Origin, Evolution, and Virulence of Porcine Deltacoronaviruses in the United States

Ma, Yuanmei; Zhang, Yu; Liang, Xueya; Lou, Fangfei; Oglesbee, Michael; Krakowka, Steven; Li, Jianrong

PdCV-positive specimens. In early January 2014, an outbreak of TGEVand PEDV-like disease was reported in several pig farms in Ohio; mortality rates ranged from 40 to 80%. Intestinal contents and feces were collected from a 7-day-old piglet with severe diarrhea and vomiting. Five grams of feces and intestine contents was homogenized in 50 ml of Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA). The suspension was centrifuged at 5,000 ϫ g for 10 min at 4°C. The supernatant was collected and filtered through 0.45-m and 0.22-mpore-size filters. The filtered intestinal contents were negative for any bacteria or fungal growth when they were cultured on LB plates and medium in both anaerobic and aerophilic conditions. RNA extraction, RT-PCR, and deep sequencing. Total RNA was extracted from feces and intestinal contents using an RNeasy minikit (Qiagen, Valencia, CA). Primers were designed to target the N gene based on the sequence of the U.S. PEDV Colorado strain (accession number KF272920), the conserved regions of the Miller M6 and M60, Purdue P115, and virulent Purdue TGEVs (accession numbers DQ811785, DQ811786, DQ811788, and DQ811789, respectively), PRCV (accession number DQ811787), and PdCV Hong Kong strain HKU15-44 (accession number JQ065042). Primers were also designed to target the VP1 gene of porcine rotavirus types A and C, porcine norovirus, and porcine sapovirus. RT-PCR was performed using a one-step RT-PCR kit (Qiagen), and the PCR products were visualized in 1% agarose gels.