Presence of specific antigens in neuronal cells infected with fixed and street rabies virus strains

Summary The presence of rabies specific antigens is investigated after infection with different rabies virus strains in neural cell lines and in the central nervous system of laboratory rodents. In fixed rabies infected cells, the rabies glycoprotein is found to be present 48 h after infection, whereas in hamsters this protein was found 5 days after an intracerebral inoculation. In contrast, rabies glycoprotein was not detectable in any of the street rabies-infected cell system by the fluorescent antibody test, although nucleoprotein was present, showing that infection occurred in these cells. Rabies glycoprotein was also undetectable in the central nervous system (CNS) of athymic nude mice which is known to be very sensitive to street rabies infection and to contain large quantities of viral material. Our results suggest that the smaller amount of rabies glycoprotein synthesized during street rabies infection are of consequence for the pathogenesis of rabies disease. The immunopathology of street rabies virus infection is certainly modulated by the failure of the viral glycoprotein to be present in large quantities on the surface of the infected cellular membrane as in the case of fixed rabies.Supported by grants from the Institut Pasteur and from INSERM (CRE 78.4.146.1 and ATP 50.77.82.009)     

Rabies encephalitis in a patient with AIDS: a clinicopathological study

A 46-year-old man was bitten by a dog in Mali; anti-rabies vaccination was incomplete. Three months later he was admitted to hospital with fever and diarrhea. Human immunodeficiency virus (HIV) serology was positive and CD4 count was 70/mm³. His status worsened rapidly with confusion hydrophobia and hypersialorrhea. Despite anti-rabies serotherapy and vaccination, he died suddenly 12 days after admission. Immunofluorescence on cerebral tissue samples established rabies encephalitis. Neuropathology showed mild encephalitis with occasional Babès nodules and rare perivascular mononuclear cuffs. Intraneuronal Negri inclusion bodies were remarkably diffuse and abundant. They were clearly demonstrated by immunocytochemistry and electron microscopy. Apoptotic neurons were identified in the brain stem and hippocampus in the vicinity of inflammatory foci. In contrast, apoptosis could not be demonstrated in non-inflammatory areas, even where Negri bodies were numerous. There was no associated HIV encephalitis or opportunistic infection. The occurrence of rabies encephalitis in AIDS represents a random association, but is probably not exceptional as rabies is endemic in many countries and the AIDS epidemic is spreading worldwide. In this case, although the incubation duration and clinical presentation were comparable to those in classical rabies, the T-cell-mediated immunosuppression may account for the weak inflammatory reaction and unusually abundant viral multiplication. This observation confirms that all those at risk for rabies, particularly immunocompromised patients, should receive complete anti-rabies treatment including vaccines and specific immunoglobulins, as soon as possible after infection. Received: 1 February 1996 / Revised, accepted: 24 April 1996     

Furious and paralytic rabies of canine origin: Neuroimaging with virological and cytokine studies

Furious and paralytic rabies differ in clinical manifestations and survival periods. The authors studied magnetic resonance imaging (MRI) and cytokine and virus distribution in rabies-infected dogs of both clinical types. MRI examination of the brain and upper spinal cord was performed in two furious and two paralytic dogs during the early clinical stage. Rabies viral nucleoprotein RNA and 18 cytokine mRNAs at 12 different brain regions were studied. Rabies viral RNA was examined in four furious and four paralytic dogs during the early stage, and in one each during the late stage. Cytokine mRNAs were examined in two furious and two paralytic dogs during the early stage and in one each during the late stage. Larger quantities of rabies viral RNA were found in the brains of furious than in paralytic dogs. Interleukin-1beta and interferon-gamma mRNAs were found exclusively in the brains of paralytic dogs during the early stage. Abnormal hypersignal T2 changes were found at hippocampus, hypothalamus, brainstem, and spinal cord of paralytic dogs. More widespread changes of less intensity were seen in furious dog brains. During the late stage of infection, brains from furious and paralytic rabid dogs were similarly infected and there were less detectable cytokine mRNAs. These results suggest that the early stage of furious dog rabies is characterized by a moderate inflammation (as indicated by MRI lesions and brain cytokine detection) and a severe virus neuroinvasiveness. Paralytic rabies is characterized by delayed viral neuroinvasion and a more intense inflammation than furious rabies. Dogs may be a good model for study of the host inflammatory responses that may modulate rabies virus neuroinvasiveness.     

Phylogenetic and epidemiologic evidence of multiyear incubation in human rabies

Eight years after emigrating from Brazil, an otherwise healthy man developed rabies. An exposure prior to immigration was reported. Genetic analysis revealed a canine rabies virus variant found only in the patient's home country, and the patient had not traveled internationally since immigrating to the United States. We describe how epidemiological, phylogenetic, and viral sequencing data provided confirmation that rabies encephalomyelitis may present after a long, multiyear incubation period, a consideration that previously has been hypothesized without the ability to exclude a more recent exposure. Accordingly, rabies should be considered in the diagnosis of any acute encephalitis, myelitis, or encephalomyelitis. ANN NEUROL 2014;75:155–160     

Rabies

Rabies is an important disease in wildlife in the United States and Canada, and dog rabies is still a major public health problem in many developing countries of the world. Rabies virus is transmitted in saliva by animal bites. Bats transmitted most recent cases of human rabies in the United States, often without known exposures. There have been recent developments in our understanding of rabies pathogenesis. Characteristic clinical features should raise the possibility of a diagnosis of rabies and initiation of appropriate diagnostic tests. Therapy of human rabies has been futile except in four patients who were immunized with rabies vaccine prior to the onset of their disease. Rabies can be prevented after an exposure in unimmunized patients with local wound cleansing and administration of rabies vaccine and human rabies immune globulin.     

Immunohistochemical study of human rabies

Rabies is a communicable disease that is almost always fatal. In its classic form, rabies is well recognized, but cases presenting with a paralytic illness mimic Landre's Guillain–Barre syndrome and in such cases the diagnosis remains in doubt. This problem is further compounded when the history of dogbite is not forthcoming. At autopsy rabies can be diagnosed by subjecting fresh tissue to virologic investigations or examining formalin‐fixed paraffin‐embedded tissue sections for the presence of characteristic inclusions; that is, the Negri bodies. However, these inclusions are not present in all cases. Hence, the need arises for a better method for diagnosis. The present study utilized immunohistochemistry as a diagnostic tool using both monoclonal and polyclonal antirabies antibodies in 20 cases of rabies encephalomyelitis. The diagnosis of rabies could be confirmed in 17 cases (85%) based on neuropathologic findings alone. In contrast, immunohistochemistry yielded positive results in all cases. Moreover, the amount of rabies viral antigen was much more abundant than could be expected from the histopathologic findings. Thus immunohistochemistry is a rapid, safe, sensitive and specific technique for the diagnosis of rabies.     

Rabies encephalitis following fox bite--histological and immunohistochemical evaluation of lesions caused by virus

Rabies caused by fox bite is uncommon, most cases being caused by bite of rabid dogs (95%). We report a 45-year-old lady with rabies encephalomyelitis caused by bite of a rabid wild fox (Vulpes vulpes), a species prevalent in the Deccan plateaus of Central India. Though foxes are known to be susceptible to rabies, literature on the pathological changes caused by fox bite rabies in humans is scarce. Unlike the mild histological alterations described in canine rabies, a florid encephalitic process evolved in fox bite rabies, in our case, with intense microglial reaction, neuronophagia and perivascular inflammatory infiltrates despite clinical manifestation as a paralytic rabies. Immunostaining using polyclonal antibodies to the rabies viral nucleocapsid antigen and to the whole virion demonstrated high viral load within neurons with extensive spread along dendritic arborization and axonal tracts. Genomic sequence analysis demonstrated close homology with canine virus strain with only minor variations.     

Rabies encephalomyelitis: clinical, neuroradiological, and pathological findings in 4 transplant recipients

BACKGROUND: Three patients received solid organ transplants from a common donor and were subsequently discharged from the hospital following an uneventful hospital course. Within 30 days, all 3 organ recipients returned to the hospital with varying symptoms that progressed to rapid neurological deterioration, coma, and death. OBJECTIVE: To describe the clinical, neuroradiological, and pathological findings of rabies virus infection in organ transplant recipients infected from a common donor. DESIGN: Case series involving a common donor and 3 organ recipients ascertained through review of clinical course and autopsy findings. A fourth case was determined by review of pending autopsy cases in which death occurred within the same time interval. Portions of postmortem central nervous system and organ tissues were frozen and formalin-fixed. Fluids and tissues were also collected for cultures, serology, and molecular studies. Postmortem fluids and tissues and antemortem fluids and tissues from all 4 transplant recipients and serum and banked lymphocyte or spleen cells from the donors were sent to the Centers for Disease Control and Prevention for further evaluation. SETTING: Transplant unit of an urban teaching hospital. RESULTS: Antemortem cerebrospinal fluid analysis for 3 of the 4 recipients was consistent with a viral etiology. Neuroimaging and electroencephalogram studies were suggestive of an infectious encephalitis or a toxic encephalopathy. Initial laboratory testing did not demonstrate an infectious etiology. Postmortem histologic analysis, immunohistochemistry, electron microscopy, and direct fluorescence antibody testing revealed rabies virus infection. Serological testing done postmortem confirmed rabies virus infection in the common donor. CONCLUSIONS: These cases demonstrate a risk for transmitting rabies virus infection through solid organ and tissue transplantation, and this diagnosis should be considered in any rapidly progressing neurological disease.     

The role of immune responses in the pathogenesis of rabies

In the absence of treatment, infection with a variety of rabies virus strains most often results in a lethal outcome. This can be averted by prompt immunization following exposure demonstrating that the development of anti-rabies viral immunity prior to extensive infection of neurons is protective. Otherwise it might be expected that immune clearance of the virus would result in neurological sequelae. Thus, the capacity of a rabies virus to induce a protective immune response is a major, negative determinant of its pathogenicity and highly pathogenic rabies viruses have characteristics that avoid triggering protective immune responses. On the other hand, there is evidence that certain aspects of immunity may contribute to the pathogenesis of rabies under certain circumstances. The relationship between rabies virus and the immune system of the host is the focus of this review.     

Rabies viral antigen in extracranial organs: a post-mortem study

Rabies is a communicable disease and a significant health hazard. Histopathological confirmation of the diagnosis depends on the demonstration of Negri bodies - characteristic intracytoplasmic inclusions. In cases where these are not seen, immunohistochemistry serves as a useful adjunct. After its establishment in the central nervous system, the rabies virus is known to reach peripheral organs by a centrifugal spread. The present study was undertaken with the aim of demonstrating rabies viral antigen (RVAg) in the extracranial organs. Eleven confirmed cases of rabies were analysed and RVAg was found in the adrenal glands, heart, gastrointestinal tract and pancreas, confirming the centrifugal spread of the virus. The detection of RVAg in the extracranial sites may serve as a useful tool in the ante-mortem diagnosis by subjecting the extracranial tissue to biopsy and subsequent immunohistochemistry.     

Electron microprobe ultrastructural localization of aluminum in rat brain

Summary A 50-year-old carpenter died in Western Pennsylvania of rabies on January 4, 1979. He had been hospitalized in an intensive care unit for 28 days. The diagnosis was made postmortem from light and electron microscopic examination of central nervous system tissue. Immunofluorescence studies confirmed the diagnosss later. No animal exposure was confirmed in this case. The clinical and neuropathologic findings of the patient are correlated. The importance of recognizing rabies and the protection of personnel who perform autopsies on these patients is emphasized. In addition, rabies should be considered in the differential diagnosis of radiculomyelitis (Guillain-Barré syndrome) and, in general, in any case of meningoencephalitis.     

Rabies: Spongiform lesions in the brain

Summary Striped skunks (Mephitis mephitis) experimentally infected with street rabies virus developed spongiform lesions that light- and electron-microscopically were indistinguishable from those found in the transmissible spongiform encephalopathies of man and animals. These previously unreported lesions were also detected in naturally occurring cases of rabies. The spongiform lesions consisted of round or oval vacuoles in the neuropil, rarely in neuronal perikarya. The most severely affected areas were the thalamus and cerebral cortex. The implications of this finding include similarities in the pathogenetic mechanisms of rabies and the traditional spongiform encephalopathies and the possibility of lesion variation due to differences in rabies viral strains. The spongiform lesions of rabies will require consideration in differential diagnosis.     

Rabies in the critical care unit: diagnostic and therapeutic approaches

Worldwide, human rabies is prevalent where there is endemic dog rabies, but the disease may present unexpectedly in critical care units when suggestive clinical features have passed. In North America transmission from bats is most common and there is often no history of a bat bite or even contact with bats. Laboratory diagnostic evaluation for rabies includes serology plus skin biopsy, cerebrospinal fluid, and saliva specimens for rabies virus antigen and/or RNA detection. Rare patients have survived rabies, and most received rabies vaccine prior to the onset of illness. Therapeutic coma (midazolam and phenobarbital), ketamine, and antiviral therapies (dubbed the "Milwaukee Protocol") were given to a rabies survivor, but this therapy was likely not directly responsible for the favorable outcome. There have been many subsequent failures of similar therapeutic approaches. There is no scientific rationale for the use of therapeutic coma in human rabies. New approaches to treating human rabies need to be developed.     

Infection of cultured rat myotubes and neurons from the spinal cord by rabies virus

Rabies virus multiplication was investigated in cultured primary rat myotubes and neurons. The susceptibility of these two cell types to fixed rabies challenge virus strain (CVS) was monitored by fluorescence and virus titration. Differentiated rat myotubes were susceptible to rabies virus infection, and showed an increasing accumulation of viral material from day one to day four. However, these cells did not release infective viral particles, nor did they accumulate infectious virions in the cytoplasm. In contrast, infected neurons released large amounts of infectious particles. Electron microscopy observation of infected myotubes showed minor alterations and the presence of typical viral inclusions in the cytoplasm without mature virions assembling viral membranes. Competition binding experiments show that alpha-bungarotoxin inhibits rabies virus infection from 10(-5) to 10(-7) M, whereas lower toxin concentrations failed to have any effect. These data do not confirm the hypothesis of a fixed rabies virus amplification step at the site of the viral entry. On the other hand, the high susceptibility of peripheral neurons to rabies virus infection is an argument for the direct uptake of virions by these cells. The restrictive viral multiplication in the myotubes is an alternative explanation for the local persistence of rabies virus at the site of inoculation.     

A patient with leg weakness

The present case was typical in many respects for neuroinvasive WNV infection. The differential diagnosis considered was appropriately comprehensive. The present case also reminds us that little or no abnormalities will be seen on imaging in many cases, and that initial serology may be negative and should be repeated beyond the acute phase ante- or postmortem. Fortunately, specific antibodies are also now available for identification of viral proteins in tissue although sensitivity of the latter may be affected by the stage of infection and sampling of areas bearing a higher viral load. West Nile Virus, along with other emerging infections, serves notice of the health care implications of humanity's globalization of ecosystems.     

Pleomorphism of fine structure of rabies virus in human and experimental brain

Identification of the Negri bodies in the brain of an 8-year-old boy who died 8 days after a paralytic illness and 20 days after a dog bite, and who had received 9 injections of Semple's anti-rabies vaccine, provided evidence that he died of acute rabies encephalitis and not of post-vaccinal allergic encephalomyelitis. The Negri bodies in the human subject and those seen in the inoculated mouse differed in their morphological structure: the former consisted of a matrix of very fine granular material bearing larger granules or strands of higher electron-density resembling nucleic acids and representing products of host cell-virus interaction; and the latter showed better defined areas of granular matrix containing tubular, bullet-shaped and elongated forms of viral structures, and nucleocapsids or capsule-deficient cores, representing the virions, emerging from them. Fine structural examination of the patient's brain and of the inoculated mouse has provided evidence of the pleomorphism of the Negri bodies and the various stages of formation of viral material and virions in them, the animal alone showing the mature virions of rabies, and proving the infectivity of the Negri bodies of the human brain.     

Update on rabies

Rabies remains an important public health problem worldwide due to endemic dog rabies in developing countries. Rabies was a re-emerging disease in the United States during the 1990s due to bat rabies virus variants. Australian bat lyssavirus also emerged in Australian bat populations and caused two human deaths. There have been important recent advances in our knowledge of the pathogenesis of rabies and in our ability to diagnose and prevent it.     

The long incubation period in rabies: delayed progression of infection in muscle at the site of exposure

The striped skunk (Mephitis mephitis) is a host of rabies in large areas of Canada and the United States. In each of two experiments, equal numbers of skunks in two groups were inoculated intramuscularly with low doses of a field strain of rabies virus (street rabies virus). In each experiment, skunks in one group surviving to 2 months were killed at this time and selected tissues were used for examination by the polymerase chain reaction (PCR) method or by immunohistochemistry for rabies antigen. Results of detailed examinations using PCR technology (experiment 1) indicated that muscle at the inoculation site contained viral RNA at 2 months postinoculation, when other relevant tissues on the route of viral migration and early entrance into the central nervous system were negative. The cellular location of virus/antigen, as determined immunohistochemically in experiment 2, was striated muscle fibers and fibrocytes. Our results indicate a major role of muscle (tissue) infection at the inoculation site in the long incubation period of rabies in skunks. These and related findings will be useful in rabies control and, if applicable to other species, will be relevant in postexposure treatment. Received: 31 July 1996 / Revised, accepted: 11 November 1996