Depoin(Psa

MiAna Vetb Schc De

SE-7d Me

1. I

bocorigPM200apphas

Veterinary Microbiology 164 (2013) 293–298

A R

Artic

Rece

Rece

Acce

Keyw

Porc

Porc

Torq

Gen

Post

synd

*

fax:

037

http

tection of a porcine boca-like virus in combination withrcine circovirus type 2 genotypes and torque teno sus virus

pigs from postweaning multisystemic wasting syndromeMWS)-affected and non-PMWS-affected farms in archivalmples from Great Britain

chael J. McMenamy a,*, John McKillen a, Irene McNair a, Catherine Duffy b,ne-Lie Blomstrom c, Catherine Charreyre d, Michael Welsh a, Gordon Allan b

erinary Sciences Division, Agri-Food and Biosciences Institute, Belfast, Northern Ireland BT4 3SD, United Kingdom

ool of Biological Sciences, Queen’s University Belfast, Belfast, Northern Ireland BT9 7BL, United Kingdom

partment of Biomedical Sciences and Veterinary Public Health, Swedish University of Agricultural Sciences Uppsala, PO Box 7028,

5007, Sweden

rial SAS, 29 Avenue Tony Garnier, 69007 Lyon, France

ntroduction

Porcine boca-like virus (PBo-likeV) occupies theavirus genus of the subfamily Parvovirinae and wasinally discovered in lymph nodes recovered from

WS-affected pigs from Sweden (Blomstrom et al.,9). This single-stranded DNA virus has a genome ofroximately 5 kb in length (Zeng et al., 2011). PBo-likeV

been associated with respiratory infections in

weanling piglets from China (Zhai et al., 2010) anddiscovered in combination with PCV2 and TTSuV inPMWS-affected pigs (Blomstrom et al., 2010). Subse-quently other novel bocaviruses have been identified inpigs including PBoV1 and 2 (Cheng et al., 2010) and PBoV3and 4 (McKillen et al., 2011). However, only PBo-likeV has,as yet, been tentatively linked to disease in swine.Currently PBo-likeV has been reported in pigs in Chinaand Northern Europe and in European wild boar (Cadaret al., 2011). PBo-likeV has been detected in PMWS-affected swine in Sweden (Blomstrom et al., 2010)therefore warranting further investigation into PBo-likeVprevalence in other geographically separate populationsaffected by PMWS. The essential infectious agent of PMWS

T I C L E I N F O

le history:

ived 10 February 2012

ived in revised form 25 October 2012

pted 1 March 2013

ords:

ine bocavirus

ine circovirus type 2

ue teno sus virus

ogroups

weaning multisystemic wasting

rome

A B S T R A C T

In this study we detail the detection and genetic analysis of a novel porcine boca-like virus

(PBo-likeV) in archival sera and tissue samples from pigs from farms in Great Britain. We

also investigate the distribution of porcine circovirus type 2 (PCV2) genotypes and Torque

teno sus virus (TTSuV) genogroups 1 and 2 in combination with this novel PBo-likeV. PBo-

likeV was detected in over 70% of all tissues investigated. Over 24% of all tissues recovered

from PMWS-affected animals had all viruses present and 25% of tissues recovered from

non-PMWS-affected pigs were positive for all 4 viruses.

Crown Copyright � 2013 Published by Elsevier B.V. All rights reserved.

Corresponding author. Tel.: +44 02890 525864;

+44 02890 525823.

E-mail address: michael.mcmenamy@afbini.gov.uk (M.J. McMenamy).

Contents lists available at SciVerse ScienceDirect

Veterinary Microbiology

jo u rn al ho m epag e: ww w.els evier .c o m/lo cat e/vetmic

8-1135/$ – see front matter . Crown Copyright � 2013 Published by Elsevier B.V. All rights reserved.

://dx.doi.org/10.1016/j.vetmic.2013.03.009

M.J. McMenamy et al. / Veterinary Microbiology 164 (2013) 293–298294

(Allan et al., 2007) is recognised as PCV2, although it hasbeen noted that PMWS may not develop through infectionwith PCV2 alone (Ellis et al., 2008). The possibility remainsthat other agents might be responsible for the develop-ment of PMWS in association with PCV2 infection. NovelPBo-likeV may be one such candidate. Studies have alsodemonstrated that TTSuV has been detected in higherincidences in PMWS-affected pigs than in non-PMWS-affected animals, where Torque teno sus virus genogroup2 (TTSuV2) is more prevalent in infected animals(Kerkarainen et al., 2006). Torque teno sus virus gen-ogroup 1 (TTSuV1) may also be a potentiating co-factor inthe development of PMWS by immunologic dysregula-tion, even though it is not considered a directly causativeagent (Ellis et al., 2008). In this study we report theincidence of PCV2 (genotypes a and b), TTSuV1 andTTSuV2 and a novel PBo-likeV in archival serum and tissuesamples recovered from pigs from farms in Great Britain(GB) between 2000 and 2004.

2. Materials and methods

2.1. Samples

103 sera and 132 tissue samples were collected fromfarms throughout GB between 2000 and 2004. Eighteensera were collected from pigs on farms considered PMWSnegative and 85 from pigs on farms considered PMWSpositive by classical methodologies (Segales and Domingo,2002). The 132 tissue samples were collected from 61 pigsof known PMWS status. Tissues included lung, liver,kidney, spleen and lymph nodes.

2.2. Viral nucleic acid extraction

Viral nucleic acids were extracted from 200 ml of sera andhomogenised tissue samples using a MagNa Pure LCautomated liquid handling system (Roche, Burgess Hill, UK).

2.3. Detection of PCV2

Fifty ml PCR reactions were carried out in duplicate,each containing 4 ml of extracted nucleic acid from each ofthe sera and tissue samples. Amplification was carried outin a DNA Engine Dyad thermal cycler (Bio-Rad LaboratoriesLtd., Herts, UK) using HotStarTaq MasterMix (Qiagen Ltd.,Crawley, UK) according to the manufacturer’s instructionswith 0.5 mM of PCV2 specific primers designed to amplifyan 814 bp amplicon which included the ORF2 codingsequence according to Allan et al., 2007. Resultant PCRproducts were visualised on 1.5% (w/v) agarose gels (MastGroup Ltd., Bootle, UK), before being excised from theagarose gel and purified using a Wizard1 SV Gel and PCRClean-UP System (Promega, Southampton, UK) accordingto the manufacturers instructions.

2.4. Detection of TTSuV1, TTSuV2 and PBo-likeV

All sera and tissue samples were assessed for thepresence of TTSuV1, TTSuV2 and PBo-likeV in real-timeusing a Roche LC480 instrument (Burgess Hill, UK).

Real-time PCR for the detection of TTSuV1 was carriedout using a set of primers 1005 (50-CGTTTGCTGCCARGCG-GACC-30) and 1007 (50-CGTCTGATTGGTTACACCCTATGCA-30) which had been previously published (Pozzuto et al.,2009). The real-time thermal cycling conditions weremodified for use as follows; the final reaction volume of25 ml included 12.5 ml of QuantiTect SYBR Green PCRMaster Mix (Qiagen, West Sussex, UK), 0.125 ml each offorward and reverse primers to give a final concentrationof 0.5 mM each, 2 ml of extracted viral nucleic acid as targetand 10.25 ml of DEPC water (Ambion, Warrington, UK).Amplification was achieved using 40 cycles of 95 8C for30 s, 60 8C for 30 s and 72 8C for 30 s followed by a melt-curve between 50 and 95 8C.

TTSuV2 was detected in the same manner, this timeusing a primers forward-2 (50-AGTTACACATAACCAC-CAAACC-30) and reverse-2 (50-ATTACCGCCTGCCCGA-TAGGC-30) previously used in the detection of TTSuV2(Kerkarainen et al., 2006). In this case amplification wasachieved using 40 cycles of 95 8C for 30 s, 52 8C for 30 s and72 8C for 30 s followed by a melt-curve between 50 and95 8C.

PBo-likeV was also detected using the Roche LC480system. Primers used in detecting PBo-likeV (SBF: 50-CGACATGCCACTTGCTAAAG-30 and SBR: 50-ACCGCCGCAA-GATTCAATATC-30) were based on sequence providedkindly by Anne-Lie Blomstrom (Swedish University ofAgricultural Sciences, Uppsala, Sweden) (data not pub-lished). Amplification was achieved using the sameconditions as used to detect TTSuV1 and TTSuV2 withthe annealing temperature modified to 50 8C for 30 s.

2.5. Analytical sensitivity of PBo-likeV primers

Four ml of nucleic acid from a selected PBo-likeVpositive field sample was amplified using Hot StarTaqMaster Mix (Qiagen, Crawley, UK) according to themanufacturer’s instructions with 0.5 mM of primers(SB1780: 50-GGCAGCATGGCTCCTAACTTGC-30 and SB2398:50-CTCCCCCTGTATGGGCTCT-30). The resultant ampliconwas 616 bp in length. Thermal cycling with this primerset was carried out using a GRI G-Storm GS2 Thermal Cycler(G-Storm, Surrey, UK). An initial 95 8C denaturation step wascarried out for 15 min before PCR cycling for a further 40cycles consisting of 30 s of denaturation at 95 8C, 30 s ofannealing at 55 8C, and a 1 min extension at 72 8C. Theresultant reactions were visualised on 1.5% (w/v) agarosegels (Mast Group Ltd., Bootle, UK). The 616 bp product waspurified using a Wizard1 SV Gel and PCR Clean-UP System(Promega, Southampton, UK) according to the manufac-turer’s instructions. Purified DNA was quantified using aJenway Genova UV/VIS spectrophotometer (BarloworldScientific Ltd., Essex, UK) to estimate DNA mg/ml. Thisinformation and the average weight of a nucleotide wereused to calculate copy number. A dilution series from 109 to100 copies/ml was then produced.

2.6. Sequencing of PCV2 and PBo-likeV

PCV2 PCR products were sequenced to determineif genotypes 2a or 2b were present using BigDye1

Tertonet ausi30 aamrea8 mof t20

SeqwacycPerGairateinsusiWa

2.7.

formbioandthethaGenGUCluNP-alsoHMandspeFJ11) (gorphyneipre

3. R

3.1.

like

Tab

Perc

sam

Sa

Ti

Se

a

M.J. McMenamy et al. / Veterinary Microbiology 164 (2013) 293–298 295

minator Kit version 3.1 (Applied Biosystems, Warring-, UK) and those primers used in amplification (Allanl., 2007). Samples positive for PBo-likeV were amplified

ng primers (SB2126: 50-GTAATAAACGACATGCCACTTG-nd SB3171: 50-AGCTTATAATAAACATGAGCACAA-30) toplify a region including the NP-1 gene. The totalction volume included 1 ml of 3.2 pmol/ml primer,l of diluted BigDye1 reaction mix and a variable volumearget DNA. The final reaction volume was made up toml with DEPC treated water (Ambion, Warrington, UK).uencing reactions were cycled as follows; the samples initially denatured at 96 8C for 1 min, followed by 25les of 96 8C for 10 s, 50 8C for 5 s and 60 8C for 4 min.forma1 DTR Gel Filtration Cartridges (Edge BioSystems,thersburg, MD, USA) were used to remove unincorpo-d dNTPs and ddNTPs according to the manufacturer’s

tructions. Resultant sequencing data was analysedng an ABI 3100 Genetic Analyser (Applied Biosystems,rrington, UK).

Analysis of PCV2 and PBo-likeV sequence

Resultant PCV2 ORF2 sequence was converted to Fastaat and analysed using GeneDoc (http://www.psc.edu/

med/genedoc) sequence analysis software to determine fully investigate genotype. Comparison was made of

nucleotide similarity of PBo-likeV NP-1 sequence tot of existing porcine bocavirus sequences submitted toBank (HQ223038; FJ872544; GU902967; GU902968;

902969; GU902970; GU902971; HQ872052) usingstalW2 (http://www.genome.jp/tools/clustalw/). Those1 sequences derived from virus detected in GB pigs were

compared to other porcine bocaviruses (HM053693;053694; JF512472; JF512473; HQ291308; HQ291309), representative strains of bocaviruses from othercies including human bocavirus (HBoV) (DQ000495;70279; FJ948861; FJ973561), bovine parvovirus 1 (BPV-DQ335247), canine minute virus (CMV) (FJ214110) andilla bocavirus 1 (GBoV1) (NC_014358; HM145750). Alogenetic tree was also created by bootstrapped

ghbour joining analysis of all samples mentionedviously using MEGA version 5.

esults

Detection of PCV2, TTSuV1, TTSuV2 and PBo-likeV

Prevalence of PCV2, TTSuV genogroups 1 and 2 and PBo-

collected from pigs from PMWS positive farms is detailedin Table 1. PCV2, TTSuV1, TTSuV2 and PBo-likeV werepresent in both PMWS-affected and non-PMWS-affectedpigs. Over 24% of PMWS-affected pigs were positive for thepresence of all viruses simultaneously, whereas this figurewas 25% in non-PMWS-affected pigs. All 4 viruses couldnot be detected simultaneously in sera from non-PMWS-affected farms and only 1% of sera from PMWS-affectedfarms proved positive for all 4 viruses concomitantly.

3.2. Analytical sensitivity of PBo-likeV detection primers

Testing dilutions of the representative PBo-likeVamplicon, demonstrated that the PBo-likeV detectionprimers were capable of detecting triplicates of thismaterial from 2 � 108 to 2 � 101 copies/reaction with adynamic range of 8 logs.

3.3. Analysis of PCV2 and PBo-likeV sequence

Details on those PCV2 genotypes that could be detectedin sera and tissue samples are included in Table 2. Thosesera samples collected from PMWS positive and negativefarms, that were possible to sequence, during the year2000 exhibited a mixture of PCV2 genotype 2a and 2b;those recovered in 2001 and 2002 were 2a in character andthose from 2003 to 2004 were 2b. PCV2 genotypesrecovered from tissues from PMWS positive and negativepigs between 2002 and 2004 were exclusively 2b in nature.

Sequence data for the NP-1 gene was derived from virusdetected in 24/132 tissues which equated to sequencederived from viral nucleic acids recovered from 12individual pigs. PCR product of sufficient strength andquality for sequencing could not be recovered from any ofthe 5 PBo-likeV positive sera samples. Individual NP-1sequences derived from tissue samples were assignedaccession numbers as follows: JN862548 (S123); JN862549

le 1

entage prevalence of PCV2, TTSuV1, TTSuV2 and PBo-likeV in tissues recovered from PMWS-affected and non-PMWS-affected pigs and from sera

ples from PMWS-affected and non-PMWS-affected pigs.

mple PMWS Status PCV2 TTSuV1 TTSuV2 PBo-likeV

ssuea PMWS +VE 100% (29/29) 41% (12/29) 79% (23/29) 69% (20/29)

PMWS �VE 97% (31/32) 63% (20/32) 63% (20/32) 72% (23/32)

Total 98% (60/61) 53% (32/61) 71% (43/61) 71% (43/61)

ra PMWS +VE 62% (53/85) 58% (49/85) 51% (43/85) 6% (5/85)

PMWS �VE 61% (11/18) 50% (9/18) 56% (10/18) 0% (0/18)

Total 62% (64/103) 56% (58/103) 52% (53/103) 5% (5/103)

Data from multiple tissues recovered from the same pig were combined.

Table 2

PCV2 genotypes detected in tissue samples from PMWS-affected and

non-PMWS-affected pigs and from sera samples from PMWS-affected

pigs.

Sera (103) 2000–2004 Tissuea (61)

2002–2004

PCV-2a 11/103 (2000–2002) 0/61

PCV-2b 16/103 (2000–2004) 57/61

No sequencing result 76/103 4/61a

Data from multiple tissues recovered from the same pig were

bined.

V in PMWS positive and negative tissue and in sera com

M.J. McMenamy et al. / Veterinary Microbiology 164 (2013) 293–298296

(S107); JN862550 (B229); JN862551 (B230); JN862552(F83); JN862553 (F91); JN862554 (S104); JN862555(S106); JN862556 (S125); JN862557 (L222); JN862558(L176); JN862559 (L608). The NP-1 gene sequence fromthe 12 pigs was �98% similar to each other at nucleotidelevel and �97% similar to those other NP-1 gene sequencesrecovered from Chinese and Swedish pigs. Comparison ofthe 12 sequences to the NP-1 region of other European andAsian porcine bocaviruses not grouped as PBo-likeV andbocaviruses from other species is detailed in Table 3. Thephylogenetic relationship between all of these samples isdetailed in Fig. 1.

4. Discussion

The detailed characterisation of PBo-likeV has beenrelatively recent (Blomstrom et al., 2009). PBo-likeVoccupies the same genera as BPV-1, CMV, HBoV and themore recently discovered GBoV1 (Kapoor et al., 2010).Recently an increasing number of porcine boca viruses andrelated variants have been detailed in Europe and Asia(Blomstrom et al., 2009; Cheng et al., 2010; McKillen et al.,2011; Shan et al., 2011; Cadar et al., 2011; Lau et al., 2011).Whereas BPV-1, CMV and HBoV (Sandals et al., 1995;Schwartz et al., 2002; Carmichael, 2004; McIntosh, 2006;Kesebir et al., 2006) have been associated with disease andGBoV1 recovered from gorillas suffering from enteritis(Kapoor et al., 2010), as yet PBo-likeV is the only porcinebocavirus substantively associated with disease in pigs(Zhai et al., 2010). Whereas PBo-likeV has been noted inNorthern European pig populations, and PBoV3 and 4detected in Northern Irish pigs (McKillen et al., 2011) todate PBo-likeV has not been noted in pigs in WesternEuropean farms.

Unlike other parvoviruses (with the exception of PPV4),bocaviruses are uniquely distinguished by the presence of

a 3rd open reading frame (ORF) located between the non-structural and capsid protein coding regions. This 3rd ORFhas been designated as NP-1 coding region. This regionprovides a suitable target for boca-specific phylogeneticanalysis. Sequence data from the NP-1 gene of 12 pigs wasanalysed to determine the phylogenic relationship withPBo-likeV sequence data from other European and Asianisolates and to compare PBo-likeV with associated porcinebocaviruses and bocaviruses that have been discovered inother species. The phylogenetic tree demonstrates theclose relationship between GB isolates and those recoveredfrom Northern Europe and Asia. Analysis of the sequencehomology confirms that these PBo-likeV isolates are atleast 97% similar based on the NP-1 gene. Analysis ofhomology of the NP-1 has suggested that in some cases thedegree of relatedness between bocaviruses of other speciesand PBo-likeV was higher than between PBo-likeV andother bocavirues recovered from swine highlighting thesequence diversity evident in porcine bocaviruses.

Previous studies have demonstrated that PBo-likeV ispresent in PMWS-affected pigs also infected with TTSuV(Blomstrom et al., 2010). Other studies have also indicatedthat pigs suffering from PMWS exhibited a 1.25 timeshigher incidence of infection with TTSuV (Kerkarainenet al., 2006) and that TTSuV was present in swine sufferingfrom PMWS and porcine respiratory disease complex(PRDC) (Taira et al., 2009; Rammohan et al., 2012). As theremay be an association between PMWS and infection withTTSuV it was reasonable to also investigate the prevalenceof PCV2 and TTSuV in combination with PBo-likeV.Investigation of archival sera and tissue samples fromGB farms has demonstrated that PBo-likeV is present in GBpigs. PBo-likeV amplified product was relatively weak insera samples in comparison to the other viruses underinvestigation, potentially suggesting low viral load. ThePBo-likeV present in the sera samples was very weak,making it difficult to recover sufficient nucleic material forsequence analysis. The limited number of PBo-likeVpositive sera samples could potentially suggest that thatPBo-likeV infection rates in the pigs from which thesesamples had been collected was very low. It is arguablethat this is unlikely considering that PBo-likeV wasprevalent into an average of 71% of tissues recoveredfrom PMWS-affected and non-PMWS-affected pigs.Although sera and tissue samples were collected fromdifferent pigs the there may be a suggestion from the seraresults that PBo-likeV exhibits a transient viraemic phase.

Unlike the levels detected in sera samples PBo-likeVwas detected at much higher levels in tissue samples, bothfrom PMWS-affected and non-PMWS-affected pigs. Onaverage 71% of tissue samples were positive for thepresence of PBo-likeV. All of the viruses investigated werepresent in both PMWS-affected and non-PMWS-affectedtissues. Over 24% of tissues from PMWS-affected pigs werepositive for the presence of PCV2, TTSuV1, TTSuV2 andPBo-likeV simultaneously, whereas 25% of non-affectedpigs were positive for the same combination of virusessimultaneously. Taken in isolation the incidence of PBo-likeV was in fact nominally lower in tissues derived fromPMWS-affected pigs. It is difficult to infer from this dataif there is in fact any potential contributory effect from

Table 3

Percentage similarity of UK PBo-likeV NP1 coding-regions to other

European and Asian porcine bocaviruses, and selected bocaviruses from

other species.

Virus Accession

no(s).

Species % similarity

to UK NP1

PBo-likeV

sequence

PBoV1 HM053693 Porcine 36%

PBoV2 HM053694 Porcine 29–35%

PBoV3 JF512472 Porcine 22–23%

PBoV4 JF512473 Porcine 25%

PBoV1-H18 HQ291308 Porcine 98–99%

PBoV2-A6 HQ291309 Porcine 35%

PBoV3-SH20F JF429834 Porcine 23–24%

PBoV4-1-SH17N-1 JF429835 Porcine 23–24%

PBoV4-2-SH17N-2 JF429836 Porcine 23–24%

PBoV3 strain 22 JF713714 Porcine 23–24%

PBoV3 strain 23 JF713715 Porcine 29%

HBoV DQ000495;

FJ170279;

FJ948861;

FJ973561

Human 28–35%

BPV1 DQ335247 Bovine 30–32%

CMV FJ214110 Canine 34–37%

GBoV1 NC_014358;

HM145750

Gorilla 28–30%

PBofromwaPCVunl

Fig.

isola

M.J. McMenamy et al. / Veterinary Microbiology 164 (2013) 293–298 297

-likeV to the PMWS status of pigs. Although tissues over 70% of pigs in this study had PBo-likeV present, it

s only present in a quarter of pigs in combination with2, the necessary causal agent of PMWS. It would seem

ikely that PBo-likeV would have a combined effect with

other viruses in contributing to the onset or progression ofPMWS. However, the possibility remains that PBo-likeVmight have an as yet undefined sub-clinical effect or havean impact as an opportunistic infection in animals alreadysuffering from detrimental infections.

1. Bootstrapped neighbour joining comparison of GB PBo-likeV NP-1 sequences to other porcine bocaviruses and bocaviruses from other species. *All GB

tes indicated by a ^.

M.J. McMenamy et al. / Veterinary Microbiology 164 (2013) 293–298298

The temporal distribution of PCV2 genotypes in thesamples tested from PMWS affected and non-affected pigstaken within this study is of interest in that, on the onehand, it reflects what has been reported elsewhere (Allanet al., 2007), but on the other does not agree with theresults reported in association with the outbreak of PMWSin other countries (Dupont et al., 2008). In Denmark adirect relationship between the introduction of PMWS intothe national herd and the introduction of PCV-2b has beenreported, leading to speculation that this genotype is morepathogenic than PCV-2a. This is further discussed in theDanish study suggesting that PCV-2a might be consideredless pathogenic due to its prevalence in non-PMWS-affected animals (Dupont et al., 2008). Other studiesconducted in Canada and Spain (Gagnon et al., 2007; Grau-Roma et al., 2008) agree that there might be evidence tosuggest that PCV-2b exhibits higher pathogenicity thanPCV-2a. However, this direct relationship cannot beapplied to the PMWS epizootic in the GB as our resultsshow that cases of diseases associated with PCV-2a, andnot PCV-2b infection were occurring between 2000 and2001. At present there is no simple answer for PCV2genotype related pathogenicity. Therefore more researchin this area is essential.

Our results also show that in GB a major shift away froma mixed distribution of PCV2 genotypes to an exclusivepredominance of PCV-2b in PMWS affected pigs occurredpost 2002. The reasons for this are unknown but reflect asimilar shift in PCV2 genotype distribution in diseasedanimals around the world post 2002.

Acknowledgement

This work was supported by the BBSRC IndustrialPartner Grant BB/F020171/1, in collaboration with Merial.

References

Allan, G.M., McNeilly, F., McMenamy, M., McNair, I., Krakowka, S.G.,Timmusk, S., Walls, D., Donnelly, M., Minahin, D., Ellis, J., Wallgren,P., Fossum, C., 2007. Temporal distribution of porcine circovirus 2genogroups recovered from postweaning multisystemic wasting syn-drome affected and nonaffected farms in Ireland and Northern Ire-land. J. Vet. Diagn. Invest. 19, 668–673.

Blomstrom, A.L., Belak, S., Fossum, C., McKillen, J., Allan, G., Wallgren, P.,Berg, M., 2009. Detection of a novel porcine boca-like virus in thebackground of porcine circovirus type 2 induced postweaning multi-systemic wasting syndrome. Virus Res. 146, 125–129.

Blomstrom, A.L., Belak, S., Fossum, C., Fuxler, L., Wallgren, P., Berg, M.,2010. Studies of porcine circovirus type 2, porcine boca-like virus andtorque teno virus indicate the presence of multiple viral infections inpostweaning multisystemic wasting syndrome pigs. Virus Res. 152,59–64.

Cadar, D., Csagola, A., Lorincz, M., Tombacz, K., Kiss, T., Spınu, M., Tuboly,T., 2011. Genetic detection and analysis of porcine bocavirus type 1(PoBoV1) in European wild boar (Sus scrofa). Virus Genes, [Epubahead of print].

Carmichael, L. (Ed.), 2004. Neonatal viral infections of pups: canineherpesvirus and minute virus of canines (canine parvovirus-1).Recent Advances in Canine Infectious Diseases (last updated:19.08.2004). International Veterinary Information Service (www.ivis.org), Ithaca, NY, USA, Document No. A0102.0804.

Cheng, W.X., Li, J.S., Huang, C.P., Yao, D.P., Liu, N., Cui, S.X., Jin, Y., Duan, Z.J.,2010. Identification and nearly full-length genome characterizationof novel porcine bocaviruses. PLoS ONE 5, e13583.

Dupont, K., Nielsen, E.O., Bækbo, P., Larsen, L.E., 2008. Genomic analysisof PCV2 isolates from Danish archives and a current PMWS case-control study supports a shift in genotypes with time. Vet. Micro-biol. 128, 56–64.

Ellis, J.A., Allan, G., Krakowka, S., 2008. Effect of coinfection with gen-ogroup 1 porcine torque teno virus on porcine circovirus type 2-associated postweaning multisystemic wasting syndrome in gnoto-biotic pigs. Am. J. Vet. Res. 69, 1608–1614.

Gagnon, C.A., Tremblay, D., Tijssen, P., Venne, M.-H., Houde, A., Elahi, S.M.,2007. The emergence of porcine circovirus 2b genotype (PCV-2b) inswine in Canada. Can. Vet. J. 48, 811–819.

Grau-Roma, L., Crisci, E., Sibilia, M., Lopez-Soria, S., Nofrarias, M., Cortey,M., Fraile, L., Olvera, A., Segales, J., 2008. A proposal on porcinecircovirus type 2 (PCV2) genotype definition and their relationshipwith postweaning multisystemic wasting syndrome (PMWS) occur-rence. Vet. Microbiol. 128, 23–35.

Kerkarainen, T., Sibila, M., Segales, J., 2006. Prevalence of swine Torqueteno virus in post-weaning multisystemic wasting syndrome(PMWS)-affected and non-PMWS-affected pigs in Spain. J. Gen. Virol.87, 833–837.

Kapoor, A., Mehta, N., Esper, F., Poljsak-Prijatelj, M., Quan, P.L., Qaisar, N.,Delwart, E., Lipkin, W.I., 2010. Identification and characterization of anew bocavirus species in gorillas. PLoS ONE 5, e11948.

Kesebir, D., Vazquez, M., Weibel, C., Shapiro, E.D., Ferguson, D., Landry,M.L., Kahn, J.S., 2006. Human bocavirus infection in young children inthe United States: molecular epidemiological profile and clinicalcharacteristics of a newly emerging respiratory virus. J. Infect. Dis.194, 1276–1282.

Lau, S.K., Woo, P.C., Yip, C.C., Li, K.S., Fu, C.T., Huang, Y., Chan, K.H., Yuen,K.Y., 2011. Co-existence of multiple strains of two novel porcinebocaviruses in the same pig, a previously undescribed phenomenonin members of the family Parvoviridae, and evidence for inter- andintra-host genetic diversity and recombination. J. Gen. Virol. 92,2047–2059.

McKillen, J., McNeilly, F., Duffy, C., McMenamy, M., McNair, I.,Hjertner, B., Millar, A., McKay, K., Lagan, P., Adair, B., Allan, G.,2011. Isolation in cell cultures and initial characterisation of twonovel bocavirus species from swine in Northern Ireland. Vet. Micro-biol. 152, 39–45.

McIntosh, K., 2006. Human bocavirus: developing evidence for patho-genicity. J. Infect. Dis. 194, 1197–1199.

Pozzuto, T., Mueller, B., Meehan, B., Ringler, S.S., McIntosh, K.A., Ellis, J.A.,Mankertz, A., Krakowka, S., 2009. In utero transmission of porcinetorque teno viruses. Vet. Microbiol. 137, 375–379.

Rammohan, L., Xue, L., Wang, C., Chittick, W., Ganesan, S., Rama-moorthy, S., 2012. Increased prevalence of torque teno viruses inporcine respiratory disease complex affected pigs. Vet. Microbiol.157, 61–68.

Sandals, W.C., Povey, R.C., Meek, A.H., 1995. Prevalence of bovine parvo-virus infection in Ontario dairy cattle. Can. J. Vet. Res. 59, 81–86.

Schwartz, D., Green, B., Carmichael, L.E., Parrish, C.R., 2002. The canineminute virus (minute virus of canines) is a distinct parvovirus that ismost similar to bovine parvovirus. Virology 302, 219–223.

Segales, J., Domingo, M., 2002. Postweaning multisystemic wasting syn-drome (PMWS) in pigs. A review. Vet. Q. 24, 109–124.

Shan, T., Lan, D., Li, L., Wang, C., Cui, L., Zhang, W., Hua, X., Zhu, C., Zhao, W.,Delwart, E., 2011. Genomic characterization and high prevalence ofbocaviruses in swine. PLoS ONE 6, e17292.

Taira, O., Ogawa, H., Nagao, A., Tuchiya, K., Nunoya, T., Ueda, S., 2009.Prevalence of swine Torque teno virus genogroups 1 and 2 in Japaneseswine with suspected post-weaning multisystemic wasting syn-drome and porcine respiratory disease complex. Vet. Microbiol.139, 347–350.

Zhai, S., Yue, C., Wei, Z., Long, J., Ran, D., Lin, T., Deng, Y., Huang, L., Sun, L.,Zheng, H., Gao, F., Zheng, H., Chen, S., Yuan, S., 2010. High prevalenceof a novel porcine bocavirus in weanling piglets with respiratory tractsymptoms in China. Arch. Virol. 155, 1313–1317.

Zeng, S., Wang, D., Fang, L., Ma, J., Song, T., Zhang, R., Chen, H., Xiao, S.,2011. Complete coding sequences and phylogenetic analysis of por-cine bocavirus. J. Gen. Virol. 92, 784–788.