Genetic Identification of Species of Bats that Act as Reservoirs or Hosts for Viral Diseases

This work was carried out in collaboration between all authors, about design the study, write the protocol and interpret the data, anchor the field study, gather the initial data and perform preliminary data analysis, research the literature searches and produce the initial draft. All authors contributed at same way, read and approved the final manuscript. ABSTRACT Introduction: Viruses have been identified as the main etiologic agents of both zoonoses and emerging infectious diseases (EIDs) and various species of wild fauna can be involved in the maintenance of these diseases. The very wide variety of bats, together with their ability to adapt to different environments and fly long distances, means that these animals are currently one of the main reservoirs for zoonoses and EIDs. For these reasons the correct identification of different bat species is essential. Aims: This paper describes the genetic identification of 56 samples isolated from different bat species. Methodology: Sequencing and phylogenetic analysis of the mitochondrial DNA cytochrome b (mtDNA cyt-b) gene. Results: Four families (Molossidae, Vespertilionidae, Noctilionidae and Phyllostomidae), twelve genera and nineteen different species of bats were identified, and the Basic Local Alignment Search Tool (BLAST) was used to confirm species identity. The phylogenetic tree constructed revealed two main clusters (1 and 2), both consist in two subclusters. Conclusions: Our results were concordant with those obtained by morphometric identification and genetic identification carried out by other authors, showing that the method described here can be used as an effective alternative to, or in combination with, morphometric identification of bats.


INTRODUCTION
Zoonoses are diseases that can be transmitted between humans and domestic or wild animals. According to World Health Organization (WHO) data, approximately 60% of infectious agents affecting humans originate in animals or products derived from them [1]. Zoonoses can be caused by bacteria, parasites, fungi, viruses or even unconventional agents. However, both classical and emerging viral zoonoses have been gaining attention because of recent episodes of diseases involving such agents around the world. Emerging infectious diseases (EIDs) are defined as diseases that have newly appeared in a population or have existed previously but are rapidly increasing in incidence or geographic range [2].
The viruses that cause zoonoses and/or EIDs belong to different families and cause a variety of diseases in humans. Rabies is one of the oldest known zoonoses and continues to cause serious public health problems. According to WHO estimates, 55,000 deaths are caused by this disease every year [1]. EIDs caused by viruses include Spanish flu, which became a pandemic and killed approximately 50 million people between 1918 and 1920, and Acquired Immunodeficiency Syndrome (AIDS) caused by the human immunodeficiency virus (HIV), which appeared in the '80s and is currently still a major public health problem34. The following viruses are also etiologic agents of emerging diseases: Wild fauna play an important role in the emergence and maintenance of these diseases; indeed, the majority (71.8%) of zoonotic EIDs that have occurred since the '40s originated in wild fauna, and the number of such diseases continues to increase [3]. Identification of the reservoirs for these diseases can help clarify how the pathogens are maintained in nature, leading to more effective disease control and avoiding indiscriminate extermination of wild animals.
In recent years bats have been associated with a significant number of EIDs and have increasingly been recognized as potential reservoirs and/or hosts for viruses that can cross barriers and infect humans and other animals [4,5,6]. The very wide variety of bats and their great ability to adapt to different environments are reflected in the many different species already identified on all continents except polar regions and ocean islands, distant from continents and their different feeding habits, which can be insectivorous, frugivorous, polinivorous, nectarivorous, carnivorous, piscivorous, hematophagous and omnivorous [7]. This is reflected in the large number of viruses from different families identified recently in bats, such as Bat adenovirus B20, Polyomavirus [8], Nipah virus [9], Menangle virus [10] and Hepatitis E-related virus [11]. Bats are also important reservoirs for viruses belonging to the genus Lyssavirus in the family Rhabdoviridae. To date 14 species have been identified in this genus, 12 of which are included in the recent list published by the International Committee on Taxonomy of Viruses [12] and two of which are recently proposed species [13,14]. Twelve of these have already been identified in bats.
The great diversity of bats and the important role they play in the spread of diseases make the need for correct identification of different bat species particularly important. Morphometric species identification is an efficient method but requires specialized personnel. Furthermore, because many laboratories receive degraded carcasses this type of identification is often impossible.
Genetic identification can be a tool in such situations, and sequencing of mitochondrial DNA (mtDNA) has been used for the genetic identification of different mammal species. By sequencing the hypervariable region of mtDNA, Carnieli et al. [15] identified which species of wild canid was involved in the maintenance of one of the rabies cycles in Brazil. The mtDNA cytochrome b (mtDNA cyt-b) gene has been used by various authors to study the ecology, evolution and systematics of bats [16,17,18,19,20], and in 2010 Larsen et al. [21], using the same gene, identified natural hybridization between various groups of bats.
The aim of the present study was to perform genetic sequencing of the mtDNA cyt-b gene of bats to genetically identify different Brazilian bat species, currently the main reservoirs in the Americas of the rabies virus and other species from the genus Lyssavirus, as well as other EIDcausing viruses.

Samples
For the genetic identification, 56 samples of livers from different species of bats that had previously been morphometrically identified were used. The samples came from different towns in the state of São Paulo, in the southeast of Brazil, and had been sent to the Rabies Diagnostic Laboratory at the Pasteur Institute of São Paulo.

DNA Extraction and Polymerase Chain Reaction (PCR)
Total DNA was extracted from the samples using the Wizard® Genomic DNA Purification kit #TM050 (Promega Corporation) in accordance with the manufacturer's instructions. Ultrapure DNase/RNase-free water was used as a negative control in all the steps from DNA extraction through PCR amplification.
Amplification of the mtDNA cyt-b gene from 5 µL of DNA was carried out in a final volume of 50 µL using Bat 05A (sense: 5'-CGACTAATGACATGAAAAATCACCGTTG-3') and Bat 14A (antisense:

Purification of the PCR Products
The amplicons were purified using the GFX TM PCR DNA and Gel Band Purification kit (GE Healthcare TM ) according to the manufacturer's instructions. After purification, the concentration of DNA samples was visually estimated by electrophoresis on 2% agarose gel using a Low Mass DNA Ladder (Invitrogen TM ) for comparison in accordance with the manufacturer's instructions.

DNA Sequencing
DNA sequencing was carried out with 4 µL of BigDye 3.1 (Applied Biosystems®), 4 µL of 5x sequencing buffer (Applied Biosystems®), 3.2 pmol of each primer (sense and antisense, as described in section 2.2), 30 to 50 ng of target DNA and DNAse/RNAse-free water to a final reaction volume of 10 µL. Reactions were carried out in a Mastercycler Gradient (Eppendorf®) under the following cycling conditions: 35 cycles at 96ºC/10 s, 50ºC/5 s and 60ºC/4 min with a ramp rate of 1ºC/s between each temperature. After each sequencing reaction the samples were purified using the HV MultiScreen 96-well filter plates and Sephadex TM G-50 fine (GE Healthcare TM ). After purification, the sequences were generated on an ABI 3130 genetic analyzer (Applied Biosystems TM ), as previously described Carnieli et al. [15].

Phylogenetic Analysis
The nucleotide sequences were edited with CHROMAS (version 2.23 Copyright 1998-2004 Technelysium Pty Ltd.), and the final sequences were aligned using CLUSTAL/W in BIOEDIT version 7.1.3.0 16 . The final sequences were used to confirm the identity of the species with BLAST. A phylogenetic tree was also constructed to determine and confirm the evolutionary relationships between the species. The tree was constructed with MEGA software (version 5.0) using the maximum likelihood method and the GTR (G + I) 5.0 substitution model 29 , and the reliability was assessed by the bootstrap method with 1,000 replicates, as previously described Carnieli et al. [15].

RESULTS
The nucleotide sequence of the mtDNA cyt-b gene was determined for 4 families, 12 genera and 19 species of bat. The number of samples for each species analyzed is shown in Table 1. The sequences identified in the study were deposited in GenBank, and the GenBank numbers are shown in Table 2.
Based on the topology of the phylogenetic tree of the species studied, two main clusters (1 and 2) could be identified. Cluster 1 consists of subcluster 1a, corresponding to the family Molossidae, and subcluster 1b, corresponding to the family Vespertilionidae. Cluster 2 consists of subclusters 2a and 2b, corresponding to the families Noctilionidae and Phyllostomidae, respectively (Fig. 1).

DISCUSSION
Bats are winged mammals that belong to the order Chiroptera.
In the present study the mtDNA cyt-b gene from 18 species belonging to the three main bat families in Brazil (Phyllostomidae, Molossidae and Vespertilionidae) and one species (Noctilio albiventris) from family Noctilionidae was sequenced, corresponding to a total of 56 samples from 19 different species.
Rabies is one of the oldest recorded zoonoses, and the main reservoirs for the disease are   Table 2 [23,25]. These findings underscore the importance of the family Phyllostomidae as a reservoir for different viruses.
The family Noctilionidae is made up of only two species in the single genus Noctilio. One of these species (Noctillio albiventris) is insectivorous and the other (Noctillio leporinus) preferably piscivorous. Although this family is of little importance as a reservoir of diseases, two specimens of the species N. albiventris were included in this study.
The family Molossidae includes insectivorous bats that have evolved the ability to fly and maneuver at speed and whose capacity to adapt to different environments makes them the most common synanthropic bats. This family is the second most numerous in this study, accounting for 42.86% of the species analyzed. The genera from this family included in the study were Tadarida, Molossus, Cynomops and Eumops, in all of which the rabies virus has previously been identified [26]. The Gossas virus, also from the family Rhabdoviridae, and the St. Louis encephalitis virus and Rio Bravo virus, from the family Flaviridae, have been identified in bats from genus Tadarida [23], while the Catu virus, from the family Bunyaviridae, has been identified in bats from genus Molossus [23].
The family Vespertilionidae is the largest family of chiropterans in terms of both diversity and geographic distribution, 407 species belonging to 48 genera and 6 distinct subfamilies having been identified to date [27,7] [23]; and other viruses from the genus Gammaretroviru [28] and Alphacoronavirus [29].
In a review of bats that act as viral reservoirs, Calisher et al. [23] reported that bats are frequently infected with viruses that cause diseases in humans and other mammals. Aguilar-Setien et al. [30] found rabies virus RNA in experimentally infected bats that did not develop clinical signs of the disease. These reports suggest the existence of unknown factors related to the immune system of bats and their ability to host and transmit different viruses.
The wide variety of EIDs, particularly viral ones, for which bats can act as reservoirs and/or hosts makes the need to study these animals all the more imperative. If the mechanism by which these viruses are maintained in nature and the role played by bats in the transmission and perpetuation of these pathogens is to be understood, correct identification of bats is fundamental. The mtDNA cyt-b gene was used in this study and allowed 19 species of bats in Brazil to be identified. The phylogenetic tree based on sequencing of this gene segregated the bats in the different families into clusters identical to those observed following morphometric identification and genetic identification described by Agnarsson et al. [31,32] and allowed identification to species level rather than just genus level.

CONCLUSION
In conclusion, our findings indicate that this genetic region is suitable for identification of bat species. Sequencing of the mtDNA cyt-b gene should therefore be used as an effective alternative to, or even in combination with, morphometric identification.

COMPETING INTERESTS
Authors have declared that no competing interests exist.