Evaluation of the LN34 Pan-Lyssavirus real-time RT-PCR assay for the detection of different variants or genetic lineages of Brazilian rabies virus (RABV)

• Grant number: 19/02637-2
• Support Opportunities: Regular Research Grants
• Start date: September 01, 2019
• End date: February 28, 2022
• Field of knowledge: Agronomical Sciences - Veterinary Medicine - Preventive Veterinary Medicine
• Principal Investigator: Enio Mori
• Grantee: Enio Mori
• Host Institution: Instituto Pasteur (IP). Coordenadoria de Controle de Doenças (CCD). Secretaria da Saúde (São Paulo - Estado). São Paulo , SP, Brazil
• Associated researchers: Karen Miyuki Asano ; Paulo César Maiorka ; Paulo Eduardo Brandão ; Sueli Akemi Taniwaki Miyagi

Abstract

Rabies is an acute progressive viral encephalitis characterized by a disorder in the central nervous system, which affects all mammals, including the human species. The direct fluorescent antibody (DFA) is considered gold standard for the rabies post mortem diagnosis, both by WHO and OIE, once that presents high sensitivity and specificity. However, some difficulties may decrease the accuracy of the results, such as the advanced degree of autolysis and low viral load in the CNS samples, quality of the laboratory supplies and even the experience of examiners in reading DFA slides. The medical decision on the rabies post-exposure prophylaxis in patients bitten by animals and the implementation of control measures of a positive outbreak is dependent of the laboratorial result. Once that the rabies presents high lethality (almost 100%), it became necessary the implementation of a confirmatory techniques, for the DFA, that are sensitive, specific, fast and accurate so that the results are always reliable. The LN34 RT-qPCR test, which is a combination of degenerate and multiples primers with locked nucleic acid (LNA) probe it is a strong candidate for the rabies diagnosis because of its high sensitivity and specificity and the easiness to detect the diversity of species present in the Lyssavirus genus. This technique amplifies and detects the beginning region of the 3' portion of the genome, composed by the leader sequence and part of the nucleoprotein gene, and this region is conserved in the different species of Lyssavirus. Therefore, this study aims the application of rabies post mortem diagnosis technique in the Pasteur Institute of São Paulo as a confirmatory test to the DFA that is highly sensitive and specific, that presents robustness, rapid and easy-to-use, from the evaluation of the LN34 Pan-Lyssavirus RT-qPCR assay using Brazilian samples of different variants or genetic lineages of RABV. To analyze the performance of the LN34 assay for the postmortem diagnosis, it will be tested 300 clinical samples (150 DFA positive samples) from the animal rabies surveillance of the Pasteur Institute. (AU)