First identification of porcine parvovirus 6 in North America by viral metagenomic sequencing of serum from pigs infected with porcine reproductive and respiratory syndrome virus

Abstract

Background: Currently, eight species in four genera of parvovirus have been described that infect swine. These include ungulate protoparvovirus 1 (classical porcine parvovirus, PPV), ungulate tetraparvovirus 2 (PPV3), ungulate tetraparvovirus 3 (which includes PPV2, porcine hokovirus, porcine partetravirus and porcine PARV4), ungulate copiparvovirus 2 (which includes PPV4 and PPV5), ungulate bocaparvovirus 2 (which includes porcine bocavirus 1, 2 and 6), ungulate bocaparvovirus 3 (porcine bocavirus 5), ungulate bocaparvovirus 4 (porcine bocavirus 7) and ungulate bocaparvovirus 5 (porcine bocavirus 3, 4-1 and 4-2). PPV6, the most recently described porcine parvovirus, was first identified in China in late 2014 in aborted pig fetuses. Prevalence of PPV6 in China was found to be similar in finishing age pigs from farms with and without evidence of swine reproductive failure.

Methods: Porcine parvovirus 6 (PPV6) was detected by sequence-independent single primer amplification (SISPA) and confirmed by overlapping and real-time PCR in the serum of porcine reproductive and respiratory virus (PRRSv) positive samples.

Results: Seven nearly complete genomes of PPV6 were identified in PRRSv genotype 2 positive serum samples submitted to state veterinary diagnostic laboratories in 2014. Further testing using overlapping and real-time PCR determined PPV6 to be present in 13.2 % of the serums tested. Additionally, PPV6 was present in samples from all of the geographic locations sampled encompassing nine states in the United States and one state in Mexico. The presence of PPV6 in serum indicates that the PPV6 infection is disseminated and not localized to a specific tissue type. Alignments of the near full length genomes, NS1, and capsid genes identified one of the five PPV6 isolates from China (98.6-99.5 % identity with the North American strains) to be the North American strains nearest relative.

Conclusions: These results are the first to report the presence of PPV6 in North America and demonstrate that the virus is found in multiple geographic areas in the United States and in Mexico. The overall prevalence of PPV6 in PRRSv viremic animals is relatively low. Further, all of the PPV6 genomes found in North America are most closely related to a PPV6 strain first identified in 2014 in healthy pigs from the Tianjin province of China.

Fig. 1

Phylogenetic relationship of seven newly identified parvovirus genomes within Parvovirinae. Phylogenetic reconstruction of nucleotide sequences of the newly identified nearly full-length genomes and 31 full-length or nearly full-length genomes downloaded from GenBank that represent the eight genera within Parvovirinae. Galleria mellonella denosivirus was used as to root the tree. The tree was reconstructed using the maximum likelihood approach and GTR + G model of sequence evolution as implemented in MEGA6. Nodal support was evaluated by 1000 bootstrap pseudoreplicates. Bootstrap values <70 % are not shown. Scale bars indicate the number of mutations along branches.

Fig. 2

Phylogenetic relationship of ORF1 of seven newly identified parvoviruses within Parvovirinae. Phylogenetic reconstruction of amino acid sequences of ORF1 (non-structural protein) using the maximum likelihood approach and the LG + G model of sequence evolution with 1000 bootstrap pseudoreplicates. Bootstrap values <70 % are not shown. Scale bars indicate the number of mutations along branches.

Fig. 3

Phylogenetic relationship of ORF2 of seven newly identified parvoviruses within Parvovirinae. Phylogenetic reconstruction of amino acid sequences of ORF2 (capsid) using the maximum likelihood approach and the LG + G + F model of sequence evolution with 1000 bootstrap pseudoreplicates. Bootstrap values <70 % are not shown. The scale bars indicate the number of mutations along branches.

Fig. 4

Map detailing the conserved and variable regions of PPV6 VP1. Conserved regions (164–256, 414–787, and 1090,1189) are highlighted in black, the variable region (1–163) is highlighted in grey and the highly variable region (103–133) is highlighted with white outlined in black.