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Influenza is generally regarded as an infection of the respiratory tract; however, neurological involvement is a well-recognized, although uncommon, complication of influenza A virus infection. The authors previously described the development of a rat model for studying influenza virus infection of the central nervous system (CNS). This model was used here to study the role of virus genes in virus replication and spread in brain. In the present work, an infectious cDNA clone of the neurotoxic WSN strain of influenza virus (rWSN) was altered by site-directed mutagenesis at five loci that corresponded to changes previously shown to confer temperature sensitivity and attenuation of the A/Ann Arbor/6/60 strain (PB1Delta 391, PB1Delta 581, and PB1Delta 661; PB2Delta 265, and NPDelta 34). Whereas rWSN and its mutated derivative (mu-rWSN) replicated equally well in MDCK cells at 37 degrees C (the body temperature of rats), rWSN grew to higher titers and infection was more widespread compared to mu-rWSN in rat brain. These results demonstrate that the five mutations that confer attenuation of the A/Ann Arbor/6/60 influenza virus strain for the respiratory system also confer attenuation for the central nervous system. Further in vivo and in vitro examination of these five mutations, both individually and in combination, will likely provide important information on the role of specific virus genes in virulence and pathogenesis.

Mumps virus is highly neurotropic, with evidence of infection of the central nervous system in more than half of clinical cases. In the prevaccine era, mumps was a major cause of viral meningitis in most developed countries. Despite efforts to attenuate the virus, some mumps vaccines have retained virulence properties and have caused aseptic meningitis in vaccinees, resulting in public resistance to vaccination in some countries. Ensuring the safety of mumps vaccines is an important public health objective, as the need for robust immunization programs has been made clear by the recent resurgence of mumps outbreaks worldwide, including the United States, which in 2006 experienced its largest mumps outbreak in 20 years. To better understand the molecular basis of mumps virus attenuation, the authors developed two infectious full-length cDNA clones for a highly neurovirulent strain of mumps virus. The clones differed at only one site, possessing either an A or G at nucleotide position 271 in the F gene, to represent the heterogeneity identified in the original virulent clinical isolate. In comparison to the clinical isolate, virus rescued from the A-variant cDNA clone grew to higher cumulative titers in vitro but exhibited similar cytopathic effects in vitro and virulence in vivo. In contrast, virus rescued from the G-variant cDNA clone, in comparison to the clinical isolate and the A-variant, was more fusogenic in vitro but replicated to lower cumulative titers and was less neurovirulent in vivo. These data suggest that nucleotide position 271 in the F gene plays a significant role in virus pathogenesis. This infectious clone system will serve as a key tool for further examination of the molecular basis for mumps virus neurovirulence and neuroattenuation.

Infection of adult rats with Borna disease virus (BDV) causes CD8 T cell-mediated meningoencephalitis. Previously, we described a complex pattern of chemokine gene expression in the central nervous system (CNS) of such rats. We now found that expression of chemokine receptor genes CXCR3, CCR5, CX(3)CR1, and CXCR4 was also upregulated, which is in agreement with the predominance in brains of adult infected rats of T cells and monocytes/macrophages that express these receptors. In contrast to these rats, neonatally infected rats (designated PTI-NB) develop a persistent CNS infection associated with neurodegenerative processes in the absence of inflammation. In brains of PTI-NB rats, sustained expression of chemokines also takes place. We therefore analyzed mRNA expression of selected chemokine receptor genes, as well as of the chemokine fractalkine in brains of PTI-NB rats. We observed a marked increase of CCR5 and CX(3)CR1 transcripts in brains of these rats. CX(3)CR1 expressing cells were predominantly microglia, and upregulation of CX(3)CR1 was mainly due to an increase in the number of CX(3)CR1 expressing microglia. Fractalkine gene expression was found to be reduced to similar extents in brains of adult and newborn infected rats. These findings might be of relevance with respect to the selective neuronal cell loss observed in brains of PTI-NB rats.

In the course of retroviral CNS infections, microglia activation has been observed frequently, and it has been hypothesized that activated microglia produce and secrete neurotoxic products like proinflammatory cytokines, by this promoting brain damage. We challenged this hypothesis in a rat model for neurodegeneration. In a kinetic study, we found that microglia cells of rats neonatally inoculated with neurovirulent murine leukemia virus (MuLV) NT40 became infected in vivo to maximal levels within 9-13 days postinoculation (d.p.i.). Beginning from 13 d.p.i., degenerative alterations, i.e., vacuolization of neurons and neuropil were found in cerebellar and other brain-stem nuclei. Elevated numbers of activated microglia cells--as revealed by immunohistochemical staining with monoclonal antibody ED1--were first detected at 19 d.p.i. and were always locally associated with degenerated areas but not with nonaltered, yet infected, brain regions. Both neuropathological changes and activated microglia cells increased in intensity and numbers, respectively, with ongoing infection but did not spread to other than initially affected brain regions. By ribonuclease protection assays, we were unable to detect differences in the expression levels of tumor-necrosis-factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and interleukin-6 (IL-6) in microglia cells nor in total brains from infected versus uninfected rats. Our results suggest that the activation of microglia in the course of MuLV neurodegeneration is rather a reaction to, and not the cause of, neuronal damage. Furthermore, overt expression of the proinflammatory cytokines TNF-alpha, IL-1beta, and IL-6 within the CNS is not required for the induction of retroviral associated neurodegeneration in rats.

Infection of newborn rats with Borna disease virus (BDV) leads to persistence in the absence of overt signs of inflammation. BDV persistence, however, causes cerebellar hypoplasia and hippocampal dentate gyrus neuronal cell loss, which are accompanied by diverse neurobehavioral abnormalities. Neurotrophins and their receptors play important roles in the differentiation and survival of hippocampal and cerebellar neurons. We have examined whether BDV can cause alterations in the neurotrophin network, thus promoting neuronal damage. We have used RNase protection assay to measure mRNA levels of the neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3), and their trkC and trkB receptors, as well as the growth factors insulin-like growth factor I (IGF-1) and basic fibroblast growth factor (bFGF), in the cerebellum and hippocampus of BDV-infected and control rats at different time points p.i. Reduced mRNA expression levels of NT-3, BDNF and NGF were found after day 14 p.i. in the hippocampus, but not in the cerebellum, of newborn infected rats. Three weeks after infection, trkC mRNA expression levels were reduced in both hippocampus and cerebellum of infected rats, whereas decreased trkB mRNA levels were only observed in the cerebellum. Reduced trkC mRNA expression was confined to the dentate gyrus of the hippocampus, as assessed by in situ hybridization. TUNEL assay revealed massive apoptotic cell death in the dentate gyrus of infected rats at days 27 and 33 p.i. Increased numbers of apoptotic cells were also detected in the cerebellar granular layer of infected rats after 8 days p.i. Moreover, a dramatic loss of cerebellar Purkinje cells was seen after day 27 p.i. Our results support the hypothesis, that BDV-induced alterations in neurotrophin systems might contribute to selective neuronal cell death.