Isolation and Characterization of Porcine Epidemic Diarrhea Viruses Associated with the 2013 Disease Outbreak among Swine in the United States

Abstract

b 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 (TCID 50 )/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.

Porcine epidemic diarrhea virus: current insights

“…The PEDV growth in cell culture was reported to be difficult, and the virus may gradually lose infectivity upon further passages in cell culture. 41 Vero (African green monkey kidney) cells support the isolation and serial propagation of PEDV in laboratory conditions; another African green monkey kidney cell line, MARC-145, was also reported to support the cell passages of PEDV. 48 However, growth of PEDV depends on the presence of trypsin supplemented in the cell culture medium.…”

“…41,55 Phylogenetic analysis of the full-length S gene inferred by a neighbor-joining method indicates that PEDV could be genetically divided into two groups, which include GI and GII. GI and GII can be further divided into subgroups Ia and Ib, and IIa and IIb, respectively ( Figure 1).…”

“…[50][51][52] Recently, several highly virulent US PEDV strains were serially propagated in Vero cell cultures for over 30 passages. 41,53 Two Chinese variants, HN1303 and YN1, have also been serially propagated in Vero cell cultures for over 130 (unpublished data) and 200 passages, respectively. 1,54 Nonetheless, low success rates (0%-14.3%) of virus isolation from PEDV-positive intestine samples or feces have been recently reported by some research teams.…”

“…1,54 Nonetheless, low success rates (0%-14.3%) of virus isolation from PEDV-positive intestine samples or feces have been recently reported by some research teams. 1,41,53 Multiple factors may affect the successful rate of virus isolation, such as the type of specimen, freshness, virus titer, substances in intestinal contents, strain-specific factors, Vero cell lines, and culture conditions.…”

“…40 In addition, the S protein is suitably used for molecular epidemiology analysis and vaccine development of PEDV. 26,36,41 The M and E proteins play an important role in viral assembly; M protein, the most abundant component among viral proteins in the envelope, is required for the assembly process by interacting with the S and N proteins. 42,43 The N protein binds to the viral genome RNA and packages into the nucleocapsid of virus particles.…”