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

Abstract

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.

Porcine reproductive and respiratory virus 2 infection of the fetus results in multi-organ cell cycle suppression

“…This study has shown, for the first time, widespread disruption in the cell cycle regulation in fetal tissues following late gestation maternal PRRSV challenge. As with many of our previous studies [ 6 , 15 , 21 , 27 ], the observed changes in gene expression were entirely restricted to highly infected fetuses. This apparent compartmentalization further demonstrates that the pathophysiological impact of PRRSV on the fetus is not a secondary effect of maternal infection, but rather the direct result of fetal infection.…”

“…Volume or tissue weight normalized PRRSV RNA concentration (target copies/µL or mg) was assessed in fetal serum and thymus as extensively described elsewhere [ 5 , 21 ]. In short, total RNA was extracted and viral RNA quantified using primer and probe sequences targeting the distal region of ORF7 and a one-step qPCR assay including a standard curve comprised of linearized plasmid.…”