Transfer of Anti-Rotavirus Antibodies during Pregnancy and in Milk Following Maternal Vaccination with a Herpes Simplex Virus Type-1 Amplicon Vector

Meier, Anita F.; Suter, Mark; Schraner, Elisabeth M.; Humbel, Bruno M.; Tobler, Kurt; Ackermann, Mathias; Laimbacher, Andrea S.

Quantitative real-time polymerase chain reaction (RT-qPCR) was performed directly from serum samples. The serum samples were 1:5 diluted in 1× PBS and heated for 3 min at 97 • C, cooled immediately on wet ice for 5 min and used directly for the RT-qPCR reaction. The Path-ID Multiplex One-Step RT-PCR Kit (Applied Biosystems, Thermo Fisher Scientific) was used for the amplification. As primer probe, we used the VetMax Swine Enteric Panel Reagent (Applied Biosystems, Thermo Fisher Scientific), a primer probe mix for detection of porcine rotavirus A-besides a primer probe for porcine rotavirus A, this kit contains also primer probes for transmissible gastroenteritis coronavirus (TGEV) and porcine epidemic diarrhea virus (PEDV)-. This primer probe mix was selected because it was the most sensitive kit. The reaction was run in a thermal cycler (CFX96 C1000 Touch, BioRad, Hercules, CA, USA) using the following settings: reverse transcription for 10 min at 48 • C followed by an inactivation/initial denaturation step of 10 min at 95 • C. Amplification was performed as 40 cycles of 15 s at 95 • C and 45 s at 60 • C. Reactions were done in duplicates. For calculation of virus dose, a standard curve was drawn using serial dilution of EDIM virus diluted in negative mouse serum and treated as described above in triplicates. Based on the resulting formula of the determined standard curve (ln(DD50) = −0.6419 × Cq + 16.565), DD 50 was calculated for each sample. Considering the applied volume of the analyzed samples, DD 50 /mL was determined (DD 50 /0.0016 mL).