Full-Length Genome Sequence of a Plaque-Cloned Virulent Porcine Epidemic Diarrhea Virus Isolate (USA/Iowa/18984/2013) from a Midwestern U.S. Swine Herd

Porcine epidemic diarrhea (PED) was recognized in U.S. swine for the first time in early 2013. A plaque-purified PED virus (PEDV) isolate (USA/Iowa/18984/2013) was obtained from a diarrheic piglet. The isolate is genetically close to other previously reported U.S. PEDVs and recent Chinese PEDVs and was virulent when inoculated into neonatal pigs.

Virus isolation was attempted from field samples submitted to the Iowa State University Veterinary Diagnostic Laboratory that were positive for PEDV by a real-time reverse transcriptase PCR (RT-PCR) assay (13) using Vero cells (ATCC CCL-81), as previously described (14). A cytopathic virus with coronavirus morphology was isolated from intestinal tissues collected from a 1.5week-old piglet from a swine farm in Iowa. In the cells that were inoculated with the virus, the virus induced syncytia and eventually cell death. The presence of PEDV in the inoculated cells was confirmed by immunofluorescence microscopy using a PEDVspecific monoclonal antibody, 6C8 (12). After 3 cell culture passages, the primary isolate was subjected to plaque purification. A plaque-cloned virus isolate was further propagated in the cells for 2 more passages, for a total of 6 cell passages (P6).
The plaque-cloned PEDV P6 isolate (USA/Iowa/18984/2013) was able to cause cytopathic effects at 12 to 18 h postinoculation (hpi) and reach a titer of 10 5.5 PFU/ml within 48 hpi. Caesareanborn colostrum-deprived piglets orally inoculated with the isolate at a rate of 10 3 PFU/ml developed severe watery diarrhea and dehydration within 24 hpi and eventually died. Microscopically, intensive immunohistochemical staining of almost all enterocytes for PEDV was observed at 18 hpi, leading to severe villous atrophy at a later time.
In conclusion, the PEDV isolate USA/Iowa/18984/2013 is a virulent strain with a genetic profile similar to those of other U.S. PEDVs reported to date. Such a representative purified virulent PEDV isolate can be a valuable reagent for studying the pathogenesis and immunobiology of PEDV and developing diagnostic reagents and kits, as well as effective vaccines.
Nucleotide sequence accession number. The complete genome sequence of PEDV strain USA/Iowa/18984/2013 was submitted to GenBank under the accession no. KF804028.

ACKNOWLEDGMENTS
We are grateful to practicing swine veterinarians for submitting samples from suspect cases to the Iowa State University Veterinary Diagnostic Laboratory and procuring additional samples. We also thank Drew Magstadt (Veterinary Diagnostic Laboratory, Iowa State University, Ames, IA) and John Neil (Ruminant Diseases and Immunology Research Unit, National Animal Disease Center, USDA/ARS, Ames, IA) for assistance in animal work and laboratory support, respectively.
The study was supported in part by funding from the Iowa Pork Producers Association (NPB no. .