Porcine reproductive and respiratory syndrome virus-induced cell death exhibits features consistent with a nontypical form of apoptosis.

Porcine reproductive and respiratory syndrome virus (PRRSV), an arterivirus, belongs to a group of RNA viruses that are cytopathic for macrophages and establish persistent infections. Apoptosis is the presumed mechanism of cell death in monkey kidney cell lines and porcine alveolar macrophages after infection with European PRRSV isolates. However, evidence from in vivo and in vitro studies using North American strains have failed to identify apoptosis in cells supporting virus replication and suggest that apoptosis is present in only uninfected bystander cells. The purpose of this study was to evaluate the mechanism of cell death following the infection of MARC-145 cells with wild-type (P6) and a cell culture-adapted (P136) strains derived from the North American isolate SDSU-23983. At 2 days after infection with P136, cytoplasmic blebbing and nuclear condensation were absent in monolayers containing almost 90% infected cells. By day 3, these infected cells detached and showed evidence of apoptosis, including nuclear condensation and inter-nucleosomal DNA fragmentation. Apoptosis in single infected floating cells was confirmed by the co-localization of FITC-anti-digoxigenin antibody, used to detect uridine-digoxigenin-labled nuclear DNA in a terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling assay, and Texas red-labeled PRRSV antibody. A majority of infected floating cells were also positive for the uptake of trypan blue, an indicator of necrotic cell death. These results demonstrate that apoptosis does occur in PRRSV infected cells, but is a late event during PRRSV replication and rapidly culminates in a necrotic-like death.

Lymphoid tissue tropism of porcine reproductive and respiratory syndrome virus replication during persistent infection of pigs originally exposed to virus in utero.

The ability of porcine reproductive and respiratory syndrome virus (PRRSV) to establish a persistent infection is the principal contributing factor to the world-wide spread of the disease. Several studies have documented the course of viral infection in postnatally infected pigs; however, very little is known regarding sites of virus replication during persistent infection of pigs exposed to PRRSV in utero. In this study, virus replication and PRRSV-specific antibody were followed for several hundred days in a group of pigs derived from three sows infected at 90 days of gestation with PRRSV isolate VR-2332. Eighty-four percent of pigs were born viremic with a mortality of 54% within 21 days after birth. At approximately 60 days sera from pigs were negative for virus by virus isolation. Analysis of virus replication in the tissues of pigs randomly sacrificed between 63 and 132 days showed no evidence of virus in lung and other non-lymphoid organs. However, virus was easily recovered from tonsil and lymph nodes and in situ hybridization identified these tissues as sites of virus replication. Even though replication was at a low level, virus was easily transmitted to sentinel pigs. By 260 days pigs became seronegative and did not transmit virus to sentinel pigs. Sacrifice of remaining pigs after 300 days showed no evidence of virus in blood and tissues. This study shows that congenital PRRSV-infected pigs can support virus replication for an extended period during which virus replication is primarily restricted to tonsil and lymph nodes.

Porcine reproductive and respiratory syndrome virus: an update on an emerging and re-emerging viral disease of swine.

Recognized in the late 1980s in North America and Europe the syndrome that caused reproductive and respiratory problems in swine was initially called "mystery swine disease" and is now termed "porcine reproductive and respiratory syndrome (PRRS)". In the early 1990 s an arterivirus, referred to as PRRS virus (PRRSV), was determined to be the etiologic agent of this disease. Since then research has progressed substantially. Most recently "porcine high fever disease" was reported in China starting in 2006 with PRRSV being a critical virus associated with high morbidity and mortality (20%) associated with this syndrome which in 2010 is still causing severe pathology in pigs in China, with spread to Vietnam and Cambodia. This volume contains a series of reviews that highlight the virus, its pathogenesis, epidemiology, immunology, vaccinology and host genetic control. This paper provides a brief historical review of PRRS and the associated PRRSV. It presents areas of research gaps that inhibit current progress towards PRRS elimination through production of effective vaccines and current plans for PRRS elimination or eradication programs. It is hoped that this discussion will stimulate further collaboration between researchers and swine veterinarians throughout the world to provide answers that enhance our understanding of PRRS and PRRSV in an effort to eliminate this economically important disease.

Peptide domains involved in the localization of the porcine reproductive and respiratory syndrome virus nucleocapsid protein to the nucleolus.

The nucleocapsid (N) protein of porcine reproductive and respiratory syndrome virus (PRRSV) is the principal component of the viral nucleocapsid and localizes to the nucleolus. Peptide sequence analysis of the N protein of several North American isolates identified two potential nuclear localization signal (NLS) sequences located at amino acids 10-13 and 41-42, which were labeled NLS-1 and NLS-2, respectively. Peptides containing NLS-1 or NLS-2 were sufficient to accumulate enhanced green fluorescent protein (EGFP) in the nucleus. The inactivation of NLS-1 by site-directed mutagenesis or the deletion of the first 14 amino acids did not affect N protein localization to the nucleolus. The substitution of key lysine residues with uncharged amino acids in NLS-2 blocked nuclear/nucleolar localization. Site-directed mutagenesis within NLS-2 identified the sequence, KKNKK, as forming the core localization domain within NLS-2. Using an in vitro pull-down assay, the N protein was able to bind importin-alpha, importin-beta nuclear transport proteins. The localization pattern of N-EGFP fusion peptides represented by a series of deletions from the C- and N-terminal ends of the N protein identified a region covering amino acids 41-72, which contained a nucleolar localization signal (NoLS) sequence. The 41-72 N peptide when fused to EGFP mimicked the nucleolar-cytoplasmic distribution of native N. These results identify a single NLS involved in the transport of N from the cytoplasm and into nucleus. An additional peptide sequence, overlapping NLS-2, is involved in the further targeting of N to the nucleolus.

Characterization of emerging European-like porcine reproductive and respiratory syndrome virus isolates in the United States.

European-like field isolates of porcine reproductive and respiratory syndrome virus (PRRSV) have recently emerged in North America. The full-length genomic sequence of an index isolate characterized in 1999, strain EuroPRRSV, served as the reference strain for further studies of the evolution and epidemiology of European-like isolates (type 1) in the United States. Strain EuroPRRSV shared 90.1 to 100% amino acid identity with the prototype European strain, Lelystad, within the structural and nonstructural open reading frames (ORFs) and 95.3% overall nucleotide identity. The 5' untranslated region and two nonstructural regions within ORF 1 were closely examined due to significant divergence from strain Lelystad. A 51-bp deletion in a region within ORF 1a, coding for nonstructural protein 2 (NSP2), was observed. Sequence analysis of the structural ORFs 2 to 7 of additional European-like isolates indicated that these isolates share 93% nucleotide identity with one another and 95 to 96% identity with the Lelystad strain but only 70% identity with the North American reference strain VR-2332. Phylogenetic analysis with published PRRSV ORF 3, 5, and 7 nucleotide sequences indicated that these newly emerging isolates form a clade with the Lelystad and United Kingdom PRRSV isolates. Detailed analysis of four of these isolates with a panel of 60 monoclonal antibodies directed against the structural proteins confirmed a recognition pattern that was more consistent with strain Lelystad than with other North American isolates.