Differentiation of a Vero cell adapted porcine epidemic diarrhea virus from Korean field strains by restriction fragment length polymorphism analysis of ORF 3

Abstract

A porcine epidemic diarrhea virus (PEDV) designated DR13 was isolated in Vero cells and serially passaged by level 100. The virus was titrated at regular intervals of the passage level. Open reading frame (ORF) 3 sequences of the virus at passage levels 20, 40, 60, 80, and 100 were aligned and compared using a computer software program. Suitability of the restriction fragment length polymorphism (RFLP) analysis for differentiating the virus from other Korean field strains was investigated. The DR13 field isolate was successively adapted in Vero cells as observed through polymerase chain reaction (PCR) and titration of the virus. RFLP analysis identified change in cleavage sites of HindIII and Xho II from passage levels 75 and 90, respectively; these RFLP patterns of ORF 3 differentiated the Vero cell-adapted virus from its parent strain, DR13, and 12 other strains of PEDV studied. The cell adapted DR13 was tested for its pathogenicity and immunogenicity in piglets and pregnant sows. The results indicated that cell adapted DR13 revealed reduced pathogenicity and induced protective immune response in pigs. Differentiation between highly Vero cell-adapted virus and wild-type virus could be the marker of adaptation to cell culture and a valuable tool for epidemiologic studies of PEDV infections. The results of this study supported that the cell attenuated virus could be applied as a marker vaccine candidate against PEDV infection.

Fig. 1

Virus titers of porcine epidemic diarrhea virus, DR13, on the course of Vero cell passage up to passage level 100.

Fig. 2

Agarose gel electrophoresis of PCR products on the S gene of porcine epidemic diarrhea virus. From left to right: lane M, 100 bp DNA ladder; lane 1, KPEDV-9; lane 2, negative control; lanes 3–8, passage levels 0, 1, 30, 60, 90, and 100, respectively.

Fig. 3

Agarose gel electrophoresis of PCR products on the ORF 3 gene of porcine epidemic diarrhea virus. From left to right: lane M, 100 bp DNA ladder; lane 1, KPEDV-9; lane 2, negative control; lanes 3–11, passage levels 0, 1, 30, 60, 70, 75, 85, 90, and 100, respectively.

Fig. 4

Sequence alignments of ORF 3 gene of porcine epidemic diarrhea virus, CV777, parent DR13, and cell-passaged DR13 at passage levels 20, 40, 60, 80, and 100.

Fig. 4

Sequence alignments of ORF 3 gene of porcine epidemic diarrhea virus, CV777, parent DR13, and cell-passaged DR13 at passage levels 20, 40, 60, 80, and 100.

Fig. 4

Sequence alignments of ORF 3 gene of porcine epidemic diarrhea virus, CV777, parent DR13, and cell-passaged DR13 at passage levels 20, 40, 60, 80, and 100.

Fig. 5

RFLP patterns of porcine epidemic diarrhea virus, DR13, on the ORF 3 gene PCR products digested with restriction enzyme HindIII. From left to right: lane M, 100 bp DNA ladder; lane 1, KPEDV-9; lane 2, negative control; lanes 3–10, passage levels 0, 1, 30, 60, 70, 75, 85, and 100, respectively.

Fig. 6

RFLP patterns of porcine epidemic diarrhea virus, DR13, on the ORF 3 gene PCR products digested with restriction enzyme Xho II. From left to right: lane M, 100 bp DNA ladder; lane 1, KPEDV-9; lane 2, negative control; lanes 3–11, passage levels 0, 1, 30, 60, 70, 75, 85, 90, and 100, respectively.

Fig. 7

RFLP patterns of 12 isolates of porcine epidemic diarrhea virus on the ORF 3 gene PCR products digested with restriction enzyme HindIII. From left to right: lane M, 100 bp DNA ladder; lane 1, DR13 at passage level 100; lane 2, parent DR13; lane 3, negative control; lanes 4–15, PEDV isolates DR11, B409, DR183, B640, DR195, GC2, B719, 1101, KJY1, KJY2, B720, and DH1, respectively.

Fig. 8

RFLP patterns of 12 isolates of porcine epidemic diarrhea virus on the ORF 3 gene PCR products digested with restriction enzyme Xho II. From left to right: lane M, 100 bp DNA ladder; lane 1, DR13 at passage level 100; lane 2, parent DR13; lane 3, negative control; lanes 4–15, PEDV isolates DR11, B409, DR183, B640, DR195, GC2, B719, 1101, KJY1, KJY2, B720, and DH1, respectively.

Fig. 9

Immune response of cell adapted DR13 and parent DR13 in 14-day-old piglets.