23 Genetic Methods for Rabies Diagnosis and Rabies Virus Typing Techniques

INTRODUCTION: Bats are one of the most important reservoirs and vectors of the rabies virus in the world. METHODS: From 1988 to 2003, the Zoonosis Control Center in São Paulo City performed rabies diagnosis on 5,670 bats by direct immunofluorescent test and mouse inoculation test. Blood samples were collected from 1,618 bats and the sera were analyzed using the rapid fluorescent focus inhibition test to confirm rabies antibodies. RESULTS: Forty-four (0.8%) bats were positive for rabies. The prevalence of rabies antibodies was 5.9% using 0.5IU/ml as a cutoff. Insectivorous bats (69.8%) and bats of the species Molossus molossus (51.8%) constituted the majority of the sample; however, the highest prevalence of antibodies were observed in Glossophaga soricina (14/133), Histiotus velatus (16/60), Desmodus rotundus (8/66), Artibeus lituratus (5/54), Nyctinomops macrotis (3/23), Tadarida brasiliensis (3/48), Carollia perspicillata (3/9), Eumops auripendulus (2/30), Nyctinomops laticaudatus (2/16), Sturnira lilium (2/17) and Eumops perotis (1/13). The prevalence of rabies antibodies was analyzed by species, food preference and sex. CONCLUSIONS: The expressive levels of antibodies associated with the low virus positivity verified in these bats indicate that rabies virus circulates actively among them.

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Digoxigenin-labeled probe for rabies virus nucleoprotein gene detection

Revista da Sociedade Brasileira de Medicina Tropical ◽

10.1590/s0037-86822006000200005 ◽

2006 ◽

Vol 39 (2) ◽

pp. 159-162 ◽

Cited By ~ 4

Author(s):

Pedro Carnieli Junior ◽

Armando Moraes Ventura ◽

Edison Luiz Durigon

Keyword(s):

Rabies Virus ◽

Diagnostic Techniques ◽

N Gene ◽

Rt Pcr ◽

Gene Detection ◽

Nucleoprotein Gene ◽

Polymerase Chain ◽

Labeled Probe ◽

The Cost ◽

Rabies Diagnosis

A digoxigenin-labeled probe was produced from the Pasteur virus strain for the detection of the rabies virus N gene. The probe hybridization was performed from amplified N gene obtained by reverse transcription polymerase chain reaction and the results by RT-PCR and hybridization showed 100% agreement. The hybridization, when carried out in products amplified by RT-PCR, increases the sensitivity of this technique even more and confers specificity to the diagnosis. The technique described in this work will be useful in rabies diagnosis laboratories, once the cost is compatible with traditional rabies diagnostic techniques.

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Effect of Postmortem Degradation on the Preservation of Viral Particles and Rabies Antigens in Mice Brains. Light and Electron Microscopic Study

Viruses ◽

10.3390/v12090938 ◽

2020 ◽

Vol 12 (9) ◽

pp. 938

Author(s):

Jeison Monroy-Gómez ◽

Gerardo Santamaría ◽

Ladys Sarmiento ◽

Orlando Torres-Fernández

Keyword(s):

Electron Microscopy ◽

Rabies Virus ◽

Immunoelectron Microscopy ◽

Degradation Process ◽

Control Group ◽

Direct Immunofluorescence ◽

Electron Microscopic ◽

Viral Antigens ◽

Viral Particles ◽

Rabies Diagnosis

Rabies diagnosis is mainly made on fresh brain tissue postmortem by means of the direct immunofluorescence test. However, in some cases, it is not possible to use this technique, given that the affected nervous tissue goes through a postmortem degradation process, due to problems in the handling and transport of the samples. For this reason, the preservation in time of the rabies virus inclusions was assessed, as well as the immunoreactivity and the ultrastructure of viral particles in tissue with postmortem degradation. Brains of mice inoculated with rabies virus and control mice were processed for conventional histology, immunohistochemistry, electron microscopy, and immunoelectron microscopy in different postmortem times. In the processed tissues for hematoxylin and eosin, the presence of eosinophilic inclusions was not observed beyond 12 h postmortem. Surprisingly, the immunoreactivity of the viral antigens increased with time, at least until 30 h postmortem. It was possible to easily recognize the viral particles by means of conventional electron microscopy until 12 h postmortem. Immunoelectron microscopy allowed us to identify the presence of viral antigens disseminated in the neuronal cytoplasm until 30 h postmortem, but immunoreactive viral particles were not observed. The rabies infection did not cause histological or ultrastructural alterations different from those in the control group as a result of the postmortem degradation. In conclusion, the immunohistochemistry is a reliable test for rabies diagnosis in samples with postmortem degradation and that have been fixed with aldehydes.

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Circulation of the rabies virus in non-hematophagous bats in the city of Rio de Janeiro, Brazil, during 2001-2010

Revista da Sociedade Brasileira de Medicina Tropical ◽

10.1590/s0037-86822012000200008 ◽

2012 ◽

Vol 45 (2) ◽

pp. 180-183 ◽

Cited By ~ 3

Author(s):

Claudius Couto Cabral ◽

Ana Carolina Nunes de Morais ◽

Alba Valéria de Almeida Barcelos Dias ◽

Marcela Garcia Araújo ◽

Wildeberg Cal Moreira ◽

...

Keyword(s):

Rabies Virus ◽

Rio De Janeiro ◽

Animal Health ◽

Control Measures ◽

Control And Prevention ◽

And Control ◽

The City ◽

Rabies Diagnosis ◽

De Medicina ◽

Janeiro City

INTRODUCTION: Rabies is one of the most known lethal zoonosis, responsible for 55,000 human deaths per year. It is transmitted to humans mainly by the bite of domestic or wild animals infected with the virus. This paper shows the circulation of this virus in non-hematophagous bats in the City of Rio de Janeiro, Brazil. METHODS: A survey was performed on the number of bats that had been sent for diagnosis by the Seção de Virologia of the Instituto Municipal de Medicina Veterinária Jorge Vaitsman and were positive for rabies. The positive animals were identified, and the isolated viruses were sent for antigenic typification with indirect immunofluorescence. The results were compared with the antigenic panel of the Centers for Disease Control and Prevention. RESULTS: During 2001-2010, the laboratory received 555 non-hematophagous bats for rabies diagnosis, with 198 (35.7%) from Rio de Janeiro City. A total of 11 (5.5%) animals were positive for this disease. Antigenic typification revealed the predominance of variant 3 in 9 (81.8%) of the isolated viruses; 1 virus was classified as variant 4 and 1 variant was identified that segregated with the viruses in insectivorous bats. CONCLUSIONS: The data obtained in this study showed the presence of the rabies virus in synanthropic populations of non-hematophagous bats in the City of Rio de Janeiro. The circulation of this agent in these animals represents a serious risk to human and animal health and requires attention and control measures by the authorities.

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Real-Time RT-PCR for the Detection of Lyssavirus Species

Journal of Veterinary Medicine ◽

10.1155/2014/476091 ◽

2014 ◽

Vol 2014 ◽

pp. 1-12 ◽

Cited By ~ 4

Author(s):

A. Deubelbeiss ◽

M.-L. Zahno ◽

M. Zanoni ◽

D. Bruegger ◽

R. Zanoni

Keyword(s):

Real Time ◽

Rabies Virus ◽

Fluorescent Antibody ◽

Fatal Outcome ◽

Antibody Test ◽

Rt Pcr ◽

Causative Agents ◽

Rabies Virus Strain ◽

Rabies Diagnosis

The causative agents of rabies are single-stranded, negative-sense RNA viruses in the genus Lyssavirus of Rhabdoviridae, consisting of twelve classified and three as yet unclassified species including classical rabies virus (RABV). Highly neurotropic RABV causes rapidly progressive encephalomyelitis with nearly invariable fatal outcome. Rapid and reliable diagnosis of rabies is highly relevant for public and veterinary health. Due to growing variety of the genus Lyssavirus observed, the development of suitable molecular assays for diagnosis and differentiation is challenging. This work focused on the establishment of a suitable real-time RT-PCR technique for rabies diagnosis as a complement to fluorescent antibody test and rabies tissue culture infection test as gold standard for diagnosis and confirmation. The real-time RT-PCR was adapted with the goal to detect the whole spectrum of lyssavirus species, for nine of which synthesized DNA fragments were used. For the detection of species, seven probes were developed. Serial dilutions of the rabies virus strain CVS-11 showed a 100-fold higher sensitivity of real-time PCR compared to heminested RT-PCR. Using a panel of thirty-one lyssaviruses representing four species, the suitability of the protocol could be shown. Phylogenetic analysis of the sequences obtained by heminested PCR allowed correct classification of all viruses used.

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Diagnosis and Analysis of a Recent Case of Human Rabies in Canada

Canadian Journal of Infectious Diseases ◽

10.1155/2002/235073 ◽

2002 ◽

Vol 13 (2) ◽

pp. 129-133 ◽

Cited By ~ 9

Author(s):

Lindsay D Elmgren ◽

Susan A Nadin-Davis ◽

Frances T Muldoon ◽

Alexander I Wandeler

Keyword(s):

Polymerase Chain Reaction ◽

Rabies Virus ◽

False Negative ◽

Strain Identification ◽

Direct Immunofluorescence ◽

Product Analysis ◽

Chain Reaction ◽

Food Inspection ◽

Polymerase Chain ◽

Rabies Diagnosis

BACKGROUND: On September 30, 2000, staff at the Canadian Food Inspection Agency's Centre of Expertise for Rabies, located at the Animal Diseases Research Institute in Ottawa, Ontario, diagnosed rabies in a child from Quebec. This was the first case of rabies in a human in Canada in 15 years and in 36 years in the province of Quebec. After spending a week in intensive care in a Montreal hospital, the nine-year-old boy succumbed to this nearly always fatal disease. The boy had been exposed to a bat in late August 2000, while vacationing with his family in the Quebec countryside.METHODS: Antemortem specimens taken from the patient were sent to the Canadian Food Inspection Agency laboratory for rabies diagnosis. Samples included saliva, eye secretions, corneal impressions, cerebral spinal fluid and skin. Specimens were examined by direct immunofluorescence microscopy, and results were confirmed using molecular biological techniques. Virus strain identification was performed by both genetic methods and phenotypic analysis with monoclonal antibodies.RESULTS: Initial results from direct immunofluorescence staining indicated that rabies virus was present in the skin biopsy specimen but not in the corneal impressions. This diagnosis of rabies was confirmed by polymerase chain reaction product analysis from several of the submitted specimens. Virus isolation from postmortem samples was not possible because fresh brain tissue was not available. Rabies virus was isolated from saliva and was determined to be similar to a variant that circulates in populations of silver-haired bats.INTERPRETATION: Intravitam diagnosis of rabies in humans is very dependent on the samples submitted for diagnosis and the method used for testing. Upon first examination, only skin specimens were positive for rabies virus antigen; using polymerase chain reaction analysis, only saliva yielded positive results for rabies virus genome. Extensive testing and retesting of specimens submitted for rabies diagnosis in humans must be done to avoid false negative results.

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Evaluation of Two Real-Time, TaqMan Reverse Transcription-PCR Assays for Detection of Rabies Virus in Circulating Variants from Argentina: Influence of Sequence Variation

Viruses ◽

10.3390/v13010023 ◽

2020 ◽

Vol 13 (1) ◽

pp. 23

Author(s):

Diego A. Caraballo ◽

María A. Lombardo ◽

Paula Becker ◽

María S. Sabio ◽

Cristina Lema ◽

...

Keyword(s):

Rabies Virus ◽

False Negative ◽

Rapid Test ◽

Rt Pcr ◽

Pcr Assay ◽

Nucleoprotein Gene ◽

Qrt Pcr ◽

Reverse Transcription Pcr ◽

Pcr Assays ◽

Rabies Diagnosis

In rabies diagnosis, it is essential to count on a rapid test to give a quick response. The combined sensitivity and robustness of the TaqMan RT-PCR assays (qRT-PCR) have made these methods a valuable alternative for rabies virus (RABV) detection. We conducted a study to compare the applicability of two widely used qRT-PCR assays targeting the nucleoprotein gene (LysGT1 assay) and leader sequences (LN34 qRT-PCR assay) of RABV genomes, in all variants circulating in Argentina. A total of 44 samples obtained from bats, dogs, cattle, and horses, that were previously tested for rabies by FAT and conventional RT-PCR, were used in the study. All variants were successfully detected by the pan-lyssavirus LN34 qRT-PCR assay. The LysGT1 assay failed to detect three bat-related variants. We further sequenced the region targeted by LysGT1 and demonstrated that the presence of three or more mismatches with respect to the primers and probe sequences precludes viral detection. We conclude that the LysGT1 assay is prone to yield variant-dependent false-negative test results, and in consequence, the LN34 assay would ensure more effective detection of RABV in Argentina.

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