Use of an internal control in a quantitative RT-PCR assay for quantitation of porcine epidemic diarrhea virus shedding in pigs.
Porcine epidemic diarrhea virus (PEDV), a member of the family Coronaviridae, has caused a devastating enteric disease in the Korean swine industry. Previously, the differences between virulent field PEDV strains and a Vero cell culture adapted PEDV DR13 strain were determined using restriction fragment length polymorphism analysis (RFLP), and PEDV shedding patterns in pigs were reported. In an extension to these studies, an internal control was constructed and quantitative analysis of virus shedding after oral inoculation was established. A parent field PEDV and a cell culture adapted PEDV DR13 were inoculated orally to colostrum-deprived 1-day-old piglets, commercial 2-week-old pigs, and sows (1-5 ml dose, 10(5.8)-10(6.0) TCID(50)/0.1 ml). PEDV shedding was monitored every day and virus levels were measured using a quantitative reverse transcriptase polymerase chain reaction (RT-PCR) method. In fecal samples from experimentally-inoculated pigs, the level of virus excreted peaked at 2 days after oral inoculation and gradually decreased thereafter. In addition, PEDV from field specimens was quantified using the same RT-PCR assay to determine shedding viral load. This suggests that measurement of PEDV shedding viral load in pigs, by quantitative RT-PCR, may be a useful tool for estimating the transmission potential of PEDV in the swine population.
Song
Dae-Sub
DS
Research Unit, Green Cross Veterinary Products, Yong-In 449-903, Korea.
Kang
Bo-Kyu
BK
Lee
Sang-Sun
SS
Yang
Jeong-Sun
JS
Moon
Hyoung-Joon
HJ
Oh
Jin-Sik
JS
Ha
Gun-Woo
GW
Jang
Yong-Suk
YS
Park
Bong-Kyun
BK
D000818:Animals; D002522:Chlorocebus aethiops; D003332:Coronaviridae / Q000235:genetics / Q000302:isolation & purification*; D003333:Coronaviridae Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D018076:DNA, Complementary; D005243:Feces / Q000821:virology; D005761:Gastroenteritis, Transmissible, of Swine / Q000175:diagnosis / Q000235:genetics / Q000821:virology*; D012150:Polymorphism, Restriction Fragment Length; D012367:RNA, Viral / Q000032:analysis* / Q000235:genetics; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary*; D013552:Swine; D013553:Swine Diseases / Q000175:diagnosis / Q000821:virology; D014709:Vero Cells; D019562:Viral Load; D017201:Virus Shedding
D018076:DNA, Complementary; D012367:RNA, Viral