A double deletion prevents replication of the pestivirus bovine viral diarrhea virus in the placenta of pregnant heifers

Abstract

In contrast to wild type bovine viral diarhea virus (BVDV) specific double deletion mutants are not able to establish persistent infection upon infection of a pregnant heifer. Our data shows that this finding results from a defect in transfer of the virus from the mother animal to the fetus. Pregnant heifers were inoculated with such a double deletion mutant or the parental wild type virus and slaughtered pairwise on days 6, 9, 10 and 13 post infection. Viral RNA was detected via qRT-PCR and RNAscope analyses in maternal tissues for both viruses from day 6 p.i. on. However, the double deletion mutant was not detected in placenta and was only found in samples from animals infected with the wild type virus. Similarly, high levels of wild type viral RNA were present in fetal tissues whereas the genome of the double deletion mutant was not detected supporting the hypothesis of a specific inhibition of mutant virus replication in the placenta. We compared the induction of gene expression upon infection of placenta derived cell lines with wild type and mutant virus via gene array analysis. Genes important for the innate immune response were strongly upregulated by the mutant virus compared to the wild type in caruncle epithelial cells that establish the cell layer on the maternal side at the maternal–fetal interface in the placenta. Also, trophoblasts which can be found on the fetal side of the interface showed significant induction of gene expression upon infection with the mutant virus although with lower complexity. Growth curves recorded in both cell lines revealed a general reduction of virus replication in caruncular epithelial cells compared to the trophoblasts. Compared to the wild type virus this effect was dramtic for the mutant virus that reached only a TCID50 of 1.0 at 72 hours post infection. Author summary Here we report on animal studies elucidating mechanisms preventing the transfer of a double deletion mutant of a pestivirus to the fetus in pregnant heifers. This mutant lacks both known factors engaged in blocking the innate immune response to pestiviral infection. As shown also in earlier studies, this mutant was not detected in the fetuses at any of the tested time points in contrast to the wild-type (wt) virus. However, similar to the wt the mutant was detected in a large variety of different maternal tissues. The only exception was the placenta where only wt but not mutant virus was detected. Using gene array analyses we showed that infection of two cell lines derived either from the maternal or the fetal site of the maternal-fetal interface with the mutant virus induces a significant antiviral gene expression response. The reaction of cells from the maternal side was more complex and virus replication in these cells was reduced, almost completly blocking the mutant virus. These results support the hypothesis that replication of the mutant virus is blocked in the placenta due to a highly active innate immune response and the prevention of replication also blocks transfer of the virus to the fetus.