Evolutionary Diversity of Bat Rabies Virus in São Paulo State, Brazil

Abstract

The history of the rabies virus dates back four millennia, with the virus being considered by many to be the first known transmitted between animals and humans. In Brazil, rabies virus variants associated with terrestrial wild animals, marmosets, and different bat species have been identified. In this study, bat samples from different regions of São Paulo State, in Southeast Brazil, were analyzed to identify their genetic variability and patterns. A total of 51 samples were collected over ten years (1999–2009) and submitted to the immunofluorescent technique using monoclonal antibodies for antigenic profile detection (the diagnostic routine used in Latin American countries) and genetic evolution analysis through maximum likelihood approaches. Three antigenic profiles were detected: one related to the rabies virus maintained by hematophagous bat populations (AgV3), part of the monoclonal antibody panel used, and two other profiles not included in the panel (called NC1 and NC2). These antigenic profiles were genetically distributed in five groups. Group I was related to hematophagous bats (AgV3), Groups II and III were related to insectivorous bats (NC1) and Groups IV and V were also related to insectivorous bats (NC2). The results presented herein show that genetic lineages previously restricted to the northwest region of São Paulo State are now found in other state regions, highlighting the need for a comprehensive genetic study of bat rabies covering geographic and temporal space, through expanded genomic analysis using a standard genomic fragment.

1. Introduction

Rabies is one of the most important viral infectious diseases and, with a history dating back four millennia, is considered by many to be the first known disease transmitted between animals and humans [1]. Rabies was initially described in humans and carnivores, but studies of bats and rabies in Brazil and Trinidad in the 1920s and 1930s showed the existence of a rabies virus aerial cycle. The existence of the aerial cycle explained epidemics that occur without the presence of carnivorous animals and how the virus continues to circulate in places where rabies in domestic animals has been controlled, showing the interrelationship of this aerial cycle with the terrestrial cycle [1,2,3].

The rabies virus (RABV) belongs to the Rhabdoviridae family and Lyssavirus genus, which contains 18 viral species [4], most of them associated with bats from the Old World. The Lyssavirus rabies species is the only one that circulates among numerous mammals, including bats, carnivores, and nonhuman primates, such as marmosets in Brazil [5,6,7]. Cross-species transmission has been observed among non-canid carnivores, bats, and other mammal species, leading to the emergence of new lineages also related to American bats [5].

Antigenic characterization studies have been conducted in several Latin American countries using a panel of monoclonal antibodies (MAbs) produced by the Centers for Disease Control and Prevention (CDC), Atlanta, USA. The use of these MAbs, from the 1980s onwards, established a new era in the knowledge of RABV reservoirs and transmissibility, resulting in immediate advances in epidemiological surveillance in those countries before laboratories implemented sequencing capability. Over the years, this tool has identified several other antigenic profiles not included in the original panel [6] established by Diaz et al. [8]. These additional profiles were detected in many countries, including Brazil [9,10,11,12,13,14,15]. In a certain way, this was already expected, considering that among the samples analyzed for establishing the panel profiles, there were no varieties of isolates from different South American bat species. This identification technique continues to be used routinely in most Latin American countries.

The genetic analysis of the N gene, with a chosen genome fragment (between position 1157 and 1476 of PV-NC_001542), not only allowed the correlation of host species with their geographic distributions but also confirmed the differences observed in the reactivity pattern in the antigenic tests among samples associated with different species of bats in different countries [9,16,17,18].

In Brazil, the first antigenic [11] and genetic [19] studies showed that, as in other Latin American countries, the two predominant antigenic variants/viral lineages were associated with viruses maintained by dogs (called AgV1 and AgV2) and the Desmodus rotundus variant (AgV3). Later, antigenic variants/viral lineages associated with terrestrial wild animals, such as foxes and wild dogs [20,21], marmosets [6], and different species of insectivorous and frugivorous bats, were identified [13,22,23,24,25,26].

Both the genetic and antigenic characterizations of bat RABV isolated in the State of São Paulo in southeastern Brazil have shown the existence of variants/lineages related to D. rotundus (AgV-3), Tadarida brasiliensis (AgV4), and Lasiurus sp. (AgV6), also circulating in other species of insectivorous and frugivorous bats, along with other antigenic profiles that were not included in the CDC monoclonal panel [6,8,10,21,22].

In this study, bat samples from different regions of São Paulo State were analyzed using evolutionary approaches to identify genetic variability of the RABV in São Paulo. These isolates are compared with isolates described in previous publications, characterized antigenically and genetically, for which sequences have been deposited in Genbank (Table A1, Appendix A).

2. Materials and Methods

2.1. Samples

This study included RABV isolates from 32 municipalities in different administrative regions of São Paulo State in Southeast Brazil (Figure 1), between 1999 and 2010.

Figure 1.

Geographic location of sample collection. Colors are related with antigenic variant/profile: red for Group I (AgV3) related to D. rotundus, orange for Group II and III (related to antigenic Non-compatible 1-NC1 profile), and dark blue for Groups IV and V (NC2). Cities where more than one genetic group and/or antigenic variant was detected are in pale blue with a graphic following determined pattern of colors. GenBank sequences used in phylogenetic tree reconstruction can be observed as red dots for Group I, orange stars for samples clustered in Groups II and III, and dark blue dots for Groups IV and V. The map was modified for this study using Inkscape software version 1.3.2 (available at www.inkscape.org accessed on 4 July 2025). The original map is available at https://pt.m.wikipedia.org/wiki/Ficheiro:SaoPaulo_MesoMicroMunicip.svg (accessed on 19 February 2025).

All the samples had been previously diagnosed as positive for rabies by means of the fluorescent antibody test (FAT) [27] and mouse inoculation test (MIT) [28], considered the gold standard at the time of receiving samples for diagnosis between 1995 and 2010. In all, 48 bat samples were studied: 13 frugivorous (11 Artibeus lituratus, 01 Artibeus planirostris, 01 Artibeus fimbriatus) and 35 insectivorous (04 Myotis nigricans, 10 Neoeptesicus furinalis, 03 Neoeptesicus diminutus, 01 Neoeptesicus sp., 05 Molossus fluminensis, 03 Molossus molossus, 01 Cynomops abrasus, 02 Nyctinomops laticaudatus, 01 Nyctinomops macrotis, 01 Lasiurus blossevillii, 01 Lasiurus ega, 01 Eumops glaucinus and 02 non-hematophagous (NH) bats not identified). Samples from one cat, one bovine, and one horse were also included, giving 51 samples in total (Table 1).

Table 1.

Sequences obtained in the study, including environmental and geographical information.

GenBank Access	ID Sample/ Year	Species	Place of Origin	Antigenic Variant/Profile	Genetic Lineage
HQ666824	IB 346/99	Artibeus lituratus	São José do Rio Preto	V-3	D. rotundus
HQ666825	IB 777/00	Cynomops abrasus	Ipiguá	NC2	Insect. Bats
HQ666826	IB 249/01	Nyctinomops macrotis	São José do Rio Preto	NC1	Insect. bats
HQ666827	IB 250/01	Nyctinomops laticaudatus	São José do Rio Preto	NC1	Insect. Bats
HQ666828	IB 636/01	Neoeptesicus furinalis *	Olímpia	NC1	Insect. Bats
HQ666829	IB 808/01	Artibeus lituratus	São José do Rio Preto	V-3	D. rotundus
HQ666830	IB 1019/01	Neoeptesicus sp. *	Cardoso	NC1	Insect. Bats
HQ666831	IB 1070/01	Neoeptesicus furinalis *	São José do Rio Preto	NC1	Insect. Bats
HQ666832	IB 62/02	Neoeptesicus furinalis *	Catanduva	NC1	Insect. Bats
HQ666833	IB 109/02	Molossus molossus	Ilha Solteira	NC2	Insect. Bats
HQ666834	IB 835/02	Myotis nigricans	Cajobi	NC1	Insect. Bats
HQ666835	IB 992/02	Artibeus lituratus	Dracena	V-3	D. rotundus
HQ666836	IB 1021/02	Molossus fluminensis *	Presidente Venceslau	V-3	D. rotundus
HQ666837	IB 1141/02	Neoeptesicus furinalis *	Santo Anastácio	NC1	Insect. Bats
HQ666838	IB 1256/02	NH bat (not identified)	Martinópolis	V-3	D. rotundus
HQ666839	IB 1371B/02	Artibeus lituratus	Presidente Venceslau	V-3	D. rotundus
HQ666840	IB 1535/02	Artibeus lituratus	Taciba	V-3	D. rotundus
HQ666841	IB 1539/02	Molossus molossus	Presidente Prudente	V-3	D. rotundus
HQ666842	IB 1782/02	Lasiurus ega	Presidente Prudente	NC1	D. rotundus
HQ666843	IB 349/03	Artibeus planirostris	Santa Fé do Sul	V-3	D. rotundus
HQ666844	IB 350/03	NH bat (not identified)	Catanduva	V-3	D. rotundus
HQ666845	IB 791/03	Artibeus lituratus	Presidente Prudente	V-3	D. rotundus
-	IB 826/03	Artibeus fimbriatus	São José do Rio Preto	V-3	ND
HQ666846	IB 168/04	Myotis nigricans	Campinas	NC1	Insect. Bats
HQ666847	IB 184/04	Nyctinomops laticaudatus	São José do Rio Preto	NC1	Insect. Bats
HQ666848	IB 550/04	Artibeus lituratus	Caçapava	V-3	D.rotundus
HQ666849	LRU 329/05	Eumops glaucinus	Araçatuba	V-3	D.rotundus
-	LRU 397/05	Artibeus lituratus	Araçatuba	V-3	ND
HQ666850	LRU 43/09	Neoeptesicus diminutus *	Pereira Barreto	NC1	Insect. Bats
HQ666851	LRU 84/09	Neoeptesicus diminutus *	Araçatuba	NC1	Insect. Bats
HQ666852	LRU 149/09	Myotis nigricans	Coroados	NC1	Insect. Bats
HQ666853	LRU 181/09	Artibeus lituratus	Penápolis	V-3	D. rotundus
HQ666856	LRU 325/09	Artibeus lituratus	Birigui	V-3	D. rotundus
HQ666857	LRU 374/09	Artibeus lituratus	Guararapes	V-3	D. rotundus
HQ666858	LRU 389/09	Molossus fluminensis *	Guararapes	V-3	D. rotundus
HQ666859	LRU 433/09	Myotis nigricans	Penápolis	NC1	Insect. Bats
HQ666860	LRU 589/09	Neoeptesicus furinalis *	Penápolis	NC1	Insect. Bats
HQ666854	LRPP 199/09	Neoeptesicus furinalis *	Dracena	NC1	Insect. Bats
HQ666855	LRPP 224/09	Neoeptesicus furinalis *	Parapuã	V-3	D. rotundus
HQ666861	LRPP 672/09	Bovine/Cattle	Narandiba	V-3	D. rotundus
HQ666862	LRU 17/10	Molossus fluminensis *	Penápolis	NC1	Insect. Bats
KU299782	LRPP 28/10	Horse	Taciba	V-3	D. rotundus
HQ666864	LRPP 43/10	Neoeptesicus diminutus *	Osvaldo Cruz	V-3	D. rotundus
HQ666865	LRU 60/10	Neoeptesicus furinalis *	Birigui	NC1	Insect. Bats
HQ666866	LRU 76/10	Neoeptesicus furinalis *	Penápolis	NC1	Insect. Bats
HQ666867	LRU 169/10	Molossus molossus	Araçatuba	NC2	Insect. Bats
HQ666868	LRU 171/10	Cat	Araçatuba	V-3	D. rotundus
HQ666869	LRU 177/10	Molossus fluminensis *	Birigui	NC2	Insect. Bats
HQ666870	LRPP 198/10	Lasiurus blossevillii	Teodoro Sampaio	NC2	Insect. Bats
HQ666871	LRU 299/10	Neoeptesicus furinalis *	Penápolis	NC1	Insect. Bats
HQ666872	LRU 300/10	Molossus fluminensis *	Penápolis	V-3	D. rotundus

ND = not done; NC = not compatible; NH = non-hematophagous; V-3 = variant 3. IB—Rabies Laboratory of “Instituto Biológico de São Paulo”; LRU—Rabies Laboratory of UNESP (São Paulo State University), Araçatuba; LRPP—Rabies Laboratory of APTA (São Paulo Agribusiness Technology Agency) of Presidente Prudente; * new taxonomy bat species classification according to [29,30,31].

2.2. Antigenic Characterization

Antigenic characterization was performed using the CDC (Atlanta, GA, USA) monoclonal antibodies (MAbs) panel according to the protocol determined by Favoretto et al. [11]. These eight MAbs against the RABV nucleoprotein, developed by CDC, can identify different RABV variants through different reactivity patterns.

2.3. Sequencing and Genetic Characterization

For genetic characterization, the initial steps (i.e., extracting RNA and obtaining cDNA) were performed using methods described in previous studies [13]. Molecular reactions were performed using primers described previously by Smith et al. [32] and Campos et al. [33] to amplify 320 base pairs from the coding and non-coding region of the nucleoprotein (between positions 1157 and 1476 of PV-NC_001542). Double-strand PCR-amplified products were purified using the ExoSAP-IT system (GE Healthcare Bio-Sciences Ltd.—USB Corporation, Cleveland, OH, USA) according to the manufacturer’s instructions, and Sanger sequencing was performed as previously described by Campos et al. [33]. The excess dideoxynucleotide terminators were removed with the Applied Biosystems Big Dye XTerminatorTM Purification Kit (Applied Biosystems, Foster City, CA, USA), following the manufacturer’s recommendations. Purified samples were subjected to electrophoresis in POP6 polymer using an ABI-PRISM model 3100 automatic sequencer (Applied Biosystems, Foster City, CA, USA). The samples were tracked automatically using the Automatic DNA Analyzer software package of the ABI-PRISM model 3100.

2.4. Phylogenetic Analysis

The obtained nucleotide sequences were pre-analyzed using the BLASTn program (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 19 February 2025) to confirm amplification of the specific product and then aligned with available GenBank sequences (Appendix A) using Geneious Prime software version 2019.2.3. The chosen GenBank sequences were selected based on full information like the host, place, and year of collection. Pairwise distances were calculated by MEGA 11 version 11.0.13 (available at https://www.megasoftware.net/ accessed on 19 February 2025) and phylogenetic trees were reconstructed using IQTREE software version 2.4.0 (available at http://www.iqtree.org/ accessed on 18 February 2025). The best model fit determined by IQTree was TIM + F + I + G4. To analyze the temporal virus variability, we used TempEst v1.5.3 (available at http://tree.bio.ed.ac.uk/software/tempest/ accessed on 19 February 2025) in the phylogenetic tree and root-to-tip method with best-fitting root in correlation function. The time-scaled phylogenetic tree was analyzed via Augur version 21.1.0 and auspice version 2.62.0 implemented on Nextstrain [34,35].

2.5. Phylogenetic and Antigenic Site Amino Acid Visualization

Phylogenetic visualization of RABV sequences was conducted using R version 4.4.1 (14 June 2024, ucrt). A phylogenetic tree in nexus format was imported using the ape package and further processed and visualized with the ggtree, treeio, and igraph packages. The final annotated phylogenetic tree was visualized with tip points colored with regard to geographic location and a scale bar indicating time (years) and genetic distance (substitutions/site). The final figure editing and layout adjustments were performed using the Inkscape program version 1.3.2. The alignment used for phylogenetic tree reconstruction was translated to amino acid and used to prepare one Figure with the region of antigenic site I present in the nucleoprotein.

3. Results

Among the fifty-one samples submitted to antigenic characterization, twenty-five (49%) were characterized as AgV-3 (RABV maintained by D. rotundus hematophagous bat populations) and twenty-six were characterized as RABV maintained by non-hematophagous bat populations (NC1 and NC2); out of these, twenty-one (41.2%) presented the antigenic profile NC1 and five (9.8%) presented the antigenic profile NC2.

The samples from this study were segregated into five different phylogenetic groups, highlighted in colors according to their genetic and antigenic patterns (Figure 2). The group called Group I showed samples with a genetic lineage associated with the virus maintained by D. rotundus hematophagous bats and antigenic variant AgV3. The virus groups isolated from insectivorous bats presented four independent phylogenetic clades, called Group II, Group III, Group IV, and Group V, with the antigenic profile NC1 in Groups II and III, and antigenic profile NC2 in Groups IV and V. Group II presented the greatest diversity of its host species, consisting of Eptesicus spp. (currently called Neoeptesicus), Eumops spp., Myotis spp., Nyctinomops spp., and Lasiurus spp. Groups VI and VII, with isolates external to the present study, presented isolates from marmosets (Group VI) and bat isolates related to Tadarida brasiliensis (Group VII).

Figure 2.

Time-scaled phylogenetic tree reconstructed using 320 nucleotides from nucleoprotein terminal gene using IQ-TREE software version 2.4.0, visualized and edited using FigTree software version 1.4.4 and plotted in RStudio version 2024.12.1+563 using ggtree version 3.14.0, treeio version 1.30.0, and igraph version 2.1.4 packages. The samples from this study can be observed in the tree in red dots; other samples from Brazil, available in GenBank, are marked in a blue pallet of colors while samples from other countries are marked in a green pallet of colors. The groups determined in this study are delineated vertically by red (Group I), orange (Groups II and III), and dark blue (Groups IV and V) shading. The groups without segregated samples from this study are shown with pink (Group VI) and gray (Group VII) shadings.

The lowest percentage of identity was observed in comparison with the clade related to marmosets (Group VI in the phylogenetic tree). Estimates of evolutionary divergence over sequence pairs between groups, obtained using the maximum likelihood method in MEGA 11, were used to calculate the distances between groups (Table A2, Appendix A), showing a range from 8.7% between Groups I and VII to 17.9% between Groups II and VI. Among the groups detected in this study, the highest within-group distances (Table A3, Appendix A) were observed in Groups II (6.7%), III (4.7%), and V (5.7%) while minor within-group distances were detected in Groups IV (1.8%) and I (2.5%).

In Figure 1, we can also observe the geographic distribution of the samples from this study and from Genbank, used for the reconstruction of the phylogenetic tree, according to the resulting groups (genetic characterization) and antigenic variants/profiles (AgV-3, NC1, and NC2).

During the analysis of the antigenic site present in the nucleoprotein, we identified genetic signatures for some groups in the phylogenetic tree. Groups I, IV, V, VI, and VII and the root group showed recognized patterns (AET, AEV, TEV, TEA, TEM, and TDV, respectively), indicating the stability of these genetic groups. On the other hand, we could not identify any pattern for Groups II (TEA, TDE, IDT, TEV, and TDV) and III (TEA, TEL, and TEV). In these groups, we found higher variability that was confirmed by tree topology and can be seen in Figure 3. The full map of the antigenic site was produced and can be accessed in the Supplementary Materials.

Figure 3.

A partial amino-acid alignment showing the presence of antigenic site I in the nucleoprotein. The color match those in the phylogenetic tree presented in Figure 2. For this figure, the sequences used in the phylogenetic tree reconstruction were employed, maintaining only the variability in the antigenic site region, with a preference for sequences from this study. The genetic signature AET is shown in red (and also highlighted with red shading) for phylogenetic Group I related to hematophagous bat species D. rotundus; AEV is shown in dark blue (and also highlighted with dark blue shading) for phylogenetic Group IV related to non-hematophagous bats; TEV is shown in a gradient of dark blue (and also highlighted in a gradient dark blue shading) for phylogenetic Group V; TEA is shown in magenta/pink (and also highlighted with pink shading) for phylogenetic Group VI related to marmosets; TEM in black (and also highlighted with gray shading) for phylogenetic Group VII related to the bat species T. brasiliensis; TDV is shown in green for the root group related to the terrestrial cycle of transmission of RABV. For phylogenetic groups II and III (highlighted with gradient orange shading), it was not possible to find one genetic signature in the antigenic site I; in fact, in these groups, the variability was diverse and is noted in different colors: TEA in orange for the major antigenic site found (which was the same genetic signature found in Group VI related to marmosets), purple for variations (TEL, TDE, IDT), dark blue for the same signature present in Group V (TEV), and green for the same signature present in the terrestrial cycle of transmission (TDV) in one sequence available at GenBank (AF396064). The amino acid letters and class are outlined above.

The Pearson correlation coefficient calculated in the analysis of temporal virus variability in this dataset was 0.18 (p = 0.015), as shown in Figure 4. Although the correlation coefficient is slightly positive, the TempEst analysis showed the stable evolution rate to the dataset during the sampling period considered in the analysis.

Figure 4.

Correlation between genetic divergence and sampling time was obtained by a root-to-tip analysis using the RABV sequences and plotted using R command line. The dots are related with clades from phylogenetic tree, the red dots are related with Group I, the orange with Group II, the brown with Group III, the blue with Group IV, the clear blue with Group V, the pink with Group VI, the clear gray with the Group VII and the dark gray dots are related with the terrestrial cycle of transmission. The dashed line (colored dark gray) represents the regression line, and the light gray area around the dashed line is the confidence interval.

4. Discussion

All 13 fruit bats of the genus Artibeus presented antigenic variant 3 (AgV-3), as did bats from the genera Molossus (04), Eumops (1), and Neoeptesicus (2) and non-identified bats (2). Similar results were observed in rabies-positive bat samples from other regions of São Paulo State [11,15,19,24,36]. Previous studies had demonstrated that the frequency of RABV in Artibeus was higher than that in Desmodus bats in the study area [36,37,38,39], which could explain how AgV-3 is present in species that do not co-inhabit with D. rotundus species. This is corroborated by the finding that the genetic lineage of D. rotundus is not exclusive to the species since this lineage has been detected in non-hematophagous bats such as the fruit bat Artibeus lituratus [40] and insectivorous bats in this study, in addition to other previous studies [13].

The samples antigenically described as NC1 in phylogenetic group II were segregated with samples from the same geographic region and with one sample (EU981922) from Uruguay, with a geographical distance of more than 1,200 miles. The samples in the phylogenetic group III were segregated with samples from the same geographical region and with one sample (AB297647) from Rio de Janeiro State, more than 300 m away, and another sample (AB618034) from Paraiba State, more than 1600 m away. This antigenic profile was previously described in São Paulo State [11,12,13,14,15,41]. These groups were previously related particularly with host species described by Oliveira et al. [42]; nonetheless, in the present study, we observed different species in the same clade/phylogenetic group. Bats play an important role in virus transmission and spread in the Americas [42,43,44], and it was clearly demonstrated in this study that insectivorous bats present a heterogeneous genetic distribution independent of host species.

The NC2 antigenic profile detected in five samples from the Molossus and Lasiurus genera was previously described in the same geographical region [7] and was observed in three monophyletic clades with high bootstrap value support (81 to 98%). In a previous study [13], two samples (GU646777 and HM854031) were segregated independently as subgroups; in the present study, these samples were segregated as part of phylogenetic groups IV and V, confirming the importance of including more sequences and information from other regions of the state and the country, as well as from other bat host species. This was also visualized on the map (Figure 1), where the geographical distribution of the variants was described. With the inclusion of new samples, a more homogeneous distribution of antigenic profiles and genetic lineages across Sao Paulo State can be observed since, previously, these profiles and lineages were restricted to the northwest region of the state [13]. Nonetheless, important data from previous publications regarding the central region of this state and from other states of Brazil could not be compared with the isolates from this study, considering that the authors sequenced a different genome region or only a coding region [15,40,42,45,46,47]. This reinforces the idea that the same region of the genome should be analyzed and standardized by researchers in future studies.

Currently, the term “antigenic variants” and the CDC MAbs panel still are used mainly in Latin America and only in a few accredited laboratories. The results obtained in the present sample’s dataset corroborated previous results [13,15] that showed that this panel, despite its valuable importance in the past, does not have sufficient resolution as high as that obtained using genetic tools to characterize RABV variants from non-hematophagous bats, leading to divergences. An approach of comparing samples from different geographical regions using antigenic and genetic characterization is no longer ideal, as previously highlighted in other studies [15]. In any case, the term ‘antigenic variant’ will not become extinct immediately since it is still the language used in Ministry of Health reports in Brazil, for example. As found in this study, the antigenic characterization was realized during sample processing almost twenty years ago, providing useful information that could be used for future studies and to better understand rabies epidemiology. Therefore, future RABV studies must be focused on genetic analyses to provide a deeper and more comprehensive understanding of the virus, explaining its epidemiology, its dynamics, and possible interventions. This could lead to significant advances in rabies surveillance, prevention, and control at both population and individual levels.

Antigenic site I was presented here using nucleoprotein amino acid alignments, and the authors observed a genetic signature that had previously been described [6]. However, for genetic groups II and III, these signatures could not be observed; in fact, they are genetically and antigenically diverse, independent of the comparison between these two approaches.

In the first RABV genetic studies in Latin America, it was established that the 320 nucleotides in the nucleoprotein carboxi-terminus region, including the nucleo- and phosphoprotein intergenic regions (non-coding) between genome positions 1157 and 1476 (based in PV genome NC_001542), could be the standard for phylogenetic studies because this region presents the large nucleotide variability, 1.9 times greater than in the coding region [17]. For phylogenetic analyses, according to Smith et al. [46], groups that present a distance higher than 5% from other groups can be considered a distinct genetic lineage. Thus, for this study, the groups in the phylogenetic tree were determined by following this consideration.

In the phylogenetic tree (Figure 3 and Figure S1), the high bootstrap values (100% for aerial cycle of transmission; 78% for genetic lineages segregated into Groups I, II, III and VII; and 81% for Groups I, II and VII) support the presence of basal genotypes of the virus. For example, sample HQ666860 from an insectivorous bat, Neoeptesicus furinalis, collected in 2009 presented a long branch in Group II (bootstrap 81%), indicating a high number of nucleotide substitutions. Group I, Group III, Group IV, and Group V showed bootstrap values of 100%, 98%, 78% and 92%, respectively; this tree topology could also explain how and why samples in the antigenic analysis from genetic groups II to V presented a different antigenic profile.

The positive but low correlation coefficient (0.18) associated with the phylogenetic analysis suggests that during the short period of sequence sampling, between the years of 1986 and 2022, there was an accumulation of diversity, but the occurrence of the common ancestor to all sequences was distant in the past. This had been previously shown by other researchers [42], and this result means a stable RABV evolution rate in the analyzed period. This result reflects the profile of zoonotic viruses such as rabies, considering only the aerial cycle, and agrees with the TMRCA (time to the most recent common ancestor) of approximately 170 years determined by de Souza et al. [40] when analyzing the D. rotundus/A. lituratus genetic lineage (both antigenically AgV3).

5. Conclusions

Despite some limitations, such as analyzing only a 320-nucleotide fragment; the limited number of sequences available in GenBank for this same fragment; the absence of relevant information such as on the date, species, and collection location of these available sequences in the GenBank; and the retrospective nature of this study performed over a decade ago, these data provide valuable insights into RABV among bats. The key findings of this study are as follows: (i) antigenic profiles and genetic lineages previously restricted to the northwest region of the state of São Paulo are now found in other state regions, (ii) future rabies studies must be focused only on genetic analysis, and (iii) there is a need for a comprehensive genetic study of bat rabies in São Paulo State and greater Brazil with diverse sample locations and expanded genomic analyses using a standard genomic fragment or full genome when possible. Moreover, focusing only on host species could lead to misleading conclusions about RABV evolution and dispersal concerning time and geography.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/v17081063/s1, Figure S1: Conventional phylogenetic tree, Figure S2: Partial amino acid antigenic site I for all sequences used in phylogenetic tree reconstruction.

Appendix A

Table A1.

Bat sample sequences used in this work [47,48,49,50,51,52,53,54].

Sequence ID	Group	Year	Host	Site Origin	Country	Genbank Access	Reference
AB201802_BR_AL4	I	2002	Artibeus lituratus	Dracena, SP	Brazil	AB201802	[24]
AB201803_BR_DR1	I	2000	Desmodus rotundus	Lindóia, SP	Brazil	AB201803	[24]
AB201805_BR_DR3	I	2001	Desmodus rotundus	São José do Barreiro, SP	Brazil	AB201805	[24]
AB201806_BR_NL1	III	1998	Nyctinomops laticaudatus	São José do Rio Preto, SP	Brazil	AB201806	[24]
AB201807_BR_NL2	II	1999	Nyctinomops laticaudatus	São José do Rio Preto, SP	Brazil	AB201807	[24]
AB201808_BR_NL3	III	2001	Nyctinomops laticaudatus	Nova Granada, SP	Brazil	AB201808	[24]
AB201812_BR_EF2	II	2001	Eptesicus furinalis	Olimpia, SP	Brazil	AB201812	[24]
AB201813_BR_EF3	II	2001	Eptesicus furinalis	São José do Rio Preto, SP	Brazil	AB201813	[24]
AB201814_BR_EF4	II	2002	Eptesicus furinalis	Catanduva, SP	Brazil	AB201814	[24]
AB201815_BR_MM1	IV	1999	Molossus molossus	Jales, SP	Brazil	AB201815	[24]
AB201816_BR_MM2	IV	2002	Molossus molossus	Ilha Solteira, SP	Brazil	AB201816	[24]
AB201817_BR_MR1	I	2002	Molossus rufus	Presidente Venceslau, SP	Brazil	AB201817	[24]
AB201818_BR_MA1	IV	2000	Molossus abrasus	Itapira, SP	Brazil	AB201818	[24]
AB297630_BR_AL6	I	2001	Artibeus lituratus	Rio de Janeiro, RJ	Brazil	AB297630	[48]
AB297631_BR_AL7	I	2004	Artibeus lituratus	Vargem Grande Paulista, SP	Brazil	AB297631	[48]
AB297647_BR_NL4	III	2004	Nyctinomops laticaudatus	Rio de Janeiro, RJ	Brazil	AB297647	[48]
AB618034_strain_MPVI	III	2007	Molossus molossus	Santo Antonio, Paraíba	Brazil	AB618034	Unpublished
AF394886_2085	V	1986	Lasiurus borealis	Walker County, Texas	USA	AF394886	[49]
AF396064_cym3941_1995	II	1995	Myotis chiloensis	-	Chile	AF396064	[50]
AY233427_Batbbt123	VII	2001	Tadarida brasiliensis	Buenos Aires	Argentine	AY233427	[44]
AY233448_Stchmbt80	III	2000	Histiotus montanus	Rio Turbio, Santa Cruz	Argentine	AY233448	[44]
AY233451_Batbbt125	V	2001	Tadarida brasiliensis	Buenos Aires	Argentine	AY233451	[44]
AY654585_Brhm4097	VI	1998	Human	Ceará	Brazil	AY654585	[6]
AY654586_Brsg4108	VI	1998	Callithrix jacchus jacchus	Ceará	Brazil	AY654586	[6]
AY654587_Brhm4138	VI	1998	Human	Ceará	Brazil	AY654587	[6]
AY877435_V920	I	1993	Bovine	Chiapas	Mexico	AY877435	[51]
DQ631835_bref8150_05	III	2005	Eptesicus furinalis	Jundiaí, SP	Brazil	DQ631835	[41]
EF363743_IP306_Portel_PA_2004	I	2004	Human	Portel, PA	Brazil	EF363743	[52]
EF363751_IP5214Viseu_PA_2004	I	2004	Human	Viseu, PA	Brazil	EF363751	[52]
EF363757_IP7541Viseu_PA_2005	I	2005	Human	Viseu, PA	Brazil	EF363757	[52]
EU293113_9001FRA	I	1990	Fox	-	French Guyana	EU293113	[53]
EU293116_9704ARG	VII	1997	bat	-	Argentine	EU293116	[53]
EU873001_brctSP4551_96	III	1996	cat	São Paulo State	Brazil	EU873001	Unpublished, Favoretto
EU981922_IP6773U_2008	II	2008	Myotis sp.	-	Uruguay	EU981922	[43]
GU552788_IP2989_2007	VII	2007	Nyctnomops laticaudatus	Joanópolis, SP	Brazil	GU552788	[25]
GU552789_IP1779_2006	IV	2006	Molossus rufus	Ribeirão Preto, SP	Brazil	GU552789	[25]
GU552790_IP1992_2005	III	2005	Histiotus velatus	Vargem Grande Paulista, SP	Brazil	GU552790	[25]
GU552791_IP6883_2006	III	2006	Histiotus velatus	Campo Limpo Paulista, SP	Brazil	GU552791	[25]
GU552792_IP3321_2005	III	2005	Histiotus sp.	Belo Horizonte, MG	Brazil	GU552792	[25]
GU552795_IP10529_2005	III	2005	Nyctnomops laticaudatus	Ribeirão Preto, SP	Brazil	GU552795	[25]
GU552796_IP4359_2007	III	2007	Molossus molossus	Campinas, SP	Brazil	GU552796	[25]
GU552798_IP8089_2005	III	2005	Nyctnomops laticaudatus	São Sebastião, SP	Brazil	GU552798	[25]
GU552807_IP8061_2006	II	2006	Eptesicus furinalis	Campinas, SP	Brazil	GU552807	[25]
GU552810_IP3056_2007	II	2007	Eptesicus furinalis	Barretos, SP	Brazil	GU552810	[25]
GU552815_IP8665_2005	II	2005	Myotis nigricans	Ribeirão Preto, SP	Brazil	GU552815	[25]
GU552820_IP4157_2005	II	2005	Myotis nigricans	Águas de Lindóia, SP	Brazil	GU552820	[25]
GU552821_IP4896_2005	II	2005	Myotis nigricans	Caçapava, SP	Brazil	GU552821	[25]
GU552824_IP2654_2006	V	2006	Lasiurus cinereus	Garça, SP	Brazil	GU552824	[25]
GU592648_brdrusp100_07	I	2007	Desmodus rotundus	São José do Barreiro, SP	Brazil	GU592648	[33]
GU646775_brmn131_03	II	2003	Myotis nigricans	Araçatuba, SP	Brazil	GU646775	[13]
GU646776_brmn45_03	II	2003	Myotis nigricans	Araçatuba, SP	Brazil	GU646776	[13]
GU646777_brmm95_03	V	2003	Molossus molossus	Nova Independência, SP	Brazil	GU646777	[13]
GU646778_brmn38_03	II	2003	Myotis nigricans	Penápolis, SP	Brazil	GU646778	[13]
GU646779_bral268_98	I	1998	Artibeus lituratus	Mirandópolis, SP	Brazil	GU646779	[13]
GU646780_bral452_99	I	1999	Artibeus lituratus	Ilha Solteira, SP	Brazil	GU646780	[13]
GU646781_bref431_04	II	2004	Eptesicus furinalis	Bilac, SP	Brazil	GU646781	[13]
GU646782_brlb46_04	II	2004	Lasiurus blossevillii	Valparaíso, SP	Brazil	GU646782	[13]
GU646783_bral311_03	I	2003	Artibeus lituratus	Penápolis, SP	Brazil	GU646783	[13]
GU646784_bral304_03	I	2003	Artibeus lituratus	Penápolis, SP	Brazil	GU646784	[13]
GU646785_brmn150_03	II	2003	Myotis nigricans	Araçatuba, SP	Brazil	GU646785	[13]
GU646786_brmn234_02	II	2002	Myotis nigricans	Bilac, SP	Brazil	GU646786	[13]
GU646787_bral625_01	I	2001	Artibeus lituratus	Birigui, SP	Brazil	GU646787	[13]
GU646788_brmn610_01	II	2001	Myotis nigricans	Sud Menucci, SP	Brazil	GU646788	[13]
GU646789_bral499_01	I	2001	Artibeus lituratus	Guararapes, SP	Brazil	GU646789	[13]
GU646790_bref126_01	II	2001	Eptesicus furinalis	Penápolis, SP	Brazil	GU646790	[13]
GU646791_bral84_01	I	2001	Artibeus lituratus	Guararapes, SP	Brazil	GU646791	[13]
GU646792_bref40_01	II	2001	Epitesicus furinalis	Bilac, SP	Brazil	GU646792	[13]
GU646793_breg01_01	II	2001	Eumops glaucinus	Bilac, SP	Brazil	GU646793	[13]
GU646794_brmn839_00	II	2000	Myotis nigricans	Bilac, SP	Brazil	GU646794	[13]
GU646795_bral566_00	I	2000	Artibeus lituratus	Penápolis, SP	Brazil	GU646795	[13]
GU646796_bref213_00	II	2000	Eptesicus furinalis	Araçatuba, SP	Brazil	GU646796	[13]
GU646818_brbv119_03	I	2003	Bovine/Cattle	José Bonifácio, SP	Brazil	GU646818	[13]
GU646828_brbv356_97	I	1997	Bovine/Cattle	Guararapes, SP	Brazil	GU646828	[13]
GU646833_brbv32_94	T	1994	Bovine/Cattle	Araçatuba, SP	Brazil	GU646833	[13]
GU646835_brdg70_93	T	1993	Dog	Araçatuba, SP	Brazil	GU646835	[13]
GU646841_brhr308_00	I	2000	Horse	Barbosa, SP	Brazil	GU646841	[13]
GU646842_bral5341	I	1999	Artibeus lituratus	Birigui, SP	Brazil	GU646842	[13]
GU646843_brmm4105	I	1998	Molossus molossus	Mirandópolis, SP	Brazil	GU646843	[13]
GU646844_brle4132	I	1988	Lasiurus ega	Glicério, SP	Brazil	GU646844	[13]
GU646845_brmr4114	I	1998	Molossus molossus	Penápolis, SP	Brazil	GU646845	[13]
GU646846_brmr4095	I	1998	Molossus rufus	Araçatuba, SP	Brazil	GU646846	[13]
GU646849_brdg5356	I	2000	Dog	Ilha Solteira, SP	Brazil	GU646849	[13]
GU646855_brct60_92	T	1992	Cat	Andradina, SP	Brazil	GU646855	[13]
GU646856_brmr298_07	I	2007	Molossus rufus	Ilha Solteira, SP	Brazil	GU646856	[13]
GU646857_brmn182_07	II	2007	Myotis nigricans	Bilac, SP	Brazil	GU646857	[13]
GU646858_brmr350_06	I	2006	Molossus rufus	Andradina, SP	Brazil	GU646858	[13]
GU646859_bral309_06	I	2006	Artibeus lituratus	Ilha Solteira, SP	Brazil	GU646859	[13]
GU646860_bral195_06	I	2006	Artibeus lituratus	Araçatuba, SP	Brazil	GU646860	[13]
GU646861_bref341_02	II	2002	Epitesicus furinalis	Birigui, SP	Brazil	GU646861	[13]
HM173087_brmn100_05	II	2005	Myotis nigricans	Guararapes, SP	Brazil	HM173087	[13]
HM173088_bral239_05	I	2005	Artibeus lituratus	Birigui, SP	Brazil	HM173088	[13]
HM854029_brdg354_95	I	1995	Dog	Birigui, SP	Brazil	HM854029	[13]
HM854030_brct100_04	I	2004	Cat	Guararapes, SP	Brazil	HM854030	[13]
HM854031_brmr178_05	IV	2005	Molossus rufus	Andradina, SP	Brazil	HM854031	[13]
HM854032_brmn391_05	II	2005	Myotis nigricans	Ilha Solteira, SP	Brazil	HM854032	[13]
HM854033_bral540_05	I	2005	Artibeus lituratus	Araçatuba, SP	Brazil	HM854033	[13]
HM014315_brmm1994_08	V	2008	Molossus molossus	São Paulo, SP	Brazil	HM014315	[14]
HM014316_brmng2449_05	III	2005	Molossops neglectus	São Paulo, SP	Brazil	HM014316	[14]
HM014317_brmr6464_05	II	2005	Myotis riparius	São Paulo, SP	Brazil	HM014317	[14]
HQ666824_bral346_99	I	1999	Artibeus lituratus	São José do Rio Preto, SP	Brazil	HQ666824	This work
HQ666825_brma777_00	IV	2000	Cynomops abrasus	Ipiguá, SP	Brazil	HQ666825	This work
HQ666826_brnl249_01	III	2001	Nyctinomops macrotis	São José do Rio Preto, SP	Brazil	HQ666826	This work
HQ666827_brnl250_01	III	2001	Nyctinomops laticaudatus	São José do Rio Preto, SP	Brazil	HQ666827	This work
HQ666828_bref636_01	II	2001	Neoepitesicus furinalis	Olímpia, SP	Brazil	HQ666828	This work
HQ666829_bral808_01	I	2001	Artibeus lituratus	São José do Rio Preto, SP	Brazil	HQ666829	This work
HQ666830_bresp1019_01	II	2001	Neoepitesicus sp.	Cardoso, SP	Brazil	HQ666830	This work
HQ666831_bref1070_01	II	2001	Neoepitesicus furinalis	São José do Rio Preto, SP	Brazil	HQ666831	This work
HQ666832_bref62_02	II	2002	Neoepitesicus furinalis	Catanduva, SP	Brazil	HQ666832	This work
HQ666833_brmm109_02	IV	2002	Molossus molossus	Ilha Solteira, SP	Brazil	HQ666833	This work
HQ666834_brmn835_02	II	2002	Myotis nigricans	Cajobi, SP	Brazil	HQ666834	This work
HQ666835_bral992_02	I	2002	Artibeus lituratus	Dracena, SP	Brazil	HQ666835	This work
HQ666836_brmr1021_02	I	2002	Molossus fluminensis	Presidente Venceslau, SP	Brazil	HQ666836	This work
HQ666837_bref1141_02	II	2002	Neoepitesicus furinalis	Santo Anastácio, SP	Brazil	HQ666837	This work
HQ666838_brbat1256_02	I	2002	Non- hematophagous bat	Martinópolis, SP	Brazil	HQ666838	This work
HQ666839_bral1371_02	I	2002	Artibeus lituratus	Presidente Venceslau, SP	Brazil	HQ666839	This work
HQ666840_bral1535_02	I	2002	Artibeus lituratus	Taciba, SP	Brazil	HQ666840	This work
HQ666841_brmm1539_02	I	2002	Molossus molossus	Presidente Prudente, SP	Brazil	HQ666841	This work
HQ666842_brle1782_02	III	2002	Lasiurus ega	Presidente Prudente, SP	Brazil	HQ666842	This work
HQ666843_braj349_03	I	2003	Artibeus planirostris	Santa Fé do Sul, SP	Brazil	HQ666843	This work
HQ666844_brbat350_03	I	2003	Non-hematophagous bat	Catanduva, SP	Brazil	HQ666844	This work
HQ666845_bral791_03	I	2003	Artibeus lituratus	Presidente Prudente, SP	Brazil	HQ666845	This work
HQ666846_brmn168_04	II	2004	Myotis nigricans	Campinas, SP	Brazil	HQ666846	This work
HQ666847_brnl184_04	III	2004	Nyctinomops laticaudatus	São José do Rio Preto, SP	Brazil	HQ666847	This work
HQ666848_bral550_04	I	2004	Artibeus lituratus	Caçapava, SP	Brazil	HQ666848	This work
HQ666849_breg329_05	I	2005	Eumops glaucinus	Araçatuba, SP	Brazil	HQ666849	This work
HQ666850_bred43_09	II	2009	Neoepitesicus diminutus	Pereira Barreto, SP	Brazil	HQ666850	This work
HQ666851_bred84_09	II	2009	Neoepitesicus diminutus	Araçatuba, SP	Brazil	HQ666851	This work
HQ666852_brmn149_09	II	2009	Myotis nigricans	Coroados, SP	Brazil	HQ666852	This work
HQ666853_bral181_09	I	2009	Artibeus lituratus	Penápolis, SP	Brazil	HQ666853	This work
HQ666854_bref199_09	II	2009	Neoeptesicus furinalis	Dracena, SP	Brazil	HQ666854	This work
HQ666855_bref224_09	I	2009	Neoeptesicus furinalis	Parapuã, SP	Brazil	HQ666855	This work
HQ666856_bral325_09	I	2009	Artibeus lituratus	Birigui, SP	Brazil	HQ666856	This work
HQ666857_bral374_09	I	2009	Artibeus lituratus	Guararapes, SP	Brazil	HQ666857	This work
HQ666858_brmr389_09	I	2009	Molossus fluminensis	Guararapes, SP	Brazil	HQ666858	This work
HQ666859_brmn433_09	II	2009	Myotis nigricans	Penápolis, SP	Brazil	HQ666859	This work
HQ666860_bref589_09	II	2009	Neoeptesicus furinalis	Penápolis, SP	Brazil	HQ666860	This work
HQ666861_brbv672_09	I	2009	Bovine	Narandiba, SP	Brazil	HQ666861	This work
HQ666862_brmr17_10	II	2010	Molossus fluminensis	Penápolis, SP	Brazil	HQ666862	This work
HQ666863_breq28_10	I	2010	Equine	Taciba, SP	Brazil	HQ666863	This work
HQ666864_bred43_10	I	2010	Neoeptesicus diminutus	Osvaldo Cruz, SP	Brazil	HQ666864	This work
HQ666865_bref60_10	II	2010	Neoeptesicus furinalis	Birigui, SP	Brazil	HQ666865	This work
HQ666866_bref76_10	II	2010	Neoeptesicus furinalis	Penápolis, SP	Brazil	HQ666866	This work
HQ666867_brmm169_10	V	2010	Molossus molossus	Araçatuba, SP	Brazil	HQ666867	This work
HQ666868_brct171_10	I	2010	Feline	Araçatuba, SP	Brazil	HQ666868	This work
HQ666869_brmr177_10	V	2010	Molossus fluminensis	Birigui, SP	Brazil	HQ666869	This work
HQ666870_brlbl198_10	V	2010	Lasiurus blossevillii	Teodoro Sampaio, SP	Brazil	HQ666870	This work
HQ666871_bref299_10	II	2010	Neoeptesicus furinalis	Penápolis, SP	Brazil	HQ666871	This work
HQ666872_brmr300_10	I	2010	Molossus fluminensis	Penápolis, SP	Brazil	HQ666872	This work
JF916647_BRLB4096_1995	V	1995	Lasiurus blossevillii	Unknown	Brazil	BRLB4096	Unpublished, Favoretto
JF916650_brmyaSP4115_1998	II	1998	Myotis albecens	Unknown	Brazil	JF916650	Unpublished, Favoretto
JF916652_bralusp041_05	I	2005	Artibeus lituratus	Presidente Prudente, SP	Brazil	JF916652	[26]
JF916655_brlbusp040_07	V	2007	Lasiurus blossevillii	Presidente Prudente, SP	Brazil	JF916655	[26]
JF916656_bralusp042_07	I	2007	Artibeus lituratus	Presidente Prudente, SP	Brazil	JF916656	[26]
JF916657_brmmusp043_07	I	2007	Molossus molossus	Presidente Prudente, SP	Brazil	JF916657	[26]
JF916659_bralusp047_07	I	2007	Artibeus lituratus	Presidente Prudente, SP	Brazil	JF916659	[26]
JF916661_bralusp049_07	I	2007	Artibeus lituratus	Presidente Prudente, SP	Brazil	JF916661	[26]
JF916662_bralusp050_07	I	2007	Artibeus lituratus	Presidente Prudente, SP	Brazil	JF916662	[26]
JF916663_bralusp052_07	I	2007	Artibeus lituratus	Presidente Prudente, SP	Brazil	JF916663	[26]
JF916664_bralusp054_07	VII	2007	Artibeus lituratus	Presidente Prudente, SP	Brazil	JF916664	[26]
JF916667_brmnusp058_07	II	2007	Myotis nigricans	Presidente Prudente, SP	Brazil	JF916667	[26]
JF916668_brmnusp061_07	II	2007	Myotis nigricans	Presidente Prudente, SP	Brazil	JF916668	[26]
JF916669_bralusp062_07	I	2007	Artibeus lituratus	Presidente Prudente, SP	Brazil	JF916669	[26]
JF916671_brefusp064_07	II	2007	Eptesicus furinalis	Presidente Prudente, SP	Brazil	JF916671	[26]
JF916672_bralusp069_07	I	2007	Artibeus lituratus	Presidente Prudente, SP	Brazil	JF916672	[26]
JF916673_bralusp071_07	I	2007	Artibeus lituratus	Presidente Prudente, SP	Brazil	JF916673	[26]
JF916674_bralusp001_08	I	2008	Artibeus lituratus	Presidente Prudente, SP	Brazil	JF916674	[26]
JF916678_brefusp008_09	II	2009	Eptesicus furinalis	Presidente Prudente, SP	Brazil	JF916678	[26]
KM594026_IP_512_09	II	2009	Eptesicus furinalis	Ribeirão Preto, SP	Brazil	KM594026	[42]
KM594027_IP_230_10	II	2010	Eptesicus furinalis	Valinhos, SP	Brazil	KM594027	[42]
KM594028_IP_346_10	II	2010	Eptesicus furinalis	Tambaú, SP	Brazil	KM594028	[42]
KM594029_IP_3208_06	III	2006	Eptesicus furinalis	Vinhedo, SP	Brazil	KM594029	[42]
KM594030_IP_1400_10	II	2010	Myotis nigricans	Campinas, SP	Brazil	KM594030	[42]
KM594031_IP_163_10	II	2010	Myotis nigricans	Caieiras, SP	Brazil	KM594031	[42]
KM594032_IP_497_10	II	2010	Myotis nigricans	Campinas, SP	Brazil	KM594032	[42]
KM594034_IP_350_10	III	2010	Nyctinomops laticaudatus	Conchal, SP	Brazil	KM594034	[42]
KM594035_IP_412_10	III	2010	Nyctinomops laticaudatus	Barretos, SP	Brazil	KM594035	[42]
KM594036_IP_542_10	III	2010	Nyctinomops laticaudatus	Ribeirão Preto, SP	Brazil	KM594036	[42]
KM594037_IP_3176_09	VII	2009	Tadarida brasiliensis	Santo André, SP	Brazil	KM594037	[42]
KM594038_IP_1586_10	VII	2010	Tadarida brasiliensis	São Bernardo do Campo, SP	Brazil	KM594038	[42]
MG458314_RV1789	I	1997	Cow	Unknown	British Est Indies	MG458314	[54]
PQ671596_RS61	VII	2022	Tadarida brasiliensis	Rio Grande do Sul	Brazil	PQ671596	unpublished

Legend: samples in red are from this study; samples in blue are from our group’s previous study [13].

Table A2.

Estimates of evolutionary divergence over sequence pairs between groups.

	Gp_1	Gp_3	Gp_2	Gp_4	Gp_5	Gp_7	Gp_6
Gp_1
Gp_3	0.140
Gp_2	0.128	0.126
Gp_4	0.139	0.103	0.138
Gp_5	0.152	0.134	0.160	0.132
Gp_7	0.087	0.117	0.118	0.111	0.136
Gp_6	0.181	0.148	0.179	0.152	0.158	0.167
outgroup	0.178	0.180	0.188	0.162	0.172	0.182	0.213

Gp 1 means group I Related to hematophagous bats-AgV3, Gp 2 means group II Related to insectivorous bats-NC1, Gp 3 means group III Related to insectivorous bats-NC1, Gp 4 means group IV Related to insectivorous bats-NC2, Gp 5 means group V Related to insectivorous bats-NC2, Gp 6 means group VI Related to marmosets, Gp 7 means group VII Related to Tadarida brasiliensis-AgV4 and outgroup represents the Terrestrial cycle of transmission-root of phylogenetic tree.

Table A3.

Estimates of average evolutionary divergence over sequence pairs within groups.

Gp 1	0.0253
Gp 3	0.0467
Gp 2	0.0674
Gp 4	0.0185
Gp 5	0.0566
Gp 7	0.0029
Gp 6	0.0051
outgroup	0.0051

Gp 1 means group I Related to hematophagous bats-AgV3, Gp 2 means group II Related to insectivorous bats-NC1, Gp 3 means group III Related to insectivorous bats-NC1, Gp 4 means group IV Related to insectivorous bats-NC2, Gp 5 means group V Related to insectivorous bats-NC2, Gp 6 means group VI Related to marmosets, Gp 7 means group VII Related to Tadarida brasiliensis-AgV4 and outgroup represents the Terrestrial cycle of transmission-root of phylogenetic tree.

Author Contributions

Conceptualization: L.H.Q., S.R.F. and A.C.A.C.; methodology, L.H.Q., A.C.A.C., M.C.L., E.M.S.C., A.A., C.d.C., W.A.P., E.C.S., M.S.L., S.V.I., D.B.A. and S.R.F.; software, A.C.A.C., M.P.C. and L.G.B.G.; validation, A.C.A.C., M.C.L., E.C.S., M.S.L., S.V.I. and D.B.A.; formal analysis, A.C.A.C., L.H.Q., M.P.C. and L.G.B.G.; investigation, A.C.A.C. and L.H.Q.; resources, S.R.F., E.L.D. and L.G.B.G.; data curation, A.C.A.C. and L.H.Q.; writing—original draft preparation, A.C.A.C. and L.H.Q.; writing—review and editing, A.C.A.C., L.H.Q., S.R.F., D.B.A., M.P.C. and L.G.B.G.; visualization, A.C.A.C., L.H.Q. and S.R.F.; supervision, L.H.Q., S.R.F. and E.L.D.; project administration, L.H.Q.; funding acquisition, L.H.Q., S.R.F. and E.L.D. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

The study was conducted in accordance with the ethics principles of the Brazilian College of Animal Experimentation (COBEA) and approved by the Animal Experimentation Ethics Committee of the School of Dentistry and Veterinary Medicine of Araçatuba, UNESP (Process No. 00858-2012 and 00902-2016).

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author(s).

Conflicts of Interest

The authors declare no conflicts of interest.

Funding Statement

This research was funded by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)—grant number 2008/00976-0 and 2007/01843-0 and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)—grant number 578281/2008-2. Data analysis and publication were supported by LGBG FAPESP 2022-13054-0. Scholarships: A.C.A.C.—CNPq 102474/2022-2 and FAPESP 2024-08821-8; M.C.L.—FAPESP (04/12793-6); E.C.S.—FAPESP (2008/08423-0); M.S.L.—CNPq technical assistance (grant number 578281/2008-2), S.V.I.—FAPESP (2008/00976-0). M.P.C. was supported by the Fund to Support Teaching, Research and Extension (FAEPEX/UNICAMP) (grant #2502/24). L.G.B.G. is supported by Young Research Project FAPESP 2022/13054-0. A.C.A.C. is currently supported by Young Research Project FAPESP 2024/10801-5.