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03eline fecal virome reveals novel and prevalent enteric viruses

rry Fei Fan Ng a,b, Joao Rodrigo Mesquita c, Maria Sao Jose Nascimento d,ikola O. Kondov a, Walt Wong a, Gabor Reuter e, Nick J. Knowles f,erardo Vega g, Mathew D. Esona h, Xutao Deng a,b, Jan Vinje g, Eric Delwart a,b,*

lood Systems Research Institute, San Francisco, San Francisco, CA, USA

epartment of Laboratory Medicine, University of California at San Francisco, San Francisco, CA, USA

epartment of Animal Science, Rural Engineering and Veterinary, Polytechnic Institut of Viseu, Viseu, Portugal

epartment of Biological Sciences, Faculty of Pharmacy, University of Porto, Porto, Portugal

egional Laboratory of Virology, ANTSZ Regional Institute of State Public Health Service, Pecs, Hungary

e Pirbright Institute, Woking, Surrey, United Kingdom

CIRD, National Calicivirus Laboratory, Centers for Disease Control and Prevention, Atlanta, GA, USA

RVLB, Rotavirus Surveillance, Centers for Disease Control and Prevention, Atlanta, GA, USA

Introduction

Cats are one of the most common pets with worldwidepulation estimated at 80400 million. Interactions withmans presumably accelerated following domestication

of cats 9500 years ago (Driscoll et al., 2007). Known felineviral pathogens that can infect humans include rabies virusand rotaviruses (Tsugawa and Hoshino, 2008). Felinepanleukopenia parvovirus was the source of the highlypathogenic canine parvovirus 2 (Hoelzer and Parrish,2010). Although more than 15 viral families have now beendescribed in cats, our understanding of feline viralinfections remains limited. Most feline viruses arecurrently identied using highly specic tests, such asPCR, antigens, or antibody assays, which will not detecthighly divergent viruses.

R T I C L E I N F O

icle history:

ceived 10 January 2014

ceived in revised form 29 March 2014

cepted 1 April 2014

ywords:

tagenomics

ome

teric virus

is catus

kobuvirus

A B S T R A C T

Humans keep more than 80 million cats worldwide, ensuring frequent exposure to their

viruses. Despite such interactions the enteric virome of cats remains poorly understood.

We analyzed a fecal sample from a single healthy cat from Portugal using viral

metagenomics and detected ve eukaryotic viral genomes. These viruses included a novel

picornavirus (proposed genus Sakobuvirus) and bocavirus (feline bocavirus 2), a variant

of feline astrovirus 2 and sequence fragments of a highly divergent feline rotavirus and

picobirnavirus. Feline sakobuvirus A represents the prototype species of a proposed new

genus in the Picornaviridae family, distantly related to human salivirus and kobuvirus.

Feline astroviruses (mamastrovirus 2) are the closest known relatives of the classic human

astroviruses (mamastrovirus 1), suggestive of past cross-species transmission. Presence of

these viruses by PCR among Portuguese cats was detected in 13% (rotavirus), 7%

(astrovirus), 6% (bocavirus), 4% (sakobuvirus), and 4% (picobirnavirus) of 55 feline fecal

samples. Co-infections were frequent with 40% (4/10) of infected cats shedding more than

one of these ve viruses. Our study provides an initial description of the feline fecal virome

indicating a high level of asymptomatic infections. Availability of the genome sequences of

these viruses will facilitate future tropism and feline disease association studies.

2014 Elsevier B.V. All rights reserved.

Corresponding author at: Blood Systems Research Institute, Univer-

y of California, San Francisco, 270 Masonic Ave., San Francisco, CA

118, USA. Tel.: +1 415 923 5763.

E-mail address: delwarte@medicine.ucsf.edu (E. Delwart).

Contents lists available at ScienceDirect

Veterinary Microbiology

jou r nal h o mep ag e: w ww .e ls evier . co m/lo c ate /vetm i c

p://dx.doi.org/10.1016/j.vetmic.2014.04.005

78-1135/ 2014 Elsevier B.V. All rights reserved.

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T.F.F. Ng et al. / Veterinary Microbiology 171 (2014) 102111 103Here we performed a metagenomics analysis of nucleiccids in viral particles enriched from the feces of a singleealthy cat from Portugal. We genetically characterizedve enteric viruses and compared their genomic featuresith those of human and animal viruses. PCR screening forese ve viruses was then carried out in fecal samplesom Portuguese cats collected in various households andcilities from both diarrheic and apparently healthynimals.

.1. Overview of the feline fecal virome

High throughput metagenomic sequencing of a felinecal sample was performed using the following steps:ltration of fecal supernatant to enrich viral particles,epletion of host nucleic acid in ltrate using nucleases,nbiased sequence-independent amplication using ran-om priming, and deep sequencing using Illumina MiSeqllander et al., 2005; Ng et al., 2012). Sequence data

evealed ve eukaryotic virus species (Table 1, Fig. 1). Non-iral reads consisted mostly of bacterial and unclassiableequence by BLAST. The eukaryotic viral sequencesetected, in decreasing number of reads, were picornavir-s > astrovirus > bocavirus > picobirnavirus > rotavirusable 1). The picornavirus generated the most sequenceeads (48,000 or 9% of total reads), while rotavirusenerated the least reads (2 or

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T.F.F. Ng et al. / Veterinary Microbiology 171 (2014) 102111104The P2 region of SakoV-A encodes the non-structural 2A13 aa), 2B (150 aa) and 2C (337 aa) proteins. The 2Aotein of SakoV-A contained an H-box/NC motif con-rved in kobuvirus and passerivirus, but not in salivirus.e H-box/NC proteins are related to cellular proteinsvolved in cell proliferation. The carboxyl terminus of the

2A protein contained a transmembrane domain related tothose of kobuvirus and passerivirus. The 2C genecontained the conserved NTPase motif GxxGxGKS(GAPGVGKS). SakoV-A 2C also contain the helicase motifDDxxQ (DDIGQ), which resembles the kobuvirus/salivirus/passerivirus motifs (DD[L/I/V]GQ).

. 1. Phylogenetic and genomic analysis of ve feline enteric viruses. The Bayesian inference trees were conducted based on (A) 3D RdRp protein depicting

ationships among representative members of the family Picornaviridae, and a table detailing genomic features of the reference genomes based on

viously published literature (Lau et al., 2012b; Sweeney et al., 2012). Symbols for protease were noted as follows: Y, presence of a protease motif in L; Y/

presence of L but L is not protease; N, absence of L. Other motifs are described in the main text (B) the complete ORF2 capsid protein of representative

roviruses (C) the complete non-structural protein (NS1) of bocaviruses, (D) the partial intermediate capsid protein of rotavirus Segment 6, and (E) the

rtial RdRp protein of picobirnavirus Segment 2. Posterior probabilities of the Bayesian analysis (>95%) are shown next to each node. Scale bar indicates

ino acid substitutions per site.

Fig. 2. Genome organization, identity plot and pairwise sequence comparison analyses of three feline virus genomes (A) feline sakobuvirus A, (B) feline

astrovirus Viseu, and (C) feline bocavirus 2. Cleavage site predictions of the sakobuvirus are shown below its genome. For each virus, three identity plots are

shown below the genome organization, comparing the coding regions for each virus with three related viruses based on phylogenetic analyses in Fig. 1. The

identity values were color-coded: green, 100%; light green, 30100%, red,

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T.F.F. Ng et al. / Veterinary Microbiology 171 (2014) 102111106The P3 region of SakoV-A encodes the 3A (78 aa), 3B (29), 3C (190 aa), and 3D (468 aa) proteins. The 3C protein,hich encodes for a protease, contains the conservedtalytic triad HEC, conserved active site motif GxCGLCG), and lacks the RNA-binding motif KFRDI. All ofese 3Cpro features are also found in kobuvirus, salivirusd passerivirus (Fig. 1). The 3D protein, which encodese RdRp, contained the conserved KDELR, GGNPSG, YGDDd FLKR motifs.Four clusters of picornaviruses, the sakobuvirus,buvirus, salivirus and passerivirus, form a monophy-ic clade (shaded, Fig. 1A). These four groups sharemmon genomic features including a high GC content, Lotein, uncleaved VP0, and an HEC catalytic triad in the

protease (Fig. 1A).

Analysis of the picornavirus non-coding elements

As determined by RACE, the 50 UTR of SakoV-A was 591cleotides long and contained a type IV internalosomal entry site (IRES) (Hellen and de Breyne, 2007).is unknown whether any of the 50 UTR may still beissing. The AUG initiation codon was postulated to be atsition 592594. The rst 21 nucleotides of 50 UTR were% identical to those at the same location of caninebuvirus (GenBank JN088541). The rst 80 nucleotideshibit potential secondary and tertiary structure (hair-ns A%3Fndash;C, data not shown) found in kobuviruses

(Reuter et al., 2009). High sequence identity was foundbetween the 30 end of the 50 UTR and similar regions inporcine kobuvirus (GenBank EU787450, genus Kobuvirus)and duck hepatitis A virus 1 (GenBank EU395440, genusAvihepatovirus) (73% and 89% identity to position 462582and 506562 of the SakoV-A genome, respectively).

The predicted secondary RNA structure of the 50 UTRIRES showed a potential hepacivirus/pestivirus-like (HP)type IV IRES with pseudoknots and conserved elements inan upstream bent hairpin-like structure in domain II (II1,II2, II3) and in domain III (III1, III2, III3, III4, IIIa, IIIb, IIId, IIIeand IIIf) (Fig. 3). The predicted lengths of the pseudoknotstem 1, stem 2, linker, and spacer are 11, 8, 2, and 9nucleotides, respectively. The conserved nucleotide motifsof branching hairpins IIId and IIIe are: UUGGGAAA510517and GCCUGAUAGGGU538549 (Fig. 3).

The 30 UTR was 157 nucleotides long. Position 77187769 shared 85% sequence identity with duck hepatitis Avirus 1 strain FZ05 (position 76037653 of GenBankJX390983). The unexpected region of RNA sequencesimilarities to duck hepatitis A virus 1 in genus Avihepa-tovirus at both extremities of the sakobuvirus genome mayreect past recombinations seen even between differentpicornavirus genera (Hellen and de Breyne, 2007). TheSaKoV-A 30 UTR is predicted to form a barbell-likesecondary RNA structure also identied in members ofthe genera Avihepatovirus, Kobuvirus, Gallivirus andPasserivirus (Fig. 3).

. 3. Predicted RNA secondary structure of the 50 and 30 UTR of the feline sakobuvirus A (FeAstV-A) and the 30 UTR of the feline astrovirus Viseu. For FeAstV-0 UTR, the type IV IRES has been annotated according to picornavirus conventions. Domains are labeled II and III; individual helical segments are labeled, II2, III1, and III2, etc.; and individual hairpins are labeled IIIa and IIIb, etc. to maintain the continuity of the current nomenclature. The positions of

nserved domains and the polyprotein AUG start codon are indicated by shaded boxes. For FeAstV-A 30 UTR, detailed structure of the barbell-likemation between nucleotide 7712 and 7775 was shown. Grey boxes indicate identical nucleotides in members of genera Avihepatovirus, Kobuvirus,

allivirus and Passerivirus. The presence of poly(Y) tract characteristic of this barbell-like structure is indicated.

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T.F.F. Ng et al. / Veterinary Microbiology 171 (2014) 102111 107.1. Astrovirus

Using deep sequencing, PCR and RACE, we obtained aomplete feline astrovirus genome (FeAstV Viseu). Astro-iruses are known to infect a wide variety of mammals andirds, and feline astrovirus has been shown to inducenteritis by experimental infection of specic-pathogen-ee cats (Harbour et al., 1987). Feline astrovirus (FeAstV)as observed by electron microscopy from cat feces withr without diarrhea (Hoshino et al., 1981; Marshall et al.,987), and FeAstV sequences have been reported in Europend in Asia (Lau et al., 2013; Harbour et al., 1987).The genome of feline astrovirus Viseu was 6780

ucleotides in length, with 49.8% GC content (GenBankF374704). Consistent with other astroviruses, three ORFsere identied, including ORF1a and ORF1b that encodeon-structural proteins, and ORF2 that encodes thetructural capsid proteins. In the ORF1a/1b overlap regionf the FeAstV genome, the conserved slippery ribosomalame shift signal (AAAAAAC) was identied. In addition, ate ORF1ab/ORF2 junction, the highly conserved promoterequence UUUGGAGNGGNGGACCNAAN7 AUGNC wasentied, in which the start codon AUG initiating ORF2

located near 30 end.Phylogenetically, FeAstV Viseu belongs to the mamas-

ovirus genogroup 1 (Fig. 1B), and is closely related to aline astrovirus (FeAstV2) reported in September 2013 inats from Hong Kong (Lau et al., 2013) and more distantly

another feline astrovirus from the United KingdomeAstV Bristol, GenBank AF056197). Using pairwiseomparison of the entire capsid protein, FeAstV Viseuhared 92.6% identity to FeAstV2 from HK, 70.8% to FeAstVristol, and 58.7% to human astrovirus 1. Among felinestroviruses, FeAstV Viseu and FeAstV2 differ most fromeAstV Bristol in the C-terminal half of the capsid protein,haring %3Flt;40% identity.According to the ICTV, the ORF2 capsid proteins are

sed to distinguish genotypes and species of astroviruses.oticeably, the N-terminal half of the feline astrovirusRF2s shared high amino acid identities among them-elves (FeAstV Viseu and FeAstV 2, 96%, Fig. 2B) and withuman astroviruses (FeAstV Viseu and HAstV1, 89%). TheeAstVs are therefore most closely related to HAstVs in theapsid amino terminal half, compared to the next closestelative, the porcine astroviruses (FeAstV Viseu andoAstV2, 65%). In addition, long stretches of near-identicalmino acid residues are observed between feline anduman astroviruses (Fig. 2B, black arrow)The carboxyl terminal half of the ORF2 capsid protein

howed much weaker conservation (Fig. 2B). A conservedotif (SRGHAE) was recognized. The 30 UTR was 84ucleotides long, and shares most nucleotide identities4%) to human astrovirus SH1 (GenBank FJ375759). RNAlding analysis revealed a double stem loop structure in the%3Fprime; UTR sequence typical for astroviruses (Fig. 3).Based on currently available genome information, theost closely related viruses to the classic humanstroviruses (mamastrovirus 1 genotype 18) are there-re feline astroviruses, with the Bristol strain slightlyloser than Viseu or the Honk Kong FeAstV2 strains (Fig. 1Bnd Fig. 2B). Past human-cat cross-species astrovirus

transmission may have led to this close phylogeneticrelationship.

2.2. Bocavirus

A new bocavirus was also detected, represented by 230sequence reads that shared 15% to its closest relative, FBoV2 istherefore a viral species distinct from FBoV. The clade offeline bocaviruses (FBoV and FBoV2) was most closelyrelated to canine minute virus, canine bocaviruses, andCalifornia sea lion bocaviruses (Fig. 1C).

2.3. Rotavirus

Rotaviruses, members of the family Reoviridae, cancause severe diarrhea in young or immunosuppressedsubjects of various host species. Their genome consists of11 segments of double-stranded RNA. Based on theantigenic VP6 protein, rotaviruses have been subdividedinto eight serological species AH by the ICTV (Matthijns-sens et al., 2012).

Here a novel rotavirus, feline rotavirus Viseu (GenBankKF792839), was detected and its presence was conrmedby PCR and Sanger sequencing. We obtained a 206-base-pair sequence fragment belonging to the Segment 6encoding the intermediate capsid VP6. This partialsequence shared its highest amino acid identity (53%) tothe group H rotaviruses (Matthijnssens et al., 2012) and

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T.F.F. Ng et al. / Veterinary Microbiology 171 (2014) 102111108wever, sequences of all of its 11 segments will bequired to determine its exact phylogenetic relationship other rotaviruses.

. Picobirnavirus

Picobirnaviruses (PBVs) are small non-enveloped vi-ses with a bisegmented double-stranded RNA genomeing et al., 2011). Picornaviruses are highly diverse andn be grouped in two major clades (GI and GII).cobirnaviruses have been reported in fecal and respira-ry samples in humans, non-human mammals, reptiles,d birds, but the pathogenicity of picobirnavirus remainsknown. Until now, no picobirnaviruses had beenported from cats or the family Felidae (King et al., 2011).A feline picobirnavirus genome was partially character-d, in which we obtained 1236 base pairs of Segment 2 thatcodes the RdRp (GenBank KF792838). The feline picobir-virus was most closely related to a GII human picobirna-rus (ACY01868, Fig. 1E), sharing 65% amino acid identity.

. Prevalence and co-infections of enteric viruses in feline

es

Viral shedding for each of the ve viruses was PCRsted in the feces from 55 cats (including the original fecalmple) from the city of Viseu, Portugal. The presence of alle viruses was conrmed in the original sample. Nineher animals were found to be shedding one to three ofese feline viruses. The most common detection wastavirus (7/55), astrovirus (4/55), bocavirus (3/55), felinekobuvirus (2/55), and picobirnavirus (2/55) and co-fections were frequent (Fig. 4). Although the samplesere collected from different households, clinics, andcilities within the same city (Suppl. Table 1), the resultingrus sequences shared >99% amino acid identities in theort targeted amplicons, suggesting that the same strainsere circulating in this city. Because highly specic PCRimers were designed to target only the genomes

identied in the original animal, divergent strains maynot have been amplied. Other viruses not found in theoriginal case would also have gone undetected. Of the 55samples, eight were from cats with diarrhea, and one of thediarrheal cat was positive for sakobuvirus (Suppl. Table 1).

3. Discussion

The global distribution of cats and their close contactswith humans underscore the need to better understand thecomposition of their virome. Five viruses were detected ina single cat feces sample, four of which were highlydivergent from previously reported viruses, demonstratingthat the feline enteric virome is still largely uncharacter-ized. Although multiple precedents exist for relatedviruses detection in cats and other carnivores it is formallypossible that some of these viral sequences are fromingested meat and do not reect viruses replicating inthese cats digestive systems. Cat inoculations, andantibody testing of cats will be required to conrm thefeline tropism of these fecal viruses.

Our data show that shedding of enteric virus(es) is verycommon, as 18% (10/55) of the cats tested PCR-positive forat least one of these ve viruses (Fig. 4 and Suppl. Table 1).Nine out of the 10 cats positive for enteric viruses appearedhealthy (Suppl. Table 1.) and co-infections were common(Fig. 4 and Suppl. Table 1). The lack of dened clinicalsymptoms in cats shedding one of the viruses supportsprior studies reporting frequent enteric viral infections inhealthy animals (Sabshin et al., 2012; Shan et al., 2011) andhumans (Ayukekbong et al., 2011; Kapusinszky et al.,2012; Wits et al., 2006). In order to determine whetherthese feline viruses may cause disease in a subset ofanimals (e.g. certain breeds or immunodecient cats),disease association studies comparing their prevalence indiseased versus epidemiologically matched healthy con-trol, or laboratory inoculation of these viruses, will berequired.

This initial characterization of the feline viromeprovides candidate pathogens for future studies ofgastrointestinal or other diseases in cats. Feline sakobu-virus is distantly related to the genera Kobuvirus andSalivirus and the proposed avian genus Passerivirus.Aichi virus in the Kobuvirus genus has been associated withhuman gastroenteritis, and animal kobuviruses have beendetected in pigs and cows with diarrhea as well as inmouse feces, canine feces, and raw sewage (Kapoor et al.,2011; Li et al., 2011; Phan et al., 2011). Human salivirus(also known as klassevirus) has been associated withdiarrhea and detected in human feces from both gastro-enteritis patients and healthy people (Greninger et al.,2010; Greninger et al., 2009; Li et al., 2009). Thecharacterization of a new feline picornavirus will allowfurther investigation into its pathology using animalmodels.

Viral pathogens can originate from reassortment andrecombination events among and/or between human andanimal viral species. Some rotaviruses infecting humansare likely to have originated from animals, and felinerotaviruses have been implicated in reassortments withhuman viral strains (Martella et al., 2010; Tsugawa and

Sakobuvirus

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otavirus

1 cat with 5 viruses

2 cats with 3 viruses

1 cat with 2 viruses

cats with rotavirus

only

1 cat with picobirnavirus

only

1 cat with Sakobuvirus only

1 cat with Astrovirus

only

55 cats were investigated

45 cats negative for theviruses investigated

. 4. Venn diagram analysis of the ve enteric viruses in fecal samples

m 55 cats. Co-infections of more than one virus were annotated.

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T.F.F. Ng et al. / Veterinary Microbiology 171 (2014) 102111 109stroviruses (FeAstV Viseu, FeAstV2 and FeAstV Bristol) aree phylogenetically closest astroviruses to the mainuman astrovirus group (mamastrovirus 1). In addition,e closest known relative to the rst feline picobirnavirusenome reported here is one of the numerous variants ofuman picobirnaviruses. These results suggest the occur-ence of past astroviruses and picobirnaviruses cathumanoonoses.This study detected an unexpectedly high number of

reviously unknown and recently described viruses in theces of a single healthy cat. An even greater number ofiruses may have been detected if the virion size limitposed by the use of 220 nm pore size lters (Section 4)as increased to allow passage of larger viral particles.iven the number of new viral species in a single catample, further viral metagenomics and PCR prevalencetudies in this globally distributed carnivore are likely toapidly increase the number of viruses associated withats. The frequent contacts of cats with humans, as well asirds, rodents, and dogs, likely expose those cats toumerous viruses with zoonotic potential. The emergencef canine parvovirus 2 from feline panleulopenia in the late970s provides one of the best understood examples ofuch cross-species adaptation (Hoelzer and Parrish, 2010),hile other parvovirus zoonoses do not appear to result inequent onward transmission in their new hosts (Allisont al., 2013). Comparisons of the viromes in closelyteracting animal species, including humans, will indicatehich viruses were likely transmitted among them beforerther mutations allowed them to fully adapt to their newost.

. Materials and methods

.1. Sample collection

A total of 55 stool samples were collected betweeneptember and December 2012 from cats housed inatteries, municipal pounds, non-prot rescue shelters,eterinary hospitals, and veterinary clinics across the cityf Viseu, Portugal (Suppl. Table 1). Feces were kept at20 8C until processing.

.2. Metagenomic sequencing

Viral particles were rst enriched from the feces of oneealthy, non-diarrheal, cat. Frozen feces were resuspended

PBS buffer, claried by brief centrifugation and semi-uried by 200 nm lter (Millipore). The ltrate wascubated with a cocktail of DNase and RNase for 1.5 h (Ngt al., 2012). Puried viral nucleic acids were extractedsing QIAamp Viral RNA Mini Kit. Reverse transcriptioneactions (RT) were performed, using an arbitrarilyesigned 20-base oligonucleotide followed by a random-ed octamer sequence at the 30 end as previouslyescribed (Ng et al., 2012). After denaturation, theomplementary strand was synthesized using the Klenowagment DNA polymerase (New England Biolabs), fol-wed by 1520 rounds of PCR amplication using a primeronsisting of only the 20-base xed portion of the 30

MiSeq platform (Illumina). Raw reads were trimmed forquality and primer, and assembled de novo into contigs.Sequences and contigs were compared to the GenBanknon-redundant protein database using BLASTx with an E-value cutoff of 104.

4.3. Acquisition of viral genomes

In order to obtain full viral genomes, primers weredesigned to amplify and sequence gaps between metage-nomic sequences. PCRs were performed as follows: 95 8Cfor 5 min, 45 cycles of [94 8C for 30 s, 58 8C minus 0.2 8C percycle for 30 s, 72 8C for 1 to 5 min], followed by 72 8C for10 min. The 50 and 30 genome extremities were ampliedusing RACE amplication kits (Invitrogen) according to themanufacturers instructions and the detailed protocolspreviously described (Ng et al., 2012). PCR amplicons weredirectly sequenced in their entirety using specic primersand Sanger sequencing.

4.4. Screening of feline fecal viruses by PCR

Stool samples were diluted (10%) in sterile PBS andcentrifuged at 8000 g for 5 min to remove solids. Nucleicacid was extracted from 140 ml of the supernatant withspin column (Viral RNA mini kit, Qiagen, Germantown,USA) according to the manufacturers instructions. Primersequences were described in Table 2. For screening felinepicornavirus, primers SakobuAF and SakobuAR targeting304 nucleotides of the 50 UTR was used. For screeningfeline astrovirus, primers FeAstVAF and FeAstVBF targeting418 nucleotides of the ORF1b were used. For bocavirus,primers FBoV2AF and FBoV2AR targeting 301 nucleotidesof the nonstructural gene were used. For rotavirus, primersFeRVAF and FeRVAR targeting 151 nucleotides of theSegment 6/intermediate capsid were used. For picobirna-virus, primers FePBVAF and FePBVAR targeting 1327nucleotides of the RdRP were used.

For feline sakobuvirus, astrovirus, rotavirus and pico-birnavirus RNA detection RT-PCR kits were used (Qiagenone-step RT-PCR Kit, Hilden, Germany). Reverse transcrip-tion was performed at 42 8C for 30 min followed by 95 8Cfor 15 min. Touchdown PCR cycling conditions consistedof: (i) for sakobuvirus, 8 cycles [30 s at 94 8C, 1 min at 63 8Cminus 1 8C per cycle, 1 min at 72 8C] and 32 cycles [30 s at94 8C, 1 min at 55 8C, 1 min at 72 8C] followed by a nal

Table 2

Primers used for the PCR screening.

Primer name Primer sequence

SakobuAF CTG GTC GTG GTG ACC GGC TG

SakobuAR AGC CGC GAC CCT ATC AGG CA

FeAstVAF GAA ATG GAC TGG ACA CGT TAT GA

FeAstVBF GGC TTG ACC CAC ATG CCG AA

FBoV2AF TCG TTC GTC TTG GAA CAT AGC

FBoV2AR CAG AGC GTG GAT CTG TCT GA

FeRVAF ATC ACC AAC GTGT TGT CTA CTG A

FeRVAR TTT TGT GAC TTC CGG ATC AGC

FePBVAF AGA CCA CCG ACC ATG TTC TC

FePBVAR CTC AAA TGT GCAA ACC GAA Aoshino, 2008). Our analysis indicates that the feline r

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T.F.F. Ng et al. / Veterinary Microbiology 171 (2014) 102111110tension for 10 min at 72 8C; (ii) for astrovirus andtavirus, 5 cycles [30 s at 94 8C, 1 min at 55 8C minus 1 8Cr cycle, 1 min at 72 8C] and 35 cycles [30 s at 94 8C, 1 min

50 8C, 1 min at 72 8C] followed by a nal extension for min at 72 8C; (iii) for picobirnavirus, 2 cycles [30 s at 8C, 1 min at 52 8C minus 1 8C per cycle, 1 min at 72 8C]d 38 cycles [30 s at 94 8C, 1 min at 50 8C, 1 min at 72 8C]llowed by a nal extension for 10 min at 72 8C. For felinecavirus DNA detection, PCR was performed with KAPAq DNA Polymerase (Kapabiosystems, MA, USA) with thecling conditions consisting of 35 cycles of 30 s at 94 8C,

s at 50 8C, and 1 min at 72 8C, followed by a naltension for 10 min at 72 8C.Amplied products of appropriate size were visualizedder UV, excised from the electrophoresis gel, puriedith the GRS PCR & Gel Band Purication Kit (GRISPsearch Solutions, Porto, Portugal) and sequenced in bothrections using the BigDye Terminator v1.1 Cyclequencing kit (PE Applied Biosystems, Foster City,arrington, UK).

. Genomic and phylogenetic analyses

For picornavirus, the RNA secondary structures of theternal ribosomal entry site (IRES) of the 50 UTR wasedicted using the Mfold program with manual correctionr picornavirus (Reuter et al., 2009). For astrovirus, theA secondary structure of the 30 UTR was also analyzed

ith the Mfold program.For sequence alignment, sequences were aligned usingafft 5.8 with the E-INS-I alignment strategy andeviously described parameters (Ng et al., 2011). Bayesianference trees were constructed using MrBayes. Thearkov chain was run for a maximum of 1 millionnerations. Every 50 generations were sampled and thest 25% of mcmc samples were discarded as burn-in. Mid-int rooting was conducted using MEGA. Identity plotsere created from sliding window analyses using pairwiseino acid sequence alignments, with protein identitieslculated between translated sequential fragments of 32-frame codons, incrementing by 8 codons.

knowledgments

This work was supported by NIH grant HL105770 to., the Blood Systems Research Institute, and thengarian Scientic Research Fund (OTKA K83013). G.R.as supported by the Janos Bolyai Research Scholarship ofe Hungarian Academy of Sciences. NJK is partiallypported by core-funding provided by the Biotechnologyd Biological Sciences Research Council (BBSRC) to Therbright Institute. We would like to acknowledge Nadiato and Gregorio Gomes for technical assistance.

pendix A. Supplementary data

Supplementary material related to this article can beund, in the online version, at http://dx.doi.org/10.1016/etmic.2014.04.005.

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Feline fecal virome reveals novel and prevalent enteric virusesIntroductionOverview of the feline fecal viromePicornavirus

Analysis of the picornavirus non-coding elementsAstrovirusBocavirusRotavirusPicobirnavirusPrevalence and co-infections of enteric viruses in feline feces

DiscussionMaterials and methodsSample collectionMetagenomic sequencingAcquisition of viral genomesScreening of feline fecal viruses by PCRGenomic and phylogenetic analyses

AcknowledgmentsSupplementary data

References