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. 2016 Mar 17;11(3):e0151921.
doi: 10.1371/journal.pone.0151921. eCollection 2016.

Metagenomic Survey of Viral Diversity Obtained from Feces of Subantarctic and South American Fur Seals

Mariana Kluge  1 , Fabrício Souza Campos  1 , Maurício Tavares  2 , Derek Blaese de Amorim  2 , Fernanda Pedone Valdez  3 , Adriana Giongo  4 , Paulo Michel Roehe  1 , Ana Claudia Franco  1
Affiliations

Metagenomic Survey of Viral Diversity Obtained from Feces of Subantarctic and South American Fur Seals

Mariana Kluge et al. PLoS One. .

Abstract

The Brazilian South coast seasonally hosts numerous marine species, observed particularly during winter months. Some animals, including fur seals, are found dead or debilitated along the shore and may harbor potential pathogens within their microbiota. In the present study, a metagenomic approach was performed to evaluate the viral diversity in feces of fur seals found deceased along the coast of the state of Rio Grande do Sul. The fecal virome of two fur seal species was characterized: the South American fur seal (Arctocephalus australis) and the Subantarctic fur seal (Arctocephalus tropicalis). Fecal samples from 10 specimens (A. australis, n = 5; A. tropicalis, n = 5) were collected and viral particles were purified, extracted and amplified with a random PCR. The products were sequenced through Ion Torrent and Illumina platforms and assembled reads were submitted to BLASTx searches. Both viromes were dominated by bacteriophages and included a number of potentially novel virus genomes. Sequences of picobirnaviruses, picornaviruses and a hepevirus-like were identified in A. australis. A rotavirus related to group C, a novel member of the Sakobuvirus and a sapovirus very similar to California sea lion sapovirus 1 were found in A. tropicalis. Additionally, sequences of members of the Anelloviridae and Parvoviridae families were detected in both fur seal species. This is the first metagenomic study to screen the fecal virome of fur seals, contributing to a better understanding of the complexity of the viral community present in the intestinal microbiota of these animals.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Sample location map.
Map indicating the location of where the samples were collected along the coast of the State of Rio Grande do Sul, Brazil (shaded). The map was extracted from the Open Street Map[15] database.
Fig 2
Fig 2. Taxonomic classification of assembled reads (>100bp).
(A) Pie charts of assembled reads based on BLASTx best E-scores (cutoff: 10e-05) against the GenBank non-redundant and viral databases. (B) Taxonomic distribution of viruses for each fur seal species.
Fig 3
Fig 3. Phylogenetic analysis of fur seal anellovirus.
(A) Schematic representation of the genome of anelloviruses using as example the torque teno virus (~3.8 kb). The blue bars represent the contigs from South American fur seal and the orange bars represent the contigs from Subantarctic fur seal. (B) Neighbor-joining phylogenetic tree based on the alignment of partial amino acid sequences (233 aa) from the ORF1 of 21 anelloviruses. Human and simian torque teno viruses were used as outgroup. The anellovirus sequences from South American fur seal identified in this study are labeled with black squares. The GenBank accession numbers of the viral sequences are shown in parentheses.
Fig 4
Fig 4. Phylogenetic analysis of fur seal parvovirus.
(A) Schematic representation of the genome of parvoviruses using as example the tusavirus (~4.4 kb). The blue bars represent the contigs from South American fur seal and the orange bars represent the contigs from Subantarctic fur seal. (B) Neighbor-joining phylogenetic tree based on the alignment of partial amino acid sequences (261 aa) from the NS1 protein of 17 parvoviruses. Seal parvovirus and California sea lion sesavirus were used as outgroup. The parvovirus sequence from Subantarctic fur seal identified in this study is labeled with a black square. The GenBank accession numbers of the viral sequences are shown in parentheses.
Fig 5
Fig 5. Phylogenetic analysis of fur seal picornavirus.
(A) Schematic representation of the genome of picornaviruses using as an example the hepatits A virus (~7.4 kb). The blue bars represent the contigs from South American fur seal and the orange bars represent the contigs from Subantarctic fur seal. (B) Neighbor-joining phylogenetic tree based on the alignment of partial amino acid sequences (219 aa) from the P1 region of the polyprotein of 13 picornaviruses. Porcine kobuvirus was used as outgroup. (C) Neighbor-joining phylogenetic tree based on the alignment of partial amino acid sequences (122 aa) from the P3 region of the polyprotein of 12 picornaviruses. Human cosavirus was used as outgroup. The picornavirus sequences from South American fur seal identified in this study are labeled with a black square. The GenBank accession numbers of the viral sequences are shown in parentheses.
Fig 6
Fig 6. Phylogenetic analysis of fur seal sakobuvirus.
(A) Schematic representation of the sakobuvirus genome using Feline sakobuvirus A (~7.8 kb—NC_022802) as a reference. The orange bars represent the contigs from Subantarctic fur seal. (B) Neighbor-joining phylogenetic tree based on the alignment of partial amino acid sequences (409 aa) from the P2 region of the polyprotein of 11 picornaviruses. Sicinivirus 1 was used as outgroup. (C) Neighbor-joining phylogenetic tree based on partial amino acid sequences (255 aa) from the 3D region of the polyprotein of 13 picornaviruses. California sea lion sapelovirus 2 was used as outgroup. The sakobuvirus sequences from the Subantarctic fur seal from this study used in phylogenetic analyses are labeled with a black square. The GenBank accession numbers of the viral sequences are shown in parentheses.
Fig 7
Fig 7. Phylogenetic analysis of fur seal picobirnavirus.
(A) Schematic representation of the genome of picobirnaviruses using as an example the human picobirnavirus (~4.2 kb). The blue bars represent the contigs from South American fur seal. (B) Neighbor-joining phylogenetic tree based on the alignment of partial nucleotide sequences (743 bp) from the RdRp gene of 17 picobirnaviruses. Human picobirnavirus GII was used as outgroup. The picobirnavirus sequence from South American fur seal identified in this study is labeled with a black square. The GenBank accession numbers of the viral sequences are shown in parentheses.
Fig 8
Fig 8. Phylogenetic analysis of fur seal rotavirus.
(A) Schematic representation of the genome of rotaviruses using as an example the group C rotavirus (~17.9 kb). The orange bars represent the contigs from Subantarctic fur seal. (B) Neighbor-joining phylogenetic tree based on the alignment of partial amino acid sequences (307 aa) from the RpRd (segment 1) of 19 rotaviruses. Sequences of groups B, G and H were used as outgroup. The rotavirus sequence from Subantarctic fur seal identified in this study is labeled with a black square. The GenBank accession numbers of the viral sequences are shown in parentheses.
Fig 9
Fig 9. Phylogenetic analysis of fur seal hepevirus-like.
(A) Schematic representation of the genome of hepeviruses using as an example the hepatitis E virus (~7.2 kb). The blue bar represents the contig from South American fur seal. (B) Neighbor-joining phylogenetic tree based on the alignment of partial amino acid sequences (182 aa) from the polyprotein of 15 hepeviruses. Hepelivirus and Fesavirus 2 were used as outgroup. The hepevirus-like virus sequence from South American fur seal identified in this study is labeled with a black square. The GenBank accession numbers of the viral sequences are shown in parentheses.
Fig 10
Fig 10. Phylogenetic analysis of fur seal sapovirus.
(A) Schematic representation of the genome of sapoviruses using California sea lion sapovirus 1 (~7.5 kb—JN420370.2) as a reference. The orange bars represent the contigs from Subantarctic fur seal. (B) Neighbor-joining phylogenetic tree based on complete nucleotide sequences from the VP1 gene of 9 caliciviruses. Human norovirus was used as outgroup. (C) Neighbor-joining phylogenetic tree based on the alignment of nearly-complete nucleotide sequences from the VP2 gene of 9 caliciviruses. Human norovirus was used as outgroup. The sapovirus sequences from Subantarctic fur seal from this study used in phylogenetic analyses are labeled with a black square. The GenBank accession numbers of the viral sequences are shown in parentheses.
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Grants and funding

The research leading to these results has received funding from the National Council for the Improvement of Higher Education (CAPES—http://www.capes.gov.br/), the National Council for Scientific and Technological Development (CNPq—http://www.cnpq.br/), and the Study and Project Funding Agency (FINEP—http://www.finep.gov.br/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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