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Detection of a porcine boca-like virus in combination with porcine circovirus type 2 genotypes and torque teno sus virus in pigs from postweaning multisystemic wasting syndrome (PMWS)-affected and non-PMWS-affected farms in archival samples from Great Britain
Veterinary Microbiology 164 (2013) 293–298
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Detection of a porcine boca-like virus in combination with porcine circovirus type 2 genotypes and torque teno sus virus in pigs from postweaning multisystemic wasting syndrome (PMWS)-affected and non-PMWS-affected farms in archival samples from Great Britain Michael J. McMenamy a,*, John McKillen a, Irene McNair a, Catherine Duffy b, Anne-Lie Blomstro¨m c, Catherine Charreyre d, Michael Welsh a, Gordon Allan b a
Veterinary Sciences Division, Agri-Food and Biosciences Institute, Belfast, Northern Ireland BT4 3SD, United Kingdom School of Biological Sciences, Queen’s University Belfast, Belfast, Northern Ireland BT9 7BL, United Kingdom c Department of Biomedical Sciences and Veterinary Public Health, Swedish University of Agricultural Sciences Uppsala, PO Box 7028, SE-75007, Sweden d Merial SAS, 29 Avenue Tony Garnier, 69007 Lyon, France b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 10 February 2012 Received in revised form 25 October 2012 Accepted 1 March 2013
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. PBolikeV 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 ß 2013 Published by Elsevier B.V. All rights reserved.
Keywords: Porcine bocavirus Porcine circovirus type 2 Torque teno sus virus Genogroups Postweaning multisystemic wasting syndrome
1. Introduction Porcine boca-like virus (PBo-likeV) occupies the bocavirus genus of the subfamily Parvovirinae and was originally discovered in lymph nodes recovered from PMWS-affected pigs from Sweden (Blomstro¨m et al., 2009). This single-stranded DNA virus has a genome of approximately 5 kb in length (Zeng et al., 2011). PBo-likeV has been associated with respiratory infections in
* Corresponding author. Tel.: +44 02890 525864; fax: +44 02890 525823. E-mail address: [email protected] (M.J. McMenamy).
weanling piglets from China (Zhai et al., 2010) and discovered in combination with PCV2 and TTSuV in PMWS-affected pigs (Blomstro¨m et al., 2010). Subsequently other novel bocaviruses have been identified in pigs including PBoV1 and 2 (Cheng et al., 2010) and PBoV3 and 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 China and Northern Europe and in European wild boar (Cadar et al., 2011). PBo-likeV has been detected in PMWSaffected swine in Sweden (Blomstro¨m et al., 2010) therefore warranting further investigation into PBo-likeV prevalence in other geographically separate populations affected by PMWS. The essential infectious agent of PMWS
0378-1135/$ – see front matter . Crown Copyright ß 2013 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetmic.2013.03.009
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(Allan et al., 2007) is recognised as PCV2, although it has been noted that PMWS may not develop through infection with PCV2 alone (Ellis et al., 2008). The possibility remains that other agents might be responsible for the development of PMWS in association with PCV2 infection. Novel PBo-likeV may be one such candidate. Studies have also demonstrated that TTSuV has been detected in higher incidences in PMWS-affected pigs than in non-PMWSaffected animals, where Torque teno sus virus genogroup 2 (TTSuV2) is more prevalent in infected animals (Kerkarainen et al., 2006). Torque teno sus virus genogroup 1 (TTSuV1) may also be a potentiating co-factor in the development of PMWS by immunologic dysregulation, even though it is not considered a directly causative agent (Ellis et al., 2008). In this study we report the incidence of PCV2 (genotypes a and b), TTSuV1 and TTSuV2 and a novel PBo-likeV in archival serum and tissue samples 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 from farms throughout GB between 2000 and 2004. Eighteen sera were collected from pigs on farms considered PMWS negative and 85 from pigs on farms considered PMWS positive by classical methodologies (Segale´s and Domingo, 2002). The 132 tissue samples were collected from 61 pigs of known PMWS status. Tissues included lung, liver, kidney, spleen and lymph nodes.
Real-time PCR for the detection of TTSuV1 was carried out using a set of primers 1005 (50 -CGTTTGCTGCCARGCGGACC-30 ) and 1007 (50 -CGTCTGATTGGTTACACCCTATGCA30 ) which had been previously published (Pozzuto et al., 2009). The real-time thermal cycling conditions were modified for use as follows; the final reaction volume of 25 ml included 12.5 ml of QuantiTect SYBR Green PCR Master Mix (Qiagen, West Sussex, UK), 0.125 ml each of forward and reverse primers to give a final concentration of 0.5 mM each, 2 ml of extracted viral nucleic acid as target and 10.25 ml of DEPC water (Ambion, Warrington, UK). Amplification was achieved using 40 cycles of 95 8C for 30 s, 60 8C for 30 s and 72 8C for 30 s followed by a meltcurve between 50 and 95 8C. TTSuV2 was detected in the same manner, this time using a primers forward-2 (50 -AGTTACACATAACCACCAAACC-30 ) and reverse-2 (50 -ATTACCGCCTGCCCGATAGGC-30 ) previously used in the detection of TTSuV2 (Kerkarainen et al., 2006). In this case amplification was achieved using 40 cycles of 95 8C for 30 s, 52 8C for 30 s and 72 8C for 30 s followed by a melt-curve between 50 and 95 8C. PBo-likeV was also detected using the Roche LC480 system. Primers used in detecting PBo-likeV (SBF: 50 CGACATGCCACTTGCTAAAG-30 and SBR: 50 -ACCGCCGCAAGATTCAATATC-30 ) were based on sequence provided kindly by Anne-Lie Blomstro¨m (Swedish University of Agricultural Sciences, Uppsala, Sweden) (data not published). Amplification was achieved using the same conditions as used to detect TTSuV1 and TTSuV2 with the annealing temperature modified to 50 8C for 30 s. 2.5. Analytical sensitivity of PBo-likeV primers
2.2. Viral nucleic acid extraction Viral nucleic acids were extracted from 200 ml of sera and homogenised tissue samples using a MagNa Pure LC automated 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 of the sera and tissue samples. Amplification was carried out in a DNA Engine Dyad thermal cycler (Bio-Rad Laboratories Ltd., Herts, UK) using HotStarTaq MasterMix (Qiagen Ltd., Crawley, UK) according to the manufacturer’s instructions with 0.5 mM of PCV2 specific primers designed to amplify an 814 bp amplicon which included the ORF2 coding sequence according to Allan et al., 2007. Resultant PCR products were visualised on 1.5% (w/v) agarose gels (Mast Group Ltd., Bootle, UK), before being excised from the agarose gel and purified using a Wizard1 SV Gel and PCR Clean-UP System (Promega, Southampton, UK) according to the manufacturers instructions.
Four ml of nucleic acid from a selected PBo-likeV positive field sample was amplified using Hot StarTaq Master Mix (Qiagen, Crawley, UK) according to the manufacturer’s instructions with 0.5 mM of primers (SB1780: 50 -GGCAGCATGGCTCCTAACTTGC-30 and SB2398: 50 -CTCCCCCTGTATGGGCTCT-30 ). The resultant amplicon was 616 bp in length. Thermal cycling with this primer set was carried out using a GRI G-Storm GS2 Thermal Cycler (G-Storm, Surrey, UK). An initial 95 8C denaturation step was carried out for 15 min before PCR cycling for a further 40 cycles consisting of 30 s of denaturation at 95 8C, 30 s of annealing at 55 8C, and a 1 min extension at 72 8C. The resultant reactions were visualised on 1.5% (w/v) agarose gels (Mast Group Ltd., Bootle, UK). The 616 bp product was purified using a Wizard1 SV Gel and PCR Clean-UP System (Promega, Southampton, UK) according to the manufacturer’s instructions. Purified DNA was quantified using a Jenway Genova UV/VIS spectrophotometer (Barloworld Scientific Ltd., Essex, UK) to estimate DNA mg/ml. This information and the average weight of a nucleotide were used to calculate copy number. A dilution series from 109 to 100 copies/ml was then produced.
2.4. Detection of TTSuV1, TTSuV2 and PBo-likeV 2.6. Sequencing of PCV2 and PBo-likeV All sera and tissue samples were assessed for the presence of TTSuV1, TTSuV2 and PBo-likeV in real-time using a Roche LC480 instrument (Burgess Hill, UK).
PCV2 PCR products were sequenced to determine if genotypes 2a or 2b were present using BigDye1
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Table 1 Percentage prevalence of PCV2, TTSuV1, TTSuV2 and PBo-likeV in tissues recovered from PMWS-affected and non-PMWS-affected pigs and from sera samples from PMWS-affected and non-PMWS-affected pigs. Sample
PMWS Status
PCV2
TTSuV1
TTSuV2
PBo-likeV
Tissue
PMWS +VE PMWS VE Total
100% (29/29) 97% (31/32) 98% (60/61)
41% (12/29) 63% (20/32) 53% (32/61)
79% (23/29) 63% (20/32) 71% (43/61)
69% (20/29) 72% (23/32) 71% (43/61)
Sera
PMWS +VE PMWS VE Total
58% (49/85) 50% (9/18) 56% (58/103)
51% (43/85) 56% (10/18) 52% (53/103)
6% (5/85) 0% (0/18) 5% (5/103)
a
a
62% (53/85) 61% (11/18) 62% (64/103)
Data from multiple tissues recovered from the same pig were combined.
Terminator Kit version 3.1 (Applied Biosystems, Warrington, UK) and those primers used in amplification (Allan et al., 2007). Samples positive for PBo-likeV were amplified using primers (SB2126: 50 -GTAATAAACGACATGCCACTTG30 and SB3171: 50 -AGCTTATAATAAACATGAGCACAA-30 ) to amplify a region including the NP-1 gene. The total reaction volume included 1 ml of 3.2 pmol/ml primer, 8 ml of diluted BigDye1 reaction mix and a variable volume of target DNA. The final reaction volume was made up to 20 ml with DEPC treated water (Ambion, Warrington, UK). Sequencing reactions were cycled as follows; the sample was initially denatured at 96 8C for 1 min, followed by 25 cycles of 96 8C for 10 s, 50 8C for 5 s and 60 8C for 4 min. Performa1 DTR Gel Filtration Cartridges (Edge BioSystems, Gaithersburg, MD, USA) were used to remove unincorporated dNTPs and ddNTPs according to the manufacturer’s instructions. Resultant sequencing data was analysed using an ABI 3100 Genetic Analyser (Applied Biosystems, Warrington, UK).
collected from pigs from PMWS positive farms is detailed in Table 1. PCV2, TTSuV1, TTSuV2 and PBo-likeV were present in both PMWS-affected and non-PMWS-affected pigs. Over 24% of PMWS-affected pigs were positive for the presence of all viruses simultaneously, whereas this figure was 25% in non-PMWS-affected pigs. All 4 viruses could not be detected simultaneously in sera from non-PMWSaffected farms and only 1% of sera from PMWS-affected farms proved positive for all 4 viruses concomitantly.
2.7. Analysis of PCV2 and PBo-likeV sequence
Details on those PCV2 genotypes that could be detected in sera and tissue samples are included in Table 2. Those sera samples collected from PMWS positive and negative farms, that were possible to sequence, during the year 2000 exhibited a mixture of PCV2 genotype 2a and 2b; those recovered in 2001 and 2002 were 2a in character and those from 2003 to 2004 were 2b. PCV2 genotypes recovered from tissues from PMWS positive and negative pigs between 2002 and 2004 were exclusively 2b in nature. Sequence data for the NP-1 gene was derived from virus detected in 24/132 tissues which equated to sequence derived from viral nucleic acids recovered from 12 individual pigs. PCR product of sufficient strength and quality for sequencing could not be recovered from any of the 5 PBo-likeV positive sera samples. Individual NP-1 sequences derived from tissue samples were assigned accession numbers as follows: JN862548 (S123); JN862549
Resultant PCV2 ORF2 sequence was converted to Fasta format and analysed using GeneDoc (http://www.psc.edu/ biomed/genedoc) sequence analysis software to determine and fully investigate genotype. Comparison was made of the nucleotide similarity of PBo-likeV NP-1 sequence to that of existing porcine bocavirus sequences submitted to GenBank (HQ223038; FJ872544; GU902967; GU902968; GU902969; GU902970; GU902971; HQ872052) using ClustalW2 (http://www.genome.jp/tools/clustalw/). Those NP-1 sequences derived from virus detected in GB pigs were also compared to other porcine bocaviruses (HM053693; HM053694; JF512472; JF512473; HQ291308; HQ291309), and representative strains of bocaviruses from other species including human bocavirus (HBoV) (DQ000495; FJ170279; FJ948861; FJ973561), bovine parvovirus 1 (BPV1) (DQ335247), canine minute virus (CMV) (FJ214110) and gorilla bocavirus 1 (GBoV1) (NC_014358; HM145750). A phylogenetic tree was also created by bootstrapped neighbour joining analysis of all samples mentioned previously using MEGA version 5.
3.2. Analytical sensitivity of PBo-likeV detection primers Testing dilutions of the representative PBo-likeV amplicon, demonstrated that the PBo-likeV detection primers were capable of detecting triplicates of this material from 2 108 to 2 101 copies/reaction with a dynamic range of 8 logs. 3.3. Analysis of PCV2 and PBo-likeV sequence
Table 2 PCV2 genotypes detected in tissue samples from PMWS-affected and non-PMWS-affected pigs and from sera samples from PMWS-affected pigs.
3. Results 3.1. Detection of PCV2, TTSuV1, TTSuV2 and PBo-likeV Prevalence of PCV2, TTSuV genogroups 1 and 2 and PBolikeV in PMWS positive and negative tissue and in sera
PCV-2a PCV-2b No sequencing result
Sera (103) 2000–2004
Tissuea (61) 2002–2004
11/103 (2000–2002) 16/103 (2000–2004) 76/103
0/61 57/61 4/61
a Data from multiple tissues recovered from the same pig were combined.
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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 PBoV2 PBoV3 PBoV4 PBoV1-H18 PBoV2-A6 PBoV3-SH20F PBoV4-1-SH17N-1 PBoV4-2-SH17N-2 PBoV3 strain 22 PBoV3 strain 23 HBoV
HM053693 HM053694 JF512472 JF512473 HQ291308 HQ291309 JF429834 JF429835 JF429836 JF713714 JF713715 DQ000495; FJ170279; FJ948861; FJ973561 DQ335247 FJ214110 NC_014358; HM145750
Porcine Porcine Porcine Porcine Porcine Porcine Porcine Porcine Porcine Porcine Porcine Human
36% 29–35% 22–23% 25% 98–99% 35% 23–24% 23–24% 23–24% 23–24% 29% 28–35%
Bovine Canine Gorilla
30–32% 34–37% 28–30%
BPV1 CMV GBoV1
(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 from the 12 pigs was 98% similar to each other at nucleotide level and 97% similar to those other NP-1 gene sequences recovered from Chinese and Swedish pigs. Comparison of the 12 sequences to the NP-1 region of other European and Asian porcine bocaviruses not grouped as PBo-likeV and bocaviruses from other species is detailed in Table 3. The phylogenetic relationship between all of these samples is detailed in Fig. 1. 4. Discussion The detailed characterisation of PBo-likeV has been relatively recent (Blomstro¨m et al., 2009). PBo-likeV occupies the same genera as BPV-1, CMV, HBoV and the more recently discovered GBoV1 (Kapoor et al., 2010). Recently an increasing number of porcine boca viruses and related variants have been detailed in Europe and Asia (Blomstro¨m 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 and GBoV1 recovered from gorillas suffering from enteritis (Kapoor et al., 2010), as yet PBo-likeV is the only porcine bocavirus substantively associated with disease in pigs (Zhai et al., 2010). Whereas PBo-likeV has been noted in Northern European pig populations, and PBoV3 and 4 detected in Northern Irish pigs (McKillen et al., 2011) to date PBo-likeV has not been noted in pigs in Western European 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 nonstructural and capsid protein coding regions. This 3rd ORF has been designated as NP-1 coding region. This region provides a suitable target for boca-specific phylogenetic analysis. Sequence data from the NP-1 gene of 12 pigs was analysed to determine the phylogenic relationship with PBo-likeV sequence data from other European and Asian isolates and to compare PBo-likeV with associated porcine bocaviruses and bocaviruses that have been discovered in other species. The phylogenetic tree demonstrates the close relationship between GB isolates and those recovered from Northern Europe and Asia. Analysis of the sequence homology confirms that these PBo-likeV isolates are at least 97% similar based on the NP-1 gene. Analysis of homology of the NP-1 has suggested that in some cases the degree of relatedness between bocaviruses of other species and PBo-likeV was higher than between PBo-likeV and other bocavirues recovered from swine highlighting the sequence diversity evident in porcine bocaviruses. Previous studies have demonstrated that PBo-likeV is present in PMWS-affected pigs also infected with TTSuV (Blomstro¨m et al., 2010). Other studies have also indicated that pigs suffering from PMWS exhibited a 1.25 times higher incidence of infection with TTSuV (Kerkarainen et al., 2006) and that TTSuV was present in swine suffering from PMWS and porcine respiratory disease complex (PRDC) (Taira et al., 2009; Rammohan et al., 2012). As there may be an association between PMWS and infection with TTSuV it was reasonable to also investigate the prevalence of PCV2 and TTSuV in combination with PBo-likeV. Investigation of archival sera and tissue samples from GB farms has demonstrated that PBo-likeV is present in GB pigs. PBo-likeV amplified product was relatively weak in sera samples in comparison to the other viruses under investigation, potentially suggesting low viral load. The PBo-likeV present in the sera samples was very weak, making it difficult to recover sufficient nucleic material for sequence analysis. The limited number of PBo-likeV positive sera samples could potentially suggest that that PBo-likeV infection rates in the pigs from which these samples had been collected was very low. It is arguable that this is unlikely considering that PBo-likeV was prevalent into an average of 71% of tissues recovered from PMWS-affected and non-PMWS-affected pigs. Although sera and tissue samples were collected from different pigs the there may be a suggestion from the sera results that PBo-likeV exhibits a transient viraemic phase. Unlike the levels detected in sera samples PBo-likeV was detected at much higher levels in tissue samples, both from PMWS-affected and non-PMWS-affected pigs. On average 71% of tissue samples were positive for the presence of PBo-likeV. All of the viruses investigated were present in both PMWS-affected and non-PMWS-affected tissues. Over 24% of tissues from PMWS-affected pigs were positive for the presence of PCV2, TTSuV1, TTSuV2 and PBo-likeV simultaneously, whereas 25% of non-affected pigs were positive for the same combination of viruses simultaneously. Taken in isolation the incidence of PBolikeV was in fact nominally lower in tissues derived from PMWS-affected pigs. It is difficult to infer from this data if there is in fact any potential contributory effect from
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Fig. 1. Bootstrapped neighbour joining comparison of GB PBo-likeV NP-1 sequences to other porcine bocaviruses and bocaviruses from other species. *All GB isolates indicated by a ^.
PBo-likeV to the PMWS status of pigs. Although tissues from over 70% of pigs in this study had PBo-likeV present, it was only present in a quarter of pigs in combination with PCV2, the necessary causal agent of PMWS. It would seem unlikely that PBo-likeV would have a combined effect with
other viruses in contributing to the onset or progression of PMWS. However, the possibility remains that PBo-likeV might have an as yet undefined sub-clinical effect or have an impact as an opportunistic infection in animals already suffering from detrimental infections.
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The temporal distribution of PCV2 genotypes in the samples tested from PMWS affected and non-affected pigs taken within this study is of interest in that, on the one hand, it reflects what has been reported elsewhere (Allan et al., 2007), but on the other does not agree with the results reported in association with the outbreak of PMWS in other countries (Dupont et al., 2008). In Denmark a direct relationship between the introduction of PMWS into the national herd and the introduction of PCV-2b has been reported, leading to speculation that this genotype is more pathogenic than PCV-2a. This is further discussed in the Danish study suggesting that PCV-2a might be considered less pathogenic due to its prevalence in non-PMWSaffected animals (Dupont et al., 2008). Other studies conducted in Canada and Spain (Gagnon et al., 2007; GrauRoma et al., 2008) agree that there might be evidence to suggest that PCV-2b exhibits higher pathogenicity than PCV-2a. However, this direct relationship cannot be applied to the PMWS epizootic in the GB as our results show that cases of diseases associated with PCV-2a, and not PCV-2b infection were occurring between 2000 and 2001. At present there is no simple answer for PCV2 genotype related pathogenicity. Therefore more research in this area is essential. Our results also show that in GB a major shift away from a mixed distribution of PCV2 genotypes to an exclusive predominance of PCV-2b in PMWS affected pigs occurred post 2002. The reasons for this are unknown but reflect a similar shift in PCV2 genotype distribution in diseased animals around the world post 2002. Acknowledgement This work was supported by the BBSRC Industrial Partner 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 2 genogroups recovered from postweaning multisystemic wasting syndrome affected and nonaffected farms in Ireland and Northern Ireland. J. Vet. Diagn. Invest. 19, 668–673. Blomstro¨m, A.L., Bela´k, S., Fossum, C., McKillen, J., Allan, G., Wallgren, P., Berg, M., 2009. Detection of a novel porcine boca-like virus in the background of porcine circovirus type 2 induced postweaning multisystemic wasting syndrome. Virus Res. 146, 125–129. Blomstro¨m, A.L., Bela´k, S., Fossum, C., Fuxler, L., Wallgren, P., Berg, M., 2010. Studies of porcine circovirus type 2, porcine boca-like virus and torque teno virus indicate the presence of multiple viral infections in postweaning multisystemic wasting syndrome pigs. Virus Res. 152, 59–64. Cadar, D., Csa´gola, A., Lo˝ rincz, M., Tomba´cz, K., Kiss, T., Spıˆnu, M., Tuboly, T., 2011. Genetic detection and analysis of porcine bocavirus type 1 (PoBoV1) in European wild boar (Sus scrofa). Virus Genes, [Epub ahead of print]. Carmichael, L. (Ed.), 2004. Neonatal viral infections of pups: canine herpesvirus 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.
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Detection of a porcine boca-like virus in combination with porcine circovirus type 2 genotypes and torque teno sus virus in pigs from postweaning multisystemic wasting syndrome (PMWS)-affected and non-PMWS-affected farms in archival samples from Great Britain Michael J. McMenamy a,*, John McKillen a, Irene McNair a, Catherine Duffy b, Anne-Lie Blomstro¨m c, Catherine Charreyre d, Michael Welsh a, Gordon Allan b a
Veterinary Sciences Division, Agri-Food and Biosciences Institute, Belfast, Northern Ireland BT4 3SD, United Kingdom School of Biological Sciences, Queen’s University Belfast, Belfast, Northern Ireland BT9 7BL, United Kingdom c Department of Biomedical Sciences and Veterinary Public Health, Swedish University of Agricultural Sciences Uppsala, PO Box 7028, SE-75007, Sweden d Merial SAS, 29 Avenue Tony Garnier, 69007 Lyon, France b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 10 February 2012 Received in revised form 25 October 2012 Accepted 1 March 2013
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. PBolikeV 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 ß 2013 Published by Elsevier B.V. All rights reserved.
Keywords: Porcine bocavirus Porcine circovirus type 2 Torque teno sus virus Genogroups Postweaning multisystemic wasting syndrome
1. Introduction Porcine boca-like virus (PBo-likeV) occupies the bocavirus genus of the subfamily Parvovirinae and was originally discovered in lymph nodes recovered from PMWS-affected pigs from Sweden (Blomstro¨m et al., 2009). This single-stranded DNA virus has a genome of approximately 5 kb in length (Zeng et al., 2011). PBo-likeV has been associated with respiratory infections in
* Corresponding author. Tel.: +44 02890 525864; fax: +44 02890 525823. E-mail address: [email protected] (M.J. McMenamy).
weanling piglets from China (Zhai et al., 2010) and discovered in combination with PCV2 and TTSuV in PMWS-affected pigs (Blomstro¨m et al., 2010). Subsequently other novel bocaviruses have been identified in pigs including PBoV1 and 2 (Cheng et al., 2010) and PBoV3 and 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 China and Northern Europe and in European wild boar (Cadar et al., 2011). PBo-likeV has been detected in PMWSaffected swine in Sweden (Blomstro¨m et al., 2010) therefore warranting further investigation into PBo-likeV prevalence in other geographically separate populations affected by PMWS. The essential infectious agent of PMWS
0378-1135/$ – see front matter . Crown Copyright ß 2013 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetmic.2013.03.009
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(Allan et al., 2007) is recognised as PCV2, although it has been noted that PMWS may not develop through infection with PCV2 alone (Ellis et al., 2008). The possibility remains that other agents might be responsible for the development of PMWS in association with PCV2 infection. Novel PBo-likeV may be one such candidate. Studies have also demonstrated that TTSuV has been detected in higher incidences in PMWS-affected pigs than in non-PMWSaffected animals, where Torque teno sus virus genogroup 2 (TTSuV2) is more prevalent in infected animals (Kerkarainen et al., 2006). Torque teno sus virus genogroup 1 (TTSuV1) may also be a potentiating co-factor in the development of PMWS by immunologic dysregulation, even though it is not considered a directly causative agent (Ellis et al., 2008). In this study we report the incidence of PCV2 (genotypes a and b), TTSuV1 and TTSuV2 and a novel PBo-likeV in archival serum and tissue samples 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 from farms throughout GB between 2000 and 2004. Eighteen sera were collected from pigs on farms considered PMWS negative and 85 from pigs on farms considered PMWS positive by classical methodologies (Segale´s and Domingo, 2002). The 132 tissue samples were collected from 61 pigs of known PMWS status. Tissues included lung, liver, kidney, spleen and lymph nodes.
Real-time PCR for the detection of TTSuV1 was carried out using a set of primers 1005 (50 -CGTTTGCTGCCARGCGGACC-30 ) and 1007 (50 -CGTCTGATTGGTTACACCCTATGCA30 ) which had been previously published (Pozzuto et al., 2009). The real-time thermal cycling conditions were modified for use as follows; the final reaction volume of 25 ml included 12.5 ml of QuantiTect SYBR Green PCR Master Mix (Qiagen, West Sussex, UK), 0.125 ml each of forward and reverse primers to give a final concentration of 0.5 mM each, 2 ml of extracted viral nucleic acid as target and 10.25 ml of DEPC water (Ambion, Warrington, UK). Amplification was achieved using 40 cycles of 95 8C for 30 s, 60 8C for 30 s and 72 8C for 30 s followed by a meltcurve between 50 and 95 8C. TTSuV2 was detected in the same manner, this time using a primers forward-2 (50 -AGTTACACATAACCACCAAACC-30 ) and reverse-2 (50 -ATTACCGCCTGCCCGATAGGC-30 ) previously used in the detection of TTSuV2 (Kerkarainen et al., 2006). In this case amplification was achieved using 40 cycles of 95 8C for 30 s, 52 8C for 30 s and 72 8C for 30 s followed by a melt-curve between 50 and 95 8C. PBo-likeV was also detected using the Roche LC480 system. Primers used in detecting PBo-likeV (SBF: 50 CGACATGCCACTTGCTAAAG-30 and SBR: 50 -ACCGCCGCAAGATTCAATATC-30 ) were based on sequence provided kindly by Anne-Lie Blomstro¨m (Swedish University of Agricultural Sciences, Uppsala, Sweden) (data not published). Amplification was achieved using the same conditions as used to detect TTSuV1 and TTSuV2 with the annealing temperature modified to 50 8C for 30 s. 2.5. Analytical sensitivity of PBo-likeV primers
2.2. Viral nucleic acid extraction Viral nucleic acids were extracted from 200 ml of sera and homogenised tissue samples using a MagNa Pure LC automated 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 of the sera and tissue samples. Amplification was carried out in a DNA Engine Dyad thermal cycler (Bio-Rad Laboratories Ltd., Herts, UK) using HotStarTaq MasterMix (Qiagen Ltd., Crawley, UK) according to the manufacturer’s instructions with 0.5 mM of PCV2 specific primers designed to amplify an 814 bp amplicon which included the ORF2 coding sequence according to Allan et al., 2007. Resultant PCR products were visualised on 1.5% (w/v) agarose gels (Mast Group Ltd., Bootle, UK), before being excised from the agarose gel and purified using a Wizard1 SV Gel and PCR Clean-UP System (Promega, Southampton, UK) according to the manufacturers instructions.
Four ml of nucleic acid from a selected PBo-likeV positive field sample was amplified using Hot StarTaq Master Mix (Qiagen, Crawley, UK) according to the manufacturer’s instructions with 0.5 mM of primers (SB1780: 50 -GGCAGCATGGCTCCTAACTTGC-30 and SB2398: 50 -CTCCCCCTGTATGGGCTCT-30 ). The resultant amplicon was 616 bp in length. Thermal cycling with this primer set was carried out using a GRI G-Storm GS2 Thermal Cycler (G-Storm, Surrey, UK). An initial 95 8C denaturation step was carried out for 15 min before PCR cycling for a further 40 cycles consisting of 30 s of denaturation at 95 8C, 30 s of annealing at 55 8C, and a 1 min extension at 72 8C. The resultant reactions were visualised on 1.5% (w/v) agarose gels (Mast Group Ltd., Bootle, UK). The 616 bp product was purified using a Wizard1 SV Gel and PCR Clean-UP System (Promega, Southampton, UK) according to the manufacturer’s instructions. Purified DNA was quantified using a Jenway Genova UV/VIS spectrophotometer (Barloworld Scientific Ltd., Essex, UK) to estimate DNA mg/ml. This information and the average weight of a nucleotide were used to calculate copy number. A dilution series from 109 to 100 copies/ml was then produced.
2.4. Detection of TTSuV1, TTSuV2 and PBo-likeV 2.6. Sequencing of PCV2 and PBo-likeV All sera and tissue samples were assessed for the presence of TTSuV1, TTSuV2 and PBo-likeV in real-time using a Roche LC480 instrument (Burgess Hill, UK).
PCV2 PCR products were sequenced to determine if genotypes 2a or 2b were present using BigDye1
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Table 1 Percentage prevalence of PCV2, TTSuV1, TTSuV2 and PBo-likeV in tissues recovered from PMWS-affected and non-PMWS-affected pigs and from sera samples from PMWS-affected and non-PMWS-affected pigs. Sample
PMWS Status
PCV2
TTSuV1
TTSuV2
PBo-likeV
Tissue
PMWS +VE PMWS VE Total
100% (29/29) 97% (31/32) 98% (60/61)
41% (12/29) 63% (20/32) 53% (32/61)
79% (23/29) 63% (20/32) 71% (43/61)
69% (20/29) 72% (23/32) 71% (43/61)
Sera
PMWS +VE PMWS VE Total
58% (49/85) 50% (9/18) 56% (58/103)
51% (43/85) 56% (10/18) 52% (53/103)
6% (5/85) 0% (0/18) 5% (5/103)
a
a
62% (53/85) 61% (11/18) 62% (64/103)
Data from multiple tissues recovered from the same pig were combined.
Terminator Kit version 3.1 (Applied Biosystems, Warrington, UK) and those primers used in amplification (Allan et al., 2007). Samples positive for PBo-likeV were amplified using primers (SB2126: 50 -GTAATAAACGACATGCCACTTG30 and SB3171: 50 -AGCTTATAATAAACATGAGCACAA-30 ) to amplify a region including the NP-1 gene. The total reaction volume included 1 ml of 3.2 pmol/ml primer, 8 ml of diluted BigDye1 reaction mix and a variable volume of target DNA. The final reaction volume was made up to 20 ml with DEPC treated water (Ambion, Warrington, UK). Sequencing reactions were cycled as follows; the sample was initially denatured at 96 8C for 1 min, followed by 25 cycles of 96 8C for 10 s, 50 8C for 5 s and 60 8C for 4 min. Performa1 DTR Gel Filtration Cartridges (Edge BioSystems, Gaithersburg, MD, USA) were used to remove unincorporated dNTPs and ddNTPs according to the manufacturer’s instructions. Resultant sequencing data was analysed using an ABI 3100 Genetic Analyser (Applied Biosystems, Warrington, UK).
collected from pigs from PMWS positive farms is detailed in Table 1. PCV2, TTSuV1, TTSuV2 and PBo-likeV were present in both PMWS-affected and non-PMWS-affected pigs. Over 24% of PMWS-affected pigs were positive for the presence of all viruses simultaneously, whereas this figure was 25% in non-PMWS-affected pigs. All 4 viruses could not be detected simultaneously in sera from non-PMWSaffected farms and only 1% of sera from PMWS-affected farms proved positive for all 4 viruses concomitantly.
2.7. Analysis of PCV2 and PBo-likeV sequence
Details on those PCV2 genotypes that could be detected in sera and tissue samples are included in Table 2. Those sera samples collected from PMWS positive and negative farms, that were possible to sequence, during the year 2000 exhibited a mixture of PCV2 genotype 2a and 2b; those recovered in 2001 and 2002 were 2a in character and those from 2003 to 2004 were 2b. PCV2 genotypes recovered from tissues from PMWS positive and negative pigs between 2002 and 2004 were exclusively 2b in nature. Sequence data for the NP-1 gene was derived from virus detected in 24/132 tissues which equated to sequence derived from viral nucleic acids recovered from 12 individual pigs. PCR product of sufficient strength and quality for sequencing could not be recovered from any of the 5 PBo-likeV positive sera samples. Individual NP-1 sequences derived from tissue samples were assigned accession numbers as follows: JN862548 (S123); JN862549
Resultant PCV2 ORF2 sequence was converted to Fasta format and analysed using GeneDoc (http://www.psc.edu/ biomed/genedoc) sequence analysis software to determine and fully investigate genotype. Comparison was made of the nucleotide similarity of PBo-likeV NP-1 sequence to that of existing porcine bocavirus sequences submitted to GenBank (HQ223038; FJ872544; GU902967; GU902968; GU902969; GU902970; GU902971; HQ872052) using ClustalW2 (http://www.genome.jp/tools/clustalw/). Those NP-1 sequences derived from virus detected in GB pigs were also compared to other porcine bocaviruses (HM053693; HM053694; JF512472; JF512473; HQ291308; HQ291309), and representative strains of bocaviruses from other species including human bocavirus (HBoV) (DQ000495; FJ170279; FJ948861; FJ973561), bovine parvovirus 1 (BPV1) (DQ335247), canine minute virus (CMV) (FJ214110) and gorilla bocavirus 1 (GBoV1) (NC_014358; HM145750). A phylogenetic tree was also created by bootstrapped neighbour joining analysis of all samples mentioned previously using MEGA version 5.
3.2. Analytical sensitivity of PBo-likeV detection primers Testing dilutions of the representative PBo-likeV amplicon, demonstrated that the PBo-likeV detection primers were capable of detecting triplicates of this material from 2 108 to 2 101 copies/reaction with a dynamic range of 8 logs. 3.3. Analysis of PCV2 and PBo-likeV sequence
Table 2 PCV2 genotypes detected in tissue samples from PMWS-affected and non-PMWS-affected pigs and from sera samples from PMWS-affected pigs.
3. Results 3.1. Detection of PCV2, TTSuV1, TTSuV2 and PBo-likeV Prevalence of PCV2, TTSuV genogroups 1 and 2 and PBolikeV in PMWS positive and negative tissue and in sera
PCV-2a PCV-2b No sequencing result
Sera (103) 2000–2004
Tissuea (61) 2002–2004
11/103 (2000–2002) 16/103 (2000–2004) 76/103
0/61 57/61 4/61
a Data from multiple tissues recovered from the same pig were combined.
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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 PBoV2 PBoV3 PBoV4 PBoV1-H18 PBoV2-A6 PBoV3-SH20F PBoV4-1-SH17N-1 PBoV4-2-SH17N-2 PBoV3 strain 22 PBoV3 strain 23 HBoV
HM053693 HM053694 JF512472 JF512473 HQ291308 HQ291309 JF429834 JF429835 JF429836 JF713714 JF713715 DQ000495; FJ170279; FJ948861; FJ973561 DQ335247 FJ214110 NC_014358; HM145750
Porcine Porcine Porcine Porcine Porcine Porcine Porcine Porcine Porcine Porcine Porcine Human
36% 29–35% 22–23% 25% 98–99% 35% 23–24% 23–24% 23–24% 23–24% 29% 28–35%
Bovine Canine Gorilla
30–32% 34–37% 28–30%
BPV1 CMV GBoV1
(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 from the 12 pigs was 98% similar to each other at nucleotide level and 97% similar to those other NP-1 gene sequences recovered from Chinese and Swedish pigs. Comparison of the 12 sequences to the NP-1 region of other European and Asian porcine bocaviruses not grouped as PBo-likeV and bocaviruses from other species is detailed in Table 3. The phylogenetic relationship between all of these samples is detailed in Fig. 1. 4. Discussion The detailed characterisation of PBo-likeV has been relatively recent (Blomstro¨m et al., 2009). PBo-likeV occupies the same genera as BPV-1, CMV, HBoV and the more recently discovered GBoV1 (Kapoor et al., 2010). Recently an increasing number of porcine boca viruses and related variants have been detailed in Europe and Asia (Blomstro¨m 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 and GBoV1 recovered from gorillas suffering from enteritis (Kapoor et al., 2010), as yet PBo-likeV is the only porcine bocavirus substantively associated with disease in pigs (Zhai et al., 2010). Whereas PBo-likeV has been noted in Northern European pig populations, and PBoV3 and 4 detected in Northern Irish pigs (McKillen et al., 2011) to date PBo-likeV has not been noted in pigs in Western European 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 nonstructural and capsid protein coding regions. This 3rd ORF has been designated as NP-1 coding region. This region provides a suitable target for boca-specific phylogenetic analysis. Sequence data from the NP-1 gene of 12 pigs was analysed to determine the phylogenic relationship with PBo-likeV sequence data from other European and Asian isolates and to compare PBo-likeV with associated porcine bocaviruses and bocaviruses that have been discovered in other species. The phylogenetic tree demonstrates the close relationship between GB isolates and those recovered from Northern Europe and Asia. Analysis of the sequence homology confirms that these PBo-likeV isolates are at least 97% similar based on the NP-1 gene. Analysis of homology of the NP-1 has suggested that in some cases the degree of relatedness between bocaviruses of other species and PBo-likeV was higher than between PBo-likeV and other bocavirues recovered from swine highlighting the sequence diversity evident in porcine bocaviruses. Previous studies have demonstrated that PBo-likeV is present in PMWS-affected pigs also infected with TTSuV (Blomstro¨m et al., 2010). Other studies have also indicated that pigs suffering from PMWS exhibited a 1.25 times higher incidence of infection with TTSuV (Kerkarainen et al., 2006) and that TTSuV was present in swine suffering from PMWS and porcine respiratory disease complex (PRDC) (Taira et al., 2009; Rammohan et al., 2012). As there may be an association between PMWS and infection with TTSuV it was reasonable to also investigate the prevalence of PCV2 and TTSuV in combination with PBo-likeV. Investigation of archival sera and tissue samples from GB farms has demonstrated that PBo-likeV is present in GB pigs. PBo-likeV amplified product was relatively weak in sera samples in comparison to the other viruses under investigation, potentially suggesting low viral load. The PBo-likeV present in the sera samples was very weak, making it difficult to recover sufficient nucleic material for sequence analysis. The limited number of PBo-likeV positive sera samples could potentially suggest that that PBo-likeV infection rates in the pigs from which these samples had been collected was very low. It is arguable that this is unlikely considering that PBo-likeV was prevalent into an average of 71% of tissues recovered from PMWS-affected and non-PMWS-affected pigs. Although sera and tissue samples were collected from different pigs the there may be a suggestion from the sera results that PBo-likeV exhibits a transient viraemic phase. Unlike the levels detected in sera samples PBo-likeV was detected at much higher levels in tissue samples, both from PMWS-affected and non-PMWS-affected pigs. On average 71% of tissue samples were positive for the presence of PBo-likeV. All of the viruses investigated were present in both PMWS-affected and non-PMWS-affected tissues. Over 24% of tissues from PMWS-affected pigs were positive for the presence of PCV2, TTSuV1, TTSuV2 and PBo-likeV simultaneously, whereas 25% of non-affected pigs were positive for the same combination of viruses simultaneously. Taken in isolation the incidence of PBolikeV was in fact nominally lower in tissues derived from PMWS-affected pigs. It is difficult to infer from this data if there is in fact any potential contributory effect from
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Fig. 1. Bootstrapped neighbour joining comparison of GB PBo-likeV NP-1 sequences to other porcine bocaviruses and bocaviruses from other species. *All GB isolates indicated by a ^.
PBo-likeV to the PMWS status of pigs. Although tissues from over 70% of pigs in this study had PBo-likeV present, it was only present in a quarter of pigs in combination with PCV2, the necessary causal agent of PMWS. It would seem unlikely that PBo-likeV would have a combined effect with
other viruses in contributing to the onset or progression of PMWS. However, the possibility remains that PBo-likeV might have an as yet undefined sub-clinical effect or have an impact as an opportunistic infection in animals already suffering from detrimental infections.
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The temporal distribution of PCV2 genotypes in the samples tested from PMWS affected and non-affected pigs taken within this study is of interest in that, on the one hand, it reflects what has been reported elsewhere (Allan et al., 2007), but on the other does not agree with the results reported in association with the outbreak of PMWS in other countries (Dupont et al., 2008). In Denmark a direct relationship between the introduction of PMWS into the national herd and the introduction of PCV-2b has been reported, leading to speculation that this genotype is more pathogenic than PCV-2a. This is further discussed in the Danish study suggesting that PCV-2a might be considered less pathogenic due to its prevalence in non-PMWSaffected animals (Dupont et al., 2008). Other studies conducted in Canada and Spain (Gagnon et al., 2007; GrauRoma et al., 2008) agree that there might be evidence to suggest that PCV-2b exhibits higher pathogenicity than PCV-2a. However, this direct relationship cannot be applied to the PMWS epizootic in the GB as our results show that cases of diseases associated with PCV-2a, and not PCV-2b infection were occurring between 2000 and 2001. At present there is no simple answer for PCV2 genotype related pathogenicity. Therefore more research in this area is essential. Our results also show that in GB a major shift away from a mixed distribution of PCV2 genotypes to an exclusive predominance of PCV-2b in PMWS affected pigs occurred post 2002. The reasons for this are unknown but reflect a similar shift in PCV2 genotype distribution in diseased animals around the world post 2002. Acknowledgement This work was supported by the BBSRC Industrial Partner 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 2 genogroups recovered from postweaning multisystemic wasting syndrome affected and nonaffected farms in Ireland and Northern Ireland. J. Vet. Diagn. Invest. 19, 668–673. Blomstro¨m, A.L., Bela´k, S., Fossum, C., McKillen, J., Allan, G., Wallgren, P., Berg, M., 2009. Detection of a novel porcine boca-like virus in the background of porcine circovirus type 2 induced postweaning multisystemic wasting syndrome. Virus Res. 146, 125–129. Blomstro¨m, A.L., Bela´k, S., Fossum, C., Fuxler, L., Wallgren, P., Berg, M., 2010. Studies of porcine circovirus type 2, porcine boca-like virus and torque teno virus indicate the presence of multiple viral infections in postweaning multisystemic wasting syndrome pigs. Virus Res. 152, 59–64. Cadar, D., Csa´gola, A., Lo˝ rincz, M., Tomba´cz, K., Kiss, T., Spıˆnu, M., Tuboly, T., 2011. Genetic detection and analysis of porcine bocavirus type 1 (PoBoV1) in European wild boar (Sus scrofa). Virus Genes, [Epub ahead of print]. Carmichael, L. (Ed.), 2004. Neonatal viral infections of pups: canine herpesvirus 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.
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