Trabalho apresentado à 23ª Revista de Educação Continuada em Medicina Veterinária e Zootecnia, 2012, São Paul

Batista, H. B. C. R.

Brandão, Paulo Eduardo

Carnieli Junior, Pedro

Carrieri, Maria Luiza

Castilho, J. G.

De Paula, F. C.

Kotait, I.

Lima, J. Y. O.

Macedo, C. I.

Menozzi, B. D.

Miranda, C. C. P.

Oliveira, R. N.

Biblioteca Digital da Produção Intelectual da Universidade de São Paulo (BDPI/USP)

  Universidade de São Paulo
 
2012
 
A review of the classification of rabies virus
lineages maintained by insectivorous bats in
Brazil
 
 
Revista de Educação Continuada em Medicina Veterinária e Zootecnia, São Paulo, v. 10, n. 2/3, p.
96, res. PT.084, 2012
http://www.producao.usp.br/handle/BDPI/44327
 
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PT.084
A REVIEW OF THE CLASSIFICATION OF RABIES VIRUS 
LINEAGES MAINTAINED BY INSECTIVOROUS BATS IN 
BRAZIL
Oliveira RN1, Castilho JG1, Batista HBCR1, de Paula FC1, Carnieli Jr P1, 
Lima JYO1, Macedo CI1, Menozzi BD2, Carrieri ML1, Kotait I1, Miranda 
CCP3, Brandão PE3 – 1Instituto Pasteur, 2Universidade Estadual Paulista 
– Faculdade de Medicina de Botucatu, 3Universidade de São Paulo – 
Faculdade de Medicina Veterinária e Zootecnia
Little was known about the importance of nonhematophagous bats in the 
epidemiology of rabies in Brazil and most of Latin America until the 1980s. 
From that decade on, as canine rabies came under control in many munici-
palities and molecular and antigenic typing was incorporated in surveillance 
programs, the importance of nonhematophagous bats in the epidemiology of 
the disease began to be appreciated in these countries. In Brazil, genetic studies 
based on gene N have shown that different lineages are circulating in insectivo-
rous bats from the species Tadarida brasiliensis, Nyctinomops laticaudatus and 
genus Myotis, Eptesicus, Molossus, Histiotus and Lasiurus. In most studies, the 
characterization of these lineages is based on only 264 nt of the carboxyterminal 
region of the viral nucleoprotein, when the ideal would be to use the complete N 
gene. The aims of the present study was to review the genetic classification of the 
RABV isolated from insectivorous bats from Brazil based on current literature, 
Genbank dataset and new partial DNA sequencing of the nucleoprotein com-
paring the phylogenetic analysis of N gene based on 1218 nucleotides (nt 203 to 
nt 1420) with that based on 264 nucleotides (nt 1157 to nt 1420), corresponding 
respectively to amino acids 45 to 450 and 363 to 450 of the viral nucleoprotein. 
Phylogenetic analysis demonstrated the existence of at least eleven lineages of 
RABV associated with different genera and species of insectivorous bats. Nine 
of these lineages have already been described in literature while two of them 
were herein characterized for the first time and associated to the genus Myotis 
and Lasiurus. There were no differences in the classification of Brazilian strains 
by comparing the two alignments used, but changes were observed in phylo-
genetic relationships between the clusters, with bootstrap values always greater 
regarding the 1218 nt tree. Two sequences of RABV from the genus Myotis from 
Uruguay and Chile did not keep the same classification after the analyses with 
the two alignment lengths. These findings should be taken into account in mo-
lecular epidemiology of rabies, as sources of infections might be determined in 
a more accurate way and also in the correct use of fragments of the N Gene for 
the classification of lineages of RABV.
PT.085
IN SITU EVALUATION OF CYTOTOXIC IMMUNE RESPONSE 
IN CENTRAL NERVOUS SYSTEM OF HUMAN RABIES 
TRANSMITTED BY DOG AND VAMPIRE BAT
Guedes F1, Fernandes ER1, Martins JF2, Pagliari C3, Duarte MIS3 – 
1Instituto Pasteur de São Paulo – Imunologia, 2Instituto de Infectologia 
Emílio Ribas, 3Faculdade de Medicina da Universidade de São Paulo
Introduction: CD8 (+) T cells and natural killer (NK) cells are immune 
effectors that, by cytokine production or cytotoxicity, help to contain a viral 
infection. Objective: To quantify and compare the T CD8 lymphocytes, 
natural killer cells and B granzyme expression in central nervous system le-
sions of human rabies transmitted by dog and vampire bat. Methods: Five 
fragments of central nervous system (CNS) were selected (cortex, hippocam-
pus, basal ganglia, cerebellum and medulla oblongata) from each specimen of 
the four human rabies cases transmitted by dog and four cases by vampire bat 
(Desmodus rotundus). The fragments were subjected to immunohistochemis-
try with antibodies for CD8, CD57 and B granzyme. For each specimen, cells 
were quantified by counting the number of immunolabelled cells in thirty 
fields considering the parenchyma. For normalizing, a x10 ocular lens was used 
with a square grid in a x40 objective marking an area field of 0.0625 mm2. Sta-
tistical analysis was performed by Graph Pad Prism version 5.0 for Windows 
(Graph Pad software, San Diego, Ca, U.S.A.) using the nonparametric Mann-
Whitney test. Samples were considering different at the 95% (p≤0.05) level of 
significance. Results: The number of CD8+ T lymphocytes in human rabies 
transmitted by dog was lower (p<0.0001) than in those with human rabies 
transmitted by vampire bat. No significant difference in the number of CD57+ 
natural killer cells (p>0.05) and the number of B granzyme-expressing cells 
(p>0.05) was observed between samples evaluated of the human rabies trans-
mitted by dog and vampire bat. Discussion and Conclusions: In the 
present study, we compared lesions in CNS of human rabies transmitted by 
dogs and vampire bats by quantitative examination of the “in situ” cytotoxic 
immune response. Rare NK cells and B granzyme-expressing cells in cerebral 
parenchyma were observed, but there were no significant statistical difference 
between the human rabies transmitted by dog and vampire bat. This could 
reflect an immune evasion mechanism triggered by rabies virus, preventing 
these cells arrive at the site of injury, or that their cytotoxic function would 
be altered. CD8+ T lymphocytes were more abundant in the human rabies 
transmitted by vampire bat, which appeared related to the viral variant type 
involved in infection, however eventually the function these cells may be im-
paired. So, we can speculate if this fact also could be due to longer survival of 
these patients compared to those bitten by dog.
PT.086
CELLULAR GROWTH IN DIFFERENT BIOREACTORS TO 
RABIES VIRUS PRODUCTION
Lantieri VS1, Medeiros FM2, Frazatti-Gallina NM2 – 1Intituto Butantan – 
Seção de Raiva, 2Instituto Butantan – Seção de Raiva
The scaling up of virus production process involves different challenges, 
mainly when is used an animal cells origin with a substrate. The growing of 
the animal cells in high densities depends on the beads and these cells present 
high susceptibility to the shear stress that occurs in the process realized in bio-
reactors. The objective of this study was to evaluate the growing of Vero cells 
in the scaling up process of rabies virus production in bioreactor. Two biore-
actors were used in this study, one of 30 L (Bio Flow 4500, NBS) and other of 
150 L (Bio Flo PRO Industrial, NBS). These bioreactors have different agitation 
systems: while the 30 L has a “Cell Lift Impeller”, the industrial, one STR, has 
pitched blade impellers. This difference was important to select the velocity 
of agitation necessary to maintain the beads in suspension and to minimize 
the shear stress and bead collisions. Vero cells added to solid microcarriers, 
Cytodex 1 (2g/L), infected with PV rabies virus (MOI 0,02) were cultivated in 
serum-free medium VP SFM AGT in the two bioreactors. Were realized seven 
cycles in each bioreactor type and the initial cellular concentration was 13-14 
cell/microcarrier. Supernatants of these cultures were harvested on days 2 and 
3 after start the cycle of production. Samples of these cultures were taken every 
day during the production cycle to determine the cellular concentration. It was 
studied too the cellular loss in the first day after the cell inoculation to analyze 
the cell difficulty for spread on the microcarriers. The averages of the values of 
cell specific grow rate found before the harvest beginning were 0.025 h-1 and 
0.023 h-1 in the industrial and 30 L bioreactors respectively. The percentage 
X X I I I  R I TA  B R A Z I L


A review of the classification of rabies virus lineages maintained by insectivorous bats in Brazil

Universidade de São Paulo

RCAAP - Repositório Científico de Acesso Aberto de Portugal

A review of the classification of rabies virus lineages maintained by insectivorous bats in Brazil

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