Porcine parvovirus (PPV) is among the most important infectious agents causing infertility in pigs. Until recently, it was thought that the virus had low genetic variance, and that prevention of its harmful effect on pig fertility could be well-controlled by vaccination. However, at the beginning of the third millennium, field observations raised concerns about the effectiveness of the available vaccines against newly emerging strains. Subsequent investigations radically changed our view on the evolution and immunology of PPV, revealing that the virus is much more diverse than it was earlier anticipated, and that some of the “new” highly virulent isolates cannot be neutralized effectively by antisera raised against “old” PPV vaccine strains. These findings revitalized PPV research that led to significant advancements in the understanding of early and late viral processes during PPV infection. Our review summarizes the recent results of PPV research and aims to give a comprehensive update on the present understanding of PPV biology.

Porcine parvovirus (PPV) is a major cause of reproductive failure in swine. The mechanisms implicated in the first steps of infection that lead to the delivery of the PPV genome to the nucleus are poorly understood. In the present work, a panel of chemical inhibitors was used to dissect the cellular mechanisms involved in establishing a PPV infection. The results demonstrated that following binding to sialic acids on cell surface glycoproteins, the virus used both clathrin-mediated endocytosis and macropinocytosis pathways to gain access into cells. Virus obtained from infected cells was present either as isolated particles or as aggregates, and these two forms could be separated by low-speed centrifugation. Isolated and purified particles strongly preferred entry by clathrin-mediated endocytosis, whereas aggregates clearly favored macropinocytosis. Subsequent endosomal acidification and traffic to the late endosomes were also shown to be essential for infection. The microtubule network was found to be important during the first 10 h of infection, whereas an intact actin network was required for almost the whole viral cycle. Proteasome processing was found to be essential, and capsid proteins were ubiquitinated relatively early during infection. Taken together, these results provided new insights into the first steps of PPV infection, including the use of alternative entry pathways, unique among members of this viral family.

The Acheta domesticus densovirus (AdDNV), isolated from crickets, has been endemic in Europe for at least 35 years. Severe epizootics have also been observed in American commercial rearings since 2009 and 2010. The AdDNV genome was cloned and sequenced for this study. The transcription map showed that splicing occurred in both the nonstructural (NS) and capsid protein (VP) multicistronic RNAs. The splicing pattern of NS mRNA predicted 3 nonstructural proteins (NS1 [576 codons], NS2 [286 codons], and NS3 [213 codons]). The VP gene cassette contained two VP open reading frames (ORFs), of 597 (ORF-A) and 268 (ORF-B) codons. The VP2 sequence was shown by N-terminal Edman degradation and mass spectrometry to correspond with ORF-A. Mass spectrometry, sequencing, and Western blotting of baculovirus-expressed VPs versus native structural proteins demonstrated that the VP1 structural protein was generated by joining ORF-A and -B via splicing (splice II), eliminating the N terminus of VP2. This splice resulted in a nested set of VP1 (816 codons), VP3 (467 codons), and VP4 (429 codons) structural proteins. In contrast, the two splices within ORF-B (Ia and Ib) removed the donor site of intron II and resulted in VP2, VP3, and VP4 expression. ORF-B may also code for several nonstructural proteins, of 268, 233, and 158 codons. The small ORF-B contains the coding sequence for a phospholipase A2 motif found in VP1, which was shown previously to be critical for cellular uptake of the virus. These splicing features are unique among parvoviruses and define a new genus of ambisense densoviruses.

The genomes of all members of the Parvovirus genus were found to contain a small open reading frame (ORF), designated SAT, with a start codon four or seven nucleotides downstream of the VP2 initiation codon. Green fluorescent protein or FLAG fusion constructs of SAT demonstrated that these ORFs were expressed. Although the SAT proteins of the different parvoviruses are not particularly conserved, they were all predicted to contain a membrane-spanning helix, and mutations in this hydrophobic stretch affected the localization of the SAT protein. SAT colocalized with calreticulin in the membranes of the endoplasmic reticulum and the nucleus. A knockout mutant (SAT ⁻ ), with an unmodified VP sequence, showed a “slow-spreading” phenotype. These knockout mutants could be complemented with VP2 ⁻ SAT ⁺ mutant. The SAT protein is a late nonstructural (NS) protein, in contrast to previously identified NS proteins, since it is expressed from the same mRNA as VP2.

The genome of a novel virus of the house cricket consists of a 2,517-nucleotide (nt) circular single-stranded DNA (ssDNA) molecule with 4 open reading frames (ORFs). One ORF had a low identity to circovirus nucleotide sequences (NS). The unique properties of this volvovirus suggested that it belongs to a new virus family or genus.

Nuclear targeting of capsid proteins (VPs) is important for genome delivery and precedes assembly in the replication cycle of porcine parvovirus (PPV). Clusters of basic amino acids, corresponding to potential nuclear localization signals (NLS), were found only in the unique region of VP1 (VP1up, for VP1 unique part). Of the five identified basic regions (BR), three were important for nuclear localization of VP1up: BR1 was a classic Pat7 NLS, and the combination of BR4 and BR5 was a classic bipartite NLS. These NLS were essential for viral replication. VP2, the major capsid protein, lacked these NLS and contained no region with more than two basic amino acids in proximity. However, three regions of basic clusters were identified in the folded protein, assembled into a trimeric structure. Mutagenesis experiments showed that only one of these three regions was involved in VP2 transport to the nucleus. This structural NLS, termed the nuclear localization motif (NLM), is located inside the assembled capsid and thus can be used to transport trimers to the nucleus in late steps of infection but not for virions in initial infection steps. The two NLS of VP1up are located in the N-terminal part of the protein, externalized from the capsid during endosomal transit, exposing them for nuclear targeting during early steps of infection. Globally, the determinants of nuclear transport of structural proteins of PPV were different from those of closely related parvoviruses.IMPORTANCEMost DNA viruses use the nucleus for their replication cycle. Thus, structural proteins need to be targeted to this cellular compartment at two distinct steps of the infection: in early steps to deliver viral genomes to the nucleus and in late steps to assemble new viruses. Nuclear targeting of proteins depends on the recognition of a stretch of basic amino acids by cellular transport proteins. This study reports the identification of two classic nuclear localization signals in the minor capsid protein (VP1) of porcine parvovirus. The major protein (VP2) nuclear localization was shown to depend on a complex structural motif. This motif can be used as a strategy by the virus to avoid transport of incorrectly folded proteins and to selectively import assembled trimers into the nucleus. Structural nuclear localization motifs can also be important for nuclear proteins without a classic basic amino acid stretch, including multimeric cellular proteins.