Plasma samples from healthy pigs were collected in Great Britain in 2001. Initially, these samples were tested for PARV4-like viruses by using previously described degenerate PCR primers (10). DNA was extracted from samples by using the High Pure DNA Isolation Kit (Roche Applied Science, Roche Diagnostics Canada; Laval, Quebec, Canada). Only 3 of the 98 plasma samples contained detectable amounts of PARV4-like viruses. To further study these porcine viruses, we obtained nearly full-length genomes from overlapping PCR fragments. Primers designed for these PCRs were PrS1: 5′-CCACACCTACCTCGCCTATAAGAATCAG-3′; PrAS1: 5′-CTCCACTTGTTCAGCACGGGATCC-3′; PrS2: 5′-CCACGAGCTGGAAGTCTTTA-3′; PrAS2: 5′-GGAGTCCGCATACCCATAACAGGCTG-3′; PrS3: 5′-GTGTACCGCAGTGGGAGCCATG-3′; and PrAS3: 5′-TTCTGGGCAACCCACTGATCAGAAGG-3′. The nearly full-length clones were sequenced by primer-walking. Genomic analysis confirmed that these viruses were related to the PARV4 viruses and were close relatives of the recently identified porcine hokoviruses (PHoVs) (10).

We also confirmed the moderate frequency of PARV4-like viremia in the previously tested pig plasma samples with a more sensitive PCR assay by using specific primers PrS4 (5′-AGTTACGGGGGACCGCTACAGTG-3′) and PrAS3. In contrast, examination of 11 commercial clotting FVIII preparations showed that all of these independent lots contained substantial amounts of PARV4-like parvovirus, whereas the level of porcine parvovirus DNA was generally lower in the corresponding samples (Figure 1). Similar to the plasma samples, long overlapping PCR fragments were amplified from the FVIII preparations to obtain nearly full-length sequences. Their analysis provided information about the evolution of PARV4-like viruses, during nearly a decade, in pigs. Sequence data were registered by GenBank (accession nos. Cl2001A: FJ982246; Cl2001B: FJ982247; Cl2001C: FJ982248; F8–1994A: FJ982249; F8–1994B: FJ982250; F8–1996A: FJ982251; F8–1996B: FJ982252; F8–1999: FJ982253; F8–2000A: FJ982254; and F8–2000B: FJ982255). Phylogenetic and molecular evolutionary analyses were conducted by using MEGA version 4 (12).

The genomes of these newly isolated PARV4-like viruses were similar to the PHoVs previously identified in Hong Kong Special Administrative Region, People’s Republic of China. Although, these new isolates showed some diversity (98%–99% identity), they differed somewhat more from the PHoVs (97%–98% identity). The viral protein (VP)-ORF was highly conserved (99%), whereas the NS-ORF showed more diversity (97%–98%). Genomic and protein-coding sequences were also compared with other PARV4-like viruses (Tables 1, 2). Phylogenic analysis using neighbor-joining and maximum parsimony methods demonstrated that PHoVs grouped together, whereas PARV4-like sequences from FVIII prepared at different times were less uniform (Figure 2). Older FVIII PARV4 contaminants (especially from 1994) were related more closely to the bovine hokoviruses (BHoVs) and to PARV4-g2. Finally, analysis of the newly identified virus genomes showed an alternative coding sequence inside of the VP gene with a recognizable relationship to small alternatively translated (SAT) proteins (13). In the porcine PARV4-like viruses, the start codon for the SAT protein was 3 nt downstream relative to the position of SAT-ATG in the human and bovine PARV4 viruses. Although the SAT protein was 67 aa in all the characterized human PARV4 viruses, porcine and bovine PARV-like viruses contained SAT proteins with 84 aa. The amino acid sequences of the SAT proteins were highly conserved in each PARV4 virus group; however, they differed greatly between PARV4 viruses belonging to different host species (Table 2).

Improved virus detection methods have facilitated the discovery of new viruses and have provided insight into the existence of a wide variety of potentially pathogenic strains in biopharmaceutical products. Plasma samples, collected from individual pigs in 2001–2002, and FVIII samples, prepared during 1994–2001, were tested for PARV4-like viruses.

Sequence analysis showed that PARV4-like viruses may have undergone some degree of selective pressure during this time because the genomes sequenced showed a greater variability than the porcine parvovirus NS sequences isolated from the same samples (J. Szelei and P. Tijssen, unpub. data). In the current study, comparison of the genomic and NS protein coding sequences indicated that viruses in the older samples were more closely related to BHoV and PARV4-g2 (Figure 2). Fewer changes were observed in the VP coding sequence (Table 2). Because VPs are responsible for the entry of parvoviruses, they usually adapt to host-specific receptor(s). The presence of PARV4-g2–like isolates in older samples and the omnipresence of PARV4-like viruses in more recent samples suggested that the porcine PARV4-like virus and human PARV4 may have similarly evolved (8). These new parvovirus isolates from Great Britain would belong to a different cluster of porcine PARV4-like viruses than the hokoviruses from Hong Kong Special Administrative Region.

Although older isolates shared more identity with BHoV and PARV4-g2, the substantial differences in the DNA sequences of PARV4-like viruses from different species (human, bovine, pig) suggested that they would have diverged a long time ago. This hypothesis was also supported by the sequence stabilization of the SAT proteins, which may play important host-specific roles in the viral exit (13). Nevertheless, the existence of a wide variety of different PARV4 strains, most of which result in chronic infections, could provide a basis for an evolutionary jump and recombination and should raise major concerns about the dangers of parenteral transmission.