Characterization of a neurologic disease induced by a polytropic murine retrovirus: evidence for differential targeting of ecotropic and polytropic viruses in the brain.

A variety of ecotropic murine leukemia viruses cause neurodegenerative disease. We describe here the clinical and histopathological features of a neurologic disease induced by a polytropic murine leukemia virus, FMCF98. Clinical disease was dominated by hyperexcitability and ataxia, and the histopathology was characterized primarily by astrocytosis and astrocytic degeneration. The viral envelope gene harbored the determinants of neurovirulence, since the chimeric virus Fr98E, which contained the envelope gene of FMCF98 on a background of the nonneurovirulent virus FB29, caused a similar disease. The disease caused by Fr98E differed from that induced by the coisogenic neurovirulent ecotropic virus FrCasE in clinical presentation, histopathology, and distribution of virus in the central nervous system. Since Fr98E contains a polytropic envelope gene and FrCasE contains an ecotropic envelope gene, these phenotypic differences appeared to be determined by envelope sequences and may reflect differences in virus receptor usage in the central nervous system.     

Brain infection by neuroinvasive but avirulent murine oncornaviruses

The chimeric murine oncornavirus FrCas(E) causes a rapidly progressive noninflammatory spongiform encephalomyelopathy after neonatal inoculation. The virus was constructed by the introduction of pol-env sequences from the wild mouse virus CasBrE into the genome of a neuroinvasive but nonneurovirulent strain of Friend murine leukemia virus (FMuLV), FB29. Although the brain infection by FrCas(E) as well as that by other neurovirulent murine retroviruses has been described in detail, little attention has been paid to the neuroinvasive but nonneurovirulent viruses. The purpose of the present study was to compare brain infection by FrCas(E) with that by FB29 and another nonneurovirulent virus, F43, which contains pol-env sequences from FMuLV 57. Both FB29 and F43 infected the same spectrum of cell types in the brain as that infected by FrCas(E), including endothelial cells, microglia, and populations of neurons which divide postnatally. Viral burdens achieved by the two nonneurovirulent viruses in the brain were actually higher than that of FrCas(E). The widespread infection of microglia by the two nonneurovirulent viruses is notable because it is infection of these cells by FrCas(E) which is thought to be a critical determinant of its neuropathogenicity. These results indicate that although the sequence of the envelope gene determines neurovirulence, this effect appears to operate through a mechanism which does not influence either viral tropism or viral burden in the brain. Although all three viruses exhibited similar tropism for granule neurons in the cerebellar cortex, there was a striking difference in the distribution of envelope proteins in those cells in vivo. The FrCas(E) envelope protein accumulated in terminal axons, whereas those of FB29 and F43 remained predominantly in the cell bodies. These observations suggest that differences in the intracellular sorting of these proteins may exist and that these differences appear to correlate with neurovirulence.     

Differences in cytokine and chemokine responses during neurological disease induced by polytropic murine retroviruses Map to separate regions of the viral envelope gene

Infection of the central nervous system (CNS) by several viruses can lead to upregulation of proinflammatory cytokines and chemokines. In immunocompetent adults, these molecules induce prominent inflammatory infiltrates. However, with immunosuppressive retroviruses, such as human immunodeficiency virus (HIV), little CNS inflammation is observed yet proinflammatory cytokines and chemokines are still upregulated in some patients and may mediate pathogenesis. The present study examined expression of cytokines and chemokines in brain tissue of neonatal mice infected with virulent (Fr98) and avirulent (Fr54) polytropic murine retroviruses. While both viruses infect microglia and endothelia primarily in the white matter areas of the CNS, only Fr98 induces clinical CNS disease. The pathology consists of gliosis with minimal morphological changes and no inflammation, similar to HIV. In the present experiments, mice infected with Fr98 had increased cerebellar mRNA levels of proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha), TNF-beta, and interleukin-1 alpha and chemokines macrophage inflammatory protein-1 alpha (MIP-1 alpha), MIP-1 beta, monocyte chemoattractant protein 1 (MCP-1), gamma-interferon-inducible protein 10 (IP-10), and RANTES compared to mice infected with Fr54 or mock-infected controls. The increased expression of these genes occurred prior to the development of clinical symptoms, suggesting that these cytokines and chemokines might be involved in induction of neuropathogenesis. Two separate regions of the Fr98 envelope gene are associated with neurovirulence. CNS disease associated with the N-terminal portion of the Fr98 env gene was preceded by upregulation of cytokines and chemokines. In contrast, disease associated with the central region of the Fr98 env gene showed no upregulation of cytokines or chemokines and thus did not require increased expression of these genes for disease induction.     

Two separate envelope regions influence induction of brain disease by a polytropic murine retrovirus (FMCF98).

The major determinants involved in neurological disease induction by polytropic murine leukemia virus FMCF98 are encoded by the envelope gene. To map these determinants further, we produced four chimeras which contained neurovirulent FMCF98 envelope sequences combined with envelope sequences from the closely related nonneurovirulent polytropic virus FMCF54. Surprisingly, two chimeric viruses containing completely separate envelope regions from FMCF98 could both induce neurological disease. Clinical signs caused by both neurovirulent chimeras appeared to be indistinguishable from those caused by FMCF98, although the incubation periods were longer. One neurovirulence determinant mapped to the N-terminal portion of gp7O, which contains the VRA and VRB receptor-binding regions, while the other determinant mapped downstream of both of the variable regions. Western blot (immunoblot) analyses and immunohistochemical staining of tissue sections indicated that the variations in neurovirulence of these viruses could not be explained by differences in either the quantitative level or the location of virus expression in the brain.     

Profound amplification of pathogenic murine polytropic retrovirus release from coinfected cells

Previous studies indicate that mice infected with mixtures of mouse retroviruses (murine leukemia viruses [MuLVs]) exhibit dramatically altered pathology compared to mice infected with individual viruses of the mixture. Coinoculation of the ecotropic virus Friend MuLV (F-MuLV) with Fr98, a polytropic MuLV, induced a rapidly fatal neurological disease that was not observed in infections with either virus alone. The polytropic virus load in coinoculated mice was markedly enhanced, while the ecotropic F-MuLV load was unchanged. Furthermore, pseudotyping of the polytropic MuLV genome within ecotropic virions was nearly complete in coinoculated mice. In an effort to better understand these phenomena, we examined mixed retrovirus infections by utilizing in vitro cell lines. Similar to in vivo mixed infections, the polytropic MuLV genome was extensively pseudotyped within ecotropic virions; polytropic virus release was profoundly elevated in coinfected cells, and the ecotropic virus release was unchanged. A reduced level of polytropic SU protein on the surfaces of coinfected cells was observed and correlated with a reduced level of nonpseudotyped polytropic virion release. Marked amplification and pseudotyping of the polytropic MuLV were also observed in mixed Fr98-F-MuLV infections of cell lines derived from the central nervous system (CNS), the target for Fr98 pathogenesis. Additional experiments indicated that pseudotyping contributed to the elevated polytropic virus titer by increasing the efficiency of packaging and release of the polytropic genomes within ecotropic virions. Mixed infections are the rule rather than the exception in retroviral infection, and the ability to examine them in vitro should facilitate a more thorough understanding of retroviral interactions in general.     

Neurovirulence of polytropic murine retrovirus is influenced by two separate regions on opposite sides of the envelope protein receptor binding domain

Changes in the envelope proteins of retroviruses can alter the ability of these viruses to infect the central nervous system (CNS) and induce neurological disease. In the present study, nine envelope residues were found to influence neurovirulence of the Friend murine polytropic retrovirus Fr98. When projected on a three-dimensional model, these residues were clustered in two spatially separated groups, one in variable region B of the receptor binding site and the other on the opposite side of the envelope. Further studies indicated a role for these residues in virus replication in the CNS, although the residues did not affect viral entry.     

Identification of the infected target cell type in spongiform myeloencephalopathy induced by the neurotropic Cas-Br-E murine leukemia virus.

The Cas-Br-E murine leukemia virus (MuLV) induces a progressive hindlimb paralysis accompanied by a spongiform myeloencephalopathy in susceptible mice. In order to better understand the pathological process leading to these neurodegenerative lesions, we have investigated the nature of the cell type(s) infected by the virus during the course of the disease in CFW/D and SWR/J mice. For this purpose, we used in situ hybridization with virus-specific probes in combination with cell-type-specific histochemical (lectin) and immunological markers as well as morphological assessment. In the early stage of infection, endothelial cells represented the main cell type expressing viral RNA in the central nervous system (CNS). With disease progression and the appearance of lesions, microglial cells became the major cell type infected, accounting for up to 65% of the total infected cell population in diseased areas. Morphologically, these cells appeared activated and were frequently found in clusters. Infection and activation of microglial cells were almost exclusively restricted to diseased regions of the CNS. Neurons in diseased regions were not discernibly infected with virus at either early or late times of disease progression. Similarly, the proportion of infected astrocytes was typically < 1%. Although some endothelial cells and oligodendrocytes were infected by the virus, their infection was not limited to diseased CNS regions. These results are consistent with a model of indirect motor neuron degeneration, subsequent to the infection of nonneuronal CNS cells and especially of microglial cells. Infected microglial cells may play a role in the disease process by releasing not only virions or viral env-gene-encoded gp70 proteins but also other factors which may be directly or indirectly toxic to neurons. Parallels between microglial cell infection by MuLV and by lentiviruses, and specifically by human immunodeficiency virus, are discussed.     

Infection of cardiomyocytes and induction of left ventricle dysfunction by neurovirulent polytropic murine retrovirus

Viral infections of the heart are a causative factor of myocarditis as well as of sudden, unexpected deaths of children, yet the mechanisms of pathogenesis remain unclear, in part due to the relatively few animal models of virus-induced myocarditis. In the current study, we examined the ability of polytropic murine retroviruses to infect the heart and induce cardiac dysfunction. In situ hybridization and immunohistochemistry analysis detected virus-infected cardiomyocytes and macrophages in the heart. A significant decrease in left ventricle function, as measured by fractional shortening, was detected in mice infected with the neurovirulent retrovirus Fr98 but not in mice infected with the nonneurovirulent retrovirus Fr54. Virus infection was not associated with consistent findings of fibrosis or substantial cellular infiltrate. Fr98-induced left ventricle dysfunction was associated with a higher virus load, increased mRNA expression of the macrophage marker F4/80, increased chemokine production, and a small number of apoptotic cells in the heart.     

Quinolinic Acid Levels in a Murine Retrovirus-Induced Immunodeficiency Syndrome

Abstract: Mice infected with the retrovirus mixture designated LP-BM5 murine leukemia virus (MuLV) develop an immunosuppressive disease. Quinolinic acid (QUIN) is an endogenous neurotoxic N -methyl- d -aspartate agonist that may contribute to the pathogenesis of HIV-associated neurologic disease. In the present study, the levels of QUIN in brain and blood were measured in mice infected with LP-BM5 MuLV and compared with those in uninfected mice and mice infected with the nonpathogenic strain of ecotropic MuLV (helper component of LP-BM5 MuLV). Infection with LP-BM5 MuLV resulted in progressive increases in blood QUIN levels beginning 2 weeks after inoculation that peaked by 16 weeks postinfection. QUIN levels were also increased in cerebral cortex, hippocampus, and striatum. In systemic tissues, QUIN levels were increased in lung, liver, and spleen. In contrast, infection with the ecotropic viral component of the LP-BM5 MuLV mixture was not associated with any changes in brain, blood, or systemic tissue QUIN levels, even though helper virus burdens were comparable to those in mice infected with LP-BM5 MuLV. Treatment of LP-BM5 MuLV-infected mice with the antiretroviral agent zidovudine (azidothymidine) significantly reduced blood and brain QUIN levels in association with reductions in viral load in brain and spleen. These observations suggest that elevated QUIN production is not attributable to productive infection with retrovirus per se but occurs in response to an agent or agents, such as cytokines, that are produced by the host in response to virus infection.     

Friend小鼠白血病病毒（Fr.MuLV）对新生金地鼠的感染性研究

一般认为,Ｆｒｉｅｎｄ小鼠白血病病毒（Ｆｒｉｅｎｄｍｕｒｉｎｅｌｅｕｋｅｍｉａｖｉｒｕｓ：Ｆｒ．ＭｕＬＶ）属同种嗜性白血病病毒（ｅｃｏｔｒｏｐｉｃＭｕＬＶ）,只感染小鼠或大鼠,分别引起小鼠发生白血病和大鼠发生呆小病;但对小鼠或大鼠以外的动物（包括人等）无感染性。为进一步探讨该病毒的感染范围,本文进行了该病毒对金地鼠的感染性研究。结果表明,该病毒可在新生金地鼠体内增殖,引起新生金地鼠出现体重减轻、脾萎缩、并发早死等呆小病样症状,病理学检查发现接种该病毒后新生金地鼠的脾脏、胸腺呈明显的病理变化。提示Ｆｒ．ＭｕＬＶ对新生金地鼠具有感染作用,并产生病毒增殖和引起呆小病样症状,为阐明Ｆｒ．ＭｕＬＶ的感染范围和致病作用提供了实验依据。     

A single amino acid change in nsP1 attenuates neurovirulence of the Sindbis-group alphavirus S.A.AR86

S.A.AR86, a member of the Sindbis group of alphaviruses, is neurovirulent in adult mice and has a unique threonine at position 538 of nsP1; nonneurovirulent members of this group of alphaviruses encode isoleucine. Isoleucine was introduced at position 538 in the wild-type S.A.AR86 infectious clone, ps55, and virus derived from this mutant clone, ps51, was significantly attenuated for neurovirulence compared to that derived from ps55. Intracranial (i.c. ) s55 infection resulted in severe disease, including hind limb paresis, conjunctivitis, weight loss, and death in 89% of animals. In contrast, s51 caused fewer clinical signs and no mortality. Nevertheless, comparison of the virus derived from the mutant (ps51) and wild-type (ps55) S.A.AR86 molecular clones demonstrated that s51 grew as well as or better than the wild-type s55 virus in tissue culture and that viral titers in the brain following i.c. infection with s51 were equivalent to those of wild-type s55 virus. Analysis of viral replication within the brain by in situ hybridization revealed that both viruses established infection in similar regions of the brain at early times postinfection (12 to 72 h). However, at late times postinfection, the wild-type s55 virus had spread throughout large areas of the brain, while the s51 mutant exhibited a restricted pattern of replication. This suggests that s51 is either defective in spreading throughout the brain at late times postinfection or is cleared more rapidly than s55. Further evidence for the contribution of nsP1 Thr 538 to S.A.AR86 neurovirulence was provided by experiments in which a threonine residue was introduced at nsP1 position 538 of Sindbis virus strain TR339, which is nonneurovirulent in weanling mice. The resulting virus, 39ns1, demonstrated significantly increased neurovirulence and morbidity, including weight loss and hind limb paresis. These results demonstrate a role for alphavirus nonstructural protein genes in adult mouse neurovirulence.     

Substitution of the U3 long terminal repeat region of the neurotropic Cas-Br-E retrovirus affects its disease-inducing potential.

The Cas-Br-E and ts-Mo BA-1 murine leukemia viruses (MuLV) induce a spongiform neurodegenerative disease with different clinical manifestations, namely, either hind limb paralysis (Cas-Br-E) or tremors, spasticity, and hind limb weakness (ts-Mo Ba-1). We constructed the chimeric NEBA-1 MuLV by replacing the long terminal repeat of Cas-Br-E MuLV with that of ts-Mo BA-1 MuLV. In SWR/J or CFW/D mice, NEBA-1 MuLV induced an ataxic neurological disease characterized by clinical signs different from those induced by both parents. Although NEBA-1 MuLV did not induce lesions in novel brain areas, the spongiform lesions were more severe in deep cerebellar nuclei and in the spinal cord than those found in paralyzed mice inoculated with Cas-Br-E MuLV. By in situ hybridization, we found that the distribution of the spongiform lesions closely correlated with the distribution of the infected central nervous system cells. In the spinal cord, a close correlation was found between the number of infected cells and the severity of the spongiform degeneration. Sequencing of the substituted ts-BA-1 MuLV fragment and comparison with homologous sequences of Cas-Br-E and Moloney MuLV showed differences mainly in the U3 tandem direct repeats. Our results show that a few modifications within the U3 long terminal repeat allow the virus to cause more severe lesions in some central nervous system regions and that the severity of the spongiform degeneration correlates with the level of viral replication.     

Changes in mumps virus gene sequence associated with variability in neurovirulent phenotype

Mumps virus is highly neurotropic and, prior to widespread vaccination programs, was the major cause of viral meningitis in the United States. Nonetheless, the genetic basis of mumps virus neurotropism and neurovirulence was until recently not understood, largely due to the lack of an animal model. Here, nonneurovirulent (Jeryl Lynn vaccine) and highly neurovirulent (88-1961 wild type) mumps virus strains were passaged in human neural cells or in chicken fibroblast cells with the goal of neuroadapting or neuroattenuating the viruses, respectively. When tested in our rat neurovirulence assay against the respective parental strains, a Jeryl Lynn virus variant with an enhanced propensity for replication (neurotropism) and damage (neurovirulence) in the brain and an 88-1961 wild-type virus variant with decreased neurotropic and neurovirulent properties were recovered. To determine the molecular basis for the observed differences in neurovirulence and neuroattenuation, the complete genomes of the parental strains and their variants were fully sequenced. A comparison at the nucleotide level associated three amino acid changes with enhanced neurovirulence of the neuroadapted vaccine strain: one each in the nucleoprotein, matrix protein, and polymerase and three amino acid changes with reduced neurovirulence of the neuroattenuated wild-type strain: one each in the fusion protein, hemagglutinin-neuraminidase protein, and polymerase. The potential role of these amino acid changes in neurotropism, neurovirulence, and neuroattenuation is discussed.     

MCP-1 and CCR2 contribute to non-lymphocyte-mediated brain disease induced by Fr98 polytropic retrovirus infection in mice: role for astrocytes in retroviral neuropathogenesis

Virus infection of the central nervous system (CNS) often results in chemokine upregulation. Although often associated with lymphocyte recruitment, increased chemokine expression is also associated with non-lymphocyte-mediated CNS disease. In these instances, the effect of chemokine upregulation on neurological disease is unclear. In vitro, several chemokines including monocyte chemotactic protein 1 (MCP-1) protect neurons from apoptosis. Therefore, in vivo, chemokine upregulation may be a protective host response to CNS damage. Alternatively, chemokines may contribute to pathogenesis by stimulating intrinsic brain cells or recruiting macrophages to the brain. To investigate these possibilities, we studied a neurovirulent retrovirus, Fr98, that induces severe non-lymphocyte-mediated neurological disease and causes the upregulation of several chemokines that bind to chemokine receptors CCR2 and CCR5. Knockout mice deficient in CCR2 had reduced susceptibility to Fr98 pathogenesis, with significantly fewer mice developing clinical disease than did wild-type controls. In contrast, no reduction in Fr98-induced disease was observed in CCR5 knockout mice. Thus, signaling through CCR2, but not CCR5, plays an important role in Fr98-mediated pathogenesis. Three ligands for CCR2 (MCP-1, MCP-3, and MCP-5) were upregulated during Fr98 infection of the brain. Antibody-blocking experiments demonstrated that MCP-1 was important for retrovirus-induced neurological disease. In situ hybridization analysis revealed that MCP-1 was expressed by glial fibrillary acidic protein-positive astrocytes. Thus, astrocytes, previously not thought to play an effector role in the disease process were found to contribute to pathogenesis through the production of MCP-1. This study also demonstrates that chemokines can mediate pathogenesis in the CNS in the absence of lymphocytic infiltrate and gives credence to the hypothesis that chemokine upregulation is a mechanism by which retroviruses such as human immunodeficiency virus induce neurological damage.     

Comparison of the Neurovirulence of a Vaccine and a Wild-Type Mumps Virus Strain in the Developing Rat Brain

Prior to the adoption of widespread vaccination programs, mumps virus was the leading cause of virus-induced central nervous system (CNS) disease. Mumps virus-associated CNS complications in vaccinees continue to be reported; outside the United States, some of these complications have been attributed to vaccination with insufficiently attenuated neurovirulent vaccine strains. The development of potentially neurovirulent, live, attenuated mumps virus vaccines stems largely from the lack of an animal model that can reliably predict the neurovirulence of mumps virus vaccine candidates in humans. The lack of an effective safety test with which to measure mumps virus neurovirulence has also hindered analysis of the neuropathogenesis of mumps virus infection and the identification of molecular determinants of neurovirulence. In this report we show, for the first time, that mumps virus infection of the neonatal rat leads to developmental abnormalities in the cerebellum due to cerebellar granule cell migration defects. The incidence of the cerebellar abnormalities and other neuropathological and clinical outcomes of mumps virus infection of the neonatal rat brain demonstrated the ability of this model to distinguish neurovirulent (Kilham) from nonneurovirulent (Jeryl Lynn) mumps virus strains. Thus, this neonatal rat model may prove useful in evaluating the neurovirulence potential of new live, attenuated vaccine strains and may also be of value in elucidating the molecular basis of mumps virus neurovirulence.     

A herpes simplex virus type 1 gamma34.5 second-site suppressor mutant that exhibits enhanced growth in cultured glioblastoma cells is severely attenuated in animals

We describe here the neurovirulence properties of a herpes simplex virus type 1 gamma34.5 second-site suppressor mutant. gamma34.5 mutants are nonneurovirulent in animals and fail to grow in a variety of cultured cells due to a block at the level of protein synthesis. Extragenic suppressors with restored capacity to replicate in cells that normally do not support the growth of the parental gamma34.5 deletion mutant have been isolated. Although the suppressor virus reacquires the ability to grow in nonpermissive cultured cells, it remains severely attenuated in mice and is indistinguishable from the mutant gamma34.5 parent virus at the doses investigated. Repairing the gamma34.5 mutation in the suppressor mutant restores neurovirulence to wild-type levels. These studies illustrate that (i) the protein synthesis and neurovirulence defects observed in gamma34.5 mutant viruses can be genetically separated by an extragenic mutation at another site in the viral chromosome; (ii) the extragenic suppressor mutation does not affect neurovirulence; and (iii) the attenuated gamma34.5 mutant, which replicates poorly in many cell types, can be modified by genetic selection to generate a nonpathogenic variant that regains the ability to grow robustly in a nonpermissive glioblastoma cell line. As this gamma34.5 second-site suppressor variant is attenuated and replicates vigorously in neoplastic cells, it may have potential as a replication-competent, viral antitumor agent.     

An attenuating mutation in nsP1 of the Sindbis-group virus S.A.AR86 accelerates nonstructural protein processing and up-regulates viral 26S RNA synthesis

The Sindbis-group alphavirus S.A.AR86 encodes a threonine at nonstructural protein 1 (nsP1) 538 that is associated with neurovirulence in adult mice. Mutation of the nsP1 538 Thr to the consensus Ile found in nonneurovirulent Sindbis-group alphaviruses attenuates S.A.AR86 for adult mouse neurovirulence, while introduction of Thr at position 538 in a nonneurovirulent Sindbis virus background confers increased neurovirulence (M. T. Heise et al., J. Virol. 74:4207-4213, 2000). Since changes in the viral nonstructural region are likely to affect viral replication, studies were performed to evaluate the effect of Thr or Ile at nsP1 538 on viral growth, nonstructural protein processing, and RNA synthesis. Multistep growth curves in Neuro2A and BHK-21 cells revealed that the attenuated s51 (nsP1 538 Ile) virus had a slight, but reproducible growth advantage over the wild-type s55 (nsP1 538 Thr) virus. nsP1 538 lies within the cleavage recognition domain between nsP1 and nsP2, and the presence of the attenuating Ile at nsP1 538 accelerated the processing of S.A.AR86 nonstructural proteins both in vitro and in infected cells. Since nonstructural protein processing is known to regulate alphavirus RNA synthesis, experiments were performed to evaluate the effect of Ile or Thr at nsP1 538 on viral RNA synthesis. A combination of S.A.AR86-derived reporter assays and RNase protection assays determined that the presence of Ile at nsP1 538 led to earlier expression from the viral 26S promoter without affecting viral minus- or plus-strand synthesis. These results suggest that slower nonstructural protein processing and delayed 26S RNA synthesis in wild-type S.A.AR86 infections may contribute to the adult mouse neurovirulence phenotype of S.A.AR86.     

Coronavirus neurovirulence correlates with the ability of the virus to induce proinflammatory cytokine signals from astrocytes and microglia

The molecular and cellular basis of coronavirus neurovirulence is poorly understood. Since neurovirulence may be determined at the early stages of infection of the central nervous system (CNS), we hypothesize that it may depend on the ability of the virus to induce proinflammatory signals from brain cells for the recruitment of blood-derived inflammatory cells. To test this hypothesis, we studied the interaction between coronaviruses (mouse hepatitis virus) of different neurovirulences with primary cell cultures of brain immune cells (astrocytes and microglia) and mouse tissues. We found that the level of neurovirulence of the virus correlates with its differential ability to induce proinflammatory cytokines (interleukin 12 [IL-12] p40, tumor necrosis factor alpha, IL-6, IL-15, and IL-1beta) in astrocytes and microglia and in mouse brains and spinal cords. These findings suggest that coronavirus neurovirulence may depend on a novel discriminatory ability of astrocytes and microglia to induce a proinflammatory response in the CNS.     

Neural Stem Cells as Engraftable Packaging Lines Can Mediate Gene Delivery to Microglia: Evidence from Studying Retroviral env-Related Neurodegeneration

The induction of spongiform myeloencephalopathy by murine leukemia viruses is mediated primarily by infection of central nervous system (CNS) microglia. In this regard, we have previously shown that CasBrE-induced disease requires late, rather than early, virus replication events in microglial cells (W. P. Lynch et al., J. Virol. 70:8896-8907, 1996). Furthermore, neurodegeneration requires the presence of unique sequences within the viral env gene. Thus, the neurodegeneration-inducing events could result from microglial expression of retroviral envelope protein alone or from the interaction of envelope protein with other viral structural proteins in the virus assembly and maturation process. To distinguish between these possible mechanisms of disease induction, we engineered the engraftable neural stem cell line C17-2 into packaging/producer cells in order to deliver the neurovirulent CasBrE env gene to endogenous CNS cells. This strategy resulted in significant CasBrE env expression within CNS microglia without the appearance of replication competent virus. CasBrE envelope expression within microglia was accompanied by increased expression of activation markers F4/80 and Mac-1 (CD11b) but failed to induce spongiform neurodegenerative changes. These results suggest that envelope expression alone within microglia is not sufficient to induce neurodegeneration. Rather, microglia-mediated disease appears to require neurovirulent Env protein interaction with other viral proteins during assembly or maturation. More broadly, the results presented here prove the efficacy of a novel method by which neural stem cell biology may be harnessed for genetically manipulating the CNS, not only for studying neurodegeneration but also as a paradigm for the disseminated distribution of retroviral vector-transduced genes.     

Chemokine Receptor Utilization by Human Immunodeficiency Virus Type 1 Isolates That Replicate in Microglia

The role of human immunodeficiency virus (HIV) strain variability remains a key unanswered question in HIV dementia, a condition affecting around 20% of infected individuals. Several groups have shown that viruses within the central nervous system (CNS) of infected patients constitute an independently evolving subset of HIV strains. A potential explanation for the replication and sequestration of viruses within the CNS is the preferential use of certain chemokine receptors present in microglia. To determine the role of specific chemokine coreceptors in infection of adult microglial cells, we obtained a small panel of HIV type 1 brain isolates, as well as other HIV strains that replicate well in cultured microglial cells. These viruses and molecular clones of their envelopes were used in infections, in cell-to-cell fusion assays, and in the construction of pseudotypes. The results demonstrate the predominant use of CCR5, at least among the major coreceptors, with minor use of CCR3 and CXCR4 by some of the isolates or their envelope clones.