Identification and nearly full-length genome characterization of novel porcine bocaviruses

Abstract

The genus bocavirus includes bovine parvovirus (BPV), minute virus of canines (MVC), and a group of human bocaviruses (HBoV1-4). Using sequence-independent single primer amplification (SISPA), a novel bocavirus group was discovered with high prevalence (12.59%) in piglet stool samples. Two nearly full-length genome sequences were obtained, which were approximately 5,100 nucleotides in length. Multiple alignments revealed that they share 28.7-56.8% DNA sequence identity with other members of Parvovirinae. Phylogenetic analyses indicated their closest neighbors were members of the genus bocavirus. The new viruses had a putative non-structural NP1 protein, which was unique to bocaviruses. They were provisionally named porcine bocavirus 1 and 2 (PBoV1, PBoV2). PBoV1 and PBoV2 shared 94.2% nucleotide identity in NS1 gene sequence, suggesting that they represented two different bocavirus species. Two additional samples (6V, 7V) were amplified for 2,407 bp and 2,434 bp products, respectively, including a partial NP1 gene and the complete VP1 gene; Phylogenetic analysis indicated that 6Vand 7V grouped with PBoV1 and PBoV2 in the genus of bocavirus, but were in the separate clusters. Like other parvoviruses, PBoV1, PBoV2, 6Vand 7V also contained a putative secretory phospholipase A(2) (sPLA(2)) motif in the VP1 unique region, with a conserved HDXXY motif in the catalytic center. The conserved motif YXGXF of the Ca(2+)-binding loop of sPLA2 identified in human bocavirus was also found in porcine bocavirus, which differs from the YXGXG motif carried by most other parvoviruses. The observation of PBoV and potentially other new bocavirus genus members may aid in molecular and functional characterization of the genus bocavirus.

Figure 1. Phylogenetic analyses of partial NS1 gene nucleotide sequences from PBoV and other bocavirus members.

The phylogenetic tree was constructed by the neighbor-joining method with 1,000 bootstrap replicates, using the MEGA 4.1 software. Black circles designate reference strains, and the others are sequences generated from the present study. PBoV1 and PBoV2 are indicated with black diamonds. MVC: minute virus of canines; BPV: bovine parvovirus. HBoV: human bocavirus.

Figure 2. The phylogenetic analyses of viruses in the Parvoviridae family.

The phylogenetic tree was constructed from nearly full-length nucleotide sequences of subfamilies Parvovirinae (genera Parvovirus, Erythrovirus, Dependovirus, Amdovirus, and Bocavirus) and Densovirinae, using the MEGA 4.1 software (neighbor-joining method with 1,000 bootstrap replicates). PBoV1 and PVoB2 are labeled with black diamonds. MVC: minute virus of canines; BPV: bovine parvovirus; HBoV: human bocavirus; Pbo-likeV: porcine boca-like virus.

Figure 3. Map of the genomic organization of PBoV and HBoV.

The map was constructed using the Geneious 4.8.5 software. Genus bocavirus has a non-structural NP1 protein encoded by an ORF in the middle of the genome, which is a unique structure present in bocaviruses, but not found in most other parvoviridae members.

Figure 4. Phylogenetic relationships of three different protein (NS1, NP1, VP1) sequences in the bocavirus genus.

The phylogenetic tree was constructed from amino acid sequences using the MEGA 4.1 software (neighbor-joining method with 1,000 bootstrap replicates). PBoV1 and PBoV2 are labeled with black diamonds, 6V and 7V are labeled with black triangles, and Pbo-likeV is labeled with an open diamond. MVC: minute virus of canines; BPV: bovine parvovirus; HBoV: human bocavirus; Pbo-likeV: porcine boca-like virus.

Figure 5. Phylogenetic relationships of the common region among PBoV, Pbo-likeV and other members of parvoviruses.

The phylogenetic tree was constructed using the MEGA 4.1 software (neighbor-joining method with 1,000 bootstrap replicates). PBoV1 and PBoV2 are labeled with black diamonds, 6V and7V are labeled with black triangles, and Pbo-likeV is labeled with an open diamond. MVC: minute virus of canines; BPV: bovine parvovirus; HBoV: human bocavirus; Pbo-likeV: porcine boca-like virus.

Figure 6. Putative phospholipase A₂ (PLA₂) motif in members of the genus bocavirus and other parvoviruses.

It was constructed using BioEdit software. The HDXXY and YXGXG motifs of PLA₂ are indicated by black circles at the bottom. The HDXXY motif is strongly conserved among parvoviruses, whereas the latter motif exists as YXGXF in bocaviruses, instead of the YXGXG motif found in most parvoviruses. MVC: minute virus of canines; BPV: bovine parvovirus; HBoV: human bocavirus; B19: human parvovirus B19; SPV: simian parvovirus; GPV: goose parvovirus; PPV: porcine parvovirus; MEV: mink enteritis virus; AAV1: adeno-associated virus-1; GmDNV: Galleria mellonella densovirus; BmDNV: Bombyx mori densovirus.