Development of PCR-based techniques to identify porcine transmissible gastroenteritis coronavirus isolates.
Sixteen isolates of transmissible gastroenteritis virus and one isolate of porcine respiratory coronavirus were characterized using RT-PCR amplification of 4 antigenic subsites in the site A epitope on the TGEV spike gene. The PCR products were digested with restriction enzymes Sau3AI and SspI and the sizes of the fragments were determined. Three different digestion patterns were observed with each enzyme. The recognition site for Sau3AI was missing in 1 isolate, was present in 13 isolates and 3 isolates had 2 sites. PCR-products with a single site had 3 different fragment sizes and the other isolates produced 2 fragments with different sizes. The SspI recognition site was not present in 5 isolates and 12 isolates had a single site that produced 2 fragments of different sizes. Based on the restriction fragment sizes, the 17 isolates were separated into 7 groups. Direct sequencing of the 455 bp nested set fragments demonstrated greater than 96% sequence homology among the 16 isolates and 100% homology in the 4 antigenic subsites in the conserved site A epitope. The groups are discussed in relation to their sequence homology and virulence. In vitro procedures have been developed to identify several porcine enteric coronavirus isolates at the strain level.
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Virology Swine Research Unit, National Animal Disease Center, USDA, Agricultural Research Service, Ames, Iowa 50010, USA.
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017934:Coronavirus / Q000235:genetics / Q000302:isolation & purification; D018352:Coronavirus Infections / Q000175:diagnosis / Q000662:veterinary; D053938:DNA Fragmentation; D004262:DNA Restriction Enzymes; D004279:DNA, Viral / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D004587:Electrophoresis, Agar Gel / Q000379:methods / Q000662:veterinary; D000939:Epitopes / Q000235:genetics; D005761:Gastroenteritis, Transmissible, of Swine / Q000175:diagnosis; D009500:Neutralization Tests / Q000662:veterinary; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D011863:Radioimmunoassay / Q000379:methods / Q000662:veterinary; D012689:Sequence Homology, Nucleic Acid; D013552:Swine; D013553:Swine Diseases / Q000175:diagnosis; D005760:Transmissible gastroenteritis virus / Q000235:genetics* / Q000302:isolation & purification*
D004279:DNA, Viral; D000939:Epitopes; D004262:DNA Restriction Enzymes