Fetal hypoxia and apoptosis following maternal porcine reproductive and respiratory syndrome virus (PRRSV) infection.

BACKGROUND: Mechanisms of fetal death following maternal PRRSV2 infection remain uncharacterized, although hypoxia from umbilical cord lesions and/or placental detachment due to apoptosis are hypothesized. We performed two experiments examining hypoxia and apoptosis in PRRSV-infected and non-infected, third-trimester fetuses to elucidate possible associations with fetal death. Fetuses were selected based on four phenotypic infection groups: fetuses from non-challenged control gilts (CTRL); low viral load fetuses (LVL; Exp 1) or uninfected fetuses (UNINF; Exp 2) from inoculated gilts; viable high viral load fetuses (HVL-VIA); and HVL meconium-stained fetuses (HVL-MEC). RESULTS: In experiment 1, paraffin embedded fetal tissues collected 21 days post maternal infection (DPI) were examined for DNA fragmentation associated with apoptosis. Positively stained foci were larger and more numerous (P < 0.05) in heart, liver, and thymus of HVL-VIA and HVL-MEC compared to CTRL and LVL fetuses. In experiment 2, group differences in gene expression within the hypoxia (HIF1a, IDO1, VEGFa, LDHA, NOS2, NOX1) and apoptosis (CASP3, CASP7, CASP8, CASP9, RIPK1, RIPK3) pathways were assessed by RT-qPCR in fetal tissues collected at 12 DPI. High viral load fetuses showed differential expression relative to the CTRL and UNINF (P < 0.05 for all). Brain tissue from HVL-VIA and HVL-MEC fetuses presented increased expression of CASP7, CASP8, RIPK3, HIF1a and IDO1. Fetal heart showed increased expression of CASP8, HIF1a, IDO and NOX1 and a decrease in NOS2 expression in infected groups. CASP7, CASP9, RIPK1 and RIPK3 were only increased in the heart of HVL-VIA while VEGFa was only increased for HVL-MEC fetuses. Thymus from HVL-MEC had decreased expression of CASP9 and there was increased IDO1 in all infected fetuses. CONCLUSIONS: There is strong evidence of apoptosis occurring in the heart, liver and thymus of highly viral load fetuses at 21 DPI. Furthermore, there was clear upregulation of apoptotic genes in the heart of high viral load infected fetuses and less prominent upregulation in the brain of PRRSV-infected fetuses, whereas thymus appears to be spared at 12 DPI. There was no strong evidence of hypoxia at 12 DPI in brain and thymus but some indication of hypoxia occurring in fetal heart.

Samples sizes required to accurately quantify viral load and histologic lesion severity at the maternal-fetal interface of PRRSV-inoculated pregnant gilts.

Porcine reproductive and respiratory syndrome virus (PRRSV) is transmitted vertically, causing fetal death in late gestation. Spatiotemporal distribution of virus at the maternal-fetal interface (MFI) is variable, and accurate assessment of viral concentration and lesions is thus subject to sampling error. Our objectives were: 1) to assess whether viral load and lesion severity in a single sample of endometrium (END) and placenta (PLC), collected near the base of the umbilical cord (the current standard), are representative of the entire organ; and 2) to compare sampling strategies and evaluate if spatial variation in viral load can be overcome by pooling of like-tissues. Spatially distinct pieces of END and PLC of 24 fetuses from PRRSV-2-infected dams were collected. PRRSV RNA quantified by RT-qPCR was compared in 5 individual pieces per fetus and in respective pools of tissue and extracted RNA. Three distinct pieces of MFI were assessed for histologic severity. Concordance correlation and kappa inter-rater agreement were used to characterize agreement among individual samples and pools. The viral load of individual samples and pools of END had greater concordance to a referent standard than did samples of PLC. Larger pool sizes had greater concordance than smaller pool sizes. Average viral load and lesion severity did not differ by location sampled, and no technical advantages of pooling tissues versus RNA extracts were found. We conclude that multiple pieces of MFI tissues must be evaluated to accurately assess lesion severity and viral load. Three pieces per fetus provided a reasonable balance of cost and logistic feasibility.

Fetal Metabolomic Alterations Following Porcine Reproductive and Respiratory Syndrome Virus Infection.

PRRSV infection in third-trimester pregnant sows can lead to fetal death and abortions, although the mechanisms triggering these effects are not well understood. Since resistant and susceptible fetuses can coexist in the same litter, we propose that there may be differential mechanisms used by some fetuses to evade infection and/or disease progression. Our objectives were to investigate possible differences in the metabolome of PRRSV-infected and non-infected fetuses, as well as the interaction of altered intrauterine growth development and PRRSV infection to elucidate possible causes of fetal death following PRRSV infection. Near-term serum samples collected from fetuses on gestation day 106, 21 days post PRRSV-2 infection, were processed by direct flow injection mass spectrometry (DI-MS) and nuclear magnetic resonance (NMR) techniques. Experiment one investigated disease progression with 24 fetuses selected from each of four phenotypic groups: fetuses from non-inoculated gilts (CTRL); fetuses from inoculated gilts that escaped infection (UNINF); infected high viral load viable fetuses (INF); and infected high viral load meconium-stained fetuses (MEC). Experiment two investigated the interaction of intrauterine growth retardation (IUGR) and PRRSV infection by analyzing differences among: non-infected normal development (CON-N); CON-IUGR; PRRS infected normal development (PRRS-N); and PRRS-IUGR. Univariate and multivariate (PCA, PLS-DA) statistics determined group differences among various contrasts, and the most important metabolites associated with disease progression and fetal development. Significant differences in the metabolome were observed, especially between PRRSV-negative fetuses (CTRL and UNINF) and MEC fetuses, while INF fetuses appear to span both groups. The two metabolites with highest variable importance in projection (VIP) scores related to disease progression were alpha-aminoadipic acid (alpha-AAA) and kynurenine (KYN), having the highest concentration in MEC and INF fetuses, respectively, compared to CTRL and UNINF. In experiment two, non-IUGR fetuses were found to have increased levels of lysoPCs, PCs and amino acids compared to IUGR fetuses, while the near complete absence of lysoPCs and PCs in IUGR fetuses, even during infection, indicate a distinctive response to infection compared to non-growth retarded fetuses. Possible markers of PRRSV fetal susceptibility, such as alpha-AAA, kynurenine and lysoPCs, are presented and discussed.

Classification of fetal resilience to porcine reproductive and respiratory syndrome (PRRS) based on temporal viral load in late gestation maternal tissues and fetuses.

Although porcine reproductive and respiratory syndrome virus (PRRSV) readily crosses the maternal fetal interface (MFI) in third trimester, fetal resilience varies within litters. The aim of this study was to characterize PRRSV-2 concentration in MFI and fetuses at five time points after experimental inoculation of late gestation gilts and use this information to classify potentially resistant, resilient and susceptible fetuses. The secondary objective was to verify the relationship between PRRS viral load and intrauterine growth retardation (IUGR). Three PRRSV-inoculated pregnant gilts and 1 sham-inoculated control were euthanized at five time points in days post infection (DPI; 2, 5, 8, 12, 14). The preservation status of each fetus was determined and MFI samples adjacent to the umbilical stump of each fetus, as well as serum, thymus, umbilical cord and amniotic fluid were collected. Viral load was quantified using probe-based reverse-transcriptase quantitative PCR (RT-qPCR) targeting PRRSV NVSL 97-7895 ORF7. Our result show the MFI was largely PRRSV infected by 2 DPI and virus was first detected in fetal sera and umbilical cord by 5 DPI, and in fetal thymus and amniotic fluid by 8 DPI. This indicates that PRRSV-2 quickly crossed the placenta and traveled toward the fetus via umbilical circulation within one week of the dam's inoculation. Fetal compromise was first observed on 8 DPI and increased progressively through to 14 DPI. However, several factors were associated with fetal resilience. The random forest model identified that 'viral load in fetal thymus' and duration of infection ('DPI') as the most important factors predicting fetal resilience and resistance. Moreover, IUGR fetuses had lower viral load and were less frequently compromised or dead compared to non-IUGR and average cohorts. Understanding the mechanisms of fetal resilience to PRRSV will improve selection strategies for replacement gilts.