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

The family Parvoviridae consists of many small, non-enveloped, single-stranded DNA viruses that infect a wide variety of species [1]. Parvoviridae is further divided into two subfamilies: the Parvovirinae that infect vertebrates and the Densovirinae that infect invertebrates. Parvovirinae is comprised of eight genera; Copiparvovirus, Tetraparvovirus, Erythroparvovirus, Bocaparvovirus, Dependoparvovirus, Amdoparvovirus, Aveparvovirus and Protoparvovirus [2]. Parvoviruses have a linear genome of four to six kilobases (kb) that contains two major open reading frames (ORFs) encoding the non-structural protein(s) (NSP) and one to four capsid proteins. The protein-coding sequence is flanked by terminal palindromes that fold into duplex hairpin structures that are necessary for DNA replication [1]. The genomic organization of PPV6 is similar to that of other parvoviruses. The nearly full length PPV6 genome consists of negative strand DNA of approximately 6100 nucleotides (nt). There are two putative ORFs separated by eight nt and flanked by 5′ and 3′ UTRs (333–364 nt and 219, respectively). ORF1 encodes a putative non-structural protein (NS1) of 662 amino acids that functions as the viral replicase. ORF2 encodes the putative capsid protein (VP1) that is predicted to be 1189 amino acids in length. Comparison of the predicted lengths of the capsid proteins of porcine parvoviruses shows the capsid protein of PPV6 to be larger than the other swine-associated parvoviruses (data not shown).

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). Prevalence studies conducted in Europe and the United States have shown that ungulate tetraparvovirus 2, ungulate tetraparvovirus 3 and ungulate copiparvovirus 2 occur in both areas with varying percentages of infected animals depending upon animal age, sample type, clinical status, presence of additional viral agents and time frame. Prevalence of parvoviruses in European pigs ranged from 6.4 % for ungulate tetraparvovirus 3, 9.7 % for ungulate tetraparvovirus 2, 6.4 % for ungulate copiparvovirus 2, and 13.44 % for ungulate bocaparvovirus 2 in samples collected between 2006 and 2011 [3]. In the United States, prevalence of parvoviruses was 14.7 % for ungulate tetraparvovirus 3, 13.6 % for ungulate tetraparvovirus 2, 4.1 and 6.6 % for two virus variants in ungulate copiparvovirus 2 (PPV4 and PPV5), 17.2 – 43.1 % for ungulate bocaparvovirus 2, and 24.2 – 31.9 % for ungulate bocavirus 5 [4‐7]. In a retrospective study of dated US samples, ungulate tetraparvovirus 3, ungulate tetraparvovirus 2 and ungulate copiparvovirus 2 positive samples were found dating back to 1998. However, the viral variant of ungulate copiparvovirus 2, PPV5, was only found in samples collected after 2006 [7]. A Chinese retrospective study of ungulate copiparvovirus 2 prevalence in samples collected between 2006 and 2010 found that ungulate copiparvovirus 2 did not circulate in China prior to 2009. After 2009, low levels of ungulate copiparvovirus 2 (2.09 %) were found in samples from clinically healthy and ill animals (0.76 and 2.09 % respectively) [8]. In 2009, the first evidence of porcine bocavirus was found in China during testing of samples from pigs on farms with post-weaning multi-systemic wasting syndrome. 38.7 % of diseased pigs and 7.3 % of pigs without clinical disease were found to have the virus. However, in 2011 a study of asymptomatic pigs from five provinces across China found a prevalence rate of 63.2 and 64.4 % for two viral variants of ungulate bocaparvovirus 2. The same year, ungulate bocaparvovirus 5 was isolated in Northern Ireland. Antibodies to this virus were found in approximately 9 % of 369 pig serum samples tested. Additionally, studies by Opriessnig et al. [5], Blomström et al. [9], and Zhai et al. [10] found that animals showing clinical symptoms of disease (i.e. PCV-2, PRRS) were more likely to be positive for ungulate protoparvovirus 1, ungulate tetraparvovirus 2,ungulate tetraparvovirus 3, ungulate copiparvovirus 2, or ungulate bocaparvovirus 2 as well.

PPV6 is the most recent parvovirus of swine to be described [11]. PPV6 was first identified by Chinese researchers in aborted pig fetuses that had tested negative for multiple viruses commonly associated with swine reproductive failure (i.e. pseudorabies, PRRSv, PCV2) [11]. Using a PCR approach, pigs of different age groups from farms in four different provinces (Beijing (BJ), Jiangsu (JS), Tianjin (TJ), and Sichuan (SC)) with and without evidence of reproductive failure were screened for the presence of PPV6. PPV6 was found in 50 to 75 % of aborted pig fetuses and piglets (respectively), 15.6 % of finishing pigs, and 3.8 % of sows. Prevalence of PPV6 on farms with and without evidence for reproductive failure was similar at 16.7 % and 13.6–21.7 % [11].

Of the eight described porcine parvoviruses, ungulate protoparvovirus 1 and ungulate bocaparvovirus 5 have been isolated in cell culture. However, only ungulate protoparvovirus 1 has been demonstrated to be a causal agent of porcine reproductive failure [12‐14]. In contrast, the newly described porcine parvoviruses have not been isolated in cell culture and their pathogenicity has not been evaluated. However, PPV6 was originally identified as the only pathogen in swine aborted fetuses in China [11]. Many of the newly described parvoviruses are found as co-infections with other well-characterized viruses such as PRRSv (the present study) and PCV2 [5] in pigs showing clinical signs of illness such as post-weaning multisystemic wasting syndrome, PCV2 associated disease or reproductive failure such as fetal death or mummification. The association of ungulate tetraparvovirus 2, ungulate tetraparovirus 3, ungulate copiparvovirus 2 and the recently described PPV6 with clinical disease remains ambiguous due to the detection of porcine parvovirus DNA in both healthy and clinically ill animals. Until these newly described parvoviruses can be cultured or derived from infectious clones the connection between these porcine parvoviruses and disease will remain undetermined.

Here we report the first identification of PPV6 from swine in the United States and Mexico. Seven nearly full-length genomes with high similarity to Chinese PPV6 were found during next generation sequencing of PRRSv positive serum samples. Prevalence of PPV6 within the PRRSv positive dataset was then determined by screening all samples by quantitative PCR (qPCR).

Serum samples positive for PRRSv by qPCR submitted to the Kansas State Veterinary Diagnostic Laboratory, the Iowa State University Veterinary Diagnostic Laboratory, and the South Dakota State University Animal Disease Research and Diagnostic Laboratory were further analyzed by viral metagenomic sequencing. The Illumina (San Diego, CA) MiSeq data yielded seven samples which produced multiple contigs that showed significant expectation scores (E < 1×10⁻⁵) by BLASTn search to PPV6 sequences described in China in late 2014 [11]. The new genomes have been deposited in GenBank (KR709262-KR709268).

These North American-derived (NA) PPV6 genomes contained two non-overlapping open reading frames (ORF) whose amino acid sequences showed homology to the Parvo non-structural 1 (NS1) superfamily and Parvo capsid by BLASTp search. This search also identified homology between ORF1 of PPV6 and the replicase proteins of other members of Parvovirinae and indicated the presence of a AAA+ viral helicase domain in PPV6. The presence of the phospholipase A2 domain (PLA₂) (amino acids HDXXY) and the calcium binding loop (amino acids YXGXG), features found in many other parvoviruses, was identified in ORF2 by a motif search in CLC Genomics Workbench 7.0 (Qiagen, Valencia, CA). The Chinese PPV6 PLA₂ domain of amino acids HDIRY was conserved in the NA-derived genomes. This domain is variable in ungulate copiparvovirus 2 viral variants as it is present in PPV5 but absent in PPV4. The calcium loop motif of YXGXR was additionally conserved in the Chinese PPV6 and the NA-derived genomes. This sequence differed from the other shared motifs of YXGXF found in ungulate bocaparvovirus 1, ungulate bocaparvovirus 5, adeno-associated dependoparvovirus A and avian adeno-associated dependoparvovirus 1 and YXGXG of the remaining parvoviruses.

Phylogenetic analysis of seven newly described parvovirus genomes recovered by viral metagenomic sequencing of PRRSv positive porcine serum samples and 31 parvovirus genomes downloaded from GenBank show that these new genomes cluster with high support with the PPV6 genomes described in late 2014 from China and should be considered new strains of PPV6 as indicated by Cotmore et al. [2] within the genus Copiparvovirus as discussed by Ni et al. [11].

Although, the samples in this study have no age data associated with them, the prevalence of PPV6 in North America is similar to the prevalence found in finishing pigs in China [11]. PPV6 viremia, as determined by the presence of viral DNA in serum, is similar to the prevalence of PPV1 (14.7 %) and PPV3 (19.2 %) in sera and is more prevalent in North America than PPV4 (5.9 %) and PPV5 (7 %) in sera and in tissues (9.3 %). However, it is drastically lower than that of ungulate tetraparvovirus 3 in sera (72 %) and lung tissue (20.7 %, 40.1 % when PCV2 is also present) [4‐7]. While the prevalence of PPV6/PRRSv co-infection is relatively low (13.2 %), the geographic distribution of PPV6 suggests that it is not localized to specific regions of the U.S. as PPV6 was detected in PRRSv positive samples from every state (Arizona, Illinois, Indiana, Iowa, Kansas, Minnesota, Nebraska, North Carolina, South Dakota) and the one region in Mexico (Veracruz) tested.

Despite the noncontiguous geographic dispersion of PPV6 in the United States and Mexico the strains found in North America are very closely related to each other with homology between strains, from different geographical locations, ranging from 98.3 to 100 % at the nucleotide level and 99.2 – 100 % at the amino acid level of ORFs1 and 2. These strains are more closely related to each other than the Chinese strains are to each other at both the nucleotide sequence and amino acid sequence of the predicted proteins, 97.1 – 99.6 % and 97.3 – 99.6 % respectively [11]. The North American strains have the greatest homology with the strain first identified from Tianjin, China having between 98.6 and 99.5 % nucleotide identity with this strain as compared to 96.9 – 97.0 % nucleotide identity to the other Chinese strains.

Among all of the strains identified both in North America and China the sequence diversity is highest in ORF2, the capsid protein. This conforms to the predicted behavior of the virus in response to the host immune system, in that the capsid protein would be under the greatest direct pressure from the host immune system. Since ORF2 mutates at a faster rate than ORF1, mapping the rate of change in ORF2 might provide a method for identifying the timeframe in which PPV6 was introduced into North America.

These results are the first to report the presence of PPV6 in North America and indicate that the virus is found in multiple states in the United States and found in approximately 13 % of pigs with PRRSv viremia. Further, all of the PPV6 genomes found in North America appear to share a single source virus very similar to the strain first identified in China from healthy pigs in Tianjin in 2014. Additional sampling of PPV6 genomes with good spatial and temporal data in North America and China may elucidate ways in which viruses such as these are transported across the globe and provide insights into improved biosecurity.