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Arch Virol DOI 10.

1007/s00705-012-1470-0

ANNOTATED SEQUENCE RECORD

Discovery of a novel circular single-stranded DNA virus from porcine faeces
¨ Alyssa Sikorski • Gerardo R. Arguello-Astorga Anisha Dayaram • Renwick C. J. Dobson • Arvind Varsani

Received: 20 July 2012 / Accepted: 30 July 2012 Ó Springer-Verlag 2012

Abstract A large number of novel single-stranded DNA (ssDNA) viruses have been characterised from various environmental sources in the last 5 years. The bulk of these have been from faecal sources, and faecal sampling is an ideal non-invasive pathogen sampling method. We characterised a novel ssDNA from a porcine faecal sample from Cass Basin of the South Island of New Zealand. The novel viral genome has two large open reading frames (ORFs), which are bidirectionally transcribed and separated by intergenic regions. The largest ORF has some degree of similarity (\30 %) to the putative capsid protein of chimpanzee stool-associated circular ssDNA virus (ChiSCV) and pig stool-associated single-stranded DNA virus (PigSCV), whereas the second-largest ORF has high similarity to the putative replication-associated protein (Rep) of ChiSCV (*50 %) and bovine stool-associated

circular DNA virus (BoSCV; *30 %). Based on genome architecture, location of putative stem-loop like elements, and maximum-likelihood phylogenetic analysis of the gene encoding the Rep protein, the novel isolate belongs to the same family of ssDNA viruses as ChiSCV and BoSCV.

Electronic supplementary material The online version of this article (doi:10.1007/s00705-012-1470-0) contains supplementary material, which is available to authorized users.
A. Sikorski Á A. Dayaram Á R. C. J. Dobson Á A. Varsani (&) School of Biological Sciences, University of Canterbury, Ilam, Private Bag 4800, Christchurch 8140, New Zealand e-mail: arvind.varsani@canterbury.ac.nz ¨ G. R. Arguello-Astorga ´ ´ Division de Biologıa Molecular, Instituto Potosino de ´ ´ ´ Investigacion Cientıfica y Tecnologica, Camino a la Presa San ´ ´ Jose 2055, 78216 San Luis Potosı, SLP, Mexico R. C. J. Dobson Á A. Varsani Biomolecular Interaction Centre, University of Canterbury, Private Bag 4800, Christchurch 8140, New Zealand A. Varsani Electron Microscope Unit, University of Cape Town, Rondebosch, Cape Town 7701, South Africa

Animals are known to be a significant reservoir for emerging human pathogens [33], and recently, diverse circular single-stranded (ss) DNA viruses have been isolated from human and farm-animal faeces. Some of these are related to well-characterized animal-pathogenic circoviruses. For example, two PCV2 variants were isolated from human faecal samples, suggesting possible zoonotic transmission [20]. New molecular tools have enabled virologists to explore ssDNA viromes that were previously unknown, and in doing so, a large number of novel ssDNA viruses have been characterized over the last couple of years. However, limited information is available on their actual hosts or host ranges. Faecal samples provide an opportunity for non-invasive sampling of animals in an environment for the purpose of describing the ‘virome’ and characterising novel viruses. Much of the viral diversity and distribution in the environment is unknown [24], but once a virus is isolated and characterized, molecular tools can be designed and used to test domestic and wild animals for such infections. Circular ssDNA viruses can infect a range of hosts. To date, novel circular ssDNA viruses have been isolated from the faecal matter of animals such as chimpanzees [6, 19], rodents [22], bovines [17], bats [13], badgers and pine martens [31], and pigs [27, 28]. As with other studies that have looked at bioaccumulators of viruses [4, 21], isolates from faecal matter may contain viruses other than those infecting the host, and may contain those that are infecting

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A. Sikorski et al. Fig. 1 Illustration of the genome organisation of PoSCV showing the potential stem-loop elements in the LIR and SIR
G A T C G C C T C G G G G G G A G C C C C A A

A G T T T

T

T A C C T

Rep
A C G A A

G A T

LIR

A A C G C C G T C G T T A T

PoSCV (2589nt)

G G C A A

Cp
SIR

insects, plants, protozoa or bacteria that have been acquired from the environment [9]. The diversity of the viral community in pigs has been explored in two recent studies; both protocols used a combination of random PCR and deep sequencing [27, 28]. ¨ Sachsenroder et al. [27] isolated a novel circular ssDNA virus, pig stool-associated single-stranded DNA virus (PigSCV). PigSCV has similarity to chimpanzee stoolassociated circular ssDNA virus (ChiSCV), isolated from chimpanzee stool samples [6], in the replication-associated protein (Rep) and capsid protein (CP) open reading frames (ORFs), but the orientation is that of a same-sense genome, and the overall sequence identity is low [27]. Shan et al. [28] isolated four full genomes of novel circular ssDNA viruses from pig faeces by inverse PCR (po-circo-like virus-21, -22, -41, and -51). The Rep sequences showed similarity to the Rep sequences of enteric amoebas Entamoeba dispar and Entamoeba histolytica, the latter of which causing amebiasis disease in humans [5], suggesting a possible origin [28]. In both studies, a small group of local pigs were used; however, a comprehensive study on the geographical diversity of viromes has not been done. Here, we characterise a novel circular ssDNA virus extracted from porcine faeces in New Zealand. The pig sample in this study was taken from a domestic penned pig from a small farm in the Cass Basin in the central South Island. The faecal samples were homogenized in SM buffer (0.1 M NaCl, 50 mM Tris/HCl, pH 7.4, 10 mM MgSO4) at a ratio of 5 ml buffer to 5 g of faecal sample. The sample was centrifuged (10,000 rpm for 10 min) and filtered through 0.45 and 0.2-lm syringe filters (Sartorius Stedim Biotech, Germany). The viral DNA was extracted using a High Pure Viral Nucleic Acid Kit (Roche, USA), and the viral genomes were amplified non-specifically by rollingcircle amplification using an Illustra TempliPhi Amplification Kit (GE Healthcare, USA) as described previously [8, 23, 25, 29, 32]. The amplified viral DNA from the pig

sample was digested with a variety of restriction enzymes, and BamHI yielded a product of *2 kb. This fragment was cloned into plasmid pUC19 (Fermentas, USA) cut with BamHI. The cloned genome was sequenced at Macrogen Inc. (Korea) by primer walking. Based on the resulting sequence, back-to-back primers (as5bhF 50 -CGA GCA TTT CCT GAA CCT TGT TAC-30 and as5bhR 50 -GCG GAT TCT TCA GAC AGT TCA G-30 ) were designed in order to amplify the full genome. The genome was amplified using Kapa HiFi HotStart DNA polymerase (Kapa Biosystems, USA) and the following PCR protocol: initial denaturation at 94 °C for 2 min followed by 94 °C for 15 s, 50 °C for 15 s, 72 °C for 1 min for 25 cycles and a final extension of 72 °C for 2 min. The resulting *2.5kb product was cloned into the pJET1.2 vector (CloneJETTM PCR cloning kit, Fermentas, USA) and sequenced by primer walking at Macrogen Inc. (Korea). The sequence contigs were assembled using DNAMAN (version 5.2.9; Lynnon Biosoft). Basic analysis of the genome revealed two large ORFs, which are bidirectionally transcribed, and five smaller ORFs ([200 nucleotides; Fig. 1). A BLASTn [1] analysis of the genome revealed some 78 % identity over 10 % of the genome (E-value of 5 9 10-10) with ChiSCV isolate GT306 (GQ351278). Based on these finding we have tentatively named the novel virus porcine stool-associated circular virus (PoSCV; GenBank accession no. JX274036). We also identified two intergenic regions (IRs): the long intergenic region (LIR) and short intergenic region (SIR), of 515 and 292 nucleotides in length, respectively. A BLASTp search of the largest ORF of PoSCV (348 residues) suggested that it may encode the CP, as it has a high degree of similarity to the putative capsid proteins of ChiSCV (GenBank accession no. ADB24817; maximum identity of 34 % over 85 % of the ORF; E-value 4 9 10-47) and PigSCV (GenBank accession no. YP_006331068; maximum identity of 26 % over 97 % of

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Novel circular single-stranded DNA virus from porcine faeces

the ORF; E-value 2 9 10-24). A BLASTp search using the second-largest ORF (243 residues) revealed a significant degree of similarity with the Rep of ChiSCV (GenBank accession no. ADB24799; maximum identity of 51 % over 88 % of the ORF; E-value 4 9 10-65) and minor similarities to the Rep (spliced Rep) proteins of bovine stoolassociated circular DNA virus (BoSCV; AEW47007; 87 % identity over 33 % of the ORF; E-value 2 9 10-19), various nanoviruses and geminivirus alpha satellites (30–35 % identity over *50 % of the ORF with E-values of 4 9 10-9 to 3 9 10-5). Pairwise comparisons of the Rep (amino acid sequence with pairwise deletion of gaps) of PoSCV with those of the ChiSCV isolates indicates that they share 49.8–51.5 % identity; however, PoSCV only shares 33.5 and 28.2 % identity with BoSCV and PigSCV, respectively. On the other hand, the Rep sequences of BoSCV and PigSCV share 29.9 % pairwise identity, and they share 30.1–31.8 % and 26.7–28.8 % identity with those of the ChiSCV isolates. Sequences of representative Reps of circoviruses, cycloviruses, nanoviruses, geminiviruses, and all novel ssDNA viruses available in GenBank (as of 14th May 2012) were aligned with the Rep of PoSCV using MUSCLE [10], and the alignment was manually edited in MEGA5 [30]. The resulting alignment was used to construct a maximumlikelihood (ML) phylogenetic tree using the LG model and PHYML (version 3) [14] with approximate likelihood-ratio test (aLRT) branch support [2]. Branches with less than 60 % aLRT support were collapsed with Mesquite (version 2.75). Phylogenetically, the Rep of PoSCV clusters with the ChiSCV isolates described by Blinkova et al. [6], the ¨ PigSCV isolate described by Sachsenroder et al. [27], and the BoSCV isolate described by Kim et al. [17]. It is interesting to note that this cluster only has viral isolates from faecal material of bovine, porcine and primate origin (Fig. 2). It is well documented that the Rep of ssDNA viruses and plasmids that replicate via rolling-circle mechanisms are highly conserved [15, 18]. Within the Rep of the PoSCV, we identified the conserved motifs rolling circle motif I (MTIPR), RCR II (HWQIRI), RCR III (YETKEGQY). We also identified putative walker A (TIDTV) and B (GNVGKSW) helicase motifs (Table 1), which are characteristic of superfamily 3 (SF3) helicases (reviewed by Rosario et al. [26]). Despite the highly conserved motifs (RCR I-III) identified in the PoSCV sequence, the overall identity of the Rep protein at the amino acid level, when compared to its closest homologue with known structure (the Rep protein nuclease domain from faba bean necrotic yellows virus, PDB id 2HWT), was only *15 %. Nevertheless, we made an attempt to generate a homology model for this protein using SWISS model [3, 7, 16] and MODELLER [11, 12] to further explore its structure. The model,

however, was of poor quality and unreliable, as judged by the low QMEAN Z-score. The sequence identity increased to *24 % when the first 83 amino acids (out of 243) were aligned, but this did not significantly improve the quality of the model. Through secondary structural analysis of the two IR nucleotides, we identified a probable stem-loop structure in each of the IRs (Supplementary Fig. 1). Comparative analysis of the LIR regions of PoSCV, ChiSCV and BoSCV did not reveal any conserved stem-loop elements. In contrast, ChiSCV and BoSCV were found to contain a stem-loop element in the SIR that is conserved in position at the end of the Rep gene, and these viruses displayed a similar organisation of repeated sequences, at least in the case of PoSCV and ChiSCV. Therefore, we conclude that the actual replication origin of PoSCV, like those of ChiSCV and BoSCV, is most probably located close to the Rep gene stop codon in the SIR (Fig. 1). This is not surprising, since they have similar genome architectures and probably represent a unique family of ssDNA viruses. Interestingly, PigSCV is the only member of the unique clade (Fig. 2) that has a unidirectional genome organisation. PoSCV has a ‘‘TAGTGTTAC’’ nonanucleotide motif, whereas those of ChiSCV and BoSCV are ‘‘AATAGTTAC’’ and ‘‘CAGTATTAC’’, respectively. Therefore, PoSCV and ChiSCV are different from the ‘‘NANTATTAC’’ nonanucleotide motifs found in nanoviruses, circoviruses and cycloviruses. Based on the genome organisation, similarities in the Rep and CP sequences, and the location of the stem-loop element, PoSCV, BoSCV and ChiSCV potentially represent a new family of ssDNA viruses with PoSCV, BoSCV and ChiSCV each representing a genus within this family. The Rep of PoSCV, BoSCV and ChiSCV shares an evolutionary relationship with PigSCV and could be an ancient recombinant; however, more similar ssDNA viruses with unidirectional genomes need to be identified and analysed in order to support this notion. However, it should be noted that it is unknown whether these viruses infect the animals which are the source of the faecal matter or instead infect other organisms associated with these animals or their food source. Infectivity assays will need to be carried out once appropriate cell lines have been established. Additionally, infectious virions need to be isolated from faecal matter to infect a cell line in order to partially fulfil Koch’s postulates. Nonetheless, molecular probes can be developed based on the sequence information for screening of similar viruses in cows, pigs and chimpanzees, which may shed light on the molecular diversity and global distribution of these viruses. Over the last couple of years, there has been a tremendous amount of activity in environmental sampling of viruses, especially ssDNA viruses. To date, a total of 51

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A. Sikorski et al.

Geminiviruses
100 95 98 97

CasCV [JQ412057] SsHADV-1 [GQ365709] Mosquito VEM virus SDBVL G [HQ335086] MmFV [JN704610] ABTV [EF546813] 100 BBTV [S56276] FBNYV [AJ132187] MVDV [AB000920] 90 SCSV [U16735]
86 98

Gemimycircular viruses

Nanoviruses

ChiSCV GM488 [GQ351276] ChiSCV GM415 [GQ351277] ChiSCV DP152 [GQ351272] 92 84 ChiSCV GT306 [GQ351278] PoSCV [JX274036] BoSCV CP11-49-3 [JN634851] SAR-B [FJ959085] RW-D [FJ959080] BBC-A [FJ959086] MS584-5 [HQ322117] R15 [JF755401] 100 V76 [JF755404] 99 V77 [JF755405] 91 87 V87 [JF755406] 99 PoC21 [JF713716] 100 PoC22 [JF713717] 97 PoC41 [JF713718] PoC51 [JF713719] 76 CB-A [FJ95]9082] 91 V-77 [JF755415] 84 TM-6C [HM228875] SDWAPI [HQ335042] 90 RW-C [FJ959079] 86 100 V-91 [JF755417] V86 [JF755416 V-64 [JF75540]7] M-13 [JF755410] V-69 [JF755403] 63 V-89 [JF755402] 89 V-72 [JF755411] 100 V-81 [JF755412] 78 93 V-84 [JF755413] V-9793 [JF755414] CB-B [FJ959083] YN-BtCV-1 [JF938078] 91 100 RW-E [FJ959081] 95 M-44 [JF755408] 76 MmCVLV [JQ085285] RW-A [FJ959077] 83 M-45 [JF755409] 85 RW-B [FJ959078] SAR-A [FJ959084] DuckCV [DQ100076] 94 78 MulardCV [AY228555] 100 MuscovyCV [AY394721] CygnusCV [EU056309] 100 MN614 [GQ404852] PCV-2 [AY424401] 73 92 MN500 [GQ404853] 71 PCV-1 [NP 065678] BFDV [AF071878] GullCV [DQ845074] CanaryCV [AJ301633] 97 80 StarlingCV [DQ172906] 80 FinchCV [DQ845075] 90 90 Raven CV [DQ146997] 91 Chimp17 [GQ404851] ColumbidCV [AF252610] 100 NGchicken38 [HQ738642] BarCV [JF279961] 100 BarCV [GU799606] 89 NG13 [GQ404856] 98 PK5510 [GQ404857] 99 TN18 [GQ404858] PKbeef [HQ738634] Faecal source NG14 [GQ404855] 94 BtCV-5 [JF938082] Chimpanzee 100 PK5222 [GQ404846] Bat TN18 [GQ404848] 99 97 PK5006 [GQ404844] Human Pkgoat11 [HQ738636] 100 98 Chimp 11 [GQ404849] Pig Chimp12 [GQ404850] NGchicken8 [HQ738643] 99 92 Rodent 99 NGchicken15 [HQ738644] 89 PK5510 [GQ404847] Badger 100 BtCV-3 [JF938080] 87 BtCV 01238 [JN377566] Cow DfCyV-1 [HQ638064] BtCV-4 [JF938081] 83 NG12 [GQ404854] 85 CyCV-TB [HQ738637] 72 PK5034 [GQ404845] 82 0.5 amino acid substitutions per site BatCyV GF4c [HM228874] BtCV-2 [JF938079] 94
83

75 ChiSCV GM476 [GQ351274] 80 ChiSCV GM495 [GQ351273]

PigSCV [JQ023166] ChiSCV GM510 [GQ351275]

Novel unclassified circular Rep encoding ssDNA viruses

Circoviruses

Cycloviruses

Fig. 2 Maximum-likelihood phylogenetic tree of all of the Rep sequences of novel ssDNA, circoviruses and cycloviruses. Branches representing ssDNA viruses isolated from faecal samples are coloured (colour figure online)

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Novel circular single-stranded DNA virus from porcine faeces Table 1 Details of ssDNA viruses isolated from faecal samples and list of conserved motifs identified in the Rep sequences of these viruses Viral isolate GenBank Source Rep motifs Motif 1 PoSCV ChiSCV-DP152 ChiSCV-GM495 ChiSCV-GM476 ChiSCV-GM510 ChiSCV-GM488 ChiSCV-GM415 ChiSCV-GT306 PigSCV CyCV-PK5006 CyCV-PK5034 CyCV-PK5222 CyCV-PK5510 CyCV-PK6197 CyCV-Chimp11 CyCV-Chimp12 CsaCV-chimp17 CyCV-NG12 CyCV-NG14 CyCV-NG13 CyCV-TN25 CyCV-TN18 CyCV-GF4 Batcirco-like po-circo-like21 po-circo-like22 po-circo-like41 po-circo-like51 RodSCV-R-15 RodSCV-V-89 RodSCV-V-69 RodSCV-V-76 RodSCV-V-77 RodSCV-V-87 RodSCV-M-44 RodSCV-M-45 RodSCV-M-13 RodSCV-V-72 RodSCV-V-81 RodSCV-V-84 RodSCV-V-97 RodSCV-M-53 YN-BtCV-1 YN-BtCV-2 YN-BtCV-3 YN-BtCV-4 YN-BtCV-5 JX274036 GQ351272 GQ351273 GQ351274 GQ351275 GQ351276 GQ351277 GQ351278 JQ023166 GQ404844 GQ404845 GQ404846 GQ404847 GQ404848 GQ404849 GQ404850 GQ404851 GQ404854 GQ404855 GQ404856 GQ404857 GQ404858 HM228874 HM228875 JF713716 JF713717 JF713718 JF713719 JF755401 JF755402 JF755403 JF755404 JF755405 JF755406 JF755408 JF755409 JF755410 JF755411 JF755412 JF755413 JF755414 JF755415 JF938078 JF938079 JF938080 JF938081 JF938082 Pig Chimpanzee Chimpanzee Chimpanzee Chimpanzee Chimpanzee Chimpanzee Chimpanzee Pig Human Human Human Human Human Chimpanzee Chimpanzee Chimpanzee Human Human Human Human Human Bat Bat Pig Pig Pig Pig Rodent Rodent Rodent Rodent Rodent Rodent Rodent Rodent Rodent Rodent Rodent Rodent Rodent Rodent Bat Bat Bat Bat Bat MTIPR MTTME MTTMQ MTTMQ MTTMQ MTTMQ MTTMQ – TSIPE LGMTK FTWND FTWNN FTWNN FTWNN FTWNN FTWNN FTWNN FTLNN FTLNN FTWNN FTLNN WTLNN WTLNN FTWNN FTLNN FTINN FTINN FTINN FTINN LTYAQ WVGTK GTLNA LTYPQ LTYPQ LTYPQ FTIPD FTSYV WVFTR WLGTK WLGTK WLGTK WLGTK FTINN LLTIP FTLNN FTWNN FTWNN Motif 2 HWQIRI HWQVRC HWQVRC HWQVRC HWQVRC HWQVRC HWQVRC – HYQCCI HYQFRG HLQGFC HLQGFC HLQGFC HLQGFC HLQGFC HLQGYV HLQGYV HLQGYL HLQGFC HLQGFC HLQGFF HLQGFC HLQGFS HLQGFC HIQGFI HIQGYL HIQGYL HIQGYF HIQGYM HLHAWV HVQFVV HLQMYV HLHAWV HLHAWV HLHAWV HWQLIC HYQGYA HIQGYA HVQFVV HVQFVV HVQFVV HVQFVV HLQGYC HWQLVV HLQGFC HLQGYA HLQGFC Motif 3 YETKEGQY YETKEGKY YETKEGKY YETKEGKY YETKEGKY YETKEGKY YETKEGKY – YCRKTDNY YVYKDRNF YCSKSGIF YCKKAGHW YCSKTGIF YCSKSGEF YCSKTGIF YCRKSGIF YCRKSGIF YCSKEGDV YCSKAGNF YCSKTGNF YCSKSGNI YCSKSGEV YCSKSGEV YCSKAGDF YCKKSGTF YCTKEDQV YCTKEDQV YCSKEGNV YCSKEDQI YVTKDGHY YCSKSETR YCMKEDTR YVMKDGDT YVMKDGDT YVMKDGDT YVWKDDTC YCKKDDCR YAMKEDTR YCTKEDTR YCTKEDTR YCTKEDTR YCTKEDTR YCKKEGDF YVWKEDTS YCSKAGNF YCSKAGEI YCKKSGDF Walker A ETGNVGKS QDGNMGKS EQGNMGKS EQGNMGKS EGGNMGKS EQGNMGKS EQGNMGKS QDGNMGKS TIGGTGKS EKGNSGKT GPPGTGKS GPPGSGKS GPPGTGKS GPPGSGKS GPPGTGKS GPPGSGKS GPPGSGKS GPSGVGKS GPTGSGKS GPPGSGKS GPPGCGKS GPTGSGKS GPTGSGKS GEPGTGKS GESGAGKT GPPGKGKS GPPGKGKS GPPGSGKS GPAGSGKT GRAGCGKS GPSGSGKS GPSGTGKS GRPGCGKS GRPGCGKS GRPGCGKS GSTGMGKS GDTGVGKT GPSGSGKS GPSGTGKS GPSGTGKS GPSGTGKS GPSGTGKS GSTGTGKS GRTGAGKS GPPGSGKS GLPGTGKS GPPGSGKS Walker B TIDTV VIDIP VIDIP VIDIP VIDIP VIDIP VIDIP VIDIP WIDLP IIDTP IIDDF IIDDF IIDDF IIDDF IIDDF IIDDF IIDDF VIDDF IIDDF IIDDF IIDDF IIDDF IIDDF IIDDF VMDDF VIDDW IIDDW VMDDY LIDDF ILDDL ILDDF IFDDF VMNDV VMNDV VMNDV VIDEF – – ILDDF ILDDF ILDDF ILDDF IIEDF VIDEF IIDDF IIDDF IVDDF This study [6] [6] [6] [6] [6] [6] [6] [27] [19] [19] [19] [19] [19] [19] [19] [19] [19] [19] [19] [19] [19] [20] [20] [22] [22] [22] [22] [22] [22] [22] [22] [22] [22] [22] [22] [22] [22] [22] [22] [22] [22] [13] [13] [13] [13] [13] Reference

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A. Sikorski et al. Table 1 continued Viral isolate GenBank Source Rep motifs Motif 1 BoSCVCP11-49-3 MmFV RodSCV-V-86 RodSCV-V-91 JN634851 JN704610 JF755416 JF755417 Bovine Badger Rodent Rodent FTWNN LTYAQ FLTIN FLTIN Motif 2 HLQGFC HLHAFV HMHGII HMHGII Motif 3 YCSKSGDF YAIKDGDV YLEKTSGT YLEKTSGT Walker A GPPGTGKS GETRLGKT GTTGSGKS GTTGSGKS Walker B VIDDF – ILDDL ILDDL [17] [31] [22] [22] Reference

ssDNA viruses have been isolated from faecal matter of seven animal species, and the majority of these are novel viruses possibly representing new viral families. Faecal sampling provides an ideal ‘natural tool’ for exploring viromes related to the animal’s gut flora and its food source. Additionally, with the advent of cheaper nextgeneration sequencing techniques, sampling animal faecal matter, which is non-invasive to the animal, may prove to be useful for basic viral surveillance.
Acknowledgments This study was partially supported by an early career grant from the University of Canterbury (New Zealand) and a University of Cape Town (South Africa) block grant awarded to AV.

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