Isolation and characterization of porcine epidemic diarrhea viruses associated with the 2013 disease outbreak among swine in the United States

Abstract

Porcine epidemic diarrhea virus (PEDV) was detected in May 2013 for the first time in U.S. swine and has since caused significant economic loss. Obtaining a U.S. PEDV isolate that can grow efficiently in cell culture is critical for investigating pathogenesis and developing diagnostic assays and for vaccine development. An additional objective was to determine which gene(s) of PEDV is most suitable for studying the genetic relatedness of the virus. Here we describe two PEDV isolates (ISU13-19338E and ISU13-22038) successfully obtained from the small intestines of piglets from sow farms in Indiana and Iowa, respectively. The two isolates have been serially propagated in cell culture for over 30 passages and were characterized for the first 10 passages. Virus production in cell culture was confirmed by PEDV-specific real-time reverse-transcription PCR (RT-PCR), immunofluorescence assays, and electron microscopy. The infectious titers of the viruses during the first 10 passages ranged from 6 × 10(2) to 2 × 10(5) 50% tissue culture infective doses (TCID50)/ml. In addition, the full-length genome sequences of six viruses (ISU13-19338E homogenate, P3, and P9; ISU13-22038 homogenate, P3, and P9) were determined. Genetically, the two PEDV isolates were relatively stable during the first 10 passages in cell culture. Sequences were also compared to those of 4 additional U.S. PEDV strains and 23 non-U.S. strains. All U.S. PEDV strains were genetically closely related to each other (≥99.7% nucleotide identity) and were most genetically similar to Chinese strains reported in 2011 to 2012. Phylogenetic analyses using different genes of PEDV suggested that the full-length spike gene or the S1 portion is appropriate for sequencing to study the genetic relatedness of these viruses.

FIG 1

Cytopathic effects and IFAs of PEDV isolates in infected Vero cells. Vero cells were infected with PEDV 13-19338E P9 and PEDV 13-22038 P9 isolates. At 24 h postinfection, cytopathic effects were recorded (A, B, and C; ×160 magnification) and cells were examined by IFA using PEDV-specific monoclonal antibody (D, E, and F; ×100 magnification).

FIG 2

EM images on original homogenate and PEDV-infected Vero cells. (A) Negatively stained PEDV particles in the ISU13-22038 intestine homogenates. Some virus particles are shown by arrows. Crown-shaped spikes are visible. Bar = 100 nm. Magnification, ×150,000. (B) Thin section of Vero cells infected with ISU13-22038 P3 PEDV at 24 h postinfection. Bar = 500 nm. Magnification, ×11,000. (C) Enlarged image of the partial cytoplasm membrane showing accumulation of released virus particles. Bar = 100 nm. Magnification, ×68,000.

FIG 3

Alignment of partial ORF1b nucleotide sequences of 33 PEDV strains with entire genome sequences available. A single-nucleotide insertion between positions 20,204 and 20,205 of some PEDV strains is shown. The amino acid codon immediately before the insertion is shown as underlined.

FIG 4

Phylogenetic analysis of the full-length genome, S gene, S1 portion, S2 portion, ORF3, E, M, and N gene nucleotide sequences of 6 U.S. PEDV from this study and 27 previously published PEDV sequences (GenBank numbers are shown in the figure). The trees were constructed using the distance-based neighbor-joining method of the software MEGA5.2. Bootstrap analysis was carried out on 1,000 replicate data sets, and values are indicated adjacent to the branching points. The viruses identified in this study are indicated by diamonds. Bar, 0.002 nucleotide substitutions per site.

FIG 4

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