Differentiation of a Vero cell adapted porcine epidemic diarrhea virus from Korean field strains by restriction fragment length polymorphism analysis of ORF 3.
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.
Song
D S
DS
Department of Veterinary Medicine Virology Lab, College of Veterinary Medicine, Seoul National University, Seoul 151-742, Republic of Korea.
Yang
J S
JS
Oh
J S
JS
Han
J H
JH
Park
B K
BK
D000818:Animals; D001483:Base Sequence; D002522:Chlorocebus aethiops; D005260:Female; D007723:Korea; D051379:Mice; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D019318:Porcine Reproductive and Respiratory Syndrome / Q000235:genetics* / Q000276:immunology; D019316:Porcine respiratory and reproductive syndrome virus / Q000235:genetics* / Q000254:growth & development / Q000276:immunology / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D013552:Swine