# Absence of accessory genes in a divergent simian T-lymphotropic virus type 1 isolated from a bonnet macaque (Macaca radiata)

Philippe Afonso, Zahra Fagrouch, Martin Deijs, Henk Niphuis, Willy Bogers, Antoine Gessain, Lia van Der Hoek, Ernst Verschoor

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Philippe Afonso, Zahra Fagrouch, Martin Deijs, Henk Niphuis, Willy Bogers, et al.. Absence of accessory genes in a divergent simian T-lymphotropic virus type 1 isolated from a bonnet macaque (Macaca radiata). PLoS Neglected Tropical Diseases, 2019, 13 (7), pp.e0007521. 10.1371/journal.pntd.0007521.pasteur-02195672

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RESEARCH ARTICLE

# Absence of accessory genes in a divergent simian T-lymphotropic virus type 1 isolated from a bonnet macaque (*Macaca radiata*)

Philippe V. Afonso¹*, Zahra Fagrouch², Martin Deijs³, Henk Niphuis², Willy Bogers², Antoine Gessain¹, Lia van der Hoek³, Ernst J. Verschoor¹*

1 Unité EPVO, Institut Pasteur, UMR 3569 CNRS, Paris, France, 2 Department of Virology, Biomedical Primate Research Centre, Rijswijk, the Netherlands, 3 Laboratory of Experimental Virology, Department of Medical Microbiology, Amsterdam UMC, University Of Amsterdam, Amsterdam, the Netherlands

© These authors contributed equally to this work.

* philippe.afonso@pasteur.fr (PVA); verschoor@bprc.nl (EJV)

<figure><img src="image_2.png" /></figure>

## Abstract

### Background

Primate T-lymphotropic viruses type 1 (PTLV-1) are complex retroviruses infecting both human (HTLV-1) and simian (STLV-1) hosts. They share common epidemiological, clinical and molecular features. In addition to the canonical *gag*, *pol*, *env* retroviral genes, PTLV-1 purportedly encodes regulatory (i.e. Tax, Rex, and HBZ) and accessory proteins (i.e. P12/8, P13, P30). The latter have been found essential for viral persistence *in vivo*.

### Methodology/Principal findings

We have isolated a STLV-1 virus from a bonnet macaque (*Macaca radiata*–*Mra18C9*), a monkey from India. The complete sequence was obtained and phylogenetic analyses were performed. The *Mra18C9* strain is highly divergent from the known PTLV-1 strains. Intriguingly, the *Mra18C9* lacks the 3 accessory open reading frames. In order to determine if the absence of accessory proteins is specific to this particular strain, a comprehensive analysis of the complete PTLV-1 genomes available in Genbank was performed and found that the lack of one or many accessory ORF is common among PTLV-1.

## Conclusion

This study raises many questions regarding the actual nature, role and importance of accessory proteins in the PTLV-1 biology.

### Author summary

Primate T-lymphotropic viruses type 1 (PTLV-1) are complex retroviruses infecting both human (HTLV-1) and simian (STLV-1) hosts. It has been shown that the persistence and pathogenesis of these viruses depend on the expression of small, accessory proteins.

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**OPEN ACCESS**

**Citation:** Afonso PV, Fagrouch Z, Deijs M, Niphuis H, Bogers W, Gessain A, et al. (2019) Absence of accessory genes in a divergent simian T-lymphotropic virus type 1 isolated from a bonnet macaque (*Macaca radiata*). PLoS Negl Trop Dis 13 (7): e0007521. https://doi.org/10.1371/journal.pntd.0007521

**Editor:** Gregory Gromowski, WRAIR, UNITED STATES

**Received:** March 22, 2019

**Accepted:** June 5, 2019

**Published:** July 8, 2019

**Copyright:** © 2019 Afonso et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

**Data Availability Statement:** The PTLV-1 complete sequence is available from the Genbank database (accession number MK639100) https://www.ncbi.nlm.nih.gov/nuccore/mk639100

**Funding:** This study received funding from the "Investissement d'Avenir" as part of a "Laboratoire d'Excellence" (LabEx) French research program: Integrative Biology of Emerging Infectious Diseases (ANR10-LBX-62 IBEID). It was also funded by an internal BPRC institutional grant. The
funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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

A bonnet macaque (a monkey present in India) was found infected with STLV-1. The genome was sequenced and found quite divergent from the other STLV-1 genomes previously described. Intriguingly, this virus does not encode accessory proteins. Analysis of other available sequences found that most strains lack at least one accessory gene. Thus the importance and the role of these proteins in the PTLV-1 biology should be revisited.

Introduction

The Primate T-lymphotropic virus type 1 (PTLV-1) constitutes a group of deltaretroviruses infecting humans (HTLV-1) or non-human primates (STLV-1). Phylogenetic studies led to the definition of PTLV-1 viral subtypes [1]. Viruses belonging to the African PTLV-1 subtypes (i.e. subtypes b, d, e, f and g) are found both in humans and non-human primates (NHPs), and the human and simian strains are undistinguishable. Continuous zoonotic spillovers of these strains occur, following severe bites by infected non-human primates, or in the context of bushmeat hunting and handling [2-4]. Two subtypes are exclusively human: PTLV-1a is found in human populations scattered throughout the globe, while PTLV-1c is found in indigenous people of the Australomelanesian continent. In contrast, a group of STLV-1 has been described in macaques and great apes in Asia [5-7]. These viruses are genetically distant from other subtypes (a fact that led some researchers to consider some of them as a separate STLV, STLV-5) [8] and have never been found in humans to date.

HTLV-1 is estimated to infect at least 5-10 million people worldwide [1]. HTLV-1 is the eti-
ological agent of many severe diseases, ranging from an aggressive lymphoproliferation, the
adult T-cell leukemia/lymphoma (ATL), to inflammatory syndromes, such as a neurodegener-
ative disease called HTLV-1 associated myelopathy or tropical spastic paraparesis (HAM/
TSP). Pathogenesis does not seem to be restricted to a certain HTLV-1 subtype; for instance,
ATL cases have been reported in patients infected with HTLV-1a, -1b or 1c [9, 10]. STLV-1 is
also oncogenic. ATL have been reported in many simian species, from Macaques to Gorilla
[11, 12].

Deltaretroviruses are complex retroviruses. In addition to the canonical *gag*, *pol*, *env* retroviral genes, the PTLV-1 genome has a series of open reading frames (ORFs) encoding regulatory and accessory proteins [13–15]. Regulatory proteins are essential for viral expression and propagation both *in vitro* and *in vivo*. In contrast, accessory proteins are optional for viral expression *in vitro*, but required for viral persistence *in vivo*. There are three regulatory proteins in PTLV-1: Tax, Rex, and HBZ. PTLV-1 purportedly encodes four accessory proteins, named P12/P8 (encoded by ORF I), P13 and P30 (encoded by ORF II).

This study reports the first STLV-1 genome from a virus infecting a bonnet macaque (*Macaca radiata*), a macaque species from India. The virus replicated well in cell culture, and this material was used for sequencing, a full genome analysis of this divergent Asian STLV-1 strain was performed. While the canonical ORFs, as well as the ORFs encoding regulatory proteins were found conserved, the ORFs encoding the accessory proteins were absent or disrupted. This latter observation is intriguing as these proteins are purportedly essential for viral persistence *in vivo*. In order to determine if the absence of accessory proteins is specific to this particular strain, a comprehensive analysis of the complete genomes of different PTLV-1 subtypes available in GenBank was performed. Except for HTLV-1a strains, strains from other PTLV-1 subtypes all lacked at least one accessory ORF. This raises many questions regarding the actual role and importance of accessory proteins in the PTLV-1 biology.
# Results

## STLV detection in a sample from bonnet macaque

The bonnet macaque Mra18C9 was housed in an animal rescue center and material was sent to us for routine diagnostic screening.

Serological tests for PTLV-1 gave conflicting results. While the serum tested negative on an in-house ELISA, it reacted positively when tested by a local hospital laboratory. These conflicting results led us to perform additional serological testing using the INNO-LIA HTLVI/II assay (Fujirebio Europe, Ghent, Belgium). The serum reacted strongly with PTLV-1/2 env gp46 and gp21 antigens, but did not react with any type-specific peptide. It was thus considered as an indeterminate sero-reactivity.

As the serological tests were inconclusive, we performed a diagnostic PCR screening on the DNA isolated from PBMCs, by using a generic nested PCR assay able to amplify a fragment of the *tax/rex* region of both PTLV-I and -II. The PCR tested positive, and BLAST analysis on the 118 bp-long fragment revealed high nucleotide identity percentage (>93%) with STLV-1 identified from Formosan macaques (*Macaca cyclopis* STLV108, GenBank accession number: KM268809), stump-tailed macaques (*M. arctoides* MarB43 and marc1, GenBank AY590142 and U76625 respectively), and long-tailed macaques (*M. fascicularis* MFA-C194, GenBank U59132).

The full-length STLV-1 genome infecting the bonnet macaque Mra18C9 was obtained by using a combination of the sequence-independent VIDISCA-454 technique, which generated 4 segments scattered along the STLV genome, and standard PCRs to bridge the sequence gaps and sequence the LTR.

## Mra18C9 STLV-1 encodes the canonical retroviral proteins, as well as the regulatory proteins

The flanking long terminal repeats (LTRs) comprise a TATA box, a polyadenylation signal, and three Tax-responsive elements (TxRE) type 1. The TxRE-2 may not be fully functional as an insertion of two nucleotides is present at position 210–211. Interestingly, the LTR was quite divergent from previously published PTLV-LTR sequences, as the nucleotide identity was lower than 80% (Table A in S1 Text).

The canonical retroviral ORFs (*gag*, *pro*, *pol*, *env*) are conserved, as well as the sequences necessary for the *gag-pro* and *pro-pol* ribosomal frame-shifts (Fig 1). Nucleotide and amino acid sequence comparisons confirm that Mra18C9 STLV-1 is a highly divergent PTLV-1 strain. At the nucleotide level, the *gag*, *pro*, *pol*, and *env* genes show not more than 80% identity with their counterparts from other PTLV, while their encoded proteins differ 10–15% in amino acid identity (Tables A-B in S1 Text).

The ORFs corresponding to the regulatory proteins Tax, Rex and HBZ are also present (Fig 1), and splice-acceptor and -donor sites are preserved. The *tax* gene sequence shares roughly 82% nucleotide identity, and the encoded Tax protein has a 88–90% amino-acid identity to other PTLV-1 sequences (Tables A-B in S1 Text). Importantly, the PDZ-binding motif of Tax, which is necessary for the interaction of Tax with cellular factors, is mutated in Mra18C9 genome: the ETEV motif is changed into an ETEI motif.

Phylogenetic analyses of the concatenated *gag-pol-env-tax* genes clearly demonstrated that Mra18C9 falls into the Asian STLV-1 group (Fig 2A). Macaque STLV-1 strains all form long branches, and do not aggregate in a monophyletic group. Instead, they form a paraphyletic group. In order to better estimate the relative position of this strain, phylogenetic analyses were performed using the commonly studied LTR and *env* sequences (Fig 2B and 2C). The
<figure><img src="image_4.png" /><figcaption>**Fig 1.** Genomic organization of STLV-1 Mra18C9. Nucleotide numbering of splicing sites and initiation/stop codon are according to the Mra18C9 proviral genome. The upper boxes represent the LTRs. The other boxes represent the ORFs. * indicate the frame-shifting sites.</figcaption></figure>

https://doi.org/10.1371/journal.pntd.0007521.g001

different phylogenetic methods (Neighbor-Joining, and a Bayesian approach) gave very similar results. Results of the Bayesian analyses are shown in Fig 2. The Mra18C9 strain was consistently found in a long branch among the macaque strains; the closest known sequences were the *Macaca arctoides* strains (when considering either LTR or env sequences) (Fig 2B and 2C) and the *Macaca mulata* strains MMU-R18 and R22 (when considering the LTR) (Fig 2B).

In conclusion, the Mra18C9 strain has a typical PTLV-1 organization, but this virus is highly divergent from other PTLV, suggesting a long and independent evolution.

## Mra18C9 STLV-1 lacks accessory genes

Sequence analysis suggests that the accessory proteins encoded by ORF-I may not be functional in the Mra18C9 strain. Indeed, the start codon of the open-reading frame is mutated (ATG > ⁶⁸²¹GCG), and multiple stop codons are found. Furthermore, the splice-acceptor site is mutated (ATK ⁶³⁸⁰CAG/CAAC > Mra18C9 ⁶⁷¹⁹TAA/CAAC) and may not be functional either. Thus, Mra18C9 does not encode the P12/P8 proteins.

The splice-acceptor domain necessary for a P30 protein-encoding mRNA is also mutated (ATK ⁶⁴⁷⁵TAG/CACT > Mra18C9 ⁶⁸¹⁴GGA/CGCT), suggesting that P30 may not be produced by the Mra18C9 strain. This is reinforced by the presence of an early stop codon in the ORF.

Similarly, the splice-acceptor domain, necessary for a P13-encoding mRNA, is also mutated (ATK ⁶⁸⁷²CAG/CAGG > MRA18C9 ⁷²²³CAG/TTGG), and therefore the mRNA may not be synthesized. In addition, the ORF is also altered: the start codon is conserved but the stop codon is mutated (TAA > ⁷⁵⁴⁹CAA). The resulting protein would then be much longer (137 amino-acid long putative protein instead of 87 aa-long), which can rigorously influence its functionality.

Collectively, our analyses indicate that the STLV-1 Mra18C9 strain completely lacks the accessory genes, which have been reported in HTLV-1a strains.
<figure><img src="image_5.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_7.png" /></figure>

**Fig 2. Phylogenetic analyses of the MraC18C9 STLV-1 strain.** The presented phylogenetic trees were all generated by a Bayesian approach. The scale bar represents nucleotide substitutions per site. Values correspond to posterior
probabilities. A—Phylogenetic tree generated by a Bayesian approach using a concatenation of *gag-pol-env-tax* genes (alignment of 5820 nucleotides). PTLV-4 sequences (GabL14, Ggo51461 and Ggo 50539) were used as outgroup. B—Phylogenetic tree generated using the LTR sequence (alignment of 701 nucleotides). The tree is unrooted and comprises most PTLV-1 complete LTR sequences available. C- Phylogenetic tree using an alignment of an *env* gene fragment of 483 nucleotides. The tree is unrooted and comprises most PTLV-1 *env* sequences available. The denomination African PTLV-1 comprises HTLV-1a, and PTLV-1b, d, e, f, and g strains. MM stands for *Macaca maura*; MMU stands for *M. mulatta*; MFA stands for *M. fascicularis*; MAC and mar stand for *Macaca arctoides*; TE stands for M. tonkeana; HS stands for *Hylobates syndactylus*; Ou stands for *Orangutan*; Mra stands for M.radiata; Cxxx strains were isolated from *Hylobates pileatus*.

https://doi.org/10.1371/journal.pntd.0007521.g002

## Many published PTLV-1 strains lack one or several accessory proteins

We wondered if the absence of accessory genes was specific to the Asian macaque STLV-1 strain isolated from *M. radiata*. For this purpose, an *in silico* analysis of PTLV-1 complete genomes available in GenBank was performed (Table 1 and Tables C-F in S1 Text).

Accessory proteins were conserved in HTLV-1a strains and in the available STLV-1b strain [12]. In contrast, the macaque STLV-1 strains lacked all of the accessory genes. The loss of accessory genes in MarB43 was previously reported [7]. All the other strains (either HTLV-1b, c; or STLV-1e, f, g) lacked at least 1 accessory protein. For instance, the HTLV-1b strains lack both P12 and P30, and as previously mentioned HTLV-1c strains lack P12 [18].

In conclusion, although accessory genes have been found important for viral infection and persistence in HTLV-1a, many PTLV-1 strains lack one or more accessory genes.

## Discussion

PTLV-1 infects a wide range of non-human primates (NHPs). We report the first strain infecting a bonnet macaque (*Macaca radiata*). Complete genome analysis revealed that it is a highly divergent strain when compared to the currently known PTLVs. This would explain the conflicting results obtained by ELISA, as well as its low reactivity (indeterminate profile) on the commercial Western blot. Phylogenetic analyses positioned the Mra18C9 strain among other

<table><caption>Table 1. Many PTLV-1 lack one or more accessory proteins.</caption><thead><tr><th rowspan="2"></th><th colspan="3">Putative protein</th></tr><tr><th>P12</th><th>P13</th><th>P30</th></tr></thead><tbody><tr><td>HTLV-1a</td><td>present (99 aa)*</td><td>present (87 aa)</td><td>present (241 aa)</td></tr><tr><td>HTLV-1b</td><td>absent</td><td>present (87 aa)</td><td>absent</td></tr><tr><td>STLV-1b</td><td>present (86 aa)</td><td>present (87 aa)</td><td>present (241 aa)</td></tr><tr><td>HTLV-1c</td><td>absent</td><td>present (87 aa)</td><td>present (240 or 242 aa)</td></tr><tr><td>STLV-1e</td><td>present (99 aa)</td><td>absent</td><td>present (253 aa)</td></tr><tr><td>STLV-1f</td><td>absent</td><td>present (87 aa) or absent</td><td>present (241 aa)</td></tr><tr><td>PTLV-1 smm</td><td>present (86 aa)</td><td>absent</td><td>present (241 aa)</td></tr><tr><td>STLV-1g</td><td>present (86 aa)</td><td>absent</td><td>absent</td></tr><tr><td>TE-4</td><td>absent</td><td>absent</td><td>absent</td></tr><tr><td>Chaplin</td><td>absent</td><td>absent</td><td>absent</td></tr><tr><td>MarB43</td><td>absent</td><td>absent</td><td>absent</td></tr></tbody></table>

Many PTLV-1 strains (Table C in S1 Text) were analyzed and the presence of P12, P13 et P30 were addressed, focusing mostly on the conservation of the splicing sites, the presence of the start codon and the absence of early stop codon (Tables D-F in S1 Text).

* although the complete sequences of HTLV-1a all encode a 99 aa-long protein, many shorter versions of the proteins have been reported [16, 17].

https://doi.org/10.1371/journal.pntd.0007521.t001
Asian macaque STLV-1 viruses. This confirms the large heterogeneity within the Asian PTLV-1 clade as was described previously [5, 6].

In phylogenetic trees, Asian macaque STLV-1 strains form very long branches when compared to African (including HTLV-1a) strains. This could suggest that these viruses have evolved independently in Asia, with their simian host, for a very long period [19]. The introduction of STLV in Asian NHP has been dated at approximately 200.000 years ago [6, 7]. Under this assumption, the viruses have coevolved for long with their host, and the phylogeny of Asian STLV-1 should mirror the evolution of Asian primates. However, this is not the case for macaque STLV-1. First, they do not form a monophyletic group; instead they form a paraphyletic group. The strains are organized as a ladder, branching deeply next to the PTLV-1 root (Fig 2A). One could argue that this particular topology results partially from genetic saturation. Moreover, among Asian STLV-1 there are only a few monophyletic groups corresponding to simian species (Fig 2B and 2C). Apart from *M. fuscata* and *M. arctoides* STLV-1, the other clades are composed of sequences of mixed origins, with STLVs from Pongides and Hylobatides that infect macaques. Even when focusing on sequences isolated from macaques, the distribution of the sequences does not follow the known Macaca phylogeny [20, 21]. Together, this points to interspecies transmission (between macaques or from macaques to orangutans or gibbons) of such viruses, as others previously suggested [5, 6]. Interspecies transmission of STLV-1 has been previously reported, although in the context of captivity [22, 23]; the evidence of such transmission *in natura* is mostly inferred by phylogenetic analysis [6, 24]. Thus, one could argue that the long branching is not only due to a long independent evolution in Asia, but also to an accelerated mutation rate for these strains, which could be related to frequent interspecies transmission.

The canonical and the regulatory proteins are present in the genome of the Mra18C9 STLV-1. The Mra18C9 STLV-1 strain is a functional, replicative virus (at least *in vitro*, as it could be amplified on SupT1 cells). However, the strain may have an attenuated phenotype. First, the Tax-responsive element 2 (TxRE2) seems disrupted due to a 2-nucleotide insertion [25]. This may lead to a lower basal transcription and reduced viral expression [26]. Second, the viral transactivator Tax has a mutated PDZ-binding motif (PBM). The Tax PBM is essential for sustained proliferation both *in vitro* and *in vivo* [27, 28]. A single mutation of the last amino acid of the PBM was shown to be sufficient to abrogate its function [29-31].

The pX region of the Mra18C9 STLV-1 strain lacks both ORF-I and ORF-II. We first hypothesized that the loss of these ORFs could render the virus less pathogenic. Indeed, accessory proteins have been shown to be important for viral persistence and pathogenesis in HTLV-1a. Mutations of accessory ORFs limit the replicative capacity of HTLV-1a in a rabbit model [32]. Similarly, in the closely related bovine leukemia virus, mutations of the homologue ORFs render the virus attenuated [33]. It was proposed that Australian HTLV-1c strains, because they lack the P12 protein, might be less oncogenic [18]. Nevertheless, ATL cases have been reported in HTLV-1c-infected individuals [9]. Moreover, while the absence of accessory proteins seems to be a general feature of macaque STLV-1, ATL cases have been reported in naturally infected macaques [11]. Thus, even in the absence of accessory proteins, STLV-1 still present an oncogenic potential.

Although STLV-1 is highly prevalent among Asian NHPs, and humans are in frequent contact with macaques and may acquire other retroviral infection, such as simian foamy viruses that are endemic in several species of Asians monkeys [34], no Asian STLV zoonotic transmission has been reported so far. One could hypothesize that the absence of ORFI and ORFII in macaque STLV-1 can limit viral transmission and propagation. Indeed, HTLV-1a P12 and P30 have been found to be essential for viral replication in human and macaque dendritic cells [35], which can play a key role in viral transmission. However, this proposition is nullified by
our thorough analysis of the different PTLV-1 subtypes. Indeed, HTLV-1b, which is a very common virus in Central Africa, persists and propagates in humans despite the absence of P12 and P30 (Table 1).

In conclusion, the role and importance of accessory proteins needs to be reconsidered in light of the analysis of the different PTLV-1 strains. Indeed, as most studies have focused on HTLV-1a, the 3 accessory proteins were found conserved and their function in viral persistence and transmission was believed to be essential [17, 32, 35]. This study indicates that some PTLV-1 can persist in the absence of one or many accessory proteins. This raises the question of a dispensable role of these proteins, or the presence of other accessory proteins yet to be identified in the other PTLV-1 subtypes.

# Methods

## Ethics statement

The Bonnet macaque was housed in an animal rescue center (animal shelter VZW, Belgium) and material was sent to the BPRC for viral diagnostic screening.

The BPRC is fully licensed by the Netherlands Food and Consumer Product Safety Authority (belonging to the Ministry of Agriculture, Nature and Food Quality) to work with animal products and perform diagnostic services for third parties (Approval no. 1926950).

## HTLV diagnosis

Sera were assayed for antibodies to PTLV-1 using an in-house developed serological test with purified, lysed HTLV-1 particles as coating antigen (Advanced Biotechnologies Inc., Eldersburg, USA) [36]. Additionally, the serum from the bonnet macaque was also tested by ELISA in a local hospital laboratory (DDL, Delft, The Netherlands), and a western blot (WB) analysis using the INNO-LIA HTLVI/II assay (Fujirebio Europe, Gent, Belgium).

Genomic DNA was isolated from whole blood using the QIAamp DNA blood mini kit (QIAGEN Benelux B.V., Venlo, The Netherlands). A 118 bp tax/rex gene fragment was amplified using the TR101/TR102 and SK43/SK44 nested primer sets, as previously described [37, 38]. Furthermore, A pan-STLV PCR was performed with primers PH1F and PH2R, as described by van Dooren et al. [39]. The amplified fragment of 192 bp fully overlapped the 118 bp fragment from the first PCR.

## Virus isolation and sequencing

The virus discovery method VIDISCA-454 was used for the analysis of cell culture supernatant from the PTLV-infected SupT1 cell culture, as previously described [40]. In brief, PBMCs were isolated on a Ficoll and stimulated for 2 days with PHA (1 ug/ml final). Next, they were co-cultured with SupT1 cells until CPE was visible (2–3 weeks). The cell culture supernatant was centrifuged to remove cell debris and treated with TURBO DNase (Ambion, Thermo Fisher Scientific, Breda, The Netherlands). Next, nucleic acids were isolated with a QIAamp Viral RNA Mini Kit (QIAGEN Benelux BV, Venlo, the Netherlands) and reverse-transcribed with SuperScript II (Thermo Fisher Scientific) using non-ribosomal random hexamers. Subsequently, second strand DNA synthesis was performed with 5 U of Klenow fragment (New England Biolabs, Bioke, Leiden, The Netherlands). Double-stranded DNA was purified by phenol/chloroform extraction and ethanol precipitation and digested with Mse I restriction enzyme (New England Biolabs). Adaptors with different Multiplex Identifier sequences (MIDs) were ligated to the digested fragments of the different samples. Before PCR
amplification, the fragments were purified with AMPure XP beads (Agencourt AMPure XP PCR, Beckman Coulter, Woerden, The Netherlands).

A 28-cycles PCR with adaptor-annealing primers was performed. The program of the PCR-reaction was: 5 min 95°C, and cycles of 1 min 95°C, 1 min 55°C, and 2 min 72°C, followed by 10 min 72°C and 10 min 4°C. After purification with AMPure XP beads, the purified DNA was quantified with the Quant-it dsDNA HS Qubit kit (Invitrogen, Carlsbad, CA, USA) and diluted to 10⁷ copies/µl. Samples were pooled and Kapa PCR (Kapa Biosystems, Wilmington, MA, USA) was performed to determine the quantity of amplifiable DNA in each pool. Subsequently, the Bioanalyser (hsDNA chip, Agencourt) was used to determine the average nucleotide length of the libraries. The pools were diluted until 10⁶ copies/µl, titrated with beads (DNA:beads ratio of 0.5:1, 1:1, 2:1 and 4:1) and used in an emulsion PCR according to the supplier's protocol (LIB-A SV emPCR kit). Sequencing was done on a 2 region GS FLX Titanium PicoTiterPlate (70x75) with the GS FLX Titanium XLR 70 Sequencing kit (Roche, Woerden, The Netherlands). Sequence reads were analyzed using the blastn and blastp algorithms (National Center for Biotechnology Information).

PCR primers were designed on basis of the four fragments that were obtained from the VIDISCA as well as from the consensus LTR sequence derived from the alignment of other PTLV-1 (Tables G-H in S1 Text). PCR reactions were performed in a final volume of 50 µl. Each amplification reaction was performed in 1x DreamTaq buffer containing 200 µM of each dNTP, 50 pmol of each primer, and 1.25 U DreamTaq DNA polymerase (Thermo Fisher Scientific). The amplification reactions were performed for 35 cycles consisting of a 30 s denaturation step at 94°C, a 30 s annealing step at 55°C and an elongation step of 150 sec at 72°C. Amplicon purification and sequencing was performed essentially as described above, but by using a primer-walking sequencing strategy. Sequences were assembled with the SeqMan Pro software (DNASTAR, Inc., Madison, USA). The resulting contig was further analyzed using the MacVector software package (MacVector, Inc., Cambridge, UK). The complete genome of STLV-1 Mra18C9 has been deposited at GenBank with accession number MK639100.

## Phylogenetic analyses

LTR (701 bases) and env (483 bases) sequences were aligned together with most sequences available on GenBank using DAMBE [41]. An alignment of concatenated gag-pol-env-tax sequences (5820 bases) was also generated with the sequences of complete PTLV-1 genomes available on GenBank.

Phylogenetic trees resulted from analyses using the neighbor-joining method performed with the PAUP* v4.0b10. The final alignment was submitted to the Modeltest program (version 3.6) to select, according to the Akaike information criterion, the best model to apply to phylogenetic analyses. The selected substitution models were: Tamura-Nei for env and concatenated gag-pol-env-tax, and general time-reversible (GTR + γ) for the LTR. To test the robustness of the tree topologies, 1,000 bootstrap replicates were performed. Bayesian approaches were inferred with the MrBayes 3.2.7 program and robustness was tested with posterior probabilities. Both methods raised similar phylogenetic tree topology.

## Analysis of ORF in PTLV-1 complete genomes

The analysis was performed following the alignment of complete genomes available in Gen-Bank (Table C in S1 Text). Splicing acceptor and donor sequences were previously described for ATK [13]. Frameshift sites had been previously identified [42].
**Supporting information**

S1 Text. Supplementary tables.

(PDF)

**Author Contributions**

**Conceptualization:** Philippe V. Afonso, Antoine Gessain, Ernst J. Verschoor.

**Data curation:** Lia van der Hoek.

**Funding acquisition:** Willy Bogers.

**Investigation:** Zahra Fagrouch, Martin Deijs, Henk Niphuis, Ernst J. Verschoor.

**Supervision:** Ernst J. Verschoor.

**Writing – original draft:** Philippe V. Afonso.

**Writing – review & editing:** Antoine Gessain, Ernst J. Verschoor.

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