Pathology of US Porcine Epidemic Diarrhea Virus Strain PC21A in Gnotobiotic Pigs

Kwonil Jung, Qiuhong Wang1

, Kelly A. Scheuer, Zhongyan Lu, Yan Zhang, and Linda J. Saif1

Author affiliations: The Ohio State University, Wooster, Ohio, USA (K. Jung, Q. Wang, K.A. Scheuer, Z. Lu, L.J. Saif); Ohio Department of Agriculture, Reynoldsburg, Ohio, USA (Y. Zhang)

Abstract

To understand the progression of porcine epidemic diarrhea virus infection, we inoculated gnotobiotic pigs with a newly emerged US strain, PC21A, of the virus. At 24–48 hours postinoculation, the pigs exhibited severe diarrhea and vomiting, fecal shedding, viremia, and severe atrophic enteritis. These findings confirm that strain PC21A is highly enteropathogenic.

A highly contagious coronavirus that causes porcine epidemic diarrhea (PED) was first reported in the United States in May 2013 in Iowa. Since then, the virus—porcine epidemic diarrhea virus (PEDV)—has spread rapidly nationwide (1,2). PEDV (family Coronaviridae, genusAlphacoronavirus) was previously reported only in Europe and Asia. The first US outbreaks caused a high number of deaths among suckling pigs and, as a consequence, substantial economic losses (1,2).

Results of PEDV pathogenesis studies using the prototype European PEDV strain, CV777, were reported in the 1980s (3,4). Strain CV777 infections caused intestinal villous atrophy with substantially reduced ratios of villous height to crypt depth (VH:CD) (3,4). Pathogenic features of CV777 are similar to those observed for Asian PEDV strains that circulated in the 1990s (4–6). To understand the progression of PEDV infection, we studied the pathogenesis of the newly emerged US strain, PC21A.

The Study

Figure 1

Thumbnail of Electron micrograph of a US porcine epidemic diarrhea virus (PEDV) particle detected in a field fecal sample collected during a 2013 outbreak of PED on a farm in Ohio, USA; the fecal sample from which PEDV strain PC21A in this study was detected was from a pig on the same farm during the same outbreak. The sample was negatively stained with 3% phosphotungstic acid. Scale bar = 50 nm.

Figure 1. . Electron micrograph of a US porcine epidemic diarrhea virus (PEDV) particle detected in a field fecal sample collected during a 2013 outbreak of PED on a farm in Ohio, USA;...

In June 2013, intestinal contents were obtained from a 1-day-old pig with diarrhea on a farm in Ohio, USA. PEDV strain PC21A was detected in the sample by reverse transcription PCR (RT-PCR) selective for the nucleocapsid gene (229–557 nt). The partial nucleocapsid gene sequence of PC21A was identical to that of 2 US PEDV outbreak strains from Colorado, USA: USA/Colorado/2013 (GenBank accession no. KF272920) and 13-019349 (GenBank accession no. KF267450). Only coronavirus-like particles were observed in the fecal sample by electron microscopy (Figure 1). The sample was negative for rotavirus groups A and C and for transmissible gastroenteritis virus/porcine respiratory coronavirus by RT-PCR (7,8).

The sample was bacteriologically sterilized by using 0.22-μm syringe filters and then prepared as inoculum. Near-term gnotobiotic pigs were delivered aseptically by hysterectomy from a specific pathogen–free sow (9). Six 10- to 35-day-old pigs were randomly assigned to a PEDV-infected group (pigs 1–5) or a negative control group (pig 6). Information about inoculation and inocula pig-passage number is described in Table 1. Pigs 1–3 and 5 were inoculated orally and/or intranasally with 6.3–9.0 log₁₀ genomic equivalents (GE) of PEDV strain PC21A; pig 4 was exposed to the virus by indirect contact with inoculated pig 3. For each sample, the quantity of PEDV RNA GE was ≈106 times higher than plaque assay results for a cell-adapted PEDV strain, PC22A. Clinical signs were monitored hourly. Pig 4 was monitored for longer-term clinical signs and virus shedding. Pigs were euthanized for pathologic examination at 3 stages of infection: acute, mid, and later stages (<24 h, 24–48 h, and >48 h, respectively, after onset of clinical signs). The Ohio State University Institutional Animal Care and Use Committee approved all animal-related experimental protocols.

Fecal or rectal swab samples were prepared as described (9). Virus RNA was extracted by using the MagMAX Viral RNA Isolation Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. Titers of virus shed in feces were determined by TaqMan real-time RT-PCR using the OneStep RT-PCR Kit (QIAGEN, Valencia, CA, USA) as reported (10), with modifications in the forward primer and probe to provide a 100% match to the US strains: forward 5′-CGCAAAGACTGAACCCACTAAC-3′ and probe FAM-TGYYACCAYYACCACGACTCCTGC-BHQ. A standard curve was generated by using the PCR amplicon (PEDN 229/557) of strain PC21A. The detection limit was 10 GE per reaction, corresponding to 4.8 log₁₀ and 3.8 log₁₀ GE/mL of fecal and serum samples, respectively.

Small and large intestine tissues, lung, liver, heart, kidney, spleen, and mesenteric lymph node were examined grossly and histologically. Mean jejunal VH:CD was measured by using PAX-it software (PAXcam, Villa Park, IL, USA) as described (11). The frozen tissues were prepared and tested by immunofluorescence staining, as described (12), for the detection of PEDV antigen, using monoclonal antibody 6C8-1 against the spike protein of PEDV strain DR13 (provided by Daesub Song, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea).