Porcine reproductive and respiratory syndrome virus infection is associated with an increased number of Sn-positive and CD8-positive cells in the maternal-fetal interface.

It is already known that porcine reproductive and respiratory syndrome virus (PRRSV) infection in lungs changes a local cell pattern and cytokine profile. However, there is no information about cellular and immunological events upon PRRSV infection in the maternal-fetal interface yet. The altered number and/or function of macrophages and NK cells in the maternal-fetal interface during infection may have a functional importance for virus replication. In addition, local cellular and immunological disbalance may also disrupt fragile homeostasis and contribute to the PRRSV-related reproductive disorders. Sialoadhesin (Sn)-positive macrophages are target cells for PRRSV and Sn overexpression has been observed upon chronic inflammatory and infectious diseases. It is also known that mouse Sn-positive macrophages in lymph nodes are able to closely interact with and activate NK cells in response to viral particles. Therefore, the main purpose of the present study was to examine if PRRSV infection is associated with altered Sn expression on endometrial and placental macrophages. In addition, CD8-positive cells (porcine endometrial NK cells were previously described as CD8(+)CD3(-) cells) were localized and quantified in the PRRSV-positive and control tissues. Tissue samples were obtained from three PRRSV-inoculated and three non-inoculated control sows at 100 days of gestation. Real-time RT-PCR showed a clear upregulation of Sn mRNA expression in the PRRSV-positive endometrium/placenta (p<0.05). Sn-, CD163- and CD14-specific immunofluorescence stainings revealed that PRRSV-inoculated sows had a significantly higher number of Sn-positive macrophages in the endometrium and placenta due to de novo Sn expression on local CD163-positive macrophages. Along with the increased number of Sn-positive macrophages an increased number of CD8-positive cells, which were mostly CD3-negative, was observed in the PRRSV-positive endometrium. The effects of the observed cellular changes on virus replication and potential contribution to placental damage and reproductive disorders are discussed.

Impact of a novel inactivated PRRS virus vaccine on virus replication and virus-induced pathology in fetal implantation sites and fetuses upon challenge.

Preventing congenital infection is important for the control of porcine reproductive and respiratory syndrome (PRRS). Recently, in our laboratory, an inactivated porcine reproductive and respiratory syndrome virus (PRRSV) vaccine has been developed. Promising results in young pigs encouraged us to test the vaccine potency to prevent congenital infection. In the present study, the performance of this experimental inactivated vaccine was investigated in pregnant gilts. An advanced protocol was used to test the PRRSV vaccine efficacy. This protocol is based on recent insights in the pathogenesis of congenital PRRSV infections. Three gilts were vaccinated with an experimental PRRSV 07V63 inactivated vaccine at 27, 55, and 83 days of gestation. Three unvaccinated gilts were included as controls. At 90 days of gestation, all animals were intranasally inoculated with 10(5) tissue culture infectious dose 50 (TCID(50)) of PRRSV 07V63. Twenty days postchallenge animals were euthanized and sampled. The vaccinated gilts quickly developed virus neutralizing (VN) antibodies starting from 3 to 7 days postchallenge (1.0 to 5.0 log2). In contrast, the unvaccinated gilts remained negative for VN antibodies after challenge. The vaccinated gilts had shorter viremia than the control gilts. Gross pathology (mummification) was observed in 8% of the fetuses from vaccinated gilts and in 15% of the fetuses from unvaccinated gilts. The number of fetuses with severe microscopic lesions in the fetal implantation sites (a focal detachment of the trophoblast from the uterine epithelium; a focal, multifocal, or full degeneration of the fetal placenta) was lower in the vaccinated (19%) versus unvaccinated (45%) gilts (P < 0.05). The number of PRRS-positive cells in the fetal placentae was higher in unvaccinated versus vaccinated gilts (P < 0.05). In contrast, the number of PRRS-positive cells in the myometrium/endometrium was higher in vaccinated versus unvaccinated gilts (P < 0.05). Fifty-seven percent of the fetuses from the vaccinated gilts and 75% of the fetuses from the unvaccinated gilts were PRRSV-positive. In conclusion, implementation of the novel experimental inactivated PRRSV vaccine primed the VN antibody response and slightly reduced the duration of viremia in gilts. It also reduced the number of virus-positive fetuses and improved the fetal survival, but was not able to fully prevent congenital PRRSV infection. The reduction of fetal infection and pathology is most probably attributable to the vaccine-mediated decrease of PRRSV transfer from the endometrium to the fetal placenta.

Micro-dissecting the pathogenesis and immune response of PRRSV infection paves the way for more efficient PRRSV vaccines.

Porcine reproductive and respiratory syndrome virus (PRRSV) is the most important infectious pathogen in pigs worldwide nowadays. Due to its genetic drift and increasing power to escape from immunity, PRRSV becomes more and more difficult to control. Based on a better knowledge of PRRSV, its interaction with the host cell, the macrophage, its pathogenesis and the immunity against this virus, new vaccines can now be constructed. This research-based development of new generation vaccines will allow swine industry to face the devastating consequences of PRRSV infections in the future. The present review summarizes the present knowledge on the pathogenesis, the immune response and the research-based vaccine development.

Complete genome characterization of a East European Type 1 subtype 3 porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome (PRRS) is a swine disease of major economic importance that causes reproductive and respiratory problems in pigs. PRRSV strains are divided into European (Type 1) and North-American (Type 2) genotypes. Within the European PRRSV genotype, three subtypes have been delineated. Full genome sequences for North American and European subtype 1 strains have been described. Here, the first complete genomic characterization of a European subtype 3 strain (Lena) is described. Amplification of Orf1a and Orf1b fragments was achieved using a set of degenerate oligonucleotides. Using RT-PCR with Lena-specific primers, the full length sequence (15001 nt) was obtained. Alignment of Lena with European subtype 1 reference strain Lelystad showed variation over the entire length (84% identity/89% similarity at amino acid level) with the most variation in Orf1a (Nsp2/NSP2) with a deletion of 29 amino acids. Phylogenetic relationships using different Orfs supported Lena's genetic distinction from European subtype 1 strains. The availability of the European subtype 3 PRRSV full genome may be important for the understanding of PRRSV evolution and the more pronounced pathogenic nature of Lena.

Pathologic and virologic findings in mid-gestational porcine foetuses after experimental inoculation with PCV2a or PCV2b.

Two major genotypes of porcine circovirus type 2 (PCV2) have been described: PCV2a and PCV2b. Previous studies mainly used PCV2a to experimentally reproduce reproductive failure in sows. This study aims to determine the clinical and virological outcome of surgical inoculation of 55-day-old immuno-incompetent porcine foetuses with PCV2a or PCV2b. Twelve foetuses were inoculated with PCV2: three with the post-weaning multisystemic wasting syndrome (PMWS)-associated PCV2a strain Stoon-1010, three with the reproductive failure-associated PCV2a strain 1121, three with the PMWS-associated PCV2b strain 48285 and three with the porcine dermatitis and nephropathy syndrome-associated PCV2b strain 1147. Four foetuses were mock-inoculated with cell culture medium. At 21 days post-inoculation eleven out of twelve PCV2-inoculated foetuses were oedematous and had distended abdomens, whereas one had a normal external appearance. All PCV2-inoculated foetuses had haemorrhages and congestion in internal organs and an enlarged liver. High PCV2 titres (>10(4.5)TCID(50)/g tissue) were found in all PCV2-inoculated foetuses, especially in the heart, spleen and liver. High numbers of PCV2-infected cells (>1000 infected cells/10mm(2) tissue) were observed in the hearts. PCR and DNA sequencing of the capsid gene recovered pure PCV2a and pure PCV2b sequences from PCV2a- and PCV2b-inoculated foetuses, respectively. All mock-inoculated and the remaining foetuses were normal in appearance and were PCV2 negative in virus titrations and indirect immunofluorescence stainings. The present study shows that PCV2a and PCV2b induce similar gross pathological lesions and replicate to similar high titres in organs of 55-day-old immuno-incompetent porcine foetuses.

Quantitative changes of sialoadhesin and CD163 positive macrophages in the implantation sites and organs of porcine embryos/fetuses during gestation.

Porcine reproductive and respiratory syndrome virus (PRRSV) crosses the placenta most easily in the last third of gestation. Further, PRRSV does not replicate in preimplantation embryos but does replicate in postimplantation embryos and fetuses. In the present study, it was aimed to find an explanation for these observations by localization and quantification of the macrophages carrying two entry mediators that play a crucial role in PRRSV replication, sialoadhesin (Sn) and CD163, in the implantation sites and organs of embryos/fetuses during gestation. Uterus and embryos or organs (liver, spleen, lungs) from fetuses were obtained from pregnant PRRSV negative sows at different days of gestation (20-35, 50-60, 70-80, 114) and the Sn(+) and CD163(+) macrophages were quantified. In endometrium and placentas, two macrophage subsets were observed: Sn(-)CD163(+) and Sn(+)CD163(+). The highest number of Sn(+) and CD163(+) macrophages was counted at 114 days of gestation. In the mid-gestation fetal placentas (50-60 days of gestation), most CD163(+) macrophages were Sn negative. The number of Sn(+) and CD163(+) macrophages in organs increased during gestation. In the liver, the Sn(+) and CD163(+) macrophages were most abundant (Sn(+): 8.1-48.7%; CD163(+): 22.0-55.0%); the lowest number of Sn(+) and CD163(+) macrophages was observed in the lungs (Sn(+): 0-15.2%; CD163(+): 4.0-19.3%). Double immunofluorescence staining revealed three macrophage subsets in the spleen: Sn(+)CD163(-), Sn(-)CD163(+) and Sn(+)CD163(+); and two macrophage subsets in the lungs: Sn(-)CD163(+) and Sn(+)CD163(+). In the liver, due to physiological presence of biotin, the double immune-fluorescence staining could not be performed. The present results show clear changes in the quantity of Sn(+) and CD163(+) macrophages in the placentas and organs of embryos/fetuses during gestation which most probably have a physiological basis. The absence of Sn on macrophages in the fetal placenta at mid-gestation might explain the difficulty for PRRSV to spread transplacentally at this stage of gestation.