key: cord-0709303-0cjgxezw
authors: Weber, M.N.; Mosena, A.C.S.; da Silva, M.S.; Canova, R.; de Lorenzo, C.; Olegário, J.C.; Budaszewski, R.F.; Baumbach, L.F.; Soares, J.F.; Sonne, L.; Varela, A.P.M.; Mayer, F.Q.; de Oliveira, L.G.S.; Canal, C.W.
title: Virome of crab-eating (Cerdocyon thous) and pampas foxes (Lycalopex gymnocercus) from southern Brazil and Uruguay
date: 2020-06-21
journal: Infect Genet Evol
DOI: 10.1016/j.meegid.2020.104421
sha: 419e82486719ec5f98e35527323b8ef7e7f55baf
doc_id: 709303
cord_uid: 0cjgxezw

Crab-eating (Cerdocyon thous) and Pampas foxes (Lycalopex gymnocercus) are wild canids distributed in South America. Domestic dogs (Canis lupus familiaris) and wild canids may share viral pathogens, including rabies virus (RABV), canine distemper virus (CDV), and canine parvovirus 2 (CPV-2). To characterize the virome of these wild canid species, the present work evaluated the spleen and mesenteric lymph node virome of 17 crab-eating and five Pampas foxes using high-throughput sequencing (HTS). Organ samples were pooled and sequenced using an Illumina MiSeq platform. Additional PCR analyses were performed to identify the frequencies and host origin for each virus detected by HTS. Sequences more closely related to the Paramyxoviridae, Parvoviridae and Anelloviridae families were detected, as well as circular Rep-encoding single-stranded (CRESS) DNA viruses. CDV was found only in crab-eating foxes, whereas CPV-2 was found in both canid species; both viruses were closely related to sequences reported in domestic dogs from southern Brazil. Moreover, the present work reported the detection of canine bocavirus (CBoV) strains that were genetically divergent from CBoV-1 and 2 lineages. Finally, we also characterized CRESS DNA viruses and anelloviruses with marked diversity. The results of this study contribute to the body of knowledge regarding wild canid viruses that can potentially be shared with domestic canids or other species.

agricultural borders, or spreading of urban communities on the natural environment, as well as the habitat fragmentation caused by the roads. These foxes seem to be tolerant to human disturbance and are frequently seen in rural areas and close to urban regions.

These wild canids have nocturnal scavenger habits and live in close proximity with domestic animals, which may be notable, as domestic host species can play a role in the transmission of infectious agents to wild animals (Alves et al., 2018a; Antunes et al., 2018; Ferreyra et al., 2009; Hübner et al., 2010) .

It is known that domestic dogs (Canis lupus familiaris) and wild canids may share viral pathogens, including the rabies virus (RABV) (Antunes et al., 2018; Rocha et al., 2017) , canine distemper virus (CDV) (Conceição-Neto et al., 2017; Ferreyra et al., 2009; Hübner et al., 2010; Megid et al., 2009) , canine parvovirus 2 (CPV-2) (de Almeida Curi et al., 2010; Hübner et al., 2010) , canine coronavirus (Alfano et al., 2019) , canine adenoviruses 1 and 2 (Dowgier et al., 2018) , and canine astrovirus (Alves et al., 2018a) .

However, the knowledge about sanitary conditions of these animals are still scarce.

The enhanced availability and application of high-throughput sequencing (HTS) technologies has facilitated the detection of known and unknown viruses (Goodwin et al., 2016; Paim et al., 2019) . Thus, the knowledge of the virus genetic diversity present in different host species can be improved. HTS sequencing has been applied in the knowledge of the virome of domestic dogs (Moreno et al., 2017; M. N. Weber et al., 2018) , but reports of its application in wild canids remain scarce. Therefore, the present study aimed to evaluate and characterize the spleen and mesenteric lymph node virome of crab-eating and Pampas foxes from southern Brazil and Uruguay using HTS. The results outline the viral agents that compound the microbiota of these wild dogs, helping to elucidate the viral population present in these wild canid species.

Journal Pre-proof

Five Pampas foxes (Lycalopex gymnocercus) and seventeen crab-eating foxes (Cerdocyon thous) run over by cars were collected by the Veterinary Pathology sector of Universidade Federal do Rio Grande do Sul and Plataforma de Salud Animal of Instituto Nacional de Investigación Agropecuaria Tacuarembó using an active search on highways between November 2017 and September 2019. Figure 1 shows the location on the foxes sampled in the present study. The mesenteric lymph nodes and spleen of the 22 animals were collected, macerated and diluted to 20% (w/v) in phosphate-buffered saline (PBS) (pH 7.2), centrifuged at low speed (1,800 x g for 30 min), filtered through a 0.45-µm filter for removal of small debris and stored at -80°C for subsequent analysis.

These organs were selected since they concentrate antigens in order to presentation for immune system, which could increase the chance of detecting some viral agents.

The authorization for the collection of the samples from Brazil was registered in SISBio/ICMBio under number 67053. Samples obtained of dead animals do not require authorization for molecular analysis in Uruguay.

The 22 wild canid spleens and mesenteric lymph nodes (17 crab-eating foxes and five Pampas foxes) were assembled into one pool containing 500 μL of each sample. A total of 11 mL was passed through a 0.22-µm filter and subsequently centrifuged on a 25% sucrose cushion at 150,000 g for 3 h at 4°C in a Sorvall AH629 rotor. The pellet respectively. The viral DNA was enriched using the Genoplex® Complete Whole Genome Amplification (WGA) kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer's recommendations. Furthermore, the viral RNA was reversetranscribed using the TransPlex® Complete Whole Transcriptome Amplification Kit (Sigma-Aldrich) following the manufacturer's recommendations. The DNA products from these enrichment protocols were pooled in equimolar amounts and purified using the PureLink™ Quick Gel Extraction and PCR Purification Combo Kit (Thermo Fisher Scientific). The quality and quantity of the DNA were assessed through spectrophotometry and fluorometry performed with NanoDrop™ (Thermo Fisher Scientific) and Qubit™ (Thermo Fisher Scientific), respectively. The DNA libraries were further prepared with 1 ng of purified DNA using the Nextera XT DNA sample preparation kit and sequenced using a MiSeq Reagent kit v2 300 (2 x 150 paired-end) at the MiSeq platform (Illumina®).

The quality of the generated sequences was evaluated using FastQC. Furthermore, the sequences with bases possessing a Phred quality score < 20 were trimmed with the aid of Geneious software (version 9.0.5). Subsequently, the paired-end sequence reads were de novo assembled into contigs with SPAdes Assembler version 3.11.1 (Bankevich et al., 2012) . All assemblies were confirmed by mapping reads to contigs produced by the best E-value. Gene and protein comparisons were performed with BLASTN and BLASTP programs (https://blast.ncbi.nlm.nih.gov/Blast.cgi).

Sequences representative of viruses belonging to circular Rep-encoding single-stranded (CRESS) DNA viruses and the families Anelloviridae, Parvoviridae and Paramyxoviridae were obtained from GenBank and aligned with the sequences identified in the present study with MAFFT software (Katoh and Standley, 2013) .

Phylogenetic trees were constructed using MEGA6 (Tamura et al., 2013) .

The 22 animals were screened individually for CPV-2 (Buonavoglia et al., 2001) , CDV (Fischer et al., 2013) , canine circovirus (CaCV) (Li et al., 2013) , and RABV (Soares et al., 2002) . Positive samples in the applied CDV-RT-nested PCR were submitted to an additional assay to amplify a fragment of CDV-hemagglutinin (An et al., 2008; Riley and Wilkes, 2015) for genotyping.

CBoV were searched applying two independent PCR protocols in the 22 individual wild canid samples, where the first one was designed against one of the contigs obtained in the HTS in order to amplify a 154-bp fragment from the nonstructural protein of CBoV- Table) . The second PCR protocol using primers CBoV-QFX1-f1 and CBoV-QFX1-r2 amplifies a 311-bp fragment of the VP2 gene of CBoV-1 (Kapoor et al., 2012) The sequences related to anelloviruses detected through HTS higher than 400 amino acids in ORF1 were investigated individually to define the host origin (Lycalopex USA). Reactions were performed in a Veriti 60-well Thermal Cycler (Applied Biosystems, Foster City, CA, USA) under the following conditions: 3 min at 95°C followed by 35 cycles of 45 s at 95°C, 45 s at 50°C and 45 s at 72°C with a final extension at 72°C for 7 min.

All positive samples in PCR and RT-PCR were submitted for Sanger sequencing to confirm the specificity of the tests. The PCR products were purified using the PureLink™ Quick PCR Purification Kit (Invitrogen, Carlsbad, CA, USA). Both DNA strands were sequenced with an ABI PRISM 3100 Genetic Analyzer utilizing a BigDye Terminator v.3.1 cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA).

Furthermore, overlapping fragments were aligned and assembled using Geneious software.

One DNA library consisting of 22 pooled wild canid spleens and mesenteric lymph nodes (17 crab-eating foxes and five Pampas foxes) was generated and sequenced using paired-end 2×150 base runs on the Illumina MiSeq platform, which generated a total of Furthermore, eukaryotic exogenous virus-related sequences with single-stranded DNA (ssDNA) genomes belonging to two viral families (Anelloviridae and Parvoviridae) and CRESS DNA viruses were observed, as well as with single-stranded RNA (ssRNA) genomes belonging to the Paramyxoviridae family (Table 1) . Moreover, the majority of the viral sequences studied shared a high degree of identity with known animal viruses (CPV-2, CDV and CBoV), while others (anellovirus-like and CRESS DNA virus) exhibited a high degree of divergence to genomes already recorded in GenBank.

Information regarding the sequences obtained is described in the following sections. All 22 samples analyzed using CaCV (Li et al., 2013) and RABV-specific PCR (Soares et al., 2002) were negatives.

Three contigs closely related to CDV were obtained in the pooled wild canid organs sequenced by HTS (Table 1) 

In the 22 pooled wild dog organs submitted for HTS, the presence of two contigs closely related to CPV-2 was observed ( Table 1) 

Three contigs more closely related to CBoV were obtained from the pooled wild dog organs submitted for HTS (Table 1) . One of the 22 (4.55%) samples was positive in both assays and was from a Lycalopex gymnocercus sampled in Brazil. The two amplification products were submitted for Sanger sequencing. The sequence obtained using the primers designed in the present study (Supplementary Table) supported by a 99% bootstrap value. This genetically divergent CBoV cluster evolved from the same common ancestor that originated the CBoV-1 and CBoV-2 sequences, as supported by an 82% bootstrap value.

J o u r n a l P r e -p r o o f Journal Pre-proof A total of 171 contigs with a closer association with Anelloviridade members were also observed ( Table 1 ). The contigs ranged between 2,415 and 886 nt in length. It was possible to obtain two complete genomes ( Figures 4A and 4B ) and five additional sequences displaying the complete ORF1 gene ( Figure 4C ).

To identify the host of origin (pampas or crab-eating fox), we applied specific PCR protocols designed using the sequences identified in HTS (Supplementary Table) . No sequence was identified in either Cerdocyon thous or Lycalopex gymnocercus. The sequences were named the putative Anelloviridae family species Cerdocyon thous torque teno virus 1 (CtTTV-1), CtTTV-2, CtTTV-3, CtTV-4, Lycalopex gymnocercus torque teno virus 1 (LgTTV-1), and LgTTV-2, where two different sequences displaying 81.21% nucleotide identity (Table 2) were classified as LgTTV-1 putative species. According to ICTV, nucleotide divergences in the ORF1 gene higher than 35% and 56% denote the same anellovirus species and genus, respectively (ICTV, http:// www.ictvonline.org/virusTaxonomy.asp). Apparently, six putative new Anelloviridae species (CtTTV-1, CtTTV-2, CtTTV-3, CtTTV-4, LgTTV-1 and LgTTV-2) belonging to five putative new genera were observed ( Figure 4C ). CtTTV-1 and CtTTV-2 can be classified in the same genus, since they present 63.15% nucleotide identity in the ORF1 gene. The relation of the sequences reported in the present study and other members of the family Anelloviridae is reported in Table 3 . LgTTV-1 LV07 ( Figure 4B ) also displayed a typical Anelloviridae organization comprised of a circular single-stranded DNA genome containing 2,352 nucleotides (nt) and a 44.4% C+G content. The sequence presents an untranslated intergenic region comprising 460 nucleotides, putative ORF1 (nt 395 to 2,095) and ORF2 genes (nt 204 to 581), and an ORF3 gene divided in two intervals (nt 204 to 578 and 1,638 to 2,090).

To analyze the sequences detected in the present study and representative sequences within the Anelloviridae family, a complete amino acid ORF1 phylogenetic reconstruction was performed ( Figure 4C ). CtTTV-1 LV05, CtTTV-2 LV15 and CtTTV-3 LV08 were closely related and apparently emerged from the same common ancestor that originated Torque teno canis virus (genus Thetatorquevirus) that was reported in domestic dogs (Lan et al., 2011) . CtTTV-4 LV23 emerged from the same common ancestor that originated unclassified anelloviruses detected in masked palm civet (Paguma larvata) (Nishizawa et al., 2018) . LgTTV-1 LV06 and LV07 apparently evolved from the same common ancestor of CtTTV-4 LV23 and the unclassified anelloviruses reported in the masked palm civet. Finally, LgTTV-2 LV13 apparently presents the same common ancestor of Etatorquevirus and Lambdatorquevirus genera members reported in domestic cats (Okamoto et al., 2002) and seals (Ng et al., 2011), respectively.

Seven contigs closely related to unclassified CRESS DNA viruses were detected by HTS in the present work (Table 1) 

The virome present in spleen and mesenteric lymph node samples obtained from 17 crab-eating foxes (Cerdocyon thous) and five Pampas foxes (Lycalopex gymnocercus) has been described using HTS and metagenomic analysis ( . We also observed the presence of highly prevalent viral agents in domestic dogs from Brazil, such as CPV-2 and CDV (Alves et al., 2018b) , that are genetically closely related to those reported in dogs of the same Brazilian region where the wild canids were sampled.

RABV was not detected in the analyzed samples using either HTS (Table 1) or RABVspecific PCR (Soares et al., 2002) . In Brazil, RABV was controlled in domestic dogs by intense public vaccination campaigns (Freire de Carvalho et al., 2018) . However, the circulation of RABV in wildlife has become a major concern for public health (Antunes The CDV species, renamed canine morbillivirus, was detected in four of the 17 crabeating foxes sampled from Brazil in the present study by applying an RT-nested PCR protocol against the CDV nucleocapsid (Fischer et al., 2013) . Two of these samples had a partial sequence within the CDV-hemagglutinin (An et al., 2008; Riley and Wilkes, 2015) sequenced and were genotyped as South America I/Europe (Figure 2) , which is the most prevalent CDV genotype in southern Brazil and Uruguay (Budaszewski et al., 2014; Fischer et al., 2016; Sarute et al., 2014) and is also reported in Argentina (Panzera et al., 2012) . Other CDV genotypes as South America 2, 3 and 4 are frequently reported in other South American countries as Argentina, Ecuador and Colombia (Espinal et al., 2014; Panzera et al., 2014 Panzera et al., , 2012 Sarute et al., 2014) . The CDV sequences detected in crab-eating foxes of the present study were assigned as closely related to CDV infecting J o u r n a l P r e -p r o o f Journal Pre-proof domestic dogs from southern Brazil in both nucleocapsid and hemagglutinin sequencing. CDV infection causes high mortality in domestic and wild dogs (Beineke et al., 2015) , and it is a common cause of wildlife population declines (Cleaveland et al., 2000; Roelke-Parker et al., 1996; Viana et al., 2015) . Additionally, CDV was determined to be the cause of death of crab-eating foxes in Brazil (Megid et al., 2009) and Argentina (Ferreyra et al., 2009) . Our data reinforce that crab-eating foxes may be wild reservoirs of CDV and suggest a possible spillover between crab-eating foxes and domestic dogs.

CPV-2 was reclassified as carnivore protoparvovirus 1 (genus Protoparvovirus, subfamily Parvovirinae, family Parvoviridae) by the International Committee of Taxonomy of Viruses (ICTV) (Cotmore et al., 2014) . In the present study, CPV-2 genome segments were found in one Pampas fox and two crab-eating foxes from Brazil.

These sequences were subtyped as CPV-2b and -2c. CPV-2b and CPV-2c strains are more frequently found in domestic dogs from Brazil, where CPV-2c substituted CPV-2b as the most prevalent CPV-2 subtype (Pinto et al., 2012) . Additionally, the CPV-2 fox sequences were closely related to those reported in domestic dogs of the same Brazilian region. Wild canids are also likely to act as reservoirs of CPV-2 infection for domestic canine populations (Truyen et al., 1998; Van Arkel et al., 2019) , and CPV-2positive serology was previously reported in Pampas and crab-eating foxes from southern Brazil (de Almeida Curi et al., 2010; Hübner et al., 2010) . CPV-2 is the most frequent canine pathogen worldwide (Alves et al., 2018b; Decaro et al., 2011; Decaro and Buonavoglia, 2012) and an important cause of severe diarrhea in puppies (Decaro and Buonavoglia, 2012). Moreover, CPV-2 is highly stable in the environment and can persist in domestic dog populations due to its indirect faecal-oral transmission and circulation in susceptible dogs (Van Arkel et al., 2019) . Our data reinforce that Pampas J o u r n a l P r e -p r o o f Journal Pre-proof and crab-eating foxes may be wild reservoirs of CPV-2. However, more studies are required to understand the impact of CPV-2 on these two wildlife species.

CBoV was detected in one Pampas fox from Brazil in the present study (Figure 3 ). This sequence was closely related to bocaparvoviruses reported in domestic dogs presenting respiratory disease (Kapoor et al., 2012) . This CBoV is genetically different from CBoV-1 and CBoV-2, which were reclassified as carnivore bocaparvovirus 2 (CBPV-2) in the genus Bocaparvovirus of the subfamily Parvovirinae and family Parvoviridae (Cotmore et al., 2014) . Apparently, the CBoV sequence from the present study is from a new species within the genus Bocaparvovirus. This putative new genus was only reported in domestic dogs from the United States (Kapoor et al., 2012) and China (Guo et al., 2016) . To the best of our knowledge, this report is the first to describe these genetically divergent CBoVs in South America and in canids other than domestic dogs.

The Anelloviridae members represent nonenveloped ssDNA viruses comprised of more than 65 species grouped in 16 genera (ICTV, http:// www.ictvonline.org/virusTaxonomy.asp), where some anelloviruses remain unassigned (Nishizawa et al., 2018) . The anellovirus-related sequences were the most abundant viral sequences detected by HTS in the present study (Table 1) Moreover, we described two complete genomes (Figures 4A and 4B ) and five additional sequences that displayed the complete ORF1 gene, comprising five putative new genera within the Anelloviridae family ( Figure 4C ). The present study expands the host range of this viral family and suggests that novel anelloviruses of marked diversity can be found in wild canids. It is important to emphasize that anellovirus pathogenicity in J o u r n a l P r e -p r o o f Journal Pre-proof canids has not been examined in depth (Lan et al., 2011; Sun et al., 2017; M. N. Weber et al., 2018) .

We also detected sequences related to Rep and capsid (Cap) proteins of different circular ssDNA viruses (Table 1) . One of these sequences presented the complete Rep protein that is used for the classification of the so-called CRESS-DNA viruses ( Figure   5 ). This sequence was genetically distinct from the other reported genomes and apparently was not classified in any assigned viral family. CRESS DNA viruses were previously described as novel circovirus-like viruses, since these viruses present Rep and Cap genes in agreement with Circoviridae members (Rosario et al., 2012b) .

Recently, with the recognition of its diversity and low degree of genome similarity with members of the family Circoviridae, the term CRESS-DNA viruses was proposed (Rosario et al., 2012a) . These viruses have been detected in samples from a number of widely different sources, including samples from humans and other mammals, fishes, and insects, as well as from the environment (López-Bueno et al., 2016; Rosario et al., 2012a; Steel et al., 2016; Matheus N. Weber et al., 2018) . Apparently, CRESS viruses were not reported in previous works that analyzed the virome of domestic (Moreno et al., 2017; M. N. Weber et al., 2018) and wild canids (Conceição-Neto et al., 2017; Lojkić et al., 2016) , which may suggest that these viruses were not abundant in those animal species.

The present study categorized the virome of crab-eating and Pampas foxes. We reported J o u r n a l P r e -p r o o f 

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Baumbach: Investigation; Methodology, Writing -review & editing

Soares: Conceptualization, Funding acquisition, Supervision, Writing -review & editing

Data curation, Funding acquisition

Formal analysis, Writing -review & editing

Formal analysis, Funding acquisition, Writing -review & editing

Methodology, Funding acquisition, Writing -review & editing

Canal: Conceptualization, Funding acquisition

Writing -review & editing

The authors are grateful to Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior 

None declared.