Differential replication of two porcine parvovirus strains in bovine cell lines ensues from initial DNA processing and NS1 expression.

Porcine parvovirus (PPV) is a small DNA virus with restricted coding capacity. The 5 kb genome expresses three major non-structural proteins (NS1, NS2 and SAT), and two structural proteins (VP1 and VP2). These few viral proteins are pleiotropic and interact with cellular components throughout viral replication. In this regard, very few cell lines have been shown to replicate the virus efficiently. Cell lines were established from a primary culture of bovine cells that allowed allotropic variants of PPV to be distinguished. Three cell lines were differentially sensitive to infection by two prototype PPV strains, NADL-2 and Kresse. In the first cell line (D10), infection was restricted early in the infectious cycle and was not productive. Infection of the second cell line (G11) was 1000 times less efficient with the NADL-2 strain compared with porcine cells, while production of infectious virus of the Kresse strain was barely detectable. Restriction points in these cells were the initial generation of DNA replication intermediates and NS1 production. Infection with chimeras between NADL-2 and Kresse showed that residues outside the previously described allotropic determinant were also partially responsible for the restriction to Kresse replication in G11 cells. F4 cells were permissive to both strains, although genome replication and infectious virus production were lower than in the porcine cells used for comparison. These results highlight the dependent nature of parvovirus tropism on host factors and suggest that cells from a non-host origin can fully support a productive infection by both strains.

Expression strategy of Aedes albopictus densovirus.

The transcription map of the Aedes albopictus densovirus (AalDNV) brevidensovirus was identified by Northern blotting, rapid amplification of cDNA ends (RACE) analysis, and RNase protection assays. AalDNV produced mRNAs of 3,359 (NS1), 3,345 (NS2), and 1,246 (VP) nucleotides. The two overlapping P7/7.4 NS promoters employed closely located alternate transcription initiation sites, positioned at either side of the NS1 initiation codon. All NS mRNAs coterminated with VP mRNA. All promoters, explored using luciferase assays, were functional in insect and human cell lines.

Organization of the ambisense genome of the Helicoverpa armigera Densovirus.

A natural densovirus (DNV) of a serious phytophagous pest, Helicoverpa armigera, was isolated. The genome of HaDNV contained 6,039 nucleotides (nt) and included inverted terminal repeats (ITRs) of 545 nt with terminal Y-shaped hairpins of 126 nt. Its DNA sequence and ambisense organization with four typical open reading frames (ORFs) demonstrated that it belonged to the genus Densovirus in the subfamily Densovirinae of the family Parvoviridae.

The Acheta domesticus densovirus, isolated from the European house cricket, has evolved an expression strategy unique among parvoviruses.

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.

Structure of Penaeus stylirostris densovirus, a shrimp pathogen.

Penaeus stylirostris densovirus (PstDNV), a pathogen of penaeid shrimp, causes significant damage to farmed and wild shrimp populations. In contrast to other parvoviruses, PstDNV probably has only one type of capsid protein that lacks the phospholipase A2 activity that has been implicated as a requirement during parvoviral host cell infection. The structure of recombinant virus-like particles, composed of 60 copies of the 37.5-kDa coat protein, the smallest parvoviral capsid protein reported thus far, was determined to 2.5-Å resolution by X-ray crystallography. The structure represents the first near-atomic resolution structure within the genus Brevidensovirus. The capsid protein has a ß-barrel "jelly roll" motif similar to that found in many icosahedral viruses, including other parvoviruses. The N-terminal portion of the PstDNV coat protein adopts a "domain-swapped" conformation relative to its twofold-related neighbor similar to the insect parvovirus Galleria mellonella densovirus (GmDNV) but in stark contrast to vertebrate parvoviruses. However, most of the surface loops have little structural resemblance to any of the known parvoviral capsid proteins.

Parvovirus 4-like virus in blood products.

Porcine plasma and factor VIII preparations were screened for parvovirus 4 (PARV)-like viruses. Although the prevalence of PARV4-like viruses in plasma samples was relatively low, viruses appeared to be concentrated during manufacture of factor VIII. PARV4-like viruses from human and porcine origins coevolved likewise with their hosts.

Comparative Genomic Analysis of Acheta domesticus Densovirus Isolates from Different Outbreaks in Europe, North America, and Japan.

The first densovirus from a cricket, Acheta domesticus densovirus (AdDNV) (Parvoviridae), was isolated in Europe in 1977 and has been studied previously. We compared seven additional AdDNV genomes isolated from 4 other European outbreaks, 2 major North American outbreaks, and a Japanese outbreak. Phylogenetic analysis suggested that the 2009 Japanese and North American outbreaks were not related.

SAT: a late NS protein of porcine parvovirus.

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.