Attenuated tissue culture-adapted and natural street rabies virus (RV) strains differ greatly in their neuroinvasiveness. To identify the elements responsible for the ability of an RV to enter the CNS from a peripheral site and to cause lethal neurological disease, we constructed a full-length cDNA clone of silver-haired bat-associated RV (SHBRV) strain 18 and exchanged the genes encoding RV proteins and genomic sequences of this highly neuroinvasive RV strain with those of a highly attenuated nonneuroinvasive RV vaccine strain (SN0). Analysis of the recombinant RV (SB0), which was recovered from SHBRV-18 cDNA, indicated that this RV is phenotypically indistinguishable from WT SHBRV-18. Characterization of the chimeric viruses revealed that in addition to the RV glycoprotein, which plays a predominant role in the ability of an RV to invade the CNS from a peripheral site, viral elements such as the trailer sequence, the RV polymerase, and the pseudogene contribute to RV neuroinvasiveness. Analyses also revealed that neuroinvasiveness of an RV correlates inversely with the time necessary for internalization of RV virions and with the capacity of the virus to grow in neuroblastoma cells.reverse genetics ͉ pathogenicity ͉ internalization
A variety of molecular genetic approaches were used to study the effect of rabies virus (RV) infection on host gene expression in mouse brain. The down-regulation of gene expression was found to be a major effect of RV infection by using subtraction hybridization. However, a combination of techniques identified approximately 39 genes activated by infection. These included genes involved in regulation of cell metabolism, protein synthesis, synaptic activity, and cell growth and differentiation. Northern blot analysis to monitor temporal activation of several of these genes following infection revealed essentially two patterns of activation: (i) an early response with up-regulation beginning within 3 days after infection and correlating with transcription of RV nuclear protein; and (ii) a late response with enhanced expression occurring at days 6 -7 after infection and associated with peak RV replication. The gene activation patterns and the known functions of their products suggest that a number of host genes may be involved in the replication and spread of RV in the brain.T he outcome of rabies virus (RV) infection is determined by the convergence of several different virus-host interactions, including those that contribute to virus replication and spread, pathogenic effects on cells, and antiviral responses. RV is particularly suitable for the study of virus-host interactions because of the small size of its genome and its high specificity for infection of neurons. Nevertheless, the mechanisms involved in the disease process are undoubtedly complex. For example, the virus, which uses the neuronal network to spread within the host, must initially replicate in neurons without causing significant functional impairment that would compromise the infection cycle (1). While there is some understanding of the roles of viral proteins in rabies pathogenesis, the contribution of host factors to RV transcription͞replication and axonal͞trans-synaptic spread remain unknown. Various technologies are now available to identify host genes that might be essential for the life cycle of RV, important in antiviral defense, or activated nonspecifically in RV infection (2-4). Because each technology is suited for the detection of different classes of genes and different levels of gene expression, we used a variety of assays, including differential display (DD), cDNA array hybridization, subtraction hybridization (SH), and restriction fragment differential display (RFDD), to assess gene expression in rabies-infected mouse brain. With these approaches, we have revealed the effects of rabies infection on select gene expression patterns in the brain. Based on known functional properties of several gene products as well as the timing of their up-regulation after infection, we discuss possible contributions of host genes to the pathogenesis of rabies. Materials and MethodsVirus Infection of Mice. Female, 6-to 8-week-old Swiss Webster mice were purchased from Taconic Farms. Mice were maintained under pathogen-free conditions and used to 10 w...
To provide a cost-effective and safe replacement for human rabies immunoglobulin (HRIG), we used DNA recombinant technology to express 3 human rabies virus-neutralizing human monoclonal antibodies (huMAbs) in a rhabdovirus vector (RhV). Infection of either baby hamster kidney cells or CHO cells, with the resulting RhV-huMAb recombinant viruses, yielded high-level production (< or =40 micro g/mL/48 h) of RhV recombinant-expressed huMAbs (rhuMAbs) that differ in both isotype and epitope-recognition specificity. A cocktail of these rhuMAbs neutralizes several fixed and street wild-type rabies viruses (RVs). Mice and hamsters treated only once with this rhuMAb cocktail after infection with a lethal dose of RV were protected. In the mouse models, the postexposure prophylaxis (PEP) efficacy obtained with the rhuMAb cocktail was comparable to that obtained with HRIG, a finding strongly suggesting that rhuMAbs should be given serious consideration for use in future PEP of humans.
This study extends the previous findings of a systemic treatment effect of a unique combination product, IGV-001, in recurrent GBM. As an autologous tumor cell treatment, results were postulated to be an immune response. This follow-on phase Ib trial for newly diagnosed GBM was designed to further test this hypothesis with broad entry criteria. While the primary objective remained safety, unexpected clinical and radiographic responses prompted termination of randomization and accrual only to the highest exposure cohort after patient 23. The protocol amendment also elevated analysis of clinical endpoints from exploratory objectives to clinical endpoint assessments joining the established radiographic assessments. The results of this trial reflect compelling improvement in PFS in the ITT population and for PFS, OS and PFS MGMT methylated subgroups who would have met entry criteria for current phase 3 newly-diagnosed GBM trials. These clinical endpoint improvements were accompanied by striking radiographic improvements with sustained meaningful quality of life.
The contribution of host factors to rabies virus (RV) transcription͞ replication and axonal͞transsynaptic spread is largely unknown. We previously identified several host genes that are up-regulated in the mouse brain during RV infection, including neuroleukin, which is involved in neuronal growth and survival, cell motility, and differentiation, and fibroblast growth factor homologous factor 4 (FHF4), which has been implicated in limb and nervous system development. In this study, we used real-time quantitative RT-PCR to assess the expression of mRNAs specific for neuroleukin, the two isoforms of FHF4 (FHF4-1a and -1b) encoded by the FHF4 gene, and N protein of RV in neurons and astrocytes isolated by laser capture microdissection from mouse brains infected with the laboratory-adapted RV strain CVS-N2c or with a street RV of silver-haired bat origin. Differences in the gene expression patterns suggest that the capacity of RV strains to infect nonneuronal cells and differentially modulate host gene expression may be important in virus replication and spread in the CNS.A lthough the predominant effect of rabies virus (RV) infection is the down-regulation of gene expression in the CNS, we have identified a subset of host genes that are induced by the infection (1). Among these are genes relevant to neuronal functions that may be involved in the replication and spread of RV, including neuroleukin (NLK) and fibroblast growth factor homologous factor 4 (FHF4). The member of a family of FHF4-1 genes, the FHF4 gene, which is predominantly expressed in the CNS in mice, encodes two isoforms, FHF4-1a and -1b, through alternative exon usage (2). The FHF4 isoforms both lack secretory signal peptides and accumulate intracellularly but differ functionally and in their cellular distribution (3). NLK has multiple functions, including acting as a growth factor to promote the survival and neurite outgrowth of motor and sensory neurons (4), as an autocrine motility factor to induce cell motility (5), as a maturation factor to mediate the differentiation of myeloid precursor cells to mature monocytes (6), and as a phosphohexose isomerase to catalyze the conversion of glucose-6-phosphate to fructose-6-phosphate (7).RV strains, such as the laboratory-adapted CVS-N2c RV strain and street viruses of silver-haired bat origin (SHBRV), often have highly disparate growth characteristics in vivo and in vitro (8-10) that may be reflected in their pathogenicity. We speculate that differences in the capacity of RV to replicate and spread is a consequence of the differential induction of factors such as NLK and FHF4-1a and -1b. To test this hypothesis, we have analyzed the expression patterns of these genes in neurons and astrocytes isolated from mice infected with CVS-N2c and SHBRV-17. Materials and MethodsVirus Infection of Mice and Tissue Preparation. All animal experiments were performed according to procedures approved by the Institutional Review Board's Animal Care and Use Committee. C3H mice 6-8 weeks old were purchased from Taconic Farm...
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