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Characterization of a new anellovirus species infecting an ocelot (Leopardus pardalis) in Brazil

Abstract

A complete genome of the first anellovirus infecting the wild felid Leopardus pardalis (ocelot) and a partial genome were assembled and annotated through high-throughput sequencing protocols followed by Sanger sequencing validation. The full-length virus obtained comprises 2,003 bp, while the partial genome comprises 1,224 bp. Phylogenetic analysis grouped these two sequences in two distinct clusters related to previously described Felidae anelloviruses. The ORF1 of the partial genome was identified as a new species provisionally called Torque teno ocelot virus, with 53.6% identity with its sister lineage. The complete genome was inferred as a new representative of the Torque teno felid virus 3 species, with 73.28% identity to the closest reference. This study expands known virus diversity and the host span of anelloviruses.

Keywords:
Virome; felid; Anelloviridae

The Anelloviridae family represents non-enveloped single-stranded viruses with circular negative-sense DNA, whose genomes range from 1.6 to 3.9 kb. The group is classified in 30 genera and 155 species by the International Committee of Virus Taxonomy (Varsani et al., 2021Varsani A, Opriessnig T, Celer V, Maggi F, Okamoto H, Blomström A-L, Cadar D, Harrach B, Biagini P and Kraberger S (2021) Taxonomic update for mammalian anelloviruses (family Anelloviridae). Arch Virol 166:2943-2953.). Anelloviruses are a group of commensal viruses that cause asymptomatic chronic infections. No pathology has been associated with them to date (Kaczorowska and van der Hoek, 2020Kaczorowska J and van der Hoek L (2020) Human anelloviruses: Diverse, omnipresent and commensal members of the virome. FEMS Microbiol Rev 44:305-313.). They are one of the major constituents of the human virome (Arze et al., 2021Arze CA, Springer S, Dudas G, Patel S, Bhattacharyya A, Swaminathan H, Brugnara C, Delagrave S, Ong T, Kahvejian A et al. (2021) Global genome analysis reveals a vast and dynamic anellovirus landscape within the human virome. Cell Host Microbe 29:1305-1315.e6.) and their known diversity drastically increased through virome investigations of non-model animals (Ng et al., 2011Ng TFF, Wheeler E, Greig D, Waltzek TB, Gulland F and Breitbart M (2011) Metagenomic identification of a novel anellovirus in Pacific harbor seal (Phocavitulinarichardsii) lung samples and its detection in samples from multiple years. J Gen Virol 92:1318-1323.; de Souza et al., 2018de Souza WM, Fumagalli MJ, de Araujo J, Sabino-Santos G Jr, Maia FGM, Romeiro MF, Modha S, Nardi MS, Queiroz LH, Durigon EL et al.(2018) Discovery of novel anelloviruses in small mammals expands the host range and diversity of the Anelloviridae. Virology 514:9-17.; Weber et al., 2020Weber MN, Mosena ACS, da Silva MS, Canova R, de Lorenzo C, Olegário JC, Budaszewski RF, Baumbach LF, Soares JF, Sonne L et al. (2020) Virome of crab-eating (Cerdocyonthous) and pampas foxes (Lycalopex gymnocercus) from southern Brazil and Uruguay. Infect Genet Evol 85:104421.; Kraberger et al., 2021Kraberger S, Serieys LE, Richet C, Fountain-Jones NM, Baele G, Bishop JM, Nehring M, Ivan JS, Newkirk ES, Squires JR et al. (2021) Complex evolutionary history of felid anelloviruses. Virology 562:176-189.; Cosentino et al., 2022Cosentino MAC, D’arc M, Moreira FRR, Cavalcante LT de F, Mouta R, Coimbra A, Schiffler FB, Miranda TDS, Medeiros G, Dias CA et al. (2022) Discovery of two novel Torque Teno viruses in Callithrix penicillata provides insights on Anelloviridae diversification dynamics. Front Microbiol 13:1002963.). In wild-life felids, anelloviruses have been identified in pumas (Puma concolor), bobcats (Lynx rufus), Canada lynx (Lynx canadensis) and caracals (Caracal caracal) (Kraberger et al., 2021). In the present study, we characterize two novel anelloviruses in a Brazilian felid, the ocelot (Leopardus pardalis).

During an annual examination to verify the health status of the captive felids at Rio de Janeiro City Zoo (RIOZOO) - Brazil, a blood sample was collected from a nine-year-old female ocelot (Leopardus pardalis). This project was performed with the authorization number 64515 from the Brazilian Institute for the Environment and Renewable Natural Resources (IBAMA). Plasma was filtered, centrifuged and the supernatant was discarded, leaving the bottom 0.2 mL to digestion with several nucleases. Viral nucleic acid was then extracted using QIAamp Mini Elute Virus Spin Kit (QIAGEN) and subjected to complementary DNA (cDNA) synthesis using Superscript III First-Strand Synthesis Supermix Kit (ThermoFisher). The second strand cDNA synthesis was performed with Klenow Fragment 3’-->5’ exo- (NEB). To get a highly efficient purification with superior quality DNA without salt carryover, the Agencourt AMPure XP Kit (Sinapse Biotecnologia) was used. To quantify double-strand DNA (dsDNA), the QuBit dsDNA High Sensitivity Assay Kit (ThermoFisher) was used, and the library was prepared using the Nextera XT DNA Library Preparation Kit (Illumina). Next-generation sequencing was conducted using the Illumina MiSeq V2 500-cycle kit. FASTq reads were processed with an in-house pipeline that included quality trimming, host genome filtering (Felis catus - #GCF_000181335.3, Panthera tigris - #GCA_000464555.1 and Panthera pardus - #GCA_001857705.1) and two rounds of BLASTx against RefSeq virus and nr (non-redundant) NCBI databases, as described by (D’arc et al., 2020D’arc M, Moreira FRR, Dias CA, Souza AR, Seuánez HN, Soares MA, Tavares MCH and Santos AFA (2020) The characterization of two novel neotropical primate papillomaviruses supports the ancient within-species diversity model. Virus Evol 6:veaa036.).

A total of 1,562 reads were assigned to the Anelloviridae family. These reads were de novo assembled using Geneious v.11.1.2, and the two biggest contigs were then submitted to BLASTn and two different anellovirus reference genomes were retrieved as best hits. A consensus sequence was used to design specific PCR primers to obtain the complete genome using genomic DNA from blood. Novel sequences were evolutionarily contextualized with a comprehensive Anelloviridae ORF1 dataset combining a dataset containing all known Anelloviridae genera (Cosentino et al., 2022Cosentino MAC, D’arc M, Moreira FRR, Cavalcante LT de F, Mouta R, Coimbra A, Schiffler FB, Miranda TDS, Medeiros G, Dias CA et al. (2022) Discovery of two novel Torque Teno viruses in Callithrix penicillata provides insights on Anelloviridae diversification dynamics. Front Microbiol 13:1002963.) and one containing 220 novel sequences from multiple Felidae species (Kraberger et al., 2021Kraberger S, Serieys LE, Richet C, Fountain-Jones NM, Baele G, Bishop JM, Nehring M, Ivan JS, Newkirk ES, Squires JR et al. (2021) Complex evolutionary history of felid anelloviruses. Virology 562:176-189.). ORF1 genes were aligned by MAFFT v7.505 (Katoh and Standley, 2013Katoh K and Standley DM (2013) MAFFT multiple sequence alignment software version 7: Improvements in performance and usability. Mol Biol Evol 30:772-780.) and TrimAL v.1.4 (Capella-Gutiérrez et al., 2009Capella-Gutiérrez S, Silla-Martínez JM and Gabaldón T (2009) trimAl: A tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics 25:1972-1973.) was used to remove non informative regions with the “gappyout” option. A maximum likelihood phylogeny was inferred with IQ-TREE v.2.0.3 (Nguyen et al., 2015Nguyen L-T, Schmidt HA, von Haeseler A and Minh BQ (2015) IQ-TREE: A fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol Biol Evol 32:268-274.) and node support was estimated by 1,000 iterations to both metrics, the SH-like approximate likelihood ratio test (Guindon et al., 2010Guindon S, Dufayard J-F, Lefort V, Anisimova M, Hordijk W and Gascuel O (2010) New algorithms and methods to estimate maximum-likelihood phylogenies: Assessing the performance of PhyML 3.0. Syst Biol 59:307-321.) and Ultrafast bootstrap (Minh et al., 2013Minh BQ, Nguyen MAT and von Haeseler A (2013) Ultrafast approximation for phylogenetic bootstrap. Mol Biol Evol 30:1188-1195.). Tree visualization was obtained in R studio v.2022.12.0+353 with the package ggtree v.3.5.3 (Yu et al., 2017Yu G, Smith DK, Zhu H, Guan Y and Lam TT-Y (2017) Ggtree: An R package for visualization and annotation of phylogenetic trees with their covariates and other associated data. Methods EcolEvol 8:28-36.). Clustal Omega v.1.2.4 (Sievers et al., 2011Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K, Li W, Lopez R, McWilliam H, Remmert M, Söding J et al. (2011) Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol 7:539.) was used to calculate an identity matrix with the most closely related Felidae anellovirus sequences. Alignment and tree files can be found in Supplementary File S1 Supplementary material The following online material is available for this article: File S1 - The tree file with maximum likelihood phylogeny inferred to contextualize the new viruses in ocelot and the fasta files to corresponding nucleotide and amino acid sequence alignment can be found in https://github.com/matheus-cosentino/2023_Novel-anelloviruses-in-ocelot. .

A 2,003-bp full-length Anellovirusgenome infecting Leopardus pardalis was assembled. This virus was provisionally named WF10 strain 1 (accession number: MK069470). Annotation was performed by aligning each ORF of the Torque teno felis virus 2 (accession number: NC_038349) against the novel genome, which allowed the inference of homologous genome regions. Three open reading frames (ORF) encoding ORF 1 (1,224-bp), ORF 2 (321-bp) and ORF 3 (468-bp) were inferred. One partial genome was also assembled, comprising an incomplete ORF1 sequence with 1,173 nucleotides and was provisionally named WF10 strain 2 (accession number: OL449682). Felidae anelloviruses can be found in two major lineages and phylogenetic analysis of ORF1 gene grouped both new strains within feline anellovirus Group 1, in distinct lineages (Figure 1A). The WF10 strain 1 (accession number: MK069470) was inferred as a sister lineage to Torque teno felis virus (MT538137) (SH-aLRT = 100 / Bootstrap = 100). Sequences shared 73.8% of pairwise identity, confirming they belong to the Torque teno felid virus 3 species in accordance with findings in Kraberger et al. (2021Kraberger S, Serieys LE, Richet C, Fountain-Jones NM, Baele G, Bishop JM, Nehring M, Ivan JS, Newkirk ES, Squires JR et al. (2021) Complex evolutionary history of felid anelloviruses. Virology 562:176-189.). It is worth noting the great genetic divergence observed within Torque teno felid virus 3, indicating that more than one viral species might be found in the present lineage upon further sampling (Supplementary File S1 Supplementary material The following online material is available for this article: File S1 - The tree file with maximum likelihood phylogeny inferred to contextualize the new viruses in ocelot and the fasta files to corresponding nucleotide and amino acid sequence alignment can be found in https://github.com/matheus-cosentino/2023_Novel-anelloviruses-in-ocelot. ) (Figure 1B).

Figure 1 -
Maximum likelihood phylogeny of the ORF1 gene of the Anelloviridae family. Phylogeny of the ORF1 gene, inferred with a dataset of an alignment of 1183 aa length and 884 sequences under the LG+F+G4 model. The tree was midpoint-rooted for visualization purposes. Node shapes colored in black represent node support for SH-aLRT and Ultrafast Bootstrap equal or superior to 75. When only SH-aLRT was superior to this threshold, node shapes were represented in gray. Conversely, when only Ultrafast Bootstrap was superior, the white color was used. Nodes with support inferior to 75 in both parameters were not marked. Tip labels are colored according to the identified host, where orange tip labels represent anelloviruses identified in Puma concolor, dark yellow in Leopardus pardalis, gray in Felis catus and light yellow in other Felidae. (A) Maximum likelihood phylogeny of the ORF1 gene of the Anelloviridae family, with clades highlighted in yellow to evidence Leopardus pardalis anellovirus. (B) Monophyletic clade of Torque teno felid virus 3 genomes identified in L. pardalis. (C) Monophyletic clade of the novel putative species Torque teno ocelot virus.

The ORF1 of the WF10 strain 2 (OL449682) was retrieved as sister to the Torque teno felid virus 1 species clade (EF538878 - KM229766) with robust node support (SH-aLRT = 98 / Bootstrap = 100) (Figure 1C). As an outgroup, Torque teno viverrid virus 3 (LC387548) was recovered with high support (SH-aLRT = 98.3 / Bootstrap = 100), indicating that the present monophyletic lineage likely descends from an ancient virus that co-diverged along the Suborder Feliformia. Based on ICTV species delimitation criteria (identity below 69%) (Varsani et al., 2021Varsani A, Opriessnig T, Celer V, Maggi F, Okamoto H, Blomström A-L, Cadar D, Harrach B, Biagini P and Kraberger S (2021) Taxonomic update for mammalian anelloviruses (family Anelloviridae). Arch Virol 166:2943-2953.), the partial viral genome denoted a new species - provisionally named Torque teno ocelot virus - with 53.72% identity to the closest reference (accession number: EF538878).

The captive L. pardalis explored by HTS protocols was coinfected by two different species of Anelloviruses, one new strain of Torque teno felid virus 3 (accession number: MK069470) and a putative novel species provisionally named Torque teno ocelot virus (accession number: OL449682). Anelloviridae co-infection by multiple unique lineages is common in healthy humans (Arze et al., 2021Arze CA, Springer S, Dudas G, Patel S, Bhattacharyya A, Swaminathan H, Brugnara C, Delagrave S, Ong T, Kahvejian A et al. (2021) Global genome analysis reveals a vast and dynamic anellovirus landscape within the human virome. Cell Host Microbe 29:1305-1315.e6.), and this phenomenon was identified in felids as well (Kraberger et al., 2021Kraberger S, Serieys LE, Richet C, Fountain-Jones NM, Baele G, Bishop JM, Nehring M, Ivan JS, Newkirk ES, Squires JR et al. (2021) Complex evolutionary history of felid anelloviruses. Virology 562:176-189.). The characterization of these new viral species and sequences in a non-model animal expands Anelloviridae host span and may assist further studies aiming to characterize the complex evolutionary history of anelloviruses (Kraberger et al., 2021Kraberger S, Serieys LE, Richet C, Fountain-Jones NM, Baele G, Bishop JM, Nehring M, Ivan JS, Newkirk ES, Squires JR et al. (2021) Complex evolutionary history of felid anelloviruses. Virology 562:176-189.; Cosentino et al., 2022Cosentino MAC, D’arc M, Moreira FRR, Cavalcante LT de F, Mouta R, Coimbra A, Schiffler FB, Miranda TDS, Medeiros G, Dias CA et al. (2022) Discovery of two novel Torque Teno viruses in Callithrix penicillata provides insights on Anelloviridae diversification dynamics. Front Microbiol 13:1002963.).

Acknowledgements

We declare no conflicts of financial or personal interests. This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico/CNPq (grant number 313005/2020-6 to AFS) and Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro/FAPERJ (grant numbers E26/211.040/2019, E26/211.355/2021 and E26/201.193/2022 to AFS). L.T.F.C. has been sponsored by a Ph.D. scholarship from CNPq/FAPERJ by the Graduate Program in Genetics at Universidade Federal do Rio de Janeiro, Brazil. We thank employees and health care workers from Fundação Rio-Zoo for sample collection. We acknowledge the use of equipments from Laboratório de Uso Comum e Apoio Técnico from the Department of Genetics, Universidade Federal do Rio de Janeiro (Rio de Janeiro, Brazil).

References

  • Arze CA, Springer S, Dudas G, Patel S, Bhattacharyya A, Swaminathan H, Brugnara C, Delagrave S, Ong T, Kahvejian A et al (2021) Global genome analysis reveals a vast and dynamic anellovirus landscape within the human virome. Cell Host Microbe 29:1305-1315.e6.
  • Capella-Gutiérrez S, Silla-Martínez JM and Gabaldón T (2009) trimAl: A tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics 25:1972-1973.
  • Cosentino MAC, D’arc M, Moreira FRR, Cavalcante LT de F, Mouta R, Coimbra A, Schiffler FB, Miranda TDS, Medeiros G, Dias CA et al (2022) Discovery of two novel Torque Teno viruses in Callithrix penicillata provides insights on Anelloviridae diversification dynamics. Front Microbiol 13:1002963.
  • D’arc M, Moreira FRR, Dias CA, Souza AR, Seuánez HN, Soares MA, Tavares MCH and Santos AFA (2020) The characterization of two novel neotropical primate papillomaviruses supports the ancient within-species diversity model. Virus Evol 6:veaa036.
  • de Souza WM, Fumagalli MJ, de Araujo J, Sabino-Santos G Jr, Maia FGM, Romeiro MF, Modha S, Nardi MS, Queiroz LH, Durigon EL et al(2018) Discovery of novel anelloviruses in small mammals expands the host range and diversity of the Anelloviridae. Virology 514:9-17.
  • Guindon S, Dufayard J-F, Lefort V, Anisimova M, Hordijk W and Gascuel O (2010) New algorithms and methods to estimate maximum-likelihood phylogenies: Assessing the performance of PhyML 3.0. Syst Biol 59:307-321.
  • Kaczorowska J and van der Hoek L (2020) Human anelloviruses: Diverse, omnipresent and commensal members of the virome. FEMS Microbiol Rev 44:305-313.
  • Katoh K and Standley DM (2013) MAFFT multiple sequence alignment software version 7: Improvements in performance and usability. Mol Biol Evol 30:772-780.
  • Kraberger S, Serieys LE, Richet C, Fountain-Jones NM, Baele G, Bishop JM, Nehring M, Ivan JS, Newkirk ES, Squires JR et al (2021) Complex evolutionary history of felid anelloviruses. Virology 562:176-189.
  • Minh BQ, Nguyen MAT and von Haeseler A (2013) Ultrafast approximation for phylogenetic bootstrap. Mol Biol Evol 30:1188-1195.
  • Ng TFF, Wheeler E, Greig D, Waltzek TB, Gulland F and Breitbart M (2011) Metagenomic identification of a novel anellovirus in Pacific harbor seal (Phocavitulinarichardsii) lung samples and its detection in samples from multiple years. J Gen Virol 92:1318-1323.
  • Nguyen L-T, Schmidt HA, von Haeseler A and Minh BQ (2015) IQ-TREE: A fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol Biol Evol 32:268-274.
  • Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K, Li W, Lopez R, McWilliam H, Remmert M, Söding J et al (2011) Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol 7:539.
  • Varsani A, Opriessnig T, Celer V, Maggi F, Okamoto H, Blomström A-L, Cadar D, Harrach B, Biagini P and Kraberger S (2021) Taxonomic update for mammalian anelloviruses (family Anelloviridae). Arch Virol 166:2943-2953.
  • Weber MN, Mosena ACS, da Silva MS, Canova R, de Lorenzo C, Olegário JC, Budaszewski RF, Baumbach LF, Soares JF, Sonne L et al (2020) Virome of crab-eating (Cerdocyonthous) and pampas foxes (Lycalopex gymnocercus) from southern Brazil and Uruguay. Infect Genet Evol 85:104421.
  • Yu G, Smith DK, Zhu H, Guan Y and Lam TT-Y (2017) Ggtree: An R package for visualization and annotation of phylogenetic trees with their covariates and other associated data. Methods EcolEvol 8:28-36.
  • Author Contributions

    LTFC conceived the study, conducted the experiments, analyzed the data and wrote the manuscript. MACC analyzed and curated the data and co-wrote the manuscript. MD conducted the experiments and analyzed the data. FRRM and RM analyzed and curated the data. AMA and FT collected and processed biological specimens. AFS and MAS conceived the study.All authors read and approved the final version.
  • Data Availability

    The aneloviruses genome and partial genome were deposited in DDBJ/ENA/GenBank under accession numbers MK069470 and OL449682.

Supplementary material

The following online material is available for this article:

File S1 - The tree file with maximum likelihood phylogeny inferred to contextualize the new viruses in ocelot and the fasta files to corresponding nucleotide and amino acid sequence alignment can be found in https://github.com/matheus-cosentino/2023_Novel-anelloviruses-in-ocelot.

Edited by

Associate Editor:

Rogerio Margis

Publication Dates

  • Publication in this collection
    04 Dec 2023
  • Date of issue
    2023

History

  • Received
    17 Feb 2023
  • Accepted
    06 Oct 2023
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