R n S J A a b c d a A R R A A K B R D E R 1 t n c m r a m a f i 2 F B ( 0 d Virus Research 165 (2012) 119– 125 Contents lists available at SciVerse ScienceDirect Virus Research jo u r n al hom epa ge: www.elsev ier .com/ locate /v i rusres abies virus distribution in tissues and molecular characterization of strains from aturally infected non-hematophagous bats usan Dora Allendorfa, Adriana Cortezb, Marcos Bryan Heinemannc, Camila M. Appolinário Hararya, oão Marcelo A.P. Antunesa, Marina Gea Peresa, Acácia Ferreira Vicentea, Miriam Martos Sodréd, driana Ruckert da Rosad, Jane Megida,∗ UNESP, Faculdade de Medicina Veterinária e Zootecnia, Departamento de Higiene Veterinária e Saúde Pública, Botucatu, SP, Brazil UNISA, Escola de Veterinária de Santo Amaro, São Paulo, SP, Brazil UFMG, Escola de Veterinária, Departamento de Medicina Veterinária Preventiva, Belo Horizonte, MG, Brazil Centro de Controle de Zoonoses de São Paulo, São Paulo, SP, Brazil1 r t i c l e i n f o rticle history: eceived 12 August 2011 eceived in revised form 20 January 2012 ccepted 22 January 2012 vailable online 13 February 2012 eywords: a b s t r a c t Bats are main reservoirs for Lyssavirus worldwide, which is an important public health issue because it constitutes one of the big challenges in rabies control. Yet, little is known about how the virus is maintained among bats, and the epidemiological relationships remain poorly understood. The aim of the present study was to investigate the distribution of the rabies virus (RABV) in bat tissues and organs and to genetically characterize virus isolates from naturally infected non-hematophagous bats. The hem- inested reverse transcriptase polymerase chain reaction (hnRT-PCR) and sequencing using primers to the ats abies virus istribution pidemiology T-PCR nucleoprotein coding gene were performed. The results showed a dissemination of the RABV in differ- ent tissues and organs, particularly in the salivary glands, tongue, lungs, kidneys, bladder, intestine and feces, suggesting other possible forms of RABV elimination and the possibility of transmission among these animals. The phylogenetic analysis confirmed that different variants of RABV are maintained by non-hematophagous bats in nature and have similar tissue distribution irrespective of bat species and tion. phylogenetic characteriza . Introduction Rabies is a zoonosis that affects mammals and is endemic hroughout the world, with the exception of the Antarctica conti- ent, islands such as Hawaii and New Zealand and some European ountries (Fooks et al., 2009). The World Health Organization esti- ates that 55,000 human deaths occur each year as a result of abies (WHO, 2007); the majority of cases are reported in Africa nd Asia (WHO, 2005). Data obtained from global mortality esti- ates suggest that every 10 min, one person dies from rabies and pproximately 300 others are exposed (Fooks et al., 2009). RABV genotype 1, the prototype of the genus Lyssavirus in the amily Rhabdoviridae, is the etiological agent of classical rabies that s responsible for the majority of human deaths worldwide (WHO, 007). Lyssaviruses are enveloped bullet-shaped viruses with a ∗ Corresponding author at: Departamento de Higiene Veterinária e Saúde Pública, aculdade de Medicina Veterinária e Zootecnia, UNESP, Distrito de Rubião Júnior, s/n, otucatu, SP, CEP-18618-970, Brazil. Tel.: +55 14 3811 6270; fax: +55 14 3811 6075. E-mail addresses: cortez.adri@yahoo.com.br (A. Cortez), mabryan@ufmg.br M.B. Heinemann), jane@fmvz.unesp.br (J. Megid). 1 ccz@ccz.com.br. 168-1702/$ – see front matter © 2012 Elsevier B.V. All rights reserved. oi:10.1016/j.virusres.2012.01.011 © 2012 Elsevier B.V. All rights reserved. single-stranded and negative-sense RNA genome that encodes five structural proteins: nucleocapsid protein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and the RNA polymerase or large protein (L) (Wunner, 2007). In Brazil during the last 10 years, human cases of rabies trans- mitted by dogs have diminished considerably as a result of control measures in domestic animals. However, the number of cases of human rabies acquired by bats has been increasing (Mayen, 2003; Travassos da Rosa et al., 2006; WHO, 1992) and bats have become the main transmitters of human rabies in Brazil (SVS/MS, 2008). Bats are now the most prominent source of rabies for humans, domestic animals and wild animals in the Americas (Kuzmin and Rupprecht, 2007). Rabies is a complex problem with economic, public health and ecological implications (Cunha et al., 2010). Environmental changes due to urban development may have con- tributed to the increase in the bat population in urban areas, not only because of the wide variety of shelters but also due to the large food supply and absence of predators (Sodré et al., 2010). Controlling and understanding bat rabies in natural populations has been a large challenge for researchers worldwide. Given bats’ nocturnal habits, ability to fly and variety of species with different habits, studying bat rabies under natural conditions is difficult, and many gaps in the understanding of rabies remain (Klug et al., 2011). dx.doi.org/10.1016/j.virusres.2012.01.011 http://www.sciencedirect.com/science/journal/01681702 http://www.elsevier.com/locate/virusres mailto:cortez.adri@yahoo.com.br mailto:mabryan@ufmg.br mailto:jane@fmvz.unesp.br mailto:ccz@ccz.com.br dx.doi.org/10.1016/j.virusres.2012.01.011 1 s Resea t f c v a c 2 d s R c i S c i 1 2 b g R 3 c A b v c n s l t i d e t t R h i t a p b b t s w a 2 2 t r a n o Twenty isolates had the ideal concentration for sequencing. The amplified DNA fragment was purified using a commercial kit (Illustra GFXTM PCR DNA and Gel Band Purification Kit® (GE 20 S.D. Allendorf et al. / Viru Colonial bats are gregarious animals. They have close social con- act, sometimes fighting or grooming each other, and even mutually eed by regurgitation of ingested blood in the case of vampire bat olonies. These interactions can lead to the transmission of the irus through infected saliva by bites and licking, inhalation of erosolized saliva, and probably by ingestion of regurgitated blood ontaminated with infected saliva (Constantine, 1988; Souza et al., 009). As a result of enhanced surveillance, the number of rabid bats etected is increasing each year, especially in non-hematophagous pecies (Cunha et al., 2010). Bats maintain circulation of specific ABV variants in urban areas, which is a significant public health oncern (Rupprecht et al., 1987; Smith et al., 1995). The transmission and pathobiology of RABV in free-ranging bats s still unclear and remains poorly understood (Carneiro et al., 2010; teece and Calisher, 1989). Although there is evidence that virus an be transmitted among bats by bite, aerosol, ingestion of virus- nfected milk or blood and transplacental infection (Constantine, 966, 1967a; Constantine et al., 1968; Sims et al., 1963; Souza et al., 009), the mechanism of virus transmission between bats has not een studied in detail (Kuzmin and Rupprecht, 2007). The high seroprevalence rates in apparently healthy bats sug- est they may be able to control natural infection (Lyles and upprecht, 2007). Aguilar-Setien et al. (2005) demonstrated that of 14 vampire bats survived for 2 years after a massive parenteral hallenge with RABV and occasionally excreted virus in their saliva. continuous exposure may explain why the virus is endemic in ats population, a equilibrium between fatalities and exposure to irus (biting, scratches) probably exists and the low virus dose ould induce a immune response and the development on virus eutralizing antibodies (VNAs) and clear the virus, while others uccumb to disease (Franka et al., 2008, 2009). The apparent low evel of virus shedding and the low mortality among experimen- ally infected bats leave the mechanisms of the transmission cycle n nature a mystery (Vos et al., 2007). However, it was demonstrated that naturally infected bats do ie from rabies (Carneiro et al., 2010; Kuzmin et al., 2008; Freuling t al., 2008). Moreover, a long and variable incubation period is hought to occur in nature, and this may be influencing the main- enance of enzootic rabies in wildlife (Jackson, 2007; Moore and aymond, 1970). Considering the great diversity of bats with different feeding abits, their ecology and widespread distribution in the country, mproving knowledge about virus circulation among bats will con- ribute to a better understanding of the bat rabies epidemiology nd will provide information to identify the real risk to the human opulation when in contact with those animals. Thus, the aim of this study was to analyze the viral distribution y molecular techniques in tissues and organs of naturally infected ats with different habits to look for the presence of virus in organs hat may be involved in RABV transmission among bats and pos- ibly to humans and other animals. Additionally, the virus isolates ere partially sequenced and subjected to genetic characterization nd molecular epidemiological studies. . Materials and methods .1. Bats A total of 26 non-hematophagous bats found by members of he general population or by health agents and sent for definitive abies diagnosis were used; some of these bats were found dead nd other moribund. Bats captured alive were humanely eutha- ized with ether. The bats were previously identified by the Center f Zoonosis Control, São Paulo-SP as Artibeus lituratus (13), Myotis rch 165 (2012) 119– 125 nigricans (4), Eptesicus furinalis (5), Eptesicus diminutus (1), Lasiurus blossevillii (1), and Lasiurus ega (2). The bats were collected between 2004 and 2009 in 7 cities in the western region of São Paulo state and all were diagnosed as positive for rabies by means of direct immunofluorescence and virus isolation by Dr. Avelino Albas.2 2.2. Samples Fragments of brain tissue, salivary gland, tongue, lung, heart, stomach, liver, spleen, kidney, bladder, intestine, and brown fat were aseptically collected and stored at −80 ◦C until processing. It was impossible to collect all organs because of the state of con- servation of the bat carcasses. The feces samples were collected from the anus and, when nec- essary, by opening the final part of the large intestine. Each sample was collected with individual instruments and washed with sterile water 3 times to avoid cross-contamination between tissues from the same animal. The positive control used for all reactions was the rabies chal- lenge virus strain (CVS), and sterile water (DNAse/RNAse-free) was used as a negative control. Every third sample, one negative control was added. 2.3. RT-PCR The viral RNA was extracted by the RTP DNA/RNA Virus Mini Kit® (Invitek, Germany) following the manufacturer’s instructions. The RT-PCR And hnRT-PCR techniques used was described by Soares et al. (2002); reverse transcription was performed with 7 �l of RNA first denatured for 5 min at 94 ◦C and chilled on ice and then added to a final volume of 20 �l containing 1 mM of each dNTP (Invitrogen, USA), 20 pmols of primer P510, 1× First Strand Buffer, 1 mM dTT, 200 units M-MLV Reverse Transcriptase (Invitro- gen, USA) and 0.01% Nuclease Free Water (IDT, USA). The samples were then incubated for 1 h at 42 ◦C. Primary amplification were carried out with 5 �l of the reverse transcripted c-DNA template in a final volume of 50 �l, containing 0.2 mM of each dNTP, 25 pmols of sense primer P510 (ATAGAGCAGATTTTCGAGACAGC), 25 pmols of anti sense primer P942 (CCCATATAACATCCAACAAAGTG), 1.5 mM of MgCl2, 1× PCR Buffer, 1.25 units of Platinum Taq DNA Polymerase (Invitrogen, USA) and Nuclease Free Water (IDT, USA). The samples that resulted negative on this primary amplification were submit- ted to a hnRT-PCR, in order to improve sensitivity. The hnRT-PCR was performed with antisense primer P784 (CCTCAAAGTTCTTGTG- GAAGA) and sense primer P510. The cycling conditions for the primary amplification were: initial heating at 94 ◦C/6 minutes (min), 35 cycles at 94 ◦C/45 seconds (s), 55 ◦C/60 s, 72 ◦C/90 s, fol- lowed by a final extending at 72 ◦C for 10 min. The thermal cycles for the nested assay were the same but with 25 cycles for amplification. Products of PCR were run in 2% agarose gel electrophoresis in stan- dard TBE stained with ethidium bromide 0.5 �g/ml and gels were observed under UV light and photographed. Primer sets P510/P942 and P510/P784 defined 455 and 295 base pairs, respectively. 2.4. Sequencing The amplicons obtained from rabies virus amplification from brain samples were used for nucleotide sequencing. All isolates were sequenced with a forward (P510) and reverse primer (P942). 2 Agência Paulista de Tecnologia dos Agronegócios, Pólo da Alta Sorocabana, Pres- idente Prudente-SP. S.D. Allendorf et al. / Virus Research 165 (2012) 119– 125 121 Table 1 GenBank accession number/isolates and bats species from which the samples were collected. Accession number/strain Bat specie JF950546/11 Artibeus lituratus JF950539/04 Artibeus lituratus JF950547/12 Artibeus lituratus JF950552/17 Artibeus lituratus JF950554/19 Artibeus lituratus JF950540/05 Artibeus lituratus JF950555/20 Myotis nigricans JF950543/08 Eptesicus furinalis JF950544/09 Artibeus lituratus JF950545/10 Artibeus lituratus JF950541/06 Lasiurus blossevillii JF950536/01 Lasiurus ega JF950548/13 Eptesicus furinalis JF950549/14 Myotis nigicans JF950550/15 Eptesicus diminutus JF950553/18 Myotis nigricans JF950542/07 Eptesicus furinalis JF950537/02 Eptesicus furinalis H L u a 2 ( a ( q T ( a r 5 n M t B 2 c g 3 a s g w i b g t Table 2 Rabies virus distribution in organs and tissues obtained by hnRT-PCR in naturally infected non-hematophagous bats. Samples Positive Negative Brain 100% (26/26) 0% Salivary gland 100% (26/26) 0% Tongue 88% (23/26) 12% (3/26) Brown fat 79% (19/24) 21% (5/24) Lung 69% (18/26) 31% (8/26) Heart 60% (15/25) 40% (10/25) Stomach 79% (19/24) 21% (5/24) Liver 52% (13/25) 48% (12/25) Spleen 33% (6/18) 67% (12/18) Bladder 79% (15/19) 21% (4/19) Kidney 58% (15/26) 42% (11/26) Intestine 58% (15/26) 42% (11/26) et al., 2005). JF950538/03 Eptesicus furinalis JF950551/16 Artibeus lituratus ealthcare, USA), visually quantified with the Low DNA Mass adder® (Invitrogen, USA) in agarosis gel, and finally, sequenced sing the BigDyeTM Terminator Kit® (Applied Biosystems, USA) on n automated sequencer (ABI model 377, Applied Biosystems). .5. Phylogenetic analysis First, the raw sequencing data were edited using BIOEDIT v.7.0.5 Hall, 1999). The complete sequence assemblies were then cre- ted with the PHRED/PHRAP (Ewing and Green, 1998) and CAP3 Huang and Madan, 1999) programs using nucleotide data with uality higher than 20 (http://bioinformatica.ucb.br/electro.html). he derived rabies sequences were aligned using BIOEDIT v.7.0.5 Hall, 1999). Phylogenetic analysis was performed at nucleotide level on the ligned data set, using sequences from the N gene and was car- ied out by the neighbor-joining algorithm implemented in Mega .0 (Tamura et al., 2011). The best evolutive model was determi- ated by Model test (Posadas and Crandall, 1988) implemented in ega 5.0. Bootstrap values were calculated on 1000 repeats and he bootstrap value cut off was 65%. The nucleotide sequence data reported are available in the Gen- ank databases under the accession numbers listed in Table 1. .6. Statistical analyses The Fisher’s Exact Test was used to obtain percentages and to ompare the results obtained in the viral distribution among fru- ivorous and insectivorous bats. . Results The results showed a wide distribution of RABV in the tissues nd organs analyzed, with a higher rate of virus positivity in certain amples. All of the bats had detectable virus in the brain and salivary lands (Table 2). The results of the viral distribution obtained by hnRT-PCR ere grouped into frugivorous and insectivorous bats for compar- son. According to the Fisher’s Exact Test, the difference observed etween these two groups was not statistically significant. The percentages of samples testing positive for virus from fru- ivorous and insectivorous bats, respectively, were as follows: in ongue, 92% and 85%; in brown fat, 82% and 77%; in lung, 62% and Feces 40% (10/25) 60% (15/25) 77%; in heart, 42% and 77%; in stomach, 92% and 64%; in liver, 38% and 67%; spleen, 43% and 27%; in bladder, 73% and 88%; in kid- ney, 77% and 38%; in intestine, 77% and 38%; in feces, 38% and 42%. All brains and salivary glands tested positive in the primary amplification (Fig. 1). Phylogenetic analysis revealed that RABV isolates were grouped into clusters according to the bat species, and there was evidence of species-specific variants. The sequences were analyzed for the identity of nucleotides and amino acids using BIOEDIT v.7.0.5 (Hall, 1999), which revealed a high similarity among different bat-related RABV from different geographic areas, most of them with 98–99% homology and some with 100% homology. The sequenced samples formed a phylogenetic tree divided into two major monophyletic lineages: frugivorous (I) and insectivo- rous (II) samples. Within these larger groups, clusters were formed according to bat species, with group I showing A. lituratus cluster- ing together with Desmodus rotundus (GenBank: BRDR18, BRDR21, BRDR14) and Tadarida brasiliensis (GenBank: IP2136 06–IP185 05/retrieved number). Group II contained subgroups 2 and 3, which were mostly collected from colonial non-migratory bats (M. nigri- cans and Eptesicus spp.), and subgroup 4, which contained strains related to migratory and solitary bats (Lasiurus spp.) (Fig. 2). An intra specific transmission has been occurring in different bat colonies. The idea can be confirmed by the sequencing results, virus isolates from the same species have similarities not only in the nucleotides but also in pathogenic characteristics observed by intracerebral and intramuscular experimental inoculation (Wang Fig. 1. Comparison of positive results obtained in samples of rabies virus naturally infected frugivorous and insectivorous bats. http://bioinformatica.ucb.br/electro.html 122 S.D. Allendorf et al. / Virus Research 165 (2012) 119– 125 Fig. 2. Phylogenetic tree based on the sequence of 442 bp corresponding to the middle of nucleoprotein gene (N) located between nucleotides 589 and 1031 of the PV virus ( hbor- s plicat u (data 4 c e A t t e GenBank number M13215.1). Phylogenetic analysis was performed using the neig hown at the nodes of the genetic clusters represent the bootstrap values for 1000 re sing Maximum Likelihood test yielded similar results to neighbor-joining method . Discussion There is a lack of information involving the virus distribution of lassic rabies virus genotype 1, in naturally infected bats. The dis- ase has been investigated on its native host by few researchers. s already suggested, in bats the virus spreads centripetally from he inoculation site to the brain and then centrifugally to other issues and organs (Freuling et al., 2009). Rabies virus in differ- nt organs were detected in our research, in agreement with other joining method with Tamaru 3 parameter and invariant sites model. The numbers es, and the RABV fixed strain was used as an outgroup. Phylogenetic tree performed not shown). studies which indentified the virus in different tissues of infected bats; despite the fact that some samples presented a higher rate of positivity no significant pattern of viral distribution was observed (Franka et al., 2008; Freuling et al., 2009; Johnson et al., 2006). We could not determine a pattern of viral distribution in the studied bat species, suggesting that is most likely to be related to others factors than with the bat specie or even with the lyssavirus genotype. According to Freuling et al. (2009), the variation on viral distribution in the organs and tissues of the infected bats might Resea b s i r ( b ( t a f a o t e p e t i v b i c t T t a i b b i s 1 q g S f s n t t s T c t ( t ( a a r e M w l s g e o e 2 u S.D. Allendorf et al. / Virus e related to the site of inoculation. This observation could be upported by another study in which Eptesicus fuscus were exper- mentally infected with European bat lyssavirus 1 (EBLV-1), the esults revealed that animals who received lower intramuscular i.m.) virus dose appeared to have a broader organ distribution than ats receiving the higher i.m dose and bats inoculated intracranially i.c.) (Franka et al., 2008). In addition, the dose and mode of infec- ion among free-ranging bats could lead to an abortive infection nd possible be responsible for the naturally occurring antibodies ound in bat populations (Davis et al., 2007; Franka et al., 2008). In our study, RABV was detected in 100% of the salivary glands nd in 88% of the tongues analyzed. These data corroborate previ- us studies that detected high virus positivity rates obtained in the ongues from different bat species (Carneiro et al., 2010; Johnson t al., 2008; Freuling et al., 2009). Although we cannot exclude the ossibility of viral contamination through infected saliva, the pres- nce of virus antigen in lingual papilla epithelium associated with he taste buds positivity in salivary gland was characterized by mmunohistochemical analysis confirming the presence of rabies irus in these tissues (Freuling et al., 2009). The release of virus by epithelial cells in the tongue proba- ly occur in infected bats as it was observed with experimentally nfected serotine bat (Eptesicus serotinus) were the viral antigen ould be indentified in lingual epithelium but not be in the secre- ory epithelial cells of the salivary gland (Freuling et al., 2009). he presence of large amounts of RABV in salivary gland and in ongue regardless of the virus genotype reinforces the knowledge nd importance of saliva as a source of RABV elimination in bats rrespective to bat species. As suggested, the transmission through ites is probably the most important mode of infection in free living ats (Franka et al., 2006). Positivity of 79% in brown fat was detected in our study, which is n accordance with several reports that detected RABV in the inter- capular brown adipose tissue in naturally infected bats (Bell et al., 962; Villa et al., 1963; Scheffer et al., 2007). In Myotis bats, the fre- uency of isolation of RABV from the brain, brown fat, and salivary lands was 92%, 30% and 17%, respectively. Sulkin et al. (1960) and ulkin (1962) experimentally infected insectivorous bats (Mexican ree-tailed bat and Myotis lucifugus) with the objective to under- tand the mechanism by which these animals serve as reservoirs in ature. The results suggested that the brown fat provides a sit for he storage of RABV during bat hibernation and for virus replica- ion. The virus isolates were quantified by RABV titration in tissue uspensions of the brain, salivary gland and brown fat of mice. hese results revealed high virus titers in the interscapular fat, with oncentrations similar to those of the brain and salivary glands. RABV transmission via an airborne route is much less common han by bites but can occur in caves where millions of bats roost Constantine, 1962; Jackson, 2007). RABV has been observed in he nasal mucosa of naturally infected Mexican free-tailed bats Tadarida brasiliensis mexicana) by virus isolation and fluorescent ntibody test, suggesting that the nasal mucosa is a portal of entry nd possible portal from which virus is expelled into the air in respi- atory mucus particles (Constantine et al., 1972). Another study xamined the viral distribution in paralyzed or recently deceased exican free-tailed bats collected from caves in Texas. Rabies virus as detected in 130 bats, in 30% which the virus was isolated from ung samples (Constantine, 1967a,b). In this study, from 26 bats tudied, the virus was detected in the lungs of 18 (69.2%), sug- esting that airborne transmission may occur in natural conditions, specially in caves with many bats and without air circulation, as bserved by Constantine (1967b). In contrast, bats exposed to aerosolized rabies virus under xperimental conditions survived and produced VNAs (Davis et al., 007). Franka et al. (2009) observed that after intranasal (i.n.) inoc- lation with EBLV-1a all animals survived, none developed VNAs, rch 165 (2012) 119– 125 123 in contrast to the bats infected parenterally, the authors suggested a possible role for innate immunity in peripheral clearance of rabies virus in bats. The lack of observed morbidity and mortality led the researchers to conclude that an aerosol route of exposure might not play a major role in transmission of rabies among free-living bats. Although not frequently reported, rabies transmission by an oral route can occur (Jackson, 2007). It has been proved that the ingestion of infected carcasses by carnivores can transmit rabies (Ramsden and Johnson, 1975). It was also demonstrated that the virus remains viable in infected skunk carcasses for 22 days at 10 ◦C and for 14 days at 24 ◦C (Schaefer, 1983) and that ingestion of infected tissue may be a mode of transmission to mammalian scav- engers (Correa-Giron et al., 1970; Ramsden and Johnson, 1975). Bell and Moore (1971) demonstrated that striped skunks may be fatally infected after eating a single rabid mouse, suggesting that bats could cause rabies infection by an oral route when carnivores feed on them. Assuming that the transmission of rabies can occur by eating infected bat carcasses, we cannot exclude the possibil- ity of rabies transmission to other carnivore species in rural and urban locations, or even to other bats. There are reports that hoary bats (Lasiurus cinereus) may prey on Pipistrelles bats (Constantine, 1967a; Bishop, 1947; Orr, 1950). Cannibalism among bats has been observed in captivity and probably occurs in nature more fre- quently than is reported (Constantine, 1967a). The high percentage of virus positivity in stomach tissue (79%) could be due to swallowing of the virus, as previously suggested by Johnson et al. (2006). Viral RNA was also detected in bat feces, kid- ney and bladder. These results suggest that bat urine as well as feces could be a source of infection and a possible mean of rabies trans- mission, as previously suggested (Constantine et al., 1972; Johnson et al., 2006; Scheffer et al., 2007). However, the epidemiological importance of these excretions must be evaluated more closely in a natural environment in which rabies virus is inactivated by unfavorable ambient conditions. The low virus positivity rate found in spleen (33%) was similar to other results reported by Carneiro et al. (2010), who suggested that these low levels could be explained by the lack of specific involvement of RABV with the lymphatic system. It has been demonstrated, through the analysis of partial nucle- oprotein gene sequences, that there are different virus variants circulating among bats (Nadin-Davis et al., 2001; Oliveira et al., 2010; Vellasco-Villa et al., 2006). The 400 nucleotides in the amino- terminal coding region of the N gene are recommended for use in phylogenetic analysis because they can determine the geographical distribution of the major virus lineages (Kissi et al., 1995). How- ever, it has been observed that in general, similar conclusions can be made about epidemiological relationships regardless of the spe- cific sequenced region of the target gene (Nadin-Davis, 2007). In our study, the sequenced region corresponded to the middle of the N gene, and phylogenetic analysis revealed that virus variants tended to form groups according to bat species. Group I, a cluster defined by bootstrap of 91, contained sam- ples from A. lituratus, vampire bats and T. brasiliensis (numbers retrieved from GenBank) that allowed us to conclude that there is a significant relationship between hematophagous and frugivorous bats. The results obtained here had already been observed by other researchers (Kobayashi et al., 2005, 2007; Shoji et al., 2004), imply- ing that an interaction had occurred between these species; the virus isolated from A. lituratus is clearly characterized as vampire bat-related RABV. There are hypotheses that try to explain the transmission of RABV between hematophagous and frugivorous bats; some researchers have suggested that it, while others posit that some vampire bats may have fed on other species of bats that shared the same roosts, especially during inclement weather, when bats may be confined (Greenhall, 1988). Virus transmission might have 1 s Resea o ( b 5 o s g p a b d i e d t v t i A f R A B B B C C C C C C C C C C D E F F F 24 S.D. Allendorf et al. / Viru ccurred by sharing the same roost, as was observed in Mexico Forment et al., 1971). Further studies should be carried out to etter understand the relationship between these species. . Conclusion These results indicate the presence of RABV in several organs f naturally infected non-hematophagous bats, particularly in the alivary glands, lungs, kidneys, bladder, intestine, and feces sug- esting other possible forms of rabies virus elimination and the ossibility of transmission among these animals. The phylogenetic nalysis confirmed that different variants of RABV are maintained y non-hematophagous bats in nature and have similar tissue istribution, irrespective of bat species and phylogenetic character- zation. However within the same bat species geographic genotypes xist, confirming a local viral circulation. It was not possible to efine a pattern of viral distribution in the analysed samples. In conclusion, knowing the characteristics of each virus con- ribute for a better understanding of the relationship between the irus, the animals and the species. The role that the infectious dose, he pathogenicity and the immune innate response plays on rabies nfection in wildlife requires more detailed studies. cknowledgement We thank Avelino Albas for providing the bats for this study and or the previous diagnosis of rabies infection. eferences guilar-Setien, A., Loza-Rubio, E., Salas-Rojas, M., Brisseau, N., Cliquet, F., Pastoret, P.P., Rojas-Dotor, S., Tesoro, E., Kretschmer, R., 2005. Salivary excretion of rabies virus by healthy vampire bats. Epidemiol. Infect. 133, 517–522. ell, J.F., Moore, G.T., Raymond, G.H., Tibbs, C.E., 1962. Characteristics of rabies in bats in Montana. Am. J. Publ. Health 52, 1293–1301. ell, J.F., Moore, G.J., 1971. Susceptibility of carnivore to rabies virus administered orally. Am. J. Epidemiol. 93, 176–182. ishop, S.C., 1947. Curious behavior of a hoary bat. J. Mammal. 28, 293–294. arneiro, A.J.B., Franke, C.R., Stöcker, A., Dos Santos, F., De Sá, J.E.Ú., Moraes-Silva, E., Alves, J.N.M., Brünink, S., Corman, V.M., Drosten, C., Drexler, J.F., 2010. Rabies virus RNA in naturally infected vampire bats, northeastern Brazil. Emerg. Infect. Dis. 16 (12), 2004–2006. onstantine, D.G., 1962. Rabies transmission by nonbite route. Public Health Rep. 77, 287–289. onstantine, D.G., 1966. Transmission experiments with bat rabies isolates. Reaction of certain carnivore, opossum, rodents and bats to rabies virus of red bat origin when exposed by bat or by intra-muscular inoculation. Am. J. Vet. Res. 27, 24–32. onstantine, D.G., 1967a. Bat rabies in the southwestern United States. Public Health Rep. 81 (10), 867–888. onstantine, D.G., 1967b. Rabies transmission by air in bat caves. In: Public Health Service Publication 1617. National Communicable Disease Center, Atlanta. onstantine, D.G., Solomon, G.C., Woodall, D.F., 1968. Transmission experiments with bat rabies isolates: responses of certain carnivores and rodents to rabies virus from four species of bats. Am. J. Vet. Res. 29, 181–190. onstantine, D.G., Emmons, R.W., Woodie, J.D., 1972. Rabies virus in nasal mucosa of naturally infected bats. Science 175, 1255–1256. onstantine, D.G., 1988. Transmission of pathogenic microorganisms by vampire bats. Public Health Greenhall. In: Schmidt, A.M.U. (Ed.), The Natural History of Vampire Bats. CRC Press, Florida, pp. 167–189. orrea-Giron, E.P., Allen, R., Sulkin, S.E., 1970. The infectivity and pathogenesis of rabies-virus administrated orally. Am. J. Epidemiol. 91, 203–215. unha, E.M.S., Nassar, A.F.C., Lara, M.C.C.S.H., Villalobos, E.C.M., Sato, G., Kobayashi, Y., Shoji, Y., Itou, T., Sakai, T., Ito, F.H., 2010. Pathogenicity of different rabies virus isolates and protection test in vaccinated mice. Rev. Inst. Med. Trop. Sao Paulo 52, 231–235. avis, A.D., Rudd, R.J., Bowen, R.A., 2007. Effects of aerosolized rabies virus exposure on bats and mice. J. Infect. Dis. 195, 1144–1150. wing, B., Green, P., 1998. Base-calling of automated sequencer traces using phred. II. Error probabilities. Gen. Res. 8, 186–194. ooks, A.R., Johnson, N., Rupprecht, C.E., 2009. Rabies. In: Barrett, A.D.T., Stanberry, L.R. (Eds.), Vaccines for Biodefense and Emerging and Neglected Diseases. Aca- demic Press, Italy, pp. 609–630. orment, W.L., Schmidt, U., Greenhall, A.M., 1971. Movement and population studies of vampire bat (Desmodus rotundus) in Mexico. J. Mammal. 52, 227–228. ranka, R., Denny, C.G., Kuzmin, I., Velasco-Villa, A., Reeder, S.A., Streicker, D., Orciari, L.A., Wong, A.J., Blanton, J.D., Rupprecht, C.E., 2006. A new philogenetic lineage rch 165 (2012) 119– 125 of Rabies Virus associated with western pipestrelle bats (Pipistrellus hesperus). J. Gen. Virol. 87, 2309–2321. Franka, R., Wu, X., Jackson, F.R., Velasco-Villa, A., Palmer, D.P., Henderson, H., Hayat, W., Green, D.B., Blanton, J.D., Greenberg, L., Rupprecht, C.E., 2009. Rebies virus pathogeneis in relationship with intervention with inactivated and attenuated rabies virus vaccines. Vaccine 27, 7149–7155. Franka, R., Johnson, N., Muller, T., Vos, L., Neubert, L., Freuling, C., Rupprecht, C.E., Fooks, A.R., 2008. Susceptibility of north American big brown bats (Eptesicus fuscus) to infection with European bat lyssavirus type 1. J. Virol. 89, 1998–2010. Freuling, C., Grossmann, E., Conraths, F.J., Schameitat, A., Kliemt, J., Auer, E., Greiser- Wilke, I., Muller, T., 2008. First isolation of EBLV-2 in Germany. Vet. Microbiol. 131, 26–34. Freuling, C., Vos, A., Johnson, I., Denzinger, A., Neubert, E., Mansfield, K., Hicks, D., Nuñez, A., Tordo, N., Rupprecht, C.E., Fooks, A.R., Muller, T., 2009. Experimental infection of serotine bats (Eptesicus serotinu) with European bat lyssavirus type 1a. J. Virol. 90, 2493–2502, doi:10.1099/vir.O.O11510-0. Greenhall, A.M., 1988. Feeding behavior. In: Greenhall, A.M., Schmidt, U. (Eds.), Nat- ural History of Vampire Bats. CRC Press, Boston, pp. 111–131. Hall, T.A., 1999. Bioedit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/nt. Nucl. Acids Symp. Ser. 41, 95–98. Huang, X., Madan, A., 1999. CAP3: a DNA sequence assembly program. Genome Res. 9, 868–877. Jackson, A., 2007. Pathogenesis. In: Jackson, A.C., Wunner, W.H. (Eds.), Rabies. Aca- demic Press, San Diego, pp. 341–381. Johnson, N., Wakeley, P.R., Brookes, S.M., Fooks, A.R., 2006. European bat lyssavirus type 2 RNA in Myotis daubentonii. Emerg. Infect. Dis. 12, 1142–1144. Johnson, N., Vos, A., Neubert, L., Freuling, C., Mansfield, K.L., Kaipf, I., Denzinger, A., Hicks, D., Nuñez, A., Franka, R., Rupprecht, C.E., Muller, T., Fooks, A.R., 2008. Experimental study of European bat lyssavirus type-2 infection in Daubentonı̌s bats (Myotis daubentonii). J. Gen. Virol. 89, 2662–2672. Kissi, B., Tordo, N., Boourhy, H., 1995. Genetic polymorphism in the rabies virus nucleoprotein. Virology 209, 526–537. Klug, B.J., Turmelle, A.S., Ellison, J.A., Baerwald, E.F., Barclay, R.M.R., 2011. Rabies prevalence in migratory tree-bats in Alberta and the influence of roosting ecol- ogy and sampling method on reported prevalence of rabies in bats. J. Wildl. Dis. 47 (1), 64–77. Kobayashi, Y., Sato, G., Shoji, Y., Sato, T., Itou, T., Cunha, E.M.S., Samara, S.I., Carvalho, A.A.B., Nociti, D.P., Ito, F.H., Sekai, T., 2005. Molecular epidemiological analysis of bat rabies viruses in Brazil. J. Vet. Med. Sci. 67, 647–652. Kobayashi, Y., Sato, G., Kato, M., Itou, T., Cunha, E.M., Silva, M.V., Mota, C.S., Ito, F.H., Sakai, T., 2007. Genetic diversity of bat rabies viruses in Brazil. Arch. Virol. 152 (11), 1995–2004. Kuzmin, I.V., Rupprecht, C., 2007. Bat rabies. In: Jackson, A.C., Wunner, W.H. (Eds.), Rabies. Academic Press, San Diego, pp. 259–307. Kuzmin, I.V., Niezgoda, M., Franka, R., Agwanda, B., Markotter, W., Beagley, J.C., Ura- zova, O.Y., Breiman, R.F., Rupprecht, C.E., 2008. Lagos bat virus in Kenya. J. Clin. Microbiol. 46, 1451–1461. Lyles, D.S., Rupprecht, C.E., 2007. Rhabdoviridae. In: Knipe, D.M., Griffin, D.E., Lamb, R.A., Strauss, S.E., et al. (Eds.), Fields Virology. Lipincott, Philadelphia, pp. 1363–1408. Mayen, F., 2003. Haematophagous bats in Brazil, their role in rabies transmission, impact on public health, livestock and alternatives to an indiscriminate reduc- tion of population. J. Vet. B: Infect. Dis. Vet. Pub. Health 50, 469–472. Moore, G.J., Raymond, G.H., 1970. Prolonged incubation period of rabies in naturally infected insectivorous bat, Eptesicus fuscus (Beauvois). J. Wildl. Dis. 6, 167–168. Nadin-Davis, S.A., Huang, W., Armstrong, J., Casey, G.A., Bahloul, C., Tordo, N., Wan- deler, A.I., 2001. Antigenic and genetic divergence of rabies viruses from bat species indigenous to Canada. Virus Res. 74, 139–156. Nadin-Davis, S.A., 2007. Molecular epidemiology. In: Jackson, A.C., Wunner, W.H. (Eds.), Rabies. Academic Press, San Diego, pp. 69–122. Oliveira, R.N., De Souza, S.P., Lobo, R.S.V., Castilho, J.G., Macedo, C.I., Carnieli Jr., P., Fahl, W.O., Achkar, S.M., Scheffer, K.C., Kotait, I., Carrieri, M.L., Brandão, P.E., 2010. Rabies virus in insectivorous bats: implications of the diversity of the nucleoprotein and glycoprotein genes for molecular epidemiology. Virology 405, 352–360. Orr, R.T., 1950. Unusual behavior and occurrence of a hoary bat. J. Mammal. 31, 456–457. Posadas, J., Crandall, K.A., 1988. Model test: testing the model of DNA substitution. Bioinformatics 14, 817–818. Ramsden, R.O., Johnson, D.H., 1975. Studies on the oral infectivity of rabies virus in carnivore. J. Wildl. Dis. 11, 318–324. Rupprecht, C.E., Glickman, L.T., Spencer, P.A., Wiktor, T.J., 1987. Epidemiology of rabies virus variants. Differentiation using monoclonal antibodies and discrim- inant analysis. Am. J. Epidemiol. 126, 298–309. Schaefer, J.M., 1983. The Viability of Rabies in Carrion., http://digitalcommons. unl.edu/cgi/viewcontent.cgi?article=1287&context=gpwdcwp. Scheffer, K.C., Carrieri, M.L., Albas, A., Santos, H.C.P., Kotait, I., Ito, F.H., 2007. Rabies virus in naturally infected bats in the state of São Paulo, Southeastern Brazil. Rev. Saúde. Publ. 41, 389–395. Sims, R.A., Allen, R., Sulkin, S.E., 1963. Studies on the pathogenesis of rabies in insectivorous bats. III. Influence of the gravid state. J. Infect. Dis. 112 (1), 17–27. Shoji, Y., Kobayashi, Y., Sato, G., Itou, T., Miura, Y., Mikami, T., Cunha, E.M., Sâmara, S.I., Carvalho, A.A., Nocitti, D.P., Ito, F.H., Kurane, I., Sakai, T., 2004. Genetic char- acterization of rabies viruses isolated from frugivorous bat (Artibeus spp.) in Brazil. J. Vet. Med. Sci. 66 (10), 1271–1273. dx.doi.org/10.1099/vir.O.O11510-0 Resea S S S S S S S S T S.D. Allendorf et al. / Virus mith, J.S., Orciari, L.A., Yager, P.A., 1995. Molecular epidemiology of rabies in the United States. Semin. Virol. 6, 387–400. oares, R.M., Bernardi, F., Sakamoto, S.M., Heinemann, M.B., Cortez, A., Alves, L.M., Meyer, A.D., Ito, F.H., Richtzenhain, L.J., 2002. A heminested polymerase chain reaction for the detection of Brazilian isolates from vampires bats and herbi- vores. Mem. Inst. Oswaldo Cruz 97 (1), 109–111. odré, M.M., Gama, A.R., Almeida, M.F., 2010. Updated list of bat species positive for rabies in Brazil. Rev. Inst. Med. Trop. Sao Paulo 52, 71–81. teece, R.S., Calisher, C.H., 1989. Evidence for prenatal transfer of rabies virus in the Mexican free-tailed bat (Tadarida brasiliensis mexicana). J. Wildl. Dis. 25 (3), 329–334. ouza, M.C., Nassar, A.F., Cortez, A., Sakai, T., Itou, T., Cunha, E.M., Richtzen- hain, L.J., Ito, F.H., 2009. Experimental infection of vampire bats Desmodus rotundus (E. Geoffroy) maintained in captivity by feeding defibrinated blood added with rabies virus. Braz. J. Vet. Res. Anim. Sci. 46, 92– 100. ulkin, S.E., Krutzsch, P.H., Allen, R., Wallis, C., 1960. Studies on the pathogenesis of rabies in insectivorous bats. I. Role of brown adipose tissue. J. Exp. Med. 112, 595–617. ulkin, S.E., 1962. Bat rabies: experimental demonstration of the ‘reservoiring mech- anism’. Am. J. Public Health 52, 489–498. VS/MS – Secretaria da Vigilância Sanitária do Ministério da Saúde, 2008. http://portal.saude.gov.be/portal/saude. amura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S., 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol., doi:10.1093/molbev/msr121. rch 165 (2012) 119– 125 125 Travassos da Rosa, E.S., Kotait, I., Barbosa, T.F.S., Carrieri, M.L., Brandão, P.E., Pin- heiro, A.S., Bergot, A.L., Wada, M.Y., Oliveira, R.C., Grisard, E.C., Ferreira, M., da Silva Lima, R.J., Montebello, L., Medeiros, D.B.A., Souza, R.C.M., Bensabath, G., Carmo, E.H., Vasconcelos, P.F.C., 2006. Bat transmitted human rabies outbreaks, Brazilian Amazon. Emerg. Infect. Dis. 12, 1197–1202. Vellasco-Villa, A., Orciari, L.A., Juarez-Islas, V., Gómez-Sierra, M., Padilla-Medina, I., Flisser, A., Souza, V., Castillo, A., Franka, R., Escalante-Mañe, M., Sauri-Gonzáles, I., Rupprecht, C.E., 2006. Molecular diversity of rabies virus associated with bats in Mexico and other countries of the Americas. J. Clin. Microbiol. 44, 1697–1710. Villa, B.R., Alvarez, B.A., Dominguez, C.C., 1963. Presencia y persistencia del virus de la rabia en la glandula inter-escapular de algunos murciélagos mexicanos. Ciência 22 (5), 137–140. Vos, A., Kaipf, I., Dezinger, A., Fooks, A.R., Johnson, N., Muller, T., 2007. European bat lyssavirus – an ecological enigma. Acta Chiropt. 9, 283–296. Wang, Z.W., Sarmento, L., Wang, Y.X.-q., Dhingra, V., Tseggai, T., Jiang, B., Fu, Z.F., 2005. Attenuated rabies virus activates, while pathogenic rabies virus evades, the host innate immune response in the central nervous system. Virology 79, 12554–21256. World Health Organization, 1992. Expert Committee on Rabies. Eight Report. WHO Technical Report Series No. 824. World Health Organization, Geneva. World Health Organization, 2005. Expert Consultation on Rabies. First Report WHO Technical Report Series No. 931. World Health Organization, Geneva. World Health Organization, 2007. Rabies vaccines, WHO position paper. Wkly. Epi- demiol. Rec. 82, 425–435. Wunner, W.H., 2007. Rabies virus. In: Jackson, A.C., Wunner, W.H. (Eds.), Rabies. Academic Press, San Diego, pp. 23–68. http://portal.saude.gov.be/portal/saude dx.doi.org/10.1093/molbev/msr121 Rabies virus distribution in tissues and molecular characterization of strains from naturally infected non-hematophagous bats 1 Introduction 2 Materials and methods 2.1 Bats 2.2 Samples 2.3 RT-PCR 2.4 Sequencing 2.5 Phylogenetic analysis 2.6 Statistical analyses 3 Results 4 Discussion 5 Conclusion Acknowledgement References