Banca de QUALIFICAÇÃO: RAPHAELA MELLO ZAMUDIO

Uma banca de QUALIFICAÇÃO de MESTRADO foi cadastrada pelo programa.
DISCENTE : RAPHAELA MELLO ZAMUDIO
DATA : 10/09/2021
HORA: 14:00
LOCAL: por participação remota
TÍTULO:

Rabies Virus Isolation in HEK-293 Cells and Identification of Viral Protein Expression in Different Cell Lines

PÁGINAS: 50
GRANDE ÁREA: Ciências Biológicas
ÁREA: Microbiologia
RESUMO:

Rabies is a zoonotic disease affecting all mammals, characterized by a generally fatal encephalomyelitis. This disease is caused by the neurotropic Rabies Lyssavirus (RABV), belonging to the family Rhabdoviridae and genus Lyssavirus. The direct fluorescent antibody test (DFAT) is considered the gold standard for rabies diagnosis. Viral isolation in cell culture (Rabies Tissue Culture Infection Test - RTCIT) can be used as a complementary test. Considering the importance of RTCIT for rabies diagnosis, the first study described here reports on the use of the HEK-293 (Human Embryonic Kidney) cell line in this technique, comparing sensitivity and specificity against the traditionally used cell, Neuro-2a (Mus Musculus Neuroblastoma). 93 central nervous system samples from different species were analyzed, for RTCIT on HEK-293, two protocols were tested (with and without viral adsorption, both tested with three different incubation times: 24, 48 and 72 hours). The HEK-293 cell showed susceptibility to RABV, with better results in the protocol with viral adsorption and 72 hours of incubation (95.35% sensitivity and 100% specificity). Analyzing the results obtained in this study, the main conclusion that can be drawn is that HEK-293 cells can be used as an alternative for RTCIT. Considering the relation between RABV and cell lines, the second study developed aimed to identify the expression of RABV proteins in different cell lines. Initially, viral titration of five different RABV genetic lineages was performed in four different cells. The cells used were Neuro-2a, BHK-21 (Baby Hamster Kidney), HEK-293 and EidNi/41 (Eidolon Helvum kidney). The genetic strains of RABV used were: genetic strain characteristic of bats (Artibeus lituratus and Myotis nigricans), of canids (Canis lupus familiaris and Cerdocyon thous) and the standard RABV Pasteur Virus (PV) sample. All samples showed statistically similar titers in HEK-293, BHK-21 and Neuro-2a cells. Only in the EidNi/41 cell, all samples showed significantly reduced titers. Significantly higher viral titers were observed only in the PV sample in all cell lines. The high susceptibility of the traditionally used Neuro-2a and BHK-21 cells to RABV is evident. Similarly, HEK-293 susceptibility was confirmed once again. However, despite the susceptibility to RABV, Eidni/41 showed significantly lower infection capacity compared to the other cells. This fact becomes instigating since EidNi/41 cells originate from bats, which play a crucial role in the epidemiological cycle of RABV. Realizing these effects and repercussions of different RABV genetic strains in varied cells, the hypothesis of distinct identification of protein expression patterns of these RABV samples in different cells is raised.

MEMBROS DA BANCA:
Presidente - Interno ao Programa - 927.634.300-82 - HELENA BEATRIZ DE CARVALHO RUTHNER BATISTA - UFRGS
Membro Titular - Examinador(a) Interno ao Programa - 2605420 - MARIA CRISTINA CARLAN DA SILVA
Membro Titular - Examinador(a) Externo ao Programa - 1764675 - CHRISTIANE BERTACHINI LOMBELLO
Membro Suplente - Examinador(a) Interno ao Programa - 2605490 - SERGIO DAISHI SASAKI