Detection Of Rabies Virus RNA Research Articles

Difficulties of life-time diagnosis of rabies. Clinical observation

The paper presents a case of rabies with lethal outcome in a fourteen-year-old adolescent. The child did not undergo postexposure prevention of rabies after a cat’s attack in connection with late addressing for medical care, just after occurrence of clinical symptoms of this disease. The child was not diagnosed “rabies” when was alive. Diagnosis was verified by detection of rabies virus RNA in autopsy material using polymerase chain reaction.

Ante mortem diagnosis of human rabies using saliva samples: Comparison of real time and conventional RT-PCR techniques

Background Rabies is an enzootic and fatal disease and is still a major problem in developing countries. Ante mortem diagnosis in human cases is necessary for medical management of the patient and to ensure appropriate post-exposure treatment of contacts. Both conventional RT-PCR and Real time PCR (TaqMan) have been described for the detection of rabies virus RNA from saliva and tissue respectively, however to date, there have been no studies comparing conventional and real time PCR assays for detection of rabies virus nucleic acid using saliva samples for ante mortem diagnosis. Objectives In this study, we evaluated the utility of conventional RT-PCR and SYBR Green I Real time PCR in the ante mortem diagnosis of rabies using saliva samples. Study design Saliva samples collected from twenty-four patients presenting with typical clinical manifestations of rabies were tested in the two assays. Results Amongst the 24 samples tested, 21 samples (87.5%) were positive by either of the two molecular methods. Of these 21, rabies virus RNA was detected in 6/21 in the conventional RT-PCR assay while SYBR Green I Real time PCR could detect RNA in 18/21 samples. Conclusion Real time PCR assay was more sensitive than conventional RT-PCR assay (sensitivity 75% versus 37%, p = 0.0189). This study highlights the utility of molecular diagnostic tests in establishing ante mortem diagnosis of rabies using saliva samples within a few hours.

Detection of rabies virus RNA isolated from several species of animals in Brazil by RT-PCR.

Brain samples from different animal species including humans: five vampire bats, 14 cattle, 12 dogs, 11 cats, two horses, one pig, one sheep and three humans collected from various geographical regions of Brazil were found to be positive for rabies by means of the fluorescent antibody test (FAT) and the mouse inoculation test (MIT). The brain samples were retested for rabies by means of the reverse transcription and polymerase chain reaction (RT-PCR) with 2 primer sets (P1/P2 and RHNI/RHNS3), which amplified full or partial regions on the nucleoprotein (N) gene of the rabies virus, respectively. Brain samples from five vampire bats, 13 cattle, one horse and one sheep failed to yield PCR products when the RHN1/RHNS3 primer pair was used, but all brain samples successfully yielded the products when the P1/P2 primer pair was used. These results suggest that Brazilian rabies virus isolates could be principally divided into two populations according to genetic difference.

Effects of post-mortem autolysis on the detection of rabies virus genomic RNA and mRNA in mouse brain by using in situ hybridization

The effects of post-mortem autolysis were studied on the detection of rabies virus RNA in the brains of mice with experimental rabies by using in situ hybridization (ISH). The brains of CVS-infected mice were subjected to autolytic periods in situ of up to 72 h. ISH was performed with 3H-labelled RNA probes for rabies virus glycoprotein gene genomic RNA and mRNA. During the post-mortem period there was progressive loss of signals for genomic RNA and mRNA, which was greater for mRNA. ISH signals in perikarya also changed for genomic RNA from a multifocal to a diffuse distribution during the post-mortem period. Rabies virus antigen was better preserved during the autolytic period. Effects of the agonal state, degradation of RNA by ribonucleases, and diffusion of RNA out of cells prior to fixation could explain the loss of ISH signals in post-mortem tissues.

Detection of rabies virus mRNA in mouse brain by using in situ hybridization with digoxigenin-labelled RNA probes

A non-isotopic method of in situ hybridization (ISH) was developed for the detection of rabies virus RNA in paraffin-embedded tissues. Digoxigenin-labelled RNA probes for rabies virus glycoprotein mRNA were used. The method had good sensitivity and low backgrounds, and there was excellent cellular localization of signals. ISH wih digoxigenin-labelled probes was compared with ISH with 3H-labelled probes. This non-isotopic method of ISH is more convenient than the radiolabelled method, and it is quicker because a long autoradiographic exposure is not required.

Detection of rabies virus RNA in the central nervous system of experimentally infected mice using in situ hybridization with RNA probes

Rabies virus is usually demonstrated in human or animal tissues using antigen-detection or viral isolation techniques. Rabies virus RNA can be demonstrated in paraffin-embedded tissues using in situ hybridization. Negative (−) sense 35S- and 3H-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.