Rabies is a zoonosis, invariably fatal, caused by the acute infection of the central nervous system of mammals caused by a virus. It is widely distributed around the world, except in regions such as Antarctica, Australia, Japan and New Zealand [1]. In Brazil, two main variants are described, one associated with the urban cycle, isolated from dogs, cats and humans, and another with the sylvatic cycle, isolated from bats and cattle [1–4]. The sylvatic cycle variants are endemics to several regions of the Brazil [1, 5–6].
Rabies virus belongs to the family Rhabdoviridae, genus Lyssavirus [1, 7]. Comprises a single-stranded, negative-sense RNA virus [8]. The viral genome contains five genes distributed in the order 3' N-P-M-G-L 5', which encode five proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and viral-dependent RNA RNA polymerase (L) [1, 9]. The glycoprotein is responsible for the adsorption of the virus to the host cell, assists in viral denudation, catalyzes the fusion of the endocytic membrane, being the major determinant of pathogenicity and is responsible for the induction of neutralizing antibodies [9–10].
Rabies lyssavirus (RABV) is one of seventeen members belonging to the genus Lyssavirus, fifteen of which have members of Chiroptera as exclusive reservoirs, showing the importance of this order as a reservoir for the genus [1, 11–12]. In addition to bats, the virus affects several species of mammalian, some with economic importance, such as cattle [1, 13–14].
In South America, rabies in herbivores occurs cyclically, reappearing every 5–7 years [6]. It is usually transmitted by the vampire bat Desmodus rotundus, the species is the most common and abundant vampire bat. Inhabits forested and desert areas, taking shelter in tree hollows, caves, culverts, abandoned mines and even civil constructions [15–16].
Given this cyclical relationship between cattle and bats in the transmission of rabies virus, the objective of this study was to analyze the phylogenetic relationship of the virus from two different hosts, bats and cattle.
Glycoprotein (G) RNA sequences obtained from the GenBank database (https://www.ncbi.nlm.nih.gov/genbank/) were selected using the following search parameters: “nucleotide, glycoprotein, rabies lyssavirus, bat, partial”, the same was done for cattle, replacing the term “bat” with “cattle”.
The European bat lyssavirus 1 (EBLV-1) virus was chosen as the outgroup. Belonging to the genus Lyssavirus, it causes an infection similar to classic rabies. To obtain the sequence, “nucleotide, glycoprotein, european bat lyssavirus 1, partial” were used as parameters, selecting a sequence with a size of 743 bp.
For data analysis, the MEGA-X software [17] (available at https://www.megasoftware.net/) was used. The sequence alignment was performed in MUSCLE, and manually corrected. Afterwards, an analysis of Maxima Parsimony was performed, generating the phylogeny. The rooting of the outgroup was verified and the support of the branches was calculated by the Bootstrap method in 1000 repetitions. With the generated tree, the 50% cutoff value was chosen for data reliability in the consensus tree.
In addition, an individual analysis of the parsimoniously informative sites was performed to assess the uniformity and difference between the sequences in relation to the host.
The analyzes involved 44 sequences, corresponding to 12 sequences for bats and 32 sequences for cattle, with sizes between 805 bp and 1572 bp. A total of 1595 base pairs were obtained. Of these, 871 sites remained conserved, 724 variable and 412 sparingly informative. 10 trees of maximum parsimony with a size of 1138 were generated. The consistency and retention indexes were 0.78 and 0.83, respectively.
Figure 1 presents the phylogeny generated from the Maximum Parsimony analysis. In the branches, bootstrap values above 50% are reproduced, indicating the reliability of the data.
Phylogenetic analyzes of the G gene showed that the virus evolved from bats to cattle. The virus strain isolated from bats has a basal position compared to the bovine strain, with the exception of 3 branches (AB247429.2, AB247426.2 and AB247427.2).
Of the 412 thrifty informative sites, it was observed, through the individual analysis of each site, that in 21 positions there was uniformity within and divergence between groups (virus extracted from bovine and bat). Table 1 presents the substitutions that took place.
Table 1
Mutations in the parsimoniously informative sites.
| Site position | Nucleotide in the bovine virus | Nucleotide in the bat virus |
| 98 | C | G | |
| 194 | T, C | T, G | |
| 281 | T, C | A, C | |
| 287 | T | G | |
| 293 | T | C | |
| 348 | A, C | A | |
| 353 | A, T | A | |
| 366 | T, G | G | |
| 455 | A, C | A | |
| 506 | T, A | G, A | |
| 569 | C, A | T, A | |
| 570 | C, A | T, A | |
| 572 | C, A | C, T | |
| 578 | C, A | C, T | |
| 623 | C, A | T, A | |
| 627 | T, A | G, A | |
| 647 | T, C | T | |
| 661 | T, C | T, A | |
| 677 | A, G | A, T | |
| 697 | A, G | A, T | |
| 749 | A, G | C, G | |
The virus-host relationship provides an extraordinary situation to study evolutionary processes. Viruses are obligate intracellular parasites and their evolution is inexorably linked to the biology of the host [12]. The biological relationships of viruses and their hosts are a delicate balance between the host's immune system and the virus' escape mechanisms.
The G gene is indicated for studies involving genetic analysis, as it is used to classify RABV isolates according to lineage, variant, host species, geographic origin and/or distribution [9–10, 18]. The glycoprotein is responsible for viral adsorption and denudation, catalyzes endocytic membrane fusion and is the main antigen of rhabdoviruses [1, 9–10, 18].
Although many mammalian species are susceptible to Lyssavirus infection, the virus can only establish transmission networks in small numbers, indicating that there are important barriers to transmission between species [19].
Bats are known to be the main reservoir for all Lyssavirus [20]. Members of the genus are thought to have evolved originally from bats and later into carnivores [21]. This study corroborates that the same may have happened for cattle. When analyzing the parsimoniously informative sites, it is suggested that some mutations are crucial for the virus to be able to infect another host, such as those described in Table 1. It is also observed a greater uniformity in the viral sequences of bovine origin when compared with those of the bat. The bat is the natural host of RABV and for this reason, the virus must present greater genetic diversity in this host and not all variants of this virus seem to be important in the infection of cattle, as can be seen in the phylogenetic tree (Fig. 1) and in the mutations observed at the parsimoniously informative sites (Table 1).
Studies have shown that the rate of nucleotide substitution varies visibly in viruses that infect bats, reflecting a combination of host and environmental factors [19], corroborating the results of the present research (Table 1). The work by Marston et al. [22] addressed the host specificities of Lyssavirus, arguing that infection of a new host requires adaptations of the virus and that several factors must be considered, both in the host and in the virus. Lissaviruses have co-evolved with specific bat species in restricted host reservoirs [22].
The vampire bat species D. rotundus is distributed throughout Latin America, occurring from northern Mexico to Argentina [15–16]. As D. rotundus feeds preferentially on the blood of cattle, the introduction and dissemination of herds provided food resources widely available in several regions of South America, which have potential for livestock production. In this way, the endemic character of rabies in cattle is maintained, giving rise to sporadic outbreaks of the disease [23]. When interventions such as vaccination and surveillance are not implemented or are not successful, the virus can establish itself in a new host [22].
No studies were found that relate the evolution of the virus from bats to cattle. However, the work conducted by Badrane et al. [24] showed that there is strong phylogenetic support indicating that Lyssavirus in bats are much earlier than those of carnivores, and that the change of hosts — bats to carnivores — occurred in the history of the Lyssavirus genus. The authors conducted studies with strains of the virus isolated from raccoons, skunks and bats. They observed that the lineages are phylogenetically related, arguing that rabies may have an independent autochthonous origin, maintained by bats, with spillover to skunks and raccoons [24–25].
The maximum parsimony tree (Fig. 1) generated had a consistency and retention indexes of 0.78 and 0.83, respectively. The consistency index was proposed to measure the level of homoplasy, the higher its value, the lower the number of homoplasies present. While the retention index measures how many of the synapormorphies are real and also considers the maximum number of changes of a character [26]. The results obtained indicate that the sequences used are suitable for the analysis performed.
Lines AB247429.2, AB247426.2 and AB247427.2 (Fig. 1) are phylogenetically closer to cattle, possibly due to mutations that occurred during the process of adaptation to the host (Table 1), indicating that these are viable lines cattle infection.
The results of this research indicated that the virus (RABV) evolved from bats to cattle. In addition, the data showed a great variation in the sequences of nitrogenous bases and that some variants of the virus seem to have greater importance in the infection of cattle. Studies of virus-host relationships are important to understand viral infections, making it possible to promote control measures to prevent future outbreaks of the disease.