Molecular characterization of rabies virus isolated from non-haematophagous bats in Brazil Caracterização molecular do vírus da raiva isolado de morcegos não-hematófagos no Brasil

Introduction: Rabies is an important zoonosis that causes thousands of deaths worldwide each year. Although the terrestrial cycle, mainly transmitted by dogs, is controlled in Brazil, the aerial cycle remains a serious public health issue, besides the economic problem. In the aerial cycle, the haematophagous bat Desmodus rotundus is the main source of infection, where several different species of non-haematophagous bats can be infected and can transmit the virus. Methods: The aim of this work was to study the epidemiological pattern of rabies using antigenic characterization with monoclonal antibodies and genetic characterization by reversetranscriptase polymerase chain reaction followed by sequencing and phylogenetic analysis of non-haematophagous bats’ and herbivorous animals’ central nervous system samples from the western region of the State of São Paulo, Brazil. Results: From 27 samples, 3 antigenic variants were identified: AgV-3, AgV-4, and AgV-6; and from 29 samples, 5 different clusters were identified, all belonging to the rabies virus species. Conclusions: Although only nonhaematophagous bats were evaluated in the studied region, the majority of samples were from antigenic and genetic variants related to haematophagous bats Desmodus rotundus. Samples from the same antigenic variant were segregated in more than one genetic cluster. This study demonstrated the diversity of rabies virus genetic lineages presented and circulating in nonhaematophagous bats in the studied region.

Rabies is an infectious disease responsible for more than 55,000 deaths worldwide and 15 million post-exposure treatments annually; this is despite the existence of effective vaccines for human and veterinary use 1 .Rabies remains to be a serious public health concern and a cause of serious economic and environmental problems.Rabies virus, a neurotropic virus from the Lyssavirus genus, Rhabdoviridae family, is the agent of the disease [2][3] .The disease is widespread and is found in all continents except Antartica 1 .All mammals are susceptible to rabies virus, mainly the Carnivora and Chiroptera orders.Dogs and, occasionally, cats are mainly responsible for the transmission of urban rabies; and in sylvatic rabies, the major host is a different species of wild mammals in different regions of the world 4 .
Although dogs remain as the principal source of rabies infection to humans, several countries, including Brazil, have been able to control this cycle of the disease 5 .However, rabies virus still circulates in several species of wild mammals that can infect domestic animal species and humans 1,[6][7] .
In terms of wild mammal hosts, bats are the main reservoirs of rabies virus, and cases of rabies caused by accidents with different bat species have been reported worldwide in the past decades.In Latin America, the haematophagous bat Desmodus rotundus is the major responsible for the aerial cycle with important role in the transmission of the disease to livestock animals 8 as well as the critical source of infection to human rabies [9][10][11] .Aggression by Desmodus rotundus is currently the main cause of human rabies in Brazil, and a great number of rabies cases on nonhaematophagous bat species have been diagnosed, although with no human cases 5 .Bat rabies, combined with recent aggression by wild canines (Cerdocyon thous) and marmosets (Callithrix jacchus) [12][13] , points out to the importance of a continuous vigilance in wild animals; to understand the epidemiology of the disease in those species and to establish adequate strategies of prevention and control.

Albas A et al -Rabies virus isolated from non-haematophagous bats in Brazil
The region of Presidente Prudente (Lat.22 0 07' 32"; Lon.51 0 23' 20") is located in the Western region of the State of São Paulo, Brazil.The terrestrial cycle the rabies transmitted by dogs is controlled in the state, but the aerial cycle, where bats are the source of infection, remains a serious issue, also in the studied region.The number of notified rabies cases in the state of São Paulo in 2010 was 32 in cattle (all due to related with bats), two in haematophagous bats, and 54 in non-haematophagous bats 5 .These data confirm the importance of the knowledge of the antigenic and genetic patterns of rabies in nonhaematophagous bats in the region.
The aim of the present study was the determination of the antigenic and genetic profiles of rabies virus circulation in the Western region of the State of São Paulo, Brazil, using antigenic and genetic characterization of samples from non-haematophagous bat species and herbivorous animals.

Samples
The studied samples were from the Polo da Alta Sorocabana Laboratory, in the City of Presidente Prudente, Western region of the State of São Paulo, Brazil.
All samples were previously diagnosed as rabies virus positive by fluorescent antibodies test (FAT) 14 and mouse inoculation test (MIT) 15 techniques; 24 samples were from non-haematophagous bat species, five from cattle, and one from horse.
Samples were submitted to virus amplification by inoculation on swiss albine mouse brains, identified, and stored at -70 o C on the NPR sample bank.A sample of the standard rabies virus (brcvsusp47/05), maintained in BHK-21 cell culture, was used as positive control, and non-infected mouse brain served as negative control.

Antigenic characterization with monoclonal antibodies
A panel of eight monoclonal antibodies against rabies virus nucleoprotein was used.The panel was produced by the Centers for Disease Control and Prevention (CDC), Atlanta, GA, USA, and pre-established for characterization of American rabies isolates by the Pan American Health Organization (PAHO) 7 .
The panel allows the identification of different reactivity patterns, related with the maintenance and transmission of rabies virus.The indirect immunofluorescence test to determine the antigenic variants was performed according to the protocol described by Favoretto et al. 12

Reverse transcriptase-polymerase chain reaction
Total RNA Extraction: fragments of brain samples were macerated with 500uL of DEPC ® H 2 O, and the RNA was obtained by using the Trizol LS Reagent ® (Invitrogen Corporation, Carlsbad, CA) following the manufacturer's instructions.
Reverse transcriptase polymerase chain reaction: the RT-PCR was conducted according to the protocol described by Campos et al. 17 , with modifications.
Four µL of the extracted RNA was added to a mix containing 8µL of enzyme buffer, 1µL of 10mM dNTP Mix, 5µL of the primers 21G and 304 (10pmol/uL), 04mM DTT, 40U of ribonuclease inhibitor (RNAse out -Invitrogen Corporation, Carlsbad, CA), 200U of reverse transcriptase-Moloney murine leukemia virus/super script and H 2 O DEPC ® .The reaction was incubated at 42 o C for 60min.
The cDNA obtained was added to a mix containing 10x reaction buffer, 2µM of MgCl 2 , 200µM of each dNTP, 50pmol of the primers SeqN and 304, 2.5U of TaqDNA polymerase and H 2 O DEPC ® to a final volume of 50µL.
Amplification was carried out using the following cycle: samples were preheated for 5mins.at 95 °C followed by 35 cycles of denaturation for 45s at 94 °C, primer annealing for 45s at 45 °C, primer extension for 90s at 72 °C, and a final primer extension for 5min at 72 °C in a Gradient Thermocycler ® (Eppendorf -Germany).All reactions were made with positive and negative controls.
The amplified products were analyzed by eletrophoresis in agarose gel 1.5% in TBE 1x buffer and ethydium bromide.The results were compared with positive and negative controls and a DNA ladder of 100bp.Positive fragments presented 765bp.
Sequencing and genetic analysis The nucleotide sequences obtained were edited and aligned using the ClustalW 18 and BioEdit v7.0.9 -Sequence Aligment Editor (1197-2007, Tom Hall, Ibis Biosciences Carlsbad, CA) programs, based in homologous sequences available in GenBank.For the similarity analysis, 320 base pairs, between the 1,157 and 1,476 positions, correspondent to the carboxyl-terminal portion of the nucleoprotein gen, were used.
Determination of the genetic distance and reconstruction of phylogenetic trees were made using MEGA Program 4.0 version 19 and Kimura 2-parameters and neighbor-joining methods for topology reconstruction.The determination of the topology reliability in the phylogenetic analysis was conducted with the bootstrap probability values calculus, by using 10,000 replicas.

Antigenic characterization
Out of the 30 samples, 27 were antigenically characterized (21 bats being 11 from frugivorous bats, nine from insectivorous bats, and one from nectivorous; five cattle and one horse).Three antigenic variants were identified (Table 1).
The AgV-4 variant, related to populations of insectivorous bats Tadarida brasiliensis, was observed in three insectivorous bats (two Eptesicus furinalis and one Myotis nigricans) and one frugivorous bat Artibeus lituratus.The AgV-6 variant, related to populations of insectivorous bat Lasiurus cinereus, was observed in one Lasiurus blossevillii insectivorous bat.

Genetic characterization
From the 30 samples, 29 (24 bats, five cattle) were genetically characterized through nucleotide sequence.All samples were characterized as Rabies virus specie, Lyssavirus genus.
The reconstruction of the phylogenetic tree, with samples from the studied region and from GenBank, revealed the presence of six clusters: group 1 to group 6; the average intrinsic distance within group was from 0.8% to 50%, and the average intrinsic distance among the six groups was from 11% to 21.9%.
Group 2 presented only one sample from the frugivorous bat Artibeus lituratus; this sample was segregated with the genetic variant maintained by the Tadarida brasiliensis bat.The average intrinsic distance within group was 0.8%, and the average intrinsic distance between groups ranged from 11% with group 1 and 18.5% with group 3.
In group 3, two Myotis nigricans insectivorous bats were segregated with samples from this bat species, with an average intrinsic distance within group of 3% and average intrinsic distance between groups ranging from 16% with group 4 and 21.9% with group 6.
Group 4 presented three samples from Eptesicus furinalis insectivorous bats, segregating with two different species of insectivorous bats (Eumops auripendulus and Nyctinomops laticaudatus) and with samples of the same species and one sample of an Artibeus lituratus frugivorous bat isolated in a different region in the State of São Paulo.This group presented an average intrinsic distance of 1.6% within group, and the average intrinsic distance between groups ranged from 12.9% with group 5 and 20.3% with group 6.

DISCUSSION
The monoclonal antibodies technique allows the characterization of rabies antigenic variants and possible reservoirs of the virus 12 .Among the 27 samples submitted for antigenic analysis, three antigenic variants were identified, all belonging to profiles from the existent panel and previously identified in Latin America by several authors 12,[20][21][22] .These variants represent rabies virus reservoirs from Latin America maintained by Desmodus rotundus (AgV-3), Tadarida brasiliensis (AgV-4), and Lasiurus cinereus (AgV-6) bat populations.
Besides the bats isolates, 6 herbivorous (five cattle and one horse) rabies positive samples were also typed; all were related to the rabies virus variant maintained by the haematophagous bat Desmodus rotundus.This bat species is the main source of infection for herbivorous animals.
The genetic lineage of 29 samples was determined by molecular biology techniques considering the carboxyl-terminal portion of the nucleoprotein gene, as previously described by Kissi et al. 23 for these studies.All samples belonged to the Lyssavirus genus, Rabies virus species.The reconstruction of the phylogenetic tree, with samples from the studied region, demonstrated that all segregated with samples available at GenBank and representative of genetic lineages related to bats species already identified [24][25][26][27] .The observed clusters were supported by high bootstrap values (91% to 100%).
The fact that genetic variant related to haematophagous bats was identified in 75.8% (22/29) of the studied samples, those samples from group 1 confirmed the results obtained with the antigenic characterization as AgV-3.This variant was also found in 90.9% of the frugivorous bats Artibeus lituratus, 50% of the insectivourous bats (one Lasiurus blossevillii, one Molossus molossus, one Eptesicus furinalis, and three Lasiurus ega), and the nectivorous bat Glossofaga soricina.Those results were also related in studies with samples isolated from bats from the State of São Paulo and other Brazilian regions [26][27] .
Epidemiological studies with samples isolated from bat species in Brazil are very rare.To compare results, only the region until the stop codon of the nucleoprotein gene was considered, as also described by Oliveira et al. 25 One sample from an Artibeus lituratus frugivorous bat was segregated with samples related to the genetic lineage maintained by Tadarida brasiliensis bats.This substitution was observed in one tyrosine found in the Pasteur Virus (PV) sample (NC_001542) by asparagine in Brazilian samples 25 ; this substitution was also observed in the studied samples that were segregated in this cluster.This group presented the lowest average intrinsic distance within group.
The genetic lineage identified in the samples from group 3 was previously related 25 to the Myotis bat genus.The same research group The authors declare that there is no conflict of interest.
All techniques were carried out according to Biosafety Proceedings Standards at the Centers for Rabies Research (NPR) in the Level 3 Biosafety Laboratory of the Clinical and Molecular Laboratory, Institute of Biomedical Sciences II (ICB II), University of São Paulo (USP), Brazil.

FIGURE 1 -
FIGURE 1 -Phylogenetic tree with maximum likelihood reconstructed with MEGA4.Bootstrap values obtained with 10,000 replicas.