised blood-brain barrier is important for clearing the virus infection from CNS, thereby preventing an otherwise lethal rabies virus infection.     

Biological basis of rabies virus neurovirulence in mice: comparative pathogenesis study using the immunoperoxidase technique.

The CVS strain of fixed rabies virus causes acute, fatal encephalomyelitis in young adult ICR mice. Variant RV194-2, which was selected from CVS virus in cell culture with a neutralizing antiglycoprotein monoclonal antibody, has a single amino acid change in the glycoprotein. The infections caused by CVS virus and RV194-2 virus were compared in mice for 14 days postinoculation of 5 x 10(7) PFU into the right masseter muscle. All CVS virus-infected mice died (mean time to death, 7.9 days), compared with a mortality rate of 8.5% for RV194-2 virus-infected mice. RV194-2 virus spread to the ipsilateral trigeminal ganglion during the first 2 days postinoculation, and both viruses spread to the ipsilateral motor nucleus of the trigeminal nerve in the pons. Both viruses spread centrifugally and caused infection of bilateral trigeminal ganglia on day 3. The viruses spread throughout the central nervous system (CNS) at similar rates, but CVS virus infected many more neurons than did RV194-2 virus. Rabies virus antigen was observed in only occasional CNS neurons after day 6 of RV194-2 virus infection. By this time, CVS virus had caused severe widespread infection. In this model, virulence depends on improved efficiency of viral spread between CNS neurons rather than the rate of spread or topographical distribution of the infection.     

The range and distribution of murine central nervous system cells infected with the gamma(1)34.5- mutant of herpes simplex virus 1.

Wild-type herpes simplex virus 1 (HSV-1) multiplies, spreads, and rapidly destroys cells of the murine central nervous system (CNS). In contrast, mutants lacking both copies of the gamma(1)34.5- gene have been shown to be virtually lacking in virulence even after direct inoculation of high-titered virus into the CNS of susceptible mice (J. Chou, E. R. Kern, R. J. Whitley, and B. Roizman, Science 250:1262-1266, 1990). To investigate the host range and distribution of infected cells in the CNS of mice, 4- to 5-week-old mice were inoculated stereotaxically into the caudate/putamen with 3 x 10(5) PFU of the gamma(1)34.5- virus R3616. Four-micrometer-thick sections of mouse brains removed on day 3, 5, or 7 after infection were reacted with a polyclonal antibody directed primarily to structural proteins of the virus and with antibodies specific for neurons, astrocytes, or oligodendrocytes. This report shows the following: (i) most of the tissue damage caused by R3616 was at the site of injection, (ii) the virus spread by retrograde transport from the site of infection to neuronal cell nuclei at distant sites and to ependymal cells by cerebrospinal fluid, (iii) the virus infected neurons, astrocytes, oligodendrocytes, and ependymal cells and hence did not discriminate among CNS cells, (iv) viral replication in some neurons could be deduced from the observation of infected astrocytes and oligodendrocytes at distant sites, and (v) infected cells were being efficiently cleared from the nervous system by day 7 after infection. We conclude that the gamma(1)34.5- attenuation phenotype is reflected in a gross reduction in the ability of the virus to replicate and spread from cell to cell and is not due to a restricted host range. The block in viral replication appears to be a late event in viral replication.     

SEC14 is a specific requirement for secretion of phospholipase B1 and pathogenicity of Cryptococcus neoformans

Secreted phospholipase B1 (CnPlb1) is essential for dissemination of Cryptococcus neoformans to the central nervous system (CNS) yet essential components of its secretion machinery remain to be elucidated. Using gene deletion analysis we demonstrate that CnPlb1 secretion is dependent on the CnSEC14 product, CnSec14-1p. CnSec14-1p is a homologue of the phosphatidylinositol transfer protein ScSec14p, which is essential for secretion and viability in Saccharomyces cerevisiae. In contrast to CnPlb1, neither laccase 1-induced melanization within the cell wall nor capsule induction were negatively impacted in CnSEC14-1 deletion mutants (CnΔsec14-1 and CnΔsec14-1CnΔsfh5). Similar to the CnPLB1 deletion mutant (CnΔplb1), CnΔsec14-1 was hypovirulent in mice and did not disseminate to the CNS by day 14 post infection. Furthermore, macrophage expulsion of live CnΔsec14-1 and CnΔplb1 (vomocytosis) was reduced. Individual deletion of CnSEC14-2, a closely related CnSEC14-1 homologue, and CnSFH5, a distantly related SEC fourteen like homologue, did not abrogate CnPlb1 secretion or virulence. However, reconstitution of CnΔsec14-1 with CnSEC14-1 or CnSEC14-2 restored both phenotypes, consistent with functional genetic redundancy. We conclude that CnPlb1 secretion is SEC14-dependent and that C. neoformans preferentially exports virulence determinants to the cell periphery via distinct pathways. We also demonstrate that CnPlb1 secretion is essential for vomocytosis.     

Perforin and gamma interferon-mediated control of coronavirus central nervous system infection by CD8 T cells in the absence of CD4 T cells

Infection of the central nervous system (CNS) with the neurotropic JHM strain of mouse hepatitis virus produces acute and chronic demyelination. The contributions of perforin-mediated cytolysis and gamma interferon (IFN-gamma) secretion by CD8(+) T cells to the control of infection and the induction of demyelination were examined by adoptive transfer into infected SCID recipients. Untreated SCID mice exhibited uncontrolled virus replication in all CNS cell types but had little or no demyelination. Memory CD8(+) T cells from syngeneic wild-type (wt), perforin-deficient, or IFN-gamma-deficient (GKO) donors all trafficked into the infected CNS in the absence of CD4(+) T cells and localized to similar areas. Although CD8(+) T cells from all three donors suppressed virus replication in the CNS, GKO CD8(+) T cells expressed the least antiviral activity. A distinct viral antigen distribution in specific CNS cell types revealed different mechanisms of viral control. While wt CD8(+) T cells inhibited virus replication in all CNS cell types, cytolytic activity in the absence of IFN-gamma suppressed the infection of astrocytes, but not oligodendroglia. In contrast, cells that secreted IFN-gamma but lacked cytolytic activity inhibited replication in oligodendroglia, but not astrocytes. Demyelination was most severe following viral control by wt CD8(+) T cells but was independent of macrophage infiltration. These data demonstrate the effective control of virus replication by CD8(+) T cells in the absence of CD4(+) T cells and support the necessity for the expression of distinct effector mechanisms in the control of viral replication in distinct CNS glial cell types.     

Antibody is required for clearance of infectious murine hepatitis virus A59 from the central nervous system, but not the liver.

Intracerebral inoculation with mouse hepatitis virus strain A59 results in viral replication in the CNS and liver. To investigate whether B cells are important for controlling mouse hepatitis virus strain A59 infection, we infected muMT mice who lack membrane-bound IgM and therefore mature B lymphocytes. Infectious virus peaked and was cleared from the livers of muMT and wild-type mice. However, while virus was cleared from the CNS of wild-type mice, virus persisted in the CNS of muMT mice. To determine how B cells mediate viral clearance, we first assessed CD4(+) T cell activation in the absence of B cells as APC. CD4(+) T cells express wild-type levels of CD69 after infection in muMT mice. IFN-gamma production in response to viral Ag in muMT mice was also normal during acute infection, but was decreased 31 days postinfection compared with that in wild-type mice. The role of Ab in viral clearance was also assessed. In wild-type mice plasma cells appeared in the CNS around the time that virus is cleared. The muMT mice that received A59-specific Ab had decreased virus, while mice with B cells deficient in Ab secretion did not clear virus from the CNS. Viral persistence was not detected in FcR or complement knockout mice. These data suggest that clearance of infectious mouse hepatitis virus strain A59 from the CNS requires Ab production and perhaps B cell support of T cells; however, virus is cleared from the liver without the involvement of Abs or B cells.     

Overexpression of tumor necrosis factor alpha by a recombinant rabies virus attenuates replication in neurons and prevents lethal infection in mice

The effect of tumor necrosis factor alpha (TNF-alpha) on rabies virus (RV) infection of the mouse central nervous system (CNS) was studied, using recombinant RV engineered to express either soluble TNF-alpha [SPBN-TNF-alpha+] or insoluble membrane-bound TNF-alpha [SPBN-TNF-alpha(MEM)]. Growth curves derived from infections of mouse neuroblastoma NA cells revealed significantly less spread and production of SPBN-TNF-alpha+ than of SPBN-TNF-alpha(MEM) or SPBN-TNF-alpha-, which carries an inactivated TNF-alpha gene. The expression of soluble or membrane-bound TNF-alpha was not associated with increased cell death or induction of alpha/beta interferons. Brains of mice infected intranasally with SPBN-TNF-alpha+ showed significantly less virus spread than did mouse brains after SPBN-TNF-alpha- infection, and none of the SPBN-TNF-alpha+-infected mice succumbed to RV infection, whereas 80% of SPBN-TNF-alpha- -infected mice died. Reduced virus spread in SPBN-TNF-alpha+-infected mouse brains was paralleled by enhanced CNS inflammation, including T-cell infiltration and microglial activation. These data suggest that TNF-alpha exerts its protective activity in the brain directly through an as yet unknown antiviral mechanism and indirectly through the induction of inflammatory processes in the CNS.     

Impaired T cell immunity in B cell-deficient mice following viral central nervous system infection

CD8(+) T cells are required to control acute viral replication in the CNS following infection with neurotropic coronavirus. By contrast, studies in B cell-deficient (muMT) mice revealed Abs as key effectors in suppressing virus recrudescence. The apparent loss of initial T cell-mediated immune control in the absence of B cells was investigated by comparing T cell populations in CNS mononuclear cells from infected muMT and wild-type mice. Following viral recrudescence in muMT mice, total CD8(+) T cell numbers were similar to those of wild-type mice that had cleared infectious virus; however, virus-specific T cells were reduced at least 3-fold by class I tetramer and IFN-gamma ELISPOT analysis. Although overall T cell recruitment into the CNS of muMT mice was not impaired, discrepancies in frequencies of virus-specific CD8(+) T cells were most severe during acute infection. Impaired ex vivo cytolytic activity of muMT CNS mononuclear cells, concomitant with reduced frequencies, implicated IFN-gamma as the primary anti viral factor early in infection. Reduced virus-specific CD8(+) T cell responses in the CNS coincided with poor peripheral expansion and diminished CD4(+) T cell help. Thus, in addition to the lack of Ab, limited CD8(+) and CD4(+) T cell responses in muMT mice contribute to the ultimate loss of control of CNS infection. Using a model of virus infection restricted to the CNS, the results provide novel evidence for a role of B cells in regulating T cell expansion and differentiation into effector cells.     

Immune-mediated protection from measles virus-induced central nervous system disease is noncytolytic and gamma interferon dependent

Neurons of the mammalian central nervous system (CNS) are an essential and largely nonrenewable cell population. Thus, virus infections that result in neuronal depletion, either by virus-mediated cell death or by induction of the cytolytic immune response, could cause permanent neurological impairment of the host. In a transgenic mouse model of measles virus (MV) infection of neurons, we have previously shown that the host T-cell response was required for resolution of infection in susceptible adult mice. In this report, we show that this protective response did not result in neuronal death, even during the peak of T-cell infiltration into the brain parenchyma. When susceptible mice were intercrossed with specific immune knockout mice, a critical role for gamma interferon (IFN-gamma) was identified in protection against MV infection and CNS disease. Moreover, the addition of previously activated splenocytes or recombinant murine IFN-gamma to MV-infected primary neurons resulted in the inhibition of viral replication in the absence of neuronal death. Together, these data support the hypothesis that the host immune response can promote viral clearance without concomitant neuronal loss, a process that appears to be mediated by cytokines.     

Lymphotoxin-alpha- and lymphotoxin-beta-deficient mice differ in susceptibility to scrapie: evidence against dendritic cell involvement in neuroinvasion

Transmissible spongiform encephalopathy or prion diseases are fatal neurodegenerative disorders of humans and animals often initiated by oral intake of an infectious agent. Current evidence suggests that infection occurs initially in the lymphoid tissues and subsequently in the central nervous system (CNS). The identity of infected lymphoid cells remains controversial, but recent studies point to the involvement of both follicular dendritic cells (FDC) and CD11c(+) lymphoid dendritic cells. FDC generation and maintenance in germinal centers is dependent on lymphotoxin alpha (LT-alpha) and LT-beta signaling components. We report here that by the oral route, LT-alpha -/- mice developed scrapie while LT-beta -/- mice did not. Furthermore, LT-alpha -/- mice had a higher incidence and shorter incubation period for developing disease following inoculation than did LT-beta -/- mice. Transplantation of lymphoid tissues from LT-beta -/- mice, which have cervical and mesenteric lymph nodes, into LT-alpha -/- mice, which do not, did not alter the incidence of CNS scrapie. In other studies, a virus that is tropic for and alters functions of CD11c(+) cells did not alter the kinetics of neuroinvasion of scrapie. Our results suggest that neither FDC nor CD11c(+) cells are essential for neuroinvasion after high doses of RML scrapie. Further, it is possible that an as yet unidentified cell found more abundantly in LT-alpha -/- than in LT-beta -/- mice may assist in the amplification of scrapie infection in the periphery and favor susceptibility to CNS disease following peripheral routes of infection.     

Critical role for protein tyrosine phosphatase SHP-1 in controlling infection of central nervous system glia and demyelination by Theiler's murine encephalomyelitis virus

We previously characterized the expression and function of the protein tyrosine phosphatase SHP-1 in the glia of the central nervous system (CNS). In the present study, we describe the role of SHP-1 in virus infection of glia and virus-induced demyelination in the CNS. For in vivo studies, SHP-1-deficient mice and their normal littermates received an intracerebral inoculation of an attenuated strain of Theiler's murine encephalomyelitis virus (TMEV). At various times after infection, virus replication, TMEV antigen expression, and demyelination were monitored. It was found that the CNS of SHP-1-deficient mice uniquely displayed demyelination and contained substantially higher levels of virus than did that of normal littermate mice. Many infected astrocytes and oligodendrocytes were detected in both brains and spinal cords of SHP-1-deficient but not normal littermate mice, showing that the virus replicated and spread at a much higher rate in the glia of SHP-1-deficient animals. To ascertain whether the lack of SHP-1 in the glia was primarily responsible for these differences, glial samples from these mice were cultured in vitro and infected with TMEV. As in vivo, infected astrocytes and oligodendrocytes of SHP-1-deficient mice were much more numerous and produced more virus than did those of normal littermate mice. These findings indicate that SHP-1 is a critical factor in controlling virus replication in the CNS glia and virus-induced demyelination.