Complete coding sequences and phylogenetic analysis of porcine bocavirus

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1 Journal of General Virology (2011), 92, DOI /vir Short Communication Correspondence Shaobo Xiao Received 27 October 2010 Accepted 10 January 2011 Complete coding sequences and phylogenetic analysis of porcine bocavirus Songlin Zeng,3 Dang Wang,3 Liurong Fang, Jun Ma, Tao Song, Ruoxi Zhang, Huanchun Chen and Shaobo Xiao Division of Animal Infectious Diseases, State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan , PR China Here we report, for the first time, the nearly full-length genome sequence of porcine bocavirus (PBoV), a recently discovered parvovirus from pigs. Phylogenetic trees based on this genome sequence showed that PBoV belongs to the branch containing the genus Bocavirus, which comprises canine minute virus (CnMV), bovine parvovirus, gorilla bocavirus and human bocavirus (HBoV), and was most closely related to the group containing CnMV. PBoV was predicted to contain three potential ORFs encoding the non-structural protein NS1, the characteristic NP1 protein and the capsid protein VP1/VP2, with an organization similar to that of known bocaviruses. Interestingly, the NS1 gene of PBoV was more similar in length to the homogeneous gene found in HBoV than to those of other known bocaviruses. In addition, highly conserved unique splicedonor and -acceptor sites were identified in the NS1 gene of HBoV and PBoV. The family Parvoviridae is classified into two subfamilies: the subfamily Parvovirinae, which infects vertebrates, and the subfamily Densovirinae, which infects arthropods. Based on a report published by the International Committee on Taxonomy of Viruses (ICTV), the subfamily Parvovirinae is divided into five genera: Parvovirus, Erythrovirus, Dependovirus, Amdovirus and Bocavirus (Tattersall et al., 2005). Recently, Hokovirus was proposed as a new genus of the subfamily Parvovirinae (Adlhoch et al., 2010; Lau et al., 2008). Bocavirus, one genus of the subfamily Parvovirinae, isa non-enveloped, autonomously replicating, single-stranded DNA virus containing a genome of approximately 5 kb in length, and has close relationships with respiratory and enteric infections in humans and animals (Allander et al., 2005; Chen et al., 1986, 1988, 2010; Kapoor et al., 2010a, b; Manteufel & Truyen, 2008; Schwartz et al., 2002). The genus Bocavirus comprises four members: bovine parvovirus (BPV), canine minute virus (CnMV), human bocavirus (HBoV) and gorilla bocavirus (GBoV), and has gained special attention because of HBoV, which causes respiratory diseases in children. In the field of veterinary medicine, BPV and CnMV were discovered in the 1960s and they share about 40 % sequence identity (Schwartz et al., 2002). 3These authors contributed equally to this work. The GenBank accession number of the nearly full-length genome of PBoV strain WUH1 reported in this paper is HQ A supplementary table of the primers used for the analysis of the complete coding sequence PBoV is available with the online version of this paper. Using random amplification and large-scale sequencing technology, a novel porcine parvovirus was recently discovered in Swedish pigs with post-weaning multisystemic wasting syndrome (PMWS) (Blomström et al., 2009). Based on partial nucleotide sequencing, this novel porcine parvovirus was proposed to be a porcine boca-like virus (PBoV-like). Subsequent studies revealed the high prevalence of this novel PBoV-like in weaning piglets with respiratory tract symptoms or PMWS, and even in pigs without PMWS (Blomström et al., 2010; Zhai et al., 2010). However, to date, the complete genomic sequence for PBoV-like has not been reported. In this study, we first detected the prevalence of this novel PBoV-like from clinical samples collected in central China. Using an established PCR, which amplifies a 496 bp fragment containing the partial VP1/VP2 genes of PBoVlike (Zhai et al., 2010), 120 clinical serum samples from clinically normal pigs from four different farms in Hubei province, China, were analysed. Nearly 40 % (47 of 120) of the samples were positive when tested by PCR (data not shown). The PCR products of eight PBoV-like-positive samples (two samples from each farm) were sequenced and aligned. Only two nucleotide-mutation sites were observed among the eight sequenced samples (data not shown) and they showed % identify with a Swedish PBoV-like reference strain (GenBank accession number FJ872544), indicating that this region is highly conserved in our tested samples. Because the 496 bp sequenced fragments were highly similar, one PBoV-like-positive sample was selected and used to obtain information about the complete genome G 2011 SGM Printed in Great Britain

2 The genome characterization of PBoV Two specific PCR primers, PBoV-1F and PBoV-1R, were first used to amplify a fragment of about 2000 bp, including the complete coding sequence of the NP-1 gene and the partial coding sequence of the putative VP1/2 gene, as described previously (Blomström et al., 2009). A 1929 bp DNA fragment was amplified and sequenced. Based on the sequences obtained, the 59- and 39-end sequences were determined using a primer walking approach, as described previously (Kapoor et al., 2010b). Briefly, each PCR extension step used one primer specific for the novel virus sequence, along with a degenerate primer able to hybridize to all known bocavirus sequences. A schematic diagram of the primer walking approach is depicted in Fig. 1(a) and all of the primers used are listed in Supplementary Table S1 (available in JGV Online). After the complete genome was assembled, each nucleotide was sequenced in triplicate to confirm the sequence. A total of 4786 bp of newly identified PBoV-like genome sequence were assembled and the nearly full-length genome of strain WUH1 was submitted to GenBank under accession number HQ To determine the phylogenetic relationship of PBoV-like with the other members of the subfamily Parvovirinae, phylogenetic analysis and alignment were performed using the neighbour-joining method with the molecular evolutionary genetics analysis (MEGA) 4 software (Tamura et al., 2007). A total of 29 nearly full-length genome sequences representing different parvovirus species of the subfamily Parvovirinae, including Parvovirus (HPV, MPV, LuIII, MVM, CPV, FPV and PPV1), Amdovirus (AMDV), Bocavirus (CnMV, BPV, HBoV1 4 and GBoV1), Erythrovirus (B19, SPV2, BPV3 and ChPV), Dependovirus (MDPV and GPV) and Hokovirus (PARV4, BHoV and PHoV) were used in this analysis. In addition, two novel porcine parvoviruses, PPV4 and Cnvirus (PPV-H1), recently discovered in piglets but not classified with any known parvovirus species of the subfamily Parvovirinae (Cheung et al., 2010; Wang et al., 2010), were also included. As shown in Fig. 1(b), the overall branching pattern of the tree was split into several distinct branches, with each branch representing an individual genus. The newly characterized PBoV-like isolate WUH1 belonged to the branch comprising the genus Bocavirus, along with CnMV, BPV, GBoV and HBoV, and showed a close mutual evolutionary relationship that was distant from other genera of the Parvovirinae. Furthermore, other identified parvoviruses from swine, including the recently discovered PPV4 and Cnvirus, formed two distinct branches within other subfamilies of the Parvovirinae (Fig. 1b), consistent with previous studies (Cheung et al., 2010; Wang et al., 2010). Because the closest genetic relative of PBoV-like belonged to the genus Bocavirus, we identified it as a porcine bocavirus (PBoV) strain. As well as encoding non-structural protein NS1 and structural protein VP1/VP2, similar to other members of the family Parvoviridae, bocavirus also contains a characteristic ORF between the ORFs encoding NS1 and VP1/ VP2 that encodes a unique NP1 protein. Using the ORF PBoV-2F PBoV-4F NS1 PBoV-1F PBoV-4R NP1 VP1/2 PBoV-F PBoV-R PBoV-1R PBoV-2R PBoV-3F PBoV-3R Parvovirus Amdovirus Bocavirus Erythrovirus Dependovirus Cnvirus Hokovirus Fig. 1. Identification of a new porcine bocavirus isolate WUH1. (a) Schematic diagram of the primer-walking approach used to determine the nearly full-length genome sequence of porcine bocavirus. The primers used are listed in Supplementary Table S1 (available in JGV Online). (b) Phylogenetic analyses of PBoV-like isolate WUH1 and other parvovirus species of the subfamily Parvovirinae were conducted using the MEGA4 software and the neighbour-joining method. Numbers at nodes indicate the value of 1000 bootstrap analyses. Bar, Base substitutions per site. The sequences were derived from GenBank entries with the following accession numbers: NC (AAV, adeno-associated virus-1), NC (AMDV, Aleutian mink disease virus), NC (B19, human parvovirus B19), EU (BHoV, bovine hokovirus), NC (BPV, bovine parvovirus), NC (BPV-2, bovine parvovirus 2), AF (BPV-3, bovine parvovirus 3), NC (CPV, canine parvovirus type), GQ (ChPV, chipmunk parvovirus), M38246 (FPV, feline panleukopenia virus), HM (GBoV, gorilla bocavirus), NC (GPV, goose parvovirus), U34255 (HPV, hamster parvovirus), NC (HBoV1, human bocavirus), NC (HBoV2, human bocavirus 2), NC (HBoV3, human bocavirus 3), NC (HBoV4, human bocavirus 4), NC (PARV4, human parvovirus 4), NC (LuIII, LuIII virus), AF (CnMV, minute virus of canines), NC (MVM, minute virus of mice), NC (MPV, mouse parvovirus), NC (MDPV, Muscovy duck parvovirus), EU (PHoV, porcine hokovirus), NC (PPV1, porcine parvovirus), HM (PPV4, porcine parvovirus 4), AB (PPV-H1, porcine parvovirus H1) and U26342 (SPV, simian parvovirus). X, Parvovirus isolates from pigs

3 S. Zeng and others finder available at the NCBI website ( nih.gov/gorf/gorf.html), three ORFs were identified in the nearly full-length genome of PBoV, as expected. The first 59 ORF of PBoV, NS1, comprised 1911 bp and encoded 636 aa. The second ORF, NP1, comprised 657 bp and encoded 218 aa. The third ORF, VP1/VP2, comprised 1872 bp and encoded 623 aa. To investigate further the genetic diversity of PBoV, the complete coding sequence of PBoV from strain WUH1 was compared with the other available human and animal bocavirus sequences. The sequence identity between different strains was calculated using the CLUSTAL W program online ( uk/tools/msa/clustalw2). The complete coding sequence of PBoV had sequence similarity at the nucleotide level to CnMV of %, to BPV of %, to GBoV of 50 % and to HBoV of %, supporting the finding that PBoV was phylogenetically related to CnMV (Fig. 1b). To examine the PBoV genome further and identify any unique features, we compared the NS1, NP1 and VP1/VP2 genes of PBoV with other available bocavirus sequences. At the amino acid sequence level, the predicted NS1 gene of PBoV showed sequence identity with CnMV of %, BPV of %, GBoV of 40 % and HBoV of %; the NP1 gene showed sequence identity with CnMV of %, BPV of %, GBoV of 32 % and HBoV of %; and the VP1/VP2 gene showed sequence identity with CnMV of %, BPV of %, GBoV of 34 % and HBoV of %. Phylogenetic trees based on the NS1, NP1 and VP1/VP2 sequences were constructed using both the nucleotide and deduced amino acid sequences. As is shown in Fig. 2, phylogenetic analysis of the nucleotide sequences of NS1 showed that PBoV belonged to the branch containing CnMV. However, phylogenetic analysis of the nucleotide sequences of NP1, a protein of unknown function that is restricted to bocaviruses, indicated that PBoV formed a distinct branch with the other available bocaviruses. Interestingly, phylogenetic analysis of the nucleotide sequence of the VP1/VP2 gene indicated that PBoV formed a distinct branch, whereas phylogenetic analysis of the amino acid sequence of VP1/VP2 indicated that PBoV belonged to the branch containing CnMV (data not shown). According to the criteria of the ICTV for the classification of bocaviruses, NS gene DNA sequence homology is defined as an important marker of different bocavirus species ( In addition, previous studies have revealed that the NS1 protein encoded by animal bocaviruses is longer than that encoded by human bocaviruses (Kapoor et al., 2010b). Although the predicted NS1 gene of PBoV had higher sequence identity with CnMV at the amino acid sequence level, PBoV NS1 encodes a shorter protein (636 aa), which is more similar in length to the NS1 protein found in HBoV, than to that in other known animal bocaviruses (Kapoor et al., 2010a, b; Qiu et al., 2007; Sun et al., 2009). Recently, Kapoor et al. (2010b) showed the presence of highly conserved potential RNA splicing signals in the NS1 gene of all HBoV species, Nucleotide sequence of the NS1 gene Nucleotide sequence of the NP1 gene Nucleotide sequence of the VP1/2 gene Fig. 2. Phylogenetic analyses of the nucleotide sequences of NS1, NP1 and VP1/VP2 were conducted using the MEGA4 software and the neighbour-joining method. Numbers at the nodes indicate the values of 1000 bootstrap analyses. $, Strain WUH1 PBoV for which a nearly full-length genome is reported in this paper. and revealed a putative elongated aa NS1 protein resulting from such a spliced transcript from HBoV. Using an online program to analyse gene structure ( mit.edu/genscan.html), we found, in all four HBoV species and in PBoV, the presence of a stretch of amino acids similar to those encoded in the C terminus of the longer NS1 proteins of other bocaviruses (Fig. 3a). The genomes of all HBoV species and PBoV were aligned to determine the presence of conserved potential RNA splicing signals and the putative second exon encoding the C-terminal region of NS1 (Fig. 3b, splice donor, branch and splice acceptor sites). The putative NS1 protein resulting from such a spliced transcript encoded a 748 aa protein and an elongated carboxy terminus that showed higher similarity to the NS1 protein of HBoV than to that of other known bocaviruses (Fig. 3c). More recently, Chen et al. (2010) demonstrated that the spliced NS1-transcript of HBoV1 was expressed as a large protein of approximately 100 kda in human alveolar epithelial A549 cells transfected with the nearly full-length clones of HBoV Journal of General Virology 92

4 The genome characterization of PBoV PBoV HBoV BPV CnMV GBoV NS1 NP1 VP1/VP2 Splice donor site Branch site Splice acceptor site NS1 protein before splicing Intron Extended NS1 protein Fig. 3. Unique genomic features of the NS1 gene of PBoV. (a) Genome structure of different species of known bocaviruses. (b) Consensus RNA splicing sequence and the presence of similar sequence motifs that are conserved among the NS1 genes of all HBoV and PBoV. Potential stop codons of the nonspliced NS1 transcripts are underlined. (c) Protein identity between the extended carboxy termini of NS1 of PBoV and the NS1 termini of other animal bocaviruses. Despite the high genetic diversity observed in all HBoV species, the PBoV genome also has highly conserved NSgene RNA splicing sites, suggesting that two different NS proteins of PBoV may be expressed in host cells. As an emerging porcine parvovirus, a high prevalence (nearly 40 %) of PBoV infection in the present study was observed in serum samples from clinically normal pigs. Our results are consistent with previous studies that indicated that 44 % of healthy pigs were PBoV positive (Blomström et al., 2010). It is interesting to note a rather high prevalence (88 %) of PBoV infection in pigs with PMWS (Blomström et al., 2010). Therefore, more studies need to be conducted to see if PBoV contributes to the development of PMWS or to other related complex swine diseases. Because a higher PBoV infection rate was observed in pigs, and to date natural PBoV has not been isolated in vitro, we tried to isolate PBoV from PBoV-positive samples in primary cells isolated from pigs and from some established cell lines in vitro. Although several kinds of cells, including porcine kidney cells (PK-15), swine testicular cells, porcine alveolar macrophages, monkey kidney cells (MARC-145) and human embryonic kidney epithelial cells (HEK293T) cells, were used to propagate this novel PBoV, no growth was detected in any of the cells tested (data not shown). Further studies are therefore required to obtain or establish a permissive cell line for PBoV infection in vitro. In summary, this is the first study to report the nearly fulllength genome sequence of porcine bocavirus. PBoV contains three potential ORFs, with a genome organized similarly to those of known bocaviruses. Phylogenetic analysis revealed that PBoV is most closely related to the group of viruses containing CnMV. The results of this study increase our understanding of animal bocaviruses and provide a foundation for novel detection, diagnosis and prevention strategies for PBoV. Acknowledgements This work was supported by the 863 project (2011AA10A208) and the New Century Excellent Talent Project (NCET ). References Adlhoch, C., Kaiser, M., Ellerbrok, H. & Pauli, G. (2010). High prevalence of porcine Hokovirus in German wild boar populations. Virol J 7, 171. Allander, T., Tammi, M. T., Eriksson, M., Bjerkner, A., Tiveljung- Lindell, A. & Andersson, B. (2005). Cloning of a human parvovirus by molecular screening of respiratory tract samples. Proc Natl Acad Sci USA102, Blomström, A. L., Belák, 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,

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