Effect of Porcine Epidemic Diarrhea Virus Infectious Doses on Infection Outcomes in Naïve Conventional Neonatal and Weaned Pigs

Thomas, Joseph T.; Chen, Qi; Gauger, Phillip C.; Giménez-Lirola, Luis G.; Sinha, Avanti; Harmon, Karen M.; Madson, Darin M.; Burrough, Eric R.; Magstadt, Drew R.; Salzbrenner, Holly M.; Welch, Michael W.; Yoon, Kyoung-Jin; Zimmerman, Jeffrey J.; Zhang, Jianqiang

A PEDV nucleocapsid (N) gene-based real-time RT-PCR was previously developed at the Iowa State University Veterinary Diagnostic Laboratory (ISU VDL) [24, 25] . On the basis of the PEDV N gene-based rRT-PCR, a quantitative rRT-PCR to determine genomic copies/ml of PEDV in a sample was developed in this study. To generate in vitro transcribed RNA standards, a fragment covering the PEDV N genebased RT-PCR products (nucleotide positions 26,679-26,885 of the PEDV USA/IN19338/2013, GenBank # KF650371) flanked by restriction sites EcoRI and HindIII at its 5' and 3' end, respectively, was cloned into the plasmid vector pIDTBlue to obtain the plasmid pIDTBlue:PED-V_N_IVT (IDT, Coralville, Iowa, USA) in which the PEDV N gene products were located downstream of the bacteriophage T7 RNA polymerase promoter. The recombinant plasmids were transformed into One Shot 1 TOP10 chemically competent Escherichia coli cells (Thermo Fisher Scientific) and propagated following the instruction manual. The plasmids were extracted using a QIAprep 1 Spin Miniprep Kit (Qiagen, Valencia, California, USA) according to the manufacturer's instructions. The plasmid DNA was linearized with HindIII, treated with Proteinase K, purified with QIAquick 1 PCR purification kit (Qiagen), and resuspended in nuclease-free water. The linearized DNA was subject to run-off in vitro transcription into RNA using a MEGAshortscriptâ„¢ T7 Transcription Kit (Thermo Fisher Scientific) followed by purification using a MEGAclearâ„¢ Transcription Clean-up Kit (Thermo Fisher Scientific) according to the instruction manuals provided with the kit. The in vitro transcribed RNA was quantified using a BioSpectrometer (Eppendorf, Enfield, Connecticut, USA).