Fetal cytokine response to porcine reproductive and respiratory syndrome virus-2 infection
Introduction
Under normal conditions the developing fetus is protected from external threats by the placental barrier, which prevents the transfer of most microorganisms present in the dam. Although fetal sterility throughout pregnancy has been challenged by evidence of a low biomass commensal microflora, this concept remains controversial [1]. There is however, little disagreement with regards to the consequence of fetal infection with pathogenic organisms. This is particularly true in swine, where despite the significant barrier posed by epitheliochorial placentation, numerous viral pathogens are known to infect the fetus and cause significant mortality and economic loss [2], [3], [4]. While some of these pathogens, such as porcine parvovirus, can be easily controlled through effective vaccination programs [5] others remain a significant burden within the swine industry.
With minimal progress toward a broadly heterologous vaccine or any other effective long-term control mechanism, the impact of porcine reproductive and respiratory virus (PRRSV) on the pig fetus remains a significant challenge for the swine industry. Much of the difficulty encountered with this pathogen stems from its capacity for manipulating the host immune system, a topic that has been extensively studied both in vitro and in vivo. However, the overwhelming majority of this work has focused on post natal swine and, as a result, comparatively little is known about how the virus interacts with the fetal immune system. The late-gestation pig fetus has been shown to be capable of mounting an immune response to PRRSV infection [6], [7]. However, in spite of this immune response, viral strains such as NVSL 97-7985 and KS06-72109 which typically cause only minor morbidity in postnatal animals [8] lead to significant mortality among infected fetuses [9]. In addition, fetuses that do survive to parturition may be congenitally infected and are at greater neonatal susceptibility to other pathogens [10] suggesting that development of the immune system is substantially compromised and unable to clear infection following in utero infection. The mechanism by which PRRSV causes such exaggerated effects following fetal infection remains elusive. With little evidence of pathology in the form of lesions found during examination of fetal tissues [11], much of the work in the area has focused on the placenta. The severity of reproductive PRRS has in the past been associated with viral replication and its effects on the placenta [12], [13], however, more recent examination into the temporal progression of placental pathology following infection has cast doubt on this relationship [14].
In humans, fetal meconium staining, and in particular meconium aspiration, has been associated with the presence of placental lesions [15] as well as hypoxia and abnormal cardiac function [16] and more generally fetal distress. In the PRRSV infected fetus, meconium staining has been associated with higher viral load in both the thymus and serum [17], apoptotic cell numbers in the maternal-fetal interface [18], as well as the presence of fetal and umbilical lesions [14], and is generally thought to be an early indicator of fetal death. In the present study, we probed the relationship between meconium staining, as an indicator of disease progression and reduced viability, and select elements of the fetal immune response. We focused on the expression of cytokines in fetal thymus which is thought to be the primary site of PRRSV viral replication [11], as well in fetal spleen. We then compared this transcriptional response to the systemic accumulation of select cytokines. Finally, we examined expression of cyclin dependant kinases due to their ability to regulate the cell cycle and post-transcriptionally interfere in the production of Interferon beta.
Section snippets
Pregnant gilt challenge model
Fetal spleen, thymus and serum were obtained from a large-scale challenge experiment [19], for which highly detailed methodology has previously been published [20], [21]. In short, a total of 130 pregnant Landrace gilts were obtained from a PRRSV free nucleus herd at day 80 of gestation and housed in a level II containment. At day 85, 111 of these animals were inoculated with a total of 1 × 105 TCID50 of PRRSV-2 strain NVSL 97-7895 delivered 50/50 intramuscularly and intranasally, with 19
Thymic cytokine gene expression
To evaluate the fetal capacity to respond to PRRSV infection and determine if this response was associated with reduced fetal viability, the relative expression of eight cytokines in the fetal thymus was measured (Fig. 1), with the expectation that expression profiles of HV-VIA and HV-MEC would be distinct from each other as well as from UNIF and CON groups. The acute pro-inflammatory cytokine tumor necrosis factor alpha (TNF) was elevated in the HV-VIA group by 1.9 fold relative to CON but
Discussion
Although the host immune response to PRRSV infection has been extensively studied in the post-natal pig, the fetal response to this virus remains inadequately characterized. The present study is, to our knowledge, the first to evaluate the immune response across a variety of phenotypes representing resistant (UNIF), resilient (HV-VIA) and susceptible (HV-MEC) fetuses following maternal infection with PRRSV. Our results show a robust fetal immune response occurs among infected fetuses, as well
Conclusions
Collectively our results show that the late gestions pig fetus is capable of mounting a robust immune response following vertical transmission of PRRSV and find this response is highly compartmentalized, noting only minor increases in select cytokines within uninfected animals. Contrary to expectation, we find limited variation in the immune response associated with disease progression and reduced fetal viability as defined by meconium staining. The primary exception was the over expression of
Author’s contributions
JAP performed fetal selection, conducted the assays, analysis and drafted the manuscript. JCSH planned and led the large scale PRRSV infection trial. DJM and JCSH supervised experimental design. All authors read and revised the final manuscript.
CRediT authorship contribution statement
J. Alex Pasternak: Conceptualization, Methodology, Software, Formal analysis, Investigation, Data curation, Writing - original draft, Visualization. Daniel J. MacPhee: Conceptualization, Resources, Writing - review & editing, Supervision, Funding acquisition. John C.S. Harding: Conceptualization, Methodology, Validation, Resources, Data curation, Writing - review & editing, Supervision, Project administration, Funding acquisition.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
The samples used in this experiment were derived from a large scale multi-institutional challenge experiment involving numerous researchers and staff from the University of Saskatchewan and University of Alberta. The authors would like to collectively acknowledge this group, particularly former HQP responsible for phenotyping fetuses. In addition, we with to acknowledge Dr. Joan Lunney for her review of the manuscript and provision of material support including access to the anti-CCL2
References (50)
The interaction between PRRSV and the late gestation pig fetus
Virus Res.
(2010)- et al.
Pathogenicity of three type 2 porcine reproductive and respiratory syndrome virus strains in experimentally inoculated pregnant gilts
Virus Res.
(2015) - et al.
Distribution of porcine reproductive and respiratory syndrome virus in stillborn and liveborn piglets from experimentally infected sows
J. Comp. Pathol.
(2001) - et al.
Porcine reproductive and respiratory syndrome virus (PRRSV) causes apoptosis during its replication in fetal implantation sites
Microb. Pathog.
(2011) - et al.
Meconium aspiration syndrome: intrapartum and neonatal attributes
Am. J. Obstet. Gynecol.
(1989) - et al.
Novel insights into host responses and reproductive pathophysiology of porcine reproductive and respiratory syndrome caused by PRRSV-2
Vet. Microbiol.
(2017) - et al.
Classification of fetal resilience to porcine reproductive and respiratory syndrome (PRRS) based on temporal viral load in late gestation maternal tissues and fetuses
Virus Res.
(2019) - et al.
The chemokine CCL5 induces selective migration of bovine classical monocytes and drives their differentiation into LPS-hyporesponsive macrophages in vitro
Dev. Comp. Immunol.
(2014) - et al.
Hijacking and exploitation of IL-10 by intracellular pathogens
Trends Microbiol.
(2001) - et al.
Cytokine expression by macrophages in the lung of pigs infected with the porcine reproductive and respiratory syndrome virus
J. Comp. Pathol.
(2010)
Transient correlation between viremia levels and IL-10 expression in pigs subclinically infected with porcine circovirus type 2 (PCV2)
Res. Vet. Sci.
Differential production of proinflammatory cytokines in the pig lung during different respiratory virus infections: correlations with pathogenicity
Res. Vet. Sci.
Identification of a translation inhibitory element (TIE) in the 3’ untranslated region of the human interferon-beta mRNA
Gene
Multiple functions of p21 in cell cycle, apoptosis and transcriptional regulation after DNA damage
DNA Repair (Amst)
Interferon-alpha delays S-phase progression in human hepatocellular carcinoma cells via inhibition of specific cyclin-dependent kinases
Hepatology
In utero infection with porcine reproductive and respiratory syndrome virus modulates leukocyte subpopulations in peripheral blood and bronchoalveolar fluid of surviving piglets
Vet. Immunol. Immunopathol.
A critical assessment of the “sterile womb” and “in utero colonization” hypotheses: implications for research on the pioneer infant microbiome
Microbiome
Experimental infection of piglets and pregnant gilts with a parvovirus
Vet. Rec.
Reproductive disease and congenital malformations caused by Menangle virus in pigs
Aust. Vet. J.
Infectious agents identified in aborted swine fetuses in a high-density breeding area: a three-year study
J. Vet. Diagn. Invest.
Biology of porcine parvovirus (Ungulate parvovirus 1)
Viruses
Altered hippocampal gene expression and morphology in fetal piglets following maternal respiratory viral infection
Dev. Neurosci.
Genetic relationships of antibody response, viremia level and weight gain in pigs experimentally infected with porcine reproductive and respiratory syndrome virus
J. Anim. Sci.
In utero infection by porcine reproductive and respiratory syndrome virus is sufficient to increase susceptibility of piglets to challenge by Streptococcus suis type II
J. Virol.
Pathogenesis and prevention of placental and transplacental porcine reproductive and respiratory syndrome virus infection
Vet. Res.
Cited by (23)
Effects of ruminal lipopolysaccharide exposure on primary bovine ruminal epithelial cells
2024, Journal of Dairy ScienceDetection of PRRSV-2 alone and co-localized with CD163 positive macrophages in porcine placental areolae
2022, Veterinary Immunology and ImmunopathologyCitation Excerpt :Control fetuses (CON) were randomly selected from six non-infected gilts. Uninfected fetuses (UNINF) had no detectable virus in fetal placenta, serum and thymus despite originating from nine dams infected with PRRSV-2 (Pasternak et al., 2020). These represented the most resistant fetal phenotype.
Effect of porcine reproductive and respiratory syndrome virus 2 on tight junction gene expression at the maternal-fetal interface
2022, TheriogenologyCitation Excerpt :This 2 x 2 factorial selection resulted in the following four phenotypically distinct groups of 11 fetuses: CTRL-IUGR, CTRL-N-IUGR, HVL-IUGR, HVL-N-IUGR (Table 1). A second subset of fetuses with brain to liver Z-scores, calculated by litter, near as possible to the median value were selected (n = 11/group) based on a previously established experimental paradigm evaluating fetal resistance, resilience and susceptibility [28]. In short, resistant fetuses were defined as those with no detectable virus in the thymus (UNIF-THY), resilient fetuses were viable but showed high viral load (HVL-VIA) and susceptible as those with equivalently high viral loads but meconium staining in the body (HVL-MEC-B).
Effects of lipopolysaccharide exposure in primary bovine ruminal epithelial cells
2020, Journal of Dairy ScienceCitation Excerpt :The cycle threshold (Ct) values of the genes of interest were normalized to the geometric mean of the 3 housekeeping genes. Changes in gene expression were calculated as fold change using the formula 2−ΔΔCt (Pasternak et al., 2020). Expression of treatments was held relative to the mean of the time-matched medium control within animal.
Molecular and Cellular Mechanisms for PRRSV Pathogenesis and Host Response to Infection
2020, Virus ResearchCitation Excerpt :Similarly, HP-PRRSV generates high levels of inflammatory cytokines including IL-1, IL-6 and TNF-α in peripheral blood (Li et al., 2017), indicating that HP-PRRSV may aggravate inflammation and damage tissues and organs. In addition, in pregnant gilts that are challenged on 85 days of gestation and euthanized 21 days postinfection, cytokine gene expressions are significantly upregulated in the thymus and spleen of the fetuses (Pasternak et al., 2020). PRRSV also upregulates cytokine in PAMs (Qiao et al., 2011) and microglia (Chen et al., 2014a).