Detection of rabies virus genomic RNA and mRNA in mouse and human brains by using in situ hybridization (original) (raw)
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Journal of Virological Methods, 1989
Rabies virus is usually demonstrated in human or animal tissues using antigendetection or viral isolation techniques. Rabies virus RNA can be demonstrated in paraffin-embedded tissues using in situ hybridization. Negative (-) sense 35S-and 'H-labeled RNA probes, specific for rabies virus nucleocapsid protein mRNA, were used for the detection of rabies virus RNA in the nervous system of mice experimentally infected with fixed and street strains of rabies virus. In situ hybridization signals were compared with rabies virus antigen demonstrated with immunoperoxidase staining. Rabies virus RNA and antigen were.also demonstrated in the same neurons using a double-labeling technique. In situ hybridization has potential applications as a diagnostic test for rabies and in studies of rabies pathogenesis.
Journal of Virological Methods, 2006
Archival formalin-fixed and paraffin-embedded brain tissues are important source for diagnosis and molecular analysis. However, nucleic acids are particularly vulnerable to degradation during tissue processing. The brain cutting process usually is performed after 1 week of brain storage in formalin followed by embedding of each particular neuro-anatomical specimen in paraffin. A simple method of deparaffinization, proteinase K digestion and RNA extraction using the Boom technique to obtain rabies RNA in unbuffered, formalin-fixed and paraffin-embedded brain tissues kept at 30 • C for 16 years is described. Reverse transcription-polymerase chain reaction (RT-PCR) can be used to identify rabies viral N gene sequences of 150 bases in length in all patients, but not from every immunohistochemical (IHC)-positive specimen. Direct sequencing of 301 bp of N gene was achieved in 4 of 7 patients. Results of sequencing a single sample of 1432 bases of N gene from a 24 h processed formalin-fixed and paraffin-embedded rabies infected brain tissue after 1 month storage were in accord with those from frozen specimen analysis. It is strongly suggested that for further molecular analysis, a piece of fresh brain tissue should be saved prior to the brain sectioning process and stored no longer than 24 h in formalin before embedding.
Detection of Rabies Virus Genes by In-Situ Polymerase Chain Reaction
Veterinary Research Communications, 2007
Rabies is diagnosed by FAT in the impression smears of brain tissues. In this study, an attempt was made to diagnose rabies using in situ polymerase chain reaction (ISPCR). A digoxigenin-labelled double-stranded probe specific for a portion of the ‘N’ gene of rabies virus was used. Positive signals were identified as blue dots in the intraneuronal and neuropil areas.
Rabies virus detection by RT-PCR in decomposed naturally infected brains
Veterinary Microbiology, 2002
The warm climate of Israel and mishandling of the cadavers during transit to the laboratory requires an accurate method for diagnosis of rabies in decomposed tissues. By using the reverse transcriptase polymerase chain reaction (RT-PCR) 10 decomposed brain samples that collected between 1998 and 2000 were diagnosed as negative by direct fluorescent antibody test (FAT), were found positive. Three of the 10 decomposed brains were confirmed as positive by isolation of rabies virus in tissue culture and by mouse inoculation (MIT) while the other seven decomposed samples were found positive only by RT-PCR. Direct sequencing and molecular analysis of a 328 bp fragment of the N gene of all the rabies sequences confirmed their geographical origin. These results demonstrated the importance of the RT-PCR in the detection of rabies virus in decomposed naturally infected brains, especially in cases when the sample is not suitable for other laboratory assays. Thus, the RT-PCR can provide a positive diagnosis; however, when a negative result is obtained due to the nature of the decomposed tissue that can be caused by technical reasons and a false negative might be the case. #
Preservation of rabies virus RNA from brain tissue using glycerine
Transactions of the Royal Society of Tropical Medicine and Hygiene, 2003
The challenge virus standard (CVS) strain and a wild isolate from a Mexican child who died of hematophagous bat (Desmodus rotundus)-transmitted rabies were injected intracerebrally into BALB/c mice. Brains obtained from infected mice were immersed in 80%, 50%, and 40% glycerine/phosphatebuffered saline (PBS). RNA was extracted from brains on days 1, 2, 3, 7, 21, and 60, and reverse transcriptase polymerase chain reaction-restriction fragment length polymorphism (RT-PCR-RFLP) tests were performed for rabies virus characterization. Storage temperature variation was recorded during the preservation period. The RT-PCR-RFLP tests were successfully performed on brain samples preserved in 50% glycerine/PBS, but not in those preserved in 80% or 40% glycerine/PBS. Temperatures ranged from 12 to 33°C and were not harmful, provided that 50% glycerine/PBS was used. We concluded that brain samples obtained and stored under field conditions (i.e. without refrigeration) for up to 60 d can arrive at a reference laboratory in an adequate condition for viral RNA analysis.
Sequence Analysis of Rabies Virus in Humans Exhibiting Encephalitic or Paralytic Rabies
Journal of Infectious Diseases, 2003
Two distinct clinical patterns, encephalitic (furious) and paralytic (dumb), have been recognized in human rabies. It has been postulated that different rabies virus variants associated with particular vectors may be responsible for these different clinical manifestations. Analysis of the glycoprotein (G), nucleoprotein (N), and phosphoprotein (P) genes of rabies viruses from 2 human cases of encephalitic rabies and from 2 human cases of paralytic rabies demonstrated only minor nucleotide differences. Deduced amino-acid patterns of the N protein were identical in both human and canine samples that came from the same geographic location, regardless of the clinical form. All differences in amino-acid patterns of the G protein were found outside the ectodomain, in either the signal peptide or the transmembrane and endodomains. None of the amino-acid differences of the P protein was within the interactive site with dynein. These findings support the concept that clinical manifestations of rabies are not explained solely by the associated rabies virus variant.
Journal of Neurovirology, 1999
Expression of major histocompatibility complex (MHC) molecules on cells of the central nervous system (CNS) plays an important role in the pathogenesis of acute viral encephalitis. We have compared the induction of MHC class I and II mRNA transcripts in mice upon infection with the virulent challenge virus standard (CVS) strain of rabies virus and avirulent rabies virus variant RV194-2. Rabies virus antigen was detected with immunoperoxidase staining and 35 Slabeled RNA probes were used to detect MHC class I and class II mRNA transcripts by in situ hybridization in infected brains. In CVS and RV194-2 infected animals, MHC class I mRNA expression occurred in the brain in neurons, glia, choroid plexus epithelial cells, ependymal cells, and in¯ammatory cells; expression was moderately higher in CVS-infected mice. In contrast, MHC class II mRNA expression was minimal in CVS-infected mice and it was markedly upregulated in CNS in¯ammatory cells upon RV194-2 infection. Both viruses induced an acute in¯ammatory reaction in the cerebrospinal¯uid (CSF), which was more pronounced in CVS-infected mice. Both viruses also induced an antigen speci®c T and B cell response detectable in lymph nodes and spleen. These studies, which show a correlation between greater expression of MHC class II mRNA in the brain following intracerebral RV194-2 infection and protection against RV194-2 infection in the brain, suggest that recovery from avirulent rabies virus infection of neural cells involves T helper cells produced and/or retained in the brain for reasons that are not entirely clear.
REVIEW ON RABIES INFECTION AND PATHOGENESIS
Rabies is a zoonotic viral disease and it is transmitted by bites of a rabid animal. The rabies virus encoded five proteins, namely glycoprotein, nucleoprotein, phosphoprotein, matrix protein and RNA dependent RNA transcriptase protein. These proteins play an important role in inducing immune against rabies infection, regulating and transcription, interaction with the cytoplasmic domain of the glycoprotein and the RNP during virus assembly and budding, RNA synthesis and capping respectively. The general characteristics of rabies virus such as morphology, transmission, host defense, virus adaptation, period of communicability and cycle of infection and replication are important to understand the diseases briefly. The pathogenesis of rabies virus is unclear, but recent progresses have been made to elucidate these phenomenon. The paths of rabies virus to the central nervous system and within the central nervous system occur by fast axonal transport. Antibodies induced by vaccination, particularly those with neutralizing activity, play prominent role in immune defense against infection. Keywords: Central nervous system, Encoded protein, Pathogenesis, Rabies virus, Ribonucleoproteins.
Osong Public Health and Research Perspectives
Despite all the efforts and increased knowledge of rabies, the exact mechanisms of infection and mortality from the rabies virus are not well understood. To understand the mechanisms underlying the pathogenicity of rabies virus infection, it is crucial to study the tissue that the rabies virus naturally infects in humans. Methods: Cerebellum brain tissue from 9 human post mortem cases from Iran, who had been infected with rabies virus, were examined histopathologically and immunohistochemically to evaluate the innate immune responses against the rabies virus. Results: Histopathological examination revealed inflammation of the infected cerebellum and immunohistochemical analyses showed an increased immunoreactivity of heat shock protein 70, interleukin-6, interleukin-1, tumor necrosis factor-alpha, caspase-3, caspase-9, toll-like receptor3 and toll-like receptor4 in the infected brain tissue. Conclusion: These results indicated the involvement of innate immunity in rabies infected human brain tissue, which may aggravate the progression of this deadly disease.
Pesquisa Veterinária Brasileira, 2011
. Detection of rabies virus nucleoprotein-RNA in several organs outside the central nervous system in naturally-infected vampire bats. Pesquisa Veterinária Brasileira 31(10):922-925. Rabies is a neurological disease, but the rabies virus spread to several organs outside the central nervous system (CNS). The rabies virus antigen or RNA has been identiϐied from the salivary glands, the lungs, the kidneys, the heart and the liver. This work aimed to identify the presence of the rabies virus in non-neuronal organs from naturally-infected vampire bats and to study the rabies virus in the salivary glands of healthy vampire bats. Out of the ϐive bats that were positive for rabies in the CNS, by ϐluorescent antibody test (FAT), viral isolation in N2A cells and reverse transcription -polymerase chain reaction (RT-PCR), 100% (5/5) were positive for rabies in samples of the tongue and the heart, 80% (4/5) in the kidneys, 40% (2/5) in samples of the salivary glands and the lungs, and 20% (1/5) in the liver by RT-PCR test. All the nine bats that were negative for rabies in the CNS, by FAT, viral isolation and RT-PCR were negative for rabies in the salivary glands by RT-PCR test. Possible consequences for rabies epidemiology and pathogenesis are discussed in this work.