Uladzimir Karniychuk

Porcine reproductive and respiratory syndrome virus (PRRSV) causes apoptosis during its replication in fetal implantation sites

Reproductive failure due to porcine reproductive and respiratory syndrome virus (PRRSV) is characterized by late-term abortions, early farrowing and an increase of dead and mummified fetuses and weak-born piglets. The mechanism of PRRSV-induced reproductive failure is poorly understood. Human pregnancies, complicated by some pathogens leading to reproductive disorders exhibit increased apoptosis in the fetal membranes. Because PRRSV-target cells are present in endometrium/fetal placentas from healthy sows and PRRSV-infected macrophages in other organs die by apoptosis, we hypothesized that PRRSV can replicate and induce apoptosis in the fetal implantation sites at the last stage of gestation. In the present study, identification, localization and quantification of the PRRSV-positive and apoptotic cells were performed in the fetal implantation sites. Three dams were inoculated intranasally with 10(5) TCID(50) PRRSV 07V063 at 90 days of gestation and sampled at 10 days post-inoculation. Two non-inoculated dams that were euthanized at 100 days of gestation served as control animals. Inoculation of the dams resulted in a viremia that lasted until the end of the study. Transplacental PRRSV spread was detected in all inoculated dams. Using immunofluorescence staining, single PRRSV-positive cells were found in the endometrial connective tissues adjacent to both PRRSV-positive and PRRSV-negative fetuses. In the fetal placental mesenchyme of the PRRSV-positive fetuses, infected cells were more abundant and spread focally. Double staining showed that all PRRSV-positive cells in the fetal implantation sites were positive for sialoadhesin and CD163. Apoptotic cells (TUNEL+) were detected in endometrium and fetal placentas of both non- and PRRSV-inoculated dams. The number of apoptotic cells was significantly higher in PRRSV-positive endometrium/fetal placentas. PRRSV caused apoptosis in infected cells since 20-61% of PRRSV-positive cells were apoptotic and in surrounding cells since 43-91% of the apoptotic cells were virus-negative. The main conclusion obtained from the present study is that PRRSV replicates in the fetal implantation sites and causes apoptosis in infected macrophages and surrounding cells at the last stage of gestation. The possible mode of PRRSV replication in the fetal implantation sites and the events that might contribute to the reproductive disorders are discussed.

Comparison of the efficacy of autogenous inactivated Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) vaccines with that of commercial vaccines against homologous and heterologous challenges

BackgroundThe porcine reproductive and respiratory syndrome virus (PRRSV) is a rapidly evolving pathogen of swine. At present, there is a high demand for safe and more effective vaccines that can be adapted regularly to emerging virus variants. A recent study showed that, by the use of a controlled inactivation procedure, an experimental BEI-inactivated PRRSV vaccine can be developed that offers partial protection against homologous challenge with the prototype strain LV. At present, it is however not known if this vaccine can be adapted to currently circulating virus variants. In this study, two recent PRRSV field isolates (07 V063 and 08 V194) were used for BEI-inactivated vaccine production. The main objective of this study was to assess the efficacy of these experimental BEI-inactivated vaccines against homologous and heterologous challenge and to compare it with an experimental LV-based BEI-inactivated vaccine and commercial inactivated and attenuated vaccines. In addition, the induction of challenge virus-specific (neutralizing) antibodies by the different vaccines was assessed.ResultsIn a first experiment (challenge with 07 V063), vaccination with the experimental homologous (07 V063) inactivated vaccine shortened the viremic phase upon challenge with approximately 2 weeks compared to the mock-vaccinated control group. Vaccination with the commercial attenuated vaccines reduced the duration of viremia with approximately one week compared to the mock-vaccinated control group. In contrast, the experimental heterologous (LV) inactivated vaccine and the commercial inactivated vaccine did not influence viremia. Interestingly, both the homologous and the heterologous experimental inactivated vaccine induced 07 V063-specific neutralizing antibodies upon vaccination, while the commercial inactivated and attenuated vaccines failed to do so.In the second experiment (challenge with 08 V194), use of the experimental homologous (08 V194) inactivated vaccine shortened viremia upon challenge with approximately 3 weeks compared to the mock-vaccinated control group. Similar results were obtained with the commercial attenuated vaccine. The experimental heterologous (07 V063 and LV) inactivated vaccines did not significantly alter viremia. In this experiment, 08 V194-specific neutralizing antibodies were induced by the experimental homologous and heterologous inactivated vaccines and a faster appearance post challenge was observed with the commercial attenuated vaccine.ConclusionsThe experimental homologous inactivated vaccines significantly shortened viremia upon challenge. Despite the concerns regarding the efficacy of the commercial attenuated vaccines used on the farms where the field isolates were obtained, use of commercial attenuated vaccines clearly shortened the viremic phase upon challenge. In contrast, the experimental heterologous inactivated vaccines and the commercial inactivated vaccine had no or only a limited influence on viremia. The observation that homologous BEI-inactivated vaccines can provide a more or less standardized, predictable degree of protection against a specific virus variant suggests that such vaccines may prove useful in case virus variants emerge that escape the immunity induced by the attenuated vaccines.

Pathogenesis and prevention of placental and transplacental porcine reproductive and respiratory syndrome virus infection

Porcine reproductive and respiratory syndrome virus (PRRSV)-induced reproductive problems are characterized by embryonic death, late-term abortions, early farrowing and increase in number of dead and mummified fetuses, and weak-born piglets. The virus recovery from fetal tissues illustrates transplacental infection, but despite many studies on the subject, the means by which PRRSV spreads from mother to fetus and the exact pathophysiological basis of the virus-induced reproductive failure remain unexplained. Recent findings from our group indicate that the endometrium and placenta are involved in the PRRSV passage from mother to fetus and that virus replication in the endometrial/placental tissues can be the actual reason for fetal death. The main purpose of this review is to clarify the role that PRRSV replication and PRRSV-induced changes in the endometrium/placenta play in the pathogenesis of PRRSV-induced reproductive failure in pregnant sows. In addition, strategies to control placental and transplacental PRRSV infection are discussed.

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.

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.

Antibody response and maternal immunity upon boosting PRRSV-immune sows with experimental farm-specific and commercial PRRSV vaccines

The porcine reproductive and respiratory syndrome virus (PRRSV) causes reproductive failure in sows and respiratory disease in pigs of all ages. Despite the frequent use of vaccines to maintain PRRSV immunity in sows, little is known on how the currently used vaccines affect the immunity against currently circulating and genetically divergent PRRSV variants in PRRSV-immune sows, i.e. sows that have a pre-existing PRRSV-specific immunity due to previous infection with or vaccination against the virus. Therefore, this study aimed to assess the capacity of commercially available attenuated/inactivated PRRSV vaccines and autogenous inactivated PRRSV vaccines - prepared according to a previously optimized in-house protocol - to boost the antibody immunity against currently circulating PRRSV variants in PRRSV-immune sows. PRRSV isolates were obtained from 3 different swine herds experiencing PRRSV-related problems, despite regular vaccination of gilts and sows against the virus. In a first part of the study, the PRRSV-specific antibody response upon booster vaccination with commercial PRRSV vaccines and inactivated farm-specific PRRSV vaccines was evaluated in PRRSV-immune, non-pregnant replacement sows from the 3 herds. A boost in virus-neutralizing antibodies against the farm-specific isolate was observed in all sow groups vaccinated with the corresponding farm-specific inactivated vaccines. Use of the commercial attenuated EU type vaccine boosted neutralizing antibodies against the farm-specific isolate in sows derived from 2 farms, while use of the commercial attenuated NA type vaccine did not boost farm-specific virus-neutralizing antibodies in any of the sow groups. Interestingly, the commercial inactivated EU type vaccine boosted farm-specific virus-neutralizing antibodies in sows from 1 farm. In the second part of the study, a field trial was performed at one of the farms to evaluate the booster effect of an inactivated farm-specific vaccine and a commercial attenuated EU-type vaccine in immune sows at 60 days of gestation. The impact of this vaccination on maternal immunity and on the PRRSV infection pattern in piglets during their first weeks of life was evaluated. Upon vaccination with the farm-specific inactivated vaccine, a significant increase in farm-specific virus-neutralizing antibodies was detected in all sows. Virus-neutralizing antibodies were also transferred to the piglets via colostrum and were detectable in the serum of these animals until 5 weeks after parturition. In contrast, not all sows vaccinated with the commercial attenuated vaccine showed an increase in farm-specific virus-neutralizing antibodies and the piglets of this group generally had lower virus-neutralizing antibody titers. Interestingly, the number of viremic animals (i.e. animals that have infectious virus in their bloodstream) was significantly lower among piglets of both vaccinated groups than among piglets of mock-vaccinated sows and this at least until 9 weeks after parturition. The results of this study indicate that inactivated farm-specific PRRSV vaccines and commercial attenuated vaccines can be useful tools to boost PRRSV-specific (humoral) immunity in sows and reduce viremia in weaned piglets.

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.

Demonstration of microchimerism in pregnant sows and effects of congenital PRRSV infection

The presence of foreign cells within the tissue/circulation of an individual is described as microchimerism. The main purpose of the present investigation was to study if microchimerism occurs in healthy sows/fetuses and if porcine reproductive and respiratory syndrome virus (PRRSV) infection influences this phenomenon. Six dams were inoculated intranasally with PRRSV and three non-inoculated dams served as controls. Male DNA was detected in female fetal sera of all dams via PCR. Male DNA was also detected in the maternal circulation. Sex-typing FISH showed the presence of male cells in the female fetal organs and vice versa. PRRSV infection did not influence microchimerism, but might misuse maternal and sibling microchimeric cells to enter fetuses.

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.

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.