Pathogenesis of canine distemper virus in experimentally infected raccoon dogs, foxes, and minks.

Canine distemper virus (CDV) infects a broad range of carnivores and causes a highly contagious disease with severe immunosuppression. The disease severity markedly varies in different species. To investigate the pathogenesis of CDV in raccoon dog (Nyctereutes procyonoides), fox (Vulpes vulpes) and mink (Neovison vison) species, three groups of CDV sero-negative animals were infected with CDV strain LN(10)1. This CDV strain belongs to the Asia-1 genotype, which is epidemiologically predominant in carnivores in China. CDV infection provoked marked differences in virulence in the three species that were studied. Raccoon dogs developed fever, severe conjunctivitis, and pathological lesions, with 100% (5/5) mortality and with high viral RNA loads in organs within 15 days post infection (dpi). In infected foxes, the onset of the disease was delayed, with 40% (2/5) mortality by 21 dpi. Infected minks developed only mild clinical signs and pathological lesions, and mortality was not observed. Raccoon dogs and foxes showed more severe immune suppression (lymphopenia, decreased lymphocyte proliferation, viremia and low-level virus neutralizing antibodies) than minks. We also observed a distinct pattern of cytokine mRNA transcripts at different times after infection. Decreased IFN-γ and IL-4 mRNA responses were evident in the animals with fatal disease, while up-regulation of these cytokines was observed in the animals surviving the infection. Increased TNF-α response was detected in animals with mild or severe clinical signs. Based on the results, we could distinguish three different patterns of disease after experimental CDV infection, e.g. a mild form in minks, a moderate form in foxes and a severe disease in raccoon dogs. The observed differences in susceptibility to CDV could be related to distinct host cytokine profiles. Comparative evaluation of CDV pathogenesis in various animal species is pivotal to generate models suitable for the evaluation of CDV-host interactions and of vaccine response.

Viral protein expression and phenotyping of inflammatory responses in the central nervous system of phocine distemper virus-infected harbor seals (Phoca vitulina).

The central nervous system (CNS) represents an important target organ of the phocine distemper virus (PDV). The aim of the present study was to characterize pathological changes in the CNS of harbor seals suffering from natural PDV-infection. The distribution of virus protein and mRNA was investigated by immunohistochemistry (IHC) and in situ hybridization, respectively. In addition, inflammatory and glial cells were characterized by IHC. Polioencephalitis with glial activation, neuronal death and perivascular mononuclear infiltrations in the cerebral cortex was the main histopathological finding. Inflammatory responses, dominated by CD3(+) T-cells and activated microglia/macrophages were associated with a prominent MHC-II upregulation within the CNS. Viral protein was found predominantly in neurofilament-expressing neurons within inflamed areas as demonstrated by immunohistochemical double-labeling. Morbillivirus nucleo-, phospho-, matrix-, fusion- and hemagglutinin-proteins were found in CNS-lesions. The expressions of viral matrix- and fusion-proteins were reduced in severely inflamed plaques. Comparison of viral protein and mRNA expression revealed a diminished amount of viral phosphoprotein preferentially associated with perivascular inflammation. In summary, CNS-lesions in PDV-infected seals are similar to canine distemper virus-induced acute polioencephalitis in dogs and measles virus inclusion body polioencephalitis in men, respectively.

Canine distemper viruses expressing a hemagglutinin without N-glycans lose virulence but retain immunosuppression.

Paramyxovirus glycoproteins are posttranslationally modified by the addition of N-linked glycans, which are often necessary for correct folding, processing, and cell surface expression. To establish the contribution of N glycosylation to morbillivirus attachment (H) protein function and overall virulence, we first determined the use of the potential N-glycosylation sites in the canine distemper virus (CDV) H proteins. Biochemical characterization revealed that the three sites conserved in all strains were N glycosylated, whereas only two of the up to five additional sites present in wild-type strains are used. A wild-type virus with an H protein reproducing the vaccine strain N-glycosylation pattern remained lethal in ferrets but with a prolonged course of disease. In contrast, introduction of the vaccine H protein in the wild-type context resulted in complete attenuation. To further characterize the role of N glycosylation in CDV pathogenesis, the N-glycosylation sites of wild-type H proteins were successively deleted, including a nonstandard site, to ultimately generate a nonglycosylated H protein. Despite reduced expression levels, this protein remained fully functional. Recombinant viruses expressing N-glycan-deficient H proteins no longer caused disease, even though their immunosuppressive capacities were retained, indicating that reduced N glycosylation contributes to attenuation without affecting immunosuppression.

Vimentin-positive astrocytes in canine distemper: a target for canine distemper virus especially in chronic demyelinating lesions?

In canine distemper demyelinating leukoencephalitis (DL), caused by canine distemper virus (CDV), astrocytes represent the main virus target. In these cells, glial fibrillary acidic protein (GFAP) is the main intermediate filament, whereas vimentin occurs early in the astrocytic lineage and is replaced gradually by GFAP. To further characterize the role of astrocytic infection in dogs with DL, an animal model for multiple sclerosis, formalin-fixed paraffin-embedded cerebella were investigated immunohistochemically and by immunofluorescence. The expression and morphological alterations of these intermediate filaments were also determined by immunofluorescence studies of CDV-infected canine mixed brain cell cultures. In acute distemper lesions, the astrocytic response was mainly composed of GFAP- and CDV-positive cells. In contrast, vimentin-positive astrocyte-like cells were present in advanced lesions, which represented the main cell type harboring the pathogen, indicating a change in cell tropism and/or susceptibility of glial cells during lesion progression in CDV encephalomyelitis. Canine cell cultures were composed of GFAP-positive astrocytes, vimentin-positive cells and other glial cells. Following infection with the CDV-R252 strain, GFAP-positive astrocytes, especially multinucleated syncytial giant cells, displayed a disrupted cytoskeleton, whereas vimentin-positive cells though more frequently infected did not show any alteration in the filament network. This indicates increased vulnerability of mature GFAP-positive astrocytes compared to immature, vimentin-positive astrocytes. The latter, however, exhibited increased susceptibility to CDV. To conclude, the present findings indicate a change in cell tropism of CDV and/or the occurrence of less differentiated astrocytes representing a permanent source for virus infection and spread in advanced lesions of DL.

Canine distemper virus infection of primary hippocampal cells induces increase in extracellular glutamate and neurodegeneration.

The canine distemper virus (CDV) belongs to the Morbillivirus genus which includes important human pathogens like the closely related measles virus. CDV infection can reach the nervous system where it causes serious malfunctions. Although this pathology is well described, the molecular events in brain infection are still poorly understood. Here we studied infection in vitro by CDV using a model of dissociated cell cultures from newborn rat hippocampus. We used a recombinant CDV closely related to the neurovirulent A75/17 which also expresses the enhanced green fluorescent protein. We found that infected neurons and astrocytes could be clearly detected, and that infection spreads only slowly to neighboring cells. Interestingly, this infection causes a massive cell death of neurons, which includes also non-infected neurons. Antagonists of NMDA-type or alpha-amino-3-hydroxy-5-methylisoxazole-4-propinate (AMPA)-type glutamate receptors could slow down this neuron loss, indicating an involvement of the glutamatergic system in the induction of cell death in infected and non-infected cells. Finally, we show that, following CDV infection, there is a steady increase in extracellular glutamate in infected cultures. These results indicate that CDV infection induces excitotoxic insults on neurons via glutamatergic signaling.

Disease manifestations of canine distemper virus infection in ferrets are modulated by vitamin A status.

The measles virus (MV) causes half a million childhood deaths annually. Vitamin A supplements significantly reduce measles-associated mortality and morbidity. The mechanisms whereby vitamin A acts against MV are not understood and currently there is no satisfactory small animal model for MV infection. We report on the development of a ferret model to study antiviral activity of vitamin A against canine distemper virus (CDV). CDV is closely related to MV at the molecular level and distemper in ferrets mimics measles in humans. We infected vitamin A-replete (control) and vitamin A-depleted ferrets with CDV and assessed the ability of high-dose vitamin A supplements to influence CDV disease. In control ferrets, CDV infection caused fever, rash, conjunctivitis, cough, coryza, and diarrhea. In contrast, control ferrets that were given 30 mg of vitamin A did not develop typical distemper after infection and exhibited only a mild rash. The supplement did not negatively affect ferret health and resulted in a 100% increase in serum and liver vitamin A concentrations. We also found that profound vitamin A deficiency is inducible in ferrets and can be rapidly reversed upon high-dose vitamin A supplementation. Vitamin A deficiency caused anorexia, diarrhea, cataracts, behavioral abnormalities, and ultimately death, with or without CDV infection. All ferrets that received vitamin A supplements, however, recovered uneventfully from CDV infection. These results replicate many aspects of the observations of vitamin A therapy in humans with measles and suggest that CDV infection in ferrets is an appropriate model for the study of the antiviral mechanism of vitamin A.

Demyelination in canine distemper virus infection: a review.

Canine distemper virus (CDV) causes severe immunosuppression and neurological disease in dogs, associated with demyelination, and is a model for multiple sclerosis in man. In the early stage of the infection, demyelination is associated with viral replication in the white matter. In acute demyelinating lesions there is massive down-regulation of myelin transcription and metabolic impairment of the myelin-producing cells, but there is no evidence that these cells are undergoing apoptosis or necrosis. Oligodendroglial change is related to restricted infection of these cells (transcription but no translation) and marked activation of microglial cells in acute lesions. Concomitant with immunological recovery during the further course of the disease, inflammation occurs in the demyelinating plaques with progression of the lesions in some animals. A series of experiments in vitro suggests that chronic inflammatory demyelination is due to a bystander mechanism resulting from interactions between macrophages and antiviral antibodies. Autoimmune reactions are also observed, but do not correlate with the course of the disease. The progressive or relapsing course of the disease is associated with viral persistence in the nervous system. Persistence of CDV in the brain appears to be favored by non-cytolytic selective spread of the virus and restricted infection, in this way escaping immune surveillance in the CNS. The CDV Fusion protein appears to play an important role in CDV persistence. Similarities between canine distemper and rodent models of virus-induced demyelination are discussed.

Relation of clinical signs to pathological changes in 19 cases of canine distemper encephalomyelitis.

In an attempt to associate the clinical neurological syndromes with the neuropathological features of canine distemper (CD), 19 spontaneous cases with neurological involvement were examined, before and after euthanasia. Seventeen dogs were less than one year of age and all except two (89.4%) were unvaccinated against CD. Various extraneural signs associated with CD encephalomyelitis (CDE) were seen in 15 dogs. Generalized or localized myoclonus was the most common sign observed (13/19). Seventeen of the dogs presented with signs suggestive of one neuroanatomical location of lesions. Of these animals, seven had signs of cerebral, two of cerebellar, four of cervical, one of cervicothoracic, two of thoracolumbar and two of lumbosacral syndrome. The diagnosis of CD was confirmed immunohistochemically (detection of CD viral antigen), serologically (neutralizing serum antibody titre > or = 16) and histopathologically (CDV inclusion bodies, type of central nervous system lesions). An association of the neuroanatomical lesion location and the histopathological findings was noted in 14 out of 17 dogs (82.3%). Myoclonus could be attributed to lower motor neuron damage in eight out of 13 dogs (61.5%).

Canine distemper infections, with special reference to South Africa, with a review of the literature.

Canine distemper virus is a member of the genus Morbillivirus of the family Paramyxoviridae that causes severe disease in dogs and a range of wild mammals. The clinical signs relate essentially to the respiratory, gastrointestinal and central nervous systems. In South Africa, infection with Ehrlichia canis and canine parvovirus may present similarly Many dogs will initially present with a wide range of central nervous system signs without any history of systemic disease. A recent South African study evaluating ante mortem diagnosis highlighted the importance of recognising clinical signs, cerebrospinal fluid IgG titres, serum IgM titres and immunocytochemistry of epithelial tissue. A 2-year retrospective evaluation of cerebrospinal fluid samples collected from dogs presented to the Onderstepoort Veterinary Academic Hospital indicates that distemper infection is common, and this disease should routinely be suspected in cases of diverse neurological disease in dogs. The South African dog population is specifically at high risk for the disease because of the large pool of unvaccinated, reproductively-active dogs that expose the wildlife resources of the country to risk of fatal disease. Outbreaks of disease in dogs continue to occur in developed and developing communities in both vaccinated and unvaccinated dogs worldwide, and have also been described in a wide range of free-ranging wildlife, including seals, dolphins and lions, and in endangered zoo animals. Modified live virus vaccines have contributed markedly to disease control in the dog population but have caused mortality in some wild carnivores. New recombinant vaccines are being developed that will be safe in wild animals. The pathogenesis of CNS demyelination has been compared to various important demyelinating diseases in humans and, amongst other things, relates to down-regulation of the oligodendrocyte gene coding for myelin synthesis and non-immunocyte CNS cell expression of type II major histocompatibility receptors. Early CNS lesions are characterised by demyelination and later lesions by perivascular round cell cuffing. Treatment is supportive.

Alteration of the leptin network in late morbid obesity induced in mice by brain infection with canine distemper virus.

Viruses can induce progressive neurologic disorders associated with diverse pathological manifestations, and therefore, viral infection of the brain can impair differentiated neural functions, depending on the initial viral tropism. We have previously reported that canine distemper virus (CDV) targets certain mouse brain structures, including the hypothalamus, early and selectively. Infected mice exhibit acute encephalitis, with late disease, characterized by motor impairment or obesity syndrome, appearing in some of the surviving mice several months after the initial viral replication. In the present study, we show viral persistence in the hypothalami of obese mice, as demonstrated by low, but still significant, levels of CDV nucleoprotein transcripts, associated with a dramatic decrease in F gene mRNAs. Given the pivotal role of the hypothalamus in obesity (eating behavior, energy consumption, and neuroendocrine function) and that of leptin, the adipose tissue-derived satiety factor acting through hypothalamic receptors, we analyzed the leptin networks in both obese and nonobese mice. The discrepancy found between the chronic and dramatic increase in blood leptin levels and the occurrence of obesity may be due to leptin resistance in the brain. In fact, expression of the long leptin receptor isoform, representing the functional leptin receptor, was specifically downregulated in the hypothalami of obese mice, explaining their inability to generate an adequate response to leptin in the brain. Intriguingly, during the acute phase of infection, its expression was increased in CDV-targeted structures in all infected mice and remained high in obese mice in all CDV-targeted structures, except for the hypothalamus. The biphasic change in hypothalamic leptin receptor expression seen during the progression of CDV-induced obesity provides a new paradigm for understanding mechanisms of neuroendocrinological, virus-induced abnormalities.

[Viruses and the neuroendocrine system: model of murine obesity induced by cerebral infection by canine distemper virus]. / Virus et système neuroendocrinien: modèle d'obésité murine induite après infection cérbrale par le virus de la maladie de Carré.

It is currently well established that the nervous, endocrine and immune systems inter-communicate using biologically active soluble factors, synthesised and produced by these three systems themselves (e.g. immunomodulator effect of hormones, effect of substances secreted by immune cells on endocrine function.). In addition, these systems jointly express receptors for hormones, peptides, growth factors and cytokines. Immuno-neuroendocrine interactions therefore underlie physiological processes and their deregulation can result in various pathological states. By entering into complex relationships with the specialized and differentiated cells of these three systems viruses can alter inter-cellular communication and result in the appearance of pathological processes directly linked to these disturbances. In order to understand the role of viruses in the genesis of neuroimmunoendocrine pathologies, we have developed a cerebral infection model using canine distemper virus (CDV). In infected mice, this paramyxovirus, closely related to the human measles virus, induces early neurological pathologies (encephalitis) which are associated with active viral replication. Mice surviving the acute phase of infection exhibit motor deficits (paralysis and turning behaviour) or obesity during the viral persistence phase, despite the fact that the virus is no longer detectable. The obesity is characterised by hyperinsulinaemia, hyperleptinaemia and hyperplasia of the adipocytes, associated with decreased expression of the OB-Rb hypothalamic leptin receptor and modulated expression of hypothalamic monoamines and neuropeptides. These results support the viral "hit and run" theory, since the initial viral impact in the hypothalamus may be the origin of the changes in later immunoneuroendocrine communication. Thus, certain human neurodegenerative or neuroendocrine diseases may have a previous viral infection aetiology without it being possible to clearly identify the agent responsible.

Inhibition of tyrosine hydroxylase expression within the substantia nigra of mice infected with canine distemper virus.

Experimental infection of mouse brain with a neuroadapted strain of canine distemper virus (CDV) leads to early acute encephalitis, followed by late neurological diseases such as motor pathologies (paralysis and turning behavior) or obesity syndrome. We have previously shown that, during the early stage of infection, CDV replicates transiently in selective structures of the brain including the substantia nigra, a structure known to play a critical role in motor control. In this study we demonstrate that CDV replication in the substantia nigra induces an early decrease in transcript level of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis. The CDV infection of neuroblastoma cell culture, constitutively expressing TH, results in downregulation of TH transcription in the absence of cell death. In the few surviving mice with motor deficiencies, a pronounced decrease in TH expression is associated with a loss of dopaminergic cell bodies in the absence of any viral transcripts and proteins, suggesting that the initial CDV infection was sufficient to trigger irreversible neurodegenerative processes.

Electroencephalographic findings of encephalitis in beagle dogs experimentally infected with canine distemper virus (CDV).

The present research study had two goals. Firstly, the effect of medetomidine/propofol on the electroencephalographic (EEG) recordings of nine SPF dogs was analysed. It was found that the basic pooled pattern of EEG recordings was characterized by a mean amplitude of 28 uV (range = 15-70 uV) and mean frequency of 1.8 Hz (range = 1-2.5 Hz), with superimposed low voltage (amplitude range = 4-20 uV; mean 14 uV) and fast activity (frequency range = 10-27 Hz; mean 18 Hz). All results were significantly constant (P < 0.05) and accurately reproducible (R = 0.70). The second goal was to investigate the value of EEG findings from dogs experimentally infected with canine distemper virus (CDV) under the same anaesthetic conditions, for the diagnosis of encephalitis. It was found that the EEG traces were characterized by high voltage, slow activity (HVSA) with superimposed low voltage, fast activity (LVFA). By comparing the mean values of amplitude and frequency for each single phase at different time intervals, it was found that only the mean value of the superimposed frequency was linearly distributed and changed significantly (P < 0.001). Thus, at least three readings should be taken from phase III of the EEG recording in order to obtain accurate values. The study has shown that electroencephalography can supply valuable information in cases of CDV encephalitis and can assist greatly in the differential diagnosis.

The effect on chick osteoclasts of infection with paramyxoviruses.

The detection of virus in osteoclasts from Pagetic patients is now well known, but it has yet to be shown convincingly that the presence of virus in Pagetic osteoclasts influences their behaviour. In this study, osteoclasts from embryonic chick tibiae were infected with canine distemper virus or measles virus and compared with mock-infected controls. Infection was confirmed using virus-specific fluorescent antibodies. It was found that virus infection did not alter osteoclast morphology or tartrate-resistant acid phosphatase (TRAP) activity. Both infected and mock-infected osteoclasts produced resorption pits on bovine bone slices; these could be divided into two distinct size classes with a computer-based measuring system. Virus infection significantly increased the proportion of the larger size class of resorption pit. These results suggest that virus infection can increase bone resorption by osteoclasts, lending further support to the hypothesis that viruses play a role in Paget's disease of bone.

Epidemiological observations on recent outbreaks of canine distemper in Tokyo area.

Recent outbreaks of canine distemper virus (CDV) infection in Tokyo area were investigated on the basis of clinical features and serological test. The affected dogs were clinically classified into two groups; dog with respiratory and gastrointestinal signs associated with central nervous system (CNS) signs, and those with CNS signs alone. Of 62 dogs examined, 34 belonged to the former and 28 to the latter. In immunoperoxidase assay, anti-Onderstepoort strain of CDV serum reacted at a low level against 2 field isolates of CDV. These results suggested the presence of different types of CDV population in the field.

[RT-PCR: a tool for the generation of clones for research on canine distemper]. / RT-PCR: Ein Hilfsmittel zur Herstellung von Klonen für die Staupeforschung.

In the present work an adapted method of the reverse transcriptase polymerase chain reaction (RT-PCR) is described. This procedure was an important tool for cloning a variety of gene sequences derived from oligodendrocytes and canine distemper virus (CDV). Among other applications, the obtained clones were used to produce probes for in situ hybridization and to sequence the inserted DNAs. Due to these molecularbiological techniques new insights were gained into the pathogenesis of CDV-induced demyelination and CDV-persistence in the central nervous system.

Serological survey of canine distemper virus infection using enzyme-linked immunosorbent assay.

For detection of antibodies against canine distemper virus (CDV), an enzyme-linked immunosorbent assay (ELISA) was developed using a crude extract from cells infected with the Onderstepoort strain of CDV as antigen. Twenty-six sera from dogs experimentally vaccinated with the Snyder-Hill strain of CDV were compared by ELISA and a standard virus neutralization (NV) test. Since a good correlation between the titers obtained by both tests was observed, ELISA was considered to be a rapid and reliable method for a serological survey of CDV infection. When a total of 167 sera from dogs suspected of canine distemper under natural conditions were examined by the ELISA, 29 of the sera (17%) were found to have low VN antibody titers and high ELISA titers. The reason for the discrepancy in the titers was discussed.

Electroencephalogram and evoked potentials in the primate model of viral encephalitis.

Squirrel monkeys with induced canine distemper virus (CDV) encephalitis showed characteristic clinical signs such as seizures or myoclonus, with EEGs showing periodic synchronized discharge (PSD). Histopathologically, there was gliosis and neuronal degeneration diffusely distributed in both the gray and white matters in the subacute phase, the lesions resembling those found in childhood acute viral encephalitis and subacute sclerosing panencephalitis (SSPE). The auditory brain-stem response (ABR) changes were remarkable in the subacute phase, and the recovery of latency was correlated with survival. The visual evoked potential (VEP) abnormalities disappeared in the acute phase and appeared again with delayed latency in the subacute stage. One monkey which did not show clinical signs but had the pathological changes showed the VEP abnormality. These data suggest that the evoked potentials are helpful in judging the prognosis, and that this model is useful for the analysis of the pathogenesis of viral encephalitis.

Canine distemper myoclonus and sleep: observation of a case.

Evolution of myoclonus was analysed electromyographically throughout quiet wakefulness, NREM sleep and REM sleep in a dog with canine distemper myoclonus. Compared with quiet wakefulness, the frequency of myoclonus was decreased and the intensity of discharges in individual myoclonic bursts was also lowered during NREM sleep. When NREM sleep shifted to REM sleep, neither of these parameters was noticeably altered. However, as REM sleep continued, the former was increased markedly and the latter was further attenuated. In general, violent motor activity was concurrent in the limbs, trunk and/or head. Thus, the increase in the frequency of myoclonus seemed to be similar in nature to the phasic event during REM sleep. This indicated that lower motoneurons producing myoclonic discharges responded well to supraspinal influences. In successive myoclonic bursts, a significant and consistent positive correlation existed between the silent period and the subsequent discharge period throughout the three different levels of consciousness, that is, the longer the silent period, the longer the subsequent episode of myoclonus. Therefore, this feature may be intrinsic to myoclonic firing of lower motoneurons in canine distemper myoclonus. These findings may support the idea that hyperexcitability of the lower motoneurons is primarily responsible for the genesis of canine distemper myoclonus.