Virology 552 (2021) 1–9

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Virology
journal homepage: www.elsevier.com/locate/virology

A plate of viruses: Viral metagenomics of supermarket chicken, pork and

beef from Brazil
Samuel Cibulski a, *, Diane Alves de Lima b, c, Helton Fernandes dos Santos d,
Thais Fumaco Teixeira e, Caroline Tochetto b, Fabiana Quoos Mayer e, Paulo Michel Roehe b
a
Centro de Biotecnologia – CBiotec, Laboratório de Biotecnologia Celular e Molecular, Universidade Federal da Paraíba – UFPB, João Pessoa, Paraíba, Brazil
b
Departamento de Microbiologia Imunologia e Parasitologia, Laboratório de Virologia, Universidade Federal do Rio Grande do Sul – UFRGS, Porto Alegre, Porto Alegre,
Rio Grande do Sul, Brazil
c
Centro Universitário da Serra Gaúcha – FSG, Caxias do Sul, Grande do Sul, Brazil
d
Departamento de Medicina Veterinária Preventiva, Universidade Federal de Santa Maria – UFSM, Santa Maria, Rio Grande do Sul, Brazil
e
Centro de Pesquisa em Saúde Animal, Instituto de Pesquisas Veterinárias Desidério Finamor (IPVDF), Departamento de Diagnóstico e Pesquisa Agropecuária, Secretaria
de Agricultura, Pecuária e Desenvolvimento Rural, Eldorado do Sul, RS, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: A viral metagenomics study was conducted in beef, pork, and chicken sold in supermarkets from Southern Brazil.
Meat virome From chicken, six distinct gyroviruses (GyV) were detected, including GyV3 and GyV6, which for the first time
Viral metagenomics were detected in samples from avian species, plus a novel smacovirus species and two highly divergent circular
Gemonoviridae
Rep-encoding ssDNA (CRESS-DNA) viruses. From pork, genomes of numerous anelloviruses, porcine parvovirus 5
Anelloviridae
Parvoviridae
(PPV5) and 6 (PPV6), two new genomoviruses and two new CRESS-DNA viruses were found. Finally, two new
Cressdnaviricota CRESS-DNA genomes were recovered from beef. Although none of these viruses have history of transmission to
humans, the findings reported here reveal that such agents are inevitably consumed in diets that include these
types of meat.

1. Introduction In 2014, an interesting study identified a series of viral genome se­

quences in beef, pork, and chicken purchased from stores in San Fran­
Meat and meat products are the source of numerous bacteria (e.g. cisco, USA (Zhang et al., 2014b). Despite no association with disease, the
Salmonella enterica), protozoa (e.g. Toxoplasma gondii) and worms (e.g. innovative workflow lead to the detection of a large number of known
Trichinella spiralis) that may cause significant illness in humans. There­ and unknown viral genomes harbored by the meats sampled on the
fore, it has been rising the overall concern of viral transmission by food occasion. Bearing in mind that microorganism’s evolution mimics the
products, especially the ones causes by meat and meat-derived. Meats lines of a poem by Gonçalves Dias (The Song of Exile): the birds that
have been pointed in charge, once is well-known that food-source ani­ chirp here are not the same as those who chirp there (…)” (Dias, 1846),
mals are themselves subject to virus infections that might led to trans­ we set up a metagenome-based study to examine the virome of meats
mission to other species along the food chain. A large number of viruses sold in supermarkets in Porto Alegre, the capital of Rio Grande do Sul,
harbored by farm or wild animals can cause zoonotic infections such as the southernmost State in Brazil. The present study revealed that
noroviruses, rotaviruses and hepatitis E virus (Spahr et al., 2018; Vil­ chicken, pork and beef samples harbor highly divergent CRESS-DNA
labruna et al., 2019). Furthermore, viral transmission between different viruses, including genomes that could not be classified at the family
animal species, including domestic and wild animals may also occur, level. In chicken and pork samples, diverse anelloviruses (gyroviruses
facilitating viral adaptation and consequently enhancing the viral host from chicken and Torque teno viruses from swine) were detected. In
range (Ma et al., 2009; Morse et al., 2012). Today, emerging viruses are addition, in all meat samples analyzed, the vast majority of viral se­
a major concern, since devastating, occasionally life-threatening epi­ quences detected were attributed to bacteriophages species hosted by
demics – e.g., COVID-19 and pandemic influenza – are caused by viruses Acinetobacter and Pseudomonas, common genera of bacteria isolated
which originally spilled over from animals to humans. from meat samples.

* Corresponding author.
E-mail addresses: E-mail address: Spcibulski@gmail.com (S. Cibulski). cibulski@cbiotec.ufpb.br (S. Cibulski).

https://doi.org/10.1016/j.virol.2020.09.005
Received 29 June 2020; Received in revised form 12 September 2020; Accepted 13 September 2020
Available online 28 September 2020
0042-6822/© 2020 Elsevier Inc. This article is made available under the Elsevier license (http://www.elsevier.com/open-access/userlicense/1.0/).
S. Cibulski et al. Virology 552 (2021) 1–9

2. Material and methods then aligned with the sequences identified in the present study using
MAFFT software, which was optimized for accurate global alignment
2.1. Chicken, pork and beef samples and virus enrichment (option “G-INS-i”) (Katoh and Standley, 2013) and further trimmed with
trimAL (Capella-Gutiérrez et al., 2009). These alignments were used to
Ten samples of chicken (drummete), pork (chop) and beef (ground generate maximum-likelihood phylogenetic trees using PhyML (Guin­
meat) were purchased in a supermarket in Porto Alegre City, Rio Grande don et al., 2010) with best fit substitution models determined by Smart
do Sul, Brazil. All samples were from animals produced within the state Model Selection (Lefort et al., 2017). Statistical significance analyses of
and processed by local slaughterhouses. Tissue fragments (~1 cm3) of tree topologies were performed with the approximate likelihood branch
each sample were aseptically excised and pooled. The three pools support test (aLRT) (Guindon and Gascuel, 2003). The GenBank acces­
(chicken, pork and beef) were processed essentially as described previ­ sion numbers of the viral sequences used in the phylogenetic analyses
ously (Cibulski et al., 2020). Briefly, pooled samples were macerated are shown on phylogenetic trees.
with sterile sand, resuspended in 10 vol of PBS (pH 7.2), followed by
vigorous vortexing for 15 min. Subsequently, the suspensions were 3. Results and discussion
centrifuged at 3,000×g for 30 min at 4 ◦ C and the supernatant filtered
through a 0.45 μm membrane (Millipore) to remove bacteria and other 3.1. Viruses identified in chicken
particulate debris. The supernatants were centrifuged at 150,000×g on a
25% sucrose cushion for 4.5 h at 4 ◦ C (in a Sorvall AH629 rotor), and the Approximately 531,000 high-quality reads were generated by met­
pellet containing viral particles was treated with a mixture of DNase, agenomic sequencing. After assembly, a total of 2,370 contigs were
RNase A and benzonase to digest non-protected nucleic acids. generated; 50.3% were viral contigs. From these, 0.75% were assigned
to eukaryotic viruses and 99.25% to bacteriophages. However, despite
2.2. Viral nucleic acids isolation, enrichment and sequencing the relatively small number of contigs associated with viral genomes
previously known to infect Eukarya, ~20% of the reads were mapped
Capsid-protected nucleic acids were extracted using a standard back to these contigs, indicating a high sequence coverage. A detailed
phenol-chloroform protocol (DNA) or TRIzol® (RNA). Viral DNA was taxonomic classification, including the numbers of reads for each
enriched by multiple displacement amplification using phi29 DNA po­ Eukarya-related viral contig recovered is this study, is provided in
lymerase (da Silva et al., 2018). RNA was converted to cDNA using Supplementary Table 1.
SuperScript III reverse transcriptase and random-amplificated (Clem Nine Eukarya-related viral contigs were identified in this meta­
et al., 2007). The DNA products resulting from enrichment protocols genome and, six contigs corresponded to gyrovirus sequences (Supple­
were purified using a Genomic DNA clean and concentrator (Zymo mentary Table 1); in four of these, full genomes were obtained (chicken
Research). DNA libraries were further prepared with 50 ng of purified anemia virus, CAV; avian gyrovirus 2, AGV2; gyrovirus 3, GyV3; and
DNA using a Nextera DNA sample preparation kit (Illumina) and gyrovirus 4, GyV4) (Fig. 1). The other two gyroviruses’ contigs corre­
sequenced using an Illumina MiSeq instrument (2x150 paired-end reads sponded to a genome of gyrovirus 6 (GyV6) and to gyrovirus 3 (GyV3
with the Illumina v2 reagent kit). 376).
Gyroviruses are small naked DNA viruses with a negative sense,
2.3. Viral metagenomic data assembly and sequence analyses single-stranded circular DNA genome of about 2.3 kb classified in the
family Anelloviridae, where it clusters close to Torque teno viruses. Based
The quality of generated sequences was evaluated using FastQC tool on information gathered from CAV (Welch et al., 2006), gyroviruses are
(Andrews, 2010). Low-quality sequences were trimmed with the aid of highly resistant to chemical and heat inactivation, which gives them the
CLC Workbench 12 software. The paired-end sequence reads were ability to persist and spread in the environment. Chicken meat has been
assembled into contigs with metaSPAdes and CLC Workbench v12. All reported to carry numerous gyroviruses, including the highly prevalent
assemblies were confirmed by mapping reads to contigs as described in CAV, the worldwide distributed, and recognized pathogen of chicken.
Lima et al. (2019) using Geneious Software (version R9). The assembled Over the last decade, twelve new putative Gyrovirus species were
contigs were examined in search for similarities to known sequences described, most of these recovered from chickens. Nevertheless, several
with BLASTx software. Sequences with E-values of 10− 3 or less were other gyroviruses have been reported in feces of humans and other
classified as likely originating from a eukaryotic virus, bacteria, phages, mammals, suggesting possible dietary sources of contamination, such as
eukaryote or unknown, based on the taxonomic origin of the sequence consumption of gyrovirus-infected chicken meats.
with the best E-value as described by Caesar et al. (2019). Three of the four full genomes recovered in the present study, i.e.
Open reading frames (ORF) predictions and genome annotations of CAV chicken meat/Brazil, AGV2 chicken meat/Brazil and GyV4 chicken
the complete near-full-length genomes were performed with the aid of meat/Brazil (Fig. 1), are highly similar (>98%) to recently reported
Geneious software and NCBI ORF finder and later identified by BLASTx/ genomes from southern Brazil (Lima et al., 2019, 2017), indicating that
BLASTn. Gene and protein comparisons were performed with BLASTn the viruses circulating in chicken within that region are closely related
and BLASTp programmes. To identify the main protein motifs, Motif (Fig. 1). The fourth complete genome recovered, GyV3, shares high nt
Scan (https://myhits.isb-sib.ch/cgi-bin/motif_scan) was applied; stem- identities with GyV3 genomes from human and cat feces (Phan et al.,
loop structures were analyzed using Mfold (Zuker, 2003). Nucleotide 2012; Zhang et al., 2014c). In addition, a VP1-coding genome fragment
and amino acid sequences were aligned using the MAFFT software related to GyV3 was identified. This fragment shares low nt and aa
(Katoh and Standley, 2013) and then visually inspected in Geneious. identity with previously reported GyV3 isolates (~72% of nt and ~75%
Rolling circle replication (RCR) and superfamily 3 helicase (SF3) motifs at amino acid level), and probably represents another genome variant,
(Gorbalenya et al., 1990; Gorbalenya and Koonin, 1993; Ilyina and since the degree of VP1 identity between other GyV3 is greater than
Koonin, 1992; Rosario et al., 2012) were analyzed from the sequence 70%. Another interesting remark is that, to date, GyV3 has only been
alignment using as reference Cressdnaviricota replication-associated identified in feces from mammalian hosts (human and cat). Therefore, it
proteins (Krupovic et al., 2020). seems very likely that GyV3 may, in fact, be a virus of avian origin – here
detected in chicken meat. The last gyrovirus contig identified in the
2.4. Construction of phylogenetic trees chicken meat virome corresponds to a segment of a GyV6 genome. GyV6
was also described in human and cat feces (Gia Phan et al., 2013; Zhang
Amino acid sequences representative of known viral families, as well et al., 2014c), both of which likely include chicken in their diets
as CRESS-DNA genomes, were obtained from GenBank (June 2020) and (although confirmatory evidence for consumption of chicken meats in

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Fig. 1. Gyroviruses recovered from chicken meat. A VP1-coding region maximum likelihood phylogenetic tree was constructed using PhyML (Guindon et al., 2010)
with best fit substitution models (GTR_G + I) determined by Smart Model Selection (Lefort et al., 2017). Statistical significance analyses of tree topologies were
performed with the approximate likelihood branch support test (aLRT) (Guindon and Gascuel, 2003). In the right, the putative genomic organization of the
assembled gyrovirus genomes. Sequences described in this study are marked in red. (For interpretation of the references to color in this figure legend, the reader is
referred to the Web version of this article.)

the sampled populations were not available in those reports). Here, meadow vole (Microtus pennsylvanicus; JF755404).
GyV6 is for the first time reported to be associated with chickens, once CRESS 77F (2,895 nt, 38.3% of GC content) showed a type II genome
more bringing up the possibility of an avian origin for such virus. (Rosario et al., 2012), with cap and rep genes bidirectionally organized
Three contigs with the complete genome sequences assigned to the and separated by two untranslated regions (Fig. 3A). In the high
recently reported Cressdnaviricota phylum (Krupovic et al., 2020) were GC-content (58%) portion of the untranslated region at the 5’ terminal
recovered from chicken virome. The first Cressdnaviricota contig evi­ of the cap gene, the nonamer CAGTATTAC was located. The Rep dis­
denced clear characteristics from representatives of Smacoviridae family. played the RCR motifs I, II and III, as well as SF3 helicase motifs A, B and
Although also detected in invertebrates (Dayaram et al., 2015), sma­ C. The coat protein was highly divergent, sharing <20% aa identity with
coviruses have been reported mainly in samples of feces from humans a CRESS-DNA virus recovered from minnow tissue (MH617562).
and chickens. This is the case of ‘Smaco 2466F’ genome (2,466 nt) here Phylogenetically (Fig. 3B), a clade within CRESSV1 was formed by
reported, which is closely related to a smacovirus detected in feces from CRESS 77F, CRESS-DNA sequences from rainbow trout (MH617109),
humans (human smacovirus 1) and chickens (chicken huchismacovirus red snapper (MH617681), CRESS 36B (from bovine meat, described in
1) (Fig. 2). Due to high degree of nt and amino acid conservation and this study) and KX259434 (wastewater) (Kazlauskas et al., 2018).
strong phylogenetic evidence, the smacoviruses recovered from chicken
and human were classified in the same species within the Huchismaco­
3.2. Viruses identified in pork
virus genus (Varsani and Krupovic, 2018). Again, these findings might
suggest that smacoviruses detected in human feces may also be of avian
Approximately 465,000 high-quality reads were produced by meta­
origin, as previously speculated for the gyroviruses above.
genomic sequencing of pork sample. After assembly, a total of 2,922
The last two genomes recovered were CRESS-DNA viruses that could
contigs were generated; 55% of these were representative of viral se­
not be allocated to any previously described viral family. CRESS 53F
quences. From these, 1.4% were assigned to eukaryotic viruses, whilst
(5,532) exhibited four ORFs encoding Rep, Cap and two hypothetical
the remaining sequences were representatives of bacteriophages. A
proteins (with homologues in other CRESS-DNA viruses) (Fig. 3A). The
detailed taxonomic classification, including the numbers of sequenced
N-terminus of Cap presented a R-rich region, whereas the Rep showed
reads of each Eukarya-related viral contig recovered in this study, is
motifs implicated in RCR (Supplementary Table 2). In addition, between
provided in Supplementary Table 1.
cap and rep genes, one stem-loop structure was putatively formed. At the
In the same way that gyroviruses were found in large amounts in
apex of this secondary DNA structure, a conserved nonanucleotide
chicken meat, Anelloviridae contigs (Torque teno sus viruses, TTSuV)
sequence (TAATATTAC) was found. This large ssDNA virus was classi­
represented the majority of the contigs recovered from pork in this
fied as type I genome (Rosario et al., 2012). Phylogenetically (Fig. 3B),
study. TTSuVs are ubiquitous entities, detected at high frequency in pigs
CRESS 53F Rep forms a sister clade with CRESSV2 sequences
and wild boars worldwide (Martínez et al., 2006; Ramos et al., 2018;
(Kazlauskas et al., 2018) and was closely related to unclassified
Teixeira et al., 2013). These Anelloviridae genomes have been classified
CRESS-DNA viruses detected in a chimpanzee (NC_030466) and in a
into two genera: Iotatorquevirus (TTSuV-1a and -1b) and

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Fig. 2. Smacoviruses and genomoviruses genomes in chicken and pork meats. (A) Genomic organization and ML phylogenetic tree of smacoviruses. Huchismacovirus
genus branch are highlighted in blue. (B) Genomic architecture of genomoviruses recovered from pork samples and phylogenetic assessment. Gemycircularvirus and
Gemykibivirus genus are highlighted in blue and purple color. ML phylogenetic trees were constructed using PhyML (Guindon et al., 2010) with best fit substitution
models (LG_G + I + F for both sequence sets) determined by Smart Model Selection (Lefort et al., 2017). Statistical significance analyses of tree topologies were
performed with the approximate likelihood branch support test (aLRT) (Guindon and Gascuel, 2003). Sequences described in this study are marked in red. (For
interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Kappatorquevirus (TTSuV-k2a and -k2b). Interestingly, members of these disease in swine, their role in pathological conditions, if any, is still
genera display a remarkable genetic diversity. In this study, eleven unclear, as TTSuVs have been detected in healthy and diseased pigs at
contigs of TTSuVs (eight of which constitute complete genomes) were high viral genome loads (Da Silva et al., 2020; Ramos et al., 2018;
identified (Fig. 4A), including six contigs assigned to Iotatorquevirus Rogers et al., 2017; Teixeira et al., 2015).
TTSuV1a (with four complete genomes), yet with high genetic vari­ In addition to the well-knows anelloviruses, contigs related to Par­
ability between genomes (76–96% of genomic identity). In addition, two voviridae (genus Copiparvovirus) were also identified (Fig. 4C). These
Iotatorquevirus TTSuV1b also recovered here displayed 23% nt diver­ contigs had high nucleotide identities with porcine parvovirus 5 and
gence between them. Phylogenetically, such iotatorqueviruses were porcine parvovirus 6 (PPV5 and PPV6) genomes. Whether these novel
close to TTSuVs previously detected in Southern Brazil (Fig. 4B) (Da parvoviruses play some role in disease in pigs remains to be determined,
Silva et al., 2020; Tochetto et al., 2020). Regarding kappatorqueviruses, since these have neither been isolated in cell culture nor evaluated in
one TTuSV-k2a genome (plus an additional partial genome) and one experimental infections (Miłek et al., 2019). To date, a few sequences of
TTSuV-k2b genome were recovered. Phylogenetically, these genomes these viruses have been publicly released; all bearing a significant de­
were closely related to kappatorqueviruses previously sequenced in gree of conservation. Phylogenetically, the PPV5 genome fragment re­
South America (Fig. 4B) (Da Silva et al., 2020; Tochetto et al., 2020). In ported here clustered with sequences recovered in China and USA;
addition, it was noticed that most of the viral reads from pork virome are whereas the PPV6 sequences reported here seem more closely related to
from TTSuVs, especially from kappatorqueviruses (Supplementary sequences recovered from pigs in USA (Fig. 4D).
Table 1). Although many studies have attempted to link TTSuVs to Two genomes of unrelated genomoviruses were detected in pork

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Fig. 3. Unclassified CRESS-DNA viruses recovered from chicken, pork and beef meat. (A) ML phylogenetic tree of unclassified Cressdnaviricota genomes discovered in
this study. ML phylogenetic trees were constructed using PhyML (Guindon et al., 2010) with best fit substitution models (rtRev_G + F) determined by Smart Model
Selection (Lefort et al., 2017). Statistical significance analyses of tree topologies were performed with the approximate likelihood branch support test (aLRT)
(Guindon and Gascuel, 2003). Sequences described in this study are marked in red. (B) Genomic architecture of new CRESS-DNA viruses recovered from chicken,
pork and beef samples. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

virome. Genomoviruses are closely related to geminiviruses and have genome identity >78% (that define a same species) (Varsani and Kru­
often been detected in metagenomic studies in samples from mammals, povic, 2017). Thus, Genomo194 (diverges >25% with your closest
birds, plants and environmental samples. The genomes reported here, counterpart) was tentatively classified as a novel species to which the
Genomo194 and Genomo200, exhibited the classical Genomoviridae ar­ name “porcine associated gemykibivirus 1” (PAGkV-1) is suggested.
chitecture (Fig. 2B) (Krupovic et al., 2016), with the Likewise, Genomo200 seems to be another new species within the
replication-associated protein containing the motifs involved in RCR Gemycircularvirus genus (diverges >30% with your closest counterpart),
(Supplementary Table 2). Phylogenetically (Fig. 2B), Genomo194 clus­ to which the name “porcine associated gemycircularvirus 3” (PAGcV-3)
tered with sequences previously detected in plants (MK947375) and is here proposed.
bison and were assigned to the Gemykibivirus genus. The second Two unclassified Cressdnaviricota genomes were also identified in
genomovirus, Genomo200, could be assigned to the Gemycircularvirus pork samples: CRESS 169P (2,465 nt, type V genome) and CRESS 173P
genus, clustering along with a genomovirus detected in mouse (2,435 nt, type I genome) (Fig. 3A) with the nonanucleotide motif
(MK032755). The species demarcation criterion in Genomoviridae is (NANTATTAC) located at untranslated region. As other Cressdnaviricota,

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Fig. 4. Porcine anellovirus and parvovirus genomes in pork samples. (A) ML phylogenetic tree (based in VP1 coding sequence) of Torque teno sus viruses. (B)
Genomic organization of iotatorqueviruses (TTSuV 1b pork 195) and kappatorqueviruses (TTSuV k2a pork 298). (C) Contigs from PPV5 and PPV6. (D) Capsid-coding
ML phylogenetic tree of PPV5 and PPV. BPV2 is used as outgroup. ML phylogenetic trees were constructed using PhyML (Guindon et al., 2010) with best fit sub­
stitution models (LG_G + I + F for both sequence sets) determined by Smart Model Selection (Lefort et al., 2017). Statistical significance analyses of tree topologies
were performed with the approximate likelihood branch support test (aLRT) (Guindon and Gascuel, 2003). Sequences described in this study are marked in red. (For
interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

the Rep of these CRESS-DNA viruses possesses the classical motifs different capsid was expected.
involved in RCR. Phylogenetically, CRESS 169P was distantly related to
CRESSV2 replication-associated proteins (Kazlauskas et al., 2018), and 3.3. Viruses identified in beef
formed a cluster with sequences of CRESS-DNA genomes recovered from
mouse tissues and from a small passerine bird from temperate Asia, Approximately 523,000 high-quality reads were obtained at meta­
Phoenicurus auroreus (MN928932). genomic sequencing. After assembly, a total of 1,739 contigs were
CRESS 173P, formed a sister clade with replication-associated pro­ generated; 51.6% were viral contigs. From these, 0.5% were assigned to
teins from Nanoviridae and Alphasatellitidae, and was closely related to eukaryotic viruses, whereas remaining contigs corresponded to bacte­
unclassified CRESS-DNA viruses recovered from Ilex paraguariensis riophage sequences. A detailed taxonomic classification, including
(NC_040708) and wastewater (KY487924). Nanoviridae is a family of numbers of sequenced reads of each Eukarya-related viral contig
plant-infecting viruses with genomes composed by multiple segments of recovered in this study, is provided in Supplementary Table 1.
circular ssDNA. Despite the similarity with nanoviruses Rep, when Cap Four contigs of CRESS-DNA viruses were identified in the beef
sequence is examined, no similarities to nanovirus coat proteins were virome (Fig. 3A). CRESS 36B (3,539 nt) is a full genome sequence of a
found. CRESS-DNA viruses evolved on several independent occasions, highly distinct CRESS-DNA virus. The coding region of Rep and Cap
and recombination seems to be an important drive of the evolution of proteins are in the same strand (negative), what places such genome as a
these viruses (Kazlauskas et al., 2019; Martin et al., 2011). Coat proteins type VI virus (Rosario et al., 2012). It possesses a conserved nonamer
are key proteins involved in entry in host cells by interaction with cell (TATTATTAT) at the top of a stem-loop structure; also, the
receptors. So, since it is a virus that apparently infects mammals, a replication-associated protein contains the RCR motif III and SF3

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helicase motifs, implicated in genome replication (Supplementary During slaughterhouse processing, meats may be contaminated by the
Table 2). Interestingly, CRESS 36B replication-associated protein lack skin, intestinal content, personnel, and processing equipment. The mi­
the RCR motif I and II (or possess highly divergent motifs). Between cap crobial load varies from 102 and 104 cfu/cm2 (Russo et al., 2006). The
and rep ORFs, a putative ORF coding for an as yet undescribed protein commonest microbial genera isolated from meat samples are Acineto­
was found. Such ORF, has been previously detected in some CRESS-DNA bacter, Pseudomonas, Brochothrix, Flavobacterium, Psychrobacter, Morax­
viruses (based in BLASTp analysis), in which it seems to code for a ella, Staphylococcus, Micrococcus, lactic acid bacteria, and
conserved peptide of unknown function. Contig CRESS 75B (2,851 nt) Enterobacteriaceae (Doulgeraki et al., 2012). Then, it is not surprising
contains the complete cap and rep coding regions; following the cap that most of the phages found in this study are hosted by bacteria that
coding region, at the apex of a hairpin structure, the nonamer TAT­ are often found in meat samples.
TATTAC was found. This genome is classified as type I virus, since its rep
and cap are encoded in opposite directions (Rosario et al., 2012) 4. Concluding remarks
(Fig. 3A); RCR and SF3 motifs were found in replication-associated
protein; Cap protein, differentially of CRESS 36B, contains at N-termi­ Viral metagenomics techniques shed to light a myriad of viral entities
nus a R-rich region. The two other viral sequences detected here were that change the current mindset about viral quantity and diversity in all
small contigs with identity to replication-associated protein (CRESS forms of life, in all Earth’s environments (Koonin et al., 2020; Mokili
852B contig) and capsid proteins (CRESS 1094B contig). These share et al., 2012; Simmonds et al., 2017). Therefore, the breathed air, the
low aa identity (58% in Rep and 31.9% in Cap) with an unclassified drinking water and the food are full of viruses. In this study, the main
CRESS-DNA virus recovered from the genital tract of cattle in China objectives were first, to evaluate which viruses are present in the most
(MH782428). consumed animal protein sources in Brazil and second, to verify if any of
Phylogenetically, CRESS 36B (Fig. 3B) was related to viruses detec­ these viruses could pose risks to human health. Not surprisingly, a large
ted in red snapper fishes (CRESS virus sp. isolate ctcd80, MH617681) number of known viruses, as well as a vast number of genome sequences
and clustered with CRESSV1 sequences (Kazlauskas et al., 2018). of new viral entities were recovered from chicken, pork and beef sam­
Interestingly, some regions of CRESS 36B genome are quite similar to ples. It was a pleasant satisfaction concerning the second main objective,
CRESS ctcd80, sharing 52.5% of global nt identity. CRESS 75B clustered since no viruses associated with human diseases was found. Moreover,
with CRESSV3 sequences (Fig. 3B) (Kazlauskas et al., 2018) and was even analyzing a small number of samples, we bring to light several viral
close related to an unclassified CRESS-DNA virus detected in haddock genomes, highlighting the still high and unexplored viral diversity in
tissues (CRESS virus sp. isolate ctcd114, MH617702). CRESS 75B and farm animals.
CRESS ctcd114 shared similar genomic architecture, with remarkable Compared with the viral composition of US meat samples (Zhang
synteny. et al., 2014b), we found certain similarities, such as the presence of
It is interesting to point that only DNA viral sequences were identi­ well-known and ubiquitous anelloviruses and parvoviruses in pork, and
fied in beef chicken and pork viromes. This may be due to limited gyroviruses in chicken samples. In addition, CRESS-DNA viruses were
sampling of tissues from presumably healthy animals. Furthermore, found in the three analyzed viromes. However, they were not phyloge­
RNA viruses seems to predominate fecal material of farm animals netically related – showing that the viruses found in Brazilian meat
(Sachsenröder et al., 2014; Shan et al., 2011; Zhang et al., 2014a). samples are not the same as those reported in North American samples.
Additionally, it is necessary to consider the lability of RNA, since the Moreover, Brazilian meat samples seem to have a greater diversity of
meat evaluated here was stored at 4 C and submitted to a complex viral species, particularly in chicken and pork. Finally, it shows that
◦

workflow to viral metagenomics study. In order to clarify the presence despite the high rates of globalization in agribusiness – which can favor
(or absence) of RNA viruses in chicken, pork and samples, total RNA was the spread of different viruses, including the potentially pathogenic ones
isolated from individual tissues, retrotranscribed and used as template to –, the viral community present in these species from different
broadly range PCRs able to detect astroviruses (Chu et al., 2008), geographic locations is significantly different. In addition to virological
coronaviruses (Lima et al., 2013), kobuviruses (Reuter et al., 2009) and data, this study highlights valuable viral metagenomic techniques that
hepatitis E virus (Jothikumar et al., 2006) – common RNA viruses with can be used in routine applications of metagenomic sequencing in
high frequency of detection in Brazilian farm animals. All samples were diagnostic context, facilitating viral detection and offering huge poten­
negative in these assays, reinforcing the viral metagenomic data. tial for tracing viruses in (foodborne) outbreaks.

3.4. Bacteriophages in chicken, pork and beef Declaration of competing interest

Bacteriophages or phages are the most abundant organisms in the The authors have no conflicts of interest to declare.
biosphere and a ubiquitous feature of prokaryotic existence (Clokie
et al., 2011). Not surprisingly, in virome studies, the phage fraction is Acknowledgments
the most abundant one (Džunková et al., 2015; Perez Sepulveda et al.,
2016). This work was supported by the National Council for Scientific and
Here, in all types of meat, bacteriophages were the dominant viruses, Technological Development (CNPq), Financiadora de Estudos e Projetos
corresponding to 99% of the obtained contigs. Sequences from Caudo­ (FINEP, grants 01.10.0783.04 and 01.12.01001.12.0113.00) and
virales order (tailed bacteriophages with dsDNA genome) were the most Fundação de Amparo à Pesquisa do Rio Grande do Sul (FAPERGS). PMR
abundant in the three metagenomes, comprehending >95% of the is a CNPq 1A Research fellow.
contigs. Viruses from Siphoviridae, Podoviridae and Myoviridae families
represented the majority of the sequences recovered (Supplementary Appendix A. Supplementary data
Figure 1). Regarding phage host, from chicken, phages from Pseudo­
monas, Acinetobacter, Bacillus and Enterobacteriaceae species were most Supplementary data to this article can be found online at https://doi.
frequent; from pork, phages associated to Pseudomonas and Acinetobacter org/10.1016/j.virol.2020.09.005.
and Enterobacteriaceae; from beef, Acinetobacter, Pseudomonas and
Staphylococcus were the most frequent. Author contributions section
Meat is among the most perishable foods and a favorable environ­
ment for the replication of microorganisms because of its high concen­ Samuel Cibulski, Diane Alves de Lima, Helton Fernandes dos Santos,
trations of nutrients and high water activity (Odeyemi et al., 2020). Thais Fumaco Teixeira and Fabiana Quoos Mayer performed

7
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experiments, analyzed data, and wrote manuscript. Paulo Michel Roehe Krupovic, M., Ghabrial, S.A., Jiang, D., Varsani, A., 2016. Genomoviridae: a new family
of widespread single-stranded DNA viruses. Arch. Virol. https://doi.org/10.1007/
and Caroline Tochetto, analyzed data and wrote manuscript. Samuel
s00705-016-2943-3.
Cibulski, Thais Fumaco Teixeira and Paulo Michel Roehe designed the Krupovic, M., Varsani, A., Kazlauskas, D., Breitbart, M., Delwart, E., Rosario, K.,
study. All authors read and approved the final version of the manuscript. Yutin, N., Wolf, Y.I., Harrach, B., Zerbini, F.M., Dolja, V.V., Kuhn, J.H., Koonin, E.V.,
2020. Cressdnaviricota: a virus phylum unifying 7 families of Rep-encoding viruses
with single-stranded, circular DNA genomes. J. Virol. https://doi.org/10.1128/
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