Full-genome sequencing of dozens of new DNA viruses found in Spanish bat feces

Abstract

Bats are natural hosts of multiple viruses, many of which have clear zoonotic potential. The search for emerging viruses has been aided by the implementation of metagenomic tools, which have also enabled the detection of unprecedented viral diversity. Currently, this search is mainly focused on RNA viruses, which are largely over-represented in databases. To compensate for this research bias, we analyzed fecal samples from 189 Spanish bats belonging to 22 different species using viral metagenomics. This allowed us to identify 52 complete or near-complete viral genomes belonging to the families Adenoviridae, Circoviridae, Genomoviridae, Papillomaviridae, Parvoviridae, Polyomaviridae and Smacoviridae. Of these, 30 could constitute new species, doubling the number of viruses currently described in Europe. These findings open the door to a more thorough analysis of bat DNA viruses and their zoonotic potential.

Importance: Metagenomics has become a fundamental tool to characterize the global virosphere, allowing us not only to understand the existing viral diversity and its ecological implications but also to identify new and emerging viruses. RNA viruses have a higher zoonotic potential, but this risk is also present for some DNA virus families. In our study, we analyzed the DNA fraction of fecal samples from 22 Spanish bat species, identifying 52 complete or near-complete genomes of different viral families with zoonotic potential. This doubles the number of genomes currently described in Europe. Metagenomic data often produce partial genomes that can be difficult to analyze. Our work, however, has characterized a large number of complete genomes, thus facilitating their taxonomic classification and enabling different analyses to be carried out to evaluate their zoonotic potential. For example, recombination studies are relevant since this phenomenon could play a major role in cross-species transmission.

Keywords: DNA viruses; bat viruses; metagenomics; viral emergence; viromics; zoonotic viruses.

Fig 1

Sampling points throughout Spain. The number of individuals captured in each area is indicated in parentheses. This map was created using R software (https://www.R-project.org/).

Fig 2

Distribution of MAVGs per bat species/pool. Viral families are shown in different colors.

Fig 3

Optimized tanglegram between a papillomavirus subclade of the ML tree obtained from concatenated E1, E2, L2, and L1 nucleotide sequences (63) and associated host species. The host species tree was downloaded from www.timetree.org. The newly described viruses are highlighted in red boxes. Bootstrap values are shown at nodes. Both trees are rooted at the midpoint.

Fig 4

ML tree of the family Polyomaviridae using 135 RefSeq LTAg amino acid sequences (NCBI TaxId: 151341). Taxonomic groups are collapsed by the genus. Only taxa belonging to the genus Alphapolyomavirus are explicitly indicated, and the group known as the VP3-less clade is highlighted in blue. Taxa are denoted by GenBank protein accession number and virus name, and novel viruses are labeled in red. Phylogenetic analysis was done using the substitution model LG + F + I + G4. SH-aLRT and bootstrap values higher than 80 and 95, respectively, are indicated with red circles. The tree is rooted at the midpoint. The scale bar indicates the evolutionary distance in amino acid substitutions per site.

Fig 5

ML tree of the Parvovirinae subfamily using 126 NS1 amino acid sequences. Taxonomic groups are collapsed by genus except for Dependoparvovirus and Protoparvovirus genera. Taxa are denoted by GenBank protein accession number and virus name, and novel viruses are labeled in red. Phylogenetic analysis was done using the substitution model LG + F + I + G4. SH-aLRT and bootstrap values higher than 80 and 95, respectively, are indicated with red circles. The tree is rooted at the midpoint. The scale bar indicates the evolutionary distance in amino acid substitutions per site.

Fig 6

ML trees of the Adenoviridae family using DNA polymerase (A) and hexon (B) amino acid sequences from 73 representative members. Taxonomic groups are collapsed by genus, except for the genus Mastadenovirus. Taxa are denoted by GenBank protein accession number and virus name, and novel viruses are labeled in red. Phylogenetic analyses were done using the substitution model LG + F + I + G4. SH-aLRT and bootstrap values higher than 80 and 95, respectively, are indicated with red circles. The tree is rooted at the midpoint. The scale bar indicates the evolutionary distance in amino acid substitutions per site.

Fig 7

ML tree of the Circoviridae family based on Rep amino acid sequence. Taxa are denoted by GenBank accession number and virus name, and viruses found in this study are indicated in red, while new species are indicated by an asterisk. Sequences were downloaded from the ICTV Circoviridae data resources (27 November 2023). In addition, 4 RefSeq sequences (NC_076479, NC_040639.1, BBI18985.1, and YP_009552020.1) were added to illustrate its similarity with novel MAVGs. Phylogenetic analysis was done using the substitution model LG + F + R6. SH-aLRT and bootstrap values higher than 80 and 95, respectively, are indicated with red circles. The tree is rooted to define monophyletic groups of each family genus. The scale bar indicates the evolutionary distance in amino acid substitutions per site.

Fig 8

ML tree of the family Smacoviridae based on 215 Rep amino acid sequences. Taxonomic groups are collapsed by genus, except for Porprismacovirus genus, and some non-illustrative clades within this genus. Taxa are denoted by GenBank accession number and virus name, and novel viruses are labeled in red. Phylogenetic analysis was done using the substitution model LG + F + I + G4. SH-aLRT and bootstrap values higher than 80 and 95, respectively, are indicated with red circles. The tree is rooted at the midpoint. The scale bar indicates the evolutionary distance in amino acid substitutions per site.

Fig 9

ML tree of the family Genomoviridae based on 94 representative amino acid sequences of Rep gene. Taxonomic groups are collapsed by genus, except for those genera where new viruses are identified. Taxa are denoted by GenBank accession number and virus name, and novel viruses are labeled in red, indicating with an asterisk those that are defined as new species. When a new species includes more than one novel MAVG (see Table S9), only one is indicated with an asterisk. Phylogenetic analysis was done using the substitution model LG + F + I + G4. SH-aLRT and bootstrap values higher than 80 and 95, respectively, are indicated with red circles. The tree is rooted at the midpoint. The scale bar indicates the evolutionary distance in amino acid substitutions per site.