Genomic characterization and high prevalence of bocaviruses in swine

Abstract

Using random PCR amplification followed by plasmid subcloning and DNA sequencing, we detected bocavirus related sequences in 9 out of 17 porcine stool samples. Using primer walking, we sequenced the nearly complete genomes of two highly divergent bocaviruses we provisionally named porcine bocavirus 1 isolate H18 (PBoV1-H18) and porcine bocavirus 2 isolate A6 (PBoV2-A6) which differed by 51.8% in their NS1 protein. Phylogenetic analysis indicated that PBoV1-H18 was very closely related to a ∼2 Kb central region of a porcine bocavirus-like virus (PBo-LikeV) from Sweden described in 2009. PBoV2-A6 was very closely related to the porcine bocavirus genomes PBoV-1 and PBoV2 from China described in 2010. Among 340 fecal samples collected from different age, asymptomatic swine in five Chinese provinces, the prevalence of PBoV1-H18 and PBoV2-A6 related viruses were 45-75% and 55-70% respectively, with 30-47% of pigs co-infected. PBoV1-A6 related strains were highly conserved, while PBoV2-H18 related strains were more diverse, grouping into two genotypes corresponding to the previously described PBoV1 and PBoV2. Together with the recently described partial bocavirus genomes labeled V6 and V7, a total of three major porcine bocavirus clades have therefore been described to date. Further studies will be required to elucidate the possible pathogenic impact of these diverse bocaviruses either alone or in combination with other porcine viruses.

Figure 1. The PBoV1 and PBoV2 genomes.

Diagrammatic representation of PBoV1-H18 and PBoV2-A6 sequences showing the position of ORFs for NS1, NP1, VP1 and VP2, compared with other bocaviruses. The black boxes represent the known spliced exon of NS1 transcripts.

Figure 2. Phylogenetic tree constructed by the neighbor-joining method with 1,000 bootstrap replicates using MEGA4.0 software.

Bootstrap values are indicated at each branching point. Scale bar indicates estimated genetic distance. Phylogenetic analysis of the nearly full-length genomes of PBoV1 and PBoV2 and 33 representative bocavirus species/strains (A). Phylogenetic analysis of nucleic acid and amino acid of NS1 (B and E), NP1 (C and F) and VP1 (D and G) ORFs of PBoV1-H18 and PBoV2-A6 and representative bocavirus species.

Figure 3. Phylogenetic tree of PBoV2-A6 related isolates in China showing the presence of two genotypes using a 439-nt VP1 gene sequence alignment.

Bootstrap value was obtained from 1000 re-samplings of the data. The PBoV1-H18 strain is included as outgroup. Scale bar indicates estimated genetic distance.