**3. Epidemiological surveillance in bats by metagenomic approaches: a powerful tool for conducting large-scale studies**

In recent years, emerging and serious infectious diseases have caused worldwide fear. It is also known that many of these diseases are caused by viruses from bats, such as Ebola, Marburg, SARS coronavirus (SARS-CoV), MERS coronavirus (MERS-CoV), Nipah (NIV) and Hendra (HEV) [20], and nowadays, SARS-CoV-2. The growing recognition of the bats importance as reservoirs for new diseases is due to the fact that they constitute 20% of known mammal species, have unique and diverse lifestyles, including the ability to fly, often presenting gregarious social structures achieving incredible abundance and densities (some cave bats reaching up to 500 individuals per square meter) and long life [21].

As more information has been obtained regarding the factors or causes of emergence, there has been an expectation that it is possible to predict the emergence of new pathogens. These and other factors have significantly increased the demand for new viral pathogens, especially at the human-animal interface in species of wild and domestic animals [22].

With the exception of studies focusing on lyssavirus, most viruses' investigations in bats have been limited to one particular zoonotic agent involved in an outbreak of geographically localized disease [23]. COVID-19 showed the need to form an

international front for active surveillance of bats different populations to detect potential zoonotic agents as well as low pathogenicity unknown viruses that can recombine/mutate and become pathogenic.

The emergence of highly pathogenic viruses such as SARS and MERS-CoV has identified coronaviruses as agents of high interest in epidemiological surveillance. In addition to concluding that SARS-CoV may have originated from bats, it is suggested that several other new viruses exist in animals and some of them pose a risk to public health [24–27].

Although great advances have been made in the knowledge of these viruses, there is much to learn about the evolution of highly pathogenic agents in reservoir animals such as bats [1]. Several studies have pointed out a great diversity of coronaviruses belonging to the genus α*-* and β*-coronavirus* of the subfamily Orthocoronavirinae that occur widely in bat species around the world, including Africa, Europe, the Americas, and Asia. Interestingly, an analysis of viruses isolated from bats in Mexico showed that host species were driving forces in the evolution of coronaviruses, and that a single bat species can contain several coronaviruses. In addition, the phylogenetic association of CoVs with the species/genus was particularly evident in allopatric populations separated by significant geographical distances [22].

Simas and Arns [28] described a metagenomic methodology using bat common specie from urban areas in the Americas, the *Tadarida brasiliensis,* in order to establish a rapid methodology for active epidemiological surveillance in bats as the best reservoir animal model. The assay aimed to identify viral agents in oral and rectal swabs collected from asymptomatic *T. brasiliensis* bats from a colony in the Campinas-SP, Brazil. From this, these researchers described the diversity and abundance of the identified viral agents and could relate phylogenetically the identified. The workflow is described in **Figure 1**.

The most important steps are the pretreatment and the validation because these can remove the host genetic material and confirm the sequences of dataset identified. For the pretreatment, the researchers used filtration and treatment with

#### **Figure 1.**

*Workflow described by Simas and Arns [28] to conduct active epidemiological surveillance from model reservoir animals like bats using metagenomic approach.*

**279**

*Metagenomics and Pandemic Viruses*

sequence dataset.

sity studies like this.

other animals even cause human infections.

and herpesviruses are widely distributed in bat populations.

has already been described [31].

zoonotic viral diseases [33].

*DOI: http://dx.doi.org/10.5772/intechopen.93687*

in studies that use the same metagenomic methodology.

DNAse and proteinase K enzymes. These procedures help to eliminate the genetic material "contaminants" and to assure the most viral genetic material into the

Using these assays, a large number of excellent quality paired-end sequences were obtained in the HiSeq 2500 Illumina platform (345,409,110 reads pairedends—76.47% Q ≥ 30). In the reading assembly procedures with the MetaVelvet and Metavir 2 genome assemblies' platforms, many viral genetic materials from several pathogenic viruses were identified. It can be noted that the different platforms used provided complementary data, indicating the need to carry out similar procedures

Although the search for similarity carried out by MetaVelvet in different databases provided a small number of viral matches (97; 2 for coronavirus), these results were validated and allowed the identification of a coronavirus with a strong phylogenetic relationship with Porcine Epidemic Diarrhea virus (PEDV), a high pathogenicity swine virus, and human coronavirus, HCoV-NL63. PEDV has been reported in many other countries, including Germany, France, Switzerland, Hungary, Italy, China, South Korea, Thailand and Vietnam and was first identified in the United States in May 2013. The US outbreak occurred in 23 states, with 2692 confirmed cases leading to major economic losses. Studies have shown that all American PEDV strains are closely related to a strain from China, AH2012 [29]. However, the identification of PEDV in wild animals common in the Americas, such as bats *Tadarida brasiliensis*, can help to understand the evolution of these agents in animal reservoirs and to understand the eco-epidemiology from the genetic diver-

Metavir 2 identified sequences of viruses associated with various pathogens in humans. Many sequences have been classified as belonging to the Herpesviridae family. Several viral agents in this family are known to cause a wide variety of human diseases, including various types of cancer. In addition, since they have a great capacity to infect many types of cells or tissues [30], bats may be serving as a reservoir for recombination and the emergence of new strains capable of infecting

Several viruses of the Order Caudovirales were also identified, most from the Siphoviridae family. These phages are capable of infecting several species of human pathogenic bacteria (*Enterobacteria*, *Shigella*, *Mycobacterium*, and *Bacillus*), so it is an indirect evidence of the presence of these bacteria also in bats. The concomitant detection of herpes and phages indicates that bats can act as important agents in the evolution of these viral agents, since the existence of recombination between them

Many betaretroviruses, viruses that cause various types of tumors in primates, sheep, and rats have been detected. Sano et al. [32] also identified several viral agents from the Retroviridae and Herpesviridae families in bats in the Philippines. Dacheux et al. [12] determined the viral diversity of five different species of French insectivorous bats (nine specimens). All of these results suggest that retroviruses

The detection of several dsRNA virus sequences, a virus group that cause gastroenteritis in children (rotavirus) and others that are pathogenic for cattle and sheep, their identification in bats contributes to the understanding of their circulation in ecosystems. Another Brazilian study also reported the presence of rotavirus in bat feces. Phylogenetic analyzes indicated the formation of a clade with sequences of bovine and human origins, suggesting recombination between the strains in animal hosts, events that precede transmission to humans in

*Genetic Variation*

to public health [24–27].

recombine/mutate and become pathogenic.

The workflow is described in **Figure 1**.

*reservoir animals like bats using metagenomic approach.*

international front for active surveillance of bats different populations to detect potential zoonotic agents as well as low pathogenicity unknown viruses that can

The emergence of highly pathogenic viruses such as SARS and MERS-CoV has identified coronaviruses as agents of high interest in epidemiological surveillance. In addition to concluding that SARS-CoV may have originated from bats, it is suggested that several other new viruses exist in animals and some of them pose a risk

Although great advances have been made in the knowledge of these viruses, there is much to learn about the evolution of highly pathogenic agents in reservoir animals such as bats [1]. Several studies have pointed out a great diversity of coronaviruses belonging to the genus α*-* and β*-coronavirus* of the subfamily Orthocoronavirinae that occur widely in bat species around the world, including Africa, Europe, the Americas, and Asia. Interestingly, an analysis of viruses isolated from bats in Mexico showed that host species were driving forces in the evolution of coronaviruses, and that a single bat species can contain several coronaviruses. In addition, the phylogenetic association of CoVs with the species/genus was particularly evident in allopat-

ric populations separated by significant geographical distances [22].

Simas and Arns [28] described a metagenomic methodology using bat common specie from urban areas in the Americas, the *Tadarida brasiliensis,* in order to establish a rapid methodology for active epidemiological surveillance in bats as the best reservoir animal model. The assay aimed to identify viral agents in oral and rectal swabs collected from asymptomatic *T. brasiliensis* bats from a colony in the Campinas-SP, Brazil. From this, these researchers described the diversity and abundance of the identified viral agents and could relate phylogenetically the identified.

The most important steps are the pretreatment and the validation because these can remove the host genetic material and confirm the sequences of dataset identified. For the pretreatment, the researchers used filtration and treatment with

*Workflow described by Simas and Arns [28] to conduct active epidemiological surveillance from model* 

**278**

**Figure 1.**

DNAse and proteinase K enzymes. These procedures help to eliminate the genetic material "contaminants" and to assure the most viral genetic material into the sequence dataset.

Using these assays, a large number of excellent quality paired-end sequences were obtained in the HiSeq 2500 Illumina platform (345,409,110 reads pairedends—76.47% Q ≥ 30). In the reading assembly procedures with the MetaVelvet and Metavir 2 genome assemblies' platforms, many viral genetic materials from several pathogenic viruses were identified. It can be noted that the different platforms used provided complementary data, indicating the need to carry out similar procedures in studies that use the same metagenomic methodology.

Although the search for similarity carried out by MetaVelvet in different databases provided a small number of viral matches (97; 2 for coronavirus), these results were validated and allowed the identification of a coronavirus with a strong phylogenetic relationship with Porcine Epidemic Diarrhea virus (PEDV), a high pathogenicity swine virus, and human coronavirus, HCoV-NL63. PEDV has been reported in many other countries, including Germany, France, Switzerland, Hungary, Italy, China, South Korea, Thailand and Vietnam and was first identified in the United States in May 2013. The US outbreak occurred in 23 states, with 2692 confirmed cases leading to major economic losses. Studies have shown that all American PEDV strains are closely related to a strain from China, AH2012 [29]. However, the identification of PEDV in wild animals common in the Americas, such as bats *Tadarida brasiliensis*, can help to understand the evolution of these agents in animal reservoirs and to understand the eco-epidemiology from the genetic diversity studies like this.

Metavir 2 identified sequences of viruses associated with various pathogens in humans. Many sequences have been classified as belonging to the Herpesviridae family. Several viral agents in this family are known to cause a wide variety of human diseases, including various types of cancer. In addition, since they have a great capacity to infect many types of cells or tissues [30], bats may be serving as a reservoir for recombination and the emergence of new strains capable of infecting other animals even cause human infections.

Several viruses of the Order Caudovirales were also identified, most from the Siphoviridae family. These phages are capable of infecting several species of human pathogenic bacteria (*Enterobacteria*, *Shigella*, *Mycobacterium*, and *Bacillus*), so it is an indirect evidence of the presence of these bacteria also in bats. The concomitant detection of herpes and phages indicates that bats can act as important agents in the evolution of these viral agents, since the existence of recombination between them has already been described [31].

Many betaretroviruses, viruses that cause various types of tumors in primates, sheep, and rats have been detected. Sano et al. [32] also identified several viral agents from the Retroviridae and Herpesviridae families in bats in the Philippines. Dacheux et al. [12] determined the viral diversity of five different species of French insectivorous bats (nine specimens). All of these results suggest that retroviruses and herpesviruses are widely distributed in bat populations.

The detection of several dsRNA virus sequences, a virus group that cause gastroenteritis in children (rotavirus) and others that are pathogenic for cattle and sheep, their identification in bats contributes to the understanding of their circulation in ecosystems. Another Brazilian study also reported the presence of rotavirus in bat feces. Phylogenetic analyzes indicated the formation of a clade with sequences of bovine and human origins, suggesting recombination between the strains in animal hosts, events that precede transmission to humans in zoonotic viral diseases [33].
