Bocaviruses (about 5 kb genome size) are small, non-enveloped, ssDNA viruses within the genus Bocavirus, subfamily Parvovirinae, family Parvoviridae [1]. They cause specific diseases (such as respiratory disease and enteritis) in humans and other animals. Bocaviruses have three open reading frames (ORF), two encoding non-structural proteins (NS1 and NP1), and one structural protein (VP1/VP2). The NP1 protein (the third ORF, ORF3) is characteristic of Bocavirus genus, its corresponding genes are located between NS1 gene region and VP1/VP2 gene region [2]. The first bocavirus was discovered in calves with diarrhea in 1961. It was named the HADEN (hemadsorbing enteric) virus of calves [3]. However, it is now called bovine parvovirus 1 (BPV1), which is different from newly discovered BPV2 and BPV3 [4]. About ten years later, minute virus of canines (MVC), also called canine parvovirus type 1 and canine bocavirus, was identified in the rectal specimens of four mature German shepherd dogs [5]. In 2005, an emerging human parvovirus, called human bocavirus (HBoV), similar to BPV1 and MVC in genome structure, was characterized in pooled specimens taken from the respiratory tracts of children with respiratory diseases [6]. Since then, another three species of human bocavirus, HBoV2, HBoV3, and HBoV4, have also been discovered [7- 10]. Porcine bocavirus (PBoV) was first described in 2009 in Swedish pigs with postweaning multisystemic wasting syndrome (PMWS) [11]. After that, diversiform PBoVs were reported by scientific researchers from different labs in several countries [12-23]. Its nomenclature was confused and irregular, which might hinder the research of PBoVs. The goal of the present study was to compare the sequences of those emerging porcine bocaviruses, perform phylogenetic analysis, and present a proposal on regarding nomenclature.

Reference sequences of porcine and other bocaviruses

In order to select useful materials for this study, genome sequences of some porcine bocaviruses and other bocaviruses described during the same period were downloaded from the GenBank database. The information is presented in (Tables 1 and 2).

JN681175

Table 1: Reference genome sequences of porcine bocaviruses in this study.

Bioinformatic analysis

Bioinformatic software can be used to evaluate sequence similarity among multiple nucleotides and amino acids, sequence mutation, deletions and insertions, sequence recombination, and genetic evolution of viruses, bacteria, and other species. In this study, DNAStar software (Clustal W method) and MEGA 5.1 software (Neighbor-joining method) were used to collect the above information regarding porcine and other bocaviruses. Sequence similarity, sequence mutation, deletion, or insertion, and sequence recombination among reference bocaviruses were obtained using DNAStar software, and its genetic evolution was determined using MEGA 5.1 software.

Genome structure of PBoVs

To better understand genome characterization of different porcine bocaviruses, structure maps (Figure 1) were drawn electronically using sequence descriptions given in the GenBank database. PBoV1: Until now, there have been only two nearly complete genomes of PBoV1 in the GenBank database and no fully complete ones, they shared a similar genome structure. ORF1 (encoding NS1 protein) of genotype PBoV1-a had 1,911 NTs and that of genotype PBoV1-b had 1,872 NTs. ORF2 (encoding VP1/VP2 protein) of genotype PBoV1-a had 1,908 NTs and that of PBoV1-b had 1,863 NTs. The first difference lay in a 3-NT insertion or deletion (not shown), and the second difference lay in a 9-NT insertion or deletion (not shown). Both of genotypes had the NP1 genes of the same size (657 NTs) (Figure 1). PBoV1-a and PBoV1-b also had the intervals of the same size nucleotides: there were 185 NTs between the area downstream of NS1 and the area upstream and there were 52 NTs between the area downstream of NP1 and the area upstream (not shown).

Figure 1: Genome structure of different porcine bocaviruses.

PBoV2 has been reported in some countries, including the USA, China, Romania, Hungary, and Cameroon [13,14; 24-26]. Descriptions of its genome are limited. Generally, the isolates from the USA and China are divided into two different genotypes, PBoV2-a and PBoV2-b, respectively. ORF2 (encoding VP1/VP2 protein) and ORF3 (encoding NP1 protein) of PBoV2-a and PBoV2-b had similar structures (2, 187 NTs for VP1/VP2 genes and 615 NTs for NP1 genes) (Figure 1). They also had intervals of the same length. There were 149 NTs between the upstream and downstream areas of PBoV2-a NS1 and 263 NTs between the upstream and downstream areas of PBoV2-b NP1 (not shown). PBoV2-b differed from other isolates in that it had a 111 NT deletion from the area upstream of the NS1 gene (not shown).

PBoV3 was discovered using viral metagenomic methods [12,27]. At present, the isolates of PBoV3 can be divided into three different genotypes, PBoV3-a, PBoV3-b, and PBoV3-c. The NS1 genes of these three genotypes are the same size (2,112 NTs) (Figure 1). In comparison to PBoV3-a (687 NTs) and PBoV3-c (687 NTs), there was a discontinuous 6-NT (3+3) deletion in the NP1 genes of PBoV3-b (693 NTs) (not shown). The VP1/VP2 genes differ from those of PBoV3-b (2,130 NTs) in a discontinuous 12 NT (9+3) deletion and 15 NT (12+3) deletion (not shown), leaving length of these VP1/VP2 genes were 2,118 NTs and 2,115 NTs, respectively (Figure 1). PBoV3-a and PBoV3-c had intervals of the same size (72 NTs) between the area downstream of NS1 and the area upstream of NP1. They also had the same number of overlapping nucleotides (17 NTs) between the area downstream of NP1 and the area upstream of VP1/VP2. However, PBoV3-b had the same number of overlapping nucleotides (17 NTs) as PBoV3-a and PBoV3-c between the area downstream of NP1 and the area upstream of VP1/VP2, but it also had a different number of interval nucleotides (69 NTs) between the area downstream of NS1 and the area upstream of NP1 (not shown).

The species PBoV4 can be divided into eight genotypes by the differences in its genome structure, PBoV4-a to PBoV4-h. PBoV4-a, PBoV4-b, PBoV4-d, and PBoV4-g had NS1 genes of 2,004 NT in length, but the others had NS1 genes of different lengths: 2,403 NT for PBoV4-c, 1,800 NTs for PBoV4-e, 1,755 NT for PBoV4-f, and 2,058 NT for PBoV4-h. PBoV4-a, PBoV4-b, PBoV4-c, and PBoV4-e had NP1 genes of 681 NT in length, and PBoV4-f, PBoV4-g, and PBoV4-h had NP1 genes of 675 NT in length. However, the length of the NP1 gene of PBoV4-d was 678 NT. PBoV4-b, PBoV4-c, and PBoV4-g had VP1/VP2 genes of 2,055 NT in length, PBoV4-a and PBoV4-c had VP1/VP2 genes of 2,052 NT in length, and PBoV4-d and PBoV4-f had VP1/VP2 genes that differed from each other (Figure 1). Almost all PBoV4 genotypes had 9 overlapping sequences between NP1 and VP1/VP2. However, there were intervals of many different sizes between NS1 and NP1. PBoV4-a and PBoV4-e both had intervals of 121 NT. PBoV4-b and PBoV4-d both had intervals of 124 NT. PBoV4-f and PBoV4-g had intervals of 129 NT. PBoV4-h had the shortest interval (107 NT). Notably, the NS1 and NP1 of PBoV4-c overlapped by 276 NT (not shown).

Nucleotide similarity of different protein-encoding regions of PBoVs

NS1 region: As shown in (Figure 1), the size of NS1 among PBoVs varied from 1,755 NT to 2403 NT. Even if the viral strains belonged to the same species, there were still differences in the size of NS1. Generally, nucleotide similarity of the NS1 region varied from 6.8% to 99.8% (Table 3). HQ223038 and HQ291308 both had 77.4% nucleotide similarity to PBoV1. For PBoV2, the truncated NS1 (lacking 111 NTs) of GQ387499 and the complete NS1 of GQ387500 had 99.8% nucleotide similarity. For PBoV3, the NS1 genes of HM053693, HM053694, and HQ291309 were 2112 bp in size, but their homologies varied from 93.8% to 94.3%. For PBoV4, the nucleotide similarity of those strains varied between 79.1% and 96.8%.

Table 2: Reference genome sequences of other bocaviruses in this study.

Table 3: Nucleotide homology (%) of NS1 regions of PBoV1-4.

NP1 region: The NP1 genes of PBoV1-4 were no more than 700 nucleotides in size. However, there were considerable changes in nucleotide similarity, from 3.1% to 99.7% (Table 4). For PBoV1, HQ223038 and HQ291308 had 99.2% nucleotide similarity, which was higher than that of NS1. For PBoV2, like NS1, the NP1 of GQ387499 and GQ387500 had considerable nucleotide similarity (99.7%). For PBoV3, HM053693, HM053694, and HQ291309 had less nucleotide similarity (from 91.6% to 93.7%) than NS1 did. For PBoV4, there was at least 95% nucleotide similarity between JF429834, JF429835, JF512472, and JF713714. JF713715 and JN831651 also had 91.7% nucleotide similarity. For PBoV4, they had about 80% nucleotide similarity.

Table 4: Nucleotide homology (%) of NP1 regions of PBoV1-4.

VP1/VP2 region: For the VP1/VP2 region, inter-species nucleotide similarity was low, ranging from 0.8% to 58.6%. More nucleotide similarity (from 93.1% to 99.8%) was detected among intra-species (PBoV1, PBoV2, and PBoV3) than PBoV4 (from 75.3% to 88.7%) (Table 5).

Table 5: Nucleotide homology (%) of VP1/VP2 regions of PBoV1-4.

Phylogenetic analysis of different protein-encoding regions of PBoVs

A phylogenetic tree of NS1 was constructed based on two PBoV1 strains, 2 PBoV2 strains, 3 PBoV3 strains, 8 PBoV4 strains, and another 25 bocavirus strains (Figure 2). PBoV1, PBoV3, and PBoV4 were clustered in the branch with CBoV1, CBoV2, FBoV, and CslBoV. However, PBoV2 was divided into one separate branch, which had farthest genetic distances from the other bocaviruses (Figure 2).With respect to NP1, PBoV1 and PBoV4 had close genetic relationships and were clustered in the same branch. PBoV3 and FBoV were placed on the same branch. Like NS1, PBoV2 was placed far from other viral species (Figure 2).With respect to VP1, PBoV1 and PBoV3 had less genetic differences from CBoV1, CBoV2, FBoV, and CslBoV and were placed on one big branch. PBoV4 had the close genetic relationship to BPV1. PBoV2 was far from other bocaviruses in the phylogenetic tree (Figure 2). With respect to VP2, PBoV1 was close to HBoV1- 4, GBoV1, and BPV1. Similarly, PBoV3 was not far from CBoV1, CBoV2, FBoV, or CslBoV. PBoV4 was far from other bocaviruses (Figure 2).

Figure 2: Phylogenetic analysis of NS1, NP1, VP1 and VP2 genes for porcine bocaviruses and other reference bocavirus.

PBoV1 was discovered in 2009, before any of the others [11]. This is why it was named PBoV1 [18,23]. Shan et al. [18] found PBoV1 circulating in China. Then, it had also been found in Romania, Uganda, and Cameroon [26,28,29]. Due to the low level of nucleotide similarity (especially in the NS1 region) and genome characterization among species, PBoV1 should be divided into two genotypes, PBoV1-a and PBoV1-b (Figure 1). In 2010, Cheung et al. [13] identified a novel porcine parvovirus different from previous parvoviruses (PPV1, PPV2, and PPV3). This virus was tentatively named PPV4. However, its genome structure and RNA profile showed it had the characteristics of bocaviruses, herein, so it was renamed PBoV2 [23,30]. Considering the major differences within the PBoV2 genome, it should include two genotypes. PBoV2-a and PBoV2-b, the second of which lacks 111 NT in its NS1 region (Figure 1).Two Chinese research teams detected two novel bocaviruses almost simultaneously. They were named PBoV1 and PBoV2 [c,18]. Based on the sequence comparison and phylogenetic analysis described herein, these two strains are named PBoV3. PBoV3 should contain three genotypes, PBoV3-a, PBoV3-b, and PBoV3-c (Figure 1). However, there was considerable nucleotide similarity in different coding regions (93.8% to 94.3% for NS1, 91.6% to 93.7% for NP1, and 93.1% to 96% for VP1/VP2) (Tables 3-5). PBoV3 and non-PBoV3 (present PBoV4) were found and reported at about the same time [12]. Many PBoV4-like genome sequences have been published [15-17,19-20]. These PBoV4-like strains could be considered as members of the PBoV4 species. Based on sequence differences, PBoV4 can be divided into eight different genotypes (PBoV4-a and PBoV4-h) (Figure 1). Moreover, to our knowledge, at present, porcine bocaviruses were cultured poorly in vitro. Therefore, it is very difficult to us to master the pathogenicity for every PBoV species. However, some epidemiological studies showed that PBoV1, PBoV2 and PBoV4 had higher prevalence in diseased pig herds than PBoV3 [11-14,22,23]. Whether the sequence diversities of viral genes (NS1, VP1/VP2) could have any impact on the virulence or infectivity of different virus species should be further studied. According to the present literatures, All the four PBoV species were reported in Europe (such as Sweden, Hungary), Asia (such as China, South Korea, Hong Kong), North America, Africa (such as Uganda, Cameroon) [31]. It suggested that PBoVs lacked of the significant geographical prevalence, which was associated with frequent international trading of live pigs and pork products. Meanwhile, there were few PBoV reports in Australia, South America, Southeast Asia, the Middle East countries. The reason was that no research team did epidemiological survey in those regions. For the origin of every PBoV species, the mystery was still not uncovered.

The current criteria for classification of bocaviruses issued by the International Committee on Taxonomy of Viruses (ICTV) defines separate species as a group of individuals with less than 95% homologous non-structural gene DNA sequence (http://www.ictvdb.org/). However, the NS1 genes of PBoVs divide them into ten species, but their NP1 genes only divide them into nine. Recently, VP1-based classification has also been proposed. PBoVs were divided into three groups: group 1 or PBoV1 (represented by PBo-likeV), group 2 or PBoV2 (PBoV1/2-CHN and PBoV2-A6), and group 3 or PBoV3 (PBoV3/4-UK, PBoV3/4-HK, and PBoV3C) [20]. However, here, we added PPV4 (boca-like virus) to the PBoV family. According to the present proposal, which is based on genome comparison and phylogenetic analysis, PBoVs can be divided into four different species, PBoV1 (represented by PBo-likeV-SWE and PBoV1-CHN), PBoV2 (PPV4-USA), PBoV3 (PBoV1/2-CHN and PBoV2-A6-CHN), and PBoV4 (PBoV3/4-UK, PBoV3/4-HK, PBoV3-USA, PBoV3C-CHN, and PBoV5-CHN). Further classification showed that these four species also contained 2-8 different genotypes, respectively. In summary, it is here suggested that PBoVs are highly genetically diversified. The proposed nomenclature of PBoVs was used in the present studies, and it may facilitate better understanding of PBoVs. If nothing else, it may facilitate the development of a standardized nomenclature system.

This work was supported by the grant (No. 2012SZJJ-005) from Institute of Animal Health, Guangdong Academy of Agricultural Sciences, and the grant (No. 2012224-26) from Guangzhou Science and Technology and Information Bureau.